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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-2-591619120410.1186/1742-4690-2-59ResearchAmino acid residues that are important for Hyal2 function as a receptor for jaagsiekte sheep retrovirus Duh Fuh-Mei [email protected] Clarissa [email protected] Michael I [email protected] A Dusty [email protected] Basic Research Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA2 Laboratory of Immunobiology, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA3 Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA4 Current address: University of Washington, Seattle, Washington 98195, USA2005 28 9 2005 2 59 59 1 9 2005 28 9 2005 Copyright © 2005 Duh et al; licensee BioMed Central Ltd.2005Duh 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 by jaagsiekte sheep retrovirus (JSRV) and by enzootic nasal tumor virus (ENTV) depends on cell-surface expression of the virus entry receptor, hyaluronidase 2 (Hyal2). Human Hyal2 binds the envelope (Env) proteins of these viruses and is functional as a receptor, but Hyal2 from mice does not bind Env nor does it mediate entry of either virus. Here we have explored the amino acid determinants that account for the difference in receptor function.
Results
Analysis of human-mouse Hyal2 chimeric proteins showed that amino acid differences responsible for the difference in Hyal2 receptor activity were localized to the central third of Hyal2. Human Hyal2 mutants containing single or double amino acid replacements with the respective mouse amino acids were generated across this region and were assayed for activity. None of the single or double mutation reduced the receptor activity of human Hyal2 by more than 10-fold, whereas mouse Hyal2 activity is reduced 1,000-fold from that of human Hyal2. While the 3-dimensional structures of mammalian Hyal2 proteins are unknown, bee venom hyaluronidase shows significant amino acid similarity to human and mouse Hyal2 and its structure has been determined. Many mutations having the largest negative effects on human Hyal2 function mapped to a small region of the bee venom hyaluronidase close to but not overlapping the active site of the enzyme, suggesting that this site represents the binding site for Env. Analysis of synonymous and non-synonymous nucleotide substitutions in the coding sequences of multiple mammalian Hyal2 proteins shows that the proteins are undergoing strong selection for amino acid conservation. We found no evidence for positive selection of amino acid changes that might reflect evolution of mammalian hosts to resist JSRV or ENTV infection.
Conclusion
These results show that the greatly reduced receptor activity of mouse Hyal2 in comparison to that of human Hyal2 is determined by multiple amino acid changes acting in concert. In particular, no one amino acid change blocks infection. However, the most important amino acids map to a small patch on a predicted 3-dimensional Hyal2 structure, which may represent the binding site for Env.
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Background
JSRV and ENTV are closely-related retroviruses that induce tumors in the lower airways and nasal epithelium, respectively, of sheep and goats [1]. Both viruses utilize the glycosylphosphatidylinositol-anchored cell-surface protein Hyal2 as a receptor for cell entry [2]. Hyal2 is a member of a family of proteins, some of which exhibit high hyaluronidase activity and are capable of rapid degradation of hyaluronan, a component of the extracellular matrix. However, Hyal2 exhibits only weak hyaluronidase activity [3] and its primary biological role in mammals is not known. Evidence for Hyal2 function as the virus receptor is provided by experiments showing that expression of sheep or human Hyal2 in cells that are not normally susceptible to infection renders the cells fully infectable by retroviral vectors bearing the JSRV or ENTV Env proteins [4-7]. For example, the titers of JSRV and ENTV vectors on mouse cells expressing human Hyal2 are >1,000-fold higher than those on cells expressing mouse Hyal2. In addition, the receptor-binding surface (SU) domains of the JSRV and ENTV envelope proteins bind with high affinity to cells expressing human Hyal2 but not to cells expressing nonfunctional receptors such as mouse Hyal2 [6,8,9], indicating that Hyal2 is the primary receptor that binds virus prior to entry.
Here we have used a transient transfection assay to analyze the amino acid differences between human and mouse Hyal2 that account for the large difference in receptor activity of the two proteins. Non-susceptible NIH 3T3 mouse cells were transfected with the Hyal2 expression constructs and receptor function was quantitated by measuring transduction of the cells with a JSRV vector. We could localize most of the difference in receptor function to the central 38% of the Hyal2 proteins. Single and double amino acid changes made throughout this region in human Hyal2 to convert the residues to those in the mouse sequence were made and tested. None of the changes reduced human Hyal2 activity more than 10-fold, but a combination of three of the most important mutations reduced the activity by 17-fold. We conclude that the difference in receptor activity between human and mouse Hyal2 is explained by the effects of multiple amino acid changes acting together. However, the most important amino acids mapped to a small surface region of the known bee venom hyaluronidase suggesting that this is the binding site for Env.
Results
Deletions of the amino or carboxy termini of human Hyal2 abrogate receptor activity
The overall structure of human Hyal2 is shown in Fig. 1. The protein is directed to the endoplasmic reticulum by a 20 amino-acid signal peptide at the amino terminus [3], deletion of which abolishes the receptor activity of human Hyal2 [5]. However, this signal can be replaced with a preprotrypsin signal sequence followed by a Flag peptide tag without affecting the receptor activity of human Hyal2 [5]. To determine if portions of the amino terminus beyond the signal sequence could be deleted without affecting receptor activity of human Hyal2, we made a series of deletion mutants that consisted of the preprotrypsin signal sequence followed by the Flag tag fused in frame to the remainder of the protein. Deletion of amino acids 1–30 of human Hyal2 (corresponding to a deletion of 10 amino acids after the signal peptide) reduced its activity to 1% of that of human Hyal2, and deletions of 40, 50, 60, 120, 180, 240, and 300 amino-terminal amino acids reduced receptor activity to undetectable levels (data not shown). Loss of receptor activity could be due to an inability of the JSRV Env to bind the truncated receptors, to defects in later steps of virus entry mediated by Hyal2, or to improper folding or processing of the deletion mutant Hyal2 proteins to the cell surface. Regardless, these data show that most of the amino terminus of Hyal2 is required for proper receptor function, with the possible exception that a subset of the first 10 amino acids after the signal peptide may be dispensable for receptor function.
Figure 1 Human Hyal2 protein features. Amino acids 1–20 constitute the endoplasmic reticulum signal peptide for human Hyal2 [3]. A glycosylphosphatidylinositol (GPI) anchor is predicted to replace all residues following amino acid 447 in human Hyal2, which contains a C-terminal hydrophobic tail at residues 456–473 that localizes the protein in the membrane prior to GPI anchor addition.
The carboxy end of Hyal2 is predicted to contain a GPI addition signal, and experimental data confirms the presence of the GPI linkage of Hyal2 to the cell surface [5]. Complete removal of the GPI addition signal (amino acids 440–473) or only the GPI addition site (amino acids 440–453) abrogated the receptor activity of human Hyal2 [5]. We have not explored whether internal deletions upstream of the GPI signal are compatible with receptor function or whether other membrane linkages might yield functional receptors, but it is clear that the carboxy end of Hyal2 is important for normal receptor function.
Analysis of chimeric mouse/human Hyal2 proteins localizes amino acids responsible for the difference in receptor activity to the central region of the proteins
Mouse and human Hyal2 are 82% identical at the amino acid level but mouse Hyal2 shows 1,000-fold lower JSRV receptor activity. In an attempt to localize the amino acids responsible for this difference we made a series of human/mouse Hyal2 chimeras and assayed them for receptor function (Fig. 2). Hyal2 was divided into four domains based on the availability of convenient restriction enzyme sites in the cDNAs at positions corresponding to amino acids 158, 305, and 354. Constructs are named based on the order of the mouse and human sequences. Construct hmmh has very low activity while construct mhhm has activity near that of human Hyal2, showing that the central regions of mouse and human Hyal2 are responsible for most of their receptor phenotype. Constructs hmhh and hhmh have activity between that of mouse and human Hyal2 showing that both the second and third domains of mouse Hyal2 contribute to the low activity of mouse Hyal2. Constructs mhmm and mmhm show that insertion of the second domain of human Hyal2 can restore most of the activity of mouse Hyal2, while insertion of the third domain of human Hyal2 is unable to increase the low activity of mouse Hyal2. In conclusion, differences between mouse and human Hyal2 in the central regions of these proteins are primarily responsible for large difference in JSRV receptor activity of the two proteins.
Figure 2 Receptor activity of mouse/human receptor chimeras. Scale drawings of human (open boxes), C3H mouse (black boxes) and chimeric Hyal2 receptors are shown. Receptor activities following transfection of NIH 3T3 mouse cells are indicated at right and are expressed as a percentage of that of human Hyal2. The top diagram shows the positions of the signal peptide (amino acids 1–20), the GPI anchor addition site (after amino acid 447), and the amino acid positions where the Hyal2 cDNAs were recombined. The human Hyal2 [GenBank:U09577.1] and C3H mouse Hyal2 [GenBank:AF302843.1] sequences used here have been described. The Hyal2 protein sequence of C3H mice is identical to that of NIH Swiss mice [GenBank:AF535140.1], and differs by one amino acid (V at position 355) from that of Czech II mice [GenBank:AF302844.1] (I at position 355).
Analysis of human Hyal2 mutants containing single and double mouse amino acid substitutions shows cooperative effects of the changes
To identify the individual mouse amino acid changes that account for the decreased activity of mouse Hyal2, we made human Hyal2 mutants containing single and double mouse amino acid substitutions throughout the critical central domain defined above (Fig. 3). Most changes had little effect on receptor activity, but one in the second domain (E189N, 44% activity) and two in the third domain (A322S, 11% activity; L327F, 19% activity) had relatively low activities. Importantly, no one amino acid replacement reduced the activity of human Hyal2 by more than 10-fold, whereas mouse Hyal2 activity is reduced 1,000-fold from that of human Hyal2. We also made a human Hyal2 mutant that contained all three of these mutations, and this mutant had 6% of the activity of human Hyal2 (average of two experiments, data not shown). We conclude from analysis of these human Hyal2 mutants and from the mouse/human chimeric Hyal2 results that the low JSRV receptor activity of mouse Hyal2 is due to the combined action of multiple amino acid changes acting in concert.
Figure 3 Alignment of Hyal2 protein sequences from different species and effect on receptor function of conversion of amino acid residues in human Hyal2 to those present in mouse Hyal2. ClustalW [23] was used to generate an alignment of ovine [GenBank:AF411974.1], human [GenBank:U09577.1], C3H mouse [GenBank:AF302843.1], and rat [GenBank:AF535141.1] Hyal2 protein sequences. Ovine Hyal2 has a three amino acid insertion following amino acid 56 that is not present in the other Hyal2 proteins. Amino acid identity (*), strong similarity (:), weaker similarity (.), and dissimilarity (no mark) are indicated below the sequences. Downward arrows indicate positions of junctions between chimeric human/mouse Hyal2 chimeras, and dots above the sequences indicate 10 base-pair intervals. Residues that differ between human and mouse Hyal2 in the region between amino acids 158 and 354 are indicated in red, and the receptor activities (% of human Hyal2 activity) of human Hyal2 proteins containing the single or double replacements with the respective mouse amino acids are shown above the amino acid residues. Where two amino acids were altered, a bracket depicts the residues that were altered.
Interestingly, the mutation R301G increased the receptor activity of human Hyal2 by 65%. A glycine is present at this position in both the mouse and the sheep receptor. The sheep receptor functions better as a JSRV receptor than does the human receptor in previous assays [6], suggesting that this glycine is important to achieve the highest receptor activity.
We also made several changes to mouse Hyal2 to see if its activity could be increased by replacement of mouse amino acids with the corresponding amino acids from human Hyal2. Based on the results above, we made the mutations F327L, N189E, and both mutations together in mouse Hyal2. All of these mutants had receptor activities (0.2% of human Hyal2, means of 2 experiments for each construct) similar to mouse Hyal2 (0.1%). The result for the N189E mutation is particularly interesting since replacement of the second domain of mouse Hyal2 with that of human Hyal2 restores nearly full receptor activity (construct mhmm in Fig. 2), and none of the human Hyal2 mutants containing single and double replacements with the corresponding mouse amino acids significantly reduced human Hyal2 activity besides the E189N replacement (Fig. 3). Again, these results indicate that alteration of receptor activity is due to multiple amino acid changes acting in concert.
Mouse Hyal2 accumulates at the cell surface at levels at least as high as those of human Hyal2
We considered the possibility that the low JSRV receptor activity of mouse Hyal2 was simply due to poor processing of mouse Hyal2 to the cell surface in comparison to human Hyal2. To test this possibility, we generated stable cell lines containing plasmids that encoded Flag-tagged human or mouse Hyal2, or a plasmid that did not contain a eukaryotic expression cassette. The plasmids were introduced into cells by cotransfection with a plasmid encoding neomycin phosphotransferase (Neo) and by selection of the cells in G418. FACS analysis using an anti-Flag monoclonal primary antibody and a fluorescently-labeled anti mouse IgG secondary antibody showed increased Flag levels on the cells containing the Flag-tagged mouse and human plasmids in comparison to the control plasmid, with the highest levels of expression on the cells expressing the Flag-tagged mouse Hyal2 (Fig. 4). We conclude that the reason that mouse Hyal2 does not act as a receptor for JSRV is not because of poor processing of the mouse protein to the cell surface.
Figure 4 Cell-surface mouse Hyal2 expression levels are at least as high as those of human Hyal2 following expression plasmid transfection. Plasmids encoding amino-terminal Flag-tagged mouse or human Hyal2, or an empty plasmid as a control, were cotransfected with pSV2neo at a 10:1 ratio into NIH 3T3 cells. One day after transfection the cells were treated with trypsin and seeded in medium containing G418. Two weeks later, cell-surface Flag-tagged protein expression was measured by FACS after incubation of the cells with an anti-Flag mouse monoclonal primary antibody followed by a goat anti-mouse IgG secondary antibody. Green indicates cells transfected with control plasmid, red indicates cells transfected with Flag-tagged human Hyal2, and orange indicates cells transfected with Flag-tagged mouse Hyal2. Control analyses included Flag-tagged human Hyal2-transfected cells that were not incubated with antibody (purple) or with secondary antibody only (blue). The percentages of cells in the indicated gate were determined and are shown in the inset. The entire experiment was repeated and the repeat values are shown in parentheses.
Mapping of human Hyal2 amino acids important for receptor function to the bee venom hyaluronidase crystal structure indicates a potential site for Hyal2/Env interaction
The receptor activities of human, rat and mouse Hyal2 proteins (high, low and none, respectively) correlate with their relative abilities to bind JSRV Env [8]. Assuming that Hyal2 receptor activity is primarily determined by its ability to bind Env, the positions of mutations in Hyal2 that affect receptor activity might define a binding site for Env on Hyal2. None of the 3-dimensional structures of mammalian hyaluronidase proteins have been determined, but the sequence of the hyaluronidase found in bee venom (Hya) is quite similar to those of the mammalian Hyal2 proteins (Fig. 5), and its crystal structure has been solved [10]. In an attempt to define a potential binding site for JSRV Env on Hyal2, we used the amino acid alignment shown in Fig. 5 to map the amino acid residues in human Hyal2 that are important for receptor activity to the crystal structure of bee venom hyaluronidase (Fig. 6). This crystal structure includes a hyaluronic acid tetramer (stick structure) which lies in a groove containing the enzyme active site (colored blue) (Fig. 6, middle structure).
Figure 5 Amino acid alignment of human Hyal2, C3H mouse Hyal2, and bee venom Hya. Proteins were aligned by using ClustalW [23]. The sequence of the mature protein is shown for Hya [10], while amino acids 1 – 377 of the precursor forms of the Hyal2 proteins are shown. Amino acid residues colored red indicate the positions at which alteration of human to mouse Hyal2 amino acids have the largest effects on the receptor activity of human Hyal2. White letters indicate amino acids shared between at least two sequences, and black or red letters indicate unique amino acids at a given position. Amino acid identity (*), strong similarity (:), weaker similarity (.), and dissimilarity (no mark) are indicated below the sequences, numbers above the sequences refer to amino acid position in the Hyal2 sequences, and dots indicate 10 base-pair intervals in the Hyal2 sequences. Downward arrows indicate positions of junctions in the chimeric human/mouse Hyal2 chimeras.
Figure 6 Mapping of amino acid residues that are important for the receptor function of human Hyal2 to the bee venom Hya structure. Amino acids of human Hyal2 were mapped to the bee venom Hya protein structure [PDB:1FCV]. Positions at which alteration of the human Hyal2 sequence to that of the mouse resulted in a decrease in receptor activity are colored red, those that resulted in an increase in receptor function are colored green. The hyaluronic acid tetramer that was present in the crystal structure and the active site of the enzyme (blue) can be seen in the middle structure. Protein structure images were generated by using PyMOL [24].
Of the seven human Hyal2 amino acids that when mutated to the mouse sequence showed the largest negative effects (colored red in Fig. 6), 4 mapped to one surface patch on Hyal2 (E307, M308, D345, and R349) (Fig. 6, left structure), two mapped under this patch (A322 and L327) (Fig. 6, right structure), and one mapped to a distant surface site (E189) (Fig. 6, right structure). Two of the residues in the surface patch are on the same loop of a helix (E307 and M308) while the other two are on adjacent loops of another helix (D345 and R349). The L327F mutation in human Hyal2 that results in a 5-fold reduction in Hyal2 receptor activity lies just underneath this surface patch with the side chain of this amino acid pointing toward the patch, and the increased size of phenylalanine compared to leucine might cause movement of the 2 helices that compose the surface patch resulting in loss of receptor activity. It is more difficult to explain the 9-fold reduction of Hyal2 receptor activity caused by the A322S mutation, but this amino acid also maps close to the surface patch and may cause distortion of the patch. This residue is unlikely to be involved directly in Env binding, even though it has a surface localization, because additional amino acids at the N- and C- termini of Hyal2 that cannot be mapped to the Hya structural model would likely interfere with Env binding to this amino acid residue (see Fig. 6, right structure).
The human Hyal2 amino acid that when mutated to the mouse sequence showed the largest positive effect (R301, colored green in Fig. 6, middle structure) maps to the surface near the active site of Hya and could interact with Env, while E189 is distant from the proposed binding patch (Fig. 6, right structure). Thus a plausible binding site for Env that involves many of the important amino acid residues in human Hyal2 is predicted by this analysis.
Comparison of mammalian Hyal2 sequences show strong pressure to conserve the protein sequence but no evidence for selective pressure to avoid JSRV infection
Viruses bearing the JSRV Env protein can infect cells from multiple mammalian species, and we considered the possibility that there might be selection during the evolution of mammals for Hyal2 variants that cannot act as receptors for these viruses. Evidence for such selection has been found for other proteins involved in virus defense, including APOBEC3G and TRIM5α [11,12]. The location of positively selected amino acid variations might provide additional data regarding the Env binding site on Hyal2. In particular, we previously found that bovine Hyal2 acts as a weak receptor for JSRV and ENTV compared to sheep or human Hyal2, but bovine Hyal2 is much more closely related to sheep Hyal2 than to human Hyal2, suggesting that there might be specific selection in cattle for resistance to transmission of JSRV or ENTV from sheep and goats [6].
Positive selection for alteration in protein function can be detected by measuring the rate of non-synonymous (Ka) versus synonymous (Ks) nucleotide changes in DNA during species evolution. Ka/Ks = 1 indicates a lack of selection for or against the protein sequence, Ka/Ks < 1 indicates negative selection, or selection for amino acid conservation, and Ka/Ks > 1 indicates positive selection for protein alteration. We examined the Ka/Ks ratios for pairwise combinations of Hyal2 proteins from sheep, cattle, pig, dog, human, mouse, and rat (see Additional files 1, 2, 3 for DNA sequences used and amino acid alignment) by using the K-estimator 6.1 program created by JM Comeron [13]. The overall Ka/Ks values ranged from 0.11 to 0.23, indicating strong selection for amino acid conservation in Hyal2 proteins from these species. We also analyzed Ka/Ks ratios using a sliding window approach to detect particular regions of Hyal2 that might be under positive (diversifying) selection, but again found no evidence for such selection (data not shown).
Strong selection for amino acid conservation can mask small numbers of amino acids undergoing positive selection for altered protein function, so we looked more closely for amino acid selection at the single amino acid level. Analysis for selection at individual amino acid sites using the Single Likelihood Ancestor Counting (SLAC) program [14-16] identified 48 negatively-selected (conserved) sites and no positively-selected sites at the 0.1 level of significance. Further analysis using the Random Effects (REL) program [14-16], that provides a less conservative analysis for selection at individual amino acid sites, found 35 negatively-selected (conserved) sites and no positively-selected sites at the default program significance setting of 50. Together these results provide strong evidence for conservation of Hyal2 protein sequence but no evidence for positive selection to resist virus infection.
Discussion
We have found that the difference in activity of human and mouse Hyal2 as entry receptors for viruses bearing the JSRV Env are determined by multiple amino acid differences between the two proteins. This is in contrast to results of a similar analysis of the activity of human and mouse CAT1 as entry receptors for ecotropic murine leukemia viruses. In the latter case, mouse CAT1 is active while human CAT1 is inactive as a receptor. Substitution of only one amino acid in the mouse receptor with the corresponding amino acid from the human receptor was enough to abrogate receptor activity, and substitution of 2 amino acid residues in human CAT1 with those of mouse CAT1 could convert human CAT1 into a functional receptor [17]. In other examples, amino acid changes resulting in receptor glycosylation were found to be responsible for the lack of receptor activity in receptor orthologs from cells that were resistant to virus infection [18,19]. In the case of mouse and human Hyal2 there are no predicted N- or O-linked glycosylation sites in the middle region of these proteins that we found was responsible for the difference in receptor activity, indicating that the difference in receptor activity is not due to a difference in glycosylation.
We used amino-terminal Flag tags to show that the mouse and human Hyal2 proteins were both processed to the cell surface. Levels of mouse Hyal2 were somewhat higher than those of human Hyal2, indicating that the lack of mouse Hyal2 receptor activity was not due to poor processing of the protein to the cell surface. We did not perform this analysis for the mouse/human Hyal2 chimeras or for the mutant Hyal2 proteins, and some proportion of reduced receptor activity we detected in some constructs could be due to improper folding and or processing of the proteins to the cell surface. We attempted to measure cell-surface processing of mouse and human Hyal2 at 2 days after Hyal2 plasmid transfection, at the same time we assay for JSRV vector transduction, but the cell-surface Flag signal was too low to allow reliable quantitation. Selection for cells expressing the transfected plasmid was required for Flag detection, and even then the signal was heterogeneous (Fig. 4). In retrospect, a better way to perform these studies might have been to Flag-tag all of the Hyal2 constructs and insert these cDNAs into a retroviral vector, to generate virus using packaging cell lines, to transduce the target cells at a multiplicity of infection such that each cell received one vector copy, and to select the cells for the presence of the vector. These selected populations would then stably express the Hyal2 constructs and could then have been assayed for susceptibility to JSRV vector transduction and for cell-surface Flag expression to provide more accurate data on cell-surface protein processing and receptor activity. Regardless, results presented here allow us to draw some strong conclusions about the determinants of Hyal2 receptor function. For example, if any of the human Hyal2 mutants containing the corresponding mouse Hyal2 residues had no activity, it would have been important to show that this result was not due simply to a processing defect. However, all mutants had activity well above the mouse Hyal2 level, and were thus functional to some extent, allowing us to conclude that multiple amino acid differences contribute to the low activity of mouse Hyal2 in comparison to that of human Hyal2.
We have mapped four of the amino acid residues that are important for human Hyal2 function as a virus receptor to a surface patch on the bee venom Hya crystal structure, and two others map to positions underneath this patch and could influence the structure of the patch. We hypothesize that this patch represents the binding site for Env, but it is also possible that this site is required for Hyal2 binding to another protein involved in virus entry. Furthermore, this structural extrapolation to the bee venom hyaluronidase may not be entirely valid and determination of a crystal structure for mouse or human Hyal2 is important to confirm these predictions. A soluble form of human Hyal2 has been made that can bind tightly to JSRV Env, as measured by surface plasmon resonance analysis, and that can block JSRV vector transduction by binding to JSRV vector virions [3]. We are currently working to crystallize this protein to allow more definitive analysis of results presented here.
Conclusion
We show that the greatly reduced receptor activity of mouse Hyal2 in comparison to that of human Hyal2 is determined by multiple amino acid changes acting in concert, and that no one amino acid change blocks infection. However, the most important amino acids map to a small patch on a predicted 3-dimensional Hyal2 structure which we hypothesize is the binding site for Env.
Methods
Cell culture and virus production
NIH 3T3 (TK-) Swiss mouse embryo fibroblasts [20], 208F Fischer rat embryo fibroblasts [21], and PJ4/LAPSN vector-producing cells [4] were grown in Dulbecco's modified Eagle medium (DMEM) with high glucose (4.5 g/L) and 7% fetal bovine serum at 37°C in a 10% CO2-air atmosphere at 100% relative humidity. PJ4/LAPSN cells produce virus containing the LAPSN retroviral vector [22] that encodes human placental alkaline phosphatase (AP) and neomycin phosphotransferase (Neo). Virus produced by PJ4/LAPSN cells contain Moloney murine leukemia virus Gag-Pol proteins and have a JSRV Env protein coat. LAPSN(PJ4) vector was produced by feeding confluent PJ4/LAPSN cells and harvesting the medium 12 to 24 h later. Virus-containing medium was centrifuged at 3,000 × g for 5 min to remove cells and debris and was frozen at -70°C until use.
Plasmid construction
All Hyal2 plasmids tested were constructed in either the pFLAG-CMV-1 mammalian expression vector (Sigma-Aldrich, Saint Louis, MO) or the pCR3.1 TOPO eukaryotic expression vector (Invitrogen, Carlsbad, CA). Each of the human Hyal2 amino or carboxy terminus deleted plasmids was cloned into the EcoRI site of the pFLAG-CMV-1 vector after PCR amplification of the desired coding regions. The human and mouse Hyal2 mutants containing single, double or triple amino acid substitutions were created by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) following recommended protocols. Human and mouse Hyal2 cDNAs share two common unique restriction sites, PflMI (located at codon 158), and BsaAI (located at codon 354). The mouse Hyal2 cDNA contains 3 BlpI restriction sites (located near codons 305, 321, and 413), which are completely absent in human Hyal2. By performing site-directed mutagenesis without altering the encoded amino acids, a BlpI site near codon 305 was created in human Hyal2 and the BlpI site near codon 321 was eliminated in mouse Hyal2. Both the human and mouse Hyal2 can therefore be divided into 4 domains by the 3 restriction sites: PflMI (codon 158), BlpI (codon 305), and BsaAI (codon 354). The chimeric mouse/human Hyal2 plasmids were obtained by exchanging restriction fragments between human and mouse Hyal2. The Hyal2 coding regions of all plasmids were sequenced after construction to confirm the expected sequences.
Receptor activity assay
For assay, NIH 3T3 mouse cells or 208F rat cells were seeded at 2.5 × 105 per well (d = 3.5 cm) of 6-well plates. The next day the cells were transfected with 4.5 μg of the test Hyal2 plasmid plus 0.5 μg of a plasmid encoding β-galactosidase (β-gal) per well by calcium phosphate coprecipitation as previously described [8]. One day after transfection the cells were trypsinized and divided 1:6 into 6-well plates. Two days after transfection the cells were exposed to serial dilutions of LAPSN(PJ4) vector in the presence of 4 μg/ml Polybrene. Four days after transfection the cells exposed to the LAPSN(PJ4) vector were stained for AP to determine the vector titer and cells not exposed to the vector were stained for β-gal expression to evaluate transfection efficiency for each plasmid. Experiments for which β-gal staining was low or variable were not included in the results. In each experiment the LAPSN(PJ4) vector titer was measured on cells transfected with an unmodified human Hyal2 expression plasmid, and results for the test plasmids are expressed as a percentage of that for the human Hyal2 plasmid (typically 104 to 3 × 104 AP+ focus-forming units (FFU) per ml following transfection of NIH 3T3 mouse cells and 103 to 2 × 103 FFU/ml following transfection of 208F rat cells with the human Hyal2 plasmid). Several Hyal2 constructs were tested in both mouse and rat cells with similar results, but most of the receptor activity assays shown were performed only in the mouse cells. Results are means of at least two independent experiments.
FACS analysis
Cell-surface expression of amino-terminal Flag-tagged Hyal2 proteins was evaluated by FACS analysis after incubation of cells with anti-Flag mouse monoclonal primary antibody followed by incubation in goat anti-mouse IgG secondary antibody. Propidium iodide was added to the cells before analysis and only cells that excluded the dye (live cells) were included in the analysis. 30,000 live cells were analyzed per sample.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
FMD designed and made all of the plasmid constructs, CD performed some initial transfection analyses, MIL helped design the plasmid constructs and the experiments, and ADM assayed the function of the wild-type, chimeric, and mutant receptors, analyzed the data, and drafted the manuscript. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
"Hyal2 DNA sequences.fasta". Bovine [GenBank:AF411973.1], ovine [GenBank:AF411974.1], pig [GenBank:AY497544.1], dog [GenBank:XM_541876.2], human [GenBank:U09577.1], C3H mouse [GenBank:AF302843.1] (same as NIH Swiss mouse), Czech II mouse [GenBank:AF302844.1, and rat [GenBank:AF535141.1] Hyal2 DNA sequences in Fasta format.
Click here for file
Additional File 2
"Hyal2 DNA sequences.phy". DNA sequence alignment of bovine, ovine, pig, dog, human, mouse (2 alleles), and rat Hyal2 DNA sequences in PHYLIP format made by using ClustalW [23] and suitable for use with K-estimator [13] software or for upload into Fast Positive and Negative Selection Detection website [16].
Click here for file
Additional File 3
"Hyal2 protein alignment.pdf". Alignment of bovine, ovine, pig, dog, human, mouse (2 alleles), and rat Hyal2 made by using ClustalW [23] software.
Click here for file
Acknowledgements
We thank Neal Van Hoeven for assistance with the FACS analysis, Roland Strong, Vladimir Vigdorovich, and Shervin Bahrami for help with the Hyal2 structural analysis, and Harmit Malik for help with the protein evolution analysis. CD and ADM were funded by NIH grants DK47754, HL36444 and HL66947. FMD and MIL were funded by the National Cancer Institute, NIH under contracts No. NO1-CO-56000 and NO1-CO-12400, and by the Intramural Research Program of the NIH, NCI. The content of the publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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Wei CM Gibson M Spear PG Scolnick EM Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1 J Virol 1981 39 935 944 6270359
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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-2-611620216110.1186/1742-4690-2-61Short Report12th international conference on human retrovirology: HTLV and related retroviruses Lairmore Michael D [email protected] Masahiro [email protected] Department of Veterinary Biosciences and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210 USA2 Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210 USA3 Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, OH 43210 USA4 Division of Virology, Niigata University Graduate School of Medical and Dental Sciences 1-757, Asahimachi-Dori Niigata, Niigata 951-8510 Japan2005 4 10 2005 2 61 61 20 9 2005 4 10 2005 Copyright © 2005 Lairmore and Fujii; licensee BioMed Central Ltd.2005Lairmore and Fujii; 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 12th International Conference on Human Retrovirology: HTLV and Related Retroviruses, was held at the Half Moon Hotel in Montego Bay, Jamaica, from June 22nd to June 25th 2005. The scientific conference, sponsored by the International Retrovirology Association, is held biennially at rotating international venues around the world. The meeting brings together basic scientists, epidemiologists and clinical researchers to discuss findings to prevent HTLV infection or develop new therapies against HTLV-mediated diseases. The Association fosters the education and training of young scientists to bring new approaches to the complex problems of HTLV research, such as translational research to bring findings from the laboratory into clinical trials that benefit HTLV-infected patients. The breadth and quality of research presentations and workshops at the 12th International Conference indicate that these goals are being accomplished. As HTLV research enters its third decade a new generation of scientists face many challenges. However, HTLV scientists and clinicians displayed exciting new approaches and discoveries during plenary talks and poster sessions. The conference encouraged research in HTLV infections and disease, fostered collaborations, and stimulated new partnerships between clinicians and scientists to encourage clinical trials and novel therapeutic interventions.
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Findings
Jamaica and the International HTLV Conference
The International Retrovirology Association in conjunction with the National Institutes of Health and the University of the West Indies, Jamaica welcomed over 300 scientists from diverse disciplines to the 12th International Conference on Human Retrovirology: HTLV and Related Retroviruses, held at the Half Moon Hotel in Montego Bay, Jamaica, from June 22nd to June 25th 2005. The Association was established in May 1994 to promote informal discussions among established human T-lymphotropic viruses (HTLV) scientists to enhance the efforts of scientists and clinicians to form interdisciplinary groups to study HTLV and its related diseases in a cooperative and creative manner. The Association sponsors its biennial general scientific conferences at rotating international venues, generally in areas with endemic HTLV infection. This unique meeting brings together basic scientists, epidemiologists and clinical researchers in a free form exchange of data to discuss approaches to prevent HTLV infection or develop new therapies against HTLV-mediated diseases. The Association has focused its international meeting on HTLV and other related human and nonhuman primate retroviruses, to promote excellent science and to facilitate the communication of scientific results. Equally important, the Association fosters the education and training of young scientists to bring new approaches to the complex problems of HTLV research, such as translational research to bring findings from the laboratory into clinical trials that benefit HTLV-infected patients. Judging from the breadth and quality of research presentations and workshops at the 12th International Conference, these goals are being accomplished.
HTLV research enters its third decade with the challenge to bring a new generation of scientists to the challenges facing the field [1]. Many of the scientists attending the meeting were from developing countries where HTLV is endemic. It was particularly relevant that the conference was held in context to the 25th anniversary of the discovery of the first identified human retrovirus, HTLV-1. Scientists in this field of research have many new discoveries to energize their work, many of which were on display during the meeting as plenary talks or during poster sessions. The conference encouraged research in HTLV infections and disease and fostered collaborations between research groups. Importantly, the conference through its many workshops provided a setting to stimulate new partnerships between clinicians and scientists to encourage the development of clinical trials and novel interventions.
Human T-lymphotropic virus type 1 (HTLV-1) and the closely related HTLV-2 were the first human retroviruses discovered [2]. HTLV-1, is a member of the deltaretrovirus genera, and infects approximately 15 to 20 million people around the world [3]. HTLV-1 infection occurs worldwide, but is particularly endemic in Central Africa, the Caribbean, and South America and southwestern Japan, while HTLV-2 is endemic among Indian tribes of South, Central, and North America. The virus causes adult T cell leukemia/lymphoma (ATLL), an aggressive malignancy of CD4+ T lymphocytes in 1 to 5% of infected individuals that is refractory to most therapies [4]. HTLV-1 is also associated with a progressive neurologic disease termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a variety of chronic inflammatory diseases including infectious dermatitis, uveitis, and arthropathy [5,6]. The following summarizes selected key presentations at the 12th International Conference on Human Retrovirology, but is by no measure a comprehensive overview of all of the exciting findings from the conference and we apologize in advance to those investigators we have not mentioned in an effort to provide a concise summary of the meeting.
Molecular Epidemiology of HTLV Infections: New Insights
Reports of well characterized cohorts by Dr. Edward Murphy of the University of San Francisco (United States) began the meeting with a clear challenge to carefully examine the relationship between viral loads and disease outcomes. Data from these groups suggest that a "set point" of viral load is determined in each individual studied based on their ability to form an effective early immune response to the infection. Risk factors for the transmission of HTLV-1 from infected mothers to their children based on HLA haplotype was presented by Dr. Robert Biggar and colleagues (National Institutes of Health, United States), who illustrated how immune recognition may determine rates of viral transmission.
A highlight of this session included Dr. Masao Matsuoka's and colleagues' (Kyoto University, Japan) recent findings related to the functional significance of an anti-sense message that codes for the protein HBZ and the role of methylation in the control of HTLV-1 gene expression. His group provided data to indicate that proviral DNA in cell lines derived from ATLL patients was highly methylated correlating with low or absent HTLV-1 gene expression. Unlike the 5' LTR of proviral DNA and the coding region and the 3' LTR was not heavily methylated in these same cells. The expression of HBZ mRNA, but not those encoding other viral genes such as tax, was detected in all the fresh ATL samples, strongly suggesting that HBZ has a significant role in leukemic cell survival in vivo. These findings were further supported subsequently in the meeting by Dr. Eric Wattel (Lyon Rhone, France) who also found correlation between proviral loads in patients with HBZ mRNA expression. In addition, it appears that HBZ expression had a growth stimulating influence on ATL cells. Transfection of HBZ expressing plasmids in an IL-2 dependent human T-cell line augmented the cell line growth and inhibition of HBZ expression by siRNA interference reduced cell growth of ATL-derived cell lines. Finally, transgenic mice containing HBZ gene under the control of a CD4 promoter exhibited an increased number of CD4+ spleen-derived T-cells. Dr. Matsuoka proposed that HBZ and Tax act synergistically to transform CD4+ T-cells eventually leading to the development of ATLL. Since HBZ, but not Tax is expressed in ATL cells in vivo, this unique anti-sense encoded nonstructural protein may represent a promising therapeutic target in ATLL patients. Later in the meeting, the HBZ encoding region was further characterized and mapped to reveal new start sites for the anti-sense transcript by Dr. M. Cavanagh and colleagues (CHUL Research Center, Canada). Collectively, these presentations provide new insights into the complex interaction between HTLV-1 and virus replication and lymphocyte survival.
An exciting summary of recently published findings about novel nucleotide sequences of human retroviruses, HTLV-3 and HTLV-4 were summarized by Dr. William Switzer of the United States Centers for Disease Control and Prevention. These researchers used polymerase chain detection to discover evidence of two related HTLVs (named HTLV-3 and HTLV-4). The first group directed by Switzer investigated 930 central Africans with contact with nonhuman primate blood through hunting and butchering. Serological tests and partial DNA sequencing of blood samples showed that two humans were infected with novel viruses, designated HTLV-3 and HTLV-4. Phylogenetic analysis showed that HTLV-3 is originated from a known monkey virus STLV-3 through cross-species transmission, while a corresponding monkey virus to HTLV-4 has not been reported yet and genetically equidistant from all known HTLVs/STLVs. The second group directed by Dr. Antoine Gessain (Pasteur Institute, France) tested serum specimens or DNA from 240 subjects from Cameroon to provide suggestive evidence that an HTLV-3 related to STLV-3 was present in individuals exposed to nonhuman primate blood or tissues. Importantly, these findings suggest that cross species transmission between nonhuman primates and humans is ongoing where people are exposed to primate blood and tissues [7,8]. A challenge to researchers in the field in the coming years will be to fully characterize these new viruses in terms of complete genomic sequences and to identify replication or pathogenic mechanisms of HTLV-3 and HTLV-4 compared to HTLV-1 and HTLV-2.
Basic Biology: Novel Mechanisms of Transformation and Disease
The study of HTLV-1 Tax initiated the concept that complex retroviruses regulate their expression by producing transactivating proteins. The mechanisms used by HTLV-1 Tax to alter cell cycle regulation or cell division, as in past meetings, continued to dominate the basic biology sessions of the 2005 meeting. Dr. Jennifer Nyborg (Colorado State University, United States) provided an overview of how the well-studied transactivating protein of HTLV-1, Tax, promotes viral transcription from the chromosomally-integrated HTLV-1 promoter. By using chromatin immunoprecipitation analysis, Dr. Nyborg's group provided data that the promoter activation by Tax1 correlated with the apparent loss of nucleosomes from the promoter and coding region. These results suggest that the Tax functions by causing nucleosome removal from the HTLV-1 promoter. Later in the meeting, by using in vitro transcription using "chromatinized" templates, Dr. Fatah Kashanchi (George Washington University, United States) presented complementary data indicating that Tax converts the randomly-assembled nucleosomes into periodic assembled ones, and this periodic assembly of the nucleosomes correlated with the activation of HTLV-1 promoter by Tax. His group has previously demonstrated that Tax recruits SWI/SNF complex containing BRG1 on the HTLV-1 promoter, and the recruitment is essential for the transcriptional activation of HTLV-1 promoter [9]. Thus, SWI/SNF containing BRG1 may play a role in the reassemble of nucleosomes with the HTLV-1 promoter. Dr. Susan Marriott (Baylor University, United States) provided new data on the effects of HTLV-1 Tax on cell cycle progression and genomic instability. Tax appears to increase genomic instability by altered key checkpoints in the cell cycle. Her work has focused on the G1 and S phases of the cell cycle.
Dr. Kuan-Teh Jeang (National Institutes of Health, United States) presented data that continued this theme of the meeting. His new findings suggest that Tax interacts with the Ran-GTP network to cause abnormal amplification of cellular centrosomes, which may be an initial event of cancer caused by the virus. Dr. Patrick Green (Ohio State University, United States) furthered this subject area by providing new information about the role of the anti-sense encoded protein, PDZ-binding motif of Tax in induction of micronuclei, a hallmark of genomic instability. Dr. Chou-Zen Giam (Uniformed Services University, United States) reported that "unscheduled" activation by the Cdc20-associated anaphase promoting complex by HTLV-1 Tax induces mitotic dysfunction and inactivation of critical regulators of mitosis. This presentations overall solidified the concept that the acquisition of genetic and epigenetic changes in Tax-expressing T cells favors the selection of cells with mutated proviral DNA in a manner to favor eventual outgrowth of the transformed cell in a way that avoids immune system recognition. Collectively, the precise role of Tax in cellular transformation was a major theme of the basic science sections of the meeting.
Dr. Warner Greene (Gladstone Institute, United States) provided a stimulating plenary talk about the role of APOBEC 3G (A3G), a cellular deoxycytidine deaminase with broad anti-retroviral activity. Dr. Greene summarized what is currently understood about A3G, which is inactive as a high-molecular-weight ribonucleoprotein complex in activated CD4 T-cells, but is active in low-molecular-weight complexes in resting CD4 T-cells. Resting CD4+ T-cells are typically resistance for HIV-1 infection, but the resistance was greatly relieved by A3G-specific siRNAs that block A3G function. Surprisingly, sequence analysis showed rare dG-dA hypermutations, a signature of A3G-mediated inhibition, in infected CD4 T-cells. Thus, A3G may have another mechanism to inhibit HIV infection. Dr. Greene suggested that deaminase activity may have a broader role and perhaps may influence HTLV-1's ability to infected CD4+ T-cells, a preferred natural target of HTLV-1 infection in vivo. In this regard, Dr. David Derse and colleagues (National Cancer Institute, United States) at the meeting reported that exogenous overexpression of APOBEC 3B (A3G) also inhibited HTLV-1 infectivity. A3G was incorporated into HTLV-1 virions, but the amount was much less than that of HIV-1 stains that lack the ability to inactive the cellular co-factor (HIV Vif defective). Interestingly, HTLV-1 does not appear to have vif-like gene in its genome, but data presented from Dr. Derse suggested that the HTLV-1 structural gene, gag contained sequences that act to inhibit A3G incorporation into HTLV-1 virions. Thus, HTLV-1 may overcome A3G anti-retroviral activity through gag-mediated exclusion of A3G. Consistently with these results are findings that indicate that there are rare dG-dA hypermutations among HTLV-1 isolates.
Clinical and Translational Research: New Approaches in Therapy
The meeting brought together a variety of clinicians seeking better treatments against ATLL and HAM/TSP. Dr. Thomas Waldmann (National Institutes of Health, United States), a pioneer in the field of therapeutic approaches against ATLL, provided a summary of the role of IL-15 in T-cell signaling and his groups recent findings involving Hu-Mik-Beta-1 (HM-beta) to block β-chain signaling following IL-15 receptor engagement. This approach may provide exciting new avenues to inhibit lymphocyte-mediated disorders such as HAM/TSP. Clinical trials using the potent immunosuppresive agent Campath-1H (anti-CD52) was presented by Dr. J.C. Morris (National Institutes of Health, United States). HTLV-1 Leukemic patients appeared to respond well to Campath-1H, despite its adverse side effects. A key factor in non-responsive ATL lymphoma patients appears to be the difficulties with drug penetration of solid tissues.
HTLV-1 Tax has been shown to interact directly with different members of the NF-κB family [10]. Interference with post-translational modifications of Tax including ubiquitylation and sumoylation by proteasome inhibition was presented by Dr. Ali Bazarbachi and colleagues (American University, Beirut, Lebanon) as effective ways to block NF-κB signaling, a key pathway of Tax-mediated cell transformation. Dr. Lee Ratner (Washington University, United States) presented new data regarding his novel transgenic mouse model. His new work indicates from this model indicates that both the canonical and non-canonical pathways of NF-κB activation are involved in resistance to apoptotic stimuli in Tax transgenic mouse derived cell lines.
Novel treatments continue to be a focus of researchers in the HTLV and related retrovirus field. Dr. Luc Willems and colleagues (Faculte Universitaire des Sciences Agronomiques, Belgium) using their established bovine leukemia virus infection of sheep model, revealed new approaches to treatment of leukemia using the drug, valproate. This treatment was shown to be effective in decreasing lymphocyte numbers and tumor regression in this model system.
Educational and Technical Workshops: Bringing Scientists together to Address Complex Problems
A particularly interactive portion of the meeting were the educational and technical workshops held offsite at the Barnett Estates, a serene setting that promoted scientific exchange. Morning workshops discussed the epidemiology, animal models, immunology, and basic virology of HTLV and related viruses. These group sessions were spirited and were lead by scientist who provided an excellent overview of their fields before leading the discussion of selected topics of interest to the group. These sessions allowed a more informal exchange of ideas and unpublished data, a goal of any scientific meeting. New virus strains including HTLV-3 and HTLV-4 characterized by molecular signatures were the subject of many conversations. Current strengths of weakness or gaps in the literature dominated many sessions as participants provided their interpretation of recent published or new data at the early plenary sessions. These workshops were extended in the afternoon to include important and clinically focused sessions based on HTLV disease associations. Thus, workshops devoted to adult T-cell leukemia/lymphoma, infectious dermatitis, and HTLV-1 neurologic disease allowed scientist to debate current approaches and how clinical trials could be evaluated and improved. Overall, these workshops provided a dynamic interaction among scientists and clinicians, which were apparent when patient case studies were included to focus the discussions.
Viral Pathogenesis: Interplay between HTLV's and Lymphocytes
Dr. Masahiro Fujii and colleagues (Niigata University, Niigata, Japan) reported data using co-transfection assays of Tax expression plasmids with luciferase reporters to test the functional differences between HTLV-1 and HTLV-2 Tax [11]. This group's results suggest that IL-2 autocrine secretion establishes benign life-long HTLV-2 infection and the distinct cytokine production regulated by the nuclear factor of activated T-cells (NFAT) is a key factor for the distinct differences in disease association between these two related viruses. Further insights into the pathogenesis of HTLV-1 infection was provided by Dr. Brian Wigdahl (Drexel University, United States) who provided provocative data indicating that Tax is not only secreted from infected cells, but can differentially modulate the function of dendritic cells and astrocytes. This work may provide a unique mechanism of neurologic damage by HTLV-1. Dr. Renaud Mahieux and colleagues (Institut Pasteur, France) continued to implicate unique molecular features that differentiate HTLV-1 Tax (Tax-1) from HTLV-2 Tax (Tax-2). Tax-2 appears to have a distinct cytoplasmic distribution compared to Tax-1. Futures studies to define the role of particular motifs that may explain the pathogenic differences between HTLV-1 and HTLV-2 will be directed at these important regulatory proteins.
Dr Claudine Pique and colleagues (Saint Louis Hospital, Paris, France) presented new findings that neuropilin 1 (NP-1), a receptor for Sematophorin 3a and vascular endothelial growth factor (VEGF), acts as a co-factor for HTLV-1 entry. NP-1 directly interacted with HTLV-1 envelope protein. There appears to be two separable domains in the HTLV-1 envelope that are required for the virus entry. While one domain is a binding surface for GLUT-1, a recently identified HTLV-1 receptor, the other was that for NP-1. NP-1, GLUT-1 and the envelope proteins appear to form a ternary complex on the cell surface. These results argued that HTLV-1 has two cellular receptors for the viral entry into host cells similar to HIV-1.
Dr. Steven Jacobson (National Institutes of Health, United States) presented new information about the intriguing findings related to regulatory T-cells (CD4+, CD25+, Foxp3+) in the immunopathogenesis of HTLV-1-associated neurologic disease. Interestingly, his research group found that Tax specifically inhibits foxp3 expression, which would be predicted to suppress the function of these important regulatory T-cells, perhaps contributing to lymphocyte-driven diseases. Dr. Charles Bangham (University of London, United Kingdom) and his research group continue to lead the field of cellular immunity against HTLV-1 infection. His presentation provided novel insights into the efficiency of cytotoxic T-cell killing as a key determinant of patient viral load outcome and data describing unique lymphocyte labeling techniques (6,6-D(2)-glucose) to monitor the kinetics of lymphocyte proliferation and death in HTLV-1-infected subjects.
The role of nonstructural proteins including proteins included in pX ORFs 1 and 2 were featured by a number of investigators. Dr. Franchini's research group (National Cancer Institute, United States) presented work indicating that p12I is recruited to the immunological synapse and inhibits signaling from the T-cell receptor. Dr. Vincenzo Ciminale (University of Padova, Padova, Italy) provided an update of his research of a novel mitochondrial localizing protein p13II in modifying lymphocyte survival and apoptotic cell death. Dr. Michael Lairmore and his colleagues (Ohio State University, United States) provided new information on the role of p30II in modifying G2 exit of the cell cycle furthering implicating this protein in lymphocyte survival.
Conclusions and Perspective
The meeting concluded with poignant remarks by one of the pioneering epidemiologist in the field of HTLV research, Dr. Nancy Mueller (Harvard, United States). She provided appropriate context to past studies of cohorts of HTLV-1 infected subjects, disease associations, and the useful applications of carefully controlled population-based studies. As the meeting came to a close, it was clear that HTLV and related retrovirus researchers faces many challenges, which confound and frustrate scientists in the HTLV field. However, the 12th International Conference on Human Retrovirology illustrated the dedication of the many scientists, clinicians, and patient advocates to address these challenges with a new determination and energy, as they all reluctantly left the gracious atmosphere of Jamaica to travel back to their homes, laboratories and offices throughout the world.
List of Abbreviations
HTLV, human T-lymphotropic viruses
HTLV-1, human T-lymphotropic virus type 1
HTLV-2, human T-lymphotropic virus type 2
HAM/TSP, HTLV-1-associated myelopathy/tropical spastic paraparesis
ATLL, adult T-cell lymphoma/leukemia
Competing interests
The authors have no competing financial or other interests involved in the data, methods, or writing of this manuscript.
Authors' contributions
Each author (MF and ML) have each met the definition of author as outlined by the Retrovirology journal. Each has made substantive intellectual contributions to the commentary. Each author has given final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content.
Acknowledgements
We thank Beverly Cranston for technical and logistic assistance in the organization of the International Retrovirology Conference 2005, other members of the conference organizing committee including Drs. Edward Murphy (University of San Francisco), Steven Jacobson (NIH), Genoveffa Franchini (NIH), Graham P. Taylor (Imperial College, UK), Barrie Hanchard and Owen Morgan (University of the West Indies, Jamaica), Michie Hisada (NIH), William Hall (University College, Dublin, Ireland), Mark Beilke (Tulane University, New Orleans, LA, USA), and Steven Foung (Stanfored University, Stanford, CA, USA). We thank the National Institutes of Health for R13 conference grants to support the International Retrovirology Conference.
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Yoshida M Jeang KT Preface to 25 years of HTLV-1 and ATL research Oncogene 2005 24 5925 10.1038/sj.onc.1208967
Gallo RC The discovery of the first human retrovirus: HTLV-1 and HTLV-2 Retrovirology 2005 2 17 15743526 10.1186/1742-4690-2-17
Proietti FA Carneiro-Proietti AB Catalan-Soares BC Murphy EL Global epidemiology of HTLV-I infection and associated diseases Oncogene 2005 24 6058 6068 16155612 10.1038/sj.onc.1208968
Takatsuki K Discovery of adult T-cell leukemia Retrovirology 2005 2 16 15743528 10.1186/1742-4690-2-16
Franchini G Nicot C Johnson JM Seizing of T cells by human T-cell leukemia/lymphoma virus type 1 Adv Cancer Res 2003 89 69 132 14587871
Osame M Pathological mechanisms of human T-cell lymphotropic virus type I-associated myelopathy (HAM/TSP) J Neurovirol 2002 8 359 364 12402162 10.1080/13550280260422668
Wolfe ND Heneine W Carr JK Garcia AD Shanmugam V Tamoufe U Torimiro JN Prosser AT Lebreton M Mpoudi-Ngole E Mccutchan FE Birx DL Folks TM Burke DS Switzer WM Emergence of unique primate T-lymphotropic viruses among central African bushmeat hunters Proc Natl Acad Sci U S A 2005 102 7994 7999 15911757 10.1073/pnas.0501734102
Calattini S Chevalier SA Duprez R Bassot S Froment A Mahieux R Gessain A Discovery of a new human T-cell lymphotropic virus (HTLV-3) in Central Africa Retrovirology 2005 2 30 15882466 10.1186/1742-4690-2-30
Kashanchi F Brady JN Transcriptional and post-transcriptional gene regulation of HTLV-1 Oncogene 2005 24 5938 5951 16155601 10.1038/sj.onc.1208973
Hall WW Fujii M Deregulation of cell-signaling pathways in HTLV-1 infection Oncogene 2005 24 5965 5975 16155603 10.1038/sj.onc.1208975
Niinuma A Higuchi M Takahashi M Oie M Tanaka Y Gejyo F Tanaka N Sugamura K Xie L Green PL Fujii M Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus J Virol 2005 79 11925 11934 16140768 10.1128/JVI.79.18.11925-11934.2005
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Respir ResRespiratory Research1465-99211465-993XBioMed Central London 1465-9921-6-1081616806710.1186/1465-9921-6-108ResearchEffects of cigarette smoke on degranulation and NO production by mast cells and epithelial cells Wei Xiu M [email protected] Henry S [email protected] Rakesh K [email protected] Gavin J [email protected] John E [email protected] H Patrick [email protected] Paul S [email protected] Inflammation Research Unit, School of Pathology, Faculty of Medicine, UNSW, Sydney, Australia2 Department of Respiratory Medicine, Prince of Wales Hospital, Randwick, NSW, 2031, Australia2005 19 9 2005 6 1 108 108 28 6 2005 19 9 2005 Copyright © 2005 Wei et al; licensee BioMed Central Ltd.2005Wei 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.
Exhaled nitric oxide (eNO) is decreased by cigarette smoking. The hypothesis that oxides of nitrogen (NOX) in cigarette smoke solution (CSS) may exert a negative feedback mechanism upon NO release from epithelial (AEC, A549, and NHTBE) and basophilic cells (RBL-2H3) was tested in vitro. CSS inhibited both NO production and degranulation (measured as release of beta-hexosaminidase) in a dose-dependent manner from RBL-2H3 cells. Inhibition of NO production by CSS in AEC, A549, and NHTBE cells was also dose-dependent. In addition, CSS decreased expression of NOS mRNA and protein expression. The addition of NO inhibitors and scavengers did not, however, reverse the effects of CSS, nor did a NO donor (SNP) or nicotine mimic CSS. N-acetyl-cysteine, partially reversed the inhibition of beta-hexosaminidase release suggesting CSS may act via oxidative free radicals. Thus, some of the inhibitory effects of CSS appear to be via oxidative free radicals rather than a NOX -related negative feedback.
nitric oxidemast cellsepithelial cellscigarette smoke
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Introduction
Cigarette smoke is a complex medium containing approximately 4000 different constituents [1] separated into gaseous and particulate phases. The components of the gaseous phase include carbon monoxide, carbon dioxide, ammonia, hydrogen dioxide, hydrogen cyanide, volatile sulphur-containing compounds, nitrogen oxides (including nitric oxide, NO), and other nitrogen-containing compounds. The particulate phase contains nicotine, water and tar [2]. Pulmonary effects of cigarette smoke include chronic obstructive pulmonary disease, increased airway reactivity, exacerbations of asthma, and an increased frequency of pulmonary infections. These effects are considered to be due to the direct actions of cigarette-derived toxins and ciliotoxins causing connective tissue destruction, hypersecretion, pooling of mucus and blebbing of membranes of endothelial cells. Cigarette smoke also reduces levels of exhaled nitric oxide in active and passive smokers, suggesting that it inhibits NO production [3-5]. Su et al [6] have shown that exposure to cigarette smoke extract inhibits the activity, protein and messenger RNA of NO synthase (eNOS) in pulmonary artery endothelial cells irreversibly. Whether alterations in NO play a role in the increased risk of pulmonary disease is not completely understood.
Mast cells play a crucial role in acute and allergic inflammation, and have high-affinity receptors for IgE (FcεRI) on their surface. Cross-linking of surface IgE molecules results in exocytosis of preformed mediators such as amines and proteases, as well as release of newly generated mediators including leukotrienes, prostaglandins and a variety of cytokines [7]. In the lungs and skin of smokers mast cells increase in absolute numbers and smoking may be associated with activation of mast cells [8,9]. They may contribute to some of the changes seen in smoking by releasing chemotactic factors, secreting proteases and other mediators. Some reports suggest that NO may be a participant in mast cell activation, but others suggest that it may also inhibit mast cell pre-formed mediator release [10,11]. Since cigarette smoke contains high levels of NO, it was hypothesised that NO may exert an inhibitory effect on degranulation, perhaps via negative feedback.
Airway epithelial cells (AEC) are important regulators of inflammation in the airway [12]. They have a function in host defence and play a significant role in airway inflammation by releasing NO, a potentially important mediator of airway inflammation [13,14], as well as releasing other mediators and recruiting inflammatory cells [12,15,16]. Cigarette smoke interferes with and inhibits the normal function of AEC by a variety of mechanisms. Some of these include decreases in the level of exhaled NO, enhanced release of pro-inflammatory cytokines, and inhibition of the airway repair process [5,17,18].
This study was designed to examine whether cigarette smoke induces dysfunction of airway mast cells and epithelial cells via the donation of cigarette-derived NO. It was hypothesized that the NO from cigarette smoke may induce negative feedback and cause a reduction in endogenous NO production from mast cells and epithelial cells. Thus, NO scavengers were added to a cigarette smoke solution (CSS). In addition, a NO donor was studied as a positive control and NO inhibitors as controls for endogenous NO production. NO generation was measured as nitrite.
A rat basophilic leukemia cell line, RBL-2H3 representing mucosal type mast cells [19], which has been extensively applied in studies of mast cell biochemistry and signalling, was used as an in vitro model of mast cells for this study. Beta-hexosaminidase was used as a marker of mast cell activation and degranulation. Primary cultures of murine epithelial cells, normal human tracheobronchial (NHTBE) and transformed alveolar epithelial (A549) cell lines were studied in parallel [20,21].
Materials and methods
Cell culture and polymerase chain reaction (PCR) reagents were purchased from Invitrogen Corporation (Sydney, Australia) and chemical reagents were bought from Sigma-Aldrich, (Sydney, Australia) unless otherwise specified. Animal tissue research was approved by the institutional animal ethics committee.
Cell Culture
The rat basophilic leukemia cell line, RBL-2H3 (ATCC, American Type Culture Collection, Rockville, MD, USA) was grown in complete Eagle Minimal Essential Medium with 15% fetal bovine serum (FBS), 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 2.0 mM L-glutamine, 50 IU/ml penicillin and 50 μg/ml streptomycin. A549 cell line (ATCC) was maintained in complete F-12 Nutrient Medium supplemented with 10% FBS, 50 IU/ml penicillin and 50 μg/ml streptomycin. Mouse airway tracheal epithelial cells (AEC), obtained from tracheas of 8–10 week-old specific pathogen-free BALB/C, were cultured and maintained as previously described [20] on collagen-coated plastic ware. Third- to fifth-passage AEC were used for experiments. Normal human tracheal bronchial epithelial cells (NHTBE, Clonetics, USA) were maintained in Bronchial Epithelial Cell Growth Medium (BEGM) Bullet Kit (CC-3170, Clonetics, San Diego, CA, USA).
Preparation of the cigarette smoke solution (CSS)
Water-soluble extract of cigarette smoke (both gas and particulate phases) was prepared as described previously [22]. Briefly, mainstream smoke from commercial cigarettes (Marlboro, Philip Morris, Australia) was drawn through 1 ml of medium by the application of a vacuum to the vessel containing the medium. Each cigarette was burned for 5 min, and 5 cigarettes were used for each millilitre of the appropriate medium for different cells. The pH of the resultant extract was titrated to pH 7.4, and diluted with medium. Solutions ranging from 0.125% to 1.0% were used in the present study in response to preliminary experiments which indicated that these were non-toxic concentrations. CSS was used within 2 hrs of preparation, and the NOx content of the CSS was in the range 1.3–2.6 mM, mean 1.76 (S.E. 0.67) mM. CSS was incubated also in control wells with media but without cells at the same concentrations and for the time periods. The final NOx content in these latter wells was subtracted from the values in the experimental wells.
Beta-hexosaminidase secretion assay
106/ml RBL-2H3 cells were sensitised with 100 ng/ml of mouse monoclonal IgE anti-DNP overnight. Cells were washed twice with phosphate buffered saline (PBS) and pre-incubated with different concentrations of CSS for a further 6 h prior to activation with either 100 ng/ml DNP-HSA antigen or 10 μmol/L of calcium ionophore A23187. Beta-hexosaminidase release from RBL-2H3 was measured by incubating 25 μl of the supernatant or lysed cell pellet with 5% Triton-100 with 25 μl of p-NAG in a 96-well plate (Nunc, Roskilde, DM) for 2 h at 37°C. The reaction was stopped with 250 μl 0.2 M glycine (pH 10.6) and the resultant change in absorbance read at 405 nm. The net percentage of release of beta-hexosaminidase was calculated by the following formula:
net percent release (%) = [S/(S+P)-Scontrol/(Scontrol+Pcontrol)] × 100,
where S, P are the mediator contents of supernatants and pellets of stimulated cells, respectively, Scontrol/(Scontrol+Pcontrol)(%) is spontaneous release of mediator without a stimulus.
Nitrite and nitrate measurements
RBL-2H3, A549, NHTBE and AEC were cultured in complete media until 90% confluent. Cells were washed with PBS and incubated with nicotine (31.25 ng/ml–400 ng/ml) or CSS as above for a further 24 h or 48 h (A549 cells), then measured as nitrite and nitrate (NOx) accumulation in media as described previously [23-25]. Briefly, nitrate was measured as nitrite after enzymatic conversion by nitrate reductase. Volumes of 20 μl NADPH, 10 μl FAD and 20 μl nitrate reductase were diluted in reaction buffer and added to yield final concentrations of 50 μmol/L, 5 μmol/L and 200 IU/L, respectively. Samples of 50 μl each were subsequently incubated for 1 hour at 37°C. Next, 10 μl of 2,3-diaminonaphthalene (DAN, 0.05 mg/ml in 0.62 M HCl) was added to each well and incubated for an additional 10 mins. The reaction was stopped by 10 μl of 2.8 M NaOH. The fluorescence of final product (1H-naphthotriazole) was measured using Perkin-Elmer Cytofluor 4000 plate reader (excitation 360/40, emission 395/25, gain 50). Nitrite concentration was calculated using a standard curve of serially diluted sodium nitrite.
RT-PCR analysis of iNOS and eNOS expression
Cells were incubated with 1%CSS at different time-points (3 h, 6 h, 24 h). Total cellular RNA was extracted using TRI-Reagent (Sigma) according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 μg total RNA with SuperScript II using Oligo (dT) as primers (Invitrogen, Carlsbad, CA, USA). PCR was performed on the reverse transcription products using specific oligonucleotide primers, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene.
PCR reactions contained 2 μL cDNA, 10 μM primers (Table 1), 1.5 mmol/L Mg, 200 μmol/L dNTPs and 0.5 IU Platinum Taq polymerase (Invitrogen) in a total reaction volume of 50 μL. PCR products were electrophoresed on 1.2% agarose gel containing 0.1% ethidium bromide. Positive and negative controls were run concurrently to exclude DNA contamination.
Table 1 PCR primers as used in Methods.
NOS isoform Primer sequence
Rat iNOS [26] sense:5'-GGACCACCTCTATCAGGAA-3', antisense 5'-CCTCATGATAACGTTTCTGGC-3';
Rat eNOS [27] sense: 5'-TACCAGCCGGGGGACCAC-3', antisense: 5'-CGAGCTGAC-AGAGTAGTA-3'.
Human iNOS [28] sense: 5'-GAGCTTCTACCT-CAAGCTATC-3', antisense: 5'-CCTGATGTTGCCATTGTTGGT-3';
Human eNOS sense:5'-GCACAGGAA-ATGTTCACC TAC-3', antisense: 5'-CACGATGGTGAC-TTTGGCTAG-3'.
Mouse iNOS (real time PCR) probe sense: 5'-CAGCTGGGCTGTACAAACCTT-3', antisense: 5'-CATTGGAAGTGAAGCGTTTCG-3', 5'-6FAM (fluorescent reporter dye, 6-carboxyfluorescein)-CGGGCAGCCTGTGAGACCTTTGA-TAMRA (quenching agent, 6-carboxytetramethylrhodamine, Applied Biosystems, CA, USA).
Rat iNOS and eNOS conditions
After initial denaturation at 95°C for 2 min, 25–35 cycles of amplifications at 94°C 30 sec, 60°C for 30 sec and 72°C for 45 sec were carried out using Perkin-Elmer 2400 thermal cycler.
Human iNOS and eNOS conditions
The thermocycle consisted of 94°C for 30 sec, 58°C (eNOS)/60°C (i NOS and GAPDH) for 30 sec and 72°C for 45 sec. The numbers of amplification cycles were 28–35 (iNOS and eNOS) and 25 (GAPDH).
Quantitative Real Time-PCR analysis
Due to the difficulty of growing large numbers of mouse AEC cells, quantitative real-time RT-PCR, which only requires small amounts of RNA, was chosen to determine mouse iNOS mRNA levels and β-actin (internal standard). Quantification of mRNA was performed by determining the threshold cycle (CT) on ABI PRISM 7700 Sequence Detector (Perkin-Elmer, Applied Biosystem). Standard curves were constructed using the values obtained from serially diluted positive control mouse iNOS plasmid.
Real time-PCR was performed in 50 μl reaction volumes containing 2X TaqMan Universal PCR Master Mix 25 μl (Roche, Branchburg, New Jersey, USA), 2.5 μl 18 μM sense/antisense primers, 2.5 μl 5 μM probe and 7 μl cDNA samples (Table 1). The following thermal profile was used: 2 min at 50°C, 10 min at 95°C and 50 cycles of 95°C for 15 sec, 60°C for 1 min.
iNOS/eNOS Western-Blot
CSS-treated RBL-2H3 and A549 cells were rinsed with PBS and isolated by scraping in ice cold radio-immunoprecipitation (RIPA) buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) with freshly added aprotinin (30 μl/mL RIPA). Cell lysate was passed several times through a 25 gauge needle to shear the DNA and incubated 30 minutes on ice. RIPA (10 μl/ml) with 10 mg/ml phenylmethylsulfonylfluoride (PMSF) was added and cell lysate was microcentrifuged at 12,000 rpm for 20 minutes at 4°C. Protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Supernatants (20 μg) were loaded on NuPAGE™ 4–12%Bis-Tris Gels (Invitrogen Corp.) and transferred to nitrocellulose membranes. The membranes were blocked overnight in 5% skimmed milk and incubated for 1 h at room temperature with primary antibodies at dilutions of 1:3000(iNOS/eNOS, BD Transduction Laboratories, San Diego, CA, USA). Peroxidase-labeled secondary antibody rabbit anti-mouse IgG was added at a dilution of 1:1000 (DAKO, CA, USA) for 1 h at RT. Membranes were developed with Enhanced Chemiluminescence Reagent (Perkin-Elmer Life Sciences, Boston, MA, USA) for 1 min and exposed for 30 sec to scientific imaging film (BioMax, Kodak, Rochester, USA).
Flow cytometric analyses of iNOS/eNOS proteins
CSS (1%) was added to RBL-2H3 and A549 cells for 24 h, which were then trypsinized and fixed in 4% formaldehyde. Cells were permeabilized in 0.15% saponin PBS/2% BSA for 1 h on ice and washed in PBS/2% BSA. Subsequently, cells were incubated in 10 μg/mL anti-iNOS or 20 μg/mL anti-eNOS (BD Transduction Laboratories) for 30 mins on ice, washed twice and added goat anti-mouse IgG conjugated with FITC (DAKO) for further 30 mins in the dark on ice. Cells were washed, resuspended in 500 μl 1% formaldehyde, and analyzed by flow cytometry (BD FACSort). Mouse IgG1 and IgG2a (DAKO) were used as negative controls for iNOS and eNOS antibodies, respectively.
Cell viability and cytotoxicity
Viability and cytotoxicity were assessed by trypan blue vital dye exclusion and lactate dehydrogenase release (LDH Kit, Sigma).
Statistical analysis
Data are expressed as mean ± SEM. Analysis was performed by one-way ANOVA with the application of Dunnett's multiple comparisons test, and a p value <0.05 was considered significant. Data are representative of at least 3 different experiments. In the case of data expressed as percentage of baseline, the ANOVA and subsequent comparisons were performed on the raw data, prior to transformation.
Results
Effect of CSS on degranulation and [NOX] generation by RBL-2H3
RBL-2H3 cells activated with either IgE/DNP or A23187 showed a concentration-dependent decrease in degranulation after incubation with CSS (0.125%–1.0%) for 6 h (Fig 1, p < 0.0001, ANOVA). Treatment with CSS decreased the percentage of beta-hexosaminidase release by up to 89% at 1.0% CSS. The CSS-induced inhibition of beta-hexosaminidase was also observed after 2 h incubation (data not shown). As shown in Figure 1, RBL-2H3 treated with CSS (0.125% to 1.0%) for 24 h resulted in a concentration-related inhibition of nitrite production (p < 0.05, ANOVA, where baseline NOx = 3.04 +/- 0.21 μM), with only a slight inhibition of nitrite at 6 h (p > 0.05, data not shown). Viability of RBL-2H3 and epithelial cells remained constant (generally >90%) before and after CSS treatment.
Figure 1 Effects of CSS on the release of beta-hexosaminidase and NOx production from RBL-2H3. Cells were passively sensitised with anti-DNP IgE, and incubated with 0.125–1% CSS for 6 hrs prior to activation with DNP. There is a CSS concentration-related decrease in the release of beta hexosaminidase. NOx formation was similarly decreased after 24 h incubation with the same range of CSS. Data are the means of 6 experiments with the mean and S.E.M. expressed as % of control, where baseline NOx = 3.04 +/- 0.21 μM, (ANOVA performed on raw data, *p < 0.001 with Dunnett's multiple comparison test compared to baseline values).
Effect of nicotine on degranulation and [NOX] in RBL-2H3, A549, NHTBE, and airway epithelial cells (AEC)
In order to investigate whether nicotine, the major component of cigarette smoking in particulate phase, is responsible for the inhibition of degranulation and production of nitric oxide, RBL-2H3 cells were incubated in nicotine solutions (62.5–250 μM) adjusted to physiological pH for 24 h. Neither degranulation nor production of nitric oxide was affected (Figure 2). Incubation of A549 cells with nicotine (31.25–400 ng/ml) for 48 h caused a significant decrease in production of NOx, but incubation of NHTBE and AEC cells with nicotine solutions (31.25 ng/ml–400 ng/ml) did not demonstrate any significant change (Table 2).
Figure 2 Effects of nicotine on the release of beta-hexosaminidase and NOx production from RBL-2H3. Cells were passively sensitised with anti-DNP IgE, incubated in nicotine solutions (62.5–250 μM) for 24 h, and then activated with DNP for beta hexosaminidae release, or accumulated NOx measured. No significant effects were seen. Data are the means of 4 experiments with the mean and S.E.M. expressed as % of control where mean (SD) baseline NOx = 3.5 +/- 0.11 μM (ANOVA performed on raw data, p > 0.05).
Table 2 Effects of nicotine on the production of NO, measured as NOx from A549, AEC, and NHTBE cell lines over 48 hrs. Data are expressed as mean (S.E.M.) % of control of 3 experiments (ANOVA performed on raw data, * p < 0.05 in A549 group; p > 0.05 in AEC and NHTBE groups; Dunnett's multiple comparison test)
NOx (Percentage of Control)
Nicotine(μM) 31.25 62.5 125 250
A549 Cell Line 67.0 ± 5.79* 65.0 ± 7.87* 69.8 ± 7.12* 69.4 ± 7.81*
AEC Cells 112.8 ± 25.06 120.2 ± 9.70 105.7 ± 20.68 108.5 ± 22.95
NHTBE Cell Line 87.9 ± 8.61 83.4 ± 10.53 87.9 ± 3.37 99.9 ± 0.07
Effects of NO pathway inhibitors on degranulation of RBL-2H3
To investigate whether the NO present in CSS may have a role in mast cell inhibition, a NO scavenger (hemoglobin) was pre-incubated for 1 h with CSS. A NO donor (SNP) was used as a positive control in studies without CSS. In addition, in case CSS stimulated the production of NO in these cells, an inhibitor of NOS (L-NMMA), and a cGMP inhibitor (ODQ) were studied in conjunction with CSS. The doses chosen were those optimised in previous work [29].
Activated or resting RBL-2H3 monolayers were incubated with: sodium nitroprusside (SNP 1 μM, 10 μM, and 100 μM); CSS plus hemoglobin (100 μM and 1 mM); or L-NMMA (1 μM, 10 μM and 100 μM) for 5 h. None of these was found to have a significant effect on basal or IgE mediated beta-hexosaminidase release, nor did hemoglobin or L-NMMA affect CSS-induced inhibition of beta-hexosaminidase release. Hemoglobin (1 mM) was pre-incubated with CSS for 1 h to neutralise NO in CSS and no effect was observed upon the degranulation of RBL-2H3. Pre-incubation of RBL-2H3 cells with the guanylyl cyclase inhibitor ODQ (10-6M) for 30 min did not reverse the CSS-induced inhibition of degranulation.
Effects of CSS on [NOX] from AEC, A549 and NHTBE cells
Incubation of A549 cells with CSS (0.125%–1.0%) for 48 h caused a significant and dose-related decrease in production of NOx . Also, 24 h incubation of NHTBE and AEC with CSS (0.25%–1.0%) resulted in a dose-related inhibition of NO production, which ranged between 47 and 67% inhbition with 1% CSS (baseline mean (SD) NOx of the cell lines were: A549 2.7 +/- 0.16 μM; mouse AEC 1.34 +/- 0.2 μM, and NHTBE 1.56 +/- 0.18 μM, Figure 3).
Figure 3 Effects of CSS on the production of NOx from A549, mouse AEC, and NHTBE. Cells were incubated with CSS for 24 h (AEC and NHTBE) or 48 h (A549). NOx production was assessed from the supernatants from each condition and cell line. Data are the means of 3 experiments with the mean and S.E.M. expressed as % of control. Baseline mean (SD) NOx of the cell lines were: A549 2.7 +/- 0.16 μM; mouse AEC 1.34 +/- 0.2 μM, and NHTBE 1.56 +/- 0.18 μM. ANOVA was performed on raw data: A549, *p < 0.05; AEC, *p < 0.05, 1% CSS compared with control; NHTBE, *p < 0.01, 1.0-0.5% CSS compared with control, *p < 0.05, 0.25% CSS vs. control; Dunnett's multiple comparison test.
Expression of NOS isoforms
Incubation of A549 cells with 1% CSS at different time-points caused a time-dependent decrease in iNOS mRNA levels (Figure 4). No iNOS mRNA was detected after 6 h CSS incubation in this cell line. The same pattern was observed in NHTBE which only expressed eNOS mRNA. eNOS mRNA was not detected in A549 cells. In RBL-2H3 cells, the eNOS mRNA band decreased at 3 h, although it returned to control levels after 24 h. Nicotine treated RBL-2H3 cells resulted in a slight decrease in eNOS mRNA expression. There was no iNOS mRNA observed in this basophilic cell line.
Figure 4 RT-PCR analysis of NOS expression. Panel A. Time course of the effect of 1% CSS upon A549 iNOS mRNA expression. Upper panel: Lane 1: control, Lane 2: 3 h, Lane 3: 6 h, Lane 4: 24 h, Lane 5: 24 h CSS exposure and then cells returned to normal media for further 24 h; bottom panel: GAPDH corresponding to each sample. PCR gels shown are representative of three separate experiments. There was a decrease in iNOS mRNA in A549 cells reaching undetectable levels at 6 h, which persisted to 24 hr, even after further incubation in normal culture media. Panel B. Time course of the effect of 1% CSS on NHTBE eNOS mRNA expression. Upper panel: Lane 1: control, Lane 2: 3 h, Lane 3: 6 h, Lane 4: 24 h, Lane 5: 24 h CSS and returned to normal media for further 24 h; bottom panel: GAPDH corresponding to each sample. PCR gels are representative of three separate experiments. Similar changes were seen in the mRNA expression of eNOS in the NHTBE line as in the A549 cells with decreased levels at 3 h which persisted throughout the study period. Panel C. Effect of 1% CSS and 100 μM nicotine on rat RBL-2H3 eNOS mRNA expression. Upper panel: Lane 1: control, Lane 2–4: CSS 3 h, 6 h, 24 h, Lanes 5–7: nicotine 3 h, 6 h, 24 h; bottom panel: GAPDH corresponding to each sample. PCR gels are representative of three separate experiments. After CSS, there was a decline in mRNA at 3 h which returned to baseline by 24 h, while exposure to nicotine showed a decrease at 6 and 24 h.
Quantitative Real Time-PCR analysis
Using the technique of real-time PCR to detect changes in AEC iNOS expression with 1% CSS, a progressive fall in copy number was seen over 24 h (Figure 5). The time-course is similar to that seen in the RT-PCR data for the A549 cells above.
Figure 5 Effects of CSS on iNOS mRNA from mouse AEC by real-time PCR. Quantitative real-time RT-PCR was performed to determine mouse iNOS mRNA levels. Quantification of mRNA was performed by determining the threshold cycle and standard curves were constructed using the values obtained from serially diluted positive control mouse iNOS plasmid, and determining the values for experimental samples from these curves. A progressive fall in copy number was seen in AEC iNOS expression with 1% CSS, over 24 h. Data points represent 3 replicates with S.E.M.
NOS protein expression by Western-Blot and flow cytometry
Using flow cytometry to detect iNOS positive cells, the number of cells expressing iNOS protein were seen to be decreased by 1% CSS in A549 cells. The ratio of positive to negative cells declined from 1.60 to 1.29 (t = 8.931, p = 0.012, paired t test, two-tailed). Similarly, eNOS positive RBL-2H3 cells were decreased after 1% CSS treatment from 2.78 to 2.24 ± 0.17. iNOS and eNOS protein levels were undetectable by immunoblot.
Effects of NAC on CSS-induced inhibition of degranulation of RBL-2H3
To investigate whether free radicals in CSS contribute to the inhibition of degranulation, RBL-2H3 cells were incubated with free radical scavenger N-acetyl-L-cysteine (NAC, 1 mM) for 30 min prior to their incubation with CSS. Compared with control CSS exposure and activation there was a significant reversal of the CSS-induced inhibition (Figure 6).
Figure 6 Effects of 1 mM NAC on 0.5 % CSS-induced inhibition of degranulation of RBL-2H3 cells. Data are the means of 5 experiments with the mean and S.E.M. expressed as % of total (ANOVA performed on raw data, Dunnett's multiple comparison test).
Discussion
Nitric oxide is a ubiquitous intracellular and intercellular signaling molecule, which plays a role in the functions of various inflammatory cells including mast cells, lymphocytes, neutrophils and macrophages [30]. NO can have deleterious or beneficial roles in inflammatory conditions depending on the setting because of its role as both an immune mediator and an effector molecule [30]. There is increasing evidence that the interaction between NO and mast cells is important in the control of the human nasal airway response, the physiological and pathological regulation in immune system, and the inhibition of gastric acid secretion [30]. Some researchers have reported that NO may modulate mast cell pre-formed mediator release[31]. For instance, an increase in cGMP levels was found to inhibit histamine release in rat peritoneal mast cells which was reversed by L-NMMA (32). Brooks et al. [32] demonstrated that NO induced by interferon-gamma could inhibit the IgE-activated secretory function of mouse mixed peritoneal mast cells. Koranteng et al. [34] reported that NO generation inhibits pre-formed mediator release in murine peritoneal mast cells, but not in other mast cells which were of a different phenotype. CSS has variable effects upon isolated mast cells [22], but in vivo has been clearly demonstrated to reduce exhaled NO and the mechanism for this reduction was therefore studied in in vitro models of airway epithelial cells and mast cells.
The effects of cigarette smoke upon the NO pathways and NOS isoenzymes are controversial and may vary according to the disease, model or location of the NOS. For example, while exhaled NO has been shown to be decreased in humans after acute cigarette exposure, iNOS mRNA expression increased in the lungs of rats exposed to cigarette smoke, while nNOS showed a longer term increase in both transcription and translation [3,5,35,36]. Cigarette smoke has been shown, however, to cause a reduction in nitrite concentration and iNOS expression in a murine lung epithelial cell line in vitro[37]. In contrast, Comhair et al showed no change in iNOS expression in airway cells from healthy subjects exposed to cigarette smoke [38]. The effects of cigarette smoke on NOS in the vasculature has shown a reduction in ecNOS in the pulmonary vessels in vitro and in vivo [6,39], genetic variation in man, [40,41] while vascular intimal thickening and up – regulated iNOS has been described in mice [42]. These seemingly contradictory effects are probably explained in part by the different tissue situations and also by variation in the constituents of the cigarette smoke. This is an important factor in the preparation of the CSS and while CSS represents the aqueous phase of the stimulus, the gaseous portion may easily contain additional stimuli which we did not study. For these reasons investigators are often exposing cells in vitro to direct cigarette smoke rather than CSS alone to more accurately simulate the in vivo situation.
The major findings of this study were that CSS inhibited mast cell degranulation, production of NO by mast cells and tracheobronchial epithelial cells, as well as expression of the dominant NOS isoform by these cells. We had hypothesised that the NOX-rich CSS might exert a negative feedback mechanism upon NO release from these epithelial and mast cells. The addition of scavengers did not inhibit the effect, nor did SNP, a NO donor mimic CSS, thus disproving this hypothesis. In addition, NOS inhibitors did not affect the response, indicating that the endogenous cellular production of NO was not involved in the response to CSS. Nicotine appeared reduce the ability of the A549 cell line to generate NOx, but this was not dose-dependent and was not seen in other cell lines. The significance of this apparently idiosyncratic response is unclear.
The effects of CSS shown here could not be attributed to the pharmacological activity of nicotine, but may to be related to oxidative free radicals as they are inhibited by N-acetyl-L-cysteine [44]. NAC, an anti-oxidant, has been studied quite extensively for its ability to exert protective effects. Because of its SH group, NAC scavenges H2O2 (hydrogen peroxide), •OH (hydroxyl radical), and HOCl (hypochlorous acid). In addition, NAC reduces cellular production of pro-inflammatory mediators [43]. CSS causes a reduction in NOS expression, and the mechanism would therefore seem to be at the level of the gene. The inhibition of the effects of CSS by NAC would appear to be congruent with the observations that NAC can reduce DNA adducts, clastogenic changes and other cellular toxic effects caused by mutagens and cigarette smoke in in vitro and in animal models, reviewed in De Vries 1993 [43]. These observations have led to the use of NAC in clinical trials in an attempt prevent or reduce the risk of recurrence of cancers by using NAC and other anti-oxidants [44]. This study did not use additional methods to confirm that the effect of CSS was via reactive oxygen species and NAC has complex attributes with actions other than by acting purely as an anti-oxidant, e.g. mucolytic activity, L-cysteine donation, and these could also play a role in modulating the effects of cigarette smoke [44].
Abbreviations
airway epithelial cells, AEC; cigarette smoke solution, CSS; endothelial nitric oxide synthase, eNOS; inducible nitric oxide synthase, iNOS; N-acetyl-L-cysteine, NAC; normal human tracheal bronchial epithelial cells, NHTBE; nitric oxide, NO; polymerase chain reaction, PCR.
Acknowledgements
This work was supported by the NHMRC, Australia, Asthma New South Wales, and a generous donation from the Lee family.
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Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-2-391618803910.1186/1742-4682-2-39ReviewData from necropsy studies and in vitro tissue studies lead to a model for allometric scaling of basal metabolic rate Painter Page R [email protected] Office of Environmental Health Hazard Assessment, California Environmental Protection Agency, P. O. Box 4010, Sacramento, California 95812, USA2005 27 9 2005 2 39 39 12 4 2005 27 9 2005 Copyright © 2005 Painter; licensee BioMed Central Ltd.2005Painter; 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 basal metabolic rate (BMR) of a mammal of mass M is commonly described by the power function αMβ where α and β are constants determined by linear regression of the logarithm of BMR on the logarithm of M (i. e., β is the slope and α is the intercept in regression analysis). Since Kleiber's demonstration that, for 13 measurements of BMR, the logarithm of BMR is closely approximated by a straight line with slope 0.75, it has often been assumed that the value of β is exactly 3/4 (Kleiber's law).
Results
For two large collections of BMR data (n = 391 and n = 619 species), the logarithm of BMR is not a linear function of the logarithm of M but is a function with increasing slope as M increases. The increasing slope is explained by a multi-compartment model incorporating three factors: 1) scaling of brain tissue and the tissues that form the surface epithelium of the skin and gastrointestinal tract, 2) scaling of tissues such as muscle that scale approximately proportionally to body mass, and 3) allometric scaling of the metabolic rate per unit cell mass. The model predicts that the scaling exponent for small mammals (body weight < 0.2 kg) should be less than the exponent for large mammals (> 10 kg). For the simplest multi-compartment model, the two-compartment model, predictions are shown to be consistent with results of analysis using regression models that are first-order and second-order polynomials of log(M). The two-compartment model fits BMR data significantly better than Kleiber's law does.
Conclusion
The F test for reduction of variance shows that the simplest multi-compartment allometric model, the two-compartment model, fits BMR data significantly better than Kleiber's law does and explains the upward curvature observed in the BMR.
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Introduction
The basal metabolic rate (BMR) has been extensively measured in mammals that are "mature, in postabsorptive condition, measured in the range of metabolically indifferent environmental temperatures, and at rest, or at least without abnormal activity" [1]. The scaling exponent β in the conventional allometric expression,
BMR = αMβ, (1)
can be estimated from data on BMR for animals of mass M as the value that minimizes the sum of squares of residuals (SSR), where a residual is defined as log(αMβ) - log(BMR). This procedure is termed least-squares logarithmic regression (LSLR). For the model of Equation (1), the procedure is equivalent to regression of the logarithm of BMR on the logarithm of M, which calculates the maximum-likelihood estimate (MLE) of β when the distribution of residuals is Gaussian. Analyses of metabolic rate data in the 19th century showed that the scaling exponent β for mammals at rest is less than 1. In the best-known 19th century study of the resting metabolic rate, Rubner [2] argued that the rate of metabolism is proportional to the 2/3 power of body mass. Rubner's 2/3-power law was widely used for metabolic scaling for several decades. In the 20th century, the law was questioned following analysis of BMR data by Kleiber [1,3] and Brody [4,5]. For data collected by these physiologists, the MLE for β determined by LSLR is close to 3/4. Based on these results, the 3/4-power law (Kleiber's law),
BMR = aM0.75, (2)
became widely used in physiology and ecology.
More recent analysis of BMR data sets that are much larger than those used initially to support the 3/4-power law has shown that the MLE for the scaling exponent is between 2/3 and 3/4 [6-11]. The largest of these data sets comprises BMR values from 619 mammalian species [11]. The 95% confidence interval (CI) for β from LSLR of their data is 0.674 – 0.701 with the MLE of 0.687. Including an adjustment for the effect of body temperature on BMR gives a MLE of 0.67. Analysis of other large data sets has also shown that the slope of the logarithm of BMR, plotted as a function of logarithm of M, increases as M increases [10,12,13].
Several theories that predict a value for the BMR scaling exponent have been critically reviewed. Dodds et al. [13] conclude their assessment of both the scaling of BMR and of theories that predict 3/4-power scaling by stating "we find evidence that there may not be a simple scaling law for metabolic rate, and if it were to exist, we also find little compelling evidence that the exponent should be α = 3/4." Agutter and Wheatley [15] conclude in their review of models that offer explanations for the allometric scaling of BMR that none of them can be universally accepted and that no model has yet addressed every relevant issue.
Critical evaluations of two prominent theories for the basis of 3/4-power scaling have been published [15,16]. The evaluation of the theory of West et al. [17], which is based on maximization of the scaling of nutrient exchange surface area in a fractal distribution network, questions their assumption that the fractal dimension of an object in 3-dimensional space can be equal to 4. The evaluation of the theory of Banavar et al. [18,19], which is based on mathematical properties of outward-directed supply-demand networks, points out that the fundamental theorem in this theory requires the assumption that nutrient uptake rates at uptake sites are statistically independent of the distance from the heart to a site. This assumption is questionable for the system of arteries and capillaries because nutrient uptake for all cells other than endothelial cells occurs through the capillary walls, which are the most distant sites in the model.
Two recently published mathematical models of BMR scaling appear to be compatible with values of the scaling exponent other than 3/4. The first is the Allometric Cascade Model [20], which is discussed below. The second is based on quantum mechanics of the electron transport system (ETS) and on resource availability [21]. In this model, parameters describing the ETS are determined by natural selection. For mammals in environments with scarce but dependable resources, the selected parameters correspond to 3/4-power scaling. For animals that have ample but temporarily available resources, parameters corresponding to 2/3-power scaling are selected.
In the Allometric Cascade Model, Darveau et al. [20] propose that the metabolic rate of a mammal can be described by the sum of power functions,
Individual power-function terms describe the scaling of the energy requirement for a specific biochemical process. Examples are the energy requirement for protein synthesis, for Ca++ transport across the cytoplasmic membrane and for Na+ transport across the cytoplasmic membrane.
While this model has been criticized for being tautological [22,23], it is clearly different from the conventional power law of Equation (1) whenever the exponents βi do not all have the same value. As shown below, the logarithm of the metabolic rate in Equation (3), plotted as a function of the logarithm of M, has a slope that increases as M increases, while this slope is the constant value β for Equation (1).
An expression for the BMR that is equivalent to Equation (3) can be derived from the conceptualization of Heusner [24] based on scaling of the mass of individual tissues and organs (e.g., bone or brain). As reviewed by Brown et al. [25], allometric scaling exponents for the mass of an organ or organ system vary considerably. For example, a MLE of the scaling exponent for bone mass is 1.09 [26], and an average of MLEs of the scaling exponent for brain mass is 0.73 [27]. The anatomical conceptualization has also been used to develop a five-compartment anatomical model (brain, liver, kidney, heart and all other organs) as an explanation for Kleiber's law [28]. The anatomical conceptualization is the basis of the metabolic compartments in the models studied in our report.
The metabolic scaling of an organ or tissue depends on both the scaling of the mass of the organ or tissue and the scaling of the metabolic rate per unit mass of the organ or tissue, i.e., the specific metabolic rate (SMR). The SMR has been measured in vitro as oxygen uptake by tissue or cell cultures from mammals of different sizes. LSLR of the data of Krebs [29] gives estimates of the scaling exponent k for SMR of -0.07 (kidney cortex), -0.07 (brain), -0.12 (liver), -0.14 (spleen) and -0.10 (lung). Estimates of k from the data of Couture and Hulbert [30] are -0.21 (liver) and -0.11 (kidney). The estimate of k from hepatocyte cell cultures is -0.18 [31].
One goal of this paper is to develop mathematical expressions for BMR that are based in part on the Heusner conceptualization and in part on results of tissue culture metabolic rate studies. A second goal is to derive predictions of the equations for BMR and to determine whether the predictions are consistent with the BMR data described above.
Assumptions and input data
The first assumption in our theory for BMR scaling is that, for each cell type contributing significantly to energy metabolism, the SMR, in the physiological state when BMR is measured, is closely approximated by a simple allometric expression . The second assumption is that, for each cell type contributing significantly to energy metabolism, the cell mass is closely approximated by a simple allometric expression . These assumptions imply that BMR scaling can be closely approximated by Equation (3), where αi = ciai and βi = k1+bi. If these assumptions are correct, Equation (3) states the tautology that the metabolic rate is equal to the metabolic rates of the tissues composing the mammalian body. This equation describes a family of scaling models with an unspecified number of parameters. Because the number of degrees of freedom is undefined, it is not possible to make a standard comparison of the goodness of fit of this general model with that of the conventional allometric power function. To evaluate whether the above assumptions can better predict the scaling of BMR, we identify relatively simple models in the family that appear to be good approximations of more complex and possibly more precise models, and we test these simple models for goodness of fit to large BMR data sets.
The scaling exponent for the mass of a number of mammalian organs or tissues is close to 1. For example, the MLE of the scaling exponent for the mass of the largest tissue, muscle tissue, calculated from the data of Weibel et al. [32] is 1.01. The MLE of the scaling exponent for tissues forming the skeleton is 1.09 [33]. MLEs of the scaling exponent for the mass of the heart, which is mostly cardiac muscle tissue, are 1.00 [34]0.99 [35] and 0.98 [36]. The MLE of the scaling exponent for the mass of the spleen, which largely comprises red and white pulp of hematopoietic origin, calculated from the data of Stahl [36] is 0.92.
The scaling exponent for the mass of skin estimated by Pace et al. [37] is 0.96. However, it would be incorrect to conclude that the mass of the most metabolically active tissue in skin has a scaling exponent of approximately 0.96. This is because skin consists of a relatively acellular tissue, the dermis, that makes up most of the mass of skin and a thin, highly cellular layer, the epidermis. Histological examination of the epidermis reveals that the thickness of metabolically active cells in the stratum Malpighi does not increase proportionally with mammalian linear body dimensions. For example, the thickness of the stratum Malpighi is approximately 10 μm and 16 μm in mice and rats, respectively, and 26 μm and 28 μm in horses and cows, respectively [38], and the scaling exponent for thickness of this layer is approximately 0.09. Combining this exponent with an estimate of the scaling exponent for the surface area of the epidermis, 0.66 [39], give the estimate 0.75 for the scaling exponent of the mass of cells in the stratum Malpighi. The scaling exponent for the dermis, which accounts for nearly all of the mass of skin, is assumed to be close to the estimate of the scaling exponent for skin, 0.96.
The scaling exponent for the mass of the gastrointestinal tract is also close to 1. However, histological examination reveals a metabolically active layer of cells forming the epithelium of the GI tract. The thickness of this layer varies from region to region in the GI tract, but for a region (e.g., colon) the thickness is nearly identical in small and large mammals [40]. Therefore, the mass of this tissue scales with intestinal surface area, which is assumed to be proportional to body surface area. Other tissues that may scale approximately with body surface area are the epithelial tissues of the mucous membranes of the eyes, mouth, pharynx and upper respiratory tract. One organ with a scaling exponent that is closer to that of body surface area than the scaling exponent for body volume, 1, is the brain with a scaling exponent of 0.73 [27].
The next step in deriving a useful approximation for Equation (3) is to replace sums of scaling terms with exponents that cluster around a central value by a single power function with an exponent that is equal to the central value. The αi-weighted average of the βi values in the cluster is a reasonable choice for the central value. However, estimates of αi are not available for most tissues. The unweighted average of the values of βi in the cluster is not used because it can be manipulated by subdividing an organ, e.g., subdividing the small intestine into duodenum, jejunum and ileum. The midpoint of the cluster is chosen as the exponent for the power function that approximates the sum of terms with similar values of βi. This midpoint is estimated as the midpoint of the values of ki plus the midpoint of the values of bi because values of ki are not available for certain tissues. For scaling of the brain and the epithelial tissues of the skin and gastrointestinal tract, the midpoint of the values of bi is 0.71. The midpoint of the values of ki is -0.14, the midpoint of the values of scaling exponents for the SMR of tissues reviewed in the introduction. Therefore, the scaling of the BMR contribution of this compartment, termed the epithelium-brain compartment, is approximately described by
BMReb = aebM0.57,
where aeb is a constant.
Estimates for the scaling exponents for adrenal, heart, muscle, spleen and bone tissues as well as those for non-epithelial tissues of the skin and gastrointestinal tract form a second cluster. Again, the central value of ki+bi is estimated as the midpoint, 1.00, of the bi values in this cluster plus the midpoint of ki values, -0.14, selected above. Therefore, the scaling of the BMR contribution of tissues in this compartment, termed the volume compartment, is approximately described by
BMRv = avM0.86,
where av is a constant.
Finally, the overall BMR scaling expression is approximated as the sum of the BMR approximation for the epithelium-brain compartment and the approximation for the volume compartment, giving the two-parameter expression
BMR = aebM0.57 + avM0.86. (4)
This two-compartment model does not include the scaling of liver and kidney tissue, which is between the scaling of the epithelium-brain compartment and the volume compartment. The liver-kidney compartment is omitted because it does not affect the asymptotic behaviour of the model and because we choose to first test the usefulness of the simplest examples of Equation (3), which are two-compartment models. In the following section we compare the goodness of fit of (4) with that of the single compartment model of Equation (1) and with that of the general two-compartment allometric model
Note that the slope, d log(BMR)/d log(M), is determined by a single parameter, the ratio aeb/av, for Equation (4) and is determined by three parameters, β1, β2 and α1/α2 for Equation (5).
The first prediction for these multi-compartment allometric models is that log(BMR) expressed as a function of log(M) has a slope that is strictly increasing. This can be seen by writing the sum of power functions in Equation (3) in order of increasing magnitude of the term scaling exponent as
where y = ln(M) and βi ≠ βj for i ≠ j. We define and rewrite the above equation as
F(y) = F1(y) + F2(y) + ... + Fn(y). (6)
We next express d ln(F)/dy as
βn- [(βn-β1)F1(y)/F(y) + (βn-β2)F2(y)/F(y) + ... + (βn-βn-1)Fn(y)/F(y)].
Because each term (βn-βi)Fi(y)/F(y) is positive and strictly decreasing as y and M increase, the above derivative is strictly increasing.
The second prediction is that the above slope approaches βn as M increases (and y increases) and approaches β1 as M decreases (and y decreases). The asymptotic behaviour for large M follows from the observation that each term (βn - βi)Fi(y))/F(y) in the above derivative goes to 0 as y and M increase. The asymptotic behaviour for small M follows from writing the derivative as
β1 + [(β2-β1)F2(y)/F(y) + (β3-β1)F3(y)/F(y) + ... + (βn-β1)Fn(y)/F(y)],
and noting that each term [(βi-β1)Fi(y)/F(y) approaches 0 as y decreases and M approaches 0.
The third prediction is that log(M) is approximately described by
log(BMR) = A + B [log(M)] + C [log(M)]2, (7)
where C is positive. To derive Equation (7), we modify the analysis of Painter and Marr [41] developed for continuous statistical distribution functions. For a specified value of M, we treat the numbers βi as discrete random variables. The probability associated with βi is pi = αi/(∑ai) assuring that ∑pi = 1. Because M is fixed, the second-order Taylor's expansion of Fi(y) about the mean value E(βi) = ∑[piβi] is
where S = (∑ai). Substitution of the Taylor's expansions of all Fi(y) into Equation (6) gives
Substitution of E(βi) for ∑[piβi] and 1 for ∑pi gives where Var(βi) denotes ∑[pi(βi - E(βi))2], the variance of βi. The approximation ln(1+x) = x, gives
ln(BMR) = ln(S) + E(βi) ln(M) + 1/2 Var(βi)[ln(M)]2.
The equivalent expression for log10(BMR) is
log10(BMR) = log10(S) + E(βi) log10(M) + 1/2 Var(βi)ln(10)[log10(M)]2 (8)
For symmetrical distributions, the approximations used to derive the above formula underestimate the second derivative. The maximum value of the second derivative of log(BMR) with respect to log(M) can be calculated by defining a second distribution fi = Fi(y)/F(y). By differentiation of lnF(y) with respect to y, it can be shown that the second derivative reaches a maximum when ∑[βifi - (∑βifi)]3 = 0, i.e., the third moment of the distribution is 0. The value of M where this occurs is obviously in the range of the values of βi. The value of the second derivative at this point is equal to the fi-weighted variance of βi values.
For the model in Equation (4), the slope is predicted to increase from approximately 0.57 to approximately 0.86 (Prediction 2), and the second derivative reaches a maximum (curvature) at the size M = Mm, where Mm satisfies aebMm0.57 = avMm0.86. At this value of M, the epithelium-brain compartment and the volume compartment contribute equally to BMR. The second derivative of log10(BMR) with respect to log10(M) at this value is the fi-weighted variance, [(0.86 - 0.57)/2]2, multiplied by ln(10). This product is 0.048. If Equation (8) is used to estimate the second derivative, the coefficient of [log10(M)]2 in Equation (7) is estimated to be 0.024.
Evaluation of model predictions
Table 1 shows that the slope from LSLR of BMR data from mammals weighing less than 0.2 kg is less than 2/3 for both the data of Heusner [10] and the data of White and Seymour [11]. For both of these data sets, the slope is greater than 3/4 for mammals weighing more than 10 kg. Remarkably, the 95% CIs for the slope of small mammals (<0.2 kg) and large mammals (>10 kg) from the White and Seymour data have no overlap. These results are similar to results of earlier investigations [10,12]. The CIs for the slope of the regression line for small mammals are consistent with Prediction 2 as are the CIs for the slope of the regression line for large mammals.
Table 1 Results of regression analysis of the logarithm of basal metabolic rate on the logarithm of body mass.
Body mass n Slope (95% CI) Reference
0.0025 – 367 kg 391 0.707 (0.691 – 0.724) 10
0.0025 – 0.200 kg 208 0.624 (0.608 – 0.717) 10
0.200 – 10.00 kg 150 0.707 (0.657 – 0.757) 18
10.00 – 367 kg 33 0.877 (0.700 – 1.06) 10
0.0024 – 326 kg 619 0.687 (0.674 – 0.701) 11
0.0024 – 0.200 kg 382 0.652 (0.613 – 0.692) 11
0.200 – 10.00 kg 206 0.718 (0.674 – 0.761) 11
10.00 – 324 kg 31 0.902 (0.706 – 1.10) 11
Second-order polynomial regression of log10(BMR) yields a coefficient of [log10(M)]2 of 0.038 with 95% CI of 0.026 - 0.049 from the data of Heusner and yields a coefficient of [log10(M)]2 equal to 0.030 with 95% CI of 0.019 – 0.042 from the data of White and Seymour. The estimate for the coefficient of the second-order term, 0.024, from Taylor's approximation and the maximum value of the second derivative, 0.048, bracket the MLE for C calculated from both the Heusner and the White and Seymour data. Curvature of similar magnitude has been noted by second-order polynomial regression of BMR data (8) and breathing rate data [42].
Table 2 lists the minimal SSR for empirical values of log(BMR) when Equations (1), (2), (4), (5) and (6) are used to predict log(BMR). The two-parameter model of Equation (4) and the three-parameter model of Equation (7) fit the data approximately equally well, and these models fit the data better than the conventional allometric scaling model described by Equation (1) does. For the optimal fit of the four-parameter model of Equation (5) to the data of White and Seymour, β1 and β2 are 0.56 and 0.91, respectively, and the MLE for α1/α2 is 0.57/0.43. The MLE estimates for β1 and β2 are close to the exponents in Equation (4) estimated from data gathered by necropsy and in vitro studies. Therefore, it is not surprising that the fit of Equation (5) is only slightly better than the fit of Equation (4).
Table 2 Minimal sum of squares of residuals (SSR) and P values from the F test for reduction of variance for models that predict the basal metabolic rate
Model SSR P* SSR/n SSR P* SSR/n
Kleiber's Law 18.87 0.0306 12.99 0.033
Equation (1) 16.62† 0.057† 0.0269† 12.35‡ 0.28‡ .0316‡
Equation (4) 15.98† 0.019† 0.0258† 11.26‡ 0.070‡ .0288‡
Equation (5) 15.90† 0.017† 0.0257† 11.17‡ 0.065‡ .0286‡
Equation (7) 15.93† 0.018† 0.0257† 11.13‡ 0.065‡ .0280‡
* P value for reduction of variance calculated using the F test. The variance in the numerator is the variance from the optimal fit of Kleiber's law.
† Calculated using data from reference 11
‡ Calculated using data from reference 10
When the F test for reduction of variance is used to compare the fit of Kleiber's law, Equation (2), to the other models using the Heusner data, none of the models fits significantly better, but the probability of the calculated F statistic for models (4), (5) and (7) is close to 0.05. When the F statistic is calculated using the White and Seymour data, the fit of models (4), (5) and (7) is significantly better than the fit of Kleiber's law using the P < 0.05 criterion.
If Equation (4) and Equation (5) are good approximations and if the parameters in these equations are accurately estimated, they should predict BMR values from body-weight data that are consistent with measured values. A relevant measure of consistency is the scaling exponent from LSLR. Table 3 lists scaling exponents from BMR prediction using body-weight data from Heusner [10] and from Kleiber [1,3]. Parameters used for the predictions are MLEs from fitting the equations to the data of White and Seymour, which is the most powerful data set for this purpose. Each of the scaling estimates from analysis of predicted BMR data is within the experimentally defined confidence interval for the scaling exponent. Note that no information on metabolic rates in the studies of Heusner and Kleiber is used in generating the BMR predictions.
Table 3 Scaling exponents from LSLR of BMR predictions using Equation (4) or Equation (5) with parameters that optimise the fit to data of White and Seymour.
Source of body-weight data BMR predictions based on:
Equation (4) Equation (5)
Heusner (10) 0.701 0.704
Kleiber (1, 3)† 0.728 0.744
† To make the data of Kleiber comparable to other data sets analysed, multiple data points for a species were replaced by a single data point calculated as the average value of body weight and the average value of BMR for the species. The MLE and 95% CI for the scaling exponent calculated from LSLR are, respectively, 0.750 and 0.728 – 0.771.
Discussion
In comparing Equation (1), the conventional allometric model, with the 2-parameter alternative, Equation (4), it is clear that the presence of positive curvature of the logarithm of BMR versus the logarithm of body mass is correctly predicted by Equation (4) and not by Equation (1). For both small mammals and large mammals, Equation (4) yields predictions for the slope of BMR data that are in the CIs for the slope determined from LSLR of large data sets. Equation (1) yields no predictions of slopes. In addition, the exponents in Equation (4) are based on published scaling relationships for organs, on well-known patterns of cell organization in animal tissues and on a repeatedly documented, although poorly understood, relationship between cell metabolic rate and body mass that was discovered by Krebs [28]. In Equation (1), the scaling exponent is a fitted parameter with little support of prior information other than past examples showing that it is a useful predictive tool.
When BMR predictions are made by Equation (4) and (5) using MLEs of parameters calculated by fitting the White and Seymour data, scaling exponents from LSLR of predictions based on body-weight data of Kleiber are greater that those from LSLR of body-weight data of Heusner. The difference between scaling exponents for these two data sets is the result of very different distributions of body weight in the two data sets. Kleiber [1,3] included only one small mammal (< 0.2 kg) in his first analysis of 13 data points and one small mammal in his second analysis (26 data points). In the data sets of Heusner [10] and White and Seymour [11], well over one-half of the mammals weigh less than 0.2 kg. Furthermore, 61 percent of the mammals in Kleiber's data sets are large (>10 kg) while only 8 percent of Heusner's mammals and 5 percent of White and Seymour's mammals are large. Thus, it appears that the 3/4-power law was "discovered" because the BMR data that came to the attention of Kleiber were largely from studies of mammals weighing more than 0.2 kg.
The allometric cascade model may give predictions for the scaling exponent of small mammals and the exponent of large mammals that are similar to the predictions of the multi-compartment model based on the anatomical conceptualization. Indeed, these two versions of the multi-compartment model are compatible, and the scaling behavior of individual organ and tissue SMRs may be derivable from the allometric cascade model when data on tissue-specific scaling of components of the cellular energy budget are available.
While the multi-compartment scaling model predicts the positive curvature of mammalian log(BMR) data, understanding of the overall slope requires an understanding of the control of cellular SMR. One potential source for a mechanistic understanding of the control of cellular metabolic rates is the model of Demetrius [21] described in the introduction.
An alternative mechanistic model for cell and tissue SMR can be developed from a tissue blood flow model similar to the pulmonary venous flow capillary pressure (PVFCP) model [43]. A key assumption of this model is that the cardiac output rate at the maximum metabolic rate (MMR) is determined by a critical value of pressure in pulmonary capillaries. When the pressure in capillaries rises above the critical pressure, pulmonary edema develops, and the rate of uptake of oxygen into blood in the capillaries decreases.
As explained in the PVFCP model, pressure in capillaries necessarily falls as the rate of blood flow in a tissue decreases (assuming that the level of constriction in veins draining the tissue does not change). Tissue fluid and the fluid in lymph come from blood in capillaries when the pressure is above the oncotic pressure (approximately 20 mm Hg). Consequently, there is a critical capillary blood pressure below which the supply of tissue fluid and lymph becomes inadequate. If it is assumed that, in the basal metabolic state, blood flow in a tissue or organ is the flow that generates this critical pressure and that tissue metabolic rate is proportional to blood flow, then tissue SMR is predicted to fall as tissue or organ size increases.
Another possible source of a correct explanation for cellular metabolic rates may come from anatomical and biochemical studies. Experimental investigations of cells from mammals of different size suggest that this control may be related to cell membranes. As reviewed by Hulbert and Else [44] cellular SMR is correlated with the polyunsaturated fatty acid content of cell membranes. In cultured hepatocytes, the cellular SMR is correlated with the surface area of inner mitochondrial membranes per gram of cell mass [45,46]. The mechanisms for controlling the polyunsaturated fatty acid composition of membranes and the surface area of mitochondria are unknown. Their discovery may complete our understanding of the scaling of the BMR.
Conclusion
The multi-compartment allometric model follows directly from observations on the scaling of tissues and organs and from observations on the scaling of tissue SMRs. The simplest multi-compartment allometric model, the two-compartment model, fits BMR data significantly better than Kleiber's law does and explains the upward curvature observed in BNR.
Acknowledgements
I thank D. Rice, K. Stewart and C. Salocks for their reviews and comments and thank D. Rice for polynomial regression analysis.
==== Refs
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Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-2-401619754910.1186/1742-4682-2-40CommentaryUncertainty principle of genetic information in a living cell Strippoli Pierluigi [email protected] Silvia [email protected] Francesco [email protected]'Addabbo Pietro [email protected] Lorenza [email protected] Federica [email protected] Luca [email protected] Raffaella [email protected] Paolo [email protected] Maria [email protected] Flavia [email protected] Center for Research in Molecular Genetics "Fondazione CARISBO", Department of Histology, Embriology and Applied Biology, University of Bologna, Via Belmeloro 8, 40126 Bologna (BO), Italy2 Department of Physics, University of Bologna, Via Irnerio 46, 40126 Bologna (BO), Italy; Sezione INFN, Bologna, Italy3 Dipartimento di Genetica e Microbiologia, University of Bari, 70126 Bari, Italy2005 30 9 2005 2 40 40 19 7 2005 30 9 2005 Copyright © 2005 Strippoli et al; licensee BioMed Central Ltd.2005Strippoli 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
Formal description of a cell's genetic information should provide the number of DNA molecules in that cell and their complete nucleotide sequences. We pose the formal problem: can the genome sequence forming the genotype of a given living cell be known with absolute certainty so that the cell's behaviour (phenotype) can be correlated to that genetic information? To answer this question, we propose a series of thought experiments.
Results
We show that the genome sequence of any actual living cell cannot physically be known with absolute certainty, independently of the method used. There is an associated uncertainty, in terms of base pairs, equal to or greater than μs (where μ is the mutation rate of the cell type and s is the cell's genome size).
Conclusion
This finding establishes an "uncertainty principle" in genetics for the first time, and its analogy with the Heisenberg uncertainty principle in physics is discussed. The genetic information that makes living cells work is thus better represented by a probabilistic model rather than as a completely defined object.
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Background
The formal problem of knowing the genome sequence in a living cell
We pose the formal problem: can the genome sequence forming the genotype of a given living cell be known with absolute certainty so that the cell's behaviour (phenotype) can be correlated to that genetic information? Firstly, the genome being the cell's DNA content [1], we define the description of the total genetic information "I" (the cell's genome sequence, forming its genotype) as a matrix comprising the linear base sequences for the distinct genomic DNA molecules in that cell (Fig. 1). For the purpose of this discussion, a living cell (prokaryotic or eukaryotic, from a monocellular or multicellular organism, germinal or somatic) is able to perform all its normal natural functions (operatively, capacity for division and/or development into an organism, and/or performance of the functions typical of its terminally differentiated state). A consensus sequence is a sequence created by choosing, for each position, the most representative base in a set of aligned DNA sequences. It should be noted that all genomic sequences provided by modern genome projects (e.g. human) [2,3] are actually consensus sequences for different homologous chromosomes (in the case of diploid cells), different cells [4], and, often, different individuals. It is worth emphasizing that there is no formal proof that such a "mean" sequence would work in a real cell. Furthermore, each living cell experiences continuous progression from one state, i.e. a pattern configuration of the system at a particular instant, to another [5], and even in a non-dividing cell the genome structure is subjected to dynamic changes over time due to DNA modifications, lesions and repair [6]. However, for the purpose of discussing the problem posed above, we assume the existence of a completely defined cell genomic DNA sequence that is determined at a certain "time zero" instant.
Figure 1 Formal representation of total cellular genetic information. Each matrix column should contain the sequence of each distinct DNA molecule strand in the cell (e.g. human sequence data), because mutations first arise only in one strand, and telomeres normally have a protruding single-strand of variable length.
We propose three thought experiments to show how "I" could be determined with absolute certainty in a living cell, assuming that, after determination of the genome sequence, the original cell is further available for tracing its behaviour, simulating or verifying predictions about its genotype/phenotype relationships, or obtaining derivative cells or organisms.
The most common method used is to isolate the cell's DNA molecules and sequence them by enzymatic or chemical manipulations. In the case of a single cell, several technical problems must be faced: it is difficult to extract the very small amount of DNA without damaging it, and the requisite in vivo or in vitro amplification of the molecules may add artifactual mutations. However, for the purpose of this discussion, we hypothesize that a suitable method could be devised. Even in this case, however, knowledge of "I" would coincide with the irreversible unavailability of the original cell to exploit that biological information.
An alternative to traditional DNA sequencing could be direct imaging of the DNA molecules, at a level of resolution sufficient to read its sequence. In principle, this method could be extended to reading the DNA sequence inside a living cell ("Star Trek" method) [7]. By definition, the wavelength used to image the DNA sequence would have to be adequate for resolution in the order of the atomic radius (~0.1 nm), so high frequency and energy (>10 keV) are physically inevitable. If a single cell were irradiated with >10 keV waves in order to image each segment of the millions or billions of base pairs constituting its DNA (10-9–10-6 J absorbed, respectively, even hypothesizing one particle for each base pair) it could not survive this irradiation, which is several orders of magnitude greater than the lethal dose (~1000 rad [8] = 10 Gy, i.e. ~10-11 J/ng). In addition, it has recently been demonstrated that secondary free electrons, even at energies well below ionization thresholds, induce single- and double-strand breaks in DNA [9], thus in any case modifying the original genetic information "I" in the cell.
Scanning probe microscopes are based on a new concept of very high-resolution imaging, and they are being studied as a method for DNA sequencing [10]. Although they do not use high-energy radiation, these instruments deploy a microscopic tip that scans the molecule surface from very close range. Their suitability for DNA sequencing depends critically on the successful preparation of DNA on a surface [10], which is again not consistent with the maintenance of cell integrity.
A different method for deriving the sequence of a DNA molecule based on assessment of its energetic state, without needing to "visualize" its molecular shape, has been discussed on purely theoretical grounds [11]. It has been shown that an uncertainty relationship emerges between temperature and the order (negative entropy) of the DNA molecule [11]. This makes it impossible to reach absolute certainty about the structure of the DNA, even if this method should become technically feasible and shown to be applicable to DNA in living cells.
The only remaining method appears to be genome sequencing of a cell with supposedly identical genetic information. This procedure will destroy the test cell, leaving an equivalent living cell available for observation. The most adequate test cell would be a direct relative of the cell to be studied (Fig. 2). However, any cell is separated from its nearest relative by at least one cell division. In this process, a copy of the genome is made and each copy is distributed to the two daughter cells. The DNA replication process is central to cell life, and it is accomplished by complexes of copying and proofreading enzymes. These proteins are molecular machines subject to the laws of thermodynamics [12], and their effectiveness cannot be 100 percent; thus, replication errors inevitably accumulate during successive cell divisions [13,14]. These errors lead to changes in the original sequence (mutations, including polymorphisms and pathogenic mutations), and the "mutation rate" for a given organism can be defined as the number of changed positions (in base pair, bp) for each cell per generation [14]. The mutation rate is, in nature, greater than 0, so in each cell a certain number of base pairs is likely to differ from those in the initial genome.
Figure 2 Determination of total genetic information of a cell genome: nearest relative analysis; in this case, even sequence identity among multiple cells from a common ancestor "C" (e.g. C2.1 and C2.3) is not formal proof of sequence identity with the other extant cells (e.g. C2.2 and C2.4). For simplicity, only the sequence of one strand is shown.
Results and Discussion
Uncertainty principle of genetic information in a living cell
In view of the above-described thought experiments, we conclude that in a genome of total size "s" (measured in bp), the average number of mutated base pairs, used as a measure of uncertainty (U) about its actual sequence in a living cell, can be quantified by:
U ≥ μs (1)
where μ is the mutation rate of the cell type under consideration. For example, in the human genome, uncorrected replication errors occur with a frequency varying between 10-9 and 10-11 per incorporated nucleotide [14], depending in particular on the type of genome region [15,16]. Considering the total length of the human genome sequence (~6 × 109 bp), the overall uncertainty in the identity of the whole sequence is between 6 and 0.06 nucleotides per replication, meaning in the latter case that one cell will have a probability of 6 percent of having one mutation per replication. For simplicity, we do not consider other possible but less frequent contributions to overall mutation deriving from the distribution, rather than replication, of nuclear or mitochondrial DNA molecules [14].
It should also be noted that any conceivable method for measuring the incorporation of nucleotides to determine the actual sequence in a living cell will similarly entail an error proportional to the mutation rate, because the accuracy of any such method is ultimately dependent on the accuracy of the DNA replication machinery.
In the case of stem cell replication, it is possible that the same original "immortal strand" is continuously retained by an undifferentiating stem cell, while the newly synthesized strand is asymmetrically distributed, at the next cell replication, to the differentiating daughter cell [17]. In this selected case the sequence of a stem lineage cell (e.g. cells C, C1.1 and C2.1 in Fig. 2) could be derived from the consensus sequence from randomly mutated differentiating daughter cells (e.g. C1.2, C2.2 and so on in Fig. 2). However, at each moment, the stem cell also retains a newly synthesized and potentially mutated strand, the sequence of which can only be known with an associated uncertainty that is, again, proportional to the mutation rate. This does not allow the matrix in Fig. 1 to be completed with absolute certainty for that cell.
The actual genome sequence in any living cell can thus be known only with a certain amount of indeterminacy, which may be very small but is always greater than 0 because of fixed physical constraints dictated by the cell structure itself and by formal limits on any process for determining DNA sequences without disrupting the cell. These limits are in turn intrinsically related to the submicroscopic scale of genetic information in nature, independently of any methodological approach or any current or future technological device. The importance of any single base pair for the phenotype cannot be over-emphasized, as exemplified, for example, by the case of human achondroplasia (short-limb dwarfism), in which a single base substitution in a single chromosome invariably has dramatic effects on skeleton growth [18] via a single amino acid change.
In addition, there is growing evidence that genomic regions other than classical gene protein-coding regions have biological function. Changes in the 5' or 3' untranslated regions of mRNAs have been recently related to disease phenotypes [e.g. [19,20]]. Many types of functional "noncoding" RNAs [21] may be transcribed from non-genic regions or from the opposite DNA strand in protein-coding genes, even in classical constitutive heterochromatin zones. For instance, yeast centromeric repeat sequences have recently been shown to be transcribed and then processed by components of the RNA interference (a sequence-specific gene silencing) pathway [22]. Finally, even mutations in coding regions previously deemed "silent" (mutations that do not affect the amino acid sequence) may have phenotypic effects via their influence on splicing accuracy or efficiency [23]. In general, organisms with larger genome sizes tend to have a greater number of deleterious mutations, and it has been estimated that, in humans, the deleterious genomic mutation rate is high [24]; it should also be noted that many phenotypic changes induced by variations in a particular genomic region could be present but could go undetected if they do not grossly affect morphology and physiology and if they are not directly, actively searched. Overally, this information clearly indicates that the relevance of small numbers of subtle mutations in a single cell may be high, particularly if this cell is the founder of a new organism or a new colony of individuals. Thus, although the connectivity of networks between genes and transcription factors and the complexity achieved by genetically encoded information-processing systems such as nervous and immune systems add further dimensions to biological complexity [25], it is important to establish whether the genetic information of a living cell may be known definitely in its entirety.
The uncertainty principle discussed here should not be confused with the critique of biological determinism, which states that, given a certain piece of biological information, we cannot confidently predict the behaviour of the whole cell or organism because of the complex relationships between genotype and phenotype [26]. Uncertainty has been also proposed in biology in respect of the full understanding of gene function. Owing to effects of gene function that are possibly important for long-term fitness within a population but very small in individuals, the formal elucidation of gene function could require experiments on an evolutionary scale, involving the whole population of the relevant species [27]. Finally, a purely qualitative uncertainty relationship has been put forward between the degree of molecular perturbation in the cells investigated and the number of biological pathways simultaneously examined by the "array" approach (able to monitor genome-wide DNA expression profiles) [28]. In these and similar discussions it is assumed that the cell genome is a known starting point and the problem lies in predicting how epigenetic changes (DNA modifications that can alter gene expression without changing DNA sequences), RNA editing (post-transcriptional RNA modification), post-translational protein modification or any other intracellular or extracellular interacting factor might affect the expression of genetic information.
Our concept applies upstream of these problems: defining intrinsic uncertainty in the knowledge of a complete, actual genotype, to be further related to a phenotypic/functional outcome. This type of uncertainty also reinforces arguments against the reductionist approach to biology, i.e. the attempt to explain complex phenomena by listing all the individual components of multicomponent systems and defining their functional properties [29]. Systems biology has recently emerged as the successor to reductionism, seeking to predict the behaviour or "emergent properties" of complex, multicomponent biological processes by trying to understand the general picture rather than the sum of the workings of the parts in isolation [29]. Although systems biology could cope with indeterminacy in the formal knowledge of the complete cell "parts list", including its complete genome sequence, its models always remain subject to an irreducible degree of unpredictability due to the sum of intractable uncertainties at each successive level of investigation from genes to the whole organism.
Possible practical implications of the uncertainty principle of genetic information in a living cell concern problems such as in silico cell modeling and the diagnostic value of specific methods. These implications will need further specific investigation and discussion.
Genomics and the physical limits of the knowledge
We have presented here the first uncertainty principle to be announced in structural genomics. This is an addition to the uncertainty principles in physics, where Heisenberg established that it is impossible to know the position and the momentum of an electron simultaneously with absolute certainty (Heisenberg's uncertainty principle) [30], and in mathematics, where Gödel showed that a great variety of logical systems contain formally undecidable propositions [31].
In the broadest sense, statements of this type all demonstrate the formal impossibility of knowing a given system at a desired arbitrary level [32], although in his 1927 article Werner Heisenberg insisted that the uncertainty he described is not due to technical or intrinsic features of the measuring process, but it is a fundamental feature of reality itself, i.e. an electron cannot in principle have a precise position and momentum simultaneously. It is interesting to note that in his 1933 lecture "Light and life" [33], Niels Bohr applied an analogous uncertainty concept in biology to argue that a living being would be killed by detailed physical investigation, so there is "complementarity" between the simultaneous existence of life and the possibility of describing it scientifically. Bohr concluded that life "must be considered an elementary fact that cannot be explained" (although in his later 1962 revisitation of the problem [34] he avoided any reference to incompatibility between scientific description and existence of life, possibly influenced by results in molecular biology obtained by his student Max Delbrück [35]). In our case, instead, uncertainty arises from the intrinsic impossibility of determining a physical quantity that nevertheless exists (the real genome sequence present at a given instant within a living cell).
However, if we consider the evolution of the state of a system, the analogy may still hold: in physics, the Heisenberg principle affects any attempt to determine the future behaviour of an atomic particle in a certain position; in genetics, the future biological behaviour of a living cell cannot be linked with absolute certainty to the positions of nucleotides in the current genome sequence. For a living cell, we can only determine a "consensus" sequence from its relatives, and this fluctuates with a certain probability around the actual sequence. Recently, the concept that an ideal "average cell" exists has been challenged in respect of gene expression, and it has been shown that, although expression at the cellular level does not require tight specifications and there is high tolerance of variation, each single nucleus is probabilistic in its expression repertoire [36].
Finally, we note that replication errors leading to spontaneous point mutations arise from transient alternative states of the DNA base functional groups (tautomeric shifts [37], base ionization [38]). Precise knowledge of the quantum jump events in the base molecule could allow subsequent copy errors to be predicted [39,40], but the Heisenberg principle does not allow this with complete certainty. In this sense, the Heisenberg principle is not only analogous to the genetic information uncertainty principle, but is profoundly relevant to the roots of the latter.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All authors contributed to define the concept that we present; they all drafted the manuscript and approved the final version.
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Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-2-411620214210.1186/1742-4682-2-41ResearchVolume of the effect compartment in simulations of neuromuscular block Nigrovic Vladimir [email protected] Johannes H [email protected] Anton [email protected] Shashi B [email protected] Department of Anesthesiology, Medical University of Ohio, Toledo, OH, USA2 Research Group for Experimental Anesthesiology and Clinical Pharmacology, University Hospital Groningen, Groningen, The Netherlands3 Department of Anesthesiology and Critical Care Medicine, Leopold-Franzens University, Innsbruck, Austria, and Department of Environmental Sciences, The Swiss Federal Institute of Technology, Zürich, Switzerland2005 3 10 2005 2 41 41 2 9 2005 3 10 2005 Copyright © 2005 Nigrovic et al; licensee BioMed Central Ltd.2005Nigrovic 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 examines the role of the volume of the effect compartment in simulations of neuromuscular block (NMB) produced by nondepolarizing muscle relaxants.
Methods
The molar amount of the postsynaptic receptors at the motor end plates in muscle was assumed constant; the apparent receptor concentration in the effect compartment is the ratio of this amount and the volume arbitrarily assigned to the effect compartment. The muscle relaxants were postulated to diffuse between the central and the effect compartment and to bind to the postsynaptic receptors. NMB was calculated from the free concentration of the muscle relaxant in the effect compartment.
Results
The simulations suggest that the time profiles of NMB and the derived pharmacokinetic and pharmacodynamic variables are dependent on the apparent receptor concentration in the effect compartment. For small, but not for large, volumes, times to peak submaximal NMB are projected to depend on the magnitude of NMB and on the binding affinities.
Conclusion
An experimental design to estimate the volume of the effect compartment is suggested.
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Background
In the majority of the pharmacokinetic-pharmacodynamic (PK-PD) models proposed to simulate neuromuscular block (NMB) [1-3], the volume of the effect compartment is postulated to be negligibly small or the compartment is postulated to contain a negligibly small amount of the muscle relaxant. The models simulate NMB based on the concentration of the muscle relaxant in this compartment using the equation of Hill. Binding of muscle relaxants to the postsynaptic receptors at the motor end plates is not considered. Because muscle relaxants produce NMB by binding to these receptors, consideration of the interaction of muscle relaxants with the receptors represents a more realistic approach and an advancement in simulations [4-6]. Donati and Meistelman [5] were the first to consider binding of muscle relaxants to the receptors. These investigators suggested that the receptor concentration in the effect compartment is 2.8·10-7 M, but the volume of the effect compartment was assumed to be negligibly small. Given a fixed amount of postsynaptic receptors, a finite receptor concentration is not compatible with a negligibly small volume of the effect compartment.
We decided to examine the role of the volume of the effect compartment in a pharmacokinetic-pharmacodynamic model for NMB and were interested in answering the following questions: (1) Is it necessary to postulate a negligibly small amount of a muscle relaxant in the effect compartment? (2) Do the projections from simulations using a small or a large volume of the effect compartment differ? If so, what are the differences? (3) Can the simulations suggest an experimental design suitable to test whether the volume of the effect compartment is negligibly small or a large volume may be more appropriate?
Methods
General approach
(1) The amount of the postsynaptic receptors at the motor end plates in muscle, in terms of mol per kg body weight, was assumed constant and the receptors uniformly diluted in the effect compartment. (2) The plasma concentrations of a hypothetical muscle relaxant after administration of an intravenous bolus dose, defined by an arbitrary multiexponential equation, are labeled target concentrations. In the simulations, the target plasma concentrations fulfill the role of the experimentally determined plasma concentrations. (3) A PK-PD model was designed a priori to include an effect compartment of an assigned volume. The pharmacokinetic parameters in the model were defined by the postulate that the concentrations in the central compartment (compartment1) fit the target plasma concentrations. (4) The muscle relaxant diffuses from the central to the effect compartment. (5) Pharmacodynamic parameters were obtained from the postulate that peak neuromuscular block from a bolus ED50 dose occurs at 4.5 minutes after injection. The peak concentration of the muscle relaxant in the effect compartment at this moment corresponds to the IC50 concentration. (6) The relationship between NMB and the free concentrations of the muscle relaxant in the effect compartment is defined by the Hill equation.
The target plasma concentrations
Muscle relaxant D was postulated to display linear pharmacokinetics. The triexponential equation that defines the time course of the molar amounts of the muscle relaxant in plasma is given by (braces indicate molar amounts):
Here, N, O, and P (P = 1 - N - O) are fractions of the dose that are eliminated from plasma with the first order rate constants λN, λO, and λP, respectively. The dose is in mol·kg-1. Division of the equation by VC, VC expressed in L·kg-1, converts the amounts in plasma to molar concentrations. VC represents the volume of the space into which the muscle relaxant is uniformly diluted at time t = 0, i.e., at the moment of bolus intravenous injection.
The values assigned to the parameters in the triexponential equation were based on the following postulates: For the hypothetical muscle relaxant D, VC approximates the volume of plasma and VSS, the volume of distribution at steady state, approximates the volume of the extracellular space. The dose that produces NMB50, i.e., ED50, is defined by the postulate that the concentration in plasma at 4.5 min after bolus intravenous injection is [D]plasma = IC50. The definition of IC50 is provided below. The following values satisfy these requirements:
N = 0.71; O = 0.192; P = 0.098
λN = 1.3 min-1; λO = 0.31 min-1; λP = 0.0231 min-1
VC = 0.044 L·kg-1 VSS = 0.28 L·kg-1
Compartmental interpretation of the triexponential decay of the plasma concentrations yields the following parameters for the standard 3-compartment pharmacokinetic model assuming a mammillary arrangement of the compartments and elimination only from compartment1[7]:
V1 = VC = 0.044 L·kg-1 k10 = 0.1848 min-1
k12 = 0.3771 min-1 k21 = 0.5581 min-1
k13 = 0.4229 min-1 k31 = 0.0902 min-1
Estimation of the receptor amount
The molar amount of receptors per kg body weight was estimated based on the following assumptions: One hundred g of muscle is represented as a cube with side length of 4.64 cm, i.e., specific density of muscle ~ 1. There is 430 g muscle per kg body weight. The muscle fibers are densely packed cylinders with the diameter of 50 μm and the length of 4.64 cm (928 rows × 928 columns of fibers in a cross section perpendicular to the length of the fibers). Each muscle fiber has one motor end plate with 2.1·107 receptors at each end plate [8,9].
The PK-PD Model
The pharmacokinetic model consists of four compartments: the central (compartment1), two peripheral (compartment2 and compartment3), and the effect compartment in mammillary arrangement with elimination from the central compartment. The model is defined in terms of the amounts of the muscle relaxant present in each compartment and the amount eliminated from the body. Transport between the central and the effect compartment is defined as diffusion according to the concentration gradient of the free muscle relaxant in both compartments. As a result, at the moment when the free muscle relaxant attains the peak concentration in the effect compartment and there is no net transport between the compartments (steady state), the concentrations in the two compartments are equal. In the model, this constraint necessitates that the transport rate constant into the effect compartment, k1e, be defined in terms of the transport rate constant from the effect to the central compartment, ke1. Hence, k1e = (Ve/V1)·ke1, where Ve and V1 represent the volumes of the effect and the central compartments, respectively. The volume of the central compartment is known (V1 ≈ VC in the triexponential function). The volume of the effect compartment was assigned different values. Hence, the amounts of D in the central and the effect compartments may be converted to concentrations. Compartment2 and compartment3 are defined only in terms of the amounts present in them.
The amount of receptors in the effect compartment is constant and independent of the volume assigned to the effect compartment. A small assigned volume results in a high receptor concentration, while the concentration is low in the large effect compartment.
For an assigned volume of the effect compartment (Ve), the pharmacokinetic parameters in the PK-PD model were estimated in a two-step procedure. In the first step, the parameter ke1 was obtained using the following constraints: dose = ED50, the amounts in plasma as defined by the triexponential equation, and the maximal NMB = 50% attained at 4.5 min after administration of the muscle relaxant. In the second step, the parameters V1, k10, k12, k21, k13, and k31 were estimated using the following constraints: dose = ED50 and ke1 fixed to the value obtained in the first step. The parameters were fitted by minimizing the sum of squared differences between the logarithms for the calculated concentrations in compartment1 and the target concentrations in plasma. The evaluations were carried out at 250 time points from t = 0 to t = 25 min and at 50 points for t = 25 to t = 50 min after administration. Goodness-of fit was expressed as the coefficient of variation (CV in % 1) of the differences between the two time profiles.
Interaction between the muscle relaxant and the postsynaptic receptors was defined in terms of the association, kassoc, and dissociation, kdis, rate constants. We assumed that each receptor possesses only a single binding site for the muscle relaxant. The ratio kdis/kassoc defines the equilibrium dissociation constant, KD. The inverse of KD defines the affinity of the receptors for the muscle relaxant.
The values of all the mentioned parameters are listed in Table 1. The set of five ordinary differential equations defining the amounts of the muscle relaxant in the four compartments and the amount of the complex with the receptors in the effect compartment is presented in the Appendix.
Table 1 Pharmacokinetic and pharmacodynamic parameters for the PK-PD model. The volume of the effect compartment (Ve) was postulated to be either small (SMALL) or large (LARGE).
Parameter Unit SMALL LARGE
ED50 mol·kg-1 2.2325·10-7
V1 L·kg-1 0.0440 0.0434
k10 min-1 0.1847 0.1795
k12 min-1 0.3769 0.3574
k21 min-1 0.5587 0.7663
k13 min-1 0.4226 0.1981
k31 min-1 0.0909 0.0581
kassoc M-1·min-1 2.4·1010
{R}total mol·kg-1 1.2921·10-10
Ve L·kg-1 4.4·10-5 9.23·10-2
ke1 min-1 0.6159 0.1477
[R]total M 2.9367·10-6 1.4·10-9
Onset time min 4.50
Calculation of NMB
The intravenous bolus dose of the muscle relaxant required to produce a half-maximal NMB, NMB50, is labeled ED50. We postulated that NMB50 is attained at 4.5 min after the bolus injection. The peak concentration of the free muscle relaxant in the effect compartment established by ED50 is IC50. At 4.5 min after injection, [D]plasma = peak [D]e = IC50. The fractional receptor occupancy by the muscle relaxant (Occ) at NMB50 is labeled OccNMB50 and assigned a value of 0.875 [10]. Because KD = [D]e·(1 - Occ)/Occ, and at NMB50 [D]e = IC50 and Occ = OccNMB50 = 0.875, it follows that IC50 = 7·KD.
Neuromuscular block (NMB) was calculated using the Hill equation, the free concentrations of the muscle relaxant in the effect compartment, [D]e, and two parameters: γ and IC50 (γ = 4 and IC50 = 7·KD, Eq 1 in Appendix).
To describe quantitatively the simulated NMB as a function of doses used to establish the peak concentrations in the effect compartment, the values for NMB calculated from peak [D]e were plotted as a function of doses of the muscle relaxant. A modified equation of Hill (Eq. 2, Appendix) was fitted to these points using the program TableCurve2D from SPSS, Chicago, IL, and the fitted estimates of the exponent γf and ED50f are reported.
All calculations were performed independently using the programs MATHEMATICA (version 5.1) from Wolfram Research, Inc., Champaign, IL, MULTIFIT and PKPDFIT written by J.H. Proost, and MATLAB (version 6.1.0.450(R12.1)) from The Mathworks Inc., Natick, MA.
Results
The estimated total molar amount of receptors at the motor end plates in muscles is {R}total = 1.2921·10-10 mol·kg-1. Receptor concentration in the effect compartment is the ratio of this amount and the volume assigned to the effect compartment.
Simulations with a small or a large volume assigned to the effect compartment
For the initial simulations, Ve was assigned the value of 0.001·VC, i.e., Ve = 4.4·10-5 L·kg-1 [11], for the small and 0.0923 L·kg-1 for the large effect compartment. The latter approximates the volume of the interstitial space in muscle. Receptor concentrations in the effect compartment were: [R]total = 2.94·10-6 M and 1.4·10-9 M for the small and large volume, respectively. The hypothetical muscle relaxant D was assigned KD = 1·10-7 M. The assignment defined ED50 as ED50 = 2.23·10-7 mol·kg-1. Optimal estimates of the pharmacokinetic parameters, including ke1, were obtained as described in the Methods section. The target amounts of the muscle relaxant in plasma and those estimated in compartment1 as well as the amounts in the small and the large effect compartments are graphically presented in the upper panel of Figure 1. The three curves for the amounts in plasma overlap. The good fit of the amounts in compartment1 to the target amounts in plasma is evident from the small values of the coefficient of variation, 0.0007% for the small and 0.7% for the large volume of the effect compartment. The peak free amount of D in the small effect compartment constitutes a small fraction of ED50, 1.38·10-4. On the other hand, the peak free amount of D in the large effect compartment accounts for a sizable fraction of ED50, 0.289 (upper panel in Figure 1). The PK-PD model that includes a large effect compartment requires intercompartmental transport rate constants different from those for the small volume of the effect compartment (Table 1). The peak receptor occupancy, Occ = OccNMB50 = 0.875, and the peak [D]e = IC50 = 7·KD, were attained at 4.50 min for either volume of the effect compartment. Hence, for both volumes the simulated peak NMB = NMB50 and occurs at 4.5 min after injection, but the time course of NMB is different between the small and large volumes of the effect compartment (lower panel in Figure 1). To reach the respective peaks at 4.50 min after the injection required ke1 that was approximately four times higher for the small than for the large effect compartment (Table 1).
Figure 1 Upper panel: The amounts of muscle relaxant D (KD = 1·10-7 M) in plasma and compartment1 and the free amounts in the effect compartment assigned a small (Ve = 4.4·10-5 L·kg-1) or a large (Ve = 9.23·10-2 L·kg-1) volume. All the amounts are normalized to the injected dose (= ED50 = 2.23·10-7 mol·kg-1). Solid and dashed lines indicate the amounts contained in the small and the large effect compartment, respectively. Filled circles denote the target amounts in plasma defined by the triexponential function. The three curves for the amounts in plasma overlap. The estimates were obtained at 0.1 min intervals. Lower panel: Time course of the neuromuscular block (NMB) by ED50 of the muscle relaxant D. NMB was calculated using Eq 1 (Appendix), [D]e for the small and large volume of the effect compartment presented in the upper panel, and by setting γ = 4.0 and IC50 = 7·10-7 M. The lines are identical to those in the upper panel for the small and large volume of the effect compartment.
The calculations were verified by calculating the sum of the amounts in the four compartments plus the amount eliminated from the body. For all times between 0 and 50 min after injection, the sum was equal to ED50. Expressed as fractions of the administered dose (= ED50), the peak amounts in compartment2 and compartment3 and the times after injection when the peaks were attained are for the small volume of the effect compartment 0.199 at 1.6 min and 0.483 at 7.3 min, respectively. For the large volume, the corresponding values are 0.158 at 1.3 min and 0.268 at 11.9 min.
Two additional observations were made during these simulations. First, exclusion of the small effect compartment from the PK-PD model only minimally influences the fit of the amounts in compartment1 to the target plasma amounts. The result is not unexpected, because the intercompartmental transport rate constants (microconstants) in the model with a small volume of the effect compartment (Table 1) are close to those in the standard 3-compartment model. Second, when the effect compartment in the PK-PD model was postulated not to contain the receptors, i.e., {R}total = 0, identical values of ke1 establish the peak free amount of D in the respective effect compartment at identical times (data not presented).
Based on the derived pharmacokinetic rate constants, NMB was simulated with different doses of D. One thousand points were selected for a 10-fold increase in doses. NMB was calculated with the peak free concentrations of D in the effect compartment using Eq 1 in the Appendix (γ = 4 and IC50 = 7·10-7 M). The relationship between NMB and the doses that produced the peak concentrations differed between the models (upper panel in Figure 2 for NMB = 0.05 to NMB = 0.95, i.e., NMB05 to NMB95). To obtain a quantitative estimate for the difference, equation of Hill (Eq 2) was fitted to both sets of points to describe the relationship between NMB and the injected doses. The fit was excellent for both sets (r2 > 0.9999, the number of points, n, = 381 for the small and n = 641 for the large volume of the effect compartment). The 95% confidence interval (95%CI) for the fitted γf was 6.819 to 6.838 for the small and 4.0040 to 4.0041 for the large effect compartment. The 95%CI for the fitted ED50f were (2.235 to 2.236)·10-7 mol·kg-1 and (2.23256 to 2.23258)·10-7 mol·kg-1, respectively.
Figure 2 Upper panel: Neuromuscular block (NMB) calculated as a function of the peak concentrations of muscle relaxant D in the effect compartment using Eq 1 presented in the Appendix (γ = 4.0 and IC50 = 7·10-7 M). The doses presented along the abscissa refer to the doses that established the peak concentrations. The range of NMB is from NMB05 to NMB95. One thousand logarithmically equidistant values were used for a 10-fold increase in doses. The volumes of the effect compartment and the lines are identical to those presented in Figure 1. Lower panel: Onset times as a function of the magnitude of NMB. Onset times are defined as the times after the bolus intravenous injection of muscle relaxant D needed to establish peak NMB, from NMB05 to NMB95. Other details are identical to those in the upper panel.
The onset times for NMB05 to NMB95 differed between the models assigned different volumes of the effect compartment (lower panel in Figure 2). The model with the large effect compartment projected that the onset times were nearly independent of the magnitude of NMB. The model incorporating a small volume of the effect compartment projected an inverse relationship between the onset times and the magnitudes of NMB. For NMB < NMB50, the onset times were longer, and for NMB > NMB50 the onset times were shorter than those projected by the model with a large effect compartment.
Simulations with different volumes assigned to the effect compartment using ED50
Next, the influence of the volume assigned to the effect compartment was examined systematically. The volumes varied from 1·10-6 to 1·10-1 L·kg-1 (11 logarithmically equidistant values). The pharmacokinetic parameters, including ke1, were estimated as previously stipulated, i.e., ED50 dose establishes peak receptor occupancy = Occ875 and peak [D]e = IC50 at 4.5 min after injection. The coefficient of variation for the fit of the concentrations of D in compartment1 to the target plasma concentrations was CV = 0.84% for the largest and = 0.0006% for the smallest volume. The values for ke1 as a function of the assigned volumes, estimated with ED50 and using the same muscle relaxant (KD = 1·10-7 M), are presented in the upper panel of Figure 3. The results demonstrate that ke1 increases markedly for the smaller values of Ve. The relative amounts of D bound to the receptors, the amounts free in the effect compartment, and the ratio of the bound to the total amount in the effect compartment with ED50 show (lower panel in Figure 3) that for all volumes the amounts of D bound to the receptors are constant. For smaller volumes the bound amounts make up nearly all of D present in the effect compartment, while for larger volumes the total amount of D is nearly completely accounted for by the free amount.
Figure 3 Upper panel: Values estimated for the transport rate constant between the effect and the central compartment, ke1, as a function of the volume assigned to the effect compartment, Ve. The dose of the muscle relaxant D = ED50 = 2.23·10-7 mol·kg-1 and KD = 1·10-7 M. Lower panel: Amounts of the muscle relaxant D (left Y-axis) bound to the receptors (filled upright triangles) and the amounts free (filled diamonds). The amounts are normalized to the injected dose presented in the upper panel. The ratio of the bound to the total amounts (empty circles; total = bound + free; right Y-axis) in the effect compartment is presented as a function of the volume assigned to the effect compartment.
Simulations using different volumes and different doses
We used the set of pharmacokinetic parameters obtained for each assigned volume, but now varied the dose using 1000 values for a ten-fold increase. The results are presented in Figure 4. Increasing doses increase the peak free concentrations of D for each volume of the effect compartment (upper panel in Figure 4). The increase is steepest for the smallest volume and the slopes decrease for the larger assigned volumes. The estimated peak free concentrations of D in the effect compartment were used to calculate NMB (IC50 = 7·10-7 M and γ = 4, Eq 1 in Appendix). The values of NMB from NMB05 to NMB95 as calculated using [D]e were plotted against the injected doses separately for each assigned volume, similarly to the results presented in the upper panel of Figure 2. The modified equation of Hill (Eq 2, Appendix) was fitted to each of these 11 sets of points to define NMB as a function of the injected doses. The fit was excellent (r2 > 0.9996 for n between 314 to 641 points). The fitted values of γf are presented in the lower panel in Figure 4. The values increase markedly for smaller volumes. The 95%CI for the eleven fitted estimates of ED50f varied between (2.225 to 2.226)·10-7 mol·kg-1 for the smallest and (2.25720 to 2.25722)·10-7 mol·kg-1 for the largest volume. These simulations permitted us to estimate the times to NMB05 and NMB95 (onset times). The results are presented in the lower panel of Figure 4. Onset times for NMB05 and NMB95 differ widely for the small volumes, but the differences progressively decrease for larger volumes of the effect compartment. The onset times for NMB05 and NMB95 are nearly identical for the largest assigned volume.
Figure 4 Upper panel: The peak free concentrations of muscle relaxant D in the effect compartment calculated with variable doses of D as a function of the volumes assigned to the effect compartment. The assigned volumes were: 1·10-6, 3.16·10-6, 1·10-5, 3.16·10-5, 1·10-4, 3.16·10-4, 1·10-3, 3.16·10-3, 1·10-2, 3.16·10-2, and 1·10-1 L·kg-1. The bold solid and the dotted lines indicate the lowest and the highest assigned volumes, respectively. Concentrations for the intermediate volumes are indicated in sequence by thin solid lines. The three dashed lines parallel with the X-axis represent the free concentration of D for NMB95 (IC95, upper line), for NMB50 (IC50, middle line) and for NMB05 (IC05, lower line). The concentrations for IC05 and IC95 were calculated based on γ = 4.0 (Eq 1, Appendix). Lower panel: Times to NMB05 (open circles) and NMB95 (filled circles, left Y-axis) as a function of the volumes assigned to the effect compartment, Ve. Neuromuscular block was calculated using Eq 1 (IC50 = 7·10-7 M, γ = 4) and the peak free concentrations of D presented in the upper panel. The values of the exponent γf (filled diamonds, right Y-axis) were obtained by fitting Eq 2 to the calculated NMB.
Simulations with different binding affinities assigned to muscle relaxants
The PK-PD model was also tested with two additional muscle relaxants using the previously defined small and large volumes of the effect compartment. One muscle relaxant, D2, was assigned a 10 times lower affinity for the binding sites at the receptors, KD2 = 1·10-6 M. The other, D3, was assigned a 10 times higher affinity, KD3 = 1·10-8 M. The assignments changed the respective kdiss, but not kassoc. Two series of simulations were performed. In the first series, all the pharmacokinetic constants, including ke1, were those defined previously for either the small or the large volume of the effect compartment and for the muscle relaxant with KD = 1·10-7 M (Table 1). A 100-fold increase in affinities was projected to require 16.3 times lower ED50 for the small volume (ED50 = 1.144·10-6 mol·kg-1 for D2 and ED50 = 7.018·10-8 mol·kg-1 for D3), but a 98.9 times lower ED50 (ED50 = 2.230·10-6 mol·kg-1 for D2 and ED50 = 2.255·10-8 mol·kg-1 for D3) for the model with a large volume of the effect compartment. For the small effect compartment, the times to reach NMB50 were 1.82 min for D2 and 34.74 min for D3. In the model with a large effect compartment, the times to NMB50 differed only minimally, from 4.50 min for D2 to 4.54 min for D3.
In the second series of simulations, we postulated that ED50 of either D2 or D3 produces NMB50 at 4.5 min after injection using either the small or the large volume of the effect compartment. The doses producing NMB50 were related to the assigned KD values, i.e., ED50 = 2.2325·10-6 mol·kg-1 for D2 and ED50 = 2.2325·10-8 mol·kg-1 for D3. These doses establish plasma concentrations at 4.5 min [D]plasma = IC50 = 7·KD = peak [D]e. In the model containing a small volume of the effect compartment, the postulate was satisfied by ke1 = 0.196 min-1 for D2 and ke1 = 4.710 min-1 for D3. For the large volume, the estimates of ke1 were 0.1475 min-1 for D2 and 0.1498 min-1 for D3.
Discussion
The simulations suggest that the volume of the effect compartment per se is not the critical parameter in a PK-PD model for nondepolarizing muscle relaxants. If the effect compartment is postulated to be void of the postsynaptic receptors, then the peak concentration of free muscle relaxant in this compartment is attained at identical times using identical transport rate constant ke1 for any volume of the effect compartment. These conclusions agree with those obtained from PK-PD models assuming a negligibly small volume of the effect compartment and not taking into account binding of a muscle relaxant to the postsynaptic receptors [1-3]. However, NMB is produced not by the free molecules of muscle relaxants in the effect compartment, but by the molecules bound to the postsynaptic receptors at the motor end plates. Therefore, consideration of binding of muscle relaxants to the postsynaptic receptors in the effect compartment is advantageous in PK-PD modeling. The present simulations confirm the conclusion from the reports [4-6] that the receptor concentration in the effect compartment is a critical parameter in PK-PD modeling. The predictions from our simulations assuming a low or a high receptor concentration differ with respect to (1) the onset times to the peak but submaximal neuromuscular block for a single muscle relaxant (lower panel in Figure 2), (2) the time course of NMB using ED50 (lower panel in Figure 1), (3) the shape of the NMB-versus-dose curves (upper panel in Figure 2), (4) the estimates of ke1 (Table 1 and upper panel in Figure 3), and (5) the estimates of ED50 and the onset times as a function of affinities assigned to the muscle relaxants for binding to the receptors (D2 and D3, Results).
In the aforementioned models [4-6], receptor concentration was an explicit model parameter. In contrast, the present model defines the receptor concentration as the ratio between the constant amount of postsynaptic receptors and the variable volume assigned to the effect compartment. This approach allows PK-PD modeling without the constraint of a negligibly small effect compartment. The earlier models taking into account receptor concentration [4-6] assumed a negligibly small volume of the effect compartment. Given a fixed amount of postsynaptic receptors, a finite receptor concentration is not compatible with the negligibly small volume of the effect compartment. This is an inherent weakness of such models.
A compartment is defined by Jacquez [12] as "an amount of a material that acts kinetically like a distinct, homogenous amount". This is the reason that the five equations defining the transport and the distribution of a muscle relaxant in the body (Appendix) were formulated in terms of amounts rather than concentrations. The total amount of a drug in the body is represented by two, three, or more such compartments. The necessity to invoke more than a single compartment arises from the physico-chemical properties of the drug in relation to those structures in the body that prevent drug's uniform dilution. Anatomical structures and/or physiologic processes represent these barriers. For muscle relaxants, small hydrophilic cations with MW < 1000 da, the principal barriers are the capillary wall and the cellular membranes. It seems plausible to postulate that muscle relaxants diffuse through the pores in the capillary wall into the surrounding interstitial spaces. Diffusion across the cellular membranes is very unlikely due to the high hydrophilicity of the molecules. Therefore and as a first approximation, muscle relaxants remain diluted in a space limited to plasma and the interstitial space. The pharmacokinetic compartments for muscle relaxants likely represent the amounts of muscle relaxants in plasma and the interstitial spaces of different tissues.
In the muscle, muscle relaxants diffuse throughout the interstitial space, including the synaptic clefts at the motor end plates. There are no anatomical barriers between the interstitial space in muscle and the synaptic clefts to prevent diffusion of muscle relaxants into the synaptic clefts [13]. These considerations qualify the interstitial space in muscle, including the synaptic spaces, as a single pharmacokinetic compartment. The volume of the interstitial space in muscle defines the volume of this compartment.
Due to the presence of the postsynaptic receptors in the synaptic clefts, the
compartment represents the effect compartment for muscle relaxants. The functional receptors are immobile and are located exclusively within the synaptic clefts. Hence, interaction between the receptors and the free molecules of a muscle relaxant occurs due to diffusion of the free molecules of the muscle relaxant to the receptors. In effect, interaction between the two partners may be represented as proceeding in a space common to both, i.e., the interstitial space in muscle. Volume of this space defines the volume of the effect compartment, Ve. We suggest that the apparent or the effective concentration of the postsynaptic receptors for the interaction with muscle relaxants is the ratio of the amount of receptors and the volume of interaction, [R]total = {R}total/Ve.
Transport of a drug between two compartments is represented in a standard pharmacokinetic model by two first-order rate constants. A modification of this approach is needed, if the transport is assumed to proceed via diffusion. Occurrence of a peak amount in a non-central compartment suggests that at that moment there is no net transport. The postulate of transport via diffusion implies that the concentration of a muscle relaxant in the central and the peak concentration in the effect compartment are identical at that moment. In the simulations, the transport rate constant out of the effect compartment into compartment1 is represented by the symbol ke1. The rate constant in the opposite direction, k1e, is expressed as a function of ke1, viz., k1e = ke1·(Ve/V1). The expression results from the postulate that the transport occurs via diffusion. We suggest that ke1 may be interpreted as the ratio of the plasma flow to the muscle and the volume of the interstitial space in muscle. For the adductor pollicis muscle and assuming plasma flow to the forearm or the hand of 0.9 to 4.7 mL·min-1·(100 g muscle)-1 [14] and the volume of the interstitial space in muscle of 15 to 22 mL·(100 g muscle)-1, the value of the transport rate constant ke1 may be estimated to between 0.041 and 0.313 min-1.
The postulate seems plausible that the molar amount of the postsynaptic receptors is a physiologic constant. The value of the constant may be 10 times lower or 10 times higher than the assigned value (Table 1) without markedly altering the results of the simulations. The postulate of a constant amount of postsynaptic receptors permits the definition of the apparent receptor concentration in the effect compartment via the relationship [R]total = {R}total/Ve.
The results of the simulations demonstrate that a PK-PD model may be constructed for a wide range of volumes assigned to the effect compartment. We examined Ve from 1·10-6 to 1·10-1 L·kg-1and the corresponding apparent concentrations of the receptors. In general, smaller volumes require higher values of ke1 (upper panel in Figure 3), are associated with smaller total amounts of the muscle relaxant in the effect compartment and larger fractions of the muscle relaxant in the bound form (lower panel in Figure 3). The smaller volumes are compatible with the intercompartmental transport rate constants close to those in the standard 3-compartment pharmacokinetic model. For volumes < 1·10-3 L·kg-1, the onset times of submaximal NMB are negatively correlated with the magnitude of NMB (lower panels in Figures 2 and 4). The onset times are also markedly dependent on the values assigned to the equilibrium dissociation constants for binding of the muscle relaxants to the receptors (muscle relaxants D2 and D3), higher affinities associated with prolonged onset times. All these findings change for Ve > 1·10-3 L·kg-1 and the receptors concentrations < 1·10-7 M (Figures 3 and 4). Specifically, the values of the rate constant ke1 become smaller and relatively independent of the assigned volumes (upper panel in Figure 3), the differences between onset times for NMB05 and NMB95 progressively disappear (lower panel in Figure 4), the affinities do not influence the onset to NMB50, and ED50 doses are proportional to KD (muscle relaxants D2 and D3, Results). It appears as if the value of Ve of about 1·10-3 L·kg-1 and the receptor concentration ~ 1·10-7 M represent the critical threshold for the difference between a "small" and a "large" volume of the effect compartment.
The results of the simulations reveal a difference in the slopes of the NMB curves when evaluated as a function of the injected doses of a muscle relaxant. As in the available pharmacodynamic models, NMB in the proposed model was calculated using the peak free concentration of a muscle relaxant in the effect compartment and two constants: γ and IC50 (Eq 1, Appendix). When the NMB, calculated using peak [D]e, was plotted as a function of the doses that produced these peak concentrations in the effect compartment, the fitted value of γf (Eq 2, Appendix) was larger than γ used in the calculation of NMB from [D]e (upper panel in Figure 2 and lower panel in Figure 4) and the fitted values of γf increase progressively for smaller volumes assigned to the effect compartment (lower panel in Figure 4). For volumes > 10-3 L·kg-1, the fitted values of γf approach the value of γ used in the calculations of NMB from [D]e (lower panel, Figure 4). The difference is due to the relationship between the peak concentrations of the free muscle relaxant in the effect compartment and the injected doses (upper panel in Figure 4). For volumes < 10-3 L·kg-1, the peak concentrations increase rapidly with increasing doses. The steeper slope implies that the difference in doses producing IC05 and IC95, corresponding to NMB05 and NMB95, respectively, is smaller the smaller the volume assigned to the effect compartment. The narrower spread of these doses leads, in turn, to higher fitted values of γf when NMB is represented as a function of the injected dose. To summarize, if Ve < 10-3 L·kg-1, then a correlation of NMB to the doses needed to establish the peak concentrations requires fitted values for γf higher than the value of γ used in calculating NMB from [D]e. For Ve > 10-3 L·kg-1, the estimates of the fitted γf approach the value of γ used in calculating NMB as a function of [D]e. Therefore, a comparison of γ, estimated in a PK-PD model and based on [D]e, with γf, obtained experimentally in a NMB-versus-dose study, provides information about the volume of the effect compartment and the receptor concentration in it. The fitted values of ED50f are rather independent of the volumes assigned to the effect compartment and the estimates are close to the a priori defined ED50 used in calculating the target plasma concentrations.
The PK-PD models are based on two sets of experimental data: the time course of the plasma concentration of a muscle relaxant and the time course of NMB. The models simulate, and are applicable only to, the concentrations in plasma and in the postulated effect compartment. The amounts or concentrations in the other compartments and the amount eliminated from the body are not verifiable from the available experimental data. These compartments are included in the current PK-PD model solely to preserve mass balance and to fit the amounts or concentrations of D in compartment1 to the target plasma amounts or concentrations. A posteriori addition of a large effect compartment to the standard 3-compartment PK model alters the simulated amounts or concentrations in compartment1 and the fit of the standard 3-compartment model to the target plasma concentrations is lost. Realization of this fact was the primary reason for the postulate of a negligibly small effect compartment in the previously introduced PK-PD model [2]. However, as demonstrated in the current simulations, a PK-PD model may include an effect compartment of any volume and contain a sizable fraction of the injected dose, if the model is designed a priori and the pharmacokinetic rate constants, including ke1, adjusted so that the amounts in compartment1 represent as closely as possible the observed amounts in plasma. The fitting process is similar to that for fitting a standard pharmacokinetic model to the observed plasma concentrations. Alternatively and without prejudging mass transport from plasma to any compartment, the amounts in plasma may be described using a multiexponential equation without detriment to the pharmacodynamic part of the model.
Conclusion
The simulations do not indicate whether a PK-PD model containing a small or a large effect compartment is more appropriate. The selection should be based on the results of prospective clinical experiments. The simulations suggest an optimal experimental design. The study needs to be conducted with several muscle relaxants. Several doses of each are selected to produce less than complete NMB, e.g., NMB10 to NMB90. The experiment needs to answer the following question: Is the onset time of submaximal NMB produced by a single muscle relaxant a function of the level of NMB? If the results with a single muscle relaxant show an inverse relationship between the level of NMB and the onset times, then the model containing a small volume of the effect compartment and a high receptor concentration is more appropriate. If the onset times are independent of the magnitude of the submaximal NMB, then the PK-PD model containing a large volume of the effect compartment and a low receptor concentration is more appropriate.
Appendix
The pharmacokinetic part of the model was formulated with the volume of the effect compartment, Ve, explicitly incorporated in the model. The following symbols are used: D for the muscle relaxant and R for the receptors. The braces denote molar amounts per kg body weight. The first and second subscript appended to the rate constants denote the number of the source and the target compartments, respectively. Subscript e denotes the effect compartment, e.g., k1e denotes the rate constant for the transport from compartment1 to the effect compartment and ke1 the transport in the reverse direction. The symbol {D}e denotes the free amount of D in the effect compartment.
DR represents the 1 : 1 complex of D with the receptors within the effect compartment. The differential equation for {DR} was derived from the differential equation for [DR] written in terms of the molar concentrations [D]e, [R]total, and [DR]. Multiplication of this equation by Ve converts the concentrations into amounts. The definition of k1e in terms of ke1, viz., k1e = ke1·(Ve/V1), results from the postulate of diffusion as the transport mechanism and implies that the peak concentration of D in the effect compartment equals the concentration in compartment1 at the same moment. The initial conditions at t = 0 are: {D}1 = dose, and {D}2 = {D}3 = {D}e = {DR} = 0.
The Hill equation for the calculation of NMB from the free molar concentrations of the muscle relaxant D in the effect compartment, [D]e, is given by:
where [D]e = peak {D}e/Ve and IC50 = 7·KD = peak [D]e when Occ = 0.875. The exponent γ was arbitrarily assigned a value of 4.0.
A different form of the Hill equation was used to fit the calculated NMB (Eq.1) as a function of the doses producing the peak [D]e. The modified equation relates NMB to the injected doses:
The values for the exponent γf and ED50f were derived in the fitting process.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All authors have contributed equally to the conception and the design of simulations, to acquisition of data and data analysis, and to the preparation of the manuscript. All authors read and approved the final manuscript.
Note
1, where [Dplasma]i is the molar concentration of D in plasma calculated from the triexponential equation at time ti, [D1]i is the molar concentration in compartment1 at the same time, and n is the number of time points (= 300).
Acknowledgements
A.A. is grateful to the Bernhard-Lang Research Association for continuous support.
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Donati F Meistelman C A kinetic-dynamic model to explain the relationship between high potency and slow onset time for neuromuscular blocking drugs J Pharmacokinet Biopharm 1991 19 537 552 1783991 10.1007/BF01062962
Proost JH Wierda JM Meijer DK An extended pharmacokinetic/pharmacodynamic model describing quantitatively the influence of plasma protein binding, tissue binding, and receptor binding on the potency and time course of action of drugs. Journal of Pharmacokinetics and Biopharmaceutics 1996 24 45 77 8827583
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Pestronk ADDBSSG Measurement of junctional acetylcholine receptors in myasthenia gravis: Clinical correlates Muscle & Nerve 1985 8 245 251 4058469 10.1002/mus.880080311
Amaki Y Waud BE Waud DR Atracurium-receptor kinetics: simple behavior from a mixture Anesth Analg 1985 64 777 780 3839351
Fisher DM Szenohradszky J Wright PM Lau M Brown R Sharma M Pharmacodynamic modeling of vecuronium-induced twitch depression. Rapid plasma-effect site equilibration explains faster onset at resistant laryngeal muscles than at the adductor pollicis Anesthesiology 1997 86 558 566 9066321 10.1097/00000542-199703000-00007
Jacquez JA Compartmental analysis in biology and Medicine 1996 3rd Dexter, MI, Thompson-Shore, Inc.
Eccles JC Jaeger JC The relationship between the mode of operation and the dimensions of the junctional regions at synapses and motor end-organs Proc R Soc Lond B Biol Sci 1958 148 38 56 13494474
Lentner C Geigy Scientific Tables 1990 V 8th West Caldwell, NJ, CIBA-GEIGY Corp. 222
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Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-2-421620216610.1186/1742-4682-2-42ResearchHybrid dynamic/static method for large-scale simulation of metabolism Yugi Katsuyuki [email protected] Yoichi [email protected] Ayako [email protected] Masaru [email protected] Institute for Advanced Biosciences, Keio University, Fujisawa, Kanagawa, 252–8520, Japan.2005 4 10 2005 2 42 42 25 4 2005 4 10 2005 Copyright © 2005 Yugi et al; licensee BioMed Central Ltd.2005Yugi 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 computer studies have employed either dynamic simulation or metabolic flux analysis (MFA) to predict the behaviour of biochemical pathways. Dynamic simulation determines the time evolution of pathway properties in response to environmental changes, whereas MFA provides only a snapshot of pathway properties within a particular set of environmental conditions. However, owing to the large amount of kinetic data required for dynamic simulation, MFA, which requires less information, has been used to manipulate large-scale pathways to determine metabolic outcomes.
Results
Here we describe a simulation method based on cooperation between kinetics-based dynamic models and MFA-based static models. This hybrid method enables quasi-dynamic simulations of large-scale metabolic pathways, while drastically reducing the number of kinetics assays needed for dynamic simulations. The dynamic behaviour of metabolic pathways predicted by our method is almost identical to that determined by dynamic kinetic simulation.
Conclusion
The discrepancies between the dynamic and the hybrid models were sufficiently small to prove that an MFA-based static module is capable of performing dynamic simulations as accurately as kinetic models. Our hybrid method reduces the number of biochemical experiments required for dynamic models of large-scale metabolic pathways by replacing suitable enzyme reactions with a static module.
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Background
Recent progress in high-throughput biotechnology [1-3] has made advances in understanding of cell-wide molecular networks possible at the systems level [4,5]. To reconstruct cellular systems using the high-throughput data that are becoming available on their components, computer simulations are being revisited as an integrative approach to systems biology. Mathematical modelling of biochemical networks has been attempted since the 1960s, and before genome-scale pathway information became available, they mostly employed numerical integration of ordinary differential equations for reaction rates [6-10]. This kind of dynamic simulation model provides the time evolution of pathway properties such as metabolite concentration and reaction rate. To create accurate simulations, dynamic models require kinetic parameters and detailed rate-laws such as the MWC model [11] and those derived using the King-Altman method [12]. However, with few exceptions such as human erythrocyte metabolism [13,14], it is virtually impossible to collect a complete set of kinetic properties for large-scale metabolic pathways. Therefore, the applicability of the dynamic method has been limited to relatively small pathways.
Another approach, such as metabolic flux analysis (MFA) using stoichiometric matrices, has been employed for large-scale analyses of metabolism [4,15,16]. Assuming a steady-state condition, MFA provides a flux distribution as the solution of the mass balance equation without the need for rate equations and kinetic parameters [16,17]. Since it is a "static" approach, the ability of MFA to predict the dynamic behaviour of metabolic pathways is limited. It provides a snapshot of a certain pathway in a single state, but is insufficient to predict the dynamic behaviour of metabolism [18]. Recently, this approach was extended to allow the prediction of dynamic behaviour. This extension, dynamic flux balance analysis (DFBA) [19], provides optimal time evolution based on pre-defined constraints, including kinetic rate equations. However, this extension was not intended to reduce the masses of information necessary for developing dynamic cell-scale simulation models. In addition, this DFBA study did not define the criteria for segmenting a whole metabolic pathway into parts defined by kinetic rate equations and a stoichiometric model. Therefore this effort does not suffice as a generic modelling approach.
Here we propose a method for dynamic kinetic simulation of cell-wide metabolic pathways by applying the kinetics-based dynamic method to parts of a metabolic pathway and the MFA-based static method to the rest. Because the static module does not require any kinetic properties except the stoichiometric coefficients, this method can drastically reduce the number of enzyme kinetics assays needed to obtain the dynamic properties of the pathway. We have evaluated the accuracy of the hybrid method in comparison to a classical dynamic kinetic simulation using small virtual pathways and an erythrocyte metabolism model.
Results
Evaluation of errors
The hybrid simulation method integrates the two types of simulation method within one model: the static module comprises enzymatic reactions without their kinetic properties and the dynamic module covers the rest of the pathway, thereby enabling the static module to be calculated in a quasi-dynamic fashion (Figure 1). At steady-state, a hybrid model of a hypothetical pathway that included an over-determined static module (Figure 2a) yielded an almost identical solution to a dynamic model of the pathway. The reaction rates were calculated by numerical integration of the rate equations. We employed the errors between the dynamic and hybrid models in the first integration step as an index to estimate the accumulation of errors in the subsequent integration steps (one-step error; see Methods for a detailed definition). The one-step error was 8.592 × 10-16 of the maximum for the reaction rates. All the metabolite concentrations in the hybrid model were identical to those in the dynamic model (Table 1). When the concentration of metabolite A was increased two-fold, the hybrid and the dynamic models displayed similar time evolutions (Figure 3a and 3b). The maximum one-step errors after this perturbation were 4.000 × 10-11 and 8.889 × 10-6 for metabolite concentrations and reaction rates, respectively (Table 1).
Table 1 Errors between the dynamic model and the hybrid model of the pathway shown in Fig. 2a. The maximum errors were measured within one numerical integration step. "Perturbation" denotes whether the errors were measured under a steady-state condition (-) or after a two-fold increase of metabolite A (+)
Perturbation Maximum error (concentration) Maximum error (reaction rate)
Boundary - 0 0
+ 8.000 × 10-11 (C) 0
Static part - 0 8.592 × 10-16 (E_CD)
+ 4.000 × 10-11 (D,E,F,G) 8.889 × 10-6 (E_CD)
The hybrid model was also as accurate as the dynamic model in the case of a simple pathway with an underdetermined static module (Figure 2b). The maximum one-step errors at steady state were 5.049 × 10-12 for metabolite concentrations and 2.837 × 10-6 for reaction rates (Table 2). The time courses after a two-fold increase in the concentration of metabolite A were very similar between the dynamic and the hybrid model (Figure 3c and 3d). The maximum errors at the first integration step after the perturbation were 3.575 × 10-7 for the metabolite concentrations and 0.00120 for the reaction rates.
Table 2 Errors between the dynamic model and the hybrid model of the pathway shown in Fig. 2b. The maximum errors were measured within one numerical integration step. "Perturbation" denotes whether the errors were measured under a steady-state condition (-) or after a two-fold increase of metabolite A (+)
Perturbation Maximum error (concentration) Maximum error (reaction rate)
Boundary - 5.049 × 10-12 (F) 5.609 × 10-12 (E_FG)
+ 3.575 × 10-7 (C) 1.323 × 10-7 (E_FG)
Static part - 7.176 × 10-15 (D) 2.837 × 10-6 (E_CD, E_DF)
+ 1.192 × 10-7 (D) 0.00120 (E_CD)
In contrast, the models did not agree as closely when (i) the static module involved enzymes of which the reactions were bottlenecks of dynamic behaviour, i.e. were not sufficiently susceptible to the boundary reaction rates, and (ii) a boundary reaction rate underwent a large change in response to changes in substrate concentrations. For example, the hybrid model of the hypothetical pathway with an over-determined static module exhibited approximately 10-fold higher one-step errors in the reaction rates of the static module when the rate constants of a boundary reaction E_BC were altered from kf = 0.01s-1, kr = 0.001s-1 to kf = 0.1s-1, kr = 0.091s-1.
Figure 1 Summary of the hybrid method. (i) In the dynamic module (V1, V2, V9, and V10), the rate equations provide the reaction rates. (ii) In the static module, the reaction rate distribution (V3, V4, V5, V6, V7, and V8) is calculated from the matrix equation at the right, which corresponds to v = S#b. S# denotes the Moore-Penrose pseudo-inverse of S. (iii) Numerical integration of all the reaction rates (V1-V10) determines the concentrations of the metabolites (X1-X13). The metabolites X5, X7, and X11 are at the boundary.
Figure 2 Hypothetical pathways for simulation experiments. Simple pathway models employed to evaluate the accuracy of the hybrid method in comparison with conventional kinetic simulation. The reactions in the boxes were replaced with a static module in the hybrid models. (a) A pathway model with an over-determined static module. (b) A model including an underdetermined static module. (c) A simple linear pathway model. (d) A pathway map of the glycolysis model [13, 20]. See Tables 4 and 5 in Additional file 1 for the abbreviations of the metabolites and the enzymes, respectively.
Figure 3 Comparisons of time courses produced by dynamic and hybrid models. The coloured lines and the broken black lines represent the time courses calculated by dynamic and hybrid models, respectively. Refer to Fig. 2 for pathway nomenclature. The hybrid model in Fig. 2a yielded similar time courses of change in the reaction rates and the metabolite concentrations to the corresponding dynamic model. (a) The reaction rates of E_BC (yellow) and E_DF (blue). (b) The concentrations of compounds D (yellow) and H (blue). The time courses of the pathway model in Fig. 2b were also in agreement with the dynamic model. (c) The reaction rates of E_BC (yellow), E_CF (green), E_CE (red), and E_CD (blue). (d) The concentrations of compounds E (yellow) and H (blue). The results of these models were also in good agreement for the erythrocyte model. (e) The reaction rates of the hybrid model differed only slightly from those of the dynamic model. The lines in blue, purple, yellow, green, and red denote the reaction rates of GSSGR, G6PDH, TK2, TA and R5PI, respectively. (f) The hybrid and dynamic models yielded almost identical time courses in the concentrations of metabolites such as X5P (yellow), GSSG (blue), and NADP (red).
Correlation between elasticity and errors
Relationships between kinetic properties and one-step errors were examined in depth using a simple linear pathway at a steady state (Figure 2c) and 2a glycolysis model [13,20] (Figure 2d). Elasticity is a coefficient defined by metabolic control analysis. It represents the sensitivity of reaction rate to changes in substrate concentration (See Eq. (4) in Methods). The one-step errors of all the reactions in the static module (E_CD, E_DE, and E_EF) were proportional to the elasticity of the boundary reaction E_BC (Figure 4a). In addition, the errors of E_CD and E_DE were negatively correlated with their own elasticities (Figure 4b, c and 4d). It was also observed in the glycolysis model that the one-step errors of reaction rates in static modules are proportional to the elasticities of the boundary reactions (Figure 4e). These results were in good agreement with the implications derived from Eq. (2), that a static module should be composed of reactions with large elasticities and boundary reactions with small elasticities.
Figure 4 Correlation between elasticity and error. (a) The error between the hybrid model and the dynamic model was positively correlated with the elasticity of the boundary reaction. (b,c,d) The elasticity of the reactions replaced by a static module was negatively correlated with the error. (e) The correlation between error and elasticity was also observed in the glycolysis model.
Application to erythrocyte metabolism
The same analysis was performed using an erythrocyte metabolism model [14] to evaluate the applicability of the hybrid method to more realistic and more complex pathways. A group of enzymes surrounded by glucose-6-phosphate dehydrogenase (G6PDH), transketolase I (TK1), transketolase II (TK2) and ribulose-5-phosphate isomerase (R5PI) was replaced with a static module (Figure 5) to verify the implications of Eq. (2), that a static module should be composed of reactions with large elasticities and boundary reactions with small elasticities. These enzymes were selected because they exhibit relatively small elasticity ratios (see Methods for definition) compared to others in this pathway. The static module is an over-determined system (eight metabolites and five reactions).
Figure 5 A pathway map of the erythrocyte model. The erythrocyte model contains 39 metabolites and 41 reactions (not all are shown here). The reactions represented by red arrows are placed in the static module of the hybrid model. The other reactions belong to the dynamic module. The abbreviations of metabolites and enzymes are described in Tables 4 and 5 in Additional file 1, respectively.
The hybrid and dynamic erythrocyte models yielded similar dynamics in response to a three-fold increase of FDP concentration (Figure 3e and 3f). The errors between the dynamic and hybrid models of the erythrocyte pathway were quantified by the procedure used for the hypothetical pathways. In a steady-state condition without an increase in FDP, the maximum error, 2.17 × 10-4, was observed in the reaction rate of 6-phosphogluconate dehydrogenase (6PGODH) (Table 3). (Note that this was true only when the gluconolactone-6-phosphate (GL6P) concentration was excluded. Owing to its small initial concentration (7.572 nM), the error in GL6P was sensitive to small changes and was associated with a large error of 0.00780.) The error in the 6PGODH rate remained the maximum error when the FDP concentration was perturbed.
Table 3 Comparisons of the dynamic model and the hybrid model of the erythrocyte pathway shown in Fig. 5. The maximum errors were measured within one numerical integration step. "Perturbation" denotes whether the errors were measured under a steady-state condition (-) or after a three-fold increase of FDP concentration (+).
Perturbation Maximum error (concentration) Maximum error (reaction rate)
Boundary - 7.796 × 10-3 (GL6P) 1.555 × 10-7 (R5PI)
+ 1.153 × 10-7 (GL6P) 3.020 × 10-5 (TK1)
Static part - 1.111 × 10-8 (GSSG) 2.170 × 10-4 (6PGODH)
+ 4.282 × 10-12 (GO6P) 2.170 × 10-4 (6PGODH)
When the boundary reaction was relocated from G6PDH, which forms a bottleneck of dynamic response in a transient state and has low elasticity at steady state, to phosphoglucoisomerase (PGI), which has a larger elasticity, the time courses calculated by the hybrid model were different from those produced by the dynamic model.
Discussion
In the simulation experiments using hypothetical pathways and an erythrocyte model, the discrepancies between the dynamic and the hybrid models were sufficiently small to prove that an MFA-based static module is capable of performing dynamic simulations as accurately as a kinetic model. The key idea behind our method is to distinguish between dependent and independent variables (reactions). Although independent reactions can be affected by other dependent/independent reactions through effectors such as ADP in the phosphofructokinase reaction, the time evolution of adjacent reaction rates are mainly determined by independent reactions which constitute bottlenecks of dynamic behaviour in the metabolic network. Therefore, static modules should consist of only such dependent reactions, whereas dynamic modules can include both independent and dependent reactions. Our hybrid method reduces the number of biochemical experiments required for dynamic models of large-scale metabolic pathways by replacing suitable enzyme reactions with a static module. The optimal conditions for this method are (a) a system with few bottleneck reactions in order to enlarge the static modules, (b) small fluctuations in the reaction rates in static modules, and (c) accurately identifiable bottleneck reactions. How can such enzymes be identified? One obvious criterion for the enzymes to be suitably modelled by a static module is not to incorporate a bottleneck reaction in a transient state. Thus, the enzymes should not reach the maximum velocity quickly or be restrained at lower activities by allosteric regulation. Although the model comprising dynamic and static modules as a whole can represent transient states, it is assumed that the reactions in the static modules achieve or nearly achieve steady states within one numerical integration step. The existence of one or more bottleneck reactions in the static module may cause inconsistencies, because the hybrid method solves algebraic equations for static modules under a steady state assumption, although metabolites will be accumulated or depleted in real cells. Therefore, bottleneck reactions must be excluded from static modules. Another situation that should be avoided involves reaction rates in static modules that are affected by changes in enzyme concentration, such as those caused by changing levels of transcriptional/ post-transcriptional control. Such reactions should be included in dynamic modules.
A similar cause of inconsistency is the reversibility of reactions. Since the hybrid method assumes that reactions in the static module are reversible, inclusion of an irreversible step may cause inconsistencies, particularly in the presence of a perturbation downstream of the irreversible step (data not shown).
The accuracy of the calculation can also be affected by a time lag. In the static module of the hybrid model, time lags between the upstream and downstream reactions are not represented because the boundary reactions affect all subsequent reactions in the static module within one integration step regardless of the number of enzyme reactions. Depending on the simulation time scale, the static module should be limited to minimize the influence of time lags. This influence can be estimated by the ratio of elasticities, which can be an important criterion for including a reaction in the static module.
The correlation between elasticity and one-step error (Figure 4) indicates that, to ensure the accuracy of the simulation, the static module of a pathway should include reactions with larger elasticities and should be surrounded by boundary reactions with small elasticities. A large elasticity indicates that the enzyme is capable of changing its reaction rate rapidly in response to changes in substrate concentrations [21]. The result shown in Figure 4 demonstrates that enzymes with large elasticity contribute to the accuracy of the static module. On the other hand, boundary reactions with small elasticities, large substrate concentrations and/or small reaction rates change their activities little in response to substrate concentrations over a short period of time; perturbations are thus dampened by boundary reactions before being transmitted to the static modules. As a result, the reaction rates in the static modules do not change much after perturbations. Such a moderate time evolution allows even reactions that are not very fast to realize a reaction-rate distribution, v, that can be calculated from v = S#b in as little as one numerical integration step. This allows the hybrid model to produce results that are in agreement with the dynamic model when the boundary reactions weaken perturbation.
The results we obtained when we relocated the boundary of the static module in the erythrocyte model support the importance of elasticity ratios. When G6PDH was included inside the static module, PGI became the new boundary reaction instead of G6PDH. The elasticity of PGI is large (elasticity = -452.496) compared to its neighbour G6PDH (elasticity = 0.0955). The relocated boundary is therefore composed of a pair of reactions that might produce unacceptable calculation errors, and in fact led to inconsistencies between the hybrid and dynamic models. Thus, the analytical conclusion presented in Eqs. (2) and (3) also holds for complex pathways, and elasticity provides a criterion for identifying groups of enzymes that can be approximated with sufficient accuracy by static modules. However, a large amount of experimental data is still required to determine the elasticities of all enzymatic reactions. In addition, the demarcation of the static module using elasticities determined by conventional biochemical experiments is unrealistic with respect to their throughput. Hence, the comprehensive determination of bottleneck reactions is the key task in the construction of large-scale metabolic pathway models using the hybrid method. Recent advances in flux measurement, quantitative metabolomics and proteomics allow large-scale measurement of flux distributions [22], intracellular metabolite concentrations and amounts of enzymes [23].
Recently, a method for high-throughput metabolomic analyses using capillary electrophoresis assisted by advanced mass spectrometry (CE-MS) and LC-MS/MS has been developed by the metabolomics group at our institute [24-27]. This technology allows us to determine the concentrations of more than 500 different metabolites quantitatively in a few hours. Furthermore, we are developing a method to calculate whole reaction rates of metabolic systems. This method has already achieved preliminary successes in determining the reaction rates of glycolysis in E. coli and human red blood cells. Pulse-chase analyses using 13C labeled molecules and the CE-MS/LC-MS high-throughput system have also been used successfully by the same metabolomics group to determine fluxes in the E. coli central carbon pathway.
Several approaches have been proposed to quantify elasticity and other coefficients of metabolic control analysis from experimental data such as flux rates, metabolite concentrations or enzyme concentrations [28-31]. Thus, the hybrid method, in combination with the 'omics' data of metabolism, enables a dynamic kinetic simulation of cell-wide metabolism.
Conclusion
Using this hybrid method, the cost of developing large-scale computer models can be greatly reduced since precise modelling with dynamic rate equations and kinetic parameters is limited to bottleneck reactions. This drastically reduces the number of experiments needed to obtain the kinetic properties required for the dynamic simulation of metabolic pathways.
Methods
Calculation procedure
The hybrid method works within one numerical integration step as follows: (i) all the reaction rates in the dynamic module are calculated from dynamic rate equations (V1, V2, V9, and V10 in Figure 1); (ii) the reaction rate distribution in the static module (V3, V4, V5, V6, V7, and V8) is derived from the balance equation Sv = b, where S denotes the stoichiometric matrix, v the flux distribution, and b the rates of the dynamic exchange reactions at the system boundary (V2, V9, and V10) that are calculated in step (i); and (iii) the concentrations of the metabolites (X1-X13) are determined by numerical integration of the reaction rates calculated in steps (i) and (ii). All the reactions in the static module are assumed to be reversible.
The calculation of the reaction rate distribution in the static module is similar to that in the MFA method. The only difference is that the exchange reactions between the dynamic and static modules are represented by kinetic rate equations instead of constant fluxes. In this study, we term a dynamic exchange reaction of a static module a "boundary reaction". Dynamic boundary reactions provide quasi-dynamic changes in the reaction rate distribution in the static module. The reaction rate distribution in the static module is calculated at every integration step that refers to the boundary reaction rates, which are determined by concentrations of metabolites inside and outside the static module. The time evolution of the metabolite concentration in the static module is calculated at every integration step by numerical integration of the reaction rates as well as the metabolites in the dynamic module.
In step (ii), the Moore-Penrose pseudo-inverse is employed to calculate the reaction rate distribution of the static module at each numerical integration step. This should result in a smaller computational cost than linear programming, which is commonly used to determine the flux distribution of the underdetermined system. When the linear equation Sv = b is determined, S#, the Moore-Penrose pseudo-inverse of S, is identical to S-1, the inverse of S. Thus, the reaction rate distribution of the static module is solved uniquely as v = S-1b. If the equation Sv = b is over-determined, v = S#b provides the least squares estimate of the reaction rate distribution [32] which minimizes |Sv-b|2. Through this procedure, the error is distributed equally among the reaction rates of the static module.
In the case of an underdetermined static module, the solution was chosen from the solution space of the balance equation Sv = b to minimize the error of the ideal reaction rate distribution specified by the user. The optimal solution vbest is represented in Eq. (1) below [see Supplementary Text 1 in Additional file 1 for the derivation]:-
vbest = i + S# (b - Si) (1)
where vbest is the closest solution to the ideal reaction rate distribution i in the solution space [Figure 6 in Additional file 1].
Evaluation of errors at steady state
To compare the accuracy of the hybrid method with the conventional dynamic kinetic method analytically, we first employed a pathway model comprising the three sequential reactions shown below. The whole pathway is assumed to be at a steady-state.
In the remainder of this report, a "dynamic model" refers to a metabolic pathway model that is represented by kinetic rate equations only. Let v1, v2 and v3 be the reaction rates of the three sequential reactions. In the hybrid model, the reaction rate v2 was represented as a static module of this pathway. When the concentration of metabolite A, the substrate of v1, is perturbed, the discrepancy between v2 in the hybrid model and v2 in the dynamic model is as described below [see Supplementary Text 2 in Additional file 1 for the derivation]:
where v2d, v2k, [A], [B], εv1A and εv1B denote the reaction rate v2 in the dynamic model, v2 in the hybrid model, concentration of metabolite A, concentration of metabolite B, elasticity of v1 with respect to metabolite A, and elasticity of v2, respectively. The variables with Δ are increments after a small time step Δt. The parameter p represents a ratio of the reaction rate in the static module to the influx, as in Δv2h = pΔv1. The ratio p is determined by the stoichiometric matrix of the pathway.
In Eq. (2), the left bracket term on the right-hand side indicates the magnitude of the perturbation transmitted to the static module. This term indicates that the error between the hybrid and dynamic models is proportional to the increment of metabolites and the elasticity of the boundary reactions. The right bracket describes the susceptibility of the reaction rate v2 to v1. When εv2B satisfies the relationship below, v2 in the hybrid model exhibits identical time evolution to the dynamic model:
Since a small Δt (<<1.0s) is usually employed for accurate simulations of metabolic pathways, Eq. (3) implies that a reaction with large elasticity can be appropriately replaced by a static module.
For more complex pathways, such a theoretical analysis is not practical because large numbers of variables and parameters might impede clear discussions. Instead, simulation experiments were performed to compare the accuracy of hybrid models with dynamic models by numerical methods.
The accuracy of the hybrid model was evaluated numerically in comparison with a conventional kinetic model of the same metabolic pathway under two conditions: a steady-state condition and a time evolution after a two-fold increase of metabolites that are catalyzed by boundary reactions. The errors under steady-state conditions were employed as controls to evaluate discrepancies in dynamic behaviour. These computer simulations were performed using the E-Cell Simulation Environment version 1.1 or 3.1.102 for RedHat Linux 9.0/i386. The errors of reaction rates and metabolite concentrations were measured as below:-
where vd and vh denote either the reaction rates or the concentrations in the dynamic and hybrid models, respectively. The values of vd and vh were taken at the first numerical integration step, in which the concentration increase influences the initial steady-state values of the reaction rates and metabolite concentrations. In this article, this is termed "one-step error". We used one-step errors to represent the discrepancies between the two simulation methods in transient dynamics.
The one-step errors were evaluated using two simple pathways; the static module of one is determined, while the other is underdetermined (Figure 2a and 2b). All the reaction rates in these simple pathways were represented as v = kf[S]-kr[P] where v, kf, kr, [S], and [P] are a reaction rate, a forward rate constant, a reverse rate constant, a substrate concentration and a product concentration, respectively. In the pathway with the over-determined static module, the rate constants were kf = 0.05s-1 and kr = 0.091s-1 for E_CD and E_CE, kf = 0.1s-1 and kr = 0.091s-1 for E_DF and E_EG, and kf = 0.01s-1 and kr = 0.001-1 for the other reactions in the pathway of Figure 2a. The initial metabolite concentrations were 1.0 mM for A, B and C, and 0.5 mM for the other metabolites. Metabolite A was increased two-fold to evaluate the errors in transient dynamics. In the pathway with an underdetermined static module, the kinetic parameters were kf = 0.01s-1 and kr = 0.001s-1 for E_AB, E_BC, and E_FG; kf = 0.1s-1 and kr = 0.098s-1 for E_CD and E_DF; kf = 0.1s-1 and kr = 0.097s-1 for E_CE and E_EF; and kf = 0.1s-1 and kr = 0.96s-1 for E_CF. The steady-state flux distribution was employed for the ideal reaction rate distribution in the static module; the ideal reaction rates were 2 μM/s for E_CD and E_DF, 3 μM/s for E_CE and E_EF, and 4 μM/s for E_CF. All the initial metabolite concentrations were 1.0 mM. The concentration of metabolite A was increased two-fold to evaluate the error.
Correlation between elasticity and error
Elasticity is a coefficient used to quantify the sensitivity of the enzyme to its substrates and is defined as below in the context of metabolic control analysis [21]:
where [S] and v denote the substrate concentration and the reaction rate of the enzyme, respectively. Correlation between the one-step errors and elasticities of each enzyme at a steady state was examined using a linear pathway and a glycolysis model [13,20] (Figure 2c and 2d, respectively). In the linear pathway model, the reaction rate v is represented by the same equation as in the two hypothetical models above. The kinetic parameters were kf = 0.01s-1 and kr = 0.009s-1 for E_AB, E_BC, and E_FG and kf = 0.1s-1 and kr = 0.099s-1 for E_CD, E_DE, and E_EF. All the initial metabolite concentrations were 1.0 mM. The two rate constants of reactions E_BC, E_CD, E_DE, and E_DF were altered within the range 0.01<kf<1.0. The value of kr was determined to satisfy kf-kr = 0.01 to sustain the initial steady-state concentrations. The concentration of metabolite A was increased two-fold to evaluate the errors. For error measurements in the glycolysis model, each enzymatic reaction was replaced, one by one, with a static module. The substrate concentrations of the boundary reactions were increased three-fold.
Application to erythrocyte metabolism
A cell-wide model of erythrocyte metabolism [14] was employed to evaluate the applicability of the hybrid method in a more realistic and complex pathway. This erythrocyte model reproduces steady-state metabolite concentrations similar to experimental data. The static region was determined using a ratio of elasticities as below:
where εb and εx denote the elasticities of a boundary reaction and of reaction X, respectively. All the elasticities of the model were calculated by numerical differentiation of each rate equation. A group of enzymes with small r values were regarded as appropriate candidates for inclusion in a static module. The concentration of fructose-1,6-diphosphate (FDP) was increased three-fold to measure the errors in dynamic behaviours.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Yugi contributed to the development and implementation of the hybrid method into the E-Cell system, and developed methods for analyzing errors at a steady state. Nakayama provided the concept of hybrid method and directed the project. Kinoshita contributed to the development of simulation models and the analyses, and Tomita is a project leader.
Supplementary Material
Additional File 1
Derivations of equations (Eqs. (1) and (2)), supplementary tables (Table 4 and Table 5) and figure (Figure 6).
Click here for file
Acknowledgements
We thank Nobuyoshi Ishii for insightful discussions; Yoshihiro Toya for the preparation of one of the small virtual pathway models; Pawan Kumar Dhar, Yasuhiro Naito, Shinichi Kikuchi and Kazuharu Arakawa for critically reading the manuscript; and Kouichi Takahashi for providing technical advice. This work was supported in part by a grant from Leading Project for Biosimulation, Keio University, The Ministry of Education, Culture, Sports, Science and Technology (MEXT); a grant from CREST, JST; a grant from New Energy and Industrial Technology Development and Organization (NEDO) of the Ministry of Economy, Trade and Industry of Japan (Development of a Technological Infrastructure for Industrial Bioprocess Project); and a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology for the 21 st Century Centre of Excellence (COE) Program (Understanding and Control of Life's Function via Systems Biology).
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Thromb JThrombosis Journal1477-9560BioMed Central London 1477-9560-3-141620217010.1186/1477-9560-3-14ReviewHeparin-induced thrombocytopenia: an update Franchini Massimo [email protected] Servizio di Immunoematologia e Trasfusione, Azienda Ospedaliera di Verona, Verona, Italy2005 4 10 2005 3 14 14 25 7 2005 4 10 2005 Copyright © 2005 Franchini; licensee BioMed Central Ltd.2005Franchini; 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.
Heparin-induced thrombocytopenia (HIT) is the most important and most frequent drug-induced, immune-mediated type of thrombocytopenia. It is associated with significant morbidity and mortality if unrecognized. In this review, we briefly discuss the main features of heparin-induced thrombocytopenia, particularly analyzing the most recent advances in the pathophysiology, diagnosis and treatment of this syndrome.
Plateletsheparinthrombosis
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Introduction
Heparin is a drug widely used for thromboprophylaxis or treatment in many clinical situations, including cardiovascular surgery and invasive procedures, acute coronary syndromes, venous thromboembolism, atrial fibrillation, peripheral occlusive disease, dialysis and during extracorporeal circulation [1,2]. However, it can cause serious adverse effects, including heparin-induced thrombocytopenia (HIT) which is a common, serious and potentially life-threatening condition [3-6]. Unfortunately, because thrombocytopenia is common in hospitalized patients and can be caused by a variety of factors [7], HIT often remains unrecognized.
Heparin-induced thrombocytopenia is defined as a decrease in platelet count during or shortly following exposure to heparin [8]. Two different types of HIT are recognized. The first, HIT type I (also called heparin-associated thrombocytopenia in the past), is a benign form not associated with an increased risk of thrombosis. The mechanism of HIT type I is still unknown but it is likely to be non-immune, probably related to its platelet pro-aggregating effect. This form of HIT affects up to 10% of patients under treatment with heparin and is characterized by a mild and transient asymptomatic thrombocytopenia (rarely less than 100,000 platelets/μL) that develops early (usually within the first two days of starting heparin) and disappears equally quickly once the heparin is withdrawn. The second form of HIT, HIT type II, is immune-mediated and associated with a risk of thrombosis. It has recently been proposed that the term "HIT type I" be changed to "non-immune heparin associated thrombocytopenia" and that the term "HIT type II" be changed to "HIT" to avoid confusion between the two syndromes [9].
In this review we briefly analyze the main characteristics of the clinically relevant, immune-mediated, second type of HIT, focusing particularly on the epidemiology, pathophysiology, clinical manifestations and treatment of this syndrome. For simplicity and also in accordance with the new recommendations, in the following the term HIT refers to HIT type II.
Incidence
Heparin-induced thrombocytopenia is the most important of the immune-mediated, drug-induced thrombocytopenias. Recent data show that up to 8% of heparinized patients will develop the antibody associated with HIT [10] and that approximately 1–5% of patients on heparin will progress to develop HIT with thrombocytopenia [11,12], suffering from venous and/or arterial thrombosis in at least one-third of cases [13,14]. In a recent analysis of 598 consecutive hospitalized medical patients treated with subcutaneous unfractionated heparin, Girolami and colleagues diagnosed five cases of HIT (0.8%) [15]. In general, the antibodies occur more frequently in patients undergoing cardiovascular surgery than those undergoing orthopedic surgery, and in post-surgical patients than in medical patients. HIT antibodies are also more frequent in patients receiving unfractionated heparin (UFH) than in those treated with low molecular weight heparin (LMWH) [16,17], although it must be highlighted that antibodies developing in patients receiving UFH frequently cross-react with LMWH [13]. In a study conducted on 665 patients undergoing elective hip arthroplasty who had been randomized to receive either UFH or LMWH for thromboprophylaxis, Warkenitin and colleagues reported that HIT occurred in 9 of 332 patients who received UFH and in none of 333 patients who received LMWH (2.7% versus 0%, P = 0.0018) [10]. In addition, development of heparin-dependent antibodies and thrombotic events associated with thrombocytopenia were more common in patients treated with UFH than in those treated with LMWH.
Pathophysiology
The mechanism underlying heparin-induced thrombocytopenia is an immune response [18,19]. The principal antigen is a complex of heparin and platelet factor 4 (PF4). Platelet factor 4 is a small positively charged molecule of uncertain biological function normally found in α-granules of platelets. When platelets are activated, PF4 is released into the circulation and some of it binds to the platelet surface. Because of opposite charges, heparin and other glycosaminoglycans bind to the PF4 molecules, exposing neoepitopes that act as immunogens leading to antibody production. In fact, patients who develop HIT produce an IgG antibody against the heparin-PF4 complex, which binds to the complex on platelet surface through the Fab region [20]. The Fc portion of the HIT antibody can then bind to the platelet Fc receptor and this interaction triggers activation and aggregation of the platelets. Activated platelets release PF4, thus perpetuating the cycle of heparin-induced platelet activation. In addition, the platelet activation leads to the production of prothrombotic platelet microparticles which promote coagulation. Finally, as a result of the presence of heparin-like molecules (heparan sulfate) on the surface of endothelial cells, the HIT antibody-PF4-heparan sulfate complexes formed on the endothelial surface may induce tissue factor expression with further activation of the coagulation cascade and thrombin generation [21,22]. Thrombocytopenia in HIT is largely due to the clearance of activated platelets and antibody-coated platelets by the reticulo-endothelial system [1]. Figure 1 illustrates the pathophysiology of HIT.
Figure 1 Pathophysiology of heparin induced thrombocytopenia.
Clinical features and diagnosis
The onset of heparin-induced thrombocytopenia may be rapid or delayed. The platelet count in patients with pre-existing heparin-PF4 antibodies from a previous exposure and sensitization to heparin may decrease within the first 3 days or even hours after re-exposure to heparin (rapid-onset HIT) [23]. However, in patients receiving heparin for the first time, the onset of thrombocytopenia usually occurs 5 to 10 days after the administration of the heparin. Conversely, in delayed-onset HIT, the thrombocytopenia occurs 5 or more days after heparin withdrawal [24].
The thrombocytopenia in HIT is usually moderate in severity, with a median platelet count being between 50 and 80 × 109/L, although the nadir platelet count can remain at a level considered normal (i.e. > 150 × 109/L) but having dropped by 50% or more with respect to the pre-heparin value. The platelet count starts to rise 2 to 3 days after discontinuing heparin and usually returns to normal within 4 to 10 days. The antibody disappears within 2 to 3 months after cessation of heparin therapy [11]. Although HIT does not invariably recur during subsequent re-exposure to heparin, future use of heparin is contraindicated [25]. Despite thrombocytopenia, bleeding is rare [2]. Contrariwise, HIT is strongly associated with thrombosis, which frequently leads to the recognition of HIT [26]. Thrombosis in HIT is associated with a mortality of approximately 20–30%, with an equal percentage of patients becoming permanently disabled by amputation, stroke or other causes [27]. Thromboembolic complications can be venous, arterial, or both and include deep venous thrombosis, pulmonary embolism, myocardial infarction, thrombotic stroke and occlusion of limb arteries [28]. However, the type and site of thrombosis depends on the patient's clinical profile. For example, deep vein thrombosis and pulmonary embolism occur very frequently in postoperative patients who are already prone to developing venous thromboembolism [10]. In fact, Warkentin and colleagues reported that the incidence of deep vein thrombosis in orthopedic patients who received heparin for thromboprophylaxis was 17.8%, but that this incidence increased dramatically to 88.9% among patients who developed HIT [10]. Similarly, patients with central venous catheters and HIT develop upper limb venous thrombosis more frequently than those without HIT [1]. In some cases thrombosis of the cerebral venous sinuses can occur, giving rise to a clinical picture of severe headache and progressive neurological deficits [29]. In contrast, arterial thrombosis occurs more frequently than venous thrombosis in HIT patients receiving heparin for cardiovascular diseases [30]. Furthermore, areas of necrosis developing at the site of heparin injections can be a manifestation of HIT, and are not necessarily associated with thrombocytopenia [31]. Platelet activation and thrombosis due to heparin-dependent, platelet-activating IgG have been shown to be the underlying pathogenic mechanisms of this complication [31]. In some cases, thrombosis may be generalized leading to a syndrome resembling disseminated intravascular coagulation [32].
Finally, in some patients with HIT resistance to heparin may occur, meaning that an increasing dose of heparin dose is required to maintain the activated partial thromboplastin time (aPTT) within the therapeutic range [13].
The diagnosis of HIT remains a clinical one, supported by confirmatory laboratory testing [5,6]. The criteria include: a) thrombocytopenia (i.e., a drop of the platelet count to below 100 × 109/L or a drop of > 50% from the patient's baseline platelet count); b) the exclusion of other causes of thrombocytopenia; c) the resolution of thrombocytopenia after cessation of heparin [1]. As regards the laboratory tests, HIT-antibodies can be demonstrated in vitro by functional tests and immunoassays [4,8]. Functional tests, which measure platelet activity in the presence of the patient's serum and heparin, include heparin-induced platelet aggregation (HIPA) and the serotonin release assay (SRA). Although the HIPA test is easier to perform and thus more commonly used, the SRA is more sensitive, albeit more complex, technically demanding and not readily available in most centers, and is therefore considered the "gold standard" [1]. The immunoassays utilize immunoenzymatic tests (enzyme-linked immunesorbent assay, ELISA) to detect the HIT antibody that binds to the PF4/heparin complex. Immunoassays are technically easier to perform than the functional assays and are also more sensitive [1]. On the other hand, comparative and prospective studies have demonstrated that functional tests are more specific than enzyme immunoassays and thus, being better at detecting the clinically significant HIT antibodies, are more helpful in the diagnosis of HIT [3].
Treatment
When HIT is suspected clinically, immediate cessation of all formulations of heparin is mandatory, but this will neither stop continuing thrombin generation nor avoid subsequent thrombotic events, which occur in as many as 40–50% of the patients over the next several days or weeks [33]. Interestingly, in a retrospective analysis of 113 patients with HIT, Wallis and colleagues [34] found that early heparin cessation (0.7 ± 0.6 days) was no more effective in reducing morbidity and mortality than was late heparin cessation (5 ± 3 days), thus indicating that heparin cessation alone is not sufficient treatment for HIT. In fact, the appropriate treatment for HIT requires immediate removal of the trigger (heparin cessation) as well as control of the thrombin storm of HIT (by providing appropriate alternative anticoagulation). Currently, three non-heparin anticoagulants that do not cross-react with HIT antibodies, danaparoid, lepirudin and argatroban, are available for alternative anticoagulation in HIT [35-41]. These drugs are immediately active and either inhibit thrombin directly or inhibit thrombin generation. As reported above, LMWH cannot be used in patients with HIT because of the strong cross-reactivity of the HIT antibody with the LMW heparin/PF4 complex. The duration of treatment for patients with HIT is not well defined. However, anticoagulation treatment is required for at least 2 to 3 months to prevent recurrence of thrombosis. Oral anticoagulation with warfarin should be initiated until substantial platelet count recovery has occurred and while the patient is receiving danaparoid or a thrombin-specific inhibitor (an overlap of at least 5 days is recommended) [33]. In fact, it has recently become known that HIT patients who are switched to warfarin alone after the discontinuation of heparin may paradoxically have worsening thrombosis and develop venous limb gangrene and skin necrosis [42]. The mechanism appears to be a warfarin-induced marked decrease in protein C before prothrombin levels are adequately suppressed [2].
Danaparoid has been successfully used as a replacement for heparin in patients with HIT [43]. This anticoagulant is composed of a mixture of three glycosaminoglycans (heparin sulfate, dermatan sulfate and chondroitin sulfate) and, via antithrombin, inhibits anti-FXa activity. In a prospective randomized study conducted by Chong and colleagues [41], danaparoid was shown to be more effective than dextran 70 in the treatment of HIT-associated venous and arterial thrombosis. In a compassionate use program, more than 460 patients with HIT-associated thrombosis were treated with danaparoid with a success rate of over 90% [44]. For treatment of HIT, danaparoid is given as an intravenous bolus dose of 2500 U followed by 400 U/hour for 4 hours, then 300 U/hour for 4 hours and subsequently 200 U/hour until anticoagulation is no longer required, adjusting the dose to maintain plasma anti-Xa levels within 0.5–0.8 U/mL. Alternatively, danaparoid can be administered subcutaneously using a bolus of 1250 U followed by 2000 U twice a day [1].
Recombinant hirudin (lepirudin), an anticoagulant protein originally produced by the medicinal leech, inhibits thrombin directly [1]. In a meta-analysis of three prospective multicenter trials including 91 patients with laboratory-confirmed acute HIT treated with lepirudin, Lubenow and colleagues [45] found that the incidence of the combined end-point of death, new thromboembolic complications and limb amputation was significantly lower in the lepirudin-treated patients than in a contemporaneous control group not treated with lepirudin. Currently recommended doses are 0.4 mg/kg as a bolus followed by 0.15 mg/kg/hour adjusting the dose to achieve an aPTT of 1.5 to 3 times the baseline value [33]. In a retrospective study of 175 lepirudin-treated HIT patients and 126 danaparoid-treated HIT patients, Farner and colleagues [46] found no significant difference in the same combined end-points between the two groups.
Argatroban, an arginine-based synthetic anticoagulant, is a direct inhibitor of thrombin that reversibly binds the catalytic site of thrombin [13,47]. A multicenter, prospective study conducted on 304 HIT patients receiving argatroban found that the above mentioned combined end-points were significantly reduced in argatroban-treated patients compared to in historical controls [40]. The recommended initial dose is 2 μg/kg/minute given intravenously and adjusted to achieve an aPTT 1.5 to 3 times the baseline value. Since argatroban is cleared by the liver, lepirudin, which is cleared through the kidneys, should be preferred in patients with liver disease. Vice versa, argatroban would be a better initial choice in patients with renal insufficiency. Thrombin-specific inhibitors also prolong the INR, but this effect is particularly pronounced with argatroban [48]. Thus, during the transition from argatroban to oral anticoagulation special precautions must be taken [49,50].
Finally, there is recent evidence that a novel synthetic heparin pentasaccharide, fondaparinux, which does not cross-react with HIT antibodies [51], can be successfully used for the treatment of patients with HIT [52,53]. However, additional controlled clinical studies are required to further evaluate the safety and efficacy of this agent in patients with HIT.
Conclusion
The analysis of the literature data reveals that heparin-induced thrombocytopenia is not only a common but also a serious complication of heparin therapy with a high rate of morbidity and mortality. Its prompt clinical and laboratory recognition is thus essential in order to stop heparin use immediately and commence an alternative anticoagulant. The low molecular weight heparinoid, danaparoid, and the thrombin-specific inhibitors, lepirudin and argatroban, have been shown to be effective in HIT patients.
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J Transl MedJournal of Translational Medicine1479-5876BioMed Central London 1479-5876-3-351617430210.1186/1479-5876-3-35ReviewT cell avidity and tumor recognition: implications and therapeutic strategies McKee Mark D [email protected] Jeffrey J [email protected] Michael I [email protected] Department of Surgery, The University of Chicago, Chicago, IL, USA2005 20 9 2005 3 35 35 29 8 2005 20 9 2005 Copyright © 2005 McKee et al; licensee BioMed Central Ltd.2005McKee 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.
In the last two decades, great advances have been made studying the immune response to human tumors. The identification of protein antigens from cancer cells and better techniques for eliciting antigen specific T cell responses in vitro and in vivo have led to improved understanding of tumor recognition by T cells. Yet, much remains to be learned about the intricate details of T cell – tumor cell interactions. Though the strength of interaction between T cell and target is thought to be a key factor influencing the T cell response, investigations of T cell avidity, T cell receptor (TCR) affinity for peptide-MHC complex, and the recognition of peptide on antigen presenting targets or tumor cells reveal complex relationships. Coincident with these investigations, therapeutic strategies have been developed to enhance tumor recognition using antigens with altered peptide structures and T cells modified by the introduction of new antigen binding receptor molecules. The profound effects of these strategies on T cell – tumor interactions and the clinical implications of these effects are of interest to both scientists and clinicians. In recent years, the focus of much of our work has been the avidity and effector characteristics of tumor reactive T cells. Here we review concepts and current results in the field, and the implications of therapeutic strategies using altered antigens and altered effector T cells.
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T cell – tumor antigen interactions
Antigens recognized by tumor reactive T cells
One of the key advances in the study of tumor immunology has been the identification of specific protein antigens recognized by tumor reactive T cells. Both MHC class I and MHC class II-restricted peptides have been identified from tumor-associated antigens (TAA) on a variety of human cancers. The identification of TAA has dramatically improved our ability to study the interactions between tumor reactive T cells and their targets, and has been the foundation of new clinical strategies to treat cancer patients [1-4].
TAA can be classified into five groups based on their origin, structure, and tissue expression. Several of the earliest identified TAA were melanoma-melanocyte differentiation antigens [5-7]. These antigens, such as MART-1, gp100, and tyrosinase, are expressed exclusively by cells of the melanocyte lineage. They are considered to be shared TAA because they are expressed by the vast majority of melanomas tested [5-10]. A second group of antigens called cancer/testis antigens are expressed by normal testis and a variety of human tumors including cells from melanoma, breast, bladder, colon, lung, head and neck, gastric, ovarian, neuroblastoma, and prostate cancers [11-14]. These antigens are not universally expressed by tumors of a particular histology, but instead are seen in only a small fraction of any tumor type [15-22]. A third group of antigens are derived form normal viral proteins, and are found exclusively on tumors that are induced by viral infection of human cells [11-13]. This category includes antigens such as EBNA-3 on Epstein Barr virus-induced lymphomas and the E6 and E7 proteins on human papilloma virus-induced cervical cancers [23-25]. The fourth group of antigens is characterized by aberrant expression in tumors relative to normal tissues [12,13]. Many of these proteins have been implicated in tumorigenesis or tumor growth and progression. Antigens such as Her-2/neu and p53, each of which may be highly over-expressed by tumor cells relative to normal tissues, fall into this category [26-32]. The final group of antigens is characterized by protein structures that contain mutations in the sequence [12,13]. These mutations alter the processing, presentation, or recognition of the epitope by the immune system. Such mutations have been described for the β-catenin and CDK4 genes, as well as others [25,33,34]. With the wide variety of antigens available for recognition by the immune system, it is not surprising that proteins expressed by many common tumors can be targeted by T cells.
To date, tumor reactive T cells have been identified that recognize dozens to hundreds of different peptide epitopes. Epitopes may be presented by MHC class I for CD8 T cell recognition, or by class II molecules for CD4 T cell recognition. Epitopes for TAA restricted by HLA A, B, C, and DR alleles have been identified [11-13]. Epitopes with the most clinical relevance are those that are restricted by the most common MHC molecules (HLA-A2, C7, A1, B44, A3, B7, and DR4). These epitopes can be targeted in treatments for the greatest number of patients [35].
T cell avidity and tumor cell recognition
Avidity describes the strength of interaction between a T cell and its target antigen. Avidity is usually measured via T cell activation by a target cell, and is a sum of several contributing components, such as T cell receptor (TCR) expression levels, TCR/peptide/MHC binding affinity, co-stimulatory molecule expression, and the extracellular microenvironment. Experimental evidence suggests that avidity may exert fine control over the response of an activated T cell by influencing the binding and signaling of TCR complexes on the T cell surface. Certain T cell responses are extremely sensitive to activation by antigen. It has been reported that one TCR/peptide/MHC interaction can lead to activation of a T cell as measured by Ca+2 mobilization, three interactions lead to target cell lysis, and ten interactions lead to full activation as measured by T cell proliferation [36]. However, other more commonly used methods for measuring T cell function, such as cytokine secretion or cytolysis, fail to detect T cell responses unless far more peptide is encountered on the target. These assays are commonly performed using peptide loaded antigen presenting cells (APC) as targets in co-culture with T cells. The avidity of a T cell population can be defined by the concentration of antigen required to elicit a T cell response after target loading. In these assays, a high avidity T cell requires less antigen (< 1 nM peptide loaded on an APC) for activation than a moderate (1–100 nM peptide loaded on an APC) or low (>100 nM peptide loaded on an APC) avidity T cell [37].
Many investigators have demonstrated a correlation between T cell avidity and target recognition of T cell populations that recognize virally infected targets, murine tumor models, and human cancers. The first reported relationship between T cell avidity and target cell recognition examined interactions between polyclonal T cell populations and the protein antigen gp160 on HIV infected target cells [38]. In this study, immunization with high doses of antigen led to expansion of T cells with low avidity, whereas immunization with low doses of antigen led to expansion of T cells with high avidity. In a second report, it was shown that if high avidity T cells were exposed to high levels of antigen on targets, activation induced T cell death resulted [39]. These studies illustrated that T cell avidity plays an important role in both T cell priming and T cell response to antigen. Zeh et al. subsequently examined whether T cell avidity also influenced recognition of antigens expressed by tumor cells using a murine melanoma model [4]. In this study, high avidity T cells were raised to the antigens TRP-2 or p15E by stimulating T cells with very low amounts of antigenic peptide. In adoptive therapy experiments, the resultant high avidity T cells were more effective at eliminating lung metastases from B16 melanoma than low avidity T cells. Similar results have been seen with human T cells. Dudley et al. examined the response of individual T cell clones that recognized the melanoma TAA gp100:209–217 [40]. In co-culture with targets, the peptide load required for response by individual T cell clonotypes varied by several logs. Furthermore, there was a correlation between the relative avidity of the T cell clonotypes and their ability to recognize tumor cells. Taken together, these mouse and human results suggest that the relative sensitivity of a T cell to antigen influences its ability to recognize tumors, and that high avidity T cells are required for efficient anti-tumor immunity.
Though it is intuitive that high avidity T cells would better recognize tumors than low avidity T cells, there are reports of T cell populations which do not follow these avidity rules. Many T cells have been raised for TAA recognition through stimulation of naïve lymphocytes by peptides selected according to known MHC binding motifs [41]. The 369–376 peptide from Her-2/neu has generated conflicting reports regarding the relationship between T cell avidity and tumor target recognition. Several groups have identified T cells that recognize Her-2/neu:369–376 peptide as well as Her-2/neu+ tumor cells [26,27,42-44]. However, others have identified high avidity T cells that recognize peptide loaded targets but not tumors. Two clinical trials of immunization with the Her-2/neu:369–376 peptide resulted in the detection of T cells reactive with peptide loaded cells but not tumor cells [45,46]. These contrasting results suggest that the relationship between avidity and target recognition in vivo is complex, and that it is likely under the influence of other significant factors. Identifying and controlling these other factors may be vital if T cells with the genetic capacity and sufficient avidity to recognize TAA are to function as potent anti-tumor effectors.
Experimental studies of TCR affinity and T cell avidity
TCR affinity is the strength of the molecular interaction between the receptor and peptide-MHC complex. TCR affinity has been proposed by some as the single most important component of T cell avidity, which is in agreement with current models of T cell activation that are based on the stability of TCR/peptide/MHC contact. However, experimental evidence can be found both supporting and opposing this point of view. For example, several groups have reported that bright tetramer staining, and thus high affinity TCR/peptide/MHC binding, correlates with high avidity T cell-target interactions [47,48]. On the other hand, other groups have found no correlation between tetramer binding and T cell avidity [49,50]. In several investigations, we have evaluated the avidity and affinity of T cells and TCR based on 1) recognition of APC's loaded with low concentrations of peptide, 2) recognition of tumor targets, or 3) an ability to signal without CD8 coreceptor binding. These studies, the results of which are detailed below, have shown that T cells with identical receptors may behave with different avidities in different circumstances.
T cell clones with identical TCR's may have different relative avidity for peptide loaded APC targets. T cell clones that recognize the HLA-A2 restricted TAA epitope gp100:209–217 were isolated from patients with malignant melanoma. DNA sequence analysis of the TCR subunits was performed on the clones, and several clones with identical receptors (sister clones) were identified. Assays measuring cytokine secretion by the sister clones after stimulation with peptide loaded APC targets or melanoma tumor targets revealed different relative avidities and differing abilities to recognize various tumor lines. These observations are not confined to melanoma reactive T cells or human TCR. Sister T cell clones recognizing the Her-2/neu:369–377 peptide have been isolated with different reactivities against the same Her-2/neu expressing target cells, and studies in animal models have also found T cells sharing the same TCR that have markedly different avidities [51].
High avidity T cells may have receptors that bind peptide-MHC complex with low affinity. A gp100:209–217 reactive T cell clone (R6C12) isolated from a patient with malignant melanoma was shown to have extremely high avidity and recognize HLA-A2+ gp100 positive tumor cells [52,53]. Despite the high avidity of the R6C12 cells, they stained poorly with gp100:209–217 tetramers, suggesting that they had low affinity receptors. Tetramer staining by these cells was enhanced using a modified gp100 peptide that more tightly bound the HLA-A2 molecule [54]. Binding of modified tetramers was easily inhibited by anti-CD8 mAb, providing further evidence that despite the high avidity of CTL clone R6C12, its TCR had relatively low affinity. We have used gene transfer studies to characterize the R6C12 TCR in more detail [55,56]. The R6C12 receptor was cloned, and the receptor was transferred to Jurkat cells using a retroviral construct. These cells, derived from a human T cell lymphoma, do not express the CD8 coreceptor. Transduced Jurkat cells recognized peptide antigen on loaded APC targets with high avidity yet failed to recognize tumor cells, suggesting that the affinity of the receptor for peptide-MHC was insufficient for T cell signaling without coreceptor binding. Subsequently, the R6C12 TCR was transferred to peripheral blood T cells from normal donors [57]. These cultures, in contrast to transduced Jurkat cells, demonstrated the high avidity of the original R6C12 T cell clone. In sum, these data showed that the high avidity of the R6C12 T cell was not due to a high affinity TCR.
Finally, low avidity T cells may have receptors that exhibit characteristics of high affinity TCR/peptide/MHC binding. We have described a tyrosinase reactive T cell with low-moderate avidity characteristics in assays using peptide loaded APC targets, but with the high affinity TCR characteristic of CD8 independence. Of note, this T cell is also capable of recognizing tumor cell targets. A T cell clone recognizing a HLA-A2 restricted epitope from tyrosinase was isolated from the CD4+ population of a patient with malignant melanoma, and the receptor was used for TCR transfer studies like those described above. Both the original human T cell clone and transduced murine 58α-β-cells, which lack human CD8, were able to recognize HLA-A2+ tyrosinase+ tumor cells, even though greater than 100 ng/ml of peptide on targets was required to stimulate IL-2 secretion in APC co-culture assays. In direct contrast to the R6C12 TCR described above, this TCR from a low avidity T cell clone binds and signals in the absence of CD8 coreceptor. Taken together, our studies suggest to us that T cell avidity does not necessarily predict the affinity of the TCR, and that T cells are likely able to modulate their avidity independent of TCR affinity.
Other factors influencing T cell recognition of targets
If T cells have the capacity to alter their antigen responsiveness by factors independent of their antigen receptor, molecular mechanisms other than the TCR must be implicated. Investigations by others have described numerous mechanisms by which T cell function may be altered in cancer patients. Mizoguchi et al. reported that T cells from mice bearing MCA 38 colon carcinoma tumors had reduced expression of CD3ζ chain expression on their surface, and that they had reduced levels of the tyrosine kinases p56lck and p59fyn [58]. Given that CD3ζ chain, p56lck and p59fyn are required for TCR-mediated signaling to occur [59], decreased expression of these molecules in tumor bearing hosts will result in impairment of T cell immunity. It was recently reported that the levels of L-arginine in the cell culture medium could regulate CD3ζ chain expression [60] and that the enzyme arginase I produced by macrophages may regulate the levels of L-arginine in cancer patients [61]. Other investigators have shown that tumor bearing mice have lower levels of the transcription factor NFκB [62]. These signaling defects have been confirmed in several mouse tumor models and in patients with colorectal carcinoma, renal cell cancer, head and neck cancers, and other malignancies [63-67]. Other metabolic pathways also appear to regulate T cell function, such as oxidative stress from hydrogen peroxide released by cells of the monocyte/macrophage lineage [68] and the level of tryptophan metabolites resulting from indoleamine 2,3-dioxygenase expression by macrophages [69,70]. Clearly, the influence of tumors on the physiology of the host may impact the ability to mount an immune response to malignancy by myriad mechanisms.
The CD8 coreceptor and its influence on the recognition of T cell targets deserve special emphasis. The CD8 coreceptor plays a critical role in the activation of some CD8+ T cells by binding to the α3 domain of MHC class I and recruiting the kinase p56lck to the CD3 complex [71]. As discussed above, the dependence upon CD8 coreceptor function by a specific T cell clone is greatly influenced by the TCR/peptide/MHC binding characteristics of the cell. Classically, CD8 is described as a T cell membrane αβ heterodimer [72,73]. Recently, a CD8 αα homodimer form has been described [74]. Transfection studies have shown that the CD8 αβ heterodimer has higher affinity for MHC class I and p56lck than the CD8 αα homodimer, and that the αβ heterodimer more efficiently mediates T cell activation [74]. The ratio of CD8 αβ to CD8 αα as well as the ability for CD8 αβ to co-localize with the TCR to lipid rafts can have a profound impact on T cell avidity [51]. Future investigations will further clarify the role of coreceptor molecules in T cell tumor recognition, and may lead to new immunotherapy strategies based in part on T cell coreceptor function.
Enhancing tumor recognition with modified TAA
Enhancing the immunogenicity of TAA by enhancing MHC-peptide binding
Tumor antigen based clinical trials have led to relatively few clinical responses [75-80]. In addition, many cancer vaccine trials show little evidence of anti-tumor immunity in the peripheral blood of patients following vaccination [78,81]. In an effort to enhance the immunogenicity of known tumor antigens, investigators have introduced modifications into the amino acid sequences of known epitopes. Amino acid substitutions at MHC anchor positions in the antigenic peptide can lead to enhanced peptide/MHC binding [82], and can enhance the immunogenicity of an otherwise weakly immunogenic peptide both in vitro and in vivo [83-86]. The melanoma epitope gp100:209-217-2M is a well-studied example of an anchor residue-substituted peptide. Substituting a methionine for the native threonine at position 2 enhances binding of this peptide to HLA-A2 9-fold. More importantly, this M substitution enhances the immunogenicity of the peptide in vitro and in vivo with the resulting T cells having the capacity to recognize tumor cells [75,83].
Modifications of weakly immunogenic peptides at MHC anchor residues can result in other desirable effects, such as enhancing a peptide's stability in solution. The stability of the weakly immunogenic HLA-A2 restricted peptide antigen NY-ESO-1:155–163 is enhanced by an amino acid substitution at an MHC anchor residue [82]. A substitution of valine for cysteine at position 9 in the peptide not only enhances binding to HLA-A2, but also prevents disulfide bridge formation, thus eliminating dimerization of the peptide in solution [85]. Similarly, a substitution of a serine or alanine for the cysteine at position 2 of the HLA-A1 restricted tyrosinase:243–251 decreases the amount of peptide required to elicit T cell responses in vitro by two to three logs [87]. This simple approach of modifying the MHC binding residues of weakly antigenic peptides represents a powerful strategy for activating T cell populations that would otherwise be unresponsive to stimulation by the native antigen.
Enhancing the immunogenicity of TAA by altering TCR contact residues
It has been shown that immunization with xenogeneic proteins can lead to enhanced immunity to the native protein. The genes encoding the human or rodent homologs of several tumor antigens have been used to vaccinate mice [41,88-90]. In these studies, the xenogeneic antigens routinely resulted in greater immune responses, leading to improved anti-tumor immunity. It was speculated that differences in the amino acid sequence between the xenogeneic antigen and the target antigen resulted in heteroclitic peptides (peptide analogs substituted at positions other than MHC contact residues) that were capable of inducing both effector and helper T cell responses. This hypothesis was directly tested using a peptide from the murine tumor antigen AH-1 [91]. Substituting an alanine for a valine at position 5 increased the binding to the TCR while having no impact on binding to the murine MHC I molecule. This substitution increased the ability of the AH-1 peptide to elicit CTL responses that protect mice from challenges with AH-1 expressing tumors [91]. These animal studies indicated that modifications to TCR contact residues can enhance the immunogenicity of peptide antigens.
Several investigations have also examined the response of human T cells to peptides modified at TCR contact residues [92-94]. One such study identified a heteroclitic peptide for the immunodominant HLA-A2 restricted epitope from human carcinoembryonic antigen, CEA:605–613. Substituting an aspartic acid for the asparagine at position 6, a TCR contact residue, enhances the capacity of this peptide to elicit CEA reactive T cells that can recognize CEA antigen on tumor cells [92]. Furthermore, clinical responses have been reported in colon cancer patients receiving a tumor vaccine comprised of autologous dendritic cells loaded with this heteroclitic CEA peptide [95]. Based on these promising results, other groups have evaluated modified peptides and identified heteroclitic peptides from several tumor antigens [82,94,96]. These modified peptides represent a promising approach for vaccinating cancer patients with otherwise weakly immunogenic antigens.
Influence of peptide modifications on the T cell repertoire
Despite the ability of modified peptides to elicit strong anti-tumor immune responses when used for vaccinating patients, these peptides have generally failed to induce effective anti-tumor immunity and tumor regression [75,77]. Among several possible explanations for these results, one must consider whether modified peptides will optimally stimulate the TAA reactive T cell repertoire in vivo. The T cell repertoire has tremendous diversity due in part to the structure of the TCR molecule. TCR α and β chains consists of a variable (V) segment, a joining (J) segment, and a constant (C) region with the β chain also containing a diversity (D) region. Germline rearrangements occurring within the TCR α and β loci during T cell development randomly join different V-J or V-D-J regions into a single transcriptional unit. The majority of the TCR diversity is the result of the random insertion or deletion of nucleotides at the junctions between the V and J segments for the α chain, and between the V and D and the D and J segments for the β chain. It is these V-J and V-D-J junctions of the α and β chains respectively that encode the putative third complementarity determining region (CDR3), the structural feature of the TCR critical for antigen recognition [97,98].
Though initial reports suggested that there was a limited TCR repertoire used by tumor reactive T cells [99-104], we and others have failed to find evidence of restricted TCR V gene usage [105-112]. When we performed a detailed analysis of the TCR V genes used by MART-1:27–35 and gp100:209–217 reactive T cells, we found that 19 (out of a possible 46) different TCR Vβ were used by the MART-1:27–35 reactive T cell clones [105,108,110,113-116], and 16 different TCR Vβ were used by gp100:209–217 reactive T cell clones (unpublished). Further, no homology was found within the CDR3 regions of the TCR β chains of MART-1:27–35 or gp100:209–217 reactive T cell clones. These observations suggest that there is likely to be considerable TCR diversity among tumor reactive T cells.
Amino acid substitutions in peptides at the TCR contact residues can influence TCR binding and alter the TCR repertoire. This was elegantly demonstrated in a study using single TCR chain transgenic mice. Animals expressing the transgene for a single TCR subunit chain on all T cells were vaccinated with the native moth cytochrome C (MCC) peptides or peptides containing non-conservative amino acid substitutions at the TCR contact residues. MCC reactive T cell hybridomas were isolated from the T cell repertoire after vaccination. By introducing a positively charged amino acid residue into the immunizing peptide, the investigators could induce the presence of negatively charged amino acids in the non-transgenic TCR chains of reactive clones [117]. Thus, alterations in the immunizing peptide influenced the animals' T cell repertoire significantly. We have seen similar changes in the TCR repertoire of patients after vaccination with peptide antigens modified at MHC anchor residues. We found that after vaccination with a gp100:209–217 peptide containing methionine instead of a threonine at position 2, T cell clones could be isolated from patients that recognized the modified peptide but not the native peptide or tumor cells [118]. One patient was identified from whom gp100:209 specific tumor reactive T cell clones could be isolated prior to vaccination. After vaccination, none of the peptide reactive T cell clones isolated from his peripheral blood were able to recognize tumor cells. These results indicate that even changes in the antigenic peptide which do not face the TCR can impact on the TCR repertoire. Given these observations, the potential effects on the T cell repertoire must be considered when contemplating vaccine strategies using substituted peptides.
Enhancing tumor recognition by modifying T cells
Generating tumor reactive T cell populations by TCR transfer
Generating an effective anti-tumor response in vivo requires the presence of T cell precursors capable of recognizing TAA. In many cancer patients, TAA reactive precursors can not be expanded from harvested tumor tissue, lymphoid tissue, or peripheral blood samples. It is not clear whether this is due to the low frequency of T cells against self-antigens, which comprise the majority of shared TAA, or due to an inability to activate or induce proliferation of reactive cells in vitro. A potential solution for these patients is to engineer tumor reactive T cells from naïve lymphocytes using gene therapy techniques. The validity of this approach has been established in pre-clinical studies briefly described above: through the use of specially designed DNA constructs, gene modification of effector T cells in vitro has enabled investigators to re-direct the specificity of T cell populations and T cell clones toward TAA. The majority of work in this area has used single chain antibody constructs bound to intracellular T cell signaling domains, although several investigators have transferred naturally occurring two-chain TCR molecules with their associated activities.
Redirecting T cell specificity through TCR gene therapy requires the transfer of naturally occurring TCR α and β chains to alternate effectors. TCR gene therapy has potential advantages over other adoptive immunotherapy strategies, such as the relative uniformity of the therapeutic agent and the precision with which the transduced T cell population can be measured before and after treatment. The feasibility of redirecting T cell specificity by TCR gene transfer was demonstrated by Dembic et al. in 1986 [119]. With the identification of the first shared tumor antigens for human melanoma in the early 1990's [120], we set out to transfer TAA recognition to a naïve lymphocyte population using this strategy. A TCR recognizing the melanoma antigenic peptide MART-1:27–35 was chosen for initial studies, since MART-1 is expressed by most melanomas and the epitope is restricted by the predominant MHC allele expressed in the United States, HLA-A2. The unique TCR α and β chain sequences from two HLA-A2 restricted, MART-1/Melan A reactive T cell clones were identified [105]. The Jurkat cell line was co-transfected with plasmids containing the α and β chain genes, and transfected cells were cloned in limiting dilution. Expression of the introduced TCR was confirmed, and the functional capacity of transfected clones with varied levels of TCR expression was determined by co-culturing the transduced population with peptide loaded target cells. Transfected Jurkat clones secreted IL-2 in response to culture with MART-1 loaded targets but not targets loaded with an irrelevant peptide. Furthermore, the functional avidity of the transfected clones correlated with the expression level of the transferred TCR. This was the first demonstration that a TAA specific TCR could be transferred with its characteristic antigen recognition to alternate T cells. Since these studies, Jurkat cells have been used to evaluate the transfer of other TCR's, including an HLA-A1 restricted TCR specific for MAGE-3 [121,122].
Next, we attempted to transfer the MART-1:27–35 reactive TCR to primary human T cells from peripheral blood [123,124]. A retroviral vector, designated A7, was constructed for transducing lymphocytes with the MART-1 receptor. To facilitate incorporation of retrovirus into the target cell genome, peripheral blood lymphocytes (PBL) were stimulated to proliferate with anti-CD3 antibody and IL-2 [125]. Transduced primary T cells were able to recognize peptide loaded targets as well as HLA-A2+ melanoma cells. Clones generated from these cultures had varied effector functions in response to co-culture with target cells. Further analysis revealed that only those that expressed the CD8 coreceptor were capable of recognizing tumor cells. Clones which expressed only the CD4 coreceptor could only recognize targets loaded with an excess of exogenous peptide, suggesting that the transferred receptor was dependent upon CD8 for full receptor function. This study verified that T cells suitable for adoptive immunotherapy could be re-directed to recognize tumor cells by TCR gene transfer. TCR's specific for a number of TAA and viral antigens associated with tumor development have been successfully introduced into T cells via retroviral gene transfer. These include TCR's specific for melanoma antigens MAGE-3, gp100, tyrosinase and CAMEL, the widely expressed oncoprotein MDM2, and the Epstein-Barr Virus protein LMP2 expressed by Hodgkin's lymphoma [57,121,122,126-130]. Recently, the transfer of a TCR into T cells with known specificity has been shown to result in individual cells reactive to both antigens [131,132]. It is therefore conceivable to engineer individual T cells with the ability to recognize multiple TAA.
The two-chain approach to TCR transfer has been modified by other investigators to address inherent problems of the approach with TCR subunit expression and pairing. When full-length TCR genes are introduced into normal T cells the native TCR α and β chains may pair with the exogenous TCR β and α chains respectively. This serves to dilute the number of functionally paired TCR's on the cell surface [133,134], and it raises the possibility that TCR's with unknown specificity could be generated, possibly leading to unexpected autoimmunity. To counter these problems, chimeric TCR genes have been generated by fusing the cytoplasmic signaling domain of CD3ζ to MAGE-1 reactive TCR α and β genes [135]. The chimeric TCR gene successfully conferred MAGE-1 reactive function to T cells following retroviral transfer. Notably, subunit genes were shown to pair exclusively to each other following retroviral transfer to T cells, preventing both dilution of functional transferred TCR and generation of TCR's with unknown specificity.
Viral vectors for TCR transfer
Several viral vectors have been investigated for human gene therapy. Adenoviruses were the first viral vectors used due to their abilities to infect both dividing and non-dividing cells and to generate very high titer viral stocks. However, adenoviral vectors lack the ability to provide long-term transgene expression and are highly immunogenic. The viral vector of choice for many gene therapy studies, particularly in haematopoietic cells, is the retrovirus. Retroviruses infect only dividing cells and incorporate into the host cell genome, resulting in long-term transgene expression. They have low immunogenicity, providing a combination of beneficial properties for their use in gene therapies. Removal of the structural genes (gag), gene encoding enzymes for nucleic acid metabolism (pol), and the envelope encoding genes (env) serves to prevent self replication of the retrovirus following infection of target cells. The transgene TCR subunits and, commonly, a gene for cell selection encoding antibiotic resistance or a cell surface marker are then inserted under the control of the LTR and internal promoters. Our laboratory now employs a vector in which segments of the LTR have been replaced with elements of the cytomegalovirus (CMV) immediate early gene promoter. This hybrid promoter allows higher transcription levels in packaging cells leading to higher retroviral titer. We use both an internal promoter and IRES to allow for transcription of TCR genes and a selectable marker [136]. Other retroviruses that have been used for transfer of genes to human cells include murine stem-cell viruses and lentiviruses. Lentiviruses are a subset of retroviruses that are more genetically complex than MMLV. Their low immunogenic properties coupled with the capability of infecting non-dividing cells have made them a candidate for use in gene therapy. Recently, several groups have demonstrated lentiviral based gene transfer to primary human T cells [137-140]. While transduction of non-dividing T cells is possible, it has been repeatedly shown that T cell activation is still necessary for high level transfer and expression of the transgene. Furthermore, while use of retroviral based gene therapy is clinically established, lentiviral based therapies are not yet approved for clinical use.
Generating tumor reactive T cell populations with chimeric antibody-receptors
Chimeric antibody receptors are another single chain alternative to TCR for redirecting T cell specificity to TAA. Chimeric immunoglobulin (cIg) receptors are composed of the heavy and light chain variable regions of an antibody fused to the transmembrane/intracellular portion of a lymphocyte signaling molecule. The most commonly used transmembrane/intracellular portions are from the Fc εRI-γ chain and the CD3-ζ chain. cIg receptors, described shortly after the development of single chain Ab molecules in the 1980's, are attractive constructs for modifying T cell specificity because their binding is not MHC-restricted, and because cIg can recognize intact surface proteins without the need for antigen processing and presentation by the target cell [141]. TCR transduced T cells, on the other hand, are more likely to demonstrate normal antigen binding and signaling behavior, which may be important for eliciting optimal CTL responses.
In 1993, Stancovski et al. reported anti Her-2/neu activity by T cell hybridomas transduced with Her-2/neu specific cIg fused to the Fc εRI-γ chain [142]. Subsequent studies by other investigators have demonstrated the efficacy of cIg receptor constructs specific for the breast cancer antigens Her3 and Her4 [143,144]. Ovarian cancer, lung cancer, melanoma, prostate cancer, and renal cell carcinoma are among the tumors that have been targeted with cIg receptor retroviral constructs by various groups [145-149]. Several groups have targeted glycoprotein molecules such as carcinoembryonic antigen (CEA) and GA733-2 that are expressed by a majority of colorectal cancers and other tumors of gastro-intestinal origin, and their cIg transduced T cells have shown efficacy in vitro and in murine models [150-154]. A comparison of CEA-directed cIg fused to the Fc εRI-γ chain or the CD3-ζ chain found that despite similar levels of transgene expression, CD3-ζ-linked cIg were able to better control the growth of CEA-expressing tumors in murine models [155]. Recently, these constructs have been further engineered to incorporate a costimulatory signaling mechanism [156-159]. Constructs containing the heavy and light chain variable regions of an antibody, the CD28 signaling domain, and the CD3-ζ chain in series were first described by Finney et al in 1998. T cells transduced with cIg containing the CD28 signaling domain have shown enhanced ability to control the growth of CEA-expressing tumors in murine models [158,160].
In summary, techniques for altering T cell-tumor interactions through gene transfer are being widely investigated. At the present time, several groups of investigators are addressing the methodologic and regulatory hurdles that must be overcome in preparing these agents for clinical use. The first clinical trials of TCR gene therapy have recently been initiated. If promising, the early scientific and clinical results of these studies may soon stimulate broad interest in TCR gene therapy for cancer and associated areas of investigation.
Conclusion
Despite the wealth of information that has been acquired pertaining to T cell recognition of tumors, we are left with far more questions than answers regarding ways by which the immune response might be manipulated to improve cancer treatment. Though the T cell repertoire is expansive, the repertoire of tumor reactive cells in any individual may be very limited, or may be difficult to activate and expand either in vitro or in vivo. The relationship between TCR affinity, T cell avidity, and T cell effector function is complex. This may account for the disparity between our success in stimulating antigen reactive precursor T cells through immunization and generating cells for adoptive therapy in vitro, and our inability to achieve a high rate of durable clinical responses. A universal approach to immunization against tumor antigens or adoptive immunotherapy may not be possible for any tumor type. Instead, combined therapeutic approaches or therapy optimized for the individual may be necessary. Current and future investigations of specific T cell – tumor interactions and novel therapeutics will determine whether broadly effective immune therapies are to be realized.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MDM conceived the design and organization for the review, participated in the immunological research included, and drafted the manuscript. JJR performed the immunological research included and participated in drafting the manuscript. MIN conceived, designed, and coordinated the immunological research included and participated in drafting the manuscript. All authors read and approved the final manuscript.
Acknowledgements
Funding for the studies and authors were supplied by NIH grant CA096775 (MDM) and by NIH grants CA090873 and CA102280 (MIN). Thanks to Sandra Cleek for helping with preparation of the manuscript.
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-3-641619427610.1186/1477-7819-3-64ResearchUsing the intraoperative hand held probe without lymphoscintigraphy or using only dye correlates with higher sensory morbidity following sentinel lymph node biopsy in breast cancer: A review of the literature Kim Suk Chul [email protected] Dong Wook [email protected] Renee M [email protected] Chun K [email protected] Samprit [email protected] Michail K [email protected] Arlene [email protected] Josef [email protected] Borys R [email protected] Department of Radiology, Division of Nuclear Medicine, The Mount Sinai School of Medicine, The Mount Sinai Hospital, New York, New York, USA2 Department of Nuclear Medicine, Albert Einstein College of Medicine of Yeshiva University, and the Montefiore Medical Center, Bronx, New York, USA3 Department of Health Policy, The Mount Sinai School of Medicine, The Mount Sinai Hospital, New York, New York, USA4 Department of Surgery, The Mount Sinai School of Medicine, The Mount Sinai Hospital, New York, New York, USA2005 29 9 2005 3 64 64 28 7 2005 29 9 2005 Copyright © 2005 Kim et al; licensee BioMed Central Ltd.2005Kim 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 no studies that have directly investigated the incremental reduction in sensory morbidity that lymphoscintigraphy images (LS) and triangulated body marking or other skin marking techniques provide during sentinel lymph node biopsy (SLNB) compared to using only the probe without LS and skin marking or using only dye. However, an indirect assessment of this potential for additional sensory morbidity reduction is possible by extracting morbidity data from studies comparing the morbidity of SLNB to that of axillary lymph node dissection.
Methods
A literature search yielded 13 articles that had data on sensory morbidity at specific time points on pain, numbness or paresthesia from SLNB that used radiotracer and probe or used only dye as a primary method of finding the sentinel node (SN). Of these, 10 utilized LS, while 3 did not utilize LS. By matching the data in studies not employing LS to the studies that did, comparisons regarding the percentage of patients experiencing pain, numbness/paresthesia after SLNB could be reasonably attempted at a cutoff of 9 months.
Results
In the 7 studies reporting on pain after 9 months (> 9 months) that used LS (1347 patients), 13.8% of patients reported these symptoms, while in the one study that did not use LS (143 patients), 28.7% of patients reported these symptoms at > 9 months (P < 0.0001). In the 6 studies reporting on numbness and/or paresthesia at > 9 months that used LS (601 patients), 12.5% of patients reported these symptoms, while in the 3 studies that did not use LS (229 patients), 23.1% of patients reported these symptoms at > 9 months (P = 0.0002). Similar trends were also noted for all these symptoms at ≤ 9 months.
Conclusion
Because of variations in techniques and time of assessing morbidity, direct comparisons between studies are difficult. Nevertheless at a minimum, a clear trend is present: having the LS images and skin markings to assist during SLNB appears to yield more favorable morbidity outcomes for the patients compared to performing SLNB with only the probe or performing SLNB with dye alone. These results are extremely pertinent, as the main reason for performing SLNB itself in the first place is to achieve reduced morbidity.
==== Body
Background
There is a trend towards minimally invasive surgery in all aspects of breast cancer management. In keeping with this trend is the increasing popularity of sentinel lymph node biopsy (SLNB), which has been shown to result in lower levels of morbidity in the staging of patients with breast cancer as compared to conventional axillary lymph node dissection (ALND) [1-20]. Therefore, it has essentially become the standard of care [21]. Furthermore, SLNB potentially provides even better staging than ALND, as SLNB offers the added benefit of allowing for a more extensive histological analysis of the SNs, by allowing for additional sections through nodes, and by making it possible to use additional immunohistochemical staining methods. These added maneuvers are not routinely performed on nodes obtained from "classical" ALND, since there are too many nodes available for examination from ALND, and testing all of them is not feasible. [22,23].
The role that lymphoscintigraphy (LS) images play in reducing morbidity during SLNB vs. using only the probe without images or using only dye has not been formally examined. Surgeons from ours and other institutions find LS and triangulated body marking (TBM) or other skin marking techniques essential during SLNB [22-26], while others do not find the images or markings helpful [27-29].
Theoretically, an accurate 3-dimentional representation of the number and location of SNs in the body in the form of LS images and TBM reference points or other skin markings, should expedite surgery by allowing a more targeted, minimally invasive approach. This should limit the amount of dissection, and therefore, the level of morbidity. This is an important issue, as the main purpose for performing SLNB is morbidity reduction.
Since no direct studies have investigated the additional reduction in sensory morbidity that LS images and TBM may provide during SLNB, an indirect assessment of this potential was performed by extracting sensory morbidity data on SLNB from studies comparing the morbidity of SLNB to that of ALND [1-20] as described below.
Methods
The computerized bibliographic databases of PubMed and Medline were searched. The key words used in the search included breast, node, and morbidity. In addition, an extensive manual search and cross-referencing from review and original articles was performed. The global inclusion criteria were prospective and retrospective studies that listed any sensory morbidity related to SLNB in breast cancer patients.
Information on study design, lymphoscintigraphy techniques, time of follow-up, and patients' characteristics were collected from the searched articles. When information was not present in the referenced text, personal communications were initiated with the authors to obtain additional information missing from the text; mainly to establish the use or non-use of LS. Based on the information from the articles and personal communications with the authors, 20 articles were found that met these initial criteria [1-20] (Table 1). Three comparisons were made, based on decreasingly stringent levels of comparative parameters described below.
Table 1 Studies with data on sensory morbidity and sentinel lymph node biopsy in breast cancer (ref 1-20).
Reference Year Journal Country Sample Size Design Lymphoscintigraphy Mean Length of Follow-up
Schrenk P et al.(1) 2000 Cancer Austria 35 Prospective Used 15.4 M
Roumen RM et al.(2) 2001 Br J Surg Netherlands 100 Prospective Used 24 M
Burak WE et al.(3) 2002 Am J Surg USA 48 Prospective Not Used 15.4 M
Haid A et al.(4) 2002 Breast Ca Res T Austria 57 Retrospective Used 18 M
Temple LK et al.(5) 2002 Ann Surg Oncol USA 171 Prospective Used 3 M, 6 M, 12 M
Swenson KK et al.(6) 2002 Ann Surg Oncol USA 169 Prospective Not Used 1 M, 6 M, 12 M
Haid A et al.(7) 2002 Eur J Surg Ocol Austria 66 Prospective Used Not specified
Leidenius M et al.(8) 2003 Am J Surg Finland 49 Prospective Used 2 W
Schijven MP et al.(9) 2003 Eur J Surg Oncol Netherlands 180 Retrospective Used 12 M
Blanchard DK et al.(10) 2003 Arch Surg USA 685 Prospective Used 2.4 Y
Veronesi U et al.(11) 2003 N Engl J Med Italy 100 Prospective Used 6 M, 24 M
Rietman JS et al.(12) 2003 Cancer Netherlands 66 Prospective Used* 6 W
Peintinger F et al.(13) 2003 Br J Cancer Austria 25 Prospective Used 1 W, 9–12 M
Baron RH et al.(14) 2004 Oncol Nurs Forum USA 197 Prospective Used 24 M
Armer J et al.(15) 2004 Lymphololgy USA 9 Retrospective Used 9 M
Ronka RH et al.(16) 2004 Acta Oncol Finland 57 Prospective Used 12.6 M
Rietman JS et al.(17) 2004 Ann Surg Oncol Netherlands 66 Prospective Used* 12 M
Langer S et al.(18) 2004 Am Surg USA 40 Retrospective Not Used 51 M
Luini A et al.(19) 2005 Breast Ca Res T Italy 244 Prospective Used 6–12 M
Ronka RH et al.(20) 2005 Breast Finland 43 Prospective Used 12 M
*Use/method of lymphoscintigraphy unclear, radiotracer used as backup method of finding sentinel node compared to primary dye based technique.
Initial comparisons
For the initial set of comparisons, parameters of morbidity assessment included; 1) pain in the arm or axilla, and 2) numbness on the operated side and/or paresthesias. A subset of the group of 3 studies not using LS were compared to a subset of the 17 studies that used LS. Studies were matched as closely as possible based on the mean time frames of morbidity assessment, to generate best matched time frames of assessing morbidity.
Excluded studies and exclusion criteria for the initial comparisons
The study by Schrenk et al [1] was initially excluded, because SLNB morbidity data were presented for a mixed group of blue dye only and combined dye/radiotracer procedures.
The study by Temple et al [5] was excluded, as a unique morbidity scoring system was employed that could not be adapted in any way for the comparisons. In addition, prevalence data was reported, but the number of patients experiencing symptoms could not be extracted.
One of the two studies by Haid et al [7] was excluded, as no data on the timing of symptom assessment were provided. Symptoms tend to decrease over time, and the time of assessment is needed for any meaningful comparisons.
The study of Ronka et al [16] presented morbidity data which were strictly confined only to the breast, excluding the axilla where SLNB is performed, and therefore was excluded.
Two studies by Rietman et al [12,17], were initially excluded in this constrained comparison because the use of lymphoscintigraphy was unclear [12,17,30-32] and portions of the data in the initial earlier study were in error [12,31,33]. In addition, there are suggestions that a dissection intensive blue dye technique was used as the main method of finding the SN over a more targeted radiotracer/probe method, which appeared to only have a secondary role in finding the SN [12,17,30-32]. These two studies [12,17] were based on the exact same population of patients but at different time points of morbidity assessment. Yet, the later study [17] fails to acknowledge the existence of the earlier study and its results [12].
Luini A et al [19] presented morbidity data on a mixed group of ALND and SLNB patients where pure SLNB data could not be extracted. The study was therefore excluded.
We also initially excluded additional studies [2,4,10,14,15,18], because pain and numbness and/or parasthesia morbidity parameters were not assessed at comparable time points to the remaining studies.
For all remaining studies, the criteria for assessing morbidity was chosen as such: either symptoms were present (yes) or absent (no). In studies where a sliding scale of intensity was the method of reporting morbidity symptoms, the percentage of patients fitting into a percent adjusted yes or no group was used [6,20].
After the initial study selection process described above, 7 studies were included in the initial comparative analysis. Among the 7 studies, 5 used LS [8,9,11,13,20] and 2 did not use LS [3,6] (Table 2).
Table 2 Initial comparisons. Comparisons of studies at specific time points that met the constrained criteria detailed in the initial comparison (see text).
Morbidity Mean Length of F/U Lymphoscintigraphy (+) Lymphoscintigraphy (-) p-value
Mor (%) Total Pt (N) References Mor (%) Total Pt (N) References
Pain ≤ 1 m 31.08% 74 8,13 56.52% 161 6 0.0004
6 m 16.00% 100 11 37.50% 152 6 0.0002
12–15.4 m 11.71% 222 9,20 28.67% 143 6 < 0.0001
Numbness/Paresthesia 6 m 2.00% 100 11 30.26% 152 6 < 0.0001
12–15.4 m 4.50% 222 9,20 22.75% 189 3,6 < 0.0001
The 7 studies were grouped into the same morbidity category when data was available. Depending on follow-up periods and sensory morbidity assessment techniques, a total of 5 time periods were established.
The resulting comparable time periods of morbidity assessments for pain were ≤ 1 month, 6 months and 12 to 15.4 months. Likewise, numbness/parasthesia morbidity assessments were at 6 months and 12 to 15.4 months (Table 2).
The statistical analysis was performed using R 2.01 software available in the public domain. Fisher's exact test was used to analyze the nominal variables in the form of frequency tables. A result was considered to be significant only if the P-value was lower than 0.05. All p-values reported are two-sided.
Secondary comparisons
The 20 studies were then reanalyzed with less stringent time of morbidity assessment criteria. Two time periods were created to include the greatest number of studies in the analysis and resulted in the time periods of ≤ 9 months and > 9 months for pain and numbness/paresthesia (Table 3). This allowed the 6 initially excluded studies from table 2[2,4,10,14,15,18], to be integrated into table 3, resulting in a total of 13 studies included in the secondary comparative analysis. Among the expanded group of 13 studies, 10 used LS [2,4,8-11,13-15,20] and 3 did not use LS [3,6,18] (Table 3). Statistical analysis for Table 3 again utilized Fisher's exact test.
Table 3 Secondary comparisons. Comparisons of studies at two time periods that met the criteria detailed in the secondary comparison (see text).
Morbidity Mean Length of F/U Lymphoscintigraphy (+) Lymphoscintigraphy (-) p-value
Mor (%) Total Pt (N) References Mor (%) Total Pt (N) References
Pain Acute (≤ 9 m) 22.41% 174 8,11,13 47.28%* 161* 6 < 0.0001
Chronic (> 9 m) 13.81% 1347 2,4,9,10,11,14,20 28.67% 143 6 < 0.0001
Numbness/Paresthesia Acute (≤ 9 m) 5.50% 109 11,15 35.58%* 160* 6 < 0.0001
Chronic (> 9 m) 12.48% 601 4,9,11,13,14,20 23.14% 229 3,6,18 0.0002
* The average morbidity at 1 month and 6 months in article 6. Pain morbidity at 1 month and 6 months are 56.52% (91/161) and 37.50% (57/152), respectively. Numbness/paresthesia morbidity at 1 month and 6 months are 40.63% (65/160) and 30.26% (46/152), respectively.
Tertiary comparisons
To further maximize the number of studies compared and be as inclusive as possible, additional studies were integrated into a tertiary analysis by relaxation of criteria. These included the study by Schrenk et al [1], where the heterogeneity of the data set as noted above was overlooked, and the two articles by Rietman et al [12,17], by discounting the issues related to them as noted above and including them in the analysis (Table 4).
Table 4 Tertiary comparisons. Comparisons of studies at two time periods that met the revised (relaxed more encompassing) criteria detailed in the tertiary comparison (see text).
Morbidity Mean Length of F/U Lymphoscintigraphy (+) Lymphoscintigraphy (-) p-value
Mor (%) Total Pt (N) References Mor (%) Total Pt (N) References
Pain Acutet (≤ 9 m) 22.41% 174 8,11,13 47.28%* 161* 6 < 0.0001
Chronic (> 9 m) 13.77% 1365 1,2,4,9,10,11,14,20 28.67% 143 6 < 0.0001
Numbness/Paresthesia Acute (≤ 9 m) 27.43% 175 11,12,15 35.58%* 160* 6 0.1254
Chronic (> 9 m) 12.56% 677 1,4,9,11,13,14,17,20 23.14% 229 3,6,18 0.0003
* The average morbidity at 1 month and 6 months in article 6. Pain morbidity at 1 month and 6 months are 56.52% (91/161) and 37.50% (57/152), respectively. Numbness/paresthesia morbidity at 1 month and 6 months are 40.63% (65/160) and 30.26% (46/152), respectively.
t This comparison remained unchanged from table 3.
Issues with the remaining articles [5,7,16,19] continued to be too significant to be reasonably included in the tertiary comparison, as no meaningful data could be extracted for the reasons already noted above. Statistical analysis for Table 4 again employed Fisher's exact test.
Among the 20 articles, there were several with similar or partly overlapping patient populations. Specifically the articles of Leidenius M et al [8] and Ronka RH et al [20], as well as Rietman JS et al [12,17] fit this pattern. However, for these articles the times of morbidity assessment were different, so no overlapping of patient populations occurred in any of the comparisons. In one group of articles, Temple et al [5] and Baron RH et al [14], the former was not included in any of the comparisons for reasons already mentioned above, so no overlapping patient population issues occurred when the latter was included in the comparisons. For the studies of Roumen RM et al [2] and Schijven MP et al [9], there potentially appeared to have been very minimal overlap of patient populations. The earlier prospective study [2] had data from 90 patients from one hospital, while the later retrospective study [9] had data on 180 patients from the same hospital as well as another hospital, with the later study [9] data collected mainly outside the timeframe of the earlier study [2]. Correspondence with the author of the earlier study [2] suggested no overlap with independent cohorts. Both studies were therefore included in the analysis.
Results
For studies that met the constrained criteria, for all comparison periods, there was less pain and numbness/paresthesia morbidity reported for studies using LS than those that did not, as shown in Table 2. The differences were highly statistically significant (< 0.001). Similar results were obtained in the secondary comparisons, in which the time of morbidity assessment was segregated into two time periods, allowing more studies to be included in the analysis, as shown in Table 3. For the tertiary comparisons in which inclusion criteria were relaxed as described above to maximize the number of studies included, all comparisons showed lower morbidity when LS was used, but only 3 out of 4 were statistically significant (see Table 4). The p-value for the numbness/paresthesia comparison at ≤ 9 months did not reach statistical significance (0.1254). Globally, for the three levels of comparison (Tables 2, 3, 4), all 12 unique comparisons, using various inclusion/exclusion criteria, showed less morbidity for the LS groups. In 11 out of the 12 unique comparisons, the p-values were highly statistically significant (p-value range < 0.0001 to 0.0004). The results for numbness/paresthesia at > 9 months shown in Table 4 are especially pertinent, as this reflects long term outcomes and includes the largest number of patients, and the greatest number of LS and non LS studies.
Discussion
The concept that using LS further reduces morbidity over that of using the probe without LS or using dye alone is intriguing to say the least. The analysis we performed suggests that a strong trend exists for additional morbidity reduction in the groups using LS.
The results from this analysis can also be conceptualized from a purely theoretical standpoint; that is: mapping out the location of the SNs in three dimensions prior to incision, knowing the true number of "hot" SNs (or lack of nodes) and the relative intensity and time of appearance to one another should facilitate a targeted surgical approach. Surgeons can plan their approach with this information and adjust their dissection based on the findings of the probe with/without concurrent use of dye.
The results are also supported by the experience of surgeons who have access to high quality LS and skin markings and understand how to use the information. They are of the opinion that both the corresponding images and markings allow for a more directed dissection [23,26].
The studies reviewed in the analysis were not directly trying to evaluate whether LS further reduces morbidity. Had they been, it can be speculated that potentially even better results could have been obtained with LS, especially as the techniques of LS, TBM/skin marking, and SLNB continue to evolve [22-26,34-41], with a renewed focus on accurate anatomical delineation and minimally invasive surgery. In addition, 9 studies that employed LS indicated that they used variable forms of skin marking. Had all studies that employed LS used an updated version of this technique of 3-dimensional SN reference, i.e. TBM, a further morbidity reduction could have potentially been achieved by directing an even more targeted surgical approach [34,36,41].
The greatest limitation of our analysis is that there are only a few studies that did not employ LS (n = 3). Most of the comparisons in the tables were with only a single non LS study [6]. Nevertheless, the results of nearly all comparisons are consistent with results from the specific comparisons that contained 3 non LS studies. The number of studies employing LS was much greater, and provides stable estimates for the proportion having the morbidities of pain and numbness/paresthesias. Because of variations in techniques, time and method of assessing morbidity, direct comparisons between studies are difficult and subject to confounding issues.
The number of nodes removed, use of radiotherapy after SLNB, use of dye in addition to radiotracer, and the harvesting of level III SNs and/or non-axillary nodes could potentially also influence the level of morbidity. These factors were not scrutinized as the information was not consistently available in the referenced articles. For example, information on the biopsy of non-axillary nodes was available only for one reference [8], where 16% (8/49) of SN biopsied were non-axillary. While our study results show an overwhelming advantage for using LS (+) over no LS (-) (essentially a 50% reduction in sensory morbidity at P < 0.0001–0.0003), we are very conservatively describing the results as only a "strong trend" or correlation for the reasons noted above.
Many clinicians dispute any advantages that LS and skin markings may provide in serving the patient during SLNB [27-29,42,43]. The reasons for this could be partly explained by the great variability in the quality of LS studies that are performed [23,27,28,34-37], and/or perhaps by difficulties on the part of the surgeons in integrating the information provided by LS and skin markings into their surgical techniques during SLNB. Surgical techniques need to be addressed in the pursuit of morbidity reduction. Training tools for surgeons have been developed that have the potential for integrating the information from LS and skin markings, with the goal of performing minimally invasive surgery [38,40].
When the surgeons have a 3-dimensional concept and image of the location of SNs prior to surgery, they are then able to fine tune their surgical approach. An example of this ability to fine tune surgical approach can be applied to a hypothetical case of an upper outer quadrant breast lesion. Initially, the surgeon may plan to attempt to reach the SN via the tumor excision site in an attempt to reduce morbidity. However, when provided with LS images and skin markings the surgeon might realize that this is not the optimal approach from a standpoint of morbidity reduction, and would be able to plan an alternative approach that further minimizes morbidity. It is conceivable that more experienced, better informed surgeons are more likely to utilize LS because of its benefits, and that these surgeons perform a more delicate dissection resulting in less morbidity. However, the exact contribution of LS vs. the skill level of the surgeons that directly contributes to this reduction in morbidity can not be independently deduced from the data in the articles.
Based on this analysis, the surgical evaluation of the sentinel node in the axilla should utilize LS, TBM or other skin marking techniques, and a probe guided dissection in order to reduce sensory morbidity.
Conclusion
Though no randomized studies exist that directly ask this question, our literature analysis suggests that lymphoscintigraphy imaging is a very useful tool in further reducing morbidity during sentinel lymph node biopsy.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SCK contributed to data analysis, design, and preparation of manuscript.
DWK contributed to data analysis, design, and preparation of manuscript.
RMM contributed to data analysis, design, and preparation of manuscript.
CKK contributed to data analysis, design, and preparation of manuscript.
SC contributed to data analysis, design, and preparation of manuscript.
MKS contributed to data analysis, design, and preparation of manuscript.
AT contributed to data analysis, design, and preparation of manuscript.
JM contributed to data analysis, design, and preparation of manuscript.
BRK developed the initial concept and contributed to data analysis, design, and preparation of manuscript.
All authors read and approved the final manuscript.
==== Refs
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==== Front
BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-1161616805710.1186/1471-2407-5-116Research ArticleSurvey of oxaliplatin-associated neurotoxicity using an interview-based questionnaire in patients with metastatic colorectal cancer Leonard Gregory D [email protected] Maurice A [email protected] Mary G [email protected] Suzanne [email protected] Nancy [email protected] Barbara [email protected] Rebecca R [email protected] Jean L [email protected] Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute-Navy Medical Oncology Program, National Naval Medical Center, Bethesda, MD 20889-5105, USA2 Medical Oncology Research Unit, Center for Cancer Research, National Cancer Institute-Navy Medical Oncology Program, National Naval Medical Center, Bethesda, MD 20889-5105 USA2005 16 9 2005 5 116 116 29 4 2005 16 9 2005 Copyright © 2005 Leonard 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
New chemotherapy regimens for patients with colorectal cancer have improved survival, but at the cost of clinical toxicity. Oxaliplatin, an agent used in first-line therapy for metastatic colorectal cancer, causes acute and chronic neurotoxicity. This study was performed to carefully assess the incidence, type and duration of oxaliplatin neurotoxicity.
Methods
A detailed questionnaire was completed after each chemotherapy cycle for patients with metastatic colorectal cancer enrolled in a phase I trial of oxaliplatin and capecitabine. An oxaliplatin specific neurotoxicity scale was used to grade toxicity.
Results
Eighty-six adult patients with colorectal cancer were evaluated. Acute neuropathy symptoms included voice changes, visual alterations, pharyngo-laryngeal dysesthesia (lack of awareness of breathing); peri-oral or oral numbness, pain and symptoms due to muscle contraction (spasm, cramps, tremors). When the worst neurotoxicity per patient was considered, grade 1/2/3/4 dysesthesias and paresthesias were seen in 71/12/5/0 and 66/20/7/1 percent of patients. By cycles 3, 6, 9, and 12, oxaliplatin dose reduction or discontinuation was needed in 2.7%, 20%, 37.5% and 62.5% of patients.
Conclusion
Oxaliplatin-associated acute neuropathy causes a variety of distressing, but transient, symptoms due to peripheral sensory and motor nerve hyperexcitability. Chronic neuropathy may be debilitating and often necessitates dose reductions or discontinuation of oxaliplatin. Patients should be warned of the possible spectrum of symptoms and re-assured about the transient nature of acute neurotoxicity. Ongoing studies are addressing the treatment and prophylaxis of oxaliplatin neurotoxicity.
==== Body
Background
Colorectal cancer is the third commonest cause of non-skin cancers in men and in women with a total of 146,940 new cases and 56,730 deaths estimated in the United States in 2004 [1]. Approximately 25% of patients present with metastatic disease. Despite improvements in adjuvant therapy, a substantial number of patients with localized disease will ultimately develop metastatic disease. The 5-year survival for patients with metastatic colorectal cancer is less than 10%. The role of chemotherapy in patients with unresectable disease as a neoadjuvant approach to make resection possible is currently being explored in clinical trials; retrospective analyses suggest that patients who can undergo complete surgical resection of hepatic metastases after neoadjuvant chemotherapy may have a survival approaching 50% [2].
The standard chemotherapy regimen used as first-line treatment of colorectal cancer for many years was bolus 5-FU modulated by (LV), but this has recently been superseded by newer combination regimens involving LV-modulated 5-FU given as a mixed bolus and continuous infusion with the addition of either oxaliplatin or irinotecan. Oxaliplatin, an organoplatinum complex, produces cytotoxicity via the formation of DNA adducts which interfere with DNA replication and transcription and lead to induction of apoptosis. The different spectrum of clinical activity and toxicity of oxaliplatin compared to cisplatin and carboplatin is believed to be due to its 1,2-diaminocyclohexane structure, which forms bulkier DNA adducts. A Phase III trial in patients with metastatic colorectal cancer comparing oxaliplatin and biweekly LV-modulated mixed bolus and infusional 5-FU (FOLFOX 4) to bolus 5-FU/LV given daily for five days every month demonstrated superiority in both response rate and progression-free survival [3]. Another Phase III trial demonstrated a significant survival advantage with FOLFOX-4 compared to irinotecan and bolus 5-FU/LV (median survival 19.5 vs 15 months, p = 0.0001) [4]. A randomized trial comparing sequential administration of biweekly LV-modulated mixed bolus and infusional 5-FU combined with either oxaliplatin (FOLFOX 4) or irinotecan (FOLFIRI) followed by the other regimen at the time of disease progression showed equivalent survival of about 21 months for both sequences [5]. The Food and Drug Administration approved FOLFOX-4 as salvage therapy of metastatic colorectal cancer in August, 2002, and as first-line therapy in January, 2004. Oxaliplatin has also shown activity in other platinum-sensitive tumors, including in some cancer patients with documented cisplatin-refractory disease [6-8].
The dose-limiting toxicity of oxaliplatin is neurotoxicity [9,10]. The neurotoxicity has two distinct manifestations: acute, transient symptoms are due to peripheral sensory and motor neuron hypersensitivity; cumulative, persistent symptoms are due to chronic peripheral sensory neuropathy [9-12]. The pathogenesis of the acute neuropathy is believed to be due to a channelopathy (a temporary dysfunction of an ion channel in the nerve membrane), while chronic sensory neuropathy may be a result of direct, cumulative neurotoxic effects resulting from platinum accumulation in the dorsal root ganglion [11-15]. A number of grading systems have been used to report oxaliplatin-associated neurotoxicity. Clinical studies involving oxaliplatin appear to have largely described the chronic sensory neuropathy, whereas information on the acute peripheral motor and sensory neuropathy is more limited. In order to obtain information on the incidence and type of oxaliplatin-associated acute and chronic neurotoxicity, we devised a questionnaire to be used in a phase I study of oxaliplatin and capecitabine
Methods
Voluntary subjects
Patients were enrolled in a Phase I study examining the safety and feasibility of administering a fixed dose of oxaliplatin with escalating doses of capecitabine to patients with advanced colorectal or small bowel adenocarcinoma. The protocol was approved by the Cancer Therapy Evaluation Program, NCI, and Institutional Review Boards of the NCI and the National Naval Medical Center. All patients gave written informed consent. As part of the trial, we collected information on neurotoxicity experienced by the patients during therapy. We previously reported information obtained using EMG and NCS as an objective assessment of the neurotoxicity [11,12]. The objective of the present analysis was to determine the incidence, type and duration of neurotoxicity using a questionnaire that was filled in by a research nurse during an interview with the subject.
Chemotherapy and dose modifications
Oxaliplatin 130 mg/m2 was infused by vein over 2-hours on day one, while a total daily dose of capecitabine ranging from 1,200 to 3,000 mg/m2 was given in two divided doses on days 1–5 and 8–12 of a planned 21-day cycle. As part of this intramural NCI research study, patients were hospitalized overnight at no charge following the first infusion of oxaliplatin to allow monitoring for acute neurotoxicity, and patients were interviewed the next morning to record any acute symptoms. Protocol therapy was continued until evidence of disease progression occurred, unacceptable toxicity developed, or the patient elected to withdraw. A neurotoxicity grading system developed by the pharmaceutical sponsor was used to describe the paresthesias/dysesthesias, which could be cold-induced or not: grade 1, symptoms of short duration that resolve and do not interfere with function; grade 2, interfering with some functions, but not with the basic activities of daily living; grade 3, pain or functional impairment that interfere with activities of daily living; grade 4, persistent symptoms that are disabling or life-threatening.
If subjects experienced acute, transient neurotoxicity that was particularly bothersome, the duration of infusion was increased to six hours in subsequent cycles. The dose of oxaliplatin was decreased by 25% if grade 2 neurotoxicity persisted between cycles. The dose of oxaliplatin was decreased by 25% for grade 3 neurotoxicity that resolved before the next cycle of therapy, while the drug was discontinued for either grade 3 neurotoxicity that persisted between cycles, or for grade 4 neurotoxicity, regardless of duration. In a subset of twelve patients, an empiric trial of carbamazepine was begun five days prior to the second dose of oxaliplatin and continuing for a total of seven days as previously reported to assess any possible impact on acute neurotoxicity symptoms and neurophysiologic studies [11].
Neurologic assessment
Subjects were asked to keep a daily diary of side-effects. A research nurse interviewed the subjects and filled out a questionnaire that characterized the incidence and type of neurotoxicity symptoms and how long they lasted. The questionnaire was divided into 3 parts addressing the upper extremity, the lower extremity and orofacial area (Tables 1, 2, 3), and was completed after each 3-week cycle of chemotherapy. Objective neurological assessment was also obtained by physical exam in all patients at the start of each cycle. The results of serial EMG and NCS tests in a subset of patients have been reported previously [12].
Results
Demographics
Eighty-six patients (56 males/30 females) were included in this assessment; 69 were Caucasian, 14 were African-American, and three were Hispanic. The median age was 56 years (range 26–74). The location of the primary tumor was colon (76 patients), rectum (8) or appendix (2). The median ECOG performance status was 1 (range 0–2). The majority of the patients had undergone prior surgical resection of their primary tumor; 90% had received prior 5-FU, and two-thirds had received prior irinotecan-based therapy.
Use and analysis of the questionnaire
The questionnaire was a tool that was designed to help capture the variety and location of potential neurotoxic side effects that the patients might experience. Patients were specifically queried about symptoms occurring the upper extremities, lower extremities, and face/mouth. Patients were asked if they had each of the specific symptoms, yes or no. If yes, then there were two parts to the assessment: first, whether the patient felt the symptoms were minimal to very prominent; second, whether the symptoms affected the daily activities hardly at all through a range that the subject was extremely bothered by the symptoms in a functional sense. The following scores on the questionnaire corresponded to the following neurotoxicity grades: 1,2 = grade 1; 3 = grade 2; 4 = grade 3; 5 = grade 4.
Neuropathy
Dysesthesia, herein defined as painful or distressing sensations experienced in the absence of stimulation, was experienced by 87.2% of patients. Paresthesia, herein defined as non-painful but abnormal sensations such as numbness or tingling, was experienced by 94.3% of patients at some point during oxaliplatin-based chemotherapy (Table 4). For most patients, the worst grade of dysesthesia and paresthesia was grade 1 (70.9% and 66.3% respectively). However, some patients experienced functional impairment affecting activities of daily living. One patient developed an ascending sensorimotor syndrome after the fourth dose of oxaliplatin that led to profound weakness in the upper and lower extremities without respiratory impairment, although it was uncertain whether this was due to oxaliplatin or due to the development of an unrelated neurological condition [16].
Dysesthesias occurred more often in the face than in the extremities, perhaps due to acute, painful cold sensitivity experienced while eating, drinking or handling a cold object (Figure 1). Paresthesias occurred more often in the hands and feet, which may be consistent with the glove and stocking distribution associated with chronic peripheral neuropathies.
The duration of the neuropathy increased as the cumulative exposure to oxaliplatin increased (Table 5). After the first cycle of chemotherapy, the median duration of dysesthesia was only 5 days, whereas it was 21 days in patients who received 12 cycles of chemotherapy. The median duration of paresthesia after cycle one was 7 days, but after cycle 12 was 21 days or longer. The proportion of patients who complained of dysesthesia of any grade after one cycle of chemotherapy (73%) was similar to the proportion of patients who complained of paresthesia (77%) (Figure 2). After six cycles of chemotherapy, 68% of patients complained of dysesthesia, whereas 92% of patients complained of paresthesia. The increased incidence of paresthesia is consistent with the development of a cumulative sensory neuropathy in most patients. The relatively stable incidence of dysesthesia may reflect that such symptoms are more commonly associated with the transient hyperexcitability phenomenon. The acute symptoms do not appear to be cumulative phenomenon. By cycles 9 and 12, the incidence of grade 3 dysesthesia and paresthesia became noticeable. Remarkably, some patients did not complain of any paresthesia or dysesthesia even after 12 cycles of therapy. By cycles three and six, oxaliplatin dose reductions for neurotoxicity were needed in 2.7% and 20% of patients. By cycle 9, 37.5% of patients required a dose reduction (4% of these had discontinued oxaliplatin); by cycle 12, 62.5% of remaining patients had discontinued oxaliplatin.
Acute neurotoxicity symptoms were recorded with particular focus on the nature of the symptoms. The commonest symptom experienced was cold sensitivity (>80%), which was often reported to be painful by patients (Figure 3). Pain with the initial bite of food was noted by about 60% of the patients, most likely attributed to masticatory spasm. Some symptoms were of very short duration (lasting only seconds or at most a few minutes), while other lasted for several hours (ptosis, sensation of leg heaviness); both short- and longer-lived symptoms could be of major concern to both patient and physician. The most distressing symptom was pharyngolaryngeal dysesthesia, which patients often described as a poor awareness that they were breathing. Other patients noted transient hoarseness or change in their voice, and some commented on visual field deficits or ocular pain. Despite the fact that symptoms resolved spontaneously over a short period of time, many patients required anxiolytics to reduce their level of distress. A subset of patients who received carbamazepine as a prophylactic measure to reduce the symptoms of acute neuropathy did not appear to derive any benefit from this approach [12].
Discussion
Oxaliplatin is an integral component of the various FOLFOX regimens, which have become a standard treatment for metastatic and node-positive colorectal cancer [3,4,17]. Encouraging results have been reported from phase II trials using oxaliplatin in combination with pemetrexed, raltitrexed or capecitabine in patients with metastatic colorectal cancer [18-24]. Enthusiasm regarding the efficacy of oxaliplatin/5-FU/LV regimens has been tempered by the associated neurotoxicity which can significantly impair the quality of life of patients and limits the optimal use of oxaliplatin. Many published trials provide limited details concerning the precise type and location of neurotoxicities experienced, and most appear to have focused on the cumulative sensory neuropathy. Data on the reported incidence of neuropathy from randomized trials, presented in Table 6, may be influenced by the toxicity scale employed, the cumulative oxaliplatin dose, and schedule of administration (3–5,1 7, 25–29). For three trials that employed 85 mg/m2 oxaliplatin given over 2 hours as first-line therapy for metastatic colorectal cancer, the incidence of grade 3 or worse neuropathy was reported to be a mean of 15.4%. A higher incidence of grade 3 neuropathy (31–34%) was reported when oxaliplatin was given at 20 mg/m2 as a 24-hour infusion daily for 5 days every 3 weeks or as 100 mg/m2 over 2 hours every 2 weeks. The incidence of grade 3 neuropathy was lower with IROX, in which 85 mg/m2 is given over 2 hours every 3 weeks. The lowest incidence of serious neurotoxicity has been reported in trials in which FOLFOX is used as second- or third-line salvage therapy, presumably because the cumulative dose of oxaliplatin is less than when employed as first-line therapy.
Although the symptoms associated with acute peripheral sensory and motor nerve hyperexcitability seem to be transient in nature, they can cause distressing symptoms in patients. We used a questionnaire that was filled out by the research nurse during a face- to face interview with the patient. All patients kept a daily calendar diary of side effects, and were interviewed the morning after the initial dose of oxaliplatin. This approach provided detailed information on the incidence, type, location and duration of neuropathy experienced by patients. Since all research subjects were admitted for their first cycle of chemotherapy, close observation for acute neurological symptoms and next-day interviews with patients was possible. These measures may have facilitated identification of symptoms or signs that perhaps would otherwise have gone unreported.
Our study demonstrated that most patients experience either dysesthesia or paresthesia at some point during their treatment, although the worst toxicity grade was grade 1 in most patients. Due to stringent dose modification guidelines, only a small proportion of patients experience serious functional impairment. However, as the cumulative dose of oxaliplatin increases, patients are more likely to experience paresthesia due to a cumulative sensory neuropathy. In patients who receive more than 6 cycles of chemotherapy (projected maximum cumulative dose on this every 3-week schedule of 780 mg/m2), the neuropathy can become persistent and affect the subject's ability to perform routine activities of daily living. Oxaliplatin-associated cumulative sensory neuropathy is slowly reversible in most patients [9,10,17].
Among the various acute, transient neuropathic symptoms experienced by patients, cold sensitivity is the most common form, but it is rarely debilitating since patients can adapt to avoid cold stimuli. The temporary loss of awareness of breathing is relatively uncommon, but is probably the most alarming symptom. It is imperative that patients are warned about these symptoms and their transient nature prior to chemotherapy administration. Such patients can talk, and their breath exhalation is audible. Acute oxaliplatin-associated neurotoxicity can result in other disturbing symptoms such as visual field cuts, blurred vision and ptosis. Administration of FOLFOX regimens require a centrally implanted venous catheter to deliver outpatient infusional 5-FU. Since our study involved oral capecitabine, patients were not required to have central venous access. However, due to the occurrence of whole arm pain in the extremity used to infuse oxaliplatin, and due to extravasation injuries, we amended our protocol to recommend placement of central venous access devices.
There is no standard treatment for oxaliplatin-related neurotoxicity. A variety of strategies have been employed to prevent or treat oxaliplatin neurotoxicity [10,30-39], including carbamazepine, gabapentin, alpha lipoic acid, amifostine, glutathione, and celecoxib. The largest experience is the retrospective analysis of the benefit of infusing magnesium and calcium prior to oxaliplatin administration in 96 patients compared to 65 patients who did not receive magnesium/calcium. The percentage of patients with grade 3 distal paresthesia was lower in Ca/Mg group (7% vs 26%, p = 0.001), and acute symptoms such as distal and lingual paresthesia were much less frequent and severe in the Ca/Mg group. The authors concluded that Ca/Mg infusions seemed to reduce the incidence and intensity of acute oxaliplatin-induced symptoms and might delay cumulative neuropathy [32]. A large, randomized trial will be required to clarify the link between acute, transient symptoms and the likelihood of development of chronic sensory neuropathy, and confirm whether strategies such as Ca/Mg infusions reduce the neurotoxicity without impacting on the anti-tumor efficacy.
Another approach involves the administration of six cycles of FOLFOX-7, followed by the use of biweekly FULV2 for 12 cycles; oxaliplatin was then re-introduced for six cycles, or earlier if disease progression occurred on FULV2 [40]. The intention is to allow recovery from neurotoxicity during the oxaliplatin-free period which may allow the re-introduction of oxaliplatin. Preliminary results demonstrate that this is a convenient regimen with no detrimental effects on efficacy.
Several cases have been reported in the literature concerning peripheral neuropathy in association with 5-FU and capecitabine therapy. Stein reported two subjects who developed symptoms of pain and weakness in the lower extremities while receiving fluorouracil and levamisole [41]. EMG revealed axonal and demyelinting polyneuropathy involving small and large fibers. Two patients treated in a phase I trial of oral 5-FU, leucovorin and eniluracil, an inhibitor of dihydropyrimidine dehydrogenase, developed delayed onset symptoms of unsteady gait and reduced sensation in the legs [42]. EMG and NCS revealed sensorimotor polyneuropathy. Saif reported two patients who experienced either right leg weakness with foot drop or perioral and upper extremity paresthesias with capecitabine [43]. EMG and NCS showed sensorimotor peripheral neuropathy in both patients. In each of these three reports, other common etiologies of peripheral neuropathy were excluded. Couch et al reported a patient given capecitabine who experience acute onset of neuromuscular symptoms, the most prominent of which was trismus, [44]. Peripheral neuropathy may thus represent an extremely rare complication of fluorouracil or capecitabine therapy. The possible contribution of capecitabine to the neurologic symptoms in the patients in the current trial is unclear. However, the neurophysiologic studies obtained shortly after administration of oxaliplatin are unique and are reminiscent of neuromyotonia.
Conclusion
The use of oxaliplatin chemotherapy is increasing and has resulted in significant improvements in outcomes in colorectal cancer. However, oxaliplatin-associated neurotoxicity can cause detrimental effects to the patient's quality of life and may require dose reduction or discontinuation. Our study provides detailed information on the incidence, type and duration of oxaliplatin neurotoxicity. Further understanding of oxaliplatin-associated neurotoxicity is necessary to warn patients of potential side effects and to facilitate strategies to prevent or treat this neurotoxicity. Optimizing the quality of life of cancer is of paramount importance. Continued research on oxaliplatin will help achieve this goal while also providing further progress with respect to clinical benefit outcomes.
Abbreviations
5-FU, 5-fluorouracil; LV, leucovorin; FOLFOX, a combination of folinic acid (leucovorin), 5-FU and oxaliplatin given on a fortnightly schedule; FOLFIRI, a combination of folinic acid (leucovorin) given with 5-FU and irinotecan on a fortnightly schedule; NCI, National Cancer Institute; EMG, electromyogram; NCS, nerve conduction study
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
GDL participated in the conduct of this trial and drafted the manuscript. MW participated in the conduct of this trial. MGQ participated in the conduct of this trial. SF participated in the conduct of this trial. NH participated in the conduct of this trial. BS participated in the conduct of this trial. RT participated in the conduct of this trial. JLG conceived of the study, coordinated its design and implementation, and wrote the final version. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Figures and Tables
Figure 1 Location of dysesthesias/paresthesias during therapy with oxaliplatin and capecitabine (n = 86 subjects).
Figure 2 Percentage of subjects with dysesthesias/paresthesias as a function of number of cycles. The number of subjects at each cycle is shown in parentheses.
Figure 3 Percentage of subjects experiencing specific types of symptoms due to acute peripheral sensory and motor nerve hyperexcitability (n = 86 subjects).
Table 1 Questionnaire documenting upper extremity symptoms of acute and chronic neuropathy experienced during or after oxaliplatin-based chemotherapy.
Upper Extremity Symptoms If you had symptoms during the last cycle....
How much of the symptoms did you have? Did the symptoms affect your daily activities?
Do you have.... Hardly any → Very much Hardly at all bothered → Extremely bothered
Tingling (pins and needles) Yes No 1 2 3 4 5 1 2 3 4 5
Numbness Yes No 1 2 3 4 5 1 2 3 4 5
Difficulty telling the difference between rough and smooth surfaces Yes No 1 2 3 4 5 1 2 3 4 5
Difficulty feeling hot things Yes No 1 2 3 4 5 1 2 3 4 5
Difficulty feeling cold things Yes No 1 2 3 4 5 1 2 3 4 5
A greater than normal sense of touch (i.e. putting on gloves) Yes No 1 2 3 4 5 1 2 3 4 5
Burning pain or discomfort without cold Yes No 1 2 3 4 5 1 2 3 4 5
Burning pain or discomfort with cold Yes No 1 2 3 4 5 1 2 3 4 5
Difficulty identifying objects in your hand (i.e. coin) Yes No 1 2 3 4 5 1 2 3 4 5
Do you have involuntary hand movements Yes No 1 2 3 4 5 1 2 3 4 5
Table 2 Questionnaire documenting lower extremity symptoms of acute and chronic neuropathy experienced during or after oxaliplatin-based chemotherapy.
Lower Extremity Symptoms If you had symptoms during the last cycle....
How much of the symptoms did you have? Did the symptoms affect your daily activities?
Do you have.... Hardly any → Very much Hardly at all bothered → Extremely bothered
Tingling (pins and needles) Yes No 1 2 3 4 5 1 2 3 4 5
Numbness Yes No 1 2 3 4 5 1 2 3 4 5
Difficulty telling the difference between rough and smooth surfaces Yes No 1 2 3 4 5 1 2 3 4 5
Difficulty feeling hot things Yes No 1 2 3 4 5 1 2 3 4 5
Difficulty feeling cold things Yes No 1 2 3 4 5 1 2 3 4 5
A greater than normal sense of touch (i.e. discomfort with socks) Yes No 1 2 3 4 5 1 2 3 4 5
Burning pain or discomfort without cold Yes No 1 2 3 4 5 1 2 3 4 5
Burning pain or discomfort with cold Yes No 1 2 3 4 5 1 2 3 4 5
Legs feel heavy Yes No 1 2 3 4 5 1 2 3 4 5
Table 3 Questionnaire documenting oral or facial symptoms of acute and chronic neuropathy experienced during or after oxaliplatin-based chemotherapy.
Oral/Facial Symptoms If you had symptoms during the last cycle....
How much of the symptoms did you have? Did the symptoms affect your daily activities?
Do you have.... Hardly any → Very much Hardly at all bothered → Extremely bothered
Jaw pain Yes No 1 2 3 4 5 1 2 3 4 5
Eyelids drooping Yes No 1 2 3 4 5 1 2 3 4 5
Throat discomfort Yes No 1 2 3 4 5 1 2 3 4 5
Ear pain Yes No 1 2 3 4 5 1 2 3 4 5
Tingling in mouth Yes No 1 2 3 4 5 1 2 3 4 5
Difficulty with speech Yes No 1 2 3 4 5 1 2 3 4 5
Burning or discomfort of your eyes Yes No 1 2 3 4 5 1 2 3 4 5
Loss of any vision Yes No 1 2 3 4 5 1 2 3 4 5
Feeling shock/pain down back Yes No 1 2 3 4 5 1 2 3 4 5
Problems with breathing Yes No 1 2 3 4 5 1 2 3 4 5
Table 4 Worst grade of toxicity across all cycles of therapy
No toxicity Grade 1 Grade 2 Grade 3 Grade 4
Dyesthesia 12.8% 70.9% 11.6% 4.7% 0%
Paresthesia 5.7% 66.3% 19.8% 7% 1.2%
Table 5 Duration of dysesthesia and paresthesias (21 day cycles)
Dysesthesias
Cycle number Median duration (days) Percent persisting ≥ 14 days Percent persisting ≥ 21 days
1 5 9.5% 3.2%
3 7 14.3% 6.1%
6 10.5 38.2% 17.6%
9 13.5 50% 37.5%
12 21 60% 60%
Paresthesias
Cycle number Median duration (days) Percent persisting ≥ 14 days Percent persisting ≥ 21 days
1 7 19.7% 6.1%
3 12.5 35.6% 18.6%
6 14 54.3% 37%
9 = 21 86.4% 77.3%
12 = 21 83.3% 83.3%
Table 6 Cumulative chronic sensory neuropathy rates in selected phase III trials involving oxaliplatin and 5-FU based combination chemotherapy
Ref. Regimen Oxaliplatin Dose: mg/m2 Planned Oxaliplatin Dose intensity: mg/m2/wk Treatment ≥ grade 3
25 OX + FU/LVa chronotherapy Both arms: 20 days 1–5 q 3 wk 20/10.5 hr CI; peak at 4:00 pm 33.3 First line 16%
Constant rate 20/24 hr CI 31%
26 OX + FU/LVb 125/6 hr day 1 q 3 wk 10:00 am to 4:00 pm 41.7 First line 10%
3 FOLFOX 4 85/2 hr day 1 q 2 wk 42.5 First line 18.2%
4 FOLFOX 4 85/2 hr day 1 q 2 wk 42.5 First line 18%
IROX 85/1.5 hr day 1 q 3 wk 28.3 7%
5 FOLFOX 6 100/2 hr day 1 q 2 wk 50 First line 34%
27 FUFOX 50/2 hr days 1, 8, 15, 22 q 5 wkc 40 First line 17%
28 FUFOX 50/x hr days 1, 8, 15, 22 q 5 wkc 40 First line 25%
CAPOX 70/x hr d 1, 8 q 3 wk 46.7 16%
17 FOLFOX 4 85/2 hr day 1 q 2 wk 42.5 Adjuvant 12%
5 FOLFOX 6 100/2 hr day 1 q 2 wk 50 Second lined 20%
29 FOLFOX 4 85/2 hr day 1 q 2 wk 42.5 Second or third linee 3%f
FOLFOX: oxaliplatin and biweekly FULV2; IROX: oxaliplatin and irinotecan;
FUFOX: oxaliplatin and weekly 24 hour infusional 5-FU/LV; CAPOX: capecitabine and oxaliplatin
a 5-FU 600 mg/m2 and LV 300 mg/m2 daily for 5 days over 12 hr with peak at either 04:00 am or over 24 hr by constant rate infusion; b 5-FU 700 mg/m2 and LV 300 mg/m2 daily for 5 days over 12 hr with peak at 04:00 am; c after the first four cycles, oxaliplatin given every 2 weeks; d patients had previously progressed on FOLFIRI; e Grade 3–4 acute, cold-sensitive paresthesia also occurred in 3% of patients.
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Stein ME Drumea K Yarnitsky D Benny A Tzuk-Shina T A rare event of 5-fluorouracil-associated peripheral neuropathy: a report of two patients Am J Clin Oncol 1998 21 248 249 9626791 10.1097/00000421-199806000-00008
Saif MW Wilson RH Harold N Keith B Dougherty DS Grem JL Peripheral neuropathy associated with weekly oral 5-fluorouracil, leucovorin and eniluracil Anti-Cancer Drugs 2001 12 525 531 11459999 10.1097/00001813-200107000-00006
Saif MW Wood TE McGee PJ Diasio RB Peripheral neuropathy associated with capecitabine Anti-Cancer Drugs 2004 15 767 771 15494638 10.1097/00001813-200409000-00005
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-1191617153010.1186/1471-2407-5-119Research ArticleInvestigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma Lee Yen-Ching [email protected] Janeanne R [email protected] Evelyn L [email protected] Michael P [email protected] Maria A [email protected] Peter G [email protected] Sally-Anne [email protected] Department of Haematology/Oncology, The Queen Elizabeth Hospital, Woodville, South Australia, Australia, 50112 Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia, Australia, 50112005 20 9 2005 5 119 119 10 5 2005 20 9 2005 Copyright © 2005 Lee 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 EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported.
Methods
RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas.
Results
All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease.
Conclusion
EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target.
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Background
Prostate cancer is the most frequent cause of cancer death in men in Australia. Although many genetic changes have been detected in human prostate cancer, the role of most of these in initiation and progression of the disease is unclear. Receptor tyrosine kinases (RTKs) couple ligand binding to downstream signalling cascades and gene transcription and are key regulators of normal cellular processes such as growth, differentiation, migration and apoptosis. RTKs are also critically involved in the development and progression of human cancers and are therefore useful targets for anti-cancer therapies [1,2]. At least 50 RTKs in 20 different families have been identified and the largest family of these contains the Eph receptors [3]. This family in humans currently includes 14 members divided into two classes, designated A and B, based on sequence homology, structure and ligand binding affinity [4,5]. The ligands for the Eph receptors are called ephrins and are anchored on the plasma membrane through either a glycosyl phosphatidylinositol (GPI) link (ephrin-A) or a transmembrane domain (ephrin-B) [6].
Ligand binding induces receptor autophosphorylation of intracellular tyrosine, threonine and serine residues and allows interactions with a variety of different proteins that regulate cell functions such as contact inhibition, cytoskeletal organisation and cell motility [7,8]. Several studies have described intricate signalling networks often important to cell proliferation, migration, survival and differentiation [reviewed in [9]] that centre around Eph receptors and their ligands.
Members of both classes of Eph receptor have been found to have increased gene expression and/or protein levels in tumours from many different human tissues [10-20]. In particular EphB4, located at chromosome 7q22.1, and initially isolated from a human hepatocellular carcinoma cell line Hep3Ba [10] has been reported by us and others to be highly expressed in many tumour tissues including colon [11,12], breast [13,14], endometrium [15,16], lung [17] and head and neck [18,19]. Robinson et al (1996) used degenerate RT-PCR oligonucleotide primers specific to two conserved motifs in the tyrosine kinase domains of 40 different kinases to amplify those kinases that are expressed in a prostate cancer xenograft model [20]. EphB4 was identified as one of the RTKs expressed in the xenograft tissue. More recently, Xia et al (2005) report that EphB4 is commonly expressed in prostate tumour tissues and cell lines and knockdown of EphB4 protein using siRNA and antisense approaches inhibited cell growth/viability, migration and invasion both in vitro and in vivo [21]. Concurrently with Xia et al's study, in this pilot study we also investigated the expression patterns of the EphB4 gene and its protein product in prostate cancer cell lines and tumour tissue samples.
Methods
Cell culture
Human prostate cancer cell lines LNCaP, DU145 and PC3 were cultured in HEPES-buffered RPMI 1640 medium (Invitrogen, Carlsbad, CA), pH 7.4. The medium was supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 160 μg/ml L-glutamine and 10% heat-inactivated foetal bovine serum (JRH Biosciences, Lenexa, KS) in 75 cm2 vented tissue culture flasks at 37°C in a 5% CO2 environment. Cells were collected at >90% confluency by trypsin digestion and centrifugation for 5 min at 1000 rpm, resuspended in phosphate buffered saline (PBS) and counted using a haemocytometer.
RNA extraction from cell lines
RNA was isolated from cell lines using Tri Reagent (Invitrogen). Growth medium was aspirated from the flask of growing cells (>90% confluent) and the cells washed with phosphate-buffered saline (PBS) before being directly lysed using 1 ml of Tri Reagent. After a five min incubation with gentle rocking, the cell lysate was removed to a 2 ml eppendorf tube. The RNA fraction was extracted using the manufacturer's recommendations.
Protein extraction from cell lines
Growth medium was aspirated from the flask of growing cells (>90% confluent) and the cells washed with phosphate-buffered saline (PBS) before being directly lysed using 1 ml of Cell Lytic M Cell Lysis Reagent (Sigma) supplemented with 5 μl Protease Inhibitor Cocktail (Sigma). Protein lysate was removed to a 1.5 ml microfuge tube and the solution mixed for 30 min at 4°C on a rotary mixer. Insoluble protein was pelleted by centrifugation at 4°C for 30 min and the supernatant containing the soluble proteins was stored at -80°C until required for Western analysis. Protein concentrations were determined using the DC Protein Assay Kit from Biorad (Sydney, NSW, Australia) following the manufacturer's protocol and using bovine serum albumin diluted from 0.1 mg/ml to 10.0 mg/ml to determine the standard curve.
Real-time RT-PCR to determine relative expression of EphB4
Total RNA (2 μg) was reverse transcribed at 37°C using 3 μl pD(N)6 primers (Invitrogen), 200 μM each deoxyribonucleoside triphosphate (dNTP) (Pharmacia, Uppsala, Sweden) and 200 U Superscript III reverse transcriptase (Invitrogen) in a reaction volume of 30 μl. Primers specific to either EphB4 or the housekeeping genes Porphobilinogen deaminase (PBGD) and Hypoxanthine phosphoribosyl-transferase 1 (HPRT 1) were used in a PCR reaction carried out in a BioRad iCycler MyiQ Real Time thermocycler or a Corbett Rotorgene 3000 (Table I). The following conditions for the PCR reaction were used: 1.5 mM MgCl2, 200 μM each dNTP, 50 ng of each primer, and 0.5 units of HotStarTaq DNA polymerase in 1 × PCR buffer (Qiagen, Melbourne, Australia). Cycling conditions included an initial denaturation at 94°C for 15 min, followed by 45 cycles of 94°C for 30 sec, 67°C or 68°C for 30 sec, and 72°C for 30 sec, with a final extension of 72°C for 7 min.
Western analysis
Proteins (50 μg) from samples extracted from prostate tumour cell lines were separated on duplicate 8% Tris-Glycine iGels (Gradipore, Sydney, Australia). The protein separated on one of the gels was visualised by coomassie staining and the protein on the duplicate gel was electrophoretically transferred to MFS nitrocellulose membrane (Adelab, Adelaide, Australia). Non-specific binding was blocked by incubation for 1 h at room temperature using a Western blocking buffer containing 1% casein in maleic acid buffer (Roche) diluted using TBS with 0.1% Tween-20 (TBS-T). EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4 (Zymed, CA) in blocking buffer. After a 1 h incubation at room temperature, the primary antibody was detected using a HRP-labelled anti-mouse secondary antibody (Roche) and the ECL Western Blotting System from Amersham Biosciences (Sydney, Australia) following the manufacturer's recommendation. Blots were exposed to Hyperfilm™ECL™ (Amersham Biosciences) for between 5 and 30 sec. The chemiluminescence was removed from the blot by incubation in a stripping solution containing 100 mM β-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl pH 6.7 for 20 min at 50°C with gentle agitation. The filter was then washed with TBS-Tween 20 before blocking and re-probing with a mouse anti-α-actin monoclonal antibody (Chemicon, Temecula, CA) then it was stripped again and probed with a rabbit anti-calnexin antibody (Sigma) for loading comparison.
Immunofluorescence of cells grown on slides
Cell were grown to >50% confluence in the wells of 8 well chambered slides. Before staining, the medium was aspirated and the cells washed gently with PBS. Cells were then fixed in 4% paraformaldehyde and washed well before non-specific binding sites were blocked with 3% serum in PBS for 20 min at room temperature. Cells were then incubated overnight at 4°C with a 1:200 dilution of the EphB4-specific rabbit polyclonal antibody H-200 (Santa Cruz Biotechnology, Santa Cruz, CA). After rinsing with PBS, the sections were incubated with an Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Molecular Probes). Immunofluorescence was visualised using a TE2000E microscope with a C1 confocal scanning head (Nikon, Tokyo, Japan).
Tissue immunohistochemistry
Four consecutive 8 μm sections of formalin-fixed paraffin-embedded tissue from 15 different patients with prostate cancer were a gift from Dr Michael Brown, Medical Oncology, Royal Adelaide Hospital, Adelaide, Australia. A fifth slide, stained to visualize the histological features of the tissue, was used to identify the foci of tumour tissue within the adjacent normal prostate tissue by a trained pathologist. The paraffin was removed by incubation in Histoclear (National Diagnostics, Atlanta, Georgia) and the section re-hydrated in ethanol before antigen retrieval by boiling in 10 mM citric acid pH 6 for 10 min. Sections were cooled, then washed in PBS before removal of endogenous peroxidase activity by incubation in 0.5% hydrogen peroxide/methanol for 30 min at room temperature. Non-specific binding sites were blocked with 3% normal goat serum in PBS for 20 min at room temperature and the Vector Laboratories (Burlingame, CA) Avidin/Biotin blocking kit following the manufacturer's instructions. The sections were then incubated overnight at 4°C with a 1:200 dilution of the H-200 EphB4-specific antibody (Santa Cruz Biotechnology). After rinsing with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories) for 30 min at room temperature followed by washing with PBS. Immunoreactivity was detected with the avidin-biotin system (Vector Laboratories) using 18.5 mM 3,3'-diaminobenzidine tetrahydrochloride (Sigma) as a chromogen for 2 min. The sections were then counterstained using Harris haematoxylin, dehydrated, cleared using SUB-X clearing solution (Surgipath Medical Industries, Inc. Richmond, IL) and mounted using Entellan New (Merck, Darmstadt, Germany). Sections were visualised and images recorded using a Nikon Eclipse E800 microscope with a Spot Camera 2.3.1 (Diagnostic Instruments) and Spot Advanced Version 3.5 software.
Results
Relative expression of EphB4 in prostate cancer cell lines
The relative expression of EphB4 in three prostate cancer cell lines was determined using reverse transcription and real time PCR using two different real-time PCR machines. Cell lines used included the androgen-dependent line LNCaP derived from a lymph node biopsy (moderately differentiated), the androgen-independent line DU145 from a central nervous system metastasis (moderately differentiated with foci of poorly differentiated cells) and the androgen-independent line PC3 from a bone metastasis (poorly differentiated) [22-24]. For each experiment performed amplification reactions were resolved on agarose gels to confirm products of the predicted size for each gene were amplified (example shown in Figure 1). In the first instance, the EphB4 gene and the house-keeping control gene PBGD were amplified in triplicate from cDNA made using three different RNA extractions for each cell line (Figure 2A) and a serial dilution of one of these cDNA preparations (Figure 2B) using a BioRad iCycler MyiQ. In the second experiment, a second housekeeping gene, HPRT 1, was amplified in addition to EphB4 and PBGD using a Corbett Rotorgene 3000 (Figure 2C). For each individual sample, EphB4 expression level was normalised to the expression of PBGD and HPRT 1 in these samples (EphB4/PBGD or EphB4/HPRT 1). Comparison of the combined data for each cell line showed that there was no statistical difference in the level of EphB4 expression in the three prostate cancer cell lines (Figure 2). In a third experiment, RNA was extracted from 6 replicates of cells grown to either full confluence or 50–80% confluence and EphB4 expression again normalised to both PBGD and HPRT 1. There was no statistically significant difference in the level of expression of EphB4 in the non-confluent and confluent cells for any of the three cell lines (data not shown).
In these experiments the ratios of EphB4/PBGD and EphB4/HPRT 1 were all close to 1 indicating that there were comparable amounts of EphB4 and PBGD/HPRT 1 templates in each cell line RNA sample. There was no statistical difference between the ratios obtained using serial dilutions of a single cDNA sample for each cell line showing that the amplification is consistent regardless of the starting amount of template. These results support the use of both PBGD and HPRT 1 as normalisation controls for the EphB4 message.
Western analysis of EphB4 protein in prostate cancer cell lines
Protein was isolated from the three prostate cancer cell lines for western analysis to determine whether the level of gene expression correlated with the amount of protein present. Duplicate samples were run on two separate gels, one of these was immunoblotted and the other stained with coomassie blue to confirm protein integrity and consistency in the amount of protein loaded. The predicted size of the mature EphB4 protein is 120 kDa and a band of this approximate size was clearly visible in each sample after immunoblotting with an EphB4-specific antibody (Figure 3A). The non-transformed breast cell line MCF10A engineered to over-express EphB4 under control of the constitutive CMV promoter was used as a positive control (labelled +ve in Figure 3). In the prostate cell lines a second band of a slightly smaller size is also clearly visible and this may represent an alternatively modified (phosphorylated/glycosylated) or spliced form of EphB4. The coomassie staining of the duplicate gel shows that similar amounts of protein from each cell line was loaded and suggests that the PC3 cell line produces more EphB4 protein than LNCaP than DU145 (Figure 3B). To confirm efficient transfer the blot was also incubated sequentially with an antibody recognizing α-actin. Although a 10 sec exposure time suggested there were similar levels of protein present, a shorter exposure showed differences in the relative amount of actin in each protein lysate. Therefore a second control antibody recognising the ER chaperone calnexin was also used. There was little difference in the relative amounts of calnexin in each sample.
Immunological analysis of expression of EphB4 in prostate cancer cell lines
The expression and localisation of EphB4 in the three cell lines grown on slides was determined using immunofluorescence with an EphB4-specific polyclonal antibody. In LNCaP and DU145 cells EphB4 appeared to be present on both the cell surface and in the cytoplasm but in PC3 cells the EphB4 appeared to be mainly cytoplasmic (Figure 4). This cytoplasmic localisation of EphB4 in tumour cells contrasts with its known localisation within sites of cell-cell contact between non-transformed epithelial cells and may suggest that active EphB4 signalling has caused endocytosis of the receptor-ligand complex in these cells. Similar results have been reported by Wu et al (2004) who also used the EphB4 antibody sc-5536 from Santa Cruz Biotechnology in their study of EphB4 expression in breast cancer cell lines and tissue samples [14]. All four breast cancer cell lines examined showed strong immunostaining in the cytoplasm.
Immunohistochemical analysis of expression of EphB4 in primary prostate tumours
To localize the expression of EphB4 in normal prostate and tumour tissue, we performed immunohistochemistry using an EphB4-specific antibody on 15 prostate tumour samples collected by transurethral resection. These patient samples were chosen because they contained foci of prostate cancer or benign prostatic hyperplasia within the normal tissue. The results for 8 representative patient sample sets are presented in Figure 5. The epithelial cells lining the ducts in the normal tissue can be clearly distinguished by the rows of regular nuclei stained blue with Harris Haematoxylin. There was either no or only very weak diffuse staining of the normal epithelial cells, stroma and endothelial cells of blood vessels indicating little immunoreactivity to the EphB4 antibody. There was no staining with an isotype matched IgG antibody or the secondary antibody alone in either normal or tumour tissues (results not shown).
Foci of prostate cancer within these samples showed brown staining indicating immunoreactivity to the EphB4 antibody and therefore the presence of the EphB4 protein. A single patient sample had a low level of staining associated with foci of basal cell hyperplasia. In this case EphB4 may be involved in a proliferative role [25]. There also appeared to be an increase in the amount of staining associated with an increase in Gleason score. There was little staining in the 5 different patient samples that contained foci of well-differentiated adenocarcinoma (2 examples are shown in Figure 5), and increased staining in the three patients with foci of moderately differentiated and the three patients with poorly differentiated adenocarcinomas. Because prostate cancer is multifocal, each sample was examined to determine if it contained foci of different grades and whether immunoreactivity differed in intensity within these. As these samples were collected during transurethral resection to debulk enlarged prostates, and were later found to contain foci of disease, only a few samples contained foci of different grades. One patient in particular had large regions classified clinically as prostatic intraepithelial neoplasia (PIN) and staining within these foci was surprisingly of equal intensity to the regions of poorly differentiated adenocarcinoma (Figure 6).
Discussion
In recent years, experiments using monoclonal antibodies with limited normal-tissue reactivity have indicated that these are good candidates for development as therapeutic agents against cancer. EphB4 is a receptor protein tyrosine kinase (RTK) that is dramatically up-regulated on many epithelial cancers, displays limited expression on normal adult tissue and would therefore appear to be a potential target for monoclonal therapies. EphB4 was identified by Robinson et al (1996) as one of several RTKs that were expressed in a xenograft model of prostate cancer [20] and until recently this was the only report of any link between EphB4 expression and prostate cancer.
During the review of this article, Xia et al (2005) presented results showing that EphB4 is increased in 66% (41/62) of prostate tumours tested with low intensity expression in only 15% (3/20) normal prostate samples [21]. Our western analysis of PC3, LNCaP and DU145 confirmed Xia et al's results [21], with higher amounts of protein present in PC3 lysates that LNCaP and DU145. In the case of DU145 this can perhaps be explained by regulation of EphB4 gene expression as real-time PCR using normalisation to two different house-keeping genes demonstrated that there was approximately 20% less EphB4 transcript expressed in the DU145 cell line than in LNCaP and PC3 (P < 0.05). However, as there was no significant difference in the activity of the gene in LNCaP and PC3, it is possible that EphB4 may also be regulated post-transcriptionally. The presence of a second band detected by western analysis with the EphB4-specific antibody may also indicate that EphB4 is regulated post-translationally and this needs to be explored in more detail.
Although Xia et al's study of cell lines also indicated an association between the level of EphB4 expression and aggressive growth, they did not report a direct correlation between increased EphB4 expression with higher grade of the tumour tissue samples. We examined a panel of 15 prostate cancer clinical specimens collected by transurethral resection. The prostate tumour samples we examined contained both normal prostate and tumour foci and this enabled a comparison of EphB4 immunoreactivity in normal prostate and tumour cells simultaneously from the same patient and the determination of whether the level of EphB4 also correlates with histological grade and/or stage of prostate carcinoma. Using immunohistochemical techniques, we showed that EphB4 is produced in increased amounts in human prostate cancer tissue compared with adjacent normal tissue and that this immunoreactivity was associated with the tumour epithelial cells themselves. There also appeared to be trend towards an increasing level of EphB4 protein in the tumours from the well-differentiated to the moderately and poorly differentiated cancers.
A positive correlation between histological grade, stage of carcinoma and level of EphB4 protein has also been reported in breast and endometrial carcinoma [13-16]. EphB4 has been reported to be elevated in breast primary infiltrating ductal carcinomas with a high grade of malignancy [13] and recently Wu et al (2004) reported that EphB4 protein expression in 94 tumour samples was positively associated with increased clinical stage and histological grade [14]. Sakano et al, (1996) suggested that the balance of EphB4 and its preferred ligand ephrin-B2 is disrupted when mammary epithelial cells become transformed [25] and loss of ephrin-B2 expression has been associated with an increase in EphB4 expression [26]. However contrary to this Berclaz et al (2002) report a highly significant correlation between EphB4 positivity and low histological grade [27]. One of the patients examined here also showed a comparable level of EphB4 staining in foci of PIN and poorly differentiated adenocarcinoma. If the PIN is a precursor to adenocarcinoma in this case, this might suggest that increased expression of EphB4 was an early event in the development of this patient's disease. Furthermore, a single focus of basal cell hyperplasia in a second patient did show a low level of EphB4 immunoreactivity and this may indicate a role for EphB4 in proliferation of these cells [25].
The results presented here suggest that elevated levels of EphB4 are relevant to prostate cancer but despite substantial evidence in the literature that EphB4 has an important role in progression of many epithelial tumours, the mechanism by which these receptors contribute to tumorigenesis is still being resolved. In only a very few cases have Eph receptors been demonstrated to have transforming potential [28-30]. In particular, a study reported by Zelinski et al (2002) showed that EphA2 expression transforms MCF10A cells as judged by conversion to a fibroblastic morphology, growth in soft agar and the ability to engraft and metastasise in a nude mouse model [29].
Although similar experiments over-expressing EphB4 in non-transformed cell lines have yet to be reported, a study by Munarini et al (2000) using transgenic mice expressing EphB4 and neuT suggested that EphB4 over-expression by itself is not tumorigenic but provides convincing evidence that it favours an invasive/metastatic phenotype [31]. Although mammary tumours were not observed in the EphB4 transgenic animals, in double transgenic animals expressing both EphB4 and neuT, tumour appearance was significantly accelerated relative to neuT-only animals, and in addition metastases were observed in the lung. It was not clear if this was an effect of EphB4 on metastasis or a result of accelerated tumour growth but these results clearly implicate over-expression of EphB4 in tumour growth and/or establishment of the invasive phenotype in the adult mammary tumours. While the single transgenic EphB4 animals did not develop tumours during this experiment it is possible that these studies were not taken out far enough to conclude that EphB4 over-expression is insufficient to induce transformation with a long latency.
Xia et al's (2005) recent experiments targeting EphB4 using siRNA to knockdown EphB4 expression in vitro have shown that EphB4 is involved in growth/viability, migration and invasion of prostate cancer cell lines and supports Munarini et al's study [21]. Furthermore, EphB4 antisense oligonucleotides were given intraperitoneally to nude mice bearing PC3 xenografts in the posterior prostate (n = 6), tumours were fewer and smaller, with increased apoptosis and reduced microvascular density than sense- or diluent-treated control animals. The reduction in microvascular density is particularly interesting given that in other epithelial tumours, EphB4-positive cells are often found in regions that are rich in capillaries [13] and it has been suggested that EphB4 over-expression in tumours may promote tumour growth by facilitating angiogenesis.
A direct role for EphB4 in tumour angiogenesis has been suggested by experiments showing that A375 melanomas form smaller tumours in the presence of soluble EphB4 [32] possibly because the soluble EphB4 interferes with binding of endogenous EphB4 on tumour cells to ephrin-B2 on endothelial cells. A role in angiogenesis would be consistent with the normal role for EphB4 in vascular development and remodelling demonstrated in mouse and Xenopus [33,34]. Noren et al (2004) contradict this finding in a study that found that increased expression of a signaling-defective form of EphB4 in breast cancer cells (dominant negative) was still able to make tumour xenografts grow more rapidly [35]. This may be because the EphB4 ectodomain exerted a chemoattractive effect on endothelial cells through ephrin-B2 expressed on these cells and promoted endothelial cell proliferation and survival resulting in more tumour vasculature. However, Noren et al applied a high concentration of soluble ephrin-B2 Fc which is promiscuous and signals other Eph receptors (particularly EphB2) but the phosphorylation of other EphRs was not determined in this study.
Conclusion
Further investigation is needed to determine the roles of EphB4 in prostate cancer development and progression. The trend of increasing EphB4 reactivity toward higher grade disease seen in this pilot study might suggest that it is more important in the later stages of the tumour development such as metastasis and further investigation of EphB4 in the development of prostate cancer is warranted. Therapies that target EphB4 may prove to be successful in preventing the metastatic spread of the disease.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YCL carried out most of the studies with the assistance of JP and ED and drafted the manuscript. JP and ED also participated in the analysis and interpretation of the data and revision of the manuscript. MR optimised the real-time PCR experiments, participated in the analysis and interpretation of the data and revision of the manuscript. MB optimised the Western analysis, participated in the analysis and interpretation of the data and revision of the manuscript. PB participated in the analysis and interpretation of the data and revision of the manuscript. SS conceived of and co-ordinated the study and helped to draft 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 would like to thank Dr Michael Brown for providing prostate tissue samples and Dr Wayne Tilley and Dr Lisa Butler for prostate cell lines. We would also like to thank Dr Jurgen Stahl for reviewing the tissue immunohistochemistry results and Dr Edwina Ashby for critical reading of the manuscript. Sally Stephenson is supported by a senior research fellowship from the Cancer Council of South Australia. This work was supported by grants from The Queen Elizabeth Hospital Research Foundation and The South Australian Medical Research Trust.
Figures and Tables
Figure 1 RT-PCR analysis of EphB4 expression in prostate cancer cell lines. RT-PCR analysis of EphB4 and PBGD expression in duplicate RNA samples from the three prostate cancer cell lines LNCaP, DU145 and PC3. Sizes of the amplified products are shown on the right (bp). An RNA sample to which no reverse transcriptase was added and a PCR reagent only (containing no template) amplification were also performed as negative controls for each stage of the RT-PCR.
Figure 2 Normalisation of EphB4 expression to housekeeping genes PBGD and HPRT1. EphB4 expression normalised to PBGD in prostate cancer cell lines LNCaP, DU145 and PC3. (A) Triplicate amplifications of EphB4 and PBGD in three samples for each cell line were analysed to determine the relative levels of EphB4 expression using the BioRad iCycler. All EphB4/PBGD ratios were close to 1 indicating that there are comparable amounts of both templates in each RNA samples. (B) Dilutions of a single RNA sample for each cell line were also amplified in triplicate with both gene primer sets to confirm that the ratio was consistent regardless of starting template concentration. (C) Triplicate amplifications of EphB4, PBGD and HPRT1 in three samples for each cell line using the Corbett Rotorgene confirmed the previous result for EphB4/PBGD and showed a similar result when EphB4 expression was normalised to a second housekeeping gene.
Figure 3 Western analysis of EphB4 in prostate cancer cell lines. Western analysis of 20 μg EphB4 protein in prostate cancer cell lines LNCaP (L), DU145 (D) and PC3 (P). (A) The immunoblot was incubated sequentially with antibodies specific to EphB4, α-actin and calnexin and exposed to autoradiographic film for the indicated times. MCF10A engineered to express EphB4 (5 μg protein lysate) was used as a positive control. The arrow indicates the expected size of each protein – 120 kDa for EphB4, 43.2 kDa for α-actin and 90 kDa for calnexin. The size of the marker proteins is shown on the left in kDa. (B) Coomassie stained duplicate gel for loading comparison.
Figure 4 Immunofluorescent staining of EphB4 in prostate cancer cell lines. Immunofluorescent staining of EphB4 in prostate cancer cell lines showing diffuse staining on the surface and in the cytoplasm of LNCaP and DU145 and in the cytoplasm only of PC3. There was no reactivity to the primary antibody (EphB4 1° only) or secondary antibody (2° only) alone and little background fluorescence (No staining).
Figure 5 Immunohistochemical staining of EphB4 in prostate cancer samples. The expression of EphB4 in human prostate carcinomas was detected using immunohistochemical staining of formalin fixed, paraffin-embedded tissue samples. Images (20× magnification) of normal (panel 2) and diseased tissue (panel 4) from eight different patient samples are shown with a Haematoxylin/eosin stained consecutive section (panels 1 and 3). The Gleason score for each disease focus is shown in the top left-hand corner of the image of each tumour focus. A single sample that contained a focus of basal cell hyperplasia, two samples with well-differentiated adenocarcinoma, two samples of moderately differentiated adenocarcinoma and three samples of poorly differentiated adenocarcinoma are shown. The brown stain from the biotinylated secondary antibody indicates the EphB4 protein. Nuclei are stained with Harris haematoxylin and appear blue. Staining of normal tissues was either absent or weak and diffuse. Increased staining of the tumour tissue appeared to correlate with increased Gleason score. There was no cross-reactivity with the secondary antibody alone (result not shown).
Figure 6 Immunohistochemical staining of EphB4 in several foci of a single prostate cancer sample. The expression of EphB4 a different foci in a single patient sample was compared by immunohistochemistry. Images from 10× magnification (A) and a region from this (as indicated by the box) at 20× magnification (B) are shown for 2 regions of normal prostate duct (1 and 2) and 8 regions with disease (3–10) from this single sample. A comparable level of EphB4 staining was seen in regions of high grade PIN (eg. focus 8 indicated PIN) and regions of poorly differentiated adenocarcinoma (eg. focus 7 indicated PDA). Regions of well-differentiated (eg focus 3) and moderately differentiated (eg focus 10) also show comparable staining.
Table 1 Primer sequences used to amplify EphB4 and the control housekeeping genes PBGD and HPRT 1 with expected size of the corresponding PCR product in base pairs (bp) and the annealing temperature used in the reaction.
Primer Sequence 5'-3' Product Annealing
EphB4 F CCC CAG GGA AGA AGG AGA GCT G
EphB4 R GCC CAC GAG CTG GAT GAC TGT G 250 bp 68°C
PBGD F CTT TCC AAG CGG AGC CAT GTC TGG
PBGD R CAT GAG GGT TTT CCC GCT TGC AGA 377 bp 68°C
HPRT F GGC TAT AAA TTC TTT GCT GAC CTG CTG
HPRT R CAA AGT CTG CAT TGT TTT GCC AGT GTC 230 bp 67°C
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-1261620213410.1186/1471-2407-5-126Research ArticleNumbers of mutations to different types of colorectal cancer Calabrese Peter [email protected] Jukka-Pekka [email protected]ärvinen Heikki J [email protected] Lauri A [email protected]é Simon [email protected] Darryl [email protected] Program in Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA2 Second Department of Surgery, Helsinki University Central Hospital, FIN-00029, Helsinki, Finland3 Jyvaskyla Central Hospital, FIN-40620, Jyvaskyla, Finland4 Department of Medical Genetics, Haartman Institute, FIN-00014, University of Helsinki, Finland5 Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA, and Department of Oncology, University of Cambridge, Cambridge, UK6 Department of Pathology, University of Southern California School of Medicine, Los Angeles, CA 90033, USA2005 3 10 2005 5 126 126 15 1 2005 3 10 2005 Copyright © 2005 Calabrese 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 numbers of oncogenic mutations required for transformation are uncertain but may be inferred from how cancer frequencies increase with aging. Cancers requiring more mutations will tend to appear later in life. This type of approach may be confounded by biologic heterogeneity because different cancer subtypes may require different numbers of mutations. For example, a sporadic cancer should require at least one more somatic mutation relative to its hereditary counterpart.
Methods
To better estimate numbers of mutations before transformation, 1,022 colorectal cancers were classified with respect to microsatellite instability (MSI) and germline DNA mismatch repair mutations characteristic of hereditary nonpolyposis colorectal cancer (HNPCC). MSI- cancers were also classified with respect to clinical stage. Ages at cancer and a Bayesian algorithm were used to estimate the numbers of oncogenic mutations required for transformation for each cancer subtype.
Results
Ages at MSI+ cancers were consistent with five or six oncogenic mutations for hereditary (HNPCC) cancers, and seven or eight mutations for its sporadic counterpart. Ages at cancer were consistent with seven mutations for sporadic MSI- cancers, and were similar (six to eight mutations) regardless of clinical cancer stage.
Conclusion
Different biologic subtypes of colorectal cancer appear to require different numbers of oncogenic mutations before transformation. Sporadic MSI+ cancers may require more than a single additional somatic alteration compared to hereditary MSI+ cancers because the epigenetic inactivation of MLH1 commonly observed in sporadic MSI+ cancers may be a multistep process. Interestingly, estimated numbers of MSI- cancer mutations were similar (six to eight mutations) regardless of clinical cancer stage, suggesting a propensity to spread or metastasize does not require additional mutations after transformation. Estimates of oncogenic mutation numbers may help explain some of the biology underlying different cancer subtypes.
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Background
Cancer is thought to arise through a multistep process involving sequential cycles of mutation and selection [1]. The identities and numbers of mutations required for transformation are uncertain, but perhaps six general cellular functions are typically altered [2]. Numbers of oncogenic mutations may also be inferred from the age-related increases in frequencies observed with many cancer types. For example, logarithms of cancer frequencies versus age typically yield straight lines, with slopes proportional to numbers of cancer mutations [3].
Colorectal cancer epidemiology is consistent with approximately five to seven oncogenic mutations before transformation [3-6]. The variability in estimated numbers of mutations may reflect a number of differences. For example, estimates vary between populations, with five to six mutations in England and six to seven mutations in Finland [4]. Recent advances in cancer genetics also reveal biologic colorectal cancer heterogeneity. Approximately 5% of all colorectal cancers have strong familial predispositions and arise in individuals with germline mutations in critical susceptibility loci [7]. Such hereditary cancers (familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC)) typically present at younger ages and should require fewer somatic mutations than their sporadic counterparts because one mutation is inherited.
Genetic instability also divides colorectal cancers into two groups [8]. Approximately 10 to 15% of sporadic cancers exhibit microsatellite instability (MSI) secondary to somatic loss of DNA mismatch repair (MMR). Most other cancers exhibit chromosomal instability (CIN) characterized by aneuploidy and loss of heterozygosity (LOH) [7,8]. CIN and MSI+ colorectal cancers have different characteristics with respect to mutated loci, tumor location, morphology, and clinical outcomes [7,8].
Numbers of oncogenic mutations may differ between cancer subtypes. Therefore, colorectal cancers arising in a population-based setting were molecularly classified as either sporadic or hereditary, and MSI+ or MSI-. Cancers were also classified with respect to clinical stage because additional mutations may be required for invasion or metastasis. Ages at cancer for each subgroup were used to infer numbers of mutations required for each type of colorectal cancer.
Methods
Specimens
MSI status was determined for 1,022 colorectal cancers sampled from nine large regional hospitals in southeastern Finland as part of a study to characterize genetic alterations in a well-defined population [9]. The cancers represent approximately 60% of all colorectal cancers removed from this population in 1994 to 1998 [9]. Germline mutations in MLH1 or MSH2 were detected by allelic specific PCR assays (for the two common Finnish MLH1 germline mutations) or by direct genomic sequencing of coding exons [9]. The data can be downloaded from the following website: . Approval for this research was obtained from the appropriate ethics committees, which are in compliance with the Helsinki Declaration.
A second data set (SEER 11 Regs Public-Use, Nov 2001 Sub (1992–1999)) was obtained from the Surveillance, Epidemiology, and End Results (SEER) Program, a population-based registry in the United States of America that records all cancers regardless of clinical treatment [10]. A total of 108,275 records were analyzed for ages at cancer selected by site (colon and rectum), race (white), histology (adenocarcinoma, ICD-0-2 codes 8000–8500), and stage (localized, regional, or distant). These cancers were not characterized with respect to HNPCC or MSI.
Quantitative analysis
Numbers of oncogenic alterations (genetic mutations or epigenetic alterations) required for transformation were estimated from ages at cancer using a Bayesian approach as previously described [11]. This method requires the use of a life table from census data: for the Finnish data set we used a Finnish life table from the World Health Organization website , for the SEER dataset we used a United States life table as described previously [11]. The model assumes the first visible clonal expansion occurs at the time of transformation and ignores the interval after transformation. The analysis ignores temporal trends, which may influence our mutation estimates.
For the SEER dataset, we also fit our model for cancer progression [11] with the inferential method described in reference 12. This method does not require a life table, but unlike our method it does require information on all the cancer cases for the population at risk. Therefore this method is appropriate for analysing the SEER dataset but not the Finnish dataset. Our method [11] is appropriate for analysing both datasets. For the SEER dataset, the two methods inferred the same number of mutations required for cancer.
Results
The presence or absence of MSI was determined for 1,022 colorectal cancers obtained from nine large regional hospitals in southeastern Finland [9]. There were 895 (87.6%) MSI- cancers and 127 (12.4%) MSI+ cancers. The MSI+ cancers were further classified as sporadic (N = 98 or 9.6% of all cancers) or HNPCC (N = 29 or 2.9% of all cancers) based on germline MLH1 or MSH2 mutations (Table 1).
Ages at cancer can be used to estimate likely numbers of oncogenic mutations required before transformation [3-6,11]. Average ages for sporadic MSI+, MSI-, and HNPCC cancers were respectively 71.5, 67.5, and 50.3 years (Figure 1A). For HNPCC cancers, estimated numbers of oncogenic mutations were between four and seven (95% credibility interval), with the most likely value of five mutations (Table 1). For MSI+ sporadic cancers, estimated numbers of mutations were between six and nine (95% credibility interval) with more likely values of seven or eight mutations. The most likely number of mutations was seven for sporadic MSI- cancers.
Duke's stage and age at clinical presentation (Figure 1B) were documented for 884 of the 895 MSI- sporadic cancers (Table 1). Average ages were 68.6 years for stage A, 69.0 years for stage B, 65.2 years for stage C, and 65.4 years for stage D. The most likely numbers of oncogenic mutations were seven for stage A cancers, eight for stage B cancers, and six for stage C or D cancers (Table 1).
Mutation number estimates with respect to clinical stage may be biased with the Finnish data because it includes only specimens with tissue available for molecular analysis. Advanced cancers may not be removed. Therefore, a similar analysis was performed on a population-based cancer registry [10] from the United States of America (SEER 11 Regs Public-Use, Nov 2001 Sub (1992–1999)), which records ages and stages at diagnosis regardless of treatment (Table 2). The average age at diagnosis was 70.5 years, consistent with an estimate of six mutations to colorectal cancer for the 108,275 white males and females with stage data. Like the Finnish cancers, ages were similar for SEER patients of different clinical stages, with an estimate of six mutations for cancers with localized, regional or distant clinical stages (Table 2 and Figure 1C).
Discussion
The exact identities and numbers of mutations required for transformation are uncertain. With simple multistage models [3-6,11], all cancers of a given type require the same number of oncogenic mutations, but stochastic differences in the times to accumulate these mutations allow individual cancers to appear at different ages. Precisely when and how quickly mutations accumulate are unknown, but a basic premise is that cancer types requiring more mutations will tend to appear later in life. Therefore, numbers of mutations may be estimated from cancer epidemiology. Colorectal cancer frequencies increase with age, and the pattern of this increase is consistent with approximately five to seven oncogenic mutations [3-6].
In this study numbers of mutations were estimated for well-defined subgroups of colorectal cancers because biological heterogeneity may confound this type of quantitative analysis. Such estimates should be considered rough guides rather than absolute values because our model does not account for all factors. Cancers were classified as MSI+ or MSI-, and MSI+ cancers were further sub-classified as either hereditary (HNPCC) or sporadic. As expected because one MMR mutation is inherited, estimated numbers of critical mutations were less for MSI+ HNPCC cancers compared to sporadic MSI+ cancers. However, sporadic MSI+ cancers required more than one additional somatic mutation compared to HNPCC cancers. Of interest, a difference of more than a single mutation has also been inferred between sporadic and FAP cancers, with estimates of three to four mutations for FAP cancers versus six for sporadic cancers [6,13], although another analysis was consistent with a difference of only a single mutation [14]. Therefore, germline mutations (APC and MMR loci) in both common colorectal familial cancer syndromes (FAP and HNPCC) appear to advance progression by more than a single mutation relative to their sporadic counterparts.
An epigenetic mechanism may help explain why sporadic MSI+ cancers require more than one additional somatic alteration relative to HNPCC cancers. Inactivation of the normal MMR allele occurs through mutation (usually LOH [15]) in HNPCC whereas MMR loss in sporadic MSI+ cancers is associated with MLH1 promoter methylation [16,17]. CpG islands may be "protected" from methylation because most are unmethylated at birth and usually remain unmethylated throughout life [18]. Epigenetic MLH1 inactivation may require at least two cis acting somatic alterations---loss of a mechanism that normally prevents methylation, followed by the accumulation of methylation at sufficient numbers of CpG sites to silence expression.
In agreement with prior studies, there were seven mutations estimated for sporadic MSI- Finnish cancers [4], and seven or eight mutations for MSI+ cancers. A requirement for more alterations before tranformation for sporadic MSI+ compared to sporadic MSI- cancers may help explain why sporadic MSI+ cancers are a minority of all colorectal cancers and occur in slightly older patients [19,20]. Although numbers of oncogenic mutations before transformation are similar between sporadic MSI+ and MSI- cancers, their identities likely differ [7,8].
Colorectal cancers also differ by their extent of spread. Progression to metastasis may involve a long sequence of potentially rate limiting steps [21]. If invasion or metastasis depends on mutations that arise after transformation, advanced cancers should require more oncogenic mutations and more time for progression (Figure 2). However, ages at diagnosis and estimated mutation numbers did not markedly differ between cancers of different clinical stages.
Equivalent numbers of mutations regardless of clinical stage are consistent with recent speculation that an invasive potential is acquired early in progression [22], albeit only rare cells actually form visible metastases. Primary breast cancer expression patterns correlate with clinical outcomes or metastases [22-25], suggesting that a propensity to spread is already present at the time of transformation. Alternatively, all cancers may have the same abilities to invade and metastasize, with clinical stage dependent on random events that occur rapidly after transformation. A short interval between transformation and detection may help limit spread because clinical surveillance tends to detect localized colorectal cancers [26-28].
Multistage models are mechanistically different from tumor progression models and more consistent with a hypothesis that mutations acquired early during progression help determine extent of invasion (Figure 3). Mutations sequentially accumulate before transformation in both models, but the adenoma-cancer sequence suggests most cancer mutations start to accumulate after the age of 50 years in adenomas [7]. Such tumor progression imposes purpose to early mutations because each additional mutation confers incremental changes to a non-invasive adenoma phenotype. Therefore, tumor progression models would likely differ between MSI+ and MSI- cancers because their biology and types of mutations are quite different [7,8].
In contrast, mutations accumulate throughout life in multistage models. Genetically engineered mice and familial cancer syndromes reveal that many oncogenic mutations are also compatible with normal phenotypes [11], allowing for the possibility that many "cancer" mutations may first accumulate in normal-appearing colon very early in life. Such pretumor progression [11] more readily allows for an invasive or metastatic cancer phenotype at transformation because genetic progression is uncoupled from tumor progression (Figure 2). Rather than incremental stepwise changes in phenotype after each new mutation, a tumor phenotype may only emerge after several initially occult mutations accumulate in a single normal appearing cell. In this way our multistage model can apply to both MSI+ and MSI- cancers despite their marked differences in types of mutations because early critical mutations (whatever they are) do not visibly change phenotype but instead accumulate in normal appearing colon. Early or advanced sporadic MSI- colorectal cancers appeared to require similar numbers of mutations, consistent with the phenotype at cancer diagnosis contingent on mutations acquired much earlier in life and present at the time of transformation. However, ascertainment bias may also be responsible for the similar frequency-age distributions of colorectal cancers of different clinical stages.
Progression to cancer has been modeled by a number of investigators with different approaches and assumptions [3-6,11-14,29,30]. In our previously reported approach there is no growth until after the last required mutation has been acquired [11]. In this paper we apply this model to cancer subtypes instead of considering colorectal cancers as a single uniform disease. Modeling is potentially more informative and specific when applied to distinct cancer subtypes because their progression pathways can differ. The ability to apply a simple multistage model to different colorectal cancer subtypes that have marked differences in final types of mutations and clinical outcomes suggests its basic underlying premise (most critical alterations first accumulate in normal colon) may be correct.
Conclusion
The biology of cancer must underlie the epidemiology of cancer. Here we illustrate that multistage models provide conceptually plausible solutions even when colorectal cancers are divided into biologically relevant and quite different subtypes. Ages at cancer are consistent with five or six somatic oncogenic mutations for hereditary (HNPCC) MSI+ cancers and seven or eight mutations for its sporadic counterpart. The apparent requirement for more than one additional somatic mutation in sporadic MSI+ cancers may reflect that MMR inactivation is commonly epigenetic, which may involve multiple steps. Ages at MSI- cancers were consistent with six or seven oncogenic mutations, with similar estimates for all clinical stages, suggesting that mutations acquired very early in life dictate the cancer phenotype at the time of transformation. Better integration of cancer epidemiology with its biology remains a further challenge.
Competing interests
The author(s) declare they have no competing interests.
Authors' contributions
Drs. Calabrese and Tavaré developed and applied the Bayesian algorithm, and edited the manuscript. Drs. Aaltonen, Mecklin, and Järvinen provided the clinical and molecular information for the Finnish cohort. Dr. Shibata helped analyze the data and wrote the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors thank the Finnish collaborators who helped collect the specimens and clinical data. ST is a Royal Society-Wolfson Research Merit Award holder.
Figures and Tables
Figure 1 Cumulative colorectal cancer frequencies and patient ages at diagnosis. A) Finnish MSI-, and hereditary (HNPCC) and sporadic MSI+ colorectal cancers. B) Finnish MSI- cancers with respect to Duke's stage. C) SEER data with respect to clinical stage.
Figure 2 Mutation timing and numbers with respect to invasiveness. If invasion or metastasis depends on mutations acquired after transformation, clinically higher stage cancers would be expected to require more time and mutations. However, if an invasive phenotype depends on mutations acquired before transformation, cancers of different clinical stages could require similar numbers of oncogenic mutations and times for progression.
Figure 3 Multistage versus tumor progression models. In multistage models, early mutations fail to confer visible changes in phenotype because they accumulate throughout life from birth. Tumors appear only after a cell has accumulated a critical number or combination of oncogenic mutations. In contrast, the adenoma-cancer sequence suggests most oncogenic mutations confer stepwise incremental changes in phenotype and accumulate much later in life in visible tumors.
Table 1 Finnish Colorectal Cancers Sample
Cancer Type Average Age (years) Number (%) Most Likely Numbers of Mutations*
4 5 6 7 8 9
HNPCC 50.3 29 (2.9) 0.08 0.43 0.39 0.09
Sporadic MSI+ 71.5 98 (12.4) 0.03 0.2 0.34 0.33 0.1
Sporadic MSI- 67.5 895 (87.6) 0.01 0.99
Sporadic MSI-
Stage A 68.6 187 (21.2) 0.09 0.75 0.16
Stage B 69.0 330 (37.3) 0.44 0.56
Stage C 65.2 246 (27.8) 0.8 0.2
Stage D 65.4 121 (13.7) 0.06 0.66 0.28
* Probabilities of the number of mutations required for cancer. Most likely values are underlined.
Table 2 SEER Colorectal Cancers
Cancer Type Average Age (years) Number (%) Number of Mutations*
All Cancers 70.5 108,275 6
Localized 70.9 44,773 (38.7) 6
Regional 70.5 42,380 (36.6) 6
Distant 69.5 21,122 (18.3) 6
* Most likely values
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de Vos tot Nederveen Cappel WH Nagengast FM Griffioen G Menko FH Taal BG Kleibeuker JH Vasen HF Surveillance for hereditary nonpolyposis colorectal cancer: a long-term study on 114 families Dis Colon Rectum 2002 45 1588 1594 12473880 10.1007/s10350-004-7244-3
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-1271620214710.1186/1471-2407-5-127Research ArticleSurvivin expression and its clinical significance in pancreatic cancer Lee Myung Ah [email protected] Gyeong-sin [email protected] Hee-Jung [email protected] Ji-Han [email protected] Jin-Hyoung [email protected] Young Seon [email protected] Kyung Shik [email protected] Dong-gu [email protected] Seung-Nam [email protected] Division of Oncology, Department of Internal Medicine, Catholic University of Medical College, Seoul, Korea2 Department of Pathology, Catholic University of Medical College, Seoul, Korea3 Department of General Surgery, Catholic University of Medical College, Seoul, Korea2005 4 10 2005 5 127 127 23 5 2005 4 10 2005 Copyright © 2005 Ah Lee et al; licensee BioMed Central Ltd.2005Ah Lee 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
Survivin, an inhibitor of apoptosis is expressed in several human cancers. Its expression is known to be associated with poor clinical outcome, but not widely studied in pancreatic cancer. We performed this study to determine the survivin expression in pancreatic cancer and its clinical significance as a prognostic factor.
Methods
We performed immunohistochemical staining for survivin, p53, and Bax in formalin-fixed, paraffin-embedded block from forty-nine pancreatic tissues. To determine the association with clinical course, we reviewed the patients' clinical record.
Results
Of the 49 cases of pancreatic cancer, 46 cases (93.9%) were positive for survivin expression. There was no significant association between survivin expression and p53 or bax. For clinicopathological parameters, perineural invasion was more common in survivin positive and venous invasion was more common in survivin negative (p = 0.041 and 0.040, respectively). Responsiveness to chemotherapy appeared to be slightly better in patients with low survivin expression.
Conclusion
Survivin expression may be associated with venous or perineural invasion, indicating metastatic route, and seems to have a potential as a predictive marker for chemotherapy. Further study of large scale is required to determine the clinical significance of survivin expression in pancreatic cancer.
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Background
Survivin has recently been identified as a novel inhibitor of apoptosis (IAP). It blocks common downstream elements of both the mitochondrial pathway and the death receptor pathway, by directly inhibiting terminal effector caspase-3, caspase-7, and caspase-9 activity[1,2]. Thus, it inhibits apoptosis pathway differently from bcl-2, which blocks mitochondrial cytochrome c release into the cytosol, resulting in the inhibition of mitochondrial apoptotic pathway. Survivin is expressed during embryonic and fetal development but is undetectable in terminally differentiated normal adult tissue. However, it is re-expressed in transformed cell lines and several human cancer cells at a frequency of 34–100 % [3,4]. As a prognostic factor, survivin expression is significantly associated with poor clinical outcome in cancers, such as neuroblastoma, colorectal cancer, breast cancer, lung cancer and esophageal cancer [5-10].
Survivin expression in pancreatic cancer has not been widely studied. Satoh et al reported that survivin was expressed in malignant portion of intraductal papillary-mucinous tumor (IPMT) but not in chronic pancreatitis, and thus proposed that survivin expression may be upregulated at an early stage of tumorigenesis by reducing cancer cell apoptosis [11]. In another study, Asanuma et al reported that survivin acts as an inducible radioresistance factor in pancreatic cancer cell line [12].
In the present study, we investigated survivin expression in pancreatic cancer and its association with clinical outcome.
Methods
Patients and specimens
Formalin-fixed, paraffin-embedded blocks from forty-nine surgically resected pancreatic tumor tissues were studied. All samples were collected under protocols approved by the local Institutional Review Board (IRB). All patients were diagnosed with pancreatic cancer at Kangnam St. Mary's hospital between May 1994 and November 2001. Patients' clinicopathological characteristics are summarized in Table 1 &2. Median age was 59 years (range, 33–76 years). According to AJCC cancer stage system (4th ed, 2002), 34 patients (69.4%) had stage I and II disease, and 10 patients (20.4%) had stage III. disease. Fourteen patients had only palliative resection and treated combination chemotherapy with epirubicin, cisplatin and 5-FU. Of 35 patients who had curative surgery, 20 patients (40.8%) underwent adjuvant treatment with radiotherapy. Recurrent cancer developed in 18 patients, and the median time to recurrence was 156 days (range, 58–1499 days).
Table 1 Patients' characteristics
No. of case
Age* 59 (33–76)
M:F 36:13
Chief Complaint Pain 20(40.8%)
Jaundice 18(36.7%)
Indigestion 6(12.2%)
Others 5(10.1%)
Location Head 35(71.4%)
Body 1(2.0%)
Tail 7(14.3%)
Body & Tail 6(12.3%)
Stage I 8(16.3%)
II 26(53.1%)
III 10(20.4%)
IV 5(10.2%)
Adjuvant treatment Yes 20(40.8%)
No 14(28.6%)
Recurrence after curative surgery 18/35+
*: median,
+: number of recurrence/total number of operable cases
Table 2 Pathological characteristics
number
differentiation well 8 (16.3%)
moderate 34 (69.4%)
poorly 3 (6.1%)
others 4(8.2%)
lymphatic invasion + 28 (57.1%)
- 18 (36.7%)
vein invasion + 10 (20.4%)
- 36 (73.5%)
perineural invasion + 34 (69.4%)
- 12 (24.5%)
Tissue microarray
To construct the tissue microarray block, small core biopsies were taken from non-necrotic, morphologically representative areas of paraffin-embedded tumor tissues and assembled on a recipient paraffin block. This was performed using a precision instrument (Micro Digital Co. Korea). The biopsied core was 3.0 mm in diameter, which was sufficient for assessing the morphological features in the tissues, and then 30 cores were assembled on a recipient paraffin block. After construction, 5 μm sections were cut and hematoxylin-eosin staining was performed on the initial slide to verify the histology.
Immunohistochemistry
Immunohistochemical staining was performed on 5 μm sections of the tissue microarray blocks. Paraffin sections were mounted on superfrost glass slides, deparaffinized and rehydrated in a graded ethanol series, and then subjected to microwave antigen retrieval. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. Sections were incubated for l hour at room temperature or at 4°C overnight with the following primary antibodies at the dilutions specified: survivin Ab-6(NeoMarkers, CA, USA) diluted 1:50, p53 (DAKO Corporation, Carpinteria, CA, USA) diluted 1:100, Bax (DAKO) diluted 1:200. Immunohistochemical staining was performed using the rabbit or mouse DAKO ChemMateTM EnVisionTM system, and the Peroxidase/DAB Kit (DAKO). Sections were then counterstained with Mayer hematoxylin and then dehydrated, cleared and mounted.
Results were interpreted by two independent pathologists who were blinded to the specific diagnosis and prognosis for each case. Tumor cells that showed distinct nuclear or cytoplasmic staining were considered positive. Immunohistochemical staining was scored on a three-tiered scale: score 0 = less than 10% of cells positive; 1 = 10 – 49% positive; score 2 = ≥50% positive.
Statistical analysis
The SPSS (version 11.0) statistical package was used to analyze the data. Using the Chi-square Test and the Fisher's exact test, the immunohistochemical profiles were compared with clinico-pathologic parameters.
Results
Expression of survivin, p53, and Bax
Of forty-nine cases of pancreatic cancer, 46 cases (93.9%) were positive for survivin expression (Fig. 1). In immunohistochemical staining for p53 and Bax, sixteen cases (32.7%) and thirty-eight cases (77.5%) were positive, respectively (Fig. 2A & B). These are summarized in Table 3. Survivin expression was not associated with p53 or Bax expression (p = 0.9969 and 0.0931, respectively, Table 4)
Figure 1 Immunohistochemical staining for surviving. Strong cytoplasmic expression for survivin in pancreatic cancer gland (A: × 100, B: × 400).
Figure 2 Immunohistochemical staining for p53 and Bax. A: Strong positive expression of p53 in cancer cell (× 400). B: Positive expression of Bax in pancreatic cancer cell(× 200).
Table 3 The result of immunohistochemical staining
Survivin p53 Bax
Score 0 3(6.1%) 33(67.3%) 11(22.4%)
Score 1 17(34.7%) 9(18.4%) 11(22.4%)
Score 2 29(59.2%) 7(14.3%) 27(55.1%)
Table 4 Survivin expression and p53, Bax in pancreatic cancer
p53 (p = 0.9969) Bax (p = 0.0931)
0 1 2 0 1 2
survivin 0 2 0 1 2 1 0
1 12 3 2 6 2 9
2 19 6 4 3 8 18
Survivin, p53, and bax expression and clinico-pathologic characteristics
Of 49 tissue specimens, only tumor tissues were obtained from 3 patients due to far advanced stage. We evaluated pathological characteristics in 46 patients. There was no correlation between disease stage and expression of survivin, p53, or bax protein (Table 5), and no correlation was found between survivin expression and p53 or bax protein expression. However, perineural invasion was more common in the survivin-positive, and venous invasion was more common in the survivin- negative group (p = 0.041 and 0.040, respectively, Table 6).
Table 5 Stage and survivin, p53, bax expression
Stage I Stage II Stage III Stage IV p value
Survivin 0 0 2 0 1 0.386
1 3 7 5 2
2 5 17 5 2
p53 0 5 18 7 3 0.207
1 1 4 3 1
2 2 4 0 1
Bax 0 4 5 2 0 0.414
1 2 6 1 2
2 2 15 7 3
Table 6 Pathological parameters and survivin expression
Lymphatic invasion (p = 0.094) Venous invasion (p = 0.040) Perineural invasion (p = 0.041)
Yes No Yes No Yes No
0 1 1 1 1 2 0
1 12 5 3 14 12 5
2 15 12 6 21 20 7
Clinical outcome and survivin expression
We evaluated the association between survivin expression and survival. We did not find any significant difference correlation between survival and survivin expression (Fig 3) because only three patients showed survivin negative. It was difficult to identify the association due to the vast majority of patients expressing survivin.
Figure 3 Overall survival. No significant difference in overall survival according to intensity of survivin expression.
Fourteen patients received epirubicin, cisplatin and 5-FU combination chemotherapy as initial treatment for advanced stage of disease. Responsiveness to chemotherapy appeared to be slightly better in patients with low survivin expression compared to those with high expression (28.8% vs.7.1%, respectively; Table 7), although there was no statistically significant correlation. We also investigated the efficacy of chemotherapy and Bax expression, but all patients who treated with chemotherapy showed 2+ in Bax expression. Therefore, we did not analyze statistical significance.
Table 7 Responsiveness to chemotherapy and survivin expression
Responder Non-responder
survivin Score 1 4 (28.8%) 4 (28.6%)
(p = 0.2098) Score 2 1 (7.1%) 5 (36. 7%)
Discussion
Survivin, an IAP, has been studied as a prognostic marker in various cancers. Adida et al reported that survivin expression in neuroblastoma correlated with unfavorable histology, aggressive and disseminated disease [5]. In colorectal cancer and breast cancer, patients with survivin-positive tumors had a decreased apoptotic index and worse survival rates than those with survivin-negative tumors [7,8]. In addition, survivin expression correlated with poor prognosis in esophageal cancer and non-small cell lung cancer [9,10].
The clinical significance of survivin expression in pancreatic cancer is not well understood. Previous studies reported that survivin was expressed at an incidence of 77%–88% [11,13]. In our study, survivin was expressed in 93.9%, and was highly expressed in most cases (59.2%). In pathological parameters, neural invasion was more common in survivin positive group. Neural invasion is a specific metastatic route in pancreatic cancer and perineural invasion is known to be a prognostic marker for the recurrence of pancreatic cancer after surgery [14]. However, venous invasion was more common in survivin negative group. Venous invasion in pancreatic cancer can be a risk factor for liver metastasis [15]. We did not explain this discrepancy, but we suggested that venous invasion may be an independent prognostic factor irrelevant to survivin expression. Recent study reported that survivin expression has different clinical significance according to expression pattern [16]. Although we did not distinguish the expression pattern, it may be another explanation for this discrepancy. It is important to predict recurrence or metastatic pattern after curative surgery because of determining adequate adjuvant treatment modality in pancreatic cancer, so further study may be warranted to investigate the association between survivin and pathological parameters.
We did not find any other association between survivin expression and clinical course due to the limitation of patients enrolled in this study. Most patients in our study were at an early stage, making it difficult to compare survivin expression according to various stage. Secondly, the vast majority of pancreatic cancer tissue in the present study expressed survivin, meaning survivin-negative group was very small. For the same reason, we could not determine that survivin expression was a prognosis factor. Recently, Kami et al reported that survivin expression may be a prognostic factor in pancreatic cancer. In that study, 63.8% of patients showed survivin expression, resulting in a good proportion of survivin-negative patients for comparison [17]. We also investigated whether survivin expression was associated with poor clinical outcome, including recurrence and survival, but no significant correlation was observed. However, It is not determined that survivin may not be associated with poor clinical outcome because of limitation of our study. It may be that further study involving great number of patients is required in order to determine the clinical significance of survivin expression in pancreatic cancer.
It is not certain whether survivin expression is a predictive marker for anticancer therapy such as radiotherapy or chemotherapy in pancreatic cancer. In a study using a pancreatic cancer cell line, Asanuma et al reported that survivin may be a radioresistant factor [12], and Kato et al suggested that survivin expression may be a predictive marker for chemotherapy as well as prognostic marker in esophageal cancer in their previous study [9]. We examined the survivin expression in fourteen patients treated with chemotherapy to evaluate the potential of survivin as a predictive marker for chemotherapy. Although the findings were statistically not significant, there was a suggestion that non-responder group showed high survivin expression. On the basis of this finding, we hypothesize that survivin is a potential marker for predicting responsiveness to chemotherapy.
The p53 mutation is frequently seen in pancreatic cancer, and its expression correlates with overall survival after pancreatectomy [18]. Previous study found survivin and p53 may play important roles in the transition from adenoma to carcinoma in situ in IPMT, and that survivin expression was higher in p53-positive pancreatic cancers [18,19]. In the present study, while 34.6% of tumors expressed p53, there was no correlation between p53 and survivin expression. Bcl-2 family proteins also play important roles in regulation of apoptosis. Previous work showed Bax expression was associated with a better prognosis and was found to be a predictive marker for adjuvant chemotherapy [20]. In the present study, while 84.6% of tumors expressed Bax, there was no correlation between its expression and survivin expression or clinico-pathological parameters. The present data did not indicate that p53 or Bax protein correlated with survivin expression in pancreatic cancer. It is not certain why this discrepancy exists, although all these proteins may block apoptotic pathway. Whether p53 or Bax expression in pancreatic cancer is related to prognosis is still under debated. In addition, some authors suggest that survivin may be independent prognostic marker not associated with p53 or Bax expression [21,22]. Therefore, we hypothesized that tumor cell may have other pathway to evade apoptosis after Bax or p53-involved steps in pancreatic cancer. It appears further large scale studies are required to investigate any correlation between survivin and cell cycle regulatory protein expression in pancreatic cancer.
Conclusion
In summary, survivin was expressed at high levels in the vast majority of pancreatic cancer. While the data did not indicate any correlation between survivin expression and clinical outcome, they may be interpreted as suggesting survivin may be a predictive marker in pancreatic cancer for anticancer therapy in pancreatic cancer. We believe further prospective studies are warranted to determine whether survivin is a prognostic or predictive marker in pancreatic cancer.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Lee MA conceived of the study and carried out design and analysis. Park GS, Lee JH, and Jung JH carried out immunohistochemical staining and reviewed the all pathological data. Kang JH, Hong YS and Lee KS participated in coordination and helped to draft the manuscript. Kim SN and Kim DG supplied tissue specimen and collect clinical data.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-5-281617153110.1186/1471-2261-5-28Research ArticleCharacteristic wave detection in ECG signal using morphological transform Sun Yan [email protected] Kap Luk [email protected] Shankar Muthu [email protected] Bioinformatics Institute, Singapore 1386712 Biomedical Engineering Research Center, School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 6397982005 20 9 2005 5 28 28 24 1 2005 20 9 2005 Copyright © 2005 Sun et al; licensee BioMed Central Ltd.2005Sun 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
Detection of characteristic waves, such as QRS complex, P wave and T wave, is one of the essential tasks in the cardiovascular arrhythmia recognition from Electrocardiogram (ECG).
Methods
A multiscale morphological derivative (MMD) transform-based singularity detector, is developed for the detection of fiducial points in ECG signal, where these points are related to the characteristic waves such as the QRS complex, P wave and T wave. The MMD detector is constructed by substituting the conventional derivative with a multiscale morphological derivative.
Results
We demonstrated through experiments that the Q wave, R peak, S wave, the onsets and offsets of the P wave and T wave could be reliably detected in the multiscale space by the MMD detector. Compared with the results obtained via with wavelet transform-based and adaptive thresholding-based techniques, an overall better performance by the MMD method was observed.
Conclusion
The developed MMD method exhibits good potentials for automated ECG signal analysis and cardiovascular arrhythmia recognition.
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Background
The detection of the major characteristic waves in ECG, namely the QRS complexes, P and T waves, is one of the essential tasks in ECG analysis. The performance of an automated ECG analysis system depends heavily on the reliable detection of these fiducial waves. The difficulties of characteristic waves detection lie in oscillations in the baseline, irregular morphology of the waveforms, and frequency overlapping among the wide-band distribution of the characteristic waves [1], etc.
A significant amount of research effort has been devoted to the automated detection of the fiducial (reference) points of the ECG characteristic waves [2-12]. Most of these methods are filtering or adaptive thresholding based, which exhibit limitation in real application. Very few algorithms work well for the detection of all fiducial points such as the onsets and offsets of the P wave, T wave and the QRS complex (also known as the ECG wave boundaries). The main drawback of filtering-based approach is that frequency variations in the characteristic waves often adversely affect its performance. The frequency distribution of QRS complexes generally overlaps with that of the noise, resulting in both false positive and false negative detections. The main problems of the thresholding techniques are their high noise sensitivity and their low efficiency when dealing with odd morphologies. Therefore, more sophisticated signal processing techniques are needed to facilitate the development of new detection schemes with higher detection accuracy.
As a nonlinear filtering technique, it has been proven that morphological dilation and erosion satisfy the causality and the additive semigroup property required by multiscale analysis [13-15] for signals of any dimension with local maxima and local minima as singular points. The fiducial points in ECG signal, such as the Q wave, R peak, S wave, the onsets and offsets of the P wave and T wave, can be regarded as such singular points [16,17]. In this paper, a new multiscale morphological derivative (MMD) transform-based technique, was developed for the detection of the fiducial points of the ECG characteristic waves. By applying a morphological derivative transform defined at different scales, noise sensitivity inherent in single scale operation can be reduced in MMD method. In addition, the problem of position deviation existed in wavelet transform-based techniques [10] can be avoided due to the nonlinearity of morphological transform. As a result, tracing across scales to locate the singular points is not needed.
The proposed multiscale morphological derivative (MMD) detector
In the present study, the signal to be processed is limited to continuous function f : 2 → with only finite oscillations on a closed interval which is differentiable everywhere except at some singular points. A singular point in the one-dimensional signal is defined as a point with derivative on the right and the derivative on the left exist with different signs.
For the singular point to be defined using multiscale morphological derivative, the derivative on the right can be represented by morphological sup-derivative , which is defined as
Similarly, the derivative on the left can be represented by morphological inf-derivative , which is defined as
Here, the notation of [18] is used to introduce morphological operators on functions. Denoting the functions, f : D ⊂ n → and gs : Gs ⊂ n → (s > 0), the two fundamental operations of multiscale morphology are:
where Dx is the translation of D, Dx = x + t:t ∈ D, sup(f) and inf (f) refer to the supremum (least upper bound) and infimum (greatest lower bound) of f, s is scale, and gs is the scaled structuring function [19]. In the discrete case where the function is a finite set of points, max() and min() are used instead of sup() and inf().
We propose a multiscale morphological derivative difference , to be defined to characterize the difference between the left and right derivatives as follows:
The scaled version of at scale s, , can then be defined as:
By choosing a flat structuring function, where gs(x) = 0, x ∈ G, where G = {x: ||x|| ≤ s} [14], the above multiscale morphological derivative transform described by Equation (6) is simplified to the following process: Choose a moving window with a length of (2s + 1) samples and find the maximum and minimum values in the window, as well as the value of the signal at the cental point f(x). Then, the MMD transform at the central point can be specified as
At a positive peak in ECG signal, its left derivative is positive and its right derivative is negative, therefore, positive peaks in the ECG signal correspond to the local minima in . At the onset or offset of a positive peak, there is an abrupt increase in its derivative value from left to right. So, the onsets and offsets correspond to the local minima in . As applied to ECG lead II signal, the R peak, Q wave an S wave correspond to the local minima of the , while the onsets and offsets of the P wave and T wave correspond to the local maxima of the . Hence the characteristic QRS complex, P wave and T wave, can be detected using the proposed MMD detector by detecting the local extrema in the MMD transformed signal.
Characteristic wave detection in ECG using the MMD detector
The MMD detector is a single lead detection method. In this paper, we only use the ECG lead II for algorithm development and testing. A similar analysis can be done to extend method to other leads. The detailed procedure for ECG characteristic wave detection using the proposed MMD detector, is described as follows:
1. ECG signal is preprocessed by morphological filtering for noise reduction and baseline correction.
2. Multiscale morphological transform is performed on the preprocessed input signal.
3. The local maxima and minima with absolute amplitude larger than a threshold, Thf, at a selected scale sm are detected (sm = 20 for MIT-BIH database and sm = 15 for QT database in this study). The local minima with absolute amplitude larger than a threshold, ThR, are detected as R peaks, where, the selection of ThR and Thf is based on an adaptive thresholding from the histogram of the MMD transformed data.
4. For each detected R peak, the first local maximum point on its left is detected as the beginning of the R wave; the first maximum point on its right side is detected as the end of the R wave.
5. The first local minimum from the left of the positive R wave is detected as the Q wave. If the minimum cannot be detected, the Q wave is judged to be missing. (There is a time interval for Q wave detection, which is set as the prior clinical value of QRS complex, here, 0.12s).
6. The first local minimum from the right of the positive R wave is detected as the S wave. Otherwise, the S wave is judged to be missing. Same time interval as for Q wave detection is set for S wave detection.
7. The subsequent two consecutive local maxima from the left of the Q wave are detected as the offset and onset of the P wave; the first and second local maxima from the right of the Q wave are detected as the onset and offset of the T wave, respectively.
The preprocessing in step 1 is performed as follows:
where fo is the original input signal; fb is the baseline drift signal; f is the signal after preprocessing; o is morphological opening operator; • is morphological closing operator; structuring elements, Bo, Bc and B, are selected based on the properties of ECG characteristic waves. Further details can be found in [20]. For each preprocessed signal, its morphological derivative at scale sm was calculated according to Equation (7) and its local maxima and minima were detected. It is known that the number of maxima or minima at a larger scale is much less than that at a lower scale. In addition, high frequency noise decays greatly at large scales so that less extrema due to noise are found at larger scale. Therefore, morphological derivative transformed signal at a larger scale was used for detecting the locations of the objective feature points. However, in order not to smooth the characteristic waves in ECG, sm should be as large as possible but less than Wwfs, where Ww is the width of the characteristic wave, and fs is the sampling frequency of ECG signal. The width of QRS is generally from 0.06s to 0.12s. The P wave and the T wave are generally longer than the QRS complex. Hence, in the proposed study, sm = 20 for MIT-BIH database and sm = 15 for QT database, were chosen. No calculation was performed at other scales since MMD operation does not cause drift of singular points across different scales.
For the detection of local maxima and minima, two thresholds ThR and Thf were used, which were adaptively computed from the histogram of the MMD transformed data. The two between-peak valleys in the histogram gave rise to the values of ThR and Thf. ThRwas used for the detection of the local minima, which correspond to R peaks; Thf was used for the detection of the local minima, which correspond to other characteristic waves.
In any single ECG beat, the R peak, Q wave and S wave correspond to adjacent local minima in the morphological derivative-transformed signal. The onset and offset of the P wave correspond to local maxima adjacent to the Q wave. In the normal cases, the onset and offset of the T wave are local maxima adjacent to S wave. Otherwise, the T wave is judged as inverted. However, for other abnormal T waves, such as the biphasic T wave, the MMD detector may falsely detect the onset and offset of the T wave.
Results and discussion
The proposed morphological approach for the characteristic wave detection in ECG signal was tested using the first ECG leads from the MIT-BIH arrhythmia database [21] and the QT database [22], which were developed with the aim to be benchmarking references for automated analysis of ECG. The MIT-BIH arrhythmia database contains 48 records (each 30 minutes long) with a sampling frequency of 360 Hz. The QT database is a mixed database with a sampling frequency of 250 Hz, which consists of 105 excerpts (each 15 minutes long) taken from other ECG databases, where, 15 from MIT-BIH Arrhythmia Database, 6 from the MIT-BIH ST Change Database, 13 from the MIT-BIH Supraventricular Arrhythmia Database, 10 from the MIT-BIH Normal Sinus Rhythm Database, 33 from the European ST-T Database, 24 from "sudden death" patients from BIH, and 4 records from the MIT-BIH Long-Term ECG Database.
For each input ECG signal, the following procedures were performed: (i) signal preprocessing; (ii) multiscale morphological derivative transform; (iii) detection of local maxima and minima in morphological derivative-transformed signals; (iv) detection of characteristic waves in the original ECG signal. Some results using MMD detector for characteristic wave detection are given in Figure 1, where (a) is for normal ECG beat, (b) is for left bundle branch block (LBBB) ECG beat, (c) is for atrial premature contraction (APC), and (d) is for premature ventricular contraction (PVC). In each subfigure, the three plots from top to bottom are: the single ECG beat selected from the MIT-BIH database; the MMD transformed signal with marked points (the onset and offset of the R wave are marked with 'Δ'; other fiducial points, such as, the Q wave, R peak, S wave, as well as the onsets, offsets, the peaks of the P wave and T wave, are marked with '*'); the automatically detected characteristic waves are in solid line.
Figure 1 Results of characteristic wave detection for single ECG beat. The three plots in each subplot from top to bottom are: original ECG signal; the MMD transformed signal at scale 20 with the detected characteristic points marked; detected characteristic waves in solid line. (a) Normal ECG beat (b) LBBB (c) APC (d) PVC.
As shown in Figure 1, the characteristic waves in normal beat are observed to be well detected. For LBBB, in spite of the appearance of a sub-R peak, the boundaries of all waves are still well detected. In APC or PVC no preceding premature P wave appears. In addition, the position of the left 'Δ ' overlaps with the '*' because the onset of the preceding T wave is submerged in the QRS complex. The position of the right 'Δ' also overlaps with the '*' and the Q wave is judged to be missing.
Figure 2 gives more results of characteristic wave detection in ECG signal series. It is obvious that all three characteristic waves (the QRS complex, the P wave, the T wave) in ECG time series with normal beats, APCs, and LBBB beats, are detected reliably. Even the onsets and offsets of inverted T waves in PVCs can be detected reliably.
Figure 2 Detection results of ECG series, from top to bottom, they are: original ECG signal; MMD transformed signal with fiducial points marked; detected characteristic waves highlighted in bold. (a) ECG series with normal beats and PVC (b) ECG series with normal beats and APC (c) ECG series with LBBBs.
More than 20000 annotated ECG beats randomly selected from MIT-BIH arrhythmia database were used to test the performance of the proposed MMD detector. An overall false detection rate of 0.35% was obtained for the QRS complex detection. In addition, more than 2500 annotated ECG beats randomly selected from the QT database were tested for evaluating the performance of the proposed MMD detector for ECG wave boundaries detection. In the QT database, a minimum of 30 beats from each of its 105 records had been manually annotated by one or more cardiologists. The annotation files were taken as reference for evaluating the performance of automated algorithms for detecting the onsets and offsets of the P wave, T wave, and the QRS complex during ECG analysis.
In order to quantify the performance of ECG characteristic wave detection by the proposed MMD technique, three parameters were used, which included the mean error (m) and standard deviation (σ) of the differences between the annotation results and the automated detection results, as well as Sensitivity (Se). m is used to determine how close the automated detection results are to the annotation results. σ gives an idea of the stability of detection. Se is defined as , for measuring the detection sensitivity, where TP is the number of true detections; FN is the number of manual annotations that are not registered in the automatic detections.
The statistical results for m, σ, and Se, for ECG fiducial points and characteristic waves detection by the proposed MMD technique were compared with the threshold-based detector (TD) [22] and the wavelet-based detector (WD) [23], and they are shown in Table 1. The accepted standard deviation tolerances from the measurements required by CSE were given in the last row of Table 1.
Table 1 Comparative Results of ECG characteristic wave detection
Technique Parameter Pon Poff QRSon QRSoff Ton Toff
MMD Se(%) 97.2 94.8 100 100 99.8 99.6
m(ms) 9.0 12.8 3.5 2.4 7.9 8.3
σ(ms) 9.4 13.2 6.1 10.3 15.8 12.4
TD Se(%) 96.2 97.0 99.9 99.9 98.8 98.9
m(ms) 10.3 -5.7 -7.3 -3.6 23.3 18.7
σ(ms) 14.1 13.6 10.9 10.7 28.3 29.8
WD Se(%) 89.9 89.9 100 100 99.1 99.2
m(ms) 13.0 5.4 4.5 0.8 -4.8 -8.9
σ(ms) 12.7 11.9 7.7 8.7 13.5 18.8
CSE σ(ms) 10.2 12.7 6.5 11.6 - 30.6
*Results on TD and WD are extracted from [10] and [11] for comparison purpose
The proposed MMD method works best for the QRS complex detection. Lower values of mean bias and standard deviation as well as higher value of detection sensitivity are observed. The average detection bias for the QRS's onset and offset are 3.5 ms and 2.4 ms respectively. The corresponding standard deviations are 6.1 ms and 10.3 ms, both of which are within the acceptable limits required by the CSE committee.
However the requirements can not be fully satisfied by the TD and WD methods although the mean bias obtained by the WD method is a little better than the results obtained by the MMD method. As for the P wave onset detection, only the MMD method can fulfill the CSE requirement while it fails for the P wave offset detection. The WD method performs better for P wave offset detection while worse for P wave onset detection compared with the MMD method. TD method fails to meet CSE requirements for both P wave onset and offset. As for the T wave offset detection, all three methods can satisfy the limit required by the CSE committee. Among them, the MMD and the WD methods perform much better than the TD method. The clinically important intervals for arrhythmia recognition, such as the PR interval (from the onset of the P wave to the onset of the R wave), the QT interval (from the Q wave to the offset of the T wave), are not related to the positions of the offset of the P wave and the onset of the T wave. Therefore, the weakness of the proposed MMD technique does not cause significant problem for arrhythmia recognition. The correlation coefficients between the results obtained by MMD method and those computed from annotation data are 0.9264 for PR interval, 0.9542 for QRS complex, and 0.9316 for QT interval.
The proposed MMD method for ECG wave boundary detection is well performed with reasonable mean bias and standard deviation values within the limits required by CSE, on over 79% of the records. In the QT database, best detection performance is observed for records from the MIT normal sinus rhythms database. Records with poor detection performance are mostly from the European ST-T database and the Supraventricular database, in which, low signal-to-noise ratio or non-homogeneous repolarization exists. In summary, it is concluded that the proposed MMD detector has acceptable performance comparable to those given by experts.
Conclusion
In this paper, a new algorithm based on multiscale morphological derivative transform, called MMD detector, has been developed for fiducial point detection and applied for ECG wave boundary detection. The MMD detector could not only work for the QRS complex, but also the onsets and offsets of the characteristic waves. The standard deviations for important ECG characteristic wave detection obtained by the proposed MMD detector were within the limits required by the CSE committee. Furthermore, the statistical results obtained by the MMD detector were compared with those obtained by wavelet transform-based and adaptive thresholding-based techniques. In overall, better performance by the MMD technique was observed, considering that less empirical parameters were needed. Therefore we conclude that the proposed MMD method exhibits good potentials in the clinical applications for automated analysis of ECG signal.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SY conceived the study, performed data analysis and drafted the manuscript. CKL and KSM guided the study, helped the analysis and interpretation of the results, and critically reviewed the manuscript. All authors read and approved the final script.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors wish to extend their sincere appreciation to Nanyang Technological University, Singapore for supporting this work. The authors also acknowledge the clinical collaboration from Singapore General Hospital.
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Sun Y Chan KL Krishnan SM ECG signal conditioning by morphological filtering Computers in Biology and Medicine 2002 32 465 479 12356496 10.1016/S0010-4825(02)00034-3
MIT-BIH arrhythmia Database Beth Israel Hospital, Biomedical Engineering Division Room
Laguna P Mark RG Goldberger AL Moody GB A database for evaluation of algorithms for measurement of QT and other waveform intervals in the ECG Computers in Cardiology 1997 673 676
Martinez JP Olmos S Laguna P Evaluation of a wavelet-based ECG waveform detector on the QT database Computers in Cardiology 2000 81 84
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1211615939510.1186/1471-2164-6-121Research ArticleFunctional genomics and expression analysis of the Corynebacterium glutamicum fpr2-cysIXHDNYZ gene cluster involved in assimilatory sulphate reduction Rückert Christian [email protected] Daniel J [email protected] Daniel A [email protected] Andreas [email protected] Sascha [email protected]ühler Alfred [email protected] Jörn [email protected] International NRW Graduate School in Bioinformatics & Genome Research, Universität Bielefeld, D-33594 Bielefeld, Germany2 lnstitut für Genomforschung, Universität Bielefeld, D-33594 Bielefeld, Germany3 Lehrstuhl für Genetik, Universität Bielefeld, D-33594 Bielefeld, Germany2005 13 9 2005 6 121 121 22 6 2005 13 9 2005 Copyright © 2005 Rückert et al; licensee BioMed Central Ltd.2005Rückert 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
Corynebacterium glutamicum is a high-GC Gram-positive soil bacterium of great biotechnological importance for the production of amino acids. To facilitate the rational design of sulphur amino acid-producing strains, the pathway for assimilatory sulphate reduction providing the necessary reduced sulfur moieties has to be known. Although this pathway has been well studied in Gram-negative bacteria like Escherichia coli and low-GC Gram-positives like Bacillus subtilis, little is known for the Actinomycetales and other high-GC Gram-positive bacteria.
Results
The genome sequence of C. glutamicum was searched for genes involved in the assimilatory reduction of inorganic sulphur compounds. A cluster of eight candidate genes could be identified by combining sequence similarity searches with a subsequent synteny analysis between C. glutamicum and the closely related C. efficiens. Using mutational analysis, seven of the eight candidate genes, namely cysZ, cysY, cysN, cysD, cysH, cysX, and cysI, were demonstrated to be involved in the reduction of inorganic sulphur compounds. For three of the up to now unknown genes possible functions could be proposed: CysZ is likely to be the sulphate permease, while CysX and CysY are possibly involved in electron transfer and cofactor biosynthesis, respectively. Finally, the candidate gene designated fpr2 influences sulphur utilisation only weakly and might be involved in electron transport for the reduction of sulphite. Real-time RT-PCR experiments revealed that cysIXHDNYZ form an operon and that transcription of the extended cluster fpr2 cysIXHDNYZ is strongly influenced by the availability of inorganic sulphur, as well as L-cysteine. Mapping of the fpr2 and cysIXHDNYZ promoters using RACE-PCR indicated that both promoters overlap with binding-sites of the transcriptional repressor McbR, suggesting an involvement of McbR in the observed regulation. Comparative genomics revealed that large parts of the extended cluster are conserved in 11 of 17 completely sequenced members of the Actinomycetales.
Conclusion
The set of C. glutamicum genes involved in assimilatory sulphate reduction was identified and four novel genes involved in this pathway were found. The high degree of conservation of this cluster among the Actinomycetales supports the hypothesis that a different metabolic pathway for the reduction of inorganic sulphur compounds than that known from the well-studied model organisms E. coli and B. subtilis is used by members of this order, providing the basis for further biochemical studies.
==== Body
Background
In many micro-organisms as well as in higher plants the metabolism of sulphur is based on the uptake and subsequent reduction of oxidised, inorganic sulphur compounds, usually sulphate. While this pathway has been studied in detail in the model organisms Escherichia coli for the Gram-negative bacteria [1] and Bacillus subtilis for the Firmicutes (low-GC Gram-positives), almost no data is available for the high-GC Gram-positives of the order Actinomycetales with the exception of the functional analysis of cysH from Mycobacterium tuberculosis [2].
The pathway for assimilatory sulphate reduction and its genetic basis in E. coli and B. subtilis can be summarised as follows: After uptake through either an ABC-type transporter, in E. coli encoded by sbp [EMBL:AAC76899] and cysUWA [EMBL:AAC75477, EMBL:AAC75476, EMBL:AAC75475] [1] or, in B. subtilis, a permease encoded by cysP [EMBL:CAB13432] [3], reduction of inorganic sulphate starts with the activation of sulphate by adenylylation, catalyzed by sulphate adenylyltransferase (EC 2.7.7.4), and yielding adenylylsulphate (APS). In E. coli this enzyme is a heteromer encoded by cysD and cysN [EMBL:AAC75794, EMBL:AAC75793] while B. subtilis uses the homomeric protein Sat [EMBL:CAB13437] (encoded by ylnB ) which is known in other organisms to be involved in dissimilatory sulphate reduction [4]. The release of sulphite from APS can occur either by direct reduction through APS reductase (CysH, EC 1.8.4.10) or by APS kinase (CysC, EC 2.7.1.25) mediated phosphorylation and subsequent reduction of the product, phosphoadenylylsulphate (PAPS), by PAPS reductase (CysH, EC 1.8.4.8). E. coli has been demonstrated to use the two-step reaction via CysHC [EMBL:AAC75804, EMBL:AAC75792] while B. subtilis CysH [EMBL:CAB13431] can also directly convert APS to sulphite [5]. In either case, sulphite is then reduced to sulphide by sulphite reductase (CysIJ [EMBL:AAC75805, EMBL:AAC75806], EC 1.8.1.2) [6]. In addition to the enzymes directly involved in the catalysis, two additional proteins are thought to be necessary for sulphate reduction in E. coli: uroporphyrin-III C-methyltransferase (CysG [EMBL:AAC76393], EC 2.1.1.107) and PAPS phosphatase (CysQ [EMBL:AAC77171]). The former is needed for the biosynthesis of the sulphite reductase cofactor siroheme, catalyzed by YlnD [EMBL:CAB13435] and YlnF [EMBL:CAB13437] in B. subtilis [7], while the latter is believed to eliminate excess amounts of PAPS which are highly toxic for the cell [1].
In a continuation of our work to elucidate the biosynthesis of L-methionine in the Gram-positive non-sporulating actinobacterium Corynebacterium glutamicum [8], the reduction of inorganic sulphur compounds was investigated in order to learn how the reduced sulphur needed for incorporation into L-methionine is formed. Based on the knowledge of assimilatory sulphur reduction and the genes described for the two model organisms E. coli and B. subtilis, we tried to identify the set of genes involved in the assimilatory reduction of inorganic, oxidised sulphur compounds in C. glutamicum. Real-time RT-PCR was then applied to analyse the operon structure of the genes found and to elucidate their regulation in cells incubated with different sulphur sources. Subsequently, RACE-PCR was used to map the transcription start points of the identified transcription units to identify the potential promoters. Finally, comparison with other completely sequenced members of the Actinomycetales was carried out in order to learn about the phylogenetic distribution of the assimilatory sulphur reduction genes.
Results
Identification of a Corynebacterium glutamicum gene cluster possibly involved in assimilatory sulphate reduction by sequence similarity and comparative genomics
In a previous investigation, the whole genome sequence of C. glutamicum [9,10] was used to elucidate the pathway for L-methionine biosynthesis [8]. In this study, we extended this work and analysed the genetic basis for the reduction of inorganic sulphate. Using genes known to be involved in the assimilatory reduction of sulphate from Escherichia coli [1], Mycobacterium tuberculosis [2], and Bacillus subtilis [3,7,11] similar coding sequences (CDS) were searched for in the C. glutamicum genome sequence.
This approach led to the identification of a cluster of four CDS (cg3118, cg3116, cg3115, and cg3114 ) in C. glutamicum which might be the orthologues of the E. coli genes cysI, cysH, cysD, and cysN (Table 1 and Figure 1), while no genes with high similarity to the E. coli genes cysC, cysJ, sbp or the cysPUWA cluster could be found (data not shown). Likewise, no clear homologues of the B. subtilis genes cysP or ylnB were detected. Closer examination of the genomic neighbourhood of the four candidate genes revealed another three CDS which are located within (cg3117 ) or directly adjacent (cg3112, cg3113 ) to the identified cluster of cys genes. To determine the boundaries of this cluster, all genes in this genomic region were compared with the genome of the closely related species Corynebacterium efficiens [12], using a bidirectional best BLAST hit method [13].
Table 1 Properties and sequence similarities of the C. glutamicum proteins possibly involved in assimilatory reduction of sulphate
C. glutamicum protein properties BLAST resultsb
CDS (protein a ) length (aa) pI mw [kDa] hit against in organismc description E-value identical/positive aa
Cg3112 (CysZ) 309 9.98 32.0 Q8FM69 C. efficiens Conserved hypothetical protein 2e-134 77/88%
Q81NU8 B. anthracis Putative membrane protein 8e-58 46/64%
Q813F4 B. cereus Hypothetical membrane spanning protein 4e-57 46/64%
Cg3113 (CysY) 241 5.44 24.7 Q8FM68 C. efficiens Hypothetical protein 1e-80 67/77%
P61817 B. megaterium Sirohydrochlorin ferrochelatase SirB 5e-16 28/45%
Q93RW8 S. coelicolor Hypothetical protein SC01858 2e-13 31/43%
Cg3114 (CysN) 433 5.08 46.9 Q8FM67 C. efficiens Putative sulphate adenylyltransferase SU 1 0.0 84/90%
Q9ADG6 S. coelicolor Sulphate adenylyltransferase SU 1 3e-lll 55/67%
Q10600 M. tuberculosis Sulphate adenylyltransferase SU 1 1e-104 50/69%
Cg3115 (CysD) 304 5.09 34.3 Q8FM66 C. efficiens Putative sulphate adenylyltransferase 7e-168 95/96%
Q9ADG5 S. coelicolor Sulphate adenylyltransferase SU 2 2e-123 71/83%
Q9X5UO S. lavendulae Sulphate adenylyltransferase SU 2 6e-123 70/82%
Cg3116 (CysH) 261 5.30 28.5 Q8FM65 C. efficiens Putative PAPS reductase 3e-129 86/89%
Q82L82 S. avermitilis Putative PAPS reductase 7e-52 56/67%
P71752 M. tuberculosis APS reductase 5e-52 54/69%
Cg3117 (CysX) 82 7.86 9.5 Q8FM64 C. efficiens Hypothetical protein 5e-36 81/89%
Q82L83 S. avermitilis Hypothetical protein 4e-05 50/57%
Q9ADG2 S. coelicolor Hypothetical protein SC06101 5e-05 50/57%
Cg3118 (CysI) 561 5.53 63.0 Q8FM63 C. efficiens Putative ferredoxin-nitrite reductase 0.0 91/95%
Q7WT38 Streptomyces sp. Nitrile/sulphite reductase 0.0 56/70%
Q82L84 S. avermitilis Putative nitrile/sulphite reductase 0.0 55/71%
Cg3119 (Fpr2) 457 4.88 50.1 Q8FM62 C. efficiens Putative ferredoxin-NADP reductase 0.0 87/93%
Q8NM28 C. glutamicum NADPH-glutamate synthase beta chain 0.0 74/86%
Q8FMB5 C. efficiens Putative ferredoxin/adrenodoxin reductase 0.0 75/85%
FPRA_MYCTU M. tuberculosis Ferredoxin-NADP reductase FprA 6e-83 39%
FPRB_MYCTU M. tuberculosis Ferredoxin-NADP reductase FprB 63e-66 36%
a by sequence similarity
b identified by BLASTP similarity search. The protein sequences derived from the C. glutamicum candidate CDS were used as queries against the UniProt database
c B. = Bacillus, C. = Corynebacterium, M. = Mycobacterium, S. = Streptomyces
Figure 1 The fpr2cysIXHDNYZ gene cluster in the C. glutamicum genome. Coloured arrows indicate genes that are part of the cluster most probably involved in assimilatory sulphate reduction. Hairpins mark potential rho-independent transcription termination signals predicted by the TransTerm software. Black bars denote binding sites for the transcriptional repressor McbR [15].
Thereby it could be shown that besides the identified cys orthologues, the localisation of cg3112, cg3113, and cg3117 is also well conserved between C. glutamicum and C. efficiens, as is that of cg3119 while directly up- and downstream of the cluster the syntenous order of genes is interrupted by insertions in C. glutamicum (data not shown). The notion that the cys gene-cluster extends from cg3112 to cg3119 is corroborated by the finding that transcriptional termination signals were predicted by the TransTerm software [14] directly behind cg3112 and cg3119 (Fig. 1). This furthermore suggests that cg3118 to cg3112 form an operon, providing another hint that also cg3112, cg3113, and cg3117 are involved in the reduction of sulphate. Additional evidence that the whole cluster is involved in sulphur metabolism was the identification of four binding sites of the transcriptional repressor McbR which controls almost all genes involved sulphur metabolism in C. glutamicum [15] in the intergenic region shared by cg3118 and cg3119 (Fig. 1).
Bioinformatic analysis of the Corynebacterium glutamicum candidate genes possibly involved in assimilatory sulphate reduction
As a first step in the analysis of the eight candidate genes similarity searches against the UniProt and PFAM databases were done. These searches led to a functional assignment for cg3114 and cg3115 as cysD and cysN respectively, most likely encoding the two subunits for sulphate adenylyltransferase known from, e.g., E. coli (Table 1).
For Cg3118 and Cg3116, the similarity searches indicated possible deviations from the enzymatic functions described for E. coli (Table 1): Cg3118 (CysI) from C. glutamicum, while moderately similar to CysI from E. coli, displays a higher degree of similarity to ferredoxin-dependent sulphite and nitrite reductases (NirA) known from other Actinomycetales and plants. In turn, based on a multiple alignment, these sequences cluster much more closely with the ferredoxin-dependent nitrite reductases from plants and cyanobacteria (e.g. Nir from Synechococcus sp.) than with CysI homologues of the a- and γ-proteobacteria (data not shown). Likewise, Cg3116 (CysH) was found to be much more similar to CysH from M. tuberculosis which has been proven to act as APS reductase (EC 1.8.4.10) [2]. Furthermore, all the sequence motifs demonstrated to be necessary for the reduction of APS [16] are present in CysH from C. glutamicum (data not shown).
For the proteins encoded by the four novel CDS cg3112, cg3113, cg3117, and cg3119, no significant similarities to proteins known to be involved in sulphate reduction were detected, and only Cg3119 shows similarities to well characterised proteins (Table 1). The gene cg3112 encodes a protein similar to putative membrane proteins of the domain of unknown function family 81 (DUF81), which consists of integral membrane proteins. The notion that Cg3112 is located in the membrane is further corroborated by the TMHMM software which predicts the presence of six transmembrane helices. The possible presence of a signal peptide, detected by SignalP, indicates a Sec-dependent insertion into the membrane (data not shown). Cg3113 is weakly similar to sirohydrochlorin ferrochelatase from B. megaterium, indicating a possible involvement in the biosynthesis of the siroheme cofactor of sulphite reductase. The small CDS encoding Cg3117 shows similarities to small hypothetical proteins in several Actinomycetales. A multiple alignment of the significant hits revealed the presence of a conserved sequence motif (CPYCx(14,19)WxCxxCxRxF) containing four conserved cysteine residues. They are reminiscent of those found in Fe-S cluster coordination sites. Finally, Cg3119 is highly similar to FprA and FprB, known to act as ferredoxin-NADP reductases in mycobacteria. FprA from M. tuberculosis has been studied on the molecular level and is now classified as NADPH-ferredoxin reductase (EC 1.18.1.2), but it is unknown in which biological processes FprA and FprB are involved in M. tuberculosis [17,18]. Interestingly, a highly similar paralogue (Cg3049) is present in the C. glutamicum genome itself. Based on the well characterised FprA protein of M. tuberculosis, cg3049 and cg3119 were therefore named fprl and fpr2, respectively.
Comparison of the putative sulphate assimilation gene cluster of Corynebacterium glutamicum with gene clusters of other members of the actinomycetes
As the cluster of genes possibly involved in sulphate assimilation had been found to be conserved between C. glutamicum and C. efficiens, the question arose whether the findings for sulphate assimilation in C. glutamicum are specific for these two organism or if the cluster and the pathway is conserved among the order of the Actinomycetales. Therefore, a search for conserved parts of the cluster in all completely sequenced genomes of the bacterial order as well as in the only partially completed genomes of Brevibacterium linens and Thermobifida fusca was conducted, using the programs GECKO [19] and TheSEED [20].
This approach revealed that most of the genes found to be involved in sulphur metabolism in C. glutamicum are also found in other members of the Actinomycetales. Notable exceptions are C. diphtheriae, Bifidobacterium longum, Leifsonia xyli, M. leprae, Propionibacterium acnes, and Tropheryma whipplei, as in all these organisms the genes needed for assimilatory sulphate reduction are completely absent. All these organisms are known pathogens or commensals of humans, animals, or plants, the most likely explanation is that these bacteria obtain the required sulphur-containing organic compounds directly from their hosts.
Concerning the conservation of the cluster, it was found to be almost perfectly conserved in C. glutamicum and C. efficiens (Fig. 2), as well as in the recently sequenced C. jeikeium [21]. In the other members of the Actinomycetales that contain the genes needed for sulphate assimilation, these genes are also clustered, but in many cases the cluster is split in two or three parts. In addition, duplications seem to have occurred, e.g., in M. avium, N. farcinica, and S. avermitilis.
Figure 2 Conservation of the C. glutamicum gene cluster possibly involved in assimilatory sulphate reduction in the Actinomycetales. Functions were inferred based on sequence similarity from BLAST searches against the UniProt database. Only those genes are displayed that were found to be clustered (at least two adjacent genes possibly involved in assimilatory sulphate reduction).
Of the four novel genes cg3112, cg3113, cg3117 and cg3119 (fpr2 ), only a cg3113-like gene was found in all Actinomycetales containing parts of the cluster (Fig. 2), adding additional evidence that it is involved in sulphate assimilation. On the other hand, it proved to be the least conserved on the amino acid level (data not shown).
cg3112 seems to be present only in the Corynebacteriaceae. This might be due to the fact that in the Mycobacteriaceae, Streptomycetaceae, and N. farcinica an ABC-type transporter replaces Cg3112.
Fpr homologues are only found to be clustered with other cys genes in Corynebacteriaceae, but at least one possible homologue is present in all Actinomycetales that contain the cluster. Another hint for the involvement in the reduction of sulphate is the co-occurrence of Cg3117 and Fpr homologues lacking a ferredoxin-domain: M. tuberculosis, M. avium, and N. farcinica all contain a two-domain protein consisting of a ferredoxin-like and a Fpr-like domain. The Corynebacteriaceae, Streptomycetaceae, and T. fusca all lack such a protein but possess a Cg3117-homologue in the cluster. As the flavoprotein subunit of sulphite reductase CysJ is absent in all Actinomycetales, the data from the comparative genomics approach provides circumstantial evidence that the enzymatic equipment for sulphite reduction in the Actinomycetales is similar to that found in plants: In these eukaryotes electrons are transferred directly from photosystem I via ferredoxin-NADP reductase and ferredoxin to the ferredoxin-dependent sulphite reductase [22]. For C. glutamicum, this leads to the hypothesis that electrons might be transferred from NADPH+H+ by Fpr2 to Cg3117 which in turn delivers them to the ferredoxin-dependent sulphite reductase.
Characterisation of the Corynebacterium glutamicum gene cluster possibly involved in assimilatory sulphate reduction by targeted gene deletion, mutational analysis, and complementation
To determine if the CDS of the identified gene cluster cg3119-3112 are involved in the assimilatory reduction of sulphate, deletion mutants of all genes were constructed. Gene-SOEing was used to create fusion products of chromosomal DNA sections of approximately 500 bp length directly up- and downstream of the target gene. The resulting fusion products were cloned into the vector pK18mobsacB and the obtained plasmids were used for targeted gene deletion.
The resulting mutants CR018 to CR025 (Table 4) were tested for their ability to grow on a solid minimal medium containing differing inorganic sulphur compounds at 2 mM concentration as sole source of sulphur. Seven of the eight mutant strains CR018 to CR024 were affected in their ability to utilise inorganic sulphur sources. The most common observed phenotype was a complete inability to grow, like strain CR024 (ΔcysI ) on medium containing sulphate or sulphite, but in some cases, growth was only severely reduced, like that of strain CR023 (Δcg3117 ) when utilising oxidised inorganic sulphur compounds (Table 2).
Table 2 Growth of C. glutamicum mutant strains on different inorganic sulphur sources on solid minimal medium
deleted growtha after 48 h on MMS with addition of 2 mM of
strain CDS (gene) sulphate sulphite thiosulphate sulphide L-cysteine
WT / + + + + +
CR018 cg3112 (cysZ ) - - + + +
CR019 cg3113 (cysY ) - - - ° +
CR020 cg3114 (cysN ) - - ° + +
CR021 cg3115 (cysD ) - - ° + +
CR022 cg3116 (cysH ) - - ° + +
CR023 cg3117 (cysX ) ° ° ° + +
CR024 cg3118 (cysI ) - - - ° +
CR025 cg3119 (fpr2 ) + + + + +
a compared to the wild-type grown on MMS containing sulphate:
+ denotes wild-type like growth
° indicates a severely reduced growth rate
- represents a complete lack of growth
All four mutants in the potential cys gene homologues (cg3114, cg3115, cg3116, and cg3118) were unable to utilise sulphate as sole source of sulphur while they could still grow on sulphide, thereby confirming that the encoded proteins are involved in the reduction of inorganic sulphur compounds. Surprisingly, the mutants lacking cysD, cysH, or cysN were also unable to grow on sulphite and displayed a reduced growth rate on thiosulphate. This observation stands in contrast to the findings made, e.g., for E. coli, where a loss of these genes does not influence the ability to utilise sulphite or thiosulphate [1].
Of the four novel genes, the mutational analysis provided strong evidence that the three previously uncharacterised CDS cg3117, cg3113, and cg3112 are involved in assimilatory sulphate reduction (Table 2). The corresponding mutant strains could either no longer grow on MMS containing only sulphate or sulphite (Δcg3113 and Δcg3112) or displayed a severely reduced growth rate (Δcg3117). Therefore, they were named cysY, cysZ, and cysX, respectively. On the other hand, a loss of fpr2 did not influence growth under the tested condition, indicating that the encoded protein is either not involved in sulphate reduction or its function can be replaced, most likely by Fpr1, the product of cg3049. Unfortunately, all attempts to verify this hypothesis failed, because deletion or disruption of fpr1 was not possible. This indicates that Fpr1 might have another, essential function in C. glutamicum.
As the genes cysI to cysZ might constitute an operon, homologous complementation of the mutant strains CR018 to CR024 (Table 4) was used to verify that the observed phenotypes were due only to the loss of the deleted gene and not caused by polar effects on genes downstream of the deletion. This was done by PCR amplification of the gene in question and subsequent cloning of the product in a suitable expression vector. The resulting plasmids pCR018e to pCR024e (Table 4) were transferred into the corresponding mutant strains and the ability of the plasmid carrying strains to utilize inorganic sulphur sources was tested as described above. In all cases, growth of the mutant strain expressing the missing gene in trans was indistinguishable from that of the wild-type, thus ruling out polar effects.
The plasmids pCR020e, pCR022e and pCR024e (carrying cysDN, cysH, and cysI, respectively) were also used to try heterologous complementation of corresponding E. coli mutant strains to get additional evidence for the assumed gene functions. Surprisingly, the complementation of E. coli JM221 (cysD-) and JM246 (cysI-) with pCR20e, respectively, failed. Only AB462 (cysC-) and JM226 (cysH-) carrying pCR022e were able to grow with sulphate as sole source of sulphur, and even these grew only slowly. These results add evidence to the hypothesis that C. glutamicum uses either a pathway differing from that in E. coli or that the enzymes can only function as a complex in C. glutamicum.
Analysis of the Corynebacterium glutamicum gene cluster fpr2cysIXHDNYZ by growth tests in liquid culture
Based on the data obtained from the in silico and in vivo analysis, a hypothesis of the possible functional role of the four novel CDS was formulated, leading to a preliminary model for the pathway for assimilatory sulphate reduction in C. glutamicum: The presence of transmembrane domains and the inability of the mutant strain to grow on sulphate and sulphite as sole sources of sulphur indicated that CysZ might be involved in the uptake of these compounds. For CysY, a function in the biosynthesis of the siroheme cofactor of sulphite reductase could be supposed as the phenotype of the CR019 (Δcg3113) mutant strain was indistinguishable from that of CR024 (ΔcysI). The small protein CysX might be involved in electron transfer, with the four conserved cysteine residues possibly being part of a Fe-S cluster like those found in ferredoxins. For Fpr2, a function in the reduction of ferredoxins could be assumed, possibly acting on CysX.
In order to substantiate these hypotheses, growth of the mutant strains CR018, CR019, CR023, and CR025 (ΔcysZ, ΔcysY, ΔcysX, and Δfpr2, respectively) was measured via real-time nephelometry in liquid minimal medium on different inorganic sulphur sources at a concentration of 2 mM. From the obtained growth curves, the generation times and the duration of the lag phases were calculated and compared to those determined for the wild-type strain (Table 3).
Table 3 Growth characteristics of C. glutamicum mutant strains on different inorganic sulphur sources at 2 mM concentration in liquid medium
generation timea [h] lag timea [h]
sulphur source WT CR018 (ΔcysZ) CR019 (ΔcysY) CR023 (ΔcysX) CR025 (Δfpr2) WT CR018 (ΔcysZ) CR019 (ΔcysY) CR023 (ΔcysX) CR025 (Δfpr2)
sulphate 2.1 ndb,c nd 6.2 2.2 8.0 nd nd 23.5 11.0
sulphite 2.2 nd nd 4.2 2.1 8.0 nd nd 16.0 11.0
thiosulphate 3.2 3.2 nd 9.5 2.9 9.5 14.0 nd 37.5 10.5
sulphide 3.8 3.8 17.6 7.4 3.2 15.0 13.5 16.5 23.5 15.5
a averages, calculated from 18 measurements (3 independent cultivations, 6 replicates per cultivation)
b significant changes are given in bold script
c nd = not determined due to lack of growth
Table 4 Bacterial strains and plasmids
Name Relevant genotype/information a Source/reference
E. coli
DH5αMCR F- endA1 supE44 mcrA thi-1 hsdR17 λ- recA1 relA1 Δ (lacZYA-argF )U169 (Φ 80dlacZΔM15 ) gyrA96 deoR Δ (mrr-hsdRMS-mcrBC ) [40]
AB462, CGSC 462 F- DE (gpt-proA )64 lacYl galK2 (Oc)hisG4 (Oc) λ- cysC9 xylA5 mtl-1 thi-1 CGSCb
JM226, CGSC 5468 F- pro-0 lac -tsx -? galT47 trp-74 his -cysH57 xyl argA9999 rpsL -(strR) mal -ilvA -mtl - CGSC, [47]
JM221, CGSC 5745 F- pro-50 lac- tsx -galT47 trp-74 his-97 cysD91 argA-rpsL--(strR) mat xyl-ilvA-mtl- CGSC, [47]
JM246, CGSC 5747 F- λ- cysI53(Am)IN(rrnD-rrnE)1 rph-1 CGSC, [47]
C. glutamicum
ATCC 13032 Wild type, Nxr ATCCc
CR018 Δcg3112 (cysZ) this study
CR019 Δcg3113 (cysY) this study
CR020 Δcg3114 (cysN) this study
CR021 Δcg3115 (cysD) this study
CR022 Δcg3116 (cysH) this study
CR023 Δcg3117 (cysX) this study
CR024 Δcg3118 (cysI) this study
CR025 Δcg3119 (fpr2) this study
CR026 cg3118::IS6100, pAT6100 this study
Plasmids
pK18mobsacB sacB, lacZa, Kmr, mcs mobilizable vector, allows for selection of double-crossover in C. glutamicum [35]
mobilizable vector, allows for selection of double-crossover in C. glutamicum
pEC-XK99E Ptrc, lacIq, Kmr inducible E. coli – C. glutamicum – shuttle expression vector [48]
pAT6100 IS61 00, Kmr [36]
pCR018d pK18mobsacB carrying cg3112 deld this study
pCR018e pEC-XK99E carrying cysZ eve this study
pCR019d pK18mobsacB carrying cg3113 del this study
pCR019e pEC-XK99E carrying cysY ev this study
pCR020d pK18mobsacB carrying cg3114 del this study
pCR020e pEC-XK99E carrying cysDN ev this study
pCR021d pK18mobsacB carrying cg3115 del this study
pCR022d pK18mobsacB carrying cg3116 del this study
pCR022e pEC-XK99E carrying cysH ev this study
pCR023d pK18mobsacB carrying cg3117 del this study
pCR023e pEC-XK99E carrying cysX ev this study
pCR024d pK18mobsacB carrying cg3118 del this study
pCR024e pEC-XK99E carrying cysI ev this study
pCR025d pK18mobsacB carrying cg3119 del this study
a r superscript indicates resistance. nx, Nalidixic acid; Km, Kanamycin
b CGSC; E. coli Genetic Stock Center, Yale University, New Haven, CT
c ATCC; American Type Culture Collection, Rockville, MD
d the postfix del indicates inserts used for targeted gene deletion
e the postfix ev indicates genes preceded by an artificial RBS
This approach confirmed the observations made during growth on solid minimal medium. In the case of CysX, it provided further evidence for the hypothesis that CysX might act like a ferredoxin: The generation time of the mutant strain CR023 (ΔcysX) is doubled to tripled (Table 3), indicating that the function of CysX can be partially replaced by another protein, most likely one of the ferredoxins found in C. glutamicum. Interestingly, under these conditions strain CR025 (Δfpr2) also delivered a phenotype, namely an increase in the lag phase during growth with sulphate or sulphite of approximately three hours. This provides circumstantial evidence that Fpr2 is indeed involved in sulphur reduction, although its loss can be compensated for.
As prior growth tests on solid minimal medium containing high concentrations of sulphate had shown that strain CR018 (ΔcysZ) could grow under these conditions (data not shown), additional tests with this strain were performed in liquid culture using varying concentrations of sulphate. These tests confirmed that the mutant strain is unable to grow with sulphate concentrations of less than 5 mM. With increasing sulphate concentration, growth is gradually restored until, at above 30 mM, it reaches almost the level of the wild-type strain (Fig. 3). This concentration-dependent restoration of growth provides strong evidence that CysZ acts as the high-affinity sulphate transporter in C. glutamicum. Furthermore it indicates that at least one other transporter for sulphate must exist which is able to function effectively only at sulphate concentrations above 5 mM.
Figure 3 Growth of the C. glutamicum wild-type and the ΔcysZ mutant strain in medium with different sulphate concentrations. Wild-type and mutant strain were grown in liquid minimal medium, cell growth was determined by real-time nephelometry. For each time point the mean of 18 measurements is displayed (3 independent cultivations, 6 parallels per cultivation). The wild-type strain is denoted with filled diamonds, open symbols are used to indicate the mutant strain.
Transcriptional analysis of the Corynebacterium glutamicum cys gene cluster
As gene order and the presence of possible transcription termination sites indicated that the genes cysI to cysZ might form an operon (Fig. 1), real-time RT-PCR was used to verify this hypothesis. To disrupt the hypothetical operon, a transposon mutant in cysI (CR026, insertion 997 bp downstream of the cysI gene start) was used, as it could be assumed that the insertion of the transposon and the vector with a size of ≈ 5.5 kb would lead to a premature termination of transcription. Using real-time RT-PCR, the mRNA levels of the seven genes in the wild-type and the mutant strain both grown with L-cysteine as sulphur source were compared. The measurements revealed an equally strong reduction of the relative mRNA levels of all seven genes in the mutant downstream of the transposon insertion site (Fig. 4). This observation and the finding that the mRNA level upstream of the insertion is only slightly increased provides strong evidence that cysIXHDNYZ indeed form an operon and that no additional promoters downstream of cysI exist.
Figure 4 Comparison of the mRNA levels of the cys genes of the C. glutamicum wild-type and a cysI transposon mutant. Total RNA was isolated from cells grown in MMS with 1 mM L-cysteine as sulphur source and the relative transcription levels were determined using real-time RT-PCR to quantify the mRNAs of the displayed genes. Small black bars inside the arrows representing the genes indicate the position of the internal fragments amplified in the real-time RT-PCR.
Real-time RT-PCR was also applied to analyse whether the clustered genes are subject to regulation by either one or more of the inorganic substrates or by the products of assimilatory sulphur reduction (namely sulphide and L-cysteine). Therefore, the mRNA levels of fpr2, cysI, and cysZ in cells incubated in MMS with different sulphur sources at 1 mM concentration was compared with those in cells incubated in MMS without added sulphur. In all cases with the exception of thiosulphate, a strong simultaneous reduction of mRNA abundance of all three genes in cells incubated with a sulphur source was observed (Fig. 5A). Transcription levels of the genes of the cluster were found to be strongly reduced in the presence of most inorganic sulphur sources while L-cysteine had a much weaker, albeit still significant effect. Surprisingly, the relative transcription levels decreased only marginally in presence of thiosulphate, even at higher concentrations of up to 5 mM (data not shown).
Figure 5 Change of the fpr2cysIXHDNYZ mRNA levels in the presence of different sulphur sources and varying amounts of sulphate. The relative mRNA levels of the fpr2, cysI, and cysZ genes in cells incubated in MMS with either (A) different sulphur sources at 1 mM concentration or (B) sulphate at varying concentrations were compared to that in cells incubated in MMS without additional S-sources using real-time RT-PCR. Small black bars inside the arrows representing the genes indicate the position of the internal fragments used in real-time RT-PCR.
This data clearly proves that the assimilation of sulphur in C. glutamicum is regulated by either the substrates or the products of the assimilatory reduction of sulphate. Furthermore, the data indicates that this regulation is different from that found in E. coli, as thiosulphate has been shown to repress transcription of the cys genes in that organism [1]. Finally, the co-regulation of fpr2 and the cys genes in C. glutamicum also provides circumstantial evidence that Fpr2 is involved in the reduction of sulphate.
The strong regulation of the three genes under study raised the question at which concentration it would occur. Therefore, the relative transcription levels in the presence of varying amounts of sulphate were measured. By this approach it could be demonstrated that already at a concentration of 100 μM sulphate the genes are as strongly repressed as in the presence of 1 mM sulphate and more while at 50 μM sulphate transcription levels are comparable to those observed in MMS without added sulphur (Fig. 5B).
Determination of the transcription start sites in front of the Corynebacterium glutamicum fpr2 and cysI genes
As the real-time RT-PCR data indicated that all seven cys genes are described from one promoter, the RACE technique was applied to identify the promoter elements in the intergenic region between fpr2 and cysI. Thereby, one transcription start point was identified for each of the two transcription units (Fig. 6). The sequences upstream were compared to the the σ70 consensus promoter [23] revealing two hexamers showing similarity to the -10 and -35 region including an optimal spacing of 17 bases, but no elements of an extended -10 region are detectable (Fig. 6). These hexamers are in 7 respectively 8 of 12 positions identical to the consensus sequence, which represents an average value for promoters in C. glutamicum, with the most conserved bases of the consensus promoter also present in the two promoters described here. An interesting finding was the observation that both promoters overlap with potential binding-sites of the transcriptional repressor McbR [15]. This strongly indicates that McbR is involved in the observed regulation.
Figure 6 The promoter/operator regions of the C. glutamicum fpr2 and cysI genes. The determined transcriptional start points of the two transcriptional units fpr2 (A) and cysIXHDNYZ (B) are marked as '+1'. Parts of the two potential promoters (-35, -10, +1) are overlined, bases in bold type in these regions indicate bases matching the C. glutamicum σ70 consensus promoter [23]. DNA motifs matching the consensus sequence of the McbR binding-site are boxed. Bases in bold italics mark potential ribosome binding-sites, open underlining arrows indicate the annotated starts of genes.
Discussion
In this report, we describe the identification and validation of a set of genes involved in the assimilatory reduction of sulphate in Corynebacterium glutamicum, including the up to now unknown genes cysX, cysY, cysZ, and fpr2.
Initial comparison of the genetic equipment for the assimilatory reduction of sulphate indicated that C. glutamicum possesses a set of genes similar to that known from E. coli [1], with exception of cysC (encoding APS kinase) and cysJ (encoding the flavoprotein subunit of sulphite reductase). On the other hand, genes typical for B. subtilis like sat [3], ylnF [7], and cysP [3] could not be found in C. glutamicum. Despite several similarities, the pathway present in C. glutamicum displays several features that distinguish it from that described for E. coli, based on the obtained data (Fig. 7):
Figure 7 Model of the pathway for assimilatory sulphur reduction in C. glutamicum.. For proteins with gene names given in black, the involvement in the reduction of sulphate has been verified experimentally, for those in grey it has been inferred from circumstantial evidence.
Uptake of sulphate is most likely mediated by a novel type of permease, CysZ, instead of the ABC-type transporter Sbp CysPTWA known from E. coli [24,25]. Although not proven by biochemical data, the observed growth deficits of the ΔcysZ mutant strain together with the bioinformatic evidence that CysZ is located in the membrane strongly support this theory. Interestingly, the obtained data also indicates the existence of at least one low-affinity sulphate transporter and one or more transporter(s) for the uptake of the other inorganic sulphur compounds (like thiosulphate) which are not clustered with the other cys genes in C. glutamicum.
Strong evidence also exists that the activation of sulphate and the subsequent reduction to sulphite is performed in only two steps in C. glutamicum: Like cysH from M. tuberculosis [2], the heterologous expression of cysH from C. glutamicum can complement E. coli cysH- as well as cysC- mutants. This and the missing of a homologue of APS kinase clearly indicate that CysH acts as APS reductase in C. glutamicum, corroborating the prediction made by Lee [26].
The final step, the reduction of sulphite to sulphide seems to differ the most from the situation found in E. coli and B. subtilis, albeit this hypothesis is backed only by circumstantial evidence: CysI from C. glutamicum and the potential homologues in the other Actinomycetales share the highest degree of similarity to ferredoxin-dependent nitrite and sulphite reductases found in plants and cyanobacteria. Together with the presence of Fpr and CysX, this leads to the hypothesis that electrons are transferred from NADPH+H+ via Fpr2 and CysX to CysI to reduce sulphite, similar to the pathway used in non-photosynthetic plant tissues [22]. Unfortunately, this hypothesis could not be proven conclusively with the used genetic methods alone. The loss of CysX clearly affected growth on all oxidised inorganic sulphur compounds, but the exact function cannot be inferred from this analysis. In case of Fpr2, the phenotypical evidence is even weaker as a mutant lacking Fpr2 is almost unaffected in the utilisation of sulphate and sulphite, except for a slightly increased lag phase. Still, the absence of a clear phenotype is easily explainable by the presence of a highly similar paralogous protein, Fpr1, that seems to be essential. The notion that Fpr2 is indeed involved in the pathway under study is also supported by the finding that it is co-regulated with the genes of the cys operon and is controlled by McbR, the global regulator of sulphur metabolism in C. glutamicum [15].
Based on the phenotype of the deletion mutant, CysY is also involved in the reduction of sulphite. The weak similarity of CysY to sirohydrochlorin ferrochelatase from B. megaterium (EC 4.99.1.4) [27] indicates an involvement in the biosynthesis of the siroheme cofactor of sulphite reductase, but again this assumption cannot be proven conclusively by genetic analysis alone.
Concerning the pathway as a whole, the data from the mutational analysis, i.e. the inability of cysH, cysD, and cysN mutants to utilise sulphite, and the failed heterologous complementation of E. coli cys mutants, suggest that the enzymes might form a multi-enzyme complex in C. glutamicum.
By analysing the mRNA levels of the eight clustered genes, it could be proven that cysIXHDNYZ constitute one single transcription unit. This unit seems to be co-regulated with fpr2, as expression of both was found to be strongly influenced to the same extent by the presence of most inorganic sulphur sources and L-cysteine.
The observed reduction of the mRNA levels of the eight genes under study in presence of various sulphur sources is reminiscent of the regulation described for E. coli [1] with exception for thiosulphate which also causes a strong anti-induction in E. coli. For C. glutamicum, it remains to be determined if the observed regulation is an anti-activation, as in E. coli, or a repression of a sulphur-starvation response. Based on the gathered data, the latter appears to be more likely, as cysI has been shown to be regulated by McbR, the global repressor of sulphur metabolism [28]. Mapping of the potential promoters of cysI and fpr2 revealed that both overlap with potential binding-sites of this master regulator [15], which corroborates the finding of Rey et al. that the transcription of the genes of the cluster is increased in a ΔmcbR mutant [15]. This strongly suggests that the observed regulation is at least in part due to the action of McbR. This hypothesis is further backed by the finding that McbR was found to be conserved in all completely sequenced Corynebacteriaceae [29] which is in accordance with the high degree of conservation of the cluster in C. glutamicum, C. efficiens, and C. jeikeium. Other candidates that might be involved in the regulation of the cluster are two genes under McbR-control, cg0012 and cg0156. The corresponding proteins both belong to the ROK/NagC-type family of regulators [29] and cg0012 has recently been proven to be involved in the metabolism of sulphur as it encodes the sulphate-inhibited activator of sulphonate utilisation, SsuR [30]. Therefore, cg0156 is a interesting candidate encoding a possible regulatory protein that should be characterised in a future study.
Although the regulatory protein(s) involved are not yet known, the regulatory network has been demonstrated to act extremely sensitive, as an external sulphate concentration of only 100 μM already leads to a strong reduction of the mRNA levels of the fpr2 and cys genes by almost 1000-fold. This might indicate that the cell needs to strictly control the amounts of reduced sulphur, which is plausible as most of these compounds are toxic.
An interesting finding was the high degree of conservation among cys gene clusters in different members of the Actinomycetales. With exception of several pathogens and commensals. As the pathway described for C. glutamicum in this study seems to be present, with slight deviations, in at least 11 other species, it stands to argue that the findings for one member of this order can be easily transferred to other members. As C. glutamicum is one of the few non-pathogenic species of the Actinomycetales, and the genes, with exception of fpr, are present in only one copy, this bacterium is well suited to study this important pathway more closely, e.g. on the biochemical level.
Conclusion
Based on sequence similarity, comparative genomics, and subsequent mutational analysis, we were able to identify a cluster of eight genes involved in assimilatory sulphate reduction. The obtained data supports a conclusive model for this pathway in C. glutamicum which differs considerably from those described for E. coli and B. subtilis, although further biochemical studies are necessary to prove the suggested functions. By using comparative genomics, we could gather strong evidence that the pathway described here might be present in at least 11 other members of the actinomycetes, thus distinguishing this group from the Gram-negative and low-GC Gram-positive bacteria in this metabolic function.
Furthermore, we could demonstrate that the eight genes under study are strongly regulated by the presence of either substrates and products of this pathway. Although reminiscent of the regulation described for E. coli, mapping of the transcription start points revealed that the C. glutamicum gene cluster might be controlled by repression of transcription rather than by anti-activation.
Methods
Bacterial strains, plasmids and culture media
The bacterial strains and plasmids used in this study are listed in Table 4. E. coli strains carrying plasmids were routinely grown on solid Antibiotic Medium No. 3 (PA) (Oxoid, Wesel, Germany) at 37°C. C. glutamicum strains were grown on solid brain-heart broth (BH) (VWR International, Darmstadt, Germany) at 30°C. Auxanography on solid medium was performed using a minimal medium (MMS), containing only trace amounts of inorganic sulphur. MMS is composed of 5% glucose, 150 mM NH4C1, 50 mM urea, 6 mM K2HP04, 1.5 mM MgCl2, 70 μM FeCl2, 50 μM MnCl2, 70 μM CaCl2, 7.5 μM ZnCl2, 1 μM CuCl2, 0.1 μM, NiCl2, 500 μg/l thiamine, and 50 μg/l biotin, for the growth test different sulphur sources were added at 2 mM concentration (calculated for sulphur). For solidification, 16 g agarose were added per litre MMS.
For growth tests in liquid medium, a modified version of MME [31], called MMES, was used, consisting of 2.5% glucose, 17 mM NaNH4HP04, 1 mM MgCl2, 60 mM K2HP04, 10 mM citric acid, 37.5 μM FeCl2, 50 μM MnCl2, 67.5 μM CaCl2, 7.5 μM ZnCl2, 1 μM CuCl2, 0.1 μM, NiC12, 500 μg/l thiamine, and 50 μg/l biotin, with addition of different sulphur sources at varying concentrations.
Sulphur sources used were sodium sulphate (Na2S04), sodium sulphite (Na2SO3), sodium thiosulphate (Na2S2O3), sodium sulphide (Na2S), and L-cysteine.
Antibiotics used for selection of plasmids and strains were nalidixic acid (50 μg/ml for corynebacteria) and kanamycin (50 μg/ml for E. coli, 25 μg/ml for corynebacteria).
DNA isolation, transfer and manipulation
Standard procedures were employed for molecular cloning and transformation of E. coli DH5α, as well as for electrophoresis [32]. Transformation of C. glutamicum was performed by electroporation using the methods of Tauch et al. [33].
Construction of plasmids
Plasmids pCR016d to pCR025d were constructed using the gene splicing by overlap extension (gene-SOEing) method described by Horton et al. [34], the PCR primers used are listed in the supplementary Table S1 [see Additional file 1]. The primary products were amplified using Pwo DNA polymerase (Roche, Mannheim, Germany). The resulting products were purified using the PCR purification kit (QIAGEN, Hilden, Germany) and used as templates for the second round of PCR. The final products were digested with restriction enzymes corresponding to the cleavage sites introduced via PCR and ligated into appropriately digested pK18mobsacB. The ligation mixture was used to transform E. coli DH5αMCR, the transformants were selected on PA plates containing 50 μg/ml kanamycin and 40 mg/l X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside).
Plasmids for complementation experiments were created by amplifying the gene of interest via PCR. The resulting constructs were cleaved using restriction sites added by the PCR primers and then ligated into plasmid pZ8-l, cut with the corresponding enzymes. The ligation mixture was used for transformation of E. coli DH5αMCR, the transformants were selected on PA plates containing 50 μg/ml kanamycin.
Site-specific gene deletion
Site-specific gene deletion was performed using the non-replicable integration vector pK18mobsacB which allows for marker-free deletion of the target gene [35]. The plasmids pCR018d to pCR025d were transferred into the C. glutamicum wild-type strain (ATCC 13032) by electroporation [33]. Tests for first and second cross-over were performed as described previously [8].
Identification of a cysI transposon mutant
Using a cloned copy of the IS6100 mobile element [36], a transposon mutant library was constructed. The mutants were gained with a modified approach that uses electrotransfer of the IS6100-based transposon vector pAT6100 into the C. glutamicum host cells. Subsequent phenotypic screening of the transposon library for cystein-auxotrophic mutants was applied, and the transposon integration loci of these mutants were determined by a plasmid rescue technique and subsequent sequence similarity analysis [37].
Relative quantification of mRNA levels using real-time RT-PCR
Bacterial cell cultures were grown to the early logarithmic phase (o.D.600 between 8 to 12) in MMS with 250 μM L-cysteine as sulphur source. Incubation took place in an Innova 4430 orbital shaker (New Brunswick, N J) at 300 rpm, using 25 ml medium in a 250 ml Erlenmeyer flask.
For each experiment, 5*109 cells were pelletised by centrifugation, washed twice with MMS without added sulphur source (preheated to 30°C) and resuspended in 5 ml MMS. To this culture the sulphur source to be analysed was added at varying concentrations and incubated for an additional 30 min.
For RNA isolation, about 109 cells per culture were harvested by 15 sec centrifugation at 16,000 g, followed by immediate removal of the supernatant and freezing of the pellet in liquid nitrogen. Preparation of total RNA from C. glutamicum cells was performed as described by Hüser et al. [38].
Primers for real-time RT-PCR were constructed to amplify intergenic regions of about 150 bp length of the genes to be analysed, designated as *_lc1/2 [see Additional file 1]. The primers were designed using the Primer Designer 4.2 software (Sci Ed Software, Durham, NC) and were purchased from SIGMA-ARK (Darmstadt, Germany).
All real-time RT-PCR experiments were performed using a LightCycler (Roche, Mannheim, Germany) with the QuantiTect SYBR Green RT-PCR Kit (QIAGEN, Hilden, Germany). PCR mixes were set up and PCR was performed as described by Koch et al. [39]. All measurements were performed for two biological replicates per condition tested and with two technical replicates per biological replicate. The amounts of the mRNAs of the genes of the cluster were normalised on total RNA and the relative change in transcription rate was determined as 2-ΔCP with ΔCP equal to the difference of the measured crossing points for the test and the control condition.
Determination of transcriptional starts with the RACE method
Total RNA was isolated from a C. glutamicum wild-type culture grown in MMS medium and subjected to sulphur starvation as described above. Primers binding downstream of the annotated translational starts of the cysI and fpr2 genes (*SP1-3 in Table 5) along with 1.5 μg of total RNA were used for cDNA synthesis. The cDNA was then modified and amplified using the 5'/3' RACE kit (Roche Diagnostics) according to the supplier's protocol. The obtained PCR products were cloned into the pCR-Blunt II-TOPO vector (Invitrogen, Karlsruhe, Germany) and transferred into E. coli DH10B cells [40]. At least four different clones per gene were selected for plasmid preparation and DNA sequencing (IIT Biotech, Bielefeld, Germany).
Real-time monitoring of cell growth using nephelometry
All strains to be tested (the wild-type strain and different mutants) were grown overnight in liquid BH medium in an Innova 4430 orbital shaker (New Brunswick, NJ) at 300 rpm. One ml of an o/n culture was pelletised, washed once with liquid MMES and transferred into a 100 ml Erlenmeyer flask containing 10 ml fresh, preheated MMES with addition of 500 μM L-cysteine as sulphur source. After o/n incubation in an orbital shaker at 300 rpm, one ml of the culture was again washed and transferred into 10 ml MMES as described above. These cultures were incubated for 6 hours in an orbital shaker at 300 rpm.
For real-time growth monitoring, 1 ml of each culture was pelletised and washed twice with MMES. For each test condition, 1 ml MMES with addition of a sulphur source at varying concentrations was inoculated with washed cells of the strain to be tested for a final o.D.600 of 0.01. Per test condition, 6 wells of a 96-well Cellstar suspension culture plate (Greiner Bio-One, Essen, Germany) were filled with 100 μl inoculated MMES per well. The plates were sealed with Breathe-Easy membrane (Diversified Biotech, Boston, MA) and growth was measured in a microplate nephelometer (BMG Lab Technologies, Offenburg, Germany). For these measurements, the parameters selected were: Incubation at 30°C, 50% laser intensity, gain of 86, 2.5 mm laser focus point, 900 sec orbital shaking with 3 mm shaking width, and measurement after each cycle with 0.2 sec measurement time per well, 0.0 sec positioning delay.
GenBank/TrEMBL accession numbers
All protein sequences from other organisms were obtained from the UniProt database [41]. The nucleotide sequences of all genes from C. glutamicum mentioned can be found via the genome entry (accession number [EMBL:BX927147]) with the ORF name as locus-tag. The amino acid sequences of the corresponding proteins are available under the following accession numbers: Cg3049 [EMBL:CAF20776], Cg3112 [EMBL:CAF20834], Cg3113 [EMBL:CAF20835], Cg3114 [EMBL:CAF20836], Cg3115 [EMBL:CAF20837], Cg3116 [EMBL:CAF20838], Cg3117 [EMBL:CAF20839], Cg3118 [EMBL:CAF20840], and Cg3119 [EMBL:CAF20841]. The accession numbers of the genome sequences used for comparative genomics are [EMBL:AE014295] (Bifidobacterium longum), [EMBL:BX248353] (C. diphtheriae), [EMBL:BA000035] (C. efficiens), [EMBL: AE016822] (Leifsonia xyli), [EMBL:AE016958] (Mycobacterium avium), [EMBL:AL450380] (M. leprae), [EMBL:AL123456] (M. tuberculosis), [EMBL:AP006618] (Nocardia farcinica), [EMBL:AE017283] (Propionibacterium acnes), [EMBL:BA000030] (Streptomyces avermitilis), [EMBL:AL645882] (S. coelicolor), and [EMBL:BX072543] (Tropheryma whipplei).
Bioinformatic analysis
Sequence similarity-based searches with nucleotide and protein sequences were performed using BLAST, the Basic Local Alignment Search Tool [42] against the UniProt database [41]. Searches using profile Hidden Markov Models (HMMs) from the PFAM database [43] were done using the HMMer software package. Transmembrane domains were searched for using the TMHMM software [44] while for the detection of signal peptides SignalP [45] was used. Multiple alignments were done using DIALIGN 2 [46]. Search for conserved gene clusters was performed using the GECKO software [19] and TheSEED [20].
Authors' contributions
CR performed the bioinformatic analyses and carried out the mutational and transcriptional studies. DJK aided the mutational analysis and performed additional growth tests. DAR carried out transcriptional studies. AA performed the mapping of transcriptional start points. SM constructed the transposon library and identified a suitable mutant. AP aided in coordination and conceived of the design of tables and figures. JK conceived and coordinated this study. All authors read and approved of the final manuscript.
Supplementary Material
Additional File 1
Supplementary Table S1.eps Supplementary Table S1 Oligonucleotides used in this study.
Click here for file
Acknowledgements
CR and DJK acknowledge a grant of the International NRW Graduate School in Bioinformatics and Genome Research. This work was further supported by Degussa AG, Germany. Sequence data for B. linens and T. fusca were produced by the US Department of Energy Joint Genome Institute .
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1271616476010.1186/1471-2164-6-127Research ArticleComparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi Parsons Marilyn [email protected] Elizabeth A [email protected] Pauline N [email protected] Jeremy C [email protected] Seattle Biomedical Research Institute, 307 Westlake Ave. N., Seattle, WA, 98109 USA2 Department of Pathobiology, University of Washington, Seattle, WA, 98195 USA3 Wellcome Centre for Molecular Parasitology, The Anderson College, University of Glasgow, Glasgow G11 6NU, UK2005 15 9 2005 6 127 127 15 7 2005 15 9 2005 Copyright © 2005 Parsons et al; licensee BioMed Central Ltd.2005Parsons 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 trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi cause some of the most debilitating diseases of humankind: cutaneous leishmaniasis, African sleeping sickness, and Chagas disease. These protozoa possess complex life cycles that involve development in mammalian and insect hosts, and a tightly coordinated cell cycle ensures propagation of the highly polarized cells. However, the ways in which the parasites respond to their environment and coordinate intracellular processes are poorly understood. As a part of an effort to understand parasite signaling functions, we report the results of a genome-wide analysis of protein kinases (PKs) of these three trypanosomatids.
Results
Bioinformatic searches of the trypanosomatid genomes for eukaryotic PKs (ePKs) and atypical PKs (aPKs) revealed a total of 176 PKs in T. brucei, 190 in T. cruzi and 199 in L. major, most of which are orthologous across the three species. This is approximately 30% of the number in the human host and double that of the malaria parasite, Plasmodium falciparum. The representation of various groups of ePKs differs significantly as compared to humans: trypanosomatids lack receptor-linked tyrosine and tyrosine kinase-like kinases, although they do possess dual-specificity kinases. A relative expansion of the CMGC, STE and NEK groups has occurred. A large number of unique ePKs show no strong affinity to any known group. The trypanosomatids possess few ePKs with predicted transmembrane domains, suggesting that receptor ePKs are rare. Accessory Pfam domains, which are frequently present in human ePKs, are uncommon in trypanosomatid ePKs.
Conclusion
Trypanosomatids possess a large set of PKs, comprising approximately 2% of each genome, suggesting a key role for phosphorylation in parasite biology. Whilst it was possible to place most of the trypanosomatid ePKs into the seven established groups using bioinformatic analyses, it has not been possible to ascribe function based solely on sequence similarity. Hence the connection of stimuli to protein phosphorylation networks remains enigmatic. The presence of numerous PKs with significant sequence similarity to known drug targets, as well as a large number of unusual kinases that might represent novel targets, strongly argue for functional analysis of these molecules.
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Background
Trypanosomatid pathogens of humans include Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, causative agents of African sleeping sickness, Chagas disease, and cutaneous leishmaniasis respectively [1]. Trypanosoma brucei lives extracellularly in the human host, primarily in the bloodstream and cerebrospinal fluid. African sleeping sickness, which is estimated to afflict 300,000–500,000 people per year in sub-Saharan Africa, with a disease burden of 1.6 million disability adjusted life years (DALYs), is invariably fatal unless treated [2]. Trypanosoma cruzi, which is found in Latin America, results in a disease burden of 650,000 DALYs. This parasite can invade most types of nucleated cells. About 30% of infected individuals progress to a chronic phase that culminates in heart disease and mega syndrome [3]. Of those infected it is estimated the 50,000 will die each year. Leishmania parasites result in a disease burden of 2.3 million DALYs, with greater than 80,000 deaths/year and cause a variety of diseases depending on the infecting species. The most dangerous manifestation is the visceral disease known as kala azar, caused by L. donovani. Kala azar is re-emerging in India in a particularly aggressive form that is resistant to standard treatment [4]. No vaccine has been approved for any of these diseases and many of the drugs in use are highly toxic and prone to the development of drug resistance. There is therefore an urgent need to identify new drug targets and the recent completion of the genome sequence of the three model trypanosomatids, T. brucei, T. cruzi and L. major, can be exploited in this regard [5-7].
During development the parasites pass through different environments. Each species is carried by a different insect vector, in which the parasite undergoes specific developmental changes that allow it to infect the human host. For example, Leishmania parasites move from the sandfly midgut up to the mouthparts, then into the human host where they invade macrophages and live within a phagolysosome. In each environment, the parasites respond with significant changes in their metabolic and protein profile. The signal transduction pathways mediating these changes remain unknown. Only a few receptor-like proteins have been identified, primarily receptor adenylate cyclases with an extracellular putative ligand binding domain and an intracellular catalytic domain [8,9]. Intermediate steps of signal transduction in the parasites have not been defined, although genomic analysis shows that they possess numerous molecules predicted to bind second messengers, as well as protein kinases and phosphatases [5]. The culmination of the signaling pathways is unlikely to be at the level of transcription, since most genes are transcribed in polycistronic units with little evidence for regulation [7,10,11]. Many changes in protein phosphorylation during the parasite developmental cycles have been documented [12-14]. The parasites also possess an integrated cell cycle that coordinates the inheritance of the single mitochondrion, flagellum, and nucleus [15,16].
Protein kinases (PKs) are key mediators of signal transduction, transmitting environmental cues and coordinating intracellular processes. Eukaryotic protein kinases (ePKs) are categorized by the amino acid sequence of their catalytic domains. Broadly, ePKs fall into two superfamilies: protein serine/threonine kinases and protein tyrosine kinases. The former are ubiquitous in eukaryotes. The latter are present in all metazoa for which the genome sequence is available, but relatively few examples have been found in unicellular eukaryotes [17,18]. However, protein tyrosine phosphorylation has been well documented in trypanosomatids [13,14,19,20]. Mammalian receptor protein kinases are generally tyrosine kinases [21], while all known plant receptor kinases are serine/threonine kinases [22,23]. Receptor kinases are activated by ligands, facilitating intercellular communication within multicellular organisms. Parasites that live in multicellular hosts could conceivably use similar mechanisms to respond to host or parasite ligands, although such reactions have not been defined at the molecular level.
Six major groups of ePKs have been defined on the basis of sequence similarity of the catalytic domains: AGC, CAMK, CMGC, TK, TKL, STE [24]. ePKs that do not fall into these groups are categorized as "Other". Within each group (including "Other"), multiple families have been defined. Interestingly, the substrate preferences break into groups along the same lines: for example AGC and CAMK kinases tend to phosphorylate motifs containing basic residues, CMGC kinases often are proline-directed, while CK1 and CK2 kinases phosphorylate motifs with acidic residues [24]. Additional features that correlate with group assignments include responses to other mediators such as ligands (receptor TK), calcium (CAMK), and certain second messengers (AGC). Relatively few protein kinases have been studied in detail with respect to expression and function in each of the trypanosomatids (see Additional file 1).
Atypical PKs (aPKs) are not closely related to ePKs at the sequence level, lacking the 11 subdomains that define ePKs. They include a variety of molecules that have been shown to have protein kinase catalytic activity in specific systems. Among the aPKs, the most well-characterized are the PIKK kinases, which have catalytic domains resembling those of lipid kinases in sequence [25]. Interestingly, the RIO and alpha groups show remnants of many of the ePK subdomain motifs [26,27]. The other atypical kinases require further study for definitive analysis of their activity.
An analysis of partial genomic sequence suggested that trypanosomatids might differ considerably from the host in signaling mechanisms, lacking typical signaling receptors with the exception of adenylate cyclases, as well as SH2 domains and transcription factors [28]. These speculations have been borne out by the completed genomic sequences of the three trypanosomatids known as the TriTryps: T. brucei [6], T. cruzi [5], and L. major [7], as briefly discussed in the T. cruzi genome paper [5]. In this report, we present a detailed examination of the TriTryp kinome.
Results and discussion
The TriTryp kinome
To identify all protein kinase genes in the three trypanosomatid genomes, we searched GeneDB [29] for all genes bearing Pfam protein kinase domains, as well as by BLAST using representatives of all major protein kinase gene families, including aPKs. All ePKs were examined for the presence of the 11 characterized subdomains, and specifically for the presence of the key lysine in subdomain 2 and aspartic acids in subdomains 6 and 7. The genomic analysis revealed 179, 156, and 171 ePKs and 17, 20, 19 aPKs in L. major, T. brucei and T. cruzi respectively. These numbers suggest that phosphorylation is an important mechanism for cellular regulation in all three trypanosomatids and are considerably larger than that described for another intracellular parasite that transits diverse environments; Plasmodium falciparum. P. falciparum possesses 65 ePKs and 20 ePK-related sequences, designated FIKK [30-32]. The latter have not yet been shown to have protein kinase activity [30]. The activation of many ePKs requires phosphorylation in the activation loop between subdomains 7 and 8. These kinases are typically marked by an RD motif within subdomain 6 [33]. In T. brucei, 130 of the 156 ePKs are RD kinases, further supporting the concept that phosphorylation networks are complex and important in these organisms.
We examined the relationship of the trypanosomatid ePKs to the groups and families of kinase domains of human, worm, fly, and yeast using the available datasets [34]. Most ePKs had a highly significant BLAST score against at least one member of the 4-kinome dataset. For example, 58% of the T. cruzi ePKs had an E-value of at least 10-40, and 77% had a score of at least 10-30 against a member of this dataset (Figure 1). Based on BLAST E-values, as well as phylogenetic inference (see below), assignments to ePK groups and families were made (Table 1, Additional file 1). We generated phylogenetic trees of the kinase domains of the entire T. brucei ePK kinome, seeding the tree with human and yeast PKs to facilitate classification (Figure 2 shows the MRBAYES tree). The trees were generally consistent with the BLAST assignments (the few that did not match are marked by an asterisk in the tree). Of note are several unique kinases that are on long branches originating near the center of the tree, indicating their high divergence from other ePKs.
Figure 1 Similarity of T. cruzi ePKs to those in the 4-kinome dataset. Full-length proteins were tested by BLAST analysis against the database of catalytic domains of all human, Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans protein kinases. The best E-value is graphed for each kinase, which are clustered according to their classification group. Non-cat: protein kinases predicted to be non-catalytic due to lack of subdomain I or catalytic residues. Different colors were used to facilitate viewing of closely spaced spots.
Table 1 Groups and families of ePKs in the TriTryps
Group Family T. brucei T. cruzia L. major Group Family T. brucei T. cruzi L. major
AGC (na)b 6 7 6 Other AUR 3 3 3
NDR 2 1 1 CAMKK 4 4 4
PKA 3 3 3 CK2 2 2 2
RSK 1 1 1 NAK 0 1 1
Total 12 12 11 NEK 20 22 22
CAMK (na) 7 7 9 PEK 3 2 3
CAMKL 7 6 7 PLK 2 2 1
total 14 13 16 TLK 2 1 1
CK1 CK1 4 7 6 ULK 1 1 1
TTBK 1 1 1 VPS 1 1 0
Total 5 8 7 WEE 1 2 2
CMGC (na) 1 1 1 total 39 42 40
CDK 11 10 11 STE (na) 7 8 10
CLK 4 5 4 STE7 2 2 2
DYRK 7 7 8 STE11 14 18 21
GSK 2 2 2 STE20 2 3 1
CDKL (MAPK-like)c 2 1 2 total 25 31 34
MAPKd 10 11 12 Unique 19 23 26
RCK (MAPK-like)c 3 3 3 TOTAL ePK 156 171 179
SRPK 2 2 2
total 42 42 45 Non-catalytic 13 16 16
a Multiple copies of TcCRK7 were counted as one gene. The number of tandem repeated CK1 genes in T. cruzi is not clear. One putative T. cruzi PEK gene is truncated by the contig end for both alleles, and is assigned on the basis of orthology in the non-catalytic sequence.
bna, not assigned to a family.
c CDKL and RCK families are related to MAPKs and CDKs [57] but are classified separately in the human kinome [21].
d One MAPK-like sequence in T. brucei and two in T. cruzi and L. major lack the typical regulatory motifs (see Table S1). One MAPK-like COG, which fell below the E-value criterion, was designated as MAPK on the basis of regulatory motifs.
Figure 2 Unrooted tree of T. brucei protein kinases. The catalytic domains of predicted functional ePKs were analyzed using MRBAYES. Bootstrap values greater than 0.95 are indicated by a dot at the node, and selected lower values are shown. No members clustered with TK or TKL kinases from human or yeast (not shown on the tree). T. brucei sequences are identified by systematic gene IDs. Selected human (Hs), S. cerevisiae (Sc) and D. melanogaster (Dm) were included to provide landmarks and these are shown in red font. Asterisks mark sequences for which the MRBAYES tree conflicted with the BLAST analysis at the group level. Kinases classified as unique through BLAST are marked with "U".
In general, the TriTryp kinomes are closely related. COGS are clusters of orthologous genes, as revealed by analysis of mutual BLASTP hits across the genomes. The majority (68%) of the ePK genes reside in COGS that contain members from each of the three species (Additional file 1). Conversely, only a small number of genes appear to be unique to a species (20 in L. major, 11 in T. cruzi, and 3 in T. brucei, Additional file 1). As with other genes in these organisms, members of these COGs are generally syntenic among the three species, furthering the concept that the molecules are orthologous. Figure 3 compares the representation in the major groups and families of human protein kinases with those of L. major and Table 1 shows the representation of groups and families among the TriTryps, with further details including systematic gene names provided in Additional file 1.
Figure 3 Comparison of L. major and human ePK classification.
Protein tyrosine kinases
A key difference between host and parasite kinomes is the complete lack of ePKs that map to the tyrosine kinase (TK) and tyrosine kinase-like (TKL) groups in the trypanosomatids. Representatives of the former in humans include receptor protein kinases such as the insulin receptor and cytosolic kinases such as src. The latter group contains ePKs such as RAF1 and TGFβR2. We also found no evidence of the receptor guanylyl cyclase (RGC) group of proteins, which are structurally related to protein kinases. These groups of ePKs are also absent in malaria parasites, which further lack the STE group of kinases. Interestingly, it has been reported recently that the genome of the unicellular protist Entamoeba histolytica encodes TKs with SH2 domains, TKLs, and a large family of putative receptor serine-threonine ePKs [18]. As E. histolytica also possesses genes encoding putative 7 transmembrane receptors and heterotrimeric G proteins, which the trypanosomatids lack [5], the mechanisms regulating cell signaling appear to be very different among the parasitic protozoa.
As noted above, phosphorylation on tyrosine is well documented in trypanosomatids. We propose that this activity is likely to be due to the action of atypical tyrosine kinases such as Wee1 and dual-specificity kinases that can phosphorylate serine, threonine, and tyrosine. Multiple members of the dual specificity kinase families (DYRKs, CLKs, and STE7) are present in the trypanosomatid genomes. Although Wee1 is functionally a tyrosine kinase, it most closely resembles serine/threonine kinases such as Chk1 and cAMP-dependent kinases in structure and primary amino acid sequence [35]. In yeast and higher eukaryotes Wee1 phosphorylates a conserved tyrosine residue in the ATP binding pocket of CDK1 (cdc2), inactivating the protein kinase. This mechanism is likely to be conserved in the three trypanosomatids, since there are two Wee1 family members in L. major and T. cruzi and one in T. brucei. In addition, CRK3, the putative functional CDK1 homologue in trypanosomatids [36-38], contains a conserved tyrosine residue in the same subdomain as the human CDK1 regulatory tyrosine [39,40]. In T. brucei, 18 other CMGC members also have this tyrosine within subdomain 1 (Additional file 2), reiterating the potential for widespread regulation of protein kinase activity via tyrosine phosphorylation. The potential conservation in regulatory mechanisms for CDK activity between yeast, mammals and trypanosomatids may not extend to all protozoa, as the putative P. falciparum Wee1 lacks a key active site residue suggesting it may not be active [31] and dual-specificity ePKs appear to be absent. In addition, no tyrosine phosphorylation has been demonstrated to date in that species. The existence of other unusual protein tyrosine kinases in trypanosomatids is an intriguing possibility given the large number of protein kinases in the trypanosomatid kinomes that cannot be easily placed into typical ePK groups or families (see below).
Serine-threonine protein kinases
Poorly represented groups: CAMK and AGC
The CAMK and AGC groups are relatively poorly represented within trypanosomatid genomes as compared to humans. The CAMK group (which includes the Ca+2/calmodulin regulated kinases and AMP-dependent kinase, AMPK) is small in trypanosomatids with only 13 CAMKs predicted to be active in T. cruzi, 14 in T. brucei, and 16 in L. major. In contrast, the human genome encodes 74 CAMKs. A phylogenetic tree of the trypanosomatid CAMK and CAMK-like unique kinase domains is shown in Figure 4. Also included are along with representatives of each family of CAMKs from human, two yeast CAMKs and a plasmodial calcium dependent CAMK. Of the 19 trypanosomatid CAMK genes identified, 13 have representatives in each species as determined by COG analysis, and supported by phylogenetic trees. An additional CAMK-like kinase, marked as unique due to its low E-value in BLAST analysis, was also conserved, as was a COG in which two of the three orthologues were predicted to be inactive. The tree shown in Figure 4 also shows a characteristic common to all trees in which groups of ePKs from the trypanosomatids were compared: the trypanosomatid ePKs falling within COGS formed a tight cluster with high confidence, and were more distantly related to ePKs from humans or yeast.
Figure 4 Phylogram of CAMK ePKs. Kinase domains from the TriTryp predicted proteins classified as CAMKs by BLAST or CAMK-like from the T. brucei tree in Figure 2 were analyzed using MRBAYES. Also included are CAMKs from all families present in humans (Hs), plus several S. cerevisiae (Sc) and a P. falciparum (Pf) kinase. Nodes with a bootstrap value greater than 0.95 are marked by a dot, while values ranging from 0.7–0.95 are indicated numerically. Similar results were obtained with PAUP*. Kinase domains are indicated by systematic gene IDs, which are abbreviated in the case of L. major to Lm in lieu of LmjF. Additionally, the invariant digits in the T. cruzi systematic names were deleted (all gene names start with Tc00.1047053). Only one T. cruzi allele was included for each gene. ψ marks a T. cruzi and T. brucei gene that are predicted to be non-functional due to a lack of a recognizable subdomain 1. The CAMK-like kinases classified as unique by BLAST are marked, as are the trypanosomatid CAMKs with EF-hand accessory domains. ScCDC28, a CMGC kinase, was used as an outgroup.
BLAST analysis against the 4-kinome dataset indicated that about half of the trypanosomatid CAMKs belong to the CAMKL subfamily, the remainder were not assigned to a specific family. The phylogenetic trees generally agreed with these predictions, and supported the further classification of two sets of trypanosomatid genes as members of the AMPK subfamily. In other organisms, AMPKs are regulated by AMP and hence are involved in metabolic sensing [41]. In addition, two sets of the predicted CAMK kinases contain EF hand sequences, which may provide for sensitivity to Ca+2 (marked in Figure 4). This juxtaposition of a protein kinase domain with EF hand motifs is characteristic of CDPKs, a group of calcium dependent protein kinases that are prominent in plants [42] and in P. falciparum [30,31,43], but which are absent in humans and yeast. However, the phylogenetic inference does not support the clustering of these trypanosomatid CAMKs with the plasmodial calcium dependent kinase CDPK1. These trypanosomatid genes are likely therefore to encode a novel class of EF-hand containing ePKs.
The AGC group includes ePKs structurally related to protein kinases that respond to second messengers: protein kinase A (responsive to cAMP), protein kinase G (responsive to cGMP), and protein kinase C (responsive to diacyl glycerol). Normalized to kinome size, trypanosomatids have approximately half as many AGC kinases as humans. The parasite genomes encode 3 AGC kinases that are related to PKA. However, T. brucei PKA appears to be activated by cGMP rather than cAMP [44]. Also within the AGC group are the NDR kinases. BLAST analysis and phylogenetic tree inference indicates that T. brucei possesses two NDR family kinases. One of these is conserved and syntenic among the trypanosomatids, while the other, PK50 (Tb10.70.2260), is specific to T. brucei. This molecule is a functional homologue of Schizosaccharomyces pombe Orb6 [45] and interacts with MOB1 to form an active kinase complex that has a potential role in cytokinesis, but not mitosis [46]. Whether the conserved NDR kinases also interact with MOB1 is not yet known. The remainder of the AGC kinases could not be assigned to a specific family by sequence alone, except for one RSK-like sequence. Phylogenetic inference of the T. brucei sequences carried out as detailed in Methods supports these general conclusions and provided no indication of trypanosomatid-specific clusters (data not shown).
Over-represented groups: CMGC and STE
The CMGC group and the STE group are relatively well represented within these trypanosomatid genomes as compared to humans. Examples of CMGC kinases include ePKs such as cyclin-dependent kinases (CDKs), MAP kinases (MAPKs), and dual specificity CLK and DYRK kinases. Trypanosomatids have a large number of these kinases (e.g., 45 in L. major as compared to 61 in humans). All of the CMGC families identified in humans are also represented in trypanosomatids, as indicated by BLAST analysis and phylogenetic inference. The CDK family is relatively large in trypanosomatids with 11 members in T. brucei and L. major and 10 in T. cruzi. This complexity may reflect the problem of dividing a highly polarized cell with an elaborate cytoskeleton and a single mitochondrion, along with an integral link between cell cycle control and life cycle differentiation. Despite the existence of a large number of CDK family members (named CRK for cdc2-related kinase), only 2 have been shown to be essential for cell cycle progression in trypanosomatids. CRK3 in complex with the CYC6 mitotic cyclin is essential for G2/M phase progression and is the functional homologue of CDK1 [36-38,47]. CRK3 in complex with CYC2 is essential for G1 progression [48,49]. A PHO80-like cyclin and a B-type cyclin control the cell cycle of the procyclic form of Trypanosoma brucei [50], while TbCRK1 is also an essential gene required for G1 phase progression [38,47,51,52]. However, the roles of CRKs in the cell cycle is complex, with functional differences between bloodstream and procyclic form T. brucei as revealed by RNAi knockdown studies [37,38,47,48]. CRK7 has the highest level of sequence identity to CDK7 of mammals. CDK7, in complex with cyclin H and MAT1, is a CDK-activating kinase (CAK) that phosphorylates the T-residue of CDKs (e.g., T160 of human CDK1). No cyclin H or MAT1 orthologues can be identified in trypanosomatids based on sequence, so it remains to be determined if CRK7 is a functional cyclin-dependent kinase or indeed if it has CAK activity. However, many CRKs, including CRK1, 2, 3, 6, 7, 8, 9 and 12, have a conserved T-loop residue, suggesting that the CRKs might be activated in vivo by a CAK activity [53].
Interestingly, T. cruzi possesses a large number of genes encoding CRK7 isoforms (counted only as one unique gene in our analyses). These are dispersed near the telomeres of many chromosomes, being adjacent to a retrotransposon hotspot protein gene. Of the 27 sequences identified, most contain all of the catalytic residues, although a few are truncated. The biological significance of this gene amplification is not known, however expansion of gene families within subtelomeric regions of trypanosomatid chromosomes is a feature of these genomes in general.
Two families of CMGC kinases phosphorylate serine/arginine rich motifs in serine-arginine rich SR proteins, which function in RNA processing and splicing in many higher organisms: SRPKs [54] and the dual specificity CLKs [55]. Two SRPKs [54] and four or five CLKs are encoded within each trypanosomatid genome. Given the major role of RNA processing and turnover in modulating gene expression in trypanosomatids, these families of CMGC kinases may be of key interest in studying parasite gene regulation. Two GSKs, which are drug targets in diabetes and neurological diseases [56], are also present.
A large number of MAPK-related genes are also present in trypanosomatids, possibly reflecting a role of 3-component MAP kinase cascades in coordinating responses to environmental cues (Table 1). Among these genes are those which are most closely related in sequence to the MAPK family, and those which are most closely related to the CDKL and RCK families. These latter families possess the residues characteristic for the regulation of MAPKs and hence are considered to be part of a MAPK superfamily by some authors [57], even though they are more similar in sequence to CDKs. Among the identified MAPK-like predicted proteins, two sets lack the predicted regulatory motifs (LmjF13.0780 and its T. cruzi orthologue; LmjF03.210 and its T. brucei and T. cruzi orthologues, Table S1) and hence must be regulated in a distinct manner. Thus the total complement of protein kinases likely to be regulated as MAPKs numbers 14 in T. brucei, 13 in T. cruzi, and 15 in L. major.
The parasites clearly find themselves in environments that vary substantially in temperature, pH, nutrients, and stresses during their developmental cycle. An elaborate phosphorylation signaling system to respond to those changes may be a key strategy of this group of organisms. Many MAPKs, and those kinases likely to regulate them (see below), appear to be involved in developmentally regulated processes in trypanosomatids. Nine MAPKs have been cloned and analyzed from L. mexicana (LmxMPK1-9) and their mRNAs abundances are developmentally regulated [58,59]. LmxMPK1 is essential for amastigote, but not promastigote proliferation [59], while LmxMPK9 is involved in regulating flagellar length, a stage-regulated function in Leishmania [60]. Three MAPKs have been analyzed in T. brucei. KFR1, an ERK-like MAPK, has been proposed to be involved in the proliferation of bloodstream form trypanosomes and is the first trypanosomatid ePK reported to be regulated by a specific extracellular molecule, interferon γ [61,62]. TbMAPK2, also ERK-like, is not essential for proliferation of the bloodstream form trypanosome, but is important for successful differentiation [63]. Mutants lacking TbMAPK2 have delayed kinetics of differentiation from the bloodstream form to the procyclic form; the resulting procyclic forms undergo cell cycle arrest. TbECK1, which has characteristics of both MAPKs and CDKs, and was named T. brucei ERK-like, CDK-like protein kinase [64], falls into the CDKL family by phylogenetic analysis (this study). This kinase appears to be essential in all life cycle stages analyzed [64]. TbECK1 has an unusual C-terminal extension and overexpression of TbECK1 lacking the C-terminal extension in procyclic trypanosomes leads to a significant reduction in growth, suggesting an important role in cell cycle control. The C-terminal extension appears to act as a cis-acting negative regulator of protein kinase. The roles of many trypanosomatid MAPKs remain to be explored.
MAPKs are activated by phosphorylation within the activation loop, typically both on a tyrosine and a threonine. This phosphorylation is mediated by MAP kinase kinases (MAP2Ks), which are members of the STE7 family, one of the three major families of STE group kinases that are generally described as upstream regulators of MAP kinase cascades. Although only two STE7 genes were assigned through BLAST analysis, phylogenetic inference revealed that five sets of orthologues cluster with good confidence into the STE7 family (Figure 5), suggesting that they may function as MAP2Ks in this organism. A previously identified Leishmania mexicana MAP2K, LmxPK4, has a potential role in parasite differentiation [65], while another LmxMKK, is involved in the maintenance of flagellar length [66]. STE11 family ePKs often function as MAP3Ks and are especially numerous in the trypanosomatids. Several of the STE11 kinases formed trypanosomatid-specific clusters in our phylogenetic analyses. Another cluster was found to be ubiquitous amongst the trypanosomatid, yeast and human kinomes. LmMRK1 (LmjF32.0120) is an essential STE11 family kinase [67]. In contrast to STE11, the STE20 family kinases, many of which function as MAP4Ks, are relatively rare in trypanosomatids. Another arm of the MAPK activation pathways is mediated by RAF1, a TKL kinase. The TKL group of protein kinases is absent in trypanosomatids. It is clear that sequence data alone cannot accurately predict specific three-component signaling pathways in the trypanosomatids – detailed biochemical analyses will be required. Nonetheless, taken together, these findings provide interesting insight into trypanosomatid-specific aspects of MAP kinase cascades. The STE group of kinases is relatively expanded in trypanosomatids, with 34 members in L. major. In contrast, it is either absent or highly abbreviated in the malaria parasite [30,31], once again highlighting the differences amongst protozoan lineages.
Figure 5 Phylogram of STE kinases. Kinase domains from the TriTryp predicted proteins classified as STE by BLAST or STE-like from the T. brucei tree in Figure 2 were analyzed using MRBAYES. Also included are STEs from all families present in humans (Hs), plus examples from S. cerevisiae (Sc), C. elegans (Ce) and D. melanogaster (Dm). Nodes with a bootstrap value greater than 0.95 are marked by a dot, while values ranging from 0.7–0.95 are indicated numerically. Similar results were obtained with PAUP*. Gene names are shown as in Figure 4. ScTPK1, an AGC kinase, used as an outgroup.
Other serine/threonine kinases
The NEK family of ePKs shows a significant expansion within trypanosomatids, having 20–22 members (compared to the 15 representatives in the human genome). The NEK kinases have been relatively little studied in model systems, but several appear to be involved in cell cycle [68] and cytoskeletal functions [69,70]. Some of the NEK kinases appear to function in cascades, with human NEK9 phosphorylating and activating NEK6 and NEK7 [71]. Indeed, all of the T. brucei NEK kinases possess the RD motif in subdomain 6, which is an indicator that phosphorylation in the activation loop is likely to be required for maximal activity. As with most of the human NEK kinases, the catalytic domain is situated at or near the N-terminus of the T. brucei NEK kinases. Phylogenetic analysis of the 20 T. brucei NEK kinases shows that the parasite kinases do not form tight clusters with the NEK kinases represented in the 4-kinome database nor with the NEK kinases of the protozoan P. falciparum (Figure 6), although in two of three phylogenetic methods implemented (MRBAYES and PHYML Likelihood), one of the T. brucei NEK kinases (Tb06.2N9.460) did cluster with a plasmodial kinase (MAL6P1.56). In contrast, several clusters of NEK kinases across yeast and metazoa were identified: e.g., ScKIN3, DmNEK2 and HsNEK2 form a highly supported clade, as do HsNEK10 and CePQN25. Of particular interest to us was the identification of a trypanosomatid-specific clade containing 12 of the T. brucei NEK kinases, which was supported by all of the methodologies. The trypanosomatid NEK kinases have perhaps a modestly higher preponderance of accessory domains compared to other trypanosomatid kinases (see below). For example, several possess a coiled coil region downstream of the catalytic domain (Tb03.27C5.650, Tb05.26K5.430, Tb10.61.2330, and Tb10.70.7860). This feature is also found in human NEK1 and NEK2. Several other trypanosomatid NEK kinases have a C-terminal PH domain, a combination not described in the NEK kinases of other species. These kinases lie within the trypanosomatid-specific clade. The roles of the trypanosomatid NEK kinases have not been studied in any detail, although at least one is known to be developmentally regulated [72] and one has a role in basal body duplication (D. Robinson, personal communication).
Figure 6 Phylogram of NEK kinases. NEK sequences from human (Hs), S. cerevisiae (Sc), C. elegans (Ce), D. melanogaster (Dm), P. falciparum (Pf) and T. brucei (Tb) were analyzed using MRBAYES. TbCK2A1 was used as an outgroup. Nodes with a bootstrap value greater than 0.95 are marked by a dot, values ranging from 0.7–0.95 are indicated numerically. Similar results were obtained with PAUP*. Two NEK kinases had recognizable PH domains that did not achieve the Pfam HMM search cutoff (Tb04.24M18.60, and Tb08.10K10.710).
Among the families of "Other" protein kinases represented in trypanosomatids, several have been shown to be involved in cell division in various organisms (AUR, Aurora; PLK, polo-like kinases; Wee1) DNA replication/repair (TLK, also ATM/ATR atypical kinases described below), and stress responses (PEK family). Activators of CAMKs (CAMK kinases, CAMKK) are also present, as are multiple CK1 and CK2 isoforms (formerly known as casein kinase I and II) [73,74]. A member of the VPS15 family (involved in vacuolar protein sorting), was also identified, although the Leishmania orthologue may not be catalytically active. One representative of the ULK family kinases was found in each trypanosomatid. ULK kinases are involved in autophagy in yeast [75] and in pattern formation and development in multicellular organisms [76].
A significant number of ePKs were classified as unique, as they showed no clear affinity to any known group or family within the 4-kinome dataset. For example, a number of T. cruzi ePKs which had significant matches to the protein kinase Pfam domain signature (Pfam 00069) did not show any distinct similarity to specific kinases in the 4-kinome dataset. Of this group, half had E-values of 10-35 or better against the Pfam domain (Figure 7). On the other hand, approximately one-third of the T. cruzi unique kinases showed relatively poor matches against the Pfam domain (E-values ≤ 10-16), but nonetheless were observed to possess a complete subdomain structure as well as the required catalytic residues. The ePKs classified as unique were the least conserved among the trypanosomatids, with 63% being absent in at least one of the three species. As such, the unique kinases are likely to represent instances of lineage-specific evolution defined by gene gain and/or loss in these organisms. Such divergent kinases may provide a set of useful protein kinase drug targets, since they have no closely related homologues in the host.
Figure 7 T. cruzi unique ePKs: similarity to other ePKs and Pfam kinase domain. The E-values for the relationship of individual T. cruzi ePKs as compared to the 4-kinome dataset and to the Pfam domain. Many of the T. cruzi ePKs show strong E-values against the Pfam kinase domain, despite their low similarity to the kinases in human, D. melanogaster a, C. elegans, and S. cerevisiae.
Membrane kinases interfacing with the environment?
Most mammalian receptor kinases belong to the tyrosine kinase group, a group which is lacking in trypanosomatids. However, in plants, most receptor kinases are serine/threonine kinases. Bearing this in mind, we searched the T. brucei genome for genes bearing the protein kinase Pfam domain plus the annotation of a transmembrane domain. Ten candidates fit the criteria (see Additional file 3), these were spread among a variety of ePK groups, with a somewhat higher representation among the STE kinases. At this juncture, there is no evidence that any domain of these molecules is displayed on the parasite surface, where it might respond to host or parasite derived ligands. Alternatively, if surface-localized, the kinase could phosphorylate host or parasite molecules to modify their environment. We note with interest previous reports of an ectokinase with a substrate profile characteristic of CK1 in Leishmania [77,78]. Intriguingly, one of the L. major CK1 genes identified in this analysis encodes a protein with a predicted signal anchor sequence (LmjF17.1780). Assessing whether any parasite protein kinases interface with the host environment is an important arena for future experimental studies.
Inactive protein kinases
Approximately 8% of the ePKs of each species are predicted to be catalytically inactive, based on the presence of mutations in essential residues (K in subdomain 2 and D in subdomains 6 and 7). Most of these possess an orthologue in at least one other trypanosomatids. Of the 13 T. brucei ePKs predicted to be catalytically inactive, 11 are mutated to a predicted non-catalytic form in each of the three species. Genome-wide, the level of amino acid sequence identity among COG members averages 61 +/- 7% between T. brucei and T. cruzi [79], with a similar level of identity for a sampling of ePKs (60% +/- 7%). The ePKs predicted to be inactive show a lower level of identity at 44% +/- 8%. Hence, while conserved, these sequences are somewhat more divergent across species.
We also estimated the synonymous (Ks) and nonsynonymous nucleotide (Ka) nucleotide substitution rates in T. brucei versus T. cruzi genes encoding ePKs predicted to be catalytically active or inactive. The Ka/Ks ratio (sometimes designated as dN/dS) can reflect the selective constraints on a gene. Ka/Ks = 1 is expected for genes evolving neutrally. Ka/Ks < 1 is thought to indicate selection to remove amino acid replacements. In the rare cases where the Ka/Ks > 1, selection for amino acid divergence is usually invoked. For a random subset of 90 active ePKs, Ka = 0.336, Ks = 4.822 and Ka/Ks = 0.077. For the inactive ePKs, these figures were 0.535, 9.639, and 0.110, respectively. These data indicate that in both sets synonymous mutations are highly preferred. Nonetheless, the Ka and Ka/Ks were significantly different in the active versus inactive datasets (p = 0.0003 and p = 0.0045, Mann-Whitney U-test, two tailed). There was no statistically significant difference in the calculated Ks scores between these two datasets (p = 0.3). These findings suggest that the encoded proteins continue to play a significant functional role within the organisms, although the predicted lack of catalytic activity indicates this role is likely to be via a distinct mechanism, such as regulation via protein-protein interaction. Indeed, a recent analysis has shown that inactive protein kinases are not an exception in metazoa and that a few have evolved novel functions, some of which might be involved in processes that enhance the complexity of regulatory phosphorylation networks [80].
Accessory domains
A characteristic of human ePKs is the presence of accessory domains. Indeed, over 50% of human protein kinases have additional Pfam domains, and more are found when criteria are relaxed [21]. We examined all of the trypanosomatid ePKs for significant matches to additional Pfam domains (Table 2). In the case of L. major, only 25 ePKs possessed additional Pfam domains that met the default cutoff. Three additional ePKs, which had orthologues in T. brucei or T. cruzi that had a significant Pfam domain, were found to possess partial motifs, and others had domains of lower significance. The accessory domains were generally conserved among members of a COG. Notably four of the five most common Pfam domains on human ePKs are absent in the trypanosomatid kinome: Ig, fn3, SH2, and SH3. Ig and fn3 domains are generally extracellular domains that interact with ligands, so their absence may not be surprising given the paucity (or absence) of receptor ePKs in trypanosomatids. SH2 domains interact with phosphotyrosine, and their absence in the trypanosomatid genomes could suggest a co-evolution with dedicated tyrosine kinases. SH3 domains bind to proline rich sequences.
Table 2 Additional Pfam domains on trypanosomatid ePKs
Pfam Tba Tc Lm TriTrypb Other kinomesb Comments
Armadillo 0 1 1 ULK NEK, STE11, ULK related to HEAT
B-box Zn finger 0 0 1 NEK nd Zn binding
C1-like 0 1 1 AGC AGC possible diacylglyerol binding
cap-gly 1 1 1 CAMK VPS15 cytoskeleton associated
cNMP binding 0 1 2 STE AGC cyclic nucleotide binding
EF Hand 2 2 2 CAMK CAMK Calcium binding
FHA 1 2 4 CAMK, STE CAMK phosphopeptide binding
FYVE 1 2 1 AGC nd Zn binding
HAMP 0 1 0 STE nd in diverse signaling proteins
HEAT 1 1 0 ULK VPS15 protein interaction
HECT 0 0 1 unique nd ubiquitin transferase
Kelch 1 0 0 unique nd propeller structure
LRR 1 1 1 CAMKK TK, TKL, plantrk protein interaction
MORN 1 1 1 STE TKL, unique unknown function
PAS 1 1 1 STE CAMK, TKL signal sensor
PH 6 7 6 AGC, NEK AGC, CAMK, STE20 Phosphatidylinositol binding
PKC-Cterm 0 0 2 AGC AGC found on protein kinases
POLO 1 1 1 POLO POLO POLO kinase region
PX 1 1 1 AGC AGC phosphoinositide binding
RWD 1 0 0 PEK PEK unknown function
TPR2 0 2 1 STE, unique plantrk protein interaction
zf-C2H2 2 1 1 unique AUR zinc binding
a The number of genes bearing the indicated Pfam domain.
b Classification of ePKs bearing the designated domain in the TriTryps or other kinomes (as detected in S. cerevisiae, Schizosaccharomyces pombe, P. falciparum, C. elegans, D. melanogaster, A. thaliana, and Oryza sativa) using the web tools at the Kinases in Genomes (KING) website [81]. The plantrk group is comprised of serine/threonine receptor kinases present in plant; nd, none detected.
Several unusual domain combinations are found in trypanosomatid protein kinases (Table 2, examples shown in Figure 8). A search of eight eukaryotic kinomes using the KinG Kinases in Genomes Resource [81] revealed that some accessory domains found in trypanosomatids that were not associated with ePK catalytic domains in other species. For example, LmjF35.4000, which is a Leishmania-specific gene, contains a unqiue ePK catalytic domain, along with three TPR motifs (which are present on some plant receptor kinases) along with a domain associated with ubiquitin transferase (HECT), a domain not seen in the sampled genomes. A T. cruzi ePK, Tc00.1047053511727.210, has a TPR motif and HAMP domain (found on various signaling proteins including histidine kinases), both of which are recognizable on the T. brucei orthologue, but not on the L. major orthologue. Others domains are associated with ePKs of different classification. For example, the very large STE kinase LmjF15.1200, has an unusual juxtaposition of two domains related to cyclic nucleotide binding and a PAS domain, associated with signal sensing. In other species, the PAS domain is found on CAMK and TKL kinases, while the cNMP domain is restricted to AGC kinases. The L. major domain structure is likely conserved in T. cruzi, although the coding region is interrupted by a contig break. No orthologue is present in T. brucei. Some other accessory domains are found on similar groups of kinases (RWD, PX). Despite the paucity of identified domains, the trypanosomatid ePKs are generally considerably larger than the 250 aa kinase domain. For example, half of T. cruzi ePKs are larger than 64 kDa, and 38 are larger than 100 kDa.
Figure 8 Domain structure of Tritryp ePKs with unusual additional domains. Examples from Table 2 are shown. ePK domains are predicted to be active.
Atypical protein kinases
The parasites possess a complement of atypical protein kinases, including representatives of all of the more well-characterized families: RIO, alpha, PIKK and PDK (Figure 9 and Additional file 4), although no functional analyses have been carried out to date on any representative aPK from trypanosomatids. The RIO family of atypical kinases is related to ePKs, but RIO proteins lack the sequences known to be involved in peptide binding in ePKs [82]. Nonetheless, the catalytic residues are present. Trypanosomatids possess two RIO proteins, which are clearly assigned to the RIO1 and RIO2 subfamilies. In other organisms both RIO1 and RIO2 are required for ribosomal biogenesis, and RIO is involved in cell cycle progression [26]. Interestingly, the similarity between human and trypanosomatid RIO2 extends into the N-terminus, where the structure of the human enzyme shows a winged helix-turn-helix motif [82]. Such helix-turn-helix motifs are often found on DNA binding proteins such as transcription factors, a class of proteins which are rare in trypanosomatids.
Figure 9 Comparison of T. brucei and human atypical protein kinase classification. See Additional file 4 for systematic gene names.
The alpha kinases, so named because they phosphorylate their substrates within alpha helices, show a small amount of sequence similarity to ePKs, with conservation of the catalytic residues in subdomains 2, 6, and 7 [27]. One set of the alpha kinases in trypanosomatids is comprised of small molecules, being little more than the 241 aa alpha domain. This type of alpha kinase is present in all three species. However, L. major possesses two additional alpha kinase genes, which are very large (>1000 amino acids). Interestingly, three of the four L. major alpha kinases are found on a 12 kb segment on chromosome 36. None of the alpha kinases appear to be fused to an ion channel, as is the case for certain vertebrate alpha kinases [27].
The PIKK kinases represent a particularly interesting family in which the protein kinase domain structurally resembles that of phosphatidylinositol 3-kinases [25]. In addition to the kinase domain, these proteins also have FAT and FATC motifs which are not found in the lipid kinases. The similarity between the trypanosomatid PIKK kinases and those in the 4-kinome dataset is highly significant, with E-values of 10-90 or better. PIKK kinases are quite large in general and those in T. brucei are no exception, ranging in predicted size from 271 to 468 kDa. The parasites possess clear homologues to the specific PIKK kinases involved in genome surveillance: ATM and ATR [83]. They also have four kinases that belong to the FRAP family (this family includes FRAP and mTOR). TOR (target of the immunosuppressive agent rapamycin) modulates translation and cell cycle in response to nutrient and growth signals [84]. Multiple drugs targeting mTOR are in trials for the treatment of various cancers [84].
The trypanosomatids contain 3 genes encoding putative pyruvate dehydrogenase kinases (PDK). In mammals, the activity of mitochondrial pyruvate dehydrogenase is tightly regulated by multi-site serine phosphorylation of the E1α subunit [85]. Despite their exclusive phosphorylation of serine residues, the PDKs lack the domains characteristic of ePKs. Rather, these kinases have two distinct domains. A C-terminal domain that shares structural conservation with the GHKL ATPase/kinase superfamily (including members of the histidine kinase family) and an N-terminal domain that resembles a histidine phosphoryl transfer domain of bacterial two component systems [86]. The presence of an active pyruvate dehydrogenase in trypanosomatids with an E1α subunit suggests that regulation of activity by phosphorylation is likely to be conserved in these species.
Conclusion
The analysis presented here shows that trypanosomatids possess a large complement of protein kinases, indicating that protein phosphorylation is a key mechanism for regulation of parasite processes. In metazoa and yeast, the ultimate targets of many signaling cascades are transcription factors, which then trigger the expression of new sets of genes. In contrast, since trypanosomatids indiscriminately transcribe most genes in large polycistronic units, signaling cascades in these organisms must function in post-transcriptional regulation. Key regulators of specific mRNA turnover are still being sought, and we propose that protein kinases are major players in these processes. We also propose that trypanosomatids, more than many other organisms, rely on the phosphorylation of the downstream molecules that perform stage-specific and cell-cycle specific functions. Phosphorylation has been shown to modulate protein turnover, localization, interaction and activity for various molecules in eukaryotes. Both ePKs and aPKs are the targets of major drug discovery efforts in chronic human diseases [56,84,87,88]. Exploiting the knowledge and resources generated in those efforts could provide new answers in the search for new drugs to combat trypanosomatid diseases. A major effort to understand the functions of individual protein kinases will allow increased focus on key molecules. We suggest that PKs closely related to human drug targets would be a useful first set to be explored. However, perhaps just as useful could be the group of unique kinases, which show little resemblance to human PKs.
Methods
Classification
All ePKs were retrieved from GeneDB [29] through a combination of searches with the protein kinase Pfam domain, BLAST analysis using diverse ePKs, and examination of COGS. L. major (version 5.0, Feb 2005); T. brucei (version 4.0, Feb 2005) and T. cruzi (version 3.0, July 2004) were the final datasets used in this study. In the case of T. cruzi, in which the genome strain is a hybrid, the two presumed alleles were identified through analysis of COGs [79], and counted as one gene, even though up to 7% sequence allelic sequence divergence occurs in this strain [5]. All ePKs were examined for the presence of the 11-subdomain structure, and the presence of lysine in subdomain 2 and aspartic acid in subdomains 6 and 7, which are required for catalysis [24,89]. Those lacking these residues were categorized as catalytically inactive. Similarly, a few ePKs lacked any sequence resembling subdomain 1, which functions in ATP binding, and were also categorized as catalytically inactive. Atypical PKs were identified by BLAST analysis using representatives of each group of aPKs from other species as queries.
Each ePK was analyzed for the presence of additional domains by hidden Markov model analysis of the Pfam database [90]. The default cutoffs were used.
All predicted ePKs from each species were analyzed by BLAST analysis against the 4-kinome dataset comprised of all human, Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans protein kinase domains [34]. Since phylogenetic inference indicated that members of trypanosomatid COGs were more closely related to each other than to ePKs of the 4-kinome dataset, all COG members were classified congruently (the sole exception we found was two kinases that shared extensive homology outside of the kinase domain, LmjF15.1200 and Tc00.1047053505977.13). Assignments required an E-value difference of 5 logs or more between groups or families of ePKs for one of the trypanosomatid orthologues. When all trypanosomatid orthologues had E-values poorer that 10-16, or had similar E-values for different groups of ePKs, those ePKs were designated "unique".
Phylogenetic inference
Due to the relatively low degree of sequence conservation at the nucleotide level within some of these families, phylogenetic inference was carried out on amino acid alignment, rather than attempting alignment at the nucleotide level. Kinase domains were identified by analysis of alignments with the Pfam protein kinase domain, and extended manually as needed. Insertions larger than 25 amino acids were identified and removed prior to subsequent analyses. The SAM (Sequence Alignment and Modeling System using Hidden Markov Model (HMM) software was used to build HMMs representing the kinase domains of the gene families discussed in this paper [91]. These trained models were then used to identify residues capable of discriminating between the various domain families. In addition, by aligning the sequences of the kinase domains to these models, we created multiple sequence alignments of these gene families. These alignments were then visually inspected to verify that all subdomains were appropriately aligned, as well as to allow removal of both gene-specific insertions (in addition to those previously removed) and deletions and phylogenetically uninformative residues. These edited HMM-generated alignments were used as the starting point for phylogenetic reconstruction of these domain families.
Phylogenetic analysis of the kinase domains of these proteins was carried out using a variety of techniques. As a preliminary step in the phylogenetic investigation of our dataset, we used the Neighbour-joining approach as defined by Saitou and Nei as implemented in ClustalX [92]. Due to the relatively high degree of divergence that might be expected within our dataset we used the correct for multiple substitutions option in our analysis. Bootstrapping was carried out on the dataset with 1,000 replicates.
These aligned amino acid sequences were also subjected to parsimony analysis using PAUP*, version 4.0b8 [93,94]. Given the relatively large number of taxa in this dataset, the use of an exhaustive search was not possible. In its place, an heuristic search strategy was employed to attempt to find the best tree by reducing the set of trees examined and just calculating the score for likely trees. It should be noted that this method is not guaranteed to identify the most parsimonious trees from the sequences. We carried out 100 random stepwise addition sequences of taxa, each with TBR swapping and MAXTREES set to 10,000. Parsimony bootstrapping on the dataset was performed with 1,000 replicates and the same settings, except that only 10 random stepwise addition sequences were used per bootstrap replicate.
The same datasets were analysed using a Maximum Likelihood approach, as implemented in the web available PHYML application [95,96]. Analysis using the WAG amino acid substitution model inferred the starting tree. The proportion of invariable sites and the gamma distribution parameter were estimated by maximizing the likelihood of the phylogeny. The number of substitution rate categories was set at four for these analyses. Non-parametric bootstrap analysis was then carried out on the original data set with 500 replicates (the upper limit available for this web service). Majority-rule consensus trees were created for each of the three methodologies outlined above.
Finally, MRBAYES was used to carry out a Bayesian analysis of our data [97]. We used the WAG amino acid substitution model (based on nuclear genes and globular protein sequences, respectively), with a gamma rate distribution estimated from the data set to infer the phylogeny of our dataset. Starting from random trees, four parallel Markov chains were run to sample trees using the Markov Chain Monte Carlo (MCMC) principle. In general, 1,000,000 generations were run; after the burn-in phase, every 100th tree was saved.
Estimation of Ka/Ks
Analysis of the synonymous/non synonymous substitution rates required construction of pairwise, codon aligned, sequence alignments. These were generated using the PAL2NAL web server [98], which converted full-length amino acid alignments and the corresponding DNA sequences into a codon-based DNA alignment. The amino acid sequence alignments were obtained using the Water programme from the EMBOSS package using default parameters [99]. Estimation of Ka/Ks (dN/dS) ratios was then carried out by maximum likelihood using the pairwise codon-based substitution model in Codeml, which is part of the Phylogenetic Analysis by Maximum Likelihood (PAML) suite of programs [100].
List of abbreviations
aPK, atypical protein kinase; COG, clustered orthologous groups; ePK, eukaryotic protein kinase, PK, protein kinase, TriTryp, the trypanosomatids Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi.
Authors' contributions
MP carried out the analysis of T. brucei and T. cruzi protein kinases and drafted the manuscript. EAW performed the phylogenetic inference analyses. PNW carried out the analysis of L. major protein kinases. JCM contributed to interpretation of the data and the writing of the manuscript. MP and JCM conceived and coordinated the study. All authors read and approved the final manuscript.
Supplementary Material
Additional file 1
Table S1, Classification, orthology and systematic IDs of trypanosomatid ePKs
Click here for file
Additional file 2
Table S2, T. brucei protein kinases with predicted tyrosine in subdomain I
Click here for file
Additional file 3
Table S3, T. brucei protein kinases with predicted transmembrane domains
Click here for file
Additional file 4
Table S4, Classification, orthology and systematic IDs of trypanosomatid atypical PKs
Click here for file
Acknowledgements
The authors thank Daniel Nilsson for assistance in COG analyses, particularly with respect to the T. cruzi genome. We also thank Peter Myler and Christiane Hertz-Fowler for advice on parasite genomics, the TriTryp Genome Consortium for their considerable effort that made this work possible and Tansy Hammarton for comments on the manuscript. We appreciate helpful comments from Gerard Manning regarding protein kinase classification. This work was supported in part by NIH R01 AI31077 (MP), the M.J. Murdock Charitable Trust (MP), the Medical Research Council (JCM) and Wellcome Trust (JCM).
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1341617658410.1186/1471-2164-6-134SoftwareGeneSeer: A sage for gene names and genomic resources Olson Andrew J [email protected] Tim [email protected] Ravi [email protected] Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA2005 21 9 2005 6 134 134 18 4 2005 21 9 2005 Copyright © 2005 Olson et al; licensee BioMed Central Ltd.2005Olson 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
Independent identification of genes in different organisms and assays has led to a multitude of names for each gene. This balkanization makes it difficult to use gene names to locate genomic resources, homologs in other species and relevant publications.
Methods
We solve the naming problem by collecting data from a variety of sources and building a name-translation database. We have also built a table of homologs across several model organisms: H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. cerevisiae, S. pombe and A. thaliana. This allows GeneSeer to draw phylogenetic trees and identify the closest homologs. This, in turn, allows the use of names from one species to identify homologous genes in another species. A website is connected to the database to allow user-friendly access to our tools and external genomic resources using familiar gene names.
Conclusion
GeneSeer allows access to gene information through common names and can map sequences to names. GeneSeer also allows identification of homologs and paralogs for a given gene. A variety of genomic data such as sequences, SNPs, splice variants, expression patterns and others can be accessed through the GeneSeer interface. It is freely available over the web and can be incorporated in other tools through an http-based software interface described on the website. It is currently used as the search engine in the RNAi codex resource, which is a portal for short hairpin RNA (shRNA) gene-silencing constructs.
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Background
"Biologists would rather share their toothbrush than share a gene name":Michael Ashburner [1]. Biologists use a variety of names for genes, based on their specialization. It is a daunting task to use gene names to locate resources such as sequences or publications. For example:
1. ABCA4, ABC10, ABCR, FFM, RMP, RP19, STGD, STGD1 are all names for the same gene; ATP binding cassette, sub family A (ABC1).
2. The same gene name can be written in different ways; cyclinD1 versus cyclin D1.
3. Researchers modify names; hPRL is used to denote the human form of PRL.
4. Names can be species specific. For example, human p53 is called tumor protein 53 (Li-Fraumeni syndrome), whereas in mouse it is called transformation related protein 53 (trp53).
5. Names can be specialization specific. The same gene is known as PUF60 in the pre-mRNA splicing field and FIR in the transcription field. It is also known by the names RoBPI and siah-bp. The D. melanogaster field knows it as hfp. To add to the confusion, yeast has a family of proteins called PUF that have similar function but are unrelated in terms of sequence homology.
In order to locate genomic resources for a given gene using a familiar name, a reference name has to first be identified from GenBank (or other databases such as Swiss-Prot, TrEMBL [2] or ENSEMBL [3]). This reference name can then be used to access resources from a variety of databases (GenBank, ENSEMBL etc.). For example, once [GenBank:NM_000546] is identified as one of the reference names for p53, then sequences and other resources can be easily accessed in GenBank. But a search for p53 in the nucleotide database at NCBI [4] results in a list of more than 5700 accessions (over 285 pages), which necessitates manual curation to find the specific accessions of interest. It is possible to narrow down the search using advanced search features, but is error-prone and inconvenient, requiring several trials before a search can be fine-tuned.
There are attempts being made to streamline and standardize the naming process through the HUGO gene nomenclature committee (HGNC [5]). Unfortunately, there are historic names from different fields and scientists still tend to use fanciful names (especially in the D. melanogaster field, where names such as crossbronx, disco-related etc. are quite common).
Thus, there is a need for a tool that allows accessing information through names that are familiar to biologists from different sub-fields. In addition, analysis of large datasets creates the need for a programming interface that allows a program to access resources and accessions. Once a programming interface is designed, a website can be designed quite easily, to provide user-friendly access to the programming interface through cgi scripts and present results in an aesthetic and ergonomic fashion.
There are other tools and approaches that partially solve some of these problems and these are discussed and compared to GeneSeer in the discussion section.
Implementation
GeneSeer retrieves and stores synonyms for the model organisms: H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. cerevisiae, S. pombe and A. thaliana from the following sources, GenBank [4], FlyBase [6], ExPASy [2,7], HUGO [5], ENSEMBL [3], UCSC [8], and Gene Ontology [9]. It also holds sequences for proteins and nucleotides as well as related information (splice variants, expression patterns etc.) about the genes. Similarities (homologies) between proteins are pre-calculated and stored in GeneSeer.
This paper is organized with the description of the software tools used in GeneSeer coming first followed by the algorithm and the underlying architecture.
Software tools
There are three critical software tools we use, a database server, a genomic sequence server and a viewer to display alignments.
Database
We store most of the data (except for sequence data) in a MySQL relational database [10]. There are separate tables for synonyms from each data source and an auxiliary table for synonyms from data sources such as micro-arrays or RNAi libraries. The data management is described in more detail in the name-translation section below.
Genome packer (Gpacker)
GeneSeer needs to be able to frequently access sections of sequences from the sequence database. A tool called Gpacker was developed to allow fast random access to any of the SOFAR (described below) sequences or assembled genomic sequences. Gpacker uses a binary system to store sequence information, using 4 bits for each base in nucleotide data (a DNA sequence requires only 2 bits per position, but if SNP data is included, then 4 bits are necessary) and 8 bits for each amino acid in protein data (to account for the 20 possible amino acids). The binary files are indexed, with the indices stored in a database. This allows for fast random access of sequences.
Light weight genome viewer (lwgv)
Many genes exhibit alternative splicing, with several splice variants created from a single locus. In addition, there are annotations of features such as repeats, SNPs, CpG islands etc. The light weight genome viewer (lwgv) is used to display the annotations of features at a given genomic locus. This tool also allows navigation to other resources on the web, such as NCBI [4] and UCSC [8], lwgv was developed for in-house use and is now publicly available at Source Forge [11].
Architecture of GeneSeer
GeneSeer has three key features, the SOFAR database, name translation tables, and homology tables. SOFAR is a non-redundant collection of transcript sequences in each genome, whose construction is described below. The name-translation tables connect synonyms to each other. The homology tables contain pre-computed similarity scores for all pairs of proteins between and within species from a non-redundant collection, based on SOFAR.
GeneSeer is designed as a hub-and-spokes system, with the SOFAR database serving as the hub and the connections to resources and names serving as spokes. Every name or sequence that is submitted gets translated to a SOFAR name, either directly via the synonym tables, or indirectly through a BLAST [12] search of the SOFAR sequence database or through the use of the homology tables. SOFAR members are linked to resources and information, both internal and external to GeneSeer. We describe the three components of this architecture below.
SOFAR – Set Of FastA Representatives
Entrez Gene [13] (formerly LocusLink) provides an indexing for coding regions of the genome. However, this indexing is not complete, since there are regions that have cDNAs associated with them, which are not in Entrez Gene. Another feature that would be useful, but not provided by Entrez Gene, is a set of non-redundant accessions to represent each locus. The term locus is used here as a synonym for a coding region of the genome.
We built a set of mRNA accessions that includes the known genes and expert-curated cDNAs called SOFAR for each organism to overcome these problems. SOFAR is the key to GeneSeer's ability to return a concise set of results for any search.
The SOFAR database for an organism starts with one coding sequence. Each subsequent coding sequence that is considered for addition to the database gets checked for similarity to sequences already in SOFAR, through a BLAST [12] search, and gets added only if it is sufficiently different. We use a criterion of 60% similarity as a cutoff for entry into SOFAR.
The order in which sequences are considered for inclusion in SOFAR is crucial. In the case of human and mouse genomes, ordered gene lists are created by first using genes from RefSeq [14], NCBI's reference sequence resource, then sequences from Entrez Gene loci but not in RefSeq and then sequences that are expert annotated but are not in Entrez Gene, such as some kinases [15] and cDNAs for other functional groups. RefSeq annotates genes according to the reliability of the underlying evidence, for example, validated ranks higher than predicted. This is used to order the RefSeq genes amongst themselves. In case of all else being equal, the sequences are ordered by length (longest first).
The arbitrary 60% similarity cutoff can cause two kinds of mis-identifications,
1. Two similar sequences that are from different loci get assigned to the same locus, while they should have separate entries in SOFAR, e.g. duplicated genes such as FUT5 and FUT6 which occur on different loci on human chromosome 19.
2. Two sequences that are from the same locus can get identified as being sufficiently different from each other and get separate entries in SOFAR (e.g. INK4a and ARF are the same Entrez Gene, CDKN2A [16], figure 6).
Figure 6 Result of a search for INK4A in H. sapiens. In the results that are returned for each locus, all the RefSeq [14] accessions at the locus are returned, but the ones in SOFAR are highlighted in green. If a locus does not have a RefSeq accession, then only the accessions in SOFAR are returned. In this case the locus is CDKN2A [16] with three accessions, two of which, [GenBank:NM_058195] (ARF) and [GenBank:NM_058197] (INK4A), are from SOFAR and highlighted in green. This is discussed in the text.
Most of the cases from the first type can be resolved using Entrez Gene indices, however there remain eighty-nine cases of SOFAR members from multiple loci, all of which are immunoglobulin genes. For example, [GenBank:M14158] and [GenBank:D86998] are from different loci, but both are genomic sequences that contain parts of an immunoglobulin gene. On searching for them in GeneSeer, it tries to BLAST these sequences against SOFAR and comes up with unrelated genes. GeneSeer is not very useful for accessing resources for genes from this family. IMGT, the international ImMunoGeneTics information system [17,18] with its specialized sequence (IMGT/LIGM-DB) and gene (IMGT/GENE-DB [19]) databases is better suited for this purpose.
It is important to note that the two genes INK4a and ARF [16] are sufficiently different that they warrant inclusion as separate entries, since they have distinct sequences and functions. In H. sapiens there are 1130 loci which have multiple SOFAR members. This is not a problem, as the SOFAR members are different from each other and we want SOFAR to contain all possible non-redundant coding sequences.
As an additional step, unique sequences from splice variants are then entered to ensure that SOFAR contains every possible transcribed 25-mer.
A SOFAR database has been built for each of the organisms in GeneSeer. In the case of D. melanogaster, the set of FBgn numbers provided by FlyBase [6], which are a unique set of genes similar to Entrez Gene, was used to create the SOFAR database.
Almost every SOFAR member has a corresponding protein, except in the case of partial coding sequences, predicted genes and non-coding RNAs. These proteins can be used to construct a SOFAR set of proteins, which is used to generate data for the homology tables and viewer described below.
The SOFAR database is useful for other purposes, such as the design of RNAi hairpin constructs that can silence single genes [20]. Designs were checked against the SOFAR database to ensure that they did not match (with up to 2 mismatches) more than one sequence in the database.
Name translations
Each of the following databases, HUGO [5], ExPASy [2,7] (Swiss-Prot and TREMBL names), ENSEMBL [3], GenBank [4], FlyBase [6] and Gene Ontology [9], comes with a list of synonyms, which were downloaded and entered into a separate table for each dataset. The Genbank data contains a mapping from gene names to Entrez Gene IDs. The bulk of the names in GeneSeer are taken directly from a file provided at NCBI's ftp site [21]. FlyBase provides additional synonyms. The set of names is extended even further by extracting names from the definition lines of sequences which cross-reference an Entrez Gene ID.
Information relevant to the individual mRNA accessions, such as coding sequences (CDS) and protein domains, is extracted from GenBank flat files and stored in additional tables. The system utilizes the Entrez Taxonomy database [22,23] for translating between taxonomy ids and organism names. Up-to-date Gene Ontolgy (GO) [9] terms and associations are also incorporated, which allows users to search for genes by GO terms. To find GO terms for genes, it is easier to use tools such as GObar [24] or AMIGO [25]. Tables of genomic alignments provided by UCSC [8] are directly imported to the GeneSeer database. These alignments are currently used to help visualize the exon-intron structure of the genes.
An auxiliary translation table is used to store names that might be specific to particular datasets such as short hairpin names from the publicly available RNAi libraries or probe names from microarrays.
The database tables remain current through regular updates. The active GeneSeer database is replaced once every month, after a new version of the database is built and tested.
Each name gets mapped to a SOFAR representative. If an accession or EST name is submitted to the system and it is not recognized, then GeneSeer downloads the sequence and uses BLAST against SOFAR to identify its name. If this fails, then a mapping to the genome is used to identify the closest locus that contains a SOFAR representative. There are cases where everything fails and nothing is returned, such cases have to be curated manually, as they are usually ESTs that might not be reliable. Everytime such a translation succeeds, the result gets cached for fast response the next time around. The cached results will not survive an update to the GeneSeer system.
Homology tables and viewer
GeneSeer can identify homologs across species and present a phylogenetic tree. A matrix of similarity scores, based on BLAST [12], is pre-calculated for all pairs of SOFAR proteins in the system. The construction of the SOFAR protein database is described above. This matrix is used to generate clusters of related proteins. The clusters are aligned, using ClustalW [26], when the user requests a tree. A phylogenetic tree is then created from this multiple alignment using PHYLIP [27], which has been modified to run in batch mode, to build rooted and unrooted trees. A custom program renders the tree in scalable vector graphics (SVG) format to allow user interaction. This tree is not the same as one that would be derived from a careful alignment of domains and might be less accurate, but it definitely allows quick identification of close homologs. The results of the homology viewer can be a starting point for a more detailed phylogenetic analysis of the proteins in a family.
The homology viewer can be accessed using the Action menu item, explore-homologs, in the results page of a GeneSeer search. The result of clicking on this link is a page that allows fine-tuning the search parameters, or eliminating a species that might not be of interest. If the threshold of BLAST similarity scores is set lower, then fewer homologs will be considered while drawing the phylogenetic tree. Limiting the cluster sizes is important as the rendering of the phylogenetic tree can take a long time if there are too many members in the family, leading to a time-out error from the browser.
Sometimes it is not possible to reach a protein in a distant species directly. In such cases, it may be possible to use an intermediate organism to make the connection, that is, the intermediate organism's protein has homology to proteins in both species of interest. An expand checkbox in the fine-tuning page described above, allows such an exploration.
Results
The GeneSeer server [28] can be accessed either using a web-browser or through a programming interface that is described below.
Gene names can be entered by hand or uploaded in the form of files containing lists. Sequences can also be uploaded in the form of fasta files. Results can be downloaded in the form of excel spreadsheets or text files and can be used to access information from NCBI, or to access splice variants and homologs. Tissue specificity of mRNA expression can also be accessed.
Since GeneSeer is accessible over http, programs can be written in almost any language to use it. We have plans to improve programmatic access, especially using semantic-web [29] based technologies such as Resource Description Framework(RDF), but improvements will be driven primarily by user-feedback and the needs of the community.
• To retrieve the SOFAR name for genes named p53 in csv format, use the URL, .
• To retrieve a list of homologs for the human gene named p53 (gene id 7157) use the URL, .
• To retrieve a list of genes with either the symbol p53 or p21 in the human genome (taxonomy id = 9606) in html format use the URL, .
GeneSeer search features
GeneSeer is flexible in the kinds of names that can be used for searching. Thus, searches can be conducted using Gene Symbols/Names, Keywords (partial terms such as casp for caspase), Keyword Symbols (partial symbols, such as erb for erbb2), OMIM ids (online mendelian inheritance in man [30]), diseases/disorders (such as diabetes), Tissue specificity (tissue expression patterns, such as genes expressed in muscle, derived from UniGene [31]), Gene Accessions, Protein Accessions, Entrez Gene IDs (from Entrez Gene [13]), UniGene Cluster IDs (from UniGene [31]), CDD Domain IDs (from conserved domain database [32]), Gene Ontology IDs (from Gene Ontology [9]), Definitions (from definition lines in GenBank [4]), HUGO IDs (from IDs defined by HUGO [5]), ENSEMBL IDs (from IDs defined by ENSEMBL [3]), SNPs (from dbSNP [33]) and Sequences (nucleic acid and protein sequences).
Search terms can be entered either individually or in a comma-separated list or by uploading files (e.g. Excel spreadsheets, fasta files, or simple text files) containing the list of terms.
The easiest option is to first use the automatic search mode. The software will try to guess what the user has provided (accession, symbol etc.) and return its best answer. The automatic mode uses a restrictive search first, such as a name search, and iteratively expands the types of searches it performs, and stops searching when it finds a result. If this is unsatisfactory, then the Go Further button can improve results. The Go Further button will continue a few more methods and return more ambiguous results. If the results continue to be unsatisfactory, then one of the specialized modes (listed above) will need to be used and modifying the search terms might also help. For example, if caspase2 does not return a result, a search with casp as a keyword will return results that will definitely include caspase 2.
Results are displayed on a webpage but can also be downloaded to a comma-separated-value (csv) file using the download .xls operation provided on the results page. The csv file can be opened in a spreadsheet program or a text editor. If possible, the returned results always show the Entrez Gene IDs for each name. GeneSeer can be used to translate names into IDs for use in other programs which prefer to use Entrez Gene IDs, such as GObar [24].
In addition, on the results page, NCBI [4] pages for the search results can be accessed. Each individual result also has an associated Action link, that allows exploration of PubMed [34], for papers related to the item), tissues, UCSC (the genome browser at UCSC for the relevant genomic region) [8], explore_homology (view an approximate phylogeny of related genes), and visualize gene (visualize the genomic region and study splice variants).
If GeneSeer fails to find any of the terms, they are listed on the results page. The failures can be re-analysed using different search methods (searching by "keyword", "symbol" or other variations). As a last resort, a bug report can be sent via the website and a human curator will resolve the issue.
Complicated queries using the search history button can also be performed: an example is the search for all caspases that are expressed in the human brain, which is explained below. A list of searches done using any computer are stored on the server, and can be accessed using the Search History link. This link can be used to access prior searches or limit search results. For example, one can search first for casp as "keyword symbol" and then for mRNAs expressed in the brain, searching by tissue for the term brain. The queries can be accessed using the search history button and can be combined using boolean logic (AND/OR/NOT/XOR) to get mRNAs that are caspases and/or/not/xor expressed in the brain.
Specific examples of GeneSeer use are given in the next section.
Discussion
We have addressed three problems in genomic research with this project,
1. Access to genomic information through gene names: Biologists have been struggling with this problem for many years, especially in the genomic era where data on sequences has been piling up at a rapid pace.
2. Mapping sequences to gene names: Data from a variety of sources can be in the form of DNA or protein sequences and it is useful to be able to get back to other resources for the gene to which the sequence fragment belongs.
3. Identification of homologs (both orthologs between species and paralogs within a species) across several species for a given gene: It is difficult to locate orthologs and paralogs, given the name of a gene in one species. It would also be useful to get a quick view of an approximate phylogeny of the set of genes returned.
We use the gene p53 to showcase some of the abilities of GeneSeer. Figure 2 shows the result of searching for p53 on GeneSeer. Figure 3 shows the alignment of the splice variants of p53 accessed through the Action menu on the page shown in figure 2. Figures 4 and 5 show a phylogenetic tree for p53 generated using the Action menu on the page shown in figure 2.
Figure 2 Results of search for p53. GeneSeer results for a search of p53. Note the concise and accurate list of genes, which is almost impossible to find on any other website/tool without human curation, based on currently available resources.
Figure 3 View of alignment of splice variants of mouse p53. A view of the alignments of p53 splice variants against the genomic sequence, using lwgv, a C-based light weight genome viewer from our lab that is freely available on Source Forge [11].
Figure 4 Homologs of p53 and a rudimentary phylogenetic tree. All the homologs of the p53 protein and a thumbnail of a phylogenetic tree constructed from their multiple alignment are shown. The thumbnail links to a bigger picture with more details, as explained in the next figure. The tree gives a rough idea of the phylogenetic relationships between the various proteins and identifies the proteins that need to be analysed further for understanding the evolution of this family.
Figure 5 Detailed view of p53 phylogeny. A more detailed view of the phylogenetic trees seen as thumbnails in the previous figure. Each protein name is followed by the Taxonomy ID for the organism, as specified by NCBI [22]. For example, 9606 is the Taxonomy ID for Homo sapiens. Here, the tree is rendered in SVG format (scalable vector graphics) where each protein name is linked to resources for that gene which appear in a pop-up window on a mouse-over. The pop-up window (the blue box in the figure) can be locked in place by a click on the left-mouse-button. A static jpeg format image is also offered on the website. The SVG image allows control of image magnification through the mouse button.
GeneSeer can handle all the examples (ABC1, cyclin D1, hPRL, p53, and PUF60) cited in the introduction. It has information on several model organisms: H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. cerevisiae, S. pombe and A. thaliana. It can help visualize splice variants, SNPs and short hairpin RNA silencing constructs (shRNAs, using names from RNAi codex [35], it returns the mRNA that contains the construct) that align with any mRNA. It allows searching for homologs for any gene across species, based on our pairwise sequence alignments of the proteins.
Another example that affords some valuable lessons is the Major Histocompatibility Complex (MHC) region in the human genome [36]. There are several genes in this region that exhibit duplications and also exhibit variations across populations. One such gene, HLA-A (accession [GenBank:NM_002116] can be found through GeneSeer. There are variants such as Aw-80 (accession [GenBank:Q09160], which is in the same locus, but will not appear as a separate gene. In fact, a search on GeneSeer will return HLA-A. To study the variants and the intricacies of this gene family (as well as the immunoglobulin family cited earlier), the best place to start is use a specialized information system such as IMGT [17]. HUGO [5] now assigns names starting with the letters HCG to genes in the MHC, one way to search for them in GeneSeer is to use HCG% and do an automatic search, but this will return several unrelated hits, a better approach, since we know HCG is going to be a part of a gene symbol, is to search by keyword symbol with this term.
GeneSeer has some limitations, that necessitate ocassional human intervention. GeneSeer is designed to err on the side of caution. It will find unique answers where possible, but will leave in all ambiguities when a unique resolution is impossible without more information. For example, the symbol nos is used for both nitric oxide synthase and Nanos in D. melanogaster, GeneSeer will return both results. The program will reduce the number of possibilities, so that a human user is not overwhelmed by information. Sometimes human judgement is required to find homologs of genes: For example, while GeneSeer will list all homologs of staufen, the human user has to decide which one is interesting from a biological standpoint and if it is a functional homologue, that is, contains the active domains of interest.
Comparison with other tools
Most of the tools we have been able to find are geared towards controlled vocabularies and aim at reducing the diversity of names. Our viewpoint is different, we start with unique sequences represented in our SOFAR databases for each species, and resolve all names to sequences in this database. We describe some of these papers and tools to give an idea of why GeneSeer is unique.
Some work has been done on identification and disambiguation of gene symbols, we consider one such report here [37] which is a thesaurus-based approach. The method underlying their approach of building a translation table using names from a variety of sources is similar, but their goal is to recognize names in documents and abstracts in order to mine texts, while GeneSeer aims to use gene symbols/names to locate genomic resources and homologs in other species. We also believe our synonym table is much more extensive, since we have identified the gaps in various synonym tables that are available, through intensive field testing.
GeneCards [38] is a database of human genes, their products and their involvement in diseases. It is designed to return concise information on the function of genes and is human-gene centric. A search for p53 results in 925 hits, which are organized into microcards (single line descriptors only) and minicards that have detailed information and are organized by relevance to the search term. The top microcard is the actual p53 gene, and the second one in this list is Mdm2, which interacts with p53. To find Mdm2 through a p53 search in GeneSeer, one would have to search by Definitions for the term p53 and it will return p53 and others that interact with p53. Searching for PUF as a keyword symbol in GeneSeer returns PUF60/RoBPI/siah-bp while GeneCards returns unrelated genes. The point of this discussion is not to find cases where GeneSeer excels, but to highlight the differences in the abilities of these programs.
Global Gene Hunter [39] is a tool that is a part of the Saccharomyces Genome Database (SGD) [40]. Given a gene name, the site runs searches on six databases, Saccharomyces Genome Database (SGD), PubMed, Entrez Gene, Protein Data Bank Homologs, UniProt [41] and MIPS [42]. The search can be limited to a subset of these databases. The results from each of these databases is returned as part of a large page, but are not organized. It suffers from the failings of the search interfaces provided by these databases and puts the onus of organizing the results on the user.
BioMinT [43], the Gene and Protein Name Synonyms Database, allows the user to find synonyms. A search for PUF60 on this site listed fourteen H. sapiens and twenty-one D. melanogaster genes and proteins, but they were essentially products of a single gene from each genome. The returned results could have been compressed further.
GeneDB [44] is a resource that provides a portal for access to data generated by pathogen sequencing at several collaborating research centers. Searching for PUF60 found 7 hits in G. morsitans, probably from the same gene, the interface is a bit inconvenient and the results were not comprehensive.
DBGET [45] holds information from a variety of databases. But a search for PUF60 failed and a search for p53 returned a long list that was not easily comprehensible.
Thus, GeneSeer is more comprehensive and has an easier interface when compared with other public tools. Its focus and goals are also a bit different from most tools that are currently available online.
Conclusion
GeneSeer is a powerful engine that is available freely over the web and can be accessed either by a web-browser or by standard programming languages. GeneSeer is an evolving project, there are many more features that can be added, such as sequence and genomic data from new organisms. We plan to add data from F. rubripes [46] in the near future. Prokaryotic gene names are in the system, but it requires additional work to develop SOFAR databases and the homology viewer. User-feedback will be used to prioritize improvements and addition of new features. We want to make GeneSeer compatible with the goals of the semantic web [29] by adding features such as RDF/xml downloads, that will allow it to be indexed and be machine readable. GeneSeer was designed with flexible use in mind, allowing it be freely incorporated into other tools. It is used in several tools developed at Cold Spring Harbor Laboratory (such as the RNAi Codex, an shRNA portal [35]).
Availability and requirements
The GeneSeer server [28] can be freely accessed over http allowing easy access from any computer with an internet connection and a web-browser. GeneSeer can also be accessed via a programming interface that has been described above.
Authors' contributions
Ravi Sachidanandam conceived the idea for the project, developed several tools and resources and wrote the manuscript. Andrew Olson is the primary implementer of the ideas, developed the website and also contributed ideas to the design of the tool. Tim Tully suggested the homology tool and identified the biological relevance of the tool.
Figure 1 Web interface for GeneSeer. The front page of the GeneSeer website.
Table 1 Results of TYRO keyword symbol search for several species. All the results returned for keyword symbol search for TYRO also known as Eph are shown here. Some proteins that interact with TYRO are also reported.
Keyword Symbol Search TYRO
Number Species Accession GeneID Definition
1 9606 [GenBank:NM_005233] 2042 Homo sapiens EphA3 (EPH A3), transcript variant 1, mRNA.
2 9606 [GenBank:NM_182644] 2042 Homo sapiens EPH receptor A3 (EPHA3), transcript variant 2, mRNA.
3 9606 [GenBank:NM_004438] 2043 Homo sapiens EphA4 (EPHA4), mRNA.
4 9606 [GenBank:NM_004439] 2044 Homo sapiens EphA5 (EPHA5), transcript variant 1, mRNA.
5 9606 [GenBank:NM_182472] 2044 Homo sapiens EphA5 (EPHA5), transcript variant 2, mRNA.
6 9606 [GenBank:NM_004442] 2048 Homo sapiens EphB2 (EPHB2), transcript variant 2, mRNA.
7 9606 [GenBank:NM_017449] 2048 Homo sapiens EphB2 (EPHB2), transcript variant 1, mRNA.
8 9606 [GenBank:NM_004443] 2049 Homo sapiens EphB3 (EPHB3), mRNA.
9 9606 [GenBank:NM_004444] 2050 Homo sapiens EphB4 (EPHB4), mRNA.
10 9606 [GenBank:NM_006182] 4921 Homo sapiens discoidin domain receptor family, member 2 (DDR2), mRNA.
11 9606 [GenBank:NM_000372] 7299 Homo sapiens tyrosinase (oculocutaneous albinism IA) (TYR), mRNA.
12 9606 [GenBank:NM_006293] 7301 Homo sapiens TYRO3 protein tyrosine kinase (TYRO3), mRNA.
13 9606 [GenBank:X72887] 7302 H. sapiens TYRO3P mRNA.
14 9606 [GenBank:NM_003332] 7305 Homo sapiens TYRO protein tyrosine kinase binding protein (TYROBP), transcript variant 1, mRNA.
15 9606 [GenBank:NM_198125] 7305 Homo sapiens TYRO protein tyrosine kinase binding protein (TYROBP), transcript variant 2, mRNA.
16 10090 [GenBank:NM_010140] 13837 Mus musculus Eph receptor A3 (Epha3), mRNA.
17 10090 [GenBank:NM_007936] 13838 Mus musculus Eph receptor A4 (Epha4), mRNA.
18 10090 [GenBank:NM_010142] 13844 Mus musculus Eph receptor B2 (Ephb2), mRNA.
19 10090 [GenBank:NM_010143] 13845 Mus musculus Eph receptor B3 (Ephb3), mRNA.
20 10090 [GenBank:NM_010144] 13846 Mus musculus Eph receptor B4 (Ephb4), mRNA.
21 10090 [GenBank:NM_022563] 18214 Mus musculus discoidin domain receptor family, member 2 (Ddr2), mRNA.
22 10090 [GenBank:NM_019392] 22174 Mus musculus TYRO3 protein tyrosine kinase 3 (Tyro3), mRNA.
23 10090 [GenBank:NM_011662] 22177 Mus musculus TYRO protein tyrosine kinase binding protein (Tyrobp), mRNA.
24 10090 [GenBank:NM_009465] 26362 Mus musculus AXL receptor tyrosine kinase (Axl), mRNA.
Acknowledgements
Vladimir Grubor gave many detailed suggestions, pointed out references (especially [1]) and helped test the tool. Michele Hastings helped make the paper more biologically relevant. Xavier Roca, Susan Janicki and Nihar Sheth helped with critical comments and suggestions. Cat Eberstark put tremendous effort into improving the figures. The DART Neurogenomics Alliance funded this project. The anonymous reviewers suggested several important improvements to the tool and the manuscript. One of the reviewers was instrumental in identifying competing tools.
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BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-5-631618535710.1186/1472-6963-5-63Research ArticleChances of late surgery in relation to length of wait lists Sobolev Boris G [email protected] Adrian R [email protected] Lisa [email protected] Robert [email protected] Department of Health Care and Epidemiology, University of British Columbia, Vancouver, Canada2 Centre for Clinical Epidemiology and Evaluation, Vancouver Coastal Health Research Institute, Vancouver, Canada3 Centre for Health Evaluation and Outcome Sciences, St. Paul's Hospital, Vancouver, Canada4 Department of Surgery, Royal Columbian Hospital, New Westminster, Canada2005 26 9 2005 5 63 63 1 6 2005 26 9 2005 Copyright © 2005 Sobolev et al; licensee BioMed Central Ltd.2005Sobolev 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 proportion of patients who undergo surgery within a clinically safe time is an important performance indicator in health systems that use wait lists to manage access to care. However, little is known about chances of on-time surgery according to variations in existing demand. We sought to determine what proportion of patients have had late coronary bypass surgery after registration on wait lists of different size in a network of hospitals with uniform standards for timing of surgery.
Methods
Using records from a population-based registry, we studied wait-list times prospectively collected in a cohort of patients registered on wait lists for coronary artery bypass grafting procedures. We compared the number of weeks from registration to surgery against target access times established for three urgency groups. The chances of undergoing surgery within target time have been evaluated in relation to wait-list size at registration and the number of surgeries performed without registration on a wait list.
Results
In 1991–2001, two in three patients were at risk of late surgery when registered on wait lists for isolated coronary bypass procedures in British Columbia, Canada. Although urgent patients had never seen a wait list with clearance time exceeding one week, the odds of on-time surgery were reduced by 25%, odds ratio [OR] = 0.75 (95% confidence interval [CI] 0.65–0.87) for every additional operation performed without registration on a list. When the wait list at registration required a clearance time of over one month, semi-urgent patients had 51% lower odds of on-time surgery as compared to lists with clearance time less than one week, OR = 0.49 (95%CI 0.41–0.60), after adjustment for age, sex, comorbidity, calendar period, hospital and week on the list. In the non-urgent group, the odds were 69% lower, OR = 0.31 (95%CI 0.20–0.47). Every time an operation in the same hospital was performed without registration on a wait list, the odds of on-time surgery for listed patients were reduced by 7%, OR = 0.93 (95%CI 0.91–0.95) in the semi-urgent group, and by 10%, OR = 0.90 (95%CI 0.87–0.94), in the non-urgent group.
Conclusion
Chances of late surgery increase with the wait-list size for semi-urgent and non-urgent patients needing coronary bypass surgery. The weekly number of patients who move immediately from angiography to the operation without registration on a wait list reduced chances of surgery within target time in all urgency groups of listed patients. When advising patients who will be placed on the wait list about the expected time to treatment, hospital managers should take into account the current list size as well as the weekly number of patients who require CABG immediately after undergoing coronary angiography.
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Background
In health systems that provide universal access to care, efforts to contain costs for stand-by hospital capacity usually result in waiting lists for surgical procedures [1]. From clinical perspective, however, delay in necessary treatment due to surgical wait lists is a major concern [2,3]. Establishing a clinically appropriate time that patients can safely wait for the operation is generally perceived as a method to prevent adverse outcomes of delay [4]. For example, priority wait lists [5] are commonly used for queuing patients with coronary artery disease requiring bypass surgery based on the severity of condition [6,7].
The proportion of patients who undergo surgery within clinically acceptable time is an important performance indicator in health systems that use wait lists to manage access to care [8,9]. Describing variations in waiting times for coronary artery bypass surgery (CABG), Katz et al suggested that the wait-list size may be an important factor in delaying surgery [10]. Indeed, if there are patients on the list, then for a patient who just arrived to be admitted within a certain time all patients ahead must have been served. Sobolev et al performed an empirical analysis of a population-based registry and found that the length of queue at registration affected the time to elective surgery [11]. Surprisingly, little is known about how the list size at registration affects the chances of undergoing elective surgical procedures within acceptable time. The common concern for evaluation purposes is, therefore, whether one can accurately estimate the proportion of late surgeries without considering the length of a wait list.
In theory, queuing procedures should ensure access to care according to urgency of treatment if implemented uniformly across a health system [8]. However, the chances of admission for elective surgery within target time can be easily altered if surgical services experience an uneven influx of more urgent case [12]. In the Canadian province of British Columbia, there are two pathways to surgical revascularization: registration on a wait list, or direct admission after coronary angiography, as described in [11]. Patients presenting with symptoms of coronary artery disease are referred to cardiologist to assess the need for coronary revascularization. The cardiologist evaluates the coronary angiogram and decides on treatment. If coronary angioplasty is not indicated, then a cardiac surgeon is consulted to assess the patients' suitability for coronary bypass surgery. Following the consultation in which surgery is indicated, surgeons register on their wait lists patients who require and decide to undergo the operation. Alternatively, patients may be admitted to a hospital cardiac ward directly from the catheterization laboratory when urgent assessment is deemed necessary. If suitable for surgery, such patients remain in hospital until the operation.
The objective of this study was to determine the proportion of patients that have had late surgery after registration on surgical wait list of different sizes. To examine the association between the length of wait lists and timely access to elective CABG surgery, we used data on registrations and waiting times for elective coronary bypass surgery collected at a provincial cardiac surgery registry in BC. We also examined the relation between the number of surgeries performed without wait-list registration and the chances of surgery within recommended time among wait-listed patients.
Methods
Data sources
Data were taken from a population-based registry set up to capture the time of registration for surgery, the time of surgery or removal from wait lists without surgery, for all patients accepted for coronary bypass surgery in the four tertiary hospitals delivering adult cardiac care to residents of BC [15]. Offices of all cardiac surgeons weekly provide information to the registry on registrations for surgery, operations performed, waitlist reconciliation (removals), and discharge summaries. Coexisting medical conditions were identified in the BC Linked Health Database Hospital Separations File via a deterministic link with the registry records.
Patients
There were 9,366 records of registration for isolated CABG added to the registry between January 1991 and December 2000. We excluded 147 records of patients who were emergency cases (44), were removed on the registration date (99), or who had missing operating room reports (4). The remaining 9,219 records had either the surgery date or the date and reason of removal from the list without surgery.
As patients who moved from angiography to surgery on an expedited basis were not added to the wait lists, they were not included in the analysis of wait-list times. These patients contributed to demand figures only.
Urgency groups
All cardiac surgeons in BC use a common guideline for prioritizing patients and assigning a target time for surgery based on angina symptoms, affected coronary anatomy, and left ventricular function impairment as described elsewhere [15]. Each patient was classified as urgent if the suggested time to surgery was three days, semi-urgent if the time was six weeks, and non-urgent if the time was 12 weeks.
Demand for surgery
For each calendar week during the study period, the demand for surgery was characterized by the existing list size and the number of direct admissions immediately after angiography. For each patient, the list size was a census of patients with higher or equal priority present at registration on the list in a hospital. Patients contributed one count to the list size for each week they remained on the list, except for the week of arrival. As operations are scheduled a week in advance, patients who underwent surgery are considered removed from the wait list in the week previous to their admission dates. The number of direct admissions was a weekly count of surgeries performed without wait-list registration.
Comorbidity
We used diagnoses reported in discharge abstracts within one year prior to registration for coronary bypass surgery. Each patient was classified as 1) presenting with no co-existing conditions, 2) presenting with congestive heart failure, diabetes, chronic obstructive pulmonary disease, cancer or rheumatoid arthritis, or 3) presenting with other co-existing chronic conditions as defined in [16].
Statistical analysis
Waiting time
Each patient had a waiting time computed as the number of calendar weeks between registration and surgery or removal for other reasons. The date at which a surgeon's office submits the operating room booking request for surgery served as the date of registration on the list. For procedures delayed beyond target access time, we studied the number of weeks to target time.
Study variables
The list size was categorized in relation to clearance time, that is, a hypothetical time within which the list will be cleared at a maximum weekly service capacity if there are no new arrivals. We divided the list size in four categories: 1) lists requiring less than a week of clearance time, 2) half a month, 3) a month, and 4) over one month. In three hospitals with the service capacity of 15 operations a week, the following numbers of patients on the list – 0 to 14; 15 to 29; 30 to 59; and over 60 – correspond to clearance time of a week, half a month, a month, over one month. In a hospital with the service capacity of 25 operations weeks, the same clearance times correspond to 0 to 24, 25 to 49, 50 to 99 and over 100 patients on the list. The weekly number of direct admissions was treated as a continuous variable.
Regression models
Primary outcome was admission to surgery within target access time. Primary comparisons were between wait-list size categories. To evaluate the effect of the list size, we estimated the odds ratios associated with list-size categories using discrete-time survival regression models for each urgency group [14]. In this service, scheduling patients for surgery has provided a weekly opportunity for admission to occur. Measured as the number of service scheduling cycles, waiting time is inherently discrete and is best measured as the number of new scheduling cycles from registration to admission or removal for other reasons. For this analysis, we performed a pooled analysis of binary regression models developed for each week on the list, treating weeks as ordered strata [13]. All cases removed from waiting lists without surgery or that exceeded target access time were treated as censored observations at one week after the target time. In multivariate analysis we adjusted for age, sex, comorbidity, period, the number of direct admissions and hospital.
We entered an indicator variable for each hospital in the models in order to obtain regression estimates for the study variables adjusted for possible variations in access management. Hospital 1 was coded as referent.
For the direct admissions, we interpret the odds ratio derived from the model as a change in the weekly odds of on-time surgery associated with one additional surgery performed immediately after angiography.
Results
Waiting outcomes
The baseline characteristics of registered patients are shown in Table 1. The most prevalent groups at registration were patients aged 60–69 (38%) and 70–79 (30%) years, men (82%), those registered in semi-urgent group (71%), those without major comorbidities (52%), and those registered in 1995–1996 (22%). Among the four hospitals, the majority of patients were registered at hospital 2 (34%). At registration most patients (43%) saw a list-size requiring one month of clearance time, whereas the minority (14%) required half a month.
Table 1 Characteristics of 9,219 subjects registered for isolated coronary artery bypass surgery in British Columbia 1991–2000
Characteristic N (%)
Age group (yr)
<50 731 (7.9)
50–59 2006 (21.8)
60–69 3526 (38.2)
70–79 2764 (30.0)
≥80 192 (2.1)
Sex
Women 1630 (17.7)
Men 7589 (82.3)
Urgency at registration
Urgent 652 (7.1)
Semi-urgent 6553 (71.1)
Non-urgent 2014 (21.8)
Major comorbidity at registration
None 4775 (51.8)
CHF, diabetes, COPD, rheumatoid arthritis, cancer 2435 (26.4)
Other conditions 2009 (21.8)
Calendar period
1991–1992 1725 (18.7)
1993–1994 1889 (20.5)
1995–1996 1997 (21.7)
1997–1998 1888 (20.5)
1999–2000 1720 (18.7)
Hospital ID at booking
1 1903 (20.6)
2 3137 (34.0)
3 2124 (23.0)
4 2055 (22.3)
Wait-list size category
1 – <1 week 1502 (16.3)
2 – half month 1276 (13.8)
3 – 1 month 3954 (42.9)
4 – >1 month 2487 (27.0)
CHF – congestive heart failure, COPD – chronic obstructive pulmonary disease
Among those who was removed before or on a target access time (TAT), 2959 (93.6%) received surgery, 37 (1.2%) died, 64 (2.0%) continued with medical treatment, 31 (1.0%) declined surgery, 22 (0.7%) were transferred to another surgeon or hospital, and 47 (1.5%) were removed for other reasons (data not shown).
Among those who was removed after TAT, 5018 (82.2%) eventually underwent surgery, 55 (0.9%) died, 112 (1.9%) continued with medical treatment, 157 (2.6%) declined surgery, 77 (1.3%) were transferred to another surgeon or hospital, 165 (2.7%) were removed for other reasons, and 475 (7.8%) were still on the wait list at 52 weeks (data not shown).
Of the 652 urgent patients, 22 (3.4%) were removed from the wait list without surgery, and in 106 (16.3%) patients the urgency was downgraded to semi-urgent or non-urgent.
Access to surgery in urgency groups
Overall, the proportion of patients who underwent surgery within the target access time was 32% (95% confidence interval [CI] 31–33%). The proportion varied significantly across urgency groups. Table 2 shows that among urgent, semi-urgent, and non-urgent patients, 21% (18–24%), 34% (33–35%), and 29% (27–31%) underwent surgery on time, respectively. The proportions of CABG performed within target time were similar in three hospitals ranging between 34 and 38%, with only 19% in hospital 3, Table 2.
Table 2 Probability of undergoing surgery within target time in relation to urgency, hospital, and wait-list size
Characteristic N % (95% CI)
Urgency at registration
Urgent 137 21.0 (17.9, 24.1)
Semi-urgent 2235 34.1 (33.0, 35.3)
Non-urgent 587 29.1 (27.2, 31.1)
Hospital ID at booking
1 695 36.5 (34.4, 38.7)
2 1079 34.4 (32.7, 36.1)
3 405 19.1 (17.4, 20.7)
4 780 38.0 (35.9, 40.1)
Wait-list size category
1 – <1 week 596 39.7 (37.2, 42.2)
2 – half month 454 35.6 (33.0, 38.2)
3 – 1 month 1163 29.4 (28.0, 30.8)
4 – >1 month 746 30.0 (28.2, 31.8)
Access to surgery by list size
The percentage of patients receiving on-time surgery decreases with the list size from 40% (37–42%) in list-size category 1 (clearance time less than one week) to 30% (28–32%) in list-size category 4 (clearance time over one month), Table 2. Among all patients, the crude odds of on-time surgery was 58% lower in list-size category 2, OR = 0.42 (0.37–0.48), 66% lower in category 3, OR = 0.34 (0.31–0.38), and 71% lower in category 4, OR = 0.29 (0.25–0.32), compared to list-size category 1 (data not shown).
For semi-urgent and non-urgent patients, Table 3 shows the association between list size and the probability of on-time surgery as measured by unadjusted odds ratios. In the semi-urgent group, the crude odds of on-time surgery were 51% lower in list-size category 2, OR = 0.49 (0.42–0.57), 56% lower in category 3, OR = 0.44 (0.39–0.50), and 43% lower in category 4, OR = 0.57 (0.49–0.65).
Table 3 Association between wait-list size and chances of on-time surgery as measured by odds ratios derived from discrete-time survival regression models
Semi-urgent Non-urgent
Crude Adjusted* Crude Adjusted*
Surgery demand OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI)
Wait-list size category
1 – <1 week list-size 1.00 1.00 1.00 1.00
2 – half month list-size 0.49 (0.42, 0.57) 0.64 (0.54, 0.75) 0.88 (0.62, 1.25) 0.75 (0.51, 1.09)
3 – 1 month list-size 0.44 (0.39, 0.50) 0.53 (0.45, 0.62) 0.39 (0.28, 0.55) 0.38 (0.26, 0.56)
4 – >1 month list-size 0.57 (0.49, 0.65) 0.49 (0.41, 0.60) 0.30 (0.21, 0.41) 0.31 (0.20, 0.47)
Direct admission† - 0.93 (0.91, 0.95) - 0.90 (0.87, 0.94)
* Adjusted for age, sex, comorbidity, calendar period, hospital, and week on the list.
† Associated with one additional surgery performed without wait-list registration
In the non-urgent group, the crude odds of on-time surgery were 12% lower in list-size category 1, OR = 0.88 (0.62–1.25), 61% lower in category 3, OR = 0.39 (0.28–0.55), and 70% lower in category 4, OR = 0.30 (0.21–0.41).
All urgent patients had a list-size category 1. Therefore the effect of the list size was examined using regression analysis.
Regression analysis
In urgent patients wait-list size was studied as continuous variable ranging between 0 (28.1%) and 10 or more (1.7%). The effect of additional patient on the wait lists at registration was not significant, OR = 0.97 (0.86–1.10), after adjustment for age, sex, comorbidity, calendar period, hospital, and week on the list.
For semi-urgent and non-urgent patients, Table 3 shows the association between list size and the probability of on-time surgery as measured by the adjusted odds ratios. In the semi-urgent group, the odds of on-time surgery were 36% lower in list-size category 2, OR = 0.64 (0.54–0.75), 47% lower in category 3, OR = 0.53 (0.45–0.62), and 51% lower in category 4, OR = 0.49 (0.41–0.60), compared to list-size category 1 (clearance time less than one week).
In the non-urgent group, the odds of on-time surgery were 25% lower in list-size category 2, OR = 0.75 (0.51–1.09), 62% lower in category 3, OR = 0.38 (0.26–0.56), and 69% lower in category 4, OR = 0.31 (0.20–0.47), compared to list-size category 1.
Every time an additional patient was operated without being registered on wait lists, for non-urgent patients registered in that week the odds of on-time surgery were reduced by 10%, OR = 0.90 (0.87–0.94). Similarly, for semi-urgent patients the odds of on-time surgery were reduced by 7%, OR = 0.93 (0.91–0.95), and for urgent patients were reduced by 25%, OR = 0.75 (0.65–0.87).
Discussion
Whether waiting times vary due to chance alone after adjustment for clinical factors and variations in demand remains an important question in health services research on access to care. However, chances of late surgery have not been previously described according to the length of wait list at registration in a multiple-list setting. This paper examines the relationship between the proportion of patients undergoing surgery within accepted standards and the length of the wait lists at registration for CABG surgery on multiple wait lists in a health system where all medically necessary services are publicly funded.
Using records from the provincial population-based registry of patients identified as needing isolated CABG, we determined the proportion of listed patients undergoing the operation within target access times across the list size categories. The list size was a simple count of patients with higher or equal priority present on the list at registration of a new patient on a given list.
We found that two in three patients were at risk of late surgery if registered on long wait lists. Our results provide evidence that list size had an effect on chances of undergoing elective surgery on time, with lower chances for longer lists in semi-urgent and non-urgent patients. When clearance time exceeded one week, half a month, or one month, the odds of on-time surgery were, respectively, 36%, 47%, and 51% lower in the semi-urgent group, and 25%, 62%, and 69% lower in the non-urgent group, compared to a shorter list, after adjustment for potential confounders. We also found an independent effect of the number of patients who were operated without being registered on wait lists.
Individual waiting times were analyzed as prospective observations beginning at the time of registration. We represented the wait for each patient by a sequence of binary indicators that indicate if the patient left the list at a certain wait-list week [13]. The likelihood function of such indicators can be factored into contributions that involve the conditional probabilities of surgery in a certain week among those remaining on the list. This observation justifies the practice of treating the binary indicators from one patient as independent Bernoulli trials [14]. To fit a pool of binary regression models developed for each patient by using the maximum likelihood method, it was assumed that binary indicators were independent across patients.
Misclassification of the recorded urgency of treatment is a concern in this analysis. Retrieved from the registry, the urgency category is a composite variable based on a variety of clinical factors. No audit has been performed to evaluate the quality of these records. The observation that higher priority patients were more likely to undergo CABG through the direct admission indicates that the degree of misclassification of priority was likely small. Another concern is that urgency of some patients were re-classified at the time of surgery. However, the timing of changes in urgency is not recorded.
The contribution of this paper to the research on access to care is three-fold. It provides evidence that for evaluation purposes one can not accurately estimate the proportion of late surgeries without considering the length of a wait list. It quantifies the effect of the queue length on the proportion of patients having late coronary bypass surgery in a network of hospitals with uniform standards for timing of surgery. It quantifies the effect of the operation performed without registration on a wait list, on the odds of on-time surgery in the patients registered on the list in that week.
Conclusion
Chances of late surgery increase with the wait-list size for semi-urgent and non-urgent patients needing coronary bypass surgery. The weekly number of patients who move immediately from angiography to the operation without registration on a wait list reduced chances of surgery within target time in all urgency groups of listed patients. Our findings have implications for policies on access to elective cardiac surgery in a network of hospitals. If queue length varies substantially from hospital to hospital, policy makers may consider re-distribution of cases across hospitals with the aim of reducing the proportion of late surgeries. Our results also suggest that an informed decision on choosing a surgeon requires cardiologists and patients to consider information about the chance of undergoing surgery beyond a target time and associated risks. When projecting the expected time to treatment for patients who will be placed on the wait list, hospital managers should take into account the current list size as well as the weekly number of patients who require CABG immediately after undergoing coronary angiography. More research is needed to evaluate whether referral patterns across hospitals depend on wait-list size.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Study concept and design: Sobolev. Data acquisition: Levy, Hayden. Analysis and interpretation: Sobolev, Kuramoto, Levy, Hayden. Drafting of the manuscript: Sobolev, Kuramoto.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This study received financial support from Canada Research Chairs Program (BS), Canada Foundation for Innovation (BS, AL), Michael Smith Foundation for Health Research (AL), Vancouver Coastal Health Research Institute (BS, LK), and St Paul's Hospital Foundation (AL). None of the sponsors had any role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or the decision to submit the paper for publication.
The following cardiac surgeons are members of the Surgical Research Committee: James Abel, Richard Brownlee, Larry Burr, Anson Cheung, James Dutton, Guy Fradet, Virginia Gudas, Robert Hayden, Eric Jamieson, Michael Janusz, Shahzad Karim, Tim Latham, Jacques LeBlanc, Sam Lichtenstein, Hilton Ling, John Ofiesh, Michael Perchin-sky, Peter Skarsgard and Frank Tyers.
The authors gratefully acknowledge the contributions of Mark FitzGerald, Martin Schechter, and Rita Sobolyeva. We are grateful to the external reviewers for thoughtful and useful suggestions.
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MacCormick AD Collecutt WG Parry BR Prioritizing patients for elective surgery: a systematic review ANZ J Surg 2003 73 633 642 12887536 10.1046/j.1445-2197.2003.02605.x
DeCoster C Measuring and managing waiting times: what's to be done? Healthcare Management Forum 2002 15 6 50 12078358
Kee F McDonald P Kirwan JR Patterson CC Love AH What is a safe waiting time for coronary artery bypass surgery? QJM 1997 90 669 676 9474347 10.1093/qjmed/90.11.669
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Sykora K Slaughter PM Young W Garlin D Naylor CD Cardiovascular Health and Services in Ontario: An ICES Atlas 1999 Institute for Clinical Evaluative Sciences 239 254
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Sobolev B Levy A Hayden R Kuramoto L Does wait-list size at registration influence time to surgery? Analysis of a population-based cardiac surgery registry Health Services Research 2005
Sobolev B Brown P Zelt D Shortt S Access to elective vascular surgery within the recommended time according to emergency referrals Clin Invest Med 2001 24 236 241 11603507
Sobolev B Brown P Zelt D Kuramoto L Waiting time in relation to wait-list size at registration: statistical analysis of a waiting-list registry Clin Invest Med 2004 27 298 305 15675109
Allison PD Discrete-Time Methods for the Analysis of Event Histories Sociological Methodology 1982 Jossey-Bass 61 98
Levy A Sobolev B Hayden R Kiely M FitzGerald M Schechter M Time on wait lists for coronary bypass surgery in British Columbia, Canada, 1991 – 2000 BMC Health Services Research 2005 5 22 32 15766381 10.1186/1472-6963-5-22
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Cardiovasc DiabetolCardiovascular Diabetology1475-2840BioMed Central London 1475-2840-4-131611149910.1186/1475-2840-4-13Original InvestigationFenofibrate Intervention and Event Lowering in Diabetes (FIELD) study: baseline characteristics and short-term effects of fenofibrate [ISRCTN64783481] The FIELD Study Investigators [email protected] The FIELD Study, C/o NHMRC Clinical Trials Centre, Mallett St Campus, University of Sydney NSW 2006, Australia2005 22 8 2005 4 13 13 29 6 2005 22 8 2005 Copyright © 2005 Keech and The FIELD Study Investigators; licensee BioMed Central Ltd.2005Keech and The FIELD Study Investigators; 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.
Objective
The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Study is examining the effects of long-term fibrate therapy on coronary heart disease (CHD) event rates in patients with diabetes mellitus. This article describes the trial's run-in phase and patients' baseline characteristics.
Research design and methods
FIELD is a double-blind, placebo-controlled trial in 63 centres in 3 countries evaluating the effects of fenofibrate versus placebo on CHD morbidity and mortality in 9795 patients with type 2 diabetes mellitus. Patients were to have no indication for lipid-lowering therapy on randomization, but could start these or other drugs at any time after randomization. Follow-up in the study was to be for a median duration of not less than 5 years and until 500 major coronary events (fatal coronary heart disease plus nonfatal myocardial infarction) had occurred.
Results
About 2100 patients (22%) had some manifestation of cardiovascular disease (CVD) at baseline and thus high risk status. Less than 25% of patients without CVD had a (UKPDS determined) calculated 5-year CHD risk of <5%, but nearly all had a 5-year stroke risk of <10%. Despite this, half of the cohort were obese (BMI > 30), most were men, two-thirds were aged over 60 years, and substantial proportions had NCEP ATP III features of the metabolic syndrome independent of their diabetes, including low HDL (60%), high blood pressure measurement or treatment for hypertension (84%), high waist measurement (68%), and raised triglycerides (52%).
After a 6-week run-in period before randomisation with all participants receiving 200 mg comicronized fenofibrate, there were declines in total and LDL cholesterol (10%) and triglycerides (26%) and an increase in HDL cholesterol (6.5%).
Conclusion
The study will show the effect of PPAR-alpha agonist action on CHD and other vascular outcomes in patients with type 2 diabetes including substantial numbers with low to moderate CVD risk but with the various components of the metabolic syndrome. The main results of the study will be reported in late 2005.
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Introduction
The cardiovascular benefits of long-term treatment using HMG-CoA reductase inhibitors (statins) have been conclusively shown in several studies (summarized in [1]) of people with and without established cardiovascular disease, which have included more than 18000 men and women with diabetes mellitus [2-8]. To date, however, no clinical trials using peroxisome proliferator-activated receptor (PPAR) alpha agonists (such as fibrates) have specifically examined cardiovascular event rate changes in large numbers of patients with diabetes mellitus. In the 6 trials of fibrates reported to date the aggregate number of patients with diabetes mellitus is just over 2000 [9-14], with VA-HIT accounting for about 40% of these [11]. Fibrates cause favorable changes to the typical lipid profile of type 2 diabetes mellitus, by raising high-density lipoprotein (HDL) cholesterol, lowering triglycerides, and reversing the tendency to formation of small, dense, low-density lipoprotein (LDL) cholesterol particles [15-18].
If PPAR alpha agonists (and other modulators of the PPAR axis) are to assume a role as mainstream agents for reducing the risk of fatal and nonfatal cardiovascular events, adequately powered placebo-controlled trials, similar to the statin trials, are needed. They should include patients with diabetes of both sexes across a wide range of age and absolute cardiovascular risk.
The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Study is a 3-country (Australia, New Zealand and Finland), 63-centre, double-blinded placebo-controlled trial evaluating the effects of fenofibrate compared with placebo on coronary heart disease morbidity and mortality in 9795 patients with type 2 diabetes [1]. The study incorporated a 6-week active-treatment run-in period, with all subjects receiving 200 mg comicronized fenofibrate before randomization to long-term placebo or active treatment. This article reports the baseline and treatment-entry characteristics of this cohort and defines the absolute risk of its study population.
Methods
The design of the FIELD study has been described in detail elsewhere [1]. The study was approved by local ethics committees at each participating institution. In brief, FIELD is a randomized double-blind, placebo-controlled, parallel-group trial. Participants are middle-aged to elderly people (50 to 75 years of age) with type 2 diabetes mellitus considered to be at risk of coronary heart disease. The first patient in FIELD was registered in November 1997 and randomized in February 1998. A total of 14 247 subjects were registered, and 13 900 were screened in clinics for eligibility at Visit 1.
Patients were recruited from 63 centres in Australia, New Zealand and Finland. Those with and without known vascular disease were eligible for participation provided that, at randomization, the usual physician considered that there was no current indication for lipid-modifying treatment. This meant that some patients meeting the FIELD eligibility criteria for lipid levels were instead treated by their usual doctors once they became aware of the patients' lipid profile at screening; therefore, such patients were not randomized and were not included in the study. Patients with or without lipid abnormalities, such as low HDL cholesterol or elevated triglycerides, were eligible if the total blood cholesterol level at screening fell between 3.0 and 6.5 mmol/L (about 115–250 mg/dL) and either the total-to-HDL cholesterol ratio was 4.0 or higher, or the triglyceride level was over 1.0 mmol/L. Patients were excluded if they had triglyceride levels over 5.0 mmol/L. Lipid entry criteria were consistent with recruiting people who would not qualify for fully subsidised lipid-modifying treatment under the government guidelines in all 3 countries. Participants could not be taking any lipid-modifying therapy at the start of the dietary run-in period. However, the protocol allows for statin or other lipid-lowering therapy to be added at any time after randomization and recommends continuing study medication. The study is thus evaluating the role of fenofibrate on a background of usual care and will provide safety data on fibrate therapy in combination with other lipid-lowering treatments. A prespecified analysis will explore the effects of fenofibrate separately among those patients taking other lipid-lowering agents during follow-up and those taking study treatment alone. Intention-to-treat methods of analysis will result in conservative estimates of any effects of fenofibrate in the event that drop-ins to statin treatment are more frequent in the placebo-treated group. Confounding with respect to other drug use should be prevented by the randomization process, which will result in good balance across the treatment groups.
Patients known to have type 2 diabetes (identified through hospital clinic records, diabetes society membership lists and various patient registers) were invited to attend a special clinic. After clinical screening of patients for eligibility and obtaining informed consent, blood was taken for biochemical measurements of fasting glucose, lipids, apolipoproteins, fibrinogen, HbA1c, insulin, renal and liver biochemistry, and homocysteine, and urine was collected for albumin-to-creatinine ratio. The LDL cholesterol level was calculated from the Friedewald formula [19]. Biochemical analysis used standard methods in 2 central laboratories (Southpath, Flinders Medical Centre, Adelaide, Australia, and the National Public Health Institute, Helsinki, Finland) which were aligned by using reference material from the Canadian External Quality Assurance Laboratory, Vancouver, Canada, traceable back to CDC, Atlanta, USA. Both laboratories participated in local external quality assurance programs. Eligibility was confirmed for consenting participants. All screened patients were asked to follow agreed national dietary recommendations for type 2 diabetes mellitus and provided with literature summarizing key dietary recommendations. Patients were asked to return after 4 weeks of dietary adherence for review of their interest in participating in the long-term trial, and for a check of laboratory eligibility (triglyceride <5.0 mmol/L, serum creatinine <130 μmol/L, alanine aminotransferase (ALT) less than twice the upper limit of normal) and to commence a single-blind placebo run-in phase of one capsule daily with breakfast for a further 6-week period. Upon the patients' return after the placebo period, consenting eligible patients were then entered into a 6-week single-blind active run-in period. During this time, they were to take comicronized fenofibrate 200 mg once daily. The placebo run-period was conducted to familiarize patients with the long-term commitment to the trial. The active run-in was conducted to provide data on the short-term effects of fenofibrate therapy on cholesterol levels for all randomized patients.
After the 16-week run-in procedures had been completed, a further blood sample was taken at the time of randomization (week 0) to determine the short-term effects of the comicronized fenofibrate on total cholesterol, HDL cholesterol, triglycerides, LDL cholesterol and fibrinogen. At this visit, patients were randomized, by a stratified adaptive randomization scheme, to receive either fenofibrate (200 mg comicronized formulation) or matching long-term placebo as one capsule daily with breakfast [1]. The final patient was randomized to the study on 3 November 2000.
All patients were then followed up through regular visits to a special clinic set in place for the purposes of the study in addition to routine care provided through the family doctor and usual diabetes clinics.
For a primary outcome of CHD events (CHD death plus nonfatal MI), it is projected that approximately 500 CHD events will have occurred when a median of 5 years of follow-up has elapsed (during the first quarter of 2005); this represents an event rate of approximately 1% per annum. By this time the trial will have 80% power to detect an observed 22% reduction in CHD events (based on the intention-to-treat method of analysis). This will also provide 90% power to detect a 25% relative reduction in CHD events (based on intention-to-treat analysis). These calculations assumed an average drop-out rate from active treatment of 10% over the course of the study but allowed for a larger drop-in rate from placebo to open-cholesterol treatment of 17%* by the mid-point of the trial and 32% by study close, in view of the possible increased uptake of statin therapy after the Heart Protection Study.7 (*This was incorrectly referred to as 10% in the FIELD design paper,1 the assumption used in the original power calculations.)
Therefore, follow-up in the study, as stipulated in the protocol, was to be for a median duration of not less than 5 years and until 500 major coronary events had occurred, unless the study was terminated prematurely on the recommendation of the independent Safety and Data Monitoring Committee.
Results
Of the 13 900 patients screened in study clinics, 75.9% (10 553) proceeded to enter the placebo run-in phase and 73.4% (10 203) the active run-in phase, and ultimately 9795 (70.5%) patients were randomized (Figure 1). The demographic characteristics are shown for the randomized and nonrandomized patients in Table 1. The study cohort included more men than women, and had a mean age in the mid-60s, and just over 2100 randomized patients had a prior history of cardiovascular disease.
Figure 1 Numbers of patients enrolled in the run-in phases of the trial.
Table 1 Baseline characteristics of the FIELD study cohort
Characteristic Category Screened, not randomized
(n = 4105) Randomized
(n = 9795)
n % n %
Sex Male 2424 59.0 6138 62.7
Female 1681 41.0 3657 37.3
Age at Visit 1 (years) <55 682 16.6 1668 17.0
55–59 802 19.5 1976 20.2
60–64 856 20.8 2196 22.4
65–69 888 21.7 2202 22.5
70+ 877 21.4 1753 17.9
Ethnicity* Caucasian 3757 91.5 9093 92.8
Indigenous 161 3.9 258 2.6
Asian 93 2.3 141 1.4
Other 94 2.3 303 3.1
Body mass index (kg/m2) (BMI) BMI < 25 805 19.6 1198 12.2
25 ≤ BMI < 30 1511 36.8 3855 39.4
30 ≤ BMI < 35 1099 26.8 2869 29.3
BMI ≥ 35 652 15.9 1866 19.1
Waist (cm) Female (mean ± SD) 98 ± 15 101 ± 14
Male (mean ± SD) 103 ± 13 105 ± 12
Waist-hip ratio ratio < 0.8 242 5.9 333 3.4
0.8 ≤ ratio < 0.9 1184 28.8 2531 25.8
0.9 ≤ ratio < 1.0 1931 47.0 4942 50.5
1.0 ≤ ratio < 1.1 645 15.7 1797 18.4
ratio ≥ 1.1 73 1.8 179 1.8
Smoking (cigarettes) Nonsmoker 1735 42.3 4000 40.8
Ex-smoker 1933 47.1 4908 50.1
Current smoker 404 9.8 887 9.1
Clinical history Prior cardiovascular disease † 1045 25.4 2131 21.8
Prior myocardial infarction 278 6.7 485 5.0
Stroke 194 4.7 346 3.5
Angina 588 14.3 1188 12.1
Hypertension 2247 54.7 5544 56.6
Claudication or peripheral vascular disease 351 8.6 711 7.2
Transient ischemic attack 155 3.8 307 3.1
Prior coronary revascularisation No CABG or PTCA 3908 95.2 9432 96.3
CABG only 122 3.0 230 2.4
PTCA only 49 1.2 102 1.0
CABG and PTCA 26 0.6 31 0.3
Diabetes management Diet only 1119 27.3 2542 26.0
Diet + OH only 2238 54.5 5874 60.0
Diet + insulin 429 10.5 569 5.8
Diet + OH + insulin 319 7.8 810 8.3
Diabetic complications ‡ Retinopathy 431 10.5 814 8.3
Neuropathy 636 15.5 1394 14.2
Nephropathy 161 3.9 279 2.9
Skin ulcers 139 3.4 299 3.1
Amputations 106 2.6 176 1.8
Diabetes diagnosis ‡ Age at diagnosis (mean ± SD) 54.8 ± 8.7 55.5 ± 8.3
Median duration of diabetes in years (quartile 1, quartile 3) 6 (2, 11) 5 2, 10
* Patients are assumed to be Caucasian unless otherwise stated. Indigenous includes Aborigines, Torres Strait Islanders, Maori and Pacific Islanders. Other includes mixed races, African-American, Indian, African, etc.
† Prior cardiovascular disease is defined as a reported history of myocardial infarction, angina (stable and unstable), coronary revascularization (CABG or PTCA), stroke, claudication or peripheral vascular disease or peripheral revascularization before randomization. The count includes 13 randomized patients and 10 nonrandomized patients who suffered or reported a cardiovascular event during the run-in period (visit 1 to visit 4 (randomization)).
‡ Diabetic complications and age at diabetes diagnosis are self-reported.
CABG = coronary artery bypass grafting; PTCA = percutaneous transluminal coronary angioplasty; SD = standard deviation; OH = oral hypoglycemic agents
There were 6051 subjects randomized from Australia, 2351 from New Zealand and 1393 from Finland. There were no differences across the 3 countries in mean ages of patients or ages at diagnosis (55.8 years, 53.0 years and 56.2 years for Australia, Finland and New Zealand, respectively). The median periods from diabetes diagnosis to randomization were 5, 7 and 5 years, respectively. The patients recruited were a mix from hospital and the community, and therefore were diverse in absolute risk. The 7664 participants who had no history of cardiovascular disease were at lower risk on the basis of interim determination using the modified Framingham equation of the UKPDS Risk Engine (Table 2) [20,21]. The prevalence rates of the various components of the metabolic syndrome (using criteria from NCEP ATP III definitions) at baseline are detailed in Table 3. A total of 84% of subjects met the ATP III criteria for metabolic syndrome. Baseline biochemistry for the entire randomized cohort and the effects of the 6 weeks of treatment with comicronized fenofibrate are shown in Table 4. On average, there was a 10% reduction in total cholesterol, a 10% reduction in LDL cholesterol, a 26% reduction in triglycerides and a 6.5% increase in HDL cholesterol after this short-term exposure to the active agent (average HDL increase was 6.1% in men versus 7.3% in women). Apolipoprotein B levels fell by an average of 14%, and the total-to-HDL cholesterol ratio by about 15%. Creatinine levels increased by an average of nearly 13% (an effect that is believed to be reversible and harmless, though not fully understood) [22,23], and fibrinogen levels fell by 11%.
Table 2 Distribution of projected 5-year risk (%, UKPDS risk engine) of coronary and other vascular outcomes for randomized patients with no history of cardiovascular disease (n = 7664)
Outcome measures Projected 5-year risk (%, from the UKPDS risk engine)
<5% 5%–<10% 10%–<15% 15%–<20% ≥20%
All CHD 24.6 37.0 20.7 9.5 8.2
Fatal CHD 56.1 26.8 9.9 4.6 2.6
All stroke 79.3 17.0 2.8 0.7 0.2
Fatal stroke 99.9 0.1 0.0 0.0 0.0
Table 3 Prevalence of various components of the metabolic syndrome among the 9795 randomized participants in the FIELD study
Men
(n = 6138) Women
(n = 3657) Total
(n = 9795)
Metabolic syndrome feature: ATPIII criteria * n % n % n %
Waist measurement (women >88 cm; men >102 cm) 3613 58.9 3034 83.0 6647 67.9
Triglycerides >= 1.7 mmol/L 3073 50.1 2020 55.2 5093 52.0
HDL (men <40 mg/dL; women <50 mg/dL) 3365 54.8 2455 67.1 5820 59.4
Hypertension (SBP >= 130 and DBP >= 85 mm Hg) 5050 82.3 3131 85.6 8181 83.5
Fasting glucose >= 110 mg/dL or diabetes 6133 99.9 3646 99.7 9779 99.8
* When 3 of the 5 listed characteristics are present, a diagnosis of metabolic syndrome is established (84% of the cohort met the criteria)
Table 4 Baseline fasting biochemistry for the 9795 randomized participants in the FIELD study
Baseline After 6 weeks of fenofibrate||
Parameter Mean SD Median Q1, Q3 Mean SD
Total cholesterol (mmol/L) 5.04* 0.69 5.03* 4.56, 5.54 4.49 0.69
HDL cholesterol (mmol/L)¶ 1.10* 0.26 1.06* 0.92, 1.24 1.16 0.29
Calculated LDL cholesterol (mmol/L) 3.07* 0.64 3.08* 2.62, 3.51 2.71 0.62
Triglycerides (mmol/L) 1.94* 0.88 1.74* 1.34, 2.34 1.37 0.63
Total-to-HDL cholesterol ratio 4.81* 1.10 4.71* 4.04, 5.49 4.08 1.13
Apolipoprotein B (g/L) 0.97* 0.17 0.97* 0.86, 1.09 0.83 0.18
Creatinine (mmol/L) 0.08† 0.02 0.08† 0.07, 0.09 0.09 0.02
Fibrinogen (g/L) 3.6† 0.8 3.6† 3.1, 4.1 3.12 0.75
Fasting glucose (mmol/L) 8.9† 2.6 8.5† 7.0, 10.3 -- --
Urinary albumin-to-creatinine ratio (mg/mmol) 5.9† 21.8 1.2† 0.7, 3.0 -- --
Homocysteine (μmol/L) 10.2§ 3.7 9.5§ 7.9, 11.6 -- --
Insulin (mU/L) 15.7‡ 24.0 12.0‡ 8.0, 18.5 -- --
HbA1c (%) 7.1† 1.4 6.9† 6.1, 7.8 -- --
* Average of Visit 2 and Visit 3.
† Average of Visit 1 and Visit 3.
‡ Visit 1 only.
§ Visit 3 only.
¶ Baseline HDLc (mean ± SD): men = 1.03 ± 0.23 mmol/L, women = 1.21 ± 0.28 mmol/L
|| All changes at six weeks were statistically significant at P < 0.0001
-- not measured
Discussion
Almost all lipid-modifying trials that have included patients with diabetes have used statins as the intervention agent. Data on the effects of statin therapy among more than 18000 persons with type 2 diabetes are now available and show important reductions in coronary and vascular events [24] . Those studies involving more than 1000 people with diabetes include Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) [5], the Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) study,[5] the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT-LLT) [4], the Heart Protection Study [7] and the most recently reported trial, the Collaborative Atorvastatin Diabetes Study (CARDS). CARDS used atorvastatin, 10 mg/day, and showed a 36% reduction in combined cardiovascular endpoints (2.46% per year for placebo and 1.54% for atorvastatin-treated) [8]. The results from a similarly structured trial (Atorvastatin Study for Prevention of Coronary Heart Disease Endpoints in Non Insulin Dependent Diabetes Mellitus, ASPEN) are pending.
Two large-scale trials of fibrate therapy have also been completed: the Veterans Low-HDL Cholesterol Intervention Trial (VA-HIT) and the Bezafibrate Infarct Prevention (BIP) trial enrolled subjects with and without diabetes mellitus. Both studies were limited to people with prior myocardial infarction and reported reductions in major cardiovascular events among participants with low HDL and high triglycerides at baseline, which were greater than with use of the same fibrate among those without dyslipidemia [11,25]. The VA-HIT trial also reported lower coronary heart disease mortality in those with diabetes receiving gemfibrozil and reduced cardiovascular events, though rates of nonfatal myocardial infarction did not change significantly [11]. A third, smaller, trial that used a fibrate, the Diabetes Atherosclerosis Intervention Study (DAIS), showed reduced progression of established coronary atherosclerosis among those randomized to fenofibrate compared with those receiving matching placebo over 3 years [10].
The identification of the PPAR transcription factor as the primary pathway through which fibrate and glitazone-agonist actions are triggered [18] has stimulated renewed interest in the antiatherogenic effects of these agents. PPAR-alpha activation by fibrates has the potential to prevent atherosclerosis via regulation of lipid metabolism and hemostatic pathways [26,27]. Consequently, there is great interest in the FIELD study because it will generate clinical information about fibrates in diabetes similar to that which is already available for the statins.
These baseline data of the FIELD study cohort without known prior cardiovascular disease show that over half such patients had a (UKPDS-determined) calculated 5-year coronary heart disease risk of less than 10% and nearly all patients had a stroke risk of less than 10% over 5 years. Part of the reason for this low-risk status may be that the duration of diabetes was only 5 years, on average, and reflecting this short duration, the median HbA1c was 6.9% for the entire cohort and the proportion of patients using no diabetes glucoregulatory therapy was just over a quarter. This probably reflects the recruitment process, in that many community-based subjects chose to enter the study in response to information provided by newsletters sent through Diabetes Australia, the Finnish Diabetes Society, the New Zealand Society for the Study of Diabetes and a New Zealand national diabetes consumer database.
Nonetheless, the cohort still had a profile with many characteristics of high cardiovascular risk. Half of the patients were obese (BMI > 30), most were male, two-thirds were over the age of 60 years, and substantial proportions had NCEP ATP III features of the metabolic syndrome additional to their diabetes mellitus, including low HDL cholesterol, either high blood pressure measurement and/or treatment for hypertension, high waist measurement and raised triglyceride. Over 2100 (22%) had established cardiovascular disease, and 39% of those without known cardiovascular disease had a projected 5-year absolute risk of a coronary event higher than 10%.
Evaluation of the effect of comicronized fenofibrate over 6 weeks immediately before randomization was included in the protocol. The purpose of this was to measure the effect of the agent in the entire cohort. After 6 weeks there were major reductions in triglycerides, lesser decreases in LDL cholesterol, and rises in HDL cholesterol. Creatinine levels rose while fibrinogen fell, several of the effects that have been observed elsewhere and with other fibrate agents [28,29].
Approximately 140 million adults were estimated to have diabetes mellitus in 1997; it is the most common endocrine disorder worldwide. Projections put diabetes prevalence by 2010 at 221 million, about 60 percent higher. Just as many people again have an elevated fasting glucose level, or impaired fasting glucose, which can progress rapidly to diabetes [30]. Without the FIELD study data, doctors would remain uncertain about the merits of using PPAR-alpha agonists, with or without concomitant statin therapy, when confronted with a patient with diabetes mellitus. The study will have special application to showing the effect of PPAR alpha agonist action on cardiovascular outcomes in those with low HDL dyslipidemic profiles. The main results of the FIELD study will be reported in late 2005.
Declaration of competing interests
Of the Management Committee of the FIELD Study:
PB, YAK and RS have received reimbursements, fees, funding, or salary in the past five years from an organization that may in any way gain or lose financially from the publication of this paper;
No authors hold or have held stocks or shares in such an organization;
No authors have other financial competing interests;
AK has the following nonfinancial competing interest: Advisory board membership.
Authors' contributions
The Writing Committee are the authors responsible for this article.
Study organization
Writing committee
R Scott, J Best, P Forder, M-R Taskinen, J Simes, P Barter, A Keech
Management Committee
P Barter*, J Best*, P Colman, M d'Emden, T Davis, P Drury, C Ehnholm, P Glasziou, D Hunt, A Keech* (study chairman and principal investigator), YA Kesaniemi, M Laakso, R Scott*, RJ Simes*, D Sullivan, M-R Taskinen*, M Whiting; J-C Ansquer, B Fraitag (non-voting sponsor representatives). * Executive Committee members
Outcomes Assessment Committee
N Anderson, G Hankey, D Hunt (chairman), S Lehto, S Mann, M Romo; LP Li (outcomes officer, in attendance),
Safety and Data Monitoring Committee
C Hennekens, S MacMahon (chairman), S Pocock, A Tonkin, L Wilhelmsen; P Forder (unblinded statistician, in attendance).
Site Principal investigators
Australia: H Akauola, F Alford, P Barter, I Beinart, J Best, S Bohra, S Boyages, P Colman, H Connor, D Darnell, T Davis, P Davoren, F Lepre, F De Looze, M d'Emden, A Duffield, R Fassett, J Flack, G Fulcher, S Grant, S Hamwood, D Harmelin, R Jackson, W Jeffries, M Kamp, L Kritharides, L Mahar, V McCann, D McIntyre, R Moses, H Newnham, G Nicholson, R O'Brien, K Park, N Petrovsky, P Phillips, G Pinn, D Simmons, K Stanton, B Stuckey, D R Sullivan, M Suranyi, M Suthers, Y Tan, M Templer, D Topliss, J H Waites, G Watts, T Welborn, R Wyndham; Finland: H Haapamaki, A Kesaniemi, M Laakso, J Lahtela, H Levanen, J Saltevo, H Sodervik, M Taskinen, M Vanhala; New Zealand: J Baker, A Burton, P Dixon, J Doran, P Drury, P Dunn, N Graham, A Hamer, J Hedley, J Lloyd, P Manning, I McPherson, S Morris, C Renner, R Scott, R Smith, M Wackrow, S Young.
Co-investigators and site coordinators
Australia: F Alard, J Alcoe, F Alford, C Allan, J Amerena, R Anderson, N Arnold, T Arsov, D Ashby, C Atkinson, L Badhni, M Balme, D Barton, B Batrouney, C Beare, T Beattie, J Beggs, C Bendall, C Bendall, A Benz, A Bond, R Bradfield, J Bradshaw, S Brearley, D Bruce, J Burgess, J Butler, M Callary, J Campbell, K Chambers, J Chow, S Chow, K Ciszek, P Clifton, P Clifton-Bligh, V Clowes, P Coates, C Cocks, S Cole, D Colquhoun, M Correcha, B Costa, S Coverdale, M Croft, J Crowe, S Dal Sasso, W Davis, J Dunn, S Edwards, R Elder, S El-Kaissi, L Emery, M England, O Farouque, M Fernandez, B Fitzpatrick, N Francis, P Freeman, A Fuller, D Gale, V Gaylard, C Gillzan, C Glatthaar, J Goddard, V Grange, T Greenaway, J Griffin, A Grogan, S Guha, J Gustafson, P S Hamblin, T Hannay, C Hardie, A Harper, G Hartl, A Harvey, S Havlin, K Haworth, P Hay, L Hay, B Heenan, R Hesketh, A Heyworth, M Hines, G Hockings, A Hodge, L Hoffman, L Hoskin, M Howells, D Hunt, A Hunt, W Inder, W Inder, D Jackson, A Jovanovska, K Kearins, P Kee, J Keen, D Kilpatrick, J Kindellan, M Kingston-Ray, M Kotowicz, A Lassig, M Layton, S Lean, E Lim, F Long, L Lucas, D Ludeman, D Ludeman, C Ludeman-Robertson, M Lyall, L Lynch, C Maddison, B Malkus, A Marangou, F Margrie, K Matthiesson, J Matthiesson, S Maxwell, K McCarthy, A McElduff, H McKee, J McKenzie, K McLachan, P McNair, M Meischke, A Merkel, C Miller, B Morrison, A Morton, W Mossman, A Mowat, J Muecke, P Murie, S Murray, P Nadorp, S Nair, J Nairn, A Nankervis, K Narayan, N Nattrass, J Ngui, S Nicholls, V Nicholls, JA Nye, E Nye, D O'Neal, M O'Neill, S O'Rourke, J Pearse, C Pearson, J Phillips, L Pittis, D Playford, L Porter, L Porter, R Portley, M Powell, C Preston, S Pringle, W A Quinn, J Raffaele, G Ramnath, J Ramsden, D Richtsteiger, S Roffe, S Rosen, G Ross, Z Ross, J Rowe, D Rumble, S Ryan, J Sansom, C Seymour, E Shanahan, S Shelly, J Shepherd, G Sherman, R Siddall, D Silva, S Simmons, R Simpson, A Sinha, R Slobodniuk, M Smith, P Smith, S Smith, V Smith-Orr, J Snow, L Socha, T Stack, K Steed, K Steele, J Stephensen, P Stevens, G Stewart, R Stewart, C Strakosch, M Sullivan, S Sunder, J Sunderland, E Tapp, J Taylor, D Thorn, D Thorn, A Tolley, D Torpy, G Truran, F Turner, J Turner, J van de Velde, S Varley, J Wallace, J Walsh, J Walsh, J Walshe, G Ward, B Watson, J Watson, A Webb, F Werner, E White, A Whitehouse, N Whitehouse, S Wigg, J Wilkinson, E Wilmshurst, D Wilson, G Wittert, B Wong, M Wong, S Worboys, S Wright, S Wu, J Yarker, M Yeo, K Young, J Youssef, R Yuen, H Zeimer, R W Ziffer; Finland: A Aura, A Friman, J Hanninen, J Henell, N Hyvarinen, M Ikonen, A Itkonen, J Jappinen, A Jarva, T Jerkkola, V Jokinen, J Juutilainen, H Kahkonen, T Kangas, M Karttunen, P Kauranen, S Kortelainen, H Koukkunen, L Kumpulainen, T Laitinen, M Laitinen, S Lehto, R Lehto, E Leinonen, M Lindstron-Karjalainen, A Lumiaho, J Makela, K Makinen, L Mannermaa, T Mard, J Miettinen, V Naatti, S Paavola, N Parssinen, J Ripatti, S Ruotsalainen, A Salo, M Siiskonen, A Soppela, J Starck, I Suonranta, L Ukkola, K Valli, J Virolainen; New Zealand: P Allan, W Arnold, W Bagg, K Balfour, T Ball, B Ballantine, C Ballantyne, C Barker, C Barker, F Bartley, E Berry, G Braatvedt, A Campbell, T Clarke, R Clarke, A Claydon, S Clayton, P Cresswell, R Cutfield, J Daffurn, J Delahunt, A Dissnayake, C Eagleton, C Ferguson, C Florkowski, D Fry, P Giles, M Gluyas, C Grant, P Guile, M Guolo, P Hale, M Hammond, M Hammond, P Healy, M Hills, J Hinge, J Holland, B Hyne, A Ireland, A Johnstone, S Jones, G Kerr, K Kerr, M Khant, J Krebs, L Law, B Lydon, K MacAuley, R McEwan, P McGregor, B McLaren, L McLeod, J Medforth, R Miskimmin, J Moffat, M Pickup, C Prentice, M Rahman, E Reda, C Ross, A Ryalls, D Schmid, N Shergill, A Snaddon, H Snell, L Stevens, A Waterman, V Watts.
Coordinating centre teams
NHMRC Clinical Trials Centre, Sydney: K Jayne, E Keirnan, P Newman, G Ritchie, A Rosenfeld (project directors), E Beller, P Forder, V Gebski, A Pillai (study statisticians), C Anderson, S Blakesmith, S-Y Chan, S Czyniewski, A Dobbie, S Doshi, A Dupuy, S Eckermann, M Edwards, N Fields, K Flood, S Ford, C French, S Gillies, C Greig, M Groshens, J Gu, Y Guo, W Hague, S Healy, L Hones, Z Hossain, M Howlett, J Lee, L-P Li, T Matthews, J Micallef, A Martin, I Minns, A Nguyen, F Papuni, A Patel, J Pearse, R Pike, M Pena, K Pinto, D Schipp, J Schroeder, B Sim, C Sodhi, T Sourjina, C Sutton, R Taylor, P Vlagsma, S Walder, R Walker, W Wong, J Zhang, B Zhong, A Keech (deputy director), RJ Simes (director); Helsinki Project Office: A Kokkonen, P Narva, E-L Niemi, A Salo, A-M Syrjanen, M-R Taskinen (director); Christchurch Project Office: C Lintott, R Scott (director).
Central laboratories
Adelaide: R Tirimacco, M Whiting; Helsinki: C Ehnholm, M Ikonen, M Kajosaari, L Raman, J Sundvall, M Tukianen.
Laboratoires Fournier SA liaison: Dijon: J-C Ansquer, B Fraitag, D Crimet, I Sirugue, Sydney: P Aubonnet.
Acknowledgements
The FIELD study is supported by a grant from Laboratoires Fournier SA, Dijon, France and is being coordinated independently by the National Health and Medical Research Council Clinical Trials Centre, University of Sydney, Sydney, Australia, and overseen by the study Management Committee.
The study is also supported by the National Health and Medical Research Council, Australia (Unit grant, Project grant, and Fellowships to A. Keech and J. Simes), without which it would not be possible. We thank the National Heart Foundation, Australia, Diabetes Australia, Diabetes New Zealand, and the Finnish Diabetes Association for endorsing the study. Investigators express their thanks to Rhana Pike for her assistance with the preparation of this manuscript. Finally, the many patients participating in the FIELD study are thanked for their untiring contributions.
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Mol PainMolecular Pain1744-8069BioMed Central London 1744-8069-1-281619120210.1186/1744-8069-1-28ResearchAcidification of rat TRPV1 alters the kinetics of capsaicin responses Neelands Torben R [email protected] Michael F [email protected] Ping [email protected] Connie R [email protected] Carol S [email protected] Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, R04PM, AP9A, Abbott Park, IL, 60064-6123, USA2005 28 9 2005 1 28 28 6 7 2005 28 9 2005 Copyright © 2005 Neelands et al; licensee BioMed Central Ltd.2005Neelands 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.
TRPV1 (vanilloid receptor 1) receptors are activated by a variety of ligands such as capsaicin, as well as by acidic conditions and temperatures above 42°C. These activators can enhance the potency of one another, shifting the activation curve for TRPV1 to the left. In this study, for example, we observed an approximately 10-fold shift in the capsaicin EC50 (640 nM to 45 nM) for rat TRPV1 receptors expressed in HEK-293 cells when the pH was lowered from 7.4 to 5.5. To investigate potential causes for this shift in capsaicin potency, the rates of current activation and deactivation of whole-cell currents were measured in individual cells exposed to treatments of pH 5.5, 1 μM capsaicin or in combination. Acidic pH was found to both increase the activation rate and decrease the deactivation rate of capsaicin-activated currents providing a possible mechanism for the enhanced potency of capsaicin under acidic conditions. Utilizing a paired-pulse protocol, acidic pH slowed the capsaicin deactivation rate and was readily reversible. Moreover, the effect could occur under modestly acidic conditions (pH 6.5) that did not directly activate TRPV1. When TRPV1 was maximally activated by capsaicin and acidic pH, the apparent affinity of the novel and selective capsaicin-site competitive TRPV1 antagonist, A-425619, was reduced ~35 fold. This shift was overcome by reducing the capsaicin concentration co-applied with acidic pH. Since inflammation is associated with tissue acidosis, these findings enhance understanding of TRPV1 receptor responses in inflammatory pain where tissue acidosis is prevalent.
kineticselectrophysiologypatch-clamppharmacologyTRPV1acidcapsaicin
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Background
The vanilloid receptor 1 (TRPV1) is a member of the transient receptor potential family (TRP) of non-selective cation channels [1]. These receptors are activated by a variety of lipids, acidic conditions and temperatures above 42°C. TRPV1 channels are tetramers composed of subunits with six transmembrane spanning domains, a pore loop between TM5 and TM6, and large N- and C-terminal intracellular domains [2]. An intracellular domain just C-terminal to TM6 has been characterized as being important in the tetramerization of the channel and is coincident, in part, with the TRP box that is common among this family of ion channels [3].
The structural features of TRPV1 suggest that the primary ligand interaction site(s) and important regulatory mechanisms for the channel are intracellular. Indeed, multiple mutagenesis studies have shown that distinct intracellular regions are necessary for the binding of the exogenous TRPV1 agonist, capsaicin [4-6] although an extracellular site may also contribute to capsaicin binding [7]. In contrast, extracellular site acidic residues have been implicated in proton activation (at pH < 6) and sensitization of TRPV1 [8]. Further evidence that TRPV1 activation mechanisms are different for capsaicin and protons is provided by site-directed mutagenesis studies that disrupt capsaicin activation of the channel but leave proton actions intact [9]. Despite these differences, there is evidence of some commonality in the gating of the channel in response to capsaicin or acidic pH activation [10].
Under pathological conditions multiple agents may simultaneously influence the activity of TRPV1 receptors. For instance, inflammation, ischemia, and infections result in elevated proton concentrations that can reduce the pH below 6 in the surrounding tissues [11]. Acidic pH has been shown to stimulate a subpopulation of sensory nerves that are also activated by capsaicin [12]. In addition, disruption of the TRPV1 gene attenuates proton-induced excitation of C-fibers [13], supporting a key role for TRPV1 in inflammatory pain. Treatment of TRPV1 receptors with capsaicin in the presence of other activators, including heat and acid results in a leftward shift of the capsaicin concentration response curve [11,14]. This suggests additive or synergistic effects of acid or heat on TRPV1 activation by capsaicin. Such effects may occur through changes in capsaicin affinity or gating.
In this study, we found that acidic conditions (pH 6.5 to 4.0) alter both the activation and deactivation rate of capsaicin-activated currents, resulting in increased potency of capsaicin for TRPV1, with no change in efficacy. In contrast, the inhibitory potency of a novel competitive TRPV1 antagonist, A-425619, was significantly lowered when the two activators were co-applied. These results highlight the breadth of TRPV1 responses to different stimuli and the concept that this channel (as well as other TRP channels) may act not only as an integrator of different physical stimuli but also as a coincidence detector that may be important in determining the resultant physiological response to endogenous activators.
Results
Activation and inactivation kinetics of TRPV1 channels in response to capsaicin or acidic pH
Whole-cell patch-clamp electrophysiological techniques were employed to characterize the responses of rat TRPV1, stably expressed in HEK-293 cells, to varying concentrations of capsaicin. Short (5 s) applications of capsaicin (0.1–10 μM) to rat TRPV1-expressing cells held at -60 mV resulted in a concentration-dependent increase of inward currents (Figure 1A). The rates of activation and deactivation of these responses were calculated using a single exponential equation (see methods). As capsaicin concentrations increased, the rate of activation dramatically increased from a τ of 1107 ± 242 ms at 0.1 μM to 582 ± 141 ms at 1 μM and 164 ± 33 ms at 10 μM (n = 4 – 6) (Figure 1B). In contrast, the deactivation rate slowed slightly with increasing concentrations of capsaicin (741 ± 56, 835 ± 54, 1121 ± 249 ms for 0.1, 1 and 10 μM capsaicin respectively; n = 4 – 6) (Figure 1B).
Figure 1 Kinetics of capsaicin-activated currents in HEK293 cells stably expressing rat TRPV1 channels. Application of increasing concentrations of capsaicin to cells voltage clamped at -60 mV result in progressively larger inward currents with faster activation rates and slower deactivation rates. (A) Representative current traces. (B) Plot of average activation and deactivation rates as a function of capsaicin concentration. Data are the mean ± SEM.
Similar experiments were performed to investigate the whole-cell current kinetics of rat TRPV1 in response to acidification of the extracellular solution. Decreasing the extracellular pH from 7.4 to 4.5 in stepwise fashion resulted in a pH dependent increase in inward current (Figure 2A). Average normalized peak currents were plotted as a function of the extracellular pH and fit with a logistic equation resulting in an EC50 of pH 5.35 and Hill slope of 1.3 (n = 6) (Figure 2B), which is similar to previous reports for acid activation of TRPV1 [11]. Activation rates increased from 546 ± 89 ms at pH 6.5 to 298 ± 62 ms at pH 4.5 (n = 3) (Figure 2C). A slight increase in the deactivation rate (70.5 ± 14.7 ms at pH 6.5 to 30.1 ± 3.7 ms at pH 4.5; n = 3 – 4) was found as pH was lowered (Figure 2C).
Figure 2 Kinetics of acid-activated currents in HEK293 cells stably expressing rat TRPV1 channels. Application of solutions with increasing proton concentrations to cells voltage-clamped at -60 mV result in progressively larger inward currents with slightly slower activation and deactivation rates. (a) Representative current traces. (b) pH response curve of average normalized currents. (c) Plot of average activation and deactivation rates as a function of pH. In contrast to the activation rates, which are similar between capsaicin and acid, the deactivation rates of acid responses are much faster than the capsaicin responses. Data are the mean ± SEM.
Effect of acidic conditions on the potency of capsaicin for rat TRPV1
Capsaicin responses were measured on the same cell at pH 7.4 (30 nM – 30 μM) and pH 5.5 (3 nM – 1 μM) to determine if there was a similar shift in the potency of capsaicin. Peak currents were normalized to the maximum response for each condition and the averages were plotted as a function of capsaicin concentration (n = 6–7). The resulting dose response curves illustrate a significant leftward shift in the EC50 for capsaicin from 640 nM (nH = 0.9) at pH 7.4 to 45 nM (nH = 1.6) at pH 5.5 (Figure 3). Peak inward currents evoked by application of 30 μM were not significantly different at pH 7.4 (1577 ± 349 pA) than at pH 5.5 (1596 ± 366 pA) (n = 7), indicating that there was no change in efficacy at this concentration of capsaicin (Figure 3, inset).
Figure 3 Capsaicin concentration-dependent activation of rat TRPV1 is shifted to the left under acidic conditions. Capsaicin potency is increased ~10 fold when the pH of the capsaicin-containing external solution is dropped from pH 7.4 (EC50 = 640 nM, closed squares) to pH 5.5 (45 nM, open circles). Currents evoked by application of pH 5.5 alone or that were required to hold the cell at -60 mV (resting holding current) were subtracted from peak responses. Data are the normalized average (mean ± SEM) of the resulting maximal currents for each condition. Inset) Peak responses of currents evoked by 30 μM capsaicin were compared at pH 7.4 and pH 5.5 on the same cell. No difference was detected in these experiments indicating that acidic conditions did not alter the efficacy of capsaicin. Data are the mean ± SEM.
The effect of acidic pH on the activation rate of capsaicin-evoked currents
The mechanism by which acidic pH increases the potency of capsaicin at the rat TRPV1 receptor was studied by co-applying the two activators either simultaneously or in a variety of sequences. Activation and deactivation rates of the resulting currents were measured. In the first set of experiments, the channel was exposed to pH 5.5 alone for 1 second, followed by 3 seconds of co-application with 1 μM capsaicin (Figure 4A). This protocol resulted in capsaicin-activated currents with much faster activation rates than occurred with the same capsaicin concentration at pH 7.4 (Figure 4B). The average activation rate was significantly increased from 847 ± 119 ms at pH 7.4 to only 149 ± 17 ms at pH 5.5 (n = 4) (Figure 4C). Moreover, the activation rate of the response to pH 5.5 increased after prior application of 1 μM capsaicin (τ = 93 ± 13 ms) compared with conditions where only pH 5.5 solutions were applied (τ = 917 ± 307 ms; n = 3), suggesting that capsaicin may increase the affinity of protons to rat TRPV1 (data not shown).
Figure 4 Acidic pH increases the activation rate of capsaicin-activated responses of rat TRPV1 channels expressed in HEK-293 cells. Comparison of the activation rate of 1 μM capsaicin-activated currents in rat TRPV1 expressing cells under control conditions (pH 7.4) and after pre-application of acidic solution (pH 5.5). (A) Representative current trace. (B) Overlay of normalized current traces illustrating increased activation rate of capsaicin under acidic conditions. (C) The activation rate of capsaicin-activated currents was significantly faster at pH 5.5 (149 ± 17 ms) than at pH 7.4 (847 ± 119 ms) (n = 4). Data are the mean ± SEM.
It has previously been reported that mildly acidic pH, which alone does not cause activation of TRPV1, produces changes in the response to capsaicin [11]. Therefore, we varied the pH of the pre-pulse to determine the effect of pH on the activation rate of capsaicin-evoked currents. Reducing the pH to 6.8 did not directly activate TRPV1 channels (Figure 2) but resulted in a subtle increase in the activation rate of capsaicin responses (Table 1). Increasing the acidity of the solution to pH 6.0 or pH 5.0 resulted in further significant increases of the activation rate of capsaicin currents (Table 1).
Table 1 Acid dependent effects on the activation rate of capsaicin-activated responses. Increasing the acidity of the external solution during a 1 second pre-pulse resulted in an increase in the activation rate of capsaicin-evoked currents in rat TRPV1 expressing HEK-293 cells. Values are the mean ± SEM of the calculated activation rate (τ) using a single exponential fit.
Condition τ (ms) n
Control (pH 7.4) 1176 ± 195 15
pH 6.8 1021 ± 128 15
pH 6.0 632.0 ± 86.5 13
pH 5.0 242.7 ± 49.7 7
The effect of acidic pH on the deactivation rate of capsaicin-evoked currents
The effect of acidic pH on the deactivation rate of capsaicin currents was tested using a paired-pulse protocol. In these experiments two short (1 s) applications of capsaicin were separated by 1–5 seconds of external solution at either pH 7.4 or pH 5.5. The 5 s interruptions are shown in Figure 5; similar results were obtained at shorter interpulse intervals. The deactivation rates of the first capsaicin responses were much slower with an acidic extracellular solution (τ = 10553 ± 1880 ms) (Figure 5B,C) than with an extracellular solution at pH 7.4 (τ = 854 ± 60 ms) (Figure 5A,C). In contrast, the deactivation rates in response to the second capsaicin application, reflecting the return to external solution of pH 7.4 in both cases, were only slightly faster following acid exposure than when the interpulse solution remained at physiological pH (τ = 944 ± 94 ms vs. 665 ± 114 ms; n = 4) (Figure 5D). These results suggest that acidic pH has a limited time-dependent effect on the channel. These effects of capsaicin, pH 5.5 or a combination of the two, on the activation and deactivation rates of evoked currents are summarized in Table 2.
Figure 5 Acidic pH decreases the deactivation rate of capsaicin-activated responses of rat TRPV1 channels expressed in HEK-293 cells. A double application protocol was used to investigate the effect of different pH's on the off rate of capsaicin. Two short applications of capsaicin (1 s) were interrupted by 1 to 5 second applications of either control external solution (pH 7.4) or acidic external (pH 5.5). A). Representative trace illustrating that the deactivation rates of both capsaicin applications are similar when the interpulse interval is at physiological pH. B) In contrast, when the interpulse interval is under acidic (pH 5.5) conditions, the deactivation rate of the initial application is substantially slower as illustrated by a representative trace. C) Comparison of the effect of external pH on the deactivation rates following the first capsaicin application. D) Comparison of the effect of external pH on the deactivation of the second capsaicin application. Data are the mean off rates (τ) ± SEM.
Table 2 Comparison of the activation and deactivation kinetics of TRPV1. Mean activation and deactivation rates of responses from TRPV1 expressing HEK-293 cells following application of capsaicin (1 μM), acidic pH (5.5) or the combination. Row 3 represents activation and deactivation rates of currents evoked by 1 μM capsaicin following pre-exposure to acidic conditions (pH 5.5). Row 4 represents the activation rates of acid-evoked currents following pre-exposure to capsaicin (1 μM). The absolute value of rate measurements had slight day-to-day variations (possibly due to relative position of the drug application tube). Therefore values in this table are from cells in which direct comparisons can be made. Values are mean ± S.E.M.
Activation Rates Deactivation Rates
τ (ms) n τ (ms) n
1 μM Capsaicin 847 ± 119 4 845 ± 60 4
pH 5.5 917 ± 307 3 56 ± 13 4
1 μM CAP (pH 5.5) 149.3 ± 17 4 10553 ± 1880 4
pH 5.5 (1 μM CAP) 93 ± 13 3 -not tested-
Note: Rates from experiments shown in Figures 4 and 5.
Additional experiments were performed to investigate the pronounced effect of acidic pH on the deactivation rate of capsaicin. A single application protocol was established in which TRPV1 channels were initially activated by 1 μM capsaicin followed by 1–8 seconds treatment with either pH 5.5 + capsaicin or pH 5.5 alone (Figure 6). When capsaicin was present throughout the protocol, the current amplitude showed little desensitization and was independent of the duration of application (Figure 6A). The deactivation phase following the termination of the co-agonist treatment had a biphasic time course with similar kinetics even as the duration of application was increased (bottom of Figure 6A). These characteristics were altered when capsaicin application was terminated but the acidic pH was maintained to the end of the protocol (Figure 6B). Under these conditions, the amplitude of the current declined in a linear fashion (dotted line, bottom Figure 6B) that was faster than when capsaicin was present but slower than when the external solution was at pH 7.4 (see Figures 1A and 5A). In addition, when the pulse duration was lengthened the biphasic current deactivation was comprised of primarily the fast deactivation component (bottom of Figure 6B).
Figure 6 Prolonged exposure of TRPV1 to acidic pH produces a time-dependent change in the deactivation rate of capsaicin-activated currents. Single pulse protocols were used to determine time dependent differences in the deactivation of capsaicin currents following prolonged exposure to acidic pH. A) Co-application of acid (pH 5.5) and capsaicin produced sustained currents independent of the duration of application. TRPV1 channels were activated by 1 μM capsaicin for 4 s, followed by 8 seconds of capsaicin + pH 5.5. The time dependence (1–8 s) of the sustained current is shown below and illustrates the reproducibility of the responses, the lack of desensitization and the parallel deactivation time-courses. B) Deactivation time constant of capsaicin response is decreased if acidic conditions persist after capsaicin application is terminated. Note the slow decline in current amplitude following removal of capsaicin, and the relatively faster deactivation rates of deactivation with longer intervals of acid pH application, presumably due to fast deactivating acid responses (see figure 2) contributing a greater proportion of the total current.
The pH dependent effects on the slowing of the capsaicin response were tested utilizing a paired-pulse protocol in which the pH of the solution was varied during the intervening segment (Figure 7). As illustrated in Figure 7A, lowering the pH to only 7.0 caused a small slowing of the off rate of the first pulse even though there was no significant change in the peak current (light gray line). The extent to which the deactivation rate was slowed was dependent on the acidity of the solution and was the greatest under conditions that produced direct channel activation (pH 6.5 and below – see figure 2B). Similar to the data shown in Figure 5B, the deactivation of the second capsaicin application was not affected by previous exposure to acid. The deactivation time of the first and second capsaicin applications was measured and the average rate was plotted as a function of the intervening pH (Figure 7B,C). The deactivation of the first capsaicin application slowed from a τ of 585 ± 43 ms at pH 7.4 to 7384 ± 929 ms at pH 5.5 (n = 7 – 8), whereas the deactivation of the second application ranged from 562 ± 40 to 689 ± 58 (n = 7 – 8) and showed no acid-dependent effects.
Figure 7 Proton concentration dependent slowing of the capsaicin deactivation rate. Two applications of capsaicin were interrupted by 5 s applications of external solutions with increasing proton concentrations. A) Representative current traces. The deactivation rate was slowed slightly by mildly acidic solutions that did not activate the channel directly but that potentiated the capsaicin response (pH 7.0 and 6.8). The slowing of the capsaicin deactivation rate was enhanced even more by application of acid that could directly activate the channel (pH 6.0 and 5.5). B) Concentration-dependent effects of pH on the deactivation-time constant of capsaicin. C) No concentration dependent effects were recorded for the deactivation rate of the second capsaicin application, indicating that previous exposure to acidic conditions does not affect subsequent capsaicin deactivation.
A single pulse protocol was utilized to further investigate the deactivation rate of the capsaicin-activated response. In these experiments a slowing of the deactivation rate of the capsaicin current occurred at a pH level (pH 6.5) that was insufficient to produce an increase in the peak current. This decrease in the deactivation rate resulted in a residual current at the end of the protocol that subsequently deactivated when the external solution was returned to pH 7.4 with a time course similar to control capsaicin responses (Figure 8A). Reducing the pH to 4.0 further slowed the deactivation rate of the capsaicin-evoked current. Both an increase in the peak current and a final deactivation with biphasic kinetics predominated by the fast deactivating acid responses are observed (Figure 8B).
Figure 8 A proton concentration unable to directly activate TRPV1 channels slows the deactivation rate of capsaicin responses. Representative traces from two different cells stimulated by a single capsaicin pulse application that further illustrates the proton concentration dependent effects on capsaicin deactivation rates. A) Representative current traces showing that the deactivation of capsaicin-activated currents is slowed when the pH of the external solution was reduced (6.5) even though this pH did not significantly potentiate the capsaicin response. B) Representative current traces illustrating that lowering the pH to more acidic conditions results in both an increase in maximal current amplitude and a striking slowing of the deactivation of the capsaicin response.
Effect of co-application of acidic pH and capsaicin on the apparent affinity of a competitive TRPV1 antagonist
A-425619 is a highly potent TRPV1 antagonist that is competitive at the capsaicin binding site [15]. Since acidic pH was found to increase the apparent affinity of capsaicin for TRPV1 by both increasing the activation rate and decreasing the deactivation rate of capsaicin-activated currents we evaluated the potency of A-425619 under various conditions. Inhibitory concentration response curves were generated for A-425619 (0.1–1000 nM) against TRPV1 channel currents activated by acid and capsaicin alone, as well as in combination. Normalized average peak currents evoked by the different activators were plotted as a function of A-425619 concentration and fitted with a logistic equation (Figure 9). Currents that were activated by the ~EC50 concentration of capsaicin (1 μM) or acid (pH 5.5) alone were inhibited with similar potency (IC50 = 25.9 nM, n = 6 and IC50 = 20.1 nM, n = 8 respectively). However, co-application of 1 μM CAP + pH 5.5, concentrations which produce near maximal response when applied together, produced a significant rightward shift in the IC50 of A-425619 to 704 nM (n = 3). In comparison, the potency of A-425619 on currents activated by co-application of 100 nM capsaicin + pH 5.5, which represents a response near the capsaicin EC75 under these acidic conditions, was 63.6 nM under these conditions (n = 4). By further reducing the capsaicin concentration to 30 nM (~EC35 under acidic conditions), the IC50 for A-425619 is 23.2 nM (n = 4). Data from the inhibitory concentration response curves for A-425619 were converted to produce a three point Schild plot. The shift in the IC50 of A-425619 (Figure 9B) at different concentrations of capsaicin was fit with a linear regression with a slope of 1.13 and a pA2 of 47.9 nM. These results are consistent with competitive mechanism of action for this antagonist (Table 3).
Figure 9 Potency of A-425619 is altered by co-application of capsaicin and acidic pH. A) A-425619 inhibits currents activated by half maximal concentrations of either capsaicin (1 μM) or protons (pH 5.5) with similar efficacy (20.1 vs. 25.9 nM). Co-application of these same concentrations of capsaicin and acidic pH results in a significant rightward shift in the IC50 of A-425619 (704 nM). Reducing the capsaicin concentration to 100 nM (near the EC75 under acidic conditions) or 30 nM (~EC35) shifts the IC50 of A-425619 back to the left (63.6 and 23.2 nM respectively). B) Schild plot of the effect of A-425619 at different capsaicin concentrations. The resulting data points were fit with a linear regression with a slope of 1.13 and a pA2 value of 47.9 nM. The linear relationship is consistent with the results that show acidic conditions increases the affinity of capsaicin for the TRPV1 channel and that A-425619 is a competitive antagonist at the capsaicin site.
Table 3 Comparison of the antagonist effects of A-425619 at TRPV1 responses evoked by different agonist(s). Values are the calculated IC50's and Hill slopes derived from fits of average normalized responses following the concentration-dependent inhibition by the TRPV1 competitive antagonist A-425619.
ACTIVATOR IC50 (nM) Hill Slope
1 μM Capsaicin 20.1 -1.1
pH 5.5 25.9 -1.2
30 nM CAP + pH 5.5 23.2 -1.1
100 nM CAP + pH 5.5 63.6 -0.95
1 μM CAP + pH 5.5 704.0 -0.93
Discussion
Recent evidence has illustrated the importance of TRPV1 channels as key integrators of painful stimuli. These receptors may be an important target for the development of novel analgesics. TRPV1 channels are directly activated by acid, heat and endogenous ligands such as NADA, OLDA and anandamide. In addition, inflammatory mediators such as bradykinin have been shown to sensitize TRPV1 through activation of protein kinases [16]. The sensitized TRPV1 channel has heightened responses, and, under these conditions, activators bind the channel with higher affinity. For instance, TRPV1 is normally activated at temperatures greater than 42°C, but after the channel has been sensitized, temperatures <35°C can result in channel activation [17]. Therefore, TRPV1 is not only responsive to multiple painful stimuli, but the additive or synergistic interactions between multiple stimuli make the receptor well suited as an integrator and coincidence detector of painful stimuli and thus underscore its potential as a significant contributor to nociception and hyperalgesia.
In this study we investigated the interaction of acid and capsaicin at TRPV1 in order to understand the mechanism by which these two activators modulate the effects of each other. We found that increasing the concentration of capsaicin or protons resulted in changes in the activation and deactivation rate of the activated currents. In addition, acid responses deactivated about 10-fold faster than capsaicin responses. These differences in deactivation rates may be due in part to the proposed different interaction sites of these two activators with the channel, i.e., the acid site has been proposed to be extracellular while capsaicin site is predominantly intracellular [18]. Alternatively, the different deactivation rates may be the result of differences in the binding affinity of the activators to the channel.
It has previously been shown that acidic conditions can alter the TRPV1 affinity for capsaicin [14] and that mildly acidic conditions that cannot directly activate the channel can alter capsaicin responses [11]. On a single channel level, acidic pH has been shown to potentiate capsaicin binding to TRPV1 as well as increase channel gating [10]. In the present study, we have confirmed these results and demonstrated the novel findings that these changes are due to a slowing of the deactivation rate and an increase in the activation rate of capsaicin-activated currents at acidic pH. In combination, these changes in kinetics result in larger current amplitudes due to a higher probability of channels being bound by agonist. These current amplitudes are similar to what is observed at higher capsaicin concentrations at neutral pH. Thus, one would expect that under acidic conditions 1 μM capsaicin would produce a similar response at TRPV1 as 10 μM capsaicin does at physiological pH, consistent with the 10-fold increase in the apparent affinity for capsaicin at acidic pH.
In contrast to the increased activation rate, the deactivation rate of capsaicin-activated currents under acidic conditions was considerably slower than the rate observed with high capsaicin concentrations at pH 7.4 (see Figure 1 and 5). It is possible that binding of protons alters the conformation of the channel in a way that traps capsaicin so it cannot be released unless the proton interaction is terminated. A recent publication has shown that acidic conditions, in combination with agonists, lock the TWIK-2 channel in an open conformation that persists even after the pH is neutralized [19]. The data in our study suggest that occupancy of the proton binding site of TRPV1 might result in an allosteric change in the receptor that prevents the release of capsaicin. However, a return to physiological pH could rapidly remove this trap and allows capsaicin to be released. The relatively fast deactivation rate of the acid response would provide opportunities for capsaicin to be released, although prolonged exposure to high concentrations of protons may dramatically reduce this probability. Further studies and kinetic modeling are needed to determine if this hypothesis can be supported.
Since TRPV1 can be activated by a variety of stimuli and these stimuli can cross-sensitize the receptor to each other, TRPV1 exhibits complex pharmacology. Cross-sensitization of the channel could potentially result in altered potency and/or efficacy of TRPV1 antagonists. Indeed, we have shown in these electrophysiological studies that application of capsaicin at acidic pH can significantly decrease the in vitro potency of A-425619, a competitive antagonist at the capsaicin-binding site of TRPV1, and that this shift becomes particularly dramatic under conditions at or approaching maximal channel activation. In contrast, A-425619 has similar potency (~20 nM) for blocking TRPV1 channel responses to half maximal concentrations of capsaicin or acid. It is possible that binding of the antagonist to the channel results in a conformational change that inhibits channel function independent of the mode of activation. Although there are multiple mechanisms for channel activation, a single site for antagonists may exist that is sufficient to prevent or modulate channel opening. This site most likely is the same or overlaps significantly with the site for capsaicin binding, since A-425619 is competitive with capsaicin [15].
Conclusion
We report the novel finding that acid alters both the activation and deactivation rates of capsaicin responses and thus provide a mechanism for the increase in potency of capsaicin, and perhaps other agonists, under acidic conditions. Since inflammation is associated with tissue acidosis, these results enhance understanding of the role of TRPV1 in inflammatory pain.
Methods
Cell Culture
HEK-293 cells stably expressing the rat TRPV1 receptor were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. Cells were plated onto glass coverslips coated with polyethyleneimine. Electrophysiological recordings were performed 1–4 days following plating.
Electrophysiology
HEK-293 cells were maintained at room temperature in an extracellular recording solution (pH 7.4, 325 mOsm) consisting of (in mM): 155 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 12 glucose. Patch-pipettes composed of boroscilicate glass (1B150F-3; World Precision Instruments, Inc., Sarasota, FL, USA), were pulled and fire-polished using a DMZ-Universal micropipette puller (Zeitz Instrumente GmbH, Munich, Germany). Pipettes (2–6 MΩ) were filled with an internal solution (pH 7.3, 295 mOsm) consisting of (in mM): 122.5 K-aspartate, 20 KCl, 1 MgCl2, 10 EGTA, 5 HEPES, 2 ATP·Mg. For experiments where pH was lowered (6.8 – 4.0) MES was used in place of HEPES in the external solution. No ASIC-like responses were evident in our TRPV1 expressing HEK cells but were recorded in HEK-293 null cells in response to application of acidic pH (unpublished observations). Standard whole-cell recording techniques were utilized for voltage-clamp studies using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, U.S.A). Coverslips plated with HEK-293 cells were placed in a perfusion chamber and following establishment of whole-cell recording conditions bath perfusion (~2 ml/min) was initiated. Application of control bath solution through a multi-barrel application device with a common 360 μm polyimide tip (Cell Microcontrols, Norfolk, VA, U.S.A), positioned ~100 μm from the cell, was continued throughout the recording except during drug application. Each drug reservoir was connected to solenoid teflon valves that were controlled by a ValveLink16 system (AutoMate Scientific, CA, San Francisco, U.S.A). Drug application protocols were established using pCLAMP (Axon instruments) software that controlled rapid valve switching through the ValveLink system. Drugs were applied by gravity feed through the drug application device for durations described in the results section. Solution exchange times were determined by applying 90% external solution to an open tip electrode positioned at the same distance as during a typical recording. Following a delay of ~50 ms from the opening of the solenoid valve, currents activated at a rate of 72 ± 9 ms with a 10–90% rise time of ~120 ms (n = 4). Similarly, the current deactivated with an deactivation rate of 84 ± 16 ms (n = 4). These values give an approximation of the limitations of the drug exchange for this system across an open tip electrode and indicate that our measurements of activation and deactivation rates of whole cell currents should not be significantly affected by solution exchange times. Prior to and following each drug application external solution was applied through the application device to ensure rapid washout. Each drug application sequence was followed by a washout period of 90 to 120 seconds.
Capsaicin concentration response curves were generated by applying increasing concentrations of capsaicin for 4 seconds followed by a 90 second washout period. These responses were evoked at pH 7.4 and pH 5.5 and the responses were normalized due to large variation in peak amplitude between cells, and the subsequent average data was fitted with a four-parameter logistic equation (see below). To determine potential changes in the efficacy of capsaicin responses a separate experiment was performed comparing peak responses to 30 μM capsaicin at pH 5.5 and pH 7.4 on the same cell. Generally there was substantial rundown of the currents during the first few applications of 30 μM capsaicin. Therefore, comparisons between the responses to pH 5.5 and pH 7.4 were made after two consecutive applications of capsaicin produced similar responses.
Inhibitory concentration response curves were performed by applying the agonist(s) for 4 seconds followed by 8 seconds of co-application of agonist and antagonist. Current levels at the end of the application period were measured and normalized to the current amplitude at the end of a 12 second control application of agonist alone. Normalized responses were then plotted as a function of antagonist concentration and fitted with a four-parameter logistic equation (see below).
Data acquisition and analysis were performed using pCLAMP 9.0 and subsequent graphs were plotted and statistical analysis done using GraphPad Prism (Graphpad Software, San Diego, CA, U.S.A). Activation and deactivation rates were calculated in Clampfit using a single exponential equation provided with the software:
Agonist and antagonist concentration-response curves were fitted by a least-squares regression to the logistic equation provide in the GraphPad software:
A Schild plot for A-425619 was constructed by converting data from inhibitory concentration response curves obtained at different capsaicin concentrations (in the presence of acidic pH) to capsaicin concentration response curves under different concentrations of antagonist. The resulting curves were fit with the logistic equation shown above and the resulting EC50 were used to calculate the log dose ratio that was then plotted as a function of capsaicin concentration. A linear regression was then used to fit these data points.
All reagents were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.) except A-425619 (synthesized at Abbott Laboratories, Abbott Park, IL, U.S.A).
Authors' contributions
TRN carried out the electrophysiological studies, conceived of the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. PH generated the stable TRPV1 cell line. MFJ and CSS participated in the design of the study. All authors read and approved the final manuscript.
Acknowledgements
We would like to thank Arthur Gomtsyan and Chih-Hung Lee for the synthesis of A-425619 and Steve McGaraughty and Robert Moreland for their critical comments on the manuscript.
==== Refs
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El Kouhen R Surowy CS Bianchi B Neelands T McDonald H Niforatos W Gomtsyan A Lee CH Honore P Sullivan J Jarvis M Faltynek C A-425619, a Novel and Selective TRPV1 Receptor Antagonist, Blocks Channel Activation by Vanilloids, Heat and Acid J Pharmacol Exp Ther 2005 314 400 9 15837819 10.1124/jpet.105.084103
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Crandall M Kwash J Yu W White G Activation of protein kinase C sensitizes human VR1 to capsaicin and to moderate decreases in pH at physiological temperatures in Xenopus oocytes Pain 2002 98 109 17 12098622 10.1016/S0304-3959(02)00034-9
Welch JM Simon SA Reinhart PH The activation mechanism of rat vanilloid receptor 1 by capsaicin involves the pore domain and differs from the activation by either acid or heat Proc Natl Acad Sci U S A 2000 97 13889 94 11095706 10.1073/pnas.230146497
Chemin J Patel AJ Duprat F Lauritzen I Lazdunski M Honore E A phospholipid sensor controls mechanogating of the K(+) channel TREK-1 Embo J 2005 24 44 53 15577940 10.1038/sj.emboj.7600494
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Nutr Metab (Lond)Nutrition & Metabolism1743-7075BioMed Central London 1743-7075-2-241616229510.1186/1743-7075-2-24ResearchImmunolocalization of RANKL is Increased and OPG Decreased During Dietary Magnesium Deficiency in the Rat Rude Robert K [email protected] Helen E [email protected] Livia Y [email protected] Angelica [email protected] University of Southern California and the Orthopaedic Hospital, 1975 Zonal Ave., GNH 6602, Los Angeles, CA 90089-9317, USA2 Department of Orthopaedic Surgery, Carolinas Medical Center, P.O. Box 32861, Charlotte, NC 28203, USA2005 14 9 2005 2 24 24 8 8 2005 14 9 2005 Copyright © 2005 Rude et al; licensee BioMed Central Ltd.2005Rude 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
Epidemiological studies have linked low dietary magnesium (Mg) to low bone mineral density and osteoporosis. Mg deficiency in animal models has demonstrated a reduction in bone mass and increase in skeletal fragility. One major mechanism appears to be an increase in osteoclast number and bone resorption. The final pathway of osteoclastogenesis involves three constituents of a cytokine system: receptor activator of nuclear factor kB ligand (RANKL); its receptor, receptor activator of nuclear factor kB (RANK); and its soluble decoy receptor, osteoprotegerin (OPG). The relative presence of RANKL and OPG dictates osteoclastogenesis. The objective of this study was to assess the presence of RANKL and OPG in rats on a low Mg diet.
Methods
RANKL and OPG were assessed by immunocytochemistry staining in the tibia for up to 6 months in control rats on regular Mg intake (0.5 g/kg) and experimental rats on reduction of dietary Mg (.04%, 25% and 50% of this Nutrient Requirement).
Results
At all dietary Mg intakes, alteration in the presence of immunocytochemical staining of RANKL and OPG was observed. In general, OPG was decreased and RANKL increased, reflecting an alteration in the RANKL/OPG ratio toward increased osteoclastogenesis.
Conclusion
We have, for the first time demonstrated that a reduction in dietary Mg in the rat alters the presence of RANKL and OPG and may explain the increase in osteoclast number and decrease in bone mass in this animal model. As some of these dietary intake reductions in terms of the RDA are present in a large segment of or population, Mg deficiency may be another risk factor for osteoporosis.
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Introduction
Severe magnesium (Mg) deficiency (0.04% of nutrient requirement, NR)[1] results in osteoporosis in rodent models characterized by decreased bone formation, increased bone resorption, and increased skeletal fragility [2-8]. This degree of Mg depletion rarely exists in humans; however, we have also found that a more moderate dietary Mg restriction, 10% of NR and 25% of NR, also results in bone loss in the rat [9,10]. We have also recently found that a diet of 50% of NR also causes a reduction in bone mass (submitted for publication). These studies suggest that an inadequate Mg intake may be a risk for osteoporosis. The RDA for Mg for adult males is 420 mg/d and for adult females is 320 mg/d [11,12]; the usual dietary Mg intake however falls below this recommendation in a large proportion of the population [11-13]. Epidemiologic studies have demonstrated a positive correlation between dietary Mg intake, and bone density and/or an increased rate of bone loss with low dietary Mg intake suggesting that dietary Mg deficiency may be a risk factor for osteoporosis [13-18].
The cause of this effect of Mg depletion on bone is unclear, although increased amounts of inflammatory cytokines have been found in bone of Mg deficient mice and rats which could increase bone resorption [7,9,10]. The final pathway of osteoclastogenesis has been proposed to involve three constituents of a cytokine system: receptor activator of nuclear factor kB ligand (RANKL); its receptor, receptor activator of nuclear factor kB (RANK); and its soluble decoy receptor, osteoprotegerin (OPG) [19,20]. RANKL is a membrane bound cytokine-like molecule that is expressed in preosteoblastic cells. It stimulates the differentiation, survival, and fusion of osteoclastic precursor cells to activate mature RANK expressed in hematopoietic osteoclast progenitors, and serves as an essential factor for osteoclastic differentiation and activation. RANKL binds to RANK with high affinity and this interaction essential for osteoclastogenesis. OPG is expressed in a variety of cell types, however in bone it is mainly produced by cells of osteoblastic lineage. OPG has very potent inhibitory effects on osteoclast formation. It acts like a decoy receptor and blocks the RANKL/ RANK interaction [19]. The relative presence of RANKL and OPG therefore dictates osteoclast bone resorption activity [21-23]. It was the objective of this study to examine the effect of reduction in dietary Mg on immunocytochemical presence of RANKL and OPG in Mg deficient vs. control animals.
Material and methods
Experimental Methods
Studies reported here were approved by the IACUC at the University of Southern California. Dietary Mg deficiency was induced for up to 6 months in 6 week old, 150–175 g female Sprague Dawley rats (Charles Rivers Laboratory, Wilmington, MA). After acclimation to the vivarium as previously described [9,10], experimental diets were instituted. Group pair feeding based on food weight was performed daily in order to keep weight gain as close as possible in the Mg deficient and control groups as bone mass is closely correlated with body mass. Distilled deionized water containing < 3 × 10-5 g Mg/L was used for hydration. Rats were fed either a normal control Mg diet of 0.05 % Mg (0.5 g/kg or 100% of NR as percent of total diet) or a Mg-deficient diet (0.04%, 25%, and 50% of NR Mg) (Harlan Teklad, Madison, WI) as previously described [10]. The dietary intake of calcium (Ca) was at or near the recommended intake for rats at 0.5 %.
At the end of each experimental period, rats were anesthetized with ketamine, 50 mg/kg, and zylazine, 10 mg/kg, intramuscularly (Phoenix Pharmaceuticals Inc, St. Joseph, MO). Blood samples from the anesthetized rats were obtained by cardiac puncture and rats were then killed by open thoracotomy. The femurs and tibias were harvested at each time point for mineral analysis, micro-computerized tomography, histomorphometry, and immunocytochemistry. Prior publications review the results in terms of serology, bone mineral density and content and the presence of inflammatory cytokines [7,9,10].
Immunocytochemical Localizations of RANKL and OPG
The tibia was isolated immediately following euthanasia and fixed in 2% NBF (Neutral Buffered Formalin) for 24 hrs and decalcified in 20% EDTA 0.1 M Tris pH 7.2–7.3 solution. After dehydration and paraffin embedding at 56°C, sections were cut so that a sagittal section including the epiphysis and the metaphysis of the tibia was obtained. As previously described, indirect immunocytochemistry [7,9,10] was used to localize RANKL and OPG; both primary and secondary spongiosa were evaluated as described below. RANKL and OPG antibodies were obtained from R&D Systems, Minneapolis, MN. The source of antibody was goat. Biotin labeled goat antibody as the second antibody and streptavidin-HRP attached to the antibody complex completed immunolocalization. Rat small intestine tissue containing Peyer's patches and spleen sections served as a positive control for both RANKL and OPG antibodies. If the result was comparable with the established positive staining using the same experimental conditions, the procedure was validated.
Localization was visualized in the light microscope using peroxidase substrate containing red dye Nova Red (Vector Labs, Burlingame, CA) and counterstained with Hematoxylin (blue)(Zymed Laboratories, South San Francisco, CA). The results were photographed in a Zeiss photo microscope (Carl Zeiss, Thornwood, NY, USA) using a 40 X objective.
Evaluation of Cytokine Localizations
Background localization was minimal compared with positive and negative controls. No difference in background localization was observed between Mg deficient and control rats. Cells and tissues stained specifically as described for the antigen in the literature [24,25]. Intensity was graded as 0 = no localization, 1 = weak localization, 2 = moderate, and 3 = strong localization. The quantitative estimate of numbers of cells staining was a = <20%, b = 20–60%, and c = >60%. The mean relative positivity was <1b = 0; 2a and 2b = 1; and 3b and 3c = 2 [24,25]. In this study, in some instances, no clear staining was observed for OPG at 4 and 6 months in animals on the 25% NR Mg diet, therefore, a minimum value of 0.01 was given to allow for calculation of RANKL/OPG ratio.
Results
Our prior studies have documented that dietary Mg intake at .04% 10%, and 25% of NR in the rat results in bone loss and an increase in inflammatory cytokines TNFα and IL1β [9,10]. Similar observations have been made at the 50% NR level (submitted for publication). As discussed in the Introduction, the final pathway of osteoclastogenesis has been proposed to involve RANKL and OPG [19-23]. We have examined the immunocytochemical presence of these two cytokines in Mg deficient vs. control animals. Examples of immunocytochemical staining are shown for RANKL in Figure 1 and for OPG in Figure 2. In rats fed a diet containing 0.4% of NR, there was an increase in staining in many cells of the bone microenvironment; RANKL was increased by 448% in osteoblasts on day 1, in lymphocytes by 157% on day 3 and 100% on day 8, and in osteoclasts by 64% on day 22. No difference was observed at 28 or 84 days of depletion. OPG, in contrast, was reduced in osteoblasts by 75% on day 3 and 149% by day 21. In mononuclear cells, OPG was reduced by 53% on day 1, 100% on day 3, and 233 % on day 8. This effect continued by day 28 when OPG was decreased in osteoblasts by 27% and in osteoclasts by 74%. At 12 weeks of Mg depletion OPG had decreased in all components of the bone environment: osteoblasts by 108%, osteoclasts by 167%, megakaryocytes by 85%, and mononuclear cells by 92%. While these percentage changes are of interest, it is the relative presence of RANKL to OPG in bone that dictates overall effect on osteoclastogenesis. The ratio of RANKL/OPG relative immunostaining was calculated at this dietary intake and is shown in Table 1; the higher the value the greater the preponderance of RANKL. As noted, it was only on or after day 22 of the experimental diet that the ratio data suggest an excess of RANKL.
Figure 1 Immunocytochemical staining of RANKL in bone from Mg deficient and control animals. A. Represents animals on a 25% NR diet and B. the control group. Note the positive staining of osteoclasts (solid arrows) and osteoblasts (open arrows) in A. while minimal staining is observed the control animals (B). C. Represents animals on a 50% NR diet and D. the control group. Again, as observed in C., Mg deficient animals have much more intense staining of RANKL of osteoclasts (solid arrows) and osteoblasts (open arrows) than is observed the control animals (D).
Figure 2 Immunocytochemical staining of OPG in bone from Mg deficient and control animals. A. Represents animals on a 25% NR diet and B. the control group. Note the minimal staining of osteoclasts (solid arrows) and osteoblasts (open arrows) in A. while positive staining is observed the control animals (B). C. Represents animals on a 50% NR diet and D. the control group. Again, as observed in C., Mg deficient animals have minimal staining for OPG in osteoclasts (solid arrows) and osteoblasts (open arrows), while much more intense staining is observed in the control animals (D).
Table 1 Ratio of RANKL/OPG in Osteoclasts and Osteoblasts in Rats on a .04% NR Diet
Osteoclast Osteoblast
Day 1 Control 1 0.44
Day 1 .04% NR 0.4 .046
Day 3 Control 0.6 0.55
Day 3 .04% NR 0.51 0.24
Day 8 Control 1 0.44
Day 8 .04% NR 0.25 0.33
Day 15 Control 1.25 0.29
Day 15 .04% NR 0.79 0.38
Day 22 Control 0.65 0.05
Day 22 .04% NR 0.12 0.25
Day 28 Control .029 0.12
Day 28 .04% NR .052 0.16
Day 84 Control .096 0.14
Day 84 .04% NR 0.256 1.66
As shown in Table 1 there is no apparent excess of RANKL relative to OPG in rats on a diet containing .04% of NR until after day 22.
Samples were obtained at 2, 4, and 6 months from animals on the 25% NR intake. The percent changes and ratio of RANKL/OPG are shown in Tables 2 and 3. RANKL appears higher at the 2 and 6 month points in osteoclasts, but is lower in osteoblasts from animals with Mg deficiency. OPG was quite suppressed in both ostoclasts and osteoblasts in Mg deficient animals. The ratio calculated, however, showed a preponderance of RANKL to OPG.
Table 2 Percent Difference in Immunocytochemical Staining for RANKL and OPG
25% NR
Osteoclast Osteoblast
RANKL OPG RANKL OPG
2 Month 25% vs Control 86 -150 -10 -113
4 Month 25% vs Control -16 -160 -232 -160
6 Month 25% vs Control 116 -700 -82 -700
As shown in Table 2, the percent relative difference in immunocytochemical staining in rats on a diet containing 25% NR Mg for RANKL in osteoclasts was increased at 2 and 6 months and decreased in osteoblasts. Staining for OPG was markedly decreased in both cell lines at all time points compared to control.
At the 50% NR dietary intake, samples were obtained at 3 and 6 months. These data are shown in Tables 4 and 5. At this dietary intake level, RANKL is increased in both osteoclasts and osteoblasts of Mg depleted rats while OPG is decreased. The ratios of RANKL/OPG again favor a relative increase in RANKL in Mg deficiency.
Table 3 Ratio of RANKL/OPG in Osteoclasts and Osteoblasts in Rats on 25% NR Mg Diet
25% NR
Osteoclast Osteoblast
2 Month Control 2.32 9.82
2 Month 25% NR 108 18.75
4 Month Control 3.41 4.88
4 Month 25% NR 50 25
6 Month Control 3.12 2.12
6 Month 25% NR 54 31
As shown in Table 3, the ratio of RANKL to OPG was increased in Mg deficient animals relative to controls in both cell types which favors osteoclastogenesis.
Table 4 Percent Difference in Immunocytochemical Staining for RANKL and OPG
50% NR
Osteoclast Osteoblast
RANKL OPG RANKL OPG
3 Month 50% vs Control +250 -152 +320 -20
6 Month 50% vs Control +240 0 +700 -100
As shown in Table 4, the percent relative difference in immunocytochemical staining in rats on a diet containing 50% NR Mg for RANKL was increased at 3 and 6 months in osteoclasts and in osteoblasts. Staining for OPG was decreased in both cell lines three of the four time points compared to control.
Table 5 Ratio of RANKL/OPG in Osteoclasts and Osteoblasts in Rats on 50% NR Mg Diet
50% NR
Osteoclast Osteoblast
3 Month Control 0.24 0.12
3 Month 25% NR 0.30 0.42
6 Month Control 0.13 0.02
6 Month 50% NR 3.13 0.32
As shown in Table 5, the ratio of RANKL to OPG was increased in Mg deficient animals relative to controls in both cell types which favors osteoclastogenesis.
Discussion
In our prior publications, we have clearly demonstrated that Mg deficiency results in bone loss and is accompanied by an increase in osteoclast number and indices of bone resorption [6-10]. It was hypothesized that since Mg depletion induces a rise in substance P with subsequent stimulation of inflammatory cytokines such and TNFα, IL1β, and IL6, that this may be the mechanism for bone loss [26]. Indeed, we have demonstrated an increase in the immunolocalization of TNFα and IL1β in the Mg deficient rat and mouse [7,9,10]. Both cytokines are known to stimulate osteoclastic bone resorption. These changes were observed to begin very early in the course of Mg depletion. In contrast, the relative change in RANKL to OPG to favor bone resorption did not occur until at least 4–6 weeks into depletion at the dietary intake of .04% NR. The relative presence of RANKL and OPG dictates osteoclast bone resorption activity as discussed above. Osteoclasts can be formed or activated in a RANKL and/or a RANKL-independent mechanism by TNFα [19-23]; therefore, TNFα and IL1β may be directly responsible for the early osteoclastic bone resorption and the decline in OPG and an increase in RANKL/OPG which follows later in the course of depletion. Responses to varying dietary intakes also appear to differ in absolute changes in RANKL and OPG. We did not assess early changes at the 25% or 50% NR, and thus we do not known if there were any changes in RANKL or OPG at these time points with these higher Mg diets. As was observed at the 3 months time point (84 days) in the .04% NR diet, there was a remarkable fall in OPG at the higher Mg diets relative to control. While RANKL was also increased in osteoclasts at both the 25% and 50% NR diets, an increase was only observed in osteoblasts at the 50% NR diet. The reason for this is unclear. The ratio of RANKL/OPG throughout favors osteoclastogenesis and suggests that this may be a major mechanism for the increase bone loss in Mg deficiency.
It is clear that dietary Mg deprivation does result in a reduction in bone mass and that there may be other reasons for decrease bone mass as well. We have also observed a decrease in osteoblastic bone formation [6,7,9,10]. This could be related to a decrease in PTH and 1,25(OH)2-vitamin D relative to control [9,10,27,28]. Several other potential mechanisms may account for a decrease in bone mass/strength during Mg deficiency. Mg is mitogenic for bone cell growth, and therefore Mg deficiency may result in a decrease in bone formation [29]. Mg also affects crystal formation; a lack of Mg results in a larger, more perfect crystal which may affect bone strength [30]. Serum IGF-1 levels have also been observed to be low in the Mg deficient rat; decreased IGF-1 may adversely influence skeletal growth[31].
A limitation of studies employing immunocytochemistry is that this approach may be influenced by observer subjectivity and bias and intra-observer variation. Our findings, however, are provocative and further assessment employing other techniques are indicated and may include assessment of cytokine gene expression using both in situ and laser capture microdissection/gene array analyses. These approaches would address the question of whether low Mg intake influences gene cytokine expression, and allow study of specific cell types such as mononuclear cells, osteoblasts and osteoclasts. We have, by in situ hybridization data, demonstrated much greater gene expression of substance P and of TNFα in rats on a 10% NR Mg diet compared to control animals (unpublished data).
Conclusion
We have, for the first time, demonstrated that a reduction in dietary Mg in the rat alters the presence of RANKL and OPG and may explain the increase in osteoclast number and decrease in bone mass in this animal model. As these dietary intake reductions in terms of the RDA are present in a large segment of or population, Mg deficiency may be another risk factor for osteoporosis.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
RKR designed the study and supervised the experimental work, analyzed data, and drafted the manuscript. HEG assisted in the study design and interpretation of the data. LYW was responsible for the care and pair feeding of the animals and interaction with the veterinarian. AF performed the immunocytochemical studies and generated images as well as preparing the bone for these experiments.
Acknowledgements
This work was supported by grant 1 R01 DK060545 from the National Institutes of Health and funds from the Orthopaedic Hospital.
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Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-3-411613140310.1186/1477-7827-3-41ReviewPeroxisome proliferator-activated receptors (PPARs) and ovarian function – implications for regulating steroidogenesis, differentiation, and tissue remodeling Komar Carolyn M [email protected] Department of Animal Science, Iowa State University, 2356 Kildee Hall, Ames, IA 50011, USA2005 30 8 2005 3 41 41 14 7 2005 30 8 2005 Copyright © 2005 Komar; licensee BioMed Central Ltd.2005Komar; 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 peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors involved in varied and diverse processes such as steroidogenesis, angiogenesis, tissue remodeling, cell cycle, apoptosis, and lipid metabolism. These processes are critical for normal ovarian function, and all three PPAR family members – alpha, delta, and gamma, are expressed in the ovary. Most notably, the expression of PPARgamma is limited primarily to granulosa cells in developing follicles, and is regulated by luteinizing hormone (LH). Although much has been learned about the PPARs since their initial discovery, very little is known regarding their function in ovarian tissue. This review highlights what is known about the roles of PPARs in ovarian cells, and discusses potential mechanisms by which PPARs could influence ovarian function. Because PPARs are activated by drugs currently in clinical use (fibrates and thiazolidinediones), it is important to understand their role in the ovary, and how manipulation of their activity may impact ovarian physiology as well as ovarian pathology.
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Introduction
Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone receptors belonging to the steroid receptor superfamily. Issemann and Green identified the first PPAR in 1990 [1], and subsequently, two other family members were discovered. To date, PPARs have been identified in a variety of species from chickens [2] and fish [3], to humans (reviewed in [4,5]).
Although a great deal has been learned about PPARs since their discovery, very little is known regarding how these factors impact ovarian function. This review describes the expression of the PPARs in the ovary, and highlights the roles of these transcription factors that may affect ovarian biology. The influence of PPARs on polycystic ovary syndrome (PCOS) is not discussed in this review. There is a large body of literature on the use of thiazolidinediones, a class of drugs that activate PPARγ, in the treatment of women with PCOS. However, because these drugs can have direct effects on the ovary independent of activating PPARγ [6], and indirectly influence ovarian biology by lowering insulin levels, it is hard to discern PPARγ-dependent versus -independent effects. Therefore, this review focuses on the potential of PPARs to impact normal ovarian function and the development of ovarian tumors.
PPARs
There are three PPAR family members: PPARα (NR1C1), PPARδ [NUC-1, fatty acid-activated receptor (FAAR), β, NR1C2], and PPARγ (NR1C3). The PPARs share a common structure with other steroid hormone receptors (Figure 1A). The N-terminal A/B domain is responsible for ligand-independent transactivation function (AF-1); the C domain contains the DNA-binding domain; the D domain – also called the hinge region, plays a role in receptor dimerization; and the C-terminal E/F domain contains the ligand binding domain (AF-2).
Figure 1 Structure, relationship and splice variants of the PPARs. A) Schematic diagram of the structure common to nuclear hormone receptors and the PPARs, indicating the relative similarities between the various regions of PPAR isotypes across species [4] [139] [140]. B) Schematic of the splice variants of the PPARs. Schematic of PPARα adapted from [7] [8] [141]. The diagram of PPARδ splice variants was adapted from [9]. Exons IA, IB, IC, ID, and 2 are non-coding. Regarding PPARγ splice variants, exons 1–6 are common to all PPARγ subtypes. PPARγ1 includes the untranslated exons A1 and A2, PPARγ2 contains the translated exon B, PPARγ3 contains the untranslated exon A2, PPARγ4 contains only exons 1–6 (adapted from [4] [10] [142]). Images not drawn to scale.
Each PPAR family member is transcribed from a specific gene. Alternative splicing and the use of different promoters give rise to different splice variants of each PPAR family member (Figure 1B). In addition to the full length mRNA for PPARα, in humans a splice variant has been identified which lacks the hinge region and the entire ligand binding domain [7,8]. This splice variant of PPARα can interfere with PPAR activity, and other nuclear receptors, by competing for coactivators [8]. Four spice variants for PPARs δ and γ have been identified. The splice variants for PPARδ give rise to one primary translation product [9]. PPARγ1, γ3, and γ4 yield the same protein product [10], whereas the protein encoded by PPARγ2 has an additional 30 (mouse) [11] or 28 (human) [12] amino acids in the N-terminus. Additional splice variants for PPARγ have been identified in monkey macrophages and adipocytes [13].
Activity of PPARs
Ligand binding
There are a multitude of agents that activate the PPARs (Table 1). Many of these agents have well established roles in ovarian biology. For example, endogenous factors that have been shown to activate the PPARs that also impact ovarian function are fatty acids and prostaglandins, and exogenous activators include herbicides, industrial plasticizers, non-steroidal anti-inflammatory drugs (NSAIDs), fibrates (a class of drugs used to treat hyperlipidemia), thiazolidinediones (TZDs; hypoglycemia drugs), polycyclic aromatic hydrocarbons, organotin compounds, and traditional medicines [5,14-21]. An example of how these exogenous PPAR agonists impact the ovary is the inhibition of ovulation and 'reversible female infertility' caused by NSAIDs [22].
Table 1 Overview of ligands, both endogenous and exogenous, for the PPAR isotypes. Asterisks denote presence in the ovary, and/or reported affect on ovarian cells.
Endogenous Ligands Source Specificity for PPAR isotype Reference
Polyunsaturated fatty acids* Diet Metabolism PPARα>PPARδ>>PPARγ [25]; reviewed in [5]
Eicosanoids* Inflammation PPARα, PPARδ, PPARγ [25]
8-HETE Metabolism PPARα
PGJ2 PPARγ>>>PPARα>PPARδ [17] [26]
PGA1 PPARδ>>PPARα,PPARγ [17] [25]
PGI2 PPARδ reviewed in [16]
Leukotriene B4 PPARα [23] [25]
Lysophosphatidic acid* Metabolism PPARγ [18]
Oxidized LDL Metabolism PPARγ reviewed in [29]
Exogenous Ligands Source Specificity for PPAR isotype Reference
Herbicides/fungicides Environment PPARγ [19]; reported in [1]
Plasticizers* Environment Industry [137]; reviewed in [15]
NSAIDS Pharmaceutical PPARγ>PPARα>>PPARδ [20] [28]
Fibrates* Pharmaceutical PPARα>>>PPARγ [25]
Polycyclic aromatic hydrocarbons Environment PPARα, PPARδ [21]
Herbal/plant compounds Traditional medicine PPARα, PPARγ>PPARδ reviewed in [14]
Genistein* Plants PPARγ [138]
Thiazolidinediones* Pharmaceutical PPARγ [23] [55]
There is some specificity observed between ligands and the PPAR subtypes. For example, fibrates (i. e. WY-14,643, clofibrate) show a high affinity for PPARα, but at higher concentrations can also activate PPARγ [4]. The thiazolidinediones (troglitazone, ciglitazone, pioglitazone, rosiglitazone) selectively activate PPARγ [4,23]. Long chain fatty acids, particularly polyunsaturated fatty acids, preferentially activate PPARα [24], but are also capable of activating PPARδ and PPARγ [5,23,25]. Prostaglandins activate all PPAR family members, with PGA1 and 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2) preferentially activating PPARδ and PPARγ, respectively [5,25,26]. Prostacyclin and its analogue, carbaprostacyclin, also binds to PPARδ (reviewed by [16,27]). Hydroxyeicosapentaenoic acids and leukotriene B4 are activators of PPARα [5,25]. Interestingly, indomethacin and other NSAIDs that inhibit the production of prostaglandins, are also able to activate PPARα and PPARγ [28]. Oxidized products of LDL (9-HODE and 13-HODE) are ligands for PPARγ (see [4]).)[29] for a review). Structural and amino acid differences in the binding pocket of the PPAR isoforms contribute to selectivity for ligand binding [30].
DNA binding
PPARs heterodimerize with 9, cis-retinoic acid receptors (RXRs) (Figure 2). PPAR interaction with RXRs can occur in the absence and/or presence of ligand [31]. The heterodimer binds to a short sequence of DNA, a PPAR response element (PPRE), present in the promoter regions of target genes. The PPRE is a direct repeat of the sequence AGGTCA, separated by one nucleotide (a DR1 sequence; reviewed in [4,5]). In addition to the PPRE, the 5' flanking region has been shown to be important for PPAR binding to DNA, especially PPARα binding. The binding affinity of the PPAR/RXR heterodimer is greatly enhanced if the nucleotide between the two hexamers is an adenine, and when there is an AA/TCT sequence 5' of the PPRE (reviewed in [4,5,32]). These DNA features result in a polarity to the bound heterodimer; PPAR binds to the upstream hexamer while RXR interacts with the lower, 3' hexamer [5,32]. The integrity of the 5' sequence offers selectivity in binding for the PPAR isotypes.
Figure 2 Mechanism of action of PPARs. PPARs heterodimerize with RXRs both in the presence and absence of ligand. After ligand binding, PPARs undergo conformational change resulting in dissociation of corepressors, and the binding of coactivators. PPAR/RXR heterodimers bind to a DR1 sequence in the promoter region of target genes (see text for details).
Cofactors
Similar to other steroid hormone receptors, there are coactivators and corepressors that associate with the PPARs. Corepressors, such as nuclear receptor corepressor (NCoR) and silencing mediator for retinoid- and thyroid-hormone receptors (SMRT), dissociate from the receptor upon ligand binding (reported in [4,33]). The conformational change that occurs upon ligand binding also facilitates the recruitment of coactivators. Two coactivators that have histone acetyltransferase activity, steroid receptor coactivator-1 (SRC-1) and CREB binding protein/p300 (CBP), can bind to PPARs in a ligand-dependent manner. The latter coactivators can also interact with PPARs in a ligand-independent manner, but only transiently (reviewed in [4]). RIP140, ARA70, and members of the DRIP/TRAP family of coactivators also bind to PPARs (see [34] for a review). Other coactivators that have been identified to interact with PPARs are: PPAR interacting protein [33], PPARγ coactivator-1 (reviewed in [35]), and PPAR binding protein (PBP; [36]). Although these coactivators also bind other steroid receptors, deletion of the PBP gene in mice results in embryonic lethality due to placental insufficiency [37], the same results seen in PPARγ null mutants [38]. These findings are consistent with the hypothesis that PBP is a required factor for PPARγ transcriptional activity. The regulated expression of these various corepressors and coactivators and their concentrations in tissues also offers selectivity in transcriptional regulation by the PPAR isotypes.
A recent intriguing finding is that the association of corepressors with PPARδ can inhibit the activity of PPARs α and γ. Shi et al. (2002) demonstrated that PPARδ repressed PPARα and γ-mediated gene transcription. This repressive activity of PPARδ involved DNA binding and association with the corepressor SMRT [39]. The authors of this study concluded that the levels of each PPAR isotype, as well as the ratio of PPARs α and γ to PPARδ in a particular tissue influences the activity of each isotype.
Post-translational modifications
The activity of PPARs are modified not only by ligand binding, but also by phosphorylation, nitration, and ubiquitination. Phosphorylation sites have been identified on both PPARs α and γ. The impact of phosphorylation on the activity of PPARs depends on: 1) the residue being phosphorylated, and 2) the kinase cascade that was activated (reviewed in [40]). A modification of PPARγ that influences its activity is nitration of tyrosine residues. Shibuya et al. (2002) demonstrated that nitration of tyrosine residues in PPARγ inhibited the translocation of PPARγ from the cytosol to the nucleus [41], thus reducing its potential to influence gene transcription. PPARs can also be ubiquitinated. Ligand binding to PPARγ induces ubiquitination of the receptor [42], and therefore its degradation. In contrast, ligand binding to PPARα stabilizes the receptor by decreasing its rate of ubiquitination [43,44].
Expression and functions of PPARs
The tissue distribution of mRNA differs among the individual PPAR family members. PPARα is an important player in regulating fatty acid metabolism [4,45], and it is expressed at relatively high levels in the liver, small intestine, kidney, heart, and brown adipose tissue [46,47]. It has also been demonstrated to play a role in inflammation (reviewed in [5,35,48]). PPARδ is ubiquitously expressed with highest levels of expression seen in the liver, kidney, and brown adipose tissue in the mouse [4,46,47,49]. A study of PPARδ null mice illustrated that this PPAR subtype is involved in development, lipid metabolism, proliferation of epidermal cells, and myelination of nerves [50]. PPARδ also plays a role in wound healing (reviewed in [51]), embryonic implantation [52,53], and adaptive responses to exercise in skeletal muscle (reviewed in [54]). The expression of the various PPARγ isoforms shows tissue specificity. PPARγ1 is the most widely expressed and is found in most tissues [4,49,55]. PPARγ2 is localized primarily to adipocytes, and PPARγ3 is also found in adipocytes, as well as colonic epithelium, and macrophages [46,56]. The distribution of PPARγ4 is unclear because it cannot be discriminated from PPARγ1 or γ3 due to the similarity between them [10]. PPARγ has been shown to be an adipocyte differentiation factor (reviewed in [57,58]), and also plays a role in glucose homeostasis, the cell cycle, carcinogenesis, lipid metabolism, and inflammation (reviewed in [35,59,60]). It has been suggested that PPARs mediate dietary regulation of gene expression due to the fact that various metabolic and nutritional agents can activate these transcription factors.
PPARs and ovarian function
Expression and activity
All three PPAR subtypes have been detected in ovarian tissue. In the rat ovary, the expression of mRNA for PPARα is found primarily in the theca and stroma, whereas mRNA for PPARδ is found throughout the ovary (Figure 3). The expression of these two PPAR isotypes remains steady throughout follicular development and the ovarian cycle in the rat [61,62].
Figure 3 Localization of mRNAs corresponding to PPARα (A, B, C) and PPARδ (D, E, F) in ovarian tissue collected from immature rats 48 hours post-eCG. Tissue sections (8 μm) were hybridized with 35S-labled antisense (A, D) and sense (C, F) riboprobes for each respective PPAR isotype. Figures originally published in [62].
PPARγ has been more extensively studied in ovarian tissue than the other two family members. It has been detected in the mouse [63], rat [49,62], pig [64], sheep [65], cow [66,67], and human [55] ovary. Using RT-PCR, PPARγ was detected in granulosa cells collected during oocyte aspiration from women undergoing treatment for in vitro fertilization [68], and in porcine theca and granulosa cells [64]. This PPAR isotype has also been reported to be in oocytes from cattle [69], zebrafish [3], and Xenopus laevis (trace amounts; [70]). In cycling rats and sheep, the expression of PPARγ is restricted primarily to granulosa cells in developing follicles [61,62,65]. However, unlike the steady expression of PPARs α and δ, the expression of PPARγ is down-regulated in response to the LH surge (Figure 4) [62,65]. The expression of PPARγ decreases only in follicles that have responded to the LH surge [71]. In the rat, expression of PPARgamma is low in newly forming luteal tissue, and higher in luteal tissue present from previous ovulations [61]. Because PPARγ is primarily expressed in granulosa cells, it may influence development of these cells and their ability to support normal oocyte maturation. PPARγ could also potentially affect somatic cell/oocyte communication not only by impacting granulosa cell develpment, but by direct effects on the oocyte. Disrupting the expression of PPARγ in the ovary therefore, could potentially affect oocyte developmental competence.
Figure 4 Localization of mRNA and protein corresponding to PPARγ in ovarian tissue collected from immature rats 0 (A, E) and 48 (B, F) hours post-eCG, and 4 (C, G) and 24 hours (D, H) post-hCG. Frozen tissue sections (8 μm) were hybridized with an antisense riboprobe corresponding to PPARγ. Figures A – D originally published in [71]. Protein corresponding to PPARγ, identified by the brown reaction product, was localized in 4% paraformaldehyde-fixed, paraffin embedded tissue using an anti-PPARγ antibody (Santa Cruz).
Results from a study by Cui et al. (2002) indicate that PPARγ plays an important role in normal ovarian function. Using cre/loxP technology, the expression of PPARγ was disrupted in the ovary, rendering 1/3 of the females sterile, and the remaining females sub-fertile [63]. Females that were sub-fertile took longer to conceive and had smaller litters. There were no differences found in the number of primordial, primary, or preantral/antral follicles, size of copora lutea, or response to exogenous gonadotropins between control animals and those with PPARγ disrupted in the ovary. On the day of estrus, levels of progesterone in animals with PPARγ disrupted in the ovary were half that found in controls. However, the differences in circulating progesterone were not significantly different between the two groups, most likely due to the small sample size (n = 4/group). Implantation sites (6) were only observed in the uterus of one of three females examined with PPARγ disrupted in the ovary, compared with 5 and 7 implantation sites observed in two control females, respectively. Because the expression of PPARγ was not disrupted in the uterus of these transgenic females, the lesion responsible for the sub- and infertility most likely lies within the ovary. The authors concluded that "...ovarian function might not be sufficient to induce implantation" [63]. The insufficient ovarian function may relate to the ability of the corpus luteum to produce enough progesterone, or produce enough progesterone in a timely manner, to support the establishment of pregnancy. In addition, estradiol production by the ovary around day 4 post-coitum is also an important player in preparing the uterus for implantation. Impaired production of estradiol by ovarian cells in the transgenic females during this critical period may also lead to reduced implantation. Although not tested in this study, the competence of the oocyte to undergo fertilization and support embryonic development might also be compromised in these genetically altered mice. Further study into the role of PPARγ in ovarian steroidogenesis and somatic cell/oocyte interactions is needed to determine the cause of the fertility problems in females with reduced ovarian PPARγ expression.
Additional studies have shown that endogenous PPARγ is active in the ovary. Granulosa cells from rats and sheep were transiently transfected with reporter constructs whose expression was driven by PPREs. Both in the absence and presence of agonists for PPARγ, there was an increase in reporter activity [65,72]. PPARγ in rat granulosa cells was also shown to bind DNA [73]. These findings demonstrate that PPARγ is functional in granulosa cells, and that endogenous ligand is also present within these cells.
Regulation of Steroidogenesis
One way PPARs may influence ovarian function is by modifying the ability of estradiol to elicit cellular responses. PPARs are able to bind to estrogen response elements – EREs, [74,75], and can act as competitive inhibitors [74]. PPARγ can also stimulate ubiquitination of estrogen receptor α, leading to its degradation [76].
The synthesis and metabolism of estradiol is also affected by the PPARs. PPARγ can inhibit the expression of aromatase, the rate limiting enzyme for the conversion of androgens to estradiol by disrupting the interaction of NF-κB with the aromatase promoter II [77]. Activation of PPARα decreased the expression and activity of aromatase in granulosa cells [78,79]. In cultured human granulosa-luteal cells [68], and granulosa cells from eCG-primed immature rats [78], activation of PPARγ reduced the expression of aromatase. PPARγ was also shown to partially mediate the suppressive effects of phthalates on ovarian estradiol production [78]. However, using a different strain of rat and culture model, agonists of PPARγ were shown to increase estradiol secretion by granulosa cells collected from gonadotropin-primed immature rats [62]. Reduced levels of aromatase in granulosa cells after activation of PPARγ was also reportedly due to increased turnover in conjunction with decreased transcription [80]. We reported previously that there was no correlation between the expression of mRNAs for PPARγ and aromatase in granulosa cells during folliculogenesis or the periovulatory period [71]. PPARs may also limit the synthesis of estradiol by reducing production of androgenic precursors by theca cells. PPARγ is expressed in the theca [61,64], primarily in the theca externa and in an inconsistent pattern [61]. Both endogenous (PGJ2) and exogenous (troglitazone) agonists of PPARγ reduced basal and LH-stimulated thecal androgen production in vitro [64,81]. One study reported that troglitazone increased mRNA for CYP17, but not the corresponding protein [64], whereas a second study showed no effect of the PPARγ agonists on mRNA for CYP17, but a decrease in its phosphorylation [81]. In both granulosa [78] and liver cells [82], agonists of PPARα stimulated the expression of 17β-hydroxysteroid dehydrogenase type IV, an enzyme that oxidizes estradiol into the less active estrone. The expression of PPARα in granulosa cells is very low [61,62] and therefore may be unlikely to modify estradiol metabolism under normal physiological conditions. Taken together, these data indicate that PPARs are able to influence estradiol production, and that age and the endocrine environment may influence how these transcription factors impact ovarian steroidogenesis.
The activation of PPARγ can also influence progesterone production by ovarian cells. In cultured human granulosa cells, activators of PPARγ inhibited basal and gonadotropin-stimulated progesterone production [83]. However, activators of PPARγ stimulated progesterone secretion by granulosa cells obtained from eCG-primed immature rats [62]. When porcine theca cells were treated with synthetic and natural ligands for PPARγ, progesterone production increased [64]. Progesterone production by bovine luteal cells treated with the endogenous ligand for PPARγ, PGJ2, increased progesterone production over a 24 hour culture period [67]. Our previous work has shown that there is an inverse relationship between the expression of mRNA for PPARγ and P450 side chain cleaveage, the rate limiting enzyme in progesterone synthesis, in granulosa cells and luteal tissue from naturally cycling and gonadotropin-treated rats [71,84]. Therefore, the effect of PPARγ on progesterone production may depend on the cell type, stage of differentiation, stage of the cycle, and/or the species studied.
Tissue Remodeling
PPARs regulate the expression and activity of proteases involved in tissue remodeling and angiogenesis which are critical processes for follicular and luteal development. Plasminogen activators (PA) and matrix metalloproteinases (MMPs) are proteolytic enzymes involved in ovarian tissue remodeling and angiogenesis [85-87]. Activation of PPARα and PPARγ decreases MMP-9 expression and its activity [88-91]. The promoters for MMP-3 [92] and MMP-9 [93] contain a PPRE, indicating that transcription of these proteases is likely directly regulated by PPARs. PPARγ activation can also reduce expression of MMP-13 and MMP-1 by interfering with AP-1 activation [94-96]. PPARγ negatively affects plasminogen activator by inhibiting its expression [97] and increasing the expression of plasminogen activator inhibitor-1 [97,98]. However, there are also reports of troglitazone treatment reducing the expression of plasminogen activator inhibitor-1 [99,100]. These findings indicate that the PPARs are capable of modulating the balance of proteolytic enzymes and their inhibitors, thereby altering tissue remodeling events. Whether PPARs regulate these processes in ovarian cells, particularly at the time of ovulation when MMP and PA activities must be tightly regulated, is an important area of investigation.
Along with the proteases, vascular endothelial growth factor (VEGF) and its receptors (Flt-1, -2) are important players in new blood vessel formation in the ovary [101,102]. The activation of PPARγ with PGJ2 inhibited the expression of Flt-1 and Flt-2 in human umbilical vein endothelial cells [97]. Activation of PPARγ with its endogenous and exogenous ligands has also been shown to inhibit VEGF-stimulated endothelial cell proliferation and migration (reviewed in [103]). However, Yamakawa et al. (2000) reported that activating PPARγ in vascular smooth muscle cells results in an increase in the expression of VEGF [104]. Therefore, the effect of PPARγ on angiogenesis may depend on agonist used, experimental model, and/or cellular differences in cofactor availability [103]. Besides its effects on angiogenesis, PPARγ may influence the ovarian vasculature by its ability to regulate endothelin-1 (ET-1) and nitric oxide synthase (NOS). ET-1 is a potent vasoconstrictor and recent studies have shown that it is also an important player in ovarian physiology, especially luteal function (reviewed in [105]). NOS synthesizes nitric oxide, a vasodilator, from arginine. Nitric oxide has been implicated as a player in luteolysis [106], ovarian cyclicity [107], ovulation [107-109], oocyte maturation [108], and follicular development [110,111]. PPARγ decreases the secretion of ET-1 from endothelial cells [112], and also inhibits the expression of NOS in macrophages [90] and vascular smooth muscle cells [113].
The ability of PPARs to affect tissue remodeling could alter folliculogenesis and luteal development, and impact ovulation. Ovarian tissue is constantly changing to accommodate the dynamic geometry of growing follicles which increase in size exponentially from the primordial to preovulatory stage. For successful release of the oocyte at ovulation, the granulosa cell layer, follicular basement membrane, theca interna and externa, ovarian stroma, tunica albuginea, and surface epithelium need to be traversed. In addition, the tissue remodeling involved in developing the increased vasculature required to support follicular development and luteal formation requires protease activity. The ability of PPARs to regulate the expression of proteases and angiogenic factors, and the fact that they are expressed in the ovary and in the case of PPARγ, modulated during the periovulatory period encompassing ovulation and luteal formation, warrant further study into how the PPARs may influence these aspects of ovarian biology.
PPARs are important mediators of inflammatory responses (reviewed in [27,114-116]). The process of ovulation has been likened to an inflammatory response [117] and prostaglandins, major regulators of inflammation, have well documented roles in ovulation as well as luteal function (see [118] for a review). The rate-limiting enzyme in prostaglandin production is cyclooxygenase-2 (COX-2). The promoter region of COX-2 contains a response element for the PPARs [119], indicating that PPARs can directly influence transcription of this gene. However, there are reports of PPARγ both stimulating [119] and inhibiting [120,121] the expression of COX-2. In rat granulosa cells, the expression of COX-2 is stimulated within 4 hours of the ovulatory gonadotropin surge [122], however, PPARγ is significantly reduced in this same time frame [62]. This inverse relationship between the expression of PPARγ and COX-2 has also been observed in the placenta [123]. The variability in reported effects of PPARγ on COX-2 expression could result from: 1) the use of different cell-types, 2) transfection with COX-2 promoter constructs that did [119] or did not [124] contain the PPRE, 3) the ability of PPARs to influence COX-2 expression by binding to its promoter region, and/or 4) by PPARγ interfering with activation of NF-κB [121]. The periovulatory expression pattern of PPARγ suggests it plays an inhibitory role in COX-2 expression in ovarian cells in vivo.
Not only can PPARs regulate COX-2 expression, but as discussed earlier, prostaglandins themselves are endogenous ligands that can activate PPARs. In addition, PGF2α can activate kinase cascades resulting in the phosphorylation of PPARγ and inhibiting its activity [125]. Cumulatively, these findings imply that there is a cyclic relationship between the presence of prostaglandins, activation and/or inhibition of PPARs and feedback to the prostaglandin synthesizing enzyme – COX-2.
PPARs, cell cycle regulation, and ovarian tumors
The minority of follicles which successfully develop to the preovulatory stage must balance cellular proliferation as well as escape from programmed cell death, or apoptosis. PPARs have well documented roles in apoptosis as well as cell cycle control (reviewed in [35,60,126,127]). For example, the gene encoding bcl-2, an anti-apoptotic factor, has a PPRE, and transfection of PPARγ increased bcl-2 protein and mRNA [128]. However, administration of troglitazone to cultured rat granulosa cells decreased levels of mRNA for bcl-2 and stimulated apoptosis [73]. Froment et al. (2003) also reported that treating granulosa cells from sheep with a PPARγ agonist decreased granulosa cell proliferation [65]. One cell cycle regulator, cyclin D2, shares a similar profile of expression to that of PPARγ, however, there are conflicting reports of how activation of PPARγ affects cyclin D2. In human leukemic cells, activation of PPARγ by troglitazone or PGJ2 resulted in a decline in mRNA and protein for cyclin D2 [129]. Like PPARγ, cyclin D2 is expressed in granulosa cells of developing follicles and down-regulated within 4 hours of the LH surge, but only in follicles that responded to the gonadotropin surge [130]. However, administration of troglitazone to cultured rat granulosa cells had no effect on cyclin D2 [72]. Thus, more work investigating the role of PPARγ in granulosa cell cycle progression is needed to address the apparent dichotomy of PPARγ inhibiting cell proliferation yet being expressed at a high level in developing follicles.
PPARγ is expressed in cells from a granulosa cell tumor [131], and up-regulated in epithelial ovarian carcinomas [132]. Interestingly, the expression of PPARγ was higher in malignant tissues than in benign tumors [132]. A study investigating the relationship between the expression of PPARγ and COX-2 in human epithelial ovarian tumors reported that there was an inverse relationship between the expression of these two factors [133]. Because over-expression of COX-2 is associated with various cancers ([133] and references therein), the authors of this latter study concluded that PPARγ and its activation may be beneficial in halting the progression of ovarian tumors.
Genetic susceptibility for developing ovarian and breast cancer is linked to the BRCA1 gene. BRCA1 is a tumor suppressor, and has been shown to be down-regulated in many cases of sporadic ovarian cancer. A study by Pignatelli et al. (2003) has shown that there is a PPRE in the promoter region for the gene encoding BRCA1, and both synthetic and endogenous ligands for PPARγ increase levels of BRCA1 in MCF-7 breast cancer cells [134]. Support for PPARγ playing a role in susceptibility to ovarian cancer in vivo comes from a study of mice heterozygous for PPARγ. Both heterozygous (PPARγ+/-) and wildtype mice were treated with the carcinogen 9, 10-dimethyl-1,2-benzanthracene (7, 12-dimethylbenz[a]anthracene). PPARγ+/- mice had increased occurrences of ovarian granulosa cell carcinomas compared with wildtype littermates and the tumors that developed in PPARγ+/- mice were more advanced than those formed in wildtype animals [135]. Taken together, these data strongly indicate that PPARγ may provide a protective effect against the development of chemically induced, as well as sporadic ovarian cancer.
PPARγ is not the only PPAR isotype with differential expression observed in ovarian carcinomas. In a subgroup of ovarian endometrioid adenocarcinomas associated with deregulated β-catenin, the expression of PPARδ was significantly elevated [136]. Because of the potential for PPARs to influence the cell cycle and apoptosis, de- or misregulation of these factors may be one mechanism associated with transformation of healthy cells into tumor cells.
Future directions
The clinical use of drugs that activate the PPARs (fibrates and thiazolidinediones) and their ability to be activated by dietary agents warrents further investigation into the role of these transcription factors regulating ovarian gene expression. The inverse expression of PPARγ and P450 side-chain cleavage, and reduction in expression of PPARγ in response to LH, suggests that down-regulation of this transcription factor is important for ovulation and luteinization of follicular cells. Investigating the impact of PPARγ on the periovulatory period could be done by overexpressing PPARγ in granulosa cells, or altering PPARγ to prevent its down-regulation by LH and determining how this affects ovulation and the differentiation of follicular cells into luteal cells. Such information would elucidate mechanisms involved in the terminal differentiation of follicular cells and potentially what may go wrong leading to sub-functional corpora lutea. Investigating the influence of PPARγ on oocyte and follicular cell growth and maturation is also needed due to its high expression in granulosa cells of developing follicles and the sub- and infertility observed in mice with PPARγ disrupted in the ovary. The use of transgenic mice lacking PPARγ in the ovary and siRNA or similar technologies to reduce expression of PPARγ in cultured cells coupled with microarray and/or chromatin immunoprecipitation analyses, will allow for the determination of genes regulated by PPARγ in the ovary. The role of PPARα in ovarian steroidogenesis also needs to be better understood. Although PPARα null-mutant mice seem to reproduce normally, because activation of this isotype, as well as PPARγ, by exogenous agents alters ovarian steroid production, it may be a player and/or have a role in orchestrating ovarian hormone production. Because PPARδ can negatively regulate the activity of the other PPARs and is co-expressed in ovarian cells with PPARs α and γ, how this isotype my modulate the activity of PPARα and/or γ needs to be determined. Altering the ratio of PPARδ to PPARγ and/or PPARα within ovarian cells and how this affects the activity of the latter PPAR isotypes will add to the knowledge of how these transcription factors are regulated in the ovary. Also, understanding what triggers the expression of the PPARs in the ovary will further elucidate how gene expression in the ovary is regulated to support its normal, cyclic function.
Conclusion
There are a variety of mechanisms by which PPARs could potentially influence ovarian function, as illustrated in Figure 5. The steady expression pattern of PPARs α and δ in the ovary during follicular development and the periovulatory period suggest that these PPAR isotypes may regulate gene expression involved in basal functioning of ovarian cells under normal physiological conditions. The ability of PPARγ to regulate ovarian function has been illustrated by agonists regulating steroid production by ovarian cells in vitro, and the sub- or infertility observed in animals with PPARγ disrupted in the ovary. The ability of metabolic factors (i.e. fatty acids) to activate PPARs allows for these transcription factors to alter gene expression in response to the nutritional status of the animal. Therefore, PPARs can mediate the influence of nutrition on female fertility. In addition, environmental exposure to agents such as phthalates and polycyclic aromatic hydrocarbons can also influence gene transcription through the PPARs.
Figure 5 Proposed mechanisms by which PPARs may impact ovarian function and female fertility. The flow chart illustrates the potential interactions between the activation of PPARs and various factors known to impact processes critical for normal ovarian function. See text for details. Stimulatory impact is indicated by a (+). The ability to both stimulate and/or inhibit is denoted by (+/-). COX-2 = cyclooxygenase 2; ET-1 = endothelin -1; LDL = low density lipoprotein; MMPs = matrix metalloproteinases; NOS = nitric oxide synthase; NSAIDs = non-steroidal anti-inflammatory drugs; PAI-1 = plasminogen activator inhibitor -1; VEGF = vascular endothelial growth factor. Asterisk (*) denotes reported targets of PPARs in the ovary.
The importance of understanding of the role(s) of PPARs in the ovary is indicated by their identification in healthy tissue, and altered expression in pathological ovarian tissues. Manipulation of these transcription factors could prove to be beneficial in either the treatment of ovarian pathologies, or as a means to regulate/improve fertility. As more is learned about the impact of PPARs on ovarian function, it will advance our understanding of the pattern of gene expression driving normal ovarian function, what goes awry leading to its dysfunction, and the role of these factors in mediating nutritional and environmental impacts on female fertility.
Acknowledgements
The critical reading of this review, insightful comments and contributions from Dr. Rebecca Robker are gratefully acknowledged.
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Toda K Okada T Miyaura C Saibara T Fenofibrate, a ligand for PPARα, inhibits aromatase cytochrome P450 expression in the ovary of mouse Journal of Lipid Research 2003 44 265 270 12576508 10.1194/jlr.M200327-JLR200
Yanase T Mu Y-M Nishi Y Goto K Nomura M Okabe T Takayanagi R Nawata H Regulation of aromatase by nuclear receptors Journal of Steroid Biochemistry and Molecular Biology 2001 79 192
Veldhuis JD Zhang G Garmey JC Troglitazone, an insulin-sensitizing thiazolidinedione, represses combined stimulation by LH and insulin of de novo androgen biosynthesis by thecal cells in vitro Journal of Clinical Endocrinology and Metabolism 2002 87 1129 1133 11889176 10.1210/jc.87.3.1129
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Liu YX Regulation of the plasminogen activator system in the ovary Biological Signals and Receptors 1999 8 160 177 10213845
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Ricote M Li AC Willson TM Kelly CJ Glass CK The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation Science 1998 391 79 82
Shu H Wong B Zhou G Li Y Berger J Woods J Wright S Cai T-Q Activation of PPARα or γ reduces secretion of matrix metalloproteinase 9 but not interleukin 8 from human monocytic THP-1 cells Biochemical and Biophysical Research Communications 2000 267 345 349 10623622 10.1006/bbrc.1999.1968
Yee J Kuncio GS Bhandari B Shihab FS Neilson EG Identification of promoter activity and differential expression of transcripts encoding the murine stromelysin-1 gene in renal cells Kidney International 1997 52 120 129 9211354
Eberhardt W Akool ES Rebhan J Frank S Beck KF Franzen R Hamanda R Pfeilschlifter J Inhibition of cytokine-induced MMP-9 expression by PPARalpha agonists is indirect and is due to a no-mediated reduction of mRNA stability Journal of Biological Chemistry 2002 277 33518 33528 12093797 10.1074/jbc.M202008200
François M Richette P Tsagris L Raymondjeans M Fulchignono-Lataud M-C Forest C Savouret J-R Corvol M-T Peroxisome proliferator-activated receptor-γ down-regulates chondrocyte matrix metalloproteinase-1 via a novel composite element The Journal of Biological Chemistry 2004 279 28411 28418 15090544 10.1074/jbc.M312708200
Fahmi H Pelletier J-P DiBattista JA Cheung HS Fernandes JC Martel-Pelletier J Peroxisome proliferator-activated receptor gamma activators inhibit MMP-1 production in human synovial fibroblasts likely by reducing the binding of the activator protein 1 Osteoarthritis and Cartilage 2002 10 100 108 11869069 10.1053/joca.2001.0485
Fahmi H DiBattista JA Pelletier J-P Mineau F Ranger P Martel-Pelletier J Peroxisome proliferator-activated receptor γ activators inhibit interleuken-1β-induced nitric oxide and matrix metalloproteinase 13 production in human chondrocytes Arthritis and Rheumatism 2001 44 595 607 11263774 10.1002/1529-0131(200103)44:3<595::AID-ANR108>3.0.CO;2-8
Xin X Yang S Kowalski J Gerritsen ME Peroxisome proliferator-activated receptor γ ligands are potent inhibitors of angiogenesis in vitro and in vivo The Journal of Biological Chemistry 1999 274 9116 9121 10085162 10.1074/jbc.274.13.9116
Marx N Bourcier T Sukhova GK Libby P Plutzky J PPARγ activation in human endothelial cells increases plasminogen activator inhibitor type-1 expression Arteriosclerosis Thrombosis Vascular Biology 1999 19 546 551
Ehrmann D Schneider D Sobel B Cavaghan M Imperial J Rosenfield R Polonsky K Troglitazone improves defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis in women with polycystic ovary syndrome Journal of Clinical Endocrinology and Metabolism 1997 82 2108 2116 9215280 10.1210/jc.82.7.2108
Zirlik A Leugers A Lohrmann J Ernst S Sobel BE Bode C Nordt TK Direct attenuation of plasminogen activator inhibitor type-1 expression in human adipose tissue by thiazolidinediones Thrombosis and Haemostasis 2004 91 674 682 15045127
Otani N Minami S Yamoto M Shikone T Otani H Nishiyama R Otani T Nakano R The vascular endothelial growth factor/fms-like tyrosine kinase system in human ovary during the menstrual cycle and early pregnancy Journal of Clinical Endocrinology and Metabolism 1999 84 3845 3851 10523040 10.1210/jc.84.10.3845
Koos RD Increased expression of vascular endothelial growth/permeability factor in the rat ovary following an ovulatory gonadotropin stimulus: potential roles in follicle rupture Biology of Reproduction 1995 52 1426 1435 7543299
Margeli A Kouraklis G Theocharis S Peroxisome proliferator activated receptor-γ (PPARγ) ligands and angiogenesis Angiogenesis 2003 6 165 169 15041792 10.1023/B:AGEN.0000021377.13669.c0
Yamakawa K Hosoi M Koyama H Tanaka S Fukumoto S Morii H Nishizawa Y Peroxisome proliferator-activated receptor-γ agonists increase vascular endothelial growth factor expression in human vascular smooth muscle cells Biochemical and Biophysical Research Communications 2000 271 571 574 10814503 10.1006/bbrc.2000.2665
Milvae R Inter-relationships between endothelin and prostaglandin F2 alpha in corpus luteum function Reviews of Reproduction 2000 5 1 5 10711729 10.1530/ror.0.0050001
Friden BE Runesson E Hahlin M Brannstrom M Evidence for nitric oxide acting as a luteolytic factor in the human corpus luteum Molecular Human Reproduction 2000 6 397 403 10775642 10.1093/molehr/6.5.397
Jablonka-Shariff A Ravi S Beltsos AN Murphy LL Olson LM Abnormal estrous cyclicity after disruption of endothelial and inducible nitric oxide synthase in mice Biology of Reproduction 1999 61 171 177 10377046
Jablonka-Shariff A Olson LM The role of nitric oxide in oocyte meiotic maturation and ovulation: meiotic abnormalities of endothelial nitric oxide synthase knock-out mouse oocytes Endocrinology 1998 139 2944 2954 9607805 10.1210/en.139.6.2944
Nakamura Y Kashida S Nakata M Takiguchi S Yamagata Y Takayama H Sugino N Kato H Changes in nitric oxide synthase activity in the ovary of gonadotropin treated rats: the role of nitric oxide during ovulation Endocrine Journal 1999 46 529 538 10580745
Matsumi H Yano T Koji T Ogura T Tsutsumi O Taketani Y Esumi H Expression and localization of inducible nitric oxide synthase in the rat ovary: a possible involvement of nitric oxide in the follicular development Biochemical and Biophysical Research Communications 1998 243 67 72 9473481 10.1006/bbrc.1997.8063
Matsumi H Yano T Osuga Y Kugu K Tang X Xu JP Yano N Kurashima Y Ogura T Tsutsumi O Koju T Taketani Y Regulation of nitric oxide synthase to promote cytostasis in ovarian follicular development Biology of Reproduction 2000 63 141 146 10859253
Satoh H Tsukamoto K Hashimoto Y Hashimoto N Togo M Hara M Maekawa H Isoo N Kimura S Watanabe T Thiazolidinediones suppress endothelin-1 secretion from bovine vascular endothelial cells: a new possible role of PPARγ on vascular endothelial function Biochemical and Biophysical Research Communications 1999 254 757 763 9920814 10.1006/bbrc.1998.0126
Ikeda U Shimpo M Murakami Y Shimada K Peroxisome proliferator-activated receptor-γ ligands inhibit nitric oxide synthesis in vascular smooth muscle cells Hypertension 2000 35 1232 1236 10856269
Kota BP Huang THW Roufogalis BD An overview on biological mechanisms of PPARs Pharmacoligical Research 2005 51 85 94 10.1016/j.phrs.2004.07.012
Delerive P Furchart J-C Staels B Peroxisome proliferator-activated receptors in inflammation control Journal of Endocrinology 2001 169 459
Genolet R Wahli W Michalik L PPARs as drug targets to modulate inflammatory responses? Current Drug Targets – Inflammation & Allergy 2004 3 361 375 15584886 10.2174/1568010042634578
Espey LL Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction Biology of Reproduction 1994 50 233 238 8142541
Knobile E Neill JD The Physiology of Reproduction 1994 Second New York: Raven Press
Meade E McIntyre T Zimmerman G Prescott S Peroxisome proliferators enhance cyclooxygenase-2 expression in epithelial cells The Journal of Biological Chemistry 1999 274 8328 8334 10075740 10.1074/jbc.274.12.8328
Subbaramaiah K Liu DT Hart JC Dannenberg AJ Peroxisome proliferator-activated receptor γ ligands suppress the transcriptional activation of cyclooxygenase-2 The Journal of Biological Chemistry 2001 276 12440 12448 11278336 10.1074/jbc.M007237200
Inoue H Tanabe T Umesono K Feedback control of cyclooxygenase-2 expression through PPARγ The Journal of Biological Chemistry 2000 275 28028 28032 10827178
Sirois J Simmons DL Richards JS Hormonal regulation of messenger ribonucleic acid encoding a novel isoform of prostaglandin endoperoxide H synthase in rat preovulatory follicles The Journal of Biological Chemistry 1992 267 11592
Dunn-Albanese LR Ackerman WE Xie Y Iams JD Kniss DA Reciprocal expression of peroxisome proliferator-activated receptor-γ and cyclooxygenase-2 in human term parturition American Journal of Obstetrics and Gynecology 2004 190 809 816 15042019 10.1016/j.ajog.2003.09.052
Staels B Koenig W Habib A Merval R Lebret M Torra IP Delerive P Fadel A Chinetti G Fruchart J-C Najib J Maclouf J Tedgui A Activation of human aortic smooth-muscle cells is inhibited by PPARα but not by PPARγ activators Nature 1998 393 790 793 9655393 10.1038/31701
Reginato MJ Krakow SL Bailey ST Lazar MA Prostaglandins promote and block adipogenesis through opposing effects on peroxisome proliferator-activated receptor γ The Journal of Biological Chemistry 1998 273 1855 1858 9442016 10.1074/jbc.273.4.1855
Hatae T Wada M Yokoyama C Shimonishi M Tanabe T Prostacyclin-dependent apoptosis mediated by PPARδ The Journal of Biological Chemistry 2001 276 46260 46267 11551955 10.1074/jbc.M107180200
Na H-K Surh Y-J Peroxisome proliferator-activated receptor γ (PPARγ ligands as bifunctional regulators of cell proliferation Biochemical Pharmacology 2003 66 1381 1391 14555212 10.1016/S0006-2952(03)00488-X
Butts BD Tran NL Briehl MM Identification of a functional peroxisome proliferator activated receptor response element in the 3' untranslated region of the human bcl -2 gene International Journal of Oncology 2004 24 1305 1310 15067355
Laurora S Pizzimenti S Briatore F Fraioli A Maggio M Reffo P Ferretti C Dianzani U Barrera Peroxisome proliferator-activated receptor ligands affect growth-related gene expression in human leukemic cells The Journal of Pharmacology and Experimental Therapeutics 2003 305 932 942 12649303 10.1124/jpet.103.049098
Robker RL Richards JS Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and P27Kip1 Molecular Endocrinology 1998 12 924 940 9658398 10.1210/me.12.7.924
Mu Y-M Yanase T Nishi Y Takayanagi R Goto K Nawata H Combined treatment with specific ligands for PPARγ:RXR nuclear receptor system markedly inhibits the expression of cytochrome P450arom in human granulosa cancer cells Molecular and Cellular Endocrinology 2001 181 239 248 11476957 10.1016/S0303-7207(00)00457-3
Zhang GY Ahmed N Riley C Oliva K Barker G Quinn MA Rice GE Enhanced expression of peroxisome proliferator-activated receptor gamma in epithelial ovarian carcinoma British Journal of Cancer 2005 92 113 119 15583697 10.1038/sj.bjc.6602244
Sakamoto A Yokoyama Y Umemoto M Futagami M Sakamoto T Bing X Mizunuma H Clinical implications of expression of cyclooxygenase-2 and peroxisome proliferator activated-receptor γ in epithelial ovarian tumours British Journal of Cancer 2004 91 633 638 15266333
Pignatelli M Cocca C Santos A Perez-Castillo A Enhancement of BRCA1 gene expression by the peroxisome proliferator-activated receptor γ in the MCF-7 breast cancer cell line Oncogene 2003 22 5446 5450 12934104 10.1038/sj.onc.1206824
Nicol CJ Yoon M Ward JM Yamashida M Fukamachi K Peters JM Gonzalez FJ PPARγ influences susceptibility fo DMBA-induced mammary, ovarian and skin carcinogenesis Carcinogenesis 2004 25 1747 1755 15073042 10.1093/carcin/bgh160
Zhai Y Wu R Schwartz DR Darrah D Reed H Kolligs FT Nieman MT Fearon ER Cho KR Role of β-catenin/T-cell factor-regulated genes in ovarian endometrioid adenocarcinomas American Journal of Pathology 2002 160 1229 1238 11943708
Lovekamp TN Davis BJ Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells Toxicology and Applied Pharmacology 2001 172 217 224 11312650 10.1006/taap.2001.9156
Dang Z-C Audinot V Papapoulos SE Boutin JA Löwik WGM Peroxisome proliferator-activated receptor γ (PPARγ) as a molecular target for the soy phytoestrogen genistein The Journal of Biological Chemistry 2003 278 962 967 12421816 10.1074/jbc.M209483200
Kliewer SA Forman BM Blumberg B Ong ES Borgmeyer U Mangelsdorf DJ Umesono J Evans RM Differential expression and activation of a family of murine peroxisome proliferator-activated receptors Proceedings of the National Academy of Science 1994 91 7355 7359
Wilson TM Brown PJ Sternbach DD Henke BR The PPARs: from orphan receptors to drug discovery Journal of Medical Chemistry 2000 43 527 550 10.1021/jm990554g
Gearing KL Crickmore A Gustafsson JA Structure of the mouse peroxisome proliferator activated receptor alpha gene Biochemical and Biophysical Research Communications 1994 199 255 263 8123021 10.1006/bbrc.1994.1222
Omi T Brenig B Kramer SS Iwamoto S Stranzinger G Neuenschwander S Identification and characterization of novel peroxisome proliferator-activated recpetor-gamma (PPAR-γ) transcriptional variants in pig and human Journal of Animal Breeding and Genetics 2005 122 45 53 16130456 10.1111/j.1439-0388.2005.00508.x
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Reprod Biol Endocrinol. 2005 Aug 30; 3:41
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Head Face MedHead & Face Medicine1746-160XBioMed Central London 1746-160X-1-11627092110.1186/1746-160X-1-1EditorialHead & Face Medicine – a new journal for 'intra-interdisciplinary' science. Why? When? Where? Stamm Thomas [email protected] Poliklinik für Kieferorthopädie, Universitätsklinikum, Westfälische Wilhelms-Universität, Münster, Germany2005 24 8 2005 1 1 1 12 8 2005 24 8 2005 Copyright © 2005 Stamm; licensee BioMed Central Ltd.2005Stamm; 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 human head and face is the target structure of a large number of medical disciplines which are subject to a continuing trend in medical science – 'ongoing fragmentation' or, to use a better established term, 'opening up new fields'. An adverse side effect of this trend is the separation of scientists, which contributes to a breakdown in communication. Specialization is necessary, but who is able to recombine the pieces of knowledge gained in different branches of science? Who is able to trace back an effect to its cause through the whole system? What is the instrument that enables scientists to think 'laterally', or across disciplines?
To be one of these instruments is the vision of Head & Face Medicine. To induce 'intra-interdisciplinary' thinking of scientists by bringing together the findings achieved by different researchers from various specialties, all exploring the same target structure – the human head and face. Head & Face Medicine's objective is to support scientists in gaining new insights from different views, to recognize patterns, to extract new thoughts, to recombine them and bring new visions to life.
Evolving tools like the internet, e-publishing, Open Access and open peer review make Head & Face Medicine a cross between a traditional journal and a data stream which can be queried, analyzed and processed with the aim of increasing medical knowledge in the area of head and face medicine. These tools represent several advantages: fast publication, increase of a paper's scientific impact and ethical superiority.
Head & Face Medicine looks forward to receiving your contributions.
==== Body
Hardly any region of the human body depends upon the synergism of a variety of medical disciplines to the same extent as the human head. To understand the complexity of the whole system 'head' it is necessary to reduce the system to its most discriminable elements and to explore their nature, because the elements realize certain functions in the whole. This philosophical tradition, the reduction principle, continues to be adhered to and developed in medical science. Attributable to this development is the ongoing fragmentation of medical disciplines into more and more sub-(sub-)specialties; or to put it more positively, one would argue: the opening of new fields.
However, an adverse side effect of this progress is the separation of scientists working in different sub-specialties, resulting in a breakdown in communication. Intensive scientific debate is common within the fields but not across them. Specialization is necessary, but where in this process are the individuals who are able to recombine the pieces of knowledge gained in narrow but deep branches of science? Who is able to trace back an effect to its cause through the whole system? What is the instrument that enables scientists to think lateral or parallel to their own discipline? Definitely, there is not one single instrument, but an important one is communication – communication on different levels of knowledge linked and cross-referenced to disciplines working on the same target structure. The traditional term of this concept is 'interdisciplinary'.
What do we really mean by 'interdisciplinary'? It is not the collaboration with a scientist of an adjacent medical branch who is not interested in your problem. Interdisciplinary is a kind of thinking, and it is initiated in an individual mind. What we really mean is 'intra-interdisciplinary'. To induce this way of thinking we need a particular dose of knowledge from the adjacent disciplines. Therefore, Head & Face Medicine has developed a particular vision.
Head & Face Medicine's vision
Progress in interdisciplinary diagnostics, therapy and research of pathologic conditions of the human head and face by raising new scientific questions which demand new ways of thinking to improve medical quality.
To make this vision come true, we need your help. We need your ideas, insights, observations, and research results, across barriers of specialization, to induce creativity and innovations. This would allow us to learn from all the different disciplines which are involved in head and face disorders, by communicating, by presenting our findings, by defending our hypotheses, and by criticizing ideas and debating methodologies across the frontiers of our own formal training. It would allow us to be interdisciplinary in all ways of thinking to enrich medical knowledge. In the inaugural issue of Head & Face Medicine, the co-founders of the journal, Ulrich Meyer and Hans-Peter Wiesmann, will illustrate their vision of 'intra-interdisciplinary', with thoughts from bio-mineralization, tissue-engineering and maxillofacial surgery.
On the way to fulfilling this mission we are grateful that it was possible to establish an international editorial board which reflects the principle of inter-disciplinarity in combination with scientific quality. All members of the editorial board are well known scientists in their respective area of expertise and have agreed to spend a vast amount of their valuable time to support our vision.
Evolution is what we need in scientific literature, not revolution [1]. With BioMed Central we found a publisher who provides a platform where we can use today's 'evolving' tools for scientific literature: e-publication, Open Access and open peer review. Let me convince you by summarizing the advantages of these tools.
"Publication delay has a harmful effect on patients' health"
It has been emphasized that some of the current processes of publication involve a considerable delay in the dissemination of clinical research, which has a significant effect on patients' health [2]. A review of AIDS trials conducted in 1998 observed a publication delay of between 1.7 and 3 years. Although an improvement, the authors found that in 2004, the publication delay for randomized clinical trials was still 20 month or longer. We agree with the authors that this is unacceptable.
Many solutions have been suggested to promote timely publication. Head & Face Medicine aims to provide the authors with a first decision within six weeks after manuscript submission. Immediately on acceptance, the scientific community can read the author's article as a provisional PDF version. Once the journal is included in PubMed (which will occur approximately 2 months after the launch of the journal), the provisional version will be sent to PubMed and included after a 48-hour delay. This will be replaced by the final full-text version when available. Head & Face Medicine's general publication process is therefore faster than that of other journals with a 'rapid' publication section [2].
"Free online availability substantially increases a paper's impact"
Irrespective of economic, moral and ethic arguments on 'Open Access' in scientific literature, what are today's facts? What is the benefit for the individual patient?
It has been shown that Open Access articles were cited 50–300% more often than non-Open Access articles from the same journal and year [3]. The same applies to non-medical science literature, where on average 336% more citations for online articles were observed. There was a clear correlation between the number of times an article is cited and the probability that the article is online [4]. The ability to locate relevant scientific results quickly will dramatically improve scientific progress and therefore improve medical quality. These important findings are cogent and make it imperative for us to turn to Open Access. Head & Face Medicine has adopted BioMed Central's Open Access Charter [5], which is the successful base of many independent journals. At this point, it must be clearly stated that no individual who is involved in developing and sustaining Head & Face Medicine has competing interests.
"Most publishing scientists didn't know much about the benefits of Open Access"
About 20% of the total number of articles published annually are Open Access [6] and it has been said that most publishing scientists didn't know much about the benefits of Open Access [3]. Here is a brief description of the Open Access policy of Head & Face Medicine and its benefits for science and the general public. For all who want to dive into the Open Access debate we recommend Peter Suber's weblog [7].
All articles of Head & Face Medicine become freely and universally accessible online, and so the author's work can be read by anyone at no cost. The authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [5]. A copy of the full text of each article is permanently archived in an online repository separate from the journal. Head & Face Medicine's articles are archived in PubMed Central [8], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [9] in Germany, at INIST [10] in France and in e-Depot [11], the National Library of the Netherlands' digital archive of all electronic publications.
Open peer review is superior to traditional closed peer review – ethically
There are many arguments for and against open peer review. At present there is no evidence that any single kind of peer review leads to higher quality reports and feedback. Head & Face Medicine uses an open system because the ethical reasons to move away from anonymity are significant. Many publishing scientists have seen brusque, incompetent and destructive reports produced in anonymity. The worst abuses are blocking or stealing of new ideas. This behavior is not acceptable and we strongly believe that transparency leads to a more respectful and constructive communication.
Head & Face Medicine supports the reviewers' academic credit for the work they do
Reviewing can seem a thankless task in anonymity. In a closed system, reviewers don't receive academic credit for the power and knowledge they invested to improve the quality of an authors work. Therefore, Head & Face Medicine posts the signed reviews in a pre-publication history, which is freely available to access from the published article. Reviewers' names and reports are therefore easily accessible via the published article, which leads to wider recognition within the scientific community. With the open system, both sides win. The authors receive a constructive, high-quality contribution with a higher chance of acceptance [12], and the reviewers improve their academic reputation. Then, if a paper is frequently cited, all parties involved have the benefit of the scientific impact.
Résumé
The vision of Head & Face Medicine is to induce 'intra-interdisciplinary' thinking by bringing together the findings of different researchers from various specialties, all exploring the same target structure. The objective is to gain new insights from different views, to recognize patterns, to extract new thoughts, to recombine them and to bring new visions to life. Scientists have the ethical duty to publish their results as soon as possible, irrespective of whether or not the findings are negative, which means challenging current dogmas, tenets or opinions of experts. The internet, e-publishing, Open Access and open peer review turn a journal like Head & Face Medicine to a cross between a traditional journal and a data stream which can be queried, analyzed and processed with the aim to improve medical knowledge in the area of head and face medicine. We look forward to receiving your contributions.
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Crane-Robinson C Evolution is what's needed, not revolution Nature 2001 411 522 11385537 10.1038/35079158
Torgerson DJ Adamson J Cockayne S Dumville J Petherick E Submission to multiple journals: a method of reducing time to publication? BMJ 2005 330 305 307 15695280 10.1136/bmj.330.7486.305
Suber P Open access, impact, and demand BMJ 2005 330 1097 1098 15891208 10.1136/bmj.330.7500.1097
Lawrence S Free online availability substantially increases a paper's impact Nature 2001 411 521 11385534 10.1038/35079151
BioMed Central Open Access Charter
Rowlands I Nicholas D Huntingdon P Scholarly communication in the digital environment: what do authors want?
Suber P Open Access News
PubMed Central
Potsdam
INIST
e-Depot
Walsh E Rooney M Appleby L Wilkinson G Open peer review: a randomised controlled trial Br J Psychiatry 2000 176 47 51 10789326 10.1192/bjp.176.1.47
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Head Face MedHead & Face Medicine1746-160XBioMed Central London 1746-160X-1-21627092510.1186/1746-160X-1-2EditorialTissue engineering: a challenge of today's medicine Meyer Ulrich [email protected] Hans-Peter [email protected] Klinik und Poliklinik für Mund-, Kiefer- und Gesichtschirurgie, Heinrich Heine Universität, Düsseldorf, Germany2 Klinik und Poliklinik für Mund-, Kiefer- und Gesichtschirurgie, Universitätsklinikum, Westfälische WiIhelms-Universität, Münster, Germany2005 24 8 2005 1 2 2 22 8 2005 24 8 2005 Copyright © 2005 Meyer and Wiesmann; licensee BioMed Central Ltd.2005Meyer and Wiesmann; 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 the last years, tissue engineering-based therapies have been introduced in clinical practice in the head and face area. The regeneration of complex tissue structures for all sites of the body is envisioned for the future. In the present situation, specialists of the different fields publish excellent research papers in specialised journals. As a result, the scientific community, seperated towards distinct sub-specialities, has difficulties in communication. To overcome this problem, the demanding, complex and interdisciplinary aspects of tissue engineering has to be approached from new ways. We have conceptualised Head & Face Medicine therefore as a thematically broad ranged journal, including all disciplines involved in the head and neck area. We hope this journal will attract basic researchers and clinicians who are involved in investigating and applying complex themes (examplified by tissue engineering) in the head and face region and will contribute to a gain in scientific information, communication, and collaboration in order to improve the outcome of patient treatments.
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Tissue engineering is a new and fast growing branch of medicine that has now been introduced in clinical practice. The regeneration of various tissues for all sites of the body is envisioned for the future. The artificial generation of hard and soft tissues offers clinicians new tools in the treatment of various diseases of the head and face region. On the other hand, tissue engineering poses significant challenges, which needed to be overcome before this treatment option can be routinely performed.
One of the reasons for the fast growth of tissue engineering is the high number of excellent research papers, published in a wide array of (material-, biological-, biomedical-, biophysical-, and clinical-based) journals, covering all aspects of basic research, preclinical testing and clinical application. Additionally, numerous high quality books are available describing in detail the different aspects of tissue engineering. Despite the fact that various journals publish excellent research papers focusing on specialised areas of tissue engineering, we decided to also include papers on such specialties in our journal, Head & Face Medicine. There were two reasons for this decision: during the phase of establishment of tissue engineering in our clinics on an experimental and clinical level, we observed that many specialists of the different fields involved in approaching tissue engineering, had difficulties in overviewing the complexity of the field. Secondly, as tissue engineering forces basic researchers, mainly having a biological, biophysical or material science oriented background, to closely collaborate with clinically oriented physicians, we found that they had difficulties in communication. The basis for the problems in 'intra-interdisciplinary' [1] research seem to be related to a great extent to the restriction of scientists to involve themselves in other fields, thereby loosing the ability to open their minds towards other scientific branches. To overcome this problem, the demanding, complex and interdisciplinary aspects of tissue engineering has to be approached from new ways.
We have conceptualised Head & Face Medicine therefore as a thematically broad ranged journal, including all disciplines involved in the head and neck area. By publishing the various aspects of, for example tissue engineering in one journal the reader can gain an overview of the whole field. Research papers are intended to provide extended information for the specialist. On the other hand, we think that interested readers from other disciplines will be able to extract the data to an extent, that enable them to understand the most relevant informations.
To induce a learning curve from the acquisition of fragmented, but detailed information, to the ability to critically overview over the complex field of tissue engineering is a vision of Head & Face Medicine. We hope this journal will attract basic researchers and clinicians who are involved in investigating and applying tissue engineering in the head and face region and will contribute to a gain in scientific information, communication, and collaboration in order to improve the outcome of patient treatments.
==== Refs
Stamm T Head & Face Medicine – a new journal for 'intra-interdisciplinary' science. Why? When? Where? Head Face Med 2005 1 1 16270921 10.1186/1746-160X-1-1
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Head Face MedHead & Face Medicine1746-160XBioMed Central London 1746-160X-1-31627091610.1186/1746-160X-1-3ReviewAdenomatoid odontogenic tumor of the mandible: review of the literature and report of a rare case Handschel Jörg GK [email protected] Rita A [email protected] André C [email protected] Stefan [email protected]übler Norbert R [email protected] Department for Cranio- and Maxillofacial Surgery, Heinrich-Heine-University, Moorenstr. 5, D-40225 Düsseldorf, Germany2 Department for Pathology, Heinrich-Heine-University, Moorenstr. 5, D-40225 Düsseldorf, Germany2005 24 8 2005 1 3 3 25 3 2005 24 8 2005 Copyright © 2005 Handschel et al; licensee BioMed Central Ltd.2005Handschel 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.
Adenomatoid odontogenic tumor (AOT) is a rare odontogenic tumor which is often misdiagnosed as odontogenic cyst. To acquire additional information about AOT, all reports regarding AOT and cited in "pubmed" since 1990 onward were reviewed. AOT accounts for about 1% until 9% of all odontogenic tumors. It is predominantly found in young and female patients, located more often in the maxilla in most cases associated with an uneruppted permanent tooth. For radiological diagnose the intraoral periapical radiograph seems to be more useful than panoramic. However, AOT frequently resemble other odontogenic lesions such as dentigerous cysts or ameloblastoma. Immunohistochemically AOT is characterized by positive reactions with certain cytokeratins. Treatment is conservative and the prognosis is excellent. For illustration a rare case of an AOT in the mandible is presented.
adenomatoid odontogenic tumorreview
==== Body
Adenomatoid odontogenic tumor (AOT) is a relatively uncommon distinct odontogenic neoplasm that was first described by Steensland in 1905 [1]. However, a variety of terms have been used to describe this tumor. Unal et al [2] produced a list containing all nomenclatures for AOT reported in the literatures. Many different names like adenoameloblastoma, ameloblastic adenomatoid tumor, adamantinoma, epithelioma adamantinum or teratomatous odontoma have been used before to define the lesion currently called AOT. In 1999 Philipsen and Reichart [3] presented a review based on reports published until 1997 which showed some interesting aspects regarding epidemiological figures of this tumor. Since then numerous case reports of AOT have been published.
Epidemiology
From the early 1990s onwards 65 single cases of AOT (excluding case series of more than 10 cases) have been published. The mean age was 13.2 years (range 3 until 28 years) and the female:male ratio was 2.3 : 1. The AOT was predominantly found in the upper jaw (maxilla:mandible = 2.6 : 1). Regarding the various case series published in the literature [e.g. [4-8]] and comparing these data with the single case reports mentioned above, it has to be reasoned that the AOT has a prevalence of odontogenic tumors between 1.2% in caucasian [5] and 9% in black african patients [4]. The tumor is most often diagnosed in the second decade of life and women are about twice as many affected than men. The AOT is over two times more located in the maxilla than in the mandible and the anterior jaw is much more affected than the posterior area. According to Philipsen and Reichart [3] the AOT appears in three clinico-topographic variants: follicular, extrafollicular and peripheral. The follicular and extrafollicular variants are both intrabony and account for approximately 96% of all AOTs of which 71% are of follicular type.
Clinical features
Clinical features generally focus on complaints regarding a missing tooth. The lesion usually present as asymptomatic swelling which is slowly growing and often associated with an unerupted tooth. However, the rare peripheral variant occurs primarily in the gingival tissue of tooth-bearing areas [9]. Unerupted permanent canine are the theeth most often involved in AOTs.
Radiographic features
The radiographic findings of AOT frequently resemble other odontogenic lesions such as dentigerous cysts, calcifying odontogenic cysts, calcifying odontogenic tumors, globule-maxillary cysts, ameloblastomas, odontogenic keratocysts and periapical disease [10]. Whereas the follicular variant shows a well-circumscribed unilocular radiolucency associated with the crown and often part of the root of an unerupted tooth, the radiolucency of the extrafollicular type is located between, above or superimposed upon the roots of erupted permanent teeth [3]. Displacement of neighbouring teeth due to tumor expansion is much more common than root resorptions. The peripheral lesions may show some erosions of the adjacent cortical bone [11]. Comparing diagnostic arruracy between intraoral periapical and panoramic radiographs Dare et al. [12] found that intraoral periapical radiographs allow perception of the radiopacities in AOT as discrete foci having a flocculent pattern within radiolucency even with minimal calcifies deposits while panoramic often do not. Those calcified deposits are seen in approximately 78% of AOT [13]. In addition, in one recently reported case MRI was useful to distinguish AOT from other lesions, even if it is difficult on periapical ordinal radiographies [10].
Pathohistological features
Remarkably, all variants of AOT show identical histology. The histological typing of the WHO defined the AOT as a tumor of odontogenic epithelium with duct-like structures and with varying degrees of inductive change in the connective tissue. The tumor may be partly cystic, and in some cases the solid lesion may be present only as masses in the wall of a large cyst [14]. Moreover, eosinophilic, uncalcified, amorphous material can be found and is called "tumor droplets". Some tumor droplets show a homogenous matrix whereas most tumor droplets reveal electron-dense plaques [15]. Interestingly, there are a few reports about pigmented cells in AOT. However, all of these reported lesions did not show macroscopically visible pigmentation. Racial pigmentation probably plays an important role in such cases [16,17].
Immunhistological features
During the last few years several studies have been published dealing with the immunhistological properties of AOT. Immunohistochemically, the classical AOT phenotype is characterized by a cytokeratin (CK) profile similar to follicular cyst and/or oral or gingival epithelium based on positive staining with CK5, CK17 and CK19 [18]. On the other hand the classical AOT is negative for CK4, 10, 13 and 18. Recently, Crivelini et al. [19] detected the expression of cytokeratin 14 in AOT and concluded that this probably indicate its origin in the reduced dental epithelium which is also positive for staining with cytokeratin 14 antibodies. Positive reactions for amelogenin in limited areas in AOT are also reported as well as in ameloblasts and in the immature enamel matrix [20].
Interestingly, Takahashi et al. [21] observed a positive staining for iron-binding proteins (transferring, ferritin) and proteinase inhibitor (alpha-one-antitrypsin) in various cells of AOT indicating their role to the pathogenesis of AOT. Finally, Gao et al. [22] studied the expression of bone morphogenic protein (BMP). Whereas cementifying fibromas, dentinomas and compound odontomas demonstrated a positive reaction, all AOT as well as ameloblastomas and calcifying epithelial odontogenic tumors were negative.
Treatment and prognosis
Conservative surgical enucleation is the treatment modality of choice. For periodontal intrabony defects caused by AOT guided tissue regeneration with membrane technique is suggested after complete removal of the tumor [23]. Recurrence of AOT is exceptionally rare. Only three cases in Japanese patients are reported in which the recurrence of this tumor occurred [24]. Therefore, the prognosis is excellent.
Case report
A 23-year-old man was referred by his general dental practitioner. One year ago the dentist diagnosed a cyst with a ectopic lower right canine tooth by an x-ray. Beside an uneventful medical history the patient presented no conspicuous intraoral clinical findings except the absence of the tooth 43. Radiologically, he showed a 3 cm unicystic radiolucent image with a comparatively clear demarcation. The tooth 43 was located on the floor of this process. No resorption of the root apices was observed (Fig. 1).
Figure 1 Panoramic radiograph before therapy. Unicystic radiolucent lesion in the lawer right jaw with a comparatively clear demarcation. The tooth 43 is located on the floor of this process. There are no resorption of the root apices.
Under general anesthesia the lesion was enucleated and afterwards filled with pelvic spongiosa. Separating the lesion from mandibular bone caused no problems. The postoperative course was uneventful.
After the operation, the specimen was fixed in 4 per cent formal saline and prepared for histological examination. Some sections were stained with haematoxylin-eosin.
Histologically, the tumor is solid and there is a cyst formation (Fig. 2). The epithelium is in the form of whorled masses of spindle cells as well as sheets and plexiform strands. Rings of columnar cells give rise to duct-like appearance (Fig. 3). Calcification is sometimes seen and may be extensive (Fig. 4).
Figure 2 Tumor with fibrous connective tissue capsule (*). Nodular aggregates of cells (#). Duct-like structures (→). (HE × 50)
Figure 3 Gland-like spaces are surrounded by cuboidal to columnar cells (→). (HE × 160)
Figure 4 Tumor with calcified areas (→). (HE × 200)
Half a year after surgery a clinical and radiographic follow-up examination was performed. There was no evidence of recurrence and no apical resorption of the adjacent teeth could be observed (Fig. 5).
Figure 5 Panoramic radiograph six months after therapy. No root resorption could be observed.
With respect to the age of the patient and the localization of the AOT in the lower jaw, the reported case is a rare example of this tumor entity. Beyond it our case supports the above mentioned general description of AOTs.
Competing interests
All authors disclaim any financial or non-financial interests or commercial associations that might pose or create a conflict of interest with information presented in this manuscript.
Authors' contributions
JH, RD and NK made substantial contribution to the conception and design of the manuscript. SB and AZ carried out the pathohistological investigations and participated in creating this part of the manuscript.
All authors were involved in revising the manuscript critically and have given final approval of the version to be published.
==== Refs
Steensland HS Epithelioma adamantinum J Exper Med 1905 6 377 389 10.1084/jem.6.4-6.377
Unal T Cetingul E Gunbay T Peripheral adenomatoid odontogenic tumor: Birth of a term J Clin Pediatr Dent 1995 19 139 142 7577734
Philipsen HP Reichart PA Adenomatoid odontogenic tumour: facts and figures Oral Oncol 1998 35 125 131 10435145 10.1016/S1368-8375(98)00111-0
Adebayo ET Ajike SO Adekeye EO Odontogenic tumours in children and adolescents: a study of 78 Nigerian cases J Craniomaxillofac Surg 2002 30 267 272 12377198
Stypulkowska J Odontogenic tumors and neoplastic-like changes of the jaw bone. Clinical study and evaluation of treatment results Folia Med Cracov 1998 39 135 141
Chattopadhyay A Adenomatoid odontogenic tumour: Review of literature and report of 30 cases from India Indian J Dent Res 1994 5 89 95 9495104
Mosqueda-Taylor A Ledesma-Montes C Caballero-Sandoval S Portilla-Robertson J Ruiz-Godoy Rivera LM Meneses-Garcia A Odontogenic tumors in Mexico: a collaboratibe retrospective study of 349 cases Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997 84 672 675 9431538 10.1016/S1079-2104(97)90371-1
Arotiba JT Ogunbiyi JO Obiechina AE Odontogenic tumours: a 15-year review from Ibadan, Nigeria Br J Oral Maxillofac Surg 1997 35 363 367 9427447 10.1016/S0266-4356(97)90411-3
Buchner A Sciubba JJ Peripheral odontogenic tumours: a review Oral Surg Oral Med Oral Pathol 1987 63 688 697 3295656 10.1016/0030-4220(87)90372-0
Konouchi H Asaumi J Yanagi Y Hisatomi M Kishi K Adenomatoid odontogenic tumor: correlation of MRI with histopathological findings Eur J Rad 2002 44 19 23 10.1016/S0720-048X(01)00428-4
Philipsen HP Reichart PA Zhang KH Nikai H Yu QX Adenomatoid odontogenic tumor: biologic profile based on 499 cases J Oral Pathol Med 1991 20 149 158 2061853
Dare A Yamaguchi A Yoshiki S Okano T Limitation of panoramic radiography in diagnosing adenomatoid odontogenic tumors Oral Surg Oral Med Oral Pathol 1994 77 662 668 8065735 10.1016/0030-4220(94)90331-X
Toida M Hyodo I Okuda T Tatematsu N Adenomatoid odontogenic tumor: report of two cases and survey of 126 cases in Japan J Oral Maxillofac Surg 1990 48 404 408 2313448
Kramer IRH Pindborg JJ Shear M WHO histological typing of odontogenic tumours 1992 2 Springer Verlag Berlin, Heidelberg, New York
Philipsen HP Reichart PA The adenomatoid odontogenic tumour: ultrastructure of tumour cells and non-calcified amorphous masses J Oral Pathol Med 1996 25 491 496 8959557
Takeda Y Sato H Satoh M Nakamura S Yamamoto H Pigmented ameloblastic fibrodentinoma: a novel melanin-pigmented intraosseous odontogenic lesion Virchows Arch 2000 437 454 458 11097374 10.1007/s004280000249
Buchner A David R Carpenter W Leider A Pigmented lateral periodontal cyst and other pigmented odontogenic lesions Oral Dis 1996 2 299 302 9171515
Larson A Swartz K Heikinheimo K A case of multiple AOT-like jawbone lesions in a young patient-a new odontogenic entity? J Oral Pathol Med 2003 32 55 62 12558960 10.1034/j.1600-0714.2003.00046.x
Crivelini MM de Araujo VC de Sousa SO de Araujo NS Cytokeratins in epithelia of odontogenic neoplasms Oral Dis 2003 9 1 6 12617250 10.1034/j.1601-0825.2003.00861.x
Abiko Y Murata M Ito Y Taira T Nishimura M Arisue M Inoue T Shimono M Kuboki Y Kaku T Immunhistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin Med Electron Microsc 2001 34 185 189 11793195 10.1007/s007950100014
Takahashi H Fujita S Shibata Y Yamaguchi A Adenomatoid odontogenic tumour: immunohistochemical demonstration of transferring, ferritin and alpha-one-antitrypsin J Oral Pathol Med 2001 30 237 244 11302244 10.1034/j.1600-0714.2001.300408.x
Gao YH Yang LJ Yamaguchi A Immunohistochemical demonstration of bone morphogenic protein in odontogenic tumors J Oral Pathol Med 1997 26 273 277 9234187
Blumenthal NM Mostofi R Repair of an intrabony defect from an adenomatoid odontogenic tumor J Periodontol 2000 71 1637 1640 11063398 10.1902/jop.2000.71.10.1637
Philipsen HP Reichart PA Nikai H The adenomatoid odontogenic tumour (AOT): An update Oral Medicine & Pathology 1997 2 55 60
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Head Face MedHead & Face Medicine1746-160XBioMed Central London 1746-160X-1-31627091610.1186/1746-160X-1-3ReviewAdenomatoid odontogenic tumor of the mandible: review of the literature and report of a rare case Handschel Jörg GK [email protected] Rita A [email protected] André C [email protected] Stefan [email protected]übler Norbert R [email protected] Department for Cranio- and Maxillofacial Surgery, Heinrich-Heine-University, Moorenstr. 5, D-40225 Düsseldorf, Germany2 Department for Pathology, Heinrich-Heine-University, Moorenstr. 5, D-40225 Düsseldorf, Germany2005 24 8 2005 1 3 3 25 3 2005 24 8 2005 Copyright © 2005 Handschel et al; licensee BioMed Central Ltd.2005Handschel 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.
Adenomatoid odontogenic tumor (AOT) is a rare odontogenic tumor which is often misdiagnosed as odontogenic cyst. To acquire additional information about AOT, all reports regarding AOT and cited in "pubmed" since 1990 onward were reviewed. AOT accounts for about 1% until 9% of all odontogenic tumors. It is predominantly found in young and female patients, located more often in the maxilla in most cases associated with an uneruppted permanent tooth. For radiological diagnose the intraoral periapical radiograph seems to be more useful than panoramic. However, AOT frequently resemble other odontogenic lesions such as dentigerous cysts or ameloblastoma. Immunohistochemically AOT is characterized by positive reactions with certain cytokeratins. Treatment is conservative and the prognosis is excellent. For illustration a rare case of an AOT in the mandible is presented.
adenomatoid odontogenic tumorreview
==== Body
Adenomatoid odontogenic tumor (AOT) is a relatively uncommon distinct odontogenic neoplasm that was first described by Steensland in 1905 [1]. However, a variety of terms have been used to describe this tumor. Unal et al [2] produced a list containing all nomenclatures for AOT reported in the literatures. Many different names like adenoameloblastoma, ameloblastic adenomatoid tumor, adamantinoma, epithelioma adamantinum or teratomatous odontoma have been used before to define the lesion currently called AOT. In 1999 Philipsen and Reichart [3] presented a review based on reports published until 1997 which showed some interesting aspects regarding epidemiological figures of this tumor. Since then numerous case reports of AOT have been published.
Epidemiology
From the early 1990s onwards 65 single cases of AOT (excluding case series of more than 10 cases) have been published. The mean age was 13.2 years (range 3 until 28 years) and the female:male ratio was 2.3 : 1. The AOT was predominantly found in the upper jaw (maxilla:mandible = 2.6 : 1). Regarding the various case series published in the literature [e.g. [4-8]] and comparing these data with the single case reports mentioned above, it has to be reasoned that the AOT has a prevalence of odontogenic tumors between 1.2% in caucasian [5] and 9% in black african patients [4]. The tumor is most often diagnosed in the second decade of life and women are about twice as many affected than men. The AOT is over two times more located in the maxilla than in the mandible and the anterior jaw is much more affected than the posterior area. According to Philipsen and Reichart [3] the AOT appears in three clinico-topographic variants: follicular, extrafollicular and peripheral. The follicular and extrafollicular variants are both intrabony and account for approximately 96% of all AOTs of which 71% are of follicular type.
Clinical features
Clinical features generally focus on complaints regarding a missing tooth. The lesion usually present as asymptomatic swelling which is slowly growing and often associated with an unerupted tooth. However, the rare peripheral variant occurs primarily in the gingival tissue of tooth-bearing areas [9]. Unerupted permanent canine are the theeth most often involved in AOTs.
Radiographic features
The radiographic findings of AOT frequently resemble other odontogenic lesions such as dentigerous cysts, calcifying odontogenic cysts, calcifying odontogenic tumors, globule-maxillary cysts, ameloblastomas, odontogenic keratocysts and periapical disease [10]. Whereas the follicular variant shows a well-circumscribed unilocular radiolucency associated with the crown and often part of the root of an unerupted tooth, the radiolucency of the extrafollicular type is located between, above or superimposed upon the roots of erupted permanent teeth [3]. Displacement of neighbouring teeth due to tumor expansion is much more common than root resorptions. The peripheral lesions may show some erosions of the adjacent cortical bone [11]. Comparing diagnostic arruracy between intraoral periapical and panoramic radiographs Dare et al. [12] found that intraoral periapical radiographs allow perception of the radiopacities in AOT as discrete foci having a flocculent pattern within radiolucency even with minimal calcifies deposits while panoramic often do not. Those calcified deposits are seen in approximately 78% of AOT [13]. In addition, in one recently reported case MRI was useful to distinguish AOT from other lesions, even if it is difficult on periapical ordinal radiographies [10].
Pathohistological features
Remarkably, all variants of AOT show identical histology. The histological typing of the WHO defined the AOT as a tumor of odontogenic epithelium with duct-like structures and with varying degrees of inductive change in the connective tissue. The tumor may be partly cystic, and in some cases the solid lesion may be present only as masses in the wall of a large cyst [14]. Moreover, eosinophilic, uncalcified, amorphous material can be found and is called "tumor droplets". Some tumor droplets show a homogenous matrix whereas most tumor droplets reveal electron-dense plaques [15]. Interestingly, there are a few reports about pigmented cells in AOT. However, all of these reported lesions did not show macroscopically visible pigmentation. Racial pigmentation probably plays an important role in such cases [16,17].
Immunhistological features
During the last few years several studies have been published dealing with the immunhistological properties of AOT. Immunohistochemically, the classical AOT phenotype is characterized by a cytokeratin (CK) profile similar to follicular cyst and/or oral or gingival epithelium based on positive staining with CK5, CK17 and CK19 [18]. On the other hand the classical AOT is negative for CK4, 10, 13 and 18. Recently, Crivelini et al. [19] detected the expression of cytokeratin 14 in AOT and concluded that this probably indicate its origin in the reduced dental epithelium which is also positive for staining with cytokeratin 14 antibodies. Positive reactions for amelogenin in limited areas in AOT are also reported as well as in ameloblasts and in the immature enamel matrix [20].
Interestingly, Takahashi et al. [21] observed a positive staining for iron-binding proteins (transferring, ferritin) and proteinase inhibitor (alpha-one-antitrypsin) in various cells of AOT indicating their role to the pathogenesis of AOT. Finally, Gao et al. [22] studied the expression of bone morphogenic protein (BMP). Whereas cementifying fibromas, dentinomas and compound odontomas demonstrated a positive reaction, all AOT as well as ameloblastomas and calcifying epithelial odontogenic tumors were negative.
Treatment and prognosis
Conservative surgical enucleation is the treatment modality of choice. For periodontal intrabony defects caused by AOT guided tissue regeneration with membrane technique is suggested after complete removal of the tumor [23]. Recurrence of AOT is exceptionally rare. Only three cases in Japanese patients are reported in which the recurrence of this tumor occurred [24]. Therefore, the prognosis is excellent.
Case report
A 23-year-old man was referred by his general dental practitioner. One year ago the dentist diagnosed a cyst with a ectopic lower right canine tooth by an x-ray. Beside an uneventful medical history the patient presented no conspicuous intraoral clinical findings except the absence of the tooth 43. Radiologically, he showed a 3 cm unicystic radiolucent image with a comparatively clear demarcation. The tooth 43 was located on the floor of this process. No resorption of the root apices was observed (Fig. 1).
Figure 1 Panoramic radiograph before therapy. Unicystic radiolucent lesion in the lawer right jaw with a comparatively clear demarcation. The tooth 43 is located on the floor of this process. There are no resorption of the root apices.
Under general anesthesia the lesion was enucleated and afterwards filled with pelvic spongiosa. Separating the lesion from mandibular bone caused no problems. The postoperative course was uneventful.
After the operation, the specimen was fixed in 4 per cent formal saline and prepared for histological examination. Some sections were stained with haematoxylin-eosin.
Histologically, the tumor is solid and there is a cyst formation (Fig. 2). The epithelium is in the form of whorled masses of spindle cells as well as sheets and plexiform strands. Rings of columnar cells give rise to duct-like appearance (Fig. 3). Calcification is sometimes seen and may be extensive (Fig. 4).
Figure 2 Tumor with fibrous connective tissue capsule (*). Nodular aggregates of cells (#). Duct-like structures (→). (HE × 50)
Figure 3 Gland-like spaces are surrounded by cuboidal to columnar cells (→). (HE × 160)
Figure 4 Tumor with calcified areas (→). (HE × 200)
Half a year after surgery a clinical and radiographic follow-up examination was performed. There was no evidence of recurrence and no apical resorption of the adjacent teeth could be observed (Fig. 5).
Figure 5 Panoramic radiograph six months after therapy. No root resorption could be observed.
With respect to the age of the patient and the localization of the AOT in the lower jaw, the reported case is a rare example of this tumor entity. Beyond it our case supports the above mentioned general description of AOTs.
Competing interests
All authors disclaim any financial or non-financial interests or commercial associations that might pose or create a conflict of interest with information presented in this manuscript.
Authors' contributions
JH, RD and NK made substantial contribution to the conception and design of the manuscript. SB and AZ carried out the pathohistological investigations and participated in creating this part of the manuscript.
All authors were involved in revising the manuscript critically and have given final approval of the version to be published.
==== Refs
Steensland HS Epithelioma adamantinum J Exper Med 1905 6 377 389 10.1084/jem.6.4-6.377
Unal T Cetingul E Gunbay T Peripheral adenomatoid odontogenic tumor: Birth of a term J Clin Pediatr Dent 1995 19 139 142 7577734
Philipsen HP Reichart PA Adenomatoid odontogenic tumour: facts and figures Oral Oncol 1998 35 125 131 10435145 10.1016/S1368-8375(98)00111-0
Adebayo ET Ajike SO Adekeye EO Odontogenic tumours in children and adolescents: a study of 78 Nigerian cases J Craniomaxillofac Surg 2002 30 267 272 12377198
Stypulkowska J Odontogenic tumors and neoplastic-like changes of the jaw bone. Clinical study and evaluation of treatment results Folia Med Cracov 1998 39 135 141
Chattopadhyay A Adenomatoid odontogenic tumour: Review of literature and report of 30 cases from India Indian J Dent Res 1994 5 89 95 9495104
Mosqueda-Taylor A Ledesma-Montes C Caballero-Sandoval S Portilla-Robertson J Ruiz-Godoy Rivera LM Meneses-Garcia A Odontogenic tumors in Mexico: a collaboratibe retrospective study of 349 cases Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997 84 672 675 9431538 10.1016/S1079-2104(97)90371-1
Arotiba JT Ogunbiyi JO Obiechina AE Odontogenic tumours: a 15-year review from Ibadan, Nigeria Br J Oral Maxillofac Surg 1997 35 363 367 9427447 10.1016/S0266-4356(97)90411-3
Buchner A Sciubba JJ Peripheral odontogenic tumours: a review Oral Surg Oral Med Oral Pathol 1987 63 688 697 3295656 10.1016/0030-4220(87)90372-0
Konouchi H Asaumi J Yanagi Y Hisatomi M Kishi K Adenomatoid odontogenic tumor: correlation of MRI with histopathological findings Eur J Rad 2002 44 19 23 10.1016/S0720-048X(01)00428-4
Philipsen HP Reichart PA Zhang KH Nikai H Yu QX Adenomatoid odontogenic tumor: biologic profile based on 499 cases J Oral Pathol Med 1991 20 149 158 2061853
Dare A Yamaguchi A Yoshiki S Okano T Limitation of panoramic radiography in diagnosing adenomatoid odontogenic tumors Oral Surg Oral Med Oral Pathol 1994 77 662 668 8065735 10.1016/0030-4220(94)90331-X
Toida M Hyodo I Okuda T Tatematsu N Adenomatoid odontogenic tumor: report of two cases and survey of 126 cases in Japan J Oral Maxillofac Surg 1990 48 404 408 2313448
Kramer IRH Pindborg JJ Shear M WHO histological typing of odontogenic tumours 1992 2 Springer Verlag Berlin, Heidelberg, New York
Philipsen HP Reichart PA The adenomatoid odontogenic tumour: ultrastructure of tumour cells and non-calcified amorphous masses J Oral Pathol Med 1996 25 491 496 8959557
Takeda Y Sato H Satoh M Nakamura S Yamamoto H Pigmented ameloblastic fibrodentinoma: a novel melanin-pigmented intraosseous odontogenic lesion Virchows Arch 2000 437 454 458 11097374 10.1007/s004280000249
Buchner A David R Carpenter W Leider A Pigmented lateral periodontal cyst and other pigmented odontogenic lesions Oral Dis 1996 2 299 302 9171515
Larson A Swartz K Heikinheimo K A case of multiple AOT-like jawbone lesions in a young patient-a new odontogenic entity? J Oral Pathol Med 2003 32 55 62 12558960 10.1034/j.1600-0714.2003.00046.x
Crivelini MM de Araujo VC de Sousa SO de Araujo NS Cytokeratins in epithelia of odontogenic neoplasms Oral Dis 2003 9 1 6 12617250 10.1034/j.1601-0825.2003.00861.x
Abiko Y Murata M Ito Y Taira T Nishimura M Arisue M Inoue T Shimono M Kuboki Y Kaku T Immunhistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin Med Electron Microsc 2001 34 185 189 11793195 10.1007/s007950100014
Takahashi H Fujita S Shibata Y Yamaguchi A Adenomatoid odontogenic tumour: immunohistochemical demonstration of transferring, ferritin and alpha-one-antitrypsin J Oral Pathol Med 2001 30 237 244 11302244 10.1034/j.1600-0714.2001.300408.x
Gao YH Yang LJ Yamaguchi A Immunohistochemical demonstration of bone morphogenic protein in odontogenic tumors J Oral Pathol Med 1997 26 273 277 9234187
Blumenthal NM Mostofi R Repair of an intrabony defect from an adenomatoid odontogenic tumor J Periodontol 2000 71 1637 1640 11063398 10.1902/jop.2000.71.10.1637
Philipsen HP Reichart PA Nikai H The adenomatoid odontogenic tumour (AOT): An update Oral Medicine & Pathology 1997 2 55 60
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CoughCough (London, England)1745-9974BioMed Central London 1745-9974-1-21627092010.1186/1745-9974-1-2ReviewAn overview of the sensory receptors regulating cough Mazzone Stuart B [email protected] Howard Florey Institute University of Melbourne Parkville VIC 3010 Australia2005 4 8 2005 1 2 2 4 4 2005 4 8 2005 Copyright © 2005 Mazzone; licensee BioMed Central Ltd.2005Mazzone; 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 cough reflex represents a primary defensive mechanism for airway protection in a variety of mammalian species. However, excessive and inappropriate coughing can emerge as a primary presenting symptom of many airway diseases. Cough disorders are characterized by a reduction in the threshold for reflex initiation and, as a consequence, the occurrence of cough in response to stimuli that are normally innocuous in nature. The current therapeutic strategies for the treatment of cough disorders are only moderately effective. This undoubtedly relates in part to limitations in our understanding of the neural components comprising the cough reflex pathway. The aim of this review is to provide an overview of current concepts relating to the sensory innervation to the mammalian airways, focusing particularly on the sensory receptors that regulate cough. In addition, the review will highlight particular areas and issues relating to cough neurobiology that are creating controversy in the field.
Cough ReceptorNociceptorRapidly Adapting ReceptorMechanosensorAirwayChemosensor
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Introduction
The basic nature of the respiratory system (i.e., inspiration of air from the surrounding environment for gas exchange), as well as the shared nature of the initial anatomical structures for the passage of food and air, places the airways and lungs under the constant threat of exposure to a variety of harmful airborne particles, organisms and other substances as well as aspirated gastric contents or accidental inhalation of foodstuffs. It is therefore not surprising that a variety of defensive mechanisms have evolved along with the normal function of the respiratory system to help protect against such threats. Airway protection relies upon specialized epithelial barriers and immune responses as well as a variety of highly co-ordinated neural reflex responses that help to limit the degree of potential harm and ultimately remove or expel the harmful substance from the airways.
Perhaps the most widely recognized neural response involved in airway protection is coughing. Coughing is generally characterized by a reflex-evoked modification of breathing pattern in response to airway irritation [1]. Reflex cough occurs when subsets of airway afferent (sensory) nerves are activated by inhaled, aspirated or locally produced substances. These afferent nerves provide modifying inputs to the brainstem neural elements controlling respiration, and consequently help generate the cough respiratory pattern [1-3]. Although widely studied for many years, there has been much debate surrounding the identity of the airway afferent nerve subtype that precipitates reflex coughing (see below). In addition, cough can also be initiated voluntarily. Little is known about the cortical pathways responsible for voluntary coughing, although they likely share similarities with those pathways responsible for voluntary breath holding and other conscious modifications of respiration. This review will focus on the current understanding of the anatomical and physiological arrangement of the sensory components responsible for reflex coughing. In addition the review will highlight how modifications of the sensory pathways from the airways could lead to inappropriate coughing in disease.
Classification of afferent nerve fiber types innervating the airways and lungs
Before describing which afferent nerve fibers are involved in reflex coughing, it seems appropriate to first provide a brief overview of the various afferent nerve subtypes that have been described in the mammalian airways. For the purposes of this review, much of the classification of airway afferents will relate to information gained from studies employing guinea pigs, the most widely utilized species with respect to airway innervation and cough. Whether studies in guinea pigs (or indeed any other experimental animal) can be directly translated to humans is a subject for additional debate. The discussion will also be restricted to only those afferent fibers that innervate the airways caudal to (and including) the larynx.
Airway sensory nerves do not form a homogeneous population. However, to date, there is no single classification scheme that adequately and unambiguously subcategorizes the various afferent nerve subtypes that have been described in the airways. Although a functional classification is commonly employed (describing the physiological responsiveness of airway afferents), subtypes can be alternatively delineated based on their origin, location in the airways, neurochemistry, electrophysiological properties or by the reflexes that are evoked secondary to afferent activation [4]. This lack of a universal classification scheme, coupled with attempts to classify an afferent subtype using only one phenotypic trait, often leads to some confusion as to the identity of a given afferent nerve type. It is therefore desirable to consider multiple characteristics when defining an airway afferent fiber.
In guinea pigs (and likely true for all mammals) airway sensory nerves can be broadly functionally classified as either primarily mechanically sensitive (low threshold mechanosensors) or primarily chemically sensitive (chemosensors or alternatively, nociceptors) (Fig 1). Low threshold mechanoreceptors are readily activated by one or more mechanical stimuli, including lung inflation, bronchospasm or light touch, but generally do not respond directly to chemical stimuli unless the stimulus acts upon airway structural cells to result in mechanical distortion of the nerve terminal [5-8]. Conversely, chemosensors are typically activated directly or sensitized by a wide range of chemicals, including capsaicin, bradykinin, adenosine, PGE2, but are relatively insensitive to mechanical stimuli [9,10]. This broad delineation, however, may not be strictly correct as at least some low threshold mechanosensors also directly respond to chemical stimuli, including acid and ATP, although these mediators may still activate the nerve terminal via mechanical mechanisms [11,12]. Subtypes of both the mechanosensors and chemosensors are readily identified (described below). Regardless of the afferent fiber, the majority of airway afferent nerves originate in the vagal sensory ganglia (nodose or jugular) [13,14]. A small population of fibers (believed to be a subpopulation of chemosensitive nerves) may have their origin in dorsal root ganglia adjacent to the upper thoracic spinal cord [15]. Little is known about the role of spinal afferents in airway defense.
Figure 1 Basic schematic classification of afferent nerve subtypes innervating the guinea pig airways. Abbreviations: RAR; rapidly adapting airway mechanoreceptor; SAR, slowly adapting airway mechanoreceptor.
Low threshold mechanosensors
Two classic types of low threshold mechanosensors have been described in the intrapulmonary airways of a number of mammalian species, namely the rapidly adapting receptors (RARs) and slowly adapting receptors (SARs) [8,9,16-20]. However, when comparing only a limited number of phenotypic traits RARs and SARs may appear indistinguishable (Table 1). Thus, RARs and SARs both originate in the nodose ganglia, terminate in the intrapulmonary airways and lung parenchyma, conduct action potentials in the Aβ-range (10–20 m/s) and are sensitive to many mechanical stimuli, including changes in lung volume, airway smooth muscle constriction and airway wall oedema [9,12,17-21]. Accordingly, RARs and SARs may both display activity when the lungs are inflated [9,16-19]. RARs and SARs are also both generally insensitive to a wide range of chemical stimuli, unless the stimulus evokes coincidental changes in airway smooth muscle tone, mucus secretion or airway wall volume [8,17,19].
Table 1 Properties of low threshold mechanosensor subtypes innervating the guinea pig airways.
SAR RAR Cough Receptor
Anatomical Characteristics:
Ganglionic Origin Nodose Nodose Nodose
Extrapulmonary Termination No No Yes
Intrapulmonary Termination Yes Yes Few
Substance P Expression No No No
TRPV1 Expression No No No
Functional Characteristics:
Conduction Velocity (m/sec) ~18 (Aβ) ~15 (Aβ) ~5 (Aδ)
Mechanical Threshold Low Low Low
Sensitive to:
Punctate Mechanical Yes Yes Yes
Capsaicin Yes1 Yes1 No
Hypertonic Saline Unknown Unknown Yes
Bradykinin Yes1 Yes1 No
Acid No Unknown Yes
Inflation (≤50 cmH2O) Yes Yes No
Deflation/Collapse No Yes No
Stretch Yes Yes No
Bronchoconstriction Yes Yes No
ATP Yes Yes No
Reflex Effects on Respiration Hering-Breuer Tachypnea Cough
1 SARs and RARs are insensitive to the direct action of these chemicals on the nerve terminal. However, chemical stimuli such as capsaicin and bradykinin can activate SARs and RARs secondary to airway smooth muscle contraction, mucous secretion or edema formation. Cough receptors are insensitive to both the direct and indirect actions of capsaicin and bradykinin. See text for references.
Nevertheless, RARs and SARs can be differentiated by comparing their individual mechanical activation profiles, mechanical adaptation properties, central termination patterns and the reflexes that each precipitate (Table 1). Thus, RARs may be activated during both inflation and deflation of the lungs (including lung collapse) [9,17]. SARs, on the other hand, display activity during tidal inspirations, peaking just prior to the initiation of expiration [9,16]. As their names suggest, RARs display rapid adaptation (i.e., a rapid reduction in the number of action potentials) during sustained lung inflations, whereas SARs adapt slowly to this stimulus [9,17]. It is important to note, however, that this rapid adaptation shown by RARs during sustained lung inflations is unlikely an electrophysiological property of the nerve terminal but rather relates to the nature of the stimulus. RARs typically adapt slowly to other types of mechanical stimuli, including dynamic lung inflations, bronchospasm and lung collapse [12,19]. Finally, activation of RARs evokes tachypnea and airway smooth muscle constriction, whereas SARs are likely the primary afferent fibers involved in the Hering-Breuer reflex, which terminates inspiration and initiates expiration when the lungs are adequately inflated [16,17]. SAR activation also inhibits cholinergic drive to the airway smooth muscle, resulting in a reduction in airway tone [8]. The different reflexes that are evoked by these afferent nerve subtypes likely reflect the distinct brainstem neurons innervated by RARs and SARs [reviewed in 22].
A third type of low threshold mechanosensor has been described in the guinea pig airways [12]. These fibers also originate from the nodose ganglia, but are primary located in the extrapulmonary airways (larynx, trachea and large bronchi) and are quite distinct to RARs and SARs (Figure 2; Table 1). Extrapulmonary low threshold mechanosensors are exquisitely sensitive to punctate mechanical stimuli (such as touch) but are insensitive to physiologically-relevant tissue stretching, changes in luminal pressure or airway smooth muscle constriction [12]. Extrapulmonary low threshold mechanosensors are also readily differentiated from their intrapulmonary counterparts by a much slower conduction velocity (~5 m/sec, Aδ-range) and a lack of sensitivity to the purinergic agonist ATP [12]. During sustained punctate mechanical stimulation, extrapulmonary mechanosensors display rapid adaptation, although again this likely reflects some property of the mechanics of the stimulus in relation to tissue surrounding the nerve terminal rather than reflecting electrophysiological adaptation [23]. Circumstantial evidence suggests that analogous fibers may be present in the extrapulmonary airways of cats, dogs and humans [2,24-30]. It is presently unknown whether this mechanosensor subtype is activated during normal breathing.
Figure 2 Photomicrographs of the guinea pig trachea showing (a) all nerve fibers immunostained for the pan neuronal marker Protein Gene Product 9.5; (b) jugular ganglia derived chemosensitive C-fiber plexus immunostained for substance P and (c-f) four representative nodose ganglia-derived low threshold mechanosensors (putative cough receptors) stained using the Fluorescent Marker (FM) 2–10. Note the clear distinction between the terminal arrangements of airway C-fibers and cough receptors. The terminal structure of guinea pig SARs, RARs and Aδ-chemosensors is presently unknown. Magnification: X40 (a), X100 (b) and X200 (c-f).
Chemosensors
Chemically-sensitive airway afferent fibers are found throughout the airways and lungs and are generally quiescent in the normal airways, becoming recruited during airways inflammation or irritation. Airway chemosensors are derived from both the nodose and jugular vagal ganglia, as well as from the dorsal root ganglia [13-15]. As described above, chemosensors are typically defined by the ability of a variety of chemicals to directly activate the nerve terminal (i.e., not secondarily to structural alterations within the tissue; Table 2). However, care needs to be taken when differentiating an airway chemosensor form other airway afferent nerve subtypes. For example, often airway chemosensors are stereotypically defined by their responsiveness to the irritant chemical capsaicin and, hence, the expression of the capsaicin receptor (TRPV1). This definition, however, is not strictly accurate, as at least some species possess capsaicin-insensitive, TRPV1-negative chemosensors [31]. Alternatively, it may be assumed that all airway chemosensors are C-fiber type nociceptors. This is also incorrect, as airway (and other visceral) chemosensors that conduct action potentials in the Aδ-fiber range have been identified (analogous to somatic Aδ-nociceptors) [13,32,33]. Furthermore, due to the overwhelming number of studies conducted in guinea pigs, chemically-sensitive fibers are often presumed to express tachykinins (substance P and/ or neurokinin A) (Fig 2). Guinea pigs are perhaps unique amongst mammals and express a high density of tachykinin-containing airway C-fibers, especially in their extrapulmonary airways [34-36]. Indeed, in the airways of most mammalian species (and in the guinea pig intrapulmonary airways) the majority of C-fiber chemosensors do not express tachykinins [35,36]. Given these reasons, airway chemosensors are sometimes thought of as high threshold mechanosensors. Within this group are fibers that are not readily excited by mechanical stimulation (bronchoconstriction, lung inflations light touch, etc), but can be activated using severe mechanical manipulations (lung hyperinflation, forceful punctate stimuli etc) and one or more chemical stimuli (capsaicin, bradykinin, adenosine etc).
Table 2 Properties of chemosensor subtypes innervating the guinea pig airways.
C-Fiber C-Fiber Aδ-Fiber
Anatomical Characteristics:
Ganglionic Origin Nodose Jugular Jugular
Extrapulmonary Termination No Yes Yes
Intrapulmonary Termination Yes Yes Few
Substance P Expression (%)1 Yes (50) Yes (90–100) No (0)
TRPV1 Expression2 Yes Yes Yes
Functional Characteristics:
Conduction Velocity (m/sec) <1 <1 ~6
Mechanical Threshold High High High
Sensitive to:
Punctate Mechanical Yes3 Yes3 Yes3
Capsaicin Yes Yes Yes
Hypertonic Saline Unknown Yes Yes
Bradykinin Yes Yes Yes
Acid Yes Yes Yes
Inflation (≤50 cmH2O) No No No
Deflation/Collapse No No No
Stretch No No No
Bronchoconstriction No No No
ATP Yes No No
Serotonin (5-HT) Yes No Unknown
Reflex Effects on Respiration Apnea4 Apnea4 Apnea4
1 Percentage of soma expressing substance P shown in parentheses [taken from ref 36]. 2 Functionally responsive to capsaicin and/or TRPV1 detected immunohistochemically. There is no data available indicating percentage of cells expressing TRPV1. 3 All airway afferents are responsive to punctate mechanical stimulation. However, the threshold for activation is approximately 100 fold higher for chemosensors compared to mechanosensors. 4 The basic respiratory reflex evoked by capsaicin is apnea or respiratory slowing, often proceeded by rapid shallow breathing. However, the precise reflex response evoked by each chemosensor subtype has not been described. See text for references.
Airway afferent nerves and cough
The identity of the afferent nerve fiber subtype that is primarily responsible for evoking reflex coughing has been the subject of much debate. Studies in experimental animals and in humans show clearly that multiple types of mechanical and chemical stimuli can (under the right experimental conditions) evoke coughing [1,12,24-30,37,38]. This would argue that multiple afferent nerve subtypes (mechanosensors and chemosensors) might be involved in the production of reflex coughing. However, not all stimuli evoke cough under all conditions [3,12]. This might suggest divergence between multiple reflex pathways or the existence of primary and secondary cough afferent pathways (discussed below).
Rapidly adapting receptors (RARs) and chemosensors
RARs have long been presumed to be the primary afferent nerve fibers that evoke defensive cough in the airways [1,4,5,39]. Indeed, it has been proposed that coughing can be initiated following the activation of RARs by airway smooth muscle constriction, mucous accumulation, mechanical irritation and even capsaicin and bradykinin application (due to the resulting airway obstruction) [1,4,17]. However, several observations argue against the role of classic RARs as the primary cough-provoking afferent fibers. For example, many stimuli that produce robust activation of RARs (e.g. thromboxane, leukotriene C4 (LTC4), histamine, neurokinins, methacholine) are ineffective or only modestly effective at evoking cough [17,28,40-42]. Moreover, in some coughing species (e.g., guinea pigs) many RARs are spontaneously active throughout the respiratory cycle and yet cough is only induced in response to very specific stimuli [8,12,14,19].
Evidence also supports a role of airway chemosensitive nerve fibers in the cough reflex. For example, stimuli that are known to activate airway chemosensors, such as capsaicin, bradykinin and citric acid, are amongst the most potent tussigenic agents in conscious animals and humans [12,26,38,43,44]. However, capsaicin and bradykinin do not evoke cough in anesthetized animals or humans, even though cough can be evoked in these same animals by mechanically probing the airway mucosa [12,25,27]. In fact, in anesthetized animals acute capsaicin challenge has been shown to inhibit breathing and, as a consequence, inhibit cough evoked by mechanical stimulation of the airways [12,25,27]. These conflicting observations have lead to suggestions that in conscious animals cough-evoked by chemosensor stimuli relies on cortical processing of the stimulus, in which the activation of a subset of airway chemosensors generate the conscious perception of airway irritation and promote the urge to cough [3]. Indeed, it is interesting that capsaicin-evoked cough can be consciously suppressed in human subjects [45]. If this hypothesis is correct, then chemosensor-mediated cough may not strictly be reflexive in nature. Rather, the perception of airway irritation may induce the conscious/ voluntary decision to cough. The true respiratory reflex response that is evoked by airway chemosensor stimulation may in fact be rapid inhibition of respiratory activity, which is observed during anesthesia and perhaps over-ridden (unless the reflex is robustly activated) by voluntary control in the conscious state.
The guinea pig 'cough receptor': Conflicts and opinions
The recent characterization of an extrapulmonary low threshold mechanosensor in the guinea pig airways (distinct to classic intrapulmonary RARs and SARs) may provide some important insights into the identity of the primary cough-provoking afferent nerve fiber. As described above, these fibers are found within the wall of the larynx, trachea and mainstem bronchi and are functionally differentiated from RARs and SARs by their sensitivity to light punctuate mechanical stimulation, but not to tissue stretch, bronchospasm, ATP and positive/ negative luminal pressures within the physiological range [12]. In addition to touch-like sensitivity, extrapulmonary mechanosensors are also activated by rapid changes in pH (e.g., as might be expected to occur following aspiration of gastric contents) [11,12]. Mechanical irritation and changes in pH are both stimuli that readily evoke cough in conscious and anesthetized animals and humans [11-13,26,46]. This sensitivity profile, their apparent ideal location for airway defense (i.e., in the large airways), the absence of this fiber subtype in species that do not cough (e.g. rats and mice) and several other anatomical and functional observations makes these extrapulmonary low threshold mechanosensors a likely candidate for the primary afferent nerve subtype that evokes reflex defensive coughing in guinea pigs. Accordingly, the term 'cough receptor' has been reintroduced to describe this guinea pig afferent nerve fiber subtype [3,12,47-49].
The identification of a unique afferent fiber subtype involved in generating cough from the guinea pig airways has generated much discussion within the field of cough research. For example, although these extrapulmonary fibers are easily distinguished from classic RARs and SARs in guinea pigs, it is unclear whether analogous fibers exist in the large airways of other species. The observation that the cough reflex can be readily evoked by light touch of the larynx, trachea or mainstem bronchi but not by bronchoconstricting agents, in dogs, cats and humans, provides circumstantial evidence that similar fibers may exist in these species [2,24-30]. It is also presently not known whether cough is the only reflex event initiated by this fiber type, nor is it certain that other fiber types can not produce coughing under some circumstances. However, this fiber type is the only sensory nerve in the guinea pig airways that once activated can initiate cough in both conscious and anesthetized animals [12]. Nevertheless, careful experimentation is required to adequately address these issues.
The appropriateness of employing the term 'cough receptor' to describe the guinea pig extrapulmonary low threshold mechanosensor has also been questioned. Although physiologists commonly describe sensory nerve fiber types as 'receptors' (e.g., muscle stretch receptors, tension receptors, RARs and SARs etc), the term 'receptor' can equally be applied to describe a pharmacological entity (e.g. a G-protein coupled receptor or a ligand-gated ion channel). Given the latter definition, and the observation that capsaicin is one of the most tussigenic stimuli available in conscious animals and humans, it is not surprising that TRPV1 (i.e., the capsaicin receptor) has been identified as a possible 'cough receptor' in guinea pigs and humans [50]. With this approach, any protein responsible for the transduction of a mechanical or chemical stimulus into electrical activity in the sensory nerve terminal (leading to cough) is a pharmacological cough receptor, and therefore a given sensory nerve is likely to have many different cough receptors. However, by defining a protein as a cough receptor it implies that this protein is therefore involved in the cough reflex irrespective of the cell type, tissue or species in which it is expressed. Using the example of TRPV1, it is unlikely that all TRPV1-expressing cells in the airways, and (perhaps with the exception of some nasal and esophageal afferent neurons) improbable that any TRPV1-expressing cells in other tissues or organs are involved in the cough reflex. Furthermore, species such as rats and mice lack the cough reflex, despite possessing numerous TRPV1-positive and capsaicin-sensitive airway afferent nerves [31,51,52].
Although these issues may seem an argument of semantics, they highlight the problems associated with the lack of any standard and widely accepted nomenclature system for defining terms and concepts employed by the field. Given that a 'cough receptor' was defined in the first instance as a putative afferent nerve subtype that evokes cough (and was not obviously intended to be employed to describe a pharmacological entity) [49], it therefore seems appropriate to define the guinea pig extrapulmonary low threshold mechanosensitive afferent nerve subtype as the only cough receptor identified to date.
Multiple and interacting cough reflex pathways
The breath-to-breath activity of intrapulmonary SARs and RARs is known to play an important role in regulating the excitability of brainstem breathing circuits [53-57]. In addition, activation of bronchopulmonary chemosensors can have profound influences on breathing pattern [12,25,27,58,59]. Given that many of the brainstem neural elements involved in breathing and coughing are shared, it seems therefore logical that alterations in the activity of most airway afferent nerves will play a role in shaping the cough motor pattern, perhaps contributing to different types of cough. For example, the basic primary defensive cough pathway (i.e., uncontrollable cough in response to an acute stimulus such as aspiration or direct mechanical probing of the airway mucosa) is likely mediated primarily by extrapulmonary low threshold mechanosensors (cough receptors). This pathway may therefore represent the primary basic defensive cough reflex pathway that serves to protect the airways from acute assaults. However, cough associated with airways obstruction or more chronic airway irritation (as would be expected to occur in airways disease) may involve the recruitment of other afferent (RAR and/ or chemosensor) pathways. In this scenario, secondary airway afferent pathways may evoke or modify cough responses via interactions with central elements of the primary cough pathway.
One of the problems faced when attempting to study central afferent interactions involved in coughing is the large gap in our understanding of airway sensory nerve integration in the brainstem. Nevertheless, recent studies in guinea pigs have provided some evidence to support the hypothesis that central interactions between cough receptor afferents and airway chemosensors may play an important role in cough reflex hypersensitivity in disease [3,60,61]. In anesthetized guinea pigs, chemosensors play a permissive (but not essential) role in cough evoked by some mechanoreceptor stimulants [62]. Furthermore, activation of normally quiescent airway chemosensors (using capsaicin or bradykinin) does not evoke cough but rather potentiates cough evoked by tracheal cough receptor stimulation [60]. Chemosensor-evoked potentiation of cough may reflect convergence of cough receptors and chemosensors onto common brainstem neurons responsible for generating cough [14,22], and shares many similarities with the interaction between cutaneous mechanosensors and chemosensors in the spinal cord, which is thought to underlie the manifestation of aberrant pain states [Reviewed in 22, 63]. Studies in guinea pigs and humans also suggest that chemosensitive afferent input from the nose or esophagus may heighten cough sensitivity via central interacting mechanisms [64-66].
Combined, these observations suggest that the recruitment of airway or other visceral chemosensors, and the subsequent increase in central cough pathway excitability, may contribute to the hypertussive states that accompany inflammatory diseases of the airways, nose and/ or esophagus. These data also indicate that it may be possible to design future therapeutic strategies that reduce the excitability of secondary cough afferent pathways, thereby treating cough hypersensitivity associated with disease without inhibiting the basic (primary) defensive cough reflex which is essential for airway protection and normal airway functioning.
Conclusion
Coughing, although essential for protecting the airways from the possible deleterious effects of acute airway irritation, can become excessive and non-productive in many airways diseases. The recent increased interest in cough reflex sensory neurobiology has unveiled a previously unrecognized complexity in the interacting roles of multiple afferent nerve subtypes in regulating this defensive reflex. However, further careful dissection of the cough sensory pathways is still required for the identification of future therapeutic targets for the effective treatment of cough disorders.
Acknowledgements
The author is funded by grants from the NH&MRC of Australia (#007188 and #350333).
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Mazzone SB Mori N Canning BJ Bradykinin-induced cough in conscious guinea pigs Am J Respir Crit Care Med 2002 165 A773
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Shinagawa K Kojima M Ichikawa K Hiratochi M Aoyagi S Akahane M Participation of thromboxane A(2) in the cough response in guinea-pigs: antitussive effect of ozagrel Br J Pharmacol 2000 131 266 270 10991919 10.1038/sj.bjp.0703553
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Coleridge JC Coleridge HM Afferent vagal C fibre innervation of the lungs and airways and its functional significance Rev Physiol Biochem Pharmacol 1984 99 1 110 6695127
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Plant MethodsPlant Methods1746-4811BioMed Central London 1746-4811-1-11627092210.1186/1746-4811-1-1EditorialPlant Methods: putting the spotlight on technological innovation in the plant sciences Forde Brian G [email protected] Michael R [email protected] Editor-in-Chief, Plant Methods, Dept. of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, UK2 Deputy Editor, Plant Methods, Dept. of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, UK2005 18 8 2005 1 1 1 21 7 2005 18 8 2005 Copyright © 2005 Forde and Roberts; licensee BioMed Central Ltd.2005Forde and Roberts; 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.
Plant Methods is a new journal for plant biologists, specialising in the rapid publication of peer-reviewed articles with a focus on technological innovation in the plant sciences. The aim of Plant Methods is to stimulate the development and adoption of new and improved techniques and research tools in plant biology. We hope to promote more consistent standards in the plant sciences, and make readily accessible laboratory and computer-based research tools available to the whole community. This will be achieved by publishing Research articles, Methodology papers and Reviews using the BioMed Central Open Access publishing model. The journal is supported by a prestigious editorial board, whose members all recognise the importance of technological innovation as a driver for basic science.
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Editorial
Technical innovation is surely one of the most important catalysts for progress in any scientific discipline – and this must be particularly true for plant biology in the current post-genomics era. In launching Plant Methods at this time our principal goal is to give technical creativity in the plant sciences the high profile platform it needs and deserves. By providing a forum for the rapid publication of novel or significantly improved techniques in plant biology, Plant Methods will aim to stimulate the development and the adoption of new and improved techniques, tools and technologies in the plant sciences. By spotlighting the technical aspects of plant biology, and providing a focal point for discussion and exchange of information, we hope that Plant Methods will also be able to serve the plant community by promoting higher and more consistent standards in the practice of plant research.
The open access policy of Plant Methods, as described in the BioMed Central Open Access Charter [1], is one that has major advantages for authors and readers alike. For authors, Open Access ensures that their work is disseminated to the widest possible audience, given that there are no barriers to access. Since authors will hold the copyright they are also free to reproduce and distribute their own work (for example by placing it on their institution's website) and may grant to anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced. It has been suggested that free online articles are more often viewed and more highly cited because of their easier availability [2]. For readers, the information available to them is not limited by their library's budget but is universally, freely and permanently accessible via the Internet. Consequently a country's economic status will not negatively influence its scientists' ability to access research. For these reasons we believe that the Open Access model represents the ideal format for achieving our objectives in launching Plant Methods.
Two main formats will be available for the publication of original work in Plant Methods: Research Papers, which will report a significant technical advance and its successful application to shed important new light on a biological problem, and Methodology Papers, which will describe an important new technique or research tool (such as a new online database or genetic stocks), but with less emphasis on the generation of new data beyond the required proof of concept. Too often researchers, particularly young researchers, are discouraged from dedicating the time needed for the development of new techniques or community resources because there is little opportunity to obtain proper recognition for their contribution. For example, it could easily take the major portion of a postdoctoral appointment to work through the cycles of trial and error needed to refine a new protocol, leaving little time to obtain the biological insights that a conventional research paper will demand. By providing a high profile outlet for methodology papers Plant Methods will encourage and reward technical creativity. Additional formats, including Reviews and Protocols, will be by invitation or by prior arrangement with a member of Editorial Board.
Our Editorial Board brings together a group of internationally renowned plant scientists who share a strong belief in the fundamental importance of technological innovation. All manuscripts submitted to the journal will be rigorously peer-reviewed and papers will be published online immediately after acceptance and soon after listed in PubMed. When a paper has been accepted, an Article Processing Charge (APC) of £410 (approximately US$720) becomes payable. This pays for the article to be freely and universally accessible in various formats online, and for the processes required for inclusion in PubMed and archiving in PubMed Central. All Plant Methods articles will be archived in PubMed Central [3], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [4] in Germany, at INIST [5] in France and in e-Depot [6], the National Library of The Netherlands' digital archive of all electronic publications. (Note that Plant Methods will not levy additional page or colour charges and any number of colour figures and photographs can be included, at no extra cost). There will be no APCs for the first 6 months after launch, and after that time those authors whose institutions subscribe to BioMed Central will still be able to publish free of charge. In cases of lack of funds, authors' requests for waivers, if made at the time of submission, will be considered on a case-by-case basis.
On behalf of the Editorial Board, we hope you will support us in our efforts to make Plant Methods a success. We very much look forward to receiving your submissions.
Competing interests
The majority of the APC is used by BioMed Central to fund the web services that host the journal and the submission and review system However, a small proportion of the APC is returned to the editorial office to support running costs associated with the journal.
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BioMed Central Open Access Charter
Lawrence S Free online availability substantially increases a paper's impact Nature 2001 411 521 11385534 10.1038/35079151
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Plant MethodsPlant Methods1746-4811BioMed Central London 1746-4811-1-21627091710.1186/1746-4811-1-2MethodologyMetabolic profiling of laser microdissected vascular bundles of Arabidopsis thaliana Schad Martina [email protected] Rajsree [email protected] Oliver [email protected] Julia [email protected] Max-Planck-Institute of Molecular Plant Physiology, Department Lothar Willmitzer, 14424 Potsdam, Germany2 UC Davis, 4321 GBSF Building Health Sciences Drive, Davis (CA) 95616, USA2005 18 8 2005 1 2 2 26 5 2005 18 8 2005 Copyright © 2005 Schad et al; licensee BioMed Central Ltd.2005Schad 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
Laser microdissection is a useful tool for collecting tissue-specific samples or even single cells from animal and plant tissue sections. This technique has been successfully employed to study cell type-specific expression at the RNA, and more recently also at the protein level. However, metabolites were not amenable to analysis after laser microdissection, due to the procedures routinely applied for sample preparation. Using standard tissue fixation and embedding protocols to prepare histological sections, metabolites are either efficiently extracted by dehydrating solvents, or washed out by embedding agents.
Results
In this study, we used cryosectioning as an alternative method that preserves sufficient cellular structure while minimizing metabolite loss by excluding any solute exchange steps. Using this pre-treatment procedure, Arabidopsis thaliana stem sections were prepared for laser microdissection of vascular bundles. Collected samples were subsequently analyzed by gas chromatography-time of flight mass spectrometry (GC-TOF MS) to obtain metabolite profiles. From 100 collected vascular bundles (~5,000 cells), 68 metabolites could be identified. More than half of the identified metabolites could be shown to be enriched or depleted in vascular bundles as compared to the surrounding tissues.
Conclusion
This study uses the example of vascular bundles to demonstrate for the first time that it is possible to analyze a comprehensive set of metabolites from laser microdissected samples at a tissue-specific level, given that a suitable sample preparation procedure is used.
laser microdissectionGC-TOF MStissue-specific analysismetabolite profilingvascular bundle
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Background
Unlike unicellular organisms, plants and animals have evolved as complex organisms that are defined by distributing special vital functions to spatially separated organs and tissues. The distinct functions of tissues and organs result from the integrated activity of individual cells. Current approaches mostly ignore this fact by analyzing samples that consist of a variety of different cell types and thus average and dilute the information obtained. Parameters that define the function and the physiological state of cells include gene and protein expression, but also the complement of low-molecular-weight compounds such as lipids, carbohydrates, vitamins or hormones that carry out much of the cell's business. Therefore, in addition to transcriptomic and proteomic studies, a comprehensive metabolite analysis with high spatial resolution is essential to fully characterize the state of a certain tissue.
To achieve this, analysis of small solutes in individual plant cells has so far been performed after extracting picoliter-sized samples with glass microcapillaries [1] and specialized techniques have been developed to access the ingredients of such small-scale samples [2-5]. However, these techniques remained labor-intensive and require specialized skills and training.
The recent development of methods that allow the collection of enough individual cells to process with standard analytical methods is a key step to integrate high spatial resolution analysis into routine laboratory work. Laser microdissection (LM) [6,7] is meanwhile well-established with animal and human samples and allows the investigation of RNAs and proteins from specific cell types [8,9]. Recently, also plants have been successfully used in such experiments and information about gene [10-12] as well as protein expression [13] was obtained.
Before any tissue can be microdissected, histological sections have to be prepared. To this end the tissue is normally fixed and subsequently either embedded in paraffin [14,15] or cryosectioned [15-17]. However, the fixation and dehydration steps included in these procedures lead to an inevitable loss of small cellular components. Therefore, the resulting tissue sections do not allow measurements of the cellular distribution of metabolites and other low-molecular-weight compounds.
The intention of the current study was to develop a tissue processing procedure that enables tissue-specific measurements of metabolites in laser microdissected vascular bundle samples from A. thaliana stems with well-established metabolic profiling techniques. A combination of metabolite profiles with the already established gene and protein expression analysis from microdissected tissue samples will allow a comprehensive description of organisms under different developmental, biotic or abiotic conditions at a tissue-specific level, a further important step towards integrative biology.
Results and discussion
Preparation and collection of cell type-specific samples
The most important stage in the analysis of specific tissues or even single cells is the isolation of the target tissue away from adjacent cells. Using microcapillaries is an established option to collect the contents from living plant cells [1,3,18]. This sampling technique results not only in very small sample sizes in the picoliter range, but is also limited mainly to surface exposed cells. In this study, we have used laser microdissection (LM) as a powerful alternative sampling method, which allows a contamination-free collection of large homogeneous cell populations also from cells located deep inside the tissue [19].
To date, LM has been widely used, mainly to study cell type-specific gene expression and less frequently for tissue-specific protein analysis in animal tissues [8,9]. Recently, this technique was introduced into plant sciences to allow cell type- specific gene expression [10-12] and protein [13] profiling. Laser microdissection coupled to laser pressure catapulting (LMPC) is a method that is applicable to plant material and allows the contact-free collection of specific cells [20]. To perform successful LMPC, dry histological sections with reasonable morphology and cell integrity are needed. While preserving good morphology is required to distinguish the different cell types, it is also crucial to retain the analytes at their in vivo localization. The latter point particularly needs to be considered for the analysis of small substances like metabolites, since it is self-evident that they can easily relocate or get lost during tissue preparation.
In principle, there are two methods for tissue treatment available that are appropriate for subsequent protein or RNA analysis. The first is paraffin-embedding [21-23], which involves numerous steps to fix and dehydrate the tissue, leading to a complete loss of low-molecular-weight compounds. The second option is cryosectioning, which is commonly used to prepare sections for molecular studies of animal tissue, but often downgrades the histological architecture. Plant tissue is particularly prone to cell morphology damage, because ice crystal formation is facilitated in water-rich tissue. To overcome this problem, plant material is usually infiltrated with cryoprotection agents prior to freezing [11,12]. But as with paraffin fixation, the use of any solvent in the tissue preparation procedure results in a loss of small substances and makes cell type-specific metabolite profiling unfeasible.
For tissue-specific metabolite profiling of vascular bundles and sections without vascular bundles, we therefore followed the strategy illustrated in Figure 1. We employed a procedure where plant material is frozen without the addition of any protective compounds, and dried without chemical dehydration to avoid the loss or relocalization of small cellular components.
Figure 1 Experimental strategy.Outline of the strategy for tissue-specific metabolite profiling in A. thaliana. Stem cross cryosections were used for LMPC. Metabolites from microdissected vascular bundles and sections without vascular bundles, respectively, were extracted and analyzed by GCMS measurements.
The morphology of the sections obtained from frozen A. thaliana stems using a cryostat was reasonable and completely sufficient to selectively excise vascular bundles by LMPC. However, getting decent morphology of stable tissue parts like vascular bundles is less complicated compared to other cell types, and it might also be more difficult to attain cryosections from more delicate plant organs like leaves or roots. Therefore cryopreservation and sectioning needs to be optimized depending on the plant species, organ and tissue type of interest.
LMPC was performed to obtain tissue-specific samples as outlined in Figure 1. The stepwise process of sample collection from cryosectioned stem material is illustrated in Figure 2. First, vascular bundles were marked and thereby selected for excision by utilizing the P.A.L.M. specialized imaging software (Figure 2a). The cutting was then performed by a focused laser beam (Figure 2b), while the final tissue collection into the cap of a microfuge tube was achieved by a defocused laser pulse (Figure 2c and 2d). The lower limit of the tissue size that can be collected depends on the magnification and the minimal width of the laser beam which is in the range of 1 μm. In principle, LMPC can be used to isolate a few [19] and even single cells that are resolved e.g. in 40 × magnification (data not shown). The cap was filled with ethanol to inactivate metabolic enzymes during sample collection. This ethanol had to be refilled every 10 min due to evaporation of the solvent, since the collection of a sample consisting of 100 vascular bundles required approximately one hour. After collecting all vascular bundles from a section, the remnant sections without vascular bundles were scraped off into ethanol using a scalpel.
Figure 2 Laser microdissection.The process of laser microdissection coupled to laser pressure catapulting (LMPC) for collecting vascular bundles from 30 μm thick cross sections obtained after freezing and cryosectioning A. thaliana stems. (a) Vascular bundles are selected on the computer screen. (b) The laser beam cuts along the markings. (c) The cut cells of interest are catapulted off the slide by a defocused laser pulse and (d) are collected into a cap of a microfuge tube.
While there are studies demonstrating metabolite measurements in single individual or only a few plant cells, the resulting small sample volumes limited the subsequent metabolite analyses to a restricted number of metabolites that could be determined by enzymatic assays [1,24] or capillary electrophoresis [4,5]. These approaches allow the determination of sugars or amino acids with extreme sensitivity but require expertise that is only accessible in a few specialized labs. By employing GC-TOF MS, we intended to take advantage of a well-established analytical standard method that, although less sensitive than the methods described above and not suitable to measure metabolites in individual cells, has the advantage of allowing to comprehensively profile a variety of compounds from different classes simultaneously [25,26]. Moreover, GC MS-based platforms for comprehensive metabolite analysis are standard techniques accessible in many labs.
Metabolite profiling
Dealing with such comparably small sample sizes as obtained by LMPC increases the danger of unwanted contaminations. Initial experiments using untreated plastic reaction tubes during all steps from tissue collection to derivatization led to GC-MS spectra from blanks that not only contained the expected artificial peaks resulting from derivatization and column bleeding, but also unexpected metabolite peaks as additional contaminants. The use of reaction tubes from different suppliers resulted in dozens of contaminant peaks in all method blank chromatograms. As a consequence, reaction tubes for all further experiments (0.5 ml plastic reaction tubes for LMPC as well as 0.2 ml glass vials for GC-MS derivatization) were washed twice with distilled water and dried. Pipette tips were rinsed once with ethanol directly before use. After sample collection, vortexing and centrifugation, supernatants were transferred to glass vials where the derivatization was carried out. These precautions drastically improved the quality of blanks and were essential for metabolite profiling of small sample amounts.
Next, the influence of the sample preparation procedure on the metabolite composition was investigated. To this end we compared samples that were (a) dissolved in ethanol directly after cryosectioning or (b) cut, transferred to slides, stored at 4°C, and used for LMPC. As expected, some differences in the metabolite profiles of the two sample types were observable: 11 of 72 metabolites identified in measurements from 5 complete stem cross sections (10 replicates) appeared to be sensitive to the sample preparation procedure. These changes were statistically significant (P < 0.05) and showed an at least 1.5 fold increase or decrease in LMPC samples as compared to cryosectioned control samples (Table 1). These observed changes are probably caused by biological and chemical processes during storage, processing and microdissection, since all these steps were carried out at room temperature without air exclusion. Therefore, samples have to run through identical procedures to allow comparisons of metabolite levels. Absolute quantities of certain metabolites have to be treated with caution. Dehydroascorbate (the oxidized form of vitamin C), for example, was found to be strongly diminished by the tissue preparation process (Table 1), putatively due to oxidation by air. In LMPC collected vascular bundles and sections without vascular bundles, dehydroascorbate was completely absent.
Table 1 List of metabolites influenced by the sample preparation procedure. Metabolite amounts in stem cross sections. To analyze the influence of tissue processing and laser microdissection on metabolite profiles, complete fresh cryosections and sections after drying and laser microdissection (consisting of vascular bundles and the remaining tissue) were compared. Metabolites with significant differences (P < 0.05) in cryosections and laser-microdissected sections (>1.5) are listed.
Metabolite CAS Ratio of fresh cryosections vs. complete laser microdissected sections
glycerol 56-81-5 0.6
urea 57-13-6 0.6
lauric acid 143-07-7 0.6
linoleic acid 60-33-3 1.6
succinic acid 110-15-6 1.7
myo-inositol 87-89-8 1.7
shikimic acid 138-59-0 2.0
ethanolamine 141-43-5 2.5
gamma-aminobutyric acid 56-12-2 2.5
glucose 50-99-7 3.9
dehydroascorbic acid 490-83-5 8.5
For tissue-specific GC-TOF MS measurements, we prepared stem cross sections of five A. thaliana plants and collected by LMPC five replicates of about 100 vascular bundles (~ 5,000 cells) from each plant into ethanol. Additionally, 10 of the remaining vascular bundle-depleted sections from each plant were scraped off the slides and also collected into ethanol. The supernatants were dried, derivatized with MSTFA and subjected to GC-TOF MS metabolite profiling. Due to the limited sample amounts available, derivatization of the metabolites was done in a volume of only 10 μl. For the same reason, the injection was carried out without sample wash steps.
Data evaluation
Using the GC-TOF MS approach, 68 metabolites could be reliably identified in vascular bundles and 65 in sections without vascular bundles. This number of identified metabolites is reasonable considering the small number of cells (~5,000) used for the measurements. The obtained data sets were investigated in more detail to find out differences and similarities in the metabolic profiles from vascular bundles and the surrounding tissue. First, we carried out a principal component analysis (PCA) to see if logical grouping in the data set could be related to maximum variance. This unsupervised multivariate data analysis generates new variables, principal components (PC), that attempt to express the overall variance in the original data. When plotting the tissue-specific metabolite data, it was immediately obvious that vascular bundles and sections without vascular bundles were well separated and had their own distinct metabolite profiles (Figure 3), indicating that the localization of metabolites was retained during sample processing.
Figure 3 Statistical evaluation of the metabolite data.Principal Components Analysis (PCA) of metabolite samples generated from A. thaliana stem tissue. The PCA score plot for principal component 1 (PC1) versus principal component 2 (PC2) is presented. The circles represent microdissected vascular bundle tissues (21 samples) whilst the triangles are samples from sections without vascular bundles (23 samples).
Student t-test comparisons between the data sets revealed that about half of the identified metabolites were significantly different and either enriched or depleted in vascular bundles compared to sections without vascular bundles (Table 2), including six metabolites which were shown to be sensitive to the sample preparation procedure (Table 1). Figure 4 shows examples of chromatograms of vascular bundles and sections without vascular bundles for the mass to charge ratio (m/z) 217. This ion trace is used to look at sugars and sugar alcohols. The insets (Figures 4a and 4b) illustrate examples of metabolites being enriched and depleted, respectively, in vascular bundles. Seventeen metabolites were significantly depleted in vascular bundles, while 16 metabolites were enriched in vascular bundles and three metabolites, oxoglutarate, glyceraldehyde and glycerone, were even exclusively found in this tissue type. We are not aware of anything in the literature that would account for these metabolites being exclusively localized to vascular bundles.
Table 2 Metabolites differentially concentrated in vascular bundles and sections without vascular bundles. Metabolites that were differentially concentrated in vascular bundles and sections without vascular bundles (with P <0.05 and ratio <0.67 or >1.5) are listed. vb: found only in vascular bundles, ** metabolites which appear to be sensitive to the sample preparation procedure (see Table 1).
Metabolite CAS Ratio of vascular bundles vs. sections without vascular bundles
stigmasterol 83-48-7 0.1
gamma-aminobutyric acid** 56-12-2 0.3
ethanolamine** 141-43-5 0.3
galactose 59-23-4 0.4
fucose 634-74-2 0.4
linolenic acid 463-40-1 0.4
glucose** 50-99-7 0.4
fructose 57-48-7 0.4
phenylalanine 63-91-2 0.5
linoleic acid** 60-33-3 0.5
leucine 61-90-5 0.5
benzoic acid 65-85-0 0.6
urea** 57-13-6 0.6
isoleucine 73-32-5 0.6
valine 72-18-4 0.6
lignoceric acid 557-59-5 0.6
mannose 3458-28-4 0.7
heptadecanoic acid 506-12-7 1.6
aspartic acid 56-84-8 1.7
malic acid 97-67-6 1.7
adipic acid 124-04-9 1.8
proline 147-85-3 1.9
sucrose 57-50-1 1.9
lauric acid** 143-07-7 1.9
raffinose 512-69-6 2.1
isocitric acid 320-77-4 2.2
oleic acid 112-80-1 2.3
myristic acid 544-63-8 2.4
glycine 56-40-6 2.8
6-amino caproic acid 60-32-2 3.5
citric acid 77-92-9 3.7
trans-squalene 111-02-4 4.4
phosphate 14265-44-2 5.2
oxoglutarate 328-50-7 vb
glyceraldehyde 367-47-5 vb
glycerone 96-26-4 vb
Figure 4 Example chromatogram.Entire chromatogram for m/z 217, an ion trace for sugars and sugar alcohols. The vascular bundle sample is shown in green and the sample without vascular bundle in red. (a) shows an example of depleted sugars in vascular bundles – fructose (retention time 564.19 s) and glucose (retention time 569.419 s) while (b) illustrates the zoomed-in sucrose peak (retention time 815.369 s) being enriched in vascular bundles.
Sugars accounted for one class among the metabolites with tissue dependent differences. While reducing sugars like glucose, fucose and fructose were depleted in vascular tissue, the non-reducing sugars sucrose and raffinose were enriched. This distribution of sugars is characteristic for phloem sap that probably accounts for a great portion of vascular bundle metabolites.
A further class of compounds that showed different levels in vascular bundles compared to the surrounding tissue was amino acids. From this group glutamate, aspartate and glutamine are the major amino acids distributed through the phloem tubes in A. thaliana [27] and therefore could be expected to be enriched in vascular bundles. In this study, we found only aspartate significantly enriched in vascular bundles compared to the surrounding tissue while glutamate and glutamine were indeed abundant in vascular bundles, but similar levels were also detected in sections without vascular bundles. These findings are unlikely to be caused by the sample treatment because none of these amino acids belonged to the group of substances influenced by the sample processing procedure (Table 1).
Conclusion
This study demonstrates for the first time that laser microdissection can be successfully applied to analyze the spatial distribution of metabolites within plant tissues. The application of a suitable sample preparation protocol, omitting any solute exchange steps, followed by LMPC makes metabolite profiling of 100 vascular bundles, equivalent to only 5,000 cells, by standard GC-TOF MS measurements feasible. In principle this method should be applicable to a wide range of cells and tissues given that sufficiently good morphology is obtained following the introduced cryosectioning procedure. In combination with the previously described RNA and protein expression profiling, cell type-specific metabolite profiling will allow a comprehensive characterization of distinct tissues, an essential step towards a thorough understanding of gene functions.
Methods
Preparation of tissue sections and laser microdissection
Arabidopsis thaliana (Col-0) plants were grown on soil in a growth chamber under short day conditions (8 h light, 20°C and 16 h dark, 16°C, 75% RH) for four weeks and then in the greenhouse under long day conditions (16 h light, 20°C, 60% RH and 8 h dark, 18°C, 75 % RH). Stems of 6 week-old plants were frozen in liquid nitrogen and transferred to a cryostat (Microm, Waldorf, Germany) cooled to -30°C. Using a scalpel, 15 mm-long stem pieces were cut. Stem pieces were glued onto the sample plate by employing Neg-50 (Richard-Allan Scientific, Kalamazoo, MI, USA), a water-soluble frozen section medium. Using the cryostat, 30 μm sections were cut and transferred to glass slides where they dried within seconds at room temperature.
For microdissection, the P.A.L.M. Laser Microbeam System (Bernried, Germany) was employed. This system consists of a low heat UV (337 nm nitrogen) laser and an inverted microscope. Cells were selected using the graphics tools of the P.A.L.M. RoboSoftware. After selection, vascular bundles were isolated by the laser microbeam and afterwards collected by laser pressure catapulting into the lid of a 0.5 ml reaction tube (Eppendorf, Hamburg, Germany) filled with 50 μl ethanol (Merck, Darmstadt, Germany), placed in a holder closely above the slide. After collection of vascular bundles, the remaining sections were scraped off the slides using a scalpel into ethanol (subsequently called sections without vascular bundles). The cell numbers were estimated from the number of vascular bundles collected and the cell number counted in one representative vascular bundle.
Both types of samples were vortexed and centrifuged for 5 min at 14.000 rpm. Supernatants were collected in 0.2 ml glass vials (Chromacol, Herts, United Kingdom) and vacuum dried. All used reaction tubes, including their lids, were washed twice with distilled water and dried before use. More details of quality control procedures during sample preparation are provided in the text main body. Five parallel plants were employed. From each plant, five replicates of 100 vascular bundles and 5 sections without vascular bundles were used for metabolite analysis. As negative controls, ethanol without microdissected material was processed in parallel to the samples.
Metabolite profiling
For GC-TOF MS (Leco Pegasus II GC-TOF mass spectrometer; Leco, St. Joseph, MI, USA) analysis, the vacuum dried samples were dissolved in 1 μl methoxamine hydrochloride (20 mg/ml pyridine) and incubated at 30°C for 90 min with continuous shaking. Then 9 μl of N-methyl-N-trimethylsilyltrifluoroacetamid (MSTFA) were added to derivatize polar functional groups at 37°C for 30 min. The derivatized samples were stored at room temperature for 120 min before injection. GC-TOF MS analysis was performed on an HP 5890 gas chromatograph with tapered, deactivated split/splitless liners containing glass wool (Agilent, Böblingen, Germany) and 1.5 μl splitless injection at 230°C injector temperature. Before each injection, the liner was rinsed with a pure MSTFA injection (1 μl). Sample injection was carried out without sample wash steps due to the limited amount of total sample volume. The GC was operated at constant flow of 2 ml/min helium and a 30 m 0.32 mm id 0.25 μm MDN35 column (Macherey-Nagel, Düren, Germany). The temperature gradient started at 80°C, was held isocratic for 2 min, and subsequently ramped at 15°C /min to a final temperature of 330°C which was held for 6 min. Twenty spectra per second were recorded between m/z 85–500. Peak identification and quantification were performed using the Pegasus software package ChromaTOF 1.61 (Leco). A reference chromatogram was defined that had a maximum of detected peaks over a signal/noise threshold of 5 and used for automated peak identification based on mass spectral comparison to a standard NIST 98 library and own customized mass spectral libraries. Automated assignments of unique fragment ions for each individual metabolite were taken as default as quantifiers, and manually corrected where necessary. All known artifact peaks caused by column bleeding or phtalates and polysiloxanes derived from MSTFA hydrolysis were manually identified and removed from the results table. Remaining metabolite data were normalized to the total area of all detected metabolites and log-transformed. Due to the utmost requirements of low sample volumes and chemical background, no further internal standards were added. Statistical analyses were performed using Matlab version 6.5 (The MathWorks, MA, USA).
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MS carried out the optimization of sample preparation for GC-TOF MS measurements, collected samples by LMPC, participated in data evaluation and drafted the manuscript. RM participated in LMPC sample collection, GC-TOF MS measurements and data analysis. OF participated in GC-TOF MS optimization, data evaluation, and manuscript drafting. JK conceived of the study, arranged its design and coordination and was involved in manuscript writing. All authors read and approved the final manuscript.
Acknowledgements
The authors thank Carola Kuhn and Professor Martin Steup from the University of Potsdam for supporting the cryostat work.
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Tomos AD Sharrock RA Cell sampling and analysis (SiCSA): metabolites measured at single cell resolution Journal of Experimental Botany 2001 52 623 630 11373310 10.1093/jexbot/52.356.623
Schuetze K Lahr G Identification of expressed genes by laser-mediated manipulation of single cells Nat Biotech 1998 16 737 742 10.1038/nbt0898-737
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Nakazono M Qiu F Borsuk LA Schnable PS Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 2003 15 583 596 12615934 10.1105/tpc.008102
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J Ethnobiol EthnomedJournal of Ethnobiology and Ethnomedicine1746-4269BioMed Central London 1746-4269-1-110.1186/1746-4269-1-1EditorialWelcome to Journal of Ethnobiology and Ethnomedicine Pieroni Andrea [email protected] Lisa Leimar [email protected] Ina [email protected] Department of Pharmacy, School of Life Sciences, University of Bradford, Richmond Building, Richmond Road, Bradford BD7 1DP, UK2 SCH Group, Department of Social Sciences, Wageningen University, PO Box 8060, NL-6700 DA Wageningen, Netherlands3 Institute of Economic Botany, The New York Botanical Garden, Kazimiroff Blvd. & 200 Street, Bronx, New York 10458, USA2005 29 7 2005 1 1 1 11 7 2005 29 7 2005 Copyright © 2005 Pieroni et al; licensee BioMed Central Ltd.2005Pieroni 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.
Ethnobiology is a multidisciplinary field of study that draws on approaches and methods from both the social and biological sciences. Ethnobiology aims at investigating culturally based biological and environmental knowledge, cultural perception and cognition of the natural world, and associated behaviours and practices. Ethnomedicine is concerned with the cultural interpretations of health, disease and illness and also addresses the health care seeking process and healing practices. Research interest and activities in the areas of ethnobiology and ethnomedicine have increased tremendously in the last decade. Since the inception of the disciplines, scientific research in ethnobiology and ethnomedicine has made important contributions to understanding traditional subsistence and medical knowledge and practice. The Journal of Ethnobiology and Ethnomedicine (JEE) invites manuscripts and reviews based on original interdisciplinary research from around the world on the inextricable relationships between human cultures and nature, on Traditional Environmental Knowledge (TEK), folk and traditional medical knowledge, as well as on the relevance of the above for Primary Health Care (PHC) policies in developing countries.
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Ethnobiology and Ethnomedicine
Ethnobiology is a multidisciplinary field of study that draws on approaches and methods from both the social and biological sciences. "Ethnobiology" has proven a rather difficult term to define since the scope of ethnobiological studies has changed considerably throughout history. One of its more recent definitions refers to the study of the reciprocal relationships between human cultures and the natural world [1]. Reciprocal relationships here refer to the human perception of the biological environment, which will ultimately influence man's behaviour, while human behaviour in turn influences – or shapes – the biological environment. This broad definition of ethnobiology encompasses ethnotaxonomy (study of the classification principles of animals, plants, soils, and ecosystems according to local peoples), ethnomedicine (study of the cultural concepts of health, disease and illness, and of the nature of local healing systems), ethnoecology (study of traditional environmental knowledge and of anthropogenic effects on the environment), ethnoagronomy (study of subsistence economies and resource management), and material culture (study of biological resources used in art and technology).
Ethnobiology aims at investigating culturally-based biological and environmental knowledge, cultural perception and cognition of the natural world, and associated behaviours and practices.
Ethnomedicine is concerned with the cultural interpretations of health, disease and illness and also addresses the health care-seeking process and healing practices.
In both ethnobiology and ethnomedicine, the documentation of the consequences of particular behaviours and practices is through cultural and biological expertise intrinsic to the fields of anthropology and biology/medicine.
Why a new journal?
Research interest and activities in the areas of ethnobiology and ethnomedicine have increased tremendously in the last decade. The number of research publications has doubled and the three international, widely recognised scientific societies involved in this area (the Society for Economic Botany, the International Society of Ethnobiology and the International Society of Ethnopharmacology) have witnessed a continuous growth in their memberships. These three societies held their first joint scientific congress in June 2004 in the UK. .
Over the last ten years there has been a phenomenal growth of interest in indigenous, folk and local knowledge and practices among international organizations and the international development community, which include the World Health Organization (WHO), the Food and Agricultural Organization (FAO) and the World Bank. This interest is rooted in the growing understanding that indigenous knowledge and practices have a substantial value in the fields of agriculture, environmental management and biodiversity conservation, and health care. There is an increasing awareness that indigenous and local knowledge should be understood and utilized in any attempt to enhance human welfare. Importantly, the UNESCO Convention for Safeguarding of the Intangible Cultural Heritage that was signed at the 32nd Session of UNESCO in Paris on 17 October 2003 includes the domain of knowledge and practices concerning nature and the universe. These developments convey a clear message that development paradigms are changing, recognizing that development must be considered in a manner that includes intellectual and spiritual traditions as well as indigenous and local peoples' rights to self-determination.
Since the inception of the disciplines, scientific research in ethnobiology and ethnomedicine has made important contributions to our understanding of traditional subsistence and medical knowledge and practice. More recently, the focus has increasingly been on the issues of human well-being and environmental sustainability. The boundaries between ethnobiology and ethnomedicine are increasingly merging to address complex questions. An example includes the fight against malaria and the need to understand the complex interrelationships between cultural practices, knowledge and the environment. This requires an understanding of how local practices in agriculture facilitate or counter the spread of the vector, how people understand the life cycle of the mosquito and the epidemiology of malaria, how malaria symptoms are culturally identified and interpreted, and what kind of traditional medical treatments are being used, as well as how effective these treatments are [2]. On the other hand, the use of medicinal plants for health care can no longer be seen separately from the threats posed on biodiversity since the decrease of biodiversity will ultimately affect the use of medicinal plants as health care resources.
There is a growing demand for access to integrated empirical studies in ethnobiology and ethnomedicine beyond the scope of the journals that are currently available. Much of the scientific literature in this interdisciplinary area can be categorized as being spread throughout a number of different scientific journals, where often ethnobiology and ethnomedicine are not the primary focus. While some journals do focus on ethnobiology, or on certain aspects of ethnomedicine [3], a widely available, easily accessible reference journal that specifically focuses on and integrates these two fields has been lacking. The Journal of Ethnobiology and Ethnomedicine will fill this void. It is our opinion that a new free-access journal in this area, sustained by a large part of the world-wide community of scholars who are working in these fields and the Editorial Board , will improve the circulation and exchange of high quality research in ethnobiology and ethnomedicine. Furthermore, we believe that the free and unhindered flow of information will not only promote a rapid increase in scientific knowledge but will also allow science to better serve the public community as a whole.
Research areas touched by the Journal of Ethnobiology and Ethnomedicine
The Journal of Ethnobiology and Ethnomedicine (JEE) invites manuscripts and reviews based on original interdisciplinary research from around the world on the inextricable relationships between human cultures and nature, on Traditional Environmental Knowledge (TEK), folk and traditional medical knowledge, as well as on the relevance of the above for Primary Health Care (PHC) policies in developing countries.
Topics include ethnobotany, ethnozoology, ethnoecology (including ethnopedology and ethnoclimatology), ethnopharmacy (ethnopharmacognosy/folk Materia Medica), ethnomedicine, ethnoveterinary, Traditional Medicines (TMs), traditional health care in households and domestic arenas, migrant health care/urban ethnobiology, pluralistic health care in developing countries, evidence-based community health, visual ethnobiology and ethnomedicine, gender studies and ethnobiology as well as other related areas in nutritional, medical and visual anthropology. Manuscripts focussing exclusively on phytopharmacological or phytochemical investigations of traditionally used biological materials do not fit in the scope of the journal, nor do papers purely about paleobotanical, agricultural or botanical studies. The journal will be particularly keen to draw the attention of external stakeholders (public health actors; international, national and local environmental and health institutions; development agencies and NGOs) to the relevance of the traditional management of the environment in biological and cultural conservation.
Potential readers of the journal are ethnobiologists, environmentalists, ethnopharmacists, physicians, veterinarians, health care specialists, medical, cognitive, and cultural anthropologists, development workers, and policy makers.
Peer review policy of the JEE
The JEE is a peer-reviewed journal. The entire reviewing process will be managed and recorded electronically. Each submission will go through a pre-evaluation process in which the Editor-in-Chief and one of the Editors (a native English speaker) will provide a cursory examination for basic English language adequacy, evaluate whether the manuscript fits the scope of the journal, and check the relevance of the manuscript to the international scientific audience of the JEE. This process will take one week.
Thereafter, authors of manuscripts deemed inappropriate will be informed. Authors of manuscripts with English language problems that fit within the scope of the journal will be informed and welcomed to resubmit their manuscripts after language editing. Manuscripts that do not fit broadly within the scope of the journal will be rejected. Manuscripts passing the initial pre-screening will go through the peer review process. This process should last no longer than four weeks (except in the cases of requests for major revisions).
The Editor-in-Chief will select one Deputy/Associate Editor as Managing Editor in charge of the peer review process for the manuscript. The Editor-in-Chief can also act as Managing Editor.
The Managing Editor will send the manuscript to two appropriate scholars who are internationally recognized experts in the specific field(s) featured in the manuscript, including at least one of the JEE Editorial Board Members. In the case that one of the experts, or both, would decline, the Managing Editor will send the manuscript as soon as possible to other reviewers. It is the Managing Editor's responsibility to manage the process within the given timescale.
Both reviews will be double-blinded peer reviews (neither reviewers nor authors will be able to see their names/identities).
Once peer reviews are received by the Managing Editor, the Managing Editor will decide the status of the manuscript, being either:
- acceptable as it is
- acceptable with minor revisions and corrections (concerning comprehensiveness, formatting and the quality of illustrations and tables
- acceptable only after major revisions (the paper will be reconsidered after more substantial additions and re-organization)
- to be rejected
Following resubmission with revisions drafted by the author(s), it is up to the Managing Editor to decide whether the manuscript should be returned to the original reviewers in order to obtain their final comments. This will depend on the nature and extent of the revisions. The Managing Editor will then make an editorial decision on the manuscript.
In case of divergent opinions between two reviewers where the Managing Editor is unable to make a final decision, he/she will ask for assistance from one of the Associate or Deputy Editors of the journal.
The Managing Editor will then send the peer reviews and the editorial decision to the Editor-in-Chief for final approval. The final decision communicated to authors will be sent out by the Editor-in-Chief.
If the Managing Editor's final decision diverges from the overall view of the Editor-in-Chief, the Editor-in-Chief will ask for the assistance of one of the Deputy Editors. The final decision will be taken by the Editor-in-Chief.
When the Managing Editor assigned to the manuscript is unable to meet the proposed deadlines for handling the manuscript, the Editor-in-Chief will select another Managing Editor for the manuscript or will act in some cases as (operative) Managing Editor.
What Open Access means
The Journal of Ethnobiology and Ethnomedicine Open Access policy changes the way in which articles are published. First, all articles become freely and universally accessible online, so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [4]. Third, a copy of the full text of each Open Access article is permanently archived in other online repositories separate from the journal. JEE's articles are archived in PubMed Central [5], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [6] in Germany, at INIST [7] in France and in e-Depot [8], the National Library of the Netherlands' digital archive of all electronic publications.
Open Access has four broad benefits for science and the general public. First, authors are assured that their work is disseminated to the widest possible audience, given that there are no barriers to access their work. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution's website. Some studies have suggested a correlation between Open Access, higher citations, and higher Impact factors [9]. Second, the information available to researchers will not be limited by their library's budget, and the widespread availability of articles will enhance literature searching [10]. Third, the results of publicly funded research will be accessible to all and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. The NIH policy on public access to research calls on researchers to release to the public articles from research supported by NIH in the USA as soon as possible, and within 12 months of final publication [11]. This policy is likely to be replicated by other major funders worldwide. Fourth, a country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones (although creating access to the internet is another matter [12]).
Competing interests
The author(s) declare that they have no competing interests.
==== Refs
Cotton CM Ethnobotany Principles and Applications 1996 John Wiley & Sons
WHO Traditional Medicine Strategy 2002–2005. Document: WHO/EDM/TRM/202.1 2002 Geneva: World Health Organization
Journal of Ethnobiology; Journal of Ethnopharmacology
BioMed Central Open Access Charter last accessed 3rd May, 2005
PubMed Central last accessed 30th April, 2005
Potsdam last accessed 7th May, 2005
INIST last accessed 10th May, 2005
e-Depot last accessed 15th May 2005
Velterop J Should scholarly societies embrace Open Access (or is it the kiss of death)? Learned Publishing 2003 16 167 169 10.1087/095315103322110932
NIH Calls on Scientists to Speed Public Release of Research Publications
Tan-Torres Edejer T Disseminating health information in developingcountries: the role of the internet BMJ 2000 321 797 800 11009519 10.1136/bmj.321.7264.797
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J Ethnobiol EthnomedJournal of Ethnobiology and Ethnomedicine1746-4269BioMed Central London 1746-4269-1-21627091910.1186/1746-4269-1-2ResearchAgricultural, domestic and handicraft folk uses of plants in the Tyrrhenian sector of Basilicata (Italy) Salerno Giovanni [email protected] Paolo Maria [email protected] Giulia [email protected] Dipartimento di Biologia, Università di Roma Tre, Viale Marconi 446, 00146 Rome, Italy2 Museo Nazionale Arti e Tradizioni Popolari, Piazza Marconi 8/10, 00144 Rome, Italy2005 29 7 2005 1 2 2 26 4 2005 29 7 2005 Copyright © 2005 Salerno et al; licensee BioMed Central Ltd.2005Salerno 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
Research was carried out into agricultural and domestic-handicraft uses in folk traditions in the Tyrrhenian sector of the Basilicata region (southern Italy), as it is typically representative of ethnobotanical applications in the Mediterranean area. From the point of view of furnishing a botanical support for the study of local "material culture" data was collected through field interviews of 49 informants, most of whom were farmers.
Results
The taxa cited are 60, belonging to 32 botanical families, of which 18 are employed for agricultural uses and 51 for domestic-handicraft folk uses. Data show a diffuse use of plants for many purposes, both in agricultural (present uses 14%; past uses 1%) and for domestic-handicraft use (present uses 40%; past uses 45%); most of the latter are now in decline.
Conclusion
60 data look uncommon or typical of the places studied. Some domestic-handicraft folk uses are typical of southern Italy (e.g. the use of Ampelodesmos mauritanicus for making ties, ropes, torches, baskets or that of Acer neapolitanum for several uses). Other uses (e.g. that of Inula viscosa and Calamintha nepeta for peculiar brooms, and of Origanum heracleoticum for dyeing wool red) are previously unpublished.
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Background
Ethnobotanical studies supporting the ethno-anthropological sciences, and of "material culture", which describe aspects of farmers' and shepherds' economy now on the point of disappearance are infrequent in Italy. Generic news is sometimes present in botanical texts concerning, for example, handicraft uses, but the plant matter of single artefacts is rarely defined; this specific matter can change from place to place and can originate peculiar local artefacts. Ethnobotanical uses of plants are often lost more easily in modern civilisation, due to industrial activity that substitutes traditional handicrafts. This study was carried out in an area, the Tyrrhenian sector of Basilicata, where, in remarkable contrast with that happens in the rest of Italy, we can still witness a large folk employment of plants and a rich and intense memory of their uses is present. Due to history, economy and tradition, this area could potentially be a precious source of information already lost in other places.
A previous study of flora, vegetation and spontaneous food plants [1] underlined the still low industrial and urban impact upon the area of study, considered a good source of information for ethnobotanical applications typical in the central Mediterranean area.
This new ethnobotanical study was therefore carried out in order to document folk agricultural and domestic-handicraft usages, now on the point of disappearance, and with a view to observing potential new economic utilizations. This enquiry aims to fill a gap concerning the employment of useful plants in a territory of southern Italy, characterised in the past by the presence and influence of cultures of various origin (first and foremost among which is that of the Greek colonists).
The research was carried out in the territory of Maratea (province of Potenza), with some scattered nuclei stretching from the coast to the slopes of Mt. S. Biagio (Massa, Brefaro) and of Trécchina, in the interior (total population 7600 inhabitants). It is located between the Campania region to the north and Calabria to the south (Fig. 1), and includes 28 km of indented coastline running from the Policastro Gulf to the plain of the Noce River. More than 70% of the territory is characterised by steep slopes and includes a calcareous and calcareous-limestone mountain ridge; the highest point is Mt. Coccovello (1505 m). The climate is of Mesomediterranean type, with rainfall concentrated in the period between October and March, with the arid months being limited to June and July. Precipitation is abundant, about 1200 mm of rainfall per year [2]. The average temperature in the coldest month (January) is about 8.02°C and the warmest month (August) about 27.9°C, with an annual average of 14°C. Roughly 1/3 of the land is suitable for agricultural use whilst, in the past, this quantity was increased by the use of terracing. Some areas are cultivated uniquely with olives and vines; other areas show a typical cultivation ("vignale") made by a mosaic of olives, vines and fruit trees. There are also arable lands and pastures. Mediterranean maquis and forests are present in the area, but also mixed woods, garrigues, shrubby areas, steppes and pastures [2,3].
Figure 1 Geographical location of the field research area in the Basilicata region, Italy.
Methods
Field data were collected during the periods April – July 2002 and March 2003. Ethnobotanical data, mostly regarding the uses of wild plants was collected using structured interviews (some folk uses of cultivated plants are also reported). The informants were people who had been living in the research area for many years. The informants interviewed were 49 (26 men, 23 women) whose ages ranged from 25 to 94, and who belonged (mainly) to families with strong links to the traditional activities of the area. Most of the interviewees (34) were more than 50 years old, of whom: 5 between 50 and 60, 11 between 60 and 70, 15 between 70 and 80, and 3 over 90 years old. Among the informants 19 were farmers, the others shepherds, building workers, restaurateurs, housewives. Interviews were carried out using fresh plant specimens, or by visiting meadows and woods with the informants to collect the plants related to folk uses. Voucher specimens were kept in the Herbarium of the University of Rome Tre. Some tape recordings are also conserved in the National Museum of Arts and Folk Traditions of Rome. In the structured interviews (whose results are reported in Table 1, Additional file 1) informants were requested to indicate, for each plant: vernacular name, folk uses (in agricultural and domestic or handicraft fields), parts used, periods of gathering, frequency or rarity in the area of a given use according to the informants, and also to specify whether the use was personal (that is practised by the informant) and/or familiar (that is practised by one or more members of that family) and/or practised by others (e.g. acquaintances). Further information, related to possible personal consumption or sale of the substance or object produced, was requested. In table 1, Additional file 1 we also indicate the citations for each use (number of informants) and the current use (indicated with °). The nomenclature of the listed species follows Pignatti [4], but we have also used Tutin et al. [5] in the classification of the plants.
Results and discussion
The plants of ethnobotanical use, as collected from the field study, are reported in Table 1, which summarise all original data from interviews and further controls in the field. The reported uses are 162. Most of the information are coincident with what was previously reported in the specific Italian consulted literature [6-16]; 56 data are however uncommon or typical of the places studied. Data show a diffuse use of plants for many purposes (Fig. 2), both in agricultural (present uses 14%; past uses 1 %) and for domestic or handicraft use (present uses 40%; past uses 45%); most of the latter are now in decline. The most meaningful and interesting uses are listed below.
Figure 2 Main typologies of agricultural and domestic-handicraft uses, according to the number of citations, in the Tyrrhenian sector of Basilicata. For each category the number of species is indicated.
A) Agricultural uses
We found 18 wild species used in supporting agricultural activities: mostly as twine and "tutore", but also as shelter from winter frosts, as twines, as graftholders, or in 'sovescio' (green manure) activities.
We found many plant species used to fix cultivated plants to "tutori"; characteristic and relevant is the use of different species to support different parts of the plant, and in different seasons. For example, in the cultivation of the vine, in winter the branches that are supposed to product the fruit-bearing shoots are fixed to the "tutore" by using Salix alba subsp. vitellina (Fig. 3). The latter is planted along the edges of the whole vineyard and it is preferred for its long, thin and particularly flexible branches [7,17]; these features are obtained with a yearly pollarding. In spring-summer, on the contrary, the budding vine shoots are tied with Ampelodesmos mauritanicus leaves, to prevent their being broken by the weight of the bunches. The practice of tying vine with Ampelodesmos [9,11] is extremely old: the Greek name, in fact, literally means "twine for vine" (ampelos = vine, desmòs = twine). In the past, the plant was also gathered for sale in the neighbouring village, where it is less frequent. Other plants to cite are Holoschoenus australis and S. junceum, used above all as twine for vegetables [7,9,18]. The custom of using vegetal twines is disappearing, and with it we are losing the knowledge of the highly specific techniques of knotting.
Figure 3 Vine tied with Salix alba subsp. vitellina branches.
One activity noted in Maratea in spring, is the use Pyrus amygdaliformis as a graft-holder for cultivated varieties of pear tree (in effect, it is the root of this pear tree that is used, since it is more robust and hardier). The practice is also recorded for Sicily [11] and Cilento (G. Salerno, unpublished data). All the uses cited are still practised in the territory; on the contrary, today the practice of using "green" manure (produced from a wide range of Leguminosae, e.g. Vicia sp. pl. and Lathyrus sylvestris) appears to be almost quite obsolete. The resumption of such a practice would be desirable as it could considerably limit the use of synthetic fertilizers. For example "tutore" are stakes, or longitudinally cut trunks, or even (unusual local use) stems not deprived of secondary ramifications to support creeping plants (e.g peas). The stems with leaves of Quercus ilex, Arundo donax and Spartium junceum are used to protect delicate cultivated plants from winter frosts (above all Citrus-trees), by creating a kind of folding screen around them.
B) Domestic and handicraft uses
In Maratea a remarkable number of plants (51) employed in local handicrafts or for domestic uses was recorded, in many cases involving the production of interesting and typical artefacts (Fig. 4, 5, 6).
Figure 4 Decorations on a "puntagliera", special collar made with Acer sp. for goats and cows (various species of this genus are used to do such collars).
Figure 5 From left to right: baskets made with Salix sp. pl., sieve ("cernicchio") and rack ("gratedde") with Ampelodesmos mauritanicus, Arundo donax and Spartium junceum, chair with Typha latifolia and basket made with Castanea sativa and Fraxinus ornus.
Figure 6 Rack ("gratedda"), tray made with Arundo donax and edge of Spartium junceum, used for drying foodstuffs.
A very high number of informants (36) were registered with regard to A. mauritanicus; a large proportion of the people of Maratea were involved in gathering its leaves [19]. Up until the 1960's, this plant had represented one of their main sources of income. Large faggots of the species were sold near the port, where the fibre was extracted from small bunches of leaves that were beaten with a small club ("mazzola"). This fibre was then twisted round itself, the other end being held firm under a foot, to obtain very long, thin ropes from which thicker ropes ("libàni") could be woven. These were then sold, for use either in mussel production or as ropes for vessels. Raw fibre was also used to stuff the small mattresses of the ships' crews. The use of Ampelodesmos, particularly as wattle, is cited by Zambardi and Iannacone [20]. Rispoli [21], describing the nearby Salerno province where the plant could be found, suggests also its industrial exploitation. Ampelodesmos leaves were also utilized e.g. to light fires and for making brooms and small brushes used for cleaning cinders from fireplaces; the small stem is called "jàccola", the name probably deriving from their being used to make torches (fiàccole) for travelling by night. A number of stems were tied together in small bunches to make the torch, a use also reported by Guarrera [9].
Another occupation that was once important but that has disappeared entirely today is charcoal production. Large amounts of firewood (up to 203), were piled up to form cones about 2 m high and then covered first with Ampelodesmos or Sambucus ebulus leaves, and then with a layer of about 10 cm of earth, after which the fire was lit through an opening at the base. Combustion lasted for several days and one could be sure that the process was finished when, making holes at the bottom of the heap, greenish smoke no longer emerged. Brief mention of charcoal production is made by Lieutaghi [13] for Fagus sylvatica, Quercus ilex, Q. petraea and Q. robur; by Pirone [14] for the mentioned species and for Quercus cerris.
The production of lime was another occupation today extinct. Lime was obtained by bringing as much as 500 quintals of calcareous rocks of the area to a very high temperature in special, large fireplaces built in hollows on steep slopes. The fuel consisted of the different shrubby species of the maquis and the undergrowth, which were cut, tied in big faggots and dried before being used. "Baking" took three days, during which combustion had to be uninterrupted, a task which involved burning up to 1500 faggots!
Q. pubescens wood was instead once the object of a flourishing activity producing railway sleepers, as well as roof and ceiling beams. Embers and cinders are removed from the breadoven by means of a small, long handled broom ("mùnnulu"), made from fresh branches of Euphorbia characias, Laurus nobilis, Ruscus aculeatus or S. ebulus. Elsewhere the "mùnnulu" was made with these plants or with Ficus carica and Sorghum bicolor [9].
Several plant species are employed in occupations related to sheep-rearing and cheese making. Shepherds crooks are generally made from shrubby species that provide a particularly hard wood [10]: Prunus spinosa, Cornus mas and Crataegus monogyna. Typical collars ("puntagliere", "collane") for goat bells, of excellent craftsmanship (Fig. 4) are made from Acer neapolitanum, A. campestre and Juglans regia wood.
In the absence of rennet, obtained from the stomach of kids or calves, milk could be curdled also with small, freshly gathered pieces of leafy branches of F. carica [9,22]; freshly-made cheeses are placed in special containers ("fuscelle","recuttari") woven, once again, from Holoschoenus australis and Carex distans stems.
The use of Origanum heracleoticum to dye wool red was previously unpublished (Lieutaghi [12] reports an analogous dye use for Origanum vulgare); the dye use of Fraxinus ornus cinder to obtain grey shades was commoner [23]. The husk of J. regia was still used to obtain brown-dark or black shades. Writing ink was prepared by squeezing the fruits of S. ebulus.
A most particular use was reported for a plant, which has now been a long-since naturalized species in Italy: Agave americana. The tough terminal prickle of the leaves and the tenacious fibres connected to it, removed together, were utilized as needle and thread for mending or sewing more or less unrefined fabrics. An analogous use is cited by Parada et al. [24]. "Spartu" (Spartium junceum) was, instead, also used for making clothes, in addition to flax. The fibre was extracted by beating and then washing small bunches of the previously boiled flexible stems (for fibre extraction see also Musacchio and Barone Lumaga [25]). This activity was carried out in the territory under study by a few families, while in other areas of southern Italy [26], and above all in Calabria, it appears to have flourished notably during the 1930s in the fascist period. During this era an autocratic economy producing food and clothes (e.g. with Spartium junceum fibers) without foreign imports existed in Italy. With the inflorescences of A. donax and Arundo pliniana particularly good brooms were made. Another type of small broom used after threshing to separate the grain of Triticum aestivum from the bran was made with Calamintha nepeta stems and leaves; this particular use is previously unpublished. A typical use in this area, but elsewhere less frequent [9] was that of flavouring home-made soaps with Salvia officinalis.
The use of Euphorbia dendroides latex once poured into streams to stun and, therefore to more easily catch fishes and eels is known also in other areas [15,26-29]. In the past, during the kill of the pig, a tool named "gammieddu" was used, made with Phyllirea latifolia or Olea europaea wood. This tool was made selecting a branch of about a meter with a shape similar to that of a boomerang; its tips were sharpened and endowed with cuts for the tendons of the hind legs of the pig; in this way the body of the pig could be hoisted, making dissection more easy.
Conclusion
Data shows that practically all agricultural uses are still practised (except for "sovescio", green manure) and that almost half domestic-handicraft uses are still presently used. The differences between present and past uses in the cited categories: e.g. ropes, textile fibres and tobacco substitutes are all past uses, while walking sticks and fuel are current uses; collars, baskets and racks, brooms are partly made also today. The rediscovery of the folk uses of plants in the area under consideration is not only of historical and scientific value, but could also represent future, economic potential for the area. Several plants could still today be involved in the production of typical and appealing artefacts. In particular, the production of typical objects that are now on the decline (collars, baskets, clothes of particular textile fibres, and generally the artefacts under sale) could regain importance in the local economy.
Supplementary Material
Additional file 1
Table 1 - Agricultural and domestic-handicraft uses of plants in the Tyrrhenian sector of Basilicata.
Click here for file
Acknowledgements
The authors thank Angelo Merante for graphics support. They express their gratitude to Giovanni Gaglianone and to all the informants, especially to Giuseppe and Caterina Sgambellone, Francesco Schettino, Carmine Pacchiano and Maria Lagatta for ethnobotanical information and their help in collecting the data.
This work is supported by the National Institute for Mountain Research (INMR-MIUR).
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Corsi G Gaspari G Pagni AM L'uso delle piante nell'economia domestica della Versilia collinare e montana Atti Soc Tosc Sci Nat, Mem, sB 1980 87 309 386
Fenaroli L Gli alberi d'Italia 1967 Milano: Martello
Guarrera PM Il patrimonio etnobotanico del Lazio 1994 Roma: Regione Lazio, Assessorato alla Cultura e Dipartimento di Biologia Vegetale Università "La Sapienza", Tipar, Roma
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Sella A Flora popolare biellese Nomi dialettali, tradizioni e usi locali 1992 Alessandria: Dell'Orso
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Zambardi M Iannacone A Eva La stramma Un artigianato in via di estinzione 1997 Venafro (Isernia)
Rispoli E Sfruttamento industriale dell' Ampelodesmos mauritanicus e possibilità di sviluppo con sistema cooperativistico Monti e Boschi 1957 7 220 228
De Simoni E Guarrera PM Indagine etnobotanica nella provincia di Teramo Quaderni Bot Amb Appl 1994 5 3 10
Tammaro F Flora Officinale d'Abruzzo 1984 Chieti: Giunta Regionale d'Abruzzo. Centro Servizi Culturali
Parada M Selga A Bonet MA Vallès J Etnobotànica de les terres gironines Natura i cultura popular a la plana interior de l'Alt Empordà i a les Guilleries 2002 Girona, Diputaciò de Girona
Musacchio A Barone Lumaga MR Fibre extraction from Spartium junceum L. (Fabaceae): ancient and present methods Delpinoa 2003 45 175 177
Bernardo L Fiori e piante del Parco del Pollino 1995 2 Castrovillari: Prometeo
Alvarez Arias BT Ichthyotoxic plants used in Spain J Ethnopharmacol 2000 73 505 512 11091006 10.1016/S0378-8741(00)00336-6
Chiovenda-Bensi C Florula Medicinale delle Cinque Terre Webbia 1960 15 631 641
Lentini F Catanzaro F Aleo M Indagini etnobotaniche in Sicilia. III. L'uso tradizionale delle piante nel territorio di Mazara del Vallo (Trapani) Atti Accad Sci Lett Arti, Palermo 1988 1 29
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J Ethnobiol EthnomedJournal of Ethnobiology and Ethnomedicine1746-4269BioMed Central London 1746-4269-1-31627091410.1186/1746-4269-1-3ResearchRole of traditional healers in psychosocial support in caring for the orphans: A case of Dar-es Salaam City, Tanzania Kayombo Edmund J [email protected] Zakaria H [email protected] Mariam [email protected] Institute of Traditional Medicine, Muhimbili University College of Health sciences, P. O. Box 65001, Dar-es-Slaam, Tanzania2005 29 7 2005 1 3 3 5 7 2005 29 7 2005 Copyright © 2005 Kayombo et al; licensee BioMed Central Ltd.2005Kayombo 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.
Orphans are an increasing problem in developing countries particularly in Africa; due to the HIV/AIDS pandemic; and needs collective effort in intervention processes by including all stakeholders right from the grass roots level. This paper attempts to present the role of traditional healers in psychosocial support for orphan children in Dar-es-Salaam City with special focus on those whose parents have died because of HIV/AIDS. Six traditional healers who were involved in taking care of orphans were visited at their "vilinge" (traditional clinics). In total they had 72 orphans, 31 being boys and 41 being girls with age range from 3 years to 19. It was learned that traditional healers, besides providing remedies for illnesses/diseases of orphans, they also provided other basic needs. Further, they even provided psychosocial support allowing children to cope with orphan hood life with ease. Traditional healers are living within communities at the grass roots level; and appear unnoticed hidden forces, which are involved in taking care of orphans. This role of traditional healers in taking care of orphans needs to be recognised and even scaling it up by empowering them both in financial terms and training in basic skills of psychosocial techniques in how to handle orphans, in order to reduce discrimination and stigmatisation in the communities where they live.
TanzaniaHIV/AIDSOrphansPsychosocial supportTraditional healers
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Introduction
Orphanhood is an increasing problem especially now with the event of HIV/AIDS in developing countries [1-4]. These orphans need, amongst other things, psychosocial support in helping them to cope with orphanhood more easily, by involving all stakeholders right from the grass roots to the national level. Orphans are children who have lost one of their parents or both under eighteen years old [3]. The national data on magnitude of orphans in south of the Saharan African countries is not readily available; and hence the present information is based on estimates. By the end of 2001 it was estimated that 14 million children worldwide had lost their mother or both parents to AIDS or related causes; and it is being projected that by 2010 will reach 35 million [1,5]. Sub-Saharan Africa is the most [2] severely affected region and accounts for more than 80 % of those orphaned as a result of AIDS. In some countries south of Sahara, orphans account for 15–17% of total population [1,5]. In Tanzania, on the otherhand, it is estimated that by the end of 2001 cumulative totals of AIDS orphans were 1.3 (4.3%) million country wide; and the number is expected to rise to 2 million by year 2005 [6].
Much has been written on socio-economic and psychosocial problems encountered by orphans whose parents or guardians have died because of HIV/AIDS [1,3,5-7]. However, major issues are; how does a child deals with a parent's/guardian's loss and what type of care a child receives before and after the death of a parent/guardian and who does that? What is the child's feeling of surviving a parent's/guardian's depression, loss of family income? Furthermore, what is a child's perception of having or not having open communication between family members and/or their community? This calls for skills of psychosocial support to help children cope with orphanhood more easily. Psychosocial support is defined as an ongoing process of meeting physical, emotional, social, mental and spiritual needs of orphans and vulnerable children, all of which are considered to be essential elements for meaningful and positive human development [3]. Family Health International [3] has developed a psychosocial model which can be used in helping children cope with orphanhood. But can this model be applied in a typical traditional setting with increasing numbers of orphans?
It has been argued earlier that Sub-Saharan Africa is the most severely affected region. But who is caring for these orphans in traditional settings? Do they have skills of psychosocial support? It has to be remembered that orphanhood is as old as mankind in history because parents have been dying and living children taken care of by a remaining parent or by elder children, an aunt or uncle or grand parents within the community [1-3,8]. Nevertheless, historical large scale of orphaning has been a sporadic, short term problem associated with war, famine or disease [1]. The word orphan in the African setting was not present. However, in recent years the problem has been aggravated by HIV/AIDS and is increasing; and it is likely to be a long term, chronic problem affecting developing countries through out the world [1,5].
Notwithstanding, studies [1,3,6,8] reviewed still show over 40% of orphans in Sub-Saharan African countries continue to be cared for by traditional structures as was the case in the past and some of them do a good job. For example in Malawi, Alliance [4] has shown how children appreciated the usefulness of their grandmother
"Our grandmother is so wonderful. She helps us in so many ways. She feeds us, dresses us and brings us up properly. When we see her, we see our mother. If she was not there we would have been scattered around other families and would not be treated in the same way. We are so grateful that she is still with us" [3]
Catherine 15, the eldest of the eight grand children cared for by Irine 80 years in Malawi
But how many grand parents are doing this wonderful job in the presence of abject poverty in many African communities?
Because of the increasing problem of orphans in some cases orphans are also cared for by elder children. For example, children as young as 7 years are forced to look after their younger brothers and/ or sisters because the extended family no longer has the capacity to absorb more orphans or because the children resist being separated and opt to stay on their own without adult supervision [7]; and this was unusual in the past. The impacts of HIV/AIDS especially in caring for orphans seem to suggest that family structures are changing. Often the middle generation of both men and women are completely absent, leaving old and young to support each other. This means that older members of the family care for the orphans; and orphans and vulnerable children are compelled to take on new roles beyond their years [1].
The questions, which remain unanswered, are; besides giving orphans basic needs can they provide psychosocial support so that these children can cope with orphanhood. We are asking this question because there is evidence that suggests currently there is stigmatisation and in some instances discrimination in food allocation, education and workload from these caregivers [1,7,9]. Is it because of the fear that they can infect their children and caregivers themselves? Or is it the increasing burden of orphan hood in poor families? All in all, the outcome of these problems is that some children leave their relatives and join Community Based Organisations (CBOs) and Non Government organisations (NGOs)[1] especially in urban areas and some go to traditional healers where their parents were being treated. However, we do not know whether it is better to stay at CBOs or NGOs or with the traditional healer as an orphan.
This study used the model of psychosocial support proposed by Family Health International [3] to learn the psychosocial support provided by traditional healers to orphans in an urban setting because there is no literature which attempt to show the role of traditional healers in supporting orphans; and it is one of traditional structures that are involved in healthcare at grass roots level.
The aim of this study was to identify if any: -
- The role of traditional healers in supporting the orphans
- How do they get the orphans?
- The basic needs they can provide to the orphans;
- Techniques used for psychosocial support for orphans;
- The problem encountered when taking the orphans.
Model of Psychosocial Support
Psychosocial support model is represented with the use of a wheel. The wheel recognizes that none of these elements would be adequate if provided without input from other elements or in a vacuum. If we remove any one segment of a wheel, the wheel would not turn and progress would be impossible. The model assumes that at the centre of the psychosocial wheel model is an awareness of cultural practices, beliefs, and rituals, which inform one about the manner in which all of the other needs are met. Culture needs to be seen as a pivotal point for enrichment of children's identity. Culture also serves as a store of knowledge, values, connectedness, belonging and traditional practice, which is regarded as being essential to the general well being of the child.
One spoke of the wheel would be the physical needs of a child. This incorporates financial needs such as food, shelter, clothing, school uniforms fees and basic health care etc. Most of these economic needs of children are combined with educational needs, for example: the need for information about hygiene, nutritional diet, how to prepare food, decision making models for health care and other skills. The simple provision of financial assistance is not what children need from a psychosocial perspective, but their needs for financial support must be met in an on-going and reliable fashion that also conveys to the child on-going concern, care and support.
A second spoke is emotional needs of children. This includes need for love, security, encouragement, motivation, care, self-esteem, confidence, trust and security, sense of belonging, guidance, understanding etc. Children need to be heard and need to learn to express their feelings in an appropriate manner. At times children's emotional needs may include assisting them to cope with especially difficult circumstances, like bereavement, loss, sexual abuse, etc.
The third spoke is the mental needs, which incorporate three aspects: formal education {schooling}, informal education {opportunities for observational knowledge, adaptation skills, which support the child in order to be able to control environment and access positive reinforcement} and general skills {life skills, general knowledge, etc} combined with motivation and application to succeed.
The fourth spoke would be the children's social needs. These are essential for integration into a community without feeling stigmatised or different; to develop a sense of belonging; form friendships and community ties; acceptance; identity; acknowledgement from peers and opportunities for social interaction. They also need to learn socially acceptable behaviour though feedback from others, how to access help and learn their limits.
The last spoke of the wheel are the children's spiritual needs. Children need a belief, which enables them to develop a hope for their future. They also need to develop trust and security in their survival. This gives them hope to keep trying. Also, this facilitates a sense of connectedness to deceased parents and ancestors. Traditional healers are among the custodians of African traditional culture [10] and therefore the study expected some of the features in caring for the orphans will be manifested in their practice.
Research Methodology
Dar-es-Salaam is the largest city in Tanzania, and according to 2002 population National Census [11], its growth rate is 4.3% with a total population of 2,497,940 of which 1,236,864 (45.9%) are females living in three Municipalities namely Ilala, Kinondoni and Temeke. Thirty three percent (33%) of the total population are children under 15 years. Notwithstanding, the total number of people living in Dar-es-Salaam today is likely to be higher than mentioned due to rural urban migration and mostly youths coming to seek urban opportunities. In Dar-es-Salaam City, like other cities in developing countries, more than 60% of the population is living in squatter settlements where healthcare and environmental health services are limited.
Dar-es-Salaam City was chosen for this study because; firstly, about one tenth of the total population of Tanzania lives in Dar-es-Salaam. Secondly, the city is composed of almost all 120 ethnic groups of Tanzania; and hence multi-ethnic city and possibly each group in one way or another is captured in this study. Third, Dar-es-Salaam city has many HIV/AIDS patients (ranking third in the nation) and orphans – an area of interest of this study. Fourth, Dar-es-Salaam has NGOs and CBOs which claim to be involved in taking care HIV/AIDS patients and orphan children.
This study is part of Traditional Medicine HIV/AIDS project, which started in 2003 September and is ongoing still to date. The Traditional Medicine HIV/AIDS project recruited 25 traditional healers. The selection of these traditional healers was based on their reputation in providing healthcare to HIV/AIDS patients and also from records of traditional healers associations. The research team visited selected traditional healers at their respective vilinge. It was during these visits the research team learned that six traditional healers were caring for orphans. The research team was interested to learn how they got these orphans; and ways of caring for orphans in traditional settings and problems encountered. Further, specifically focus was on psychosocial support offered to these children.
As shown earlier the psychosocial model proposed by Family Health International [3] was used in this study. An in-depth interview questionnaire in Kiswahili language was developed as a research instrument for interviewing traditional healers who were caring for orphans whose parents had died because of HIV/AIDS. Kiswahili is the national language of Tanzania; and most of urban people are well versed with it as a means of communication. The questionnaire focused on their role of taking care of orphans and seeing how they fit in to the psychosocial model like how they got them, what things did they offer to orphans so that they can cope with orphanhood, techniques of psychosocial support (if any) and problems encountered when taking care of orphans. The research team itself with the use of notes and a tape recorder did the data collection. The collected data were screened and then transcribed from Kiswahili to English. The data collected were summarized and codes were identified for grouping the information according to issues raised in the introduction. The grouped information per code were re-summarized for getting the finally report. The summary of the final report of findings is presented below.
Results
The role of traditional healers in supporting the orphans
The study findings show that traditional healers were not only treating patients using traditional remedies, but also involved in caring for orphans using traditional psychosocial support by providing with them the basic needs and psychosocial support as shown in the psychosocial support model. Out of 25 traditional healers who were in the Traditional Medicine HIV/AIDS Project in first phase, 6 (24%) were involved as well in taking care of orphans. Five of these traditional healers were males aged between 35–50 years, and one female aged 46 years. The total number of orphans recorded from these traditional healers in study period, whose parents died because of HIV/AIDS, was 72 children ranging from 3–19 years. There distribution by age and sex is shown in Table 1. Most of these orphans (38.9%) were at age of 5–9, followed by age 15–19 (26.4%).
Table 1 Distribution of Orphans Who are Under the Care of Traditional Healers by Age Group and Sex
Age group Sex Total
male females
0–4 6 (8.3%) 5 (6.9%) 11(15.2%)
5–9 10 (13.9%) 18 (25%) 28 (38.9%)
10–14 6 (8.3%) 8 (11.1%) 14 (19.4%)
15–19 9 (12.5%) 10 (13.9%) 19 (26.4%)
Total 31 (43%) 41 (56.7%) 72 (100%)
Three traditional healers expressed that some of children were denied by relatives after death of all parents for fear that these children may infect their children through playing or exchange of things they eat like sweets, sweet potatoes or cassava that are being sold along the streets. Further, these children were abused in words by their respective relatives that their parents died of AIDS because of their bad behavior or promiscuity. All these together lead children to move away and in this way reach traditional healers, CBOs and NGOs and some end up on street.
Table 2, on the other hand, shows that 32 (44.4%) of these orphans had lost their fathers, 25 (34.7%) mothers and 15 (20.8%) both parents. Above twenty-seven percent (27.8%) had been screened and found to be HIV positive; and were between the age of 15–19 years.
Table 2 Number of orphans by kind of parents they have lost
Number of children By sex Kind of parent who has died Total
father mother Both parents
male 15 (20.8%) 11 (15.3%) 7 (9.7%) 33 (45.8%)
Female 17 (23.6%) 14 (19.4% 8 (11.1%) 39 (54.2%)
Total 32 (44.4%) 25 (34.7%) 15 (20.8%) 72 (100%)
The Secretary General of Tanzania Traditional Health Practitioners Association (TATHEPA) reported that "We are getting these children always and we have no where to send them because of their extended family generally lives far away from Dar- es -Salaam; and we have decided to stay with them as part of our family; and our main role as traditional healers is to support them in whatever we can so that they can cope with orphan hood; and where possible we attempt to visit relatives and have a discussion of how to assist these orphans".
How did they get the orphans?
The study explored ways in which traditional healers got orphans whom they were caring for and found out that they obtained them in three ways. First, some of orphans were from traditional healers' relatives and were asked to look after them after the death of parents as it is in traditional African culture. Two, other orphan children were from neighbours who came for treatment after being infected with HIV/AIDS. Children from neighbours lived at their respective relatives but during the day came to play at traditional healers' compounds as a place for socialising with other children. It was here where sometimes these orphans got their lunch and even dinner. Three, some of the orphans were abandoned by relatives at traditional healers' vilinge as they came to seek healthcare; and hence these healers have to accommodate them as part of their families.
During interviews traditional healers reported that while caring for these orphan children common problems observed from these children were repeated illness especially those who were HIV positive, anger, guilt conscience, fear, sometimes isolation from playing mates and feeling that they were being oppressed. Further, it was reported that some of the children were crying now and then especially young ones "baba yangu yuko wapi "(where is my father) or mama yangu yuko wapi (where is my mother), sleepless nights and nightmares were common. Several attempts to fulfill both basic needs and psychosocial supports were made to comfort them.
The basic needs Traditional healers can provide to orphans
Even though traditional healthcare practitioners do not earn much money in traditional medicine practice, the research team from this study learned that traditional healers attempted to provide basic needs to orphans who were under their control to help them cope with orphanhood life in their early stage of life. For example, the traditional healers in this study reported having given many things to these children in the process of making them feel at home and cope with orphanhood. Among them were basic needs like food, clothes, school uniforms and exercise books, etc for those who were in school; medical care and safety by making them part of their families. They ate together and allowed them to play with their children; and hence fitting well in first spoke and fourth spoke of the wheel in the psychosocial support model. Nevertheless these traditional healers encountered some problems as stressed by one traditional healer that
"we do not meet all what these children want to live happily because of our poor economic situation. Some of the children stay with us for a while and leave if we fail to meet their demands. Some of these children join street children we see in Dar"
Techniques used for psychosocial support of orphans
During interviews on traditional healers' practices the research team noted they have different traditional methods that are useful for helping orphans cope with orphanhood and live more easily. For example, three traditional healers reported that when children face problems like crying, talking at night were signs that they were seeing "shadows" of their respective parents and grand parents who have died; and had to give them remedies that can remove the parents' and grand parents' "shadows". Remedies may be something to wear either on the hand or around the neck. Whereas, two traditional healers, reported that they had to bath children with herbal remedies. The remedies were vitalised by words of healers through short litany of prayer asking parents and grand parents by naming them to leave the child so that s/he can stay comfortable. In addition, they comforted the orphans by telling stories in the evening; and citing several examples of orphans who have managed to cope with orphan hood and had achievement in life. All these activities reported in this section fits best second and third spoke of the wheel of psychosocial support model. All in all these symbols were meant to help children to forget what happened in the past and build a positive attitude towards living as orphans.
Besides the above, while the research team was visiting vilinge of traditional healers, they had an interesting experience where one of six traditional healers interviewed had 54 orphans. Of these 31 (57.4%) were girls and 23 (42.6%) being boys. Eleven (20.4%) of these have lost both parents, and 30 (55.5%) have lost their fathers and rest have lost their mothers. Eight (14.8%) of these children were HIV/AIDS positive.
This healer reported that she developed an interest in taking care of orphans after loosing her two bothers that left her with seven children to look after according to the African traditional culture. While practicing traditional medicine to HIV/AIDS patients she felt that orphans had a desperate life. She decided to collect other orphan children from her community and provided with them activities which help them to forget the past; and they were basically geared to psychosocial support as shown in the psychosocial model. For example, the healer involved the orphans in poetic theatre, theatre; drama, choir, whereas the others dealt in comics and traditional dances. These groups met twice a week for practice and performed when invited to a social gathering function where they received payment. The poetic theatre group has managed to record one single CD. The healer stressed, "These activities help orphans to forget the past and build a new outlook"
The problem encountered when taking the orphans
Despite traditional healers efforts to help orphans to cope with orphanhood they faced several challenges which need government and NGO support. The common challenges reported by all six traditional healers interviewed were: firstly, limited resources to meet children's basic needs and make them live happily. Secondly, repeated illness especially those who were HIV positive and health care for health problems which can be managed effectively in formal health services. Thirdly was the accommodation particularly for healers who have rented rooms for their families. Fourth was the inadequate food for feeding surplus numbers of people in their families. Finally tracing back relatives for orphans being abandoned at traditional healers' vilinge when these children become very seriously sick.
Discussion
The findings from this present study shows that traditional healers have a significant role in caring for orphans in communities using a holistic approach even though they are hardly reported in the literature. Grand parents who are caring for orphans appear to be overwhelmed with the orphans, leading them to sell even their assets or borrow money to meet daily needs at home [2] because of abject poverty. Again many NGOs and CBOs that are involved in taking care of orphans in Tanzania [6] and elsewhere in developing countries follow strategies of traditional methods of focusing on basic needs like food, clothing, shelter and medical care and going to school [1]. But caring for orphans needs to go beyond this by taking consideration of psychosocial support that helps these children to cope with life. It has to be remembered that death of parents affects children socially and psychologically and has a great impact on the orphans in daily life [7,12,13]. People caring for orphans need to capture this aspect as well to make them cope with life more easily.
In the present study, the analysis shows that traditional healers use a holistic approach when handling orphans by taking into account both basic needs and psychosocial support to make a whole support package. The analysis of traditional healers' activities when caring orphans, underscores The International Federation of Red Cross and Red Crescent Societies [13] that key components of psychosocial support for orphans are ongoing interaction and presence with the aim of bolstering feelings of security and hope and eventually attempting to meet nearly all essential components in each of the spokes of psychosocial model. This support endeavours to help the affected orphans strengthen their personal coping capacities, hope, security, trust and reinforce support from family members and friends [3,7].
Furthermore, the analysis from the traditional healers interviews supports the Family Health International [3] proposed model on psychosocial support in caring orphans on the role of culture as pivotal. The "shadows" from parents and grand parents who have died reflects the belief of traditional African culture that people who have died are alive in an "other world" and have an influence on people who are living in our world; and that is why they believed that they come in the form of "shadows" to their beloved ones. To remove the "shadows" is done through rituals which are accompanied with litany of names of people who have died asking them to stop coming to orphans because they create fear and make them fail to cope with orphanhood [14]. Moreover, the analysis from traditional healers' findings appears to cement the psychosocial support model argument that culture needs to be seen as a pivotal point for the enrichment of children's identity and coping with orphan hood.
Besides the above, traditional healers are becoming creative; and without knowing found themselves doing psychcosocial activities with orphans. For instance, the art comforting orphans like giving them living examples for some of people who have lost their parents and managed to make there way through; and story telling in the evening reduces time of thinking of the past. Also healer who collected the orphans and engaged them in social activities like poetic theatre, theatre comics, traditional dances, had strong impact on psychosocial support to these children. These activities help children to integrate into a community without feeling stigmatised or different. Also, these activities help to develop a sense of belonging, form friendships and community ties. Above, all these things help to develop acceptance; identity; acknowledgement from peers and opportunities for social interaction, trust, security and hope for the future.
Traditional healers do not only end up in psychosocial support of orphans but also provided materials like foods, clothes, and exercise books and allowed them to play with their children; where necessary they even provided bus fares to schools. All these aspects make them think that they are also being valued. These supports fit in the other spokes of the wheel of Family Health International's [3] proposed model on psychosocial support. However, as shown in the findings these traditional healers cannot meet all basic needs for orphans because they do not have enough financial resources. Financial resources they use are fees collected from patients, which are again limited.
All in all the findings in this study seem to suggest that traditional healers are a hidden force unnoticed in taking care of orphans in communities. With increasing number of orphans due to HIV/AIDS, this experience suggests that orphan children are best cared for within families and communities from the same cultural milieu including traditional healers. However, this needs creativity and empowerment of community structures that are involved in caring for orphans by giving them basic training on psychosocial techniques of how to handle orphans as stipulated in Family Health International's [3] proposed model on psychosocial support. This can be done by involving community groups like faith based congregations, and CBOs and NGOs who are first in line for households caring for vulnerable children [3]. Emphasis should be on psychological support which will help children to cope with orphan hood like sports, poetic theatre, setting income generating activities, providing spiritual, emotional and psychological support. In addition the government should support these types of healers through credit schemes.
Acknowledgements
The authors express their thanks to the Tanzania HAIV/AIDS Commission for the financial support. Further to Charlie Cray for assisting in editing this work. Above all our thanks go to the traditional healers who participated in this study. The opinion expressed herein are those of authors and do not necessary reflect the views of Tanzania Commission of HIV/AIDS.
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Subbarao K Mattimore A Plangemana K Social Protection of Africa's Orphans and other Vulnerable Children in Tanzania, Issues and Good Practice Region the World Development African Region Human Development Working paper Series 2001
Ainsworth Martha Kathleen Beegle Godlike Koda "The Impact of Adult Mortality on Primary School Enrollment in Northwest Tanzania." Draft 2000 World Bank, Development Economics Research Department, Washington, D.C
Family Health International Compilation of Psychosocial Training Materials for the Emotional Well Being Evaluation of Orphans and Vulnerable Children redcrossinforcedk/graphic/2003referencecentre/docman/5hivAIDS/compilation PSTrainorphansdoc 2001
International HIV/AIDS Alliance and Family AIDS Caring Trust Support community Action on AIDS in Developing countries Expanding community based support for orphans and vulnerable children; Workshop report 2002
Smart R Policies for Orphans and Vulnerable children; A framework for moving A head. A report of the project implemented by the Future Group International in collaboration with the Centre for development and population activities CEDPA and Research Triangle 2003
Axios International A programme on Orphan and Vulnerable Children in AIDS Affected in Tanzania 2001
Orphans and Vulnerable Children 2003 Annual Report Strengthening Community Partnerships for the Empowerment of Orphans and Vulnerable Children (SCOPE-OVC) Program 2003
International HIV/AIDS Alliance and Helping Age International Forgotten families older people as carers of orphan and vulnerable, Help Age International 2003
The Joint United Nations Programme on HIV/AIDS (UNAIDS) Report on the Global AIDS Epidemic, UNAIDS information Centre, Geneva, Switzerland 2004
Kayombo EJ The Role of culture and Indigenous Knowledge in forest conservation: A case of Ijombe/Ntangano Ward in Mbeya Region Paper presented on the 2nd National Workshop on Plant Genetic Resources (PGR) and Resources Biotechnology in Arusha 6th – 10th May, 2002
The 2002 Tanzania Population Census Bureau of Statistics Planning Commission Tanzania 2002 Dar-es-Salaam: Government Printers
William J An overview of strategic issues on Finding a way forward. Displaced Children and Orphan Fund war Victims Fund Paper presented at the workshop on Sustainable care and support for children made vulnerable and orphaned by AIDS Hosted by the Center for Interdisciplinary Research on AIDS (CIRA) Co-sponsored by CT Voices for Children and CIRA's Law Policy and Ethics Core, Washington DC USAID 2002
The International Federation of Red Cross and red Crescent Societies Health and community care 2004
Kayombo EJ Sylvester Ntomchukwu Madu, Peter Kakubeire Baguma, Alfred Pritz Tambiko as Healing Therapy in Tanzania Psychotherapy in African First Investigations 1996 World Council for Psychotherapy Vienna 71 81
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-5-251620214310.1186/1472-6750-5-25Methodology ArticleNymphal RNAi: systemic RNAi mediated gene knockdown in juvenile grasshopper Dong Ying [email protected] Markus [email protected] Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Detroit, MI 48202, USA2 Center of Developmental Biology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA2005 3 10 2005 5 25 25 22 6 2005 3 10 2005 Copyright © 2005 Dong and Friedrich; 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
Grasshopper serves as important model system in neuroscience, development and evolution. Representatives of this primitive insect group are also highly relevant targets of pest control efforts. Unfortunately, the lack of genetics or gene specific molecular manipulation imposes major limitations to the study of grasshopper biology.
Results
We investigated whether juvenile instars of the grasshopper species Schistocerca americana are conducive to gene silencing via the systemic RNAi pathway. Injection of dsRNA corresponding to the eye colour gene vermilion into first instar nymphs triggered suppression of ommochrome formation in the eye lasting through two instars equivalent to 10–14 days in absolute time. QRT-PCR analysis revealed a two fold decrease of target transcript levels in affected animals. Control injections of EGFP dsRNA did not result in detectable phenotypic changes. RT-PCR and in situ hybridization detected ubiquitous expression of the grasshopper homolog of the dsRNA channel protein gene sid-1 in embryos, nymphs and adults.
Conclusion
Our results demonstrate that systemic dsRNA application elicits specific and long-term gene silencing in juvenile grasshopper instars. The conservation of systemic RNAi in the grasshopper suggests that this pathway can be exploited for gene specific manipulation of juvenile and adult instars in a wide range of primitive insects.
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Background
Including some of the most persistent and devastating agricultural pests such as the African migratory locust Schistocerca gregaria, acridid orthopterans represent a group of insects with significant economic impact and a target of major research efforts [1-3]. Species of the grasshopper genus Schistocerca serve also as powerful model system in neurosciences, development and evolution [4-7]. The development of methods facilitating molecular manipulation of these primitive insects is thus of wide interest. However, until now successful approaches for a specific interference with gene expression have not been reported. Here we report results from investigating the conservation of the systemic RNAi pathway in the juvenile (nymphal) form of the grasshopper species Schistocerca americana.
RNA interference (RNAi) or the degradation of specific mRNA species in response to cytosolic presentation of sequence identical dsRNA molecules is a widespread phenomenon among eukaryotes. Following its discovery in C.elegans [8], RNAi has been widely adopted as powerful loss of function gene analysis tool. Components of the intracellular RNAi pathway machinery like the dsRNA processing enzyme Dicer, and the RNA induced silencing complex (RISC) have been found in many eukaryote model organisms [9]. Less is known yet regarding the conservation of mechanisms facilitating the amplifying and systemic nature of RNAi in C. elegans. Systemic RNAi describes the fact that extracellular application of dsRNA via body cavity injection, soaking, or feeding leads to global and persistent gene silencing in treated individuals and their progeny [10,11]. The systemic RNA pathway involves cellular dsRNA uptake and very likely also cellular amplification and release of dsRNA [9].
Recent molecular genetic efforts in C.elegans identified the systemic RNA interference-deficient-1 (sid-1) gene as essential and sufficient for the systemic induction of RNAi [12,13]. Sid-1 encodes a seven helix transmembrane protein which has been shown to function as a channel for the uptake of dsRNA molecules and may also facilitate the release of dsRNA from cells [13]. Homologs of sid-1 with the capacity to enhance the systemic uptake of dsRNA have been reported from humans but not yet from other organisms [13,14]. However, reports of gene knockdown following systemic application of dsRNA in phylogenetically distantly related animal species such as flatworms, annelids and insects suggests that the systemic RNAi pathway is widely conserved [15-18]. In the red flour beetle Tribolium castaneum for example, injection of dsRNA in the body cavity of the parental females (parental RNAi) or final instar larvae (larval RNAi) leads to induction of specific gene silencing in embryos and pupae respectively [17,18]. Efforts to trigger similar gene silencing effects in the fruit fly Drosophila melanogaster have failed [19]. Intriguingly, the lack of systemic RNAi competence correlates with absence of a sid-1 homolog from the Drosophila genome, while sid-1 homologous sequences are present in the EST database of the systemic RNAi competent insect species (see below). Experimental and phylogenetic evidence thus identify sid-1 as a conserved facilitator of systemic RNAi.
Results
Ubiquitous expression of a sid-1 dsRNA channel protein gene homolog in grasshopper
Considering the causal relationship between sid-1 expression and systemic RNAi competence, we investigated the presence of sid-1 in Schistocerca. Parts of a sid-1 homologous grasshopper gene (Sa_sid-1) were isolated by degenerate PCR from nymphal cDNA using a set of nested primer pairs designed against the C-terminal region of sid-1, which is strongly conserved between sid-1 of C.elegans and sid-1 homologous sequences identified in EST databases of human and honey bee (Additional File 1). Subsequent RT-PCR experiments with a single, specific primer combination amplified grasshopper sid-1 from cDNA of mid-stage embryos, nymphal head or abdomen, and adult eye, leg and ovaries (Additional File 2). Whole mount in situ hybridization with a Sa_sid-1 probe against mid-stage embryos revealed ubiquitous expression at uniform levels (not shown). In combination, these results demonstrated that grasshopper possesses a homolog of sid-1, which is expressed in ubiquitous and uniform manner.
Systemic RNAi induced knockdown of the grasshopper eye coloration gene vermilion
To be able to test if grasshopper nymphs are conducive to RNAi mediated gene silencing, we cloned part of the S.americana ortholog of the Drosophila eye coloration gene vermilion (Sa_v) (Additional File 3). The v gene encodes tryptophan oxygenase, which catalyzes the degradation of tryptophan to kynurenine, the primary substrate of the ommochrome pigment synthesis pathway [20]. v mutant Drosophila are characterized by brighter red coloration in the adult compound eye due to lack of the dark ommochrome pigment and the presence of a second, pteridine based pigment [21]. Grasshopper compound eyes also contain red pteridine and dark ommochrome pigments, which are coexpressed in five vertical stripes (Fig. 1a–e) [22]. Each stripe is formed during one of the five nymphal instars. The number of stripes at a given instar thus corresponds to the number of previously completed instars rendering eye coloration a convenient readout for assaying the temporal dynamics of gene perturbation. Sa_v dsRNA was prepared by bidirectional in vitro transcription from template DNA prepared by PCR amplification with primers targeting the vector internal transcription start sites. This strategy resulted in 698 bp long dsRNA covering the entire cloned tryptophane oxygenase open reading frame region (see Additional File 3). Sa_v dsRNA was diluted in injection buffer and injected into the dorsal heart vessel of freshly hatched first instar nymphs. In three independent injection experiments, stripes one and two, which are formed during the first and second nymphal instar, developed a persistent brick-red to light brown coloration in over 15% of injected animals (Table 1). This coloration contrasted with the dark brown to black color of the corresponding stripes in control animals and also of the posterior region of the eye, which is formed earlier during embryogenesis (compare panels 1a-c with f-h in Fig. 1). The phenotypically visible reduction of ommochrome formation in first and second eye stripes was indicative of reduced Sa_v gene activity following dsRNA injection in the first instar nymph. The persistence of ommochrome pigmentation in the posterior partition of the eye was consistent with the known long half life of ommochrome pigment granules [23].
Without exception, the third stripe formed in Sa_v phenotypic animals emerged in darker coloration than the stripes one and two, matching the coloration of eye stripes in non-treated animals at this stage of development (compare panels c and d with panels h and i in Fig. 1). The difference between stripe three and the previously formed stripes one and two suggested that Sa_v expression returned to normal levels by beginning of the third instar. This implied that the duration of Sa_v RNAi mediated gene knockdown was limited to the first two nymphal instars, which corresponds to 10 to 14 days under the culture conditions chosen. Stripes one and two remained distinct through the fifth nymphal instar (Fig. 1i). Lighter coloration and a narrower width of stripes one and two were still noticeable in adult animals suggesting that differentiated retina cells do not or just very slowly replenish ommochrome content (Fig. 1j).
No additional differences were detected in affected animals besides the transient change in eye coloration. Animals injected with dsRNA covering 300 basepairs of the Enhanced Green Fluorescent Protein-1 (EGFP-1) gene developed like non-injected animals and did not exhibit detectable morphological abnormalities (Table 1). In summary these results demonstrated that a single dsRNA surge in the grasshopper nymphal hemolymph elicits specific gene knockdown over a time course of 10–14 days.
Quantitation of systemic RNAi mediated knockdown efficiency
To confirm the Sa_v gene knockdown and measure its efficacy we investigated the relative levels of v expression in phenotypic animals by qRT-PCR (Fig. 2). These experiments revealed a two fold decrease of expression levels in second instar nymphs which rose to a 1.5 fold decrease in third instar phenotypic nymphs. Consistent with the re-initiation of normal pigmentation in the third stripe of fourth instar nymphs, Sa_v expression levels at this stage were not reduced compared to non-treated animals. Instead, slightly increased expression levels were observed which may reflect increased transcriptional activation of Sa_v in response to a high tryptophan titer in the transiently silenced animals. Overall, the course of Sa_v expression level changes in affected animals was consistent with the time window of eye coloration change.
Discussion
The present study represents the first example of targeted and specific gene expression manipulation in grasshopper. It takes advantage of the evolutionary conservation of the systemic RNAi pathway in this species. To demonstrate and investigate efficacy and specificity of this gene silencing pathway we chose a widely used target gene, the v encoded tryptophan oxygenase, which catalyzes the first step in the biosynthesis of ommochrome eye colour pigments. Our results confirm the suitability of the ommochrome synthesis pathway for the evaluation of lack of function gene manipulation methods [24,25]. Even though qRT-PCR indicated a remaining 50% of normal expression levels in affected animals, a resulting change in eye coloration could be clearly and easily detected in life animals and monitored over time. The ommochrome biosynthesis pathway thus represents a sensitive and convenient real time readout of gene dose reduction. Evolutionary conservation, ease of interference and the normal viability of hypomorphic animals under laboratory conditions render v and the ommochrome pathway a widely applicable target to evaluate loss gene function methods in insects.
Our data also represent the first demonstration of systemic RNAi induction in the juvenile stage of a primitive insect. It has been previously reported that localized application of dsRNA into the cavity of the cercus appendage in cockroach nymphs silenced target gene expression in cercal sensory neurons but not in the more distantly located neurons of the ventral nervous system [26]. These observations implied the existence of dsRNA uptake mechanisms but indicated limitations in either the reach of injected dsRNA or the accessibility of different tissues. The present results in grasshopper nymphs demonstrate the induction of gene silencing at far distance from the entry point of dsRNA injection. Insect tryptophan oxygenase is expressed in many tissues including fat body, Malpighian tubules, gut and epidermis [23]. The qRT-PCR data specifically reveal a change of Sa_v expression levels in tissues of the head. The change of eye coloration, however, is most likely due to the non-autonomous effect of v activity reduction which leads to a drop of heamolymph levels of kynurenine, the starting substrate of the ommochrome synthesis pathway. Additional evidence for systemic RNAi competence of a wide range of grasshopper tissues comes from the observation of ubiquitous expression of sid-1 in embryonic and postembryonic tissues. Consistent with this, additional experiments in our laboratory targeting cell autonomous eye selector genes result in specific perturbations of compound eye formation (Dong&Friedrich, in preparation).
The penetrance of systemic RNAi induced v knockdown in grasshopper is much lower than that reported for honey bee (95%) and flour beetle (100%) [15,17]. Likewise, the temporal perdurance of the silencing effect seems shorter that in honey bee (25 days) and flour beetle (>4 months) [15,17]. It remains to be explored if these differences are caused by species specific differences in the systemic RNAi machinery or technical aspects. The larger body volume of grasshopper nymphs compared to the juvenile instars of honey bee and flour beetle is likely to lead to greater dilution of the injected dsRNA. Penetrance and perdurance of gene knockdown in the grasshopper may be increased by raising the amounts of injected dsRNA or by improving its delivery efficiency. The same strategies may also increase the efficiency of RNAi mediated knockdown which in the case of Sa_v reached only 50% reduction of transcription levels. The relationship between mRNA expression levels and RNAi induced phenotypes has not been quantitatively investigated in insects, which makes it difficult to compare our results with that in other systems [15,17]. However, it is reasonable to assume that approaching gene expression levels of heterozygous null mutants may not yield informative phenotypes with every gene examined. Nonetheless, additional data in our laboratory reveal that the expression of upstream regulators of grasshopper eye development can be reduced sufficiently enough to obtain informative phenotypes suggesting wider applicability (Dong&Friedrich, in preparation). Furthermore, the detection of sid-1 expression grasshopper ovaries raises the possibility that parental RNAi can be induced, which may reduce mRNA levels in the embryo more efficiently.
Conclusion
Our data demonstrate conservation and expression of the dsRNA channel protein gene sid-1 in grasshopper, a primitive insect. Consistent with this, systemic application of dsRNA sequence identical to the grasshopper eye coloration gene v leads to specific, long-term and phenotypically visible reduction of endogenous grasshopper v gene activity demonstrating the conservation of the systemic RNAi pathway in grasshopper. In the light of these results we are confident that the conservation of the systemic RNAi pathway paves the way for the molecular dissection of development or neuronal processing in this primitive insect model system. The systemic RNAi pathway may also harbour potential for the development of species specific, and hence ecologically friendly pest control methods.
Based on the conservation of sid-1 in primitive and higher insects, it is reasonable to assume that the systemic RNAi pathway is conserved in a wide range of primitive insect species. Recent reports of parental RNAi induced gene knockdown in the milkweed bug Oncopeltus fasciatus and the two-spotted cricket Gryllus bimaculatus support this conclusion [27,28]. Nymphal RNAi, the systemic RNAi induced gene knockdown in juvenile stages of primitive insects, is therefore likely to develop into a valuable experimental tool analogous to the larval RNAi technique established for higher insects [17].
Methods
Animal culture
Schistocerca americana nymphs were obtained from an in house laboratory culture maintained as described [7].
Molecular cloning
For RT-PCR cloning experiments, total RNA was isolated from first instar nymphal heads with the RNAqueous Kit (Ambion). cDNA template was prepared from total RNA with random decamer or Oligo(dT) primers using the RETROscript™ kit (Ambion). Two rounds of PCR amplification were carried out with nested pairs of degenerate primers targeting conserved gene regions determined by alignment of published sid-1 homologs from distantly related species (see Additional data file 1) (primer sequences available on request). PCR fragments were cloned into the pGEM-T vector system (Promega) and plasmid DNA purified with the Perfectprep Plasmid Mini kit (Eppendorf) for sequencing with the BigDye Terminator Sequencing kit (Applied BioSystems). Sequence reads were prepared on an Applied Biosystems ABI Prism 3700 sequencer in the Applied Genomics Technology Center of Wayne State University. Sequence analysis and alignments were carried out with MacVector 7.2 (Oxford Molecular Group).
Sid-1 expression analysis
Whole mount in situ hybridization to examine the regulation of sid-1 expression in grasshopper embryos at 40% of development was carried out as described [7]. Life cycle and tissue specificity of sid-1 expression was determined by RT-PCR with specific primers (sequences available on request) on cDNA prepared from embryos at 40% of development, second instar nymphal head and abdomen, and adult eye, leg muscle and ovaries. RNA preparations were obtained by use of the RNeasy Mini kit (Qiagen) followed by DNA removal with Rnase-Free Dnase set (Quiagen).
Grasshopper nymphal RNAi
dsRNA was prepared by bidirectional in vitro transcription from PCR templates following published protocols [17]. Freshly hatched first instar nymphs were immobilized by exposure to -20°C for 15 min. Injection solution containing 4 μl dsRNA at 5 μg/ul concentration, 0.5 μl 10 × injection buffer (5 mM KCl, 1 mM KPO4) and 0.5 μl Phenol Red was backfilled into a needle pulled from 10 ul glass capillary tubes (Idaho Technology). 0.05–0.1 μl of dsRNA solution was injected into the dorsal midline between either the third thoracic and the first abdominal segment or the first and second abdominal segment using Eppendorf microinjector 5242 and a Leica compound microscope. Injected individuals were kept in mass culture during the first instar. Phenotypic animals identified in the second instar were cultured individually until reaching adulthood.
qRT-PCR
RNA was extracted from the head of single individuals as described above. Primer combinations against grasshopper genes v and armadillo (acc# AF408424), which was used as normalizer, were designed using the Primer3 software (primer sequences available on request). Optimization and measurement reactions were carried out using the SYBR green kit (ABgene) and the Promega Mx3000PTM Real Time PCR system following manufacturers instructions. Data analysis was done with the Promega Mx3000PTM Real Time PCR system software package. For each time point investigated, two phenotypic individuals were compared to two non-treated animals. Each bar in Figure 2 represents the average difference of one experimental animal to two reference animals. Each animal was represented by three independent reactions.
Authors' contributions
YD designed the experimental strategy, performed all experimental work and helped drafting the manuscript. MF conceived the study and completed the manuscript.
Supplementary Material
Additional File 1
Alignment of sid-1 orthologous genes. Ce_sid-1 = Caenorhabditis elegans sid-1 (acc# NP_504372), Hs_sid-1 = human sid-1 gene (acc# NP_060169), Am_sid-1 = Apis mellifera sid-1 (acc# XP_395167), Sa_sid-1 = Schistocerca americana sid-1 (acc# AY879097). Amino acid residues identical in more than one species are boxed.
Click here for file
Additional File 2
RT-PCR analysis of sid-1 expression. sid-1 expression analysis. Lane 1: 1 kb DNA ladder (Invitrogen). Lane 2: Total embryo at 40% of embryonic development. Lane 3: Second instar nymphal head. Lane 4: Second instar abdomen. Lane 5: Adult eye. Lane 6: Adult leg muscle. Lane 7: Adult ovary. Lane 8: Negative control reaction.
Click here for file
Additional File 3
Alignment of vermilion homologous genes. Dm_v = Drosophila melanogaster v (acc# NP_511113), Tc_v = Tribolium castaneum v (acc# AAL15466), Sa_v = Schistocerca americana v (acc# AY879098). Amino acid residues identical in more than one species are boxed.
Click here for file
Acknowledgements
We thank Bethany Strunk and Lori Pile for assistance with real time PCR. This work was supported by National Science Foundation grant DBI-0091926.
Figures and Tables
Figure 1 Time course of eye coloration change in Sa_v knockdown grasshopper nymphs. Anterolateral views of wild type (a-e) and Sa_v knockdown (f-j) grasshopper eyes at different stages of development. Anterior is to the right and dorsal up. Numbers denote instar during which the corresponding vertical eye stripe has been formed. (a,f) second instar. (b,g) third instar. (c,h) fourth instar. (d,i) fifth and final nymphal instar. (e,j) adult eye. Note bright red brown coloration of stripes one and two in panels g and h compared to dark brown coloration in the embryonic cap (ec), the pigmented compound eye area formed in the embryo. Also in the adult eye of the Sa_v knockdown animal shown in panel j, stripes one and two are more lightly coloured than the later, more anterior stripes.
Figure 2 qRT-PCR quantitation of systemic RNAi induced Sa_v gene knockdown. Bars represent relative Sa_v expression levels of phenotypic grasshopper nymphs in the second, third, and fourth instar compared to expression levels in non-treated reference animals.
Table 1 Penetrance and specificity of systemic RNAi induced Sa_v gene knockdown
Target gene Total number of injected nymphs Percentage of surviving individuals Percentage of animals showing phenotype
Sa_v 92 75.0% 15.9%
Sa_v 114 86.8% 16.2%
Sa_v 51 56.9% 20.7%
EGFP-1 80 71.3% 0%
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Kang L Chen XY Zhou Y Liu BW Zheng W Li RQ Wang J Yu J The analysis of large-scale gene expression correlated to the phase changes of the migratory locust PNAS 2004 101 17611 17615 15591108 10.1073/pnas.0407753101
Sword GA To be or not to be a locust? A comparative analysis of behavioral phase change in nymphs of Schistocerca americana and S.gregaria J Insect Physiol 2003 49 709 117 12837323 10.1016/S0022-1910(03)00092-1
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-5-261620215510.1186/1472-6750-5-26Research ArticleUltra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy Trieschmann Lothar [email protected] Alexander Navarrete [email protected] Katja [email protected] Sandra [email protected] Elke [email protected]ätzl Hermann [email protected]öhm Gerald [email protected] ACGT ProGenomics AG, Weinbergweg 22, D-06120 Halle (Saale), Germany2 Institute of Virology, Technical University of Munich, Biedersteinerstrasse 29, D-80802 Munich, Germany2005 4 10 2005 5 26 26 3 5 2005 4 10 2005 Copyright © 2005 Trieschmann 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 definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the post mortem detection of the pathological form of the prion protein (PrPSc) in brain tissue. Infectivity studies indicate that PrPSc may also be present in body fluids, even at presymptomatic stages of the disease, albeit at concentrations well below the detection limits of currently available analytical methods.
Results
We developed a highly sensitive method for detecting prion protein aggregates that takes advantage of kinetic differences between seeded and unseeded polymerization of prion protein monomers. Detection of the aggregates was carried out by flow cytometry. In the presence of prion seeds, the association of labelled recombinant PrP monomers in plasma and serum proceeds much more efficiently than in the absence of seeds. In a diagnostic model system, synthetic PrP aggregates were detected down to a concentration of approximately 10-8 nM [0.24 fg/ml]. A specific signal was detected in six out of six available serum samples from BSE-positive cattle.
Conclusion
We have developed a method based on seed-dependent PrP fibril formation that shows promising results in differentiating a small number of BSE-positive serum samples from healthy controls. This method may provide the basis for an ante mortem diagnostic test for prion diseases.
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Background
A group of fatal transmissible neurodegenerative diseases, including Creutzfeld-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) and scrapie, is caused by an unusual infectious agent that has been termed prion [1]. Prions consist of an aberrant isoform (PrPSc) of the normal cellular prion protein (PrPC). PrPC is a cell surface glycoprotein expressed in neurons [2] and other cell types [3,4]. The precise physiological function of the cellular prion protein is not known yet. PrPSc differs from PrPC in its higher content of β-sheet structure [5,6], its partial resistance to protease digestion [7], and its tendency to form large aggregates [8]. PrPSc propagates by converting the cellular prion protein to the PrPSc conformation [9]. PrPSc aggregates accumulate predominantly in the central nervous system (CNS), and definitive diagnosis of prion diseases currently relies on the post mortem detection of PrPSc in CNS tissue by immunohistochemistry, Western blotting, or ELISA [10]. Transmission studies indicate that prions may also be present in blood, potentially allowing for ante mortem diagnosis, but the sensitivity of the currently available analytical methods is insufficient for the detection of the extremely low prion titers that can be expected in body fluids [11].
Here, we report the development of a method based on kinetic differences between seeded and unseeded aggregation of prion protein that allows the detection of PrP aggregates in blood down to attomolar concentrations by flow cytometry.
Results and discussion
Detection of synthetic prion protein aggregates in serum or plasma
Kinetic differences between seeded and spontaneous polymerization of peptide monomers can be used for the detection of amyloid β-protein aggregates in the cerebrospinal fluid of Alzheimer's disease patients [15]. Here, we extend the principle of seeded polymerization to the detection of prion protein aggregates.
While trying to establish conditions for the labeling of synthetic prion protein aggregates with a fluorescently labeled prion protein probe, we observed that the formation of prion protein aggregates proceeds much less efficiently in serum or plasma (not shown) than in PBS (Fig. 1). This inhibition is probably caused by interactions of the prion protein probe with serum proteins.
Next, we found that the addition of preformed prion protein aggregates to plasma can partially overcome this inhibition (Fig. 2). The preformed aggregates presumably function as seeds that facilitate the formation of new aggregates in the inhibitory environment of plasma. The seeds stimulated the formation of prion protein aggregates at all concentrations tested, from 5 nM [120 ng/ml] to 10-8 nM [0.24 fg/ml] (Fig 2C). The average ratio of event counts in seeded samples to those in samples without seeds was 6.4. The number of events, however, was not proportional to the seed concentration, but remained relatively constant over the whole concentration range. Thus, the seed-dependent formation of prion protein aggregates can be used to detect extremely low amounts (down to the attomolar range) of spiked prion protein aggregates in blood.
Analysis of serum from clinical-stage, BSE-positive cattle
Studies demonstrating the transmission of prion diseases by blood transfusion suggest that prions are present in the blood of afflicted animals and people, even at pre-symptomatic stages of the disease [16-18]. We used the method of seed-dependent fibril formation to analyze serum from six confirmed cases of clinical-stage, BSE-positive cattle and four controls. Based on the spiking experiments described above, our hypothesis was that any PrPSc aggregates present in serum may act as seeds for the formation of easily detectable amounts of labeled PrP aggregates, whereas in the absence of seeds the formation of PrP aggregates would be inhibited. The serum samples from BSE-positive cattle and controls from healthy cattle were incubated with 10 nM of a FITC-labeled bovine PrP probe at 37°C for 20 h with continuous shaking, followed by analysis in a flow cytometer. All six BSE-samples could be clearly distinguished by a population of events that was absent in the controls (Fig. 3A–J, green dots in region R3; quantification in fig. 3K).
Conclusion
We have developed a method based on seed-dependent PrP fibril formation that shows promising results in differentiating a small number of BSE-positive serum samples from healthy controls. More samples need to be tested in order to validate its potential as an ante mortem diagnostic test for BSE and other prion diseases.
Methods
Biological fluids
Serum samples from six confirmed cases of BSE in cattle and four control animals were obtained from BFAV, Insel Riems, Germany. Control plasma was obtained from a blood bank.
Labeling of prion protein
Recombinant full-length bovine PrP was produced as described previously [12,13]. The purified protein was labeled with a FITC-labeling kit (Roche) according to the manufacturer's instructions.
Preparation of fibrils from recombinant prion protein
25 μM of unlabeled bovine prion protein in PBS containing 0.2 % SDS was incubated for 10 min at room temperature, followed by a twentyfold dilution with PBS. For fibril formation, the diluted reaction mixture was incubated for 48 h at room temperature [14].
PrP fibril formation in serum or plasma
Recombinant FITC-labeled bovine prion protein was incubated in 150 μl serum or plasma at a concentration of 5 or 10 nM for 5–10 min. at 20°C, shaking at 550 rpm in an Eppendorf thermomixer, followed by an increase of the temperature to 37°C h at constant shaking speed. The incubation was continued for 20 h. Samples were then analyzed by flow cytometry.
Flow cytometry
Analysis of the samples was carried out on a FACSVantage flow cytometer (BD Biosciences) at room temperature, measurement time was 30 sec per sample.
Authors' contributions
LT participated in the design of the study, carried out the measurements and drafted the manuscript. ANS participated in the analysis of the data. EM prepared the recombinant protein. KK and ST were also involved in protein expression and purification. HS participated in the design and coordination of the study. GB conceived of the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was kindly supported by grant No. 0312711A from the BMBF (Bundesministerium für Bildung und Forschung) in the context of the German National TSE Research Platform. The authors gratefully acknowledge the help of the TSE Research Platform, Munich, and the BFAV Riems, Germany, with respect to the kind gift of biological material in the context of this study.
Figures and Tables
Figure 1 Inhibition of PrP aggregation in serum. FITC-labeled recombinant bovine prion protein (concentration 10 nM) was incubated at 37°C for 20 h with continuous shaking, either in 150 μl PBS (left panel) or in the same volume of serum (right panel), followed by flow cytometry. The measurements are depicted in a Fluorescence 1 (FL1-H) vs. Fluorescence 2 (FL2-H) dot-plot. The number of counts in the area containing specific signals (R2) is given in the figures. Aggregate formation in serum is strongly inhibited.
Figure 2 Seed-dependent PrP aggregate formation in plasma. FITC-labeled recombinant prion protein (5 nM) was incubated in plasma as described in the methods section for 20 h either in the absence (panel A) or presence (panel B) of 10-8 nM PrP aggregates. Panel C: quantification of measurements shown in A and B, and of measurements (not shown) with different seed concentrations. The measurements are depicted in a Fluorescence 1 (FL1-H) vs. Side-Scatter (SSC) dot-plot. Aggregate formation (signal in region R1) was strongly enhanced by all seed concentrations tested, from 5 nM to 10-8 nM.
Figure 3 Analysis of serum from BSE-positive cattle. FITC-labeled recombinant prion protein (10 nM) was incubated in 150 μl of the serum samples as described in the methods section and analyzed by flow cytometry. The measurements are shown in a Fluorescence 1 (FL1-H) vs. Side-Scatter (SSC) dot-plot. All six BSE-samples (A-F) can be differentiated from the controls (G-J) by a population of events in region R3 (green dots). Panel K: Quantification of measurements shown in panels A-J.
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Kretzschmar HA Prusiner SB Stowring LE DeArmond SJ Scrapie prion proteins are synthesized in neurons Am J Pathol 1986 122 1 5 3079955
Cashman NR Loertscher R Nalbantoglu J Shaw I Kascsak RJ Bolton DC Bendheim PE Cellular isoform of the scrapie agent protein participates in lymphocyte activation Cell 1990 61 185 192 1969332 10.1016/0092-8674(90)90225-4
Manson J West JD Thomson V McBride P Kaufman MH Hope J The prion protein gene: a role in mouse embryogenesis? Development 1992 115 117 122 1353438
Pan KM Baldwin M Nguyen J Gasset M Serban A Groth D Mehlhorn I Huang Z Fletterick RJ Cohen FE Prusiner SB Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins Proc Natl Acad Sci U S A 1993 90 10962 10966 7902575
Caughey BW Dong A Bhat KS Ernst D Hayes SF Caughey WS Secondary structure analysis of the scrapie-associated protein PrP 27–30 in water by infrared spectroscopy Biochemistry 1991 30 7672 7680 1678278 10.1021/bi00245a003
Prusiner SB Groth DF Bolton DC Kent SB Hood LE Purification and structural studies of a major scrapie prion protein Cell 1984 38 127 134 6432339 10.1016/0092-8674(84)90533-6
Prusiner SB McKinley MP Bowman KA Bolton DC Bendheim PE Groth DF Glenner GG Scrapie prions aggregate to form amyloid-like birefringent rods Cell 1983 35 349 358 6418385 10.1016/0092-8674(83)90168-X
Prusiner SB Prions Proc Natl Acad Sci U S A 1998 95 13363 13383 9811807 10.1073/pnas.95.23.13363
Kretzschmar HA Ironside JW DeArmond SJ Tateishi J Diagnostic criteria for sporadic Creutzfeldt-Jakob disease Arch Neurol 1996 53 913 920 8815857
Brown P Cervenakova L Diringer H Blood infectivity and the prospects for a diagnostic screening test in Creutzfeldt-Jakob disease J Lab Clin Med 2001 137 5 13 11150018 10.1067/mlc.2001.111951
Proske D Gilch S Wopfner F Schätzl HM Winnacker EL Famulok M ion-protein-specific aptamer reduces PrPSc formation Chembiochem 2002 3 717 725 12203970 10.1002/1439-7633(20020802)3:8<717::AID-CBIC717>3.0.CO;2-C
Gilch S Wopfner F Renner-Muller I Kremmer E Bauer C Wolf E Brem G Groschup MH Schätzl HM Polyclonal anti-PrP auto-antibodies induced with dimeric PrP interfere efficiently with PrPSc propagation in prion-infected cells J Biol Chem 2003 278 18524 18531 12637572 10.1074/jbc.M210723200
Post K Pitschke M Schafer O Wille H Appel TR Kirsch D Mehlhorn I Serban H Prusiner SB Riesner D Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation Biol Chem 1998 379 1307 1317 9865603
Pitschke M Prior R Haupt M Riesner D Detection of single amyloid beta-protein aggregates in the cerebrospinal fluid of Alzheimer's patients by fluorescence correlation spectroscopy Nat Med 1998 4 832 834 9662376 10.1038/nm0798-832
Llewelyn CA Hewitt PE Knight RS Amar K Cousens S Mackenzie J Will RG Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion Lancet 2004 363 417 421 14962520 10.1016/S0140-6736(04)15486-X
Peden AH Head MW Ritchie DL Bell JE Ironside JW Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient Lancet 2004 364 527 529 15302196 10.1016/S0140-6736(04)16811-6
Hunter N Foster J Chong A McCutcheon S Parnham D Eaton S MacKenzie C Houston F Transmission of prion diseases by blood transfusion J Gen Virol 2002 83 2897 2905 12388826
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-5-271620215710.1186/1472-6750-5-27Methodology ArticleA system for precise analysis of transcription-regulating elements of immunoglobulin genes Cheng Emily Y [email protected] Cathy [email protected] Maribel [email protected] Marc J [email protected] Department of Immunology, University of Toronto, Toronto, Canada2 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada2005 4 10 2005 5 27 27 8 6 2005 4 10 2005 Copyright © 2005 Cheng 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
Precise analysis of expression-regulating elements, such as enhancers and insulators, requires that they be tested under reproducible, isogenic conditions. The commonly used methods of transfecting DNA into cell lines and selecting for drug resistance lack the requisite precision, as they yield cell lines in which varying numbers of gene copies have inserted at varying and undefined sites. By contrast, recombination-mediated cassette exchange (RMCE), by which a site-specific recombinase is used to place a single copy of a transgene at a constant chromosomal site of a cell line, offers the necessary precision. Although RMCE is generally applicable, many regulatory elements of interest are tissue-specific in their function and so require cell lines in the appropriate ontogenetic state.
Results
As reported here, we have used RMCE in a mouse B hybridoma cell line to establish a system with several additional advantages. To avoid the non-physiological features of prokaryotic DNA, this system uses the immunoglobulin μ heavy chain (IgH) gene from the hybridoma as the reporter. Expression can be measured simply by bulk culture assays (ELISA, Northern blot) and single cell assays (flow cytometry). Expression of the IgH reporter gene varies only 1.5 fold among independent transfectants, and expression is greatly (> 50 fold) increased by inclusion of the IgH intronic enhancer.
Conclusion
This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci.
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Background
Transcription-regulating elements such as enhancers, insulators and silencers are commonly detected by their effects on the expression of transfected genes, i.e., by comparing the expression obtained from transfected DNA that either bears or lacks a candidate DNA segment. Ideally, such comparisons would measure expression in a normal cellular environment and under circumstances in which the only variable is the structure of the transfected gene. However, the commonly used methods do not meet these criteria. Thus, in the case of "transient" transfections, expression is measured one or two days post transfection from extrachromosomal DNA, sometimes at very high copy number. In "stable" transfections, the transfected DNA typically inserts as an array of multiple copies; the insertions occur at undefined and irreproducible chromosomal sites, the copy number varies idiosyncratically, and the multiple copies are in both orientations. These features – insertion site, copy number, and orientation – can affect expression of the transfected DNA and obscure the analysis of regulatory elements. For example, independent transfectants bearing a gene for either the immunoglobulin μ or κ chain showed a 1000 fold range in expression [1,2]. This variation was probably due in part to the effects of neighboring elements at the insertion site [3]. However, the presence of multiple transgene copies in the array is also problematic. On the one hand, the enhancers might act multiplicatively and thus make a weak enhancer appear many fold stronger than reality. On the other hand, repeated copies of the transgene can induce gene silencing, thus leading to an underestimate of enhancer strength [4]. Also, because the array of transfected DNA contains transcription units in tandem and in both orientations, the enhancer lies both 5' and 3' of at least some promoters and in both orientations. This complexity has often obscured whether enhancers in fact function independently of their position and orientation. Finally, many reporter cassettes are derived from bacterial genes, and features such as the relatively high CpG content of non-vertebrate DNA might impose non-physiological requirements on expression.
To analyze regulatory elements in a reproducible (isogenic) context two methods have been used: homologous recombination (HR) and recombination-mediated cassette exchange (RMCE). Expression in such isogenic cell lines typically varies less than two fold [3,5-7]. Although HR has the important advantage that elements are assessed in the normal context, HR carries the disadvantage that the normal locus sometimes contains redundant or counteracting elements that obfuscate analysis. RMCE is useful for analyzing how a specific gene functions at an alternative site. To use RMCE, a selectable/counter-selectable cassette (the target cassette) flanked by site-specific recombination substrates (LoxP or FRT) is placed in the genome, generally at an undefined site [8]. A vector bearing a reporter cassette that is likewise flanked by recombination sites is then co-transfected with a vector expressing the cognate site-specific recombinase (Cre or Flp, respectively). In this way the target cassette is replaced with the reporter cassette, thus always placing the reporter cassette in the same genomic context.
In our earlier work using targeted recombinants to study the role of regulatory elements in the endogenous IgH locus of the mouse, our analysis was impeded by the presence of redundant enhancers [9]. RMCE offered the possibility of overcoming the redundancy problem, thus allowing individual activating elements to be characterized with precision. Because the enhancer and promoter of immunogolobulin genes require B cell-specific transcription factors, the existing RMCE-bearing cell lines could not be used. We therefore established an RMCE system in a mouse (hybridoma) B cell line. As reported here, this system yielded the expected reproducibility and thus allows sensitive, precise measurement of the effects of regulatory elements on gene expression. However, the system did present some unanticipated problems: The predominant product of the counter-selection was not a replacement, and even among the cells with replacements, a significant fraction had undergone additional changes that would obscure or confuse analysis. The additional tests and procedures that we developed to ensure analysis of only those transfectants with the desired replacement are also described here.
Results and discussion
The RMCE system described here made use of the Cre recombinase and its cognate LoxP sites and was based on a system developed previously [3]. In this system the selectable/counter-selectable marker was the HygR-TK fusion gene, which confers resistance to hygromycin (HygR) and sensitivity to gancyclovir (GanS). In the target vector, which is here denoted as pH/T (Fig. 1), the LoxP sites (denoted L1 and 1L) that flank the HygR-TK fusion gene are inverted with regard to each other in order to prevent Cre-mediated excision of the target cassette [3].
Construction of recipient cell line
As noted above, previous work indicated that the enhancer and promoter of immunoglobulin genes require B cell-specific transcription factors. For this reason we established RMCE in a derivative of the mouse B hybridoma cell line, Sp6, which expresses the immunoglobulin μ heavy chain and κ light chain genes at high level and assembles these chains into IgM specific for the hapten, trinitrophenyl (TNP). In designing a system for assessing regulatory elements in the immunoglobulin heavy chain (IgH) locus, we considered that it might be advantageous to use an immunoglobulin heavy chain gene as the reporter. Using the μ gene as a reporter required that the recipient cell line lack a functional μ gene. For this purpose we used a cell line, Z10, that was derived from the Sp6 hybridoma and had deleted the μ gene (see Materials and Methods). μ expression in Z10 could then be measured by ELISA (IgM), flow cytometry (intracellular μ) as well as by Northern blot of μ RNA.
In preparing a derivative of Z10 bearing the target cassette, we sought to minimize the non-physiological DNA that would adjoin the μ reporter and so excised the target cassette with enzymes that cut close to the LoxP sites. Previous work showed that ~40 nucleotides are often excised from the ends of transfected DNA [10], The target vector was therefore cut with Pvu II at sites 152 and 197 nucleotides outside the LoxP sites, with the expectation that cutting in this manner would usually result in transfectants which retained both LoxP sites. As described in Methods, DNA of the target vector was electroporated into the Z10 cell line at a relatively low concentration to reduce the occurrence of tandem or multiple insertions. Transfectants were selected in hygromycin and subcloned. We then confirmed that both LoxP sites were present, using PCR with primer pairs that flanked each of the two LoxP sites, and tested for single copy insertions by Southern blot (data not shown).
Construction of vectors bearing target and replacement cassettes
To prepare vectors bearing the reporter cassettes, a truncated μ gene from the Sp6 hybridoma was inserted between (inverted) LoxP (Fig. 1). In the vector pVOC the μ gene lacked all the intronic activating elements. The pVMEM'C vector was constructed by inserting the core enhancer (E) and flanking matrix attachment sites (M, M') into pVOC at their normal position. As described below, we used PCR to examine whether the μ gene reporter was intact after replacement. Because the switch region, with its numerous short repeats, could give a variable PCR product and thus interfere with the assessment of the reporter, both replacement vectors were constructed without the switch region.
As illustrated, the target and replacement vectors were identical outside the LoxP sites, except that the HinD III site shown to the right of the LoxP (1L) site in the pH/T vector was changed to a Nhe I site for use in the replacement vectors. This change allowed us to test simply whether the incoming μ gene had become linked to flanking sequences derived from the target vector. Because the two LoxP sites were in opposite orientations, the reporter cassette could recombine in both the "forward" and "reverse" orientations, as illustrated (Fig. 1c, d). The orientations were distinguished by PCR using different primer combinations (see below).
Several potential target cell lines, each bearing a single copy of the target cassette, were then tested for RMCE, i.e., they were co-transfected with the Cre expression vector and a replacement vector. GanR cells were selected and examined for the replacement cassette. Although none of the candidates yielded replacements with high frequency, one recipient, denoted Z10HyTK2-1, was generally better than the others and was selected for further work.
Another unexpected finding was that of 11 IgM-producing replacements obtained with Z10HyTK2-1, 10 were in the reverse orientation. We do not understand why one orientation was so strongly favored. However, our subsequent work indicated that expression of the μ gene in this orientation was enhancer-dependent and therefore suitable for analyzing enhancer function. The protocol described below was designed to detect replacements only in the reverse orientation and solely on the basis of DNA structure.
Introduction of the reporter cassette
The recipient cells bearing the target cassette were grown continually in hygromycin to eliminate cells that had spontaneously lost expression of the HygR-TK cassette. To introduce the replacement cassette, 107 cells were electroporated in the presence of 50 μg Cre-expression plasmid and 50 μg replacement plasmid. As indicated in Methods, the electroporated cells were divided among multiple flasks to obtain independent replacements. Cells were then incubated in normal medium for 5 days to allow the intracellular pool of HygR-TK RNA and protein to decrease sufficiently for cells to become resistant to gancyclovir.
Transfectants emerged as large colonies after ~9 days at a frequency of ~1–2 × 10-5 per surviving cell. DNA from these colonies was isolated and subjected to the following four tests to identify proper replacements. The fractions given below for colonies with the indicated features are based on results using several vectors of the same general structure as those shown in Figure 1.
a) Test for a μ gene in the reverse orientation. PCR using primers 1 & 7 detected colonies in which the cells had acquired the μ gene in the reverse orientation (~15% of the total number of GanR colonies, thus ~1 replacement per 106 surviving cells).
b) Test for μ gene replacement of the target cassette. The PCR product (primers 1 & 7) was incubated with HinD III and Nhe I to distinguish whether the μ gene had replaced the HygR-TK gene or inserted elsewhere in the genome. Cutting with only HinD III indicated that the only μ gene in the reverse orientation had replaced the target cassette; cutting with only Nhe I indicated that the only μ gene in the reverse orientation had randomly inserted; cutting with both enzymes indicated that both events had occurred. (4/48 of the μ-containing colonies had random insertions rather than replacements).
c) Test for intact μ gene. For those colonies with a replacement, the DNA was analyzed with two PCRs, using primer pairs 4 & 5 and 6 & 3. These PCRs generated two overlapping DNA segments spanning the entire replacement cassette and flanking LoxP sites. The two PCR products were digested with Xba I and Xba I + HinD III, respectively, and yielded fragments ranging in size from 0.140 kb to 2.9 kb. By comparing these fragments with the digestion products of the original vector we could detect even small alterations. (2/44 of the replacements had undergone a detectable change).
d) Test whether the colony also acquired a randomly inserted μ gene in the forward orientation. For colonies with an intact replacement cassette in the reverse orientation, the DNA was further analyzed by PCR using primer pairs 4 & 6 and 5 & 3. (8/47 of the replacements had an additional, randomly inserted μ gene in the forward orientation).
Analysis of μ expression
Using the foregoing protocol we isolated eight independent replacements with vector pVMEM'C and seven with pVOC. Expression of the μ gene was estimated with the IgM-specific ELISA, and the cell lines showing the highest and lowest level of IgM were then analyzed for μ mRNA by Northern blot (Fig. 2) and by flow cytometry of intracellular μ (Fig. 3). In the absence of the enhancer, expression of the μ gene was undetectable with each of the three assays. Each assay indicated that the single copy of the intronic enhancer increased expression of the μ gene by at least 50 fold. As quantified by Northern blot and by ELISA, the reproducibility in expression among independent replacements bearing the μ gene with the intronic enhancer was ~1.5 fold, which is similar to what was reported using this RMCE system and a β-galactosidase reporter in MEL cells [3]. Flow cytometry indicated that expression was homogeneous in the population.
The RNA measurements indicated that the μ gene bearing the intronic enhancer was expressed at ~8% of the level that it was expressed in the original Sp6 hybridoma. According to the measurements by ELISA and flow cytometry (geometric mean) expression of the μ gene in the replacement cassette was ~20% and 40%, respectively, of the endogenous locus. We consider that the Northern blots are the most accurate, as the quantification of the μ and κ RNA by phosphorimager was linear over a large range and normalizing with the μ/κ ratio eliminated variations in input. The difference in μ gene expression between the cassette and the endogenous locus suggests that the endogenous locus includes elements – perhaps the 3' enhancers – that contribute to endogenous expression and were lacking in the replacement cassette.
Conclusion
The potential advantages of site-specific recombination for constructing isogenic cells were reported many years ago [11,12], and several more refined systems involving the Cre/LoxP system of bacteriophage P1 and Flp/FRT system of yeast have been developed [8]. As reported here, we adapted one such system to a mouse hybridoma B cell line. The system yielded the expected reproducibility (~1.5 fold), and it was therefore a great improvement over other systems in which expression of the μ gene varies 1000 fold. However, using the RMCE system as described here demanded substantially more time and labor than other systems. The extra work was required for two general reasons. First, the frequency of replacement (~15% of the GanR cells) in our system was lower than what was found previously: > 90% for two lines derived from MEL cells; 10% and 50% for two lines derived from ES cells [3]. Second, our analysis revealed that in a significant fraction (10/47; ~20%) of the cells, the μ gene was not intact or there was an additional, ectopic insertion of the replacement vector. Identifying these cases required specific PCR tests.
Most measurements of the strength of the IgH and other enhancers have been made with transgene arrays, and, as noted in the Background, several features of the array have made it difficult to infer enhancer strength from these measurements. By contrast, our measurement of the effect of a single copy of the IgH intronic enhancer showed that this enhancer increases expression by at least 50 fold. This same system can likewise be used to measure the effectiveness of other regulatory elements – both B cell-specific and tissue-nonspecific – such as promoters and insulators. Inasmuch as this system is amenable to flow cytometry, it will also be useful for studying the molecular basis of variegated expression.
The fact that the reporter is a fully mammalian gene increases the relevance of the measurements. Moreover, because the reporter gene was derived from the endogenous gene of a hybridoma cell line and is expressed in a closely related cell line, the expression of the reporter and the endogenous μ genes can be compared directly to assess the effects of omitting or including individual elements of the endogenous locus in the reporter cassette.
Methods
RMCE vectors
The Cre expression vector and LoxP vector were obtained from E. Bouhassira [3]. The structure of the replacement vectors are described in the text and in Figure 1; the nucleotide sequences of the replacement and target vectors are given in the additional files 1–3.
Solutions
PCR lysis buffer contained 10 mM Tris, pH 8.0, 2.5 mM MgCl2, 50 mM KCl, 200 μg/ml gelatin. After autoclaving, this solution was made 0.45% NP40 and 0.45% Tween 20.
Primers
Numbers 1–7 correspond to the arrows in Figure 1b. Primer 9 was used in place of primer 4 in the initial screening of the target cell lines. Primers 8 and 10 were in the hygromycin-TK target cassette and were paired with primers 7 and 9.
1) (EC35) 5'-GTC TAC GAG GCA AGT GTT-3'
2) (EC68) 5'-AGA CAG TGA CCA GGA GAA GCA A-3'_
3) (CC1L) 5'-GCA CCC CAG GCT TTA CAC TTT ATG-3'
4) (CCL1) 5'-TAA AAC GAC GGC CAG TGA ATT CTC-3'
5) (CCM3) 5'-ATC ATG GAA AGC CAT CCC AAT GGC-3'
6) (CCM4) 5'-TCG TGG CCT ACA ACA CAG GTA TAG-3'
7) (EC69) 5'-AAC AGC TAT GAC CAT GAT TAC G-3'
8) (EC70) 5'-GGA GAT GGG GGA GGC TAA CTG A-3'
9) (EC71) 5'-GAG CCA TAA CTT CGT ATA ATG T-3'
10) (EC73) 5'-GGT CGT TGG GCG GTC AGC CAG G-3'
Isolation of recipient cell line Z10hytk2-1
The μ gene in the replacement vector was derived from the Sp6 hybridoma cell line, which secretes IgM(κ) specific for the hapten trinitrophenyl (TNP). A mutant of Sp6 lacking the μ gene but expressing the κ light chain was used to assay expression of a transfected μ gene. We planned originally to use a thymidine kinase (TK)-deficient mutant, as in this case bromodeoxyuridine (BrdU) might be used in place of gancyclovir to select replacements. To isolate such a mutant, the TK-deficient cell line igm692-R1, in which the gpt gene had been inserted 3' of the endogenous μ gene by gene targeting, was grown in thioxanthine to select Gpt-deficient cells and thus enrich for μ-deficient cells [6,13]. This enrichment was by itself insufficient, so the thioxanthine population was further enriched using the "suicide selection" for IgM deficient mutants [14]. Survivors of this enrichment were cloned and tested for colonies that lacked the μ gene. One such colony, denoted Z10, was used to create the recipient cell line bearing the target cassette. Thus, 1 μg DNA of the target vector was electroporated into 107 Z10 cells. HygR transfectants were selected in 400 μg/ml hygromycin and subcloned. We then tested whether both LoxP sites were present using PCR with primers that flanked the LoxP sites (primer pairs 7 & 8 and 9 & 10 for sites L1 and 1L, respectively). The positive colonies were then tested for single copy insertions by Southern blot. The colonies were not sensitive to BrdU, presumably because this compound is not a good substrate for the fusion protein, and gancyclovir was therefore used. One cell line, denoted Z10hytk2-1, had a comparatively low frequency of spontaneous GanR cells (1–5 × 10-5) and was used in further efforts to improve the protocol and for testing expression of μ replacements.
Transfection of replacement vector and selection of GanR colonies
The conditions for electroporation and preparation of cells have been described previously [7]. 107 recipient cells (Z10HyTK2-1) were washed and resuspended in 0.75 ml PBS and then electroporated with 50 μg Cre expression vector and 50 μg replacement vector, using two pulses at 700 v/0.4 cm and 25 μF. The cells were added to 50 ml normal media and divided among two flasks. Three such electroporations were used for each vector, thus yielding six independent replacements. Survival was measured the following day and was usually ~20%. Incubation in normal medium was continued for 5 days, diluting the cells as needed but ensuring that the cell count remained above 2 × 106/culture. On day 5, cells were plated at 5000 cells/200 μL/well in medium supplemented with 9 μM gancyclovir. On day 14 (9 days after plating) large colonies were evident. These large colonies appeared at a frequency of ~1 × 10-5/cell plated for the electroporated cells and ~2 × 10-6/cell plated for the control cells. Small colonies appeared in both electroporated and control plates at a frequency of ~2 × 10-4/cell plated. Replacements were found only for large colonies.
In an effort to increase the frequency of replacements among the GanR population of cells, we tried numerous variations in procedure: higher concentrations of hygromycin to select the cells with the target cassette; higher concentrations of gancyclovir to select replacements; different incubation protocols in normal and selective media in the periods before and after transfection. These variations had little, if any, effect.
Isolation of genomic DNA for PCR analyses
Cells (> 104) were diluted in 1 ml cold PBS and harvested by centrifugation for 1 min at 14000 RPM. The cell pellet was resuspended in 25 μL PCR lysis buffer containing 60 μg/ml proteinase K. This material was incubated for 1 hr at 56°. It was then incubated for 15 min at 95° to inactivate the proteinase K, cooled on ice and used for PCR immediately.
PCR conditions for testing
Presence of both LoxP sites (L1 and 1L) in the recipient cell line
This PCR was done with 1 U Jumpstart Taq (Sigma) in 20 μl Jumpstart buffer, including 0.8 mM dNTP's, 200 ng DNA, and 0.2 μM primers (primer pairs 7 & 8 for 1L; 9 & 10 for L1). Initial denaturation was 94° for 5 min, followed by 36 cycles of denaturation at 94° for 30 sec, annealing at 66° for 30 sec, extension at 72° for 30 sec, and a final extension at 72° for 10 min.
Presence of μ gene in forward orientation (Primers 2 & 7)
This PCR was done with 1 U Tsg+ polymerase in 20 μl Tsg buffer including 1.0 mM dNTP's, 100 ng DNA, and 0.2 μM primers (primer pair 2 & 7). Enzyme was added when samples reached 94° for "hot starts." Initial denaturation was at 94° for 3 min, followed by 30 cycles of denaturation at 94° for 30 sec, annealing at 68.5° for 30 sec, extension at 72° for 30 sec, and a final extension at 72° for 10 min.
Presence of μ gene in reverse orientation (Primers 1 & 7)
The 20 μl reaction mix in the Pfx amplification buffer contained 1U Platinum Pfx, 3X Platinum Pfx enhancer, 2 mM MgSO4, 2.4 mM dNTP's, 0.6 μM primers. The amplification protocol was an initial denaturation at 94° for 2 min, followed by 32 cycles with denaturation at 94° for 45 sec, annealing at 54° for 30 sec, extension at 68 for 30 sec.
Integrity of replacement in reverse orientation (Primers 4 & 5 and 6 & 7). Each reaction tube (25 μl) contained 2 U Roche Expand Long Template enzyme mix, buffer #3, 0.3 μM primers, 2.0 mM dNTP's, 300 ng genomic DNA. Following an initial incubation at 93° for 2 min, the protocol was 10 cycles of denaturation at 93° for 15 sec and extension at 68° for 10 min; for the next 25 cycles the extension time was increased by 20 sec/cycle, with a final extension for 7 min.
Presence of extra copy of μ gene (Primers 4 & 6 and 5 & 7). These PCR's used the same conditions as for primers 1 & 7, above.
Analysis of μ expression
ELISA's and Northern blots were done by standard methods. Flow cytometry of intracellular IgM has been described [7].
List of abbreviations
GanR, GanS, gancyclovir resistant, sensitive; HR, homologous recombination; HygR, hygromycin resistant; IgH, immunoglobulin heavy chain; RMCE, recombination-mediated cassette exchange; TNP, trinitrophenyl.
Authors' contributions
EC and MB constructed the vectors; EC isolated the mutant cell line; EC and CC isolated and analyzed the transfectants; MS provided advice and supervision.
Supplementary Material
Additional File 1
The additional (document) files denoted pH/T, pVMEM'C, and pVOC are the nucleotide sequences of the corresponding vectors.
Click here for file
Acknowledgements
This work was supported by grants from the Canadian Institutes for Health Research.
Figures and Tables
Figure 1 Structure of the target and reporter cassettes. In these drawings, the backbone of the vector is represented as a thin line, the cassettes as thick lines, with major exons as rectangles, the LoxP sites as triangles (L1 in the "forward" orientation, 1L in the "reverse" orientation). The three-stranded line represents the chromosomal DNA. The primer sites are indicated by the numbered arrows; numbers refer to the primers listed in Materials and Methods. The figure is not to scale. a) The vector (pH/T) bearing the target cassette. b) The vectors bearing the μ gene replacement cassettes. c) Replacement yielding the μ gene reporter in the forward orientation. d) Replacement yielding the μ gene reporter in the reverse orientation.
Figure 2 Analysis of μ expression by Northern blot. As described in the text, multiple independent replacements were isolated for each vector, and the concentration of IgM in culture supernatant of these cell lines was measured by ELISA. RNA from the cell lines with the highest and lowest IgM concentration was analyzed by Northern blot and probed with segments of the μ and κ genes. The intensity of the bands was quantified by phosphorimager, and the μ/κ ratio, normalized to the value for Sp6 hybridoma, is indicated below each lane. The IgM concentration for each cell line, also normalized to the value for Sp6, is listed below each lane.
Figure 3 Analysis of μ expression by flow cytometry. The indicated cell lines were stained for intracellular IgM using μ-specific, FITC-labeled antibody and analyzed by flow cytometry. Intensity of staining is represented on the horizontal axis, cell number on the vertical. The geometric mean (M) for the staining is noted in each panel.
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-5-291622131210.1186/1472-6750-5-29Methodology ArticleEfficient expression of transgenes in adult zebrafish by electroporation Rambabu K Murali [email protected] S Hari Narayana [email protected] N Madhusudhana [email protected] Center for Cellular and Molecular Biology, Hyderabad-500 007, India2 Reliance Life Sciences Pvt. Ltd. Fosbery Road, Sewree, Mumbai 400 033, India2005 13 10 2005 5 29 29 3 7 2005 13 10 2005 Copyright © 2005 Rambabu 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
Expression of transgenes in muscle by injection of naked DNA is widely practiced. Application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. Zebrafish is a precious model to investigate developmental biology in vertebrates. In this study we investigated the effect of electroporation on expression of transgenes in 3–6 month old adult zebrafish.
Results
Electroporation parameters such as number of pulses, voltage and amount of plasmid DNA were optimized and it was found that 6 pulses of 40 V·cm-1 at 15 μg of plasmid DNA per fish increased the luciferase expression 10-fold compared to controls. Similar enhancement in transgene expression was also observed in Indian carp (Labeo rohita). To establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in A2 cells and adult zebrafish after electroporation. The relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. GFP fluorescence in tissues after electroporation was also studied by fluorescence microscopy.
Conclusion
Electroporation after DNA injection enhances gene expression 10-fold in adult zebrafish. Electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. Enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish.
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Background
In vivo gene delivery into skeletal or cardiac muscle by direct injection of naked DNA is a convenient method to express proteins [1,2]. The efficiency of this procedure was improved substantially by applying electrical pulses at the site of injection [3,4]. Electroporation enhances transgene expression in tissues by causing electrical breakdown of membranes combined with electrophoresis of DNA into cells [5]. Since its discovery, electrotransfer of DNA into muscle has become a very popular method of gene delivery due to easy access of the muscle tissue, long life span of the muscle cell, abundant blood supply and its suitability for the production of proteins as systemic therapeutic agents [4]. Electroporation is routinely applied to a portion of the muscle, leading to transfection of cell layers around the site of injection. Recently, in ovo or in utero electroporation into the neural tubes and electroporation in tissue explants or solid tumor has provided promising results [1,5].
Zebrafish has proven to be a useful vertebrate complement to the mouse [6]. Easy husbandry, large supply of eggs and transparent early stages of development make zebrafish a convenient model organism to study vertebrate development [7]. Transgenic zebrafish have been routinely produced to understand the gene function by injecting the transgene into the eggs and following its expression in cell lineages [8]. Methods for large-scale generation of transgenic zebrafish were explored [9]. Electroporation was successfully demonstrated on dechorinated eggs of three fish species [10]. Recently, electroporation of the neural tube of the embryo was demonstrated to investigate gene regulation in later stages of zebrafish development [11]. Methods to express transgenes in adult fish are recently being investigated. Muscular injection of naked DNA, particle bombardment using gene gun and electroporation of the fish fin [12,13](.(are two reports on transfection methods applied to adult fish. We attempted electroporation in fully developed zebrafish (>3 months old) after DNA injection into the muscle.
Electroporation using forceps electrodes placed on either side of the body of a fish resulted in substantial enhancement of reporter gene expression.
Results and discussion
Microinjection of naked DNA into the fish muscle was sufficient for expression of transgene in muscle cells [12,14,15]. To improve the efficiency of transgene expression further, we have attempted electroporation subsequent to muscle injection. Tweezer type electrodes were used to apply pulses by holding the sides of the fish between the electrode faces. Electroporation parameters, such as number of pulses, pulse duration, voltage strength and shape of the pulse have critical bearing on the transfection efficiency [5]. Each of these parameters is required to be optimized for each tissue since the biochemical and physical disposition of tissues to electroporation is known to be different [5]. In all our experiments, square pulses of 60-ms duration were used. Initially we monitored the dependence of number of pulses on reporter gene expression at a fixed voltage of 40 V·cm-1. Luciferase activity was maximum at 6 pulses and decreased at lesser or higher number of pulses. The maximal expression of reporter gene was nearly ten fold higher than that obtained without electroporation (Fig. 1A). Next, we investigated the effect of voltage on transgene expression by keeping the number of pulses constant. Voltage applied at 40 V·cm-1 was found to maximize the expression of reporter gene with 6 pulses (Fig. 1B). The bell shaped dependency of reporter gene expression on number of pulses and voltage applied suggests that there is a significant influence of these parameters on cell entry of DNA.
In addition to the electroporation parameters, the amount of plasmid DNA used for electroporation is known to have strong bearing on the reporter gene expression [16,17]. At 10 μg of plasmid DNA per fish, we see significant increase in reporter gene expression on electroporation and above 10 μg of plasmid DNA there is no further change in the gene expression (Fig. 1C). Below 10 μg of plasmid DNA, we could not detect luciferase activity. In an earlier report muscle injection of plasmid DNA without electroporation showed maximum expression at 5 μg of DNA into adult zebrafish [14]. We have also tested these electroporation conditions on another fish species, Indian Carp (Labeo rohita). Six pulses at 40 V·cm-1 increased the reporter gene expression in the Indian carp by more than eight fold (significant at p < 0.01) indicating that electroporation enhances reporter gene activities in the Indian carp also (Fig. 1D).
Transient gene expression from different promoters in zebrafish (in vivo) and in A2 cell lines (in vitro)
To compare the strength of promoters on reporter gene expression in adult zebrafish and cell lines, we have constructed three plasmids each containing CMV (pCMV-Luc) or human EF-1α (pBOS-Luc) or Xenopus EF-1α (pESG-Luc) promoter with luciferase as the reporter gene. Strength of promoters was tested in A2 cell lines after transfecting the cells with each of the plasmids using commercial lipofecting agent, Lipofectamine (Table 1). pCMV-Luc and pESG-Luc showed equal reporter gene activity in A2 cells and it was 10-fold higher than pBOS-Luc. We have estimated the reporter gene activity under the control of these three promoters in adult zebrafish using electroporation conditions described earlier i.e., 6 pulses of 60 ms duration at 40 v·cm-1 with 10 μg of plasmid (Table 1, n = 5). We plotted reporter gene activities obtained with the three plasmids in adult zebrafish along with the activities obtained at 1:1 charge ratio in A2 cells lines. The relative strengths of the promoter were similar in zebrafish and in A2 cell lines. In zebrafish CMV and Xenopus EF-1α promoters showed 2–4 fold higher reporter gene activity compared to human EF-1α promoter (Table 1). EF-1α promoter derived from an amphibian demonstrates higher strengths for expression than the human homologue in zebrafish. However, among the three promoters the folds increase in luciferase expression with human EF-1α promoter seems to be stronger in zebrafish than A2 cell lines. In an earlier study on transient expression by naked DNA injection into the muscle of adult zebrafish, CMV promoter was shown to be more efficient than SV 40 promoter. In the same study the expression observed in muscle tissue of adult zebrafish was similar to the expression in RTH-149 and RTG-2 cell lines [14]. These experiments suggest that in vivo transient gene expression using electroporation in adult zebrafish would be a convenient way to study the strengths of promoters.
Expression of GFP in zebrafish on electroporation
We investigated the histological expression of GFP upon electroporation of pCMVGFP in zebrafish. The fish were killed two days after injection and the frozen section of the mid-, head- and tail region were taken (as shown in Fig. 2). In the absence of electroporation, green fluorescence was faintly visible in the tissue at the site of injection (Fig. 2A). Fluorescence was undetectable in control fish, which did not receive any injection. Significant GFP fluorescence was observed at the site of injection (mid-region) upon electroporation (Fig. 2C). In addition to the mid-region we could also observe GFP fluorescence, however less extensive, in tail and head region of the fish. We have estimated the number of GFP positive cells from the phase contrast and nuclear stained (DAPI) images of the same sections. Based on 50–70 cells per section and sections from three different fish, we observed 8%, 10% and 6% GFP positive cells in head-, mid- and tail-region, respectively, in non-electroporated fish. The GFP positive cells increased to 15%, 70% and 25% in head, mid- and tail-region, respectively, upon electroporation. Higher expression of GFP in electroporated samples confirms the enhanced luciferase activities observed in Fig. 1. In a related study, particle bombardment in adult zebrafish resulted in GFP expression was observed in skin epithelial cells, muscle cells, neuron-like cells etc., whereas muscle injection resulted in transfection of several muscle bundles [12].
Appearance of fluorescence far from the site of injection is intriguing. It may be possible that immediately after the injection the DNA accesses other tissues before the application of pulses. We observed that average time between muscle injection and electroporation was 20 s. Electroporation affects the permeability of cells away from the site of injection and thereby enhances transgene expression in those tissues. In addition the diameter of the disc on the tweezer type electrode is 7 mm may electroporate a larger area of the fish. Sudha et.al reported expression of GFP in non-muscle cells when the injections were made in the muscle [12].
Electroporation in zebrafish was used extensively on eggs and was also used for the functional analysis of the role of developmental genes in the neural tube of zebrafish embryo and larvae [11,18]. Electroporation was employed on the dechorinated zebrafish eggs and nearly 25% of fish showed transgene expression [9]. Muscle injection of DNA and particle bombardment using gene gun were attempted in adult zebrafish and persistent GFP expression for 50 days was demonstrated [12]. Electroporation of fins is the only one reported case of electroporation in adult zebrafish. Fins were electroporated to deliver plasmid carrying gene for GFP and also used as a method to disrupt Fgf signaling pathway during the process of regeneration [13]. Enhanced transgene expression upon electroporation in adult zebrafish would be useful in expression of proteins in the muscle and also in investigating tissue-specific expression of promoters and DNA vaccination.
Conclusion
Simplicity of muscular injection and application of electroporation on the zebrafish offers a protocol to achieve high expression of transgenes. Low voltage conditions, availability of commercial tweezer electrodes and safety of the procedure on fish allows to test the in vivo properties of regulatory gene sequences. In addition to muscular injection of DNA, electroporation would require an additional minute to administer. Observed correlation between in vivo (zebrafish) and in vitro (A2 cell line) transfection efficiencies with three plasmid constructs suggests that in vivo transfections could be routinely performed in zebrafish. High level of gene expression in adult zebrafish may offer a simple experimental system for functional analysis of gene sequences in zebrafish.
Methods
Fish
Adult zebra fish (about 3–5 months old), purchased from a local fish farm, was maintained as per the guidelines given in the Zebra fish Book (Wethersfield, 1994).
Materials and methods
Procedures for cloning and purification of plasmids were followed as reported in manuals [19]. pCMV-Luc was a gift from Dr. Robert Debs. Dr. Suresh Kumar of National University of Singapore provided plasmids pESG and pBOS-H2b/GFP. Luciferase gene from pGL3-Luc (Promega) was excised using appropriate restriction enzymes (Hind III/Xba I) and inserted into appropriately cut pESG to make pESG-Luc. We made pBOS-Luc from pBOS-H2b/GFP by excising GFP using EcoRI/NotI sites and inserting appropriately cut luciferase gene from pESG-Luc.
Cell culture
A2 cells (from Xiphophorus xiphidium) were grown at 28°C in DMEM medium (Sigma Co., USA), containing 15% fetal bovine serum, penicillin (50 μg/ml) streptomycin (60 μg/ml) and Kanamycin (100 μg/ml), in 5% CO2 environment and were propagated by transferring into fresh medium after every 48 h. A2 cells were plated in 96-well plate and allowed to reach a confluency of 60% – 70% at the time of transfection. To form a transfection complex Lipofectamine and plasmid were incubated for 30 min in serum free DMEM. Lipid:DNA complexes were incubated with cells for about 3 h, after which the medium was replaced with 10% serum containing DMEM medium. Cells were incubated further 24 h before estimating the reporter gene activity. After 24 h medium was removed and cells were washed with PBS and lysed with 50 μl lysis buffer (250 mM Tris-Cl pH 8.0 containing 0.5% NP40) for 10 min. at room temperature. 5 μl of cell lysate was used for protein estimation by modified Lowry's method [20]). Luciferase activity was monitored as per the instructions provided in the kit supplied by Promega and the light counts were recorded using a luminometer (LUMAC Biocounter M2000). We assayed reporter gene activity with these three plasmids in A2 cell line at various charge ratios of lipid to DNA. With all the three constructs, the maximum reporter gene expression was obtained at a charge ratio of 1:1 and reporter gene activity decreased either on increase or decrease of charge ratio from 1:1 (Data not shown). Such charge-ratio dependent reporter gene activity profiles have routinely been reported for several cell lines and transfecting agents [21].
DNA injection and electroporation in zebrafish
Tweezer type electrodes (Tweezertrodes, 0.7 cm large 2 cm long, BTX, USA) were used to electroporate DNA into the mid-region of adult zebrafish. We have tested several types of needle, both metal and glass, for leak proof delivery of small volumes of DNA sample. Syringe needles more than 26 gauge were good for injection but resulted in occasional leak of the solutions, and hence discontinued. Borosilicate capillaries with Kwik-Fil feature (OD/ID, mm – 1.0/0.75, WPI, UK) drawn into fine tips using a needle puller (Narishige Model PC-10) were used in this study. The volume of the capillaries was calibrated by using a concentrated blue dye (Blue dextran) and the optical density of the solution was measured by a spectrophotometer. Suction and delivery of DNA solution was performed by applying pressure through mouth using a tube. The fishes were briefly taken out of the water and were given an intra-muscular injection of approximately 20 μl in the mid dorsal region on one flank of the fish. The fish were restrained on a soft wedge and held softly with the tweezer type electrodes for electroporation. Immediately after injection, within 20 s, electrical pulses were delivered to the fish by holding it between the tweezer electrodes using electric pulse generator (Electro Square porator ECM 830, BTX, San Diego, CA). The entire operation of injection and electroporation was completed in less than 45 sec. The fishes were immediately released into water tanks containing 5 μg/ml gentamycin to avoid bacterial infections.
Injection of DNA alone did not cause any mortality in fish; however, upon electroporation the mortality was between 10–20%. Mortality, estimated from the surviving fish population after 48 h, was dependent on the voltage applied. The mortality was observed to be 6%, 15 %, 25% and 30% at 20, 40, 60 and 80 V·cm-1 respectively. For each data point ten fishes were used. After accounting for the mortality, each point in the graph represents data from at least six fishes. t-test was performed between the adjacent values in each of the figures and we found that except for the reporter gene expression at 2 and 4 pulses in Fig. 1A all values were statistically significant at p < 0.001 when compared with the adjacent values. The fish were monitored for any morbidity or death for two days. After two days the fish were killed by decapitation. The mid part of the fish was removed and homogenized in lysis buffer (Tris.phosphate, pH7. 8 25 mM; DTT, 2 mM; 1,2-diaminocyclohexane N,N,N',N'-tetracetic acid, 2 mM; glycerol, 10% and Triton X-100, 1%). Luciferase activity was monitored in the lysates as described above.
Expression of GFP in fish tissues
Zebrafish was injected with 10 μg of pCMVGFP and electroporated by applying 6 pulses and 40 V-1·cm. After two days the longitudinal cryosections of 7 μm thickness were made from the muscle tissue in which DNA was injected. The sections were placed on gelatin-coated slides and were examined under blue light (490 nm) using a Zeiss Axiovison fluorescent microscope, and photomicrographs were taken a Contax 16MT camera. Zebrafish. The injection was given at mid-dorsal region approximately at 60% of the total length from the head. The sections for the head region and tail region were taken at least 1 cm away on either side from the point of injection.
Authors' contributions
KMR has made the plasmid constructs and estimated the reporter gene activities. SHR has established the zebrafish culture and performed the injections. NMR has designed the electroporation experiments and drafted the manuscript.
Acknowledgements
The project was supported by a grant from Department of Biotechnology, India (BT/AAQ/03). The authors thank Dr. Kshitish Majumdar for his support and suggestions in the work. We are grateful to acknowledge the facilities and advise of Dr. Satish Kumar in electroporation experiments.
Figures and Tables
Figure 1 Influence of various parameters of electroporation on reporter gene activities in adult zebrafish and Indian Carp. Electrical pulses were applied following intra-muscular injection of plasmid DNA: A, Pulse number; B, Voltage strength; C, Amount of plasmid DNA present in fixed volume with (open circles) and without (closed circles) electroporation. D, Effect of electroporation (40 V·cm-1 and 6 pulses) on 2–3 month old Indian carp. Controls received only the plasmid in injection. Relative luminescence units were normalized for the amount of protein. Each data point is an average of values obtained from 6–8 fish and each experiment was repeated three times.
Figure 2 Effect of electroporation on GFP expression in zebrafish. Fluorescence images from control (A) and electroporated samples (B, C and D). (A) is an image taken from the tissue at the site of injection. B, C and D represent the images obtained from head, mid-region and tail respectively. Fluorescence from tissue sections from head and tail of control fish is similar to (A), hence not shown. At least 20 fish were electroporated in different days with pCMVGFP. We observe some variation in extent of GFP expression but the expression was always maximum in the mid-region. The presented data is from one fish.
Table 1 Luciferase expression from three promoters in adult zebrafish after electroporation and in A2 cell lines transfected with Lipofectamine
Plasmid Relative luminescent counts per mg (Mean ± coef. variation)
Zebrafish (in vivo) A2 cell line (in vitro)
pCMV 599 ± 18 5.50 × 104 ± 11
pESG 1020 ± 11 4.4 × 104 ± 11
pBOS 234 ± 47 6 × 103 ± 3
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Ogura T In vivo electroporation: a new frontier for gene delivery and embryology Differentiation 2002 70 163 171 12147136 10.1046/j.1432-0436.2002.700406.x
Sambrook J Fritsch EF Maniatis T Molecular Cloning A Laboratory Manual 1989 2 Cold Spring Harbor Laboratory Press
Markwell MA Haas SM Tolbert NE Bieber LL Protein determination in membrane and lipoprotein samples: manual and automated procedures Methods Enzymol 1981 72 296 303 6796803
Miller AD Cationic Liposomes for Gene Therapy Angewandte Chemie International Edition 1998 37 1768 1785 10.1002/(SICI)1521-3773(19980803)37:13/14<1768::AID-ANIE1768>3.0.CO;2-4
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Clin Pract Epidemiol Ment HealthClinical Practice and Epidemiology in Mental Health : CP & EMH1745-0179BioMed Central 1745-0179-1-171618536410.1186/1745-0179-1-17ResearchLong-term consequences of traumatic experiences: an assessment of former political detainees in romania Bichescu Dana [email protected] Maggie [email protected] Evangelia [email protected] Adrian [email protected] Thomas [email protected] Frank [email protected] Department of Psychology, University of Konstanz, Fach D-25, D-78457 Konstanz, Germany2 Department of Psychology, University of Iasi, Blvd. Carol I, Nr. 11, Iasi, 700506 Romania3 vivo, Cassela Postale no. 17, Castelplanio Stazione, I-60032 Ancona, Italy2005 26 9 2005 1 17 17 27 6 2005 26 9 2005 Copyright ©2005 Bichescu et al; licensee BioMed Central Ltd.2005Bichescu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Research has suggested that organized violence and torture have long-term psychological effects that persist throughout the lifespan. The present survey aimed at examining the prevalence of posttraumatic stress disorder (PTSD), and other disorders and symptoms, all present in old age, as long-term consequences of politically motivated violence in a comparison design.
Methods
A group of former political detainees (N = 59, mean age 73.5 years) who had been arrested by the Romanian communist regime were compared to an age- and gender-matched control group (N = 39). PTSD was assessed using a structured clinical interview (CIDI). The investigation of the clinical profile was further accomplished by self-rating measures for anxiety, depression, and health-related functioning, as well as by clinician-administrated interviews for substance abuse, dissociation, and somatization symptoms.
Results
Lifetime prevalence of PTSD was 54%. In the case of participants left untreated, PTSD persisted, often over four decades, such that current PTSD was diagnosed still in a third of the survivors. Other clinical conditions such as somatization, substance abuse, dissociative disorders, and major depression were also common among the former political detainees and often associated with current PTSD.
Conclusion
Our findings suggest that political detention may have long-term psychological consequences that outlast the changes in the political system.
==== Body
Background
Research on victims of organized violence and torture has suggested that prolonged severe traumata produce long-term psychological effects that persist even into old age. Many comparative epidemiological studies have been conducted to investigate the long-term consequences of organized violence, but there is still a lack of information regarding psychological and physical status of the survivors in their late life. In particular, there is little information about the consequences of subjection to political persecution in the formerly communist countries of Eastern Europe. The extensive review of empirical support for the co-occurrence of complex symptoms led to the conclusion that this was mainly the consequence of prolonged, repeated trauma that usually occurred during captivity, such as in prisons, concentration camps, or labor camps [1]. It has been argued that traumatized individuals may suffer from various combinations of symptoms over time and that this pattern of symptoms reflects the adaptation to psychological trauma on the cognitive, affective and behavioral levels, particularly when this has occurred early in life [2].
Information about the persistence of posttraumatic sequelae into old age comes mostly from investigations on Holocaust survivors and on POW-s in World War II. Irrespective of the particular type of assessment used to determine mental health consequences, most studies have found that survivors suffered from long-lasting impairment even later in life [3]. In addition, more recent studies that applied the diagnostic criteria for PTSD reported the persistence of posttraumatic symptoms and high rates of related co-morbid disorders in many victims who developed PTSD in the aftermath of the trauma, for 40–50 years after the end of World War II and up into old age [4-10]. Prospective research has also focused on the late effects of combat exposure, showing relatively few PTSD symptoms in survivors [11].
However, these results may be influenced by the participants' selection procedures. Earlier clinical investigations of Holocaust consequences late in life have also underlined an increase in the severity of depressive and somatic symptoms as an outcome of the unsuccessful emotional processing of the trauma [12]. These data have lately been sustained by other studies, which have suggested that late life might be a period of increased vulnerability in the aftermath of severe trauma [10,13].
Research concerning posttraumatic consequences of organized violence has lately focused mainly on refugees. In addition to the very relevant studies on refugees, the importance of research on victims of organized violence who continued to live in their countries of origin has been emphasized [14], as the only way in which the effects of organized violence could be separated from the ones of living in exile. More recently, increasing attention has been paid to psychological disturbances in the aftermath of political imprisonment specific to former East Germany [15-17]. Long-term consequences such as PTSD, anxiety, depression, dissociation and vegetative complaints have been found.
In countries with a history of dictatorship, terror, and institutionalized violence, both political opponents and arbitrary victims fell prey to severe abuse. In Romania, political imprisonment and deportation procedures, commonly used under King Carol's dictatorship and during the Nazi era, were reinforced beginning with August 23, 1944, when Romania started the war against Germany and the Communists came to power under the supervision of the Soviet state which had administrative control over Romania at that time. Whereas the earlier political coercion had been directed against Jews and individual political opponents, under the Communist regime it extended to the whole Romanian society, becoming part of everyday life and generating pervasive fear and terror, which affected social structures as well as individual behaviour [18]. Between 1944 and 1947, most legionary members and political personalities were imprisoned, executed or expatriated. The information regarding detainment forms during this period is scarce and contradictory [19]. On April 13, 1948, a constitution of the Soviet type was adopted. Thousands of people were arrested during the following days, including members of the Communist Party that were not "clean". To liquidate the regime's opponents, the secret police, i.e. a new instrument of repression with unlimited power, was created. The juridical and economic systems were reformed, along with the nationalization of the private properties. Any kind of behaviour that did not meet the rules set by the regime was violently repressed [20], and the Gulag-style system of forced-labor camps and political prisons resulted in a high number of deaths. Beginning with 1949, the imprisonment regime became much harsher: the prisoners were unmercifully beaten, they were not allowed to lie down during the day, and food was very little and bad [19]. Beginning with 1950, the system of forced-labor camps was established. Testimonies say that political dissidents, opponents to the new system, and other arbitrary victims were subjected to extermination practices such as forced labor, starvation, and torture (e.g. [21]). Another characteristic of the Communist system of political detainment was the regular transfer from one prison to another, so as to prevent any type of contact among the detainees, make them loose contact with the outside world, or for administrative reasons, e.g. need of working force in certain labor camps [22,23]. During this period, after further ulterior Communist propaganda, denunciation flourished and numerous sudden changes in political orientation occurred [23]. Since the change of political leadership in 1965, a mitigation of extreme policy stated that the political detention regime be officially ended. The following period, until 1989, was mainly characterized by other means of control, repression and censorship, during which the "memory of terror" ensured the political systems' efficient functioning, and political imprisonment was seldom used, being disguised under the procedures applied to common convicts [22,23].
The present study documents a clinical psychological investigation that was conducted in Romania on a sample of Romanian former political detainees now in their late sixties and seventies. A control group of elderly people that have not undergone political persecution served to explore the cause of the symptoms. Our aims were the following: (1) to explore the severity of traumatic conditions during detention; (2) to investigate the range of PTSD, the health-related functioning, anxiety, depression, substance abuse, somatization, and dissociation within this population in a controlled comparison; (3) to analyze the interrelationships between PTSD diagnosis and the clinical profile of the participants; (4) to compare clinical conditions between the former political prisoners who received psychological support and those who did not.
Methods
Participants
The study design included two groups: a political detainee group and a control group. The group of political detainees consisted of N = 59 (1 female) Romanian survivors of the Communist political imprisonment practice and persecution, who had been political activists or just arbitrary victims. Regardless of their political affiliation, time of imprisonment, or actual need for help, participants in this group were Romanian citizens arrested after 1948, according to the decision of the Military Court, some arrest warrant, the administrative order given by the Ministry of Internal Affairs or even without any written order, simply by means of the abuse of the police state organs. All possessed a legal status as former political prisoners, according to documents that prove their imprisonment. Study announcements were made at the Romanian Association of Former Political Detainees (AFDPR) in Iasi, Suceava, Botosani, and Brasov, an anti-Communist organization militating for the rights of the former political prisoners, and at the Medical Rehabilitation Clinic (MRCT) in Iasi, a branch of the IRCT (International Rehabilitation Council for Torture Victims) in Moldavia, which offers medical and psychological support for victims of political persecution and torture. The psychological support sessions were conducted by a psychiatrist at the clinic headquarters, and consisted of a regular group treatment procedure in which the participants were able to share their stressful experiences and to discuss their problems. From the 83 former political detainees (71 male and 12 female) who were approached and invited to participate, after informed consent, 58 (82%) of the men and 1 (8%) of the women agreed to enter the study. We have no explicit information on the reasons for non-participation. According to the employees of the two institutions, many women had been sexually abused during prison and they might not be willing to report such experiences, which is consistent with the analysis of written testimonies on this topic [23]. Twenty-eight of the participants were responders to the announcements at AFDPR and 31 of them responded to the announcements at MRCT. Participating survivors who were recruited from the MRCT received current medical and psychological assistance, as mentioned above. Our sample was representative in terms of age distribution, education and occupational status of the former political prisoners registered at the Association of Former Political Detainees and at the Medical Rehabilitation Clinic.
The control group (N = 39, 3 female) was a general population sample recruited from a city clinic where mostly old age pensioners from Moldavia were under treatment for rheumatoid and arthritic problems. These persons had responded to the hospital's employees' announcements for participants in the research study. From the control group, one participant reported a history of torture and four and a half years of political imprisonment between 1980–1985, because of his origins (his parents had been political opponents arrested during the Communist regime), another reported ordinary conviction to two-and-a-half- month imprisonment due to his refusal to become a member of the Communist party, and a third one reported having been persecuted because he had studied abroad. With these few exceptions, in the other cases of control subjects, their life during the Communist era had had a normal course, without interferences of the state authorities. For matching purposes, exclusion criteria were age and gender.
Overall, the two groups were comparable in terms of institutional social contacts, most socidemographic characteristics, WWII veterans, psychiatric history of the family, former psychiatric illness, and traumatic experiences other than those related to political imprisonment and persecution (Table 1). The mean age of the group of former political detainees was 73.5 years (range 60–87, SD ± 6.9), and the mean age for the control group was 71.4 years (range 59–88, SD ± 7.0). Compared to the control group, former political detainees had reached a higher educational level. Among the traumatic experiences not related to political imprisonment, war-zone exposure, accidents, exposure to natural disasters (e.g., earthquake, storm, flood, fire), witnessing trauma, physical assault, traumatic events involving a close relative or friend were reported. Only one case of sexual abuse was recorded. A significant higher percent of former political detainees in comparison with controls reported physical assault. All participants signed an informed consent containing a detailed description of the research purposes. The study was approved by the Konstanz University Ethical Review. The interviews were carried out in July–October 2001 and in August–November 2002 at the subsidiaries of the institutions participants had been recruited from.
Table 1 Sociodemographic and other characteristics of former political detainees and control subjects1
Variables Political detainees (n = 59) Control subjects (n = 39) p
Gender n.s.
Male 98% (58) 92% (36)
Female 2% (1) 8% (3)
Highest education level < 0.001
Primary school 0% (0) 8% (3)
Secondary school 2% (1) 28% (11)
High school 25% (15) 28% (11)
Technical college 15% (9) 10% (4)
University 58% (34) 26% (10)
Marital status n.s
Single 3% (2) 0% (0)
Married 83% (49) 87% (34)
Divorced 2% (1) 3% (1)
Widowed 12% (7) 10% (4)
Occupational status n.s.
Employed 14% (8) 23% (9)
Retired 86% (51) 77% (30)
WWII veterans 17% (10) 18% (7) n.s.
Psychiatric history of the family 14% (8) 18% (7) n.s.
Former psychiatric illness 3% (2) 10% (4) n.s.
Other traumas 3.4 (1.5) 2.8 (1.3) n.s.
War zone exposure 86% (51) 82% (32) n.s.
Accident 61% (36) 59% (23) n.s.
Natural disaster 46% (27) 49% (19) n.s.
Witnessing injury 31% (18) 23% (9) n.s.
Sexual abuse 2% (1) 0% (0) n.s.
Physical assault 59% (35) 21% (18) < 0.001
Trauma that happened to someone close 56% (33) 44% (17) n.s.
1Frequencies for percentages and standard deviations for means are presented in parentheses
Instruments and Procedure
An extensive evaluation of the psychological status for all participants was conducted by self-report and diagnostic clinical interview measures. A clinical psychologist and two master-degree students in psychology who had been specifically trained in the use of diagnostic interviews carried out assessments. A translation/back-translation technique concerning contents, criteria, semantic, notional and procedural correspondence was used to develop Romanian versions of the instruments [25], except for the cases in which adapted and validated Romanian versions were available, e.g., Mini Mental State Inventory (MMSI) and Beck Depression Inventory (BDI).
The Assisted Self-Report Section
Every subject completed a series of self-report surveys with the assistance of an interviewer. The next sequence for the completion of these items was the following:
(1) Demographic characteristics of the participants were recorded by the interviewer.
(2) Mini Mental State Inventory (MMSI; [26]). Based on the evaluation of mental functioning, subjects presenting substantial cognitive impairment were excluded.
(3) Short Form-12 Health Survey (SF-12; [27]). This questionnaire was used for the assessment of the health-related functioning within daily living. The second item within the questionnaire had to be adjusted to the local living conditions. This item aims at showing to what extent moderate activities such as "moving a table, pushing a vacuum cleaner, bowling or playing golf" are limited by health. The list of activities was modified and became "moving a table, pushing a vacuum cleaner, taking a short walk or riding the bicycle" (as bowling and golf are not typical activities in Romania). We obtained an abbreviated physical and mental health profile consisting of two summary measures describing the health-related quality of life, Physical Component Summary (PCS) and Mental Component Summary (MCS).
(4) State-Trait-Anxiety Inventory (STAI; [28]). In this study, this instrument was used to assess the intensity of anxiety in the present situation, i.e., at the time of the interview, and to determine the current general level of anxiety.
(5) Beck Depression Inventory (BDI; [29]). We used this questionnaire for screening the current co-morbid depression symptoms and for the diagnosis of depression disorders according to DSM-IV criteria.
(6) An adjusted version of the Persecution and Maltreatment Checklist[17,30] This is a semi-structured interview that was developed to assess the traumatic conditions during political imprisonment in Eastern Germany. By using a pre-testing and a reviewing procedure, the instrument was cross-culturally adjusted to the conditions of political imprisonment in Romania. As the development, structure and function of the political imprisonment practice in all Central and South-East European communist regimes has followed the soviet model [24], we considered this instrument to be adequate for our research purposes. Historical research and the review of written testimonies have demonstrated that the system of political detention in Romania was characterized by the use of additional specific procedures [18,19,22]. Consequently, we reviewed the survey by adding new items referring to mock executions, forced labor, torture, forced standing, and blindfolding. Next, we pre-tested the instrument with potential respondents who were survivors of political imprisonment, to make sure that the questions were clear, reasonable, and free of undue burdens. This proved that the items were adequate to their experiences during political detention and revealed the common use of another procedure, confinement in overcrowded cells. Accordingly, we added a new question concerning this procedure. The interview included questions referring to the severity of traumatic experiences during two periods (pre-trial detention and punitive imprisonment). We collected information across several maltreatment categories (solitary confinement; confinement in overcrowded cells; darkroom confinement; special confinement, i.e., arrest; blindfolding; physical maltreatment, e.g., unsystematic beating, torture, forced labor, sleep deprivation, forced standing, exposure to extreme temperatures, starvation, refused medical help; psychological maltreatment, e.g., threats, offenses; witnessing physical torture; witnessing psychological torture; witnessing death; spies among detention colleagues; death threat; mock executions). Each item was rated for the application of a specific maltreatment category (yes or no) and for self-ratings of associated distress on a seven-point scale, ranging from 1 "not at all disturbing" to 7 "extremely disturbing". Scores lower than 4 were regarded indicative of low distress, whereas 4 was considered the cut-off value, indicating a moderate level of distress. Scores ranging from 5 to 7 were considered to indicate a high level of distress. A supplementary open question regarding personal experiences was asked for each category.
The Diagnostic Interview Section
Classically structured surveys were used in the assessment for a broad range of psychiatric disorders. Post-traumatic symptoms and co-morbid symptoms were obtained by means of one interview.
(1) PTSD-section of the Composite International Diagnostic Interview (CIDI; [31]). Trained clinical interviewers assessed current PTSD diagnosis according to the DSM-IV criteria. Participants in the clinical group reported imprisonment experiences as most traumatic. Consequently, posttraumatic symptoms in this group were always determined in relation to maltreatment experiences during imprisonment from the event-checklist mentioned above. Taking into account the complexity of prolonged trauma such as repeated or continuous torture, a separate approach of the experiences that had occurred during imprisonment would have been rather artificial. The instrument also assesses lifetime PTSD. Additionally, lifetime traumatic experiences were screened by using the event list mentioned at the beginning of the interview. In order to be sure that the symptom structure was being examined, the CIDI-typical skip rules were not applied; rather, each respondent was asked the full set of questions.
(2) Alcohol and substance abuse/dependence, somatization symptoms, former psychiatric illness, and psychiatric history of the family were evaluated by the corresponding sections of the Anxiety Disorders Interview Schedule for DSM-IV (ADIS-IV; [32]).
(3) Finally, the Structured Clinical Interview for DSM-IV Dissociative Disorders (SCID- D; [33]), was used for the diagnosis and assessment of dissociative symptoms and disorders.
Data Analysis
Group differences for continuous variables were evaluated by means of Student t test. Group comparisons for categorical variables were calculated by χ2-tests. Logistic regression analysis was accomplished to study relationships between education and diagnoses, between education and symptoms, between current PTSD and dissociative disorders, between psychological support and current diagnoses and symptoms.
We also examined the inter-correlations between the PTSD-section of CIDI and the STAI, BDI, and SF-12 scales. To this purpose, the symptom clusters' frequencies from the PTSD-section of CIDI (which reflect, respectively, the symptoms of re-experiencing, avoidance, and arousal) were summed to create an overall frequency of posttraumatic symptoms. Pearson correlation coefficients (r) were performed with this PTSD scale score using the total anxiety scores (STAI scale), combined with the total depression scores (BDI scale) and the PCS and MCS measures (SF-12 scale).
Results
The History of Repression and Exposure to Traumatic Conditions during Detention
Forty-eight (81.4%) of the former political detainees reported that they had been subjected to state persecutions a few months before imprisonment: surveillance, sanctions, and interrogations. The most frequent reason for imprisonment was the membership within an anticommunist organization followed by anticommunist propaganda (Table 2). The average age at the time of arrest was 20.0 years (range 16–55, SD ± 9.39). Following arrest, there was a period of remand with an average duration of 4.6 months (range 0–24, SD ± 4.4). This time period consisted of interrogations that usually took place at the closest secret police station from the persons' residence. The pre-trial detention was followed by a formal military trial and the victim was convicted to several years of political prison for allegedly committed crimes against the communist regime.
Table 2 The distribution of reasons for imprisonment within the group of former political detainees
Former political detainees (n = 59)
Reason for imprisonment n %
Membership in an organization/political party 19 32.20
Membership in a legionary organization 8 13.56
Rebellion against the Communist system 6 10.17
Anticommunist propaganda 14 23.73
Support of political opponents 2 3.39
Victims of arbitrariness 10 16.95
On the average, imprisonment lasted 68.7 months (range 13–198, SD ± 45.7). The imprisonment occurred in 4.0 prisons on the average (range 1–12, SD ± 2.4). Traumatic conditions were more frequently reported during punitive detention (M = 10.3, range 1–18, SD ± 5.0) as compared to pretrial detention (M = 7.8, range 1–15, SD ± 3.6; t(58) = 4.7, P < .001). The characteristics of traumatic conditions during pretrial and punitive detention, and the associated level of distress are presented in frequency order in Figure 1. Among the procedures of physical torture, the most common was the falanga, i.e. the beating of the feet' soles (39% during pre-trial detention, respectively 36% during punitive detention). All measures have been associated with high distress (as measured on a seven-point scale). When asked to specify the most stressful aspect of pre-trial detention, 24 respondents (40.7%) reported physical maltreatment, 19 (32.2%) of them reported psychological maltreatment, and 6 (10.3%) of them reported isolation. During punitive detention, 11 respondents (18.6%) reported starvation, 9 (15.3%) of them reported forced labor, and 8 (13.6%) of them reported physical maltreatment as the most stressful aspects of punitive detention.
Figure 1 Prevalence of traumatic conditions during detention and associated distress *, as reported by former political detainees (N = 59). * Associated distress was measured on a seven-point scale.
On the average, 43.9 years (range 14–53, SD ± 7.2) had passed between the time of release from prison and the present investigation. Three of our participants had been imprisoned again in the 80ies, the last of them being released in 1991. Most of the former political detainees (64.4%) reported that they had been subjected to further persecutions after being released from imprisonment, such as professional disadvantages (e.g., forced to work below their level of qualification, not allowed to continue studies and to receive higher degrees), oppression (e.g., forced displacement, surveillance and regular interrogations), and other kinds of discriminations. These post-prison repressive events lasted until the end of 1989, when a rapid political change put an end to the communist regime.
Disorders, Symptoms and Results on Self-Report Scales
All survivors of political persecution and 62% (N = 18) of the control group reported traumatic events that fulfilled the stressor criterion (A-criterion) for PTSD. Table 3 shows the diagnoses and the self-reported psychological problems separately for former political detainees and for controls. The two groups differed significantly on PTSD and on most of the other clinical indicators. 31% (N = 18) of political detainees fulfilled the diagnostic criteria of current PTSD at the time of the assessment. The lifetime PTSD prevalence in survivors was 54%; no cases of delayed onset of PTSD were recorded. In contrast to controls, somatization symptoms, substance abuse (alcohol and tobacco) and dissociative disorders were also frequently reported by survivors, whereas a current major depression was only significant for one-tailed testing (Table 3). Such a difference was supported by significantly elevated depressions scores (BDI) in survivors of the imprisonment, compared to controls who scored within the normal range. Among somatization symptoms, pain and pseudoneurological symptoms were particularly common. The most frequently diagnosed dissociative disorder among former political detainees was depersonalization disorder (N = 12, 20.3%). Both groups scored high on state and trait anxiety (STAI) as self-rating scales, whereby former political detainees had significantly higher scores than control subjects on state anxiety. Using a logistic regression, there was no significant association between education and any of the clinical conditions. The educational level measured in the number of school years had no covariate effect on the self-report measures except for anxiety rates (state-trait anxiety: F(1,94) = 8.3, p < 0.01, state anxiety: F(1,94) = 4.4, p < 0.05, trait anxiety: (F(1,94) = 11.4, p < 0.001).
Table 3 Psychiatric diagnoses and ratings on self-report scales of former political detainees and comparison subjects
Political detainees (n = 59) Control subjects (n = 39) Analysis
Clinical condition n % n % χ2 p
PTSD, current 18 30.5 1 2.6 11.7 < .001
PTSD, lifetime 32 54.2 3 8.3 22.2 < .001
Major depression (current) 16 27.2 5 12.8 2.9 <.1
Dissociative disorders 20 33.9 4 10.3 7.1 < .01
Somatization symptoms 28 47.5 7 17.9 8.9 < .01
Substance abuse 22 37.2 5 12.8 7.0 < .01
Self-rating scale Mean SD Mean SD t p
Beck Depression Inventory 12.5 9.3 8.2 6.7 2.4 < .05
State-Trait Anxiety Inventory 84.1 15.8 78.7 14.9 1.7 n.s.
State anxiety 41.3 10.2 37.0 8.8 2.1 < .05
Trait Anxiety 42.9 7.2 41.7 7.3 0.8 n.s.
PCS (SF-12) 38.7 10.5 36.5 9.7 1.0 n.s.
MCS (SF-12) 46.1 11.9 44.0 12.5 0.8 n.s.
Only two (3%) of the former political detainees had no PTSD symptoms, while the majority (76%) reported more than six symptoms. The most frequent specific symptoms within the group of survivors were recollections of trauma (78%), distress when reminded of trauma (68%), bad dreams or nightmares about the trauma (64%), and avoidance of trauma thoughts (63%). The least common symptoms were restricted affect (19%) and limited expectations concerning the future (22%). The comparison of mean frequencies of current PTSD symptom clusters within the group of former political group pointed towards a significantly higher count of intrusive and avoidance as compared to hyperarousal symptoms [t(58) = 3.3; p < .01; t(58) = 2.3; p < .05].
Within the group of survivors, the PTSD score correlated positively with STAI-anxiety (r = 0.38, p < 0.01), with BDI-depression (r = 0.44, p < 0.001), and negatively with SF-12-physical health (r = -0.42, p = 0.001). Also, the depression score (BDI) was positively correlated with STAI-anxiety (r = 0.36, p < 0.01) and negatively correlated with SF-12-mental health (r = -0.34, p = 0.01). Current PTSD associated with dissociative disorders (OR (2.4, 28.3) = 8.3, p < 0.001).
Although there was no significant difference regarding lifetime PTSD between these two groups, a significantly higher prevalence of current PTSD was noted among former political detainees who did not receive psychological support (Table 4). Dissociative disorders and substance abuse were also more frequently diagnosed among those who had not received psychological assistance. Logistic regression analyses showed that psychological support was predictive of current PTSD diagnosis (OR (0.1, 0.5) = 0.3, p < 0.05), of dissociative disorders (OR (0.1, 0.9) = 0.3, p < 0.05), and of substance abuse (OR (0.1, 0.8) = 0.2, p < 0.05).
Table 4 Differences between former political detainees who have and who have not had psychological support
Diagnoses Psychological support (n = 31) No psychological support (n = 28) Analysis
n % n % χ2 p
PTSD, current 5 16 13 46 -6.4 < .01
PTSD, lifetime 17 55 15 54 0.1 n.s.
Dissociative disorders 7 23 13 46 -3.7 < .05
Somatisation symptoms 14 45 14 50 -0.6 n.s.
Substance abuse 8 26 14 50 -3.7 < .05
Major depression 9 29 7 25 0.1 n.s.
Self-rating scale Mean SD Mean SD t p
Depression (BDI) 10.3 5.7 14.8 11.8 1.9 n.s.
Anxiety (STAI) 84.8 17.0 83.5 14.6 1.4 n.s.
PCS (SF-12) 39.9 9.6 37.2 11.3 1.0 n.s.
MCS (SF-12) 45.9 12.3 46.4 11.6 -0.2 n.s.
Discussion
The present study is based on the clinical assessment of a sample of 59 survivors of the political detention regime in Romania in their late life, and it aimed at exploring long-term psychological consequences of traumatic conditions during detention within this group. Participants in our sample were young or very young at the time of arrest. As compared to other studies on political victims of former communist regimes [15,17], our results show that the exposure to traumatic conditions within the Romanian sample was more severe in terms of subjection to traumatic conditions (exposure to a mean of 8.6 forms of maltreatment) and detention duration (5 years and 9 months on average). Traumatic conditions such as forced standing, threats and offences, starvation followed by solitary confinement, unsystematic beatings, and sleep deprivation were very frequent during both pre-trial and punitive detention. During punitive detention, these traumatic experiences were commonly accompanied by confinement in overcrowded cells, work in labor camps, exposure to extreme temperatures, and torture witnessing. In addition, political detainees were very often moved from one prison to the other. These data suggest that the Romanian political detention regime was an system based mainly on methods of gradual physical and psychological weakening, which was also demonstrated by means of historical analysis [22]. This conclusion is also consistent with the political detainees' accounts of their most disturbing experiences.
The investigation of posttraumatic symptoms in our study was related to the experience of political imprisonment. Our results add evidence to the qualification of political detention among traumatic consequences with complex long-term consequences [1]. The rate of current PTSD among participants was 31%, whereas the lifetime PTSD prevalence was evaluated at 54%, confirming the hypothesis that PTSD can be a long-term consequence of political detention late in life. These results are similar to those of research on political victims of former Eastern Germany [15,17], as well as to those of studies dealing with psychological effects of the POW experiences in World War II and of the Holocaust after 40–50 years [4,10]. However, in contrast with the studies on former political prisoners from East Germany, most of the participants within the present group of survivors consisted of political activists or people who had expressed their anticommunist beliefs, in one way or another. Although less common, others were arbitrary victims. Consequently, we could not prove the notion of a low rate of PTSD among victims of political imprisonment and torture who had been political activists [14,34]; further persecutions after the release from prison may have contributed to the maintenance of the disorder.
Somatization symptoms (48%) were frequently diagnosed among former political detainees. The prevalence of other disorders such as substance abuse (37%), dissociative disorders (34%) and major depression (27%) was also high. High associations between the current PTSD diagnosis and the prevalence of dissociative disorders confirm their co-morbidity with PTSD, which has previously been found in aged survivors as well [11,35]. Also, PTSD score related to depression, supporting findings on Holocaust survivors [8], and to anxiety and physical health scores. The association of PTSD with most of the other clinical conditions in our survivor group validated our assumption that political imprisonment leads to chronic PTSD and other long-term consequences. Other possible contributors to the prevalence of these co-morbid symptoms and disorders are persistent persecution after release, and lack of rehabilitation and of social support. The high co-morbidity supports the notion of long-term complex severe psychological effects of prolonged, repeated trauma, and indicates that a current PTSD diagnosis would not capture the severe psychological harm present in such cases [1,2]. Moreover, this result is consistent with the studies conducted after a long post-trauma time interval on Holocaust survivors in old age, which have indicated that persistent posttraumatic symptoms may contribute to additional psychological and physical disturbances [10,13].
Some of our participants received psychological support within a clinical context. This group had the same life-time prevalence of PTSD as those left untreated (54%), but exhibited a significant lower rate of current PTSD, substance abuse, and dissociative disorders. Further analyses showed that a lack of psychological support was associated with current PTSD, dissociative disorders and substance abuse. As the assignment to these two groups was not done at random, we cannot ascertain a causal relationship. Nevertheless, as compared to recovery from PTSD in the treated group, which reached a worth-mentioning 70%, spontaneous remission in the untreated sample was very low (less than 15%), suggesting that, once it has developed, PTSD is likely to persist over the whole lifespan. However, treatment seemed not to produce a reduction on the level of intrusion, somatization, and major depression. Possibly, psychological support was efficient only in the reduction of posttraumatic hyperarousal and avoidance, substance abuse, and dissociation. Alternatively, it might be that those who recovered from PTSD sought treatment for their remaining mental health problems, i.e., intrusions, somatization and the collateral depressive symptoms. Given that intrusions and somatization both result from implicit memories that are not sufficiently integrated with explicit autobiographical recollection [36], a trauma-focused treatment would probably be needed [37].
Limitations of the Study
Recruitment strategies may not have allowed for the estimation of the epidemiological prevalence. First, the participants recruited from one institution did not look for treatment, whereas the other ones had already been offered psychological assistance. However, other recruitment strategies without an institutional frame would have not allowed for the selection of a sufficient number of participants, since most victims are still extremely suspicious when asked to report their experiences [38]. Secondly, 82% of the men and only 8% of the women identified as survivors agreed to participate. The scarce participation of women in this study may explain why the present sample is representative of former political detainees from the Moldavian region of Romania in terms of age distribution, education and occupational status, but not of gender distribution. If highly traumatizing experience such as sexual violence or persistent high anxiety levels precluded subjects from participation in the study, the actual PTSD rate may thus be even higher. In addition, it may well be possible that, in a country with a 75-years life expectancy, a higher fraction of former detainees with severe mental disorders died by the age of seventy, as opposed to those less affected by trauma [39].
The educational level of control subjects was lower than that of survivors. This difference was predictable, since a remarkably high educational level was a distinctive feature of the political detainees population, given that the bourgeoisie and the intelligentsia were the favorite target groups for political persecutions during the former Communist regimes [24]. The covariate effects of education showed that this factor explained the similarity of the two groups with respect to the high levels of anxiety. In addition, hospitalization of controls for rheumatoid and arthritic problems may explain the lack of a difference with respect to physical problems between the two groups. The two groups also differed with respect to exposure to physical assault. The unusual high number of such experiences reported among former political detainees might be an aspect of their subjection to political violence, as suspected by most of them, although they had no proof of it. Yet, none of the participants rated experiences of physical assault as the most traumatic experience. However, the inclusion of a culturally and sociodemographically equivalent comparison group helps validating our findings.
We also acknowledge that the long period of time of 43.9 years on the average, which had passed since the trauma until the moment of the interview, may have affected the validity of the data collected retrospectively with the Persecution and Maltreatment Checklist. On the other hand, the traumatic experiences, reported by the respondents involve "hot" memory, i.e., episodes with strong emotional and sensory components that seem to be highly consistent and long-lasting [36,40].
Conclusion
The present study shows that PTSD and other mental disorders developed significantly in former political detainees of the Romanian communist regime as a consequence of trauma, maltreatment and other stress factors that acted during the political imprisonment. Across four decades, the disorder persisted in severe forms, particularly in the former political detainees who did not receive psychological assistance. Thus, political detention and post-detention life conditions are likely to have negative long-term effects on the mental health status of former political detainees that may last even into the latest stages of life when left untreated. A high prevalence of co-morbid disorders and symptoms, including somatic complaints, add to the suffering of these survivors. Obviously, these victims need psychotherapeutic help and care that includes rigorous research concerning political persecution. However, in view of a highest efficiency, public recognition and political rehabilitation would also be necessary requirements. Further research on survivors of political persecution in the former Communist countries of Eastern Europe may add helpful data in understanding the psychological effects of this phenomenon.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DB conducted the study in Romania, performed the statistical analysis, and drafted the manuscript. MS designed the study and coordinated the studies activities in Romania. ES participated in the analyses. AN participated in the design of the study and its coordination. TE designed the study and revised the manuscript. FN designed the study and revised the manuscript. All authors read and approved the final version.
Acknowledgements
Research was supported by the Hans-Böckler-Foundation and by the Deutsche Forschungsgemeinschaft (El 101/18). We thank Christina Robert for comments on earlier versions of the ms.
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Harm Reduct JHarm Reduction Journal1477-7517BioMed Central London 1477-7517-2-161616475810.1186/1477-7517-2-16ResearchA situational picture of HIV/AIDS and injection drug use in Vinnitsya, Ukraine Barcal Katerina [email protected] Joseph E [email protected] Kostyantyn [email protected] Larisa Vasiliyevna [email protected] Department of Epidemiology, School of Public Health, The University of Alabama at Birmingham, Birmingham, Alabama, USA2 Division of Preventive Medicine, Department of Medicine, The University of Alabama School of Medicine, Birmingham, Alabama, USA3 Department of Infectious Diseases and Epidemiology, Vinnitsya National Pirogov Memorial Medical University, Vinnitsya, Ukraine4 Vinnitsya Regional Narcological Dispensary, Vinnitsya, Ukraine2005 15 9 2005 2 16 16 2 12 2004 15 9 2005 Copyright © 2005 Barcal et al; licensee BioMed Central Ltd.2005Barcal 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
New and explosive HIV epidemics are being witnessed in certain countries of Eastern Europe, including Ukraine, as well as a rapid and dramatic increase in the supply, use, and negative public health consequences of illicit drugs. A majority of registered HIV cases in Ukraine occur among injection drug users (IDUs), large numbers of whom report HIV risk behaviors such as needle sharing. The purpose of this study was to apply the World Health Organization's Rapid Assessment and Response on Injection Drug Use (IDU-RAR) guide to create a situational picture in the Vinnitsya Oblast, Ukraine, a region with very scarce information about the HIV/AIDS and injection drug use (IDU) epidemics.
Methods
The IDU-RAR uses a combination of qualitative data collection techniques commonly employed in social science and evaluation research to quickly depict the extent and nature of the given health problem and propose locally relevant recommendations for improvement. The investigators focused their assessment on the contextual factors, drug use, and intervention and policy components of the IDU-RAR. A combination of network and block sampling techniques was used. Data collection methods included direct observation, review of existing data, structured and unstructured interviews, and focus group discussions. Key informants and locations were visited until no new information was being generated.
Results
The number of registered HIV cases in Vinnitsya has increased from 3 (1987–1995) to 860 (1999–10/2004), 57 of whom have already died. Ten percent of annual admissions to the area's Regional Narcological Dispensary were for opiate disorders, and the number of registered IDUs rose by 20% from 1999 to 2000. The level of HIV/AIDS awareness is generally poor among the general population but high among high-risk populations. Both HIV/AIDS and injection drug use carry a strong stigma in the community, even among medical professionals. There was very little evidence of primary HIV/AIDS prevention efforts, and IDU prevention efforts focused on promotion of anti-drug messages in the schools.
Conclusion
Given that Ukraine has sparse resources to be devoted to this problem, action recommendations should be prioritized, realistic, and initially targeted to persons in greatest need. The following action recommendations are prioritized by the following categories: First priority: Voluntary Counseling and Testing; Second Priority: Prevention and Education; and Third Priority: Harm Reduction and Treatment. They are provided in this sequence based on what response can realistically be implemented first with limited additional resources and can make the greatest immediate impact. The persons at greatest risk, HIV positive persons and IDUs, should be attended to first.
HIV/AIDSIDUdrug abuseUkraineRapid Assessment and Response Guide
==== Body
Background
New and explosive HIV epidemics are being witnessed in certain countries of Eastern Europe: Russia, Moldova, Belarus, and Ukraine [1-6]. According to the Ukrainian Centre for AIDS Prevention, a cumulative total of 71,359 HIV cases and 4,851 deaths were registered in Ukraine by October, 2004 [11]. The actual prevalence is believed to be much higher, with the true number of existing cases estimated at a staggering 180,000–590,000. Such a figure would make Ukraine the most affected country in the region [7].
Political independence in Ukraine and surrounding Eastern European countries in the 1990s has been associated with a rapid and dramatic increase in the supply, use, and negative public health consequences of illicit drugs [8-10,16]. While little reliable epidemiological data on opiate injection drug use (IDU) is available from Ukraine, existing data defines it as significant concern. There were 83,868 officially registered cases of drug addiction in Ukraine by the end of 2002 [23]. The existing registration system does not distinguish IDU from other types of drug use, but one author suggests that 96% of all drug dependent registered patients injected drugs [12]. According to experts' opinions, the existing number of injection drug users (IDUs) exceeds official registry data by a factor of 5 to 7 times, depending on the particular region [13].
The majority of reported HIV infections in Ukraine were diagnosed in IDUs from 1987 to 2004 [14,15]. A total of 40,809 IDU-related HIV cases in Ukraine were reported from 1987 to 2004, which comprises 71.7% of all adult cases [11]. These figures represent only registered cases and therefore underestimate the number of diagnosed cases. An estimated 180,000 to 590,000 persons in Ukraine are infected with HIV, with IDU the primary source of transmission [7]. HIV risk behaviors such as needle sharing at the time of last injection were reported by 47% of IDUs [17], and only 29% of IDUs have reported consistent use of clean needles [13].
The proportion of total cases acquiring HIV through opiate IDU in certain areas of Ukraine dropped from 72.7% in 1997 to 56.7% in 2004, suggesting expansion of the epidemic to the general population [6-11]. Data indicate that an HIV epidemic fuelled by heterosexual transmission is emerging, and its expansion will depend on the size of bridge populations that link high-risk groups with the general population [4]. In comparison, of all diagnosed AIDS cases as of December 2000 in the United States, 25% occurred among IDUs [18]. Although the extent of illicit drug use is probably more limited than the extent of many other social problems in the countries of the former Soviet Union, the extreme growth and relevance to HIV/AIDS make it a primary area of concern to more than 200 million people living there [8].
Purpose
At the time of this study, information about the HIV epidemic came mainly from the most heavily affected Ukrainian cities, such as Odessa, Kharkiv, and Mykolaiv [1]. The attempts in these and several other cities to control the epidemic were documented in the areas of youth-based HIV education and social marketing, harm reduction programs (such as needle exchange), and narcological hospitals. However, there was very little information about the HIV/IDU situation in smaller cities and rural areas of the country. It was not clear how the epidemic progressed in other parts of the country and whether any prevention or treatment efforts existed outside of the major cities. The purpose of this study was to create a situational picture of the injection drug use and HIV/AIDS and IDU epidemics in the Vinnitsya Oblast (a semi-urban community in central Ukraine with scarce information about the nature and extent of the epidemics) and propose action recommendations for positive change in education, service, and research.
This project builds on a successful three-year joint collaboration between public health, HIV/AIDS, and drug addiction researchers and clinicians from the University of Alabama at Birmingham (UAB), The Regional Narcological Dispensary (RND) in Vinnitsya, Ukraine (a local government-run alcohol and drug addiction hospital), and the Vinnitsya National Pirogov Medical University (PMU). This collaboration was initiated by the UAB John J. Sparkman Center for International Public Health Education and an International Clinical, Operational and Health Services Research and Training Award (ICOHRTA) awarded by the National Institutes of Health Fogarty International Center to UAB in 2001.
The fieldwork phase of this study was completed from May through July of 2001.
Methods
This study utilized rapid assessment and response (RAR) methodology developed by the World Health Organization (WHO) in 1998. The RAR method uses a combination of qualitative data collection techniques commonly employed in social science and evaluation research to quickly depict the extent and nature of the given health problem and propose locally relevant recommendations for improvement. One of the key principles of RAR is that data are collected from different sources, which allows continuous examination of the reliability and consistency of the data and enables investigators to make better informed decisions about what evidence should be sought in the next stage of the assessment. RAR is designed to rapidly assess a current problem situation (e.g. IDU) in a community. This information is then used to make informed decisions about the development of interventions needed to reduce the adverse health and social consequences of the targeted condition. The WHO Rapid Assessment and Response guide on injection drug use (IDU-RAR) [19] was deemed appropriate for this study because it has been used successfully in resource-limited settings within the United States and around the world [20], including two Ukrainian cities – Odessa [1] and Kharkiv [13].
Due to limited financial resources and a short time frame, we directed our focus to three areas of the IDU-RAR when assessing the IDU situation: Contextual Assessment, Drug Use Assessment, and Intervention and Policy Assessment. The Contextual Assessment identifies factors that influence the current and potential situations regarding drug injection and its adverse health consequences, as well as opportunities for the development of interventions. Key areas for assessment include factors for spread of IDU, exacerbation versus amelioration of adverse health consequences of injecting, and factors that are likely to hinder or enable the development of interventions. The Drug Use Assessment focuses on the nature and extent of drug use – who is injecting drugs and where this occurs, as well as trends in injection drug use over time. Finally, the Intervention and Policy Assessment is designed to assess existing interventions and policy responses aimed at reducing drug use and its consequences while allowing the assessment team to examine their effectiveness and develop recommendations. Our study also included an assessment of the HIV/AIDS situation, which incorporated the nature and extent of the epidemic, HIV/AIDS awareness, attitudes toward individuals living with HIV/AIDS, and existing prevention/control measures.
Mapping of the Vinnitsya Community
As a first step of the rapid assessment, the principal investigator met with key collaborators in Vinnitsya to assemble an assessment team and identify key informants. Immediately following the initial consultation, a conceptual map of the Vinnitsya community was developed. The map captured key locations for the needs assessment including major gathering points for IDUs, areas where drugs are sold, needle exchange sites, treatment facilities, transportation routes, and other key locations. The investigator traveled to all key locations to develop a physical map, noting activities that would help to provide some insight into the nature of IDU.
Sampling
In order to create a representative sample of informants, a combination of network and block sampling techniques was used. The network sampling method involved a chain of referrals initiated by the key informants. All key informants were asked to provide a list of individuals who would be able to offer additional information on a given area of the needs assessment. These individuals were then contacted for further information and asked to provide another list of informants. This approach was used until no new information was being generated. The block sampling technique was similar to the networking sampling, but instead of using key informants as the starting point, the needs assessment team traveled to key locations (identified by the mapping exercise) to identify new informants. As an example, the leading investigator frequently traveled to a local market in the Vishinka district (identified as a gathering point for IDUs) to develop rapport with IDUs and gain access to new informants who would be willing to provide information on the IDU situation in Vinnitsya.
Rapid Assessment Implementation
Data were collected using a combination of rapid assessment methods, including direct observation, review of existing data (including statistical data from government reports, annual reports from non-governmental organizations [NGOs], local research studies, and media), structured and unstructured interviews, and focus group discussions. An important component of the needs assessment was the triangulation of information, or cross-checking of collected data through the multiple sources. This allowed for collection of more representative data with higher confidence in its accuracy.
Results
Extent of the HIV/AIDS epidemic
The first occurrences of HIV infection in the Vinnitsya region were officially registered with the Sanitation and Epidemiological Service between 1987 and 1995. At this point, only 3 HIV infections were registered. By 1996, the number of new HIV infections rose to 46 (for a total of 49 HIV infections), including the first officially registered AIDS case. Between 1999 and 2001, there was a sharp increase in the number of new HIV infections in the region (at approximately 130%) that brought the total number of HIV infections to 431. At the end of 2001, a total of 112 new HIV infections, 26 AIDS cases, and 17 AIDS-related deaths were recorded for the Vinnitsya region. The regional Sanitation and Epidemiological Service statistics for the year 2001 revealed that the largest proportion of HIV infections occurred through injection drug use (83.9% of the total), followed by sexual transmission (16.1% of the total). Individuals in the 20–29 age group accounted for the largest proportion of HIV infections (69.6 %). Within this age group, the male to female ratio was 3 to 1. The most heavily affected areas within the Vinnitsya Oblast were Gaisyn, Ladyzhyn, and the city of Vinnitsya with 29.5%, 20.5%, 15.1% of the total number of new HIV infections in 2001 respectively. By October 2004, there were 783 cases of HIV infection and 57 AIDS deaths registered in Vinnitsya Oblast. In 2004 for the first time heterosexual route of transmission has prevailed with 54.1% of newly registered cases [24].
Information gathered from NGOs and local researchers suggests similar trends. However, accurate prevalence rates at the present time are difficult to assess due to lack of testing resources and fear and mistrust among those who may be at risk of having HIV/AIDS.
HIV/AIDS awareness
Data collected through personal interviews with key informants and focus group discussions suggest that residents of Vinnitsya have heard of HIV/AIDS, but the level of knowledge and perception of risk vary among different groups. A focus group discussion with students from local universities in Vinnitsya revealed that young people in the community were likely to perceive HIV/AIDS as something that is found exclusively in the West and thus is not a real threat to their community. Many of the students were surprised to learn that HIV cases were reported in Vinnitsya and admitted that they were not as well informed about the disease as they needed to be. Discussions with IDU at a needle exchange program in the Vishenka District, on the other hand, revealed a much different outlook on the HIV/AIDS situation. These individuals were well aware of the presence of HIV in the community, and some even knew someone who had already contracted the virus. They felt that they were well informed about HIV/AIDS and how to protect themselves against the virus. Interviews with Vinnitsya city service providers and representatives from local NGOs confirmed that the level of HIV/AIDS awareness among high-risk populations was high. The same level of HIV/AIDS awareness, however, did not appear to extend to the rural communities. According to information from rural health providers working with IDUs, many of their patients lacked information about HIV/AIDS and did not perceive themselves as being at risk. A director of a government program focusing on the social welfare of youth in the Vinnitsya region also attested to low HIV/AIDS awareness among young adults in the rural communities.
Attitudes toward individuals living with HIV
Vinnitsya residents living with HIV face a great deal of stigma and lack a widespread support system within their community. According to infectious disease treatment providers at a local hospital in Vinnitsya, HIV positive patients tend to stay secluded and often are very concerned that other people not learn about their infection. It is not unlikely for someone diagnosed with HIV infection to seek treatment and consultation with a physician during evening hours or at other times when they would be less likely to be seen by other people at the medical facility. Though many physicians seemed compassionate toward HIV positive patients (particularly infectious disease specialists working with HIV patients), direct observations and interviews with medical staff revealed that HIV positive individuals were stigmatized within treatment facilities. Test results that were to be kept confidential were openly shared in patient charts, and HIV patients were placed in isolation blocks. Interviews with nurses and other clinical staff revealed that some of the staff were reluctant to treat HIV infected patients. HIV positive patients at a local narcological dispensary stayed in an isolation block consisting of a small single-bed patient room, a nurse's cabinet, and a small waiting area. Anyone in the facility could easily determine who was staying in the isolation block and why he or she was there (as the restricted area was clearly labeled). It was not until recently (at the time of writing this paper) that the isolation block was discontinued and persons diagnosed with HIV/AIDS mainstreamed into the hospital.
Aside from counseling provided by some physicians who work with HIV-positive patients, there was very little evidence of routine counseling and follow-up for individuals who are tested for HIV. An important concern expressed by a physician at a hospital-based HIV/AIDS center was the lack of a support system that would allow patients diagnosed with HIV to speak openly about their condition and gain more information on living with the virus.
HIV/AIDS prevention
Prevention activities found in the area were classified according to the three categories: primary (prevention of occurrence), secondary (diagnostics and treatment), and tertiary (minimization of adverse consequences). With the exception of a school-based educational program and some media efforts, there was very little evidence of primary HIV/AIDS prevention. The school-based program, organized by a local NGO, was implemented at only one of the 40 local elementary schools. The program (modeled after the American Project Hope) utilized innovative educational methods, through which children took part in skill building exercises (including self-efficacy building) and learned through peer-education. This program, however, did not have a built-in evaluation or measures to ensure sustainability. The local media were known to disseminate HIV/AIDS information through newspaper articles, but none of the information concentrated on the prevention of HIV/AIDS. At the time of the study, Peace Corps volunteers based in Vinnitsya were planning to develop and implement an HIV/AIDS education program through UNAIDS-Kyiv, which would serve as a great opportunity to create more educational programs at local schools in Vinnitsya and surrounding rural areas.
Primary prevention efforts carried out by a local NGOs reached out to high-risk populations, including commercial sex workers and IDUs, in the city of Vinnitsya and surrounding smaller cities. Outreach workers at needle/syringe exchange sites distributed condoms and information about HIV/AIDS and other sexually transmitted diseases. Educational booklets included information on safer sex practices and where to go for treatment. IDUs participating in the needle-exchange program were referred to a local hospital that had a special hepatitis and HIV/AIDS center within its facility. Some referrals to substance abuse treatment also were made. The program provided opportunities for HIV, HBV, and HCV testing free of charge. This effort, however, was fully funded through a time-bound grant, and it was not clear whether it would be sustained.
The AIDS Center at the local hospital provided HIV infected individuals with necessary medical attention and some counseling services and linked them with other sources of information. There were limited resources, however, for secondary prevention activities, such as screening, analysis, and HIV/AIDS disease monitoring. Availability of antiretroviral therapy was limited due to high cost. Patients had to make arrangements to buy their own medications. This situation was likely to change with the Ukrainian government's intention to provide subsidized antiretroviral therapy in the near future, supported by the grant from the Global Fund. The AIDS Center also worked closely with local NGOs to recruit individuals from high-risk populations for testing and treatment.
Injection drug use
During the Soviet era, IDU was largely hidden and mostly limited to individuals from wealthy families. The drugs that were consumed during that time usually included "clean" drugs such as morphine and heroin. Substance abuse treatment providers who treated patients during the Soviet era indicated that the nature of drug use in Vinnitsya has changed dramatically over the past decade. In the years following Ukraine's independence, drug use has increased rapidly and shifted from purer, more expensive drugs to drugs that can be made at home. According to recovering IDUs from a local church, raw materials became easily accessible, and preparation procedures were passed around like cooking recipes. One especially potent amphetamine-like injectible drug, commonly referred to as "vint," was prepared using readily available chemicals that could be purchased at drug stores and local pharmacies. Though this particular recipe came at a price, IDUs could easily learn how to prepare a homemade opiate solution ("hanka" or "shirka") from their peers.
Individuals who were new to IDU could purchase ready-filled syringes or small medicine containers with the opiate solution at a local market. According to informants at a market in the Vishinka District of Vinnitsya city, drug dealers (often IDUs themselves) were able to do their business there without getting much attention from law enforcement while having good access to new customers. An inexperienced person might be invited to the dealer's apartment or a nearby garage, as attested by social workers at a local needle exchange program, where the dealer would help him or her to inject the drug.
There were no reliable epidemiological studies available to estimate the prevalence rates of opiate IDU in Vinnitsya and surrounding areas. Ten percent of the annual admissions to the area's only RND for drug and alcohol treatment were diagnosed with opiate disorders, but the actual prevalence rate in Vinnitsya was estimated to be more than ten times that figure or 2,000 persons with opiate disorders (personal communication with Pavel Slobodyenyuk, M.D, 2004). Dr. Slobodyenyuk also reported that the number of registered IDUs from rural areas during the year 2000 rose by 20% from the previous year. According to reports from the regional Sanitation and Epidemiology Service, rural areas were the most heavily affected in terms of opiate IDU and reportedly include the regions of Koziatyn, Zhmerynka, Trostyanets, Ladyzhyn and Illinitsi, as well as various small towns such as Hnivan and Vapniarka.
Factors that encourage the spread of IDU
Results from this study indicated a combination of numerous factors that encouraged the spread of opiate IDU, including Ukraine's economic situation, social changes following the country's independence, easy access to poppy plants, and lack of knowledge. As Ukraine transitioned from the old (i.e. Soviet) system, many people were left without jobs or ones that pay on average US $30–50 a month as revealed by community informants. The sale of hanka quickly became a prosperous business. The drug production was not difficult, the raw material was grown in the area, and the demand for opiates was increasing. The cultivation of poppies has been part of the Ukrainian culture for many generations. Consequently, there was fairly easy access to poppies in the Vinnitsya region, especially in the rural areas. IDUs were able to gather poppy straws directly from the fields or purchase them from babushkas (grannies) at the local market. A 1-ml dose of hanka sold for about 5.00 grivnas (about $0.95 US) during the poppy season and for about 8.00 grivnas (about $1.50 US) out of season according to local IDUs. Selling a liter of hanka in a day allowed one to make more than ten times the income an average person makes in a month.
There did not appear to be a single driving force behind the increase in drug use among young adults. Discussions with IDUs at local treatment facilities and needle exchange programs revealed that people turn to drugs for different reasons. Some people sold drugs to make money while others were drawn to use IDU out of boredom, curiosity, or an attempt to be part of a social circle. As graduates from universities and technical colleges, young adults often had to travel to larger cities to find employment. When they first arrived they would try to find a social circle and a place to belong. This was how many people were introduced to people who inject drugs, according to IDUs and substance abuse treatment providers. They would then introduce their friends to IDU upon returning home and consequently form small IDU communities. Furthermore, the consumption of alcohol is a very important part of the Ukrainian culture. With the economic situation under strain, people turned to alcohol to escape the harsh realities of everyday life. IDU is slowly becoming the mechanism of abuse among young people who wished to do the same. Though information collected through different sources did not contradict this observation, more research is needed to confirm that there is indeed an association between alcohol use and illicit drug use.
Another important factor that was indicated as a possible facilitator of drug use, and one that should also be explored through further research, is the changing social environment among school-aged children. As the economy began to crumble, many parents were forced to take on additional jobs to compensate for low income. Their children were under less supervision, and some community representatives speculate that they became more vulnerable to exploring drugs. Extracurricular activities such as sports and various special interest clubs were free-of-charge during the Soviet era. Following the fall of the Soviet Union, however, local schools no longer had the necessary financial resources to sponsor extracurricular activities, and thus children were left with more free time and less to do. These are factors that may play a role in the increasing IDU epidemic. However, more research is needed in this area.
While young people from the Vinnitsya community were exposed to various forms of pro-drug messages (including the cultural acceptance of alcohol abuse) on practically a daily basis, the effort to counteract the influence of these messages by informing the public about the dangerous consequences of substance abuse was minimal. Aside from the recent implementation of school-based anti-drug programs and a special radio program that reaches out to the Vinnitsya city residents, there was very little evidence of anti-drug propaganda (i.e. in terms of social marketing and regulation). Furthermore, not much was said about the association between alcohol abuse and HIV/AIDS. This was especially true in the rural areas, where the information was needed the most.
Drug use prevention and treatment
The promotion of anti-drug messages in schools tended to be the most favored primary prevention approach toward controlling the spread of IDU in this area. The RND has been active in launching several school-based anti-drug prevention programs in all 40 local schools in Vinnitsya, as well as schools in the surrounding areas. Most schools and teachers were surprisingly open to this type of intervention. Students were also quite open and interested in the topics presented and actively asked and answered questions. These programs, however, did not appear to be carried out to their fullest potential. They were not theory-based or standardized in their delivery. The focus of the sessions was primarily on IDU and excluded more common substances such as tobacco and alcohol. The programs were mostly didactic with no experiential components. Teaching materials and information were not always age appropriate, and there were no hand-out materials or resources to obtain help or further information about drug abuse. Furthermore, the programs did not have a built-in evaluation system.
Another school-based primary prevention program implemented by a local NGO, titled Project Hope, was modeled after a project that started in the United States and has been implemented in many areas around the world – including Moscow and three Ukrainian cities (Kharkiv, Odessa, and Kremenchoug). The goal of the program was to teach children in 1st–4th grades self-efficacy skills (i.e. how to listen, hear, speak, understand and act with the attitude of "I'm free" and "I know how to live") through fairy tales, games, and various fun activities. Teachers also were provided with guides on how to conduct interactive sessions and ideas on how to get parents involved. Older children were encouraged to participate in a voluntary Children's Club, in which children taught each other about the negative consequences of drug, alcohol and tobacco use and sexual activity, as well as sexually transmitted diseases. This program, however, was being implemented at only one school in the Vinnitsya area, and no information on its efficacy has been presented.
The local media also were involved in primary prevention. The public radio aired a special program on drugs, during which various substance-abuse specialists talked about the consequences of drug abuse and how to deal with addiction. The program also included individuals who had personal experience with substance abuse and were willing to share their story with others. The goal of the program was to provide people with the necessary information to encourage young people not to take drugs or encourage individuals who use drugs to seek treatment. The local newspaper also has created a special section with information about drug use (i.e. general information, consequences, and resource contact information). Most of this information, however, was limited to individuals who live in Vinnitsya and did not reach the rural areas, where the information also was needed.
In the area of tertiary IDU prevention, a local NGO has been carrying out a needle/syringe exchange program (NEP/SEP) at various sites in the Vinnitsya area over the past three years. There were two NEP/SEP sites in Vinnitsya, as well as sites in Kalynovka, Ladyzhyn, Zhmerynka, and Haysin, with one being planned for Kaziatin. IDUs in the area were able to exchange used needles and syringes for new, sterile needles and syringes at no cost. The goal of the program was to reduce the potential harm of IDU by reaching out to individuals who put themselves at risk for physical injuries, nerve damage, and infection with sexually transmitted diseases and various blood-borne viruses including HIV, Hepatitis B and Hepatitis C. Educational booklets distributed included information on safer injection practices (to avoid serious injuries such as nerve damage), safer sex practices, and information on where to go for treatment.
Though the authorities have supported harm reduction programs such as this, needle exchange was not yet fully accepted within the community. There was still some doubt as to the effectiveness of such a program, and there was even some suspicion that NEP/SEP served as a place to purchase drugs. According to the social workers from the Vyshenka District NEP, the local police had been suspicious that the social workers were involved in the drug trade. The police had been known to send in undercover agents, for instance, to see whether they could purchase drugs or poppy straws from the social workers. The program was advertised through "word of mouth" due to the stigma placed on IDUs. There tended to be a lack of coordination between the NGO and other organizations in the community, all of which would help to make the program more effective. No empirical information on the effectiveness of this program has been presented to the research or local community over the three years of program operation.
The RND (a 160-bed inpatient, government supported hospital) is the only facility for the diagnostics and medical treatment of alcohol and drug related disorders in Vinnitsya. Located in the south Leninsky district, it is staffed by medical doctors, psychologists, social workers, and nurses. It also carries out the expert assessment of substance use disorders for local road police and the criminal justice system and provides educational lectures to local and rural elementary schools. There were more 24 thousand persons "registered" as patients of the RND. The patient population was primarily male and approximately 60 percent from the city of Vinnitsya and 40 percent from rural areas. Most patients were being treated for alcohol-related disorders. Only 10 percent were opiate IDUs, despite the increasing demand for treatment of this problem. Treatment was organized into three stages: medical, psychotherapy, and rehabilitation. Primary emphasis was placed on medical treatment.
Though the RND is a public health facility, the government covers only a small portion of the total cost of treatment, which barely covers staff salaries. Treatment in public institutions is considered free, which means that patients do not pay any hospital or physician charges. However, since the breakdown of the Soviet system, the state budget does not make provisions for medication. Consequently, patients were responsible for covering the cost of medicines and various medical supplies, which usually added up to about 150 grivnas ($28) for the minimum length of stay – an amount that exceeded some people's monthly salary. The RND provided treatment primarily for the patients' physical dependency (medical detoxification) with very little aftercare, and it was plausibly reported that many patients have short remission times and return to alcohol or drug abuse just a few months following the completion of the treatment. Each patient is recommended to follow up with a psychologist or a regional narcologist after discharge; however no monitoring system is in place, which prevents valid outcome assessment. According to the treatment providers' personal experience, very few patients do see their psychologist or narcologist before the next full-blown relapse. Factors that may hinder the effectiveness of the treatment include: high cost and short length of treatment, lack of evidenced-based interventions, lack of outpatient treatment and drug use monitoring, and absence of narcotic substitution medication therapies. There are essentially no eligibility criteria for admission; any resident of Vinnitsya oblast could enter the RND if diagnosed with a substance use disorder. High costs of medication and little faith in the efficacy of available treatment among potential patients further decrease the attractiveness of drug treatment in Vinnitsya.
There was a great amount of prejudice toward IDUs both within the community and among medical professionals that presented another barrier to the effective treatment of IDUs. Interviews with medical practitioners revealed discriminatory attitudes toward IDUs. They were perceived to be criminals and/or individuals who lack moral values. Furthermore, IDUs were not the most "liked" patients, even at the RND, where patients with alcohol dependency were said to get preferential treatment. Alcohol dependency may be more acceptable due to the strong cultural role of alcohol in this society. Some medical professionals viewed IDUs as "hopeless cases" or as individuals who are "impossible to treat." It is commonly believed that IDUs generally were not willing to change their lifestyle and came for treatment only in order to lower their tolerance level (as it becomes too expensive for them to inject) or avoid a prison sentence. Furthermore, some health professionals explained that IDUs are so mentally disturbed (due to brain damage) that they are no longer receptive to any treatment. Direct observations at the treatment center revealed that it was not unlikely to see a physician on duty refuse treatment to HIV positive IDUs and send them to another facility (i.e. an infectious disease hospital). The director and staff of the RND, however, were highly compassionate and motivated to improve the state of treatment services for alcohol and drug related disorders at the RND.
Recommendations
A response designed to positively impact mortality and morbidity associated with HIV and IDU in this part of the world must be swift and comprehensive. Given that Ukraine has sparse resources to be devoted to this problem, action recommendations should be prioritized, realistic, and initially targeted to persons in greatest need. The following action recommendations are prioritized by the following categories: First priority: Voluntary Counseling and Testing; Second Priority: Prevention and Education; and Third Priority: Harm Reduction and Treatment. They are provided in this sequence based on what response can realistically be implemented first with limited additional resources and can make the greatest immediate impact. The persons at greatest risk, HIV positive persons and IDUs, should be attended to first.
The existing ICOHRTA has a four-year history of training health care providers, medical students, and drug addiction specialists and has a relationship with existing agencies capable of responding to the HIV and IDU epidemic. As such, we recommend using the resources and staff of ICOHRTA for initiating and sustaining the implementation of the following priorities. IROHRTA resources and staff can link in-country stakeholders with educational materials, risk assessments, skill building protocols, and evidence-based prevention and intervention protocols, training. They also can offer assistance with writing grants to procure funds for HIV testing supplies, condoms, bleach kits, educational materials, research, and salary support for prevention staff.
First Priority: Voluntary Counseling and Testing
Voluntary counseling and testing for HIV (VCT) is often used as the first step in addressing HIV transmission and prevention. Most people who have HIV do not know they have it. Furthermore, many people at risk are afraid to be tested because of the stigma associated with IDU and HIV and fear of testing positive. One way to address the AIDS epidemic is to give people an opportunity to know their HIV status so that they can take precautions to avoid further spread and seek treatment if they are infected [25,26]. Appropriate agencies should be identified by ICOHRTA investigators for training and implementation of VCT.
Identification of those in need of help through effective outreach, motivational enhancement, risk assessment, and VCT must be the first priority to make an immediate impact on those at greatest risk in Vinnitsya. Vinnitsya has various locations appropriate for VCT sites. For example, the RND, the Infectious Disease Hospital, PMU, the STEPS program (an outpatient drug and alcohol addiction treatment center), and the NEP NGO are existing organizations that could offer VCT services. Training of agency staff is recommended in effective peer outreach strategies, risk assessment, motivational enhancement, and VCT protocols. Immediate diversion of agency funds and applications for small grants are recommended to fund the purchasing of HIV test kits. Successful outreach and VCT will immediately break down the first barrier to assessing the problem through anonymous testing, epidemiological prevalence surveys, and provision of prevention, education, and treatment to those most in need.
Second Priority: Prevention and Education
As a result of effective outreach and VCT, four high risk populations will emerge, in order of severity: HIV positive IDUs, HIV positive non-IDUs, HIV negative IDUs, and HIV negative non-IDUs. The second priority is the prevention of HIV transmission among high risk populations through education, skills development, and distribution of free condoms with support from the ICOHRTA. Secondary prevention or the prevention of the spread of HIV by persons who are HIV positive IDUs or HIV non-IDUs should be given the greatest attention. Since only persons with HIV can spread HIV, reducing risk behaviors among persons who are HIV positive can make the most immediate impact on transmission rates.
HIV positive IDUs should be targeted first for secondary prevention by VCT providers via education about their risk of transmitting HIV to others and how to prevent transmission through abstinence or reduce it through condom use. They should first have access to free condoms and be trained in correct condom use and then interpersonal sexual negotiation skills to practice safer sex or abstinence. HIV positive IDUs should then be educated about the HIV transmission risks associated with IDU and the dangers associated with sharing needles, syringes, paraphernalia, or drug solutions. Drug and alcohol use in general reduces the practice of safer sex and drug use behaviors, but IDU is the most risky behavior due to the opportunity to spread the virus by sharing needles, syringes or other contaminated drug use paraphernalia. Therefore, training in needle and syringe cleaning with bleach should be given high priority.
Persons who are HIV positive may be suffering from despair and depression and be less motivated than high risk populations to practice safer sex or IDU behaviors. It is recommended that VCT prevention staff be adequately trained in the areas of coping with HIV diagnosis, apathy, depression, and even suicidal ideation. Finally, prevention staff should work on identification and notification of sexual and IDU partners. HIV positive IDUs represent a significant risk to their sex and drug use partners. HIV positive IDUs should be encouraged to inform partners of their HIV status so the partners can have the opportunity to practice HIV prevention themselves.
The next two high risk populations in order of severity are HIV negative IDUs and HIV negative non-IDUs. They should be targeted next for prevention and education. Many of the prevention strategies proposed above for HIV positive persons apply to these groups. HIV negative IDUs should be given the first priority because of their risky practice of IDU. They should be provided with the abovementioned programs to prevent drug use prevention, teach sexual negotiation skills and provide condoms. They also should be encouraged to return for re-testing after three months. Finally, HIV negative non-IDUs are the next target population. This group should be assessed for unsafe sex behaviors and non-IDU risky drug and or alcohol use. They should be provided with condoms, HIV prevention education, risk reduction skills, and information about the use of drugs and alcohol and practicing unsafe sex.
Third Priority: Harm Reduction and Treatment
Once high risk populations are identified and tested for HIV, and prevention and education have been implemented, attention should be given to reducing harm from existing risky behaviors and enhancing opportunities for treatment. The philosophy of harm reduction, as opposed to abstinence, should guide the initial delivery of tertiary prevention. While abstinence from both sex and IDU behaviors is the only safe way to prevent HIV transmission, this advice is often met with resistance, takes a long time to achieve, and is not realistic for many persons. The most commonly practiced harm reduction method to limit the spread of HIV among IDUs is implemention of NEP/SEP. Vinnitsya has had an NEP funded by a NGO for the past several years. This NEP reaches out to the community from two street sites, offering clean needle exchange and alcohol swabs. The concept of NEPs in Vinnitsya is not fully accepted by the police, and the NEP sites are regularly monitored for any illegal practices. It is recommended that the ICOHRTA investigators meet with the leader of this NGO and discuss ideas of offering free condoms and prevention literature, making referrals for treatment, site expansion, and additional funding. Strategies should be discussed about how to link the NEP with treatment providers in the community to refer persons needing and motivated to seek out drug addiction treatment or medical services related to IDU or HIV/AIDS.
Treatment for IDU and HIV/AIDS is limited in Vinnitsya. However, since the beginning of the ICOHRTA approximately four years ago, training, research opportunities, and grant funding for treatment of both IDU and HIV/AIDS has increased. Assessment of drug addiction treatment model preferences was assessed among providers and patients of the RND in preparation for the technology transport of behavioral, HIV prevention, motivational enhancement, and relapse prevention interventions [21]. The National Institute of Drug Abuse (NIDA) is funding this transport of interventions for IDU and HIV risk under the leadership of the second author through training in evidenced-based psycho-social and behavioral treatments, increased use of family support, and ultimately sustaining a raised standard of care at the RND. Finally, there is an increase in antiretroviral treatment of HIV/AIDS in Vinnitsya as a result of grants to the fourth author. It is recommended that the ICOHRTA continue to support such efforts and increase in-country investigators' independence in conducting community-based developmental research that will immediately raise the standard of care for IDU and HIV/AIDS, encourage further innovation, and ultimately have a positive effect on reducing the prevalence of HIV/AIDS.
Conclusion
It has been stated that in order to control the HIV/AIDS epidemic, one must first understand the cultural, political, economic, and religious context in which populations, individuals, and their behaviors are situated [22]. An increase in IDU in Ukraine within the last decade, for instance, may be explained by complex factors such as the country's economic crisis, rapid social change, and increased poverty and unemployment [7]. A high number of out-of-treatment IDUs within Ukraine's society may be explained by the lack of effective substance abuse treatment, limited number of HIV prevention programs for IDU, and/or stigma toward individuals who take part in substance abuse [14]. The findings of this study support these theories with respect to HIV/AIDS and IDU in the city of Vinnitsya, Ukraine.
Though the Vinnitsya Oblast is not the most severely affected region in terms of HIV infection, the assessment of the current state of the HIV/AIDS in this area of the country does shed some light on what is going on there. First, we have learned that there is a great shortage of resources for testing of HIV/AIDS (especially in the less populated areas) and low HIV/AIDS awareness in the general population. It is therefore possible that the statistics from these areas may be inadequate due to underreporting. Thus, there may not be such a large difference between the heavily affected regions (i.e. Odessa, Mykolaiv, Dnipropetrovs'k, and Donets'k) and other regions of the country. The most heavily infected regions may simply have better reporting systems and resources for HIV testing.
Second, while the statistics for Ukraine as a whole seem to indicate that there is a shift toward heterosexual transmission of HIV, a great majority of the HIV infections reported prior to 2004 in the Vinnitsya region were among IDUs. It seemed plausible, that the HIV/AIDS epidemic in the Vinnitsya region was a few years behind. In fact, there were no officially registered HIV infections in this region until the year 1996, and the current distribution of HIV infections by risk group actually resembles the national statistics for that same year. Unexpectedly, the majority of newly registered HIV cases in Vinnitsya in 2004 were attributed to heterosexual transmission (54.1%). It can be explained either by a true shift in the epidemic dynamics or by improved surveillance after the establishment of the Regional AIDS Centre; however, there are no sufficient data to document either explanation. In any case given that the great bulk of existing HIV cases are among IDUs it is reasonable to conclude that high, significant problems lie ahead without enhanced prevention and treatment efforts.
There also is a shift in the nature of drug use and the demographics of individuals who abuse drugs in this part of the country. Currently, individuals who abuse drugs tend to be younger, and the drugs tend to be more potent and riskier than they were when Ukraine was a part of the Soviet Union. Drug abuse appears to start with the use of alcohol and smoking of cannabis among children as young as 10 years of age, which may help to explain the earlier onset of IDU. Konoplya, a strong cannabis-based substance that was not as common among teenagers during the Soviet era (perhaps due to a stricter regulation at the borders), may serve as a gateway drug, leading the way to experimentation with more risky drugs (i.e. homemade stimulants and opiates). Because many IDUs make the opiate solution themselves in non-sterile home-laboratories, they have control over its strength; face a greater chance of contaminating the solution (i.e. by using unclean works and solvents); and present great risks of HIV transmission through needle and drug sharing. Prevention and treatment efforts have been attempted with limited success due to inadequate resources, training, and the burgeoning IDU problem in the area. Avenues for change in the drug addiction prevention and treatment fields are open due to caring and motivated health care providers and public health officials. In order to control the HIV/AIDS and IDU epidemics, one needs to consider a great deal of factors that fuel these epidemics. Changes may have to be made at the levels of individuals, services, communities, environments, and policies. Consequently, it will take a collective effort to make significant progress.
List of Abbreviations Used
ICOHRTA – International Clinical, Operational and Health Services Research and Training Award
IDU – Injection Drug Use
IDUs – Injection Drug Users
IDU-RAR – Rapid Assessment and Response in Injection Drug Use
NEP/SEP – Needle/Syringe Exchange Program
NGO – Non-governmental Organization
PMU – Vinnitsya National Pirogov Medical University
RND – Regional Narcological Dispensary
UAB – University of Alabama at Birmingham
WHO – World Health Organization
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Katerina Barcal, MPH conceived of the study and its design, the collection of data, organizing the data for qualitative analysis, drafting the article and making final edits, and giving final approval for publication.
Joseph E. Schumacher, PhD mentored Ms. Barcal in the study conceptualization, design, data analysis, manuscript preparation, giving final approval for publication.
Kostyantyn Dumchev, MD, MPH participated in the design and methodology of the study, collection of the data, organization of the data, editing the manuscript, and giving final approval for publication.
Larisa Vasiliyevna Moroz, MD, PhD participated in the acquisition and interpretation of the data, editing the manuscript, and giving final approval for publication.
Acknowledgements
This research was supported by the Sparkman Center for International Public Health Education and the International Clinical, Operational and Health Services Research Training Award from the Fogarty International Center of the National Institutes of Health (1 D43 TW05815-01). It would not have been possible to conduct the rapid assessment without the help and contribution of our collaborators and various individuals from the Vinnitsya, Ukraine area including health professionals from the Vinnitsya National Pirogov Memorial Medical University (PMU) and the Regional Narcological Dispensary (RND). We would especially like to thank Dr. Vasiliy Maximivich Moroz, President of the PMU, for inviting us to Vinnitsya and allowing us to conduct this research through the medical university. Dr. Pavel Slobodyanyuk, Director of the RND, and his staff allowed us to learn more about their center through observation, staff shadowing, and interviews. We would also like to acknowledge the contributions of Dr. Igor Matkovskiy, Dr. Vitaliy Polonets, and Natalia Vlasova for passing on their practical knowledge of the drug abuse and HIV/AIDS problem and needs in this beautiful city. And finally, we appreciate the support of the mayor of Vinnitsya, Oleksandr Dombrovsky, who is dedicated to solving the problem of drug abuse and HIV/AIDS in this city.
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Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-3-521619119610.1186/1477-7827-3-52ReviewSmoking and reproduction: The oviduct as a target of cigarette smoke Talbot Prue [email protected] Karen [email protected] Department of Cell Biology and Neuroscience, Interdepartmental Graduate Program in Environmental Toxicology, University of California, Riverside, CA 92521, USA2005 28 9 2005 3 52 52 2 8 2005 28 9 2005 Copyright © 2005 Talbot and Riveles; licensee BioMed Central Ltd.2005Talbot and Riveles; 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 oviduct is an exquisitely designed organ that functions in picking-up ovulated oocytes, transporting gametes in opposite directions to the site of fertilization, providing a suitable environment for fertilization and early development, and transporting preimplantation embryos to the uterus. A variety of biological processes can be studied in oviducts making them an excellent model for toxicological studies. This review considers the role of the oviduct in oocyte pick-up and embryo transport and the evidence that chemicals in both mainstream and sidestream cigarette smoke impair these oviductal functions. Epidemiological data have repeatedly shown that women who smoke are at increased risk for a variety of reproductive problems, including ectopic pregnancy, delay to conception, and infertility. In vivo and in vitro studies indicate the oviduct is targeted by smoke components in a manner that could explain some of the epidemiological data. Comparisons between the toxicity of smoke from different types of cigarettes, including harm reduction cigarettes, are discussed, and the chemicals in smoke that impair oviductal functioning are reviewed.
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A. Background
Exposure to cigarette smoke may be either active or passive, and the type of smoke inhaled in each case has a different origin. Mainstream smoke is the smoke that an active smoker inhales with each puff, while sidestream smoke, the main component of environmental tobacco smoke, burns off the end of a lit cigarette and is the smoke inhaled by passive smokers. While the association between inhalation of mainstream smoke and cardiovascular disease and cancer has been established for many years, the impact of smoking on reproduction is recognized, but less well characterized and less well known [1]. Epidemiological studies have repeatedly shown that women of child bearing age who actively inhale mainstream smoke have higher rates of infertility, spontaneous abortion, ectopic pregnancy, tubal infertility, increased time to conception, and intrauterine growth retardation than nonsmokers [2-15]. Increases in infertility and ectopic pregnancy in smokers could be due to impairment of oviductal functioning. In patients with primary tubal infertility, 39% were smokers when they started trying to conceive in contrast to only 16% in the non-smoking group (OR = 2.7) [10]. Heavy smoking (> 5 pack-years) increased the odds ratio to 4.2, and similar dose related effects have been repeatedly observed [11,16].
The realization that sidestream smoke exposure adversely affects human health is even more recent [17]. In 1992, the Environmental Protection Agency published a monograph summarizing evidence that exposure to environmental tobacco smoke can produce adverse effects on cardiovascular and lung health and encouraged broader investigation in this area [17]. Subsequently, a number of studies have addressed the effect of passive smoking on various aspects of human health including reproduction and have concluded that adverse reproductive outcomes, such as delayed time to conception and reduced birth weight, do occur as a consequence of exposure to environmental tobacco smoke during pregnancy [18-30]. Moreover, an in vitro fertilization lab recently concluded that while fertilization rates and embryo quality were similar in smokers and non-smokers, implantation and pregnancy rates were adversely affected by both active and passive smoking when compared to non-smoking controls [31].
Recent reviews have addressed issues of cigarette smoke exposure and various facets of reproduction including delayed time to conception, ovarian effects and premature menopause, implantation failure, fetal growth restriction and growth retardation, placental abnormalities, reduced fecundity, congenital abnormalities, and effects on male reproduction [32-34]. However, most prior reviews have not considered smoke's interaction with the oviduct, an organ vital to reproduction. The purpose of this paper is to review the functions of the oviduct, in particular those that involve movement of gametes and embryos, and to evaluate evidence that exposure to mainstream or sidestream cigarette smoke can negatively impact oviductal functioning and thereby adversely affect reproductive outcomes. We will also consider evidence that commercial cigarettes, including harm reduction and light cigarettes, contain toxicants that impair oviductal functioning, and we will discuss the specific chemicals in smoke that impair oviductal functioning. Some of these chemicals adversely affect oviductal processes at extremely low doses, are often considered safe, and are added to cigarettes and other consumer items.
B. Functions of the oviduct
The oviduct, which is divided anatomically into the infundibulum, ampulla, and isthmus, plays important roles in mammalian reproduction (Fig. 1) [35-41]. The infundibulum is responsible for picking-up the oocyte cumulus complex following ovulation and moving it into the ampulla where fertilization occurs. Simultaneously, the oviduct moves sperm in the opposite direction from a reservoir near the uterotubal junction toward the ampulla [42]. The oviduct also provides a suitable microenvironment for capacitation of spermatozoa, fertilization, preimplantation development, and transport of the preimplantation embryos to the uterus. The movement of the embryo through the oviduct to the uterus is carefully timed by ovarian hormones and signals from the embryos [43]. While smoke exposure could affect any of these processes, most current evidence links smoke to effects on oocyte cumulus complex pick-up and embryo transport, which will be reviewed in more detail in the following sections.
Figure 1 Schematic diagram showing the three anatomical regions of the oviduct (infundibulum, ampulla, and isthmus) and the regions of the oviduct where oocyte cumulus complexes and preimplantation embryos can be found. Oocyte cumulus complexes are ovulated from ovaries (#1), picked-up by the outer surface of the infundibulum (#2), and moved toward the ostium (unlabeled arrow) by ciliary beating then into the ampulla for fertilization (#3). Fertilized eggs and embryos are transported through the isthmus to the uterine cavity where they then can implant in the uterine wall.
(1) Oocyte cumulus complex pick-up by the infundibulum
The infundibulum is the portion of the oviduct closest to the ovary and is responsible for picking up the oocyte cumulus complex following its ovulation from a mature ovarian follicle [44,45]. The oocyte cumulus complex consists of a centrally located oocyte, which is in turn surrounded by the zona pellucida, corona radiata, and cumulus cells (Fig. 2) [46-48]. The complex contains 5,000–8,000 cumulus cells, depending on the species, and these are separated from each other by an extracellular matrix, which plays an important role in the pick-up process. The structure and distribution of the extracellular matrix between cumulus cells has been well characterized in a number of species including humans [46,49-52]. Biochemically, the matrix is rich in hyaluronan (hyaluronic acid) [53-55], which is cross-linked by inter-alpha trypsin inhibitor [56-58]. TSG-6 (the secreted product of the tumor necrosis factor-stimulated gene 6) also binds to hyaluronan in the cumulus matrix [59-61]. The importance of these matrix components to reproduction is demonstrated by the TSG-6 knockout mouse which fails to assemble a cumulus matrix and is infertile [62].
Figure 2 Schematic diagram of an oocyte cumulus complex after ovulation from an ovarian follicle. The oocyte and polar body are contained within the zona pellucida. Immediately outside the zona, cells are densely packed to form the corona radiata outside of which are numerous cumulus cells. The cumulus cells are widely separated from each other by spaces filled with an extracellular matrix (matrix is shown in Figure 5).
Oocyte pickup by the infundibulum is a complex process that involves both ciliary beating and adhesion between the oviductal epithelium and the oocyte cumulus complex [63-73]. Both the inner and outer surfaces of the infundibulum are covered with ciliated epithelium (Fig. 3) [74]. Following ovulation, the oocyte cumulus complex travels along the outer surface of the infundibulum and enters the oviduct through the ostium (Fig. 3) [45,75]. The complex then rapidly moves to the ampulla where fertilization occurs. Although infundibular smooth muscle may contract during the pick-up process, it does not appear to be necessary for pick-up, which still occurs when muscle contraction is inhibited with isoproterenol [76].
Figure 3 Scanning electron micrograph showing a hamster oocyte cumulus complex, colorized blue, entering the ostium of an infundibulum. The outer and inner surfaces of the infundibulum are covered with cilia (inset).
Huang et al., developed an in vitro method for measuring oocyte pickup rate using hamster infundibula [71]. At room temperature, oocyte pickup rate averaged 55.2 + 10.6 um/sec and was dependent on the viscosity of the culture medium and temperature. Moreover, complexes were observed to move along particular pathways on the surface of the infundibula depending on where they were placed. This in vitro bioassay has subsequently evolved to allow measurement of smooth muscle contraction [77] and adhesion of the oocyte cumulus complex to the infundibulum [72].
The hamster infundibular explant has also been used to analyze the process of pick-up in hamsters in conjunction with video microscopy [45]. While small particles such as Lycopodium spores can move over the infundibular surface in the currents created by ciliary beating [45,78], the large mass of the oocyte cumulus complex does not allow it to move in the fluid currents created by ciliary beating alone. In addition to ciliary beating, adhesion between the cumulus cell matrix and the tips of the cilia is necessary to move the complex over the surface of the infundibulum [45,72]. The cumulus matrix attaches the complex to the infundibulum, and as the cilia beat in the direction of the ostium, the oocyte cumulus complex glides over the surface of the infundibulum until it reaches and enters the ostium. Figure 4 (Additional file 1) links to a video showing the movement of a hamster oocyte cumulus complex over the surface of an infundibulum. Additional videos of this process can be viewed at . In hamsters, the oocyte cumulus complex is larger in diameter than the opening of the ostium, and in order for the complex to enter the oviduct, it goes through a "churning" process that compacts the matrix between the cumulus cells making the complex small enough to pass through the ostium [45]. During churning, the oocyte is sometimes squeezed from the center of the complex to the periphery. Pick-up of a human oocyte cumulus complex has been observed in vivo using transvaginal hydrolaparascopy and involves adhesion of the complex to the tumescent fimbria of the infundibulum with ciliary beating drawing the complex into the ostium [75].
Figure 4 Micrograph showing a hamster oocyte cumulus complex, stained blue, on the outer surface of an infundibulum. Click the link to view a video of this complex being picked up by the oviduct. Reprinted from Molec Biol Cell 10:5–9, 1999 (with permission). See also
Adhesion plays an essential role in the pick-up process (Fig. 5) [66,72]. Oocytes denuded of cumulus cells are not picked up [66], and when matrix is not secreted by the cumulus cells, the complex fails to attach to the infundibulum and it is not moved into the oviduct [72]. Polycationic compounds can block oocyte cumulus complex pick-up apparently by blocking transient adhesion between the tips of the cilia and the complex [67]. Interestingly, peritoneal fluid from women with endometriosis contains a macromolecule (< 100,000 kDa) that when assayed with hamster infundibula in vitro coats the cilia on the surface of the infundibula and blocks adhesion and hence pick-up of the human oocyte cumulus complex by the hamster infundibulum [79,80]. Transmission electron microscopy revealed that adhesion during complex pickup occurs specifically between the cumulus matrix and the crowns at the tips of the infundibular cilia [72]. An in vitro assay using vacuum from a low flow peristaltic pump has been developed to measure adhesion between the oocyte cumulus complex and infundibulum [72]. This assay was used to show that factors that either increase or decrease adhesion can interfere with the pick-up process. If the matrix of the oocyte cumulus complex is made less sticky by compacting it or treating it with poly-l-lysine, the complex cannot adhere tightly enough to the infundibulum to be successfully picked up [72]. Conversely, if adhesion is increased, for example by treating complexes or the oviduct with the lectin wheat germ agglutinin, ciliary beating is not strong enough to transiently detach the complex and move it to the ostium. Thus successful pick-up requires a delicate balance between proper strength of adhesion of the complex to the infundibulum and ciliary beating towards the ostium.
Figure 5 Micrographs showing adhesion between the oocyte cumulus complex and the infundibulum. (A) Stereoscopic micrograph of an oocyte cumulus complex, colorized blue, being pulled off the surface of an infundibulum using forceps. The matrix of the complex adheres to the infundibulum. Complexes do not adhere to most other surfaces. (B) Scanning electron micrograph of cumulus matrix adhering to cilia on the outer surface of an infundibulum. The matrix was left behind by an oocyte cumulus complex that was picked-up by the infundibulum.
The ampulla serves as a reservoir for the oocyte cumulus complex, and hormonally controlled oviductal secretions play an important role in creating a suitable microenvironment for fertilization and initial preimplantation development [37,44,81,82]. After entering the female reproductive tract, sperm are stored in a reservoir near the uterotubal junction [42]. As some sperm leave the reservoir and move through the isthmus of the oviduct, they become fully capacitated and their motility becomes hyperactivated [38,83,84]. Hyperactivation is thought to be critical to fertilization as it allows sperm to detach from the oviductal epithelium, move in the lumen of the oviduct, and penetrate through the extracellular matrices surrounding the oocyte [84]. Sperm meet the oocyte cumulus complex in the ampulla where fertilization normally occurs, and after fertilization, the preimplantation embryo undergoes cleavage as it is transported through the ampulla and the isthmus to the uterus for implantation [47]. Movement through the ampulla may involve both ciliary beating and smooth muscle contraction. When sections of the ampulla were surgically reversed in their orientation, few rabbits became pregnant [85]. In cases where pregnancy did occur, muscle contraction apparently overcame ciliary beating toward the ovary, showing that the cilia in the ampulla normally play an important role in controlling movement into the isthmus [85]. The isthmus of the oviduct is essential for normal reproduction, as its removal results in infertility [86].
(2) Transport of preimplantation embryos to the uterus
A number of factors can influence the transport of preimplantation embryos through the ampulla and isthmus of the oviduct. Interestingly, the oviduct can distinguish between unfertilized oocytes and preimplantation embryos. which are transported at different rates, with embryos reaching the uterus one day earlier than unfertilized oocytes [87]. The production by embryos of platelet-activating factor (PAF), which mediates signaling to the oviduct, accelerates the passage of preimplantation embryos, but not oocytes, through the oviduct [88]. PAF may affect transport by increasing ciliary beating [89]. Human embryos likewise release PAF in vitro, and human oviducts synthesize both the PAF receptor and PAF acetylhydrolase, which degrades PAF, further supporting a role for PAF in the embryo-oviductal dialogue [90]. When rat embryos of different ages were transferred to the oviduct of pregnant females, older embryos reached the uterus before younger ones, again suggesting differential transport rates of embryos that depend on age [91]. These data from hamsters and rats support the idea that embryo transport is at least, in part, subtly controlled by the embryos themselves. Other factors such as maternal age and parity also influence embryo transport [92]. In hamsters, transport to the uterus occurred faster in young nulliparous females than in nulliparous or multiparous adult females. In the group of young females, but not the adults, development of the embryos was also highly synchronous.
Transport of preimplantation embryos through the oviduct is accomplished by smooth muscle contraction and ciliary beating [76,93]. However, the relative contributions of these two processes are not yet completely understood, and it is probable that both play roles in transport. The ampulla and isthmus are both lined by ciliated cells, which beat in the direction of the uterus [76]. The relative number of cilia decreases and the thickness of the muscle layers increases proceeding toward the uterine end [94], suggesting that cilia become relatively less important in the isthmus. Muscle contraction produces oscillating movements in the isthmus [64,95,96] that result in a net transport of preimplantation embryos towards the uterus [97]. Nitric oxide synthase inhibitors increased muscular activity in rats, and this was accompanied by increased rate of movement of eggs or microspheres in the oviduct [97]. These data support the idea that that muscle contraction can modulate (speed or slow) transport through the oviduct. Muscle contractions may also be important in keeping embryos grouped together as they are transported through the isthmus [98].
Muscle contraction in the oviduct is regulated by a variety of factors; however, the interplay of these factors with each other is not yet well understood. The oviductal muscles are innervated by the sympathetic nervous system [99,100]. Stimulation of α adrenergic receptors promotes contraction of the oviductal muscles, while stimulation of β receptors inhibits contractions [100-102]. Alternating contraction and relaxation produce the oscillatory movements involved in embryo transport. However, the adrenergic neurons may not be the primary means for controlling embryo transport since experimental depletion or inhibition these neurons does not prevent transport nor decrease fertility [35]. Chemicals produced by the oviduct itself or the preimplantation embryo can also modulate muscle contraction and may play the lead role in embryo transport. Oviductal muscle responds to sex steroids and prostaglandins. Endogenous estrogens stimulate oviductal muscle contraction, while progesterone relaxes it [103]. Likewise the prostaglandins PGF and PGE contract and relax oviductal muscle respectively [104-106]. Human oviduct smooth muscle also produces the prostaglandin prostacyclin which decreases muscle contractility and may affect embryo transport [107]. Oviductal smooth muscle also contains a nitric oxide system [108] that promotes muscle relaxation [109]. Inhibition of nitric oxide syntheses in rats increases oviductal motility and results in accelerated movement of embryos through the oviduct [97]. In additions to prostaglandins, the oviduct produces, endothelin-1 [110] and angiotensin II [111] which are involved in modulating oviductal muscle contraction and regulation of embryo transport. Recent data from the cow suggest that tumor necrosis factorα from the oviductal epithelium, immune cells of the oviduct, or even the embryo itself stimulates the release of these effectors from the oviductal epithelial cells [111]. A newly uncovered transport regulatory mechanism involves the cannabinoid receptor CB1 [112]. When CB1 is genetically or pharmacologically silenced, embryos are retained in the oviduct. This effect can be reversed by isoproterenol, a β adrenergic agonist. These data suggest that cannabinoid signaling is important in coordinating oviductal muscle contraction, and is necessary for proper embryo transport. While this review deals with conventional cigarette exposure, these results with the CB1 receptor suggest that exposure to marijuana for either recreational use or pain relief could affect embryo transport and hence female fertility. It is clear from the preceding that the regulation of embryo transport through the oviduct is complex and may depend on multiple regulatory mechanisms, some of which are just now being identified.
(3) Biological importance of pick-up and transport by the oviduct
Timing of oocyte pick-up and embryo transport is critical as the preimplantation embryos must arrive in the uterus during the window when implantation can occur [113,114]. If the oocyte is not picked up by the oviduct or if the embryo moves through the oviduct too quickly or too slowly, implantation may fail to occur or may be ectopic. In rats, embryo transport was accelerated by treatment with methoxychlor, an estrogenic pesticide, and the embryos failed to implant in the uterus [115]. Likewise women treated with ergonovine maleate, a powerful stimulant of oviductal contraction, showed decreased conception rates when the drug was delivered immediately post coitus [116]. Interference with embryo transport can adversely affect fertility and in humans lead to ectopic implantation.
C. Evidence that the oviduct is a target of cigarette smoke
While epidemiological studies have been clear in identifying increased reproductive risks for women who smoke both actively and passively (Section A), the reasons that smoke causes reproductive problems are usually not understood. Some of the risk factors for women smokers, such as ectopic pregnancy, failure to conceive, increased time to conception, could be due to effects of smoke on the pick-up and transport by the oviduct. We will next examine the in vivo and in vitro evidence supporting the idea that the oviduct is targeted by cigarette smoke.
(1) In vivo evidence that the oviduct is a target of smoke
Direct inhalation of whole smoke has been shown in several studies to adversely affect the oviduct. Oviductal motility is altered in humans [117] and in rabbits [118] by inhalation of mainstream smoke. Inhalation of mainstream or sidestream smoke by hamsters, at serum cotinine levels that were within the ranges found in active and passive human smokers (mainstream = 72.8 and sidestream = 14.9 ng cotinine/mL) produced blebbing of the oviductal epithelium at the ultrastructural level and decreased the ratio of ciliated to secretory cells in the ampulla [119]. In a related study on hamsters, inhalation of either mainstream or sidestream smoke at levels that produced serum cotinine levels equivalent to those in human smokers (mainstream = 50–250 and sidestream = 18–80 ng cotinine/ml) slowed preimplantation embryo transport through the oviduct [120]. In addition, muscle contractions of the ampulla were significantly inhibited in vivo during smoke exposure, supporting the conclusion that embryo transport rates were slowed by inhibition of smooth muscle contraction [120]. While smooth muscle contraction rates did increase after smoke exposure stopped, they did not return to control levels, showing that inhibition of contraction by smoke is not immediately completely reversible.
Several in vivo studies using animal models have established that the oviduct is a target of nicotine, a major constituent of cigarette smoke. When administered to mice in drinking water, nicotine (108 μmol/L) significantly decreased Na and K ion concentrations in the oviductal epithelium [121]. In addition, nicotine injected subcutanenously (2.5 mg twice daily) into rats produced a significant increase in lactate dehydrogenase levels in flushings of the oviduct in early pregnancy [122]. While a change in the ionic composition of the oviductal epithelium or its secretions might adversely affect adhesion of the oocyte to the oviductal surface and the oocyte pick-up process, the relationship between these nicotine-induced changes and oviductal functioning has not yet been established experimentally. Nevertheless, these studies do demonstrate that nicotine exerts effects on the oviductal epithelium.
Two additional studies on rats indicate further effects of nicotine on oviductal functioning. When pregnant rats were treated with pharmacological doses of nicotine (2.5 mg injected subcutaneously twice daily), preimplantation embryo transport was inhibited [123]. In addition, nicotine (5 mg/kg), when injected subcutaneously twice daily in pregnant rats, both retarded embryonic development and reduced blood flow to the oviduct [124]. Reduction of oviductal blood flow decreases smooth muscle contraction, which in turn can delay embryo transport [124,125]. In other studies, nicotine slowed oviductal contraction in vivo in the Rhesus monkey, which may prevent implantation [126]. Oral nicotine administration through drinking water (108 μmol/L) also interfered with oocyte maturation, fertilization, and early pregnancy in mice [121]. Collectively these data show that nicotine affects the composition and secretions of the oviductal epithelium, adversely affects preimplantation development, retards movement of embryos through the oviduct, and reduces blood flow to this organ.
In a study involving gamete intrafallopian transfer (GIFT), no differences were found among active, passive and non-smokers in number of oocytes retrieved; however the number of live births after GIFT was significantly lower for active smokers (10.5%) than for passive smokers (23.1%) or non-smokers (33.3%) smokers [127], which could indicate an effect of smoke on the human oviduct.
Taken together these in vivo studies demonstrate that the oviduct responds to exposure to both whole mainstream and sidestream smoke and to nicotine and that the transport of preimplantation embryos can be inhibited by cigarette smoking, apparently by an inhibition of oviductal smooth muscle contraction. In vivo studies have not yet been undertaken to determine if oocyte cumulus complex pick-up is slowed in smoke exposed animal models or humans.
(2) In vitro evidence that smoke affects oviductal functioning
In vitro models have facilitated studies on smoke's effect on the oviduct and have further supported the conclusion that the oviduct is responsive to chemicals in cigarette smoke (Fig. 6, additional file 2). A hamster infundibular explant model [71] has been used to simultaneously measure ciliary beat frequency, adhesion, oocyte pick-up rate, and muscle contraction, before, during, and after exposure to smoke or its constituents [70,128-133]. In general, in vitro studies show that mainstream and sidestream smoke solutions adversely affect proper functioning of the oviduct. Mainstream and sidestream smoke solutions made from University of Kentucky 2R1 research cigarettes in a medium lacking bovine serum albumin (BSA) inhibited ciliary beat frequency in a dose dependent manner [128]. When BSA was included in the medium, mainstream solutions continued to inhibit beat frequency, while sidestream solutions either had no effect or slightly stimulated beat frequency, suggesting that the presence of BSA influenced how sidestream smoke affected beat frequency [129]. Interestingly, in both mainstream and sidestream solutions containing BSA, oocyte pickup rate was inhibited in a dose-dependent manner with sidestream smoke often being more inhibitory than mainstream smoke (Fig. 6). Since ciliary beat frequency was either not affected or slightly stimulated in sidestream smoke, these data show that smoke can inhibit oocyte pick-up rate by affecting factor(s) other than ciliary beat frequency. Oocyte pickup rate was more sensitive to the gas than the particulate phase of mainstream and sidestream smoke solutions [129].
Figure 6 Micrographs showing oocyte cumulus complex pick-up by a hamster infundibulum of a control (6B) and a sidestream smoke treated (6A) preparation. Click the link to view a video of this experiment. During approximately 10 seconds of observation, the control complex moves toward the ostium while the smoked treated complex does not move. Reprinted from Molec Biol Cell 10:5–9, 1999 (with permission). See also
Since pick-up rate decreased in sidestream smoke when beat frequency increased, oocyte pick-up rate must depend on factor(s) other than ciliary beat frequency [129]. Since adhesion of the oocyte cumulus complex to the tips of the cilia is important in oocyte pick-up [66,68,72], the effect of smoke solutions on adhesion was measured in vitro using the hamster infundibular model. Both mainstream and sidestream solutions inhibited oocyte cumulus complex pick-up rate and increased adhesion of the cumulus to the oviduct [133]. As was shown previously using wheat germ agglutinin [72], increasing adhesion by exposure to smoke leads to a decrease in pick-up rate since the complex can not be moved by the cilia if it adheres too tightly to the oviduct. These effects on adhesion and pick-up rate were observed when only the oocyte cumulus complex was pretreated with smoke solution or when only the infundibulum was pretreated, indicating that both the cumulus matrix and oviduct are targets of smoke treatment [133]. The oviduct was more sensitive to treatment than the oocyte cumulus complex, perhaps because smoke pretreatment affected both ciliary beat frequency and adhesion of infundibula but only adhesion of oocyte cumulus complexes. These data indicate that factors that increase adhesion of the oocyte cumulus complex to the cilia can decrease pick-up rate and explain why both mainstream and sidestream smoke solutions decrease pick-up rate even when ciliary beat frequency is increased by treatment with sidestream smoke.
The above studies were all done using non-filtered 2R1 research brand cigarettes manufactured at the University of Kentucky. A subsequent study examined the effects on oviductal functioning of smoke solutions from a filtered research brand cigarette (1R4F), various traditional filtered and non-filtered commercial cigarettes (Marlboro Red, Marlboro Light, Camel filtered, Camel unfiltered, Kool, and Kool with the filter removed), and three brands of harm reduction cigarettes (Advance, Omni and Omni Light) [134]. Harm reduction cigarettes have recently been introduced commercially and are claimed to contain fewer carcinogens than traditional commercial brands [135]. All of the cigarettes tested (research, traditional commercial, harm reduction) adversely affected oviductal functioning, and the effects were in general stronger on oocyte pick-up rate and smooth muscle contraction than on ciliary beating. Sidestream smoke generally produced a stronger effect in all assays than mainstream smoke solution. Smoke from the 1R4F cigarettes, which more closely resemble the commercial brands smoked today than the 2R1 cigarettes, was considerably more inhibitory in the pick-up rate and muscle contraction assays than the 2R1s. Except for mainstream smoke from Marlboro Lights and Kools, all traditional brand smoke solutions reduced pick-up rate by more than 60%. Except for mainstream smoke from Marlboro Lights and Camel filtered, all smoke solutions from traditional brands reduced muscle contraction by more than 80%. Smoke from harm reduction cigarettes reduced pick-up rate by 50–80% and reduced muscle contraction by 30–98% depending on the type of smoke and brand. These data show that the adverse effects observed on oviducts with 2R1 research cigarettes are also produced by filtered research cigarettes and by filtered, non-filtered and light commercial brands. Moreover, harm reduction cigarettes, while reduced in levels of carcinogens, still contain toxicants that can impair oviductal functioning.
Pick-up rate could also be altered by action of smoke on the oocyte cumulus complex, in particular the matrix which is required for adhesion to the cilia [72]. Both mainstream and sidestream smoke solutions from 2R1 cigarettes caused more dispersal of hamster cumulus cells during in vitro incubation than control medium lacking smoke, and oocyte pick-up rate was slowed when oocyte cumulus complexes were pretreated with smoke prior to measuring pick-up rate [133]. In addition,in vitro exposure of porcine oocyte cumulus complexes to nicotine, cadmium, and anabasine, all components of cigarette smoke, suppressed FSH induced expansion of the cumulus and decreased synthesis and accumulation of hyaluronic acid in the cumulus matrix [136]. These studies show that the matrix of the oocyte cumulus complex is also a target of cigarette smoke and damage to the matrix can affect pick-up of complexes by the oviduct.
D. What chemicals in cigarette smoke impair oviductal functioning?
(1) Chemicals most studied in smoke
Cigarette smoke is a complex suspension that contains over 4,000 chemicals distributed between a gaseous and particulate phase [17]. Most studies on cigarette smoke and its components have focused on nicotine [137-139], carcinogens [140,141], polycyclic aromatic hydrocarbons (PAHs) [142-144], such as benzo-a-pyrene, tobacco-specific nitrosamines [145,146], carbon monoxide [147,148], tar [149-151], metals (cadmium, lead) [152,153], and ten to fifteen other compounds including, phenol, acrolein, acetaldehyde, hydrogen cyanide, and formaldehyde [17,154-164]. Most of these chemicals have been linked to cancer or cardiovascular and lung disease. Of the 4,000 compounds found in cigarette smoke, at least 50 are known carcinogens [141]. PAHs and tobacco-specific N-nitrosamines are major contributors to lung cancer [154,161,165,166], while tar and carbon monoxide are major contributors to cardiovascular disease and chronic obstructive lung diseases [161,163,167]. PAHs have also been shown to initiate or promote atherosclerosis in avian [168-170] and mammalian [171] models. The PAH benzo(a)pyrene, induces atherosclerosis by stimulating proliferation of vascular smooth muscle cells that migrate into the vessel lumen [172,173].
(2) Chemicals in smoke that affect the oviduct
Most of the above-mentioned chemicals that are commonly studied in smoke have not been studied with respect to their effects on the oviduct. An exception is nicotine, which did alter oviductal epithelium secretion and ion composition, embryo transport, embryo development, and oviductal blood flow in several in vivo studies (Section C1) and cumulus expansion in vitro [136].
(a) Ciliotoxic chemicals
Numerous studies have shown that cigarette smoke contains chemicals that are toxic to cilia of the mammalian respiratory system [174-178], the amphibian palate [179], Paramecium [180,181], and the gills of mussels, clams, and mollusks [182,183]. Moreover, nicotine increased ciliary beat frequency in the ferret trachea [184], formaldehyde inhibited respiratory cilia in the rabbit and pig [185]; and hydrogen cyanide, acrolein, and acetaldehyde inhibited ciliary beating in the clam [182]. Using an in vitro infundibular bioassay, the individual smoke constituents, which had been previously shown to be ciliotoxic in non-oviductal systems [175,176,182], were tested specifically for their effect on oviductal cilia [186]. Potassium cyanide (KCN), acrolein, phenol, acetaldehyde, and formaldehyde all inhibited ciliary beat frequency in a dose dependent manner in vitro [186]. However, only KCN was present in cigarette smoke solutions in a high enough concentration to account for the effect seen in vitro. KCN also inhibited oocyte pick-up rate. Nicotine did not inhibit ciliary beat frequency of the hamster oviduct, except at extremely high doses (Talbot, unpublished data).
(b) Pyridines, pyrazines, and phenols
Since cigarette smoke contains over 4000 compounds [17], there are likely other chemicals present in mainstream and sidestream cigarette smoke that can adversely affect oviductal functioning. To identify such chemicals, mainstream smoke solution from 2R1 cigarettes was fractionated by passage through 12 different solid phase extraction cartridges [77]. The eluates from each cartridge were screened using the hamster infundibular bioassay, and three cartridges were identified that retained inhibitory activity in the ciliary beat frequency, oocyte pick-up rate, and smooth muscle contraction bioassays. The chemicals in the eluates of each cartridge were then identified using GC-MS, and authentic standards of the identified compounds were purchased from commercial sources and tested independently to determine their activity in each of the three infundibular bioassays.
Pyridines, pyrazines, and phenols were the three major groups of chemicals identified in the inhibitory eluates (Table 1) [77,131,132]. Several other types of compounds including quinolines, indoles, and cyclopenten-1-ones were also present [132]. Within all groups, chemicals were identified that were highly inhibitory in all three bioassays, and some of the chemicals had LOAELs (lowest observed adverse effect levels) in the nano, pico and femtomolar range (Table 1). In general, if a chemical were inhibitory, it acted in all three bioassays, although the potency and efficacy for a particular chemical varied among the assays. Some of the compounds that were identified in this screen (Table 1) were previously thought to be safe and are included on the FEMA GRAS list (Flavor and Extract Manufacturers' Association – Generally Regarded As Safe) and the FDA EAFUS list (Everything Added to Food in the United States). Some of these chemicals are added to tobacco to flavor it (Table 1). For example, 3-ethylpyridine, which was inhibitory in all three bioassays at picomolar doses, is on the list of 599 chemicals added to tobacco in the United States [187]. Of the seven pyrazines tested, six are on the FDA EAFUS list, and in all but three assays, the pyrazines had LOAELs in the nanomolar or picomolar range. Indole and isoquinoline were the most toxic of all chemicals tested with LOAELs in the femtomolar range, except for isoquinoline which had a picomolar LOAEL in the ciliary beat frequency assay.
Table 1 Chemicals in Cigarette Smoke that Impair Oviductal Functioning 1
LOAELs (M)2
CHEMICALS Oocyte Ciliary Beat Contraction FEMA FDA ADDED5
Pick-up Rate Frequency Rate GRAS3 EAFUS4
PYRIDINES
2-ethylpyridine 9.35 × 10-12 9.35 × 10-12 9.35 × 10-12
4-methylpyridine 9.50 × 10-11 9.50 × 10-11 9.50 × 10-11
2-methylpyridine 9.35 × 10-11 9.35 × 10-11 9.35 × 10-10
4-ethenylpyridine 9.30 × 10-11 9.30 × 10-9 9.30 × 10-11
3-ethylpyridine 9.33 × 10-10 9.33 × 10-11 9.33 × 10-10 √ √ √
Nornicotine 6.85 × 10-9 6.85 × 10-8 6.85 × 10-8
beta-nicotyrine 6.33 × 10-9 6.30 × 10-8 X6
2,4,6-trimethylpyridine 8.25 × 10-8 8.25 × 10-6 8.25 × 10-8
2,4-dimethylpyridine 9.34 × 10-7 X6 9.34 × 10-9
2,3-dimethylpyridine 9.34 × 10-7 9.34 × 10-7 X6
4,4-bipyridine 8.78 × 10-6 8.78 × 10-7 8.78 × 10-4
2,5-dimethylpyridine 9.34 × 10-5 X6 9.34 × 10-5
3,4-dimethylpyridine 1.76 × 10-5 X6 1.76 × 10-4
pyridine 1.27 × 10-5 1.27 × 10-3 1.27 × 10-3 √ √ √
3-methylpyridine 1.23 × 10-5 Xd 1.23 × 10-2
2,2-bipyridine 8.74 × 10-4 8.74 × 10-2 8.74 × 10-2
cotinine 2.84 × 10-2 2.84 × 10-5 X6
nicotine 9.01 × 10-2 X6 6.70 × 10-2
PYRAZINES
2-methoxy-3-methylpyrazine 10-12 10-9 10-12
pyrazine 10-11 10-12 10-9 √ √
2-methylpyrazine 10-11 10-12 10-11 √ √ √
2-ethylpyrazine 10-11 10-12 10-12 √
2,5-dimethylpyrazine 10-11 10-8 10-9 √ √ √
2,3,5-trimethylpyrazine 10-10 10-9 10-9 √ √ √
2,6-dimethylpyrazine 10-9 10-6 10-7 √ √ √
PHENOLS, INDOLES, OTHERS
Indole 10-14 10-13 10-15 √
Isoquinoline 10-13 10-12 10-13 √
4-Ethylphenol 10-12 10-11 10-12 √ √
Quinoline 10-11 10-13 10-11 √
4-Methylphenol 10-11 10-12 10-11 √ √
2-Methylphenol 10-11 10-9 10-11 √ √
5-Methylindole 10-11 10-10 10-10
2,6-Dimethoxyphenol 10-11 10-10 10-9 √
Hydroquinone 10-10 10-10 10-10
3-Methyl-2-cylcopenten-1-one 10-10 10-7 10-10
2,4-Dimethylphenol 10-10 10-9 10-9
2-Methoxyphenol 10-10 10-8 10-8
2-Cyclopenten-1-one 10-9 10-7 10-9
4-Methoxyphenol 10-8 10-7 10-7
2-Ethylphenol 10-8 10-5 10-7
2,5-Dimethylphenol 10-7 10-8 10-6
Benzene 10-7 10-8 10-6
Phenol 10-2 10-1 10-2 √
1Compiled from References 131, 132, and 135.
2LOAEL = Lowest observed adverse effect level in the oocyte pick-up rate, ciliary beat frequency, and muscle contractions assays.
3Chemicals known to be on the FEMA GRAS list. Others may also be on this list.
4Chemicals on the FDA EAFUS list (Everything Added to Food in the United States).
5Chemicals that are added to cigarettes by American tobacco companies.
6No effect at the highest dose tested.
Many of the compounds in Table 1 were also screened using a chick chorioallantoic membrane (CAM) assay that measures growth of the CAM and chick embryo [188,189]. In the CAM assay, many pyridines and pyrazines inhibited CAM growth dramatically, even at very low doses, and in some cases they also inhibited embryo growth. It is interesting that the chemicals in Table 1 were inhibitory in assays that measure diverse biological processes (ciliary beat frequency, oocyte pick-up, smooth muscle contraction, growth). It is not yet known if inhibition occurs by distinct mechanisms or if a basic underlying mechanism, such as inhibition of ATP production, was affected. Nor is it known if the chemicals act directly or indirectly, but given the extremely low effective doses of some of the chemicals, activation of a signaling cascade is possible.
These data from the oviduct and CAM studies indicate a need for further toxicological testing on the chemicals in Table 1, especially since some of them are used routinely in consumer products including food, cigarettes and cosmetics. These data also indicate a need for additional studies on the chemicals in smoke in general. The solid phase cartridge screen found about 40 oviductal toxicants, most of which were not previously recognized as smoke toxicants. It is probable that there are other toxicants in cigarette smoke that have not yet been identified as harmful nor studied in detail.
E. Concentrations of smoke toxicants in cigarettes and in human smokers
The data on oviductal toxicants beg the question – what are the concentrations of these compounds in cigarette smoke and in actual active and passive smokers? Some of the oviductal toxicants, such as nicotine, have been studied extensively, and concentrations are well documented in both cigarettes and smokers [17,190-192]. The LOAEL concentrations of 2-ethylpyridine, 2-methylpyridine, and 3-ethylpyridine are about 10,000 to a million times lower than the concentration of these chemicals in mainstream and sidestream smoke from commercial cigarettes and cigars [77,193]. However, some of these toxicants, such as 3-ethylpyridine, have not previously been recognized as harmful, and little is known about their concentrations, in smokers. Many chemicals were inhibitory in the infundibular bioassays at nano and picomolar doses suggesting that they could be effective in vivo at extremely low doses that would be difficult to detect and measure. Concentrations of cigarette smoke components, such as phenolic compounds, vary among different brands of cigarettes [194]. For example, different types of cigarettes, such as Indian Bidi cigarettes, have higher concentrations of phenols than traditional commercial cigarettes [157]. Concentrations of chemicals also vary between various research brand cigarettes [195]. Finally, the source of the smoke also affects chemical concentration. While mainstream and sidestream smoke have similar chemicals the relative amounts of particular chemicals can vary significantly between the two types of smoke [17].
Cigarette smoke chemicals or their metabolites must gain access to the circulatory system and reach their target organs to exert their toxicity. Studies have measured nicotine, cotinine (a metabolite of nicotine), and other cigarette smoke components in reproductive tissues, although little is known about concentrations in the oviduct per se. Interestingly, levels of cigarette toxicants in reproductive tissues or fluids can be significantly higher than in serum. For example, pregnant rabbits injected with tritiated nicotine had 5–11 times higher nicotine concentrations in uterine fluid than in plasma [196]. Both nicotine [197] and phenols [198] have been detected in the cervical fluid of smokers, and nicotine concentrations in the cervical fluid (66–2620 ng/ml) were significantly higher than in serum (0.1–39 ng/ml) [199].
Cigarette smoke components have also been detected in the follicular fluid of smokers [200,201]. Cotinine, a biomarker for smoke exposure, was higher in the follicular fluid of active smokers (mean = 285.69 ng/ml; range = 62.21–595.00) than passive smokers (mean = 29.65 ng/ml; range = 20.91–45.75) and nonsmokers (mean = 3.71 ng/ml; range = 1.20–15.62) [200]. Cadmium, like other smoke components, does reach and accumulate in the follicular fluid of smokers (7.93 ng/mL) [202], and cadmium concentration in the ovary was elevated in smokers (150 ng/g) versus nonsmokers (115 ng/g) [203]. While cadmium apparently does not interfere with embryo transport through the oviduct in the rat [204], it is another example of a smoke toxicant that can accumulate in reproductive organs and which, in laboratory animals, can adversely affect reproduction [205]. While many of the toxicants that affect the oviduct have not been quantified in smokers and there is little known about their concentrations in the oviduct, those chemicals that have been studied, appear to reach the reproductive organs and are often found in higher concentration in reproductive tissues and fluids than in serum or urine.
F. Summary
The oviduct, while seemingly a simple organ, is exquisitely designed to convey gametes in opposite directions virtually simultaneously and to provide a suitable environment for preimplantation development and transport of embryos to the uterus for implantation. It is vital for reproductive success. Factors that interfere with its functioning can adversely affect fertility. The oviduct serves as a useful model to evaluate the effect of cigarette smoke and its components on a reproductive organ and in a more general sense on a variety of biological functions. While most work on smoke's effect on the oviduct has been done on ciliary beat frequency, oocyte pick-up rate, cilia-oocyte cumulus complex adhesion, and smooth muscle contraction, other parameters of oviductal functioning could be added to this array of bioassays. For example, monitoring the synthesis and secretion of the oviductal proteins may give further insight into how smoke affects oviductal functioning and secretory processes in general. The oviductal assays have been useful in identifying numerous smoke toxicants many of which were not previously recognized as harmful and some of which are widely used in consumer products. Further studies on the safety of these chemicals are needed. Commercial brands of cigarettes contain toxicants capable of shutting down oviductal functions in vitro and interestingly sidestream smoke is often more inhibitory than mainstream smoke. Harm reduction cigarettes, while apparently reduced in carcinogens, still contain chemicals that impair basic biological processes including ciliary beating, oocyte pick-up, and smooth muscle contraction. The effects of smoke on the heart and lungs is widely known and well documented. The effects of smoke on the oviduct, which are just recently becoming more widely recognized, demonstrate that organs remote from the site of inhalation may be adversely affected by chemicals in smoke are consistent with the idea that all organs are targets of smoke [1]. Active and passive smokers of reproductive age should be made aware of the possible dangers in smoking and how smoking could affect their reproductive ability.
Supplementary Material
Additional File 1
Video movie showing a stained oocyte cumulus complex (blue) being pick-up by a hamster infundibulum. The complex adheres to the surface of the oviduct and is pulled along towards the ostium by ciliary beating. Reprinted from Molec Biol Cell 10:5–9, 1999 (with permission).
Click here for file
Additional File 2
Video movies showing a control (right) and smoke treated infundibulum. The oocyte cumulus complex (blue) on the control oviduct moves over the surface of the infundibulum and is picked up at the normal rate. In the smoke exposed preparation, the oocyte cumulus complex barely moves during the same interval of time. Reprinted from Molec Biol Cell 10:5–9, 1999 (with permission).
Click here for file
Acknowledgements
We gratefully acknowledge the Tobacco-Related Disease Research Program of California which supported parts of the work reviewed in this article and our many associates who helped conduct the work. We are also grateful to Dr. Ray Talbot for his help in preparing the table.
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RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-2-601619427710.1186/1742-4690-2-60ResearchDicistronic MLV-retroviral vectors transduce neural precursors in vivo and co-express two genes in their differentiated neuronal progeny Derrington Edmund A [email protected]ópez-Lastra Marcelo [email protected] Jean-Luc [email protected] LaboRétro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, Lyon 69364 Cedex 07, France2 Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Pontificia Universidad Católica de Chile, Marcoleta 391, Santiago, Chile2005 29 9 2005 2 60 60 7 7 2005 29 9 2005 Copyright © 2005 Derrington et al; licensee BioMed Central Ltd.2005Derrington 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.
Dicistronic MLV-based retroviral vectors, in which two IRESes independently initiate the translation of two proteins from a single RNA, have been shown to direct co-expression of proteins in several cell culture systems. Here we report that these dicistronic retroviral vectors can drive co-expression of two gene products in brain cells in vivo. Injection of retroviral vector producer cells leads to the transduction of proliferating precursors in the external granular layer of the cerebellum and throughout the ventricular regions. Differentiated neurons co-expressing both transgenes were observed in the cerebellum and in lower numbers in distant brain regions such as the cortex. Thus, we describe an eukaryotic dicistronic vector system that is capable of transducing mouse neural precursors in vivo and maintaining the expression of genes after cell differentiation.
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Background
The brain constitutes one of the most important organs for gene therapy. Considerable interest resides in the development of vector-based therapies for many of the brain diseases, either to allow the expression of exogenous genes to compensate for a metabolic deficit, to express a growth factor and thus inhibit neural degeneration or to target suicide genes to cancer cells. An alternative approach has been the development of cellular vectors [1-3]. Uncommitted neural precursor cells can be isolated, transduced and grafted into host brains. They adapt to novel environments by stable integration and the expression of location-appropriate phenotypes in host. This opens new avenues for the use of neural stem cells as cellular vectors for gene therapy in the central nervous system (CNS) [2-6]. Both endogenous and transplanted stem cells spontaneously migrate to the site of lesions where they integrate to repopulate the damaged tissue [7-9].
Retroviral vectors based on the γ-retrovirus murine leukemia virus (MLV) are of particular interest for the transduction of neural precursor cells either ex vivo to generate cellular vectors, or in vivo to directly target endogenous neural precursors. They specifically target proliferating cells [10], integrate into the host genome and are conserved in cellular progeny [11]. This has made MLV-vectors a tool of choice to trace lineages and assess the function of specific genes in rodent CNS in vivo [12-14]. In previous studies we established that dicistronic MLV-based retroviral vectors efficiently transduced cells derived from a transformed human neural stem cell line [15], or cells from a primary culture of neural precursors [16]. Here we report that dicistronic MLV-based vectors can deliver and maintain expression of marker genes during neural differentiation in the CNS of new born mice.
Vector producer cells were injected in the region of the developing cerebellum, where the generation of neurons from proliferating precursors continues after birth [17-21]. At early periods post-injection, transduced cells were observed in the external granular layer (EGL) of precursors and migrating towards the internal granular layer (IGL). At later time differentiated neurons were observed scattered about the IGL or in patches. Analysis of other brain regions demonstrated a large number of transduced cells in the ependymal walls throughout the ventricular system and in the subventricular zone. Thus, our results show that dicistronic MLV-based vectors co-expressing two marker transgenes, human placental alkaline phosphatase (PLAP) and neomycin phosphotransferase (Neo) [22,23], transduce proliferating neural precursors in vivo and can penetrate throughout the ventricular system when producer cells are grafted to host animals. Moreover, transduced neural precursors maintain expression of both transgenes after differentiation into neurons demonstrating that the activity of both internal ribosome entry segments (IRES) used in their design is not altered in vivo by neural differentiation.
Results
Location of MLV-vector producer cells
The cerebellum of newborn mice constitutes an accessible model system and was used as target to evaluate the capacity of dicistronic MLV retroviral vectors (Fig. 1) to transduce neural precursors in vivo. At early ages the cartilage of the skull has not undergone calcification, is very thin and can be easily pierced with a needle; thus surgery is not required prior to injection in the brain parenchyma. At this early stage however, stereotactic guidance of the injection needle is not practical because of the difficulty of maintaining a newborn mouse head in a steady coordinated position. Therefore, upon injection of recombinant virus producing cell lines it was important to determine the location of the cells at different times post-injection. The principal site where producer cells were found 5 days post injection (dpi) corresponded to the hindbrain beneath the cerebellum (Fig. 2) with smaller clusters of cells following the needle trace up to the surface of the brain. The micrographs shown (Fig. 2C to 2G) were produced from adjacent coronal sections of caudal cerebellum and the underlying hindbrain, in the regions indicated (Figure 2A and 2B). Injected cells were easily distinguished from the surrounding tissue on the basis of transgene expression shown by immunofluorescence for Neo (Fig. 2C) or PLAP (Fig. 2F). PLAP activity was also revealed by histochemistry (Fig. 2D and 2E). Injected producer cells were also identified by staining DNA (Fig. 2G) because their nuclei appeared to fluoresce more brightly than brain cell nuclei and were elongated as opposed to the round nuclei typical of neural cells. Injected producer cells were not restricted to the injection site since histochemically labelled producer and control cells were also found at different sites including the 4th ventricle and its lateral recesses and in the perimedian sulcus (Fig. 3A,B and 3G; PLAP staining) and trapped in the subarachnoid space, between the meninges on the surface of the brain parenchyma, particularly in the region of the basal artery (Fig. 3C,D and 3F). Similar distributions were obtained with pREV-HW3 vector producing cell lines (Fig. 3A,B,C and 3D), and control helper cells transfected with pREV-HW1 (Fig. 3E,F,G and 3H). The latter vector lacks the viral packaging sequence and is thus not incorporated into recombinant vector particles [22]. The absence of stained cells in the region of the lateral ventricles indicates the failure of graft cells to migrate such great distances upstream of the injection site (Fig. 3H). Taken together these observations are consistent with a significant infiltration of injected cells into the cerebrospinal fluid (CSF) and their being carried via the CSF throughout the ventricular system and into the subarachnoid space. At later times (10 dpi and later) producer cells were no longer observed. We do not know if this is because of the suboptimal conditions for their survival in the brain or whether they were actively eliminated. The latter hypothesis appears less likely because at this early stage in postnatal life immunotolerance of self is still being acquired. Furthermore the brain is an immunoprivileged organ well isolated from the immune system. Lastly, we did not identify any evident signs of inflammation such as activated macrophages or infiltrating lymphocytes in the brain parenchyma.
Figure 1 Schematic representation of dicistronic MLV vectors. Vectors used in this study have been previously described [22, 23]. MLV E+ corresponds to the enhanced packaging region of MLV and the internal ribosome entry signal (IRES). pREV-HW3 and pEMCV-CBT4 contain two IRESes [22, 23]. For both vectors the first is that of MLV and drives expression of human placental alkaline phosphatase (PLAP). In the pREV-HW3 the second IRES is that of REV-A [22], while in pEMCV-CBT4 it is that of EMCV [66]. In both vectors the second IRES drives the expression of neomycin phosphotransferase (Neo). The pREV-HW1 lacks the packaging sequence and the IRES of MLV and thus it cannot generate recombinant virus [22]. The pREV-HW1 missing the MLV Psi/IRES sequences was used as an internal negative control.
Figure 2 Location of injected MLV-vector producer cells. Injected helper cells were found mainly in the hind brain beneath the caudal cerebellum. The red line in A indicates the position in the rostro-caudal axis, and the red rectangle in B shows the location of the photomicro graph presented in C. The sections in D, F and G correspond to the region bordered by the white rectangle shown in C. E shows the region bordered by the white rectangle in D at higher magnificationInjected cells were easily distinguished from the surrounding tissue on the basis of transgene expression shown by immunofluorescence for Neo (C) or PLAP (F). Brightly stained cells express transgene. Alternatively PLAP activity could be revealed by histochemistry in which case the dark cells express PLAP (D and E). It was also possible to identify injected producer cells by staining DNA as shown in field G which corresponds to the same field as D stained with bis-benzimide. The nuclei of injected cells appeared to fluoresce more brightly than brain cell nuclei and were elongated in contrast to the round nuclei typical of neural cells. Macroscopically, injected cells appeared as disorganized patches in the surrounding brain parenchyma. PMS, perimedian sulcus. Scale bars indicate 200 μm (C and D), 20 μm (E, F and G)
Figure 3 Dissemination of injected MLV vector producer cells from the injection site. Panels A, B, C and D are micrographs from brain sections of an animal injected with pREV-HW3 vector producer cells, while panels E, F, G and H are micrographs from brain sections of an animal injected with helper cells transfected with pREV-HW1. Panel B shows the region of A bordered by a black rectangle at greater magnification. PLAP histochemical staining shows cells in different sites including the 4th ventricle and its lateral recesses and in the perimedian sulcus (A, B and G, respectively) and trapped in the subarachnoid space, between the meninges on the surface of the brain parenchyma, particularly in the region of the basal artery (C, D and F). Similar distributions were obtained with vector producing cells (A, B, C and D) and helper cells transfected with pREV-HW1 (E, F, G and H). PMS, perimedian sulcus. Size bars indicate 200 μm (A and G), 100 μm (B), 125 μm (C and D), 450 μm (E), 360 μm (F) and 250 μm (H).
Transgene expression in differentiated cerebellar cells in vivo
We next sought to determine the location of cells transduced by the dicistronic MLV vector in vivo. At 4 dpi histochemical staining for the PLAP, reporter gene under the control of the MLV IRES, revealed transduced cells mainly in the EGL (Fig. 4A and 4B). Labeled cells in the EGL were often clustered along the edge of the parenchyma of the cerebellum and were morphologically fusiform (Fig. 4A and 4B), however patches of staining were also observed in which it was difficult to distinguish discrete cells (Fig. 4C). At 15 dpi PLAP histochemical staining revealed transduced cells in patches and scattered about the parenchyma of the cerebellum (Fig. 4D,E and 4F). The majority of cells were found in the IGL (Fig. 4E and 4F) but cells were also found in and astride bands of cerebellar white matter (Fig. 4D). The high density of the labeling made it difficult to attribute a phenotype to individual cells on the basis of morphological characteristics. However, on the basis of their locations in fibre tracts and in the IGL, PLAP expressing cells probably included both neurons and glia. In the regions of the IGL where labeled cells were more parsimoniously scattered it was possible to distinguish cells with a clearly neuronal morphology (Fig. 4F).
Figure 4 In vivo transduction of cells using pREV-HW3 vector in the cerebellum. 4 days post injection, histochemical staining of PLAP in brain sections shows labeled cells in the region occupied by neural precursors in the external granular layer of the cerebellum, observed as discrete cells (A and B) or patches of staining (C). Panel B shows the region bordered by the black rectangle in A at higher magnification. 15 dpi histochemistry revealed transduced cells in patches and scattered about the parenchyma of the cerebellum (D, E and F). The majority of cells were found in the internal granular layer (E and F) but cells were also found in and astride bands of cerebellar white matter which is indicated by a black arrow (D). Discrete cells often exhibit a clearly neuronal morphology (F), the thin arrow indicates a cell with the morphology of a granular neuron, the arrow head indicates a cell with the morphological characteristics of a cerebellar golgi neuron. PCL, purkinje cell layer. Scale bars are 200 μm (A), 100 μm (B, C, D), and 40 μm (E, F)
Immunohistochemistry for PLAP revealed intensely labeled cells in the IGL (Fig. 5A). An antiserum directed against the HU antigen which is specifically expressed only by post-mitotic neurons in the brain [24,25] allowed the unambiguous identification of some of the PLAP expressing cells as neurons (Fig. 5B, arrows). Cells expressing PLAP were also identified on the peripheral extremity of the section (Fig. 5A,B and 5C, magenta arrowhead), however, HU staining in this region is at background levels. These stained cells, at this stage (5 dpi), may correspond to undifferentiated precursors, producer cells or transduced meningeal cells. At 15 dpi double labeling for PLAP and Neo revealed patches of cells in the internal granular layer that expressed both transgenes (Fig. 5D,E and 5F). Similar data where obtained when vector pEMCV-CBT4 was used (data not shown) indicating that the MLV-based double IRES vectors can direct expression of two distinct gene products in cerebellar neurons.
Figure 5 In vivo transduction of neural cells. Immunohistochemical labeling for PLAP (A) and the neuron-specific HU antigen (B) was used to identify pREV-HW3 transduced neurons in the cerebellum (5 dpi). Examples of cells expressing both antigens are indicated by white arrows. Cells expressing PLAP were identified on the peripheral extremity of the section (magenta arrowhead in A, B and C). Transduced cells were also found in the brain parenchyma (example indicated by white arrowhead). In all the regions examined transduced cells co-expressed both proteins as illustrated by immunodetection of both PLAP (D) and Neo (E) in cells in the IGL of the cerebellum (14 dpi). Co-expression of both antigens, PLAP (G) and Neo (H), also occurred in cells in the ventricular region such as in the ependymal walls of the 3rd ventricle (G, H and I). DNA is stained with bis-benzimide (C, F and I). PCL, purkinje cell layer; IGL, internal granular layer. Scale bars correspond to 150 μm (C and F), 75 μm (I)
Taken together these results show that MLV-IRES vectors are able to transduce precursor cells in the CNS in vivo and that the IRESes of different viruses such as MLV, EMCV and REV-A remain functional in differentiated neurons in the animal.
Transduction of cells in different brain regions
Although the primary target for transduction was the cerebellum the spread of the dicistronic MLV vector to neural cells in other brain regions was also evaluated. Numerous transduced ependymal cells were observed in the ventricular walls and in the 3rd and 4th ventricles (Fig. 5G,H and 5I). A significant sub-population of transduced cells was also observed in the lateral ventricles (Fig. 6A–G) a site very distant from the site of injection of the producer cells. Whereas many transduced cells appeared to be in the ependymal wall, some of them appeared to be localized in the adjacent brain parenchyma (Fig. 6A arrow heads). These cells did not express HU antigen (Fig. 6B). Double labeling for PLAP (Fig. 6E) and GFAP (Fig. 6F) showed that the PLAP transgene was predominantly co-localized with GFAP in cells and processes (Fig. 6E and 6F). These data suggest that the PLAP expressing cells most probably correspond to ependymocytes, tanycytes and perhaps neural precursor cells interposed among the ependymocytes or in the subependymal zone [26-28]. In either case it is clear that the vector must infiltrate and permeate CSF efficiently, because no evidence of helper cells was seen in the brain parenchyma so far from the injection site in any of the control animals (e.g. Fig. 3H).
Figure 6 In vivo transduction of cells in other brain regions. Immunohistochemical staining reveals pREV-HW3 transduced cells in and adjacent to the ependymal walls throughout the ventricular system including in the lateral ventricles (A, B, C and D). PLAP expressing cells are identified in the ependymal wall, and in the adjacent brain parenchyma (arrow heads in A). Transduced cells are not reactive to anti-HU antibody (B). Double labeling for PLAP (E) and GFAP (F) showed that the PLAP transgene predominantly co-localizes with GFAP in cells (arrow in E and F) and processes (arrow heads). Analysis of the overlying cortex showed cells co-expressing PLAP (H) and HU (I) in the most superficial layers of cortex (arrows in H and I). Many of the transduced cells in this region did not express HU (white arrow heads in H, I, J and K). Note the absence of HU staining in small piece of attached meninges (magenta arrow head in H, I, J and K). DNA staining with bis-benzimide is shown C, G and J and phase contrast of the section in A, B and C, and the section in H, I and J, are shown in D and K, respectively. Scale bars correspond to 75 μm (C, G) and 100 μm (J).
Several neural precursor cell populations may be susceptible to transduction by the dicistronic MLV vectors in the ventricular zone. Ependymal cells, which have been reported to be neural precursors [29], are still proliferating quite quickly in early postnatal brain [30]. Radial glia, which may be precursors of both neurons and glia [31-35], contact the ventricular surface and proliferate in the ventricular region until postnatal day 7 [36]. Lastly, the slowly proliferating GFAP-labeled subependymal neural stem cells, which survive and continue to proliferate and generate neurons in the adult brain, have been proposed to require contact with the ventricular surface to become neurogenic [27]. Having identified this sub-population of potential precursor cells we sought cells with mature phenotypes that may represent their progeny. In the most superficial layers of the cortex, which will be formed from the latest neurogenerative mitoses of cortical precursor cells, we were surprised to find rare transduced neurons, co-expressing PLAP and HU (Fig. 6H–I). Other non-neuronal cells labeled with the transgenes were also observed in forebrain (Fig. 6H,I,J and 6K).
These results showed that rather large numbers of transduced non-neuronal cells were found in and adjacent to the ependymal walls throughout the ventricular system including in the lateral ventricles (Fig. 6) and that the viral IRESes were active in these cells in vivo.
Discussion
MLV-based double-IRES vectors pREV-HW3 and pEMCV-CBT4 were found to direct co-expression of two gene products in a variety of cell types [15,16,22,23]. Overall transgene expression driven from the MLV-based double-IRES vectors is the consequence of two distinct processes, transcription and translation initiation, both of which are tightly regulated by the host cell. Indeed, important limitations of MLV-vectors are cell-type-specific promoter silencing [13,37,38], and modulation of IRES activity. IRES activity can be regulated by diverse physiological processes such as cell cycle [39-42], cellular stress [43-46], cell transformation [47], cell death [48-51] and cell differentiation [52-56]. Previous studies suggested that the activity of the MLV IRES present in both vectors, could be modulated by oligodendrocyte differentiation [16]. The possibility of in vivo IRES regulation due to cell differentiation prompted us to extend our previous ex vivo studies [15,16], and evaluate the feasibility of using double-IRES MLV vectors in the CNS.
Results show that upon injection of producer cells in the cerebellum of newborn mice, the generated MLV-vectors transduce host cells throughout the postnatal brain ventricular system. Transduced neural precursors and their progeny could be revealed by histochemistry for PLAP or immunohistochemistry and large patches of transduced neurons could be identified expressing both transgenes 15 dpi. In double labeling studies, most cells that were PLAP positive also stained for Neo. Considering that MLV-vector producer cells appeared to survive less than 10 days in the developing brain these observations demonstrated transduction of proliferating precursor cells and the maintenance of IRES activity in neurons with each of the combinations of IRESes tested, namely MLV and REV-A (pHW3) and MLV and EMCV (pCBT4). Therefore, and consistent with previous observations [15,16], down-regulation of transgene expression was not observed in neurons generated from precursors transduced in vivo.
Transduced cells could be identified in the ependymal walls throughout the ventricular system. The 3rd and lateral ventricles lie upstream of the injection site with respect to the flow of cerebrospinal fluid. Thus, the MLV vector is capable of diffusing via the cerebrospinal fluid and targets proliferating cells in this region of the brain. Diverse cell populations identified as sources of neuronal and glial precursors are potential targets for MLV-recombinant vector in early postnatal brain [27-29,34,35]. For example, the proliferation of ependymal cells, which form the interface between the CSF and the brain parenchyma, progressively slows down during postnatal development to a very low basal level at postnatal day 12 which then remains stable in the absence of injury [30,57]. Mitotic radial glia are also in contact with the ventricular surface in the lateral ventricles during early postnatal development [36]. Subventricular astrocytes also contact the ventricular surface in adult brain and proliferate slowly [27].
In the ventricular regions the majority of transduced cells express GFAP, which is is weakly expressed by ependymal cells and tanycytes and more strongly expressed by astrocytes and the GFAP labeled "type B" cells that constitute multipotent neural precursors [28,58]. Radial glia are also GFAP positive [59]. 15 dpi, transduced cells were observed in the brain parenchyma close to the ependymal wall. In the more superficial layers of the cortex, where the most immature post mitotic neurons reside, a few transduced neurons, identified by their expression of the HU antigen, as well as non-neuronal cells could be identified.
The current approaches for gene therapy of monogenetic diseases in mature organisms are confronted by several problems including: (1) adult tissues may be poorly infected by conventional vector systems dependent upon cell proliferation for optimal infection; (2) immune responses, whether pre-existing or developing after vector delivery, may rapidly eliminate transgenic protein expression and prevent future effective intervention. Early gene transfer, in the neonatal or even fetal period, may overcome some or all of these obstacles [60]. Therefore, the experimental approach described herein, might be useful in the development of new approaches to gene therapy in young organisms.
Conclusion
In summary, we describe an eukaryotic dicistronic vector system that is capable of transducing mouse neural precursors in vivo andmaintaining the expression of genes after cell differentiation. Human placental alkaline phosphatase (PLAP) and neomycin phosphotransferase (Neo) used in this study as reporter genes can be replaced by other genes of interest to make these dicistronic vectors a novel tool to trace lineages and assess the function of specific genes in rodent CNS in vivo. Vectors might also be ideally suited to targeting suicide genes to proliferating cells, such as tumor cells, that spread and infiltrate via the CSF [61,62].
Materials and methods
Vectors, helper cells, titration
Plasmid vectors pEMCV-CBT4, pREV-HW1 and pREV-HW3, shown schematically in Fig 1, have been previously described [22,23]. NIH-3T3 cells, and the NIH-3T3 based retroviral packaging cell line GP+E-86 [63], were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) with 10% newborn calf serum at 37°C in presence of 5% CO2. MLV vectors were produced by transfection of GP+E-86 cells with pREV-HW3 or pEMCV-CBT4 constructs as previously described [22]. Vectors, produced by GP+E-86, were titrated on NIH-3T3 [15,16,22]. The negative control pREV-HW1 was produced using the same procedure as above.
Grafts
Postnatal day 1–2 mice (OF1 strain) were injected with 1–5 × 104 producer cells in a 2 μl volume in the region of the developing cerebellum as follows. Producer cells harboring the recombinant vector, or control cells (non-transfected helper cells, transduced 3T3 cells or cells transfected with the pRev-HW 1 vector described by López-Lastra et al., (1997) which cannot be packaged, see Fig. 1), were resuspended by trypsinization, washed once in medium and twice in PBS, counted and resuspended in PBS. Cell suspension was pumped from a Hamilton syringe to fill a fine plastic catheter connected to a second Hamilton syringe needle. The second needle was manually pierced to a depth of 1.5 – 2 mm through the cranial cartilage into the region of the developing cerebellum, behind the cerebral hemispheres which were visible through the skull. Then, 2 μl of cell suspension was slowly pumped into the brain using the Hamilton syringe over a 10 second period. The needle was held in place for another 10 seconds and then carefully removed. The young were then replaced with their mothers and maintained with free access to food and water until the time of sacrifice. Animals were killed by anoxia in CO2, decapitated and their brains were carefully removed and fixed by immersion in 4% paraformaldehyde in PBS for 12 – 15 h. Brains were then cryoprotected by immersion in 30% sucrose and frozen by immersion in isopentane over dry ice. Brains were then cut into serial sections of 16 μμm thickness using a Leitz cryomicrotome and recovered on gelatin-coated glass microscope slides. All experiments involving animals were performed in accordance with the French regulations and were approved by the animal experimentation committee of the Ecole Normale Supérieure, Lyon.
Histochemical staining
For placental alkaline phosphatase (PLAP) histochemical staining, cells were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde. After two washes in PBS, they were incubated at 65°C for 30 min in PBS. Tissue sections were incubated for 1 hour at 65°C. Cells or tissue sections were washed twice with AP buffer (100 mM Tris-HCl pH 9.5, 100 mM NaCl, and 5 mM MgCl2) and incubated for 5 hr in staining solution (0.1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 1 mg/ml nitroblue tetrazolium salt (NBT), and 1 mM levamisole) at 22°C. Brain regions of histochemically and immunostained cells were identified by extrapolation from a rat brain histological atlas [64].
Immunohistochemistry
Tissue sections were rinsed in PBS then incubated for 30 min in 20 mM ammonium acetate. Sections were washed twice in PBS then incubated for 30 min in a blocking solution of PBS containing 5% BSA, 1% normal goat serum and 0.2% Tween 20 prior to staining with antibodies. This same solution served to dilute all the antibodies. Double-labelling was performed by simultaneous staining with antibodies produced in different species which were then revealed using fluorochrome-conjugated goat antibodies with appropriate species specificity. Thus, PLAP was revealed using a murine monoclonal antibody (diluted 1/200) purchased from DAKO (Glostrup, Denmark). Neo was revealed using an affinity purified rabbit polyclonal antibody (diluted 1/100) generated by immunizing rabbits with peptides VENGRFSGFIDCGRL and MIEQDGLHAGSPAAC conjugated by their carboxy terminus to keyhole limpet haemocyanin. GFAP which labels astrocytes [65], a population of neural stem cells [27,58] developing ependymocytes [58] and radial glia [59] was detected by a polyclonal rabbit anti-cow GFAP antiserum (diluted 1/200), purchased from DAKO. Neurons were detected using an anti-HU antiserum generously donated by Dr. J. Honnorat and Dr. M-F. Belin (diluted 1/1000). The HU antigen comprises a group of nucleic acid binding proteins located in the nucleus and cytoplasm of post-mitotic neurons [24]. All antibodies have been tested in cell culture and on various control tissues and give appropriate patterns of specific labeling. The neural cell type-specific markers did not label helper/producer cells in vitro. Primary incubations were for 2 h at room temperature or at 4°C overnight. After washing sections 5 × 10 min in PBS, bound antibodies were revealed with FITC-conjugated goat anti-human immunoglobulin antibodies or Cy3-conjugated goat anti-rabbit IgG antibodies and either Cy3- or FITC-conjugated goat anti-mouse IgG antibodies. Anti-immunoglobulin antibodies were all at a final dilution of 1/400 in blocking buffer containing bis-benzimide (1 μg/ml) to stain DNA. Controls included no primary antibodies and non-transduced brain. Slides were washed 3 times in PBS, mounted with moviol and analyzed with a Zeiss Axioplan fluorescence microscope.
Authors' contributions
ED participated in the design of the study, conducted animal injection, maintained and handled animals, tissue sections, histochemical staining, immunohistochemistry, and drafted the manuscript. MLL participated in the design of the study, developed the retroviral vectors, generated the vector producing cell lines, aid in recombinant virus titration, and helped to draft the manuscript. JLD participated in the design of the study, was responsible for supervising and coordinating the study, and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
The authors wish to thank Christelle Daudé for her expert technical assistance. This work was supported by grants from the ANRS, the MGEN and ARC (number 5466) to J-L Darlix, and the Pontificia Universidad Católica de Chile (DIPUC 2004/06E and 2005/14PI) to M. López-Lastra. E.A. Derrington was supported in part by a fellowship from the ANRS.
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PLoS PathogPLoS PathogppatplpaplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 1625459710.1371/journal.ppat.001001905-PLPA-RA-0058R3plpa-01-02-06Research ArticleAllergy - ImmunologyInfectious DiseasesMicrobiologyEubacteriaMus (mouse)Homo (human)In VitroAnthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs the Adaptive Immune Response Anthrax Lethal Toxin Kills Dendritic CellsAlileche Abdelkrim ¤Serfass Evan R Muehlbauer Stefan M Porcelli Steven A Brojatsch Jürgen *Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of AmericaYoung John EditorThe Salk Institute for Biological Studies, United States of America* To whom correspondence should be addressed. E-mail: [email protected]¤ Current address: Department of Oncology, Georgetown University Medical Center, Lombardi Cancer Center, Washington, District of Columbia, United States of America
10 2005 28 10 2005 1 2 e197 6 2005 23 9 2005 Copyright: © 2005 Alileche et al.2005This 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.Many pathogens have acquired strategies to combat the immune response. Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen-activated protein kinase pathways, rendering dendritic cells (DCs) and T lymphocytes nonresponsive to immune stimulation. However, these cell types are considered resistant to killing by LT. Here we show that LT kills primary human DCs in vitro, and murine DCs in vitro and in vivo. Kinetics of LT-mediated killing of murine DCs, as well as cell death pathways induced, were dependent upon genetic background: LT triggered rapid necrosis in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs. This is consistent with rapid and slow killing of LT-injected BALB/c and C57BL/6 mice, respectively. We present evidence that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune-evasion strategy of the bacterium, and contribute to anthrax disease progression. We also established that genetic background determines whether apoptosis or necrosis is induced by LT. Finally, killing of C57BL/6-derived DCs by LT mirrors that of human DCs, suggesting that C57BL/6 DCs represent a better model system for human anthrax than the prototypical BALB/c macrophages.
Synopsis
Dendritic cells (DCs) are specialized white blood cells that identify and present antigens to immune cells, T cells, in order to mount an immune response targeted against specific pathogens. DCs are critical to a host's defense against infection. Previous work has shown that the anthrax bacterium disables many immune cells, including DCs, through the action of a released toxin, lethal toxin. Here the authors show that lethal toxin efficiently kills both human and murine DCs. The means by which DCs were killed by the anthrax toxin were notably distinct and dependent on their genetic background. Human DCs, as well as those derived from the murine strain C57BL/6, died over the course of 72 h via activation of apoptosis, or programmed cell death. DCs from BALB/c mice, however, died rapidly of a necrotic cell death following toxin exposure. As human and C57BL/6 DCs share an identical response to anthrax toxin, C57BL/6 mice appear to provide an excellent model for human anthrax. The study's findings suggest that specific targeting of DCs by the anthrax toxin impairs the immune response of the infected host, and the authors believe that this strategy promotes spread of the bacterium and disease progression.
Citation:Alileche A, Serfass ER, Muehlbauer SM, Porcelli SA, Brojatsch J (2005) Anthrax lethal toxin-mediated killing of human and murine dendritic cells impairs the adaptive immune response. PLoS Pathog. 1(2): e19.
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Introduction
Bacillus anthracis, the causative agent of anthrax disease [1], releases lethal toxin (LT), which is sufficient to cause death in mice even in the absence of the bacterium [2]. Broad cytopathic and lethal effects associated with B. anthracis infection can be reproduced by LT injection of mice [3,4]. Lethal toxin consists of two components: protective antigen (PA) and lethal factor (LF). PA binds to specific cell surface receptors and mediates endocytosis of LF, a metalloprotease [5,6]. LF cleaves six of seven mitogen-activated protein kinase (MAPK) kinases (MAPKKs), thereby disrupting MAPK signaling pathways (reviewed in [7,8]). Despite ubiquitous uptake by mammalian cells, LT kills only a few cell types, including murine macrophages [3,4]. Susceptibility of murine macrophages to LT killing is strain-specific, and is controlled by a region of Chromosome 11 that encodes the kinesin-like motor protein Kif1c [9,10].
B. anthracis appears to combat the host immune response by inactivating MAPK pathways, which renders immune cells, including dendritic cells (DCs), nonresponsive to immune stimulation [11–13]. This may weaken the immune response by limiting the production of inflammatory cytokines [12,14–16]. However, drastic cytokine induction has been shown in several murine strains following LT injection [3,17]. These findings call into question the in vivo relevance of LT-mediated nonresponsiveness. DCs, which are considered resistant to LT-mediated cell killing [12], are key elements of the adaptive immune response, and are responsible for uptake and presentation of microbial antigens [18]. Upon maturation, DCs migrate to secondary lymphoid organs where they stimulate naïve T cells [18].
Here we report that murine and human DCs are efficiently killed by LT. The mechanism of LT-mediated DC killing is dependent on genetic background: LT triggers rapid necrotic cell death in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs, as well as human DCs. Finally, we show rapid DC depletion in LT-treated BALB/c mice, suggesting direct immune impairment and in vivo relevance.
Results
Killing of Human DCs by Anthrax LT
LT-treated DCs were reported to be non-responsive to LPS stimulation, as well as impaired in their abilit7y to present antigens [12]. To determine whether the impairment of DCs could be due to cytopathic effects mediated by LT, we generated immature monocyte-derived dendritic cells (MoDCs) from human peripheral blood monocytes. MoDCs were found to be CD11c+, CD80+, CD86+, HLA-DR+, DEC-205+, CD14−, and CD16− (Figure 1), consistent with markers found on immature human DCs [19]. MoDCs were challenged with a dose of LT (500 ng/ml PA and 250 ng/ml LF) cytotoxic to murine J774A.1 macrophages [20]. In contrast to previous findings, we found that human MoDCs were efficiently killed by LT [12]. LT-treated MoDCs from five different subjects showed a consistent increase in annexin V binding, while remaining propidium iodide-negative 48 h after LT challenge (Figure 2A and 2B). Induction of cytopathic effects was further supported by a drop in MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) activity (Figure 2C). At 72 h post-LT exposure, the MTT signal declined to levels only marginally above those observed in MoDCs treated with the apoptosis inducer camptothecin (Figure 2C). No cytopathic effects were observed in MoDCs treated with PA or LF alone (unpublished data). Ultrastructural analysis of LT-treated human MoDCs revealed signs of apoptotic cell death, including chromatin condensation, pycnotic nuclei, and cytoplasmic vacuolization, along with membrane preservation (Figure 2D). Apoptosis induction was further supported by TUNEL staining of LT-treated MoDCs (Figure 2E). These results are consistent with reports of apoptosis induction following LT challenge of human endothelial cells, phorbol myristate acetate-stimulated monocytic cell lines, and lipopolysaccharide (LPS)-stimulated murine macrophages [13,21–23].
Figure 1 FACS Profile of Immature Human MoDCs
MoDCs were derived from human peripheral blood monocytes. Expression of HLA-DR, CD11c, CD14, CD16, CD80, CD86, and DEC-205 was assessed by flow cytometry. The data were collected from two subjects and are representative of similar experiments. Filled histograms represent isotype-matched controls.
Figure 2 Killing of Human DCs by LT
(A) MoDCs from a representative human subject were treated with LT (500 ng/ml PA and 250 ng/ml LF) or 10 μM camptothecin (Campt.) as a positive control, and annexin V/PI staining was measured by flow cytometry 48 h post-LT exposure.
(B) Percentages of annexin V-positive untreated and LT-treated MoDCs from five different subjects were determined by flow cytometry 48 h post-LT exposure.
(C) MTT assay of LT or camptothecin-treated human MoDCs. Mean + standard deviation from three independent experiments are shown.
(D) LT-treated human MoDCs show signs of apoptotic cell death, as analyzed by electron microscopy 48 h post-LT exposure. Bars: 1 μm.
(E) Human MoDCs were TUNEL-positive 48 h post-LT exposure. Untreated and LT-treated MoDCs were subjected to TUNEL and Hoechst staining.
Control of LT Killing of Murine DCs by Genetic Factors
To test whether murine DCs were also susceptible to LT killing, we generated immature bone marrow-derived dendritic cells (BMDCs) from BALB/c and C57BL/6 mice [24]. BMDCs were CD11b+, CD11c+ (over 80%), and CD14−, and they expressed low levels of CD80 and CD86 (Figure 3A and 3B), consistent with surface markers found on in vitro-generated immature murine DCs [25,26]. To ensure that we were working with immature DCs, we treated these DCs for 18 h with LPS, and confirmed maturation by measuring surface levels of CD86. As expected, LPS exposure for 18 h generated a significant up-regulation in CD86 surface expression levels (Figure 3B). Exposure of DCs to PA or LF (1 μg/ml) alone did not result in CD86 up-regulation (unpublished data).
Figure 3 FACS Profile of BMDCs
(A) BMDCs were derived from a BALB/c mouse and analyzed on day 10. Expression of CD11b, CD11c, CD14, and CD80 was assessed by flow cytometry. The data are representative of four similar experiments in BALB/c and C57BL/6 mice.
(B) BALB/c and C57BL/6-derived BMDCs were stimulated by LPS for 18 h, and CD86 expression was measured by flow cytometry. Filled histograms represent isotype-matched controls.
BMDCs from BALB/c and C57BL/6 mice were exposed to LT, and cell viability was determined at several time points post-LT exposure (Figure 4). The MTT signal dropped significantly in BALB/c-derived BMDCs within 2–4 h of LT challenge. Cell viability fell in C57BL/6-derived BMDCs as well, but only after 2–3 d of LT exposure (Figure 4A). Accordingly, BALB/c and C57BL/6-derived BMDCs underwent morphological changes 2 h and 2 d post-LT exposure, respectively, as determined by phase contrast microscopy (unpublished data). In addition to immature BMDCs, we isolated splenic DCs with a mature phenotype from BALB/c mice using magnetic beads. These splenic DCs died within 4 h of LT exposure as determined by morphological changes and MTT assays (unpublished data), suggesting that LT susceptibility of murine DCs is independent of their maturation state.
Figure 4 BALB/c- and C57BL/6-Derived DCs Differ in Their Response to LT
(A) BALB/c and C57BL/6-derived BMDCs were treated with LT (500 ng/ml PA and 250 ng/ml LF), and cell survival was determined by MTT assay.
(B) Caspase-3 activation of LT-treated BALB/c and C57BL/6 DCs as determined by a colorimetric caspase-3 cleavage assay. A representative experiment is shown.
(C) The caspase inhibitors Z-VAD-FMK (10 μg/ml) and BOC-D-FMK (40 μg/ml) prevent caspase-3 activation in LT-treated BALB/c BMDCs.
(D) The caspase inhibitors Z-VAD-FMK (10 μg/ml) and BOC-D-FMK (40 μg/ml) do not prevent LT killing of BALB/c BMDCs as determined by MTT assay.
(E) C57BL6, but not BALB/c DCs, were TUNEL-positive post-LT exposure. BALB/c and C57BL/6-derived BMDCs were treated with LT (500 ng/ml PA and 250 ng/ml LF), and were stained using a TUNEL reaction and a Hoechst counterstain.
(F) BALB/c and C57BL/6-derived BMDCs were analyzed by electron microscopy 4 and 48 h post-LT exposure, respectively. Bars, 1 μm.
Our findings are consistent with the in vitro sensitivity of murine macrophages to LT killing, which is also dependent on genetic background [3,4,27]. BALB/c-derived macrophages are efficiently killed by LT with rapid kinetics, while C57BL/6-derived macrophages are resistant to rapid LT killing [13]. Similarly, in vivo studies have shown rapid disease progression in LT-treated BALB/c mice, and a delayed onset of lethal effects in LT-treated C57BL/6 mice [3,4].
As apoptotic pathways were activated in LT-treated human DCs (see Figure 2D and 2E), we tested for signs of apoptosis in murine BMDCs following LT exposure. C57BL/6 BMDCs showed clear signs of apoptosis, as indicated by TUNEL staining and nuclear condensation (Figure 4E and 4F). In contrast, no chromatin condensation, TUNEL staining, or DNA fragmentation was detected in LT-treated BALB/c BMDCs (Figure 4E and 4F), which is consistent with rapid necrotic cell death, as has been observed in LT-treated BALB/c-derived macrophages [13,23]. Strikingly, LT exposure triggered caspase-3 activation in murine BMDCs derived from both BALB/c and C57BL/6 mice. Consistent with the cell killing kinetics described above, caspase-3 activation occurred rapidly in BALB/c-derived BMDCs (1 h post-LT exposure), and slowly in C57BL/6-derived BMDCs (24 h post-LT exposure) (Figure 4B and 4C). We used the pan-caspase inhibitors Z-VAD-FMK and BOC-D-FMK to block caspase activation. Both caspase inhibitors blocked caspase-3 activation (Figure 4C), but failed to block LT killing of BALB/c-derived DCs (Figure 4D), indicating that caspase activation was not required for induction of cytopathic effects in these cells. Experiments using caspase inhibitors were limited to BALB/c DCs, as both Z-VAD-FMK and BOC-D-FMK showed high toxicity in murine DCs when used for more than 12 h.
These results revealed that the genetic background of murine DCs determines LT killing kinetics and cell death pathway activation. The difference in the LT killing kinetics between BALB/c and C57BL/6 BMDCs or human DCs was not caused by a discrepancy in LT uptake, MAPKK-3 cleavage, or LT sensitivity. MAPKK-3 cleavage, the earliest indicator of LF action, occurred in BALB/c BMDCs, C57BL/6 BMDCs, and human MoDCs at similar time points, as determined by Western blotting (Figure 5A). The small divergences in MAPKK-3 cleavage did not correlate with differences in LT killing kinetics, suggesting that these kinetics were not determined by the rate of MAPKK-3 cleavage, and were presumably caused by downstream events.
Figure 5 MAPKK Cleavage Kinetics and LT Susceptibility of BALB/c- and C57BL/6-Derived BMDCs
(A) MAPKK-3 cleavage occurs at similar rates in murine and human DCs treated with LT (500 ng/ml PA and 250 ng/ml LF), as determined by Western blot analysis using anti-MKK3 and anti-actin (control) antibodies.
(B and C) Relative LT susceptibility of BALB/c- (B) and C57BL/6-derived (C) BMDCs. BALB/c and C57BL/6 BMDCs were subjected to PA (500 ng/ml) and varying concentrations of LF. After 4 h (BALB/c DCs) and 72 h (C57BL/6 DCs), cell viability was determined by MTT assays. Representative experiments are shown.
To determine the LT sensitivity of BALB/c and C57BL/6-derived BMDCs to LT killing, we treated these cells with increasing LF concentrations, while keeping PA constant at 500 ng/ml. Both BALB/c and C57BL/6-derived BMDCs exhibited similar susceptibility to LT, with the main increase in LT killing occurring between 1 and 10 ng/ml of LF (Figure 5B and 5C). Taken together, our results indicate that the difference in LT killing cannot be explained by divergences in MAPKK cleavage or LT sensitivity.
Control of LT Killing of Murine DCs by Proteasomal Activity
LT killing of BALB/c-derived murine macrophages is controlled by the proteasome system [20,28]. To determine whether BALB/c-derived DCs also require proteasomal activity for LT killing, we added the proteasome inhibitors MG132 or Velcade (bortezomib) to BALB/c-derived BMDCs at different time points following exposure to LT. Both proteasome inhibitors completely blocked LT killing when added simultaneously with LT to BALB/c-derived DCs (Figure 6). Strikingly, the MTT signal recovered completely from an initial drop when proteasome inhibitors were added to BALB/c BMDCs up to 1 h post-LT exposure (Figure 6). Partial recovery was obtained when the proteasome inhibitors were added 2 h post-LT treatment (Figure 6). These results suggested that proteasomal degradation of protective factors controls a late step in LT killing of BALB/c DCs. Experiments using proteasome inhibitors were limited to BALB/c DCs, as both MG132 and Velcade were highly toxic to DCs when applied for more than 8 h. It is conceivable that slow LT killing of C57BL/6-derived cells is due to increased levels or stability of putative protective factors. Proteasomes may also control LT-induced morbidity and mortality in LT-treated mice.
Figure 6 Proteasome Inhibitors Block LT-Mediated Killing of BALB/c-Derived DCs
Proteasome inhibitors MG132 (10 μM) (A), or Velcade (0.1 μM) (B) were added either simultaneously with LT or 1 or 2 h post-LT exposure, and cell viability was determined by MTT assay. Mean + standard deviation of four independent experiments are shown.
In Vivo Depletion of DCs and Loss of T Cell Activating Function Following LT Exposure
To determine whether murine DCs were also killed by LT in vivo, we injected 10-wk-old female BALB/c mice intraperitoneally with LT (200 μg PA and 200 μg LF), and analyzed bulk splenocytes from these mice using flow cytometry. We selected BALB/c mice, the commonly recognized prototype strain for anthrax research, for our in vivo experiments. Consistent with our in vitro data, levels of splenic DCs dropped rapidly in LT-treated BALB/c mice (Figure 7). Levels of DCs declined to 10% of those found in untreated control mice 3 h after LT challenge, and remained at this level for at least 24 h. Levels of splenic macrophages were also reduced, reaching 20% of control levels 24 h post-LT challenge (Figure 7B). As expected, levels of B and T cells, which are resistant to LT killing, did not drop after LT exposure (Figure 7B). These results showed that LT killing of DCs was not restricted to the cell culture system, as it also occurs in vivo. LT-treated DCs have been described as impaired in their abilities to respond to cytokines and to present antigens [12]. Our findings indicate that significant impairment of the immune response in LT-treated mice is caused by rapid DC depletion.
Figure 7 In Vivo Depletion of DCs and Loss of T Cell Activating Function Following LT Exposure
(A) Murine DCs and macrophages are killed by LT in vivo. Ten-week-old BALB/c mice were injected intraperitoneally with LT, and the percentage of DCs and macrophages in the spleen were determined by flow cytometry. A representative experiment is shown.
(B) Specific depletion of DCs and macrophages in LT-treated BALB/c mice injected intraperitoneally with either PBS or LT (200 μg PA and 200 μg LF). Levels of splenic DCs (CD11c+, MHC class II+), macrophages (CD11b+, MHC class II+), and circulating B cells (B220+) and T cells (CD3+) were determined by flow cytometry. Percent changes are shown by number of cells derived from LT-treated BALB/c mice (three mice per time point), relative to PBS-treated control mice (three mice per control experiment).
Discussion
Here we show for the first time, to our knowledge, that anthrax LT is highly toxic to human, BALB/c, and C57BL/6 DCs, even at very low concentrations. In a previous report, Tournier et al. [29] showed that infection of C57BL/6 and BALB/c DCs by anthrax spores disrupts the cells' ability to produce cytokines. The authors also reported a loss of viability in BALB/c DCs treated with a combination of B. anthracis spores and purified LT. Our results show that purified LT alone was sufficient to reproduce these observations.
Our findings on DC killing challenge an earlier report [12], which described DC impairment without cell killing. Agrawal et al. [12] specifically reported no cytopathic effects in LT-treated C57BL/6-derived DCs, as measured by Trypan blue exclusion. This is not surprising, as LT-mediated killing of C57BL/6 BMDCs occurred without significant membrane perturbation. Additionally, they reported no caspase-3 activation in C57BL/6 DCs at 6 and 48 h post-LT exposure. We found that a window of caspase-3 activation occurred between 18 and 32 h (see Figure 4B). At the time points reported in their study, we also found no increase in active caspase-3.
We show that genetic background determines the type of cell killing induced by LT: LT triggered rapid necrosis in BALB/c-derived DCs and slow apoptosis in C57BL/6-derived DCs. A genetic polymorphism containing a putative susceptibility factor at the Kif1c locus on Chromosome 11 has been identified that correlates with the sensitivity of macrophages to LT killing [9,30]. We believe that the strain-specific cell death pathways we report are also controlled by this genetic locus. It remains to be shown how genetic factors determine whether apoptotic or necrotic pathways are induced by LT.
DC killing precedes lethal effects in LT-treated BALB/c and C57BL/6 mice by approximately 24 h, and therefore constitutes an early event in anthrax pathogenesis [3]. The reason for selective LT targeting of antigen presenting cells, including macrophages and DCs, is unknown. Murine C57BL/6 DCs and human DCs share slow killing kinetics and induction of apoptosis following LT exposure. Therefore, C57BL/6-derived cells appear to be a better model system for human anthrax than the prototypical BALB/c-derived macrophages, which rapidly undergo necrotic cell death after LT challenge. Induction of distinct cell death pathways suggests that LT-mediated cell killing is controlled by strain-specific regulators and inhibitors. Extensive investigation has led to the discovery of multiple potent apoptosis inhibitors, which might block LT-mediated apoptosis, and possibly anthrax disease progression, in C57BL/6 mice and human subjects. Additionally, these findings might be important for therapeutic applications. It is conceivable that agents that block LT killing are strain-specific, and that drugs blocking LT-mediated killing of BALB/c-derived cells might be inefficient in targeting LT killing of C57BL/6-derived and human cells and vice versa. Based on our findings, it is now possible to differentiate, in LT-treated C57BL/6 mice, cells that are directly killed by LT, presumably by apoptosis induction, from cells killed by indirect means via induction of necrotic pathways.
B. anthracis infections appear to challenge the immune system in two phases. During the early phase of infection, subtoxic concentrations of LT disrupt MAPK signaling pathways, rendering immune cells nonresponsive to cytokine and immune stimulation [12,13]. We present evidence that this phase is followed by LT killing of DCs, which presumably occurs when the concentration of LT reaches cytotoxic levels. This likely causes additional impairment of adaptive immunity, and represents an immune-evasion strategy of the bacterium. DCs as well as macrophages are primary entry sites of B. anthracis [29,31,32], and early replication of the bacterium occurs exclusively within these cells. The slow LT killing kinetics found in C57BL/6 and human DCs may promote bacterial proliferation.
Specific targeting of DCs by LT should prevent stimulation of the adaptive immune response, rendering hosts highly susceptible to other microbial pathogens. Depletion of DCs also plays a critical role in human and murine sepsis [33]. Additionally, it is possible that LT killing of DCs could be exploited in therapeutic applications of the toxin. LT's potential for medical applications is not novel; sublethal LT treatment was employed in nude mice containing human melanoma xenograft tumors, resulting in tumor regression without any cytotoxic side effects [34]. Due to the high specificity and sensitivity of DCs to LT, exposure of DCs to specific toxin concentrations could represent a useful approach to transiently suppress the immune response, if cytotoxic effects of LT on other cell types could be effectively controlled or eliminated.
Materials and Methods
Cell culture and materials.
C57BL/6 and BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, Maine, United States). Human MoDCs were cultured in RPMI-1640 containing 2 mM L-glutamine and supplemented with 1% nonessential amino acids, 10 mM Hepes, 1% penicillin-streptomycin, and 10% FCS (R10 medium). The pan-caspase inhibitors Z-VAD-FMK and BOC-D-FMK (Calbiochem, San Diego, California, United States) and the proteasome inhibitor MG132 (Oncogene, Boston, Massachusetts, United States) were reconstituted in DMSO and used at a concentration of 10 μg/ml, except for BOC-D-FMK, which was used at 40 μg/ml. The proteasome inhibitor Velcade (bortezomib) was generously provided by Dr. Roman Perez-Soler (Albert Einstein College of Medicine). Recombinant anthrax LF was obtained from List Biological Laboratories (Campbell, California, United States). PA was generously provided by Dr. Steven Leppla (National Institutes of Health). PA and LF were reconstituted in water at 500 ng/ml and 250 ng/ml, respectively [35]. Recombinant LF and PA were produced in B. anthracis and were free of LPS contamination as indicated by the manufacturer, and by a lack of CD86 up-regulation on immature cells, as measured by flow cytometry.
Generation of human DCs from peripheral blood.
Immature DCs were prepared from human PBMCs as previously described [36]. Briefly, PBMCs from buffy coats were isolated by centrifugation on Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, United Kingdom) at 600 g for 20 min. After washing three times with PBS to remove platelets, PBMCs were resuspended in 60 ml of R10 medium (containing 10% FCS and 6.6 ng/ml human GM-CSF [Leukine; Berlex, Montville, New Jersey, United States]). After 30 min at 37 °C, the medium was removed and adherent cells were cultured in R10 medium containing human GM-CSF at 6.6 ng/ml and human IL-4 (PeproTech, Rocky Hill, New Jersey, United States) at 10 ng/ml for 3–4 d, to produce immature DCs. The identity of these cells was confirmed by flow cytometry using anti-CD11c, CD14, CD16, CD80, CD86, HLA-DR, and DEC-205 antibodies.
Generation of murine bone marrow-derived and splenic DCs.
BMDCs were prepared as previously described [24]. 10 ml syringes fitted with 25 gauge needles and filled with mR10 medium (RPMI-1640, 10% FCS, 10 mM Hepes, 1% penicillin-streptomycin, and 55 μM beta-mercaptoethanol) were used to flush bone marrow cells from the femurs and tibias of C57BL/6 and BALB/c mice. Red blood cells were lysed using RBC lysing buffer (Sigma, St. Louis, Missouri, United States), and the remaining cells were washed with PBS. Cells were plated in bacterial plates in mR10 medium containing 20 ng/ml murine GM-CSF (PeproTech). On days 3, 6, and 8, cells were resuspended in fresh mR10 containing 20 ng/ml murine GM-CSF. On day 10, nonadherent cells were resuspended in mR10 containing 5 ng/ml murine GM-CSF, for maintenance of differentiated DC populations. The identity of cells was assayed by flow cytometry using anti-CD11b, CD11c, CD14, CD80, CD86, and MHC class II antibodies. Splenic DCs were isolated using anti-CD11c magnetic beads according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Spleens from BALB/c mice were treated with collagenase D, cells were incubated with anti-CD11c microbeads, and splenic DCs were enriched using MS separator columns.
Cell death and viability assays.
Cell viability was measured by analysis of MTT cleavage to formazan by succinate dehydrogenases in living cells [37]. For the colorimetric MTT assay, cells were exposed to LT (500 ng/ml PA and 250 ng/ml LF), and the MTT solution (5 mg/ml MTT in PBS) was added directly to wells and incubated at 37 °C for 4 h. The dye was solubilized with acidic isopropanol (25 mM HCl and 0.5% SDS in isopropanol), and the absorbance was measured at 570 nm.
For analysis of caspase activation, we used a colorimetric caspase-3 assay, performed as recommended by the manufacturer (R&D Systems, Minneapolis, Minnesota, United States). LT-treated cells were cultured in 24-well plates and lysed in the wells after removing the culture medium. Cleavage of the substrate was measured on an LS-50 fluorescence spectrometer (Perkin Elmer, Wellesley, California, United States). TUNEL assays were performed as described previously [38].
Western blotting.
Cells were cultured in 24-well plates and treated with LT. Western blotting was performed as described previously [39]. In brief, cells were lysed in the wells after removing culture medium. For lysis, the caspase-3 lysis buffer (R&D Systems) was supplemented with a cocktail of protease inhibitors (Roche, Basel, Switzerland). Cellular lysates were centrifuged, and supernatants were mixed with SDS sample buffer and denatured at 100 °C for 3 min. Size fractionation was performed on 50 μg of protein from each sample on BioRad SDS-Tris HCl polyacrylamide gels (BioRad, Hercules, California, United States), and transferred to PVDF membranes (Amersham). Membranes were probed with anti-MEK-3 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California, United States; #Sc-960) and anti-actin monoclonal antibody (Sigma; #Ac-40). Polyclonal HRP-conjugated anti-rabbit antibodies were used as secondary antibodies (Santa Cruz Biotechnology; #Sc-2313), and blots were developed using ECL Plus solution (Amersham).
Transmission electron microscopy.
Samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, and postfixed with 1% osmium tetroxide, followed by addition of 1% uranyl acetate. After dehydration through a graded series of ethanol washes, samples were embedded in LX112 resin (LADD Research Industries, Williston, Vermont, United States). Ultrathin sections were cut on a Reichert Ultracut UCT, stained with uranyl acetate followed by lead citrate, and viewed on a JEOL 1200EX transmission electron microscope at 80 kV.
Flow cytometry.
Human DCs and murine BMDCs were analyzed by flow cytometry using FACScan and FACScalibur flow cytometers (BD Biosciences, Palo Alto, California, United States). For each staining condition,106 cells were washed once with buffer A (PBS containing 1% BSA and 0.05% NaN3). After blocking for 10 min with heat-inactivated fetal calf serum, primary antibody was added for 30 min on ice. Subsequently, cells were spun down, washed in buffer A and stained with secondary antibodies when necessary. The following antibodies were purchased from BD Biosciences: FITC-conjugated anti-CD14 (clone M5E2), anti-CD16 (clone 368), and anti-CD80 (clone L307.4); and PE-conjugated CD11c (clone B-Ly6) and anti-CD86 (clone 2331). The following antibodies were produced from hybridoma lines that were cultivated in our laboratory: anti-HLA-DR (clone L243; obtained from ATCC, Manassas, Virginia, United States), anti-DEC-205 (clone Mg38; obtained from Dr. R. Steinmann, Rockefeller University), and anti-CD58 (clone TS2/9; obtained from ATCC). FITC-conjugated goat F(ab′)2 anti-mouse IgG + IgM, fluorescein conjugate (Biosource, Camarillo, California, United States) was used as a secondary antibody when necessary. In all cases, antibody staining was compared to the appropriate isotype control.
In vivo assay.
Ten-week-old female BALB/c mice were injected intraperitoneally with LT [3,4]. Mice were sacrificed 0, 3, 6, and 24 h after LT injection. Spleens were harvested and treated with 400 U/ml collagenase D (Roche) to release DCs. Levels of splenic DCs (CD11c+, MHC class II+), macrophages (CD11b+, MHC class II+), and circulating B cells (B220+) and T cells (CD3+) were determined by flow cytometry using fluorescently labeled antibodies to surface markers.
SA Porcelli was supported by an Irma T Hirschl Career Scientist Award and by grant AI063537 from the National institutes of Health (NIH) National Institute of Allergy and Infectious Diseases. Flow cytometry studies were supported by FACS Core resources of the Albert Einstein College of Medicine Center for AIDS Research. SM Muehlbauer was supported by the NIH Medical Scientist Training Grant T32GM007288. J Brojatsch was supported by grant AI057158 from the Northeast Biodefense Center.
Competing interests. The authors have declared that no competing interests exist.
Author contributions. AA, SMM, and JB conceived and designed the experiments. AA, ERS, and SMM performed the experiments. AA, ERS, SMM, SAP, and JB analyzed the data. SAP contributed reagents/materials/analysis tools. JB wrote the paper. AA, ERS, SMM, and SAP edited the paper.
Abbreviations
BMDCbone marrow-derived dendritic cell
DCdendritic cell
LFlethal factor
LPSlipopolysaccharide
LTlethal toxin
MAPKmitogen-activated protein kinase
MoDCmonocyte-derived dendritic cell
PAprotective antigen
==== Refs
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PLoS PathogPLoS PathogppatplpaplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 1625459810.1371/journal.ppat.001002005-PLPA-RA-0124R2plpa-01-02-05Research ArticleEpidemiology - Public HealthEvolutionInfectious DiseasesGenetics/Population GeneticsYeast and FungiLow Effective Dispersal of Asexual Genotypes in Heterogeneous Landscapes by the Endemic Pathogen Penicillium marneffei
Endemism and Asexuality in
Penicillium marneffeiFisher Matthew C 1*Hanage William P 1de Hoog Sybren 2Johnson Elizabeth 3Smith Michael D 4White Nicholas J 5Vanittanakom Nongnuch 6
1 Department of Infectious Disease Epidemiology, St Mary's Hospital Campus, Imperial College London, London, United Kingdom
2 Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands
3 Mycology Reference Laboratory, Health Protection Agency, Bristol, United Kingdom
4 Department of Microbiology, Taunton and Somerset Hospital, Taunton, United Kingdom
5 Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
6 Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
Taylor John EditorUniversity of California, Berkeley, United States of America* To whom correspondence should be addressed. E-mail: [email protected] 2005 28 10 2005 1 2 e205 8 2005 28 9 2005 Copyright: © 2005 Fisher et al.2005This 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.Long-distance dispersal in microbial eukaryotes has been shown to result in the establishment of populations on continental and global scales. Such “ubiquitous dispersal” has been claimed to be a general feature of microbial eukaryotes, homogenising populations over large scales. However, the unprecedented sampling of opportunistic infectious pathogens created by the global AIDS pandemic has revealed that a number of important species exhibit geographic endemicity despite long-distance migration via aerially dispersed spores. One mechanism that might tend to drive such endemicity in the face of aerial dispersal is the evolution of niche-adapted genotypes when sexual reproduction is rare. Dispersal of such asexual physiological “species” will be restricted when natural habitats are heterogeneous, as a consequence of reduced adaptive variation. Using the HIV-associated endemic fungus Penicillium marneffei as our model, we measured the distribution of genetic variation over a variety of spatial scales in two host species, humans and bamboo rats. Our results show that, despite widespread aerial dispersal, isolates of P. marneffei show extensive spatial genetic structure in both host species at local and country-wide scales. We show that the evolution of the P. marneffei genome is overwhelmingly clonal, and that this is perhaps the most asexual fungus yet found. We show that clusters of genotypes are specific to discrete ecological zones and argue that asexuality has led to the evolution of niche-adapted genotypes, and is driving endemicity, by reducing this pathogen's potential to diversify in nature.
Synopsis
Scientists believe that micro-organisms are spread around the planet on currents of air, a hypothesis that is known as “ubiquitous dispersal”. While fungi release huge quantities of widely dispersed spores, it is not known why many species remain endemic to specific regions around the globe. Research by the authors suggests an answer to this conundrum, by investigating the genetic structure of a fungus, Penicillium marneffei, that causes disease in people with damaged immune systems. This research has shown that P. marneffei spores can be dispersed over a wide distance, but fail to penetrate the new environments that they find themselves in. This appears to be because the fungus has largely dispensed with sexual reproduction, which means that its ability to adapt to new challenges is limited. The authors use DNA typing to show that different “clones” of the fungus are associated with different environments, and suggest that adaptation to these environments is constraining the organism's ability to successfully disperse in nature. This may explain why P. marneffei is endemic to a relatively small area of southeast Asia, and the authors go on to suggest that the long-term consequence of this strategy may be the eventual extinction of the organism.
Citation:Fisher MC, Hanage WP, de Hoog S, Johnson E, Smith MD, et al. (2005) Low effective dispersal of asexual genotypes in heterogeneous landscapes by the endemic pathogen Penicillium marneffei. PLoS Pathog 1(2): e20.
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Introduction
In eukaryotes, sexual recombination is an almost universal phenomenon [1,2]. However, in many microbial eukaryotes, such as fungi, sexual reproduction is facultative, so loss-of-meiosis mutations are effectively neutral. Such mutations will, on average, be fixed by genetic drift in approximately 2Ne generations (where Ne is the effective population size [3]), leading to the possibility that populations and species may lose sexual competence. Modern ecological opinion holds that populations of microbial eukaryotes are huge owing to continent-wide dispersal via airborne spores; this is known as the “ubiquitous dispersal hypothesis” [4,5]. If this hypothesis is correct, then the large value of Ne for these organisms will negate drift as a significant force in their evolution, and mating competence will be preserved. However, recent studies of aerially dispersed fungi have shown the full continuum of population genetic structures, from global panmixia (e.g., Aspergillus fumigatus [6]) through to geographic endemicity and pronounced biogeographic population structure (e.g., Histoplasma capsulatum [7], Coccidioides immitis [8], Blastomyces dermatitidis [9], and Penicillium marneffei [10]). To understand why these fungi vary so widely in their ability to undergo successful long-distance aerial dispersal, we need to critically evaluate the ubiquitous dispersal hypothesis.
Theoretical and empirical work has shown that sex acts to increase the rate of adaptation to new environments by increasing the variance in fitness between offspring [11,12]. A corollary of this theory is that we can make a prediction that reduced sexual competence, and hence reduced recombination rates, between species are expected to correlate with a reduced potential to disperse within heterogeneous environments. Loss of sex may therefore explain why endemic species occur in the face of widespread aerial dispersal.
We investigated this hypothesis by subjecting the human pathogenic fungus P. marneffei to an analysis designed to describe the effects of (i) space, (ii) reproductive mode, and (iii) host on this species's population genetic structure within Thailand. The fungus is endemic to a narrow band of southeast Asia [10] and has a simple haploid lifecycle whereby filamentous soil mycelia give rise to aerially borne conidia [13]. If inhaled, P. marneffei disseminates as an intracellular yeast, and, as a consequence, the fungus has recently emerged as a major opportunistic infection of HIV/AIDS patients in this populous region (Figure 1). P. marneffei is morphologically asexual and is a member of the estimated one-fifth of fungal species that have no known sexual state. Interestingly, surveys of native southeast Asian rodent populations have shown that P. marneffei is associated strongly with bamboo rats, but not other rodent species [14–16]. To test the hypothesis that different lineages of P. marneffei may have adapted to infect different hosts, we acquired bamboo rat isolates from several provinces across Thailand. We further acquired isolates of P. marneffei from a cohort of AIDS patients from across Thailand and used high-resolution multilocus genotyping to analyse these, and the bamboo rat, isolates.
Figure 1 Co-Emergence of AIDS and Human Penicilliosis in Northern Thailand
Temporal emergence of HIV (antenatal data, 1990–2000 [38]) and human P. marneffei–associated penicilliosis (1985–2001; Maharaj Hospital, Chiang Mai) for the Chiang Mai region, northern Thailand.
Results
Naturally Occurring Diversity in Isolates from Humans and Bamboo Rats
Genetic diversity in our study populations was high and of the 169 isolates subjected to analysis, we were able to assign each isolate into one of 97 unique multilocus genotypes, known as microsatellite types (MTs). Epidemiological connectivity between human and bamboo rats was explored using the program eBURST [17]. This approach searches for groups of related MTs, then ascertains patterns of descent within groups. Under a model of simple asexual reproduction, founding genotypes will rise in frequency over time and subsequently diversify by the accumulation of mutations into a complex of closely related genotypes, known as a clonal complex. Such complexes are identified by eBURST as groups of genotypes that are linked by differing at only one out of the 21 multilocus microsatellite typing (MLMT) loci (known as single locus variants). Analysis of our MLMT dataset by eBURST showed that while 83 MTs were specific to P. marneffei that had been recovered from HIV-positive patients, five MTs were found to infect both humans and bamboo rats (Figure 2). This is direct evidence that P. marneffei isolates that are capable of infecting both humans and rodents do occur in nature. eBURST identified 13 clonal complexes in our dataset of MLMT genotypes. Of the MTs that are shared between humans and bamboo rats, two (MTs 10 and 29) were designated as the primary founders of the clonal complex within which they occur. For the seven isolates comprising MT 29, six were recovered from the bamboo rat species Rhizomys sumatrensis, from two sampling sites in northern Thailand separated by 50 km. These data show that the MT 29 genotype is over-represented in at least two spatially separated rodent populations. This rat-associated founder genotype has diversified into a clonal complex with at least four descendants that are found infecting humans (Figure 2B). Therefore, there is no evidence within our data for fungal lineages that are specific to bamboo rats, and hence no indication that co-evolution is occurring between host and pathogen. In this case, any observed species-specific clustering of genotypes is most likely due to the spatial clustering of genetic diversity in P. marneffei.
Figure 2 Spatial Distribution of Isolates and Genetic Diversity in Thailand
(A) Spatial distribution of sampled isolates in Thailand. Isolates that are found to co-infect both humans and bamboo rats are shown in red, and the clone range of each of these co-infecting MTs is shown as coloured regions. Inset shows the geographic variation of the PC1, which accounts for 18.8% of the total genetic variation. Spatial interpolation of the PC1 values (with colours representing the predicted degree of difference between sample points) illustrates gradients in allele frequencies between northern, eastern, and south-central Thailand.
(B) eBURST analysis of the Thailand P. marneffei dataset, with isolates coloured as noted in (A). The single green point refers to an isolate that was recovered from soil. Each clone is represented by a point, the area of which is representative of the frequency of the clone. Clones that differ from one another at a single locus, and are thus inferred to be directly related (single locus variants), are joined by lines.
Spatial Components of Genetic Diversity
To investigate whether there is a spatial component to P. marneffei's population genetic structure, we calculated a genetic distance matrix for all pairwise combinations of isolates by using an allele-sharing index (D
AS [18]). This genetic distance is based on allele-frequency data and calculates multilocus pairwise distance measurements as 1 − (the total number of shared alleles at all loci/n), where n is the number of loci compared. An associated geographic distance matrix was calculated as the distances between the coordinates of each bamboo rat sampling site and/or patient address [19]. Principal components analysis (PCA) was performed on the total matrix of individual genetic distances, and spatial relationships were visualised by plotting associated values in a geographical information system (GIS). This analysis showed that the first principal component (PC1) explains 18.8% of the total observed genetic diversity. We visualised the PC1 values as a continuous surface, by the use of an inverse-distance-weighted kriging procedure (Figure 2A). Kriging is a computational procedure that interpolates between our sample points using least-squares estimation to arrive at a surface of expected values, based on our known principal component values. That our surface shows spatial correlations between PC1 values clearly demonstrates that there is a broad division of genotypes into northern, eastern, and southern populations of P. marneffei.
We further investigated this broad spatial pattern by calculating the maximum spatial distance covered by each of the observed P. marneffei MTs. This “clone distance” was measured as the geodesic line between the spatial latitudinal and longitudinal coordinates of the most distantly related representatives of each MT. Analysing the spatial distribution of each MT using our GIS showed that 22% of P. marneffei MTs were recovered from more than one site, corresponding to an average radial distance of 28.6 km (95% confidence interval [CI] 20.8–36.2), and an average geographic clonal coverage of 1,975 km2 (95% CI 1,506–2,444; Table 1). We investigated whether these spatially “patchy” P. marneffei clones are randomly dispersed in two-dimensional space by calculating the autocorrelation coefficient, r, for all pairwise combinations of individual-by-individual multilocus genetic distances (D
AS) correlated against geographic distance [20]. Isolates that are more related to one another than is expected from a randomly mating population are characterised by a positive value for r, and if isolates are spatially structured, then r will exhibit a negative correlation with distance. These correlograms confirmed the results from our PCA by showing that there is strong, and significant, positive spatial genetic structure that undergoes rapid decay at greater scales (Figure 3). The autocorrelation coefficient observed between bamboo rat isolates showed that (i) relatedness was significantly higher than that found in clinical isolates at the same local scale (p < 0.01) and (ii) the extent of non-random genetic structure was significantly less for bamboo rat isolates (109 km compared to 367 km for human isolates). We interpret these data as showing that the high mobility of humans, relative to bamboo rats, is reflected in an increased probability of infection beyond the scale of the local population of P. marneffei.
Table 1 Dispersal Distance and Clone Range for MTs Common to Both Humans and Bamboo Rats
Figure 3 Correlograms Showing the Correlation Coefficient r as a Function of Distance for Human Clinical Isolates and Bamboo Rat Isolates
Data for (A) human clinical isolates and (B) bamboo rat isolates. Values for r are shown as solid lines with 95% CIs calculated by bootstrapping the dataset. Also shown are the 95% CIs around the null hypothesis of randomly distributed genotypes of P. marneffei, calculated over 1,000 permutations of the data, shown as dotted lines.
Relative Contributions of Asexual versus Sexual Reproduction
The occurrence of 13 clonal complexes (see Figure 2B) suggests that there is a significant asexual component to the population genetic structure of P. marneffei. We quantified the relative contributions of recombination and mutation to the generation of diversity in this dataset by examining the changes that have generated single locus variants. Changes that incorporate unique alleles are most likely due to mutation and those that utilise pre-existing alleles are most likely due to recombination. In order to prevent our estimate being biased by loci under unusual selective pressures, we excluded the highly diverse or uniform loci from the analysis. Defining recombination and mutation as above, we found that 97% of alleles were found within a single clonal complex. Using this value, we were able to estimate from eBURST that the ratio of recombination to mutation within our dataset was 1:4.7. This value is less than estimates of the same ratio for many bacterial species [21], and is surprisingly clonal for a fungal species. Moreover, our estimate is very conservative, because the mode of mutation in microsatellites by strand-slippage mispairing will inevitably create alleles that are identical by size, but not by descent. As a consequence, a proportion of the changes scored as recombination will in fact be mutations.
To analyse the extent of multilocus linkage disequilibrium, we controlled for the effects of spatial structure by using our GIS to restructure the dataset into populations that comprised at least 20 isolates falling within a single circle of radius 109 km (corresponding to our best estimate for the scale of a local population). This procedure identified two groups of isolates with centres in Chiang Mai and Ubon Ratchathani. We calculated a standardised measure of multilocus disequilibrium,
[22], and compared this value to those calculated from a comparable MLMT analysis of two related morphologically asexual fungal pathogens exhibiting endemic distributions, Coccidioides immitis and C. posadasii [23]. This analysis showed that
was highly significant for P. marneffei, even when we took into account direct clonal reproduction by removing identical genotypes. Interspecific comparisons of
showed that P. marneffei was significantly more asexual than either of the two Coccidioides species (Table 2). As an additional test of the hypothesis of clonality, a maximum parsimony approach was used to find the shortest tree that fitted the P. marneffei data, then randomisation protocols were used to simulate 1,000 randomly mating populations. In all cases, the observed tree was significantly shorter than the null distributions, demonstrating significant structure in our data (Figure S1). As we have controlled for spatial population structure by including only isolates from a small geographic area, and have considered them together with the evidence from the eBURST analysis, the most likely cause of this observed multilocus genetic disequilibrium is evolution by clonal descent.
Table 2 Multilocus Linkage Disequilibrium for P. marneffei and Coccidioides spp.
Associations between Genetic Diversity and Ecological Heterogeneity
It is clear from the above analyses that the population genetic structure of P. marneffei has two components: one caused by a low effective rate of dispersal and the other by a low effective rate of recombination. The adaptation of P. marneffei to an aerial mode of transmission, manifested by its ability to produce essentially unlimited infectious conidia, in the same size range as microbes (2–3 μm), presents a conundrum. P. marneffei is highly abundant, as is shown by the high prevalence of infection in both human and bamboo rat populations in endemic areas. Furthermore, widespread dispersal evidently occurs, as we have been able to demonstrate infection of humans and bamboo rats with identical clonal genotypes over a 47- to 577-km range (Table 1). However, this dispersive capacity has not been translated into widespread gene flow, and significant population genetic structure occurs.
Under neutral community models [24], differences in geographic biodiversity are attributable primarily to differences in dispersal rate. However, if the relative fitness of genotypes varies between regions as a consequence of strong disruptive selection, then locally adapted genotypes will tend to outcompete any incoming immigrants even in the face of frequent dispersal. A key parameter that will therefore determine the relative fitness of isolates between different regions is the strength of local selection, i.e., the degree of environmental heterogeneity. We tested whether environmental heterogeneity correlates with genetic diversity by performing ecological niche modelling based on our primary occurrence data using Genetic Algorithms for Rule-Set Prediction (GARP [25]). This algorithm uses digital maps to summarise environmental variables, then identifies associations between point occurrence data and their ecological dimensions. Rule sets that define a niche with the highest predictive accuracy (based on known occurrences) are subsequently used to generate digital maps with areas of predicted niche presence and absence (for details see [26]).
We modelled southeast Asia's ecology using the BIOCLIM 2.5 minute dataset [27], the Food and Agricultural Organization digital soil map, and remotely sensed digital vegetation maps of southeast Asia with a 1-km2 resolution (Figure S2). Three categories of genotype were used as occurrence points, corresponding to the northern, eastern, and southern Thailand genotypes determined by PCA (see Figure 2A, inset). Projecting the rule sets from these analyses clearly illustrates non-overlapping predictions for the distributions of the three genotype classes (Figure 4). This analysis shows that environmental heterogeneity exists within Thailand at the scales investigated here, and that there is a clear association between genetic and ecological diversity. The rule sets postulate a wider distribution for the southern genotype class, as the genotypes in this class are associated with a set of environmental variables that are more widely distributed than is found associated with the northern and eastern Thailand genotypes. Interestingly, the southern Thailand genotypes are predicted to occur within Viet Nam, a hypothesis that should be examined as Viet Nam is currently undergoing an emergence of human penicilliosis in association with the epidemic of HIV within this region.
Figure 4 Predicted Distributions for Three Classes of Genotype: Northern, Eastern, and Southern Thailand
The “overlap index” represents the synthesised output from 20 optimal GARP models. See Figure S2 for data sources.
Discussion
Genuinely asexual fungi are rare and few have passed the population genetic tests that we have presented here; the genetic footprint of recombination appears to be ubiquitous in nature [28]. While we are not ruling out the presence of recombination in P. marneffei, the low frequencies that we have measured will tend to decrease additive fitness variance within populations [1], leading to the evolution of a “rugged” fitness landscape with multiple peaks and valleys. In a heterogeneous environment, variance in fitness between clonal lineages is expected to lead to populations that are adapted to local conditions [11,12]. In this case limited dispersal then becomes an inevitable consequence of asexuality, as invading clones are outcompeted by better adapted local conspecific competitors because of the decreased ability of invaders to diversify within novel environments [11]. In such a system, spatial metapopulation structure will further increase biases in the frequencies of mating-types and loss-of-meiosis mutations by increasing the rate of genetic drift. Eventually, Ne is expected to become depressed to the extent that mating competence within populations will be irrevocably lost [29,30]. Our work shows that P. marneffei is perhaps the most asexual fungus yet found. We provide a theoretical mechanism to account for P. marneffei's extreme geographic endemism and fragmented population genetic structure, by arguing that the clonality we observe is driving niche adaptation in a heterogeneous environment. Further, our conclusions clearly falsify the ubiquitous dispersal hypothesis for P. marneffei, and data on the population genetic structure of other endemic fungi, as well as bacteria, suggest that our results will have broader relevance to microbial systems [28,31,32]. Further tests of our hypothesis will rely on experimental measurements of the relative fitness of asexual and sexual fungal lineages in controlled, heterogeneous environmental backgrounds. Such investigations will ultimately show whether microbial endemicity is a signpost of genomic decay, heralding the onset of a species's eventual extinction.
Materials and Methods
P. marneffei isolates were acquired as cultures from HIV/AIDS patients who were attending regional central hospitals in Chiang Mai (northern Thailand), Ubon Ratchathani (eastern Thailand), and Bangkok (south-central Thailand). Isolates were georeferenced with latitudinal and longitudinal coordinates corresponding to the recorded patient address. Our study obtained 146 epidemiologically unlinked human isolates of P. marneffei from patients widely distributed across Thailand (see Figure 2A). We further acquired 23 isolates from three species of bamboo rat, Rhizomys pruinosus, R. sumatrensis, and Cannomys badius; these isolates had all been found infecting the liver and spleen of healthy adult rodents [14,15]. Isolates were grown on slants containing Sabouraud's agar medium. Following homogenisation of mycelia, DNA was extracted using a Qiagen (Valencia, California, United States) DNeasy Tissue kit according to the manufacturer's instructions. The purified DNA was then used as a template to amplify 21 microsatellite loci using fluorescently labelled primers and a PCR multiplexing protocol [33]. Subsequently, PCR products were visualised on an Applied Biosystems (Foster City, California, United States) model 3700 capillary sequencer and the alleles were scored using Genotyper software (Applied Biosystems). All genotypes were assigned a specific MT identifier according to the P. marneffei MLMT scheme [34,35]; associated data and the MTs are accessible via the Internet at http://pmarneffei.multilocus.net/ (Table S1).
Supporting Information
Figure S1 Distribution of Tree Lengths for Northern Thailand
Using the program PAUP* 4.0, maximum parsimony was used to find the shortest tree(s) that fitted the data. In order to test whether the observed data contained a greater phylogenetic signal when compared to sexual populations, the datasets were artificially recombined 1,000 times, and the lengths of their most parsimonious trees were compared to those found for the observed dataset. This process was repeated for datasets in which all identical genotypes had been removed (clone-corrected data).
(84 KB PPT)
Click here for additional data file.
Figure S2 Point Occurrence Data and Environmental Layers Used in GARP Analysis
GARP searches for non-random associations between points of known occurrence compared to the overall study region (in this case southeast Asia). The algorithm selects optimal models after a number of iterations, based on a training subset of the data, then uses the remaining data to test model quality. The routines used here were run in Openmodeller (http://openmodeller.sourceforge.net/), and the output visualised using ArcMap 8.3 (ESRI, Redlands, California, United States). Three categories of genotype were used as occurrence points, corresponding to the northern, eastern, and southern Thailand genotypes determined by PCA (see Figure 1). Because our data are address-based, and therefore only corresponds to an estimate of where the patient (or bamboo rat) was infected, we resampled each environment within an area for a circle of radius 28.6 km (corresponding to our estimate of clone dispersal distance) around the coordinate of our point sample. This was repeated 20 times for each sample point, resulting in the point distributions seen in maps 1–3. Twenty-one digital environmental layers were obtained (above). These correspond to the Food and Agricultural Organization's Digital Soil Map of the World and Derived Soil Properties [36] (map 4), vegetation cover and classification, derived from the SPOT-4 remote sensor [37] (map 5), and 2.5 minute WORLDCLIM layers [29] (maps 6–24), corresponding to annual mean temperature (map 6), mean diurnal range (mean of monthly [maximum temperature − minimum temperature]) (map 7); isothermality (map 8), temperature seasonality (standard deviation × 100) (map 9), maximum temperature of warmest month (map 10), minimum temperature of coldest month (map 11), temperature annual range (map 12), mean temperature of wettest quarter (map 13), mean temperature of driest quarter (map 14), mean temperature of warmest quarter (map 15), mean temperature of coldest quarter (map 16), annual precipitation (map 17), precipitation of wettest month (map 18), precipitation of driest month (map 19), precipitation seasonality (coefficient of variation) (map 20), precipitation of wettest quarter (map 21), precipitation of driest quarter (map 22), precipitation of warmest quarter (map 23), and precipitation of coldest quarter (map 24).
(507 KB PPT)
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Table S1 MLMT Allele Summary for 169 P. marneffei Isolates
(18 KB PDF)
Click here for additional data file.
This study was funded by a Wellcome Trust Biodiversity Fellowship to MCF. We would like to acknowledge the many people who assisted in collecting these isolates, specifically V. Wuthiekanun and A. Rajanuwong in Ubon Ratchathani, and C. Khamwan at Maharaj Hospital, Chiang Mai.
Competing interests. The authors have declared that no competing interests exist.
Author contributions. MCF conceived and designed the experiments. MCF and NV performed the experiments. MCF and BH analyzed the data. SdH, EJ, MDS, NJW, and NV contributed reagents/materials/analysis tools. MCF wrote the paper.
Abbreviations
CIconfidence interval
GARPGenetic Algorithms for Rule-Set Prediction
GISgeographical information system
MLMTmultilocus microsatellite typing
MTmicrosatellite type
PC1first principle component
PCAprincipal components analysis
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Finlay BJ 2002 Global disperal of free-living microbial eukaryote species Science 296 1061 1063 12004115
Paoletti M Rydholm C Schwier EU Anderson MJ Szakacs G 2005 Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus
Curr Biol 15 1242 1248 16005299
Kasuga T White TJ Koenig G McEwen J Restrepo A 2003 Phylogeography of the fungal pathogen Histoplasma capsulatum
Mol Ecol 12 3383 3401 14629354
Fisher MC Koenig GL White TJ San-Blas G Negroni R 2001 Biogeographic range expansion into South America by Coccidioides immitis mirrors New World patterns of human migration Proc Natl Acad Sci U S A 98 4558 4562 11287648
McCullough MJ DiSalvo AF Clemons KV Park P Stevens DA 2000 Molecular epidemiology of Blastomyces dermatitidis
Clin Infect Dis 30 328 335 10671337
Cooper CR Jr Haycocks NG 2000
Penicillium marneffei: An insurgent species among the penicillia J Eukaryot Microbiol 47 24 28 10651292
Buckling A Wills MA Colegrave N 2003 Adaptation limits diversification of experimental bacterial populations Science 302 2107 2109 14684817
Goddard MR Godfray HC Burt A 2005 Sex increases the efficacy of natural selection in experimental yeast populations Nature 434 636 640 15800622
Chariyalertsak S Sirisanthana T Supparatpinyo K Praparattanapan J Nelson KE 1997 Case-control study of risk factors for Penicillium marneffei infection in human immunodeficiency virus-infected patients in northern Thailand Clin Infect Dis 24 1080 1086 9195061
Ajello L Padhye AA Sukroongreung S Nilakul CH Tantimavanic S 1995 Occurrence of Penicillium marneffei infections among wild bamboo rats in Thailand Mycopathologia 131 1 8 8532047
Chariyalertsak S Vanittanakom P Nelson KE Sirisanthana T Vanittanakom N 1996
Rhizomys sumatrensis and Cannomys badius, new natural animal hosts of Penicillium marneffei
J Med Vet Mycol 34 105 110 8732355
Gugnani H Fisher MC Paliwal-Johsi A Vanittanakom N Singh I 2004 Role of Cannomys badius as a natural animal host of Penicillium marneffei in India J Clin Microbiol 42 5070 5075 15528698
Feil EJ Li BC Aanensen DM Hanage WP Spratt BG 2004 eBURST: Inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data J Bacteriol 186 1518 1530 14973027
Bowcock AM Ruiz-Linares A Tomfohrde J Minch E Kidd JR 1994 High resolution of human evolutionary trees with polymorphic microsatellites Nature 368 455 457 7510853
Peakall R Smouse PE 2001 GenAlEx V5: Genetic analysis in excel. Population genetic software for teaching and research [computer program] Canberra Australian National University
Smouse PE Peakall R 1999 Spatial autocorrelation analysis of individual multiallele and multilocus genetic structure Heredity 82 561 573 10383677
Feil EJ Spratt BG 2001 Recombination and the population structures of bacterial pathogens Annu Rev Microbiol 55 561 590 11544367
Agapow P-M Burt A 2005 Multilocus v1.2.2 [computer program] Available: http://www.bio.ic.ac.uk/evolve/software/multilocus/ . Accessed 3 October 2005.
Fisher M Koenig G White T Taylor J 2002 Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis
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Anderson RP Lew D Peterson A 2003 Evaluating predictive models of species' distributions: Criteria for selecting optimal models Ecol Modell 162 211 232
Stockwell D Peters D 1999 The GARP modelling system: Problems and solutions to automated spatial prediction Int J Geogr Info Sci 13 143 158
Hijmans RJ Cameron SE Parra JL Jones PG Jarvis A 2004 WORLDCLIM, version 1.3 [online database] Available: http://biogeo.berkeley.edu/worldclim/worldclim.htm . Accessed 3 October 2005.
Taylor J Jacobson D Fisher M 1999 The evolution of asexual fungi: Reproduction, speciation and classification Annu Rev Phytopathol 37 197 246 11701822
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Fisher MC Aanensen D de Hoog S Vanittanakom N 2004 Multilocus microsatellite typing system for Penicillium marneffei reveals spatially structured populations J Clin Microbiol 42 5065 5069 15528697
Taylor JW Fisher MC 2003 Fungal multilocus sequence typing—It's not just for bacteria Curr Opin Microbiol 6 351 356 12941403
Fisher MC de Hoog GS Vannittanakom N 2004 A highly discriminatory multilocus microsatellite typing system (MLMT) for Penicillium marneffei
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Stibig HJ Beuchle R Achard F 2003 Mapping of the tropical forest cover of insular southeast Asia from SPOT4-Vegetation images Int J Remote Sens 24 3651 3662
World Health Organization 2005 Global health atlas [online database] Geneva World Health Organization Available: http://globalatlas.who.int/globalatlas/default.asp . Accessed 5 October 2005.
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PLoS PathogPLoS PathogppatplpaplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 1625459910.1371/journal.ppat.001002105-PLPA-OP-0163plpa-01-02-08OpinionsOpen-Access Science: A Necessity for Global Public Health OpinionColoma Josefina Harris Eva *Manchester Marianne Opinions Editor*To whom correspondence should be addressed. E-mail: [email protected] Coloma and Eva Harris are in the Division of Infectious Diseases, School of Public Health, University of California, Berkeley, California, United States of America, and at the Sustainable Sciences Institute, San Francisco, California, United States of America.
10 2005 28 10 2005 1 2 e21Copyright: © 2005 Coloma and Harris.2005This 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. Citation:Coloma J, Harris E (2005) Open-access science: A necessity for global public health. PLoS Pathog 1(2): e21.
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The world of scientific research and scholarly publishing is undergoing a profound transformation in large part due to the rapid development of information and communication technologies. The Internet and the advent of faster networking capabilities now allow virtually unlimited access to information, remote data gathering, real-time integration of data into databases and models, and online purchasing of research supplies. At the same time, it has opened new possibilities for researchers to communicate with colleagues and with the society in general. However, although some investigators in the developing world are keeping pace with this new reality, the majority are largely excluded from this transformation because of their limited access to scientific information. Particularly relevant to the area of pathogen research, the vast majority of infectious diseases in humans, animals, and plants occurs in the developing world, and efficient communication between local scientists in developing countries and the global community will facilitate advances in knowledge and control of these pathogens. Here we discuss open access and socially responsible philosophies in relation to scientific training, publishing, and intellectual property, and give examples of how we can help keep the developing world fully informed about these new models.
The first step is training partners in developing countries in laboratory techniques and epidemiological skills that complement their existing programs, as well as developing their proficiency in grant proposal writing. A number of organizations have undertaken workshops on such topics around the world, including the World Health Organization (http://www.who.int/en/), the Pan American Health Organization (http://www.paho.org), the Fogarty International Center of the National Institutes of Health (http://www.fic.nih.gov), and the Sustainable Sciences Institute ([SSI]; http://www.ssilink.org), which we represent. SSI is a San Francisco–based organization that helps scientists in underprivileged environments gain access to the resources, technologies, skills, and knowledge they need to conduct cutting-edge research with an impact on public health and to effectively communicate their results [1]. Importantly, these organizations make the content of their courses freely available to participants in the workshops and others, similar to an open-access policy.
Performing research is not the only issue at stake; the scientific community evaluates career advancement and stature primarily by the number and quality of published research articles in peer-reviewed journals, and few investigators in developing countries receive training on how to convert their work into publishable manuscripts. To address this problem, several organizations have begun conducting manuscript-writing workshops in developing countries, including CARE–Centers for Disease Control (http://www.careusa.org/careswork/whatwedo/health/cdc.asp), SSI, and others [1–3]. The objective is to provide the skills needed to transform existing data into publishable material, and increase the likelihood of a manuscript being accepted for publication in a reputable scientific journal. Publications boost investigators' scientific visibility in the international community, help them gain ownership of their research, and improve their chances of competing successfully for external funds. In addition, the findings in published manuscripts have the power to inform and influence local and regional public health policy. SSI has conducted four manuscript-writing workshops throughout Latin America, and the enthusiastic response indicates that this is indeed fulfilling an unmet need.
Dissemination of research is an integral part of the scientific process, and scientific journals are the classical channel used to achieve this. Medical societies, universities, research institutions, and libraries in the developed world have traditionally covered the costs of most journal subscriptions, shielding individual scientists from the steep increases in subscription prices. For example, subscriptions to scientific journals are reported to have increased 227% between 1986 and 2002 (see Graph 2 and Table 2 in [4]). However, scientists in developed countries have remained largely oblivious to this financial threat to scholarship and indifferent to the costs because they have enthusiastically embraced the desktop accessibility to journals.
In the developing world, scientists face a greater challenge to remain informed about the progress in their fields of research. Although they are disproportionately affected by infectious diseases, they are excluded from the relevant information that might help them cure, control, and manage the effects of these diseases. Institutions and libraries have been unable to keep up with increases in journal pricing, so individual researchers have had to cover the costs of the few journals to which they subscribe; however, most researchers simply cannot afford to do this and remain excluded from access to information and from the possibility of publishing in a widely distributed journal. In the last few years, initiatives such as the Health InterNetwork Access to Research Initiative (http://www.who.int/hinari/en,
http://www.healthinternetwork.net) have made access to online journals, although not always current, a possibility for those researchers with internet access—constituting a large step toward reducing the medical and scientific information gap between rich and poor countries. Nonetheless, the digital divide is still great in many regions. Latin America, for instance, is a region with islands of progress, where only a few privileged researchers have access to state-of-the-art scientific and technological infrastructures. Most national research and education networks are based on low-speed commercial internet services; there is limited global connectivity and great disparity in the levels of development within and between countries. As a result, the region as a whole is not positioned to participate effectively in global research.
Although many of us base our research on crucial collaborations with researchers in developing countries, we often do not fully understand the limitations they face every day when they try to access information or communicate their findings. As a result, it is often difficult for them to compete at the same level with counterparts in a privileged environment. We have failed to support our colleagues abroad, ignoring their isolation because we are encouraged to publish our research in high-profile journals, which will increase our competitiveness and access to further funding. This individualistic and self-perpetuating cycle can only be broken if researchers and funding agencies alike shift their mindset. The first principle we should all support is that publicly funded research should be made publicly available through the most appropriate open-access channel. Funding agencies and reviewers need to give researchers credit, not penalize them, for efforts to publish in new open-access media. Even with strict peer review, new electronic journals will not immediately attain the same impact status as traditional print journals, but they will have a greater reach and a larger global influence. In fact, it has been shown that online accessibility increases the citation rate and, thus, the impact of a journal by 157% [5]. This new online publishing venue might be the only way that scientists in the developing world conducting highly relevant research can make their data available to the world. It might also be the only means for them to obtain the most recent and relevant information for their research.
Recent developments in Europe and the United States led by the European Science Foundation (Open Archives Initiative, http://www.openarchives.org), the Public Library of Science (http://www.plos.org), and PubMed Central (http://www.pubmedcentral.nih.gov) have focused on an open-access initiative that attempts to establish a norm of no-cost dissemination of author-copyrighted articles. Since 1999, the Open Archives Initiative has developed and promoted standards to facilitate the dissemination of content, enhanced access to electronic print archives to promote the concept of electronic preprint, and created generic mechanisms to harvest data from repositories. Fortunately, there are numerous examples of systems that provide software allowing access to the research literature online through author-driven and institutional archiving, helping construct a global digital library of scientific information.
Finally, global access to scientific knowledge and the fruits of that knowledge also extend to the products that result from scientific endeavors. This, necessarily, treads into the hotly contested area of intellectual property. But, in fact, win–win solutions exist that allow profits to be generated from inventions and at the same time ensure that these products are available “at cost” in developing countries. For example, a new trend of licensing models is under way at the University of California (UC) Berkeley, where several innovations with practical use have been subjects of this novel initiative. SSI helped set the stage for this concept by developing an agreement with the UC Berkeley Office of Technology Licensing to obtain the right to market one of UC Berkeley's patents “at cost” in the developing world. This was swiftly followed by several widely publicized agreements involving royalty-free licenses for developing countries' use of products—including the Gates-funded project for artemisinin synthesis in Escherichia coli being conducted at UC Berkeley, the Institute for OneWorld Health, and Amyris Biotechnologies, and an agreement between UC Berkeley and Samoa for development of plant-derived pharmaceuticals. Importantly, a new movement for “socially responsible licensing” has been born, led by UC Berkeley, Harvard University, Massachusetts Institute of Technology, Yale University, University of Minnesota, and Columbia University, to share ideas on bringing access to technology and medical treatments to the developing world.
We believe the whole spectrum of scientific endeavor should be as open access as possible, from training in laboratory and epidemiological techniques, proposal writing, and manuscript-writing skills to open-access publishing and socially responsible intellectual property policies. In this way, a new door of opportunity can be opened so that the fruits of our scientific breakthroughs are disseminated worldwide and benefit global public health.
Figure 1 Annual Collection of Serological Samples, Managua, Nicaragua, 2005
The collection of samples, monitored here by Eva Harris (right), is part of the UC Berkeley/SSI pediatric dengue cohort study in Managua, Nicaragua. Objectives of this study include investigating the natural history and risk factors for dengue virus transmission and disease, obtaining biological samples in parallel with clinical and epidemiological information, and establishing a site for a future phase III trial of a safe tetravalent dengue vaccine.
(Photo: Alejandro Belli)
Figure 2 Josefina Coloma (left) and Eva Harris (right) in their laboratory at the School of Public Health, UC Berkeley
(Photo: Jennifer Kyle)
Abbreviations
SSISustainable Sciences Institute
UCUniversity of California
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References
Harris E 2004 Scientific capacity building in developing countries EMBO Rep 5 7 11 14710175
Glew RH 2002 The true meaning of technology transfer. Electron J Biotechnol 5 Available: http://www.ejbiotechnology.info/content/vol5/issue1/issues/04 . Accessed 3 October 2005.
Churchill RE 2002 Preventing data drain: An international project Sci Ed 25 115 117
Kyrillidou M Young M 2005 ARL statistics 2001–2002 Washington (D.C.) Association of Research Libraries Available: http://www.arl.org/stats/pubpdf/arlstat02.pdf . Accessed 6 October 2005.
Lawrence S 2001 Free online availability substantially increases a paper's impact Nature 411 521 Available: http://www.nature.com/nature/debates/e-access/Articles/lawrence.html . Accessed 3 October 2005.
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AIDS Res TherAIDS Research and Therapy1742-6405BioMed Central London 1742-6405-2-81620737110.1186/1742-6405-2-8ResearchHIV-1 reverse transcriptase mutations that confer decreased in vitro susceptibility to anti-RT DNA aptamer RT1t49 confer cross resistance to other anti-RT aptamers but not to standard RT inhibitors Fisher Timothy S [email protected] Pheroze [email protected] Vinayaka R [email protected] Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA2 Division of Cardiovascular Diseases, Merck Research Laboratories, Rahway, New Jersey 07065, USA2005 5 10 2005 2 8 8 15 7 2005 5 10 2005 Copyright © 2005 Fisher et al; licensee BioMed Central Ltd.2005Fisher 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.
RNA and DNA aptamers specific for HIV-1 reverse transcriptase (RT) can inhibit reverse transcription in vitro. RNA aptamers have been shown to potently block HIV-1 replication in culture. We previously reported mutants of HIV-1 RT with substitutions N255D or N265D that display resistance to the DNA aptamer RT1t49. Variant viruses bearing these mutations singly or in combination were compromised for replication. In order to address the wider applicability of such aptamers, HIV-1 RT variants containing the N255D, N265D or both (Dbl) were tested for the extent of their cross-resistance to other DNA/RNA aptamers as well as to other RT inhibitors. Both N265D and Dbl RTs were resistant to most aptamers tested. N255D mutant displayed mild resistance to two of the DNA aptamers, little change in sensitivity to three and hypersensitivity to one. Although all mutants displayed wild type-like ribonuclease H activity, their activity was compromised under conditions that prevent re-binding. This suggests that the processivity defect caused by these mutations can also affect RNase H function thus contributing further to the replication defect in mutant viruses. These results indicate that mutants conferring resistance to anti-RT aptamers significantly affect many HIV-1 RT enzymatic activities, which could contribute to preventing the development of resistance in vivo. If such mutations were to arise in vivo, our results suggest that variant viruses should remain susceptible to many existing anti-RT inhibitors. This result was tempered by the observation that NRTI-resistance mutations such as K65R can confer resistance to some anti-RT aptamers.
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Background
The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is a multifunctional enzyme, capable of several discrete activities required for viral replication [1]. These essential activities include DNA- and RNA-dependent DNA polymerase (DDDP and RDDP), ribonuclease H (RNase H), strand transfer and strand displacement activities. Native HIV-1 RT is a heterodimer of p66 and p51 subunits, of which the p66 subunit contains both the polymerase and RNase H domains. The p51 subunit is derived by proteolytic cleavage of the p66 subunit and is thought to play both an architectural role in the context of the p66/p51 heterodimer as well as facilitate template·primer binding [2].
Due to its essential role in synthesizing the double-stranded proviral DNA from single-stranded HIV-1 RNA genome, the HIV-1 RT is a major target of current antiviral therapies directed against HIV-1. Current anti-HIV drug regimens, termed highly active antiretroviral therapy (HAART), typically consist of a combination of at least three antiretroviral drugs, with two or more nucleotide reverse transcriptase inhibitors (NRTIs) being a staple of most regimens [3,4]. In addition to NRTIs, which are both competitive inhibitors and chain-terminators, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) consist of structurally dissimilar hydrophobic compounds that bind to a hydrophobic pocket on the RT adjacent to, but distinct from, the active site, which accommodates dNTPs and NRTIs. While HAART regimens have decreased both the mortality and morbidity of HIV-infected individuals, several factors contribute to drug failure. The highly error-prone nature of HIV-1 RT [5,6] combined with a robust rate of viral replication [7,8] provides the virus with an ideal context for the emergence of resistant variants. In addition, the significant toxicity associated with the current crop of anti-HIV drugs often leads to noncompliance, which in turn results in treatment failure [9]. For these reasons, there is a high level of interest in the development of more potent anti-HIV inhibitors that are both less likely to lead to drug-resistant variants and display less toxicity in patients.
Among a number of anti-HIV agents being developed for potential use in the treatment of AIDS are nucleic acid-based inhibitors that can serve as useful complementary therapies [10]. Of these, three nucleic acid-based approaches have recently been shown to have potent influence on HIV replication. In one, using a long antisense env RNA approach, strong inhibition of HIV replication was observed in cultured T cells [11]. This approach combined with a lentiviral vector completed the phase I clinical trials and is about to enter phase II trials [12]. The second approach, RNA interference (RNAi), uses a natural cellular pathway for gene silencing via small interfering RNAs [13-16]. The third approach is based on DNA and RNA aptamers that are derived by the iterative process of SELEX, to bind to specific protein targets [17] and has been recently shown to be effective in blocking HIV replication [18-20].
Tuerk and Gold first reported the isolation of RNA aptamers targeting HIV-1 RT using an iterative selection process of binding, washing and eluting the RNAs from a random library of RNA sequences [21]. Subsequent reports showed that both DNA and RNA aptamers generated against HIV-1 RT [22,23] are highly specific (do not bind to FIV or MuLV RTs), bind tightly to HIV-1 RT (Kd in the range of 0.05 to 50 nM) and competitively inhibit its polymerase activity. The crystal structure of an HIV-1 RT complexed with an anti-RT aptamer confirmed that the aptamer RNA is bound by the template·primer cleft of HIV RT [24]. Since these aptamers compete with template·primer for the template-binding cleft, they have been termed template analog RT inhibitors (TRTIs) [25]. In order to test the utility of anti-RT aptamers as inhibitors of HIV replication, we previously expressed RNA aptamers specific to HIV-1 RT in Jurkat T cells and showed that the tightest binding aptamers were able to potently block the infection and the subsequent spread of HIV-1 in cell culture [19]. In addition, five of the nine different clades of HIV-1 tested and all of the RTI and PI-resistant isolates tested were also severely inhibited [19]. The block was found to be in the early steps of reverse transcription. A subsequent report, using single cycle infection experiments involving one RNA aptamer (1.1), has confirmed the strong inhibition of HIV-1 replication by anti-RT aptamers [18].
It has been suggested that resistance to aptamers in vivo may be difficult due to the presumed need for multiple mutations required to disengage the interactions via the large interface between the inhibitor and HIV-1 RT [19]. In order to address this notion, we previously used a phenotypic screen based on the in situ detection of RNA-dependent DNA polymerase activity of HIV-1 RT expressed within bacterial colonies, and isolated two variants of recombinant HIV-1 RT bearing the substitutions N255D or N265D, both of which displayed in vitro resistance to the DNA aptamer RT1t49 [25]. The mechanism of resistance to these aptamers appeared to be based on the loss of affinity to the aptamer and the level of resistance increased from a range of 2- to 11-fold for single mutations to ~150-fold when the two mutations were combined. When the mutant RT sequences were incorporated into molecular clones of HIV-1, the resulting HIV virions were compromised for infectivity in single cycle infection assays and for virus replication in multi-day cell culture replication experiments [25]. Thus, despite the biochemically robust enzymatic activity that allows one to measure drug-susceptibility levels of the mutant RTs, it appeared that the aptamer-resistance mutations tend to target biologically crucial sites. In support of this view, we have further demonstrated that all three mutants (the N255D, N265D and the double mutant (Dbl) RTs containing both mutations) are defective for processive DNA-dependent DNA polymerase activity (DDDP), although N265D retained processive polymerization activity on RNA templates [26].
The data available demonstrate the utility of aptamers in inhibiting HIV-1 replication. In addition to their exquisite specificity, high level of resistance to anti-RT aptamers appears to require multiple mutations, which affect the polymerase activity of the enzyme. Although resistant virus particles could be produced from molecular clones with mutant RTs, the mutant viruses displayed reduced replication competence and thus lacked a competitive edge in the presence of a large complexity of virus population. It is important to know whether the aptamer-resistant RTs retain their sensitivity to other classes of anti-RT drugs. In the present communication, we have further evaluated the enzymatic properties of the aptamer-resistant RTs. First, we measured the breadth of cross-resistance to other anti-RT inhibitors, including several standard NRTIs and NNRTIs and otherDNA and RNA aptamers specific to HIV-1 RT. Second, we have investigated biochemical defects that may be responsible for their reduced replication fitness. These are important questions concerning the potential of anti-RT aptamers as a viable treatment option. We find that these mutants are resistant to several additional DNA aptamers, thus suggesting a common contact point on HIV-1 RT to this new class of nucleic acid-based anti-RT inhibitors. Importantly, we find that the aptamer-resistant mutations retain wild-type susceptibilities to all NRTIs and NNRTIs tested. Furthermore, amongst a series of NRTI-resistant HIV-1 RT variants, only the K65R RT mutant displayed a significant (5-fold) level of resistance to RT1t49. Our results, combined with previous reports, demonstrate that mutations conferring resistance to the DNA aptamer, RT1t49 in vitro affect the RNase H domain in addition to previously shown effect on polymerase domain, both of which are essential for efficient viral DNA replication.
Results
Cross-resistance of DNA aptamer RT1t49-resistant mutants of HIV-1 RT to other inhibitors
We investigated whether the aptamer-resistance mutations, N255D and N265D, would affect the sensitivity of HIV-1 RT to other DNA and RNA aptamers directed to HIV-1 RT [21,23]. RT1t49 and 5 other DNA aptamers representing each of the six classes of DNA aptamers described by Schneider et al. [23] and a single RNA aptamer 1.1 (termed Rknot 1.1 here) were selected. Using a steady-state nucleotide incorporation assay, a similar pattern of resistance to that of RT1t49 was observed with DNA aptamers RT26, RT4, and RT6 (Table 1). In each of these cases, the N265D mutation conferred a greater degree of resistance compared to the N255D mutation. In addition, the presence of both mutations led to an even greater degree of resistance (6- to 27-fold) to aptamers in this group. In contrast, both N255D and Dbl mutant RTs were hypersensitive (10-fold) to DNA aptamer RT8, while the N265D mutant displayed wild type levels of sensitivity (Table 1). However, with respect to the DNA aptamer RT10 and the single RNA aptamer tested (Rknot1.1), the N255D mutant was similar to wild type, while both N265D and Dbl mutants were significantly resistant. The similarity between resistance profiles of N255D and N265D mutant RTs to both DNA aptamers (RT1t49, RT26, RT4, RT6) suggest that the residues N255 and N265 are important contacts for several classes of DNA aptamers.
Table 1 Resistance of Purified RTs to DNA and RNA Aptamers. Assays were performed as described previously [34]. Data represent mean ± SEM of three independent experiments.
WT N255D N265D Dbl
aTRTI bIC50, nM cRatio IC50, nM Ratio IC50, nM Ratio IC50, nM Ratio
RT1t49d
RT26f 1.6
4.0 ± 0.05 1
1 7.9
7.6 ± 0.1 4.9
1.9 17.4
11.2 ± 0.1 10.9
2.8 245
24 ± 0.1 153
6
RT4f 38 ± 1.2 1 80 ± 3.7 2.1 1015 ± 16 27 > 1000 > 27e
RT6f 19.6 ± 0.1 1 26 ± 0.7 1.3 87 ± 2.2 4.4 142 ± 3.4 7.2
RT8f 19.5 ± 0.3 1 2.0 ± 0.02 0.1 17.2 ± 0.2 0.9 3.0 ± 0.02 0.1
RT10f 82 ± 2.5 1 57 ± 1.4 0.7 923 ± 8.9 11 509 ± 4.2 6
Rknot 1.1f 1.4 ± 0.02 1 0.8 ± 0.01 0.8 2.5 ± 0.04 2 4.5 ± 0.08 4
aDescribed in references 23 and 25.
bConcentration of aptamer at which 50% of the activity was inhibited.
cFold increase or decrease over the IC50 for the wild type (WT) RT.
dReproduced from Fisher et al. [25] eAt the highest concentration of aptamer tested (1000 nM), the Dbl mutant retained 70% of its activity, thus the actual IC50 would be much higher.
fAptamer sequences: RT1t49: 5' ATCCGCCTGATTAGCGATACTCAGAAGGATAAACTGTCCAGAACTTGGA3'
RT26: 5'ATCCGCCTGATTAGCGATACTTACGTGAGCGTGCTGTCCCCTAAAGGTGATACGTCACTTGAGCAAAATC ACCTGCAGGGG3'
RT4:5'ATCCGCCTGATTAGCGATACTTTAGCAAAGTTGAAGCCGGACTAACAAGCTCTACGACTTGAGCAAAATCA CCTGCAGGGG3'
RT6: 5'ATCCGCCTGATTAGCGATACTCAGGCGTTAGGGAAGGGCGTCGAAAGCAGGGTGGGACTTGAGCAAAATCA CCTGAGGGG3'
RT8:5'ATCCGCCTGATTAGCGATACTAGCCAGTCAAGTTAATGGGTGCCATGCAGAAGCAACTTGAGCAAAATCA CCTGCAGGGG3'
RT10:5'ATCCGCCTGATTAGCGATACTTATTTGCCCCTGCAGGCCGCAGGAGTGCAGCAGTACTTGAGCAAAATCA CCTGCAGGGG3'
Rknot 1.1: 5'GGGAGAUUCCGUUUUCAGUCGGGAAAAACUGAA3'
We next tested cross-resistance of these variant RTs to conventional RT inhibitors such as NRTIs and NNRTIs. Each of the single mutants, N255D and N265D, and the double mutant RTs were tested for their sensitivity to a selected set of NRTIs (AZTTP, ddATP, ddCTP, d4TTP and 3TCTP) or the NNRTIs (nevirapine and delavirdine). Interestingly, neither the single mutations nor the double mutants altered the susceptibility of HIV-1 RT to any of these RT inhibitors (Table 2).
Table 2 Sensitivity of aptamer-resistant RTs to NRTIs and NNRTIsAssays were performed as described in the text. Data represent mean ± SEM of three independent experiments.
WT N255D N265D Dbl
Inhibitor aIC50, μM bRatio IC50, μM Ratio IC50, μM Ratio IC50, μM Ratio
AZTTP 1.83 ± 0.25 1 2.67 ± 0.09 1.45 1.74 ± 0.28 0.9 2.43 ± 0.26 1.3
ddATP 0.93 ± 0.18 1 1.07 ± 0.11 1.2 0.84 ± 0.04 0.9 0.91 ± 0.07 1
ddCTP 0.88 ± 0.20 1 0.69 ± 0.07 0.8 0.72 ± 0.17 0.8 0.96 ± 0.09 1.1
3TCTP 4.37 ± 0.87 1 2.51 ± 1.04 0.6 5.02 ± 1.22 1.1 2.69 ± 0.95 0.6
d4TTP 0.79 ± 0.05 1 0.83 ± 0.14 1 0.64 ± 0.12 0.8 0.91 ± 0.10 1.2
Nevirapine 0.10 ± 0.01 1 0.06 ± 0.02 0.6 0.09 ± 0.03 0.9 0.07 ± 0.01 0.7
Delavirdine 0.37 ± 0.02 1 0.64 ± 0.03 1.7 0.36 ± 0.01 1 0.31 ± 0.01 1
aConcentration of inhibitor at which 50% of the activity was inhibited.
bRatio of this enzyme's drug susceptibility to that of wild type.
Some NRTI-resistant RTs display low-level resistance to the DNA aptamer, RT1t49
Similar experiments were performed to determine the effectiveness of the DNA aptamer, RT1t49 in inhibiting the polymerase activities of several NRTI-resistant mutants of HIV-1 RT. Variants of HIV-1 RT shown to confer resistance to AZT (T215Y/M41L) and ddI and ddC (L74V) were sensitive to inhibition by RT1t49 (Table 3). In contrast, mutations shown to confer resistance to multiple NRTIs, including E89G, K65R and M184V displayed low levels of resistance to RT1t49 (2–5 fold), with K65R displaying the highest level of resistance (5-fold). K65R is known to cause resistance to all clinically approved NRTIs except AZT in patients. However, in vitro biochemical experiments do show some resistance to AZTTP and it has been suggested this is due to K65R decreasing the rate of AZTMP excision. The residues E89 and K65 are located in template grip region of palm and the β3-β4 hairpin loop of fingers regions respectively. Both these regions are known to contact different parts of the template·primer molecule. Thus, these results suggest that the RT1t49 aptamer may make contact with several of the key regions of RT involved in template·primer contact.
Table 3 Sensitivity of NRTI-resistant RTs to the DNA aptamer RT1t49Assays were performed as described previously [34]. Data represent mean ± SEM of three independent experiments.
Enzyme IC50, nM Ratio
WT 1.5 ± 0.03 1
E89G 4.9 ± 0.06 3.3
K65R 8.0 ± 0.05 5.3
L74V 0.86 ± 0.02 0.6
M184V 3.2 ± 0.05 2.1
T215Y/M41L 2.1 ± 0.04 1.4
aConcentration of inhibitor at which 50% of the activity was inhibited over the IC50 for wild type (WT) RT
Anti-HIV RT aptamer-resistant RT mutants are defective for RNase H-mediated cleavage
We next tested the impact of aptamer resistance mutations on RNase H activity associated with HIV-1 RT. Previous studies have shown that alanine substitutions at several residues within the minor groove binding track (MGBT) [27] affect not only RT processivity, but also the specificity of RNase H-catalyzed removal of the polypurine tract (PPT) primer [28]. Both N255 and N265 are located in the α H helix of HIV-1 RT, and are therefore in close proximity to the MGBT. Both the polymerase-dependent and RNA 5'-end-directed RNase H activity of wild type and aptamer-resistant RTs were tested. Under conditions that prevent the RT from rebinding the substrate RNA.DNA duplex, the aptamer-resistant RTs were found to be deficient in both polymerase-dependent and RNA 5'-end-directed RNase H activities (Figure 1A and 1B). In this case, RT was pre-bound to the DNA.RNA substrate before reactions were initiated by adding both MgCl2 and heparin as a competitive trap. Therefore any cleavage products formed were the result of a single binding event.
Figure 1 RNase H cleavage of RNA.DNA hybrids by wild type (WT) and mutant RTs in the presence of a heparin challenge. A. Polymerase-dependent RNase H clevage. The substrate, as diagrammed at the top, consisted of a 142nt heteropolymeric RNA (thin line) annealed to a 30nt DNA primer (thick line). Arrows indicate the expected sites of cleavage. Reactions were performed in the absence of dNTPs and in the presence of a heparin trap. Control reactions were performed in which either no enzyme was added (C), or an RNAse H-defective mutant (E478Q) was added (RNase H-) (see Methods secion). Cleavage products were resolved on a denaturing 6% polyacrylamide gel. The sizes of the resultant radiolabeled products are represented to the left of the gel panels (including a minor product). B. RNA 5'-end-directed RNase H cleavage. The substrate was a 41nt heteropolymeric RNA annealed to a 47nt DNA template. Reaction conditions were otherwise identical to those in panel A, and are described under 'Materials and Methods' section. Cleavage products were resolved on a denaturing 12% polyacrylamide gel. The sizes of the resultant radiolabeled products are represented to the left of the gel panels.
Polymerase-dependent RNase H cleavage by wild type RT results in the formation of a 102-nt product (Figure 1A, lane 1). The smaller 94-nt product is the result of subsequent 3' → 5' directional nucleolytic activity of HIV-1 RT RNase H [29,30]. Under identical conditions, each of the aptamer-resistant RTs failed to produce significant amounts of either 102-nt or 94-nt products (Figure 1A, lanes 2–4). While there appeared to be a limited cleavage by both N255D and Dbl mutants, products formed were altered in size compared to wild type products (Figure 1A, lane 1 vs. lanes 2 and 4). These results indicate that although the N255D and Dbl mutant RTs possess residual polymerase-dependent RNase H activity under single cycle cleavage conditions, the specificity of cleavage under such conditions has not been retained.
Similar reactions were carried out to determine the effect of aptamer resistance mutations on HIV-1 RT RNA 5'-end-directed RNase H activity (Figure 1B). Following completion of minus strand DNA synthesis, RNA fragments left behind are removed by this activity in order to facilitate plus strand DNA synthesis. Both wild type and aptamer resistant RTs were incubated with the RNA:DNA substrate before reactions were initiated by adding MgCl2 and heparin trap. Wild type RT efficiently cleaved the RNA:DNA substrate, resulting in the expected 18-nt cleavage product in addition to several smaller products that are the result of processive cleavage. In contrast, reactions in which aptamer-resistant RTs were included resulted in minimal cleavage products (Figure 1B, lanes 2–4). Together, these results indicate that both aptamer resistance mutations N255D and N265D result in a severe reduction of HIV-1 RT mediated RNase H cleavage under challenged conditions.
The observed defect in Figure 1 appears to be due to a loss in substrate affinity and not due to defect in the RNase H catalytic activity of these mutant RTs. To determine whether these mutant RTs retained RNase H catalytic activity, we measured the polymerase-dependent RNase H cleavage by wild type and mutant RTs in the absence of a trap. As shown in Figure 2, within a 5-min reaction time, wild type RT made the expected 102- and 94-nt products. Unlike the previous challenged RNase H reactions (Figure 1), N255D, N265D, and Dbl mutant RTs were able to make comparable amounts of polymerase-dependent RNase H cleavage products (Figure 2). Both the overall amounts and size distribution of cleavage products were similar between wild type and mutant RTs under these conditions. Thus, the aptamer-resistance mutations do affect RNase H under conditions that require re-binding.
Figure 2 Comparison of polymerase-dependent RNase H activities of wild type (WT), and mutant RTs. HIV-1 RT and template·primer substrates were combined and time course reactions were performed with a 5'-end labeled 142nt RNA template and 30nt DNA primer for 0, 10, 30, 60, 120 and 300 seconds. Cleavage products were resolved by denaturing 6%. The product sizes are indicated to the left of the panel.
Discussion
Our results highlight several key features of the aptamer-resistant RTs bearing the mutations N255D, N265D or both (Dbl). First, each mutant displayed cross-resistance to three of the 7 anti-RT aptamers tested (Table 1). Interestingly, with three of the aptamers (RT26, RT4 and RT6), the pattern of resistance was very similar to that seen with RT1t49 in that the reduction in susceptibility was small in the case of RTs containing single mutations, and it was greater for the Dbl mutant. As shown previously, the level of resistance of each of the RTs to RT1t49 directly correlated with the dissociation constants for this aptamer. In the absence of changes in affinity to normal template·primer substrate, this suggests that the affinity of the aptamer to the RT determines the degree of inhibition achieved [25]. Therefore, our results indicate that N255 and N265 are important contact points by which HIV-1 RT interacts with each of these aptamers. In earlier work, Schneider et al. [23] classified the 30 different DNA aptamers they obtained by SELEX into six families based on primary sequence and the presence of specific secondary structures (e.g., stems, loops etc.) [23]. In spite of the dissimilarity in primary and secondary structures of the different RT-binding aptamers, it is thought that they all generate very similar 3-dimensional structures allowing them to interact with a similar binding surface on the RT protein. Additional evidence in support of this is the presence of the characteristic interrupted helices present in all RT-binding aptamers. The observation that N255D and N265D mutations confer resistance to aptamers in multiple classes suggests that these aptamers all bind HIV-1 RT in a similar manner.
The cross-resistance patterns suggest some distinct differences among the anti-RT aptamers. For example, the lack of change in sensitivity of N265D mutant to aptamer RT8 (Table 1) suggests that the residue N265 may not play a key role in binding to RT8. It is also interesting that N255D mutant displays a 10-fold hypersensitivity to RT8. We surmise that N255 residue may be involved in binding to RT8 – however, abrogation of this interaction by the N255D substitution may result in a conformational change in the RT8 or RT, which may lead to better interaction with another part of RT thus increasing its affinity to the mutant RT. Our previous work shows that changes in sensitivity to inhibition by aptamers for N255D and N265D mutant RTs directly correlate with their binding affinities to the aptamer [25]. A similar 10-fold hypersensitivity of Dbl mutant to RT8 appears to reflect the observation that the effect of N255D is dominant over that of N265D in the context of both mutations.
A long-term goal of testing anti-RT aptamers is to develop them as anti-HIV agents to be administered to individuals who have drug failure due to chronic anti-retroviral treatment or for those under supervised treatment interruption [10]. Thus, it is highly desirable that aptamers are able to suppress even drug resistant viruses. Clinically relevant aptamers can be introduced via gene therapy into hematopoietic cells of HIV-infected patients undergoing antiviral therapy. Therefore, these anti-HIV aptamers will be expressed intracellularly as RNA. In this report, we have used a DNA aptamer (RT1t49) as a model to test this notion. Our results show that most NRTI-resistant RTs display only mild resistance to aptamers (1 to 2-fold) (Table 3). However, both E89G [31], which rarely occurs among clinical isolates as a primary mutation and the more commonly encountered K65R, both display a modest level of resistance to RT1t49 (3- to 5-fold). However, both of these mutant enzymes have been shown to have altered properties with respect to their interaction with template·primer. The K65R and E89G mutants have been reported to display reductions of 50% and 32% in their dissociation constants [[32,33],196,215]. Therefore, it is likely that the increased IC50 of these enzymes to inhibition by the aptamer RT1t49 is an indirect result of their decreased dissociation from template·primer. The results of RT1t49 susceptibility testing (Table 3) with the ddI/ddC-resistant L74V, 3TC-resistant M184V and the AZT-resistant T215Y/M41L RTs are in agreement with our previously published efficacy tests using Jurkat T cell lines expressing each of the three selected anti-RT RNA aptamers, in which all the RNA aptamers were able to efficiently suppress replication of drug-resistant HIV [19].
Testing the wild type and the aptamer-resistant mutants of HIV-1 RT for inhibition by a variety of NRTIs and NNRTIs revealed that even if aptamer-resistance were to arise in vivo, such viruses can be efficiently suppressed by conventional antiretrovirals (Table 2). These results would be relevant to a scenario when aptamers are to be administered to HIV-infected individuals, possibly via hematopoietic stem cell therapy followed by bone marrow transplantation. In the event that aptamer-resistant variants would arise in such patients, standard RTIs can still be used to treat such patients.
The above observation, however, was tempered by the fact that some of the NRTI-resistance mutations, such as E89G and K65R conferred a significant degree of resistance to RT1t49 (3 to 5-fold). On the one hand, these results suggest that pre-existing NRTI-resistance mutations, due to altered affinities to template·primer can confer co-resistance to aptamers or that mutations such as K65R could arise in response to aptamer therapy. On the other hand, the resistance data provides insights into indirect means by which aptamer-RT interactions can be altered. Aptamer resistance can result from either a direct disruption of contact of the mutated residue with the aptamer or from an indirect effect on the conformation of a neighboring amino acid residue, increasing the template·primer affinity thus indirectly leading to altered susceptibility to the aptamer.
Although resistance to aptamers can be generated by specific mutations, our earlier work shows that these mutations alone reduce the virus infectivity by 12- to 30-fold over wild type in a single round of infection using an LTR-lacZ reporter cell line [25]. In addition, during a multi-day replication experiment using CD4 T cells in culture, all three viruses were unable to replicate and spread through the culture [25]. Both N255 and N265 are adjacent to the residues that form the MGBT of HIV-1 RT. The MGBT has been shown to be critical for translocation of the enzyme along the template·primer during polymerization [27]. In addition, as shown by our earlier studies, both N255D and N265D mutations affected the DNA-dependent DNA polymerase processivity, while N255D was also defective for RNA-dependent DNA polymerase processivity [26]. Our current results show that while the gross RNAse H activity is unaffected under conditions that allow re-binding (Figure 2), the processive RNAse H activity (under conditions that prevent re-binding) is affected for all three mutants (Figure 1). Thus, these mutations appear to diminish the ability of HIV-1 RT to associate with and utilize its nucleic acid substrate, therefore resulting in multiple functional defects that contribute to loss of replication fitness for the aptamer-resistant viruses. We believe that this may help explain our inability to select for resistant variants using cell lines expressing RNA aptamers (P. Joshi and V. Prasad, unpublished observations).
Conclusion
The results presented in this report attempt to unravel the wider significance of the only two mutations previously known to specifically alter sensitivity to anti-HIV-1 RT aptamers. The mutations N255D and N265D both conferred resistance to two of the 5 new DNA aptamers (with the exception of RT8) and 1 RNA aptamer tested suggesting that the N255 and N265 residues probably serve as contact points for most aptamers. Thus, it is likely that selection with the other aptamers may also lead to these same mutations. Interestingly, the mutations N255D or N265D do not affect sensitivity to any of the NRTIs or NNRTIs tested which is a useful feature if the same mutations were to arise in response to treatment with anti-RT aptamer RNAs via gene therapy in the future. Previous results showed that these two mutations, when reconstituted into molecular clones of HIV, lead to replication defective viruses. The effects documented here, on RNase H function, combined with defects in the processive synthesis of DNA previously shown, provide additional rationale for the loss of replication competence for such viruses.
Methods
Polymerization assays
Sensitivity to inhibition by aptamers, NRTIs and NNRTIs
The sensitivity of wild type and mutant RTs to DNA and RNA aptamers, NRTIs and NNRTIs was measured in standard RT reactions essentially as described earlier [34] with the exception that 16S rRNA (Roche Diagnostics, Indianapolis, Indiana) annealed to VP200 (5'-TAACCTTGCGGCCGTACTCCCC-3') was used as template·primer. Reaction mixtures (50 μl) contained 24 nM template·primer, 80 mM KCl, 50 mM Tris-Cl (pH 8.0), 6 mM MgCl2, 1 mM dithiothreitol (DTT), 0.1 mg/ml BSA, 10 μM [α-32P] dGTP or TTP, 25 μM each of the remaining three dNTPs and a range of concentrations of DNA and RNA aptamers. Reactions, initiated by the addition of 25ng of each RT (corresponding to 10, 79, 16 and 28 units respectively for wild type, N255D, N265D and Dbl) were incubated at 37°C for 15 min. IC50 values of each inhibitor for a given RT variant were determined by fitting results from at least three independent experiments to a dose-response curve using nonlinear regression (GraphPad Software Inc., San Diego) using the following equation:
RNase H Assays
Challenged, polymerase-dependent and RNA 5'-end-directed cleavages
To measure the ability of enzymes to cleave RNA:DNA duplexes as the result of a single binding event, a heparin trap was added to bind any unbound enzyme or enzyme dissociated from the duplex following cleavage. Polymerase-dependent reactions included a 30-nt DNA primer annealed to a 142-nt RNA template [35]. For RNA 5'-end-directed reactions, a 41-nt RNA primer was annealed to a 47-nt DNA template. In both cases, RNA was 5'-end labeled using [γ-32P]ATP (3000 Ci/mmol) in the presence of T4 polynucleotide kinase. Final reaction mixtures (25 μl) contained 25 mM Tris-HCl (pH 8.0), 1 mM DTT, 34 mM KCl, 6 mM MgCl2, 0.5 mM EDTA, 4 nM substrate, 4 mg/ml heparin, and 0.85 nM. The reactions were initiated with MgCl2, incubated for 15 min at 37°C, and then terminated with 25 μl stop solution. Polymerase-dependent and RNA 5'-end-directed cleavage products were resolved using denaturing 6 and 12% PAGE, respectively followed by phosphorimager analysis. Control reactions were carried out using an RNase H-defective mutant of RT, E478Q [36] showing no cleavage of the RNA:DNA duplex
Unchallenged, polymerase-dependent cleavages
Similar to challenged reactions, for unchallenged polymerase-dependent RNAse H reactions, a 30-nt DNA primer was annealed to a 142-nt RNA template [35]. The RNA template was 5'-end labelled using [γ-32P]ATP (3000 Ci/mmol) in the presence of T4 polynucleotide kinase. Reactions (100 μl) were performed under the following conditions: 3.4 nM RT, 4 nM 5'- [32P]-labeled 142-nt RNA template annealed to a 30-nt DNA primer, 25 mM Tris-HCl (pH 8.0), 1 mM DTT, 34 mM KCl, 6 mM MgCl2, and 0.5 mM EDTA. RT was preincubated with the RNA:DNA substrate in the absence of MgCl2 for 5 min at 37°C. Reactions were initiated by the addition of MgCl2, and at various time points (0, 30s, 60s, 120s) an aliquot (25 μl) was removed and combined with 25 μl stop solution to stop cleavage. Cleavage products were analyzed by denaturing 6% PAGE. RNase H-directed cleavage was detected by drying the gels followed by phosphorimager analysis.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TF carried out the drug sensitivity studies for all the aptamers using recombinant purified wild type and mutant RTs that he previously purified, performed RNase H assays and prepared the initial draft of the manuscript. PJ carried out RT inhibition studies with all the NRTIs and NNRTIs. VP conceived of the study, participated in its design and coordination and helped to generate the final manuscript. All authors read and approved the final manuscript.
Acknowledgements
The authors wish to thank W. C. Drosopoulos for reading the manuscript, R. A. Bambara for providing the plasmids for generating T7 RNA transcripts used in RNase H assay and the late Dr. Reaching Lee for providing the purified E478Q mutant RT. Research described in this report was supported by a Public Service grant to VRP (NIH RO1 AI30861). TSF acknowledges support from an institutional pre-doctoral training grant (NIH T32 GM07491).
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Schatz O Cromme FV Gruninger-Leitch F Le Grice SFJ Point mutations in conserved amino acid residues within the C-terminal domain of HIV-1 reverse transcriptase specifically repress RNAse H function FEBS Lett 1989 257 311 314 2479577 10.1016/0014-5793(89)81559-5
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BMC BiochemBMC Biochemistry1471-2091BioMed Central London 1471-2091-6-211622130510.1186/1471-2091-6-21Research ArticleNonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease Walasek Paula [email protected] John F [email protected] Department of Chemistry, University of Waterloo, 200 University Avenue, Waterloo, Ontario, Canada N2L 3G12005 12 10 2005 6 21 21 11 8 2005 12 10 2005 Copyright © 2005 Walasek and Honek; licensee BioMed Central Ltd.2005Walasek and Honek; 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 alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region.
Results
To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM), into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme.
Conclusion
Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease.
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Background
The metzincin superfamily of proteases includes the matrixins, astacins, reprolysins, snapalysins, leishmanolysins and serralysins [1,2]. These zinc-dependent endoproteases may act on a large number of substrates, or they may be highly specific, targeting only one or a few proteins or oligopeptides. There are over 700 different enzymes that have been classified as metzincins and they are produced by organisms ranging from bacteria to plants and animals [3]. As diverse as the members of this superfamily are, surprisingly they all share a structurally similar catalytic site which includes a common zinc binding motif and an absolutely conserved methionine-containing 1,4-tight β turn, termed the "Met-turn" in their active sites [4].
Many of the metzincin proteases are of medical significance. The matrixins, or matrix metalloproteinases (MMPs) include the collagenases, gelatinases and stromelysins [5,6]. Members of this subfamily not only degrade extracellular matrix for tissue remodeling and maturation, but they have also been found to release and activate several growth factors and modulate chemotactic signals [5]. In disease processes, however, they have been implicated in promoting tumour invasion and metastasis by degradation of matrix barriers, exacerbating periodontal disease, and destroying aggrecan and collagen in rheumatoid arthritis. For this reason, much interest has arisen in mechanistic and inhibitor studies of MMPs [7-9]. The astacins comprise a subfamily of enzymes involved in digestion, developmental regulation, and peptide processing. The reprolysins or adamalysins are mainly responsible for the hemorrhagic effects and tissue necrosis typical of snake bites [2]. These effects are caused mainly by destruction of the extra-cellular matrix surrounding capillaries in conjunction with the inhibition of platelet aggregation by disintegrins. The N-terminal catalytic domains of members of this family are usually zinc and calcium dependent, while the C-terminal portion may be disintegrin, cysteine-rich, or lectin type domains with hemorrhagic or cell adhesion properties. Mammalian adamalysins play important roles in reproduction, myogenesis and cytodifferentiation as well as in disorders such as asthma, cardiac hypertrophy, and endotoxic shock [10-13]. The snapalysins are secreted proteases from several Streptomyces organisms and the membrane-bound leishmanolysins originate from a number of protozoan parasites. The serralysins, of which the alkaline protease from P. aeruginosa (AprA) is a member, are secreted by several pathogenic, gram-negative bacterial species [14]. They are, on average, 50 kDa proteins consisting of 2 domains; the C-terminal domain binds up to eight calcium ions in a mainly structural role, but also contains the secretion signal. The catalytic site is located in a cleft on the N-terminal domain (Figure 1)[15]. The physiologic substrate(s) of these enzymes are not known with certainty, but are thought to be proteins of the extracellular matrix and possibly immune response elements such as interferon gamma and complement proteins [16,17].
Figure 1 Ribbon representation of alkaline protease from P. aeruginosa (AprA). The active site zinc ion is shown in blue and the conserved methionine in yellow spacefill just below the zinc. Calcium ions in the C-terminal β-roll domain are green. Figure generated using WebLab Viewer Pro 3.7 using PDB entry 1KAP [15].
To add a further dimension to the techniques applied to the analysis of protein structure and function, the incorporation of nonnatural amino acids into proteins is currently of great interest. One aspect of this approach is the incorporation of amino acids containing fluorine which would be applicable to not only probing subtle steric interactions in proteins (Van der Waals' radius of F is 1.47 Å vs. 1.20 Å for H), but also in applying 19F spectroscopy to this area [18,19]. In order to probe the methionine position in this class of proteins (specifically AprA), the fluorinated analogue, S-difluoromethyl-L-homocysteine (L-difluoromethionine, (DFM)), was utilized to replace the methionine in the Met-turn and to determine its effects on catalytic activity and thermal stability.
Results
N-terminal catalytic fragment
Attempts to overproduce the N-terminal catalytic fragment of AprA which consisted of residues T40 to N258 of the 479 residue protein led to the production of aggregates in spite of attempts to increase soluble protein expression by control of temperature, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration, co-expression of folding chaperones (GroES and GroEL) as well as secretion and fusion to a NusA solubility tag [20-23]. In several attempts at resolubilisation of these inclusion bodies (see Methods for details), semi-soluble aggregates of the truncated AprA were detected by gel filtration chromatography (Superose-12) having molecular weights higher than the exclusion limit of the resin (>2 × 106 Da). Due to the difficulty of isolating and handling sufficient quantities of this truncated version of AprA, attention was then focused on overproduction of the full-length mature form of AprA which corresponds to residues G10 to V479.
Mature full-length AprA and DFM incorporation
The gene for the full-length mature form of AprA [24-26] which corresponds to residues G10 to V479 was isolated on to pET22b and overexpressed in E. coli BL21 (λDE3), resulting in inclusion body formation. Isolation of the protein aggregates followed by solubilisation and refolding produced an active, stable and soluble enzyme of 49 500 Da, according to mass spectrometric analysis (Figure 2 and 3). L-DFM was synthesized as previously described [27,28] and incorporated into AprA using the methionine auxotrophic E. coli strain, B834 (λDE3). The resulting inclusion bodies were refolded in an identical manner as the non-labelled protein, and the expected 49 535 Da protein, as determined by electrospray mass spectrometry (ESMS), was isolated (Figure 2 and 4).
Figure 2 SDS-PAGE gel summary of the refolding of mature full-length AprA. S represents the supernatant of the crude lysate, W1 is the solution from the first wash of the inclusion body pellet, RF is the refolded protein and HS is the refolded protein after the high salt wash and DA is the refolded DFM-AprA.
Figure 3 Positive ion mode electrospray mass spectrum of the refolded, full-length, mature AprA. The major peak in the spectrum (49 500 Da) corresponds exactly to the recombinant protein without the initiator methionine. The peak at 49 534 Da may represent a calcium adduct. The peak at 49 632 Da corresponds to AprA retaining the N-terminal methionine.
Figure 4 Positive ion mode electrospray mass spectrum of DFM-AprA. The calculated molecular weight of the labelled protein with the initiating residue was 49 702 Da, and without, 49 535 Da. Possible sodium adducts (2Na+) are seen for both species at 49 747 Da and 49 581 Da.
Kinetic analysis and differential scanning calorimetry
Utilizing a previously reported spectrophotometric method for kinetic analysis of AprA, the mature, full-length recombinant protein showed similar kinetic properties for hydrolysis of Z-Arg-Arg-p-nitroanalide to those reported for the mature wild-type protein, isolated directly from P. aeruginosa growth media [29]. The kcat values were determined to be 1.6 ± 0.1 min-1 versus 0.6 ± 0.1 min-1 and the Km values were 12.6 ± 2.4 μM vs. 6 ± 1 μM for the enzyme produced by the cognate organism and the recombinant protein respectively.
Slightly improved assay conditions were found by increasing the amount of enzyme from 25 μg per assay to 100 μg in order to improve sensitivity, and by replacing the assay buffer from 5 mM TRIS to 20 mM HEPES to avoid possible metal binding effects of the TRIS buffer. The kinetic parameters of non-labelled and DFM-labelled AprA proteins were compared under the new conditions and found to be similar. The Km was not significantly altered (15.4 ± 0.9 μM for the unlabelled protein vs. 14 ± 2 μM for the DFM-incorporated), and the kcat was only slightly affected by DFM incorporation, showing a decrease from 0.57 ± 0.02 min-1 to 0.44 ± 0.02 min-1.
Due to the proximity of the Met214 residue to the metal ligands which ligate the zinc ion (Figure 5), the effect of substitution of this Met with the larger DFM residue on thermal denaturation was investigated. Replicate differential scanning calorimetry experiments were performed on both the mature full-length AprA and the DFM-labelled mature full-length AprA (DFM-AprA) in 20 mM HEPES (pH 7.8). The Tmax for unlabelled and DFM-labelled AprA were determined to be 58.7 ± 0.1°C and 58.6 ± 0.2°C respectively (Figure 6).
Figure 5 Active site of AprA showing the environment of the conserved methionine (M214). A: The three histidine ligands (labelled) are shown along with the zinc ion (in green). The spacefill representation (B) shows the closely packed arrangement around the methionine residue, as well as its proximity to the catalytic zinc and the three histidine residues. Protons (white) were added to the M214 methyl group for clarity. Based on structure from PDB entry 1KAP [15].
Figure 6 Differential scanning calorimetry data of both the unlabelled AprA and the DFM-incorporated AprA. All scans were performed at 1°C per minute in 20 mM HEPES (pH 7.8) and buffer scans were subtracted from sample runs.
19F NMR spectroscopy
DFM-labelled mature full-length AprA was analyzed by 19F nuclear magnetic resonance (19F NMR) spectroscopy without proton decoupling to produce a complex set of 19F resonance multiplets centered at approximately -93.8 ppm. Paramagnetic line broadening with addition of gadolinium(III)-N,N,N',N' -ethylenediaminetetraacetic acid ((GdEDTA)-) complex resulted in line broadening of all 19F resonances. Proton-decoupled 19F NMR experiments resulted in collapse of the 19F multiplets, however complete resolution of the N-terminal DFM from the internal (DFM214) 19F resonances was still incomplete at this field strength (564.5 MHz) (Figure 7).
Figure 7 19F NMR spectra of DFM-labelled AprA. The 564.7 MHz 19F NMR spectrum of DFM-labelled AprA in D2O with approximately 40 mM HEPES (A) and with the addition of 2 mM Gd(EDTA)- (B). Proton-decoupled spectrum of DFM-labelled AprA (C).
Discussion
AprA is a member of the metzincin superfamily of metalloproteases and is one of several proteases secreted from Pseudomonas aeruginosa into tissue of infected patients [16,30,31] (Figure 1). It appears that a combination of AprA, elastase (LasB) and LasA protease and protease IV work in concert to produce tissue invasion [32,33]. AprA is biosynthesized in P. aeruginosa as a 479 residue polypeptide which is secreted via a C-terminal signal sequence directly into the extra cellular medium where a 9 amino acid N-terminal inhibitory propeptide is autocatalytically removed [26]. A critical aspect to the structure-function of AprA and the metzincins in general is the active-site arrangement of the metal ligands and the still incompletely understood methionine-containing 1,4-tight β turn at the base of the active site present in all members of the metzincin superfamily. Although much is known concerning the cellular biochemistry of a number of metzincins, detailed knowledge of the function of the components of the active site is still lacking. For example, methionine residues in proteins are less frequent in occurrence than many other residues (third least abundant after tryptophan and cysteine [34]) and it can frequently be replaced with other hydrophobic amino acids such as leucine [35,36]. However the surprising persistent presence of methionine in over 700 members of this class of protease has been noted[3,4,37,38]. Attempts to define the contribution that this seemingly quintessential amino acid plays in this class of enzyme have indicated that, depending upon the specific superfamily member studied, some flexibility in replacement may be possible, at least in vitro. For example, in the case of MMP-2 (gelatinase A), enzymes with replacements of the active site Met with either Leu or Ser have only minor perturbations in catalytic activity [39]. In the case of protease C from Erwinia chrysanthemi, replacement of the active site methionine (Met226) by leucine, alanine or isoleucine resulted in enzyme mutants sufficiently stable to be isolated, but this was not so with M226H, M226S or M226N mutations. However the enzyme kinetic properties of the M226L, M226A and M226I mutants were compromised and crystallographic studies indicated some alteration of the zinc binding histidines which might explain the effects of these mutations on the kinetic properties of the enzyme [40].
Substitution of the active site Met by selenomethionine (SeMet) in MMP-8 (human neutrophil collagenase) was also found to be somewhat perturbing. In the catalytic domain of MMP-8, SeMet appeared to decrease the protein's resistance to urea denaturation [41]. This loss of stability was accompanied by a considerable decrease in activity and an increase in Km towards the substrate (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3- [2,4-dinitrophenyl]-L- 2,3-diaminopropionyl)-Ala-Arg-NH2. In a previous study using the substrate (dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2), however, kcat and Km values were unaffected by SeMet replacement in MMP-8 [42,43].
Evidently additional investigations into the properties of the metzincins through modification of the methionine position may be of importance. However the usual ensemble of potential replacements of amino acid residues through site-directed mutagenesis is limited in general to replacement with the other nineteen amino acids. In order to explore the effects of alterations of the critical methionine in the metzincin class of proteases, the introduction of a sterically and electronically subtle methionine analogue DFM, was the focus of the present study. For these studies the alkaline protease from Pseudomonas aeruginosa was initially investigated as it is representative of the metzincin family of proteases, is important in tissue invasion by this pathogen and has only two methionines in its structure, the N-terminal methionine and the Met-turn methionine, M214. The presence of only two methionine residues in this metzincin (and only the active site methionine if a mature secreted AprA is produced) facilitates bioincorporation of DFM into the protein without the use of complex cell free in-vitro translation system approaches [44-46]. As well, this choice of target protein for a preliminary study avoids complications with additional bioincorporation of the analogue into other methionine positions which may be present in other members of the metzincin superfamily.
DFM is a subtle derivative of methionine that has been shown to be useful as a 19F-NMR probe in proteins [28,47]. Unlike leucine and isoleucine, this unbranched analogue may only produce a moderate increase in the steric properties of the methyl group due to the incorporation of two fluorine atoms. In addition, fluorination could profoundly affect the electron density of the sulfur atom [27]. As can be seen in Figure 5, the thiomethyl group of M214 is in close proximity to H176, H180, and H186 and may contribute to the proper positioning of these metal ligands. Hence it was of interest to explore the effects of this nonnatural amino acid on the catalytic activity and thermal stability of the modified protein by introduction of L-difluoromethionine into the critical methionine position in the Met-turn.
Previous studies on other metzincins such as stromelysin, MMP-8, and astacin have shown that the truncated enzymes containing only the catalytic domain of these enzymes could be expressed and were sufficiently stable to be studied [48-50]. In the current study, initial attempts were therefore focused on the overproduction of the smaller N-terminal catalytic domain of AprA (T40 to N258). Although evidence for cellular production of this fragment was observed, almost complete aggregation of this fragment occurred under a variety of experimental conditions designed for its control (Results). Previous reports by Guzzo and co-workers have shown that even minimal recombinant expression (1 μg/mL) of soluble, intact AprA protein required the co-expression of the entire secretion apparatus (AprD, AprE, AprF) [51]. When the structural gene for AprA was expressed alone, however, intracellular accumulation of AprA was barely detectable, most likely due to rapid degradation inside the cell [52]. In other reports, significant quantities of the enzyme could be found intracellularly, but only in the form of inclusion bodies, which are known to be highly resistant to degradation [24,53,54].
However successful production of mature full length (G10 to V479) AprA, was accomplished without the co-expression of the entire secretion apparatus. Although insoluble protein aggregates were produced, it was possible to resolubilise and refold the overexpressed protein, yielding approximately 40 mg of high purity enzyme per 1 L of culture (Figure 2). The ESMS analysis revealed that although the majority of the protein isolated from this protocol had Mr = 49 500 Da (full length mature AprA, G10-V479), a small quantity of inseparable enzyme with an Mr = 49 632 Da had maintained its N-terminal methionine (Met-G10-V479) from the cloning procedure (Figure 3). Kinetic analysis of this recombinant mature AprA compared favourably with values previously reported [29] for the wild type AprA secreted by P. aeruginosa (kcat/Km of 2140 ± 540 M-1s-1 and 1800 ± 300 M-1s-1 for wild type and recombinant enzymes respectively).
An almost 100% incorporation level of the fluorinated probe into AprA was readily accomplished by induction of protein expression in minimal media supplemented with 2 mM DFM. The labelled protein was also expressed as intracellular inclusion bodies which were resolubilised and refolded using the same protocol as the unlabelled recombinant protein, yielding approximately 16 mg of nearly homogeneous protein per 1 L of culture (Figure 2). The ESMS analysis indicated that, unlike the unlabelled recombinant AprA, a majority of the DFM incorporated protein maintained the initiating residue (Mr = 49 702 Da), while this DFM was cleaved in only about one quarter of the sample (Mr = 49 535 Da) (Figure 4). The interesting observation that removal of the N-terminal DFM residue occurs to a lesser extent compared to methionine in this position indicates that the processing of fluorinated methionines by methionine aminopeptidase is a less efficient process. Related to this observation is a recent report by Budisa and coworkers indicating the lack of removal of trifluoromethionine from the N-terminus of a mutant green fluorescent protein[55]. It may be that the increased steric size of the fluorinated methyl groups in these analogues alters the correct positioning of the peptide bond of the substrate in the active site of the methionine aminopeptidase which could reduce hydrolytic efficiency by this enzyme. In any event, the presence of the N-terminal methionine (or DFM) is not expected to affect catalytic activity or thermal stability of the protein as the N-terminus is physically remote from the active site. The kinetic characteristics of the protein mixture with and without incorporated DFM were determined under the revised conditions described in the methods and results sections. The Km was not significantly altered by replacement with DFM, the Vmax and therefore kcat were only slightly impaired, resulting in a decrease in kcat/Km from 620 ± 30 M-1s-1 to 530 ± 50 M-1s-1.
In order to compare the effects of DFM on thermal stability of the protein, preliminary differential scanning calorimetry studies were undertaken on AprA and DFM-AprA. Under the conditions utilized in these experiments, the thermal denaturations of AprA and DFM-AprA were found to be irreversible. As shown in Figure 6, the thermal stability in 20 mM HEPES (pH 7.8) was not significantly altered upon fluorination of the Met-turn methionine. The Tmax for unlabelled and DFM-labelled AprA were determined to be 58.7 ± 0.1°C and 58.6 ± 0.2°C respectively. This would indicate that the introduction of two fluorine atoms into the methyl group, which is in close contact to the critical histidines in the active site, can be accommodated by the protein such that the catalytic properties and thermal stability of AprA are not drastically perturbed.
Due to the lack of background 19F resonances in most biological systems, the 100% natural abundance of the 19F isotope and its sensitivity to NMR detection of 83 % to that of 1H, the application of 19F spectroscopy to studies in protein structure and function has been an important biophysical technique that has been shown to give critical information on the environment surrounding the fluorine nucleus [19,56-58]. Previous studies on DFM incorporation into the transglycosylase of bacteriophage λ and the leucine-isoleucine-valine (LIV) binding protein from E. coli, have detected separate sets of resonances for each DFM residue present in each protein and their presence has been useful in detection of ligand binding [28,47]. As the two fluorine atoms in DFM are diastereotopic due to the presence of the chiral centre of the α-carbon of methionine, complex 19F resonances can result. In fact, it has been shown that a complex octet of 19F resonances is observed when a DFM residue is located in a conformationally restricted protein environment [28]. It was therefore of interest to investigate the 19F NMR spectroscopy of DFM-AprA. The 1H-coupled 19F NMR spectrum of DFM-AprA (Figure 7A) is a composite of the 19F resonances not only from the active site DFM at position 214, but also a contribution from the presence of protein molecules still maintaining the N-terminal DFM residue. Interestingly the two resonances overlap in the 564 MHz spectrum. It has previously been shown that application of paramagnetic line broadening agents, such as Gd(EDTA)- will broaden the 19F NMR resonances from fluorine nuclei in a protein in a distance-dependent manner and can serve to indicate which 19F resonances originate from more solution exposed residues [56,58]. To possibly distinguish the signal contributions from each DFM, paramagnetic line broadening experiments utilizing Gd(EDTA)- were undertaken. These experiments (Figure 7B) indicated that both DFM residues (DFM1 and DFM214) were equally susceptible to the line-broadening agent, indicating that close approach to the active site DFM by Gd(EDTA)- was also possible in solution. Based on the crystal structure of AprA [15,59], the active site is open to solvent and could be expected to reasonably accommodate the rather large Gd(EDTA)- molecule resulting in little discrimination between the two DFM residues. Although proton decoupling simplified the 19F spectrum (Figure 7C), the resonances from the two DFM residues were still unresolved; the complexity of which may also result from contributions of the restricted environment of the active site DFM. Attempts to remove the N-terminal DFM with recombinant E. coli methionine aminopeptidase [60] under a variety of conditions resulted in complex mixtures due to proteolytic degradation by AprA. Additional attempts to produce a variant of AprA lacking the N-terminal DFM residue by introduction of the wild-type N-terminal self-cleaving propeptide sequence or a thrombin cleavage site resulted in extremely poor isolated yields of these forms of the protease. Further studies will be focused on the application of this NMR probe to investigating conformational changes in the active site upon ligand and inhibitor binding.
Conclusion
The 19F biophysical probe, DFM, was successfully incorporated into the critical Met-turn of AprA and resulted in minimal catalytic or structural alteration of the enzyme. These findings are encouraging for the further application of DFM as a subtle probe for the investigation of the metzincin family of proteases.
Methods
Materials
Restriction endonucleases were purchased from New England BioLabs (Pickering, ON). The Expand™ and Pwo polymerase kits as well as the alkaline phosphatase (from calf intestine) were Roche (Laval, QC) products and the Taq polymerase was from Qiagen (Mississauga, ON). The thrombin (from bovine serum) was purchased from Amersham Biosciences (Baie d'Urfé, QC). The peptide substrate, Z-Arg-Arg-pNA, was purchased from Bachem (King of Prussia, PA). The VP-DSC differential scanning microcalorimeter and Origin™ software (Version 5.0) were from MicroCal™ (Northampton, MA). 19F NMR data were collected on Bruker Avance (Milton, ON) NMR spectrometers (500 and 600 MHz). Unless otherwise noted, all reagents and chemicals were of the highest commercial grade available and were used without further purification. L-Difluoromethionine was prepared by a previously reported method [28].
Cloning and bacterial expression
The genes of interest were amplified by PCR and digested using the primers and restriction endonucleases listed in Table 1. With the exception of pNusAPA, all PCR products were ligated in to appropriately digested pET22b(+) (Novagen Inc). The NusA fusion protein was produced from a pET43.1b(+) based vector (Novagen Inc). New constructs were confirmed by DNA sequencing (Mobix, Inc) in both directions and the protein products were analyzed by electrospray mass spectrometry (positive ionisation mode).
Table 1 Primers and endonucleases used in the cloning of the various constructs. Restriction endonuclease cut sites are underlined and the regions of the primers homologous to the gene of interest are in bold.
Construct Primers Restriction endonucleases Protein product
pAPA' N-terminal: 5' CCA GAA TTC CAT ATG ACC GTC GAC CAG GCG GCG GAG C 3'
C-terminal: 5' CCA GGA TCC TTA GTT GGC CCC GTA GAG CTT CTG G 3' NdeI
BamHI AprA catalytic fragment (APA')
pPelAPA' N-terminal: 5' CCG AAT TCC ATG G CC GTC GCC GTC GAC CAG GCG GCG GAG C 3'
C-terminal: Same as pAPA' NcoI APA' with pelB secretion signal
pNusAPA' N-terminal: 5' G GGC GGG GAC GAA TTG GTC AAT GGC 3'
C-terminal: Same as pAPA' Blunt end ligation at N-terminus: no digestion required APA' with NusA solubility tag and His6 tag
pWApr N-termnal: 5' CCA GAA TTC CAT ATG GGT CGT AGC GAT GCG TAT ACC 3'
C-terminal: 5' CCA GGA TCC TCA GAC GAC GAT GTC GGC CTG G 3' NdeI
BamHI Mature AprA without pro-peptide
Unlabelled proteins were produced in the E. coli strain BL21 (λDE3), while strain B834 was used for DFM incorporation. A 10 mL starter culture (LB broth with 50 mg/mL carbenicillin) was inoculated from frozen stock and incubated overnight at 37°C with shaking. For the production of unlabelled proteins, the entire starter was used to inoculate 1 L of LB broth (with 50 mg/mL ampicillin) and the culture was grown to an OD600 of 0.6 to 0.8. IPTG was added directly to a final concentration of 1 mM and the culture was incubated at 37°C with shaking for an additional 4 hours. A similar procedure to that described by Vaughan et al. [28] for DFM incorporation into phage lysozyme was used to produce the L-DFM labelled AprA protein. In brief, a 10 mL starter culture of E. coli strain B834 harbouring plasmid pWApr was used to inoculate 1 L of M9 buffer (pH 7.2: 47.6 mM Na2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, 18.7 mM NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2) supplemented with 0.4 % glucose, 50 μg/mL ampicillin, and 0.1 mM L-Met. The culture was grown at 37°C with shaking to an OD600 of approximately 0.7 and then harvested by centrifugation for 10 minutes at 5000 g. The cells were washed in M9 buffer and then resuspended in M9 supplemented with 0.4 % glucose and 50 μg/mL ampicillin. After incubating at 37°C with shaking for 30 minutes, 2 mM L-DFM and 1 mM IPTG were added, and the culture was incubated (37°C with shaking) for a further 8 hours.
Refolding of the N-terminal catalytic domain
The expressed N-terminal catalytic domain segment that was initially produced was extracted and solubilised in much the same manner as the full-length protein described below. However, several variations to the refolding buffer were made in attempts to recover the soluble, active catalytic fragment. A range of pH conditions from 7.2 to 8.0 was used. ZnSO4 was added to the refolding buffer with concentrations ranging from 0.1 μM to 0.1 mM. Glycerol (20 %) was also added to the refolding buffer and the L-arginine content was varied from 0.8 M down to 0.2 M. The arginine was replaced completely in several other attempts which instead involved 1 % Brij-35, 1 M non-detergent sulfobetaine 201 (NDSB-201), or 1 M KCl with 20 % glycerol. Finally, the dialysis buffer was also varied to include 10–20 % glycerol, 0.05 % Brij-35 or 0.05 % Triton-X 100.
Solubilisation and refolding of the full-length AprA protein
Induced E. coli BL21 (λDE3) cells harbouring plasmid pWApr were harvested in a 10 min spin at 5000 g, washed in M9 buffer (47.6 mM Na2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, 18.7 mM NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2) then resuspended in lysis buffer (50 mM TRIS (pH 7.8), 1 mM EDTA, 0.1 % Triton-X-100) and disrupted by eight 20 second rounds of continuous sonication with cooling. The crude cell lysate was centrifuged for 10 minutes at 6000 g, and the pellet was washed twice in lysis buffer and then 3 times in lysis buffer without Triton.
The resulting inclusion body pellet was dissolved in a small amount of solubilisation buffer (50 mM TRIS, pH 7.8, 1 mM EDTA, 6 M guanidine hydrochloride) and incubated at 37°C for 1 hour. The solution was then centrifuged for 10 minutes at 13000 g and a 4 μL aliquot was diluted to up to 1 mL in solubilisation buffer for an absorbance measurement at 280 nm. Protein concentration was estimated using a predicted extinction coefficient of 67 990 M-1cm-1 calculated by the ProtParam tool in ExPASy [61]. Additional solubilisation buffer was used to dilute the protein solution to 10 mg/mL. This solution was then passed through a 0.22 μm syringe filter and added dropwise with stirring to ice cold refolding buffer (50 mM TRIS, pH 7.8, 1 mM CaCl2, 0.8 M L-arginine), resulting in a 100-fold dilution (final refolding protein concentration: 0.1 mg/mL). After an overnight incubation with stirring at 4°C, the protein was dialysed against two changes of buffer with metals (20 mM HEPES, pH 7.8, 1 mM CaCl2, 0.1 mM ZnSO4), then one without CaCl2 or ZnSO4, but with 300 mM NaCl. Finally, the protein was concentrated using an Amicon pressure filtration device with a 10 kDa cut-off filter and the high salt buffer exchanged to 20 mM HEPES (pH 7.8).
Mass spectrometry
Electrospray mass spectrometry of purified protein samples was carried out on a Micromass Q-TOF Global Ultima mass spectrometer in positive ion mode. Spectra were analyzed using the MaxEnt1 algorithm in the MassLynx (V4.0, Micromass Limited/Waters, Milford, Massachusetts) software. Buffer salts were removed from protein samples using a Nanosep microcentrifuge concentrator with a 10 kDa cut-off. Once in Milli-Q (Waters Corp.) purified water, samples were diluted with a solution of 50 % acetonitrile and 0.1 % formic acid.
Activity assay
Initial determinations of kinetic parameters using the substrate Z-Arg-Arg-p-nitro-analide were carried out under the conditions described by Louis et al. for wild type alkaline protease [29], using substrate concentrations ranging from 3 μM to 200 μM. The rate of p-nitroanaline released was calculated using the extinction coefficient of 8800 M-1cm-1 at 410 nm [29]. In order to avoid possible metal chelating effects of the TRIS buffer and to increase assay sensitivity, 20 mM HEPES buffer was used in place of the 5 mM TRIS, and 100 μg of protein was used for each assay. Kinetic parameters, Km and Vmax, were determined by non-linear regression using the GraFit5™ software (Erithacus Software Limited).
Differential scanning calorimetry
A protein concentration of 0.5 mg/mL was used for each melting temperature determination. Initially, 50 mM TRIS (pH 7.8) was used as the buffer for these experiments, however, this was eventually replaced by 20 mM HEPES. Therefore, protein samples in 20 mM HEPES (pH 7.8) and reference buffer (20 mM HEPES, pH 7.8) were degassed under vacuum just prior to analysis. Scanning was performed continuously between 15 and 90°C at a rate of 1°C per minute with a 1 minute hold in between each up and down scan. An initial buffer-buffer scan was performed in order to establish a thermal history for the system. The subsequent buffer-buffer scan was then used as a reference. Once the temperature was below 35°C again, the protein solution was loaded into the sample cell and scanned several times.
Data were analysed using the Origin5.0(™) software in the following manner. The reference buffer-buffer scan was subtracted from the subsequent protein scan, the data were normalised for protein concentration, and the temperature (Tmax) at the resulting peak was determined. Plots of heat capacity, Cp, versus temperature were then plotted. (Tmax may be defined as the apparent temperature of maximum excess specific heat absorption [62,63]. The Tmax values for both the mature wild type AprA and the DFM-incorporated proteins were determined in triplicate, averaged and the standard deviation determined.
19F NMR spectroscopy of DFM-labelled AprA
Protein was first dialysed against 5 mM HEPES (pH 7.8) and then lyophilised and stored at -20°C until needed. Approximately 4 mg of lyophilised protein was made up in 500 μL of D2O for analysis. 19F NMR (564.5 MHz) data were collected using a Bruker Avance 600 MHz spectrometer fitted with a 5 mm dual 19F/1H probehead tuned to 19F. Standard parameters were11 261 Hz sweep width, 1.45 second acquisition time and a 0.5 second relaxation delay. A 1 Hz line broadening was applied. Spectra were recorded at 277 K and referenced to an external sample of trifluoroacetic acid (set at-76.53 ppm) or a solution of 0.05 % trifluorotoluene in CDCl3 (set at -63 ppm) as external references. Gd(EDTA)- line broadening experiments were realized by adding 13 μL and later 26 μL of a stock solution of Gd(EDTA)- (80 mM GdCl3, 500 mM EDTA, pH 7.1 with NaOH) to the sample.
Authors' contributions
PW carried out the cloning and protein isolation and characterization. Both authors drafted the manuscript. JFH conceived of the study, and participated in its design and coordination. Both authors read and approved the final manuscript.
Acknowledgements
The authors would like to gratefully acknowledge the financial support of the University of Waterloo and NSERC (Canada). The authors would also like to thank Dr. J. Lam for the gift of P. aeruginosa strain PA01, Dr. E. Daub and Ms. I. Bruni for initial work on AprA, Drs. T. Lowther and B. Matthews for the plasmid encoding the E. coli methionine aminopeptidase and Dr. Mark Vaughan for helpful discussions. The authors thank Dr. R. Smith for mass spectrometry support and Ms. J. Venne and V. Robertson for NMR analysis.
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-2431620213010.1186/1471-2105-6-243Research ArticlePhylogenetic detection of conserved gene clusters in microbial genomes Zheng Yu [email protected] Brian P [email protected] Richard J [email protected] Simon [email protected] Bioinformatics Graduate Program, Boston University, Boston, MA, USA2 New England Biolabs, Beverly, MA, USA3 Department of Biomedical Engineering, Boston University, Boston, MA, USA4 Center for Advanced Genomic Technology, Boston University, Boston, MA, USA2005 3 10 2005 6 243 243 12 4 2005 3 10 2005 Copyright © 2005 Zheng et al; licensee BioMed Central Ltd.2005Zheng 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
Microbial genomes contain an abundance of genes with conserved proximity forming clusters on the chromosome. However, the conservation can be a result of many factors such as vertical inheritance, or functional selection. Thus, identification of conserved gene clusters that are under functional selection provides an effective channel for gene annotation, microarray screening, and pathway reconstruction. The problem of devising a robust method to identify these conserved gene clusters and to evaluate the significance of the conservation in multiple genomes has a number of implications for comparative, evolutionary and functional genomics as well as synthetic biology.
Results
In this paper we describe a new method for detecting conserved gene clusters that incorporates the information captured by a genome phylogenetic tree. We show that our method can overcome the common problem of overestimation of significance due to the bias in the genome database and thereby achieve better accuracy when detecting functionally connected gene clusters. Our results can be accessed at database GeneChords .
Conclusion
The methodology described in this paper gives a scalable framework for discovering conserved gene clusters in microbial genomes. It serves as a platform for many other functional genomic analyses in microorganisms, such as operon prediction, regulatory site prediction, functional annotation of genes, evolutionary origin and development of gene clusters.
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Background
In microorganisms, it is often seen that genes tend to locate in conserved proximity in a number of genomes forming conserved gene clusters [1]. Further analyses often uncover biologically meaningful relationships between genes with conserved proximity [1-3]: they are often co-transcribed as operons [4], or co-regulated as part of a larger biochemical network [5-7]. Examples include the widely present DNA restriction and modification gene pairs [8], which provide a way of defending against bacteriophage and other foriegn DNA, and the two component systems which respond to changes in environmental conditions [9]. Thus, delineation of conserved gene clusters will help reveal functional relationships between genes within them [10]. In addition, the conserved gene clusters provide an invaluable resource for corroborating the growing number of co-expressed gene sets obtained from microarray based mRNA expression experiments.
The increasing accumulation of genome sequence data has facilitated the practice of finding conserved gene clusters since both similarity and synteny information can be easily obtained [11]. Various computational methods for finding clusters have been described [1,6,12-16]. An important issue has been the estimation of significance of the observed conserved proximity. It is generally accepted that phylogenetic distances between genomes largely determine the significance: conservation is deemed more significant when a cluster appears in distantly related genomes than in closely related ones. The rationale is that the greater the length of time a gene cluster has persisted, the more it has resisted dissolution by recombination events, and the stronger the selective pressure to maintain it. Previous published methods have incorporated phylogenetic information into the estimation of significance by using either 16S RNA distance [1] or statistical methods [12]. However, few efforts have focused on the development of a full evolutionary model to describe conserved gene clusters. Empirical approaches include grouping closely related genome into clades [7] or choosing a subset of genomes based on knowledge of the evolution of microorganisms [14]. Although the latter approaches are efficient in finding non-trivial conserved gene clusters, they do not scale automatically, which is problematic with more and more sequenced genomes.
Moreover, sequenced organisms are often close relatives of known model organisms or pathogens selected for biomedical reasons, so phylogenetic coverage can be rather sparse and biased. For example, there are many more close relatives of Escherichia coli than those of Helicobacter pylori in the genome database. Thus, simply counting the number of genomes where a gene cluster is conserved often overestimates the significance of conservation of a cluster observed in microorganisms such as E. coli.
Delineation of evolutionarily conserved gene clusters must take into account a stochastic model of evolution of these clusters. In this paper we used a simplified stochastic generative evolutionary process where an organism inherits a gene cluster from its immediate ancestor with a probability proportional to the evolutionary distance in the tree. This model facilitates an efficient computation of the probability that a particular pattern of conservation in many genomes is observed. In particular, observing a large number of occurrences in closely related genomes will not carry the same significance as the occurrence of a cluster in a wider evolutionary phylum.
We define a tree-based probabilistic conservation score and show that it provides a quantitative measure the strength of proximity constraints in genome evolution and serves as a better predictor of functional links among genes than more naïve methods. It also sheds insight into the relationship between evolutionary development and the functional selection.
Results
We applied our computational pipeline to 127 microbial genomes which were obtained from the NCBI website (2/2004). Pairwise genome comparisons of translated open reading frames were performed using BLASTP [17] and putative orthologs between genomes were identified as reciprocal best hits (see Methods). Conservation scores Cu and Cd were assigned to each gene (see Methods). Intuitively, Cu measures the strength of the conservation between the gene under consideration and its upstream neighboring genes while Cd does the same with its downstream neighboring genes on the chromosome. The higher the Cu or Cd of a gene is, the more significant its conservation with its upstream or downstream neighbors. Conserved gene clusters were then detected using a conservation score cutoff (see Methods section). The complete results of our method on all microbial genomes included can be accessed at [18]. Results using more than 200 genomes available now are being incorporated into the database.
Accounting for genome phylogeny can correct for databases bias
During evolution, genomes undergo frequent changes such as the rearrangement and exchange of genes. For those organisms that have diverged recently, such changes have had little time to occur so that vertically inherited synteny cannot be distinguished from functionally induced proximity information. The false positive rate could therefore drastically increase as more and more genomes from the same taxonomic group are included in the comparison. Early methods ignored the phylogenetic relationship between genomes and assumed an independence between them, which could blur the distinction between gene clusters truly constrained by selection pressures and those that merely reflect vertical inheritance. The phylogenetic method we describe here is designed to integrate genome phylogenetic information into consideration and alleviate the common problem of overestimation. For illustration we devised an experiment and compared the performance of our method with the more naïve method where each genome is treated equally, which we refer to hereafter as "simple counting method". First, we chose E. coli K12 as the query genome and a group of 6 reference genomes including 3 closely related ones (E. coli O157H7, E. coli CFT073 and E. coli O157H7 EDL933) and 3 distantly related ones (Mycobacterium tuberculosis CDC1551, Staphylococcus aureus Mu50 and Bacillus subtilis). We then examined the genomic region of the entCEBA operon in E. coli K12 [19] and used two methods to calculate the upstream conservation score (Cu) as defined in the methods section. In the simple counting method, when a gene and its upstream gene are also neighbors in a reference genome, the score for that gene will be incremented by 1. Figure 1a and 1b show the upstream conservation score profiles obtained from the simple counting method and our phylogenetic method respectively. In Figure 1a, the scores of the entCEBA operon genes barely stands out from those of the flanking genes while in Figure 1b there is a clear peak corresponding to the entCEBA operon. The signal to noise ratio, as estimated from the peak value and the base line of the Cu profile, is about 3:1 for the phylogenetic method (Figure 1b) but only about 1.3:1 for the simple method (Figure 1a).
Figure 1 Improvement of signal-to-noise ratio of the phylogenetic method over the simple method. The upper conservation scores (Cu) profiles for the genomic region surrounding ent operons in Escherichia coli are shown. (a) the simple method; (b) the phylogenetic method.
The signal to noise ratio increases as more genomes are added to the reference set. The Cu and Cd profiles are shown in Figure 2 for the same genomic region around the entCEBA operon as we progressively include more genomes in the reference set. While the scores of the genes in the operon increase as more reference genomes are included, the conservation scores of the flanking genes increase much more slowly (Figure 2). Notice that Cu for entC and Cd for entA do not increase as much as other ent genes since they are the boundary genes of the ent operon. Figure 2 shows that the phylogenetic method provides an improved framework for detecting conserved gene clusters as the number of genomes in the database increases.
Figure 2 Conservation score (Cu and Cd) profiles for the entCEBA genomic region using increasing number of reference genomes.
Conserved gene clusters inferred from orthology or similarity
It is known that using reciprocal best BLAST hits is a conservative method of identifying putative orthologous genes between two genomes. The advantage of using this conservative method is that any pairs identified are very likely to be true orthologs and share similar molecular and physiological function. However, for genes that have undergone fast evolution and for paralogous gene groups, which are quite common in bacterial genomes, the reciprocal relationships are usually obscured [20]. As a result, significantly conserved clusters or parts of a cluster may be missed if pairwise reciprocal connections are used. This is illustrated in Figure 3, which shows the upstream conservation score (Cu) profiles for a genomic region containing the fhu operon [21] of E. coli. Circles are calculated from the similarity data (BLAST E-value < 1E-5) and the squares are from the orthology data. For most genes where the orthology relationship is clear, scores calculated from the similarity data are equal to those from the orthology data (Figure 3). For those genes with no clear orthologs in other genomes, scores from similarity data are higher than those from orthology data. For instance, the Cu of the fhuC gene from similarity data exceeds the threshold (5.0 used in this paper), but its Cu from the orthology data does not. This is because fhuA, the immediately upstream gene of fhuC, belongs to a paralogous gene family with 9 members in E. coli and its reciprocal best hit cannot be found in other genomes. As a result, the gene pair (fhuA, fhuC) is not identified as a widely conserved orthologous pair. On the other hand, only using the similarity data may introduce a higher false positive rate in making predictions about functional dependence, as discussed below.
Figure 3 Upstream conservation score calculated from orthology and similarity data for a genomic region surrounding fhuABCD operon in Escherichia coli.
Application in operon prediction
To test the predictive value of our method for inferring operons, we took a compiled list of E. coli operons from the RegulonDB [22]. A total of 345 operons with multiple genes were extracted from the RegulonDB dataset. Genes that are not in the RegulonDB set and have intergenic regions of larger than 100 nucleotides on both sides comprise the negative set (1342 genes). Using different cutoffs, we calculated the sensitivity (Sn) and the specificity (Sp) by:
Sn=TPTP+FN,Sp=TNFP+TN
MathType@MTEF@5@5@+=feaafeart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqWGtbWucqWGUbGBcqGH9aqpdaWcaaqaaiabdsfaujabdcfaqbqaaiabdsfaujabdcfaqjabgUcaRiabdAeagjabd6eaobaacqGGSaalcqWGtbWucqWGWbaCcqGH9aqpdaWcaaqaaiabdsfaujabd6eaobqaaiabdAeagjabdcfaqjabgUcaRiabdsfaujabd6eaobaaaaa@447F@
where TP and FP are the number of genes that are correctly or wrongly predicted to be in operons, and TN and FN are the number of genes that are correctly or wrongly predicted not to be in operons. Notice that genes in operons must reside on the same strand while genes in clusters detected by our system may come from both strands. Nevertheless, the high percentage of conserved clusters that overlap with operon dataset suggests our ability to identify clusters that are conserved due to the transcriptional selection pressure and demonstrates the potential use of the system in the task of operon prediction.
Figure 4 compares the performance of the two methods on the operon dataset by showing the receiver operating characteristic (ROC) curves. Our method using either orthology data or similarity data consistently outperforms the simple counting method, which treats each observation with equal weight (Figure 4). As a result, when using 5.0 (P-value < 2.4E-4, default cutoff) as the threshold for making operon predictions using orthology data, our method gives 65% sensitivity and 85% specificity in E. coli (Figure 4). For the same cutoff value (e.g., 4.0 or 5.0), the results using similarity data give better sensitivity but worse specificity than the results using orthology data (Figure 4). Higher specificity is achieved when a larger cutoff is used. For instance, when a reported cluster has a score (based on orthology) larger than 6.0 (P-value < 1.7E-4), there is an 89% chance that this cluster corresponds to an operon.
Figure 4 Receiving operating characteristic (ROC) curves of different methods on the RegulonDB operon dataset. Curves are color-coded for different methods. Points using cutoffs of 4.0 and 5.0 for our method are highlighted on the curve.
Statistics of conserved gene clusters across genomes
Table 1 shows the statistics of conserved gene clusters in a number of microbial genomes where we have applied our method. Microbial genomes appear to have a highly specialized distribution of conserved clusters (Table 1). It appears that in general 10–40% of all the genes in a genome lie in conserved gene clusters (based on orthology data). Among the identified clusters, there are very few that are widely conserved across all species or conserved with invariant gene content (Y.Z. et al, unpublished results). Although it appears that in several cases the high content of conserved gene clusters occurs in relatively small genomes (Table 1), the overall correlation between the genome size and the proportion of conserved gene clusters is not significant (r2≈0.1).
Table 1 Statistics of conserved gene clusters in a number of microorganisms
Genome Total genes Total genes in clusters Total detected clusters Percentage Average cluster size
Chlamydophila pneumoniae J138 1070 209 58 0.20 3.6
Mycobacterium tuberculosis CDC1551 4187 547 161 0.13 3.4
Sinorhizobium meliloti 6205 841 230 0.14 3.7
Clostridium acetobutylicum 3672 707 166 0.19 4.3
Mycobacterium tuberculosis H37Rv 3918 543 156 0.14 3.5
Staphylococcus aureus Mu50 2748 757 174 0.28 4.4
Aeropyrum pernix 2694 160 44 0.06 3.6
Clostridium perfringens 2723 633 147 0.23 4.3
Mycoplasma genitalium 480 153 40 0.32 3.8
Agrobacterium tumefaciens C58 5301 805 221 0.15 3.6
Deinococcus radiodurans 3102 389 117 0.13 3.3
Mycoplasma pneumoniae 688 167 47 0.24 3.6
Streptococcus pneumoniae R6 2043 546 151 0.27 3.6
Agrobacterium tumefaciens C58 UWash 5402 832 224 0.15 3.7
Escherichia coli K12 4289 1313 287 0.31 4.6
Mycoplasma pulmonis 782 168 52 0.21 3.2
Streptococcus pneumoniae TIGR4 2094 534 147 0.26 3.6
Escherichia coli O157H7 5361 1327 288 0.25 4.6
Neisseria meningitidis MC58 2025 457 129 0.23 3.5
Streptococcus pyogenes 1696 501 136 0.30 3.7
Aquifex aeolicus 1553 178 57 0.11 3.1
Sulfolobus solfataricus 2977 244 65 0.08 3.8
Archaeoglobus fulgidus 2407 250 73 0.10 3.4
Nostoc sp 6129 284 88 0.05 3.2
Sulfolobus tokodaii 2826 242 65 0.09 3.7
Bacillus halodurans 4066 952 219 0.23 4.3
Borrelia burgdorferi 1709 214 57 0.13 3.8
Case studies of conserved gene clusters
Conserved gene clusters, once identified, can be used to make functional predictions for the genes within them, and to hypothesize interactions between their products. For example, in the genome of Mycobacterium tuberculosis CDC1551, we found a four-gene cluster encoding putative homologs of (1) mraZ (MT2224), (2) mraW/yabC (MT2223), (3) ftsL/mraR (MT2222), and (4) ftsI/pbpB (MT2221). This cluster is widely conserved among both gram-positive and gram-negative bacteria, and in E. coli these four genes comprise the start of a known operon of cell division and cell envelope genes [23]. FtsI and FtsL are known to be essential for the assembly of the cell division septum in E. coli. MraW has been shown to be a methyltransferase whose substrates are localized to the cell envelope [24]. Furthermore, it has been shown that lack of S-adenosylmethionine (SAM) leads to a cell division defect in E. coli, with one possible explanation being that SAM serves as a methyl donor in a required methylation event [25]. The location of mraW within this cluster suggests it may encode the methylase involved in such an event, possibly modifying the FtsL and/or FtsI proteins. Indeed, there is experimental data to suggest this [23], although it has not yet been conclusively shown.
In addition to conserved gene clusters that are widely distributed in many genomes, we also find statistically significant conserved gene clusters that are present in only a few species. Instead of the number of genomes, the large evolutionary span across genomes contributes to the significance of the conservation. Due to their limited occurrence, these clusters are difficult to find in the database using conventional methods. Figure 5 gives two intriguing examples: one is conserved in 4 genomes (Figure 5a) and the other is conserved in only 3 genomes (Figure 5b). In both examples the large evolutionary span is reflected by the fact that they are conserved in both bacterial and archaeal species.
Figure 5 Examples of gene clusters that are conserved in only a few genomes.
The gene cluster in Figure 5a was originally found and characterized in Sinorhizobium meliloti and was shown to synthesize a special type of siderophore, rhizobactin 1021, involved in iron uptake [26]. It is largely conserved in another three genomes: one is an archaea, Halobacterium sp., and the other two are bacteria, Nostoc sp. and Bacillus halodurans. Notice that this cluster is absent in other Bacilli species (Bacillus subtillis, etc.) despite their closeness to Bacillus halodurans. On the other hand, Bacillus subtilis is known to possess the dhb operon responsible for synthesizing a different type of siderophore, 2,3-dihydroxybenzoate [27], and this operon is absent in Bacillus halodurans. Figure 5a suggests that the other three microorganisms may possess the ability to synthesize rhizobactin 1021 or a siderophore with a similar structure, reflecting a requirement for iron in the life cycles of these species.
Figure 5b depicts a three gene cluster that is conserved in Pirelulla sp., Methanosarcina acetivorans and Oceanobacillus iheynesis. One of the three genes is annotated as a cellulosomal protein. A BLAST search reveals its similarity to cotH, which has been characterized in Bacillus subtilis and is essential for spore coat assembly [28]. The other two are conserved hypothetical genes in Genbank with no functional information although they are both predicted to be transmembrane proteins (by TMpred). The conservation in Figure 5b suggests the two unknown genes are functionally related to cotH. We note that these conserved gene clusters that appear in just a few genomes with a large evolutionary span could be instances of horizontal transfer.
Functional enrichment in conserved gene clusters
If conserved gene proximity indeed implies relatedness, we expect to see functional enrichment in the list of gene clusters found by our method. We used the 18 functional codes of COG [29] as a crude measure of this, counting gene pairs in which both members belong to the same COG category. For 39 genomes examined, we determined the fraction of gene pairs found by our method (m/n), then performed a one-tailed Fisher's exact test to determine the probability of observing at least m gene pairs with the same functional code among n pairs selected randomly from all gene pairs in the genome. Gene pairs including at least one gene with no functional information (i.e., having no assigned category, or designated as general function or unknown function) were excluded from the analysis.
A partial list of the results of this analysis is shown in Table 2, with the fraction of enriched clusters and the associated P-values in columns 2 and 3, respectively. In all cases, these P-values are extremely significant (P << 0.01), indicating that the gene pairs obtained by our method are much more likely to be functionally related than expected by chance.
Table 2 Statistics of functional enrichment
Phylogeny Method Counting Method
Organism Fraction P-value Fraction P-value P-value ratioa
Aeropyrum pernix 39/45 6.33E-12 40/46 2.21E-12 0.349
Aquifex aeolicus 80/107 1.23E-32 81/111 8.71E-32 7.08
Bacillus subtilis 329/503 1.86E-44 363/601 4.10E-38 2.20E+06
Buchnera sp. 167/246 1.28E-31 181/307 1.90E-23 1.48E+08
Chlamydia trachomatis 95/132 3.94E-24 123/295 1.88E-03 4.77E+20
Chlamydophia pneumoniae 104/137 5.03E-30 135/327 4.29E-03 8.53E+26
Deinococcus radiodurans 173/221 4.07E-57 167/218 5.56E-52 1.37E+05
Escherichia coli K12 439/750 8.92E-48 606/1553 0.19 2.13E+46
Halobacterium sp. 127/150 7.60E-38 125/145 6.27E-39 0.0825
Helicobacter pylori 26695 120/151 1.11E-37 122/158 4.92E-36 0.443
Lactococcus lactis 182/277 2.79E-28 196/319 4.19E-25 1500
Methanococcus janaschii 68/83 5.82E-22 64/79 5.91E-20 102
Mycobacterium tuberculosis 203/293 1.05E-42 226/347 1.09E-41 10.4
Mycoplasma pulmonis 75/98 3.61E-11 77/103 1.44E-10 3.99
Neisseria meningitidis MC58 150/222 1.25E-51 156/241 9.14E-51 7.31
a Ratio is P-value for functional enrichment by the phylogeny method divided by P-value for functional enrichment by the counting method [i.e., (column 3) / (column 5)].
Using the same methodology, we divided the gene pairs into colinear (on the same strand) and divergent (on opposite strands) pairs to see if the bulk of functionally enriched pairs were of one type or the other. The P-values for colinear pairs were similar to those for all pairs, suggesting the vast majority of gene pairs captured by our method might be operons (data not shown). On the other hand, there are only limited numbers of divergent pairs and the P-values for divergent pairs are often insignificant (data not shown).
Functional enrichment analysis has been also applied to the results from the simple counting method, using a cutoff of 10 organisms (of 127 total). The results are shown in Table 3, columns 4 and 5. Although there is no equivalency between them (one is an absolute number, whereas the other is a log-odds score), the number of gene pairs captured by the two methods with their respective cutoffs were similar for many of the genomes examined (Table 3). In most cases, the simple counting method also yielded significantly functionally enriched gene pairs, as indicated by the P-values in column 5. For most genomes, the P-values of the simple counting method are much smaller than those from the phylogeny method (column 6 of Table 2). This is especially true for those genomes which have many close relatives in the database, e.g., E. coli and other Enterobacteriaceae. In those cases, we also observe a large increase in the number of pairs obtained by the simple counting method. We expect this increase to consist largely of functionally unrelated false positives that are conserved because of close phylogenetic distance.
We recognize the limitations of using COG codes to capture functional relationships, and our results will certainly include both false positives and exclude false negatives. For example, consider a functionally related gene pair consisting of a transcriptional regulator and the gene it regulates. These genes would likely be assigned different COG codes, so such a functional relationship would not be captured by this analysis. Therefore, to independently verify our results with COG, we performed a similar analysis using KEGG pathway information in E. coli, the organism with the largest number of KEGG pathway assignments. The overrepresentation statistics for E. coli using KEGG are similar to our results using COG and thus support the above discussion (data not shown).
Discussion
There is a general problem when comparing sequenced genomes because the samples of such genomes presently available are not uniformly distributed across the microbial kingdoms. Rather, several groups of related bacteria, such as the Enterobacteriaceae, are over-represented. This means that the significance of a feature found in organisms from this group must be interpreted with caution because it may be conserved by simple lineage effects rather than by functional selection. Most papers dealing with this problem either ignore it or simply remove the multiple members of the family and settle for a single representative example. Both methods necessarily lead to inaccuracies in estimating significance. In this paper, we have attempted to overcome this problem by using a method that takes into account the phylogenies of the individual members of related families. We show that it results in a reduction in the false positive rate over the simple counting method. We have not attempted to compare it to the selective sampling method, where one organism is used to represent a phylum, because given the wide variability usually observed within a phylum, the results from that approach will vary widely depending upon which genome is selected as the exemplar.
We have applied our new phylogenetically informed method to the problem of detecting conserved gene clusters. Such clusters are generally believed to reflect conservation of biological function in that often the gene products from the various genes in the cluster are involved in closely-related pathways. This may include the traditional operons known to encode the biosynthetic pathways of intermediary metabolism, or they may reflect the fact that enzymes responsible for post-translational modification will sometimes affect neighboring gene products. Other functional connections may also be found in these clusters. This can be a powerful tool in making predictions about gene products that might otherwise be recalcitrant to direct similarity analysis. Examples here include the restriction-modification enzymes where the DNA methyltransferases often show reasonable degrees of similarity that enable them to be identified, whereas the genes for the restriction enzymes evolve rapidly and usually cannot be identified on the basis of sequence similarity. Nevertheless, the genes are usually clustered. In addition, the current methodology is not necessarily restricted to the conserved synteny between genes. It may be applied to conserved synteny of other functional elements in genomes such as cis-elements, riboswitches, etc.
In our current methodology we have made several simplifying assumptions of which the most important might be to ignore horizontal gene transfer. While this is not a problem if an entire cluster is transferred horizontally, it does become a problem for those "hitchhiking" genes that may be transferred with the cluster. Nevertheless, the results we report seem promising and we do not view this as being a serious limitation at the present time.
The mode of phylogenetic inference is an important consideration in the type of analysis presented here. The basis for the tree underlying the analysis is not restricted to shared gene content, as we have employed here, but could alternatively be based on 16S RNA sequences, gene content, gene order, genome statistics, or coding sequences. Our current method is oblivious to "local" changes such as rearrangements inside the cluster, or differential selection within individual coding sequences. For example, it is possible that genes in the cluster might have different gene trees from each other. The choice of the best phylogenetic methodology in this context is an important follow-up and we feel that the final solution will combine the benefits of multiple methodologies in a single system.
Conclusion
The methodology and results that we present here should be generally useful in any situation where the functional significance of conserved genes or clusters is being investigated, for example, as a way of cross-validating co-expressed genes inferred from many microarray experiments or as a starting point for the assembly of gene networks. As more and more genomes are sequenced, the approach described here should be generally applicable and it will scale well computationally.
Of particular significance in this paper has been our finding that many of the clusters we have observed contain unknown genes with no biological function currently assigned. In these cases it seems reasonable to hypothesize that the product of the unknown gene has a function closely related to the functions of the known genes. Such a function may be a key enzymatic step in a biosynthetic pathway, a key regulatory function or an important post-translational modification. We have assembled a database of conserved gene clusters, called GeneChords [18], with a simple user interface to permit its rapid query. This will be described in detail in a separate publication.
Methods
Once a genome is chosen for analysis, our algorithm consists of the following steps:
a) For each gene pair (as defined below) in the genome, a tree-based conservation score is computed;
b) For each gene in the genome, two neighbourhood conservation scores are computed, measuring the strength of proximity constraint of the gene with its upstream or downstream neighboring genes;
c) Adjacent genes with conservation scores between them exceeding some threshold are joined into conserved clusters.
The details are described in the following sections.
Definition of conserved neighboring gene pairs
For simplicity let us consider the problem of identification of a conserved gene pair, the building block of our gene clusters. We define a gene pair as two genes with no more than k open reading frames separating them along the chromosome (k = 1 in this paper). In contrast to the operon identification procedure [12], the two genes in a pair do not need to reside on the same strand of the chromosome. When orthologs of a gene pair form a pair in other genomes, the pair is considered conserved. Orthologous genes are often detected as reciprocal best hits (BLAST E-value < 1E-5) between the two genomes by sequence comparison software, such as BLAST [17]. However, evolutionary events such as gene duplication followed by diversification could obscure the reciprocal relationship, resulting in relatively high false negative rates in the identification of orthologous genes. To relieve this concern, we loosen the criteria to include genes with high similarity (BLAST E-value ≤ 1E-5) (see Results). However, unless specifically pointed out, the results presented are calculated on the basis of orthology data.
Computation of conservation scores using phylogenetic information
We assign a score to a conserved gene pair by computing the probability a particular pattern of conservation is observed in analyzed genomes based on a stochastic model of evolution. Before introducing the detailed implementation, we first lay out the theoretical foundation of the method and point out our assumptions.
As an example, consider the rooted bifurcating phylogenetic tree shown in Figure 6. The leaf nodes Q, A, B, C and D represent extant genomes, and internal nodes X0, X1, X2 and X3 are inferred ancestor genomes. Let us assume a gene pair in Q is conserved in A, B and C but not D. We model evolution as a stochastic process represented as a probabilistic graphical model in the form of a tree [30]. We associate a binary random variable with each node in the tree, assigning it a value 1 if the specific gene pair is conserved in the genome and 0 if absent. The values of leaves in the tree are determined by our initial gene cluster identification procedure.
Figure 6 A simple genome phylogenetic tree.
We assume that the probability of a genome acquiring a gene cluster given its absence in the ancestor is negligible, i.e., P(child = 1|parent = 0) = 0. This is a simplifying assumption which considers the predominance of vertical inheritance and omits the negligible probability of independent formation of identical clusters. Under this assumption the most recent common ancestor of all the leaves that are assigned to 1 is also set to 1. Accordingly, in the Figure 6 tree model, X0 is set to 1. We compute the significance of the conservation based on the probability of observing the specific gene cluster given that the closest common ancestor has it, that is,
P(conservation) = P(Q = 1, A = 1, B = 1, C = 1, D = 0|X0 = 1)
In our initial model we do not take into account the genomes that do not possess the cluster, although it is not difficult to do with the appropriate assumption on the conditional probability of loss of a cluster in a descendant of an organism that has it. Thus a leaf node D that lacks the cluster is dropped from further calculation. Now we have
P(conservation) = P(Q = 1, A = 1, B = 1, C = 1|X0 = 1)
The tree in Figure 6 can also be interpreted as a Bayesian network in a tree form [31,32]. In particular, the vertical inheritance along any path in the tree is a generative probabilistic process, and the probability that a child inherits a gene pair is only dependent on its immediate evolutionary ancestor. More specifically, we associate a conditional probability table with each edge of the tree enumerating the probability of the presence or absence of a gene pair in a genome given the state of its immediate ancestor. According to the tree model, we have
P(Q=1,A=1,B=1,C=1|X0=1)=∑X2P(Q=1,A=1|X2)⋅P(X2)∑X3P(B=1,C=1|X3)⋅P(X3)
MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=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@78B1@
Assuming independence between the siblings, the above can be rewritten as
P(Q=1,A=1,B=1,C=1|X0=1)=∑X2P(Q=1|X2)⋅P(A=1|X2)⋅P(X2)∑X3P(B=1|X3)⋅P(C=1|X3)⋅P(X3)
MathType@MTEF@5@5@+=feaafeart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=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@88FE@
Note that this derivation is a simple generalization of the forward algorithm [30,32,33]. According to our vertical inheritance assumption, the probability for a child to have a gene pair is approximately zero if the immediate ancestor does not have the gene pair. After applying this assumption and evaluating the equation recursively, the above formula reduces to
P(Q = 1, A = 1, B = 1, C = 1|X0 = 1) = P(Q = 1|X2 = 1)·P(A = 1|X2 = 1)·P(B = 1|X3 = 1)·P(C = 1|X3 = 1)·P(X2 = 1|X0 = 1)·P(X3 = 1|X0 = 1)
More generally, for a gene pair found in a set of genomes and a given genome phylogeny tree T, the following simple relation holds
P(Q=1,A=1,B=1,...)=∏X,Y∈TP(X=1|Y=1),
MathType@MTEF@5@5@+=feaafeart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqWGqbaucqGGOaakcqWGrbqucqGH9aqpcqaIXaqmcqGGSaalcqWGbbqqcqGH9aqpcqaIXaqmcqGGSaalcqWGcbGqcqGH9aqpcqaIXaqmcqGGSaalcqGGUaGlcqGGUaGlcqGGUaGlcqGGPaqkcqGH9aqpdaqeqbqaaiabdcfaqjabcIcaOiabdIfayjabg2da9iabigdaXiabcYha8jabdMfazjabg2da9iabigdaXiabcMcaPaWcbaGaemiwaGLaeiilaWIaemywaKLaeyicI4SaemivaqfabeqdcqGHpis1aOGaeiilaWcaaa@52BD@
where Y is an immediate ancestor of X in T.
Taking the negative logarithm of both sides, we have
log(P(Q=1,A=1,B=1,...))=∑X,Y∈Tlog(P(X=1|Y=1))
MathType@MTEF@5@5@+=feaafeart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacyGGSbaBcqGGVbWBcqGGNbWzcqGGOaakcqWGqbaucqGGOaakcqWGrbqucqGH9aqpcqaIXaqmcqGGSaalcqWGbbqqcqGH9aqpcqaIXaqmcqGGSaalcqWGcbGqcqGH9aqpcqaIXaqmcqGGSaalcqGGUaGlcqGGUaGlcqGGUaGlcqGGPaqkcqGGPaqkcqGH9aqpdaaeqbqaaiGbcYgaSjabc+gaVjabcEgaNjabcIcaOiabdcfaqjabcIcaOiabdIfayjabg2da9iabigdaXiabcYha8jabdMfazjabg2da9iabigdaXiabcMcaPiabcMcaPaWcbaGaemiwaGLaeiilaWIaemywaKLaeyicI4SaemivaqfabeqdcqGHris5aaaa@5D84@
Thus the probability of conservation of a gene cluster in a given tree is simply the sum of log conditional probabilities of all the branches on paths leading to the genomes where the gene pair is present.
We further assume that log(P(X = 1|Y = 1) is proportional to the length of the branch that connects X and its parent Y, that is, log(P(X = 1|Y = 1)) ~ d(X,Y). Thus,
log(P(Q=1,A=1,B=1,...))∼∑X,Y∈Td(X,Y),
MathType@MTEF@5@5@+=feaafeart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacyGGSbaBcqGGVbWBcqGGNbWzcqGGOaakcqWGqbaucqGGOaakcqWGrbqucqGH9aqpcqaIXaqmcqGGSaalcqWGbbqqcqGH9aqpcqaIXaqmcqGGSaalcqWGcbGqcqGH9aqpcqaIXaqmcqGGSaalcqGGUaGlcqGGUaGlcqGGUaGlcqGGPaqkcqGGPaqkcqWI8iIodaaeqbqaaiabdsgaKjabcIcaOiabdIfayjabcYcaSiabdMfazjabcMcaPaWcbaGaemiwaGLaeiilaWIaemywaKLaeyicI4SaemivaqfabeqdcqGHris5aOGaeiilaWcaaa@545D@
where Y is the parent of X. The summation of all the tree branches in the phylogenetic tree is proportional to the logarithm of the overall probability.
In our implementation, the genome phylogenetic tree is built by using the genome distance metric based on the shared gene content suggested by Snel and coworkers [34]. We first calculate the pairwise distance between genomes by d = -ln(s), where s = (number of shared orthologs)/(average of total gene numbers in two genomes). In this implementation, Escherichia coli and Salmonella typhi, have a distance of 0.35, while Escherichia coli and Bacillus subtilis, which are much more distantly related, have a larger distance of 1.18. The genome phylogenetic tree is then constructed from the pairwise distance matrix using the neighbor joining algorithm [35].
For each gene pair, a genome phylogenetic tree is built on the genomes that have the pair, the conservation score of the gene pair is the summation of all the branch lengths in the tree. Notice that another nice property of the score is that it is independent of the query genome.
Detection of long conserved gene clusters
We now extend the methodology by using the gene pair conservation to detect longer conserved gene clusters. For the gth gene on the query chromosome, we estimate its upstream conservation score (Cu) and downstream conservation score (Cd) by:
Cu(g)=∑i=1k+1s(g−i,g)Cd(g)=∑i=1k+1s(g,g+i)
MathType@MTEF@5@5@+=feaafeart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakqaabeqaaiabdoeadnaaBaaaleaacqWG1bqDaeqaaOGaeiikaGIaem4zaCMaeiykaKIaeyypa0ZaaabCaeaacqWGZbWCcqGGOaakcqWGNbWzcqGHsislcqWGPbqAcqGGSaalcqWGNbWzcqGGPaqkaSqaaiabdMgaPjabg2da9iabigdaXaqaaiabdUgaRjabgUcaRiabigdaXaqdcqGHris5aaGcbaGaem4qam0aaSbaaSqaaiabdsgaKbqabaGccqGGOaakcqWGNbWzcqGGPaqkcqGH9aqpdaaeWbqaaiabdohaZjabcIcaOiabdEgaNjabcYcaSiabdEgaNjabgUcaRiabdMgaPjabcMcaPaWcbaGaemyAaKMaeyypa0JaeGymaedabaGaem4AaSMaey4kaSIaeGymaedaniabggHiLdaaaaa@5D93@
where each s(g-i, g) is the conservation score assigned to gene pair (g-i, g) calculated using the method above; k is the maximum number of intervening genes allowed in a conserved gene pair (k = 1 in this paper). As a result, each gene will be associated with two numerical scores, measuring the extent of conservation between itself and its upstream or downstream neighboring genes respectively.
The statistical significance of the conservation scores is inferred from a bootstrap simulation. For each genome, the null distribution is computed by calculating the conservation scores of the randomly shuffled genome. The P-value cutoff is set at about 1E-4 in this paper, which corresponds to a conservation score of about 5.0 for most genomes. Notice that P-values are related to genome size since genes in very small genomes may have higher chance of forming conserved gene clusters. For instance, conservation score of 5.0 corresponds to a larger P-value (3.5E-3) in a small genome Mycoplasma genitalium than in E. coli (2.4E-4).
For genes that are at the boundaries of the cluster, only one of the conservation scores will exceed the threshold, which provides a convenient way of detecting the boundaries. We detect the maximal conserved gene clusters by scanning the genomes sequentially. The gene with Cd over the threshold, but not for its upstream genes, marks the start of a new cluster. The gene whose downstream genes have Cu scores below the threshold mark the end of the cluster. All the genes between are considered as part of the cluster.
More sophisticated dynamic programming procedures, or single-linkage clustering algorithm to identify maximal conserved gene clusters are also possible.
The main program was written in C and used the LEDA library for the manipulation of trees. Scripts for generating genome BLAST data and for analyzing data were written in Perl. GeneChords was built with PostGreSQL. All scripts are available upon request from the authors.
Authors' contributions
YZ and SK devised the method. YZ carried out the implementation of the method and database. BPA carried out the functional enrichment analysis. All authors participated in interpreting the results and writing the manuscript. RJR and SK conceived the study. All authors read and approved the final manuscript.
Acknowledgements
We thank the genome sequencing teams who made their data publicly available. We thank Drs. Eugene Koonin, Peer Bork, Russell Greiner and David Lipman, Steven Salzberg and five anonymous reviewers for many insightful comments. Y.Z. thank Kevin Wiehe, Jennifer Beane, Elisabeth George, and David Sternlichit, Paul Strominger, Yulei Fu for their early involvement in building the database interface and the Java based user interface. This work is supported in part by NSF grants DBI-0239435 and ITR-048715 and NHGRI grant #1R33HG002850-01A1 (to S.K.). B.A. and R.J.R. are supported by New England Biolabs.
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Wolf YI Rogozin IB Kondrashov AS Koonin EV Genome alignment, evolution of prokaryotic genome organization, and prediction of gene function using genomic context Genome Res 2001 11 356 372 11230160 10.1101/gr.GR-1619R
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Zheng Y Szustakowski JD Fortnow L Roberts RJ Kasif S Computational identification of operons in microbial genomes Genome Res 2002 12 1221 1230 12176930 10.1101/gr.200601
Snel B Bork P Huynen MA The identification of functional modules from the genomic association of genes Proc Natl Acad Sci U S A 2002 99 5890 5895 11983890 10.1073/pnas.092632599
Snel B Lehmann G Bork P Huynen MA STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene Nucleic Acids Res 2000 28 3442 3444 10982861 10.1093/nar/28.18.3442
Lin LF Posfai J Roberts RJ Kong H Comparative genomics of the restriction-modification systems in Helicobacter pylori Proc Natl Acad Sci U S A 2001 98 2740 2745 11226310 10.1073/pnas.051612298
Galperin MY Nikolskaya AN Koonin EV Novel domains of the prokaryotic two-component signal transduction systems FEMS Microbiol Lett 2001 203 11 21 11557134 10.1016/S0378-1097(01)00326-3
Yanai I Mellor JC DeLisi C Identifying functional links between genes using conserved chromosomal proximity Trends Genet 2002 18 176 179 11932011 10.1016/S0168-9525(01)02621-X
Bowers PM Pellegrini M Thompson MJ Fierro J Yeates TO Eisenberg D Prolinks: a database of protein functional linkages derived from coevolution Genome Biol 2004 5 R35 15128449 10.1186/gb-2004-5-5-r35
Ermolaeva MD White O Salzberg SL Prediction of operons in microbial genomes Nucleic Acids Res 2001 29 1216 1221 11222772 10.1093/nar/29.5.1216
Kolesov G Mewes HW Frishman D SNAPping up functionally related genes based on context information: a colinearity-free approach J Mol Biol 2001 311 639 656 11518521 10.1006/jmbi.2001.4701
Rogozin IB Makarova KS Murvai J Czabarka E Wolf YI Tatusov RL Szekely LA Koonin EV Connected gene neighborhoods in prokaryotic genomes Nucleic Acids Res 2002 30 2212 2223 12000841 10.1093/nar/30.10.2212
Rogozin IB Makarova KS Wolf YI Koonin EV Computational approaches for the analysis of gene neighbourhoods in prokaryotic genomes Brief Bioinform 2004 5 131 149 15260894 10.1186/1471-2105-5-131
Strong M Mallick P Pellegrini M Thompson MJ Eisenberg D Inference of protein function and protein linkages in Mycobacterium tuberculosis based on prokaryotic genome organization: a combined computational approach Genome Biol 2003 4 R59 12952538 10.1186/gb-2003-4-9-r59
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
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Sonnhammer EL Koonin EV Orthology, paralogy and proposed classification for paralog subtypes Trends Genet 2002 18 619 620 12446146 10.1016/S0168-9525(02)02793-2
Fecker L Braun V Cloning and expression of the fhu genes involved in iron(III)-hydroxamate uptake by Escherichia coli J Bacteriol 1983 156 1301 1314 6315685
Salgado H Gama-Castro S Martinez-Antonio A Diaz-Peredo E Sanchez-Solano F Peralta-Gil M Garcia-Alonso D Jimenez-Jacinto V Santos-Zavaleta A Bonavides-Martinez C Collado-Vides J RegulonDB (version 4.0): transcriptional regulation, operon organization and growth conditions in Escherichia coli K-12 Nucleic Acids Res 2004 32 D303 6 14681419 10.1093/nar/gkh140
Hara H Yasuda S Horiuchi K Park JT A promoter for the first nine genes of the Escherichia coli mra cluster of cell division and cell envelope biosynthesis genes, including ftsI and ftsW J Bacteriol 1997 179 5802 5811 9294438
Carrion M Gomez MJ Merchante-Schubert R Dongarra S Ayala JA mraW, an essential gene at the dcw cluster of Escherichia coli codes for a cytoplasmic protein with methyltransferase activity Biochimie 1999 81 879 888 10572301 10.1016/S0300-9084(99)00208-4
Newman EB Budman LI Chan EC Greene RC Lin RT Woldringh CL D'Ari R Lack of S-adenosylmethionine results in a cell division defect in Escherichia coli J Bacteriol 1998 180 3614 3619 9658005
Lynch D O'Brien J Welch T Clarke P Cuiv PO Crosa JH O'Connell M Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti J Bacteriol 2001 183 2576 2585 11274118 10.1128/JB.183.8.2576-2585.2001
Hoffmann T Schutz A Brosius M Volker A Volker U Bremer E High-salinity-induced iron limitation in Bacillus subtilis J Bacteriol 2002 184 718 727 11790741
Naclerio G Baccigalupi L Zilhao R De Felice M Ricca E Bacillus subtilis spore coat assembly requires cotH gene expression J Bacteriol 1996 178 4375 4380 8755863
Tatusov RL Koonin EV Lipman DJ A genomic perspective on protein families Science 1997 278 631 637 9381173 10.1126/science.278.5338.631
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-6-2461622129810.1186/1471-2105-6-246SoftwareT.I.M.S: TaqMan Information Management System, tools to organize data flow in a genotyping laboratory Monnier Stéphanie [email protected] David G [email protected] Tim [email protected] Federico [email protected] International Agency for Research on Cancer, F-69372, Lyon, France2 Department of Epidemiology, Harvard School of Public Health, Boston, MA 02115, USA3 Menzies Research Institute, University of Tasmania, Hobart 7001, Australia4 German Cancer Research Center (DKFZ), D-69120, Heidelberg, Germany2005 12 10 2005 6 246 246 2 5 2005 12 10 2005 Copyright © 2005 Monnier et al; licensee BioMed Central Ltd.2005Monnier 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
Single Nucleotide Polymorphism (SNP) genotyping is a major activity in biomedical research. The Taqman technology is one of the most commonly used approaches. It produces large amounts of data that are difficult to process by hand. Laboratories not equipped with a Laboratory Information Management System (LIMS) need tools to organize the data flow.
Results
We propose a package of Visual Basic programs focused on sample management and on the parsing of input and output TaqMan files. The code is written in Visual Basic, embedded in the Microsoft Office package, and it allows anyone to have access to those tools, without any programming skills and with basic computer requirements.
Conclusion
We have created useful tools focused on management of TaqMan genotyping data, a critical issue in genotyping laboratories whithout a more sophisticated and expensive system, such as a LIMS.
==== Body
Background
The completion of the human genome sequence has brought a wealth of data on genetic variation, mostly in the form of single nucleotide polymorphisms (SNPs).
As a consequence, SNP genotyping has recently become a major activity for studies of disease susceptibility and pharmacogenetics. While techniques for ultra-high throughput (hundreds of millions of genotypes scored per year) are becoming available, the vast majority of genotyping laboratories around the world are equipped with technology suited for low- to high-throughput (up to a few million genotypes scored per year). The 5' nuclease assay, also known as TaqMan, is one of the main approaches currently used for genotyping [1,2].
Small genotyping laboratories are rarely equipped with sophisticated Laboratory Information Management Systems (LIMS) to follow the flow of information on samples and genotyping results. In a typical workflow (Figure 1 represents the workflow in our laboratory at the Genome Analysis Team, International Agency for Research on Cancer), many files and many different formats are managed. For example, DNA stocks are usually stored in 96-well plates but genotyped in 384-well plates. Sample preparation and handling are often done with different kind of robots (e.g. instruments for DNA extraction, liquid handling robots for set-up of PCR reactions, plate cranes linked with TaqMan instruments), each one with its own input and output file format.
Figure 1 Data flow in a SNP genotyping laboratory. Data flow is divided into management of samples, genotyping assays and genotyping results (dotted outlines). In bold, names of macros described in the text. All operations can be interfaced with a local database storing e.g. details about names of samples in plates, sequences of primers and probes used as genotyping assays, genotyping results.
We have developed a software suite for genotyping laboratories, aimed at improving data workflow, saving time and preventing errors linked to managing data by hand. Previously, other groups have developed tool focusing on management of data workflow [3,4]. Differently from previous solutions, we propose a set of independent tools, with a focus on ease of use. Users can download some of the macros or the whole suite, according to their needs. In addition, previously developed programs were tailored to linkage analysis projects, while TIMS is aimed at genotyping in the context of population-based association studies, and therefore it includes several new functions that allow to treat more easily large numbers of sample plates, and to generate input files for downstream software that is not relevant to linkage analysis projects.
Implementation
TIMS is a suite of tools written in Visual Basic. Given that genotyping data are usually represented in tabular format, we have chosen to use the version of Visual Basic embedded in Microsoft Excel. This offers the advantage of being available to most computer users, and allows interoperability between Windows and MacOS. The different tools have the format of Visual Basic macros, and they are used at different steps of the workflow (Figure 1):
1. Sample processing
We used a simple plate map format to document the localization of DNA samples (one file per plate). Often, both 96-well and 384-well plates are used in a laboratory, which creates the necessity of conversion between the two formats.
Macro "96to384" is used to create the map of a 384-well plate, starting from the map of four 96-well plates. Two ways of interleaving sample location from 96-well to 384-well plates are proposed ("Z" or "N", reading from left to right or from top to bottom). The user has to provide a list of files corresponding to 96-well plates to be read by the program and treated in batch. An optional function is offered to the user, to create automatically all the Taqman input files from the newly created 384-well plates. This is the function of the macro "PlateToTaqMan", which transforms the plate maps into files that can be directly used as TaqMan input files, in batch for 96-well plate and 384-well plates.
Reciprocally, knowing the composition of a 384-well plate in advance, a user may want to reconstruct the four 96-well DNA source plates. The macro "384to96" gives the structure of the 96-well plates to start with, in order to have the required 384-well plate.
2. Results management
The program "ResultsFileBuilder" builds an Excel file, ready to receive the genotyping results. The format of the result file is compatible with our previously published software [5]. The macro copies the list of samples (including information on plate number and location within plates) from a series of plate map files.
The output of the TaqMan instrument is a text file, with genotype information that has to be parsed to make use of it. Another macro, called "TaqMan2Results", is used to transfer the results from the Taqman output file into the results file described above.
Several functions have been built in this macro:
- The possibility to compare analysis of the same assay read by two users. In our laboratory, we routinely perform double blind reading of each TaqMan plate, for quality control. In this case, the macro imports and compares all genotype calls from a batch of plates, read by both users. Any discrepancies are highlighted and the operator is prompted to resolve them manually.
- Transfer of all results in the results file. We have defined a sample as the concatenation of three pieces of information: sample_id, plate name and position of the well in the plate. This has been introduced in order to allow for the existence of samples duplicated on purpose.
- Duplication of a subset of samples, on the same plate or on different plates, is often used for quality control purposes. Knowing which samples have been duplicated for quality control, the macro looks for the results obtained for the first genotype and for the control genotype (identified in the TaqMan input and output files as "Qc_sample_id"), in order to compare them. The macro generates a quality control report file, where it flags all differences between samples and their controls, as well as their position in plates, to make the quality control checking easier.
3. Analysis
A macro called "Count" has also been created to make some basic statistical analysis of the results. For each SNP existing in the results file, it counts the frequency of each allele and of each genotype, and checks Hardy-Weinberg equilibrium, using a chi-square test.
Finally, we have added a macro "Translate", which converts the results from the four letters representing nucleotides into numbers. In further analysis, some pieces of software require numbers instead of letters for genotyping data, for instance Haploview [6] and/or TagSNPs [7,8]. The macro creates the starting point file for those applications. The user has still to arrange the polymorphisms according to their physical order and keep only the SNPs of interest depending e.g. on their frequencies and the blocks structure in the input file (TagSNPs software).
Conclusion
We have created a set of user-friendly tools to make laboratory life easier and processing of genotyping data safer and quicker. The use of Microsoft Excel Visual Basic allows access to a wide range of users, working on PC or Mac computers.
This set of programs can be particularly helpful in laboratories where a full-fledged LIMS is not available. They complement (and can in the future be interfaced with) our database, which stores information on polymorphisms under study and their corresponding genotyping assays [9].
The macros we are presenting have been tailored to the instruments we have in our laboratory. However they can easily be modified in order to suit specific requirements, e.g. genotyping chemistries different from the 5' nuclease assay that can run on an Applied Biosystems Sequence Detection System, such as Amplifluor [10], or MGB Eclipse [11]. To this end, the source code of the macros can be freely consulted (see Additional file 1) and altered by using the Visual Basic Editor embedded in MS Excel.
Availability and requirements
Project name
TIMS, Macroshack.
Project home page
Operating system(s)
The software has been thoroughly tested in Excel 97 and 2000 under various versions of Windows (English versions), Excel 98 of MacOS 9 and Excel X of MacOS X (English versions).
Programming language
Visual Basic. The source code of all programs is accessible by use of the Visual Basic Editor included in MS Excel.
License
GNU General Public License.
Any restrictions to use by non-academics?
None.
Authors' contributions
FC has mainly conceived the tasks of the macros.
All the authors have contributed to writing the code.
SM and FC have prepared the first draft of the manuscript, and all co-authors contributed to the final draft.
Supplementary Material
Additional File 1
Source code of software described in article. Visual Basic source code of macros described in the article. Each macro is composed of several modules or parts of Visual Basic code. Variables are defined by the key word "Dim" and comments are introduced after " ' ". Each piece of code is functional if embedded between "Sub NameOfTheSub" And "End Sub".
Click here for file
Acknowledgements
Stéphanie Monnier was recipient of a Special Training Award of the International Agency of Research of Cancer. The authors thank the members of the Genome Analysis Team of IARC for extensive testing of the software tools.
==== Refs
Morin PA Saiz R Monjazeb A High-throughput single nucleotide polymorphism genotyping by fluorescent 5' exonuclease assay Biotechniques 1999 27 538 544 10489614
Ranade K Chang MS Ting CT Pei D Hsiao CF Olivier M Pesich R Hebert J Chen YD Dzau VJ Curb D Olshen R Risch N Cox DR Botstein D High-throughput genotyping with single nucleotide polymorphisms Genome Res 2001 11 1262 1268 11435409
Li J-L Deng H Dong-Bing L Fuhua X Chen J Gao G Recker R Deng H-W Toward High-Throughput Genotyping: Dynamic and Automatic Software for Manipulating Large-Scale Genotype Data Using Fluorescently Labeled Dinucleotide Markers Genome Res 2001 11 1304 1314 11435414 10.1101/gr.159701
Hampe J Wollstein A Lu T Frevel H-J Will M Manaster C Schreiber S An Integrated system for high throughput TaqMan™ based SNP genotyping Bioinformatics Application Note 2001 17 654 655
Cox DG Canzian F Genotype transposer: automated genotype manipulation for linkage disequilibrium analysis Bioinformatics 2001 17 738 739 11524375 10.1093/bioinformatics/17.8.738
Barrett JC Fry B Maller J Daly MJ Haploview: analysis and visualization of LD and haplotype maps Bioinformatics 2005 21 263 265 15297300 10.1093/bioinformatics/bth457
Stram DO Leigh Pearce C Bretsky P Freedman M Hirchhorn JN Altshuler D Kolonel LN Henderson BE Thomas DC Modeling and E-M estimation of haplotype-specific relative risks from genotype data for a case-control study of unrelated individuals Hum Hered 2003 55 179 190 14566096 10.1159/000073202
Stram DO Haiman CA Hirschhorn JN Altshuler D Kolonel LN Henderson BE Pike MC Choosing haplotype-tagging SNPs based on unphased genotype data using preliminary sample of unrelataed subjects with an example from the Multiethnic Cohort Study Hum Hered 2003 55 27 36 12890923 10.1159/000071807
Landi S Gemignani F Monnier S Canzian F A database of single nucleotide polymorphisms and a genotyping microarray for genetic epidemiology of lung cancer Exp Lung Res 2005 31 223 258 15824023 10.1080/01902140490495624
Petkov PM Ding Y Cassell MA Zhang W Wagner G Sargent EE Asquith S Crew V Johnson KA Robinson P Scott VE Wiles MV An efficient SNP system for mouse genome scanning and elucidating strain relationships Genome Res 2004 14 1806 1811 15342563 10.1101/gr.2825804
Afonina IA Reed MW Lusby E Shishkina IG Belousov YS Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence Biotechniques 2002 32 940 949 11962616
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BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-5-1311621910510.1186/1471-2407-5-131Study ProtocolRandomized phase II – study evaluating EGFR targeting therapy with Cetuximab in combination with radiotherapy and chemotherapy for patients with locally advanced pancreatic cancer – PARC: study protocol [ISRCTN56652283] Krempien R [email protected] MW [email protected] PE [email protected] S [email protected] H [email protected] C [email protected] B [email protected] P [email protected] S [email protected] KK [email protected] A [email protected] MW [email protected] J [email protected] Department of Radiation Oncology, University of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany2 Department of Radiation Oncology, German Cancer Research Center (dkfz), 69120 Heidelberg, Germany3 Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany4 Department of Medical Physics, German Cancer Research Center (dkfz), 69120 Heidelberg, Germany5 Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany2005 11 10 2005 5 131 131 29 7 2005 11 10 2005 Copyright © 2005 Krempien et al; licensee BioMed Central Ltd.2005Krempien 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
Pancreatic cancer is the fourth commonest cause of death from cancer in men and women. Advantages in surgical techniques, radiation therapy techniques, chemotherapeutic regimes, and different combined-modality approaches have yielded only a modest impact on the prognosis of patients with pancreatic cancer. Thus there is clearly a need for additional strategies. One approach involves using the identification of a number of molecular targets that may be responsible for the resistance of cancer cells to radiation or to other cytotoxic agents. As such, these molecular determinants may serve as targets for augmentation of the radiotherapy or chemotherapy response. Of these, the epidermal growth factor receptor (EGFR) has been a molecular target of considerable interest and investigation, and there has been a tremendous surge of interest in pursuing targeted therapy of cancers via inhibition of the EGFR.
Methods/design
The PARC study is designed as an open, controlled, prospective, randomized phase II trial. Patients in study arm A will be treated with chemoradiation using intensity modulated radiation therapy (IMRT) combined with gemcitabine and simultaneous cetuximab infusions. After chemoradiation the patients receive gemcitabine infusions weekly over 4 weeks. Patients in study arm B will be treated with chemoradiation using intensity modulated radiation therapy (IMRT) combined with gemcitabine and simultaneous cetuximab infusions. After chemoradiation the patients receive gemcitabine weekly over 4 weeks and cetuximab infusions over 12 weeks. A total of 66 patients with locally advanced adenocarcinoma of the pancreas will be enrolled. An interim analysis for patient safety reasons will be done one year after start of recruitment. Evaluation of the primary endpoint will be performed two years after the last patient's enrolment.
Discussion
The primary objective of this study is to evaluate the feasibility and the toxicity profile of trimodal therapy in pancreatic adenocarcinoma with chemoradiation therapy with gemcitabine and intensity modulated radiation therapy (IMRT) and EGFR-targeted therapy using cetuximab and to compare between two different methods of cetuximab treatment schedules (concomitant versus concomitant and sequential cetuximab treatment).
Secondary objectives are to determine the role and the mechanism of cetuximab in patient's chemoradiation regimen, the response rate, the potential of this combined modality treatment to concert locally advanced lesions to potentially resectable lesions, the time to progression interval and the quality of life.
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Background
Pancreatic cancer is the fourth commonest cause of death from cancer in men and women [1,2]. Surgical therapy currently offers the only potential monomodal cure for pancreatic adenocarcinoma [3]. However only a few patients present with tumors that are amenable to resection, end even after resection of localized cancers, long term survival is poor. At presentation, only 20% of patients with pancreatic adenocarcinoma have resectable cancers, 40% have locally advanced tumors, and 40% have metastatic disease [5].
However, long-term (5-year) survival rates – even for patients undergoing "complete" resection – are below 20% [4,5]. Loco-regional recurrence and/or metastatic disease develop in the majority of patients who undergo pancreatic resection. Relapse occurs within 9–15 months after initial presentation and patients have median life expectancies of only 12–15 months without adjuvant therapy [4]. The 5-year survival rate of patients with resected pancreatic adenocarinoma is approximately 10% [6]. The statistics for the 80 to 90 % of patients who present with locally advanced and metastatic pancreatic cancer are even more dismal. Rarely do such patients achieve a complete response to treatment; median survival is 5–10 months and 5-year survival is near zero [7].
Both distant and local/regional patterns of recurrence are common, and this suggests that most patients have occult metastatic disease or local/regional (or both) at the time of resection. Postoperative chemoradiationtherapy (CRT) has been shown to improve survival in patients with resected pancreatic adenocarcinoma [8-10], although there is debate over whether radiotherapy is a beneficial component [5,11]. The problems with the postoperative adjuvant approach include the fact that at least 25% of patients do not actually receive adjuvant therapy because of complications of surgery or patient refusal [10,12]. A primary advantage of preoperative therapy is therefore the assurance that CRT is received by all patients in a timely fashion. Other benefits are the delivers of radiation to well-oxygenated tissues and the avoidance of radiation to fixed loops of intestine within the operative field. Another rationale for neoadjuvant treatment is that occult metastatic disease is given the opportunity to manifest, thus allowing patients to avoid the morbidity of resection or laparotomy. Finally, the potential for preoperative CRT to convert locally advanced lesions to resectable lesions could greatly increase the number of patients with pancreatic cancer who might be offered a chance of cure. Several trials could show that dose escalation in radiation therapy using either EBRT [8] or IORT [13,14] resulted in improved local control in combination with potentially curative resection. The efficacy of external beam irradiation (EBRT) in pancreatic cancer is limited by the inability to deliver adequate doses of irradiation secondary to the dose tolerance limits of small bowel, spinal cord, stomach, kidney, and liver [15]. Further, the use of combined modality approaches in pancreatic cancer is associated with increased gastrointestinal toxicity [16]. Technical developments like intensity-modulated radiation therapy (IMRT) have the potential to significantly improve radiation therapy of pancreatic cancers by reducing normal tissue dose, and simultaneously allow escalation of dose to further enhance locoregional control [17].
To achieve long-term success in treating this disease it is therefore increasingly important to identify effective neoadjuvant/adjuvant multimodality therapies.
Concurrent chemoradiation is the standard of care for locally advanced non metastatic pancreatic cancers. Median survival rates vary between different trials depending on their selection criteria between 7 and 12 month, while 1 year overall survival is between 30% and 45% [18].
Systemic chemotherapy, a mainstay of pancreatic cancer treatment, essentially has been ineffective until recently when gemcitabine became available [19]. In a phase III trial comparing gemcitabine versus 5-flurouracil in advanced pancreatic cancer, patients who received gemcitabine showed a modest improvement in response rate, a marginal survival advantage, and most important, superior clinical response [20]. Therefore gemcitabine became the standard treatment of advanced pancreatic cancer. Despite these results, however median survival duration in patients with advanced pancreatic cancer continues to be less than 6 month.
Still, advantages in surgical techniques, radiation therapy techniques, chemotherapeutic regimes, and different combined-modality approaches have yielded only a modest impact on the prognosis of patients with pancreatic cancer. Thus there is clearly a need for additional strategies. One approach involves using the identification of a number of molecular targets that may be responsible for the resistance of cancer cells to radiation or to other cytotoxic agents. As such, these molecular determinants may serve as targets for augmentation of the radiotherapy or chemotherapy response. Of these, the epidermal growth factor receptor (EGFR) has been a molecular target of considerable interest and investigation, and there has been a tremendous surge of interest in pursuing targeted therapy of cancers via inhibition of the EGFR [21,22].
The overexpression of EGFR has been demonstrated in a number of human tumor types, including head-and-neck cancers, colon cancer, breast cancer, gliomas, lung cancer and pancreatic cancer [23,24]. The rationale for investigating of EGFR inhibitors as radiation sensitizers in cancer therapy is based on the following observations: (1) positive correlation between EGFR expression and cellular resistance to radiation in many cell types [23]; (2) the degree of radioresistance correlates positively with the magnitude of EGFR overexpression [25]; (3) cell survival and repopulation during a course of radiotherapy are influenced by activation of EGFR/transforming growth factor alpha that is induced after exposure to radiation [26]; and (4) inhibition of EGFR signaling-enhanced radiation sensitivity [27,28].
Cetuximab is a monoclonal antibody that specifically binds to the EGFR, thereby inhibiting downstream signal transduction pathways [29]. It has been shown in vivo and in vitro to enhance radiosensitivity, to promote radiation induced apoptosis, to decrease cell proliferation, to inhibit radiation-induced damage repair, and to inhibit tumor angiogenesis [23].
Phase I studies have shown that cetuximab has tolerable toxic effects. Acneiform rash is the most common toxic effect. Besides skin toxicity cetuximab has the potential to cause allergic reactions, including anaphylaxis. However, this has not been shown to be a significant clinical problem [23].
Phase I and II clinical studies on EGFR antibodies given as a single agent were performed in patients with advanced NSCLC, ovarian, head and neck, prostate, and colorectal cancer. Stable disease with tolerable side effects was seen in about 20% of the patients [23]. Several phase I-III trials testing the effect of different EGFR inhibitors combined with radiotherapy or radiochemotherapy are currently ongoing. The so far most important trial testing EGFR inhibition in combination with radiotherapy was a randomized phase III trial testing radiotherapy alone versus radiotherapy plus cetuximab [30]. 424 patients with loco-regionally advanced squamous cell carcinoma or head-and-neck cancers were randomized into curative radiotherapy plus/minus cetuximab. Cetuximab was applied once at a dose of 400 mg/m2 in the week prior to radiotherapy (week1) and weekly during the course of radiotherapy at a dose of 250 mg/m2 before irradiation. Locoregional tumor control rates after 1 and 2 years were 69% and 56% for patients treated simultaneously with cetuximab versus 59% and 48% for patients who received radiotherapy alone (p = 0.02). Overall survival rates at 2 and 3 years after treatment were 62% and 57% for cetuximab treated patients and 55% and 44% for patients with irradiation alone (p = 0.02). Median survival times were 54 months (95%C.I.36;58) and 28 months (21;38), respectively.
A recently completed phase II trial of cetuximab in combination with gemcitabine for patients with advanced pancreatic cancer showed promising results [31]. In that trial 41 patients received cetuximab with gemcitabine and were evaluated for efficacy and toxicity. The toxicity profile of this combination was consistent with that of gemcitabine, except for acneiforme rash induced by cetuximab. A partial response rate of 12.2% (5 patients) and stable disease rate of 63.4% was observed in this study. Medial time to progression and medial survival duration were 3.8 months and 7.1 months, respectively, while 1-year survival rates and progression-free survival rates were 31.7% and 12%, respectively. This 1-year survival is considerably better than that achieved using gemcitabine alone as documented in a previous phase III trial [20]. Recently a phase three study could demonstrate the benefit of the combination of an EGFR tyrosine kinase inhibitor in combination with chemotherapy in pancreatic cancer [32]. A total of 569 patients with advanced pancreatic cancer were randomized to receive standard dose gemcitabine, 1000 mg/m2 iv weekly in 7 out of 8 weeks, than weekly 3 out of four weeks plus either erlotininb 100 mg daily (n = 285) or placebo (n = 284). Combined erlotinib therapy with gemcintabine resulted in a 24% improvment in survival as compared to placebo (p = 0.025) with corresponding 1-year survival rate of 24% and 17% (erlotinib and placebo arm, respectively).
Regarding the benefits of EGFR-targeted therapy in the combined modality treatment using either irradiation or chemotherapy, it is increasingly becoming clear that EGFR-targeted therapy is an important novel strategy for the treatment of pancreatic cancers. Since chemoradiation is the standard of care for locally advanced non metastatic pancreatic cancer, there has been considerable interest in gaining increased experience with this therapy in combination with chemoradiation in an effort to evaluate the efficacy and the toxicity profile of this regimen.
Methods/design
Trial organization
PARC has been designed by the Trial Center of the Department of Radiation Oncology, University of Heidelberg. The trial is carried out by the Department of Radiation Oncology together with the German Cancer Research Center (DKFZ) and Department of Surgery. The trial is an investigator initiated trial. Trial medication (cetuximab) is supplied by Merck KGaA, Darmstadt, Germany.
Coordination
The trial is co-ordinated by the Department of Radiation Oncology in cooperation with the DKFZ and the Department of Surgery at the University of Heidelberg. The Dept. of Radiation Oncology is responsible for overall trial management, trial registration (International Standard Randomized Controlled Trial Number [ISRCTN 56652283], ), database management, quality assurance including monitoring, reporting and for the scientific program of all trial related meetings).
Investigators
Patients will be recruited by the Department of Radiation Oncology at the University of Heidelberg. Due to the multi-modal nature of the trial, all investigators are experienced oncologists from the fields of radiation oncology, hematology/oncology, and general surgery at the University of Heidelberg co-operating in this trial.
Adverse events committee
This committee consists of 3 independent physicians (medical oncologist, radiation oncologist and surgeon) and decides on the final diagnostic classification of critical clinical events. For all serious adverse events the documentation and relevant patient data are verified by the co-ordinating personnel before submitting the data to the Adverse Events Committee for diagnostic classification.
Analysis of safety related data is performed with respect to frequency of:
• Serious Adverse Events and Adverse Events stratified by organ-system
• Adverse Events stratified by severity
• Adverse Events stratified by causality.
Patient toxicities will be assessed using the NCI Common Toxicity Criteria (CTC). Toxicity will be evaluated pretreatment, weekly during chemoradiation /chemotherapy, prior to each course of infusional Cetuximab and at follow-up. Unacceptable toxicity is defined as unpredictable, or irreversible Grade 4 toxicity. Decisions regarding weekly chemoradiation treatment, chemotherapy dose-adjustment, and cetuximab dose-adjustment will be made using the guidelines below and based on hematological parameters (ANC and platelets) monitored weekly during chemoradiation before each dose of cetuximab and gemcitabine.
Medication supply
All chemotherapeutic agents are prepared and provided by the pharmacy of the University Hospital Heidelberg. Cetuximab is provided by Merck KGaA, Darmstadt, and is stored by the pharmacy of the University Hospital Heidelberg. Medication will be prepared for each patient specifically and delivered just prior to administration to the Department of Radiation Oncology.
On-site monitoring
During recruitment of patients monitoring on site is performed according to good clinical practice (GCP) guidelines. The data management will be performed by the Trial Center of the Department of Radiation Oncology, University of Heidelberg. The medical monitoring will be done by two independent oncologists not involved in conducting this trial.
Ethics, informed consent and safety
The final protocol was approved by the ethics committee of the University of Heidelberg, Medical School (L-283/2004, Paul-Ehrlich-Institute (PEI) registration number 1205/01). This study complies with the Helsinki Declaration in its recent German version, the Medical Association's professional code of conduct, the principles of Good Clinical Practice (GCP) guidelines and the Federal Data Protection Act. The trial will also be carried out in keeping with local legal and regulatory requirements. The medical secrecy and the Federal Data Protection Act will be followed.
Written informed consent is obtained from each patient in oral and written form before inclusion in the trial and the nature, scope, and possible consequences of the trial have been explained by a physician. The investigator will not undertake any measures specifically required only for the clinical trial until valid consent has been obtained.
Patient selection
PARC focuses on hospitalized patients over 18 years of age treated with pancreatic head resection for pancreatic adenocarcinoma during an 18-months period started in August 2004. Men and women over eighteen years of age with locally advanced pancreatic adenocarcinoma will be screened for participation in the study. A detailed overview of all eligibility criteria is given in Table 2.
Table 2 Eligibility Criteria
Inclusion criteria Exclusion criteria
• Age equal or greater than 18 years
• Primary inoperable locally advanced pancreatic adenocarcinoma
• No evidence of metastatic disease.
• Hb >10.0 g/%, WBC >3,000 cells/mm3, platelets >100,000 cells/mm3.
• Performance status: Karnofsky ≥70.
• No acute infections at the time of therapy initiation.
• Patient must be able to give informed consent
• Patient has given informed consent • Active infection
• Liver function impairment
• Pregnancy or breastfeeding.
• Metastatic disease
• Other severe systemic disease
• Second malignancy (except carcinoma in situ of the cervix uteri, basal cell carcinoma of the skin after adequate oncologic treatment)
• Any other experimental treatment 4 weeks before study inclusion
• Known positive HACA (human antichimeric antibody)
• Known allergy against extrinsical proteins
• Previous antibody therapy
• Allergy against iv contrast agent (for CT-scans)
• Previous chemo- and/or radiation treatment or EGFR-inhibitor therapy for pancreatic cancer
• Lack of compliance
• Inability to follow the instructions given by the investigator or the telephone interviewer (insufficient command of language, dementia, lack of time)
• Lack of informed consent
Study design
The PARC study is designed as an open, controlled, prospective, randomized feasibility phase II trial meant to evaluate the efficacy and toxicity of chemoradiation in combination with cetuximab for patients with locally advanced pancreatic cancer. Compared are chemoradiation with simultaneous cetuximab versus chemoradiation with simultaneous/sequential cetuximab. The treatment is offered to a heterogeneous group of people under clinical circumstances, covering a wide age range, for both sexes and with heterogeneous characteristics / co-morbidities.
One year after inclusion of the first patient an interim analysis will be performed. The study design will not be changed prior to agreement of the ethics committee.
Study objectives
The primary objective is to evaluate the feasibility and the toxicity profile of this regimen and to compare between two different methods of cetuximab treatment schedules (concomitant versus concomitant and sequential cetuximab treatment) in combination with chemoradiation therapy with gemcitabine and intensity modulated radiation therapy.
Secondary objectives are to determine the role and the mechanism of cetuximab in patient's chemoradiation regimen, the response rate, the potential of this combined modality treatment to concert locally advanced lesions to potentially resectable lesions, the time to progression interval and the quality of life.
Randomization and standardized treatment scheme
A block-randomization-list is generated via computer system (SAS Version 8.2, SAS Institute Inc., Cary, USA). The sealed randomization list is stored in the investigator file. Patients are randomized using sealed opaque envelopes in the independent study center at the Department of radiation oncology until informed consent is attained and diagnostic procedures rule out any contra-indication for participation in this trial.
After randomization and pre-treatment evaluation treatment must begin within 2 weeks.
All patients will receive a combination with radiotherapy, gemcitabine weekly and cetuximab weekly.
Study arm A
Cetuximab will be given as loading dose 400 mg/m2 over 120 minutes on day 1. On day 8,15,22,29, 36 (5 doses) cetuximab 250 mg/m2 over 60 minutes will be given simultaneously with radiation. Non-steroidal anti-inflammatory drugs and steroids will be given before cetuximab.
Gemcitabine 300 mg/m2 over 60 minutes will be given on day 12,19,26,33,40 (5 doses) 2 hours after radiation therapy. Sequential chemotherapy with gemcitabine weekly 1000 mg/m2 over 60 minutes will be continued after finishing radiotherapy on day 47, 54, 62. The timing of these courses will be adjusted in patients who have treatment interruptions.
External beam radiation is to be given concurrently with chemotherapy and cetuximab with a total dose of 54 Gy in 25 fractions over 5 weeks. Patients are to be treated using an integrated intensity modulated radiation therapy (IMRT) boost concept, which allows the use of different single doses for the gross target volume (GTV) and the clinical target volume (CTV) in one fraction. GTV includes only the gross tumor volume, whereas CTV includes the primary tumor and the regional lymphnodes including the hepatoduodenal ligament, origins of the celiac axis and superior mesenteric artery. The median total dose for the GTV is to be 54.0 Gy (single dose 2.16 Gy) and for the CTV 45.0 Gy (single dose 1.8 Gy). The dose constraints for stomach, duodenum, small intestine, colon are 45 Gy in the maximum, mean dose for kidneys should be below 10 Gy, only one third of the kidneys should receive more than 20 Gy. KonRad™ (Siemens Oncology Systems, Concorde, USA) will be used for inverse treatment planning. Treatment will be performed using step-and-shoot IMRT and stereotactic target point localization with 7 coplanar fields and 50 to 65 segments. Average treatment time will be 10 minutes. Patients are to be fixed during therapy by individual immobilization devices.
Study arm B
Cetuximab will be given as loading dose 400 mg/ m2 over 60 minutes on day 1. On day 8,15,22,29, 36 (5 doses) cetuximab 250 mg/m2 over 60 minutes will be given simultaneously with radiation. Non-steroidal anti-inflammatory drugs and steroids will be given before cetuximab.
Sequential cetuximab 250 mg/ m2 over 60 minutes will be given weekly beginning on day 46, over 3 month (12 doses)
Gemcitabine will be given as in study arm A.
External beam radiation will be given as in study arm A.
Restaging using computed tomography will be performed 5 weeks after completion of radiotherapy and at the end of sequential cetuximab administration.
The treatment protocol is outlined in figure 1.
Figure 1 PARC treatment scheme.
Investigation schedule and follow-up
All patients (study arm A or B) must have appropriate lab and radiographic studies (CXR; bone scintigraphy, abdominal ultrasound, CT abdomen; CBC; platelet count; BUN; creatinine; bilirubin, CA 19-9, and CEA) conducted prior to study enrolment to meet eligibility criteria.
During days 1–70 [study arm A] or days 1–130 [study arm B] patients will be assessed with laboratory evaluation: complete blood count and blood chemistries weekly. Laboratory parameters in both study arms will be evaluated before each dose of gemcitabine, in study arm B during cetuximab weekly before cetuximab.
Vital signs (blood pressure and pulse rate) and temperature are controlled daily during treatment. Patients are evaluated prior to receiving chemoradiation or chemotherapy. Patients enrolled in study arm A and B are evaluated weekly by the radiation oncology team during treatment. The team will check patients at each visit for symptoms due to therapy; a physical examination and complete safety labs should be performed. The quality of life questionnaire will be filled out during weeks 1, 9 and 17.
During post-chemoradiation, infusional cetuximab weekly (Study Arm B) patients will be evaluated by a physician prior to treatment and every 2 to 3 weeks with clinical assessment and laboratory parameters including a CBC, electrolytes, BUN, and creatinine.
In the post-treatment period patients will be seen every 3 months by the radiation oncology department for the first 2 years, every 4 months for the third year, and every 6 months during the 4th and 5th post-treatment years.
The aggregate clinical, laboratory, and imaging evaluations required per protocol as well as the timing of the optional quality of life questionnaire are outlined in table 1.
Table 1 Investigation Pretreatment Chemoradiation / Chemotherapy weekly Each Course of Infusional Gemcitabine/ Cetuximab follow-up
Clinical examination X X X X
Laboratory tests Xa X X X
Tumor markers Xb X X X
CT Abdomen X Xc
CXR, PA, LAT X X X
Renal function Xd
Pregnancy Testf X
SAE X X X
QOL Survey Xe X X X
a. Includes Electrolytes, BUN, Creatinine, SGOT, Alk Phos, Total Bilirubin, Albumin, Glucose, Calcium, auto-antibodies
b. Includes CEA and CA 19-9
c. A CT scan will be performed 4 weeks after chemoradiation and than every 3 months.
d. Includes CT with i.v. contrast
e. QLQ-C30, QLQ-Pan 26
f. If indicated
The follow-up will be continued for two years. Follow-up data of overall survival will be evaluated annually.
Assessment of quality of life
Measurement of quality of life is one of the secondary objectives of the trial. Overall survival, return to previous employment as well as persistence of symptoms, the ability to perform appropriate activities and to care for oneself are criteria applied in the three questionnaires used in this study.
EORTC QLQ-C30 is a general measure of qualitiy of life in cancer patients. It incorporates nine multi-item scales: five functional scales (physical, role, cognitive, emotional, and social); three symptom scales (fatigue, pain, and nausea and vomiting); and a global health and quality-of-life scale [33]. Specific symptoms (dyspnoea, insomnia, anorexia, constipation, diarrhoea, and financial impact) are measured as six single items. This instrument has been used extensively with a variety of cancer patients and was able to discriminate between individuals with metastatic and non-metastatic disease, as well as between patients at different stages of illness. The scale has good internal consistency (alpha > 0.70), and good test re-test reliability (0.80 to 0.90) [34].
To assess disease-specific symptoms for patients with pancreatic cancer the pancreatic specific module (QLQ-PAN26) [35] that has been designed to use long with the general measure is used in this study.
Evaluation of the role of cetuximab
An investigation of the effects and mechanism of cetuximab will be performed. Cetuximab has been shown in vivo and in vitro to enhance radiosensitivity, to promote radiation induced apoptosis, to decrease cell proliferation, to inhibit radiation-induced damage repair, and to inhibit tumor angiogenesis [23,24,36]. In view of the encouraging results achieved by using cetuximab in combination with other antineoplastic therapies, studies are now needed to define the molecular and immunologic mechanism(s) of this modality [30,31]. If the mechanism of action of cetuximab is more clearly understood it can be applied more selectively and its therapeutic index will be enhanced. Treatment related primary and acquired chemo- radioresistance presents a significant hindrance for all current therapy regimes in pancreatic cancer patients [23,24]. Multiple factors such as genetic instability of tumors and high inter- and intratumoral heterogeneity contributes to the hardly predictable therapy resistance [22]. To understand patterns of therapy response genome expression profiling and detection of genetic polymorphisms enables to identify key mechanisms in systems biology. Microarray technology will be used to identify predictors for therapy response or failure. The objectives are to correlate and potentially predict therapy response to cetuximab in combination with gemcitabine and radiotherapy using tumor genomic fingerprints. Tissue will be obtained either prior to neoadjuvant therapy by biopsy or during surgery. In order to perform the genomic approach patients biopsies are correlated and RNA and DNA isolation will be performed. After expression profiling the most promising differentially expressed genes are validated using real-time quantitative polymerase chain reaction. To predict the efficacy of neoadjuvant trimodal therapy additionally patients blood is collected before, during, and after neoadjuvant therapy to detect and correlate well known tumor and angiogenesis marker (VEGF, bFGF, IL8 etc.) using antibody chips.
Statistical considerations and sample size estimation
The primary endpoint in this study is the feasibility and safety of the trimodal combination therapy with gemcitabine based chemoradiation and cetuximab. Secondary endpoints are overall survival period, measured from the date of therapy start. The one-year survival rates after chemoradiation with gemcitabine is 42 % [20,37]. The sample size calculation is based on the assumption of an increase of one-year survival rates up to 67% due to the triple therapy [30-32].
Assuming an accrual period of 24 months and a follow-up of 42 months, testing for a difference in hazard (hazard ratio ≠ 1) on level α = 0.05 and with a power of 80% a study sample size of 58 patients (29 patients per study arm) is needed. Taking into consideration the estimate of approximately 15% of patients which will not complete the treatment, a total number of 66 patients should be randomized.
The overall survival between both therapy arms will be compared using the Chi-square test. The overall survival will be summarized by Kaplan-Meier estimate and differences in therapy protocols will be analyzed by univariate Cox-regression.
Various secondary endpoints will be evaluated in this study as well, time to progression, measured from date of therapy start, will be summarized by Kaplan-Meier estimate. Further tumor response after 3, and 6 months and secondary operability will be calculated
One year after inclusion of the first patient an interim analysis will be carried out. The main evaluation will be performed two years after the last patient's enrolment.
There will be explicit stopping rules in place to terminate the trial early in the unlikely event that an unacceptably high rate of treatment related deaths (TRD) is observed. TRD will be monitored using the design of Thall and Simon [38]. A non-informative Beta prior distribution (i.e., B (0.015, 0.085) for TRD rate is assumed. The trial will be stopped if at any point during the trial there is a greater than 90% probability that the true TRD rate is greater than 0.05. Each patient will subsequently be evaluated and, an independent safety board will be consulted in making decision.
In view of the poor prognosis of the patient group, there will be no explicit stopping rules based on the overall number of toxicities, since even high rates of reversible toxicities seem acceptable if there is a large survival gain. Patients can withdraw from study participation at any time. Patients are taken off the study if unacceptable toxicity appears. Unacceptable toxicity is defined as unexpected serious side effects or irreversible Grade 4 toxicity. Patients who withdraw from the study may be treated with 5-FU and folinic acid or with gemcitabine. The decision will be based on the individual reasons for withdrawing from the study.
Discussion
About 20–40% of patients present with a locally advanced pancreatic cancer which is not curable by resection [3,4]. The aim of primary chemoradiation in this situation is to achieve a local response with the aim prolonged survival and of preventing local tumor complications. Further downstaging or downsizing may enable secondary respectability. Chemoradiation in locally advanced pancreatic cancer results in significantly prolonged survival with 1-year survival about 40% compared to chemotherapy alone with 1-year survival about 20% [37]. Long-term survival is poor, rarely do such patients achieve a complete response to treatment; median survival is 5–10 months and 5-year survival is near zero [7].
Advantages in surgical techniques, radiation therapy techniques, chemotherapeutic regimes, and different combined-modality approaches have yielded only a modest impact on the prognosis of patients with pancreatic cancer [4]. Thus there is clearly a need for additional strategies. One approach involves using the identification of a number of molecular targets that may be responsible for the resistance of cancer cells to radiation or to other cytotoxic agents. As such, these molecular determinants may serve as targets for augmentation of the radiotherapy or chemotherapy response. Of these, the epidermal growth factor receptor (EGFR) has been a molecular target of considerable interest and investigation, and there has been a tremendous surge of interest in pursuing targeted therapy of cancers via inhibition of the EGFR [23,36].
Regarding the benefits of EGFR-targeted therapy in the combined modality treatment using either irradiation or chemotherapy, it is increasingly becoming clear that EGFR-targeted therapy is an important novel strategy for the treatment of pancreatic cancers [30-32]. Since chemoradiation is the standard of care for locally advanced non metastatic pancreatic cancer, there has been considerable interest in gaining increased experience with this therapy in combination with chemoradiation in an effort to evaluate the efficacy and the toxicity profile of this regimen.
Being a center focusing on pancreatic diseases and especially malignancies we therefore planned and conduct such a trial.
The PARC study is an open, randomized controlled trial investigating the survival of patients with primary non-metastatic locally advanced pancreatic cancer after trimodal therapy with gemcitabine-based chemoradiation and EGFR-targeting therapy with the monoclonal antibody cetuximab. The role and the mechanism of cetuximab in patient's chemoradiation regimen are evaluated. The toxicity, the disease-free interval and the quality of life are assessed. Different factors are tested for a potential role as predictive marker.
The results of the PARC trial will definitely advance clinical and scientific knowledge on the treatment of locally advanced pancreatic adenocarcinoma.
Abbreviations
CRO Clinical Research Organisation
CRF Case Report Form
CTC Common Toxicity Criteria
CTV Clinical Target Volume
dkfz German Cancer Research Center
EBRT External Beam Radiation Therapy
EGFR Epidermal Growth Factor Receptor
GCP Good Clinical Practice
GTV Gross Target Volume
IMRT Intensity Modulated Radiation Therapy
IORT Intra-Operative Radiation Therapy
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
The first two authors contributed equally. RK, MWM, SH, MWB and JD planned, coordinated and conducted the study. Medical care is covered by CT, BD, PB, KKH, and AA. RK, MWM, KKH and HF recruited patients and provided randomization. SN, CT, BD, PB, and AA took part in conducting the study. Scientific program is planned by RK and MWM and carried out by RK, MWM, PEH, AA and HF. SN provided technical assistance and quality control in radiation treatment planning and delivery. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Trial medication (Cetuximab) is supplied by Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany
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Aaronson NK Ahmedzai S Bergman B Bullinger M Cull A Duez NJ Filiberti A Flechtner H Fleishman SB de Haes JC The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology J Natl Cancer Inst 1993 85 365 76 8433390
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Fitzsimmons D Johnson CD George S Payne S Sandberg AA Bassi C Beger HG Birk D Buchler MW Dervenis C Fernandez Cruz L Friess H Grahm AL Jeekel J Laugier R Meyer D Singer MW Tihanyi T Development of a disease specific quality of life (QoL) questionnaire module to supplement the EORTC core cancer QoL questionnaire, the QLQ-C30 in patients with pancreatic cancer EORTC Study Group on Quality of Life Eur J Cancer 1999 35 939 41 10533475 10.1016/S0959-8049(99)00047-7
Xiong HQ Molecular targeting therapy for pancreatic cancer Cancer Chemother Pharmacol 2004 54 S69 77 15316751
Treatment of locally unresectable carcinoma of the pancreas: comparison of combined-modality therapy (chemotherapy plus radiotherapy) to chemotherapy alone. Gastrointestinal Tumor Study Group J Natl Cancer Inst 80 751 5 1988 Jul 20 2898536
Thall PF Simon R Practical Bayesian guidelines for phase IIB clinical trials Biometrics 1994 50 337 49 7980801
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BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-6-351622566810.1186/1471-2121-6-35Research ArticleThe novel protein KBP regulates mitochondria localization by interaction with a kinesin-like protein Wozniak Marcin J [email protected] Martina [email protected] Cornelia [email protected] Hans-Ulrich [email protected] Reiner [email protected] Medical Clinic IV, Otfried-Müller Str.10, Tübingen, Germany2 University of Manchester, Manchester, UK3 Boehringer Ingelheim Pharma KG, Biberach an der Riss, Germany2005 14 10 2005 6 35 35 18 4 2005 14 10 2005 Copyright © 2005 Wozniak et al; licensee BioMed Central Ltd.2005Wozniak 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
Members of the Kinesin-3 family of kinesin-like proteins mediate transport of axonal vesicles (KIF1A, KIF1Bβ), distribution of mitochondria (KIF1Bα) and anterograde Golgi to ER vesicle transport (KIF1C). Until now, little is known about the regulation of kinesin-like proteins. Several proteins interact with members of this protein family. Here we report on a novel, KIF1 binding protein (KBP) that was identified in yeast two-hybrid screens.
Results
KBP was identified by using the yeast-two-hybrid system with an amino-terminal fragment of KIF1C as a bait that is strongly homologous to KIF1B. Here we investigated the interaction of KBP and KIF1B. The full length proteins coimmunoprecipitated after overexpression and in untransfected 293 cells. Immunofluorescence experiments revealed that KBP was mainly localized to mitochondria, as has been described for KIF1Bα. Overexpression of a deletion mutant or reduction of the KBP protein level using an anti-sense construct led to an aggregation of mitochondria. Such an effect is probably due to the lower activity of KIF1Bα in the absence of KBP, as was revealed in motility assays.
Conclusion
KBP is a new binding partner for KIF1Bα that is a regulator of its transport function and thus represents a new type of kinesin interacting protein.
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Background
Intracellular transport in cells is mediated by three different types of motor proteins that travel along filamentous tracks. Actin filaments are used by the myosin family of proteins, while transport along microtubules (MT) is mediated by either kinesin-like proteins (KLPs) or dyneins. The functional difference between these two kinds of molecular motors lies in the polarity of movement. Dyneins transport their cargoes toward the minus end of MT, while most KLPs transport cargo towards the minus and plus end of MT, depending on the position of the motor domain at the amino- or carboxyl-terminus of the protein (for reviews see [1,2]). The common feature of all KLPs is a high degree of homology in their motor domain, a 340 amino acids region that contains MT- and ATP-binding sites and classifies them into one of 14 families of the kinesin superfamily [3]. Human and mouse genomes encode 45 KLPs [4] whose functions classically are transport of cargo, like protein rafts, lysosomes, chromosomes or various membrane vesicles. However, KLPs also can zipper, cross-link and influence the stability of MT for building and maintaining the mitotic and meiotic spindle apparatus (for reviews see [5-7].
Our previous research identified the protein KIF1C that localizes to the Golgi apparatus. Using a dominant-negative mutant we have shown that KIF1C is involved in transport of vesicles from the Golgi to the ER [8]. Together with KIF1A and KIF1B, KIF1C belongs to the Kinesin-3 family. The three proteins show a high degree of sequence homology outside their amino-terminal motor domain and share a so called U104 domain. The biological function of this domain, also known as forkhead homology-associated domain [9] has not yet been described in KLPs but it may be involved in protein-protein interactions regulated by phosphorylation. Moreover, KIF1C has been implicated in the susceptibility of mouse macrophages to anthrax lethal toxin [10].
The other member of the Kinesin-3 family, KIF1B, is expressed as two main splicing variants that share 660 amino-terminal amino acids and have different roles in intracellular transport. The α-isoform associates with mitochondria [11], while the β-isoforms [12,13]. are involved in transport of synaptic vesicles and lysosomes in non-neuronal cells [14]. A missense mutation in the ATP binding domain of the motor may be causal for the development of Charcot-Marie-Tooth disease type 2A [15].
For the Kinesin-3 KLPs several binding proteins were identified. KIF1A binds to Liprin-α [16], and KIF1Bα interacts with the PDZ domain of the glucose transporter 1 binding protein, indicating a possible additional target of this KLP [17]. In addition, PDZ domains of PSD-90, PSD-97 and S-SCAM are responsible for interaction with KIF1Bα as well [18]. We reported previously on the binding of protein tyrosine phosphatase D1 [8] and 14-3-3 proteins [19] to KIF1C. Here we describe the identification of a novel KIF1Bα interacting protein, KBP (KIF1 binding protein). We show that KBP colocalizes with mitochondria and that it interacts with KIF1Bα. Moreover, we present evidence that this new protein plays a role for the regulation of mitochondrial distribution by regulating the KIF1Bα activity.
Results
Identification and cloning of KBP
We have previously identified two proteins that bind to KIF1C, PTPD1 and members of the 14-3-3 protein family [8,19]. To search for proteins that bind to KIF1C between the motor domain and the PTPD1 binding region, we employed the yeast two-hybrid system. Two screens were made, first with amino acids 435 – 622 as a bait, to identify possible U104 binding proteins, and second with amino acids 261 – 800 as a bait. Only the second screen with the larger bait resulted in possible interacting proteins. Out of 5 × 106 screened transformants, 10 independent isolates of the same cDNA were found to interact.
The open reading frame of the identified cDNA encodes a protein of 621 amino acids (Fig. 1A), with a calculated molecular mass of 71.8 kDa. A search in the Conserved Domain Database showed weak homology to tetratricopeptide repeat domains (amino acids 95–241) and revealed a very acidic region (amino acids 54–64). The new protein was called KBP (KIF1 binding protein). A protein BLAST search revealed that the KBP protein sequence had been identified before and exists in the HUGE Protein Database as KIAA1279.
Figure 1 Expression analysis of KBP. A, amino acid sequence of KBP, probable TPR repeats are boxed. B, expression of KBP mRNA in various human tissues was analysed by Northern blot hybridization using the complete cDNA as a probe. The size marker is indicated in kilobase. C, expression of KBP in human, mouse and hamster cell lines. Triton cell lysates were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted using the KBP antiserum. Size markers are indicated in kDa.
Expression of KBP
To estimate the transcript size and prove that the identified clone encodes the complete open reading frame, as well as to check the tissue distribution, Northern blot analysis using the whole cDNA as a probe was performed. A band of 2.6 kb was detected in all examined tissues, with the highest expression found in heart and skeletal muscle (Fig. 1B), which is similar to the expression pattern of KIF1B, as shown by Nangaku et al. [11] and Chen et al. [20]. Next, we constructed a glutathione-S-transferase-KBP (GST-KBP) fusion protein and raised a rabbit polyclonal antiserum against it. When probing lysates from various cell lines with this antibody, a protein of approximately 72 kDa was detected (Fig. 1C). This is in agreement with the predicted molecular mass, and the size of the protein in human cells corresponded with the size of transiently overexpressed protein after transfection, indicating that we indeed cloned the complete open reading frame.
Localization of KBP in NIH3T3 cells
To elucidate the function of KBP, we performed immunofluorescence experiments utilising affinity-purified antibodies. Immunostaining of NIH3T3 cells revealed that KBP localized mainly on vesicular structures throughout the cell, which were reminiscent of mitochondria (Fig. 2A). In the same cells, mitochondria were detected with MitoTracker (Fig. 2B), and the merged pictures show that KBP mainly localized to mitochondria (Fig. 2C). These results suggested that the main cellular binding partner for KBP is found at the mitochondria.
Figure 2 Intracellular localization of KBP. The localization of KBP was determined by immunofluorescence analysis in untransfected NIH3T3 (A) cells, compared to MitoTracker staining (B) and the pictures subsequently merged (C). Bars, 10 μm.
The KLP KIF1Bα is highly homologous to KIF1C, especially within the region used for the yeast two-hybrid screen, and colocalizes with mitochondria [11]. To study the possible interaction between KBP and KIF1Bα we cloned the cDNA of human KIF1Bα and raised antibodies against the carboxyl-terminal part of the protein, a region not present in the KIF1Bβ isoforms. Our antiserum did not cross-react with the homologous KIF1C and recognized an endogenous protein that was of the same size as the cloned one (Fig. 3A). We then tested the antiserum in an immunofluorescence experiment using NIH3T3 cells and, as expected, detected mitochondria-like structures (Fig. 3B). Staining along MTs was also visible (Fig. 3B – top). In a parallel staining, the cells were probed with MitoTracker, and merging of the pictures indicated an identical pattern. This experiment confirmed that we had cloned a human ortholog of the mitochondria binding α-isoform of the KIF1B protein. In addition, the experiment suggested that KBP and KIF1Bα colocalize and that colocalization likely is mediated through a direct interaction of the two proteins.
Figure 3 Expression of human KIF1Bα and interaction with KBP. A, lysates of 293 cells overexpressing KIF1Bα, KIF1Bα-myc and KIF1C or of untransfected 293 cells (10 fold amount of protein) were separated by SDS-PAGE, transferred to nitrocellulose and blotted with antiserum raised against the carboxyl-terminal part of KIF1Bα. B, affinity purified antibodies against KIF1Bα (top) and MitoTracker (middle) were used to stain NIH3T3 cells. The merged picture is shown at the bottom. Bar, 20 μm. C, KIF1Bα was immunoprecipitated from lysates of 293 cells overexpressing KBP-VSV either alone or together with KIF1Bα. Analysis was done as described and blotting performed with VSV monoclonal antibody. To confirm expression of the proteins, the filter was reprobed with KIF1Bα antibody, while KBP-VSV expression was detected in a separate aliquot of the cell lysates. D, untransfected BHK cells were lysed, proteins immunoprecipitated with the indicated antibodies against GST (control), KBP and KIF1Bα and analysed as before by blotting with anti-KIF1B antibody. Molecular mass markers are shown in kDa.
This interaction of KBP and KIF1Bα was confirmed by co-immunoprecipitation. Transfected 293 cells expressed either a VSV-epitope tagged form of KBP or KBP-VSV plus KIF1Bα. Cells were lysed, the proteins immunoprecipitated using anti-KIF1B serum, separated by SDS-PAGE, transferred to nitrocellulose and blotted with VSV antibody. As shown in Fig. 3C, KBP co-immunoprecipitated with KIF1B when both proteins were overexpressed. Finally, we tested the interaction of KBP and KIF1B in untransfected BHK cells (Fig. 3D). After immunoprecipitation of KBP, KIF1B was detected as a co-immunoprecipitated protein on a Western blot. In conclusion, KBP interacts and colocalizes with KIF1Bα.
Deregulation of KBP leads to aggregation of mitochondria
KIF1Bα was identified as a motor protein responsible for the intracellular transport of mitochondria [11]. Since KIF1Bα and KBP associated with each other and both proteins localized to mitochondria the question arose whether KBP could be a link between the motor protein and this organelle or whether it could play a role in mitochondria distribution. To investigate this question we used two approaches: overexpression of a deletion mutant of KBP that can not interact any more with KIF1Bα, and reduction of the KBP expression level using an anti-sense construct. For the first approach, we employed a deletion mutant of KBP lacking amino acids 250–281. This region was chosen because experiments using GST-fusion proteins indicated that the first 305 amino acids are sufficient for interaction, whereas a mutant lacking the first 214 amino acids was still able to bind KIF1B (data not shown). KBPΔ250–281 was not able to bind to a GST-KIF1Bα(270–641) fusion protein in a pull-down assay, as shown in Fig. 4A. When the KBP mutant was overexpressed in NIH3T3 cells followed by KBP antibody/MitoTracker staining (Fig. 4B) a mitochondrial aggregation was observed in the transfected cells. Overexpression of intact KBP did not affect mitochondrial localization or the organization of the microtubule network (data not shown).
Figure 4 Expression of KBPΔ250–281 leads to aggregation of mitochondria. A, 1 μg of GST-KIF1Bα fusion protein was incubated with equal amounts of lysates from either KBP or KBPΔ250–281 expressing 293 cells. As a control, GST-Grb2 fusion protein was used. The proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with GST-KBP antiserum (left panel). To confirm expression of KBP and the KBP deletion mutant, an equal amount of each lysate was loaded on the gel and analysed with KBP antiserum (right panel). Molecular mass markers are shown in kDa, and an arrow indicates the position of KBP. B, NIH3T3 cells transiently overexpressing the KBPΔ250–281 mutant protein were costained with KBP antibody (upper picture) and MitoTracker (lower picture). Trf, transfection; bar, 10 μm.
To support these data and to confirm a function of KBP in mitochondrial distribution in an additional and independent approach, we cloned the KBP cDNA in a reversed orientation into the cytomegalovirus promoter based expression vector. Transfection of this construct into 293 cells reduced the expression of endogenous KBP to an overall 30%, when compared to untransfected cells and as detected by blotting analysis (Fig. 5A). Based on FACS-analysis of transfected cells, we assume a transfection efficiency of some 80% with our protocol. This would predict a down-regulation of the KBP protein level to less than 20% in an individual transfected cell. Transfected cells were marked for immunofluorescence inspection by including a plasmid encoding HA-epitope tagged 14-3-3γ in the transfections at a 1:10 ratio. 14-3-3 proteins are very abundant in cells and, as concluded from experiments with 293 cells [19], their expression level is only increased two- to threefold after transfection using optimal amounts of DNA. Therefore, we did not expect an impact of exogenous 14-3-3γ-HA on the mitochondrial distribution process. After transfection, cells were double-stained with monoclonal anti-HA antibodies to identify the transfected cells and with MitoTracker to investigate effects of a reduced KBP expression on mitochondria. In Fig. 5B,C, two cells are in the frame of the picture, and the upper cell has been transfected and therefore stained with the HA-antibody. In C, staining of the mitochondria revealed the regular distribution in the untransfected cell, but an aggregated status in the transfected cell that would contain a reduced amount of KBP. To confirm that this effect was not the result of 14-3-3γ-HA overexpression, NIH3T3 fibroblasts were transfected with the 14-3-3γ-HA expression plasmid alone and anti-HA antibody/MitoTracker staining was performed. Fig. 5D,E show that overexpression of 14-3-3γ-HA did not cause the mitochondria to aggregate. In addition, when similar experiments were conducted using overexpression of GFP similar results were obtained (Fig. 5F–I). To exclude the possibility that mitochondrial aggregation is a result of MT depolymerization or changes in the cell shape, the NIH3T3 cells were transfected with the anti-sense KBP construct and costained with β-tubulin antibody and MitoTracker. In this experiment, mitochondrial clumping was used as a marker of transfection. As seen in pictures J to L of Fig. 5 the clustering of mitochondria was not accompanied by either MT depolymerization or cell shrinking. To conclude, both experiments clearly show the importance of KBP for the distribution of mitochondria in cells.
Figure 5 KBP is essential for a regular distribution of mitochondria in NIH3T3 cells. A, a scheme of the anti-KBP plasmid is depicted. The reduction of endogenous KBP in 293 cells after transient transfection with the anti-KBP plasmid is shown. (B, C) NIH3T3 cells were transiently transfected with anti-sense KBP and 14-3-3γ-HA constructs in a 10:1 ratio, respectively, and costained with HA monoclonal antibody (B), and MitoTracker (C). As a control, NIH3T3 cells were transiently transfected with 14-3-3γ-HA only (D, E). Similar as in B-E, cotransfection with GFP was performed (F-I). Alternatively, anti-sense KBP transfected cells were stained with antibodies against β-tubulin (J) and with MitoTracker (K). Arrows in J, K indicate transfected cells. The merged picture of J and K is shown in (L). Trf, transfection; bars, 20 μm.
Mitochondria aggregation and the role of KBP
The above results did not answer the question whether KBP is a mitochondrial receptor for KIF1Bα and is mediating the interaction KIF1Bα/mitochondria. We therefore analysed the localization of KIF1Bα in cells with a reduced KBP expression. As described before, cells were transfected with the anti-sense KBP construct and transfected cells identified by staining with MitoTracker. In addition, cells were stained with KIF1Bα antibody. As seen in Fig. 6A (top), a perinuclear localization of mitochondria indicates transfection with the anti-sense KBP construct. The bottom picture shows perinuclear localization of KIF1Bα and colocalization with aggregated mitochondria. This suggests that KIF1Bα is still associated with the mitochondria after down-regulation of KBP and that KBP does not function as an adapter.
Figure 6 KIF1Bα colocalizes with aggregated mitochondria. A, NIH3T3 cells were transfected with anti-sense KBP and costained with MitoTracker (upper picture) and KIF1Bα polylonal antibody (lower picture). Bar, 10 μm. B, 1 μg of KIF1Bα and KIF5B GST-fusion proteins were bound to glutathione Sepharose and incubated with equal aliquots of the same lysate from KBP expressing 293 cells. As a control, GST-Grb2 fusion protein was used. The proteins were separated, transferred to nitrocellulose and immunoblotted with GST-KBP antiserum. Molecular mass markers are shown in kDa, and an arrow indicates the position of KBP. Trf, transfection. C, KBP was transiently overexpressed either alone or together with KIF1Bα-MYC or KIF1Bα350-1135-VSV. Proteins were immunoprecipitated with either VSV or MYC antibody and KBP was detected with the specific antibody. The filter was reprobed with KIF1B antibody, and KBP expression was confirmed as before.
In addition to KIF1B, conventional kinesin (KIF5B) also was described to mediate transport of mitochondria [21]. To confirm a specific role of KBP for KIF1Bα and not KIF5B, we tested the interaction between these proteins. Therefore, we constructed a plasmid encoding a GST fusion protein encoding amino acids 265–639 of KIF5B, corresponding to the previously used GST-KIF1Bα. Proteins were purified from bacteria and incubated with glutathione-sepharose and lysates from 293 cells transiently overexpressing KBP. As indicated in Fig. 6B, only GST-KIF1Bα associated with KBP. Neither GST-KIF5B nor GST-Grb2 (as a control) were able to bind KBP. The GST-fusion proteins are visible since the anti-KBP antiserum was generated using a GST-KBP fusion protein and indicate that similar amounts were used for the pull-down.
The above finding suggests that KBP does not bind the KIF1Bα's carboxyl-terminus, which was shown to mediate cargo interactions [16,22]. To identify the KBP-binding site on KIF1Bα we generated a mutant with an amino-terminal truncation of 349 amino acids (KIF1Bα350-1135-VSV) which was not able to bind KBP (Fig. 6C). Together with the data presented in Figure 6B this confines the binding of KBP to KIF1Bα between amino acids 270 and 350 at the carboxyl-terminus of the motor domain. Thus, KBP plays an important role in mitochondria distribution in cells but it is neither a mitochondrial receptor nor a general adapter protein for mitochondria transporting KLPs.
KBP improves KIF1B motility
KBP does not mediate the association of KIF1Bα with mitochondria since it is still found there when KBP is depleted. We therefore hypothesized that KBP could either increase the time of association of KIF1Bα with MT, or it could influence the motility of the KIF1Bα motor domain. To investigate this hypothesis we used latex beads in in vitro motility assays. Extracts were prepared from untransfected HeLa cells or from HeLa cells overexpressing KIF1Bα together with KBP or the KBP deletion mutant (Fig. 7A). Extracts from untransfected cells served as control for unspecific moving. The unspecific movements could be attributed to the large amount of kinesin heavy chain present in the extract (Fig. 7A), which bound beads. The KIF5B can be inhibited by SUK4 antibody [23], and indeed treatment of extracts with the antibody strongly reduced motility in HeLa extracts. Inhibition of unspecific movements could be further improved by coating the beads with KIF1B antibody. Both treatments together reduced the amount of moving beads by about 90% (Fig. 7B, bars 1,2). Upon overexpression of KIF1Bα together with KBP the number of moving beads was significantly higher than in extracts from untransfected cells. This number was not affected by additional co-overexpression of the KBP deletion mutant (Fig. 7B, bars 3 and 4). Therefore, we assume that more than 60% of the observed movements was caused by the overexpressed KIF1Bα. When analysing the velocities of moving beads, we noticed that the deletion mutant slightly decreased the velocity of the beads as compared with the wild type (Table 1). Most striking was the strong reduction of the distance traveled by beads in extracts coexpressing KBPΔ250–281, most of which was shorter than 1 μm, while in extracts coexpressing wild type KBP the beads traveled longer than 2 μm (Fig. 7C; for average values see Table 1). Taken together, our results indicate that KBP increases the motility of KIF1Bα.
Figure 7 KBP increases the motility of KIF1Bα. A, extracts were prepared from HeLa cells that were either untransfected or transfected with KIF1Bα and KBP or KBPΔ251–281. The cells were cracked and cytosolic extracts separated by SDS-PAGE (20 μg of total protein) transferred onto nitrocellulose and blotted with KIF1B (top panel), KBP (midddle panel) and ubiquitous kinesin antibodies (bottom panel). B, uncoated or KIF1B antibody coated beads moving on MT sedimented on coverslips in flow cells were counted. Movements of beads were observed using VE-DIC in HeLa extracts as described in the Methods section. Each bar represents the number of beads counted during 5 minutes of observation on 10 planes in 3 independent experiments, where each plane was observed for 30 sec. C, distances traveled by beads in a particular extract were determined using RETRAC software. The total number of analysed beads (n) is indicated below each histogram.
Table 1 Summary of data from motility assays. Average values for velocities and distances traveled by beads are shown.
Lysate Max velocity Distance traveled along MT
[μm/s] St. error [μm] St. error
KIF1B /KBP (n = 61) 0.82 0.022 2.32 0.15
KIF1B/KBPΔ250–281 (n = 67) 0.69 0.02 1.07 0.098
P-value 2.92E-05 1.25E-09
Discussion
In the present study we report on the identification of the new KIF1 binding protein KBP. The association of KBP with KIF1Bα was verified in vitro, in 293 cells transiently overexpressing both proteins and in untransfected BHK cells. Anti-sense experiments revealed that KBP is required for a proper distribution of mitochondria in the cell. Finally, we showed that KBP increases KIF1Bα motility in vitro.
The bait used in the yeast two-hybrid screen that led to the identification of KBP contained the sequence from the motor domain to the PTPD1 binding region (amino acids 261–800). Additional data from binding experiments of KBP to KIF1C and KIF1Bα deletion mutants allowed us to narrow down the binding sequence of KIF1B/C for KBP to the carboxyl-terminal region of the motor domain. A high amino acid sequence homology of 90% between KIF1C and KIF1Bα in this region supports the conclusion that KBP can bind to both proteins, and a manuscript describing the role of KBP for KIF1C is in preparation. It is important to note that the binding region of KBP is located close to the K-loop, a region in KIF1 KLPs that increases the affinity of the motor domain for MT [24]. For KIF1B, several splice variants involving or located next to the K-loop are known [13], and it will be interesting to check whether the presence of these variants has an effect on interaction with KBP [12]. An interaction with the KIF1Bβ variant would open a role for KBP as a player in neurodegenerative diseases. Recently, the role of the K-loop in a 679 amino acid construct of the rat orthologue of KIF1C was analysed [25]. In in vitro experiments this protein was not chemically processive, the K-loop increased the affinity for MT and stabilized a weak binding mode with ADP in the active site. The hypothesis was formed that rat KIF1C could work in teams of motor proteins, which could generate processive movement of attached organelles. This would be a similar mechanism as proposed for KIF1A [22]. In our motility assays we observed that KBP increased the motility of beads in extracts from KIF1Bα overexpressing cells. When the motor protein was overexpressed with KBP, it traveled average distances of 2–3 μm and maximally 6 μm, while with KBPΔ251–281 we observed KIF1Bα moving an average of 1 μm. These experiments do not contradict the data of Rogers et al. and Klopfenstein et al. since it is possible that KBP facilitates grouping of motor proteins and thus increases the motility. However, it also indicates an alternative mechanism where associated proteins like KBP could increase the motility of single molecules of KIF1B.
Numerous proteins have been identified that interact with KLPs [26,27]. These proteins may determine the localization of the KLP complex or which cargo is transported. Very little, however, is known about associating proteins that regulate either the cargo binding of the KLP or its activity. Such a KLP associating protein that is required for the KLP function has been described in yeast. The MT minus-end directed motor protein Kar3p is important for meiosis I in S. cerevisiae, and a knock out leads to an arrest in prophase I [28]. The associating protein Cik1p targets Kar3p to MT, implicating this as a reason why loss of Cik1p also impairs meiosis, although the effect is less severe [29]. Several studies describe a regulatory role of the carboxyl-terminus in conventional kinesin processivity. Kinesin light chains were reported to inhibit the binding of the heavy chain to MT [30]. Further, the kinesin tail domain by itself has an ATPase inhibiting role that is relieved by cargo binding [31-33]. One function of the light chains and the carboxyl-terminus of the heavy chain may thus be to ensure that kinesin is active only in the presence of its cargo. However, most of these studies have been done in vitro and cannot account for effects of regulating proteins.
The drastic consequences of the loss of KBP expression for mitochondria localization could also indicate that KBP is targeting KIF1Bα to the mitochondria. However, KIF1Bα is attached to mitochondria also after depletion of KBP (Fig. 6A). In addition, KLPs normally bind their cargo with carboxyl-terminal sequences, whereas KBP interacts with KIF1Bα amino-terminal sequences. Therefore, we do not think that KBP is important for the cargo – kinesin connection. Alternatively and mentioned before, KBP may regulate the KIF1Bα activity, i.e., in the absence of KBP, KIF1B is not or less able to move mitochondria.
Only two widely expressed KLPs have been reported to transport mitochondria, KIF1Bα and KIF5B, while KIF5A and KIF5C are expressed predominantly in neuronal tissues [34]. KIF5B did not interact with KBP, and it should not be affected by KBP depletion. Thus, even if there is some expression of KIF5B, a major role of KIF1Bα for mitochondria transport in NIH3T3 cells is likely. Recently, the Drosophila protein MILTON was described that associates with the KIF5 ortholog kinesin heavy chain and may play a role similar to KBP since its mutation impairs MT-plus end directed mitochondrial transport in axons and synapses [35]. In addition to KLPs, dynein motor proteins probably can transport mitochondria in cells [36], and this transport would occur in a MT minus-end direction. When force moving mitochondria to the MT plus-ends is reduced, a perinuclear localization of the mitochondria would result. On the other hand, a complete depolymerization of MTs by nocodazole treatment led to a diffusion of mitochondria throughout the cytoplasm [21], demonstrating the specificity of the consequences of KBP depletion.
Conclusion
We have shown earlier that protein tyrosine phosphatase D1 and members of the 14-3-3 protein family interact with KIF1C, implying its regulation by phosphorylation. In this manuscript, we have identified the new protein KBP as a KLP interacting protein that is required for KLP activity. This suggests that vesicle transport in cells may be subject to regulation at the level of KLP – microtubule interaction and implicates KBP as a physiological mediator.
Methods
Yeast two-hybrid screen
Two fragments of the KIF1C cDNA encoding amino acids 435–622 (corresponding to the U104 domain) or amino acids 261–800 were cloned into the LexA fusion protein vector pBTM116 and transformed into the Saccharomyces cerevisiae strain L40 (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ [37]), generating the L40 LexA-KIF1CU104 and L40 LexA-KIF1C298–840 strains, respectively. A brain cDNA library fused to the GAL4 activation domain in the pACT2 vector (Clontech) was transformed into the L40 LexA-KIF1CU104 and L40 LexA-KIF1C298–840 strains and 5 × 106 transformants were screened for interaction as described [38]. Yeast plasmid DNA was isolated from His+ β-Gal+ colonies, rescued into Escherichia coli HB101, retransformed into L40 LexA-KIF1C298–840, and yeasts assayed for β-galactosidase activity and growth on medium with complete supplement lacking amino acids Trp, Leu, Ura, Lys, and His (Bio101). The specificity of the interaction between KIF1C298–840 fragment and potential candidates was proven by transforming the candidate plasmid also into a L40 LexA and a L40 LexA-laminin strain.
DNA sequence analysis
Clones interacting specifically with KIF1C in the two-hybrid screen were subjected to sequencing by the dideoxynucleotide chain termination method. Non-redundant databases (GenBank, EMBL, DDBJ and PDB) were searched for homologous sequences using the BLAST program [39,40].
Northern blot analysis
A multiple tissue Northern blot was obtained from Clontech. Hybridization was performed at 67°C with 5 × 106 cpm/ml of 32P-random primed DNA probe as described by the manufacturer.
cDNA cloning and protein purification
For transient expression in 293 and NIH3T3 cells, all cDNAs were cloned by standard procedures into the cytomegalovirus immediate early promoter-based expression plasmid pRK5 [41]. To clone a KIF1Bα encoding cDNA, the 3'-part of the open reading frame was received from clones FH12320 (1941–3721 bp of this clone) and HJ02790 (1–685 bp of this clone) known in HUGE Protein Database as genes KIAA1448 [42] and KIAA0591 [43], respectively. The 5'-part was amplified in a PCR reaction using a human brain cDNA library as a template. A cDNA fragment encoding the amino acids 265–639 of the human version of KIF5B was amplified with specific primers in a similar way. The corresponding region of KIF1Bα (amino acids 270–641) was cut out from the KIF1Bα cDNA using restriction enzymes, and both fragments were cloned into a pGEX vector. The corresponding fusion proteins were produced in Escherichia coli BL21 (Novagen) or 298 F' strains and purified as described [44].
Antisera
The KIF1B and KBP rabbit polyclonal antisera were raised against glutathione-S-transferase (GST) fusion proteins of KIF1B (amino acids 743–1153) and KBP (full length), respectively. Monoclonal antibodies used were derived from the following hybridoma: kinesin heavy chain – H2; phosphotyrosine – 4G10; β-tubulin – KMX1; vesicular stomatitis virus (VSV) epitope tag – p5d4; KIF5 – SUK4; hemagglutinin (HA) epitope tag – 12CA5.
Cell lines and cell culture
293 cells were grown in F12/Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 mM glutamine. BHK, HeLa, NIH3T3 and C2C12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 mM glutamine.
Transient expression, cell lysis, and immunoprecipitation
Transfection of 293 cells and NIH3T3 cells was performed using the method of Chen and Okayama [45]. The cells were lysed in 1 ml of lysis buffer/10-cm plate (1% Triton X-100, 50 mM HEPES, pH 7.5, 10% glycerol, 150 mM NaCl, 1.5 mM EGTA, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM sodium orthovanadate, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride) and the lysates were precleared by centrifugation at 13,000 g for 15 minutes at 4°C. The lysates were adjusted for equal protein concentration, the appropriate antibody (3 μl of serum or 2 μg of purified antibody) and either Protein A- or Protein G-Sepharose were added, and the lysates were incubated for at least 3 hours at 4°C on a turning wheel. The immunoprecipitates were washed with HNTG buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 10 mM NaF, 1 mM sodium orthovanadate), separated on an 8 or 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and incubated with the appropriate primary antibody. After three washes, the membranes were incubated with secondary horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies (Sigma). After three washes they were visualized using the ECL system. Before reprobing, blots were incubated for 30 minutes in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 0.1% β-mercaptoethanol at 55°C.
In vitro binding assays
For in vitro binding assays, 293 cells transiently overexpressing KBP were lysed and incubated for 4 hours at 4°C with GST or GST-KIF1Bα/KIF5B fusion proteins immobilized on glutathione-Sepharose. After washing with HNTG buffer, the proteins were separated by SDS-PAGE and analysed by Western blotting.
Immunofluorescence
NIH3T3 cells were grown on uncoated glass coverslips, fixed for 20 minutes in ice-cold methanol at -20°C, washed with phosphate-buffered saline and incubated for 10 minutes with 0.1% NaBH4 and 0.1 M glycine in phosphate-buffered saline to block autofluorescence. Nonspecific antibody binding was blocked for 45 minutes with PBS with 0.045% fish gelatin containing 5% normal goat serum and 1% bovine serum albumine. Incubation with primary antibody was done for 1.5 hours at 37°C after dilution in PBS/gelatin containing 5% normal goat serum. The affinity purified KBP and KIF1Bα antibodies were used at concentrations of 10 ng/μl and 20 ng/μl, respectively. The β-tubulin antibodies were diluted as recommended by the manufacturer, and the monoclonal VSVantibodies at a concentrations of 20 ng/μl. After five washes with PBS/gelatin, primary antibody binding was detected with isotype-specific secondary antibody conjugated with either DTAF (goat anti-rabbit, Dianova) or Alexa (Alexa Fluor 546 goat anti-mouse and goat anti-rabbit and Alexa Fluor 488 goat anti-mouse, Molecular Probes). The coverslips were mounted in PermaFluor (Immunotech). To allow unbound protein to diffuse out, extraction of cells with 0.5% Triton X-100 was performed prior to fixation for 5–10 seconds [46,47]. MitoTracker (Molecular Probes) staining was done according to manufacturer's recommendations.
Pictures were taken with a Leica TCS NT/SP Laser Scanning Confocal Microscope supplied with HCX PL APO immersion oil objective of magnification 63× and aperture 1.32–0.62. The pictures were prepared using TCS software supplied by the manufacturer.
Cell extracts and motility assays
Cell extracts were prepared in BRB80 buffer (80 mM Pipes, pH 6.8, 2 mM MgCl2, 1 mM EGTA) supplemented with protease inhibitors, through multiple passing through cell cracker (HGM, Heidelberg, Germany). The nuclei were spun down and the supernatant was spun again at 120,000 g to separate cytosol from membrane fraction The concentration of the cytosolic fraction was adjusted to 1 mg/ml and supplemented with 1 mM DTT, 20 μM taxol, 2 mg/ml BSA, 5 mg/ml casein and 1/20 volume energy mix (150 mM creatine phosphate, 20 mM ATP, 20 mM MgCl2, 2 mM EGTA, pH 7.7).
For motility assays with latex beads, 1 μl of beads (2.5% solid-latex, carboxylate 0.1 micron microspheres; Polysciences, Inc., Warrington) was left untreated or incubated for at least 30 minutes with 4 μl of KIF1B antibody solution (3 mg/ml). Then the antibody/beads suspension was diluted 1:6 in BRB80, 1 mM DTT, 20 μM taxol, 2 mg/ml BSA, 5 mg/ml casein and 1/20 volume energy mix. One μl of beads was mixed with 9 μl of cell extract and floated into a flow cell with coverslips coated with microtubules. The beads were left for 5 – 7 minutes in a humid chamber to sediment onto the microtubules. To inhibit unspecific KIF5B driven movements, SUK4 antibody was incubated with extracts for 10 minutes at a final concentration of 0.25 mg/ml, and then the extracts were used in the assay. Flow cells were prepared as described earlier [48], and microtubules were isolated from pig brain as described [49].
Motility was observed using video-enhanced differential interference contrast microscopy (VE-DIC) in real time (Olympus optics BX60 microscope, equipped with DIC optics; Japan, Tokyo). To determine the rates and distances, RETRAC software (Dr. N. Carter, Marie Curie Research Institute, Oxted, Surrey, UK) was used.
List of abbreviations
ER: endoplasmic reticulum
GST: gluthatione-S-transferase
HA: hemagglutinin
KBP: KIF1-binding protein
KLP: kinesin-like protein
MT: microtubules
VSV: vesicular stomatitis virus
Authors' contributions
MJW performed most experiments and contributed to the manuscript, MM did the yeast-2-hybrid screens, CD contributed plasmid constructs and antibodies, HUH participated in the study design, and RL conceived the study, contributed to the experiments and wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We are grateful to K. Kapp and K. Wojtachnio for critically reading the manuscript, to Viki Allan for helpful suggestions for the in vitro motility assays, and to the Kazusa DNA Research Institute for providing clones FH12320 and HJ02790. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to R.L.
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BMC Dev BiolBMC Developmental Biology1471-213XBioMed Central London 1471-213X-5-211619755110.1186/1471-213X-5-21Research ArticleDifferential range and activity of various forms of the Hedgehog protein Dawber Rebecca J [email protected] Stephen [email protected] Bram [email protected] France [email protected] den Heuvel Marcel [email protected] MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK2 Cell and Molecular Biosciences, University of Newcastle, Newcastle-upon-Tyne, UK3 Cell Biology, UMC Utrecht, Utrecht, The Netherlands4 Department of Biological Sciences, University of Essex, Colchester, UK2005 30 9 2005 5 21 21 29 9 2005 30 9 2005 Copyright © 2005 Dawber et al; licensee BioMed Central Ltd.2005Dawber 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 Hedgehog (Hh) family of secreted proteins act as extracellular messengers to control and coordinate growth and differentiation. The mechanism by which Hh protein travels across a field of cells, and results in a range of specific effects relating to the distance from the source, has been the subject of much debate. It has been suggested that the range and activity of the pathway can be linked to modifications of the Hh protein, specifically the addition of lipid groups at N- and C-terminal sites.
Results
Here we have addressed the potency of different forms of Hh protein by expressing these in Drosophila, where we are able to precisely establish pathway activity and range in naïve but responsive tissues. As expected, a construct that can produce all forms of Hh recapitulates endogenous signaling potencies. In comparison, expression of a form that lacks the cholesterol moiety (HhN) leads to an extended range, but the product is less effective at inducing maximal Hh responses. Expression of a point mutant that lacks the N-terminal palmitate binding site shows that the palmitoylation of Hh is absolutely required for activity in this system.
Conclusion
We conclude that the addition of the cholesterol moiety limits the range of the protein and is required for maximal activity, while addition of palmitate is required for all activity. These findings have implications for understanding how Hedgehog proteins move, and thus their potential at influencing distant sites, and concomitantly, how modifications of the signaling protein can affect the efficacy of the response in exposed cells.
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Background
Hedgehog (Hh) proteins are a family of secreted signalling molecules that act as extracellular messengers, signalling between cells to control and coordinate growth and differentiation. Hh proteins govern growth and patterning events in a wide variety of developmental contexts in vertebrates and invertebrates, and mutations in components of the Hedgehog signalling pathway are implicated in many human disorders including cancers [1-4]. In Drosophila, the single Hh protein is required at multiple developmental stages and is responsible for patterning embryonic segments [5,6] as well as adult structures such as wings, legs and eyes [7-9]. The response to Hh can vary considerably with the amount of signal received, for example, in the vertebrate ventral neural tube, motor neuron and interneuron generation depends on the graded activity of Sonic hedgehog (Shh) [10]. One key question is how one signal can elicit such a range of responses.
In most cases explored so far in Drosophila, Hedgehog transduces its signal through the zinc finger transcription factor Cubitus interuptus (Ci), which can exist in several different forms [11,12]. In the wing imaginal disc, Ci is expressed throughout the anterior compartment [13], in a complementary pattern to hh itself. In the absence of Hh signal, through a mechanism which is not fully understood, the Hh receptor Patched (Ptc) prevents Smoothened (Smo) from localising to the plasma membrane [14]. Under these conditions, Ci is present in a complex with a variety of proteins including the kinesin-like protein Costal2, (Cos2) [15,16], the serine-threonine kinase Fused (Fu) [17], and the PEST protein Suppressor of Fused (Su(Fu)) [17]. This complex is associated with microtubules. Through interactions with other proteins including Protein Kinase A (PKA) and the putative ubiquitin kinase Slimb [18,19], Ci is cleaved to generate a 75 kDa transcriptional repressor, which represses transcription of target genes including hh and decapentaplegic (dpp) [20]. On binding of Hh to Ptc, Smo relocates to the plasma membrane [14], Fused becomes phosphorylated and the Ci-Cos2 complex is disrupted and dissociates [15]. Full length Ci is stabilized in the cytoplasm and can then be translocated to the nucleus, where it acts as a transcriptional activator of target genes including dpp and ptc.
The amount of nuclear Ci appears to be tightly regulated through cytoplasmic/nuclear shuttling and degradation. Hh signalling increases the rate of Ci nuclear import [21,22] while rapid nuclear export also plays a major role in controlling nuclear Ci levels [22]. Ci activation, distinct from Ci stabilization and nuclear import, occurs in response to maximal Hh signalling [23-25]. Ci levels are further regulated by the action of Debra, which mediates the polyubiquitination of full-length Ci, leading to its lysosomal degradation [26]. The result of these molecular pathways regulating Ci activity is visualized at the boundary of Hh-expressing and receiving cells. A broad stripe of cells expressing the full length form of Ci is seen close to the anterior-posterior (A-P) boundary; Ci levels are high in the cytoplasm in these cells. However, very close to the Hh expressing cells, where Hh signalling is maximal and hence Ci is maximally activated, shuttling of Ci into the nucleus and its subsequent rapid export and degradation lead to low cytoplasmic levels of Ci.
Hh proteins undergo a variety of post-translational modifications, some of which have been shown to modulate biological activity. The protein is unique in that it can be dually lipid modified: both an ester-linked carboxy-terminal cholesterol moiety [27] and an amide-linked amino-terminal palmitate [28] have been found associated with the Hh signalling protein. Unprocessed Hh protein undergoes auto-proteolytic cleavage under the control of a catalytic domain in the C terminal region, releasing the active amino-terminal domain. Cleavage is accompanied by covalent bonding of cholesterol to the N-terminal moiety [29,30]. This can be followed by addition of palmitate to the amino terminus. This palmitoylation requires the action of the Skinny Hedgehog acyltransferase, also known as Sightless or RASP [31-33]. The efficient addition of palmitate has been suggested to depend on prior addition of cholesterol [34]. Initial experiments using active Shh protein relied on a construct only encoding for the amino-terminal domain in bacteria. This peptide is active in certain assays, but whether it has full range activity is difficult to establish in in vitro systems. More recent experiments indicate that this form of Hh, which is not cholesterol modified, has low level Hh activity in vertebrates [35]. It has not been clearly established whether or not this form is palmitoylated.
Despite lipid modification, which one might expect to limit its movement, Hh moves beyond the cells where it is expressed. Hh directly induces expression of Dpp and Ptc in a band of 5–10 cells beyond the boundary of Hh expression [36-38]. There are two to three cells where Ci levels are low due to rapid degradation and an additional five to seven cells where cytoplasmic levels are high. The action of Dispatched (Disp), a protein homologous to Ptc, is required for secretion of Hh from sending cells [39]. Hh signal reception in receiving cells causes up-regulation of Ptc, the receptor protein for Hh, which also sequesters Hh and thus forms a sink for Hh protein. Non-cholesterol modified Hh expressed in Drosophila has been reported to have a broader range of Hh action than normal [30]. Contrastingly, in vertebrates, it has been argued that cholesterol modification is required for long range signalling activity [35,40] and Burke et al suggest that normally processed Hh has a greater range of action than unmodified Hh in Drosophila [39]. However, their data do not show the effects of cholesterol-modified Hh expression in comparison with unmodified Hh.
In light of this conflicting evidence, we wanted to determine the effects of Hh modifications on Hh movement and efficacy in inducing target gene expression. Further, and related to this, a key question in signal transduction is how a single molecule can elicit a wide range of responses. Does Hh act through a simple gradient-threshold mechanism, or are other functional factors involved? Specifically, in the light of Hh lipid modifications, do different forms of Hh protein play discrete roles in patterning? To address these questions, we generated clones ectopically expressing various differently modified forms of Hh protein and examined their effects on target gene expression and wing disc patterning. We find that un-cholesterol-modified Hh (HhN) has a greater range of action than normally modified Hh. We further show that the palmitoylation step is crucial for Hh activity, and is likely to occur even in the absence of prior cholesterol addition. We also demonstrate that Hh lacking the cholesterol moiety is less effective than normal wild-type Hedgehog at inducing expression of target genes that require the highest levels of Hh. We discuss the implications for Hh signalling and its effects on target tissues.
Results
We generated clones of cells expressing various different forms of Hedgehog (Hh-FL, HhN, HhCtoS, HhNCtoS) in an otherwise wild-type wing disc, using a Flp-out cassette to drive expression of the constructs [37]. We expect the Hh full-length (Hh-FL) protein to be processed to yield cholesterol- and palmitate-modified N-terminal signalling domain, HhNp (and other forms if these indeed are present in nature), plus the Hh C-terminal moiety. The HhN form generates the N-terminal directly, rather than by cleavage of immature full-length Hh, and will not have cholesterol added, but should still be modified by palmitoylation. In contrast, the HhCtoS form is mutated at the residue where palmitate is added, but should still undergo cleavage and cholesterol addition. Lastly, a HhNCtoS form cannot have either of the lipid modifications.
We used various targets of Hh signalling to assess the extent and degree of pathway activation: Engrailed, which requires high pathway activation for maximal induction [9,41], Collier, with a lower threshold for induction [42], and Ci which behaves in different ways depending on the signal intensity, accumulating in response to Hh moderate signal, but showing lowered cytoplasmic levels in response to maximum Hh signal. Figure 1(a–d) shows En and Ci expression; note that at the A-P boundary (purple line), in cells receiving maximal Hh signal, hyperactivated Ci and En are expressed in the same cells (1d). Figure 1(e–f) shows wild-type Collier expression. Notice that Col expression (red) just overlaps En expression (blue) in two cells but extends anteriorly from these in response to lower levels of Hh. Figure 1(g–i) shows Hh and En expression, revealing that an area of En expressing cells exists more anterior to the Hh marked domain: these are the cells producing En in response to high Hh signal, the same set of cells as in overlap of Ci and En in Figure 1d.
Figure 1 Wild-type expression patterns for Cubitus interruptus, Engrailed, Collier and Hedgehog. Ci in red (c and d), En in blue (c, d, f and i), Col in red (f) Hh in green (i). a. Ci expression; purple line denotes antero-posterior boundary. b. En expression. c. Merge of a and b. d. Enlargement of region equivalent to that boxed in c. Note the overlap between cells expressing En (blue) and Ci (red) at the antero-posterior boundary. e. Collier expression. f. Higher power image showing Col and En. Note the slight overlap at the A/P boundary. g. Hh expression in the wing pouch h. En expression. i. Merge of g and h. It can be seen that there are cells that express En (blue) which do not express Hh (green).
We did not use Dpp as a key marker of Hh signal because no antibody is currently available that consistently recognizes the protein. Use of, for instance, dpplacZ inevitably does not reveal as accurate a picture as an antibody, particularly as β-galactosidase shows perdurance in descendants of cells that have previously expressed the construct but have since ceased expression [43].
Ectopic Expression of full length Hh recapitulates the A-P boundary in the wing disc
Use of antibodies to detect Hh target proteins reveals that clones of cells expressing Hh-FL, within the anterior presumptive pouch, cause changes in localization and expression of these targets (fig 2). Within the clone, where cells are labelled for LacZ (target group 0, fig 2e), Ci accumulation is reduced (fig 2f,j) and En expression is strongly induced (fig 2g,k). The same decreased Ci and increased En is seen for two to three cell diameters outside the clone (labelled as target group 1 in fig 2g). These cells also activate Collier (Col) expression (fig 2m–t). In cells further from the clone, at 4–8 cell diameters removed (target group 2, fig 2f), Ci accumulation is increased relative to the surrounding cells (fig 2f,j). Col is also expressed in these cells (fig 2r). However, these group 2 cells do not express En (fig 2g,k).
Figure 2 Clones of cells expressing full-length Hh recapitulate the full range of Hh responses. a, e, i, m and q: lacZ, clone marker; b, f, j: Ci; c, g and k: En; n and r: Col; o and s: Hh. D, h, l, p and t: merged images across rows (d, h and l: clone markers in green; En in blue and Ci in red. P and t: clone marker in blue, Col in red and Hh in green). a–d, Ci (red) is down-regulated and En (blue) is up-regulated in and around clones expressing Hh-FL. The anterior pouch of these discs is overgrown (a–d). e–h. Higher magnification of clone similar to those in a–d, showing target cell groups 0 (cells within clone), 1 (2–3 cell diameters immediately outside clone) and 2 (cells further from clone), (see text for details). i–l It can be seen clearly that Ci is down-regulated within and around the clone (in target groups 0 and 1) but is up-regulated further from the clone (target group 2), whereas En is up-regulated in and immediately around the clone (in target groups 0 and 1, but not 2). m–p. Collier is up-regulated in and around clones. It can also be seen that these cells express Hh (green). q–t. Higher magnification images of clones like those shown in m–p. It can be seen that Col is up-regulated in and around the clone (target groups 0, 1 and, at a lower level, 2)
This situation is similar to that seen at the A-P boundary in wild-type wing discs, where cells receiving the highest levels of Hh signal show reduced Ci accumulation (fig 1a–d); this corresponds to the region of hyper-activated Ci. These same cells show En expression activation (see fig 1d). A few cell diameters further from the boundary, there is no En expression, but Hh signal is sufficient here to result in accumulation of full length Ci and to activate col expression.
Isolated clones expressing Hh-FL have little molecular effect on the rest of the disc, outside the area as described above. There is a slight increase in Ci levels throughout the anterior of the disc, but the band of elevated Ci near the A-P boundary, a representation of moderate Hh pathway activation, is still clearly visible, indicating the Hh signalling pathway in the remainder of the disc is not (or only very mildly) affected. As expected, clones in the posterior of the wing disc, where Hh is normally expressed, have little or no effect on disc patterning. However, where Hh-FL expressing cells fall within the anterior presumptive pouch and hinge and affect target gene expression as described above, they also cause overgrowth of the disc, with the anterior presumptive wing pouch enlarged compared with a relatively normal sized posterior compartment and presumptive notum.
Ectopic expression of the Hh amino-terminal fragment induces a distinct set of target genes and at a longer range
The auto-proteolytic cleavage of native Hh protein gives rise to the active N-terminal portion decorated with cholesterol. It is still unclear if a form of Hh exists in nature that has undergone the cleavage reaction but is not decorated with cholesterol. To address this question, we used clonal analysis of the dispatched (disp) gene. It has been shown that the disp gene product is required for the secretion of the cholesterol-modified form of Hh protein [39,44]. By generating large somatic clones mutant for disp, we made wing discs where the whole posterior compartment of the wing pouch was disp deficient; these cells therefore cannot secrete any cholesterol-modified form of Hh protein. We then analyzed these discs to see if cells, which will not receive cholesterol-modified Hh, can still show any Hh response. In Figure 3, a disp clone abutting the A-P boundary contained within the posterior compartment is shown. It is clear that cells in the anterior are able to respond to a Hh signal by generating high cytoplasmic levels of Ci (see arrow, fig 3a). However it is also clear that no maximal response of Hh signalling is generated, as the hyperactivated Ci band directly anterior to the A-P boundary is not seen. Therefore, it appears that although these cells are not receiving any cholesterol-decorated Hh, they are still able to respond to a form of Hh which can be secreted by disp mutant cells. This is likely to be a form of Hh that lacks the cholesterol moiety.
Figure 3 Moderate level Hh signalling despite large disp clones. Anti Ci (a) or in green (c), anti-myc (clone marker) (b) or in red (c). Even when large disp clones fill almost the entire posterior presumptive pouch (marked by lack of anti-myc staining (b)), some form of Hh is still able to up-regulate Ci accumulation near the A/P boundary (a). Arrow indicates region of Ci accumulation. Note that there is no region of hyper-activated Ci (low level of Ci as shown in Figure 1) in this part of the disc.
In order to analyse the downstream effects of a form of Hh that lacks cholesterol, we generated clones of cells expressing a construct that can only produce the cleaved amino-terminal domain of Hh protein (HhN). Ectopic HhN expression within the anterior compartment of the wing disc does not lead to lower levels of Ci in or just outside the clone (fig 4b,f). HhN-expressing clones cause some activation of En within and around the clone (fig 4g), but at a much lower level than that seen with Hh-FL-expressing cells. Importantly, while En is only very mildly up-regulated within clones of cells expressing HhN, the effect extends much further away from the clone than with HhFL, causing two broad but weak stripes of En to extend throughout the anterior pouch in many discs (fig 4c,g). In addition, across the whole disc, Ci levels are dramatically increased (fig 4b,f), so that the band of increased Ci expression normally present at the A-P boundary can no longer be seen (compare Figure 4 with Figure 1). To analyse these apparent long-range effects further, we studied expression of the target of Hh signalling which is normally activated in the area of moderately high Hh signal and high Ci accumulation – Collier – in the anterior pouch area. In this case, the difference between Hh-N and Hh-FL expressing clones is even more obvious. The presence of even quite small groups of HhN-expressing cells in the anterior causes Col expression to extend in two strong stripes through most of the anterior pouch (fig 4j,n).
Figure 4 Clones of cells expressing HhN, which lacks the cholesterol moiety, generate moderate Hh responses, over an increased range. A, e, i and m: clone marker (LacZ); b and f: Ci; c and g: En, j and n: Col; k and o: Hh. D, h, l and p: merged images across rows (d and h: clone marker in green, Ci in red, En in blue; l and p: clone marker in blue, Col in red, Hh in green). a–d Ci is up-regulated ubiquitously in disc containing HhN expressing clones, so that the normal strip of elevated expression near the A/P boundary is lost. The entire anterior part of the disc is overgrown (compare with fig 1a–d). e–h. Higher magnification of clones. There is no local effect on Ci (no change within target groups 0–2) although Ci expression is high generally. En is weakly up-regulated in most of the wing pouch. There is very little up-regulation confined to the target group cells. i–l Col is up-regulated in and around clones but expression also extends in two broad stripes through much of the pouch. It can also be seen that the clone cells do express Hh. m–p. Higher magnification images of clones like those shown in i–l. It can be seen that Col is up-regulated in and around the clone (target groups 0, 1 and 2) but is also up-regulated in broad domains extending away from clones. These domains encompass much of the anterior wing pouch but exclude the wing margin.
As with full length Hh, clones of cells expressing HhN in the posterior compartment of the wing disc have no obvious phenotypic effect. Clones expressing HhN in the anterior, like those expressing Hh-FL, cause overgrowth. However, the overgrowth, whilst still confined to the anterior, is often more extensive, and includes the presumptive hinge and notum, and therefore covers the full anterior disc.
Thus it appears that Hh-FL is able to produce the most extreme transformation of cell identity, from anterior to posterior, and recreate maximal Hh signalling, however it can do so only at limited distance. In contrast, HhN is only able to induce lower-threshold responses, but is able to do so at a greater distance from the source.
Palmitate modification of Hh is required for function
The mature form of Hh is unique in being dually lipid modified; in addition to the ester-linked carboxy terminal cholesterol moiety [30], there is also an amide-linked amino-terminal palmitate [28]. It has been shown that palmitate addition is required for maximal Hh activity [45,46]. To investigate the requirement for palmitoylation, we created a transgene that drives expression of a modified Hh protein in which the critical Cysteine residue, the attachment site for the palmitate, has been altered into a Serine residue. The resulting Hh-CtoS protein cannot be palmitoylated, but theoretically should still be cleaved and modified by cholesterol addition.
Expression of Hh C-S has no discernable effect on Ci accumulation or on expression of the Hh target genes en and col (Fig. 5). There is no effect on growth or disc morphology. We have shown, using anti-Hh antibody, that the clonal cells do indeed express Hh (Fig. 5k,o). Thus palmitate addition is clearly required for signalling by Hh in this assay.
Figure 5 Ectopic expression of HhCtoS has no effect on growth or on any of the markers tested. A, e, i and m: clone marker (LacZ), b and f: Ci, c and g: En, j and n: Col; k and o: Hh. D, h, l and p, merged images across rows (d and h: clone marker in green, En in blue, Ci in red; l and p: clone marker in blue, Hh in green and Col in red). a–d, Clones have no effect on growth, on Ci or on En levels. e–h. Higher magnification of clones. Ci and En expression across disc is normal. i–l Col is unaffected by clones, although they do express Hh (green). m–p. Higher magnification images of clones like those shown in i–l.
To determine whether this requirement was linked to cleavage of Hh, we also expressed a form of HhN terminal portion that could not be palmitoylated (HhNCtoS). Ectopic expression of the double mutant HhNCtoS has no effect on disc growth, patterning or expression of target genes, confirming that the addition of palmitate is crucial for function (data not shown). Moreover, the fact that this form has no activity, whilst the HhN mutant is active, suggests that at least some of the HhN as expressed in this study must be modified by palmitoylation. If it were not, HhN would be predicted to behave similarly to HhNCtoS.
Discussion
There has been a debate in the literature as to whether addition of cholesterol increases or decreases the efficacy of the Hh signal. Much of the conflicting data has been obtained in vertebrate systems, where there are complications depending on whether the tissue tested has already been exposed to native Hh proteins, and whether it is competent to show the full range of responses. The advantage of using Drosophila imaginal tissues for this analysis is that it is possible to express Hh forms in tissue that is naïve, i.e. is not itself generating or receiving Hh, but which is fully responsive. Our results show that ectopic expression of a construct able to encode full-length wild-type Hh protein results in the full range of Hh responses. In contrast, expression of a construct encoding Hh lacking the cholesterol moiety (HhN) mimics moderate Hh signal, and is unable to reproduce maximal responses. Further, and importantly, HhN has a much longer range than wild-type Hh when expressed under the same conditions. We also show that palmitoylation is required for activity, both for the cholesterol-modified form of Hh and for HhN. The implications of these findings are considered below.
Palmitate is required for activity
Our results show that palmitoylation of Hh is required for activity. The CtoS mutation replaces the N-terminal cysteine residue, where the palmitate residue is attached, with a serine residue, disabling the modification. Expression of HhCtoS in wing imaginal disc tissue has no effect on disc growth, shape, or on expression of any of the markers for Hh signalling tested. We have shown that the construct does indeed express Hh, as indicated with anti-Hh antibody, but it has no activity. Thus palmitate is absolutely required for efficacy of Hh signalling in imaginal tissues. This is supported by the finding that the product of the skinny hedgehog gene, ski (also known by other names), responsible for palmitoylation of Hh, is required in Hh-expressing cells for production of Hh signalling activity and patterning in both embryos and in imaginal discs [31-33]. Similar results to those shown here have been obtained previously [47].
Further, we find that the double mutant HhNCtoS has no activity, whilst HhN displays a subset of Hh functions, suggesting that HhN must possess the palmitate (or similar) moiety, otherwise it would be inactive as is the double mutant. This somewhat contradicts previous work which suggested that cholesterol is required for subsequent efficient addition of palmitate [34]: our results indicate that activity is dependent on the residue where palmitate is added, regardless of cholesterol modification. The exact role of palmitate is unclear. It may be required for efficient binding of Hh to its receptor Ptc, alternatively, it could be required earlier for secretion of Hh from the producing cells. Our results are consistent with recent findings on the vertebrate Hh proteins [46].
Cholesterol and activity
Our results demonstrate that cholesterol modification of Hh is required for maximal signal activity. We believe that we use the best possible criteria for defining maximal versus moderate activity of Hh signal, namely those found naturally in the wing disc. We define maximal activity in terms of two main criteria. One is strong activation of En, which is well documented as a response to highest Hh signal and which occurs normally in the cells at the A/P boundary [9,41], which do not themselves express Hh but will receive the highest possible Hh signal; these cells express Ci (see fig 1). The other criterion is lowered levels of cytoplasmic Ci in regions where Ci normally accumulates, which has also been shown previously to occur in cells receiving maximal Hh input [21,22,48] and, again, which occurs naturally in cells at the A/P boundary (fig 1). This effect is believed to be a result of Ci hyper-activation, resulting in rapid nuclear import and subsequent degradation. Ectopic expression of wild-type Hh, which will result in production of cholesterol- and palmitate-modified HhNp (plus other naturally occurring forms), results in strong induction of En and lowered cytoplasmic Ci levels within and around the clone, whereas ectopic expression of HhN, which lacks cholesterol, only causes very weak induction of En and high accumulation of Ci in the cytoplasm.
This requirement for cholesterol for maximum efficacy of Hh signalling could be due to a number of factors, the most obvious being more efficient binding and/or inactivation of the Hh receptor Ptc or higher concentration of Hh near the producing cells. The first is an attractive hypothesis, as Ptc contains a sterol-sensing domain [49], involved in binding cholesterol and shown to be important in vesicle trafficking and protein localisation (reviewed [50]). Thus cholesterol-modified Hh may bind to Ptc with higher affinity than HhN, and indeed this has been suggested for Shh in vivo [51]. Alternatively, cholesterol-modified Hh might bind Ptc with similar efficiency to HhN but be more efficient at 'inactivating' Ptc, allowing increased activation of Smo and hence of the signalling pathway. The other attractive hypothesis is that cholesterol-modified Hh may be more concentrated near the producing cells than is its cholesterol-lacking relative, either because it is more 'sticky', tending to remain bound to the membrane of producing cells; this could be due to Hh's binding to proteoglycans and Shifted (see [52,53] and [54]). Alternatively, this could be due to cholesterol-decorated Hh associating more efficiently into lipid-rich multi-molecular particles. The presence of such particles containing Hh, and also containing lipophorins, the fly homologue of lipoproteins, has recently been demonstrated [55]. Given the lipophilic nature of these complexes and their similarity to lipoproteins, it is likely that cholesterol would promote association of Hh with these particles, increasing its concentration locally and perhaps also its signalling efficacy. Thus cholesterol addition may increase the potency of Hh signal by concentrating Hh near the producing cells or via increased interaction with Ptc through its sterol-sensing domain or through a combination of the two.
Cholesterol and range
HhN that lacks the cholesterol moiety travels further from the producing cells than normally processed Hh. This is most clearly seen by the broad stripes of Col activation induced through much of the anterior pouch in response to even small clones of HhN producing cells. This is in marked contrast to the effects of Hh-FL expressing clones, which only induce Col in and around the clone. It is further demonstrated by the high ubiquitous levels of Ci seen throughout the anterior pouch in discs expressing HhN; the native stripe of elevated Ci is impossible to distinguish in these discs. En is only very weakly activated in response to ectopic HhN expression, yet En too appears in two broad stripes occupying much of the presumptive anterior wing pouch, albeit at very low levels. We also observe similar weak but ubiquitous expression of Dpp (data not shown), with HhN expressing clones; while HhFL expressing clones activate Dpp in, and immediately around, the clone (data not shown; all assayed using dppLacZ). This longer range of travel of Hh in the absence of cholesterol may be due to the same reasons that cholesterol-decorated Hh is more efficient at inducing maximal signalling responses; namely, either that cholesterol increases binding affinity to Ptc or that it serves to concentrate Hh near the producing cells. If cholesterol facilitates binding to Ptc, then in its absence Hh would be expected to travel further before eventually binding Ptc and being internalised; if cholesterol serves to concentrate Hh near the producing cells then lack of cholesterol will mean that it can diffuse or travel further from its source.
Cell proliferation
Hh proteins have been shown to influence growth directly through control of cyclins [56,57]; however indirect control via target genes, which in their turn influence proliferation, is also required for proper tissue growth and patterning [58,59]. We observe a difference in cell proliferation in the wing induced by HhN as compared to HhFL, and we propose that this difference may be due to a difference in the ability to induce downstream targets. Wing discs containing clones expressing HhFL show strong overgrowth in the anterior pouch, but this overgrowth is largely confined to the pouch, having little effect on presumptive hinge and notum regions. In contrast, disc with HhN expressing clones show overgrowth throughout the anterior, including the notum, and generally show a greater degree of overgrowth. It has been shown that dpp, a direct transcriptional target of Hh, induces proliferation [59]. By examining the effects of Hh expression on dpp-lacZ (data not shown) we observe that only HhFL can induce strong dpp expression in and around the clone; HhN induces dpp-lacZ more weakly, but does so throughout the whole anterior compartment (pouch and notum). Therefore we propose that the difference in overgrowth is due to far-ranging Hh possibly directly inducing growth in HhN expressing discs, whereas localised activation of Dpp may contribute to the more limited overgrowth in the case of HhFL. These observations have some implications on tissue overgrowth induced by Hh proteins. The question of whether tumour growth is a direct consequence of Hh itself or an indirect effect via other target genes might be relevant for treatment objectives.
Hh signalling: gradient or different Hh forms?
The work presented here raises some issues as to how Hh signalling exerts its effects across a field of cells. Does Hh signalling function within the definition of a simple concentration gradient or elicit a range of responses via a combination of different Hh forms contributing to different aspects of patterning? For this mechanism to work, it would imply that HhN exists in vivo. One piece of evidence suggests that it does. Disp has been shown to be required for secretion of cholesterol modified Hh [39], whereas HhN can be secreted even in the absence of disp [44]. We show here that large disp mutant clones, which would therefore be expected to prevent any secretion of cholesterol-decorated Hh, abolish all maximal Hh response at the A/P boundary (there is no longer a stripe of lowered Ci at the A/P boundary), however, discs containing such clones still show moderate Hh responses, i.e. elevated Ci close to the A-P boundary (see fig 3), very similar to the level of response seen when HhN clones are induced. As no cholesterol-modified Hh will be secreted from the disp clone, this moderate Hh response is presumably due to secretion of HhN although this is indirect evidence based on the response only.
Therefore we would like to propose that in nature a non-cholesterol-modified form of Hh is made and this participates in complexes with the cholesterol-modified form in creating a gradient of Hh activity. We postulate that there may be complexes with different ratios of the two forms. The cholesterol form reduces the range of these complexes but induces highest activity. Complexes with more cholesterol-modified Hh will have short range but induce maximal responses at the A-P boundary, whereas those with increased amounts of HhN will be responsible for the more moderate response further from the source of Hh. It is interesting to note here that clusters of Hh protein have been shown to exist in nature [46]. The different modifications of Hedgehog protein might affect its presence in particular clusters.
Conclusion
There has been some debate in the literature as to the role of cholesterol in Hh signalling: by expressing differing forms of Hh in tissue which is both naïve in terms of previous Hh signal and responsive to that signal, and by careful choice of markers to examine signalling levels, we believe that we have the best possible system to address this dilemma, and we find that cholesterol potentiates the ability of Hh to induce maximal response, while limiting its range. Our results indicate that the morphogen-like behaviour of Hh protein is not solely dependent on a concentration-led induction of activity, but rather might be the result of different forms of Hh protein with differing activities and diffusion rates signalling across a field of cells.
Methods
Fly strains
Flp-out constructs are as described by Basler and Struhl (1994). Crosses were of the general form HsFlp/HsFlp; UAS-hh × actin>y+>Gal4 UAS lacZ, or HsFlp/HsFlp; UAS-hh × actin>y+>Gal4 UAS ubiGFP (> depicts FRT sites).
X-chromosomal HsFlp, actin>y+>Gal4 UAS-lacZ, and actin>y+>Gal4 UAS-GFP containing stocks were obtained from Bloomington. A single heat-shock was delivered to larvae in plastic vials submerged in a 37°C waterbath for one hour in second larval instar (approximately 3 days after egg laying). UAS-hhFL, UAS-hhN and UAS-hhCtoS constructs were generated by BH using standard PCR and specific primers to generate the mutant constructs. Transgenic lines were obtained in this laboratory. The FRT82B dispatched377 chromosome stocks were obtained from K Basler.
Dpp-lacZ is an insertion of the Exelexis P[dpp-lacZ.Exel1.2] stocks, in which the dpp disk enhancer described in St. Johnston et al., 1990 [60] drives lacZ expression. This is a homozygous viable and fertile insertion on the third chromosome, obtained from Bloomington.
All flies were reared at 25°C and fed on standard media.
Generation of Hedgehog constructs
Products of PCR amplified hh fragments were cloned into pGEM-T (Promega) and sequenced to verify the constructs. The sequences were then cloned into the pUAS vector and transgenic lines generated.
Full length hh was obtained as clone and used as template for the following PCR reactions. The primers we used for PCR were (all shown 5' to 3'):
5'hhN: ATGGATAACCACAGCTCA (used to generate all hh constructs)
3'hhC: TCAATCGTGGCGCCAGCT (used to generate full length coding sequence)
3'hhN CtoS: GCATGCCAGTCCGGACAC (reverse primer to generate 5' end of hh for CtoS mutation)
5'hhN CtoS: CACAGCTCTGGTCCCGGGCGA (forward primer to generate 3' end of hh for CtoS mutation)
3'hhN:CTCGAGTTAGCCGTGCACGTGG (used to generate hhN constructs at 3' end).
Antibody staining of imaginal discs
Dissection, fixation and antibody stainings were performed as follows. Larval heads were cut off in 1 × PBS on ice. They were inverted and fixed twice in 1 × PBS, 4% paraformaldehyde, firstly for 20 minutes on ice, then following 2 washes in 1 × PBS, fixed again for ten minutes at room temperature. The inverted heads were then washed five times with 1 × PBS and washed a further five times for 5 min each with 1 × PBS, 3% normal serum, matched to the secondary antibodies, and 0.5% saponin (PBT). They were incubated overnight at 4°C with the primary antibodies in PBT and washed again five times with PBT, the next day. Secondary antibodies were used at the manufacturer's recommended concentrations for 2 hr in PBT at room temperature, after which the heads were washed again five times with PBT. Discs were dissected from the heads in PBT and mounted in VectaShield (Vector Laboratories) on glass slides.
Primary antibodies were obtained as follows: Anti-Collier antibody was obtained from M. Crozatier, anti-Hh antibody from I. Guerrerro, anti-Engrailed, 4F11, from N. Patel and anti-Cubitus interuptus, 2A1, which recognises full length Ci, from R. Holmgren. Mouse anti-B galactosidase antibody was from Promega, rabbit anti-B Galactosidase, Rabbit and Goat anti-GFPs were from Abcam. Secondary antibodies were either FITC, TRITC and Cy5 labelled, from Jackson ImmunoResearch, raised in donkey, or Alexa-labelled from Molecular Probes, raised in goat.
Confocal microscopy was performed on a Zeiss LSM Meta-510 and images were processed with the accompanying Zeiss software and in Adobe Photoshop.
Abbreviations
A-P – antero-posterior
Ci – Cubitus interruptus
Col – Collier
disp – dispatched
dpp – decapentaplegic
En – Engrailed
Fu – Fused
Hh – Hedgehog
HhFL – full-length Hh
HhN – N-terminal Hh, lacking the cholesterol moiety
HhCtoS – Hh with the cysteine residue, required for palmitoylation, mutated to a serine
HhNp – normally processed Hh N-terminal region
PKA – Protein Kinase A
Ptc – Patched
ski – skinny hedgehog (also known as sightless or RASP)
Smo – Smoothened
Su(fu) – Suppressor of fused
Wg – Wingless
Authors' contributions
RJD carried out the genetics, immunohistochemical studies and drafted the manuscript. SH and BH generated and injected the constructs. FD performed the Dispatched analysis and MvdH conceived the study, and participated in its design and coordination.
Acknowledgements
We would like to thank the following people for generously providing antibodies against Drosophila proteins: Michelle Crozatier, Isabel Guerrero, Robert Holmgren and Nipam Patel. We thank Zillah Deussen for the excellent technical help. All of the contributing authors were funded by the Medical Research Council UK.
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BMC DermatolBMC Dermatology1471-5945BioMed Central London 1471-5945-5-101622567010.1186/1471-5945-5-10Research ArticleCollecting a set of psoriasis family material through a patient organisation; clinical characterisation and presence of additional disorders Inerot Annica [email protected]äck Charlotta [email protected] Fredrik [email protected] Tommy [email protected] Lena [email protected]öm Jan [email protected] Gunnar [email protected] Department of Dermatology, Sahlgrenska University Hospital/Sahlgrenska, SE-413 45 Göteborg, Sweden2 Department of Clinical Genetics, Sahlgrenska University Hospital/Ostra, SE-416 85 Göteborg, Sweden2005 14 10 2005 5 10 10 3 2 2005 14 10 2005 Copyright © 2005 Inerot et al; licensee BioMed Central Ltd.2005Inerot 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 the present study was to describe the clinical characteristics of a population of psoriatics sampled from a patient organisation and not from hospitals or out-patient clinics. Furthermore, we wanted to compare siblings with and without psoriasis regarding the occurrence of other diseases.
Methods
At the end of 1991, we initiated a project which aimed to study genetic factors leading to psoriasis. Firstly, we sent questionnaires to all the members of the Swedish Psoriasis Association. We then examined 1,217 individuals (570 with psoriasis) from 310 families, in their homes in the southern part of Sweden. All the available family members were examined clinically and asked about the course of the skin disease and the occurrence of other diseases. The eight hundred members of the proband generation were divided into two groups, with or without psoriasis, and their clinical features were compared.
Results
Most individuals in this study population had a mild form of psoriasis. The siblings with psoriasis had joint complaints significantly more frequently than their siblings without the skin disease and those with joint complaints had more widespread skin disease. Among the other studied concomitant diseases (iritis, heart or hypertension disease, endocrine disease, inflammatory bowel disease and neurological disease), we were not able to find any difference. Seventy-seven of 570 persons were found to be in remission (13.5%). Females had a mean onset 2.5 years earlier than males. We were not able to find any correlation between the extent of the skin disease and age at onset. Twice as many persons with joint complaints were found among those with psoriasis than among those without, 28% versus 13%. Almost half (48%) the psoriatics who also had joint complaints had psoriasis lesions on their nails. Endocrine disorders were found in 9% of those without any allele for Cw6, but only in 1% of those who had Cw6. In fact, none of 183 Cw6 carriers had diabetes, as compared to the population prevalence of 3–5% in Sweden.
Conclusion
With the exception of joint complaints, persons with psoriasis, collected from a patient organisation, did not have an increased frequency of (studied) co-existing diseases.
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Background
Psoriasis is a common, genetically determined inflammatory, proliferative disease of the skin, in which the immune system is involved in the pathogenesis. Among individuals with psoriasis, a substantial number also have some involvement of the joint system, but for other concomitant diseases the evidence of a connecting relationship to psoriasis is not clear.
Examinations of persons with psoriasis in census studies have been made by Lomholt and Hellgren [1,2]. Many studies with examination descriptions come from hospitals or out-patient clinics [2-6]. The recent study by Gudjonsson et al [7] describing clinical characteristics in 369 patients with familial psoriasis was also community based. Questionnaire studies of psoriasis were introduced by Farber et al. [8] and have been followed by many others from different countries [9-12].
At the end of 1991, we initiated a project designed to elucidate the genetic factors involved in the pathogenesis of psoriasis [13]. We sent questionnaires to all the members of the Swedish Psoriasis Association, of which it is estimated that approximately 10% of all Swedish psoriatics are members. Individuals from the families were examined and blood samples were collected. Based on this patient sample, we have previously reported on the identification of a novel psoriasis susceptibility locus on 3q21 (PSORS5; MIM 604316) through a genome wide scan [14]. Furthermore, we have created a locus-specific SNP map and we have utilised it to narrow down the size of the candidate region to < 250 kb [15]. A candidate gene, SLC12A8, is currently under investigation. The aim of the present study was to describe the clinical characteristics of this population of psoriatics. In contrast to other studies, they were sampled from a patient organisation and not from hospitals or out-patient clinics and are therefore more likely to reflect the true clinical distribution of the disease. There have been reports suggesting a relationship between psoriasis and an increased risk of cardiovascular disease [16], as well as several lifestyle factors, such as smoking, obesity and diabetes [5,17,18]. We therefore wanted to compare siblings with and without psoriasis regarding the occurrence of other diseases.
Methods
The members of the Swedish Psoriasis Association constitute the study base. Approval was obtained from the local Ethics Committee. One of us (AI) examined 1,217 individuals from 310 different families. An analysis of the inheritance pattern from families of members of the Swedish Psoriasis Association was the reason for selecting families in which the inheritance could fit an autosomal recessive trait. We therefore selected families in which the parents did not have the skin disease (which is 64% of the material) and in which there were (in addition to the proband) one or more siblings with psoriasis. Persons living outside the Göteborg area were visited in their homes, or, if they preferred, we met them at a doctor's office in their neighbourhood. Blood samples were drawn at the same time. The majority of the examinations took place in September to May 1991–1998, in the southern part of Sweden up to a level about 150 km north of Stockholm. In families with two or more siblings, the examination rate for all family members was 93%. Some parents were not alive and a few individuals did not wish to take part in the study. Another reason for incomplete families was members living in another part of the country. The size of the families that were examined is shown in Table 1.
Table 1 Examined persons in all 310 families
Size of sibship Families * Proband generation Parent generation
N N with pso N total N with pso N total
1 78 73 78 6 36
2 99 154 198 11 154
3 70 140 210 8 127
4 32 77 128 3 54
5 17 49 85 0 28
6 6 19 36 1 9
7 4 13 28 2 7
8 1 3 8 0 2
9 1 4 9 0 0
10 2 7 20 0 0
Total 310 539 800 31 417
*All families are not complete. This is most obvious in the small families, where only half of the individuals were examined. These families were not ideal for our genetic study, but still represent the study population in this clinic examination study.
All individuals were inspected on the scalp, the face, the elbow area, the umbilical and perianal areas, the knees, hands and feet and also in other areas where they were aware of having any skin changes. The distribution of psoriasis was recorded if the person had any active psoriasis at examination on the scalp, face, trunk, anogenital area, arms, legs, hands, feet or nails, i.e. in nine different locations. The subjects were also asked about other medical problems. In addition to spontaneous information, they were asked especially about joint complaints (both peripheral joints and back involvement), iritis, problems with heart disease or hypertension, endocrine disease (mainly diabetes or thyroid disorder), inflammatory bowel disease or neurological disease. We felt it was not possible to obtain reliable information about psychiatric or malignant disorders when meeting several members of the family at the same time in a home environment for a relatively short time. Because of the age difference between the two generations, we have chosen to compare these diseases among the groups with and those without psoriasis in the proband generation only.
In 312 of the 800 individuals in the proband generation, we had information about the presence or absence of the allele Cw6 at the HLA-C locus. In previous studies from our group [19,20], psoriatics have been analysed for the presence of the Cw6 allele using a PCR-based typing method, as previously described.
Statistical analysis
The data were analysed using the chi-squared test with Yate's correction (comparison of two different groups). A p value of < 0.05 was considered significant. Some other statistical method was used when appropriate; this is mentioned in the text when applicable.
Clinical diagnosis
For the majority of the probands, a dermatologist had already established the diagnosis. When the patient was free of skin changes at the examination, the diagnosis was based on the medical history, including the name of the dermatologist who made the diagnosis. The diagnostic criteria were the same as those used in clinical practice. Sharply demarcated, infiltrated, red skin lesions with silvery white scaling were considered to be psoriasis. Isolated nail lesions were not considered sufficient for the diagnosis. If unclear, the diagnosis was not set. Pustular psoriasis was not included, if the person did not also have psoriasis vulgaris.
Results
Correctness of diagnosis
During the first year of examination, three probands turned out not to have psoriasis, although they thought they had it and had joined the patient organisation. They and their family members have been excluded from the study. Apart from these three, all the probands had been given a correct diagnosis. We found 51 persons with psoriasis, where the proband had originally said that his or her relatives did not have psoriasis; twenty-five were siblings, 11 mothers and 15 fathers. The sex of one-third of the probands giving these reports was male (17/51), which is the same proportion as in the whole material (111/310). Among the relatives, there were three persons whose siblings were thought possibly to have psoriasis by the probands, but this could not be verified at the examination. Three other siblings were reported by the probands to have psoriasis vulgaris, but it turned out to be psoriasis pustulosa. All the other relatives reported by the probands as having psoriasis could be verified as having the disease.
Age of onset and duration of psoriasis
In the proband generation, the ages at examination ranged from 16 to 79 years, with a mean of 40.5 years. The mean age for the 539 individuals with psoriasis was 40.2 years and the corresponding figure for the 261 persons without psoriasis was 41.3 years. There was no significant difference in age between men and women. Age of onset varied from the first year of life to 51 years of age. Females had a mean onset 2.5 years earlier than males (18.0 versus 20.5). The gender difference in age at onset was increased to 4 years when analyzing only HLA-Cw6-positive individuals and no difference was found for the HLA-Cw6-negative. The duration of psoriasis varied from not being aware of the diagnosis up to the age of 67 years (a woman aged 74, already examined in 1991), the mean duration being 21.0 years.
Remission
In some instances, the person who was being studied did not have enough changes in the skin at the time of examination for the dermatologist to be able to make the diagnosis at that time-point, even though information from previous examinations (mostly by dermatologists) or pictures from earlier outbreaks indicated psoriasis. In these cases, the person was diagnosed as having psoriasis, but that it was currently in remission. Seventy-seven of 570 persons were found to be in remission, (13.5%; 95% CI: 13.5+/-2.8%), 43 women and 34 men, average age 40.2.
Guttate psoriasis
Only 15 individuals (including two sibling pairs) spontaneously described a history of guttate psoriasis. One woman had had three guttate outbreaks. The highest age at the time of the outbreak was 40 years.
Twins
Eleven pairs of twins were examined, four monozygotic and seven dizygotic. Two of the monozygotic pairs were discordant with respect to psoriasis (ages: 51 and 57 years) and three of the dizygotic pairs were discordant in the same respect (ages: 29, 43 and 49 years).
Concomitant diseases
Joint complaints
If a person had or had had significant pain or aching localised to one or several joints (including back pain), he/she was classified as having "joint complaints", without trying to make a specific diagnosis. In a few persons who denied experiencing symptoms from joints, joint deformation like that caused by joint inflammation was seen at examination and these persons were also classified as having "joint complaints". There were more than twice as many persons with joint complaints among those with psoriasis than among those without, 28% versus 13%, and the difference was significant (p < 0.001). Among female psoriatics, 33% had joint complaints, while 21% of the men had joint complaints (p < 0.01). Nail changes were more common among psoriatics with joint complaints.
Iritis
In this case, there were very small numbers, with no difference between the groups.
Heart or hypertension disease
Considering that the mean age of these groups is around 40, low values would be expected and this is also what we found. There was no significant difference between the groups.
Endocrine disease
Four per cent reported a history of endocrine disorders, with no difference between those with and without psoriasis. Of the 32 persons with endocrine disorders, 19 had diabetes mellitus, 13 thyroid dysfunction (one of them had both disorders) and one had a disease of the parathyroid glands. The prevalence of diabetes mellitus among those people with psoriasis was 2%, while among siblings without psoriasis the corresponding figure was 4%. This is not a statistically-significant difference.
Inflammatory bowel disease
Of all the people in the proband generation, 19 individuals (3.5%) claimed to have inflammatory bowel disease. No difference was found between the two groups in the proband generation.
Neurological disease
Around 3% reported a history of some neurological disease. There was no significant difference between the groups in the proband generation.
Information about diseases above are summarised in Table 2.
Table 2 Concomitant diseases at examinations in the proband generation
Siblings with psoriasis = 539 Siblings without psoriasis = 261
N = 800 "Yes" % "No" MV "Yes" % "No" MV
Joint complaints 149 27.7 389 1 35 13.4 226 0
Iritis 8 1.5 530 1 3 1.1 258 0
Heart or hypertension disease 36 6.7 499 4 21 10.4 181 59
Endocrine disease 20 3.7 516 3 12 5.9 190 59
Inflammatory bowel disease 13 2.4 523 3 6 3.0 196 59
Neurological disease 20 3.7 516 3 4 2.0 198 59
Numbers under "Yes" and "No" refer to individuals. For details see text. MV = missing value.
Alopecia areata and vitiligo
Alopecia areata at examination or in the medical history was found in six persons, three of whom had psoriasis. Vitiligo was found in nine persons; only one of them had psoriasis.
Location and extent of psoriasis, gender differences
The overall impression was that most people had a mild form of the skin disease. The examined probands (n = 302) had an average number of 4.4 different locations for their skin disease (men 5.1, women 3.9). Comparing these means produced a significant difference (p < 0.001) (Table 3).
Table 3 Severity of psoriasis, measured as number of locations affected at examination
Males Females
N sum of locations N sum of locations
average/median average/median
302 members/probands 110 5.1/5 192 3.9/4
237 siblings/not members 135 2.8/3 102 2.3/2
All 539 with psoriasis 245 3.9/4 294 3.4/3
Male members of the psoriasis association have a more extended skin disease than females. Non members with psoriasis have fewer locations affected.
Men and women in the proband generation who were not themselves members of the Psoriasis Association had a lower average number of psoriasis locations, 2.8 and 2.3 respectively (NS). Persons with joint complaints had more widespread skin disease than those without joint complaints. This difference was highly significant (p < 0.001), comparing medians (5.0 and 3.0) (rank sum test).
We were not able to find any correlation between the extent of the skin disease and age, even if we divided the material into men and women.
The most common finding was psoriasis on the arms and legs. Less common locations were the feet and the face. The anogenital area was affected in 24.3% of all the examined persons (Figure 1).
Figure 1 Body distribution of psoriasis. Body distribution of psoriasis among the 570 individuals with the diagnosis. In addition to the 539 persons in the proband generation, 31 in the parent generation, who also had psoriasis, have been included.
Men had psoriasis of the face more frequently than women. This difference was significant (p < 0.01). Men also had psoriasis more frequently on the scalp and in the anogenital area. The p-value for this difference was 0.03 for both scalp and anogenital location. The rest of the locations were distributed with no gender difference. To look for heredity in psoriasis when it came to severity, we compared 132 sibling pairs. We were not able to find any correlation between siblings from the same family and the severity of the skin disease, measured as the number of locations with involvement. Nor were we able to find any relationship between the extent of the disease and the presence or absence of an allele for Cw6.
Location of psoriasis and the occurrence of concomitant disease
Almost half (48%) of those psoriatics who also had joint complaints had psoriasis lesions on their nails, compared with 31% in the group without joint complaints. The chi-squared test showed that this was a highly significant difference (p < 0.001). A significant difference (p < 0.001) was also seen if there was anogenital psoriasis. A statistically-significant difference was also found if there was psoriasis on the trunk or the legs (p = 0.02). For the rest of the locations, we were not able to find any significant differences, after examining all the recorded locations and concomitant diseases.
HLA-Cw6
For 312 of all 800 in the proband generation, we had information about the presence or absence of the Cw6 allele at the HLA-C locus in the HLA region on chromosome 6 p. When examining other concomitant diseases, we found a difference in the prevalence of endocrine disease. This group of disorders was found in 9% of those without any allele for Cw6, but only in 1% of those who had Cw6. This difference is highly significant (p = 0.002), but it is based on very small numbers (11 of 119 without Cw6 and two of 183 with Cw6). Both persons with Cw6 and endocrine disorders had thyroid disease and neither had diabetes mellitus. Among the 436 for whom we had no information about the Cw6 status, but information about endocrine disease, the prevalence was 4%.
Discussion
The psoriasis family material examined in this study was recruited from a well-informed patient organisation, i.e. the Swedish Psoriasis Association, and not, as in most other studies, from hospitals or out-patient clinics. These individuals are more likely to be representative of most persons with psoriasis than patients from hospitals. Furthermore, this family study of psoriasis is the largest in which all the individuals have been examined by one dermatologist and no previous study has compared the occurrence of concomitant diseases in siblings without psoriasis. The distribution of psoriasis was about the same as that reported by Lomholt [1,3] and Fleischer et al. [12] and partly also like that reported by Hellgren [2] and Farber and Nall [21]. The majority of the individuals had a mild disease and an estimation of the PASI score for the average psoriasis person in this study would probably not exceed 4 (theoretical range 0–72). We have used a simple method of recording any active psoriasis lesion in nine different body regions, instead of using the PASI score [22]. When it came to the extent of the skin disease, we found a difference between men and women who were members of the Swedish Psoriasis Association. However, among the siblings, we were not able to find a significant difference between men and women. As the siblings are seldom members of the association, it is probable that they are a better reflection of the country's population. Lomholt [1] found no difference between men and women when visiting people in their homes, but Molin [3], who investigated 300 former in-patients with psoriasis, found that men had a more severe skin disease, when it came to both the extent of lesions and the course. Fleischer et al. [12], who used a self-administered psoriasis area severity index score, found a significant difference between men and women, with men having a higher score. In a questionnaire survey of the Psoriasis Society of the Greater Helsinki area, Könönen et al. [9] also found more severe and more frequent skin lesions in men. In a study from Island based on 369 psoriatics, Cw6-positive patients was found to have more extensive plaques and overall disease severity [7]. However, we were not able to find any correlation between the prevalence of Cw6 and the extent of the skin disease, in agreement with previous observations on 64 patients by Ikähemo et al. [23]. A possible reason why we did not find any difference in (disease) severity correlated to the age at onset or Cw6 may be because the individuals with psoriasis were homogeneous in that most of them had very mild disease.
The overall gender difference in age at onset was 2.5 years in accordance with many other studies [3,21]. However, this difference was increased in the Cw6-positive group (4 years) and was not found at all in the Cw6-negative group in accordance with the observations of Gudjonsson et al [7].
There are not many reports of periods of remission in psoriasis. We found that 13.5% (77/570) were free of disease at examination. Lomholt [1] found 11% (28/252), Nevitt and Hutchinson [24] 9% of 75 examined and, in a Danish study of twins [25], 13 of 48 (26%) were free from skin lesions at examination.
We felt it was not possible to obtain reliable information about psychiatric or malignant disorders when meeting several members of the family at the same time in a home environment for a relatively short time. Some individuals reported atopic dermatitis in the group both with and without psoriasis, but we did not ask specific questions about this diagnosis and we found this information unreliable for analysis.
Eleven pairs of twins among 800 siblings are in the range of what can be expected. About half of them were concordant with respect to psoriasis, among both monozygotic and dizygotic pairs. This is too small a number to draw any conclusions about the heredity of psoriasis.
It is difficult to compare the frequency of co-existing or concomitant diseases with that in other studies, as the study design varies a great deal. Probably the most important factor that can result in very different figures is the selection of the study population. The study by Lindegård [26] reflected the population of psoriatics requiring hospitalisation in the 1970s and this cannot be compared with the main population of psoriatics at the present time. In a small sample of 33 consecutive psoriatics in our own investigation, only two had been hospitalised because of their skin disease. It is our impression that this is also reflected in the whole of our material.
The problem of defining a concomitant disease adds to the selection problem when attempting to compare different studies. This is illustrated by the many different prevalence figures relating to joint complaints and/or arthritis in connection with psoriasis [27,28]. We found that 28% of psoriatics and 13% of their siblings without the skin disease had joint complaints, including back pain. In our study, women had significantly more joint complaints than men, which has previously been shown by Farber et al. [8]. We cannot rule out the possibility that there might be individuals with rheumatoid arthritis in our "joint complaints" group, especially among women. The fact that joint complaints are more common if the person has psoriatic nail lesions has been known for many decades [29]. Previous investigations have also shown a relationship between the occurrence of joint complaints and the presence of psoriasis on the buttocks and in the groin [11] and between a genital localisation of psoriasis in men and arthritis [3]. Our observation that there are more joint complaints if there are skin lesions in the anogenital area lends further strength to these observations. We also found that there was an association between the extent of psoriasis (number of locations) and joint complaints, as previously observed by several investigators [3,9,12,30]. Lomholt [1] found only four persons with psoriasis arthritis among 253 native Faroese with psoriasis. He found no one with diabetes mellitus and stated that, in his clinical practice, he also very seldom found diabetes together with psoriasis. The average age of his studied population was around 10 years less than that of the proband generation in our investigation. We now know that the presence of Cw6 is associated with the early onset of psoriasis [19,20]. and we can therefore speculate that many of the psoriatics in Lomholt's study could have had an allele for Cw6. In our material, none of the persons with a known presence of Cw6 had diabetes mellitus. If Cw6 protects from diabetes mellitus, this could perhaps be a reason why psoriasis vulgaris has stayed in the population. However, it is of importance to add that Cw6 might not be the primary association in the HLA region. It has not been made clear whether the association reflects the fact that Cw6 is the true disease-causing allele or is due to linkage disequilibrium with a nearby located gene. Like Farber et al. in 1968 [8], we found a 2% prevalence of diabetes mellitus in the proband generation with psoriasis. In a later report from the same author [8] comprising more than 5,600 patients, a total of 3.5% of the psoriasis patients reported having diabetes mellitus. The prevalence of diabetes mellitus in the general population in Sweden is reported to range from 3–5% [31,32]. In our study, thyroid disorders were found in 13 individuals (= 1.6%). By way of comparison, a study in Malmö [33] found thyroid disorders in 7.9% of a population aged around 45–50 years (men 1.5%, women 14.1%).
In the large hospital-based data register from Kiel, Henseler & Christophers found more diabetes, heart failure and obesity in psoriatics, compared with age-matched patients with other skin diseases [5]. We were not able to verify this in our study population. Our findings are in agreement with what was found in the case of psoriasis out-patients in a large register study from Sweden [16]. When it came to hypertension and heart disease, we found a prevalence of 6–10% in the proband generation (mean age: 40). In the case of unspecified "heart disease", it is difficult to find figures for comparison in previous studies among the general population, but, in a study of about 7,000 middle-aged men, Eriksson & Lindgärde [33] found that 5% were receiving drug treatment for hypertension. Psoriasis in-patients both in Finland (1973–1984) [17] and in Sweden (1964–1995) [16] had an increased mortality rate compared with the general population. In Finland, analyses of the causes of death revealed excess mortality related to alcohol and smoking. In the parent generation, only five persons (1.2%) reported having inflammatory bowel disease, compared with 3.5% in the proband generation. It is known [34] that this group of diseases has increased in the population and the age at onset is often below 40 years, but the prevalence in the general population is reported to be below 1% [34-36].
In conclusion, we have analysed a large set of family material comprising psoriasis patients, healthy siblings and their parents collected through a patient organisation. The clinical features and the presence of other diseases were recorded and compared. All the individuals in the study were examined by the same dermatologist. It is likely that this study design provides a better representation of psoriasis in general than when patients collected from hospital cohorts are used. The patient material characterised in this study has been used for the identification of the PSORS5 gene locus and it will be the subject of further genetic and clinical study in the near future.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
GS initiated the psoriasis genetic project and together with JW and TM designed the study. CE performed the genetic analysis. AI examined the persons in the families and drafted the manuscript. All authors contributed and approved the final manuscript
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The psoriasis genetic project has been supported by the Swedish Psoriasis Association, the Welander Foundation, the Swedish Medical Research Council (project no. B96-19X-11246-02B) and Glaxo-Wellcome Research Foundation and Development Ltd.
We thank Tommy Johnsson for statistical guidance.
==== Refs
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BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-5-491620970510.1186/1471-2148-5-49Research ArticleLate Quaternary loss of genetic diversity in muskox (Ovibos) MacPhee Ross DE [email protected] Alexei N [email protected] Dick [email protected] Alex D [email protected] Division of Vertebrate Zoology, American Museum of Natural History, New York, New York 10024, USA2 Laboratory of Mammals, Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, 199034, St. Petersburg, Russia3 Cerpolex/Mammuthus, Gudumholm 41, NL-2133 HG Hoofddorp, Netherlands4 GSF-National Research Center for Environment and Health, Institute of Molecular Virology, Ingolstaedter Landstr.1, 85764 Neuherberg, Germany2005 6 10 2005 5 49 49 1 7 2005 6 10 2005 Copyright © 2005 MacPhee et al; licensee BioMed Central Ltd.2005MacPhee 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 modern wildherd of the tundra muskox (Ovibos moschatus) is native only to the New World (northern North America and Greenland), and its genetic diversity is notably low. However, like several other megafaunal mammals, muskoxen enjoyed a holarctic distribution during the late Pleistocene. To investigate whether collapse in range and loss of diversity might be correlated, we collected mitochondrial sequence data (hypervariable region and cytochrome b) from muskox fossil material recovered from localities in northeastern Asia and the Arctic Archipelago of northern North America, dating from late Pleistocene to late Holocene, and compared our results to existing databases for modern muskoxen.
Results
Two classes of haplotypes were detected in the fossil material. "Surviving haplotypes" (SHs), closely similar or identical to haplotypes found in modern muskoxen and ranging in age from ~22,000 to ~160 yrbp, were found in all New World samples as well as some samples from northeastern Asia. "Extinct haplotypes" (EHs), dating between ~44,000 and ~18,000 yrbp, were found only in material from the Taimyr Peninsula and New Siberian Islands in northeastern Asia. EHs were not found in the Holocene muskoxen specimens available for this study, nor have they been found in other studies of extant muskox populations.
Conclusion
We provisionally interpret this evidence as showing that genetic variability was reduced in muskoxen after the Last Glacial Maximum but before the mid-Holocene, or roughly within the interval 18,000-4,000 yrbp. Narrowing this gap further will require the recovery of more fossils and additional genetic information from this interval.
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Background
The large-mammal (megafaunal) diversity of North America and Eurasia was considerably reduced by extinctions that occurred around the time of the Pleistocene/Holocene transition (i.e., ca. 10,000 years ago) [1]. Interestingly, some megafaunal species (e.g., woolly mammoth, Mammuthus primigenius) became extinct at this time in North American and Eurasian continental areas but survived on nearby islands for thousands of years thereafter [2,3]. Some other species whose ranges likewise straddled the Old and New Worlds became extinct on one continent, but not the other, at the end of the Pleistocene (e.g., horse, Equus caballus; saiga, Saiga tatarica). A third pattern, involving a much lengthier decline, characterizes Ovibos moschatus (tundra muskox), the subject of this paper (fig. 1, 2).
Figure 1 Sketch map of Holarctic region in polar view, noting places mentioned in text and specimens sampled for mtDNA analysis. Dark gray area, muskox range ca. AD 1870 [6]; C, cape; Cr, creek; I, island; L, lake; R, river. During the late Pleistocene, Ovibos ranged from western Europe to North America via Beringia. In the Old World, presence of muskox fossils on islands north of mainland Asia presumably indicates that Ovibos was able to extend its range during full glacial times to the subaerial parts of the continental shelf (dashed line). In the New World, in addition to central Alaska, unglaciated areas in the western Arctic Archipelago may have continuously supported muskoxen also, although the finite 14C fossil record for these islands does not begin until about 6800 yrbp [40].
Figure 2 Finite 14C date distributions for fossil specimens of Ovibos. (A) northern North America and Greenland (N = 72; X = 3222 yr, SD = 4313 yr) and (B) Arctic periphery of Asia east of Kara Sea (N = 38; X = 22,522 yr, SD = 11,145 yr), collated from the literature [32,40,41]. In addition to a notable difference in dating intensity, with many more published 14C dates being available for New World Ovibos, there is also a considerable difference in sample statistics. Skewed date distribution for New World Ovibos reflects long-term effect of former icecaps on amount of habitable area [42]. In northeastern Asia, where only small, local (cordilleran) icecaps formed, potential range was less affected and Ovibos was presumably continuously present during the last 40–50,000 years. Star in B denotes 7,000 yr gap between "last" Pleistocene and "first" Holocene dated occurrences of Ovibos in Asia. Slight offset in "period of heightened extinction" in New World vs. Old World, amounting to approximately 500–1000 yr, reflects a moderate difference in "last" occurrence dates for mainland mammoths in the two areas [32].
According to the available fossil record, the lineage terminally represented by O. moschatus arose in Eurasia but entered North America at least as early as the early Pleistocene [4-6]. At roughly the same time several other closely-related Asian ovibovine lineages (e.g., Soergelia, Praeovibos) also entered North America, where they evidently prospered before disappearing later in the Pleistocene. By the start of the Holocene, muskox diversity had been reduced to surviving O. moschatus; the species is now native only to the islands of the Arctic Archipelago and Greenland (fig. 1), if late 20th-century efforts to reintroduce muskoxen into various parts of their ancient range are ignored [6].
This lengthy history of muskox diversification in Quaternary Holarctica stands in sharp contrast to the extremely limited genetic diversity characterizing modern O. moschatus. Groves' [7,8] analysis of sequence data for cytochrome b (cyt b) and the mitochondrial hypervariable region in 37 individuals of O. moschatus from various localities in Alaska, Canada, and Greenland revealed only marginal genetic differentiation within extant populations. Groves [7] identified a total of 8 hypervariable region haplotypes, but these differed individually by only 1 or 2 nucleotide substitutions in approximately two-thirds of the cases, for a maximum of 9 differences – a total of 1.3% variation across 697 bp (base pairs), including insertions and deletions. Extreme genetic homogeneity is also indicated by microsatellite analysis [[9], but see also [10]] and the nuclear Mhc DRB3 gene [11]. As several different populations were sampled for these studies, in the absence of any other plausible explanation it seems reasonable to conclude that living muskoxen simply lack genetic diversity. The nuclear and mitochondrial data therefore complement each other and imply that muskoxen either passed through a long period of very small effective population sizes, or experienced one or more genetic bottlenecks in very recent times. The investigation reported here was prompted by the possibility that ancient DNA (aDNA) analyses might shed some light on the complex history of extirpation, replacement, and differential survival that have evidently affected muskoxen populations during the late Quaternary. Ancient DNA analysis has been successfully applied to late Pleistocene and Holocene (including the early 20th century) remains by various groups [e.g., [12-22]]. Because of existing uncertainty concerning species boundaries among some late Quaternary members of tribe Ovibovini, the terms "ovibovine" and "muskox" will be used to make general reference to such taxa, without endorsing any particular view as to their taxonomic distinctiveness or geographical distribution.
Results
Our results permit empirical recognition of two haplotype groupings within late Quaternary muskoxen. The first consists of haplotypes recovered from Pleistocene samples collected at various localities in northeastern Asia, including islands north of the mainland (fig. 1, 3). These haplotypes, which differ from all New World material so far examined as well as from the few known Asian specimens that are of Holocene age, appear to have completely disappeared and may therefore be grouped as "EHs" (extinct haplotypes). All other known haplotypes, which include those found in all living muskoxen, are very similar inter se and thus comprise a second grouping, "SHs" (surviving haplotypes).
Figure 3 Timeline showing distribution of specimens yielding sequence information. Symbols indicate whether a given specimen exhibited an "extinct" (EH) or a "surviving" (SH) haplotype. Specimens yielding substantial sequence data are in bold. SHs (i.e., haplotypes represented in extant O. moschatus) were found in all New World samples tested, as well as some Old World samples. EHs have been found so far only in Asian samples.
Sequence verification
A total of 9 fossil samples yielded reproducible sequence covering cyt b and the hypervariable region of the mtDNA genome; with the exception of OMTai14, each fragment from these samples was successfully amplified at least twice (additional fig. 1, 2, 3; table 1). One other sample (OMBolL15564) yielded consistent results for cyt b but the HV sequence could not be accurately scored (table 1, additional fig. 4). Sequences were then determined via multiple clones produced from each PCR product, a standard aDNA protocol for insuring correct sequence determination [12,22] (additional fig. 1, 2, 3). In addition, several samples were independently extracted by different individuals, amplified, and cloned. Clones were sequenced in one or another of the three separate laboratories involved in this investigation (AMNH, New York; Medigenomix GmbH, Munich; SR&D GmbH, Martinsried).
Table 1 Information on bone samples utilized in aDNA studies, with 14C age estimates
Haplotype Grouping
a
aDNA Sample Designation Specimen Institutional Number
b
Age
c
Dating Lab Number
d
Cyt b Reps
e
HV Reps
e
Specimen Locality
EH group OMTai95 CME 2000/125 44,760 ± 1700 B177044 1x/1x 1x/0x skull Lake Taimyr, Taimyr Pen.
OMBolL15564 ZIN 15564 40,270 ± 450 B193436 2x/2x 4x/3x skull Bol'shoi Lyakhovski I., New Siberian Is.
OMTai39 CME 2000/002 22,550 ± 100 B148652 2x/2x 2x/2x metapodial Lake Taimyr, Taimyr Pen.
OMTai14 CME unnumb. 22,360 ± 80 B156194 1x/1x 2x/2x metapodial Lake Taimyr, Taimyr Pen.
OMTai46 CME 2000/102 19.230 ± 80 B148627 2x/2x 2x/3x atlas Lake Taimyr, Taimyr Pen.
OMTai38 CME 2000/126 18,310 ± 70 B148628 2x/2x 2x/2x horn sheath Lake Taimyr, Taimyr Pen.
SH group OMWra2 ZIN unnumb. 22,280 ± 120 B193434 0x/2x 0x/2x unspecified Wrangel I., Chuckchi Sea
OMYak12 ZIN unnumb. 15,610 ± 80 B193435 0x/1x 0x/2x pelvis Anabar R., Yakutia
OMTai31670 ZIN 31670 3,790 ± 80f LU 52 0x/0x 1x/2x skull Cape Chelyuskin, Taimyr Pen.
OMYuk01 CMN 36137 3,280 ± 90h I10985 2x/2x 2x/2x skull Sixtymile Creek, Yukon
OMTai23658 ZIN 23658 2,970 ± 40g B157714 2x/2x 2x/5x skull Cape Chelyuskin, Taimyr Pen.
OMArA09 CMN 17674 2,520 ± 100h I3577 0x/0x 0x/1x horncore Banks I., Arctic Archipelago
OMArA03 CMN 34515 2,400 ± 40 B179009 2x/2x 2x/2x skull Bathurst I., Arctic Archipelago
OMYuk02 CMN 17678 1,020 ± 40 B179008 0x/1x 0x/1x horn core Herschel I., Yukon
OMArA05 CMN 49941 700 ± 70h TO2703 2x/2x 2x/2x humerus Axel Heiberg I., Arctic Archipelago
OMArA04 CMN 17676 160 ± 40 B195369 2x/2x 2x/2x horncore Banks I., Arctic Archipelago
OMModC1 male zoo animal (modern) - 1x/1x - hair Tierpark Hellabrunn, Munich
Notes. – aEH, extinct haplotype group; SH, surviving haplotype group (see text). For clarity, in table EH and SH samples are separated by a horizontal line; italics distinguish Pleistocene samples.
bInstitutional abbreviations: CME, Cerpolex/Mammuthus Expeditions, Khatanga; CMN, Canadian Museum of Nature, Ottawa; ZIN, Zoological Institute, Russian Academy of Sciences, St. Petersburg.
cIn 14C yrbp ± 1 σ (not δ13C corrected).
dRadiocarbon dating lab abbreviations: B, Beta Analytic, Inc.; TO, IsoTrace Laboratories, University of Toronto; I, Isotopes (Teledyne); LU, Geological Institute, Leningrad State University.
eEntries indicate whether sequence was obtained for each of 2 overlapping fragments; number indicates number of times bases in fragments were separately covered by PCRs (e.g., 2x/2x indicates each overlapping fragment was amplified and sequenced twice independently).
fC.R. Harington (pers. commun.), correcting date of "3800 ± 200 yrbp" reported by [43].
gAlso dated by Harington [44], who reported an age estimate of 2910 ± 95 yrbp.
hAs reported by Harington [44].
For example, to determine the cyt b sequence for sample OMTai38, two PCRs were performed for the first fragment and second overlapping fragment and five clones sequenced for each PCR product (additional fig. 2). The sequences summarized in tables 3, 4, 5 are thus the consensus of independent replications and multiple-clone sequences for each individual PCR fragment (with a few exceptions), and are unlikely to be laboratory artifacts (additional fig. 1, 2, 3). For this particular sample, all clones from the 3' fragments were identical with one exception: in clone 1.2, there is a C-T transition which most likely represents a polymerase error. In the 5' fragment, all five clones of the first PCR were identical. In the second fragment three classes of sequence were obtained, represented by clones 2.3 and 2.4; these were identical to the clones from the first PCR and matched the 3' fragment in the overlap. The second and third class of sequences (2.1, 2.2, and 2.5) were different from all other clones obtained in the study, unique to the second PCR amplification, and did not match the 3' fragment in the overlap. Blast searching the sequence revealed perfect or near perfect homology for both divergent sequences with other bovid species such as the domestic sheep, Ovis aries. These likely represent nuclear mitochondrial DNA sequences (Numts) and thus the sequence represented in the two independent fragments was scored as the bona fide mtDNA sequence, the consensus of which was used for further study.
Table 3 DNA sequence variation in the cyt b gene
Source Sample 324 346 366 425 436
Modern SH-OMMod01 T A T A C
SH-OMMod02 . . . . .
SH-OMMod03 . . . . .
SH-OMMod04 . . . . .
SH-OMMod05 . . . . .
SH-OMModCl . . . . .
New World SH-OMYuk01 . . . . .
SH-OMArA03 . . . . .
SH-OMArA04 . . . . .
SH-OMArA05 . . . . .
Old World SH-Wra02 nd nd . . .
SH-OM23658 . . . . .
EH-OMTai14 C C C . T
EH-OMTai38 C C C . .
EH-OMTai39 C C C . T
EH-OMTai46 C C C . .
Notes. – Sample names and symbols as in table 2. Positions numbered from first base of ATG start codon of cyt b. Extinct haplotypes italicized; "nd", not determined.
Table 4 Sequence comparisons between Holocene and Pleistocene samples
Cyt b HV region
Holocene Pleistocene Holocene Pleistocene
Number of individuals 43 5 44 4
Number of haplotypes 1 3 4 2
Haplotype diversity 0 0.667 ± 0.204 0.215 ± 0.081 0.667 ± 0.204
Nucleotide diversity 0 0.00585 ± 0.00179 0.00149 ± 0.00062 0.0113 ± 0.00346
Transitions/Transversions 0 3/1 4/0 3/2
Notes. – Samples were divided into two populations for comparison: > 10,000 yr old (Pleistocene) and < 10,000 yr old (Holocene).
Table 5 Summary of variable sites for 1,180 bp of cyt b sequence, OMTai39 compared to 5 modern muskox
Sample 37 85 136 174 213 246 324 345 365 435
SH-OMMod01 T T T C G A T A T C
SH-OMMod02 . G . . . . . . . .
SH-OMMod03 . G . . . G . . . .
SH-OMMod04 A . . . . . . . . .
SH-OMMod05 . . C T . . . . . .
EH-OMTai39 . . . . A . C C C T
Notes. – Sample names and symbols as in table 2. Positions numbered from A of ATG start codon. Extinct haplotype italicized.
This example represents a relatively extreme case, as most PCRs yielded identical or nearly identical sequences that differed by minor among-clone variations that can be explained by DNA polymerase/sequencing errors or DNA damage in the templates (for example, note the clone sequences for OMTai39 in additional fig. 2). Where a position or several positions were not determinable after two independent amplifications, additional PCRs were performed, clones sequenced, and the majority base scored as the bona fide mtDNA sequence for each position of each sequence. In other words, each base of each sequence was covered by multiple PCRs and multiple clone sequences to determine the majority base for a given position as in Krings et al. [12].
In addition, six other samples (OMTai95, OMWra2, OMTai31670, OMYuk02, OMArA09, and OMYak12) yielded some sequence information, but the amount of DNA recovered was insufficient to permit two independent replications for each fragment and/or retrieve all four fragments of interest in this study (see table 1).
Overlapping PCR reactions were used to identify possible Numts and to prevent mischaracterizing Numts as organellar mtDNA [14,23]. In some species DNA extracted from hair may yield Numts at a higher frequency than do other tissues [24], making it a useful proxy for gauging the possibility of Numt contamination. A hair sample (OMModCl, table 1) from a living muskox was amplified, products cloned, and multiple sequences determined using several of the primer combinations used in this study. Sequences were consistent in the overlapping regions and matched DNA from other modern muskoxen, indicating that the primers used in this study do not favor Numts over organellar mtDNA (additional fig. 1, 2).
Cytochrome b
A 114-bp segment of cyt b was successfully retrieved from most of the working fossil samples (table 3); in three cases (OMTai14, 39, and 46) it was possible to amplify a 376 bp fragment. All specimens identified as SHs that yielded cyt b sequence were identical. Only partial sequence data could be recovered for OMWra2 and OMYak12 (table 3). However, for comparable runs of sequence, these specimens were identical to all other SHs.
Owing to institutional sampling restrictions and the low quality of retrieved DNA, analysis of OMTai95 had to be based on one repetition of each of the two overlapping fragments. Although results for this specimen are therefore tentative, its sequence does not differ from that of OMTai38 and OMTai 46 (not shown). Thus, despite some individual variation, in all cases samples could be classed unambiguously as either EH or SH.
Specimens in the EH group differed uniformly from those classed as SH by three fixed differences (table 3, positions 324, 346, and 366). There were also some internal differences within the EH group. OMTai14 and OMTai39 possessed an additional base substitution not seen in other members (position 436), and OMBolL15564 expressed a unique A to G substitution at position 425. Given the difficulties encountered in determining the mitochondrial HV region sequence for this last sample, this result was excluded from analysis.
The Holocene samples (N = 43) exhibited no variations. Only one haplotype was retrieved (table 4), which is consistent with Groves and Shields' [8] evidence for limited among-individual variation at the cyt b locus in living muskoxen. By contrast, the Pleistocene samples yielded 2 haplotypes among 4 sequences and thus greater among-individual haplotype and nucleotide diversity in a much smaller sample.
As a proxy for resolving the magnitude of differences among cyt b haplotypes, 1,180 bp of sequence in overlapping fragments was determined for one of the best-performing specimens, OMTai39 (table 5) and aligned by eye against 5 modern muskox cyt b sequences deposited in GenBank (accession numbers U17862 and U90300-U90303, here designated as OMMod01-05). Among modern muskoxen, within-group differences in cyt b sequences range from 1 to 4 base substitutions (table 5). By comparison, OMTai39 differed from all modern samples by 5–7 substitutions. Domestic sheep (Ovis aries) [GenBank: NC001941] and goat (Capra hircus) [GenBank: NC005044] were used as outgroups for phylogenetic analysis. The takin Budorcas taxicolor was not included in the analysis as Groves and Shields [8] have shown that the relationship between this species and muskox is not demonstrably closer than that of other caprines. When submitted to phylogenetic testing using maximum parsimony, neighbor joining, and maximum likelihood methods, OMTai39 placed in all analyses outside modern muskoxen cyt b sequences (bootstrap support, 80–98%) supporting the distinctiveness of Pleistocene vs. Holocene haplotypes (fig. 4).
Figure 4 Cyt b (1,180 bp) from a representative EH specimen (OMTai39) compared to modern Ovibos moschatus (OMMod01-05). Maximum parsimony tree using goat and sheep as outgroups. Calculated bootstrap support is shown for parsimony/neighbor joining/maximum likelihood at each node. Although analysis returned an evaluation of "ns" (no statistical support) for a given grouping in only one instance, significance of results should not be overemphasized. Nevertheless, it is parsimonious to conclude that (1) all muskoxen form a cluster as against sheep and goat, and (2) moderns form a structureless subcluster as against the extinct haplotype represented by OMTai39.
Hypervariable region
Samples from Yukon and islands in the Canadian Arctic Archipelago (OMYuk01, OMArA03, OMArA04, and OMArA05) displayed minor haplotype variation affecting 2–4 positions in the studied portion of the hypervariable region (additional fig. 1). No fixed differences were found between these samples and sequence data on modern muskoxen in GenBank [7](cf. OMMod01-08, OMModCl, table 5).
One position in the GenBank samples was not observed in any of the fossil sequences, nor was it found in a modern muskox sequence (OMModC1) that was acquired specifically to test results from this study (table 6, position 168). Because all of the sequences presented here are consensus results, generated from multiple PCRs and multiple clones sequenced on both strands, the difference in the database for modern samples is best interpreted as a sequencing artifact in the published sequences.
Table 6 Summary of variable positions in the hypervariable region of modern and ancient muskoxen
Source Sample 2 104 144 145 148 166 168 178
Modern SH-OMMod01 C C G C C T C T
SH-OMMod02 . . . . . . . .
SH-OMMod03 . . . . . . T .
SH-OMMod04 . . . . . . T C
SH-OMMod05 . . . . . . . .
SH-OMMod06 . . . . . . . .
SH-OMMod07 . . . . . . T .
SH-OMMod08 . . . . . . T .
SH-OMModCl . . A . . . - .
New World SH-Yuk01 . . A T . . - .
SH-OMArA03 . . . . . . - .
SH-OMArA04 . . . . . . - .
SH-OMArA05 . . . . . . - .
Old World SH-OMWra2 nd . . . . . - .
SH-OMYak12 nd . . . . . - .
SH-OMTai23658.A . . . . . . - .
SH-OMTai23658.B . . A . . . - .
EH-OMTai14 G T A . . . - .
EH-OMTai38 . T A . T A - .
EH-OMTai39 G T A . . . - .
EH-OMTai46 . T A . T A - .
Notes. – Sample names from table 1. Base positions numbered from first readable base from 5' amplified fragment. Identity to first sequence designated by a dot; differences indicated by base sequence. Deletions indicated by "-". For some samples, specific bases were not covered; these are designated "nd" (not determined). [Accession numbers for modern sequences are GenBank: NC005044, U47063, U47065, U47067, U47069, U47071, U47073, U47075.] Extinct haplotypes as determined for cyt b and HV region are italicized.
OMTai23658, one of two Holocene samples from Taimyr, yielded two unique haplotypes for the hypervariable region, identified here as A and B (table 6, position 144). Multiple PCRs, cloning, and sequencing of clones by two independent laboratories established that these haplotypes differ by 1 base, the difference likely being due to heteroplasmy (additional fig. 1; see also [26]). Haplotype A differed by 0–2 positions and haplotype B by 1–3 positions from sequences recovered for other fossil muskoxen from the Canadian Arctic Archipelago. The difference between both haplotypes of OMTai23658 and modern muskoxen was 1–3 substitutions.
All of the other successfully sequenced samples from Taimyr (OMTai14, OMTai38, OMTai39, and OMTai46) are of Pleistocene age (> 10,000 years ago). They were found to differ by 0–3 substitutions from one another but by 2–6 positions from modern muskoxen and the Canadian fossil samples. In addition, all the Pleistocene Taimyr samples presented 1 fixed difference not observed in any of the other samples studied (table 6, position 104), while OMTai38 and OMTai46 shared 2 base changes not found in any other specimen group (table 6, positions 148 and 166). OMTai14 and OMTai39 shared one position not found in any other specimen group (table 6, position 2).
The DNA yield from one other sample, OMBolL15564 from the New Siberian Islands, was low. However, it yielded reproducible EH sequence for cyt b. This is consistent with the sample's origin (near Taimyr) and age (late Pleistocene). Unfortunately, the HV region yielded several different sequences per amplification, including sequences that were similar to SH sequences, EH sequences, and ones that diverge from all others encountered in the course of this study (additional fig. 4). Since results for this specimen were highly inconsistent, we suspect some form of sample contamination but cannot identify the likely source. This required that we remove it form further analysis.
As may be seen in table 4, the Pleistocene sequences had more transversions (2 of 5 mutations) and slightly higher haplotype and nucleotide diversity per site measured than did Holocene sequences. However, given the almost 9-fold higher representation of Holocene compared to Pleistocene sequences, the statistics should be treated with caution. Higher among-sequence diversity (i.e., 2 haplotypes from 4 sequences as opposed to 4 from 44) for Holocene samples supports the concept that genetic diversity in muskoxen has been restricted. This is also consistent with the results obtained for cyt b (table 4).
To further clarify among-sequence relationships, we depict the sequence network suggested by our data in figure 5: nodes reflect missing intermediates and correlate with the number of mutational steps between sequences. As expected, all haplotypes of the SH group are very closely related. Most of the extinct haplotypes recovered differ somewhat among themselves, and all Taimyr EHs differ more substantially from SHs than do SHs among themselves. The distance between the extinct haplotypes and the closest SH sample is two to three mutational steps. The network supports the distinctiveness of most of the HV haplotypes of Pleistocene age.
Figure 5 Minimum spanning network for hypervariable region sequences. The network is derived from the alignment shown in table 6; though indicated, the indel was omitted as it is a likely sequencing artifact in the database. Identical sequences are grouped together; EH samples are indicated by shading. The mutational change and sample mean radiocarbon age ranges (in yrbp) are shown next to each branch.
Discussion
In view of the small number of specimens analyzed, conclusions have to be tentative. The conclusion best supported by the data is that, compared to modern muskoxen, late Quaternary Ovibos populations were less homogeneous for the loci sampled in this investigation. In particular, one group of haplotypes (EHs) occurring in fossil specimens from northeastern Asia differs from haplotypes in existing databases (SHs). The actual number of differences is not great (see Results), but their existence is thrown into sharp relief by the virtual lack of variation in the modern wildherd. In view of the multiple safeguards utilized to insure accuracy and repeatability (extraction of several samples in independent laboratories, sequencing of multiple independent clones from two or more amplifications, using overlapping PCR fragments to determine sequences, and rejection of suspect or ambiguous results), we feel that we can exclude laboratory artifacts or DNA damage as the source of these differences.
When the extinct haplotypes might have disappeared is not known. In our sample, the latest accepted record (of 5) is 18,310 ± 70 yrbp (radiocarbon years before present), which tells us that populations in which these haplotypes occurred survived at least into the Late Glacial Maximum, but not whether they lasted until the period of intensified megafaunal extinction at the end of the Pleistocene approximately 10,000 years ago (although in view of the extensive end-Pleistocene radiocarbon record for Ovibos in Asia [32], we regard this as more likely than not).
Another conclusion that is supported by the limited evidence currently available is that the SH group was present in mid-Holocene Asian muskoxen. This inference is based on the fact that OMTai23658 from Taimyr yielded sequence data indistinguishable from modern. (We had less success with the only other Eurasian Holocene sample available to us, OMTai31670. For the sequences covered it was initially found to be identical to modern muskox, but repeated efforts to replicate results were unsuccessful.) Given available data, however, it is premature to conclude that EHs were definitely not present as late as the middle Holocene. If in the future other specimens of Holocene age come to light in Asia, it would be well worthwhile to sample them for aDNA studies.
A last point of interest is that Ovibos, one of the few high-latitude megafaunal mammals to have survived into recent times, has clearly done so with reduced genetic variability. As already noted, at what point before the present this variability was lost cannot be satisfactorily established with existing data. For species for which aDNA sequence information is substantially better, such as bears and bison, there is molecular evidence for repeated loss and turnover of haplotype diversity during the late Quaternary (cf. [19,20,27]). Although no aDNA investigation of the giant Irish deer, Megaloceros, has yet been published, Stuart et al. [28] have recently shown that the radiocarbon record for this taxon reveals dramatic changes in population distributions during the late Pleistocene, followed ultimately by total collapse in the mid-Holocene. Thus, the data presented here, in combination with studies performed on other megafaunal species, is consistent with a scenario in which genetic diversity in muskoxen was reduced prior to the Pleistocene/Holocene transition.
It would also be of great interest to know whether late Quaternary small mammals (few of which became extinct) were affected by population crashes at the same time as larger species were. Extensive work on the mitochondrial genetics and phylogeography of extant lemmings by Fedorov and coworkers [29,30] suggests that, in these rodents at least, bottlenecking and catastrophic loss of genetic diversity did not occur in late glacial/postglacial time. The robustness of this inference could be tested with aDNA methods similar to the ones utilized here, as there are large samples of reasonably well-dated fossil lemming material in the paleontological collections of various museums (cf. [31]).
Conclusion
In summary, the combined genetic and chronometric evidence presented in this paper establishes that Ovibos moschatus was genetically more diverse in the late Pleistocene than it is today. Precisely when haplotypes not found in extant muskoxen were lost, and under what conditions, are matters that cannot be well constrained at present. However, the data decisively show that now-extinct haplotypes persisted in Asia to a point only a few thousand years earlier than the accepted onset of the end-Pleistocene extinctions. It is reasonable to predict that, with better sampling, it may eventually be possible to show that populations of Ovibos moschatus, far from passing through this period of heightened extinction unscathed, were in fact brought down to small numbers over much of their original range, perhaps almost to the vanishing point [32]. Why muskoxen should have survived nevertheless in these circumstances, while many other high-latitude species did not, is yet another puzzle within the overarching mystery of late Quaternary extinctions.
Methods
Samples
Despite their wide holarctic distribution during the late Quaternary, ovibovines are comparatively rare as fossils except in those few areas (e.g., Taimyr Peninsula, parts of Yukon; fig. 1) where conditions were evidently optimal for them during much of the late Pleistocene. We obtained our specimens from two broad areas, designated "New World" and "Old World" (respectively, northern North America including the Arctic Archipelago, and the Arctic periphery of Asia east of the Kara Sea). A total of 31 muskoxen samples were subjected to DNA extraction and PCR amplification using various primer combinations. Of these, 16 samples worked with varying degrees of success; table 1 and fig. 3 provide information on these specimens regarding locality, age, and other relevant data (see also additional figs. 1, 2, 3, 4). The 15 remaining samples, from Alaska, Russia, and the Canadian Arctic Archipelago, did not produce amplifiable DNA on extraction and are not shown. It is particularly unfortunate that none of the Alaskan material yielded DNA. There may be an age effect: Mateus et al. [33] showed that muskoxen were common in Alaska earlier in the Pleistocene, but became very rare after 40,000 yrbp (which is the upper practical limit of radiocarbon dating). Designations combine taxon acronym/geographical origin/sample number: thus OMArA05 stands for Ovibos moschatus/Arctic Archipelago/sample 5.
Radiocarbon dating record
All specimens listed in table 1 had either been subsampled for 14C dating as part of this study or had been dated previously by other workers. Age estimates are cited in radiocarbon years before present without δ13C correction. All Beta Analytic estimates are accelerator dates on the collagen fraction of submitted specimens and typically have low associated standard errors. Other dates quoted in the table are based on older liquid scintillation technology which required large samples. However, accuracy and precision are not necessarily poorer in the latter case: OMTai23658 has been dated by both methods, and mean age estimates are statistically indistinguishable at 2 sigmas (table 1).
All samples yielded 13C/12C ratios within normal ranges for the materials dated (usually bone), and no difficulties with any samples were reported by the radiocarbon laboratory. Radiocarbon age estimates in excess of 40,000 yrbp are generally considered to be of questionable accuracy, and specimens so dated could be considerably older [37]. Although EH samples are generally older than SH samples, there is overlap around the time of the Last Glacial Maximum 21,000 calendar years ago [38], indicating that haplotype groups were coeval. Greater temporal overlap would probably have been found had sample sizes been larger.
Fig. 2 collates published finite 14C records for the study areas in the Old and New Worlds. Note that the distribution of dates in the New World record is quite asymmetrical, with few early dates and a heavy concentration in the late Holocene. A similar asymmetry is seen in fig. 3, which depicts the date distribution of specimens used in this study. The explanation concerns substantial differences in the amount of habitable range available to muskoxen during the period covered by radiocarbon dating. Because almost all of Canada and Greenland were covered by icecaps in the last (late Wisconsinan) cold phase, few early muskox dates would be expected from this area. Following icecap retreat, the muskoxen record increases markedly, especially after the mid-Holocene with return of a significant biomass of vascular plants at high latitudes [39]. By contrast, the Arctic periphery of Asia was not significantly glaciated, and muskox were evidently present throughout this zone during the latest Pleistocene, but rare or absent thereafter until ~4,000 yrbp when they made a brief reapperance.
DNA extraction, PCR, and sequencing
All fossil samples were collected by the first author using an electric drill with stainless steel "keyhole" coring drill bits that were cleaned and air dried after each use. Samples were placed in individual plastic containers, labeled, and stored while in the field in cool conditions and on return in a -20°C freezer in an isolated lab. Protocols for DNA extractions, PCR conditions, PCR reamplification, cloning strategy, colony PCR and sequencing, have been described in detail in Krings et al. [12] and Greenwood [34], with the variant that (for most samples) DNA extractions were conducted with NucleoSpin® DNA-Trace Kits in combination with NucleoSpin® funnel columns (Macherey & Nagel), Gene Clean Kit (Qbiogene), or GOLD Forensic DNA Kit (peqlab) according to manufacturer protocols and eluted in 55 to 60μl 10 mM Tris-HCl, pH 8.5. Reamplifications of PCR products when necessary were done either by direct amplification from of an aliquot of the first PCR or subsequent to gel extraction of the primary amplification band using a Qiagen Gel Extraction Kit. A buffer control was taken through each extraction procedure with no sample added and used as a PCR negative control in each PCR reaction to control for contamination. PCR primers, combinations used, and sequences are shown in table 2. Modern hair and blood DNA were prepared in a separate laboratory and never brought to laboratories conducting aDNA work so as to avoid potential contamination. Both hair and blood were prepared using Qiagen QIAamp DNA Mini Kits and PCR performed using Roche Expand Taq or Promega Taq polymerase according to manufacturer instructions. PCR conditions used were 30 cycles of 94°C 30 seconds, 50°C 30 seconds and 72°C 30 seconds. Products were cloned and clones sequenced as described in Greenwood (2002).
Table 2 Primers used in reconstructing cyt b and HV sequences
Target Primer combination Primer sequence Notes
Main cyt b primers L1 + H1 L1: CCTATACTACGGATCATACA
H1: AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA
L2 + H2 L2: GGGGGAATTCTTCTACTCA
H2: GCTGAGAGGAGGTTAGTGA
Additional cyt b primers L3 + H3 L3: CGAAGCTTGATATGAAAAACCATCGTTG
H3: GTAGAATTAGGCAGATACCT a
L4 + H4 L4: TCAAACATCTCATCATGATG
H4: GTAGAAGAATTACCCCGATG a
L5 + H5 L5: CTCCCATTTATCATCGTAGC
H5: TTGATCGTAGGATTGCGTAT a
L6 + H6 L6: TCACATTAAACCAGAGTGAT
H6: GGCATTAGCACCAGGATGAT a
L2 + H7 H7: TGGATCCTGTTTCGTGGAGG3 b
L7 + H1 L7: AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA c
Main HV region primers L1 + H1 L1: AAAGGATCCTAAACTACTCCCTGAATT
H1: AAAGGATCCATCATGCGTTGTTGCGT
L2 + H2 L2: AAAGAATTCCCAGTATCAAATCTACC
H2: AAAGGATCCGCTTGTGTTACATATGTCT
Additional HV region primers L3 + H3 L3: CACCCAAAGCTGAAGTTCTA
H3: GTTGTTGCGTGTGGAGTAGG d
Notes. – For certain samples, some primer pairs were more successful than others. Additional primer pairs were applied to a few samples during the course of the study as noted below. The Numt test for sample OMModCl was performed using primer combinations: cyt b L1 + H1, L2 + H2, L7 + H1, HV region L1 + H1, L2 + H2. All primers are shown 5' to 3'.
aPrimers used to amplify nearly full length cyt b from OMTai39.
bAdditional primer combination used on OMTai38, 39 and 46.
cAdditional primer combination used on OMTai14 and 46.
dAdditional primer combination used on OMTai14, 39, and 46.
Negative water controls and in many cases mock extraction controls were included in experiments to control for contamination. Extractions, amplification, and sequencing for samples OMTai23658, OMTai39, OMTai46, OMYuk01, OMArA03, OMArA04, and OMArA05 were performed independently at SR&D and Medigenomix; in each case the same sequences were obtained. Each amplified region was covered at least twice if possible; multiple clones were sequenced to determine the correct sequence and to avoid mis-scoring errors due to DNA damage [35]. In the few cases in which available material was insufficient for double coverage, the sequence result is regarded as tentative. The number of replications for each PCR for each sample is indicated in table 1. Sequences were also derived from overlapping PCR fragments as previously described [12] (see additional fig. 1, 2, 3). [All consensus sequences have been deposited with the following accession numbers in GenBank: AY839538-AY839551 (cyt b); AY839552-AY839563 (hypervariable region); and AY83956 (the only substantially complete cyt b sequence retrieved in this study for OMTai39).]
Phylogenetic analysis
Maximum parsimony, neighbor joining, and maximum likelihood analyses were executed using PAUP 4.0b10 [36] with Ovis (sheep) and Capra (goat) as outgroups for the nearly-complete cyt b sequence of OMTai39 (fig. 4). Although 100 bootstrap replicates were performed for each tree generated, the limited variability present in the data requires that no great weight be placed on the results of such experiments (i.e., chances are high that the few variable sites will be included in the bootstrap and will therefore nearly always replicate the same tree). Nevertheless, a strictly parsimonious interpretation of the distribution of certain fixed differences (see text) indicates that sequence data can be used to diagnostically distinguish extinct from surviving haplotypes. Networks for the hypervariable region sequences derived from the alignment shown in table 6 were drawn by hand (fig. 5).
Authors' contributions
RDEM participated in the design of the study, coordinated the drafting of the paper with the other authors, acquired and interpreted radiocarbon dates, and helped with phylogenetic analysis; ANT participated in the drafting of the paper and provided data and information from Russian sources; DM managed acquisition of specimens for sequence analysis in Russia and Canada, and participated in the drafting of the paper; and ADG participated in the design of the study, oversaw all lab work, sequence alignment, and phylogenetic analysis, and participated in drafting the paper. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Alignment of hypervariable region clones from overlapping PCR fragments used to determine the sequences for each specimen analyzed. The consensus sequence of OMModCl was used as a reference. Dots indicate identity to the consensus sequence. Differences are indicated. Base pair sequences that could not be determined for a given clone are marked "N". Gaps are indicated by "-". The first clone provides the name of each sample according to the labels provided in table 1. The numbers indicate PCR replication + clone number: thus 1.1 indicates PCR 1, first clone sequence.
Click here for file
Additional File 2
Alignment of cyt bclones from overlapping PCR fragments used to determine the sequences for each specimen analyzed. Symbols as in additional fig. 1.
Click here for file
Additional File 3
Alignment of clones from overlapping PCR fragments used to determine 1,180 bp of OMTai39 cyt b. Symbols as in additional fig. 1. The reference is a modern muskox sequence taken from GenBank. Four additional modern muskox sequences are included in the alignment.
Click here for file
Additional File 4
Clone sequences derived from multiple amplifications of sample OMBolL15564. All symbols are the same as in additional fig. 1, 2, 3. The modern DNA consensus sequence from additional fig. 1 is used as a reference sequence.
Click here for file
Acknowledgements
The authors gratefully acknowledge financial support by grants from the National Science Foundation OPP 0117400 (RDEM, ADG), the Niarchos Fund (RDEM), and the Evelyn Stefansson Nef Foundation (RDEM). The authors wish to thank Lars Giesen (Medigenomix GmbH, Germany), Simone Watzinger and Jörg Hauf (Scientific Research and Development GmbH, Germany) for excellent technical assistance. For access to specimens and other favors, we thank Richard Harington, Alison Murray, Kieran Shepherd (all of the Canadian Museum of Nature, Ottawa), Meng Jin (American Museum of Natural History, New York), and Bernard Buigues (Cerpolex/Mammuthus Project, Khatanga). We are grateful to Mike Bunce, Eske Willerslev, Hendrik Poinar, and especially Claudia Englbrecht for helpful discussions. Patricia Wynne executed the base map used for figure 1 and Laura Carter helped with the editing of the final manuscript.
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BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-5-511620971110.1186/1471-2148-5-51Research ArticleTracking Alu evolution in New World primates Ray David A [email protected] Mark A [email protected] Department of Biological Sciences, Biological Computation and Visualization Center, Center for Bio-Modular Multiscale Systems, Louisiana State University, Baton Rouge, LA, 70803, USA2 Department of Biology, West Virginia University, Morgantown, WV, 26506, USA2005 6 10 2005 5 51 51 22 6 2005 6 10 2005 Copyright © 2005 Ray and Batzer; licensee BioMed Central Ltd.2005Ray and Batzer; 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
Alu elements are Short INterspersed Elements (SINEs) in primate genomes that have proven useful as markers for studying genome evolution, population biology and phylogenetics. Most of these applications, however, have been limited to humans and their nearest relatives, chimpanzees. In an effort to expand our understanding of Alu sequence evolution and to increase the applicability of these markers to non-human primate biology, we have analyzed available Alu sequences for loci specific to platyrrhine (New World) primates.
Results
Branching patterns along an Alu sequence phylogeny indicate three major classes of platyrrhine-specific Alu sequences. Sequence comparisons further reveal at least three New World monkey-specific subfamilies; AluTa7, AluTa10, and AluTa15. Two of these subfamilies appear to be derived from a gene conversion event that has produced a recently active fusion of AluSc- and AluSp-type elements. This is a novel mode of origin for new Alu subfamilies.
Conclusion
The use of Alu elements as genetic markers in studies of genome evolution, phylogenetics, and population biology has been very productive when applied to humans. The characterization of these three new Alu subfamilies not only increases our understanding of Alu sequence evolution in primates, but also opens the door to the application of these genetic markers outside the hominid lineage.
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Background
SINEs (Short INterspersed Elements) are powerful tools for systematic and population biologists [1-8]. Examples of phylogenies elucidated using the SINE method include the use of SINEs to support the hypothesis that cetaceans (whales, dolphins and porpoises) form a clade within Artiodactyla [9], clarification of relationships between cichlid fishes [10-12] and the resolution of the human-chimpanzee-gorilla trichotomy [5]. Although applications of SINE elements to resolve population dynamics have been limited to humans [13-19] and, to a lesser extent, cichlid fishes [11,20,21], these studies have been very successful in revealing patterns of variation and there is every reason to believe that they can be as productively applied to other species.
One reason for the success of SINEs as phylogenetic and population genetic markers is that their mode of evolution is unidirectional [3,4,7,8,22]. This characteristic allows for a confident inference that the ancestral state is the absence of the SINE at each locus. Because there is no known mechanism for the specific removal of SINEs from any genome [4,23], individual SINEs are generally thought to be homoplasy-free characters [4,7,17,22-25].
Alu elements are primate-specific SINEs of ~300 bp. These elements have been extremely successful at propagating in primate genomes as evidenced by the fact that they make up ~10% of the human genome by mass [23,26]. Distinct subfamilies of Alu elements in the human genome have been described in detail [17,18,23,27-32]. Examination of these young subfamilies has provided us with clues as to the mobilization dynamics and evolution of Alu elements in the hominid lineage. Characterization of Alu mobilization in non-human primates has not been as complete. The ascertainment of lineage-specific subfamilies of Alu elements would increase our understanding of mobile element evolution in these organisms and allow for the development of SINE-based studies of population and evolutionary patterns.
We recently used Alu insertion loci to clarify various relationships among platyrrhine (New World monkeys, NWM) and cercopithecid (Old World monkeys) primates [33,34]. These projects produced examples of Alu insertions present in a wide variety of lineages along the primate tree. We have performed a phylogenetic analysis of the Alu sequences themselves (focusing on the platyrrhine-specific insertions) in order to characterize the evolutionary history of Alu lineages that have been or currently are retrotransposition competent in some non-human primates.
Results and discussion
Platyrrhine-specific Alu sequences were obtained from the data sets used in Ray et al. [34] When available, the sequences from multiple taxa at a particular locus were aligned and a consensus sequence generated to create an approximation of the sequence of the original insertion. A total of 48 platyrrhine-specific insertions were collected. All selected sequences were examined for the presence of target site duplications (TSDs). The presence of these TSDs along with the absence of each marker in hominid and cercopithecid taxa (and from the genomes of other platyrrhine primates in many cases) serves to verify that the elements are the result of retrotransposition events and not segmental duplications. To trim potentially long branches and to verify the ability of the approach to recover previously established relationships among reference sequences, we added the consensus sequences for Alu elements specific to hominids (AluYa5, AluYa5a2, AluYb8, AluYb9, AluYc1, AluYc2, AluYd3, AluYd6, and AluYe5) [18,30-32,35-37]. We also included the canonical Alu consensus sequences for the Jb, Sc, Sg, Sp, Sq, Sx, and Y subfamilies [38-40] and rooted the tree on AluJb based on previously established relationships [40-42].
The methods used to identify informative loci among cercopithecid taxa primarily involved a linker-PCR strategy using two Alu selection primers [33]. Unfortunately, this introduced a sequence bias toward particular subfamilies of recently integrated or lineage specific Alu elements. The strategy used to identify informative platyrrhine loci, on the other hand, used a combined computational-experimental approach. Over half of the loci identified were derived from Bacterial Artificial Chromosome (BAC) sequences and thus no bias was introduced. In addition, a wide variety of primers was used in the linker-PCR approach; as a consequence, the bias was reduced for experimentally-derived loci. Because of the bias in the data derived from the cercopithecids, we have not included these Alu sequences in the analyses. For platyrrhine Alu lineages, however, more confident inferences can be made.
Tree topologies recovered using Bayesian and parsimony criteria were generally congruent (Fig. 1). Minor differences in the placement of some sequences are observed but the well-supported clades recovered by the Bayesian analysis are often present in the parsimony consensus trees with reasonable support (>75%). However, bootstrap support on the parsimony-based cladogram was not as high for several of the major nodes found on the Bayesian tree. We suspect that this is due to the hybrid (partially gene converted) nature of 31 sequences that share diagnostic features of both AluSc-derived and AluSp derived elements (see below for a full discussion). Given the assumptions inherent in parsimony-based analyses (i.e. incremental sequence-based changes) hybrid elements that accumulated a whole suite of character states as a unit and that define other lineages in the data set would be expected to cause significant problems. Supplemental analyses with the hybrid elements removed confirmed this suspicion by raising support values at some nodes over 20 points (data not shown). The reduced tree-search method used is also thought to recover lower bootstrap support values than more traditional methods [43]. For these reasons, we have chosen to base our major conclusions on the topology and support values present on the Bayesian tree.
Figure 1 A) Majority-rules consensus of 10,000 trees generated using a Bayesian approach. Support values greater than 0.85 are indicated on relevant nodes. The major platyrrhine clades (A, B, C, and D) are indicated. Within clade D, members of subfamily AluTa10 are underlined. B) Majority-rules consensus tree of 107,470 equally parsimonious trees generated as described in the Methods section. Bootstrap values for nodes with greater than 50% support are indicated. Sequences representing well-supported clades from the Bayesian tree are also indicated.
Within that tree, the established relationships between canonical Alu consensus sequences were recovered as expected. The AluJb subfamily is basal to the remaining Alu sequences and relationships between the various AluS subfamilies are similar to the results of Kapitonov and Jurka [39]. Among the New World primate Alu sequences all but three platyrrhine-specific sequences fall within a well supported AluSc-AluY derived clade. This topology suggests that at there may have been three Alu lineages active at the time of the platyrrhine-catarrhine divergence around 35–40 million years ago [44]: an AluY progenitor; AluSp; and, AluSc. The three platyrrhine-specific Alu insertions that clustered outside the major platyrrhine AluSc/AluY-derived clade were 'All_NWM_Locus_1', 'All_NWM_Locus_15', and 'All_NWM_Locus_31'. Each of these insertions is present in all tested platyrrhine taxa, suggesting that they occurred before the radiation of the New World monkeys into three recognized families, (Cebidae, Atelidae and Pitheciidae). The Alu sequence at 'All_NWM_Locus_1' appears to be derived from an AluSp source gene. Direct observation of the Alu sequence confirms the presence of several AluSp diagnostic sites in this element (see supplemental alignments). Based on our analyses, the sequence for 'All_NWM_Locus_15' appears to be derived from an AluY progenitor. There is no significant support for the node, however, and it should be noted that this is merely a suggestion based on the topology of the tree. Thus, an AluY progenitor, AluSp, and AluSc were all active around the time of the split. The source of the sequence at 'All_NWM_Locus_31' is unclear given the differences in placement between the Bayesian and parsimony analyses. RepeatMasker [45] lists the element as belonging to the AluSg lineage. Thus, it may represent a fourth lineage that was active early in the evolution of New World monkeys.
A majority of the Alu sequences specific to various New World monkeys are most closely related to an AluSc and there are four well-supported clades within this group. Clade A is represented by two sequences that were found only in members of Pitheciidae. The insertions 'Callicebus_83' and 'Pithecia_46', were specific to their respective Pitheciid genera, and they share eight exclusive non-CpG mutations when compared to AluSc and other AluSc-like sequences (Bayesian support = 1.00). The close relationship between these sequences was also recovered in the parsimony analysis. While we will not assign them to a new subfamily based on only two sequences, we suggest that they are good candidates for a Pitheciid-specific lineage.
A second clade (B) within the putative AluSc-derived group was also highly supported (0.99) and was represented an insertion identified in all platyrrhine primates ('All_NWM_Locus_26'), as well as in two Atelid taxa ('Lago_and_Atel_20') and in all members of Cebidae and Atelidae ('Cebid_Atelid_Locus_14'). These sequences may represent an AluSc-derived subfamily. However, this cluster was based on only a few sequences and on shared mutations at CpG sites; thus, it should be interpreted cautiously. An alternative is that these and the other elements in this group represent true AluSc insertions that have continued to accumulate in platyrrhine genomes throughout their evolution. This is not unlikely given the recent observations of potentially polymorphic AluSc loci [46] and relatively recent AluSx insertions in humans [47]. The 'stealth' model of Alu evolution and dispersal reported by Han et al. [48] also predicts low levels of activity for older Alu subfamiles. AluSc may represent a hardy subfamily that has remained active at a low level for long periods of time in a variety of primate genomes.
Clade C (support = 0.99) comprises five sequences characterized by 11 shared mutations (including a 7-base duplication) that distinguish them from AluSc. Sequences in this clade are distributed among members of families Pitheciidae and Atelidae. One interpretation of this pattern is the emergence of the source gene prior to the expansion of a Pitheciid-Cebid clade but after the divergence of Atelid taxa. This hypothesis is unlikely, however, given the results of Ray et al. [34] in which it was made clear that family Pitheciidae was the first to diverge from the early platyrrhine groups. We suggest instead that the source gene emerged after the divergence of platyrrhine and catarrhine primates but before the platyrrhine radiation 17–20 mya [49,50], and that none of these elements was recovered for Cebid taxa due to sampling error. Additional work will be required to test this hypothesis.
Clade D is the largest of the clearly definable platyrrhine Alu clades, comprising 31 sequences from all three platyrrhine families. It is well-supported (1.00) and is distinguished by numerous shared mutations among its members. Of the new subfamilies described here, this lineage is particularly interesting because of its apparently unique origin. Close examination of the sequences reveals four shared AluSc diagnostic mutations at the 5' end of the elements; however, at the 3' end of the elements, there are five additional diagnostic sites characteristic of the AluSp subfamily. Examples of 'hybrid' elements have been described previously [17,25,29], but these represented individual instances involving the gene conversion of Alu elements already present in the genome. That does not appear to be the case here.
The presence of 31 distinct elements harboring this combination of AluSc and AluSp diagnostic mutations (plus three additional shared mutations) suggests that there is a recently active source gene with these characteristics. We propose that a source gene (most likely derived from AluSc) existed early in platyrrhine primate evolution and that the 3' end of the element was subjected to a gene conversion event via any of the three potential models described by Kass et al. [51]. Starting somewhere between bases 199 and 226 and continuing to the end of the element, the conversion event resulted in the replacement of the sequence of the source gene with sequence from an AluSp-like element (Fig. 2). The result was a 'fusion' element that remained active and may still be active in the genomes of several platyrrhine primates.
Figure 2 Multiple sequence alignment of three canonical reference sequences (AluJo, AluSc, and AluSp) with the new consensus sequences described in this work. Identical sequence residues are indicated by ".". Indels events are indicated by "-". Diagnostic mutations characteristic of AluSc and AluSp that are shared by the new consensus sequences are shaded. Substitutions distinguishing all AluT subfamily members from AluSc are boxed.
This group of elements can be further subdivided into two subfamilies based on additional shared diagnostic mutations in what appears to be the more recently derived subfamily. In addition to the AluSp and AluSc derived sites and the three additional distinguishing sites, 21 elements share four unique mutations. Thus, clade D can be subdivided into two subfamilies consisting of 10 and 21 elements, respectively (see supplemental alignments).
These two subfamilies share two diagnostic positions with the previously mentioned clade C 5' to the appearance of the AluSp indicative sites. Thus, we believe that these three groups of sequences represent a new platyrrhine-specific subfamily we dubbed AluT. We chose this designation based on the nomenclature proposed by Batzer et al. [38] in which younger subfamilies are assigned later letters of the alphabet. This is followed by a lowercase letter designating the order of publication, and a numerical designation indicating the number of diagnostic sites that differentiate it from the subfamily consensus. Because this group was similar to and apparently derived from AluSc, AluT was most appropriate. It is distinguished from AluSc by the two aforementioned diagnostic mutations and can currently be divided into three subfamilies; AluTa7, AluTa10, and AluTa15 (Fig. 2). For reference, we have included a hypothetical AluT consensus sequence based on the diagnostic sites shared by the Ta5, Ta10, and Ta15 consensi and the presumed ancestral sequence, AluSc, in figure 2.
Represented by 21 sequences, AluTa15 was only found in Cebid taxa (Aotus, Callithrix, and Siamiri). AluTa10 is represented by ten sequences and was recovered in members of all three platyrrhine families. The distribution of this subfamily of elements among platyrrine taxa and the pattern of shared diagnostic sites suggest that the AluTa10 family expanded earlier in platyrrhine evolution and may have given rise to the AluTa15 subfamily. A larger sample based solely on elements derived from unbiased methods will be required to test this hypothesis and is currently underway.
Conclusion
The identification of three (potentially four) new subfamilies that are unique to platyrrhine primates represents a step forward in our understanding of the evolution of Alu elements in the genomes of non-hominid primates. Further, this is the first report of a unique mechanism of Alu subfamily generation. Until now, the evolution of Alu subfamilies could easily be described using the sequential accumulation of diagnostic mutations. For example, the hominid Alu subfamily AluYb currently consists of four variants, Yb7, Yb8, Yb9, and Yb11 [30,31,52]. Patterns of sequence variation clearly illustrate the hierarchical nature of sequence evolution in this family. Yb9 exhibits all of the diagnostic mutations defining AluYb7 and AluYb8 as well as its own signature mutation. AluYb11 follows suit by exhibiting all of the AluYb9 mutations plus two others. This pattern is confirmed using age estimates that suggest AluYb7 is the oldest and AluYb11 is the youngest. The AluTa10 and AluTa15 subfamilies represent the first documented cases of a recently active 'fusion' element in which the diagnostic mutations were not accumulated gradually over time; instead, they represent the sudden incorporation of several signature mutations by way of a gene conversion event. Thus, a new mechanism of Alu subfamily generation, though previously considered possible [29], has now been substantiated in the genome.
On a more practical level, a number of questions raised in other taxonomic analyses of New World monkeys can now be better addressed [1,34,53-60] given the data presented here. We can confidently assign subfamily status to certain individual Alu elements in platyrrhine genomes. Thus, we are able to target particular Alu subfamilies with known expansion timeframes to address branching patterns for particular primate lineages. This technique has previously proven valuable. For example, by combining a targeted analysis of the AluYe5 subfamily with sequence database searches for additional informative loci, we were able to confidently address the human-chimpanzee-gorilla trichotomy [5]. Application of similar techniques to other primates can easily be adapted by using the linker protocols described in Ray et al. [34], Xing et al. [33] and Roy et al. [61] and by computational analyses of existing sequence data.
At the population level, the amplification dynamics of Alu elements have been well characterized in humans and even in chimpanzees, but have not been investigated extensively in other primates. This is unfortunate given their utility in studies of genome evolution in humans and chimpanzees [62-64], population biology in humans [13,15,16,27,65-74], and phylogenetic analysis at all levels of the primate tree [2,5,6,33,34,41,75]. Knowledge of these subfamilies will aid in the development of markers useful for all of the above tasks. For example, given the endangered status of many New World taxa, the existence of easy-to-ascertain markers (via a single PCR) to identify species-specific Alu insertions in tissues of unknown origin will be a boon to conservation biologists and to population geneticists. Similar genetic systems have already proven useful in other taxa ranging from humans to waterfowl [76-78]. As one simple example, we now use many of the Alu loci used in this study to verify the identity of cell lines in our laboratory. Using a single PCR to amplify a taxon-specific Alu insertion is quick and efficient when compared to methods that involve morphological analysis (if possible on a tissue sample) or amplification and sequencing of DNA.
In this study we have identified diagnostic mutations for platyrrhine specific subfamilies. The identification of particular Alu lineages is the critical first step in identifying polymorphic elements in a primate taxon [17,18,31,61]. By identifying the subfamilies that are specific to particular taxa, researchers are now better able to use previously established techniques that take advantage of diagnostic mutations to identify useful markers at various taxonomic levels. The essentially homoplasy free nature of SINE markers makes them in some ways superior to other commonly used markers for population genetics [3,4,10,12,22,34]. Thus we see this as the beginning of a series of studies in which the SINE method of population genetic analysis will be expanded beyond our own species.
Methods
Insertion/deletion (indels) events play a significant role in defining Alu subfamilies. For this reason, the phylogenetic method we used to reconstruct relationships was based primarily on the Bayesian method implemented by MrBayes, Ver. 3.1 [79,80]. We chose this method because of its robustness and its ability to take advantage of information present in the form of insertion/deletion events in the alignment. We partitioned the data into two sets, sequence data and gap data. For partition one, sequence parameters were estimated from the data The second partition was generated using indels that were present in two or more sequences. These were coded as present (sequence) or absent (gap). For the second data partition, we estimated rates of indel occurrence from the data and corrected for ascertainment bias by setting the coding option to 'variable' as per the MrBayes manual.
Two simultaneous Markov chain Monte Carlo analyses were performed using one cold and three heated chains (temperature set to default 0.2) for each analysis. We ran the analysis for 7.5 million generations, sampling the trees every 100 generations. At ~6.13. million generations, the standard deviation of split frequencies consistently reached a value of <0.01, indicating that both analyses had begun converging on similar trees. We discarded the first 6.5 million generations as burn-in and generated a majority-rules consensus tree. Nodes with probability values of 0.85 to 0.89 were considered to have low support, 0.90 to 0.94 to have moderate support and nodes greater than 0.95 to be highly supported [80].
As a comparison, we also performed a parsimony analysis of the data in PAUP* v4.0b10 [81]. Non-CpG dinucleotides were weighted at six times the value of CpG dinucleotides [82] and gaps were treated as a fifth character state. The size of the data set made a bootstrap analysis using a full heuristic search for each replicate impractical. For this reason, we employed a reduced tree-search bootstrapping method as described by DeBry and Olmstead [43] to ascertain support for nodes.
The sequences from each clearly defined clade (see Results and Discussion) were collected and examined for shared mutations that presumably represent diagnostic mutations or positions characteristic of mobile element subfamilies. Consensus sequences for each of these groups were constructed. For non-CpG sites, a simple majority-rules approach was taken to obtain the consensus for the site. Alu elements, however, are rich in CpG dinucleotides that are known to mutate at a 6-fold higher rate than non-CpG sites [82]. These sites tend to be highly variable and could represent a problem when determining the identity. We addressed this issue by examining types of variation at potential CpG sites and by referring to the presumed ancestral sequences. First, dinucleotide sites exhibiting high diversity that comprised primarily both CpA and TpG dinucleotides were assumed to be highly mutable CpG sites that decayed as the result of the spontaneous deamination of 5-methylcytosine. When it remained unclear whether or not the site should be considered a CpG dinucleotide, we referred to the AluSc or AluSp consensus sequences to determine the likely ancestral state for the site and made the appropriate assignment.
Sequence alignments used for phylogenetic analysis and for the generation of consensus sequences are available online as additional files.
List of abbreviations
NWM – New world monkeys
Mya – million years ago
hLRT – hierarchical likelihood ratio test
AIC – Akaike Information Criterion
Indels – insertion/deletion events
Authors' contributions
DAR initiated the study, collected and aligned all of the sequences used in the project, performed all analyses, interpreted the data, and prepared the manuscript. MAB provided input on the analysis and interpretation of the data and on all versions of the manuscript.
Supplementary Material
Additional File 1
Multiple sequence alignment of all Alu sequences used in this study. Clades of sequences described in the text are delimited and the newly described consensus sequences are included.
Click here for file
Acknowledgements
We thank S. Sen, J. Walker, M. Konkel, and S. Herke for comments on earlier versions of the manuscript. This research was supported by National Institutes of Health RO1 GM 59290 (MAB), National Science Foundation BCS-0218338 (MAB) and EPS-0346411 (MAB), and the State of Louisiana Board of Regents Support Fund (MAB).
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BMC Emerg MedBMC Emergency Medicine1471-227XBioMed Central London 1471-227X-5-71622567210.1186/1471-227X-5-7Research ArticleA meta-analysis of controlled trials of recombinant human activated protein C therapy in patients with sepsis Wiedermann Christian J [email protected] Nicole C [email protected] Division of Internal Medicine II, Department of Medicine, Central Hospital of Bolzano, L. Böhler Street 5, I-39100 Bolzano/Bozen (BZ), Italy2 Divison of General Internal Medicine, Department of Internal Medicine, Medical University of Innsbruck, Anich Street 35, A-6020 Innsbruck (Tyrol), Austria2005 14 10 2005 5 7 7 24 5 2005 14 10 2005 Copyright © 2005 Wiedermann and Kaneider; licensee BioMed Central Ltd.2005Wiedermann and Kaneider; 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
Meta-analysis of two randomised controlled trials in severe sepsis performed with recombinant human activated protein C may provide further insight as to the therapeutic utility of targeting the clotting cascade in this syndrome.
Methods
In search for relevant studies published, two randomized clinical trials were found eligible.
Results
The studies, PROWESS and ADDRESS, enrolled a total of 4329 patients with risk ratio (RR) and 95% confidence interval (CI) data for effect on 28-day mortality relative to control treatment of 0.92 (0.83–1.02) suggesting that recombinant human activated protein C is not beneficial in severe sepsis. In PROWESS, 873 of 1690 patients presented with low risk, and 2315 of 2639 patients in ADDRESS as defined by APACHE II score < 25. In this low-risk stratum, no effect of recombinant human activated protein C administration on 28-day mortality was observed. This observation appears to be consistent and homogenous. Heterogeneity between the two studies, however, was seen in patients with APACHE II score ≥ 25 in whom recombinant activated protein C was effective in PROWESS (n = 817; RR 0.71, CI 0.59–0.85) whereas a tendency toward harm was present in ADDRESS (n = 324; RR 1.21, CI 0.85–1.74). Even though the overall treatment effect in this high-risk population was still in favour of treatment with recombinant activated protein C (n = 1141; RR 0.80, CI 0.68–0.94), the observed heterogeneity suggests that the efficacy of recombinant human activated protein C is not robust. Not unlikely, the adverse tendency observed could have become significant with higher statistical power would ADDRESS not have been terminated prematurely.
Conclusion
This meta-analysis, therefore, raises doubts about the clinical usefulness of recombinant activated protein C in patients with severe sepsis and an APACHE II score ≥ 25 which can only be resolved by another properly designed clinical trial.
==== Body
Background
Although coagulation abnormalities may partly underlie the physiologic derangements of the sepsis syndrome, anticoagulant therapies have produced mixed results on survival in clinical studies. According to a recent meta-analysis, anticoagulants as adjuvant therapy do not appear to improve outcome in sepsis and are associated with a significant risk of bleeding complications [1]. To the extent that their treatment effect is dependent upon disease severity, the safety and efficacy of these agents may be enhanced by refinement in techniques of clinical stratification [2-4].
Recombinant human activated protein C (rhAPC) was approved for use in severely septic patients based on the results of the phase III PROWESS trial [5]. However, concerns were raised regarding rhAPC's inconsistent effects, incomplete understanding of its mechanism of action, and its safety in particular subgroups [2]. Approval was contingent among others upon agreement on post-approval clinical trials [6]. In the meanwhile, one additonal large-scale randomised controlled clinical trial has been completed, the ADDRESS study [7]. A new warning has been added to the prescribing information for rhAPC based upon exploratory analysis of the ADDRESS clinical trial database [8,9]. Because a significant portion of patients included in ADDRESS met inclusion and exclusion criteria of the PROWESS subgroup of patients for whom the current prescription labelling was given, we hypothesized that a meta-analysis of the two clinical trials of rhAPC in sepsis may provide further insight as to the therapeutic utility of targeting the clotting cascade in this syndrome with rhAPC.
Methods
Approval of rhAPC was based on the PROWESS phase III trial [5]. Additional data on 28-day all-cause mortality and safety among adult patients with severe sepsis who were treated with rhAPC are available from the ENHANCE prospective, single-arm, multicenter clinical trial (only the US data of the trial have been published so far, [10]), and from the ADDRESS study. The single-arm ENHANCE study could enter meta-analysis only if historical controls would be used which does, however, not fulfil criteria usually applied in such type of analysis. Therefore, this analysis will focus on two studies only, i.e. PROWESS and ADRESS. Data from the PROWESS study are available from its original publication and additional material provided for the registration process of the FDA. Data from the ADDRESS have not yet been published in detail.
The primary objective of the ADDRESS trial was to demonstrate that rhAPC can reduce mortality at 28 days in adult patients with severe sepsis who have a lower risk of death, compared to placebo and conventional care. It originally planned to investigate the effectiveness of rhAPC in 11,000 adult severe sepsis patients who have a lower risk of death (APACHE score > 25; less than 2 organ failure). The trial was terminated prematurely because of futility to reach the primary study goal. The trial's data were presented at the annual meeting of the European Society of Intensive Care Medicine 2004 [7].
As approval of rhAPC is restricted to patients with severe sepsis and a high risk of death, focus of this re-analysis was on patient disease severity strata with APACHE II score < 25 and those with ≥25. Cochran-Mantel-Haenszel tests stratifying for study or for disease severity were performed together with the corresponding risk ratios (RR) and 95% confidence intervals (CI) for effect of rhAPC on 28 day mortality. For comparison pooled analyses and analyses of individual studies were performed by Fishers exact test. The homogeneity of treatment effects between strata or between studies was assessed with the Breslow-Day test.
Results and discussion
PROWESS and ADDRESS investigated the therapeutic effects of rhAPC against placebo in patients with severe sepsis early in their disease and at a dose of 24 μg/kg/hr for a total infusion duration of 96 hours. Collectively, the studies enrolled 4329 patients (PROWESS: 1690, and ADDRESS: 2639). The RR (95% CI) for effect on 28-day mortality of rhAPC, relative to control treatment, was 0.92 (0.83–1.02) with a nominal p-value of 0.095 (Cochran-Mantel-Haenszel test). Observed heterogeneity between the two studies (Breslow-Day test, p = 0.01) suggest possible differences in treatment effects of rhAPC which may be due mainly to the higher frequency of low risk patients in ADDRESS.
In PROWESS, 48.3% (817 of 1690) of patients presented with APACHE II score ≥ 25 whereas in ADDRESS 12.3% (324 of 2639) were in this group (Tab. 1). In these patients with APACHE II score ≥ 25 inhomogeneity was observed (Breslow-Day test, p = 0.005); whereas in PROWESS, a treatment effect in favour of rhAPC was seen, a tendency toward harm was present in ADDRESS. The observed lack of homogeneity for the treatment effect with rhAPC in the two trials raises doubt regarding the robustness and the generalizability of the result. The overall effect in this patient group with higher risk remains beneficial for rhAPC. Possibly the difference between the two studies' results is due to differences in study design with a higher number of patients of this stratum in PROWESS than in ADDRESS. Another cause may lie in rhAPC's efficacy itself which has not been uniformly supported given the limitations of PROWESS [2].
Table 1 Meta-analysis of effects of rhAPC on 28-day mortality in patients with severe sepsis and APACHE II score of ≥25 at study entry
Clinical Trial N rhAPC Control RR 95% CI p-Value
PROWESS 817 30.9 43.7 0.71 0.59 – 0.85 0.0002 1
ADDRESS 324 29.7 24.5 1.21 0.85 – 1.74 0.32 1
Total 1141 30.6 38.3 0.80 0.68 – 0.94 0.007 1,2,3
1 two-sided Fishers exact test
2 Cochran-Mantel-Haenszel test, p = 0.006
3 Breslow-Day test homogeneity, p = 0.005.
In PROWESS, 51.7% (873 of 1690) of patients presented with APACHE II score < 25 whereas in ADDRESS 87.7% (2315 of 2639) were in this group. In the low risk stratum no effect of rhAPC administration neither in favour nor against intervention was observed (Tab. 2). This observation appears to be consistent and homogenous (Breslow-Day test, p = 0.8).
Table 2 Meta-analysis of effects of rhAPC on 28-day mortality in patients with severe sepsis and APACHE II score of <25 at study entry
Clinical Trial N rhAPC Control RR 95% CI p-Value
PROWESS 873 18.8 19.0 0.99 0.75–1.30 1.0 1
ADDRESS 2315 16.8 16.0 1.05 0.88–1.27 0.6 1
Total 3188 17.3 16.8 1.03 0.89–1.20 0.7 1,2,3
1 two-sided Fishers exact test
2 Cochran-Mantel-Haenszel test, p = 0.007
3 Breslow-Day test homogeneity, p = 0.007.
Results of this meta-analysis are interesting, firstly as they confirm in a large patient population the lack of efficacy of rhAPC in low-risk severe sepsis, and secondly suggest that beneficial treatment effects in high-risk patients may also not be so robust because patients with APACHE II score ≥ 25 did not derive benefit in ADDRESS. The adverse tendency observed could probably have become significant with higher statistical power would ADDRESS not have been terminated prematurely. Even though speculative, this might be an important aspect derived from this analysis with consequences for the clinical use or rhAPC.
The Phase II trial testing the regimen of rhAPC applied in PROWESS and ADDRESS was small with 131 patients with severe sepsis only that were studied at 4 different doses and for two different infusion periods [12]. Relevance of the findings from this study in context of the present analysis, however, is low because of patient characteristics. The predominant portion of patients had single (60%) or two-organ (33%) failure and the APACHE II score was low (mean ± SD, 17.3 ± 5.8). As this was almost exclusively a low risk patient population with a lack of data available on patients with APACHE II > 25, and due to the different dose levels which makes the study population even smaller, the Phase II study was not included in the current analysis.
Because of post-approval study obligations for the US American Food and Drug Association (FDA), Eli Lilly recently had to add a warning to the label of rhAPC (Xigris) [8]: "Among the small number of patients enrolled in PROWESS with single organ dysfunction and recent surgery (surgery within 30 days prior to study treatment), all-cause mortality was numerically higher in the Xigris group (28-day: 10/49; in-hospital: 14/48) compared to the placebo group (28-day: 8/49; in-hospital: 8/47). In a preliminary analysis of the subset of patients with single organ dysfunction and recent surgery from a separate, randomized, placebo-controlled study (ADDRESS) of septic patients at lower risk of death (APACHE II score <25 or single sepsis-induced organ failure at any APACHE II score), all-cause mortality was also higher in the Xigris group (28-day: 67/323; in-hospital: 76/325) compared to the placebo group (28-day: 44/313; in-hospital: 62/314). Patients with single organ dysfunction and recent surgery may not be at high risk of death irrespective of APACHE II score and therefore may not be among the indicated population. Xigris should be used in these patients only after careful consideration of the risks and benefits."
The European Agency for the Evaluation of Medicinal Products (EMEA) also tightened the Xigris label for the European Union including the recommendation that the product only be used in high-risk patients, mainly in situations when therapy can be started within 24 hours of the onset of organ failure and that it should only be used by experienced doctors in institutions skilled in the care of patients with severe sepsis [9].
A primary therapeutic target of rhAPC is the prevention of thrombin formation. The conflicting effects pf rhAPC in PROWESS and ADDRESS in higher risk patients raises concerns regarding original FDA and EMEA analyses suggesting that risk of death alters the effects of rhAPC in sepsis. This questions also arises in the context of other antithrombotic agents such as antithrombin III and tissue factor pathway inhibitor which can only be answered in prospectivem randomized controlled trials.
Conclusion
This meta-analysis confirms that rhAPC is not effective in low-risk patients with severe sepsis, and it suggests that also beneficial treatment effects in high-risk patients may not be robust. It supports recent restrictive regulatory decisions in the European Union and the United States of America on Xigris and is in line with most recent developments from clinical trials, namely that Lilly has had to stop early a pediatric clinical study of Xigris after interim results showed that it was ineffective and might pose a safety risk in this population [11]. This meta-analysis raises doubts about the clinical usefulness of recombinant activated protein C in patients with severe sepsis and an APACHE II score ≥ 25 which can only be resolved by another properly designed clinical trial.
Competing interests
CJW received fees for speaking by Ely Lilly Austria, ZLB-Behring Marburg, Germany, and Baxter Vienna, Austria.
Authors' contributions
CJW was responsible for data acquisition, statistical analysis, and data interpretation. NCK contributed to data interpretation and was involved in drafting and writing of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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Freeman BD Zehnbauer BA Buchman TG A meta-analysis of controlled trials of anticoagulant therapies in patients with sepsis Shock 2003 20 5 9 12813361 10.1097/01.shk.0000068327.26733.10
Haley M Cui X Minneci PC Deans KJ Natanson C Eichacker PQ Activated protein C in sepsis: emerging insights regarding its mechanism of action and clinical effectiveness Curr Opin Infect Dis 2004 17 205 11 15166822 10.1097/00001432-200406000-00006
Cohen J Guyatt G Bernard GR Calandra T Cook D Elbourne D Marshall J Nunn A Opal S on behalf of a UK Medical Research Council International Working Party New strategies for clinical trials in patients with sepsis and septic shock Crit Care Med 2001 29 880 886 11373487 10.1097/00003246-200104000-00039
Wiedermann CJ Hoffmann JN Ostermann H Briegel J Strauss R Keinecke H Jürs M Kienast J High-dose antithrombin III in the treatment of severe sepsis with a high risk of death Intensive Care Med 2004 30 S90
Bernard GR Vincent JL Laterre PF LaRosa SP Dhainaut JF Lopez-Rodriguez A Steingrub JS Garber GE Helterbrand JD Ely EW Fisher CJ JrRecombinant human protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group Efficacy and safety of recombinant human activated protein C for severe sepsis N Engl J Med 2001 344 699 709 11236773 10.1056/NEJM200103083441001
de Jonge E Drotrecogin alfa (Eli Lilly) IDrugs 2002 5 363 8 15565519
Abraham E Laterre PF Garg R Levy H Talwar D Trzaskoma BL Francois B Guy JS Bruckmann M Rea-Neto A Rossaint R Perrotin D Sablotzki A Arkins N Utterback BG Macias WL Administration of Drotrecogin Alfa (Activated) in Early Stage Severe Sepsis (ADDRESS) Study Group Drotrecogin alfa (activated) for adults with severe sepsis and a low risk of death N Engl J Med 2005 353 1332 41 16192478 10.1056/NEJMoa050935
accessed March 28, 2005
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Bernard GR Margolis BD Shanies HM Ely EW Wheeler AP Levy H Wong K Wright TJ Extended Evaluation of Recombinant Human Activated Protein C United States Investigators Extended evaluation of recombinant human activated protein C United States Trial (ENHANCE US): a single-arm, phase 3B, multicenter study of drotrecogin alfa (activated) in severe sepsis Chest 2004 125 2206 16 15189943 10.1378/chest.125.6.2206
accessed May 2, 2005
Bernard GR Ely EW Wright TJ Fraiz J Stasek JE JrRussell JA Mayers I Rosenfeld BA Morris PE Yan SB Helterbrand JD Safety and dose relationship of recombinant human activated protein C for coagulopathy in severe sepsis Crit Care Med 2001 29 2051 9 11700394 10.1097/00003246-200111000-00003
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BMC Emerg MedBMC Emergency Medicine1471-227XBioMed Central London 1471-227X-5-81623618110.1186/1471-227X-5-8Research ArticleEarly tracheostomy in closed head injuries: experience at a tertiary center in a developing country – a prospective study Chintamani [email protected] Jotinder [email protected] JP [email protected] Pranjal [email protected] Pawan [email protected] Binita [email protected] RS [email protected] Dinesh [email protected] Department of surgery, Vardhman Mahavir Medical College, Safdarjang Hospital, New Delhi-110023, India2005 14 10 2005 5 8 8 30 4 2005 14 10 2005 Copyright © 2005 Chintamani et al; licensee BioMed Central Ltd.2005Chintamani 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 factor contributing to the high mortality in patients with severe head trauma is cerebral hypoxia. The mechanical ventilation helps both by reduction in the intracranial pressure and hypoxia. Ventilatory support is also required in these patients because of patient's inability to protect the airway, persistence of excessive secretions, and inadequacy of spontaneous ventilation. Prolonged endotracheal intubation is however associated with trauma to the larynx, trachea, and patient discomfort in addition to requirement of sedatives. Tracheostomy has been found to play an integral role in the airway management of such patients, but its timing remains subject to considerable practice variation. In a developing country like India where the intensive care facilities are scarce and rarely available, these critical patients have to be managed in high dependency cubicles in the ward, often with inadequately trained nursing staff and equipment to monitor them. An early tracheostomy in the selected group of patients based on Glasgow Coma Score(GCS) may prove to be life saving.Against this background a prospective study was contemplated to assess the role of early tracheostomy in patients with isolated closed head injury.
Methods
The series consisted of a cohort of 50 patients admitted to the surgical emergency with isolated closed head injury, that were not considered for surgery by the neuro-surgeon or shifted to ICU, but had GCS score of less than 8 and SAPS II score of more than 50. First 50 case records from January 2001 that fulfilled the criteria constituted the control group. The patients were managed as per ATLS protocol and intubated if required at any time before decision to perform tracheostomy was taken. These patients were serially assessed for GCS (worst score of the day as calculated by senior surgical resident) and SAPS scores till day 15 to chart any changes in their status of head injuries and predictive mortality. Those patients who continued to have a GCS score of <8 and SAPS score of >50 for more than 24 hours (to rule out concussion or recovery) underwent tracheostomy.
All these patients were finally assessed for mortality rate and hospital stay, the statistical analysis was carried out using SPSS10 version.
The final outcome (in terms of mortality) was analyzed utilizing chi-square test and p value <0.05 was considered significant.
Results
At admission both tracheostomy and non-tracheostomy groups were matched with respect to GCS score and SAPS score.
The average day of tracheostomy was 2.18 ± 1.0038 days.
The GCS scores on days 1, 2, 3, 4, 5, 10 between tracheostomy and non-tracheostomized group were comparable. However the difference in the GCS scores was statistically significant on day 15 being higher in the tracheostomy group.Thus early tracheostomy was observed to improve the mortality rate significantly in patients with isolated closed head injury
Conclusion
It may be concluded that early tracheostomy is beneficial in patients with isolated closed head injury which is severe enough to affect systemic physiological parameters, in terms of decreased mortality and intubation associated complications in centers where ICU care is not readily available. Also, in a selected group of patients, early tracheostomy may do away with the need for prolonged mechanical ventilation.
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Background
An important factor responsible for the high mortality in patients with severe head trauma is cerebral hypoxia. Mechanical ventilation is often required because of patient's inability to protect the airway, persistence of excessive secretions, and inadequacy of spontaneous ventilation [1]. Tracheostomy plays an integral role in the airway management of such patients, but its timing remains subject to considerable practice variation [1-4]. The complications associated with prolonged endotracheal intubation are increasingly being recognized and include injury to the larynx, trachea, and patient discomfort [5-13,16-18]. In addition, endotracheal intubation often requires the administration of systemic sedation, with attendant complications. Finally the incidence of ventilator-associated pneumonia is related directly to the duration of mechanical ventilation – a complication that carries significant morbidity and mortality [19-22].
Paradoxically, although tracheostomy is frequently recommended in closed head injury patients, few studies have been carried out to assess the importance in this group of patients. Many studies recommend early tracheostomy to avoid serious oropharyngeal and laryngeal injury occurring from prolonged translaryngeal intubation though limited data is available to define the impact of early tracheostomy on duration of mechanical ventilation and hospital stay [1,4,5,9].
In a developing country like India, where even a tertiary care center like ours, is short of appropriate ICU facilities for patients with severe closed head injuries for all patients, these critical patients have to be managed in high dependency cubicles in the ward, often with inadequately trained nursing staff and equipment to monitor them.
We had observed that patients with severe head injury very frequently required prolonged intubation, primarily for airway protection after initial few days and due to fluctuating changes in airway reflexes patients often struggled with endotracheal tubes and required frequent use of sedatives.
With the underlying belief that early tracheostomy is beneficial, we searched the literature to find those objective factors (GCS and SAPS) [1,3] which can be used early to separate those patients who will ultimately require tracheostomy from those that will not.
Methods
Settings
The study was performed at a major tertiary care trauma centre in New Delhi, India. This 2200-bed hospital has an 8-bed medical/surgical ICU staffed by full-time, on-site intensivists 24 hours a day and 7 days a week. The hospital has a designated trauma service, including a consultant surgeon, available 24 hours a day. Medical care in the ICU is provided by the ICU team, with the trauma team being responsible for surgical aspects of care. In addition there is a 4-beded neuro-ICU for post-operative neurosurgical patients staffed by neurosurgeons. The surgical ward has 37 beds with a 6-bed high dependency cubicle.
The study was carried out following review with Institutional Review Board from July 2003 to November 2004. The series consisted of consecutive 50 patients who fulfilled the criteria of the study and underwent tracheostomy. First 50 case records from January 2001 that fulfilled the criteria of the study constituted the control group.
This study consisted of cohort of all patients admitted to surgery emergency with isolated closed head injury, that were not considered for surgery by the neuro-surgeon or shifted to ICU, but had GCS score of less than 8 and SAPS II score of more than 50 (predictive mortality more than 50%, calculated at ). The patients were managed as per ATLS protocol and intubated if required at any time before decision to perform tracheostomy was taken. Patients underwent NECT scan to find out any intracranial bleed and were managed as per the advice of neurosurgeon.
Those patients who continued to have a GCS score of <8 and SAPS score of >50 for more than 24 hours (to rule out concussion or recovery) were to undergo a tracheostomy with a standardized technique by the surgical resident. The following patients were excluded from the study
1) Children less than 12 years of age
2) Patients who underwent neurosurgical intervention, as these patients were later shifted to neuro-ICU
3) Patients who were shifted to the ICU as ventilator and / or ICU bed were available
4) Patients with poly trauma with other injuries besides closed head injuries, so as to constitute a homogenous cohort.
The control group was selected from previous admission records and only those cases were included in whom complete data so as to calculate serial SAPS II score were available, besides fulfilling the above criteria. This was to achieve a score matched control group as comparable to study group as possible and in order to eliminate confounding factors.
All these patients were assessed for mortality rate and hospital stay and 30 days (from the time of admission) mortality was taken in to consideration. These patients were also serially assessed for GCS (worst score of the day as calculated by senior surgical resident) and SAPS scores till day 15 to chart any changes in their status of head injuries and predictive mortality.
Surgical resident in the ward performed tracheostomy by the standardized technique using a high volume, low pressure cuffed tracheostomy tube.The patient was shifted to high dependency cubicle for monitoring and 2–3 hourly suction of the tracheostomy by the staff nurse. The nurse-patient ratio in the high dependency cubicle is 1:6. If patient required ventilatory support a small KV ventilator was arranged from ICU to supply 40% oxygen at a fixed rate and airway pressure. If that was not available the patients were put on continued Ambu-bag ventilation till a ventilator was arranged. Other resuscitation was carried as per the instruction of the ICU resident and neurosurgical resident, including use of sedatives.
All patients were to undergo flexible laryngoscopy within 24 hours of tracheostomy by an ENT senior resident for evaluation of endotracheal injury and at the time of extubation or tracheostomy removal.
A central venous access was established and daily routine investigations were sent for these patients. As and when the patient stabilized (SAPS II score <50), patient was placed in the general ward with patient-nurse ratio of 1:15. A naso-gastric tube was placed and feeding started initially along with intravenous supplement and gradually only liquid diet through naso-gastric tube, providing 2200Calories a day was started. This was done till the patient gained adequate consciousness or oropharyngeal reflexes. The patient was also placed on water mattress with nursing attendants log rolling the patients every hour to prevent bedsores. The patients' attendants were gradually introduced to nursing care of the patient and taught precautions regarding hygiene and feeding. Staff nurses or surgical residents did tracheostomy care and dressing of the bedsores on a regular basis.
Special care was taken to prevent constipation and early switch from Foley's catheter to condom catheter drainage was done. Catheter care was done as long as Foley's catheter was in-situ.
Complications like fever, cough, blocked tracheostomy tube with sputum, bedsores and or any other source of sepsis were dealt with as per standard protocol of culture sensitivity and appropriate antibiotic cover.
The patients were discharged only after
1) They regained adequate consciousness (GCS >13);
2) Patients not only improving in the GCS score but had a good SAPS II score Meanwhile attendants were adequately trained in patient care and patient gradually weaned off tracheostomy.
Patients were divided into two groups: those that under went tracheostomy (T group) and those that did not (NT group). All the data was collected and comparisons between the two groups for continuous variables are expressed as means ± standard error of the mean, and were compared using two-tailed t-tests for unequal variance. Categorical variables are expressed as absolute and relative frequencies, and were compared using χ2 tests. P ≤ 0.05 were considered statistically significant.
Results
Out of the 9071 patients admitted with head injury during the period of our study, 542 had isolated closed head injuries, of which 56 patients satisfied the criteria of our study. 6 patients could not be included due to incomplete case records. The remaining 50 constituted the cases, i.e. those undergoing early tracheostomy (group T). Of the 1125 case records of patients admitted between January 2001–December 2003 which were studied, the first 50 satisfactory case records were taken as the control (group NT). In this study the distribution of patients in the two groups was matched with respect to confounding factors. The average age in the tracheostomy group (T) was 34.54 ± 12.7737 years and in the non-tracheostomy group (NT) was 32.98 ± 11.1435 years. Using two-tailed t-test for unequal variance p value was 0. 5209, which is not significant. Similarly sex distribution in the two groups was matched. The male: female ratio in the T group was 37:13 and in the NT group was 33:17. The two groups were analyzed using chi-square test. The cell chi value was 0.11, degree of freedom was 1 and the p value was 0.4571, which is not significant.
The comparison of GCS and SAPS was done for Days 1, 2, 3, 4, 5, 10 and 15 between tracheostomy and the non-tracheostomy group. The GCS score on day 1 of admission was 4.06 ± 1.0278 (T) and 4.14 ± 0.9382 (NT); comparing the two groups using two-tailed t-test for unequal variance, p value was 0.6882, which is not significant [<0.05 taken as significant]. Similarly SAPS score on day 1 of admission was 59.34 ± 2.7247 (T) and 58.94 ± 2.2127 (NT); comparing the two groups using two-tailed t-test for unequal variance, p value was 0.6017, which is not significant. Thus at admission both tracheostomy and non-tracheostomy groups were matched with respect to GCS score and SAPS score.
The average day of tracheostomy was 2.18 ± 1.0038 days.
The GCS scores on days 1, 2, 3, 4, 5, 10 between tracheostomy and non-tracheostomy group were comparable, the p value being not significant between the two groups. However GCS scores were statistically significant on day 15 being higher in the tracheostomy group indicating improvement with tracheostomy (Table 1, Figure 1). Similarly the SAPS score on days 1, 2, 3, 4, 5 were comparable between tracheostomy and non-tracheostomy groups, the p value being not significant between the two groups. However, SAPS score on days 10 and 15 were statistically significant, being lower in the tracheostomy group indicating improvement with tracheostomy (Table 2, Figure 2).
Table 1 Comparison of sequential G.C.S. between tracheostomised and non-tracheostomised groups.
Tracheostomy Non Tracheostomy p Value
Day 1 4.06 ± 1.0278 4.14 ± 0.9382 0.6882
Day 2 5.04 ± 1.1825 4.74 ± 1.1800 0.2117
Day 3 5.82 ± 1.6815 5.40 ± 1.8617 0.2560
Day 4 6.62 ± 1.9596 6.21 ± 2.3211 0.3681
Day 5 7.12 ± 2.7472 6.91 ± 2.9671 0.7324
Day 10 8.35 ± 3.1983 7.00 ± 3.4935 0.0723
Day 15 9.97 ± 3.0477 7.20 ± 3.3506 0.0009
Figure 1 The Glasgow coma score of the two groups from day 1 to days 15.
Table 2 Comparison of sequential S.A.P.S. between tracheostomised and non-tracheostomised groups.
Tracheostomy Non Tracheostomy p Value
Day 1 59.34 ± 2.7247 58.94 ± 2.2127 0.6016
Day 2 59.24 ± 2.4294 58.30 ± 2.3515 0.7243
Day 3 57.88 ± 3.4850 56.97 ± 2.1617 0.1303
Day 4 55.70 ± 3.8587 55.97 ± 2.9302 0.6965
Day 5 54.04 ± 4.4856 55.25 ± 3.7422 0.1961
Day 10 49.48 ± 6.1954 52.20 ± 5.8929 0.0460
Day 15 43.86 ± 8.1665 49.83 ± 7.2391 0.0028
Figure 2 The SAPS score of the two groups (tracheostomized and non-tracheostomized) from day 1 to days 15.
Besides the improvement in the GCS and SAPS scores the end point of the study was to see whether tracheostomy led to decrease in mortality rate when compared with similar controls and whether there was reduction in hospital stay in tracheostomised patients. The final outcome (in terms of mortality) was analyzed utilizing chi-square test. The cell chi was 1.30 and 1.141, the degree of freedom was 1 and p was less than 0.05, which is significant (Table 3). The mortality in the T group was 36% compared to that of 58% in the NT group. This also compares favorably with the predictive mortality of >50% with SAPS II. Thus doing early tracheostomy improved significantly the mortality rate in patients with isolated closed head injury.
Table 3 Chi square table for outcome in the tracheostomised and non-tracheostomised groups.
Discharged Expired
32 18
Tracheostomy 26.5 23.5
1.141 1.30
21 29
Non Tracheostomy 26.5 23.5
1.141 1.30
Degree of freedom = 1
Overall cell chi = 4.88
p = 0.0275 (significant)
Hospital stay was analyzed separately for the patients who were discharged or those who died during the course of stay in hospital. The average day of mortality was 19.4130 ± 7.0476 (T) and 18.55 ± 9.7469 (NT); comparing the two groups using two-tailed t-test for unequal variance, p value was 0.6244, which is not significant. Similarly average day of discharge was 19.78 ± 6.9261 (T) and 18.66 ± 9.7834 (NT); comparing the two groups using two tailed t-test for unequal variance, p value was 0.5157, which is not significant (table 4). Performing early tracheostomy did not change length of hospital stay significantly in either patients who were discharged or those who expired during hospital stay. Probably this was so because abundant caution was taken to see that the patient is weaned off the tracheostomy and the attendants are adequately trained to perform nursing care of these patients. The very limited rehabilitation facilities meant that the patients had to undergo rehabilitation while they were hospitalized, prolonging further the hospital LOS.
Table 4 Comparison of hospital stay in the tracheostomised and non-tracheostomised groups.
Tracheostomy Non Tracheostomy p Value
Day of discharge 19.78 ± 6.9261 18.66 ± 9.7834 0.5157
Day of death 19.41 ± 7.0476 18.55 ± 9.7469 0.6244
Discussion
A tracheostomy is a proven adjunct in the care of head injury patients [5,8,9,12]. Tracheostomy provides an early airway protection and seems to decrease the need for prolonged mechanical ventilatory support. Secondly, severe head injury patients require a prolonged time for recovery and the airway reflexes are rarely optimal [3]. The association between the duration of intubation and risks of laryngotracheal injury is another important consideration in the timing of tracheostomy.
The tracheostomy tube facilitates pulmonary toilet and oral hygiene and has been shown to reduce the incidence of ventilator-associated pneumonia [5]. Furthermore, a tracheostomy tube is less noxious for the patient emerging from coma and sedation can be more easily weaned off. In addition, tracheostomy reduces significantly the physiological dead space of ventilation and thereby the work of breathing. This is even more beneficial in a patient having labored breathing or deteriorating respiration and may prevent the use of mechanical ventilation altogether. Because of these benefits, early tracheostomies have been shown to reduce hospital stay. However to realize the benefits of early tracheostomy without performing unnecessary tracheostomies, appropriate patients must be identified early at the time of admission. Early oxygenation and ventilatory abnormalities can predict the need for tracheostomy [19]. However, head injury patients primarily require airway protection and not necessarily ventilatory support for pulmonary failure.
A study by Major et al [1] showed that using objective scores such as GCS (less than seven) and SAPS (more than fifteen) score could aid in identifying those patients who will eventually require a tracheostomy for prolonged airway protection after blunt head trauma with high positive predictive value. However, they used day 4 scores of GCS and SAPS to determine the need for tracheostomy and performed the tracheostomy only after day 5. This way they not only excluded patients who were extubated but even those who died early, thus removing an important cohort who could benefit from tracheostomy. This skewed mortality data as severe head injury is associated with early mortality. In addition, these patients were admitted in ICU and were intubated already as per ATLS standards. Positive predictive value for GCS and SAPS score was 71%, and negative predictive value was 83%. Gurkin et al [6] found two admission criteria (GCS <9 and ISS >24) to be predictive of the need for tracheostomy in those patients that remained intubated on day 7. Rodriguez et al [9] noted a significant decrease in ventilator days and ICU days and hospital length of stay in patients who underwent tracheostomy within five days of intubation.
In a study by Sugerman et al [4] several major trauma centers refused to participate in early tracheostomy (5 to 7 days) trial because they felt strongly, like we do, that either all severly injured patients should undergo tracheostomy within 2 to 3 days after injury or tracheostomy was not necessary for as long as 3 to 4 weeks after injury. In their study they found that head injury patients who underwent early tracheostomy had higher APACHE III scores but there was no difference in ICU LOS, pneumonia and death in these patients as compared to patients with late tracheostomy.
Lesnik et al [5] retrospectively reviewed 101 adult patients with blunt injuries, 32 had tracheostomy within first 4 days and 69 underwent tracheostomy after 4 days. The author found that mean duration of ventilatory support was 6.0 days in early tracheostomy group versus 20.6 days in the late tracheostomy group (p < 0.001).
In a study by Bouderka et al a prospective study was conducted in patients with admission GCS of 8 or less, cerebral contusion on CT scan and GCS score of less than 8 on day 5. These patients were randomized into early tracheostomy and prolonged intubation. Besides demographic data admission scores SAPS, ICU stay duration of mechanical stay was compared. They concluded that early tracheostomy decreased total days of mechanical ventilation (p = 0.02). They agreed that choosing their criteria had the limitation of high mortality during the first week of hospitalization. They suggested that most of the patients did not require mechanical ventilatory support but were intubated mainly for airway protection. Early tracheostomy may provide an early alternative for airway protection and assist in early termination of mechanical ventilatory support and there fore reduce hospital stay for these patients.
In our opinion using admission data to support a decision several days later is flawed. This diminishes the benefits of early tracheostomy and makes the decision more straightforward and increases the chances of laryngotracheal injuries due to intubation.
Early tracheostomy may assist in early termination of mechanical ventilation and therefore, reduce the hospital stay and mortality. Currently, the decision to proceed to early tracheostomy is based on the attending trauma surgeon's preference. The consensus conference of 1989 recommended conversion to tracheostomy if the anticipated need for mechanical ventilation is more than 21 days [15]. Such practice was based on earlier reports showing high tracheal stenosis rates with tracheostomy as compared with endotracheal intubation [13]. However, the incidence of tracheal stenosis has decreased substantially with recognition of its aetiology and improvements in tracheostomy materials, design and management, particularly with the use of high-volume, low-pressure cuffs. Also, the complications associated with prolonged endotracheal intubation are increasingly being recognized, including injury to the larynx and trachea [16-18], and patient discomfort. In addition, endotracheal intubation often requires the administration of systemic sedation, with attendant complications.
Despite evidence to support the utility of early tracheostomy, few recommendations exist to facilitate identification of appropriate patients.
In a study by Arabi et al [2], 136 patients underwent tracheostomies, of which only 29 were early (seven days). The duration of mechanical ventilation was significantly shorter with early tracheostomy (mean ± standard error: 9.6 ± 1.2 days versus 18.7 ± 1.3 days; P < 0.0001). Similarly, ICU LOS was significantly shorter (10.9 ± 1.2 days versus 21.0 ± 1.3 days; P < 0.0001). Following tracheostomy, patients were discharged from the ICU after comparable periods in both groups (4.9 ± 1.2 days versus 4.9 ± 1.1 days; not significant). ICU and hospital mortality rates were similar. Using multivariate analysis, late tracheostomy was an independent predictor of prolonged ICU stay (>14 days). The very low mortality seen in the patients we studied may be explained by selection of proper candidates for tracheostomy, excluding those patients who were unlikely to survive. Hospital LOS in these patients was prolonged, reflecting their severe injuries that required lengthy rehabilitation periods.
Strengths of our study include prospective data collection ensuring complete data. The cohort was homogenous in that the decision for tracheostomy was not affected by other injuries (maxillofacial, neck) which may mandate early tracheostomy. Similarly the outcome in either group remained uninfluenced by systemic injuries. Our study included all patients with severe head injury (including those with early mortality) by virtue of doing early tracheostomy, unlike other studies [1-4,10,11] which excluded such patients by doing tracheostomy on day 5 onwards. By taking SAPS II as a criterion, we tried not to overdo tracheostomies by including only those patients in whom the head injury was severe enough to affect systemic physiological parameters. Lastly, we have taken serial scores as criteria for tracheostomy rather than admission scores. However data extraction and analysis was retrospective. Because the database was not designed specifically to examine tracheostomy practices, certain issues were not documented, such as comparison with intubation and the type of tracheostomy done. Also, we did not compare the morbidity in the two groups. In addition, the study was conducted from one centre. A large multicentre randomized controlled trial in which patients are randomized to early versus late tracheostomy would be the ideal way to test the impact of procedure timing on resource utilization.
Conclusion
We conclude that early tracheostomy is beneficial in patients with isolated closed head injury, severe enough to affect systemic physiological parameters, in terms of decreased mortality and intubation associated complications in centers where ICU care is not readily or easily available. Also, in a selected group of patients, early tracheostomy may actually do away with the need for prolonged mechanical ventilation.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
CM, the principal author designed the study and was the surgeon in charge monitoring the management of these patients, JK contributed to the designing of study and statistical analysis., PK, JP, BP, P kalra, were the residents in charge of the management of these cases.RSM, DB were the senior surgeons also involved in the management of the cases.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The study had been reviwed and approved by the ethical committee of the hospital
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Major KM Hui T Wilson MT Gaon MD Shabot M/M Margulies DR Objective indication for early tracheostomy after blunt head trauma Am J of Surg 2003 186 615 619 14672767 10.1016/j.amjsurg.2003.08.012
Arabi Y Hddad S Shirawi N Al Shimemeri A Early tracheostomy in intensive care trauma patients improves resource utilization: a cohort study and literature review Crit Care 2004 8 R347 R352 15469579 10.1186/cc2924
Bouderka MA Fakhir B Bouaggad A Hmamouchi B Hamoudi D Harti A Early tracheostomy versus prolonged endotracheal intubation in severe head injury J Trauma Inj Infect and Crit Care 2004 57 251 254 10.1097/01.TA.0000087646.68382.9A
Sugerman HJ Wolfe L Pasquale MD Rogers FB O'Malley KF Knudson M DiNardo L Gordon M Schaffer S Multicenter, randomized, prospective trial of early tracheostomy J Trauma 1997 43 741 747 9390483
Lesnik I Rappaport W Fulginiti J Witzke D The role of early tracheostomy in blunt, multiple organ trauma Am Surg 1992 58 346 349 1596033
Gurkin MA Parikshak M Kralovich KA Indicators for tracheostomy in patients with traumatic brain injury Am Surg 2002 68 324 328 11952241
Santos PM Afrassiabi A Weymuller EA Risk factors associated with prolonged intubation and laryngeal injury Otolaryngol Head Neck Surg 1994 111 453 459 7936678
Armstrong PA McCarthy MC Peoples JB Reduced use of resources by early tracheostomy in ventilator-dependent patients with blunt trauma Surgery 1998 124 763 766 9780999 10.1067/msy.1998.91224
Rodriguez JL Steinberg SM Luchetti FA Gibbons KJ Taheri PA Flint LM Early tracheostomy for primary airway management in the surgical critical care setting Surgery 1990 108 655 659 2218876
Kluger Y Paul DB Lucke J Cox P Colella JJ Townsend RN Raves JJ Diamond DL Early tracheostomy in trauma patients Eur J Emerg Med 1996 3 95 101 9028753
Teoh WH Goh KY Chan CL The role of early tracheostomy in critically ill neurosurgical patients Ann Acad Med Singapore 2001 30 234 238 11455734
D'Amelio Lf Hammond JS Spain DA Sutyak JP Tracheostomy and percutaneous endoscopic gastrostomy in the management of the head injured trauma patient Am Surg 1994 60 180 185 8116977
Stauffer JL Olson DE Petty TL Complication and consequences of endotracheal intubation and tracheostomy: a prospective study of 150 critically ill patients Am J Med 1981 70 65 76 7457492 10.1016/0002-9343(81)90413-7
Lanza DC Parnes SM Koltai PJ Fortune JB Early complications of airway management in head-injured patients Larygoscope 1990 100 958 961
Plummer AL Gracey DR Consensus conference on artificial airways in patients receiving mechanical ventilation Chest 1989 96 178 180 2500308
Whited RE A prospective study of laryngotracheal sequelae in long-term intubation Laryngoscope 1984 94 367 377 6700353
Lindholm CE Prolonged endotracheal intubation Acta Anaesthesiol Scand Suppl 1969 33 1 131 5459079
Colice GL Stukel Ta Dain B Laryngeal complications of prolonged intubation Chest 1989 96 877 884 2791687
Johnson SB Kearney PA Barker DE Early criteria predictive of prolonged mechanical ventilation J Trauma 1992 33 95 100 1635112
Le Gall JR Lemeshow S Saulnier F A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study JAMA 1993 270 2957 2963 8254858 10.1001/jama.270.24.2957
Ross BJ Barker DE Russell WL Burns RP Prediction of long-term ventilatory support in trauma patients Am Surg 1996 62 19 25 8540640
Vincent JL Lobo S Struelens M Ventilator associated pneumonia: risk factors and preventive measures J Chemother 2001 1 211 217 11936368
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BMC Emerg MedBMC Emergency Medicine1471-227XBioMed Central London 1471-227X-5-81623618110.1186/1471-227X-5-8Research ArticleEarly tracheostomy in closed head injuries: experience at a tertiary center in a developing country – a prospective study Chintamani [email protected] Jotinder [email protected] JP [email protected] Pranjal [email protected] Pawan [email protected] Binita [email protected] RS [email protected] Dinesh [email protected] Department of surgery, Vardhman Mahavir Medical College, Safdarjang Hospital, New Delhi-110023, India2005 14 10 2005 5 8 8 30 4 2005 14 10 2005 Copyright © 2005 Chintamani et al; licensee BioMed Central Ltd.2005Chintamani 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 factor contributing to the high mortality in patients with severe head trauma is cerebral hypoxia. The mechanical ventilation helps both by reduction in the intracranial pressure and hypoxia. Ventilatory support is also required in these patients because of patient's inability to protect the airway, persistence of excessive secretions, and inadequacy of spontaneous ventilation. Prolonged endotracheal intubation is however associated with trauma to the larynx, trachea, and patient discomfort in addition to requirement of sedatives. Tracheostomy has been found to play an integral role in the airway management of such patients, but its timing remains subject to considerable practice variation. In a developing country like India where the intensive care facilities are scarce and rarely available, these critical patients have to be managed in high dependency cubicles in the ward, often with inadequately trained nursing staff and equipment to monitor them. An early tracheostomy in the selected group of patients based on Glasgow Coma Score(GCS) may prove to be life saving.Against this background a prospective study was contemplated to assess the role of early tracheostomy in patients with isolated closed head injury.
Methods
The series consisted of a cohort of 50 patients admitted to the surgical emergency with isolated closed head injury, that were not considered for surgery by the neuro-surgeon or shifted to ICU, but had GCS score of less than 8 and SAPS II score of more than 50. First 50 case records from January 2001 that fulfilled the criteria constituted the control group. The patients were managed as per ATLS protocol and intubated if required at any time before decision to perform tracheostomy was taken. These patients were serially assessed for GCS (worst score of the day as calculated by senior surgical resident) and SAPS scores till day 15 to chart any changes in their status of head injuries and predictive mortality. Those patients who continued to have a GCS score of <8 and SAPS score of >50 for more than 24 hours (to rule out concussion or recovery) underwent tracheostomy.
All these patients were finally assessed for mortality rate and hospital stay, the statistical analysis was carried out using SPSS10 version.
The final outcome (in terms of mortality) was analyzed utilizing chi-square test and p value <0.05 was considered significant.
Results
At admission both tracheostomy and non-tracheostomy groups were matched with respect to GCS score and SAPS score.
The average day of tracheostomy was 2.18 ± 1.0038 days.
The GCS scores on days 1, 2, 3, 4, 5, 10 between tracheostomy and non-tracheostomized group were comparable. However the difference in the GCS scores was statistically significant on day 15 being higher in the tracheostomy group.Thus early tracheostomy was observed to improve the mortality rate significantly in patients with isolated closed head injury
Conclusion
It may be concluded that early tracheostomy is beneficial in patients with isolated closed head injury which is severe enough to affect systemic physiological parameters, in terms of decreased mortality and intubation associated complications in centers where ICU care is not readily available. Also, in a selected group of patients, early tracheostomy may do away with the need for prolonged mechanical ventilation.
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Background
An important factor responsible for the high mortality in patients with severe head trauma is cerebral hypoxia. Mechanical ventilation is often required because of patient's inability to protect the airway, persistence of excessive secretions, and inadequacy of spontaneous ventilation [1]. Tracheostomy plays an integral role in the airway management of such patients, but its timing remains subject to considerable practice variation [1-4]. The complications associated with prolonged endotracheal intubation are increasingly being recognized and include injury to the larynx, trachea, and patient discomfort [5-13,16-18]. In addition, endotracheal intubation often requires the administration of systemic sedation, with attendant complications. Finally the incidence of ventilator-associated pneumonia is related directly to the duration of mechanical ventilation – a complication that carries significant morbidity and mortality [19-22].
Paradoxically, although tracheostomy is frequently recommended in closed head injury patients, few studies have been carried out to assess the importance in this group of patients. Many studies recommend early tracheostomy to avoid serious oropharyngeal and laryngeal injury occurring from prolonged translaryngeal intubation though limited data is available to define the impact of early tracheostomy on duration of mechanical ventilation and hospital stay [1,4,5,9].
In a developing country like India, where even a tertiary care center like ours, is short of appropriate ICU facilities for patients with severe closed head injuries for all patients, these critical patients have to be managed in high dependency cubicles in the ward, often with inadequately trained nursing staff and equipment to monitor them.
We had observed that patients with severe head injury very frequently required prolonged intubation, primarily for airway protection after initial few days and due to fluctuating changes in airway reflexes patients often struggled with endotracheal tubes and required frequent use of sedatives.
With the underlying belief that early tracheostomy is beneficial, we searched the literature to find those objective factors (GCS and SAPS) [1,3] which can be used early to separate those patients who will ultimately require tracheostomy from those that will not.
Methods
Settings
The study was performed at a major tertiary care trauma centre in New Delhi, India. This 2200-bed hospital has an 8-bed medical/surgical ICU staffed by full-time, on-site intensivists 24 hours a day and 7 days a week. The hospital has a designated trauma service, including a consultant surgeon, available 24 hours a day. Medical care in the ICU is provided by the ICU team, with the trauma team being responsible for surgical aspects of care. In addition there is a 4-beded neuro-ICU for post-operative neurosurgical patients staffed by neurosurgeons. The surgical ward has 37 beds with a 6-bed high dependency cubicle.
The study was carried out following review with Institutional Review Board from July 2003 to November 2004. The series consisted of consecutive 50 patients who fulfilled the criteria of the study and underwent tracheostomy. First 50 case records from January 2001 that fulfilled the criteria of the study constituted the control group.
This study consisted of cohort of all patients admitted to surgery emergency with isolated closed head injury, that were not considered for surgery by the neuro-surgeon or shifted to ICU, but had GCS score of less than 8 and SAPS II score of more than 50 (predictive mortality more than 50%, calculated at ). The patients were managed as per ATLS protocol and intubated if required at any time before decision to perform tracheostomy was taken. Patients underwent NECT scan to find out any intracranial bleed and were managed as per the advice of neurosurgeon.
Those patients who continued to have a GCS score of <8 and SAPS score of >50 for more than 24 hours (to rule out concussion or recovery) were to undergo a tracheostomy with a standardized technique by the surgical resident. The following patients were excluded from the study
1) Children less than 12 years of age
2) Patients who underwent neurosurgical intervention, as these patients were later shifted to neuro-ICU
3) Patients who were shifted to the ICU as ventilator and / or ICU bed were available
4) Patients with poly trauma with other injuries besides closed head injuries, so as to constitute a homogenous cohort.
The control group was selected from previous admission records and only those cases were included in whom complete data so as to calculate serial SAPS II score were available, besides fulfilling the above criteria. This was to achieve a score matched control group as comparable to study group as possible and in order to eliminate confounding factors.
All these patients were assessed for mortality rate and hospital stay and 30 days (from the time of admission) mortality was taken in to consideration. These patients were also serially assessed for GCS (worst score of the day as calculated by senior surgical resident) and SAPS scores till day 15 to chart any changes in their status of head injuries and predictive mortality.
Surgical resident in the ward performed tracheostomy by the standardized technique using a high volume, low pressure cuffed tracheostomy tube.The patient was shifted to high dependency cubicle for monitoring and 2–3 hourly suction of the tracheostomy by the staff nurse. The nurse-patient ratio in the high dependency cubicle is 1:6. If patient required ventilatory support a small KV ventilator was arranged from ICU to supply 40% oxygen at a fixed rate and airway pressure. If that was not available the patients were put on continued Ambu-bag ventilation till a ventilator was arranged. Other resuscitation was carried as per the instruction of the ICU resident and neurosurgical resident, including use of sedatives.
All patients were to undergo flexible laryngoscopy within 24 hours of tracheostomy by an ENT senior resident for evaluation of endotracheal injury and at the time of extubation or tracheostomy removal.
A central venous access was established and daily routine investigations were sent for these patients. As and when the patient stabilized (SAPS II score <50), patient was placed in the general ward with patient-nurse ratio of 1:15. A naso-gastric tube was placed and feeding started initially along with intravenous supplement and gradually only liquid diet through naso-gastric tube, providing 2200Calories a day was started. This was done till the patient gained adequate consciousness or oropharyngeal reflexes. The patient was also placed on water mattress with nursing attendants log rolling the patients every hour to prevent bedsores. The patients' attendants were gradually introduced to nursing care of the patient and taught precautions regarding hygiene and feeding. Staff nurses or surgical residents did tracheostomy care and dressing of the bedsores on a regular basis.
Special care was taken to prevent constipation and early switch from Foley's catheter to condom catheter drainage was done. Catheter care was done as long as Foley's catheter was in-situ.
Complications like fever, cough, blocked tracheostomy tube with sputum, bedsores and or any other source of sepsis were dealt with as per standard protocol of culture sensitivity and appropriate antibiotic cover.
The patients were discharged only after
1) They regained adequate consciousness (GCS >13);
2) Patients not only improving in the GCS score but had a good SAPS II score Meanwhile attendants were adequately trained in patient care and patient gradually weaned off tracheostomy.
Patients were divided into two groups: those that under went tracheostomy (T group) and those that did not (NT group). All the data was collected and comparisons between the two groups for continuous variables are expressed as means ± standard error of the mean, and were compared using two-tailed t-tests for unequal variance. Categorical variables are expressed as absolute and relative frequencies, and were compared using χ2 tests. P ≤ 0.05 were considered statistically significant.
Results
Out of the 9071 patients admitted with head injury during the period of our study, 542 had isolated closed head injuries, of which 56 patients satisfied the criteria of our study. 6 patients could not be included due to incomplete case records. The remaining 50 constituted the cases, i.e. those undergoing early tracheostomy (group T). Of the 1125 case records of patients admitted between January 2001–December 2003 which were studied, the first 50 satisfactory case records were taken as the control (group NT). In this study the distribution of patients in the two groups was matched with respect to confounding factors. The average age in the tracheostomy group (T) was 34.54 ± 12.7737 years and in the non-tracheostomy group (NT) was 32.98 ± 11.1435 years. Using two-tailed t-test for unequal variance p value was 0. 5209, which is not significant. Similarly sex distribution in the two groups was matched. The male: female ratio in the T group was 37:13 and in the NT group was 33:17. The two groups were analyzed using chi-square test. The cell chi value was 0.11, degree of freedom was 1 and the p value was 0.4571, which is not significant.
The comparison of GCS and SAPS was done for Days 1, 2, 3, 4, 5, 10 and 15 between tracheostomy and the non-tracheostomy group. The GCS score on day 1 of admission was 4.06 ± 1.0278 (T) and 4.14 ± 0.9382 (NT); comparing the two groups using two-tailed t-test for unequal variance, p value was 0.6882, which is not significant [<0.05 taken as significant]. Similarly SAPS score on day 1 of admission was 59.34 ± 2.7247 (T) and 58.94 ± 2.2127 (NT); comparing the two groups using two-tailed t-test for unequal variance, p value was 0.6017, which is not significant. Thus at admission both tracheostomy and non-tracheostomy groups were matched with respect to GCS score and SAPS score.
The average day of tracheostomy was 2.18 ± 1.0038 days.
The GCS scores on days 1, 2, 3, 4, 5, 10 between tracheostomy and non-tracheostomy group were comparable, the p value being not significant between the two groups. However GCS scores were statistically significant on day 15 being higher in the tracheostomy group indicating improvement with tracheostomy (Table 1, Figure 1). Similarly the SAPS score on days 1, 2, 3, 4, 5 were comparable between tracheostomy and non-tracheostomy groups, the p value being not significant between the two groups. However, SAPS score on days 10 and 15 were statistically significant, being lower in the tracheostomy group indicating improvement with tracheostomy (Table 2, Figure 2).
Table 1 Comparison of sequential G.C.S. between tracheostomised and non-tracheostomised groups.
Tracheostomy Non Tracheostomy p Value
Day 1 4.06 ± 1.0278 4.14 ± 0.9382 0.6882
Day 2 5.04 ± 1.1825 4.74 ± 1.1800 0.2117
Day 3 5.82 ± 1.6815 5.40 ± 1.8617 0.2560
Day 4 6.62 ± 1.9596 6.21 ± 2.3211 0.3681
Day 5 7.12 ± 2.7472 6.91 ± 2.9671 0.7324
Day 10 8.35 ± 3.1983 7.00 ± 3.4935 0.0723
Day 15 9.97 ± 3.0477 7.20 ± 3.3506 0.0009
Figure 1 The Glasgow coma score of the two groups from day 1 to days 15.
Table 2 Comparison of sequential S.A.P.S. between tracheostomised and non-tracheostomised groups.
Tracheostomy Non Tracheostomy p Value
Day 1 59.34 ± 2.7247 58.94 ± 2.2127 0.6016
Day 2 59.24 ± 2.4294 58.30 ± 2.3515 0.7243
Day 3 57.88 ± 3.4850 56.97 ± 2.1617 0.1303
Day 4 55.70 ± 3.8587 55.97 ± 2.9302 0.6965
Day 5 54.04 ± 4.4856 55.25 ± 3.7422 0.1961
Day 10 49.48 ± 6.1954 52.20 ± 5.8929 0.0460
Day 15 43.86 ± 8.1665 49.83 ± 7.2391 0.0028
Figure 2 The SAPS score of the two groups (tracheostomized and non-tracheostomized) from day 1 to days 15.
Besides the improvement in the GCS and SAPS scores the end point of the study was to see whether tracheostomy led to decrease in mortality rate when compared with similar controls and whether there was reduction in hospital stay in tracheostomised patients. The final outcome (in terms of mortality) was analyzed utilizing chi-square test. The cell chi was 1.30 and 1.141, the degree of freedom was 1 and p was less than 0.05, which is significant (Table 3). The mortality in the T group was 36% compared to that of 58% in the NT group. This also compares favorably with the predictive mortality of >50% with SAPS II. Thus doing early tracheostomy improved significantly the mortality rate in patients with isolated closed head injury.
Table 3 Chi square table for outcome in the tracheostomised and non-tracheostomised groups.
Discharged Expired
32 18
Tracheostomy 26.5 23.5
1.141 1.30
21 29
Non Tracheostomy 26.5 23.5
1.141 1.30
Degree of freedom = 1
Overall cell chi = 4.88
p = 0.0275 (significant)
Hospital stay was analyzed separately for the patients who were discharged or those who died during the course of stay in hospital. The average day of mortality was 19.4130 ± 7.0476 (T) and 18.55 ± 9.7469 (NT); comparing the two groups using two-tailed t-test for unequal variance, p value was 0.6244, which is not significant. Similarly average day of discharge was 19.78 ± 6.9261 (T) and 18.66 ± 9.7834 (NT); comparing the two groups using two tailed t-test for unequal variance, p value was 0.5157, which is not significant (table 4). Performing early tracheostomy did not change length of hospital stay significantly in either patients who were discharged or those who expired during hospital stay. Probably this was so because abundant caution was taken to see that the patient is weaned off the tracheostomy and the attendants are adequately trained to perform nursing care of these patients. The very limited rehabilitation facilities meant that the patients had to undergo rehabilitation while they were hospitalized, prolonging further the hospital LOS.
Table 4 Comparison of hospital stay in the tracheostomised and non-tracheostomised groups.
Tracheostomy Non Tracheostomy p Value
Day of discharge 19.78 ± 6.9261 18.66 ± 9.7834 0.5157
Day of death 19.41 ± 7.0476 18.55 ± 9.7469 0.6244
Discussion
A tracheostomy is a proven adjunct in the care of head injury patients [5,8,9,12]. Tracheostomy provides an early airway protection and seems to decrease the need for prolonged mechanical ventilatory support. Secondly, severe head injury patients require a prolonged time for recovery and the airway reflexes are rarely optimal [3]. The association between the duration of intubation and risks of laryngotracheal injury is another important consideration in the timing of tracheostomy.
The tracheostomy tube facilitates pulmonary toilet and oral hygiene and has been shown to reduce the incidence of ventilator-associated pneumonia [5]. Furthermore, a tracheostomy tube is less noxious for the patient emerging from coma and sedation can be more easily weaned off. In addition, tracheostomy reduces significantly the physiological dead space of ventilation and thereby the work of breathing. This is even more beneficial in a patient having labored breathing or deteriorating respiration and may prevent the use of mechanical ventilation altogether. Because of these benefits, early tracheostomies have been shown to reduce hospital stay. However to realize the benefits of early tracheostomy without performing unnecessary tracheostomies, appropriate patients must be identified early at the time of admission. Early oxygenation and ventilatory abnormalities can predict the need for tracheostomy [19]. However, head injury patients primarily require airway protection and not necessarily ventilatory support for pulmonary failure.
A study by Major et al [1] showed that using objective scores such as GCS (less than seven) and SAPS (more than fifteen) score could aid in identifying those patients who will eventually require a tracheostomy for prolonged airway protection after blunt head trauma with high positive predictive value. However, they used day 4 scores of GCS and SAPS to determine the need for tracheostomy and performed the tracheostomy only after day 5. This way they not only excluded patients who were extubated but even those who died early, thus removing an important cohort who could benefit from tracheostomy. This skewed mortality data as severe head injury is associated with early mortality. In addition, these patients were admitted in ICU and were intubated already as per ATLS standards. Positive predictive value for GCS and SAPS score was 71%, and negative predictive value was 83%. Gurkin et al [6] found two admission criteria (GCS <9 and ISS >24) to be predictive of the need for tracheostomy in those patients that remained intubated on day 7. Rodriguez et al [9] noted a significant decrease in ventilator days and ICU days and hospital length of stay in patients who underwent tracheostomy within five days of intubation.
In a study by Sugerman et al [4] several major trauma centers refused to participate in early tracheostomy (5 to 7 days) trial because they felt strongly, like we do, that either all severly injured patients should undergo tracheostomy within 2 to 3 days after injury or tracheostomy was not necessary for as long as 3 to 4 weeks after injury. In their study they found that head injury patients who underwent early tracheostomy had higher APACHE III scores but there was no difference in ICU LOS, pneumonia and death in these patients as compared to patients with late tracheostomy.
Lesnik et al [5] retrospectively reviewed 101 adult patients with blunt injuries, 32 had tracheostomy within first 4 days and 69 underwent tracheostomy after 4 days. The author found that mean duration of ventilatory support was 6.0 days in early tracheostomy group versus 20.6 days in the late tracheostomy group (p < 0.001).
In a study by Bouderka et al a prospective study was conducted in patients with admission GCS of 8 or less, cerebral contusion on CT scan and GCS score of less than 8 on day 5. These patients were randomized into early tracheostomy and prolonged intubation. Besides demographic data admission scores SAPS, ICU stay duration of mechanical stay was compared. They concluded that early tracheostomy decreased total days of mechanical ventilation (p = 0.02). They agreed that choosing their criteria had the limitation of high mortality during the first week of hospitalization. They suggested that most of the patients did not require mechanical ventilatory support but were intubated mainly for airway protection. Early tracheostomy may provide an early alternative for airway protection and assist in early termination of mechanical ventilatory support and there fore reduce hospital stay for these patients.
In our opinion using admission data to support a decision several days later is flawed. This diminishes the benefits of early tracheostomy and makes the decision more straightforward and increases the chances of laryngotracheal injuries due to intubation.
Early tracheostomy may assist in early termination of mechanical ventilation and therefore, reduce the hospital stay and mortality. Currently, the decision to proceed to early tracheostomy is based on the attending trauma surgeon's preference. The consensus conference of 1989 recommended conversion to tracheostomy if the anticipated need for mechanical ventilation is more than 21 days [15]. Such practice was based on earlier reports showing high tracheal stenosis rates with tracheostomy as compared with endotracheal intubation [13]. However, the incidence of tracheal stenosis has decreased substantially with recognition of its aetiology and improvements in tracheostomy materials, design and management, particularly with the use of high-volume, low-pressure cuffs. Also, the complications associated with prolonged endotracheal intubation are increasingly being recognized, including injury to the larynx and trachea [16-18], and patient discomfort. In addition, endotracheal intubation often requires the administration of systemic sedation, with attendant complications.
Despite evidence to support the utility of early tracheostomy, few recommendations exist to facilitate identification of appropriate patients.
In a study by Arabi et al [2], 136 patients underwent tracheostomies, of which only 29 were early (seven days). The duration of mechanical ventilation was significantly shorter with early tracheostomy (mean ± standard error: 9.6 ± 1.2 days versus 18.7 ± 1.3 days; P < 0.0001). Similarly, ICU LOS was significantly shorter (10.9 ± 1.2 days versus 21.0 ± 1.3 days; P < 0.0001). Following tracheostomy, patients were discharged from the ICU after comparable periods in both groups (4.9 ± 1.2 days versus 4.9 ± 1.1 days; not significant). ICU and hospital mortality rates were similar. Using multivariate analysis, late tracheostomy was an independent predictor of prolonged ICU stay (>14 days). The very low mortality seen in the patients we studied may be explained by selection of proper candidates for tracheostomy, excluding those patients who were unlikely to survive. Hospital LOS in these patients was prolonged, reflecting their severe injuries that required lengthy rehabilitation periods.
Strengths of our study include prospective data collection ensuring complete data. The cohort was homogenous in that the decision for tracheostomy was not affected by other injuries (maxillofacial, neck) which may mandate early tracheostomy. Similarly the outcome in either group remained uninfluenced by systemic injuries. Our study included all patients with severe head injury (including those with early mortality) by virtue of doing early tracheostomy, unlike other studies [1-4,10,11] which excluded such patients by doing tracheostomy on day 5 onwards. By taking SAPS II as a criterion, we tried not to overdo tracheostomies by including only those patients in whom the head injury was severe enough to affect systemic physiological parameters. Lastly, we have taken serial scores as criteria for tracheostomy rather than admission scores. However data extraction and analysis was retrospective. Because the database was not designed specifically to examine tracheostomy practices, certain issues were not documented, such as comparison with intubation and the type of tracheostomy done. Also, we did not compare the morbidity in the two groups. In addition, the study was conducted from one centre. A large multicentre randomized controlled trial in which patients are randomized to early versus late tracheostomy would be the ideal way to test the impact of procedure timing on resource utilization.
Conclusion
We conclude that early tracheostomy is beneficial in patients with isolated closed head injury, severe enough to affect systemic physiological parameters, in terms of decreased mortality and intubation associated complications in centers where ICU care is not readily or easily available. Also, in a selected group of patients, early tracheostomy may actually do away with the need for prolonged mechanical ventilation.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
CM, the principal author designed the study and was the surgeon in charge monitoring the management of these patients, JK contributed to the designing of study and statistical analysis., PK, JP, BP, P kalra, were the residents in charge of the management of these cases.RSM, DB were the senior surgeons also involved in the management of the cases.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The study had been reviwed and approved by the ethical committee of the hospital
==== Refs
Major KM Hui T Wilson MT Gaon MD Shabot M/M Margulies DR Objective indication for early tracheostomy after blunt head trauma Am J of Surg 2003 186 615 619 14672767 10.1016/j.amjsurg.2003.08.012
Arabi Y Hddad S Shirawi N Al Shimemeri A Early tracheostomy in intensive care trauma patients improves resource utilization: a cohort study and literature review Crit Care 2004 8 R347 R352 15469579 10.1186/cc2924
Bouderka MA Fakhir B Bouaggad A Hmamouchi B Hamoudi D Harti A Early tracheostomy versus prolonged endotracheal intubation in severe head injury J Trauma Inj Infect and Crit Care 2004 57 251 254 10.1097/01.TA.0000087646.68382.9A
Sugerman HJ Wolfe L Pasquale MD Rogers FB O'Malley KF Knudson M DiNardo L Gordon M Schaffer S Multicenter, randomized, prospective trial of early tracheostomy J Trauma 1997 43 741 747 9390483
Lesnik I Rappaport W Fulginiti J Witzke D The role of early tracheostomy in blunt, multiple organ trauma Am Surg 1992 58 346 349 1596033
Gurkin MA Parikshak M Kralovich KA Indicators for tracheostomy in patients with traumatic brain injury Am Surg 2002 68 324 328 11952241
Santos PM Afrassiabi A Weymuller EA Risk factors associated with prolonged intubation and laryngeal injury Otolaryngol Head Neck Surg 1994 111 453 459 7936678
Armstrong PA McCarthy MC Peoples JB Reduced use of resources by early tracheostomy in ventilator-dependent patients with blunt trauma Surgery 1998 124 763 766 9780999 10.1067/msy.1998.91224
Rodriguez JL Steinberg SM Luchetti FA Gibbons KJ Taheri PA Flint LM Early tracheostomy for primary airway management in the surgical critical care setting Surgery 1990 108 655 659 2218876
Kluger Y Paul DB Lucke J Cox P Colella JJ Townsend RN Raves JJ Diamond DL Early tracheostomy in trauma patients Eur J Emerg Med 1996 3 95 101 9028753
Teoh WH Goh KY Chan CL The role of early tracheostomy in critically ill neurosurgical patients Ann Acad Med Singapore 2001 30 234 238 11455734
D'Amelio Lf Hammond JS Spain DA Sutyak JP Tracheostomy and percutaneous endoscopic gastrostomy in the management of the head injured trauma patient Am Surg 1994 60 180 185 8116977
Stauffer JL Olson DE Petty TL Complication and consequences of endotracheal intubation and tracheostomy: a prospective study of 150 critically ill patients Am J Med 1981 70 65 76 7457492 10.1016/0002-9343(81)90413-7
Lanza DC Parnes SM Koltai PJ Fortune JB Early complications of airway management in head-injured patients Larygoscope 1990 100 958 961
Plummer AL Gracey DR Consensus conference on artificial airways in patients receiving mechanical ventilation Chest 1989 96 178 180 2500308
Whited RE A prospective study of laryngotracheal sequelae in long-term intubation Laryngoscope 1984 94 367 377 6700353
Lindholm CE Prolonged endotracheal intubation Acta Anaesthesiol Scand Suppl 1969 33 1 131 5459079
Colice GL Stukel Ta Dain B Laryngeal complications of prolonged intubation Chest 1989 96 877 884 2791687
Johnson SB Kearney PA Barker DE Early criteria predictive of prolonged mechanical ventilation J Trauma 1992 33 95 100 1635112
Le Gall JR Lemeshow S Saulnier F A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study JAMA 1993 270 2957 2963 8254858 10.1001/jama.270.24.2957
Ross BJ Barker DE Russell WL Burns RP Prediction of long-term ventilatory support in trauma patients Am Surg 1996 62 19 25 8540640
Vincent JL Lobo S Struelens M Ventilator associated pneumonia: risk factors and preventive measures J Chemother 2001 1 211 217 11936368
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16225679
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PMC1266360
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CC BY
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BMC Endocr DisordBMC Endocrine Disorders1472-6823BioMed Central London 1472-6823-5-81621910610.1186/1472-6823-5-8Research ArticleThe spectrum of thyroid dysfunction in an Australian hepatitis C population treated with combination Interferon-α2β and Ribavirin Tran Huy A [email protected] Tracey L [email protected] Robert G [email protected] Hunter Area Pathology Service, John Hunter Hospital, Locked Bag Number 1, Hunter Mail Region Centre, Newcastle, New South Wales 2310, Australia2 Hepatitis C Service, Gastroenterology Department, John Hunter Hospital, Locked Bag Number 1, Hunter Mail Region Centre, Newcastle, New South Wales 2310, Australia3 Drug And Alcohol Unit, Hunter Area Health Service, John Hunter Hospital, Locked Bag Number 1, Hunter Mail Region Centre, Newcastle, New South Wales 2310, Australia2005 12 10 2005 5 8 8 13 5 2005 12 10 2005 Copyright © 2005 Tran et al; licensee BioMed Central Ltd.2005Tran 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 aims to assess the pattern of thyroid response to combination Interferon-α2β (IFN-α) and Ribavirin (RBV) anti-viral therapy in an Australian hepatitis C cohort. These include the prevalence of thyroid dysfunction (TD) including hyperthyroidism and hypothyroidism and their possible predictors, the common overall pattern of thyroid function tests whilst receiving therapy and TD outcomes, and the correlation with HCV status outcome.
Methods
A retrospective analysis of all medical records was performed to assess thyroid function in Hepatitis C Virus (HCV) patients who were treated at the Hunter Area hepatitis C treatment center between 1995 and March 2004. The centre is part of the John Hunter hospital, a major tertiary referral centre in New South Wales, Australia.
Results
There were 272 cases available for review. The prevalence of TD is 6.7 percent and is made up predominantly of females (80 percent). There were 3 (1.1 percent) cases of hyperthyroidism with 2 (67 percent) females. Thirteen out of fifteen (80 percent) cases of hypothyroidism were females with the overall prevalence of 5.5 percent. The majority of hypothyroid patients still required Thyroxine supplement at the end of follow up.
Conclusion
Ninety three percent of HCV treated patients have intact thyroid function at the end of treatment. The predominant TD is hypothyroidism. The predominant pattern of thyrotoxicosis (TTX) is that of thyroiditis although the number is small. Graves' like disease was not observed. People with pre-existing thyroid auto-antibodies should be closely monitored for thyroid dysfunction, particularly hypothyroidism.
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Background
Hepatitis C infection is one of the major epidemics afflicting young adults with more than 150,000 known infected cases in Australia and a notification rate of ~16,000 cases per year in 2002 [1]. In the United States, HCV remains the most common chronic blood-born infection [2,3]. The effective management of the new cases is critically important because, without treatment, approximately 14,000 will develop chronic HCV infection, 6,500 will develop HCV-related cirrhosis, 175 liver failure and 50 with hepatocellular carcinoma. The treatment typically involves the combination of IFN-α and RBV therapy. This is an effective therapy with a 'cure' rate of up to 70% depending on genotype as judged by the negative HCV Ribonucleic Acid (RNA) polymerase chain reaction (PCR) detection [4]. However, no treatment is free from complication and the use of IFN-α is well documented to be associated with TD, the commonest autoimmune disorder associated with IFN-α therapy. This study looks at the combined effect of IFN-α and RBV in an exclusively Australian group of patients with hepatitis C to determine the pattern of thyroid behaviour with direct regards to eu-, hyper- and hypo-thyroidism. The prevalence of hypothyroidism prevalence and outcome of treatment, in terms of HCV RNA clearance, in direct relationship to thyroid condition are both determined.
Methods
Patients
The cases of 272 patients who received combination therapy between 1995 and end of March 2004 at the John Hunter hospital Hepatitis C service were reviewed. All other causes of chronic hepatitis were excluded. No patient had dual Hepatitis B and C. Baseline characteristics of all studied subjects are included in Table 1. Family history of thyroid disease is not available other than in patients who subsequently developed TD.
Table 1 Baseline characteristics of 272 patients who received combination IFN-α and RBV therapy for HCV
Demographics
Mean age (years) 42 ± 8
Males 150 (55%)
Caucasians 204 (75%)
Asians 22 (8%)
Weight (kg) 79 ± 18
HCV Genotype
1 136 (50%)
2 22 (8%)
3 103 (38%)
4 11 (4%)
Liver Function Tests (RR)
Albumin (36–48 g/L) 41 ± 2
Serum Bilirubin (2–20 μmol/L) 15 ± 6
Alanine Aminotransferase (< 45 U/L) 133 ± 58
γ-Glutamyl Transpeptidase (1–30 U/L) 98 ± 46
Prothrombin time (11–18 seconds) 15 ± 3
Haematological Parameters (RR)
Haemoglobin (115–165 g/L) 142 ± 16
White cell counts (4.0–11.0 × 106/mL) 7.1 ± 2.0
Platelets (150–400 × 109/mL) 168 ± 49
Laboratory assays
Serum autoantibodies to anti-thyroglobulin (anti-Tg) and anti-thyroperoxidase (anti-TPO) were measured by agglutination (Serodia-ATG and Serodia-AMC, Fujirebio, Inc., Tokyo, Japan). Titre of less than 1:400 was considered normal for both. Thyroid Stimulating Immunoglobulin (TSI) was measured using cell culture and radio-immunoassay. This is an in-house bioassay using Chinese Hamster Ovary (CHO) cells in culture to detect the presence of thyroid stimulating activity. The CHO cells are transfected with the TSH receptor genes and thus are responsive to TSI. Thyroid-stimulating activity is measured by evaluating the intracellular release of cyclic Adenosine Mono-Phosphate induced by the patient's serum immunoglobulin on the CHO cells. The results are reported as units/mL (U/mL). TSI should be absent in the normal population. A TSI level of <10 is considered negative, 10–50 as weakly, 50–100 as moderate and >100 U/mL as strongly positive.
Third generation serum thyrotropin (TSH), serum free tetra- and free tri-iodothyronine (fT4 and fT3) were determined by two-site sandwich immunoassay using an automated chemiluminescent system (Diagnostic Products Corporation, Immulite 2000). The reference range (RR) for TSH was 0.4–4.0 mU/L, fT4 10.0–26.0 and fT3 3.5–5.5 pmol/L. The coefficients of variations (CV) were 5.0 % and 5.1 % at TSH concentrations of 4.0 mU/L and 10.0 mU/L respectively. For fT4, the CV was 6.5% at 10.0 pmol/L and fT3 8.9% at 3.5 pmol/L.
Therapy
All patients were treated with combination IFN-α and RBV therapy. The duration of treatment depends on the HCV genotypes; genotypes 2 and 3 were treated for 24 weeks and types 1 and 4 for 48 weeks respectively. Treatment was continued to the end for the latter irrespective of the HCV RNA status at 24 weeks. The dosage for IFN-α was 3 MIU thrice a week with RBV dose ranging from 1000 to 1200 mg daily according to bodyweight.
Thyroid function assessments
All patients received routine thyroid function tests (TFT) at the start of treatment and at monthly intervals. If there was any concern, then the frequency was increased as clinically indicated. Most, particularly the thyrotoxic group, but not all patients were referred to the Endocrine service for review. Thyroxine supplement was prescribed for hypothyroidism when deemed clinically appropriate. All patients were followed up for a period of 12 months after the completion of anti-viral therapy. Some were followed additionally for a longer period in the Endocrine outpatient service. Thyroid autoantibodies were not routinely performed until the patient developed hypo- or hyperthyroidism on clinical and/or biochemical grounds.
TTX was defined as having TSH of < 0.1 mU/L, fT4 levels > 26.0 and/or fT3 levels > 5.5 pmol/L respectively.
Hypothyroidism, including subclinical hypothyroidism, was defined as having TSH levels > 4.0 mIU/L. Thyroxine supplement was, however, given when deemed clinically appropriate regardless of the degree of TSH elevation.
Thyroid dysfunction
Thyroid dysfunction (TD) was defined as having hypo- or hyper-thyroidism, (clinically and/or biochemically based)
Statistics
Data are presented as percentage and mean ± Standard Error of Mean (SEM).
Results
Incidence of thyroid dysfunction in HCV treated group
The majority of patients (93%) had no TD at the end of treatment. Amongst the 272 patients, there were a total of 18 (6.7%) cases of TD: 3 hyper-thyroidism and 15 hypo-thyroidism.
There were 3 cases of pre-existing hypothyroidism detected at baseline and thus were excluded from the study. All cases were detected at the initial assessment for the treatment of hepatitis C and were shown to have auto-immune hypothyroidism requiring thyroxine supplement. This gave a pre-treatment hypothyroidism prevalence of 1.1% (3/275). Hypothyroidism was seen in 15 (5.5%) with 12 (80%) females after treatment. Thirteen (87%) patients required thyroxine therapy.
TTX was observed in 3 (1.1%) patients whose characteristics are listed in Table 2. Two (67%) were females and all required endocrinology attention. None had any thyroid imaging studies and thus the diagnosis was made on clinical grounds, auto-antibody detection and disease behaviour.
Table 2 The pattern of TTX in patients receiving combination IFN-α and RBVNo thyroid nuclear or ultrasonic imaging was available for all thyrotoxic cases
Subjects 1 2 3
Gender F F M
Age 26 35 36
HCV Genotype 1a 3a 1g
Regimen duration (weeks) 48 24 24
Adverse reactions Myalgia, lethargy, emotional lability None Insomnia, rash
Baseline ALT 184 142 104
ALT at time of thyroid disease 33 28 17
Duration of therapy prior to thyroid disease (weeks) 16 8 32
Symptoms of thyroid disease Anxiety and depression Non-specific Non-specific
Signs No goitre No goitre No goitre
Peak fT4 (pmol/L) 56.7 57.0 27.0
Peak fT3 (pmol/L) 15.1 15.3 12.8
Thyroid autoantibody status • Anti-Tg <1
• Anti-TPO 1:409,600
• TSI <1 • Anti-Tg <1
• Anti-TPO 1:409,600
• TSI <1 • Anti-Tg <1
• Anti-TPO <1:100
• TSI <1
Thyroid outcomes Hypothyroidism on replacement therapy Hypothyroidism on replacement therapy Thyroid condition resolved with IFN-α dose reduction
Hepatic outcomes PCR negative PCR negative PCR negative
Thyroid response to combination Interferon-α2B and RBV therapy
1.1 Euthyroid patients
Patients who were treated with IFN-α and had normal TFTs were divided into 2 groups according to duration of treatment: at 24 (n = 117) and 48 (n = 136) weeks respectively. Both demonstrated a drop in TSH levels at about 4 months after entry into therapy. Interestingly, none of these patients lowered their TSH level below 0.4 U/L. This was followed by a return to pre-treatment levels and remained so in the follow-up period. Results are illustrated in Table 3 and Figure 1.
Figure 1 The normal patterns of TSH response in hepatitis C patients during and after receiving combination IFN-α and RBV therapy in the two treated groups at different durations.
Table 3 TSH data for the 6 and 12 month treated cohorts who did not develop TD
Time (months) 0 2 4 6 16
Mean TSH ± SEM (mU/L) for the 6 month treated cohort n = 117 1.62 ± 0.11 1.51 ± 0.12 1.29 ± 0.08 1.56 ± 0.13 1.76 ± 0.11
0 4 8 12 16
Mean TSH ± SEM (mU/L) for the 12 month treated cohort n = 136 1.65 ± 0.15 1.31 ± 0.08 1.58 ± 0.17 1.57 ± 0.14 1.71 ± 0.16
1.2 Hypothyroid patients
All hypothyroid patients had non-specific symptoms and relevant data are listed in table 4. Free T3 levels were not routinely measured in the presence of hypothyroidism. Anti-TPO antibodies were detected in 6 out of 15 hypothyroid patients (40%) with titres ranging from 1: 6,400 to 1: 409,600. The rest had titres <1:400. Baseline autoantibodies were not routinely requested at the initial assessment.
Table 4 Data for hypothyroid patients receiving combination therapy for Hepatitis C infection
Number of subjects 15 (9 treated for 48 weeks and 6 for 24 weeks)
Gender 3 M : 12 F
Mean age 44 ± 2
Positive family history of thyroid disease (in first degree relatives) 8/18 (44%)
Duration of IFN-α before thyroid disease developed (months) 4.8 ± 1.2
Mean TSH levels (mU/L) 29.7 ± 8.8
Mean fT4 levels (pmol/L) 3.5 ± 0.9
The presence of TPO-autoantibodies 6/15 (40%)
Thyroxine requirement at the 12-month follow-up 13/15 (87%)
PCR negativity at the end of treatment 15/15 (100%)
1.3 Hyperthyroid patients
All 3 thyrotoxic patients had suppressed TSH levels with varying levels of fT4 and fT3. The time to development of TTX was variable but most occurred whilst receiving treatment. Anti-TPO antibodies were elevated in patients 1 and 2 (2 out of 3 cases) and both behaved in a thyroiditis fashion. Hypothyroidism developed rapidly and Thyroxine supplement was subsequently required within 1 month. In patient 3, despite the negative antibody results, the TTX responded to a reduction in IFN-α dosage suggesting an IFN-α/autoimmune mediated mechanism. TSI was undetectable in all 3 patients.
All (100%) patients with thyroid dysfunction cleared their hepatitis C infection based on the aforementioned criteria against a background overall response rate of ~75% to the combination anti-viral therapy in our cohort.
Discussion
This is the first report to characterise thyroid responses to combination anti-viral treatment in an Australian cohort with hepatitis C infection. The prevalence of TD is ~7% of which 83% is hypothyroidism. The predominant gender is female at 80%. This is consistent with previous reports relevant to this condition [5-7]. The prevalence range is broad reflecting the differences in the definition of TD, some are biochemically based, others clinically based or both.
Overall, 93% (254/272 cases) of patients have normal thyroid outcome. In the majority of cases, the TSH levels remain within reference range throughout. Levels decrease slightly at 4 months but return to pre-existing level with the continuation of treatment irrespective of the duration of therapy. The range of TSH values is narrow ranging between 1.15 at the 4-month nadir and 2.03 at 16 months in 95% of cases whilst undergoing therapy. FT4 levels were not routinely measured in the presence of normal TSH levels which remain the single best assessment of thyroid function in the presence of intact hypothalamo-pituitary-thyroid axis [8], expected in this sub-group. Whilst it is tempting to attribute the decrease in TSH level during treatment to IFN-α and RBV, it is likely that non-thyroidal illness is the main underlying pathogenesis. Other co-morbidities associated with combination treatment (but particularly IFN-α) include fever, headache, depression and neuropsychiatric disturbance etc... all contribute to the changes in TFTs [9].
In TTX, the number is too small to arrive at any definitive conclusions. Only the destructive thyroiditis form is observed in this study. Goitre is absent in all cases. In patients 1 and 2, this type of hyperthyroidism rapidly converts to hypothyroidism in the presence of high auto-antibody titre and subsequently required permanent Thyroxine supplement. In patient 3, the TTX was mild and resolved with IFN dose reduction. This was presumed to be IFN-α/autoimmune mediated despite the absence of auto-antibodies. This pattern has been observed elsewhere [10-12]. Unfortunately, radioactive iodine uptake scans were not performed so that the diagnoses could be unequivocally confirmed. TTX resembling Graves' disease was not observed. In this scenario, TTX is uncommon and thus each case should be dealt with on its own merits. The index of clinical suspicion should remain high whilst on therapy. Management is best done in a specialist Endocrine clinic, with destructive thyroiditis being more common, often resulting in permanent hypothyroidism.
Hypothyroidism is clearly the commoner cause of TD in combination therapy. It has been suggested that the virus itself may play a role in the development of hypothyroidism in IFN-naïve patients as the prevalence is higher than the general population [13]. Also, if IFN-α alone is thought to cause hypothyroidism, the prevalence of hypothyroidism should be higher than in IFN-naïve patients and this is the case of this report with a 5 folds increase after treatment. However, this observation has not been consistent in the general literature, see Table 5[14-19]. To further add to the complexity of the situation, hypothyroidism is also more frequent in patients having combination therapy of IFN-α and RBV [20] (as opposed to IFN-α treated alone). Whilst HCV is well known to induce a higher prevalence of auto-antibody, this does not necessarily translate into hypothyroidism (either clinical or subclinical) [14]. It would have been additionally interesting to characterize, in parallel, the pattern of thyroid function in our HCV patients who did not receive HCV treatment for direct comparison. Unfortunately these data are not available.
Table 5 The prevalence of hypothyroidism in the IFN-naïve and IFN-treated HCV patients compared with the general populations. The prevalence of elevated TSH levels in the various population groups include: Australia ~2.0% [17], the United States of America ~7.3% [18], Spain ~2.0% [18], Japan ~1.3% [18], Great Britain ~7.3% [18] and Italy ~1.7% [19]
Studies N = number of subjects Prevalence of hypothyroidism (before IFN) Prevalence of hypothyroidism (after IFN)
Marcellin et al. [5] N = 74 0 7.2
Preziati et al. [15] N = 78 0 22.8
Imigawa et al. [14] N = 58 0 3.4
Marazuela et al. [7] N = 207 4.7 2.8
This work N = 272 1.1% 7.5%
Baudin et al. [16] N = 68 0 7.3
Antonelli et al. [13] N = 630 13% N/A
The pathogenesis remains poorly understood but IFN-α is thought to be related to have a direct inhibitory effect on thyrocytes preventing hormonogenesis and secretion. Another postulate is immunostimulation in the presence of hepatitis C infection. This is thought to include activation of lymphocytes and natural killer cells, increased production of Tumor Necrosis Factor, IFN-α, Interleukin and other cytokines and increased production of immunoglobulins [21]. All lead to the development of thyroid auto-antibodies with complete destruction and consequently permanent hypothyroidism in genetically susceptible individuals. Although this does occur in hepatitis B IFN-α treated patients, the prevalence of hypothyroidism is much lower. This suggests that the hepatitis C virus or its genome plays an integral part the development of thyroid dysfunction [10]. The virus itself has been postulated to induce thyroid auto-antibodies by ways of generating high endogenous IFN levels triggering off autoimmune thyroid disease in susceptible individuals, similar to Coxsackievirus. This virus and others have been shown to induce a higher level of endogenous IFN-α levels which have been associated with other auto-immune diseases such as type 1 diabetes [22]. When IFN-α is administered exogenously, another layer of complexity is added. It is possible but purely speculative that exogenous IFN-α synergises with the endogenous source, thus exaggerating the effect on the thyroid thus causing additional hypothyroidism.
Whatever the underlying pathophysiological process is, the major factors contributing to hypothyroidism includes the female gender with a relative risk ranging between 3–7 times [23] and the presence of anti-TPO antibodies (Ab). These factors feature prominently in our report although the prevalence of anti-TPO Ab is lower. The time to onset of biochemical hypothyroidism means at about 5 months, similar to other cohorts [6].
When IFN-α triggers destructive thyroid disease in genetically susceptible individuals, some may recover but the majority does not in our study. Thyroxine requirement therefore is expected to be long term as illustrated in 87% (13/15) of our cases. All 6 (100%) patients where anti-TPO antibodies are detected, are on thyroxine at the completion of follow up. The symptomatology of hypothyroidism is non-specific and often overlaps with IFN-α side-effects. The diagnosis often requires biochemical testing with some patients receiving thyroxine supplement at mildly elevated concentrations of TSH and/or low/normal fT4 levels. Where thyroxine was deemed necessary and thus given, all were continued with thyroxine supplement at 12-month follow-ups suggesting that thyroid damage is permanent although longer follow-up is preferred.
All patients with TD cleared their HCV infection up until end of follow-up. The number is too small to make any conclusion about the advantage of having thyroid dysfunction when it comes to eradicating the virus. Hypothetically, these patients may have developed an exaggerated response which helps to clear the virus and in the process induces thyroid dysfunction, an unfortunate but acceptable side-effect of the treatment modality. Whilst this is highly plausible, it has not been supported by other investigators [24].
Conclusion
IFN-α in combination with RBV therapy, by and large, does not cause thyroid dysfunction in the majority of HCV patients undergoing such treatment. Patients undergoing such therapy can expect a small decrease in TSH levels, which remained in the RR however, without going into frank hyper- or hypothyroidism. The incidence of TD in HCV patients receiving combination anti-viral therapy is generally small with hypothyroidism being the most common. All patients should be assessed thoroughly for relevant risk factors and monitoring for dysfunction at the appropriate time frame. Hyperthyroidism is uncommon and thus is best managed in a specialised Endocrine clinic. The important points derived from this study are summarized in Table 6.
Table 6 Major summary points from the study
1. The majority of patients with HCV undergoing combination IFN-α and RBV therapy have normal thyroid function.
2. The commonest cause of thyroid dysfunction is hypothyroidism, a ratio of 4:1 compared with hyperthyroidism.
3. The average time from the start of anti-viral treatment to hypothyroidism is ~4–5 months, suggesting that this is the critical time to carry out thyroid testing.
4. Predisposing risk factors include female gender, family history of thyroid disease and existing thyroid auto-antibodies, especially anti-TPO antibodies. In this situation, initial TSH should be performed to exclude pre-existing hypothyroidism.
5. Hyperthyroidism is less common and each should be managed on its own merits in a specialised Endocrine service.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TLJ gathered, provided the data and participated in the discussion and drafting of the manuscript. RGB participated in the scientific discussion and drafting on the manuscript. HAT conceived the study, participated in its design, assisted with data collection, coordinated and helped to draft the manuscript. All authors read and approved the revised manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We are very much indebted to Geoffrey Kellerman for his constructive and analytical comments of this manuscript.
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Sievert W Crofts N, Dore G, Locarnini S An overview of antiviral therapy for chronic hepatitis C infection 2001 Hepatitis C: an Australian perspective. Melbourne: IP Communications 140 154
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Marazuela M Garcia-Buey L Gonzalez-Fernandez F Garcia-Monzon D Arranz A Borque MJ Moreno-Otero R Thyroid autoimmune disorders in patients with chronic hepatitis C before and during interferon-α therapy Clin Endocrinol 1996 44 635 642 10.1046/j.1365-2265.1996.751768.x
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Fernandez-Soto L Gonzalez A Escobar-Jimenez F Vazquez R Ocete E Olea N Salmeron J Increased risk of autoimmune thyroid disease in hepatitis C vs hepatitis B before, during, and after discontinuing interferon therapy Arch Intern Med 1998 158 1445 1448 9665354 10.1001/archinte.158.13.1445
Wong V Fu Xi-Li A George J Cheung NW Thyrotoxicosis induced by alpha-interferon therapy in chronic viral hepatitis Clin Endocrinol 2002 56 793 798 10.1046/j.1365-2265.2002.01553.x
Minelli R Valli MA Di Secli C Finardi L Chiodera P Bertoni R Ferrari C Barilli AL Coiro V Saccanti Jotti G Delsignore R Is steroid therapy needed in the treatment of destructive thyrotoxicosis induced by alpha-interferon in chronic hepatitis C? Horm Res 2005 63 194 199 15860921 10.1159/000085538
Antonelli A Ferri C Pampana A Fallahi P Nesti C Pasquini M Marchi S Ferrannini E Thyroid disorders in chronic hepatitis C Am J Med 2004 117 10 13 15210382 10.1016/j.amjmed.2004.01.023
Imagawa A Itoh N Hanafusa Oda Y Waguri M Miyagawa J-I Kono N Kuwajima M Matsuzawa Y Autoimmune endocrine disease induced by recombinant interferon-alpha therapy for chronic active type C hepatitis J Clin Endocrinol Metab 1995 80 922 926 7883851 10.1210/jc.80.3.922
Preziati D La Rosa L Covini G Marcelli R Rescalli S Persani L Ninno ED Meroni PL Colombo M Beck-Peccoz P Autoimmunity and thyroid function in patients with chronic active hepatitis treated with recombinant interferon alpha-2a Eur J Endocrinol 1995 132 587 593 7749499
Baudin E Marcellin P Pouteau M Colas-Linhardt N Le Floch JP Lemonnier C Benhamou JP Bok B Reversibility of thyroid dysfunction induced by recombinant alpha interferon in chronic hepatitis C Clin Endocrinol 1993 39 657 661
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Carella C Mazziotti G Morisco F Rotondi M Cioffi M Tuccillo C Sorvillo F Caporaso N Amato G The addition of ribavirin to interferon-α therapy in patients with hepatitis C virus-related chronic hepatitis does not modify the thyroid autoantibody pattern but increases the risk of developing hypothyroidism Eur J Endocrinol 2002 146 743 749 12039693 10.1530/eje.0.1460743
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Chehadeh W Weill J Vantyghem M-C Alm G Lefebvre J Wattre P Hober D Increased level of interferon-α in blood of patients with insulin-dependent diabetes mellitus: Relationship with Coxsackievirus B infection J Infect Dis 2000 181 1929 1939 10837172 10.1086/315516
Prummel FM Laurberg P Interferon-alpha and Autoimmune Thyroid Disease Thyroid 2003 13 547 551 12930598 10.1089/105072503322238809
Dalgard O Bjoro K Hellum K Myrvang B Bjoro T Haug E Bell H Thyroid dysfunction during treatment of chronic hepatitis C with interferon alpha: no association with either interferon dosage or efficacy of therapy J Intern Med 2002 251 400 406 11982739 10.1046/j.1365-2796.2002.00974.x
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BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-5-321622130710.1186/1471-230X-5-32Research ArticleSerum hyaluronate as a non-invasive marker of hepatic fibrosis and inflammation in HBeAg-negative chronic hepatitis B Montazeri Ghodrat [email protected] Arezoo [email protected] Mehdi [email protected] Negin [email protected] Farhad [email protected] Ashraf [email protected] Mohammad Hossain [email protected] Farhad [email protected] Shahram [email protected] Reza [email protected] Digestive Disease Research Centre, Tehran University of Medical Science, Tehran, Iran2 Gastrointestinal and Liver Disease Research Center, Iran University of Medical Sciences, Tehran, Iran2005 12 10 2005 5 32 32 29 3 2005 12 10 2005 Copyright © 2005 Montazeri 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
HBV infection is a serious global heath problem. It is crucial to monitor this disease more closely with a non-invasive marker in clinical trials. We aimed to evaluate the predictive value of serum hyaluronate for the presence of extensive liver fibrosis and inflammation.
Methods
28 healthy volunteers and 65 patients with HBeAg negative chronic hepatitis B were enrolled. Liver biopsies scored according to Ishak system. Association of serum hyaloronate with liver fibrosis and inflammation were assessed, and cut off points for serum hyaluronate levels were identified by receiver operating characteristics (ROC) curves and their values for prediction of fibrosis and inflammation were assessed.
Results
In patients with CHB serum hyaluronate had the most significant correlation and predictive values for the liver fibrosis and inflammation comparing to the other variables. At the cut off point of 126.4 ngm/ml it could discriminate extensive fibrosis from milder ones with sensitivity of 90.9% and specificity of 98.1%. With the same value it could discriminate extensive inflammation from their milder counterparts with sensitivity of 63.6% and specificity of 92.6%.
Conclusion
Serum hyaluronate was the best predictor of extensive liver fibrosis and inflammation and it could discriminate subgroups of patients with chronic hepatitis B. It could be used as a non-invasive test to monitor these patients more closely with developing anti viral agents in clinical trials.
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Background
HBV infection is a serious global health problem. Of the 2 billions people who have been infected with HBV, more than 350 millions are chronically infected world wide [1,2]. Chronic HBV infection is the major cause of end stage liver disease, 25% of them will die prematurely of liver cirrhosis or hepatocellular carcinoma [3]. It is crucial to monitor the course of this disease more closely, especially with developing antiviral agents in clinical trials [4]. Liver biopsy is currently considered the gold standard for evaluation of liver fibrosis [5]. However it has several limitations like sampling error, post biopsy pain and death [6-9]. It is expensive procedure and could be exceedingly dangerous in cases of advanced liver disease with prolonged prothrombin time and low platelet count [10]. Liver function tests are essential parts of assessing liver damage, but have poor correlation with histology [11-13]. Because of limitations in conventional approaches, several non invasive tests have been developed for this purpose. Among them, serum hyaluronate as a direct marker of liver fibrosis appears to be the most promising one [14-17]. The studies have shown that hyaluronic acid increase in acute liver failure [18], primary billiary cirrhosis [19], alcoholic liver disease [20] and chronic hepatitis C [15].
Most of those studies have been performed in patients with chronic hepatitis C. The value of such markers in patients with in chronic hepatitis B remained unclear. We aimed to find out the utility of serum hyaluronate to evaluate the presence of extensive liver fibrosis and inflammation in patients with chronic hepatitis B.
Methods
Study population
Population groups under study consist of 28 normal volunteers, 65 patients with HBeAg negative chronic hepatitis B. We defined chronic hepatitis B as subjects who met at least two of three following criteria: 1) serum level of aminotransferases above 1.5–10 times of upper limit of normal. 2) total score of 4 or more according to Ishak (modified HAI) scoring system, and 3) viral DNA load of more than 1.77 pg/ml (equivalent to 500,000 copies/ml) using a signal amplification technique (Naxcor assay). Patients of chronic hepatitis B were selected from an open labelled Lamivudine trial from January 2001 to January 2004. All patients with chronic hepatitis B were negative for HBe antigen. They were all negative for HDV Ab, HCV Ab and HIV Ab. None of them had any evidence for decompensated cirrhosis or any other cause for chronic liver disease. Control group were selected from healthy volunteers. Five ml of peripheral of blood was taken from each patient in fasting state, one hour before performing liver biopsy and in complete rest. serum was isolated and kept at -70 degrees centigrade for measurement of serum hyaluronate. The same procedure was applied for normal volunteers.
Assessment of liver histology
Liver biopsy was performed by automatic true cut biopsy needle. Length and width of each sample were at least 10 mm and 1.4 mm respectively and contained 4 or more portal spaces. The liver histology was scored according to Ishak (modified HAI) scoring system [21]. Maximum grade (inflammation) was scored 18, and maximum stage (fibrosis) was scored 6. Stage 3 or more was considered as significant fibrosis, and stage 5 or 6 was considered as cirrhosis. All biopsies were reviewed by a single pathologist who was unaware of patients' clinical records.
Measurement of hyaluronic acid
Hyaluronic acid test kit was provided by Corgenix Inc. (Colorado, USA, under licence of Chugai diagnostic science Co.). Serum hyaluronate was measured by ELISA according to instruction manual of manufacturer. All samples used in duplicate. Briefly 100 μl of serum or reference solution (diluted 1:10) was added to each HABP (hyaluronic acid binding protein) coated micro well, incubated for 60 minutes, after washing 100 μl HRP-conjugated-HABP solution was added and incubated for 30 minutes. Eventually 100 μl of stopping solution (0.36 N sulphuric acid) was added. Optical density (OD) was read at 450 nm. Water was used as zero blank. Standard curve was produced by using ODs of 50,100, 200, 500, and 800 ng/ml of reference solution. Linear regression equation of standard solutions was produced. Values of all samples were computed on the basis of regression equation of standard solutions.
Statistical considerations
Statistical analyses were performed by using the SPSS, version 10.1, software package (SPSS, Inc., Chicago, IL). Data are reported as means ± standard deviation (SD). Patients with CHB were arbitrary divided into subgroups according to the degrees of fibrosis (stage 2 or less vs stage 3 or more) and inflammation (grade 0–8 vs grade 9 or more). The associations between factors (age, AST, ALT, alkaline phosphatase, platelet count, albumin, total bilirubin, prothrombin time (PT), and serum hyaluronate) with liver fibrosis or inflammation were assessed by Spearman correlation test. Then, independent factors associated with extensive fibrosis or inflammation were assessed by the regression analysis. Subsequently, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of hyaluronate to differentiate between subgroups of patients with chronic hepatitis was calculated using receiver operating characteristics (ROC) curve with different cut-off points. P values less than 0.05 were considered statistically significant.
Results
Twenty eight normal volunteers consist of 17 male and 11 female with the mean age of 32.0 ± 5.2 (range: 20–44) years old. Sixty five patients with chronic hepatitis B, consist of 54 male and 11 female with the mean age of 39.8 ± 12.3 (range: 18 – 76) years old. All of them had HBeAg negative CHB. Fifty four out of 65 patients with chronic hepatitis B had mild fibrosis (stage 0–2) and 11 (17%) had extensive fibrosis (stage 3–5).
Serum hyaluronate levels of the populations under study were shown in table 1. Normal volunteers had mean ± SD of serum hyaluronate level of 20.4 ± 15.4 ng/ml. The upper limit of normal (as defined by the 95th percentile of the variable) in the normal volunteers was 58.6 ng/ml. Patients with chronic hepatitis B as a single group had higher serum hyaluronate level than normal volunteers (73.4 ± 124.8 vs 20.4 ± 15.5 ng/ml respectively; P = 0.002). Serum hyaluronate level of cases with extensive fibrosis was significantly higher than those with mild fibrosis (309.7 ± 143.5 vs 24.7 ± 31.9 respectively; p < 0.001).
Fifty four out of 65 of CHB patients with mild inflammation (grade 0–8) had hyaluronate level of 39.6 ± 74.2 ngm/ml, while eleven of them with severe inflammation (grade = 9) had serum hyaluronate level of 236.5 ± 188.7 ngm/ml (p = 0.006) (Table 1).
Correlation of the variables with the level of fibrosis, and inflammation were assessed by Spearman correlation test. Serum hyaluronate had the most significant correlation with the level of fibrosis comparing to the other variables (Correlation coefficient = 0.58, p < 0.001) (Figure 1).
Next to that was correlation of serum hyaluronate to the level of inflammation which was highly significant comparing to the remaining variables (correlation coefficient = 0.42, p < 0.001). (Table 2).
Logistic regression analysis was carried out to find the independent variables associated with extensive liver fibrosis and inflammation in two separate analyses. Stage and grade were selected as dependent factor and other variables (age, hyaluronic acid, AST, ALT, alkaline phosphatase, platelet count, albumin, total billirubin and PT) as independent factors. Serum hyaluronate was the only independent factor associated with extensive liver fibrosis (P = 0.005), and inflammation (P < 0.001) in patients with chronic hepatitis B.
The ability of serum hyaluronate as a non-invasive test to discriminate levels of fibrosis and inflammation was assessed by ROC curve. Within the subgroups of chronic hepatitis B, serum hyaluronate at the cut-off point of 126.4 ngm/ml could discriminate mild (stage 0–2) from extensive fibrosis (stage 3–5) with an area under ROC curve (AUC) of 0.98, sensitivity of 90.9%, specificity of 98.1%, PPV of 90.9% and NPV of 98.1% (Figure 2). On the same basis serum hyaluronate could differentiate mild (grade 0–8) from extensive inflammation (grade ≥ 9) with an AUC of 0.86, the sensitivity of 63.6%, specificity of 92.6%, PPV of 63.6% and NPV of 92.6% (Figure 3).
Discussion
In this study we have shown that::1) Sub groups of patients with HBeAg negative chronic hepatitis B who had extensive fibrosis and inflammation had also high serum hyaluronate level. The differences were statistically significant comparing to normal values and to the patients with milder liver involvement. 2) Serum hyaluronate had more significant correlation with severity of liver fibrosis and inflammation amongst the other variables. 3) In the regression analysis serum hyaluronate had best predictive value for liver fibrosis and inflammation in patients with chronic hepatitis B. 4) At cut-off point of 126.4 ngm/ml it could differentiates extensive fibrosis from milder ones with sensitivity of 90.9% and specificity of 98.1%. At the same cut-off point, it could differentiate extensive inflammation from milder ones with sensitivity of 63.6% and specificity of 92.6%.
Liver biopsy is the method of choice in evaluating fibrosis and inflammation in patients with parenchymal liver disease [5,17], but it has several limitations which include false negative result of 24% especially in macro nodular cirrhosis [6,7], post biopsy pain and discomfort [9], high cost [10], and death rate of 0.015% [8]. In the past decade, several investigators focused on developing non-invasive test for liver fibrosis [16,17,22]. None of them proved to be perfect. Guechot et al showed that hyaluronate had better correlation to the degrees of fibrosis than PIIIP in chronic liver disease [23]. In the study by Oberti et al, serum hyaluronate level was considered the most sensitive test for screening in viral hepatitis B, and C [14]. Imbert-Bismuth et al suggested that, by fibro test 50% of liver biopsies could be avoided in patient with chronic hepatitis C [24]. However fibro test was reanalysed in treated hepatitis C, but its complex equation will limit its usefulness in clinical practice [25]. Thus, due to expense and complexity of Fibrotest-Actitest, it could not be utilized on routine basis in developing countries. Myers et al used Fibrotest and Actitest to decriminate.mild from extensive fibrosis and inflammation in patients with chronic hepatitis B [26]. In Spite of the interesting results, it is difficult to compare it with our data. However, hyaluronic acid may have a lower sensitivity for minimal fibrosis (as in chronic hepatitis C) as well as an absence of independent assessment of both fibrosis and activity as given by FibroTest-Actitest. We were looking for a direct marker of fibrogenesis to be cheap, reproducible and simple. The price of hyaluronic acid test is about 10 US Dollars in Iran as well as in Europe. In this regard serum hyaluronate appears to be an appropriate choice.
Hyaluronate is a polysaccharide with molecular weight ranging from 4 × 103 to 8 × 106 Daltons. It forms constituent of extracellular matrix in all connective tissues [27-29]. It is mainly produced by mesenchymal cells and cleared by hepatic sinusoidal endothelial cells through a high affinity receptor (Kid = 6 × 10-11 M) with a maximum capacity of 104 molecules/cells [30-33] It has short half life and increases by age [18].
Alcohol, viruses, auto immune diseases, and inborn errors of metabolism could increase production of hyaluronate by activating hepatic stellate cells and decrease clearance by hepatic sinusoidal capilarization [34]. Sinusoidal capilarization could be associated with shunting of blood which is an additional factor for increase of serum hyaluronate in this condition [35]. It was shown that serum hyaluronate increase in alcoholic liver disease [5,20,35], primary billiary cirrhosis [19] and in patients with hepatitis C [12,36]. In addition, it could be increased in rheumatoid disease due to overproduction by synovial cells [34,38]. It also increases in renal failure because of disturbed clearance of low molecular weight hyaluronate by the kidneys [39].
Aetiology of liver damage in our patients was only HBV infection. None of them had received any alcohol in their life time. None of them had any evidence for renal failure or any other disease which could explain their liver disease except HBV. Bloods were taken in complete physical rest and fasting state in order to rule out other interfering factors like eating and physical activity. Our normal level was 20.4 ± 15.5 ngm/ml which is in agreement with manufacturer levels (28.5 ± 24) and what we reported by the other investigators with the same age groups [15,16,20,40].
The proposed cut-off points in different studies were not the same. The cut-off point will depend on the aetiology of liver disease and on the level of sensitivity and specificity that an investigator is looking for [14,15,23]. Our study showed that the cut-off point of 126.4 ngm/ml could differential extensive fibrosis from milder ones with sensitivity of 90.9% and specificity of 98.1% and at the same level could differentiate extensive inflammation from milder ones with sensitivity of 63.6% and specificity of 92.6% in patients with chronic hepatitis B. Also, it should be noted that the cut-off value for a given variable depends upon the sample in which it has been identified. In order to be reproducible, a cut-off value should be obtained in a sample representative of the population with the disease.
Interestingly enough our results showed that, serum hyaluronate had best predictive value for the fibrosis and inflammation comparing to the other variables. This is in agreement with report of Ding H et al in which serum hyaluronate reflect both inflammation and fibrosis in HBV infection [41]. Currently it is believed that serum hyaluronate is a marker of liver fibrosis rather than inflammation. To what extent increased level of hyaluronate is due to inflammation alone without fibrosis, is difficult question to answer at the moment. It is because of complex interrelation of fibrogenesis and inflammatory process in vivo, which makes separation of pure fibrosis and inflammation impossible in a clinical setting. Elucidation of this complex issue requires further work. As stated above, regression analysis showed that only serum hyaluronate was associated with significant liver fibrosis. It should not be misinterpreted that other important factors (e.g. age, serum albumin, etc) are not associated with liver fibrosis in CHB. Since, the association of such a powerful factor (e.g. serum hyaluronate) was so close to the outcome (liver fibrosis) that the effects of other factors were excluded from the regression model.
Our study has some limitations. First and the most important limitation is that our study is considered a training study, and our data should be validated in another set of patients with chronic hepatitis B. Secondly, number of our patients with extensive fibrosis was relatively small. Further works are underway to validate this test, and also to find out other markers of liver fibrosis and inflammation in patients infected with hepatitis B virus.
Conclusion
In conclusion our data indicated that serum hyaluronate had significant correlation and predictive value for the presence of significant liver fibrosis and inflammation comparing to the other variables. In addition it was able to discriminate extensive fibrosis and inflammation from their milder counterparts in patients with HBeAg negative chronic hepatitis B with good sensitivity and specificity. We think serum hyaluronate is a useful non-invasive test to monitor these patients more frequently in clinical trials.
List of abbreviations
HBV: Hepatitis B virus
CHB: Chronic hepatitis B
ROC: Receiver Operating Characteristics
AUC: area under ROC curve
HBeAg: Hepatitis B e Antigen
ALT: Alanine aminotransferase
TGF-β: Transforming growth factor Beta
HDV: Hepatitis D Virus
HCV: Hepatitis C Virus
HABP: hyaluronic acid binding protein
OD: Optical Density
PPV: Positive Predictive Value
NPV: Negative Predictive Value
Competing interests
This study was supported by local funds from digestive disease research center, Tehran University of medical sciences and has had no external financial support
Authors' contributions
GM has been involved in the design and conception of the study, supervision of the work, and writing the manuscript. AE, NN, FM, SS contributed in the design of the study, and acquisition and analysis of data. MM carried out liver biopsies, and contributed in analysis and interpretation of data, and drafting the manuscript. AM carried out all laboratory assays. MHD contributed in analysis, and interpretation of data, and writing the manuscript. FM carried out liver biopsies and contributed in acquisition of data. RM contributed in the design of the study, supervised the study, and contributed in drafting 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:
Figures and Tables
Figure 1 Box plot of serum hyaluronate level in relation to the Ishak fibrosis score. The box represents the interquartile range, the whiskers indicate the highest and lowest values, and the circles represent outliers. The line across the box indicates the median value.
Figure 2 Receiver operating characteristics curve (ROC) of serum hyaluronate for discrimination of mild (stage 0–2) from significant (stage 3 or more) of fibrosis. Hyaluronate at cut off point of 126.43 ngm/ml could differentiate mild from severe fibrosis in patients with chronic hepatitis B.
Figure 3 Receiver operating characteristics curve (ROC) of hyaluronate for discrimination of mild (grade 0–8) from significant (grade ≥ 9) inflammation. Hyaluronate at cut off point of 126.43 ngm/ml could differentiate mild from severe inflammation in patients with chronic hepatitis B.
Table 1 Level of Hyaluronate in subgroup of chronic hepatitis B comparing to normal control
Patient Groups Subgroups n Mean ± SD P value*
Normal control 28 20.4 ± 15.4
Chronic hepatitis B subgroups (degree of fibrosis) Stage 0–2 54 24.7 ± 31.9 0.69
Stage 3–5 11 309.7 ± 143.5 <0.001
Chronic hepatitis B subgroups (degree of inflammation) Grade 0–8 54 39.6 ± 74.2 0.10
Grade ≥ 9 11 236.5 ± 188.7 0.003
*Statistical difference between serum hyaluronate of different groups compared with normal controls.
Table 2 Correlation between factors and stages of liver fibrosis on Spearman correlation test
Factor Correlation coefficient P value
Age 0.27 0.03
PT 0.30 0.02
Platelet counts - 0.24 0.05
AlkP 0.44 <0.001
Serum Hyaluronate 0.58 <0.001
Serum Albumin - 0.44 <0.001
Total Bilirubin 0.17 0.19
AST - 0.04 0.73
ALT - 0.12 0.36
Abbreviations: PT: Prothrombin time; AST: Serum aspartate aminotransferase; ALT: Serum alanine aminotransferase; AlkP: Serum alkaline phosphatase.
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BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-5-331622344910.1186/1471-230X-5-33Case ReportFamilial clustering of Leiomyomatosis peritonealis disseminata: an unknown genetic syndrome? Halama Niels [email protected] Silke A [email protected] Isam [email protected] Division of Gastroenterology, Department of Internal Medicine, Medical College of Ohio, 3000 Arlington Avenue, Toledo, Ohio 43614, USA2005 13 10 2005 5 33 33 13 5 2005 13 10 2005 Copyright © 2005 Halama 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
Leiomyomatosis peritonealis disseminata (LPD) is defined as the occurrence of multiple tumorous intraabdominal lesions, which are myomatous nodules. LPD is a rare disease with only about 100 cases reported. The usual course of LPD is benign with the majority of the patients being premenopausal females. Only two cases involving men have been reported, no syndrome or familial occurrence of LPD has been described.
Case presentation
We describe a Caucasian-American family in which six members (three men) are diagnosed with Leiomyomatosis peritonealis disseminata (LPD) and three deceased family members most likely had LPD (based on the autopsy reports). Furthermore we describe the association of LPD with Raynaud's syndrome and Prurigo nodularis.
Conclusion
Familial clustering of Leiomyomatosis peritonealis disseminata (LPD) has not been reported so far. The etiology of LPD is unknown and no mode of inheritance is known. We discuss possible modes of inheritance in the presented case, taking into account the possibility of a genetic syndrome. Given the similarity to other genetic syndromes with leiomyomatosis and skin alterations, we describe possible similar genetic pathomechanisms.
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Background
Leiomyomatosis peritonealis disseminata (LPD) is a rare smooth muscle tumor, which is characterized by multiple subperitoneal or peritoneal nodules. These myomatous nodules vary in size and can be found on any omental or peritoneal surface of the abdomen, often additional leiomyoma of the uterus are present. No clear correlation between LPD and uterine leiomyomas could be found so far, cases with and without uterine leiomyomas have been reported. Typical histological features are fusiform smooth muscle cells in interdigitating fascicles with no or low-grade cell atypia or mitotic figures [1,2]. Some studies have shown additional features like marked vascularity, without evidence of vascular invasion [3]. Ultrastructural investigations demonstrated abundant intracellular contractile fibers, a basement membrane surrounding every cell, mitochondria at the nuclear poles, stretched oval-shaped nucleoli with an uniform pattern and multiple pinocytic vesicles on the cell surface [4,5]. These findings resemble a developmental stage between fibroblast and mature smooth muscle cell. Immuno-histochemical testing revealed positive results for SMA (smooth muscle antigen), vimentin and desmin [6].
Incidental detection of these abdominal nodules during diagnostic imaging studies occurs frequently and most of the patients are asymptomatic. Symptoms -if any- are often abdominal masses, distension or increasing abdominal girth. Most of the patients present to the gastroenterologist or the gynecologist for further evaluation. Only one case showed an acute abdomen due to acute bowel obstruction as presenting symptom [7].
Differential diagnosis of carcinomatosis peritonealis or abdominal disseminated malignancy presents a major diagnostic problem and only biopsy and histological analysis can reveal the underlying disease. Only two cases of males with LPD have been reported, the majority of patients with LPD are premenopausal woman with high-estrogen states (e.g. pregnancy, oral contraceptive use or the presence of an estrogen-producing neoplasm). The underlying mechanism of LPD development is not known, but involvement of estrogens as etiologic factors has been postulated. Animal studies showed that prolonged elevated levels of estrogen can cause metaplasia of mesenchymal stem cells into fibroblasts, leiomyocytes or endometrial stroma [8]. These animals developed disseminated leiomyomatous peritoneal lesions, similar to LPD. High levels of estrogen and progesterone receptors are found on LPD cells, suggesting potential hormonal responsiveness of these tumors. Reducing excess estrogen levels, removal of estrogen exposure or chemical castration with gonadotropin-releasing hormone agonists can result in regression of the tumor masses [9], cases with successful surgical removal of nodules have also been reported. No definitive therapeutical concept has evolved so far. Furthermore two histological confirmed LPD cases with estrogen- and progesteron-receptor negative tumors have been reported, one in an elderly man and one in a 27 year old woman. The number of LPD cases reported so far is approximately 100, the first case being reported in 1952 by Willson and Peale. So far no familial clustering or involvement of LPD in any syndrome has been found, however genetic syndromes like leiomyomatosis of the esophagus (X-linked Alport syndrome, [10]) or genitourinary tract (multiple cutaneous and uterine leiomyomatosis, MCUL, [11]) are well known.
In this manuscript we describe a Caucasian-American family with several individuals of different generations affected by LPD, half of them being males. Furthermore the additional presence of two clinical traits (Raynaud's syndrome and Prurigo nodularis) was found in all LPD-patients, from whom precise clinical data was obtainable. In addition, we have conducted a review of the literature specifically involving LPD and similar syndromes to evaluate the modes of inheritance. We review these findings and comment on the possible molecular mechanisms leading to the occurrence of LPD (or a genetic syndrome involving LPD) in this family.
Case presentation
The propositus in this family (IV6) is a 45 year old Caucasian female who was diagnosed with LPD at the age of 24. Childhood was unremarkable for medical conditions, starting at the age of 17 she had recurrent episodes of prolonged abdominal pain with nausea and vomiting. During multiple admissions to the emergency department, abdominal X-rays showed repeatedly intestinal obstruction. No further investigation regarding the cause for this abdominal pain was undertaken until the age of 24. During an abdominal gynecological laparatomy -conducted because of recurrent abdominal pain and dysmenorrhoe- multiple polypoid pedunculated lesions on the large bowels were seen (see Figure 1), biopsy was not carried out because the lesions were well vascularized. On surgical abdominal exploratory laparotomy about 50 fibromatous tumors were found and removed from the serosal surfaces, small and large bowels, omentum and from the uterus. Histopathological analysis of these tumors revealed benign spindle cell tumors, classified as leiomyoma. Electron microscopy showed peculiar nodules consisting of mature fibroblasts, abundant collagen with a few admixed myofibroblasts. At the age of 29 the patient had another episode of severe abdominal pain, nausea and vomiting and abdominal distension, partial small bowel obstruction was seen again on abdominal X-rays. The patient had no further episode of abdominal discomfort until now and presented to us for further evaluation with abdominal bloating and discomfort. Other recorded medical conditions include Raynaud's disease (diagnosed at the age of 19), Prurigo nodularis with lichenification of the skin (onset of symptoms in adolescence, see Figure 2) and recently diagnosed postural orthostatic tachycardia syndrome (diagnosed at age 44).
Her clinical exam of the abdomen revealed nothing except abdominal distension, no masses or tenderness were noted. Inspection of the skin revealed dry lichenificated skin on all extremities with prominent itching nodules (see Figure 2). Laboratory findings were normal at admission.
The mother of the patient (III10) is now at age 82 and was diagnosed with Leiomyomatosis peritonealis disseminata at the age of 25. She also had recurrent episodes of severe prolonged abdominal pain and underwent exploratory laparotomy due to a ruptured appendix. Incidentally a large number of tumorous lesions was found, which were later on classified as leiomyomas. Interestingly this patient also was diagnosed with Raynaud's disease and has Prurigo nodularis-like skin lesions. The sister of this patient (III13) died at age 16 of peritonitis and sepsis. On post mortem examination intestinal obstruction due to multiple tumorous lesions was found as origin of peritonitis. Another brother (III17) died of peritonitis and gangrene of the bowels at the age of 20, resulting from intestinal obstruction of multiple pedunculated tumors (as reported by operating surgeon, 1939).
The grandfather (II3) of the propositus never was diagnosed LPD but has a past medical history of abdominal problems, including intestinal obstructions. Unfortunately no further information was available.
Three more members of the family are affected by LPD and furthermore have been diagnosed with Raynaud's disease and show Prurigo nodularis with skin lichenification. All three patients (IV2, IV3, IV4) had abdominal problems (e.g. prolonged severe abdominal pain) in their late teenage years and underwent explorative laparotomy, which showed more than 50 tumors in every case. The receptor status for progesteron- and estrogen-receptors was negative in these three patients. Patient V1 is the 10 year old son of patient IV2 and already had a complete intestinal obstruction, on explorative laparotomy more than 100 pedunculated tumors have been found in the abdominal cavity.
Patients IV5, IV7 and V2 reportedly have or had abdominal problems, but no confirmed diagnosis was available. No information on medical conditions, post mortem examinations or confirmed diagnoses was available for patients I1 and I2, grand-grandparents of the propositus (IV6).
Discussion
Leiomyomatosis peritonealis disseminata (LPD) is a rare disease and only a relatively small number of cases have been reported so far. No epidemiologic data is available and cases reported have been from different ethnical groups, without any clear ethnical predominance. Except two cases, all cases describe female patients, so occurrence of LPD in males is extremely rare. No familial cases have been reported so far and no link to environmental influences has been found, except for excessive hormonal exposure [8].
Correct diagnosis is difficult and can only be achieved through invasive diagnostics together with histopathological analysis. Patients usually present with unspecific abdominal problems, e.g. abdominal discomfort or pain, rectal or vaginal bleeding (in cases with uterine leiomyomas) or symptoms of intestinal obstruction. Imaging studies revealed tumorous lesions in some cases [13], but in the propositus of this presented case no evidence of LPD could be found by any kind of imaging studies (ultrasound, computed tomography or magnetic resonance imaging). Biopsies can provide the material for a histopathologic diagnosis. However vascularized tumors can cause severe bleedings on biopsy, so that biopsies taken on explorative laparotomy seem to warrant greater safety and furthermore allow for assessing the extent of LPD or exclusion of other diagnoses. Differential diagnosis include peritoneal disseminated metastasis, malignancy or other forms of leiomyomas (e.g. benign metastasizing leiomyoma, a uterine leiomyoma associated with a smooth muscle mass in a solid organ). LPD is regarded as a benign disease, but in about 2–5% of the reported cases a progression towards malignancy has been reported [14-17]. Malignant transformation from leiomyoma to sarcoma was found on histopathological findings. However the majority of cases take a benign course, sometimes LPD resolves with hormone therapy or does not relapse after surgical resection. Some cases show recurrent growth of leiomyomas and recurrence of symptoms. The occurrence of LPD without intrauterine leiomyomas, in postmenopausal women [18], LPD with estrogen- and progesterone-receptor negative tumors [16] and the occurrence in men led to the idea, that LPD is a new disease entity [16,19].
In our reported case six siblings are affected by LPD, the two branches of the family links through the grand-grandfather of the propositus (IV6, see Figure 3, indicated by small arrow). There is no consanguinity and environmental influence is not very likely: different generations have been raised in different parts of the country and no other occurrence of LPD outside these families is reported. Assuming a genetic background of the familial clustering and taking the family pedigree into account we propose an autosomal-dominant model with varying degrees of penetrance. LPD appears in subsequent generations, skipping some generations and appears both in men and women. However other modes of inheritance cannot be excluded completely (e.g. genomic imprinting), but appear less likely. For an autosomal-recessive model a disease allele would have to be common in the population, requiring far more cases of LPD.
There is no known similar genetic disorder. However there are some similarities which can be taken into consideration. Mutations in the fumarase hydratase (FH) gene, an enzyme which is involved in the mitochondrial tri-carboxylic acid cycle, are known to cause leiomyomas [20,21]. Especially the dominantly inherited susceptibility to multiple cutaneus and uterine leiomyomas appears as hereditary leiomyomatosis. Furthermore, smooth muscle tumors have been linked to mutations in collagen genes [10]. Interestingly also skin lesions [22] have been found to be associated with collagen gene alterations. An alteration of a collagen gene as a common basis for familiar LPD seems possible. As a hypothesis, these two genes may be involved in the development of familiar LPD through unknown mutations. However we have no further data to verify these hypotheses.
Conclusion
Leiomyomatosis peritonealis disseminata is a rare disease and familial clustering has never been reported and can be considered a unique appearance so far. Nevertheless, other cases may have never been diagnosed, because these leiomyomas can present only with minor symptoms or can go completely undetected and without symptoms. Leiomyomatosis peritonealis disseminata presents a hard to diagnose disease and is rarely considered as differential diagnosis.
This familial clustering of LPD should be genetically analyzed to find the underlying genetic pathomechanism. Only further diagnostics of all affected family members can reveal all aspects of this syndrome. Careful examination of all living family members, skin biopsies of all affected family members and a complete workup regarding Raynaud's disease could be future steps in a detailed investigation.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All authors contributed equally to this work. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Written consent was obtained from the patient or their relatives for publication of the patient's details.
Figures and Tables
Figure 1 Intraabdominal picture of a leiomyoma. Intraabdominal picture taken on endoscopic laparoscopy, showing a pedunculated tumor on the bowels (large white arrows indicate the "head" and small white arrows indicate the "trunk" of the leiomyoma).
Figure 2 Skin lesions. Right lower extremity with multiple itching nodules, excoriations from scratching and general lichenification of the skin (inset: enlarged lesion with excoriation and reddish halo).
Figure 3 Family pedigree. Family pedigree (squares indicating males, circles indicating females, slashed symbols indicating deceased family members, single line indicates family members with abdominal symptoms but no diagnosis of LPD, double crossed lines indicate family members with diagnosis of LPD, question marks indicate family members without any medical information), additional diagnosis of Raynaud's disease and Prurigo nodularis is present in family members II10, IV2, IV3, IV4 and IV7.
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Tomlinson IPM Alam NA Rowan AJ Barclay E Jaeger EE Kelsell D Leigh I Gorman P Lamlum H Rahman S Roylance RR Olpin S Bevan S Barker K Hearle N Houlston RS Kiuru M Lehtonen R Karhu A Vilkki S Laiho P Eklund C Vierimaa O Aittomaki K Hietala M Sistonen P Paetau A Salovaara R Herva R Launonen V Aaltonen LA Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomatoma and papillary renal cell cancer Nature Genetics 2002 30 406 410 11865300 10.1038/ng849
Alam NA Bevan S Churchman M Barclay E Barker K Jaeger EE Nelson HM Healy E Pembroke AC Friedmann PS Dalziel K Calonje E Anderson J August PJ Davies MG Felix R Munro CS Murdoch M Rendall J Kennedy S Leigh IM Kelsell DP Tomlinson IP Houlston RS Localization of a gene (MCUL1) for multiple cutaneous leiomyomata and uterine fibroids to chromosome 1q42.3-q43 Am J Hum Genet 2001 68 1264 1269 11283798 10.1086/320124
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BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-6-471619119410.1186/1471-2156-6-47Research ArticleReconstructing recent human phylogenies with forensic STR loci: A statistical approach Agrawal Suraksha [email protected] Faisal [email protected] Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow (UP) 226014 India2 Department of Biotechnology, Bundelkhand University, Jhansi (UP), India2005 28 9 2005 6 47 47 29 5 2005 28 9 2005 Copyright © 2005 Agrawal and Khan; licensee BioMed Central Ltd.2005Agrawal and Khan; 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
Forensic Short Tandem Repeat (STR) loci are effective for the purpose of individual identification, and other forensic applications. Most of these markers have high allelic variability and mutation rate because of which they have limited use in the phylogenetic reconstruction. In the present study, we have carried out a meta-analysis to explore the possibility of using only five STR loci (TPOX, FES, vWA, F13A and Tho1) to carry out phylogenetic assessment based on the allele frequency profile of 20 world population and north Indian Hindus analyzed in the present study.
Results
Phylogenetic analysis based on two different approaches – genetic distance and maximum likelihood along with statistical bootstrapping procedure involving 1000 replicates was carried out. The ensuing tree topologies and PC plots were further compared with those obtained in earlier phylogenetic investigations. The compiled database of 21 populations got segregated and finely resolved into three basal clusters with very high bootstrap values corresponding to three geo-ethnic groups of African, Orientals, and Caucasians.
Conclusion
Based on this study we conclude that if appropriate and logistic statistical approaches are followed then even lesser number of forensic STR loci are powerful enough to reconstruct the recent human phylogenies despite of their relatively high mutation rates.
Short tandem repeatsForensicPhylogenyNeighbor-joiningMaximum likelihoodPC plot
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Background
Short Tandem Repeats (STR), with a repetitive sequence ranging from 2–6 base pairs are amongst the most polymorphic markers reported till date. They exhibit substantial allelic variability due to high rate of germline mutations [1]. The STR loci have a uniform and dense distribution throughout the genome and exhibit high level of relatively stable polymorphism [2]. All these features makes them an ideal candidate for diverse applications including forensic applications [3], individual identification, paternity/maternity detection [2], fine scale genetic mapping [4] and inter and intra group phylogenetic reconstruction [5].
However, a specific set of STR can be employed for specific applications and this specificity is solely based on the properties of STR loci involved and their suitability to the particular application. STR loci used for forensic purposes are the one that possess numerous observed alleles, high level of heterozygosity, high polymorphism information content and high power of exclusion. On the contrary, STR loci preferred for the phylogenetic analysis of the human populations are those which have substantial lower allelic counts and carries signature alleles for specific populations [6,7]. Still, there are few studies in which there is some overlap between the sets of forensic STRs and those exclusively studied for phylogenetic investigation. However, this overlap is not extensive and is without any definitive rationale or design.
There are two school of thoughts regarding the use of forensic STR in phylogenetic studies. According to one view, the requirement of extremely high level of intra group variation along with high mutation rates in forensic systems indicates a rapid diffusion of genetic variation and thus, confers a greater risk of failure in detection of convergent evolution among some populations [8]. Other perception is that random noise generated by allelic variability in forensic systems is not strong enough to veil the evolutionary signals generated by these STR loci. Furthermore, fine scale resolution of forensic STR may prove handful in delineating genetic difference and affinities between closely related ethnic groups [6].
In the present study, we have made an attempt to explore the utility of forensic STR loci in inferring phylogenetic relationships. To approach this goal, we have compiled a geographically targeted and racially diverse set of 21-population database from forensic literature obtained from Wolfgang Huckenbeck and Hans-Georg Scheil's website "The Distribution of the Human DNA-PCR Polymorphisms" [9] while the north Indian Hindus from the state of Uttar Pradesh were genotyped in our own lab. Forensic STR loci for which the allele frequency data was compiled were Tho1, vWA, FES. F13 and TPOX. They all carries tetrameric core repeat sequence and reside on different chromosomes and are amongst the most reputed one in forensic system. The choice of these loci is exclusively on the basis that these markers have been studied in all the 21 populations hence a precise phylogenetic analysis could be performed [Table 1].
Table 1 Population compiled for database of five forensic STR loci
S.No. Populations No. of samples analyzed *
TPOX FES vWA F13A Tho1
1 Basque 768 627 615 208 859
2 Poland 703 643 572 334 488
3 Germany 2876 7683 13667 3438 7373
4 Italy 11388 4827 7135 1677 4900
5 Portugal 1239 2091 4720 1158 4639
6 Spain 1782 2325 3361 1864 4037
7 Canadian Caucasians 435 321 428 435 435
8 Middle Eastern Arabs 165 132 149 127 173
9 US Caucasian 562 597 759 587 765
10 Slovenia 235 235 779 236 560
11 Austria 153 153 1946 1056 1816
12 China 658 435 1146 137 2503
13 Japan 1491 397 1743 668 2905
14 Philippine 498 103 376 133 528
15 Taiwan 716 100 600 149 764
16 Sharawasi Africans 59 59 99 59 59
17 Cameroon 65 65 65 65 65
18 Moroccan Arabs 127 199 193 75 271
19 Lisongo Africans 32 60 30 30 30
20 US Afro-Americans 580 679 797 691 793
21 North Indian Hindus ** 1000 1000 1000 1000 1000
Source of data:* [9]
** Present study
Phylogenetic assessment was carried out through two different approaches – genetic distance and maximum likelihood along with a statistical Bootstrapping procedure involving 1000 replicates. The ensuing tree topologies and PC plots were then compared with those obtained in earlier phylogenetic investigations. The main question that we have tried to address in this meta-analysis is whether a limited number of forensic STR can predict accurate human phylogenies, if the data is evaluated using proper statistical approaches.
Results
Allele frequency distribution
Analysis of five STR loci- Tho1, vWA, FES, F13 and TPOX has revealed high level of diversity among North Indian Hindus. Total 7–8 alleles were found (7 each for Tho1, vWA, F13 and TPOX and 8 for FES). All the loci were in Hardy-Weinberg equilibrium. Table 2 shows allele frequency distribution at all the five loci among North Indian Hindus. High allelic variability was further depicted by high-observed Heterozygosity (0.68 at Tho1-0.76 at vWA), high PIC (0.66 at F13-0.74 at Tho1) and high power of exclusion (0.28 at F13-0.38 at Tho1). All these criterions are indicative of the fact that these STR loci are useful and informative tools for all types of forensic applications.
Table 2 Different statistical analysis done on allele frequency data of five STR loci which are important criterion for a good forensic loci
Tho1 VWA FES F13 TPOX
Allele3 0.06
Allele 4 0.06
Allele 5 0.01 0.20
Allele 6 0.28 0.40
Allele 7 0.16 0.04 0.26 0.02
Allele 8 0.08 0.01 0.02 0.40
Allele 9 0.17 0.08 0.12
Allele 9.3 0.30
Allele 10 0.06 0.27 0.08
Allele 11 0.36 0.32
Allele 12 0.18 0.04
Allele 13 0.02 0.02
Allele 14 0.14 0.04
Allele 15 0.04
Allele 16 0.34
Allele 17 0.14
Allele 18 0.30
Allele 19 0.02
Allele 20 0.02
Observed Heterozygosity 0.680 0.760 0.760 0.740 0.730
PIC 0.738 0.714 0.725 0.660 0.669
Power of exclusion 0.381 0.354 0.371 0.283 0.308
Average heterozygosity 0.734
Mean PIC 0.700
Total exclusionary power 0.875
Phylogenetic assessment
Phylogenetic analysis carried out in 21 populations is depicted by two enrooted radial phylograms (NJ and ML) as shown in Figure 1a and 1b and a PC plot was plotted based on the allele frequency variation [Figure 1c]. The edge lengths displayed in these phylograms indicated that the amount of evolutionary change occurred along each branch. The scores next to the nodes characterize the number of bootstrap replicates (out of 1000) exhibiting these specific bifurcations.
Figure 1 Phylogenetic reconstruction based on (a) Neighbor-joining (NJ) tree with 1000 bootstrap replicates; (b) Maximum Likelihood (ML) tree with 1000 bootstrap replicates and PC plot analysis based on allele frequency differences.
NJ phylogenetic tree depicts three different basal groups corresponding to three ethnic groups- African, Caucasian and Orientals. The Caucasian cluster has three monophyletic units- Austria/German, Basque/Portuguese and USA/Canadian Caucasians. Total 8 out of the 14 nodes have bootstrap values more than 50%, most important among them is (i) division between Africans and other two groups of Caucasians and Orientals-863, (ii) the division between Caucasian and other two groups-960 and (iii) the division between Orientals and other two groups-995.
ML phylograms also displayed comparable topology to that of NJ tree. It has also depicted three basal nodes with clear demarcation of Africans, Caucasians and Orientals. Similar to NJ tree, all the three basal monophyletic topologies in ML tree have bootstrap values more than 50% i.e. 931, 790 and 995 respectively for Africans, Caucasians and Orientals. In both the phylogenies, NJ and ML, North Indian Hindus clustered with the Caucasians albeit with low bootstrap value of 456.
First and second Principal component that together constitutes 70.4% of the total variability (PC1-39.3% and PC2-31.1%) were plotted and presented in Figure 1c. There is a significant separation of the Orientals from Caucasians on Y-axis (PC2) and with that of Africans on X-axis (PC1). While Caucasians and Africans reveal relatively clear division along both X-axis (PC1) and Y-axis (PC2), with Middle Eastern Arabs and Moroccan Arabs positioning near to each other. Furthermore, the African populations have clustered into two sub-groups corresponding to the Central and North Africans. North Indian Hindus have clustered with Caucasians.
Discussion
Five forensic STR loci are found highly successful in providing fine resolution for the reconstruction of recent human evolutionary histories. All three approaches used for phylogenetic reconstruction (NJ and ML tree topologies and PC-plot analysis) have depicted strong racial partitioning and deciphering the accurate phylogenetic information about North Indian Hindus which is in accordance with those derived from other more renounced phylogenetic markers as well as historical evidences [10-12].
The phylograms (NJ and ML) generated from present data set were calculated from CONTML and NJ algorithms, where CONTML works upon the conjecture that random action of genetic drift is the solitary basis of the differences between allele frequencies in different population groups [13]. On the contrary, the NJ algorithm construct a branching array from a matrix of genetic distances calculated from Nei's formula assuming that both genetic drift and mutation causes allele frequency differences [14].
Both phylograms, NJ and ML have more or less similar basal cluster patterns among the three geo-ethnic groups indicating that component of genetic drift instead of mutation is the major player in these distant estimates. Both the trees have longer African branch than any other group. Such a patristic separation is also visible in PC-plot analysis [Figure 1c]. The African populations have been clustered into central (Cameroon and Lisongo) and North African (Moroccan Arabs and Saharawasi Africans) groups. Such clustering has also been reported by Cavalli-Sforza et al, 2003 based Fst genetic distance based on polymorphisms of 120 protein-coding genes [15] and Y-chromosome binary haplogroup [16,17]. This sub clustering further strengthens the utility of the 5 STR loci in deciphering the accurate phylogenies even within the same geographical region. Middle Eastern Arabs display a branch nearer to Caucasians and to some extent near to Moroccan Arabs suggesting strong Caucasian element along with African admixtures suggestive of the Demic expansion of the middle east genes, agriculture innovations and languages into north west Africa [16,17], which is further supported by the near medial position of Arabs in the PC-plot. Recently, Y-chromosome SNP analysis by Al-Zahery et al. 2003 [18] has also revealed similar pattern in other Middle Eastern populations. European branching pattern differs slightly between the two trees, but still both are resolving completely with Basque, Spaniards and Portuguese having a separate cluster from that of German/Austrian branch. North Indian Hindus clustered with Caucasians in both the phylograms, which is in agreement with the findings of earlier studies. Bamshad et al. 2001[12] based on mtDNA HVR-I and HVR-II sequencing and Y-chromosome haplotypes has shown that North Indian populations reveal high frequency of west Eurasian haplogroups. North Indian Hindus are basically Indo-Aryan speakers, who invaded from the steppes of central Asia and then settled in the Indus valley, in northwestern India [10].
The major finding of the present study is the productivity of a limited set of 5 forensic STR loci in resolving the human phylogenies in a similar manner as reported elsewhere on a much higher number of loci. Further, the study also highlights the utility of combined use of varied statistical approaches in reaching a definitive conclusion. Our study scores a point over some of the successful reports like that of Bowcock et al., 1997[19] which has shown substantial phylogenies but with much larger sets of STR – 30 STR loci. Similarly, Perez-lezaun et al. 1999 [20] has used 20 STR loci and computed Fst based distances depicting similar separation of inter and intra ethnic groups. Even though, in the same study, phylogenetic tree based on DSW distance exhibited a defused picture having trifurcation two Caucasoid and one African group.
Various attempts of phylogenetic reconstruction using forensic STR loci have also been done in recent past like that of Budowle and Chakraborty, 2001[21] who studied 13 CODIS loci, but their phylogenetic assessment was confined to simple NJ and UPGMA trees and distance measures which yields single output tree. To overcome this, we have incorporated both the phylogenetic approaches i.e. distance and optimal criterion along with statistical bootstrapping which yields 1000 trees and then built a consensus tree. In this regard, a successful attempt was made by Rowold et al. 2003 [6], by compiling 10 geographically and racially different populations on five forensic STR loci. However, incorporating different set of STR loci, we have been able to compile larger population database of 21 populations.
Overall, the analysis of five forensic STR loci have depicted a strong racial phylogeny indicating that high heterozygosity and/or numerous observed alleles do not necessarily interfere with the phylogenetic information content of the locus, provided that frequency distribution of the populations is significantly different. Significantly, larger number of alleles increases the chances of the presence of signature alleles in segregating populations. Despite all the potential problems associated with forensic STR loci including that of high mutation rates, successfully resolution the genetic difference between inter and intra geo-ethnic groups suggesting that if well-defined statistical approaches are followed, then even a smaller number of forensic STR loci are powerful enough in reconstructing human phylogenies.
Methods
Populations (North Indian Hindus)
A total of 1000 unrelated individuals were randomly selected. Regional addresses and detailed computerized lists were prepared before sample collection. Random numbers were generated with the help of computer and samples were collected from the different collection sites of Uttar Pradesh- Lucknow, Kanpur, Faizabad, Basti, Gonda and Agra. Whole blood was obtained by venipuncture and collected in EDTA vacutainer tubes. Three-generation pedigree charts were prepared to assure un-relatedness in all the samples. The ethical committee of the institute approved the study and blood samples were taken after obtaining informed consent from the subjects.
DNA extraction and STR genotyping
DNA was extracted by phenol chloroform method as described by Comey et al. 1993 [22] and purified by ethanol precipitation. All the five STR loci were detected by PCR. PCR amplification was performed using flanking primers described elsewhere [20]. The amplified product was separated and detected on 9% PAGE using silver staining.
Population database
20 geographically targeted populations were selected from forensic literature [9], while the data of north Indian Hindus was generated from our lab (Agrawal et al., unpublished data). The criterion of selection was to cover the major geographical and geo-ethnic groups i.e. African, Caucasoid and Orientals. All the populations selected have allele frequency data for five STR loci. In order to embrace a large sample size and to overcome the predicament of some studies focusing only on 2 or 3 STR loci, allele frequency profile of different STR loci analyzed in different populations samples but of the same geographic or ethnic origin has also been included. However, wherever possible, a care has been taken to include the allele frequency profile of the same set of sample for different markers. For example, same 65 samples of Cameroon population has been used for allele frequency data of 5 STR, whereas a large pooled sample size has been used for other groups like Germany, Portugal, Italy, China, Japan etc. In order to avoid the discrepancies, number of samples for each population genotyped for different STR loci and source of allele frequency data is shown in Table 1. Maximum of the populations compiled in the database are pooled samples from different parts of that country. Notably, Sardinians are excluded from Italians, Azores from Portugal and Canary Island from Spain. Middle Eastern Arabs included Arabs mainly from Saudia Arabia, Qatar and Yemen.
Statistical analysis
Allele frequencies were calculated by a simple gene count method. A total analysis was executed based upon the allelic frequency distribution of the five STR. Heterozygosity, HWE, PIC and power of exclusion was calculated using Cervus v1 [23]. Further, Statistical analysis was executed based upon the allelic frequency distribution of the five STR. A 1000 replicate bootstrap data was generated from SEQBOOT option in PHYLIP version 3.5c [13]. Distance values were estimated using Nei's formula [14], and a phylogeny was inferred by the neighbor joining (NJ) option in PHYLIP version 3.5c [13]. Phylogenetic reconstruction was also done based on maximum likelihood (ML) and the STR frequency distribution (CONTML in PHYLIP version 3.5c)[13]. Finally, a principal component (PC) analysis was generated by POPSTR and first and second PC was plotted as described elsewhere [24].
Authors' contributions
SA has conceptualized the paper provided important intellectual inputs in intrepretation of data and preparation of the manuscript and FK carried out statistical analysis and drafted the manuscript. Both authors read and approved the final manuscript
Acknowledgements
This work was supported by Indian Council of Medical Research (ICMR) New Delhi. Authors are thankful to Sanjay Gandhi Post Graduate Institute of Medical Sciences Lucknow for providing various lab facilities and other assistance. We thank Ms. Sudha Talwar, Ms. Manorama Tripathi and Mr. Atul Pandey for providing technical support.
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BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-6-1391620215210.1186/1471-2164-6-139Research ArticleIn silico characterization of the family of PARP-like poly(ADP-ribosyl)transferases (pARTs) Otto Helge [email protected] Pedro A [email protected] Fernando [email protected] Katharina [email protected] Friedrich [email protected] Friedrich [email protected] Institute of Immunology, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.2 DNAX Research Institute, Palo Alto, CA 94304, USA.3 Dana-Farber Cancer Institute, Harvard University, Boston, MA 02115, USA.4 Depts. of Molecular Biology and Protein Engineering, Genentech, SF, CA 94080, USA.5 Department of Integrative Biology, Brigham Young University, Provo, UT 84602, USA.2005 4 10 2005 6 139 139 13 5 2005 4 10 2005 Copyright © 2005 Otto et al; licensee BioMed Central Ltd.2005Otto 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
ADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which mono(ADP-ribosyl)transferases (mARTs) and poly(ADP-ribosyl)transferases (pARTs) transfer the ADP-ribose moiety from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins.
Results
Using a combination of database search tools we identified the genes encoding recognizable pART domains in the public genome databases. In humans, the pART family encompasses 17 members. For 16 of these genes, an orthologue exists also in the mouse, rat, and pufferfish. Based on the degree of amino acid sequence similarity in the catalytic domain, conserved intron positions, and fused protein domains, pARTs can be divided into five major subgroups. All six members of groups 1 and 2 contain the H-Y-E trias of amino acid residues found also in the active sites of Diphtheria toxin and Pseudomonas exotoxin A, while the eleven members of groups 3 – 5 carry variations of this motif. The pART catalytic domain is found associated in Lego-like fashion with a variety of domains, including nucleic acid-binding, protein-protein interaction, and ubiquitylation domains. Some of these domain associations appear to be very ancient since they are observed also in insects, fungi, amoebae, and plants. The recently completed genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7. The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for twelve pARTs. Simpler eucaryotes generally contain fewer pARTs: two in the fly D. melanogaster, three each in the mosquito A. gambiae, the nematode C. elegans, and the ascomycete microfungus G. zeae, six in the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana. GenBank contains two pART homologues from the large double stranded DNA viruses Chilo iridescent virus and Bacteriophage Aeh1 and only a single entry (from V. cholerae) showing recognizable homology to the pART-like catalytic domains of Diphtheria toxin and Pseudomonas exotoxin A.
Conclusion
The pART family, which encompasses 17 members in the human and 16 members in the mouse, can be divided into five subgroups on the basis of sequence similarity, phylogeny, conserved intron positions, and patterns of genetically fused protein domains.
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Background
ADP-ribosylation is a posttranslational protein modification in which the ADP-ribose moiety is transferred from NAD onto specific amino acid side chains of target proteins [1-4]. ADP-ribosylation was originally discovered as the pathogenic principle of Diphtheria toxin, a multidomain secreted protein which inactivates elongation factor 2 by ADP-ribosylation after translocation into eucaryotic cells [5]. Subsequently, numerous other bacterial toxins were shown to ADP-ribosylate target proteins in host cells. Moreover, endogenous toxin-like ADP-ribosylating enzyme activities were detected in eucaryotic cells. Several of these enzymes were purified to homogeneity, sequenced, expressed as recombinant proteins, and crystallized.
Sequence and structural analyses revealed the existence of two distinct families of toxin-related ADP-ribosyltransferases in mammals [6,7]: The RT6 family of GPI-anchored and secretory mono-(ADP-ribosyl)transferases (mARTs) catalyzes mono-ADP-ribosylation of cell surface and secretory proteins [8]. The PARP family of nuclear and cytoplasmic poly(ADP-ribosyl)transferases (pARTs) catalyzes poly-ADP-ribosylation of nuclear and cytosolic proteins [9-12]. While mARTs have been implicated to mediate signalling functions of extracellular NAD, pARTs have been shown to play important roles in DNA repair and maintenance of genome integrity [8,9,12].
In this paper we use the term pART (poly ADP-ribosyltransferase) rather than the more established term PARP (poly-ADP-ribosyl-polymerase) for various reasons. Firstly, to emphasize the structural and functional similarities of the poly- and mono-ADP-rib syltransferase subfamilies. Secondly, with respect to the biochemical classficiation of enzymes the term transferase is more appropriate than polymerase: ADP-riboslytransferases belong to the family of glycosyltransferases; the term polymerase is more commonly used for template-dependent DNA or RNA synthesizing enyzmes. Thirdly, use of the term PARP would have confounded comparison of our results with those of the recent review by Ame et al. [11], who used the term PARP and a numbering system without regard to structural similarities among gene family members.
The 3D-structures of rat ART.2 (PDB accession number 1og3), chicken PARP-1 (1a26, 3pax), mouse PARP-2 (1gs0), and numerous ADP-ribosylating toxins uncovered a common NAD binding fold with a conserved core of five β strands arranged in two abutting β sheets [13-19]. These two β sheets form the upper and lower jaws of a Pacman-like active site crevice (Figure 1). Remarkably, only a single amino acid residue, the catalytic glutamic acid residue at the front edge of the fifth conserved β-strand, is strictly conserved in all known 3D structures of enzymatically active mARTs and pARTs. In a seminal study, Collier and co-workers pinpointed the corresponding glutamic acid residue in PARP-1 (before its 3D structure was solved) on the basis of barely detectable sequence similarity to Diphtheria toxin [20,21]. More recently, the 3D structures of anthrax lethal factor, VIP2, and iota toxin have been discovered to harbour ART-domains that lack a corresponding glutamic acid residue and may represent inactivated enzymes [16,22,23].
Figure 1 Schematic illustration of the distinguishing structural features of the PARP-1/DT vs. the ART2/VIP2 subfamilies of ADP-ribosyltransferases. Two abutting sheets of anti-parallel β strands form the upper and lower jaws of a Pacman-like NAD-binding crevice in all known structures of ADP-ribosyltransferases. The distinguishing structural features of the PARP/DT and ART2/VIP2 subfamilies are depicted schematically on top and are highlighted in the structures of chicken PARP-1 (3pax), diphtheria toxin (DT) (1tox), an archael tRNA:NAD 2'-phosphotransferase (TpT) (1wfx), rat ART2 (1og3) and B. cereus VIP2 toxin (1qs2) below. The structures are depicted from the "front view" with a full view of the ligands bound in the active site crevice. The ligands NAD and 3MB are colored cyan and are depicted as stick models. The central four β-strands (from top to bottom: β 5, β 2, β 1, β 3, colored orange) are conserved in all mARTs and pARTs. The β strands at the edges of the respective sheets (β 4 and β 6, colored pink) show greater structural variation than the central β strands. The H-Y-E motif residues are depicted in red and their side chains are shown as sticks. The glutamic acid residue at the front edge of β 5 is the critical catalytic residue in both diphtheria toxin and PARP-1 – a corresponding glutamic acid residue is observed also in the 3D structures of rat ART2 and numerous bacterial mARTs. Diphtheria toxin (1tox), pseudomonas exotoxin A (1aer), PARP-1 (3pax), and PARP-2 (1gs0) share the following structural features which are not conserved in either rat ART2 (1og3) or most other bacterial mARTs: the orientation of β 6, the alpha helix between β 2 and β 3 (colored yellow) and the conserved histidine and tyrosine amino acid residues in β 1 and β 3. The loop between β 4 and β 5 (colored magenta) is thought to play a role in the recognition of target proteins and ADP-ribose polymers. Distinguishing features of ART2, VIP2, iota toxin (1gir), and the C3 exoenzymes (1g24, 1ojz) include three conserved alpha helices upstream of β strand 1, a seventh β strand that displaces β strand 6 and an R-S-E- motif instead of the H-Y-E motif of PARP-1 and DT. (Note that the depicted ART2 structure carries a site directed mutation of the catalytic glutamic acid residue E189I). The recently determined 3D structure of the tRNA:NAD 2'-phosphotransferase (1wfx) bears striking resemblance to that of DT and PARP-1 and carries an H-H-V variant of the H-Y-E motif. Note that the structure of the diphtheria toxin catalytic domain shown here in complex with NAD is truncated C-terminally at the proteolytic cleavage site that separates this domain from the translocation domain. The PARP-1 catalytic domain shown here is truncated N-terminally at the position of the phase 0 intron that separates this domain from a neighboring helical domain. The TpT catalytic domain is truncated N-terminally at the point of fusion to a winged-helix domain.
Comparative structure and amino acid sequence analyses revealed that PARP-1 and PARP-2 share additional secondary structure and conserved amino acids with Diphtheria toxin and Pseudomonas exotoxin A, which evidently are not conserved in other mARTs (Fig. 1) [6,7]. These additional elements include a sixth β strand, an alpha helix between β strands 2 and 3, and a trias of amino acids, the so-called H-Y-E motif, encompassing a histidine resdiue in β strand 1, a tyrosine residue in β strand 3 and the catalytic glutamic acid residue at the front edge of β strand 5. These features, highlighted in the 3D structures of PARP-1 and Diphtheria toxin in Figure 1, clearly distinguish the structures of PARP-1, PARP-2, and DT/ETA from those of a second major ART subfamily that includes rat ART2 and the Bacillus cereus VIP2 toxin. Distinguishing features of the ART2/VIP2 subfamliy include a seventh β strand that displaces β strand 6, three conserved alpha helices preceding β strand 1, and an R-S-E trias of amino acid residues in place of the H-Y-E motif of PARP-1 and DT. Interestingly, the recently reported 3D-structure of a prototype member of the family of tRNA:NAD 2' phosphotransferases (TpT) [24] revealed a striking resemblance to the structures of the PARP-1/DT subfamily rather than to those of the ART2/VIP subfamily, including the sixth β strand, the alpha helix between β strands 2 and 3, and a variant H-Y-E motif (H-H-V). These enzymes catalyze removal of a splice junction 2' phosphate from ligated tRNA. This reaction resembles the reaction catalyzed by ARTs but yields ADP-ribose 1"-2" cyclic phosphate rather than ADP-ribosylated proteins [25].
The remarkable degree of plasticity of ART amino acid sequences poses a challenging problem for genome data base mining [7] and even the most sensitive database search programs fail to connect all known members of the ART gene family. Notwithstanding, the results of such in silico analyses can provide important insight into the structural and phylogenetic relationship of ART subfamilies. We have previously demonstrated that the known members of the mART gene family in the human and mouse could be faithfully connected with many known bacterial ADP-ribosylating toxins, but not with pARTs or Diphtheria toxin [26,27]. These analyses also pointed out the presence of mART-encoding genes in the genomes of many but not all other model organisms. Of note, no mART-encoding genes could be detected in plants, fungi, or archaea. Here we provide an in depth analysis of the pART gene family.
Results and discussion
Identification of human and mouse pART family members in the EST database
The human and mouse pART gene family members were identified using a combination of data base search tools. The human and mouse EST databases as well as the nonredundant GenBank database (nr) were screened with tBLASTn using as queries the amino acid sequences of the catalytic domains of the known and newly identified pART family members. Whenever possible, the full coding sequence of the catalytic domain and of the adjacent regions was assembled using the sequences of published cDNAs and overlapping ESTs. Screening of the EST and nr databases was initiated in 1997 and was repeated in regular intervals. The coding sequences were extended when suitable new sequences became available. When the sequences of the human, mouse and rat genomes were published in 2000, 2001, and 2004, respectively, the EST database searches were complemented with corresponding tBLASTn and BLASTn searches of the genome sequences [28-30]. Thereby, 17 pART family members were identified in the human. These genes were designated pART1-pART17. Numbering reflects the degree of amino acid sequence similarity to PARP-1 (= pART1) and the degree of similarity within each of the pART subgroups. An orthologue for each of these genes was detected in the mouse and in the rat, with the sole exception of pART7.
A complete list of human pART family members, including the common names and aliases of known genes, is presented in Figure 2. Based on the degree of amino acid sequence similarities, conserved intron positions, and fused protein domains, the mammalian pART family can be divided into five major subgroups. Group 1 (pART1-pART4) contains PARP and its closest relatives, PARP-2, PARP-3 and VPARP. Group 2 (pART5, pART6) contains tankyrase 1 and tankyrase 2. Group 3 (pART7-pART10) contains four proteins including the recently described B-Aggressive Lymphoma Protein (BAL = pART9) [31] and a myc-interacting protein with PARP activity (PARP-10) [32]. Group 4 (pART11-pART14) contains four proteins including the recently described Zinc-finger Antiviral Protein (ZAP = pART13) [33] and TCDD-inducible PARP (TiPARP) [34]. Group 5 (pART15-pART17) contains three proteins of unknown function.
Figure 2 Chromosomal localizations and exon compositions of the human and mouse pART family members. A) pART family members are sorted by subgroup on the basis of similarities in amino acid sequence, intron positions and associated protein domains. Color-coding of subgroups is as follows: 1 = red, 2 = pink, 3 = orange, 4 = green, 5 = grey. This color-coding is used in subsequent figures. Official gene designations, common aliases and accession numbers are shown. Exon compositions and lengths of open reading frames are given for the longest known or predicted gene transcripts. Available full length cDNAs from the Mammalian Gene Collection (MGC) are indicated with their respective accession numbers. MGC cDNAs which apparently do not contain the full open reading frame are indicated in parentheses. Hs = Homo sapiens, Mm = Mus musculus. B) Chromosomal localizations of pART genes were determined by tBLASTn searches of the respective genome sequences using the amino acid sequences of the catalytic domains of individual pARTs. Members of the five pART family subgroups are color-coded as in A).
The steady growth in the number of matching ESTs obtained for each of the human pART gene family members over the past 6 years is illustrated in additional file 1 ("Representation of pART gene transcripts in the database of expressed sequence tags"). By October 2004, each human pART except pART7 was represented by more than 100 ESTs. Interestingly, each pART except pART7 is represented by more ESTs than poly (ADP-ribose) glycohydrolase (PARG), the single known enzyme capable of removing poly-ADP-ribose from pART target proteins. The large number of ESTs corresponds to a large variety of tissues found to contain pART ESTs and presumably reflects an ubiquitous pattern of gene expression, i.e. akin to that of the house keeping enzymes hypoxanthine-guanine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (GAPD). For comparison, the members of the mART gene family (ART1-ART5), which exhibit highly restricted patterns of expression, are each represented by much fewer ESTs than the pARTs. As of January 2005, the mammalian gene collection contains annotated full-length cDNA sequences for 10 of the 17 human pARTs and for 12 of 16 mouse pARTs (Fig. 2).
Chromosomal localizations and exon/intron structures of the human and mouse pART gene family members
The results of tBLASTn and BLASTn searches of the human, mouse, and rat genome sequences yielded the chromosomal localization and the exon/intron structure of each pART gene family member. The chromosomal localizations of the pART genes are represented schematically in Figure 2. All human and mouse pART orthologues lie in regions of conserved synteny. There are three conserved pART gene clusters containing two related paralogues (pARTs 8 and 9; pARTs 12 and 13; pARTs 15 and 17). However, the two most closely related pairs of pARTs (pARTs 5 and 6; pARTs 16 and 17) each are located on different chromosomes. All other pARTs are distributed as single copy genes on different autosomes. In the human genome, the cluster containing pARTs 8 and 9 also contains pART7. Additional file 2 illustrates the local chromosomal environment of this pART gene cluster on human chromosome 3q and the syntenic region on mouse chromosome 16B3. The local order of genes is similar in the human and mouse. However, the region corresponding to pART7 is missing in the mouse. The corresponding region is also missing in the rat genome (not shown).
The total number of exons in each pART gene is depicted in Figure 2 and the exon structure of the catalytic domain is illustrated schematically for the human pARTs in Figure 3. All intron positions within the coding region are fully conserved in human and mouse orthologues. With the sole exception of pART4 (VPARP), the catalytic domain is encoded by the 3' terminal exons. Remarkably, in all pART genes, with the exception of pART4 (VPARP) and pART14 (TiPARP), the exons encoding the catalytic domain are separated from the rest of the respective coding exons by a phase 0 intron shortly upstream of the codon for the first residue of the conserved H-Y-E catalytic site motif, the conserved histidine in β 1 (Fig. 3). For most pARTs, the amino acid sequences encoded by exons upstream of this phase 0 intron do not show any detectable similarities, except for members of a particular subgroup. We used the position of this phase 0 intron in pART1 to delineate the N-terminal border of the catalytic domain (e.g., see the green labeled end of the PARP-1-model in Figure 1 and the dashed rectangle in Figure 3B).
Figure 3 Schematic diagram of the exon/intron structures of the regions encoding the catalytic domain of pART family members. A) Exon/intron structures were determined by BLASTn searches of the human genome sequence with individual pART cDNA sequences. Only the exons corresponding to the catalytic domain of PARP-1 are shown. The coding region is marked in red, the 3' untranslated region (utr) is marked in white, and a blue bar marks the region corresponding to the catalytic domain. Exons are represented as boxes with the width of each box reflecting the size of the respective exon (the 3' utr is not drawn to scale). Exon numbers are given with exon 1 corresponding to the exon encoding the presumptive initiation methionine. In all cases except pART4 (VPARP) the catalytic domain is encoded by the 3' terminal exons. Exon sizes (or size of coding region in case of the 3' exons) in basepairs are indicated on top of the boxes. Introns are depicted as triangles and are not drawn to scale. Intron sizes in base pairs are indicated on top of the triangles. The position of each intron with respect to the reading frame is indicated in the triangles (0 = between codons, +1 = between codon positions 1 and 2, +2 = between codon positions 2 and 3). Conserved exon boundaries are marked by colored arrows. Codons corresponding to the H-Y-E motif in the NAD binding crevice of DT and PARP-1 (see Fig. 1) are marked by yellow circles. B) The catalytic domain as delineated in this paper is indicated by the dashed rectangle. For each pART the cDNA coding region within the catalytic domain is marked by a straight line, regions extending beyond this domain in the 5' direction (and in the 3' driection in case of pART4) are marked by dashed lines. The positions of the codons corresponding to the H, Y, E residues in the NAD-binding crevice are indicated by vertical lines. Intron phases are indicated by circles (phase 0), boxes (phase 1), and triangles (phase 2). Numbers indicate the distance in codons between the conserved histidine in β 1 and the next upstream phase 0 intron. Color-coding of conserved introns corresponds to that shown in A). Nonconserved introns are indicated in blue (filled) icons.
The exon/intron structures of the pART catalytic domains reveal a number of intriguing features (Fig. 3). The region encoding the catalytic domain is disrupted by a remarkable variety of introns with the number of introns varying from one in subgroup 3 and in pART14 to six in pARTs 16 and 17. The catalytic domain of pART1 (PARP-1) and those of its closest relatives in subgroup 1 are disrupted by three (pARTs 3 and 4) or four (pARTs 1 and 2) introns. Strikingly, not one of these 14 intron positions is conserved. The catalytic domains of the two closely related tankyrases in subgroup 2 each are interrupted by three conserved introns. In subgroup 3, the catalytic domains of pARTs 7–10 each contain a single conserved intron. The pARTs of subgroup 4 (pARTs 11–14) share a single conserved intron in their catalytic domains, pARTs 11–13 share a second conserved intron in the catalytic domain, which is missing in pART14. The pARTs of subgroup 5 (pARTs 15–17) share two conserved introns in their catalytic domains, pARTs 16 and 17 share four additional conserved introns in the catalytic domain, which are missing in pART15.
Conserved structural features revealed by multiple amino acid sequence alignments and secondary structure predictions
PSI-BLAST is a powerful, position sensitive iterative program designed to detect distantly related proteins in the protein database [35]. Initial matches in the first iteration correspond to those detected by classic BLASTp searches and typically reveal proteins with an amino acid sequence identity to the query sequence of > 30%. PSI-BLAST then derives a position specific scoring matrix from the aligned protein sequences obtained in the first iteration, which is then used for the subsequent search of the protein database. This process is repeated in an iterative fashion until no further matches are detected and the search 'converges'. We performed PSI-BLAST searches of the protein database using as query the amino acid sequences of the catalytic domain of each member of the pART gene family. Figure 4 schematically illustrates the tiling paths of PSI-BLAST searches obtained with the stringent default threshold setting (0.005 for the expect value) for a representative member of pART family subgroups 1, 3, 4 and 5. Typically, the other members of the same subgroup were detected in the first iteration and obtained the highest scores. The pARTs of other subgroups were usually detected within two additional iterations, except in case of pART15. Here, five iterations were required to detect all pART family members.
Figure 4 Representative tiling paths of PSI-BLAST searches initiated with the catalytic domain amino acid sequences of selected pART family members. PSI-BLAST searches were initiated with the catalytic domain amino acid sequences of the pARTs indicated on top as query sequences with the default threshold setting for the expect value of 0.005. Matching sequences from selected model organisms are indicated at the iteration in which they first appeared above threshold. pART subgroups are color coded as in Figure 2. Accession numbers of the indicated pARTs are listed in Figures 2 and 9. Species of origin is color-coded in the two letter abbreviation of the organism as follows: Homo sapiens (Hs) red, Drosophila melanogaster (Dm) and Anopheles gambiae (Ag) purple, Caenorrhabditis elegans (Ce) blue, Chilo iridescent virus (Ci) and Bacteriophage Aeh (Ba) brown.
The amino acid sequence alignments generated by PSI-BLAST typically contained the highest degree of sequence similarity in secondary structure motifs corresponding to the NAD-binding cores in the known 3D structures of chicken PARP-1 (1a26) and mouse PARP-2 (1gs0). Separate multiple amino acid sequence alignments were generated with T-Coffee for each of the pART subgroups using the orthologous sequences from human and mouse [36]. PSIPRED was used to predict secondary structure units and GenTHREADER was used to predict the optimal alignment of pART amino acid sequences with the 3D structures of chicken PARP-1 and mouse PARP-2 [37]. In all cases, predictions and alignments yielded consistent results with respect to the sole alpha helix and five of the six β-strands of the PARP-1 catalytic domain (see additional files 3, 4, 5, 6, 7: "Multiple amino acid sequence alignments, secondary structure predictions and threading results for pART subgroups 1–5"). The small β strand (β 4) at the upper edge of the active site crevice was aligned and predicted congruently only for subgroups 1–4, and could not be predicted with confidence for the most distant relatives of PARP-1 (pARTs 15–17). Regions corresponding to connecting loops showed significant sequence identities only for members of a particular pART subgroup. Most likely, these regions fold similarly only in closely related pART family members.
A striking result of the alignment analyses is that the H-Y-E catalytic site motif is fully conserved only in subgroups 1 and 2 (pARTs 1–6). All other pARTs show deviations from this motif. The histidine in β 1 is conserved in 9 of the 11 members of subgroup 3–5, the tyrosine in β 3 is conserved in all family members, yet the presumptive catalytic glutamic acid at the N-terminal end of β 6 is exchanged in each of the pARTs 7–17.
Moreover, the amino acid sequence of the loop immediately upstream of β 5 and the active site glutamic acid residue deviates markedly from those of PARP-1 and PARP-2 in most other family members except for the tankyrases (pARTs 5 and 6). A growing body of evidence indicates that this region influences the target specificity of pARTs and mARTs [38-40]. In the 3D structure of PARP-1 with carba-NAD (3pax), the ligand was found to interact with this loop outside of the active site crevice, and it was proposed that this may reflect the binding of the ADP-ribose polymer in the target protein [14].
The results of the secondary structure prediction and threading analyses were used to refine a multiple amino acid sequence alignment of the catalytic domains of all human pART family members. The resulting alignment is shown in Figure 5. The conserved secondary structure units corresponding to the catalytic NAD binding core (the six beta strands and one alpha helix marked in Figure 1) are indicated schematically below the alignment. The corresponding amino acid residues are highlighted in the alignment. Intron positions are projected onto the amino acid sequence in Figure 5. The positions of conserved introns are marked by colored arrows below the alignment. Note that the alignment diverges most strongly both in length and in sequence in the loops immediately downstream and upstream of β 3.
Figure 5 Multiple amino acid sequence alignment of the catalytic cores of the human pART family. The multiple sequence alignment was generated with T-Coffee and manually adjusted using the results of the PSI-BLAST, PSIPRED, and GenTHREADER analyses. Numbers at the sequence ends indicate the number of additional residues upstream and downstream of the alignment shown. Residues corresponding to the H Y E motif in the NAD binding crevice of diphtheria toxin are in red and marked by asterisks. The conserved β sheets and alpha helix are shaded in green and yellow. Conserved intron positions are marked in the multiple alignment using the same color-coding as in Figure 3. Conserved intron positions are indicated also above the alignment with arrows. Non-conserved intron positions are marked in blue in the alignment.
Figure 6A shows a condensed version of the alignment in which the diverging intervening loops are indicated only by the number of amino acid residues. These 66 amino acid residues can be superimposed well in the 3D structures of PARP-1, PARP-2, DT, and ETA. The respective amino acid sequences of DT, ETA and the putative Chilo iridescent virus pART are also shown for these regions. Figure 6B shows the calculated amino acid sequence identities of the pART family members in this region. The percentage amino acid sequence identity in the aligned core region is higher among members of a particular subgroup than between members of different subgroups, lending support to the subgroup assignments. For each pART, the next most closely related paralogue is a member of the same subgroup. Note that two pairs of pART paralogues show very close sequence similarity: pARTs 5 and 6 (94% identity in the aligned core region) and pARTs 16 and 17 (86% identity). This close similarity is reflected also in the conserved exon intron structures of the respective pART pairs (see Fig. 3).
Figure 6 Structure based amino acid sequence alignment of the catalytic cores of the pART gene family. A) The alignment is restricted to those regions corresponding to the conserved secondary structure units of PARP-1 and DT as highlighted in Figure 1. The H Y E motif is marked by asterisks and is highlighted in red. Black numbers indicate amino acid residues from the N- and C-terminal ends of the protein and within the loops connecting the structure units shown. For proteins with known 3D structures the pdb accession number is given and the residues corresponding to respective secondary structure units are underlined. 1tox = diphtheria toxin; 1aer = pseudomonas exotoxin A, 3pax = chicken PARP-1 (pART1), 1gs0 = mouse PARP-2 (pART2). Human and mouse pARTs are indicated by colored numbers. The sequence of the putative pART from Chilo iridescent virus is also shown for comparison (ci). B) Pairwise percentage sequence identities were calculated for the 66 amino acid residues shown in A), which correspond to the conserved core secondary structure units in Figure 1.
Comparison of mouse and human pART orthologues shows that seven of such pairs exhibit 100% sequence identity in the aligned core region (pARTs 1, 5, 6, 11, 14, 16, and 17) and six show > 90% identity (pARTs 2, 3, 4, 10, 12, and 15). The mouse and human orthologues of pARTs 8, 9, 13 show the least degrees of sequence identity in this region (82%, 82%, and 70%, respectively) (Fig. 6B).
Phylogenetic analysis of the amino acid sequences of the catalytic cores of pARTs resulted in three very similar trees when using Maximum Parsimony (PAUP), Maximum Likelihood (PhyML), and Bayesian Markov Chain Monte Carlo (MrBayes) optimization criteria (Figure 7). All topologies showed moderate to high support values for the recovered relationships. All trees recovered five basic clades corresponding to the subgroups 1–5. The results indicate that pARTs of subgroups 1 and 2 are more closely related (sistergroups) to one another than to members of the other subgroups. A similar relationship is seen for pARTs of subgroups 3 and 4. Note that the putative Chilo iridescent virus pART clusters with the mammalian pARTs of subgroup 1, suggesting that this large double stranded DNA virus may have acquired its pART by horizontal gene transfer.
Figure 7 Phylogram of the evolutionary relationship of the pART family. Evolutionary relationships of the amino acid sequences in the catalytic core of the pARTs shown in Figure 6 are illustrated as a maximum a posteriori phylogram (MAP) of Bayesian Markov Chain Monte Carlo analysis (pP = 0.92). Posterior probabilities were converted into percentages and are shown above the branches. Members of the five pART family subgroups are color-coded as in Figure 2: subgroup 1 = red, 2 = pink, 3 = orange, 4 = green, 5 = grey. Hs = Homo sapiens, Mm = Mus musculus.
The pART catalytic domain has become genetically fused to a wide spectrum of protein domains
With the exception of closely related members within a subgroup, the amino acid sequence similarity between pART family members breaks off upstream of β 1. Interestingly, loss of sequence similarity correlates well with the presence of a phase 0 intron upstream of β 1. All pART family members except pART4 and pART14 contain such a phase 0 intron 26–64 codons upstream of the conserved histidine in β 1 (Fig. 3B).
Using the sequences flanking the catalytic domain of each pART family member as queries, we performed further PSI-BLAST analyses and searches of the Conserved Domain Database [41]. The results, summarized in Figure 8, reveal that each of the 17 human pARTs with the possible exception of pART15 is a multi-domain protein. Strikingly, the pART catalytic domain is associated – in a Lego like fashion – with a broad spectrum of known protein domains. In all family members except pART4 the catalytic domain represents the C-terminal domain.
Figure 8 Schematic diagram of the domain structures of human pARTs and pARTs from distantly related organisms. Recognizable protein domains in the pART family are represented by the icons defined on the right. The domain structures of human pARTs (on the left, numbered Pacman icons) and related pARTs from other species are illustrated schematically. Potential DNA binding domains are boxed in red, potential ubiquitylation motifs are boxed in green. Members of the five pART family subgroups are grouped within colored boxes using the color-coding as in Figure 2: subgroup 1 = red, 2 = pink, 3 = orange, 4 = green, 5 = grey. Amino acids corresponding to the HYE catalytic site motif of DT and PARP-1 are shown in the mouths of the Pacman icons. Black numbers indicate protein lengths in number of amino acids. Species of origin is color-coded in the two letter abbreviation of the organisms as in Figures 4 and 9: Drosophila melanogaster (Dm) and Anopheles gambiae (Ag) purple, Caenorrhabditis elegans (Ce), Dictyostelium discoideum (Dd), Entamaoeba histolytica (Eh), and Gibberella zeae (Gz) blue, Arabidopsis thaliana (At) green, Chilo iridescent virus (Ci) and Bacteriophage Aeh (Ba) brown. Protein database accession numbers for the illustrated pARTs are listed in Figures 4 and 9. On the right, the approximate size of each domain is indicated in number of amino acid residues. The accession numbers of the respective domain families in the pfam, cd, and smart databases are indicated. In case of zinc finger (zf) containing domains, the number of recognizable zinc fingers is indicated by colored bars within the icon.
A number of associated domains occur in two or more human pART family members. Note that domain sharing generally is restricted to members of a particular pART subgroup. For example, all members of subgroup 1 contain a helical domain preceding the catalytic domain, whereas this domain is missing in members of other pART subgroups. The two members of subgroup 2 share SAM and ankyrin-repeat domains. Three of four pARTs in subgroup 3 share A1pp domains [42], all members of subgroup 4 share WWE domains, and two members of subgroup 5 contain a second, truncated pART domain, reminiscent of the duplicated inactive ART domain found in the VIP2 and iota mART toxins [16,23].
Several pARTs carry recognizable zinc-fingers containing putative RNA-, DNA-, or ubiquitin-binding domains (pART1, pART2, pART10, pART12, pART13). This indicates that the genetic fusion of a pART catalytic domain with zinc-fingers has occurred repeatedly in evolution.
Representation of pARTs in other model organisms
We also used PSI-BLAST to screen the protein database for recognizable pART family members in other organisms using as queries the amino acid sequences of catalytic domains of each of the 17 human pARTs (Figure 9). The order in which PSI-BLAST picked up putative pART sequences from the database in successive iterations was similar for different members of a particular pART subgroup but differed markedly for members of different subgroups (see additional file 8: "Representative tiling paths of PSI-BLAST searches initiated with the catalytic domain amino acid sequences of selected pART family members"). In many instances, PSI-BLAST detected pART sequences from distantly related organisms in earlier iterations than the human pART paralogues from other subgroups.
Figure 9 pARTs in distantly related species. pART relatives were identified by PSI-BLAST searches as in Figure 4. Matching sequences from other organisms were sorted by group on the basis of sequence similarity and associated domains. Accession numbers are given for pARTs from Homo sapiens (human), Mus musculus (mouse), Gallus gallus (chicken), Tetraodon nigroviridis (puffer fish), Drosophila melanogaster (fruit fly), Anopheles gambiae (malaria mosquito), Caenorhabditis elegans (nematode), Dictyostelium discoideum (slime mold), Gibberella zeae (ear root microfungus), Entamaoeba histolytica (amoeba), Arabidopsis thaliana (cress plant), Chilo iridescent virus and Bacteriophage Aeh1 (viruses), Pseudomonas aeruginosa, Corynebacterium diphtheriae and Vibrio cholerae (bacteria). Lower case letters in black indicate the pART designations used in Figure 8.
Figure 9 summarizes the matches of pART-related proteins found in model organisms with completed genome sequences. On the basis of amino acid sequence similarity, conserved intron positions and/or conserved associated domains, pARTs from other vertebrates including fish and chicken, generally can be assigned to a particular human pART orthologue. In contrast, pARTs of lower eucaryotes can be assigned to a subgroup but not to a particular vertebrate pART.
pART homologues were found in many model organisms from the animal, plant, fungi, and protist kingdoms. The recently completed genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7 [43]. The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for all pARTs except for pARTs 2, 3, 7, 10, and 17 [44]. Simpler eucaryotes generally contain fewer pARTs (two in the fruit fly D. melanogaster, three each in the malaria mosquito A. gambiae, the nematode C. elegans, and the ascomycete G. zeae; six in the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana).
Remarkably, the yeast S. cerevisae and the archaea lack detectable pARTs. Only two matches were found in the viral proteome: these derive from two double stranded DNA viruses: the insect virus Chilo iridescent virus and the bacteriophage Aeh1. Although PSI-BLAST initially failed to connect the pART family with Diphtheria toxin and Pseudomonoas exotoxin A, these toxins were readily connected with the eucaryotic pARTs when using as query a chimera, e.g. of Diphtheria toxin and Chilo iridescent virus pART in which the sequences of three of the conserved structure units highlighted in Figures 1 and 6A were interchanged. These searches uncovered a DT/ETA-like putative ADP-ribosyltransferase in V. cholerae, but no other proteins in the microbial proteome in GenBank.
Of note, none of the known R-S-E motif bacterial or vertebrate mARTs were ever connected by PSI-BLAST with the DT/ETA/pART group. In several cases, however, we observed intriguing matches just slightly below threshold (in the region surrounding the conserved H in β 1) to members of the family of RNA:NAD 2' phosphotransferases. These enzymes catalyze a reaction during tRNA splicing that is similar to the reaction catalyzed by ARTs, but in which ADP-ribose is transferred to the 2'-phosphate in immature tRNA rather than to an amino acid residue in a protein [25]. The 3D-structure of a prototype member of this gene family, indeed, reveals a structure closely resembling that of PARP-1 and Diptheria toxin (see Fig. 1), providing strong support for the relevance of the matches detected by PSI-BLAST.
For the pART homologues shown in Figure 9 we also analyzed the sequences flanking the pART catalytic domain for associated conserved domains. The results reveal that many pARTs, even from very distantly related organisms, share domain associations found in human and mouse pARTs. Some of these are illustrated in Figure 8. For example, the association of regulatory, BRCT, and DNA binding domains observed in pART1 (PARP-1) is found also in similar proteins encoded by fruit fly, nematode, microfungi and cress plant genomes. Tankyrase-like association with ankyrin repeats is found in pARTs from the fruit fly and nematode. The association of a pART catalytic domain with an A1pp domain, as seen in human pART subgroup 3, is found also in a pART from the slime mold Dictyostelium discoideum. The combination with a WWE domain, as seen in human pART subgroup 4, is found also in putative pARTs from cress plant. A domain corresponding to the unknown upstream region of the smallest human pART (pART15) is observed also in a pART from the malaria mosquito Anopheles gambiae, and a duplicated truncated pART catalytic domain as in pARTs 16 and 17 is observed also in a pART from the microfungus Gibberella zeae. These results indicate that many of the domain combinations observed in human and mouse pARTs represent evolutionary ancient inventions.
Some pARTs of distantly related proteins are associated with domains not found in any of the human pARTs. A striking example is that of G. zeae pARTc, which most closely resembles human pARTs 16 and 17, but is associated with a second potential catalytic, ubiquitin ligase domain (Fig. 8). A similar pART is found also in the related microfungus Aspergillus nidulans [GenBank: EAA66581]. These microfungal pARTs are the only examples found so far, in addition to vertebrate pART4, where a distinct domain(s) is genetically fused to the C-terminal end of the pART catalytic domain. The large domain(s) associated with the putative pART from bacteriophage Aeh1 does not bear any resemblance to pART-associated domains in vertebrates but shows distant similarity to viral coat proteins. The only organism containing an isolated pART domain reminiscent of the isolated ART domain found in verbetrate mARTs [27] is the Chilo iridescent insect virus. This "naked" viral pART catalytic domain contains the H-Y-E motif of PARP-1 and DT. It will be interesting to determine whether this protein exhibits the predicted pART activity.
A striking example of domain shuffling is observed in one of the three C. elegans pARTs: like the human tankyrases (pARTs 5 and 6), Ce.pARTc contains ankyrin repeats, but also harbors the regulatory and WGR domains typical of human group 1 pARTs instead of the SAM domain found in human pARTs 5 and 6 (Fig. 8). A similar variation of domains as in Ce.pARTc is found also in one of the ten pARTs of D. discoideum (Dd.pARTb).
Finally, we addressed the question whether the striking differences in exon/intron compositions of the closest PARP-1-homologues in groups 1 and 2 might be reflected in similar differences in pART orthologues of distantly related species. To this end we determined the exon/intron structures of distant pART orthologues by BLASTn searches of the respective genome databases using cDNA sequences as queries; and compared the results with those obtained for human pART genes. The results are illustrated schematically in Figure 10, with conserved intron positions highlighted. As in case of most other genes, the pART genes of 'lower' animals, protists, and plants in general contain fewer and shorter introns than the human homologues. However, some of the introns found in human pART genes are found also in homologues of distantly related organisms. For example, all six introns observed in D. melanogaster pARTb are found in corresponding positions also in human pART5 (tankyrase 1); yet human pART5 contains 14 additional introns not found in the fruit fly pART. The other pART of the fruit fly shares two of its five introns with human pART1 (PARP-1). The three pARTs of the nematode C. elegans show a different, only partially overlapping set of conserved introns: Ce.pARTa shares seven of its nine introns with human pART1, Ce.pARTb shares three of its four introns with human pART2, whereas Ce.pARTc does not seem to share any of its introns with pART5, despite the similar domain organization on the protein level (see Fig. 8). The pARTs from the model plant Arabidopsis thaliana contain a fairly high number of introns, however only very few intron positions correspond to ones found also in human pARTs. For example, At.pARTa which is most closely related to human PARP-1 in terms of amino acid sequence similarity and organization of conserved protein domains, evidently does not share any of its 18 introns with human pART1. Strikingly, however, the introns found in the catalytic domain of this pART exhibit conserved positions with two different human pARTs: two of the four intron positions in the catalytic domain of At.pARTa are found in corresponding positions in human pART5 (tankyrase), another intron is found at a corresponding position in human pART2 (Fig. 10), whereas the fourth intron is not found in any human pART. At.pARTb which is most closely related to human pART2 in terms amino acid sequence similarity and domain organization, shares one of its 17 introns with human pART2. Note further, that in only two cases (Chilo iridescent virus pART and pARTa of the fruit fly), the pART catalytic domain lacks introns, i.e. is encoded by a single exon as in case of the vertebrate mARTs [27].
Figure 10 Schematic diagram of the exon/intron structures of pART family members of distantly related organisms. A) Exon/intron structures were determined by BLASTn searches of the genome browsers using the pART cDNA sequences. The positions of codons corresponding to the H Y E motif in the NAD-binding crevice of diphtheria toxin are marked by yellow circles. The position of the conserved glycine and arginine pair of residues within the WGR domain is marked in blue. Coding regions for catalytic and other domains are indicated by colored bars. Conserved introns are marked by colored arrows. B) The diagram contains only those introns that are conserved in at least two distantly related species. Color-coding of the introns corresponds to that shown in A). The position of codons encoding/corresponding to the H, Y, E residues in the NAD binding crevice are indicated by vertical lines. The position of each intron with respect to the codon is indicated by circles (phase 0 introns), boxes (phase 1 introns), and triangles (phase 2 introns). Coding regions for catalytic and other selected domains are indicated by colored lines as in A).
Discussion
The results of our study illustrate the great power and utility of the public genome databases and database search programs. Moreover, they provide important novel insights into the molecular structure and evolution of the pART gene family.
Our results differ in some details from those of a recent report by Ame and coworkers [11]. These discrepancies can be explained by errors in the draft sequence of the human genome available at the time of the previous report. For example, the database entry AK023746 given by Ame et al. for PARP-5c evidently represents a truncated cDNA for pART6 (alias tankyrase 2 or PARP-5b). This entry contains two point mutations and a 65 bp deletion in the 3' utr vs. the cDNA and genomic sequences of pART6. Blast analyses of the high quality sequence of the human genome and of the EST database with the AK023746 sequence provide no evidence for a distinct copy of this gene in the human genome. We conclude that the PARP-5c gene identified by Ame et al. represents an allelic variant or cloning/sequencing error rather than a genuine pART gene family member; i.e. that the total number of human pART genes is 17 rather than 18 suggested in the previous report. Large discrepancies exist also in the number of amino acids assigned in the two reports for pART7/PARP-15 (444 vs. 989) and for pART16/PARP-8 (854 vs. 501). The earlier database entries for PARP-8 (XM_018395) and PARP-15 (XM_093336) have hence been removed as a result of standard genome annotation processing because these entries evidently contained frameshift mutations and/or fused cDNA sequences that led to erroneous amino acid assignments. Similarly, the small differences in assignments for five other PARPs/pARTs can be accounted for by differences in the draft vs. high quality sequence of the human genome (Ame et al./our study): pART2/PARP2 (583/570), pART3/PARP3 (540/533), pART10/PARP10 (1020/1025), and pART14/PARP7 (657/680).
We assigned the 17 human pARTs into five distinct subgroups (Fig. 2). This assignment is supported by several independent lines of evidence: Firstly, members of a particular subgroup show higher amino acid sequence identities to one another than to members of other subgroups (Fig. 6). This is reflected in the tiling paths of PSI-Blast searches, where members of the same subgroup were detected in the first iteration, whereas members of other subgroups generally were detected in later iterations (Fig. 4). Secondly, members of a particular subgroup typically share one or more associated domains not found in members of other subgroups (Fig. 8); pARTs 8, 10 and 15 pose exceptions to this rule. Thirdly, members of a particular subgroup typically share one or more intron positions not found in members of other subgroups (Fig. 3); pARTs 1–4 pose notable exceptions to this rule. Fourthly, when genes of two or more pARTs are physically linked in a cluster on the same chromosome, they belong to the same subgroup – possibly reflecting regional duplications (Fig. 2). Finally, results of all phylogenetic analysis converged in topologies with clearly distinct clades for each of the subgroups (Fig. 7). Members of subgroups 1 and 2 evidently are more closely related to one another than to other subgroups (Figs. 6 and 7). Similarly, members of subgroups 3 and 4 are sister-groups to one another, indicating a close relationship.
Members of the pART family are found fused to a striking variety of associated domains (Fig. 8). It is not farfetched to hypothesize that the associated domains direct the respective pARTs to subcellular structures and/or target proteins. Genetic fusion of group 1 and group 2 pARTs with DNA-binding domains is in line with their established roles in DNA-repair, chromosome remodeling, and mitotic spindle formation [9,11,12]. Moreover, the SAM and ankyrin domains of pARTs 5 and 6 have been shown to mediate interactions with target proteins in telomere-associated protein complexes [45]. Similarly, the C-terminal domain of pART4 evidently plays a role in targeting pART4 to the major vault particles [46]. A flurry of domains implicated in the ubiquitination pathway point to a possible connection between ubiqutitination and ADP-ribosylation. Indeed, it has recently been reported that ADP-ribosylation of TRF1 by tankyrase (pART5) results in the release of the protein from telomers and its subsequent ubiquitination [47]. Strikingly, pARTs from the microfungi G. zea and A. nidulans provide examples for the genetic fusion of two enzyme domains catalyzing these post-translational protein modifications into a single polypeptide.
So far, only a single example of a 'naked' pART catalytic domain akin to the isolated catalytic domain of the vertebrate ecto-ARTs 1–5 [27] was recovered from the public database. This putative pART from Chilo iridescent virus clusters with the mammalian pARTs of subgroup 1 (Fig. 7), suggesting that this large double stranded DNA virus [48] may have acquired its pART by horizontal gene transfer.
The definition of the pART catalytic domain proposed in this paper is somewhat smaller than that commonly used in the field [11]. We used the position of the common phase 0 intron upstream of the first conserved β sheet to set the N-terminal end of the catalytic domain (e.g. see Figs. 1 and 3B). The pARTs of subgroup 1 are extended N-terminally of this position by an alpha helical domain (Fig. 8) which is often included as part of the PARP-1 catalytic domain. However, since other pART family members lack this region, we propose to omit it from the proper pART catalytic domain. Moreover, this N-terminal delineation of the catalytic domain corresponds well to the N-terminus of the 'naked' pART of Chilo iridescent virus as well as to those of Diphtheria toxin and Pseudomonas exotoxin A after proteolytic processing of the signal sequence or translocation domain (Fig. 1).
With the exception of pART4, the group 1 pARTs are extended upstream of this helical region by another domain named after its conserved motif of tryptophane (W) – glycine (G) – arginine (R) residues. This WGR domain is found also in poly-A-polymerases, its function is unknown. Many group 1 pARTs from distantly related organisms, e.g. plants, insects, nematodes, and microfungi, also contain these two domains. Interestingly, in Drosophila melanogaster pARTa these three domains (WGR, helical, catalytic) are encoded by a single, large exon (Fig. 10). Human pARTs 5–17 lack the WGR and helical domains. However, pART5/6 (tankyrase)-like pARTs from C. elegans (Ce.pARTc) and D. discoideum (Dd.pARTb) contain the WGR and helical domains whereas a SAM domain is found at this position in human pARTs 5 and 6 (Fig. 8).
A puzzling finding is the lack of conservation of the classic H-Y-E motif found in the catalytic cores of PARP-1, PARP-2, Diphtheria toxin and Pseudomonas Exotoxin A (Fig. 1). This motif is conserved only in members of subgroups 1 and 2. All other human pARTs carry notable variations from this motif. In particular, all other pARTs carry a replacement of the glutamic acid residue in β 5, i.e. the residue that was shown to be critical for the catalytic activities of DT, PARP-1 and many other pARTs and mARTs [6,7,20,21]. In six cases, this glutamic acid is replaced by an isoleucine residue, in two cases by leucine, and in one case each by threonine, valine, or tyrosine. Enzyme activity has been reported recently for two of the six pARTs that carry an H-Y-I motif instead of the H-Y-E motif (pARTs 10 and 14) [32,34]. Thus, it is not unlikely that the four other pARTs carrying the H-Y-I motif turn out to be active enzymes (pARTs 11, 12, 16, and 17). Mouse pART8 also carries an H-Y-I motif, whereas its human orthologue, like pART7, carries an H-Y-L variant motif. H-Y-I and H-Y-L variant motifs are also found in pARTs from the slime mold (Dd.pARTg) and amoeba (Eh.pARTf) (Fig. 8). Human pART15 carries an H-Y-Y variant motif, which is conserved in its orthologues from mouse and the malaria mosquito (Fig. 8). It will be interesting to determine whether and how site directed mutagenesis of the H-Y-E motif in pARTs 1–6 to the variant motifs of pARTs 7–17 – and vice versa – affects their enzyme activities. Moreover, it remains to be determined whether the most striking variation of the H-Y-E motif – to Q-Y-T in human and mouse pART9 is compatible with enzyme activity.
The results of our PSI-BLAST and PSIPRED analyses (Figs. 4, 5, 9 and additional files 3, 4, 5, 6, 7, 8) support the conclusions that the pART gene family described here and the mART gene family described in our previous study [27] constitute two distinct ART subfamilies, and further, that the family of tRNA:NAD 2'-phosphotransferases [24,25] constitutes a branch that is more closely related to the pART subfamily than to the mART subfamily. Our results illuminate the power and limits of PSI-BLAST searches: PSI-BLAST readily connected members of the pART subfamily in many different species, while DT, ETA and TpTs were found at or below the threshold. In contrast PSI-BLAST searches never connected pART family members with members of the mART subfamily or vice versa. The results of PSI-BLAST searches, thus, are in accord with insights gained from the known 3D structures of representative ADP-ribosyltransferases (Fig. 1), i.e. that certain conserved structural features clearly distinguish these two subfamilies. Is it possible that some of the pART gene family members described here actually possess mono-ADP-ribosyltransferase rather than poly-ADP-ribosyltransferase activity? Given the structural similarity to DT/ETA this is a possibility. Moreover, it cannot be excluded that some family members may have lost enzyme activity and have acquired a new function. In any case, the respective proteins clearly are more closely related to the pART than to the mART gene family, in line with the nomenclature proposed here. Have all ARTs encoded in the human genome been identified? A number of ADP-ribosylation reactions have been described in mammalian cells that cannot yet be accounted for by the ARTs identified in this study or our previous study, e.g. mono-ADP-ribosylation of actin, rho, glutamate dehydrogenase, and of the alpha and beta subunits of heterotrimeric G proteins [3,4,8]. Given the fact that the pART subfamily described here and the mART subfamily described in our previous study [27] could not be interconnected by PSI-BLAST, it reamins an intriguing possibility that other ART subfamilies in the human genome still await to be identified.
Conclusion
The family of proteins containing a PARP-like catalytic domain consists of 17 members in the human and 16 in the mouse, rat, and pufferfish. The vertebrate pART family can be divided into five subgroups on the basis of sequence similarity, phylogenetic relationships, conserved intron positions, and patterns of genetically fused protein domains. The four members of group 1 and the two members of group 2 each contain a conserved trias of residues (H-Y-E motif) also observed in Diphtheria toxin and Pseudomonas exotoxin A. The eleven other pART proteins carry variants of this motif (six H-Y-I, two H-Y-L, and one each Q-Y-T, Y-Y-V, H-Y-Y). All human pARTs are multi-domain proteins in which the pART catalytic domain is associated in a Lego-like fashion with other putative protein-protein interaction, DNA binding and ubiquitination domains. In all but one case (pART4) the catalytic domain represents the C-terminal end of the multi-domain protein. Most of the domain associations observed in human pARTs appear to be very ancient inventions since they can be found also in insects, plants, microfungi, and amoeba.
Methods
Database searches
Protein databases were searched using PSI-BLAST [35]. Genome databases were searched using BLASTn and tBLASTn [49]. Tissue distributions of pART-ESTs were analyzed using Electronic Northern calculations at the GeneCard website [50].
Structure and sequence analyses
Amino acid sequence alignments were performed with T-Coffee [36]. Secondary structure predictions were performed with PSIPRED [37]. Threading of amino acid sequences onto known 3D structures in PDB were performed with GenTHREADER [37]. Sequence analyses were performed using DNA-Star software, 3D-images were prepared with PyMol [51] software.
Phylogenetic analyses
Phylogenetic analyses were applied to the 36 catalytic core amino acid sequences using the dataset in Figure 6. Phylogenetic analyses were performed on the computational cluster of the College of Biology and Agriculture at Brigham Young University by using maximum parsimony and Bayesian Markov chain Monte Carlo approaches . The topologies were reconstructed using equally weighted maximum parsimony (MP) analysis as implemented in PAUP* 4.0b10 [52], maximum likelihood (ML) with simultaneous adjustment of topology, and branch length as implemented in PhyML [53], as well as Bayesian methods coupled with Markov Chain Monte Carlo inference (BMCMC, MrBayes) [54]. The best fit likelihood model for amino acid evolution was determined based on the lowest Akaike Information Criterion (AIC) or Bayesian Information Criterion (BIC) score as implemented in ProtTest 1.2.6 [53,55,56].
The MP analysis was run using 5000 random addition replicates and tree bisection-reconnection branch swapping. Nonparametric bootstrap values were calculated for MP and ML analyses (10.000/100 bootstrap replicates, 100/1 heuristic random addition replicates) to assess confidence in the resulting relationships. ML analysis was run implementing the RtREV+I+G+F model of amino acid evolution (AIC= 4907.73; -lnL= 2800). The a priori information obtained by ProtTest 1.2.6 was incorporated into the BMCMC analysis. Bayesian phylogeny estimation was achieved using random starting trees, run for 3 × 106 generations, with a sample frequency of 1000, and ten chains (nine heated, temperature= 0.2). Analyses were repeated three times to check for likelihood and parameter mixing and congruence. Likelihood scores were plotted against generation time to determine stationery levels. Sample points before reaching stationery were discarded as "burn-in". Repeated analyses were compared for convergence on the same posterior probability distributions [57]. The maximum a posteriori tree (MAP) is presented in this paper, showing to percentage converted posterior probabilities (pP%).
Abbreviations used
ART = ADP-Ribosyltransferase, BLAST = basic local alignment search tool, 3MB = 3-methoxybenzamide, NAD = nicotinamide adenine dinucleotide, PDB = protein database.
Authors' contributions
This study was initiated in the summer of 1997 while FKN was a visiting scientist in FB's lab at DNAX. Initial database searches were performed by FKN and FB, later searches by HO, PAR, and FKN. KD performed the phylogenetic analyses. FKN supervised the study with essential contributions by FH. HO prepared the figures and FKN wrote the paper. The results represent the partial fulfillment of the requirements for the graduate thesis of HO.
Supplementary Material
Additional File 1
Representation of pART gene transcripts in the database of expressed sequence tags The public EST database was screened for ESTs encoding pARTs using tBLASTn and the amino acid sequences of the catalytic domain of known pART family members as queries at the dates indicated on top. Accession numbers of the corresponding Unigene clusters are indicated. Blank fields indicate lack of detectable ESTs encoding the respective pART catalytic domain. Tissue distribution analyses were performed for each cluster by "electronic Northern" analyses. For each family member, the two tissues with the highest numbers of ESTs are indicated. Tissue abbreviations: BMR bone marrow, BRN brain, HRT heart, MSL muscle, PNC pancreas, PST prostate, KDN kidney, LNG lung, LVR liver, LYN lymph node, SPC spinal chord, SPL spleen, TMS thymus, UTR uterus
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Additional File 2
Schematic illustration of the local human and mouse chromosomal environments of the pART subgroup 3 gene cluster The figure schematically illustrates the local chromosomal environment of the syntenic cluster of pART genes and neighboring genes on human chromosome 3q (top) and mouse chromosome 16B3 (bottom). The order and orientation of all genes in the depicted cluster is conserved. Known transcripts in GenBank are indicated schematically with their respective accession number. Exons are indicated by boxes. The direction of transcription is marked by arrows. Grey vertical bars correspond to a scale of 10.000 base pairs. The figure was modified from the respective online UCSC human and mouse genome browsers .
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Additional File 3
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 1 A multiple sequence alignment was generated for the catalytic domains of pARTs 1–4 with T-Coffee. Each residue in the sequence is reported as a single letter code. Secondary structure units in the 3D structures of chicken PARP-1 (1a26) and mouse PARP-2 (1GS0) are indicated on top of the alignment. Positions with identical residues in all sequences are marked by asterisks, similarities are marked with colons and periods below the alignment. Residues corresponding to the H Y E motif in the NAD binding crevice of diphtheria toxin are marked in red. Intron positions are projected onto the multiple alignment and are marked in grey (phase 0), blue (phase 1), and yellow (phase 2). Secondary structure predictions were generated for human pART1 with PSIPRED and are indicated in blue below the alignment (pr1); the confidence of the prediction is indicated in orange (highest confidence = 9). Secondary structure units are abbreviated as follows: H = helix; B = residue in isolated beta bridge; E = extended beta strand; G = 310 helix; I = pi helix; T = hydrogen bonded turn; S = bend.
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Additional File 4
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 2 A multiple sequence alignment was generated for the catalytic domains of pARTs 5 and 6 with T-Coffee. Residues, identities, intron positions, and secondary structure units are marked as in additional file 3. Indicated secondary structure predictions were generated for human pART5 (pr5) with PSIPRED.
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Additional File 5
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 3 A multiple sequence alignment was generated for the catalytic domains of pARTs 7–10 with T-Coffee. Residues, identities, intron positions, and secondary structure units are marked as in additional file 3. Indicated secondary structure predictions were generated for human pART7 (pr7) with PSIPRED.
Click here for file
Additional File 6
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 4 A multiple sequence alignment was generated for the catalytic domains of pARTs 11–14 with T-Coffee. Residues, identities, intron positions, and secondary structure units are marked as in additional file 3. Indicated secondary structure predictions were generated for human pART11 (pr11) with PSIPRED.
Click here for file
Additional File 7
Multiple amino acid sequence alignments, secondary structure predictions, and threading results for pART subgroup 5 A multiple sequence alignment was generated for the catalytic domains of pARTs 15–17 with T-Coffee. Residues, identities, intron positions, and secondary structure units are marked as in additional file 3. Indicated secondary structure predictions were generated for human pART15 (pr15) and for human pART16 (pr16) with PSIPRED.
Click here for file
Additional File 8
Representative tiling paths of PSI-BLAST searches initiated with the catalytic domain amino acid sequences of selected pART family members PSI-BLAST searches were initiated with the query sequences indicated on top at a threshold setting for the expect value of 0.005 as in Figure 4. pART subgroups are color coded as in Figure 2. Matching sequences from the slime mold (D. discoideum, blue) and from a model plant (A. thaliana, green) are indicated at the iteration in which they first appeared above threshold. The respective pART homologues from these species were arbitrarily numbered (pARTa-j) in the order in which they were detected in the search that was initiated with human pART1 (PARP-1). Protein data base accession numbers are listed in Figure 9. pARTs indicated in black include short possibly truncated coding sequences of pART homologues that could not be assigned to a particular subgroup with certainty.
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Acknowledgements
This work was supported by grant No310/3 from the Deutsche Forschungsgemeinschaft to FKN. HO was a grantee of the Studienstiftung des Deutschen Volkes. KD is funded by the NSF grants DEB-0120718 and DEB-9983195. DNAX is fully funded by the Schering Corporation. We thank Sahil Adriouch, Bernhard Fleischer, Stefan Kernstock, and Stefan Rothenburg (University Hospital Hamburg) for critical reading of the manuscript.
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BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-5-831622567110.1186/1471-2334-5-83Research ArticleThe 3' region of Human Papillomavirus type 16 early mRNAs decrease expression Vinther Jeppe [email protected] Maiken W [email protected] Karen [email protected] Bodil [email protected] Institute of Molecular Pathology, University of Copenhagen, Blegdamsvej 3C, Bldg. 6.2, DK-2200 Copenhagen N, Denmark2 Institute of Molecular Biology, University of Copenhagen, Universitetsparken 15, Bldg. 10, DK-2100 Copenhagen Ø, Denmark3 BioCentrum-DTU, Technical University of Denmark, Building 208, DK-2800 Lyngby, Denmark2005 14 10 2005 5 83 83 17 6 2005 14 10 2005 Copyright © 2005 Vinther et al; licensee BioMed Central Ltd.2005Vinther 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
High risk human papillomavirus (HR-HPV) infects mucosal surfaces and HR-HPV infection is required for development of cervical cancer. Accordingly, enforced expression of the early HR-HPV proteins can induce immortalisation of human cells. In most cervical cancers and cervical cancer cell lines the HR-HPV double stranded DNA genome has been integrated into the host cell genome.
Methods
We have used a retroviral GUS reporter system to generate pools of stably transfected HaCaT and SiHa cells. The HPV-16 early sequences that are deleted upon integration of the HPV-16 genome was inserted into the 3' UTR of the reporter mRNA. Pools containing thousands of independent integrations were tested for the steady state levels of the reporter mRNA by Real Time PCR and reporter protein by a GUS enzymatic activity assays. In addition, we tested the cellular distribution and half lives of the reporter mRNAs. The integrity of the reporter mRNAs were tested by northern blotting.
Results
We show that the 3' region of the HPV-16 early mRNAs (HPV-16 nucleotide (nt.) 2582–4214) act in cis to decrease both mRNA and protein levels. This region seems to affect transcription from the exogenous minimal CMV promoter or processing of the reporter mRNA. The observed repression was most pronounced at the protein level, suggesting that this sequence may also affect translation. For the HPV types: 2, 6, 11, 13, 18, 30, 31, and 35 we have investigated the regulatory effect of the regions corresponding to the HPV-16 nt. 3358–4214. For all types, except HPV-18, the region was found to repress expression by posttranscriptional mechanisms.
Conclusion
We find that the 3' region of HPV-16 early mRNAs interfere with gene expression. It is therefore possible that the deletion of the 3' part of early HPV-16 mRNAs occurring during cervical oncogenesis could contribute to transformation of cells through deregulation of the viral oncogene synthesis. Moreover, we find that the corresponding region from several other HPV types also repress expression, suggesting that the repression by this region may be a general feature of the HPV life cycle.
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Background
Human Papillomaviruses (HPV) form a large family of homologous circular double stranded DNA viruses that infect the cutaneous and mucosal surfaces of humans. Genital infections with HPV are sexually transmitted and occur frequently in women. Although the majority of infected women will clear the infection within a relatively short time period, some develop a persistent infection [1,2]. This initial clearance may depend on immunological or non-immunological mechanisms [3]. The genital HPV types can be divided into high and low risk types. The high risk HPV (HR-HPV) types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) are classified as carcinogenic to humans [4], because epidemiological and molecular studies have shown that infection with one of these HR-HPV types is necessary, but not sufficient for development of cervical cancer [5]. Only a small fraction of persistent HR-HPV infections will progress to high grade cervical lesions and cervical carcinoma. At the molecular level the development of cervical cancer is driven by enforced expression of two oncoproteins (E6 and E7) encoded by the HR-HPV types [6]. HR-HPV E6 and E7 target a large number of cellular proteins involved in normal cellular regulation and in this way facilitate cellular and viral DNA replication in differentiating post mitotic keratinocytes [7,8]. This is crucial for amplification of the viral genome and completion of the viral lifecycle. In the normal viral lifecycle E6 and E7 are expressed at low levels and preferably in the differentiating cells. Probably the low expression of the viral genes helps the virus to avoid detection by the host immune system. The regulation of E6 and E7 expression is complex and occurs on many levels. Interestingly, in most cervical cancers and cervical cancer cell lines the HR-HPV double stranded DNA genome has been integrated into the host cell genome [9-12]. There seems to be little or no preference for integration in specific genes, which suggests that insertional mutagenesis generally is not involved in the generation of cervical cancer cells [13-15]. Conversely, investigations have shown that in a high percentage of cervical cancers and cell lines derived from cervical cancers the circular viral genome has been linearized in the sequence that encode the HPV E1 and E2 proteins before integration (figure 1) [9,14,16,17]. These findings demonstrate that integration in this exact region of the HPV genome confers a selective advantage to cells during cervical oncogenesis and strongly suggest that this region could be of major importance for regulation of the E6 and E7 oncogenes during the normal viral lifecycle. Many studies have addressed the nature of this selective advantage, and several different mechanisms appear to be involved. First, breakage and integration of the HPV genome in E1 or E2 will eliminate expression of the viral proteins E1, E2, E5, L1 and L2. The elimination of E2 expression is thought to be of particular importance for malignant progression. It has been shown that the HPV promoter region contains four E2 binding sites and binding of HPV E2 proteins to these sites repress viral transcription [18,19]. Therefore abrogation of the E2 expression probably leads to increased transcription of E6 and E7. Also, the HR-HPV E2 proteins can induce apoptosis independently of other HPV proteins [20,21]. Furthermore, it is possible that the loss of HPV protein expression facilitates evasion of the immune system, because many women have immunoreactivity towards E2 epitopes that are conserved between high and low risk HPV types [22]. Second, integration may have a direct effect on transcription from the HPV promoter. It is possible that regulatory elements present in the cellular sequence adjacent to the integrated viral sequence stimulate transcription from the viral promoter. Also several Matrix Attachment Regions (MARs) are present in the HPV-16 genome and it has been found that the HPV-16 MARs repress transcription from a reporter construct when in the episomal form. However, when an identical construct was stably integrated into the cellular genome the same regions were found to stimulate transcription [23]. Third, as a consequence of integration the 3' part of the HPV mRNA will be deleted and replaced with sequences derived from the host cell (see figure 1). Thus, in these cells E6 and E7 will be expressed from a chimeric HPV/cellular mRNA. It has been shown that such chimeric mRNAs are present at higher steady state levels than the normal HPV mRNAs [17]. Expression of the E6 and E7 oncogenes from the integrated HPV genome requires that a polyadenylation signal located in the host cell DNA is used for processing of the chimeric mRNA. Investigations have shown that the HPV-31 polyadenylation signal functions quite inefficiently [24] and usage of an efficient cellular polyadenylation signal may therefore enhance the expression of the E6 and E7 oncogenes. Another possibility is that the stability or the translation of the chimeric mRNA is enhanced compared to the normal HPV mRNA. It has been demonstrated that the A/U rich 4005–4213 region from HPV-16 decreased the half-life of a reporter messenger, when inserted downstream of β-globin reporter construct [17]. In another study the same region was shown to decrease the steady state level of the early HPV mRNA by 1.6 fold [25]. This indicates that the expression of E6 and E7 may be enhanced through the deletion of the U rich region.
Figure 1 HPV-16 integrations during oncogenesis. In normal HPV-16 infections the HPV-16 genome is present as an episome. The mRNAs that encode the HPV-16 E6 and E7 oncogenes are transcribed by the main promoter, spliced from the 880 splice donor and uses the early polyadenylation signal. During cervical oncogenesis the HPV-16 genome is integrated into the cellular genome. This integration occurs in the E1/E2 region and leads to generation of chimeric mRNAs that encode E6 and E7. Most often the chimeric mRNAs are of the A type that are spliced to a cellular splice acceptor [14].
All HPV-16 mRNAs that encode E6 and E7 contain the sequence from the splice acceptor located at nt. 3358 to the early polyadenylation signal located at nt. 4215 and this sequence may be involved in regulation of the HPV-16 E6 and E7 expression in the normal HPV life cycle. Furthermore, the large majority of chimeric mRNAs created by integration of the HPV-16 genome in cervical cancer cells are spliced from the 880 splice donor located shortly downstream of the E7 reading frame to a cellular splice acceptor (figure 1) [14]. Thus, the elimination of the HPV-16 sequence from nt. 3358–4214 may also be important for regulation of E6 and E7 during the normal HPV-16 lifecycle and could contribute to selection of cells containing integrated HPV-16 genomes during cervical oncogenesis. Here, we demonstrate that the HPV sequence downstream of the HPV-16 nt. 2582 and 3358 splicesites decrease mRNA steady state level and may also affect mRNA translation. Homologous sequences from other HPV types were also found to decrease gene expression.
Methods
Cloning of reporter construct
The HRSp-GUS retroviral reporter vector [26,27] was generously provided by professor Helen Blau, Stanford University School of Medicine, California. HPV sequences were PCR-amplified from plasmid templates (pBR322-HPV-2, pSV010-HPV-6, pBR322-HPV-11, pBR322-HPV-13, pBR322-HPV-16, pBR322-HPV-18, pBR322-HPV-30, pBR322-HPV-31 and pBR322-HPV-35 (short BamHI fragment)) with pfu polymerase (Promega) and primers from MWG Biotech, Ebersberg, Germany. The following regions were amplified : HPV16: 2582–4214, 3358–4214, 4063–4214, 4063–4310, HPV-18: 3378–4234, HPV-31: 3295–4137, HPV-6: 3325–4377, HPV-11: 3325–4370, HPV-35: 3301–4158, HPV-2: 3299–4387, HPV-13: 3332–4331, HPV-30: 3206–4216. Primers included restriction sites for cloning into Apa I / PinA I digested pHRSp-GUS or into Xho I / PinA I digested pHRSp-GUS (HPV-16 nt. 4063–4310). Plasmid DNA for transfection was prepared with Qiagen kits and all inserts were sequenced.
Cell culture and retroviral infections
The SiHa cell line was obtained from American Type Culture Collection (ATCC number HTB-35) and professor Norbert Fusenig, DKFZ, Heidelberg, kindly provided the HaCaT cell line. The PG13 amphotropic packaging cell line was a gift from PhD Lene Pedersen, Aarhus University, Denmark. 8,6 cm dishes with PG13 cells (grown in 10% neonatal calf serum) were transfected with pHRSp-GUS reporter constructs using the LipofectaminePLUS reagent (Invitrogen). 72 hours post transfection Puromycin (Sigma P7255) was added to a final concentration of 2,5 μg/ml for selection of stably transfected cells. The cells were transferred to T175 flasks that were allowed to grow to confluence. Viral supernatants were harvested by changing the media to 10 % Foetal calf serum (Invitrogen) and incubating the cells for 24 hours at 32°C, 5% CO2. The supernatants were filtered through a 0,45 μm syringe filter (Satorius) and Polybrene was added to a final concentration of 8 μg/ml. For each construct three 8.6 cm dishes of HaCaT or SiHa were each infected 4 hours with 4 ml of viral supernatant. 72 hours after transduction the infected cells were selected by adding 2,5 μg/ml Puromycin (Sigma P7255) to the cell media. The number of colonies was evaluated for each infection (pool), so that at least 1000 different colonies were present in each pool. For each construct three independent infections were performed.
Quantification of mRNA levels by Real time RT-PCR
1 × 106 HaCaT or SiHa cells from the different cell pools (three for each construct) were plated in 8.6 cm cell culture dishes and after 48 hours total RNA was isolated with the Qiagen RNeasy kit according to the instructions of the manufacturer. 10 μg RNA were treated with Amplification grade RNase free DNase I (Invitrogen). Real Time PCR reactions were run on a MJ Research DNA Engine Opticon™. Primers pairs specific for the GUS (5'-GTGATGATAATCGGCTGATG-3' and 5'-CCTGCGTCAATGTAATGTTC-3') and PURO (5'-AGGGCAAGGGTCTGGGCA-3' and 5'-TCGGCGGTGACGGTGAAG-3') coding sequences were designed. The GUS primer set was used with an annealing temperature of 58°C and the PURO primer set with an annealing temperature at 63°C. The QuantiTect SYBR Green RT-PCR Kit from Qiagen was used to assemble 25 μl RT-PCR/PCR reactions in triplicate. The following PCR profile was used: 30 min. at 50°C, 15 min. at 95°C, (15 s at 94°C, 30 s at 58 or 63°C, 1 min at 72°C) × 40 followed by recording of a melting curve. The threshold cycle Ct for individual reactions was determined with the Opticon software. All templates were checked for DNA contamination by omitting the reverse transcriptase in control reactions. The GUS mRNA level relative to the PURO mRNA level for each construct was calculated with the 2-ΔΔCt method and expressed relative to the empty vector [28]. The GUS mRNA levels were normalized to the PURO mRNA levels to ensure that differences between constructs in the efficiency of retroviral infection does not influence the results [27]. ΔCt values for individual pools were calculated and the ΔCt average for the empty vector was treated as an arbitrary constant and used to calculate ΔΔCt values for all pools. The mRNA levels for individual pools were calculated by evaluating the 2-ΔΔCt term. The results for the three independent pools for each construct were averaged and the standard error of the mean was calculated. For mRNA half-life experiments Actinomycin D (Sigma A9415) was added to the medium to a concentration of 2 μg/ml and total RNA was harvested from the cells at t = 0, 1, 3, 6, 9, 15 hrs. Aliquots containing equal amounts of total RNA were used as templates for Real Time PCR analysis with the GUS specific primerset. The GUS mRNA levels for each timepoint were calculated from the Ct values, averaged and expressed relative to the mRNA level at the time of Actinomycin D addition. For examination of nuclear/cytoplasmic mRNA ratios SiHa cells were trypsinised and 2 × 106 cells were used for preparation of cytoplasmic RNA with the RNeasy kit from Qiagen. The nuclear pellets were washed three times in RLN buffer (Qiagen) and used for isolation of nuclear RNA with the Qiagen RNeasy procedure for total RNA. The mRNA levels were determined with Real time PCR. The GAPDH mRNA was detected with the following primer set (5'-GAAGGTGAAGGTCGGAGTC-3') and (5'-GAAGATGGTGATGGGATTTC-3').
Northern blot analysis
Total RNA (app. 7,5 μg) from the SiHa cell pools stably transfected with the different constructs was separated on a MOPS 1% agarose gel, transferred to a BrightStar Membrane (Ambion) and crosslinked with the autocrosslink setting in a Stratalinker (Stratagene) according to the recommendations in the NorthernMax kit from Ambion. Nt. 6–131 and nt. 538–1037 of the GUS reporter mRNA were cloned into pBluescript II SK (+) and used for preparation of radioactively labelled probes by in vitro transcription with Radioactive labelled UTP [α-32P, 800 Ci/mmol] and the T7 Maxiscript kit (Ambion) according to the recommendations of supplier. The crosslinked membrane was hybridised over night with 1 × 106 cpm probe per ml of ULTRAhyp solution at 72°C and washed the following day according to the instructions in the NorthernMax protocol. Finally, the membrane was exposed to Biomax MR film (Kodax) for 72 hours at 4°C.
Quantification of total protein and GUS protein
1 × 106 HaCaT or SiHa cells from the different cell pools (three for each construct) were plated in 8.6 cm cell culture dishes. After 48 hours cells were scraped in lysis buffer: 100 mM potassiumphosphate (pH 7.8) and 0.2 % Triton X-100 with DTT added to 0,5 mM immediately before use. Membrane debris was pelleted by centrifugation (15000 g, 4°C) for 3 min. and the supernatant was collected and stored at -80°C. The Coomassie®Plus Reagent (PIERCE) was used to determine the total protein concentration of the extracts. The level of GUS protein in the extracts was determined with the FluorAceTM β-glucuronidase Reporter Assay kit from BIORAD. All cell extracts were diluted 1:10, 1:100, 1:1000 or 1:10000 to ensure that the measurements were within the linear range of the assay. Fluorescence was counted on a Wallac VICTOR2 Multilabel Counter. The GUS protein levels were normalized with the total protein levels and expressed relative to the HRSp-GUS vector. For each construct the three independent pools were averaged and the standard error of the mean was calculated. Translational was evaluated for the different constructs by normalizing the GUS protein levels with the total levels of the GUS mRNA (not normalized to PURO) as determined by Real Time RT-PCR.
Results
We have investigated the effect of posttranscriptional regulation, including translation, on the expression of the HPV-16 early genes with the HRSp-GUS vector that has been designed for investigation of the regulatory properties of UTR sequences [26,27] (see figure 2a). The main advantage of this vector is that the reporter mRNAs are expressed at low levels from one or a few retroviral integrations. Expression from integrated reporter constructs is a good model for the integrated oncogenic HPV DNA that is found in advanced cervical lesions and furthermore the low expression ensures that transacting factors are not titrated out. In the reporter vector we have inserted different HPV-16 sequences 3' to the GUS coding sequence to determine how they influence the steady state level of the reporter mRNA and reporter protein (figure 2a). All steady state levels were determined as the average of data collected from three independent cell pools each containing several thousand clones of stably transfected SiHa and HaCaT cells. Total RNA from different clones was used for Northern Blot analysis with two probes specific for the GUS sequence. One of these probes anneals to the first 120 bases of the reporter mRNA to ensure that all spliced reporter as well as the full-length mRNAs would be detected. The probes only detected the expected full-length mRNAs and no shorter spliced forms. For the nt. 2582–4214 reporter construct no mRNA was detectable (figure 2b). We find that the inclusion of the A/U rich HPV-16 sequence from nt. 4063 to 4214 in the reporter construct has little inhibitory effect on steady state level of the reporter mRNA (figure 2c). In the SiHa cell line the reporter mRNA is expressed to app. 90 % of the level of the empty vector, whereas the HPV sequence seems to have a stabilizing effect in the HaCaT cell line, although we have large differences between our pools for this reporter construct. The region from the HPV-16 nt. 3358 splice acceptor to the early polyadenylation signal is present in all HPV-16 mRNAs that encode E6 and E7. We find that HPV-16 nt. 3358–4214 region shows a significant repression of the steady state level mRNA both in the SiHa and HaCaT cell line when inserted into the HRSp-GUS vector (figure 2b). The HPV-16 genome also contains splice acceptors at nucleotides 2582 and 2709 and we therefore investigated the steady state level of a construct with the HPV-16 nt. 2582–4214 sequence inserted. In the SiHa cell line this construct further decreased the steady state level of the reporter mRNA and in the HaCaT cell line only extremely low levels of GUS mRNA were detected (figure 2b and 2c).
Figure 2 Effects of HPV-16 sequences on the steady state level of GUS mRNA and GUS protein. a) Schematic representation of the HRSp-GUS retroviral reporter vector [26,27] and the location of the GUS northern probes and the Real Time PCR amplicon. b) Northern Blot analysis of mRNA from SiHa cell pools (only GUS probe 1 experiment is shown). Blotting of mRNA from the HaCaT cell pools is not shown, but yielded similar results. c) GUS mRNA steady state levels. For each construct the steady state levels of the GUS mRNA and PURO mRNA were determined with real-time RT-PCR. The GUS mRNA levels were normalized with the PURO mRNA. The data plotted is the average of results from three independent pools of infected SiHa or HaCaT cells expressed relative to the empty HRSp-GUS construct. The error bars indicate the standard error of the mean. d) Reporter protein produced per reporter mRNA. For each construct the steady state GUS activity and total protein levels were determined. The GUS activities were normalized with the total protein levels. Protein/RNA ratios of the different reporter mRNA was evaluated by calculation of the ratio between the steady state levels of GUS protein to GUS mRNAs (not normalized for PURO) The data plotted is the average of results from three independent pools of infected SiHa or HaCaT cells expressed relative to the empty HRSp-GUS construct. The error bars indicate the standard error of the mean. e) Cellular distribution of Reporter mRNAs. The ratio of cytoplasmic to nuclear RNA for the GUS mRNA (left), PURO mRNA (middle) and GAPDH mRNA (right) in the different reporter cell lines.
It has previously been demonstrated that the HPV-31 early polyadenylation signal functions inefficiently [24] and we therefore made a construct with the HPV-16 early polyadenylation region included instead of the bovine Growth Hormone (bGH) polyA region present in the HRSp-GUS vector (figure 2a). In our experiments usage of the HPV-16 early polyadenylation signal (HPV-16 nt. 4063–4310) does not significantly reduce the steady state levels of the reporter mRNA compared to the similar construct containing the bGH polyadenylation region (HPV-16 nt. 4063–4214) (figure 2b). This indicates that in SiHa and HaCaT keratinocytes the HPV-16 early polyadenylation signal functions reasonably efficiently.
Although it is quite possible that the expression of the HPV-16 E6 and E7 proteins is regulated at the protein level by the sequences downstream of the reading frames, this has to our knowledge never been investigated in any detail. We determined the steady state levels of the GUS reporter protein normalized to total protein levels for the different constructs. The normalized protein levels reflect the full regulatory influence of the inserted sequences, including effects on mRNA processing, mRNA transport, mRNA stability and translation. To assess the influence that the inserted HPV sequences have on the amount of protein produced per mRNA we calculated the ratio between the steady state levels of GUS protein (normalized to total protein) and the steady state levels of the GUS mRNA (normalized to total RNA)(figure 2d). This normalization reveals that in neither cell line the HPV-16 nt. 4063–4214 region changes the protein/mRNA ratio, whereas the HPV-16 nt. 2582–4214 region greatly reduces the protein/mRNA ratio. The HPV-16 nt. 3358–4214 region significantly reduces the protein/mRNA ratio in the SiHa cell line, but not in HaCaT cells. Inclusion of the HPV-16 polyadenylation region instead of the bGH polyadenylation region in the HPV-16 4063–4310 construct should give mRNAs with native HPV-16 3' ends. Compared with the similar mRNA that contains the bGH polyadenylation region (HPV-16 nt. 4063–4214), this mRNA produces less protein in the HaCaT cell line, whereas no significant difference is present in SiHa cells (figure 2d). Only the cytoplasmic pool of mRNA is available for translation and it is therefore possible that the reduction in protein/mRNA ratio that we observe is caused by an altered distribution of the reporter mRNAs. We therefore isolated cytoplasmic and nuclear mRNA fractions and determined the mRNA concentrations by real time PCR. None of the inserted HPV-16 sequences affect the distribution of the GUS reporter mRNA or the PURO and GAPDH controls (figure 2e).
The effects that we observe on mRNA steady state levels for the nt. 3358–4214 and 2583–4214 constructs could be the result of decreased stability of the reporter mRNAs or decreased transcription. To differentiate between these two possible mechanisms, we determined the half-lives of the two reporter mRNAs (figure 3b). The nt. 3358–4214 and 2583–4214 reporter mRNAs did not have significantly lower half-life than the nt. 4063–4214 and control mRNAs, which indicates the reduced steady state level is caused by inhibition of transcription.
Figure 3 Decay of reporter mRNAs. SiHa cells expressing the indicated constructs were treated with Actinomycin D to inhibit transcription. mRNA levels of the different GUS reporter mRNAs normalized to total RNA was determined with real-time RT-PCR at the indicated time points. At least three different measurements were made for each timepoint and the data plotted is the average relative to the mRNA level at the time of Actinomycin D addition. The error bars indicate the standard deviation.
The genomic organisation of the different HPV types is highly conserved. They all have two polyadenylation signals that divide the genome into an early and a late region. Moreover all HPV types express E6 an E7 from mRNAs that are spliced to a splice acceptor in the E2 reading frame analogous to the HPV-16 nt. 3358 splice acceptor. We therefore decided to determine if the negative effect on translation that we observe with the HPV16 nt. 3358–4214 region is a general regulatory feature of different HPV types. The region from the E2 splice acceptor to the early polyadenylation signal from HPV types: 2, 6, 11, 13, 16, 18, 30, 31 and 35 was therefore cloned into the 3' UTR of the pHRSp-GUS vector and the steady state level of reporter mRNA and protein was determined after transduction of SiHa cells (figure 4a). To ensure that the reporter constructs expressed the correct reporter mRNA we performed a Northern Blot analysis with both the probes shown in figure 2a. All pools expressed GUS mRNAs of the expected length (figure 4b), except for the HPV-18 construct that expressed an additional, shorter GUS mRNA of unknown structure. We find that all the tested HPV sequences, except the ones derived from 18 and 31, decrease the steady state level of the reporter mRNA compared to the empty vector (figure 4c). The largest repression was observed with the HPV-2 construct. For all types except HPV-35 and HPV-18 the repressive effect of the inserted HPV sequence also decreased the translation (figure 4d). These results therefore indicate that the posttranscriptional repression of the region from the E2 splice site to the early polyadenylation signal is a general feature of different HPV types.
Figure 4 Effects of HPV 3' early regions on the steady state level of GUS mRNA and GUS protein. a) Schematic representation of the HPV sequences that were cloned into the retroviral HRSp-GUS reporter vector [26,27]. b) Northern Blot analysis of mRNA from SiHa cell pools (only GUS probe 1 experiments shown). c) GUS mRNA steady state levels. For each construct the steady state levels of the GUS mRNA and PURO mRNA were determined with real-time RT-PCR. The GUS mRNA levels were normalized with the PURO mRNA. The data plotted is the average of results from three independent pools of infected SiHa cells expressed relative to the empty HRSp-GUS construct. The error bars indicate the standard error of the mean. d) Protein produced per reporter mRNA For each construct the steady state GUS activity and total protein levels were determined. The GUS activities were normalized with the total protein levels. The protein/RNA ratios of the different reporter mRNA was evaluated by calculation of the ratio between the steady state levels of GUS protein to GUS mRNAs (not normalized for PURO). The data plotted is the average of results from three independent pools of infected SiHa cells expressed relative to the empty HRSp-GUS construct. The error bars indicate the standard error of the mean.
Discussion
In this study we have investigated if the HPV-16 early genes are regulated at the posttranscriptional level. We find that the HPV-16 nt. 2582–4214 region very efficiently reduce the steady state level of a reporter mRNA and the amount of protein that is produced from the mRNA when compared to the empty reporter vector HRSp-GUS vector (see figure 2). Moreover, a truncated construct containing the HPV-16 nt. 3358–4214 region also decreased the steady state level of reporter mRNA and in one of the two cell lines tested reduced the protein/mRNA ratio. In contrast the HPV-16 nt. 4063–4214 region encompassing the short U rich HPV-16 early 3' UTR only, did not affect expression. The reporter assay that we have used in our experiments is based on observation of steady state levels of the different reporter mRNAs and of reporter protein. It is possible that splice acceptors present in the inserted sequences could stimulate splicing from cryptic splice donors in the reporter sequence and this would make our results very difficult to evaluate. For that reason we used Northern Blot analysis to confirm that all the reporter mRNAs have the expected unspliced structure. All constructs, except the HPV-18 construct, produced only the expected full length unspliced reporter mRNA (figure 2b and 4b). The 2582–4214 construct did not express sufficient amounts of reporter mRNA (full length or spliced) to be detected by Northern Blot. Thus, the lowered steady state levels of reporter mRNA that we observe could reflect a decrease in reporter mRNA half-life, a decreased rate of transcription or inhibition of export of mRNA. Surprisingly, we found that none of our reporter mRNAs containing HPV-16 sequence had a decreased half-life (see figure 3). This indicates that the inserted HPV-16 sequence influence the transcription from the minimal CMV promoter included in the pHRSp-GUS vector or affect the nuclear export of the mRNAs. It is known that HPV-16 E5 region contain a strong matrix attachment region [23,29] and it is possible that this region or other unidentified enhancer elements influence the CMV promoter. Moreover, it is well established that the late mRNAs from several different papillomavirus types contain sequences that interfere with mRNA export [30-33]. Often these sequences bind factors involved in splicing such as U2AF65 [32] and the U1 snRNA [30]. The HPV-16 nt. 3358–4214 and 2582–4214 regions both contain a splice donor located at HPV-16 nt. 3632 and the HPV-16 nt. 2582–4214 region also contain the splice acceptor at HPV-16 nt. 3358. Both these splice sequences diverge from the consensus sequences, but are used during the viral lifecycle and could therefore potentially contribute to the repression that we observe by inhibiting mRNA export and mediation of rapid degradation within the nucleus. Still, we investigated the distribution of the different reporter mRNA (figure 2e) and found that the HPV-16 sequences does not change the distribution of the reporter mRNAs. This suggests that the decrease in steady state mRNA levels is caused by inhibition of the minimal CMV promoter. Alternatively ineffective mRNA processing of the reporter constructs may lead to very rapid degradation (cotranscriptional) of the reporter mRNA that cannot be detected in our RNA half-life experiments.
Jeon and Lambert have previously reported that the very A/U rich 4005–4213 region from HPV-16 very efficiently decreases the stability of a reporter mRNA [17]. The 3' UTRs from short-lived mRNAs often contain destabilizing A/U-rich elements (ARE) [34], but the HPV-16 A/U rich sequence does not contain any of the canonical ARE sequences (AUUUA). AREs and a few other RNA sequences have been found to be highly overrepresented in mRNAs with high turnover rate, but they are not strong predictors of the mRNA turnover rate [35], This emphasizes that the ability of a RNA sequences to destabilize mRNAs is highly context dependent and requires cooperative binding of several factors. In our experiments the HPV-16 sequence from nt. 4063–4214 does not influence the steady state level or decrease the half-life of the reporter mRNA (figure 2a and 3). Schwartz and coworkers did not find any repressive effect of the HPV-16 nt. 4005–4213 region on protein expression in their reporter system [36] and weak inhibitory effect (1.6 fold inhibition) on the mRNA level [25]. It is possible that the upstream nt. 4005–4063 sequence is necessary for the repression observed by Jeon and Lambert and Schwartz and coworkers. Another possibility is that the reporter systems and cell lines used in the different studies influence the regulatory properties of this HPV-16 region.
It has been reported that the early polyadenylation region from HPV-31 is app. 20 fold less efficient than the SV-40 late polyadenylation region and that this difference could be reduced to a 5 fold difference by introduction of an efficient binding site for the cleavage stimulatory factor CstF-64 to the HPV-31 polyadenylation region [24]. Our experiments indicate that the efficiency of the HPV-16 early polyadenylation region from nt. 4215 to 4310 is comparable with the efficiency of the bGH polyadenylation region present in the HRSp-GUS vector (compare 4063–4214 with 4063–4310, figure 2c). Clearly, these findings may reflect a difference in the efficiency of the HPV-16 and HPV-31 early polyadenylation regions or the efficiency of the bGH and SV40 polyadenylation regions. Interestingly, Schwartz and coworkers have recently shown that the U-rich sequence present in the HPV-16 3' UTR enhances polyadenylation from the early HPV-16 polyadenylation signal [25]. The HPV-31 early 3' UTR is less U-rich than the HPV-16 early 3' UTR and may therefore not enhance polyadenylation in the same way. Still, different cells and reporter systems were used in the different studies.
In addition to decreased reporter mRNA steady state levels we also observe that the mRNAs containing the HPV-16 nt. 2582–4214 and nt. 3358–4214 sequences produce less protein per mRNA than the control mRNA. Only cytoplasmic mRNA is available for translation and since we have measured total mRNA concentration, inhibition of mRNA export and accumulation of reporter mRNAs in the nucleus would result in a decreased protein/mRNA ratio in our experiments. We find that the ratio of nuclear to cytoplasmic mRNA is unaltered by the HPV-16 sequences (figure 2e), which indicates that the inserted HPV sequences may influence translation.
A productive HR-HPV infection relies on carefully regulated expression of the HPV gene products. The expression is coupled to the differentiation of the infected cells, so that the viral proteins are expressed at low levels in the basal and suprabasal cell layers, but are expressed at higher levels in the more superficial cells that have exited the cell cycle [37,38]. This strategy most likely helps to keep the HPV infection hidden for the immune system. The repression that we observe by the HPV-16 nt. 3358–4214 and nt. 2582–4214 regions shows that HPV-16 exploits posttranscriptional mechanisms probably to keep the expression of the E6 and E7 proteins at a suitably low level. This is analogous to the repression of the late structural proteins of HPV-16, which also partly occur on the level of mRNA processing, export, stability and translation [30-32,39,40].
We next wondered if posttranscriptional repression could be a general feature of early mRNAs from different HR-HPV types and possibly all HPV types. The HPV types tested include the high risk types HPV-16, HPV-18, HPV-31, and HPV-35 and also the low risk types HPV-6, HPV-11 and HPV-13, which frequently infect the genital tract and cause condylomata acuminata, but are practically absent in high grade lesions and in cervical cancers. In addition, we have tested HPV-2, a benign HPV type with a preference for cutaneous tissue and HPV-30, which is a rather rare HPV type occasionally present in genital infections. We found that the region from the E2 splice acceptor to the early polyadenylation signal from all the tested types except HPV-18 repress expression of the reporter by decreasing mRNA steady state levels or reducing the amount of protein produced or both (figure 4). Like the HPV-16 sequence these HPV sequences therefore contain elements that interfere with transcription and/or processing of the mRNAs to reduce the steady state level of the reporter mRNAs. Moreover, most of them also reduce translation and it is therefore very likely that repression by this region of the HPV genomes in a general feature of the HPV life cycle. The HPV-18 sequence seems to increase expression of the reporter through increased mRNA steady state level and enhanced protein production, but since we find that the HPV-18 construct in addition to the expected full length reporter mRNA also express a shorter mRNA this may be the reason for the increased expression. The finding that the 3' end of most HPV E6 and E7 mRNAs repress expression by posttranscriptional mechanisms is interesting, since it suggests that posttranscriptional repression of the early HPV genes may be a general feature of many HPV types and possibly has evolved to facilitate evasion of the host immune system. Still all the HPV types tested are part of the A supergroup and more distantly related types could be regulated very differently.
Conclusion
In conclusion, we demonstrate that the HPV-16 nt. 2582–4063 region represses expression at the mRNA and possibly also the protein level. This regulation could be involved in the regulation of the HPV-16 E6 and E7 oncogenes during the normal HPV lifecycle. Also for the HPV types 2, 6, 11, 13, 16, 30, 31, 32 and 35 the region from the E2 splice acceptor to the early polyadenylation signal represses expression of the reporter construct. This indicates that regions downstream of these splicesites function in the HPV lifecycle to repress the expression of the early HPV proteins including the E6 and E7 oncoproteins. For the HR-HPV types deletion of this region through genomic integration occurring in the E1 or E2 open reading frames could contribute to enhanced expression of E6 and E7 and increase the oncogenic potential of the infected cells during cervical oncogenesis.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
JV designed the study, carried out cloning, northern blotting and drafted the manuscript. MWR carried out the retroviral infections, Real Time RT-PCR and GUS assays, mRNA half life investigations, fractionations of the cytoplasmic and nuclear mRNAs and helped to draft the manuscript. KK participated in the mRNA half live investigations. BN participated in the design and coordination of the study and helped to draft 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 Professor Helen Blau, Stanford University School of Medicine, California, for the generous gift of the pHRSp-GUS vector. Also, we are grateful for plasmids containing the different HPV genomes that we obtained from professors Harald zur Hausen, DKFZ, Heidelberg, (HPV-2, HPV-11, HPV-16, HPV-18), Wayne Lancaster, Wayne State University School of Medicine, Detroit, (HPV-31), Gérard Orth, Institut Pasteur, Paris, (HPV-13, HPV-33), Attila Lorincz, Digene Corporation, (HPV-35), Tom Broker, University of Alabama, Birmingham (HPV-6). The HaCaT cell line was very kindly provided by professor Norbert Fusenig, DKFZ, Heidelberg. Furthermore we thank Solvej Jensen for excellent technical assistance.
Our Real Time PCR machine is funded by: The Carlsberg Foundation, Dir. Ib Henriksens Foundation, Fonden Til Lægevidenskabens Fremme, Frænkels Mindefond, Fonden af 17.12.1981 and Sven Hansen og hustru Ina Hansens Fond. In addition this work was supported by Fabrikant Einar Willumsens Fond, The Novo Nordisk Foundation, Oda og Hans Svenningsens Foundation and Gerda og Aage Haensch's Foundation. KK was supported by a scholarship from The Danish Cancer Society and JV by The Danish Medical Research Council.
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BMC Med EducBMC Medical Education1472-6920BioMed Central London 1472-6920-5-351622566610.1186/1472-6920-5-35Research ArticleAmbulatory teaching: Do approaches to learning predict the site and preceptor characteristics valued by clerks and residents in the ambulatory setting? Delva M Dianne [email protected] Karen W [email protected] John R [email protected] Marshall [email protected] Department of Family Medicine, Queen's University, Kingston, Ontario, Canada2 Faculty of Education, Queen's University, Kingston, Ontario, Canada2005 14 10 2005 5 35 35 1 6 2005 14 10 2005 Copyright © 2005 Delva et al; licensee BioMed Central Ltd.2005Delva 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 a study to determine the site and preceptor characteristics most valued by clerks and residents in the ambulatory setting we wished to confirm whether these would support effective learning. The deep approach to learning is thought to be more effective for learning than surface approaches. In this study we determined how the approaches to learning of clerks and residents predicted the valued site and preceptor characteristics in the ambulatory setting.
Methods
Postal survey of all medical residents and clerks in training in Ontario determining the site and preceptor characteristics most valued in the ambulatory setting. Participants also completed the Workplace Learning questionnaire that includes 3 approaches to learning scales and 3 workplace climate scales. Multiple regression analysis was used to predict the preferred site and preceptor characteristics as the dependent variables by the average scores of the approaches to learning and perception of workplace climate scales as the independent variables.
Results
There were 1642 respondents, yielding a 47.3% response rate. Factor analysis revealed 7 preceptor characteristics and 6 site characteristics valued in the ambulatory setting. The Deep approach to learning scale predicted all of the learners' preferred preceptor characteristics (β = 0.076 to β = 0.234, p < .001). Valuing preceptor Direction was more strongly associated with the Surface Rational approach (β = .252, p < .001) and with the Surface Disorganized approach to learning (β = .154, p < 001) than with the Deep approach.
The Deep approach to learning scale predicted valued site characteristics of Office Management, Patient Logistics, Objectives and Preceptor Interaction (p < .001). The Surface Rational approach to learning predicted valuing Learning Resources and Clinic Set-up (β = .09, p = .001; β = .197, p < .001). The Surface Disorganized approach to learning weakly negatively predicted Patient Logistics (β = -.082, p = .003) and positively the Learning Resources (β = .088, p = .003).
Climate factors were not strongly predictive for any studied characteristics. Role Modeling and Patient Logistics were predicted by Supportive Receptive climate (β = .135, p < .001, β = .118, p < .001).
Conclusion
Most site and preceptor characteristics valued by clerks and residents were predicted by their Deep approach to learning scores. Some characteristics reflecting the need for good organization and clear direction are predicted by learners' scores on less effective approaches to learning.
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Background
Medical care is increasingly delivered in the ambulatory care setting. Since learning in context improves learning it is appropriate and necessary that medical training occurs in the future practice setting [1,2]. Teachers must organize the setting and approach teaching in this context in order to maximize learning.
We developed a survey instrument to assess the site and preceptor characteristics that clerks and residents believe to be most valuable to their learning in the ambulatory setting using validated questionnaires and study group consensus [3]. Many items such as giving feedback and discussing clinical reasoning were valued by most learners, although there were some differences for learners from different specialties or at different training levels. A few site and preceptor characteristics, such as teaching in the patient's presence, thought to be important for learning were not valued. Learner preferences may be appropriate guides for teaching, but it is not clear that these preferences always support effective learning. Studies in higher education suggest that the approach to learning affects learning outcomes. The conceptual models of approaches to learning include both motivation for learning and the strategies to fulfill the motivation and are influenced by the context for learning. It is widely accepted that the deep approach to learning which includes an integrative approach to understanding, leads to improved learning outcomes while surface (or reproducing) approaches which depend on rote memorization, are less effective [4]. Learners adopting surface approaches to learning may prefer site and preceptor characteristics that support their surface learning; instructors who follow those preferences may inadvertently undermine their own teaching!
Bowen and Irby, reviewed the quality and costs of education in the ambulatory setting and described what is known and the gaps in our knowledge [5]. Many studies have been conducted in single institutions and in the traditional ambulatory specialties. Bowen and Irby suggest that a conceptual model examining the teacher-learner-setting framework is needed to understand the effectiveness of the ambulatory setting for learning.
Challenges in examining the effectiveness of a learning environment include the reliability of the evaluation method, confounding factors and the ability to generalize beyond single groups or institutions. One way to examine the complex interaction of the environment and the approach taken by learners in that environment is by measuring the approaches to learning in the workplace [6].
The approach to learning, course perceptions and personal factors are known to interact to affect undergraduate learning [7-11]. Course demands influence university students to adopt surface, deep, or achieving approaches to learning. A deep approach to learning is motivated by an intrinsic desire for learning and involves strategies to form an integrated and personal understanding. In contrast, the surface approach to learning is motivated by fear of failure and is associated with rote memorization and a perception of heavy workload demands in the course. In studies of practicing physicians and clinical trainees (clerks and residents) we confirmed that perception of the workplace climate is associated with the approach to learning [12,13]. Perceptions of heavy workload are associated with surface disorganized approaches to learning (figure 1). A deep approach to learning is associated with perceptions of choice and independence and a supportive, receptive workplace climate (figure 1).
Figure 1 Definitions of Workplace Learning Factors (sample items).
Our assumption in this study was that a deep approach to learning could be viewed as a surrogate for more effective learning and that a surface disorganized approach was less effective in most circumstances. Students who adopt a deep approach to learning may value characteristics of the learning environment and teaching that support this approach which differ from students who adopt less effective approaches to learning. It would seem appropriate then for teachers to attend to those site and preceptor characteristics that support a deep approach to learning. The purpose of this study was to answer the question: Do approaches to learning and perception of the workplace climate predict the site and preceptor characteristics valued by clerkship students and residents in the ambulatory setting?
Methods
All medical clerks (n = 532) and residents (n = 2939) at the five medical schools in Ontario were invited to respond to a survey regarding the site and preceptor characteristics most valued in the ambulatory setting and to complete the Workplace Learning Questionnaire. The study was approved by the Queen's University Research Ethics Board.
Students rated 24 site characteristics and 38 preceptor behaviours on a Likert scale from 1 (very important for learning) to 5 (not at all important for learning) or D (detrimental for learning). (sample items in Table 1) The Workplace Learning Questionnaire developed by Kirby and colleagues was modified to include reference to preceptors as well as supervisors [14] The questionnaire includes 30 items pertaining to approach to learning and 15 items pertaining to perception of workplace climate (Figure 1). Responses are made on a 5 point Likert scale, ranging from 1(agree strongly) to 5 (disagree strongly).
Table 1 Definitions of site and preceptor factors (sample items rated from most important to least important)
Precptor Factor Definition
Professional Role Modeling Models professional behviours with staff and patients (Demonstrates effective interactions with support staff)
Teaching Quality and efforts to provide good teaching (Discusses clinical topics in an organized way)
Learning Climate Open and caring towards students and patients (Makes student feel like a valued member of the practice)
Feedback The provision of timely and constructive feedback. (Gives constructive feedback)
Direction Provides specific instruction on the student's role and is focused (Outlines specific task(s) to be done during a clinical encounter)
Patient Presence Teaching with the patient present (Reviews case in the patient's presence)
Health Care System Interaction Preceptor teaches about resource use. (Teaches use of community resources)
Site Characteristic Factor
Office Management Teaching skills related to the running of a practice (Teaching of time management skills)
Patient Logistics Opportunity to see a number and variety of patients (Opportunity to see an adequate number of patients)
Objectives Defines and meets objectives (Clearly defined site objectives for the rotation)
Learning Resources Availability of resources in the clinic (Library resources available in the clinic)
Clinic Set-up Proximity and educational orientation of the clinic (Close proximity of clinic to campus)
Preceptor Interaction Effective teachers who are available and willing to demonstrate (Preceptors readily available)
Questionnaires were mailed in bulk to the undergraduate and postgraduate medical schools and forwarded to all clerks and residents with addressed and stamped return envelopes. Responses were anonymous and a separate card was returned for entry into a draw for a personal digital assistant or equivalent monetary prize. An email reminder was sent four weeks later and follow up mailings were sent 8 and 20 weeks after the initial mailing.
Multiple regression analysis was used to predict the preferred site and preceptor characteristics as the dependent variables by the average scores of the approaches to learning and perception of workplace climate scales as the independent variables. Hierarchical modeling did not alter the results.
Results
Of the population surveyed, 1642 responded (47.3%). Participants with 10% or more missing data (those who did not respond to items (54) and those who responded "not applicable" (27)) were excluded, leaving 1561 respondents for analysis. Missing values for the remaining participants were replaced with item means. The distribution of respondents was compared to the distribution of clerks and residents in Ontario. The proportions of women, junior residents, family medicine residents and McMaster trainees responding were higher and Toronto trainees lower than the population surveyed [3,13].
Factor analysis revealed 7 preceptor factors and 6 site factors valued in the ambulatory setting [3]. These are defined in Table 1 with sample scale items. The factor structure and reliability of the Workplace Learning questionnaire were confirmed for the population [14]. Scales are presented in italics in the following discussion to emphasize that the associations are between scale scores.
Higher scores on the Deep approach to learning scale were associated with placing higher value on all of the preceptor and site characteristics valued by clerks and residents (table 2). Preceptor Direction was more strongly predicted by Surface Rational and Surface Disorganized scales than by the Deep scale (table 2).
Table 2 Prediction of valued preceptor characteristics by approaches to learning and perception of workplace climate. (β coefficients)
Professional Role Modelling Teaching Learning Climate Feedback Direction Patient Presence Health Care System Interaction
Deep .234** .224** .207** .155** .076* .091** .180**
Surface Rational .077* .179** .101** .111** .252** .118** .104**
Surface Disorganized .064 .013 .035 .015 .154** .072 .048
Choice/Independence -.046 -.056 .016 -.095* .023 .066 .016
Workload .094** .043 .088* .010 .070* .012 .043
Supportive Receptive .135** .054 .065 .025 -.008 -.042 .058
Adjusted R2 .094 .092 .079 .036 .132 .036 .061
*p < .01, **p < .001
The Deep scale predicted valued site characteristics of Office Management, Patient Logistics, Objectives and Preceptor Interaction (table 3). The Surface Rational scale predicted valuing Learning Resources and Clinic Set-up. The Surface Disorganized scale weakly negatively predicted Patient Logistics and positively Learning Resources.
Table 3 Prediction of valued site characteristics by approaches to learning and perception of workplace climate. (β coefficients)
Office Management Patient logistics Objectives Learning Resources Clinic Setup Preceptor Interaction
Deep .113** .119** .161** .068 .078* .086**
Surface Rational .128** .080* .190** .090** .197** .087**
Surface Disorganized .031 -.082* -.004 .088* .070 .010
Choice/Independence .047 -.017 .003 .020 .076 -.064
Workload .071 .056 .074* .051 .081** .068
Supportive Receptive .065 .118** -.010 .064 .023 .050
Adjusted R2 .054 .044 .076 .033 .083 .021
*p < .01, **p < .001
Climate factors were not strongly predictive (tables 2 and 3). Role Modeling and Patient Logistics were predicted by Supportive Receptive climate. Perception of heavy workload (Workload) predicted valuing Direction, Clinic Set-up and Objectives. Perception of Choice/Independence in the workplace weakly and negatively predicted valuing Feedback.
Discussion
In this analysis of a multi-site, multi-level and multi-specialty study we found that higher scores on the Deep approach to learning scale predicted all of the learners' preferred preceptor characteristics, supporting the implementation of these in the ambulatory setting. The Deep approach to learning was in general more strongly associated with the valued preceptor factors than the valued site characteristics except for Direction.
Giving direction was more valued by learners with higher scores on the Surface Rational and Surface Disorganized scales. One interpretation may be the need for these learners to know what is expected of them. Although struggling learners may require this support, too close direction may interfere with the development of independence and life-long learning skills. We have found that physicians who take a predominantly deep approach to learning are self-motivated for learning and prefer independent methods for continuing medical education [12]. In contrast, the physicians who score highly on surface approaches to learning are motivated by extrinsic factors such as regulating authorities or fear of a lawsuit [12]. As Samuel Johnson wrote, "when a man knows he is to be hanged in a fortnight, it concentrates his mind wonderfully" but it is likely a costly and ineffective approach for independent life-long learning. Relying too heavily on "what is on the exam" or what the preceptor wants does not allow for self-assessment and may lead to less effective outcomes.
Learners' approaches to learning are not one or another, but more or less of the approaches measured and will vary with the context. In this study we did not find a significant influence in the models of the perception of the workplace climate on valued site or preceptor characteristics. We did confirm earlier finding that the approach to learning and perception of the workplace climate are associated [6,12,13]. Thus, it may be that when the learning climate is stressful, learners may shift from a preferred approach to learning, to one that helps them survive. The surface rational approach may be necessary in a busy clinic when there is little time for reflection. In this setting, the organization or clinic characteristics may become more salient to the learner.
McManus has shown that approaches to learning are predicted by personality and learning styles [15]. Personality traits tend to be stable but learning styles and approaches to learning can be modified by formal education [16-18]. It may be important to encourage the struggling or dependent learner to become more self-directed. As workload is associated with surface approaches to learning, the challenge in the ambulatory setting will be to pace the patient volume and variety of problems to provide adequate time for learning with the aim of supporting deeper approaches to learning.
Dolmans and colleagues used path analysis to determine the importance of some variables influencing the effectiveness of student rotations at out-patient clinics in one medical school [19]. Input variables, such as organizational quality, number of students contemporaneously involved and available space, and process variables, such as patient mix and supervision, were analyzed in relation to one output variable: the students' evaluation of the effectiveness of the rotation. Supervision was the most important influence on the student's perception of the effectiveness of the rotation. This finding is consistent with the stronger prediction of valued preceptor characteristics than site characteristics in our study.
Dolman's model focused on organizational variables and did not take into account differences in the students and how they learn. Biggs' model suggests that the student's approaches to learning are input variables that interact with the organizational variables to affect what students do to learn [8]. The trend for site characteristics to be more strongly predicted by the Surface Rational approach to learning reflects the desire for good organization by some learners. Our findings extend Dolman's model to help teachers understand the different values placed on site and preceptor characteristics in the ambulatory setting by learners employing different approaches to learning.
This study has several limitations. First, the predictive power of approach to learning on valued site characteristics is small, suggesting that other factors we have not measured, such as personality, may be important in determining characteristics valued by the learners [17]. Ability, expectations and prior knowledge may also be important. Secondly, approaches to learning are influenced by many factors and do not represent an outcome of the learning experience. Thirdly, deep learners may be effective learners in any setting. It is not know if adapting the setting or changing preceptor behaviours to those valued by these learners will be helpful for learners who take less effective approaches. Further work is required to assess whether changes in preceptor or site characteristics affect learning outcomes or shift the learner's approach to learning.
Conclusion
Most site and preceptor characteristics valued by clerks and residents were predicted by their Deep approach to learning scores validating these preferences. Preceptor characteristics are likely more important for learning than site characteristics. Some characteristics reflecting the need for good organization and clear direction are predicted by learners' scores on less effective, surface approaches to learning. These findings provide insight on the needs of more vulnerable learners.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All authors participated in the conceptual planning and design of the study. DD and JK provided the statistical analysis and data interpretation. DD prepared the manuscript and all authors participated in interpretation and manuscript revision.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The project was supported by a Research in Medical Education grant from the Canadian Institute of Health Research and Association of Canadian Medical Colleges. The authors would like to thank Jason Schmelzle for careful data entry and Rachelle Sequin for project management.
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McManus IC Keeling A Paice E Stress, burnout and doctors' attitudes to work are determined by personality and learning style: A twelve year longitudinal study of UK medical graduates BMC Medicine 2004 2 29 15317650 10.1186/1741-7015-2-29
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
Biggs JB Rihn BA Kirby JR The effects of intervention on deep and surface approaches to learning Cognitive strategies and educational performance 1984 Orlando FL: Academic Press 279 293
Dolmans DH Wolfhagen HA Essed GG Scherpbier AJ Van Der Vleuten CP Students' perceptions of relationships between some educational variables in the out-patient setting Med Educ 2002 36 735 41 12191056 10.1046/j.1365-2923.2002.01280.x
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BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-5-571620737410.1186/1471-2180-5-57Research ArticleThe two authentic methionine aminopeptidase genes are differentially expressed in Bacillus subtilis You CongHui [email protected] HongYan [email protected] Agnieszka [email protected] Gang [email protected] YiPing [email protected] Anne-Marie [email protected] Antoine [email protected] National Laboratory of Protein Engineering and Plant Genetic Engineering, The College of Life Sciences, Peking University, 100871, Beijing, P.R. China2 Institut Pasteur, Unité de Génétique des Génomes Bactériens, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France3 HKU-Pasteur Research Centre, Dexter H. C. Man Building, 8 Sassoon Road Pokfulam, Hong Kong, S.A.R., P.R. China4 CEA Saclay, Laboratoire Stress Oxydants et Cancer, DSV/DBJC/SBMS, Bat 142, 91191 Gif sur Yvette Cedex, France2005 5 10 2005 5 57 57 1 8 2005 5 10 2005 Copyright © 2005 You 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
Two putative methionine aminopeptidase genes, map (essential) and yflG (non-essential), were identified in the genome sequence of Bacillus subtilis. We investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability in B. subtilis.
Results
In silico analysis of MAP evolution uncovered a coordinated pattern of MAP and deformylase that did not correlate with the pattern of 16S RNA evolution. Biochemical assays showed that both MAP (MAP_Bs) and YflG (YflG_Bs) from B. subtilis overproduced in Escherichia coli and obtained as pure proteins exhibited a methionine aminopeptidase activity in vitro. Compared with MAP_Bs, YflG_Bs was approximately two orders of magnitude more efficient when assayed on synthetic peptide substrates. Both map and yflG genes expressed in multi-copy plasmids could complement the function of a defective map gene in the chromosomes of both E. coli and B. subtilis. In contrast, lacZ gene transcriptional fusions showed that the promoter activity of map was 50 to 100-fold higher than that of yflG. Primer extension analysis detected the transcription start site of the yflG promoter. Further work identified that YvoA acted as a possible weak repressor of yflG expression in B. subtilis in vivo.
Conclusion
Both MAP_Bs and YflG_Bs are functional methionine aminopeptidases in vitro and in vivo. The high expression level of map and low expression level of yflG may account for their essentiality and dispensality in B. subtilis, respectively, when cells are grown under laboratory conditions. Their difference in activity on synthetic substrates suggests that they have different protein targets in vivo.
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Background
Ribosome-mediated protein synthesis is always initiated with either methionine (in eukaryotes) or N-formylmethionine (in prokaryotes and eukaryotic organelles) [1]. However, after removal of the N-formyl group from the polypeptide by peptide deformylase (DEF, EC 3.5.1.88), the N-terminal methionine of a large number of proteins is cleaved by methionine aminopeptidase (MAP, EC 3.4.11.18) [2,3]. The efficiency of removal of the initiator methionine is defined by a highly conserved local substrate specificity, which is determined by both methionine and its adjacent residue. MAP hydrolytically removes the N-terminal methionine only when the penultimate residue bears a small and uncharged side chain (Gly, Ala, Ser, Thr, Pro, Val, or Cys) [4-9], and residues downstream of the penultimate residue have little impact on the reaction. All MAPs studied to date were reported to be cobalt-dependent metalloproteases [3]. However, some reports showed that MAPs exhibited activity in the presence of other divalent ions, such as Zn(II), Fe(II) or Mn(II) [10-12]. According to their sequence homology, MAPs are grouped into two subtypes, type I and type II [13,14]. An insertion of approximately 60 residues in the C-terminal domain of type II methionine aminopeptidases is the only difference between the two types [14]. Eukaryotes possess at least two map genes, of both type I and type II [3], while there is only one map gene in most prokaryotic genomes, either type I (Archaea) or type II (Bacteria) [15].
MAP is distributed throughout living organisms, where it plays an important physiological role. The deletion of the single map gene in prokaryotes such as Escherichia coli [16] or Salmonella typhimurium [17] is lethal. In yeast, the deletion of any one of the two map genes (type I or type II) causes a slow growth phenotype, while deletion of both genes is lethal [18]. Why might MAP activity have an essential role in living cells? Previous studies showed that many proteins needed to have their N-terminal methionine removed to have normal biological activity, proper subcellular localization and eventual degradation (reviewed in [19]). As a result, it is possible that MAP is essential because of the essentiality of its downstream targets. Besides essentiality, MAP changes the dynamics of the sulfur containing metabolites, which may have an important role in the homeostasis of the cell.
In general, there is only one map gene in the genome of prokaryotes, with the exception of a cyanobacteria strain, which has three functional methionine aminopeptidases [20]. Surprisingly, in the genomes of Bacilli, two or even more putative MAP genes can be detected by sequence alignment. In B. subtilis, two putative genes responsible for MAP activity, map and yflG, can be identified [21]. Kobayashi et al. [22] reported that map is an essential gene in B. subtilis while yflG is not, an apparent paradox if yflG codes for a methionine aminopeptidase. In an attempt to better understand the functional role of MAP, we studied the evolutionary trend of MAPs in silico and demonstrated that both the map and yflG genes from B. subtilis code for methionine aminopeptidases in vitro and in vivo; furthermore, the finding of a high expression level for map in parallel with a low expression of yflG in B. subtilis, may account for their essentiality and dispensability, respectively.
Results
Sequence alignment and evolutionary pattern analyses
In the genome sequence of B. subtilis, two genes could code for methionine aminopeptidases, map and yflG, respectively [21] (Figure 1). The sequence similarity and identity between E. coli MAP (MAP_Ec) and B. subtilis MAP (MAP_Bs) is 65% and 46%, respectively. Similarity between MAP_Ec and YflG from B. subtilis (YflG_Bs) is 55% and their identity is 34% and similarity between MAP_Bs and YflG_Bs is 58% and their identity is 36%. However, the sequence of YflG_Bs differs slightly from the PROSITE consensus for type I MAPs (PS00680), which is [MFY]-x-G-H-G- [LIVMC]-[GSH]-x(3)-H-x(4)-[LIVM]-x-[HN]-[YWVH]. In particular it displays one extra residue next to the conserved GHG metal-binding motif. Interestingly, MAP_Bs is more similar to MAP_Ec and YflG_Bs is more similar to the only putative methionine aminopeptidase of Staphylococcus aureus (labelled as "YMAP" in Figure 1). Overall the proteins are very similar in their physico-chemical properties (they have closely similar length and are slightly acidic). Moreover, as shown in Figure 1, the metal (presumably cobalt) binding site (highlighted residues) is conserved. It was therefore important to investigate whether both proteins had methionine aminopeptidase activity.
This difference in evolution of proteins considered as essential in related organisms (Firmicutes) is intriguing, and we explored the evolution pattern of map in a study meant to identify persistent genes in bacteria [23]. When matched with the evolution of 16S RNA, the map gene does not follow a linear course of evolution, as do most ribosomal proteins [23], but shows an erratic pattern (Figure 2). This suggests that MAP might be involved in different biological processes besides its role as methionine aminopeptidase according to the organisms. This prompted us to explore the evolution of MAP with proteins that might be functionally related, using the criteria we used to identify persistent genes. We defined persistent genes as the genes present as orthologs in more than 85% of the genomes of a clade (here 26 Firmicutes), after removing obligatory endosymbionts from the list (see Methods). The protein divergence (sequence similarity divided by sequence length difference) of MAP in B. subtilis and its orthologs from other Firmicutes were compared with bacterial evolutionary distance (measured by 16S rRNA distance). Similar analyses were performed with a general protease, ClpP, DEF, ribosomal protein S4, RpsD and YkrB, a second deformylase present in B. subtilis (Figure 2). Interestingly, in contrast to the expected evolutionary pattern of RpsD, MAP exhibited the same erratic evolutionary way as Def and YkrB, the two functional deformylases in B. subtilis, suggesting co-evolution of methionine aminopeptidase and deformylase.
Enzyme activity in vitro
It is often recognized that gene function identification solely based on sequence comparisons could be misleading [24,25]. Therefore, we first determined whether map and yflG were authentic MAP genes by overproducing the proteins in E. coli and characterizing their enzymatic activity. Both products of map and yflG genes were purified to more than 90% homogeneity. Mass spectrometry showed that the molecular mass of MAP_Bs and YflG_Bs was 27409.17 ± 0.92 Da and 27209.35 ± 2.14 Da, respectively. Interestingly, both proteins purified from E. coli retained their initial methionine, consistent with the nature of the second residue, isoleucine in both cases.
As seen in Table 1, both enzymes exhibited a methionine aminopeptidase activity in vitro with the synthetic peptide substrates tested. They differed widely, however, in the extent of their activity: the specific activity of YflG_Bs was about 90- and 20-fold higher than that of MAP_Bs with the tetrapeptide MGMM and tripeptide MAS as the substrates, respectively. Both enzymes preferred the tetrapeptide MGMM as the substrate. With MAS and MG as the substrates, MAP_Bs activity was 76.9% and 46.2% of that with MGMM as the substrate, respectively, while YflG_Bs retained only 17.7% and 0.6% relative activity, respectively, with these substrates.
The effect of divalent ions (0.2 mM) on enzyme activity was investigated (Figure 3). MAP_Bs had significant activity with both Mn2+ and Co2+. MAP_Bs retained appreciable activity (26–50%) after replacement of Co2+ with Cd2+, Zn2+, Ca2+, Mg2+, Cu2+, Sr2+ and Ba2+, but not Ni2+. In contrast, YflG_Bs showed a strong preference for Co2+ with a decrease in activity to below 20% with any other divalent ions tested.
Subcellular localization
To investigate whether MAP_Bs and YflG_Bs had any difference in their subcellular localization, we constructed N-terminal green fluorescent protein (GFP) fusions of both proteins. As previously reported, the catalytic domain of the methionine aminopeptidase is located in the C-terminal part of the polypeptide chain [20,26-29], therefore N-terminal GFP fusions of both proteins were not expected to interfere with their functions. In addition, we also constructed C-terminal GFP fusions of both MAP_Bs and YflG_Bs, but because of the possible degradation of the fusion proteins in vivo or some unknown reasons, we failed to detect any fluorescence in either case (data not shown). Cells from strains BSIP8001 (MAP::GFP fusion) and BSIP8002 (YflG::GFP fusion) in the mid-exponential growth phase were collected and visualized by fluorescence microscopy, respectively. Both GFP-MAP_Bs (Figure 4A) and GFP-YflG_Bs (Figure 4B) proteins distributed evenly all over the cells. As was expected, the intensity of the fluorescence of both proteins increased with the increasing concentration of xylose used to induce expression of the GFP fusions (0.1%- 1% (w/v)) (data not shown).
MAP-defective mutants are rescued by MAP_Bs or YflG_Bs supplied in trans
The E. coli mutant EM9 contains an engineered map gene, whose expression is under the control of isopropyl β-D-thiogalactoside (IPTG), so that the strain cannot grow unless IPTG is added to the growth medium [16]. Using the replicative vector pBAD, which holds an arabinose-inducible promoter, the map and yflG genes from B. subtilis were individually introduced into strain EM9. As shown in Figure 5, the transformants could complement the map defect when IPTG was omitted (leading to a MAP deficiency) if a high concentration of arabinose was supplied, in the case of plasmids carrying either B. subtilis map or yflG genes. Lower concentrations of arabinose only partially supported growth. This demonstrated that both map and yflG from B. subtilis can supply the MAP function to E. coli in vivo.
In B. subtilis, map is an essential gene, while yflG is not [22]. However, with a back-up map or yflG copy under the control of IPTG provided in multi-copy plasmid pDG148 (30–50 copies using the pUB110 replicon from B. subtilis [30]), map::lacZ disruptants in the chromosome of B. subtilis were obtained (BSHP7046 and BSHP7037). Nevertheless, as witnessed in both cases by the systematic formation of variegated blue white streaked colonies on X-gal plates, the clones were extremely unstable, leading to the loss of transformants after a few generations (data not shown).
map expression is considerably higher than yflG expression
The fact that map is essential while yflG is not [22], together with the ability of yflG gene to rescue MAP_Bs function when supplied at a high level, points to substantial differences in the expression level of the two genes. To test this possibility, the expression of map and yflG genes in B. subtilis in vivo was studied using lacZ as the reporter gene.
Strains BSHP7042 and BFS4611 were grown in minimal medium and were used to monitor the expression of map and yflG, respectively. The map gene showed the higher promoter activity (Figure 6). Its activity increased gradually during the log phase of growth and a nearly four-fold increase was detected (109–379 U/mg) between early exponential and stationary growth phases. In contrast, the yflG gene showed very low activity under all the conditions tested (4–8 U/mg) (Figure 6). In summary, the map gene promoter showed 50 to 100-fold higher activity than that of yflG using lacZ as the reporter gene.
Promoter localization of the yflG gene
In the B. subtilis genome, the yflG gene is located upstream of the yflH gene, followed by a putative Rho-independent transcription terminator. RT-PCR experiments demonstrated that yflG and yflH belonged to a common transcription unit, making an operon (data not shown). Furthermore, lacZ transcription fusion with yflH gene (strain BSHP7043) showed the same activity changes as that of yflG gene during all the conditions tested (data not shown), substantiating the RT-PCR results. In order to identify the promoter of the yflG-yflH operon, we carried out primer extension analysis. Two primers complementary to two different regions inside the yflG gene were used yielding identification of the same transcription start point. As shown in Figure 7, the start is located 31 nt upstream of the ATG translation start codon and regions weakly similar to consensus -35 (TTCCTA) and -10 (TAAGCT) regions are found upstream of this start point, separated by 18 nt, in an AT-rich region. It is difficult at this point to correlate the structure of this promoter with the poor expression of yflG in all conditions tested.
YvoA showed a slight repression effect on yflG expression
As the expression of yflG in B. subtilis in vivo was very low, we endeavoured to uncover conditions to enhance its expression. No significant difference was found when the carbon, nitrogen and sulfur sources were changed, and no difference was found in conditions of sporulation or germination (data not shown). As genes proximal in the chromosome often code for proteins with related functions, the neighbourhood of yflG was analysed. Gene nagP, which encodes a putative phosphoenolpyruvate-dependent transport system N-acetylglucosamine-specific enzyme IICB component, is located upstream of yflG and transcribed divergently (Figure 8A). It could share common control elements. Exploring the corresponding intergenic region with a sliding window 19 nt-long we uncovered sequence AATTGGTATAGATCACTAG which has a very significant counterpart (AGCTGGTCTAGATCACTAG) upstream of the nagAB operon, encoding two N-acetylglucosamine metabolic genes and a putative transcriptional regulator (GntR family) gene, yvoA (Figure 8A). This prompted us to investigate whether YvoA was a possible regulator for yflG. Using a lacZ fusion, we monitored the expression of yflG in a yvoA-disrupted strain, BSIP8004.
As shown in Figure 8B, the promoter of yflG showed a low but consistently higher activity in the yvoA gene disrupted strain BSIP8004 when compared with the wild type parent BFS4611 (two-fold higher activity). This suggested that YvoA could contribute to the regulation of yflG expression.
Discussion
In prokaryotes, it is usually accepted that there is only one gene responsible for methionine aminopeptidase. However, the B. subtilis genome program predicted the existence of two methionine aminopeptidase genes, map and yflG [21]. We have shown that both MAP_Bs and YflG_Bs are functional methionine aminopeptidases in vitro (Table 1 and Figure 3). Furthermore, gene rescue experiments showed that both map and yflG genes expressed on replicative plasmids could supply the MAP function in E. coli and B. subtilis, demonstrating that both MAP_Bs and YflG_Bs are functional methionine aminopeptidases in vivo (Figure 5). Interestingly, Kobayashi et al. [22] reported that disruption of the map gene in B. subtilis is lethal, while the deletion of yflG is not. In the present study, we obtained B. subtilis map mutants when map or yflG genes were expressed in multi-copy replicative plasmids under the control of an IPTG induced promoter (strains BSHP7046 and BSHP7037). This observation suggests that the single copy of the yflG gene expressed in the chromosome could not supply the MAP function in B. subtilis, substantiating that under our conditions its expression is extremely low, making MAP the only significant methionine aminopeptidase available. This is corroborated by the lacZ fusion studies showing that the map gene is expressed some 50–100-fold higher than yflG under all conditions tested. Despite the weak expression of yflG, however, we detected a sigma A promoter using primer extension (Figure 7). We further showed that this expression is modulated by YvoA (Figure 8), suggesting a possible connection between N-acetylglucosamine metabolism and methionine aminopeptidase. In this respect it is worth noting that in E. coli glucosamine-6-phosphate deaminase activity is modulated by the amino-terminal methionine of the enzyme [31]. We tried several growth conditions involving N-acetylglucosamine as a carbon or nitrogen source, or both, but did not find any modulation of YflG activity (data not shown). We also explored expression of yflG::lacZ fusions during sporulation and germination. No enhancement was observed (data not shown). As a consequence, we surmise that the high expression level of map accounts for its essentiality in B. subtilis.
The published MAP_Ec activity [6,11] is about 5–10 times higher than that of MAP_Bs in our assay conditions. This may be related to the observation that the map gene in E. coli does not belong to a highly expressed ribosome protein operon, in contrast to the situation in B. subtilis [32]. As a result, the relative expression level of map in E. coli might be lower than that of the map gene in B. subtilis. For still unknown reasons it might be impossible for the yflG gene to function as the main methionine aminopeptidase in B. subtilis in vivo in spite of the high activity of YflG_Bs in vitro. As shown in Table 2, the enzyme activity of MAPs in crude cell extracts of E. coli (BL5), B. subtilis (168) and yflG disrupted B. subtilis strain (BFS4611) showed not much difference in vitro under all the condition tested. Different expression levels of map genes in a genome carrying more than one methionine aminopeptidase genes have been previously suggested to be the main reason for their different physiological roles in vivo, but no direct evidence was provided [20,33]. Methionine aminopeptidases in vivo are active on proteins, i.e. on complex polypeptides [34,35] and not on the short peptides in our in vitro assays. It could well be that specific targets require a specific aminopeptidase. A similar conclusion holds for other apparently paralogous genes. For example, there are also two functional peptide deformylases in B. subtilis, but only one is probably the predominant deformylase with concomitant high gene expression [36].
The finding of the co-evolutionary parallel trend of methionine aminopeptidase and deformylase in Firmicutes (Figure 2) is consistent with functional relationship in vivo, which correlates well with the fact that deformylase removes all N-formyl groups as a prerequisite for the subsequent function of MAP [33]. This co-evolution may be the signature of protein-protein interactions [37,38], an intriguing conjecture, which seems to be supported by the consistent duplication of deformylases in genomes with more than one methionine aminopeptidase (Table 3).
Conclusion
We proved that both MAP_Bs and YflG_Bs are functional methionine aminopeptidases in B. subtilis and we suggested that map gene is essential because of its high expression level, while yflG is nonessential possibly because of its low expression level making that it can not take over or compensate the function of map when map is not expressed, or because of specific targets dedicated to only one of the two MAPs. Conservation of several map-like genes in Bacilli suggests involvement in a process specific to this group of organisms, which may involve also peptide deformylase.
Methods
Bacterial strains, plasmids, and culture conditions
E. coli and B. subtilis strains as well as plasmids used in this work are listed in Table 4. E. coli TG1 and XL1-Blue were used for cloning experiments (TG1 for single cross-over recombination or replicative plasmids propagation and XL1-Blue for double cross-over recombination) and E.coli BL5 for IPTG-induced protein overproducing. B. subtilis 168 was used as the wild type strain in this study. E. coli cells were grown in Luria-Bertani (LB) medium [39] or in M63 minimal medium (KH2PO4, 4.4 mM; K2HPO4, 8 mM; (NH4)2SO4, 15 mM; MgSO4, 2 mM; ferric citrate, 34 μM; VitB1, 0.0001% (w/v); fructose, 0.4% (w/v); sodium citrate, 0.3 mM; CaCl2, 50 μM; MnCl2, 5 μM; ZnCl2, 12 μM; CuCl2, 2.5 μM; CoCl2, 2.5 μM; Na2MoO4, 2.5 μM) supplemented with a final concentration of arabinose from 0.02% to 0.5% (w/v) when needed. B. subtilis cells were grown in SP medium or in minimal medium (K2HPO4, 8 mM; KH2PO4, 4,4 mM; glucose, 27 mM; Na3-citrate, 0.3 mM; L-glutamine, 15 mM; L-tryptophan, 0.244 mM; ferric citrate, 33.5 μM; MgSO4 or L-methionine, 1 mM; MgCl2, 0.61 mM; CaCl2, 49.5 μM; FeCl3, 49.9 μM; MnCl2, 5.05 μM; ZnCl2, 12.4 μM; CuCl2, 2.52 μM; CoCl2, 2.5 μM; Na2MoO4, 2.48 μM). Antibiotics were added to the following concentrations when required: ampicillin, 100 μg/ml; spectinomycin, 80 μg/ml; chloramphenicol, 5 μg/ml; phleomycin, 5 μg/ml; kanamycin, 100 μg/ml for E. coli and 5 μg/ml for B. subtilis. IPTG was added at 1 mM concentration or as stated when needed. Solid media were prepared by adding 1.5% agar (w/v) to the respective liquid media. Bacteria were grown at 37°C. The optical density (OD) of bacterial cultures was measured at 600 nm. All experiments were performed in accordance with the European regulation requirements concerning the contained use of Genetically Modified Organisms of Group-I (French agreement N° 2735).
DNA techniques
DNA purification, restriction enzyme digestion, ligation and transformation of E. coli were performed according to standard protocols [40]. For cloning purpose, YieldAce or Pfu DNA polymerase (Stratagene) was used. All the DNA sequences in the plasmids constructed in this work were determined using the dideoxy-chain termination method and Thermo Sequenase Kit (Amersham Pharmacia Biotech). B. subtilis cells were transformed with plasmid DNA following the two-steps protocol described previously [41]. Transformants were selected on LB plates containing corresponding antibiotics and IPTG when needed.
Plasmid and strain constructions
In order to clone the genes from B. subtilis for protein producing in E. coli, both map and yflG genes were amplified by PCR from genomic DNA of B. subtilis 168 and cloned into expression vector pET24b(+) (Novagen) using NdeI and XhoI restriction sites. The amplified fragments included nucleotides +1 to +744 and nucleotides +1 to +747 relative to the translational start point of map and yflG, respectively. The resulting constructs, plasmids pIPP8003 and pIPP8004, were transformed into E. coli BL5 giving strains ECIP8003 and ECIP8004 for overproducing MAP and YflG, respectively.
To construct map and yflG GFP fusion plasmids and strains, both map and yflG genes were amplified by PCR from genomic DNA of B. subtilis 168 and cloned into N-terminal GFP fusion vector pSG1729 [42] using XhoI and EcoRI restriction sites. The amplified fragments included nucleotides +1 to +744 and nucleotides +1 to +747 relative to the translational start point of map and yflG, respectively. The resulting plasmids, pIPP8001 and pIPP8002, were linearized by the ScaI restriction enzyme and were transformed into B. subtilis 168. Clones were selected for the amylase deficient phenotype after double cross-over recombination at amyE site [42], giving strains BSIP8001 and BSIP8002, respectively.
To obtain map and yflG expression plasmids for further map gene rescue analysis in a conditional map gene deletion E. coli strain, EM9 [16], both map (nucleotides -30 to +744 relative to the map translation start point) and yflG (nucleotides -21 to +754 relative to the yflG translation start point) genes were amplified by PCR from genomic DNA of B. subtilis 168 with the creation of BamHI and BglII sites. Both purified PCR fragments were cleaved by BamHI and BglII restriction enzymes and inserted between the same two sites in plasmid pBAD containing arabinose inducible promoter (a modified pBAD-αβγ plasmid containing an additional BamHI cloning site was used after removing a BamHI and BglII insert [43]), producing plasmids pHPP1017 and pHPP1018, respectively. The proper orientation was checked by the ability of cloned fragment to be recovered by double digestion using BamHI and BglII restriction enzymes. Plasmids pHPP1017, pHPP1018 and the pBAD vector were transformed into E. coli strain EM9 individually and selected on LB plates containing ampicillin, chloramphenicol and IPTG, giving strains ECHP1005, ECHP1006 and ECHP1007, respectively.
To obtain a back-up map and yflG copies for further conditional map disruptant construction in B. subtilis, the map gene (nucleotides -43 to +744 relative to the map translation start point) and yflG gene (nucleotides -54 to 733 relative to the yflG translation start point) was amplified by PCR from genomic DNA of B. subtilis 168 using primers introducing a SalI cloning site at the 5' end and a SphI cloning site at the 3' end, respectively. Both fragments were then inserted into the SalI and SphI sites of the IPTG-inducible replicative vector pDG148 [44], producing plasmid pHPP7021 and pHPP7022, respectively. The wild type strain B. subtilis 168 was transformed with these plasmids, giving strains BSHP7045 and BSHP7038, respectively.
To disrupt the map gene, a 246 bp-long fragment within the map gene (nucleotides +142 to +388 relative to the map translation start point) was amplified by PCR from genomic DNA of B. subtilis 168 using primers introducing an EcoRI cloning site at the 5' end and a BamHI cloning site at the 3' end of the fragment. This fragment was then inserted into the EcoRI and BamHI sites of plasmid pJM783 [45], which contains a promoterless lacZ reporter gene, producing plasmid pHPP7025. The plasmid was introduced into the chromosome of BSHP7045 and BSHP7038 strains by a single cross-over event, giving strains BSHP7046 and BSHP7037, respectively. To ascertain that they were correctly inserted, clones were checked by PCR.
To construct a map transcriptional fusion with the lacZ gene, a DNA fragment downstream from the map gene (nucleotides from + 423 relative to the translation start point to the gene's stop codon) was amplified by PCR from genomic DNA of B. subtilis 168 using primers introducing an EcoRI cloning site at the 5' end and a BamHI cloning site at the 3' end of the fragment, then inserted into the EcoRI and BamHI sites of plasmid pJM783 [45], producing plasmid pHPP7017. The plasmid was introduced into the chromosome of B. subtilis 168 by a single crossover event, giving strain BSHP7042.
The yflG transcriptional fusion with the lacZ gene (yflG::lacZ disruptant, strain BFS4611) was constructed within the framework of the European Union and Japanese project for the functional analysis of the genome of B. subtilis, where more than 2000 genes have been disrupted by fusion with lacZ reporter gene [22,46,47].
To construct a yflH transcriptional fusion with the lacZ gene, a DNA fragment downstream from the yflH gene (nucleotides from + 32 to +310 relative to the translation start point) was amplified by PCR from genomic DNA of B. subtilis 168 using primers introducing an EcoRI cloning site at the 5' end and a BamHI cloning site at the 3' end of the fragment, then inserted into the EcoRI and BamHI sites of plasmid pJM783 [45], producing plasmid pHPP7018. The plasmid was introduced into the chromosome of B. subtilis 168 by a single cross-over event, giving strain BSHP7043.
To obtain the disruptant yvoA strain compatible with BFS4611, the BFS817 strain (yvoA::lacZ disruptant, from the European Union and Japanese consortium, see above) was transformed with the ScaI linearized pEC23 plasmid (which carries a kanamycin resistance gene, M. Simon and P. Stragier, unpublished) for replacement with the kanamycin resistance gene of the lacZ and Erm genes belonging to the pMutin plasmid (erythromycin resistant) integrated into the genome, by homologous recombination. The resulting clones were checked for their inability to grow on erythromycin and chloramphenicol. The resulting strain was named BSIP8003. The chromosomal DNA of BSIP8003 was prepared and used to transform the BFS4611 strain, producing the double yvoA and yflG disrupted strain BSIP8004.
Expression and purification of MAP_BS and YflG_Bs proteins
The strains ECIP8003 and ECIP8004 were grown at 37°C in LB medium containing kanamycin (25 μg/ml) and induced with 0.5 mM IPTG for four hours. Cell pellets were harvested by centrifugation and stored at -20°C. Cells were suspended in 20 mM Tris-HCl (pH 8.0) and sonically disrupted. The cellular debris was removed by centrifugation at 12,000 g for 40 min. The supernatants were applied to a DEAE-sephacel column chromatography (Amersham). The protein (MAP_Bs or YflG_Bs) was eluted with 0.1 M NaCl in the same buffer. Protein concentration was determined by the Bradford's method [48] using protein assay kit (Bio-Rad Laboratories). The purity of isolated proteins was determined by SDS-PAGE [40]. Ion mass spectrometry was used to measure the molecular mass of each purified protein as well as checking the existence of the first methionine in each of the proteins.
Enzyme assays
The methionine aminopeptidase assay was performed as described by Arie Ben-Bassat et al. [6] with small modifications. 10 μl protein solution was added to 90 μl of the substrate solution (4 mM peptide in 0.1 M K2HPO4 (pH7.5), and 0.2 mM CoCl2), then incubated at 37°C for 10 min. The reaction was stopped by incubation at 100°C for 2 min. After addition of 900 μl of the colour development mixture (containing 0.2 mg of L-amino acid oxidase, 0.03 mg of horseradish peroxidase, and 0.2 mg of o-dianisidine dihydrochloride in 0.1 M Tris-HCl (pH 7.4)), the tube was incubated at 37°C for 10 min, then the absorbance at 440 nm was recorded. Standard curve with known concentration of L-methionine in the colour development buffer was plotted for quantitative analysis. The absorbance of 1 μmol of methionine per ml at 440 nm is equivalent to 8.6 [6]. One Unit of activity was defined as 1 μmol of methionine produced per min under the assay conditions used. Preference of metal ions was tested by changing the cobalt in the substrate solution into other divalent metal ions at a final concentration of 0.2 mM.
Amylase activity was detected after growth of B. subtilis strains on Tryptose Blood Agar Base (TBAB, Difco) supplemented with 10 g/l hydrolyzed starch (Sigma). Starch degradation was detected by sublimating iodine onto the plates [49].
β-galactosidase specific activity was measured as described by Miller [50]. One Unit of β-galactosidase activity was defined as the amount of enzyme that produced 1 nmol of o-nitrophenol per min at 28°C. Specific activity was expressed in Units per mg protein. The experiments were performed in triplicates.
Subcellular localization analysis
To visualize GFP-MAP_Bs and GFP-YflG_Bs fusions locations in living cells, BSIP8001 and BSIP8002 strains were grown in minimal medium with 0.5% fructose as the sole carbon source supplemented with xylose to different final concentrations (0.1% to 1% (w/v)). Cells from the mid-exponential growth phase were collected and visualized by fluorescence microscopy. Microscope Axiovert 135 TV (ZEISS, Germany) was used in the experiment and fluorescence filter sets used to visualize GFP were obtained from Chroma Technology Corp (USA). Cells were visualized on 1% agarose (w/v) slides [51] using Axiovision 4 system (Allied High Tech Products, Inc.) with exposure time of 3–10 s.
Genetic rescue experiment
The growth of EM9, ECHP1005 (containing B. subtilis map gene), ECHP1006 (containing B. subtilis yflG gene) and ECHP1007 (containing pBAD vector alone), was compared on M63 minimal medium plates (37°C, overnight) in the presence or absence of IPTG (1 mM) with or without arabinose (0.02 %, 0.1 % and 0.5 %).
RNA isolation and manipulation
Total cellular RNA was extracted from B. subtilis 168 cells growing in minimal medium to an OD600 of 0.5 using High Pure RNA Isolation Kit from Roche. The RNA concentration was determined by light absorption at 260 nm and 280 nm. 2 μg of RNA were loaded onto 1.2% agarose gel to check the RNA purity and integrity. RT-PCR experiments were performed using RT-PCR System (Promega) as specified by the manufacturer.
Primer extension analysis was carried out using reverse transcriptase AMV (Roche) as described [52]. Two oligonucleotides (+33 to +62 and +97 to +126 relative to translation start point of yflG) were used for the identification of yflG promoter. The same primers were used for the generation of sequence ladders. Reaction products were separated on 7% denaturing polyacrylamide gel containing 8 M urea. DNA sequences were determined using Sanger's dideoxy chain-termination method with "Thermo Sequenase radiolabeled terminator cycle sequencing kit" from Amersham Pharmacia Biotech.
DNA sequence and in silico analysis
DNA sequences were analyzed using the DNA Strider software [53]. The program BLAST [54] was used to search for homologous sequences in the database. The program CLUSTALW was used for multiple alignment [55]. B. subtilis sequences were analyzed using the SubtiList database [56,57]. Accepted consensus sequences were extracted from PROSITE [58,59].
Evolutionary pattern analyses
First, MAP orthologs (orthologs were defined using the BBH (Bi-directional Best Hit) strategy, i.e. if gene a of genome A is the most similar hit of gene b in genome B and vice versa, gene a and b are regarded as a pair of orthologs [60]) were retrieved between each pair of bacterial genomes. Secondly, the BBHs, with protein sequence similarity >50% and protein length difference <1.3, of the gene map in B. subtilis were collected. Thirdly, the orthologs of each of the BBHs were collected in all the genomes (a total of 206 bacterial genomes in EMBL as of July 1st, 2005), after recursive identification until the members in this cluster is no longer extended. Last, the protein divergence (sequence similarity divided by sequence length difference) of MAP in B. subtilis and its orthologs from other Firmicutes were compared with bacterial evolutionary distance (measured by 16S rRNA evolution). Same analyses were carried out on ClpP, Def, RpsD and YkrB.
Authors' contributions
CHY carried out MAPs enzyme activity analysis, subcellular observation, expression level comparisons, and drafted the manuscript. HYL did genetic rescue experiment in E. coli and primer extension analysis. AS designed the initial experiments, completed the genetic rescue experiment in B. subtilis, constructed lacZ fusion mutants of B. subtilis, and wrote part of the manuscript. GF did the evolutionary pattern analyses. YPW helped to analyse the experimental data and draft manuscript. AMG supervised the MAPs enzyme activity analysis, and helped to draft the manuscript. AD designed and supervised the whole study.
Acknowledgements
The initial stage of the work was supported by the HKU-Pasteur Research Centre (SAR Hong Kong, China) and we would like to thank HF Kung and KY Yuen for their interest at that early moment. CH You was supported by EGIDE (France). We would like to thank JD Huang for his kind gift of pBAD-αβγ vector, Chiron Culture Collection Center for conditional map gene deletion E. coli mutant, EM9, U Mechold for kind suggestions to both the experiment and manuscript drafting, and YL Yap for contribution in silico analyses and discussion.
Figures and Tables
Figure 1 Sequences alignment between MAP from E. coli, B. subtilis, and S. aureus. The first line shows the protein sequence of MAP from B. subtilis (MAP_Bs), the second shows MAP from E. coli (MAP_Ec), the third shows YflG from B. subtilis (YflG_Bs) and the last shows YflG/MAP from Staphylococcus aureus (YMAP_Sa). Identical amino acids are shadowed in dark and conservative amino acids are shadowed in grey. The highlighted residues stand for those conserved amino acids important for MAP activity (bold and italic residues are conservative basic ones, while bold only are acidic ones). [MFY]-x-G-H-G-[LIVMC]-[GSH]-x(3)-H-x(4)-[LIVM]-x-[HN]-[YWVH] (where H is a cobalt ligand) is the conserved motif within the protein sequences (placed under the alignment).
Figure 2 Evolutionary trend analyses of MAP, ClpP, Def, RpsD and YkrB. Map, ClpP, Def, RpsD and YkrB were picked up from B. subtilis. Their BBHs from other Firmicutes were detected. Their protein divergence (sequence similarity divided by sequence length difference) in B. subtilis and their orthologs from other Firmicutes were compared with bacterial evolutionary distance individually (measured by 16S rRNA evolution).
Figure 3 Relative activity of (A) MAP_Bs and (B) YflG_Bs in the present of different ions. MGMM (4 mM) was used as the substrate. All ions were tested at 0.2 mM concentration. Each of their relative activity was defined as 100% in the presence of Co2+ (MAP_Bs: 2.6 U/mg; YflG_Bs: 232.0 U/mg).
Figure 4 Localization of GFP-MAP_Bs and GFP-YflG_Bs fusions. A. Localization of GFP-MAP_Bs in strain BSIP8001 grown in minimal medium with 0.5% xylose (w/v); B. Localization of GFP-YflG_Bs in strain BSIP8002 grown in minimal medium with 1% xylose (w/v).
Figure 5 Genetic rescue experiments. The bacteria were streaked on M63 minimal solid medium and the plates were incubated at 37°C overnight. The concentration of IPTG was 1 mM. The order of the plates in each picture described below is from left to right and from top to bottom. A. EM9 host strain on M63 with IPTG; EM9 on M63 without IPTG. B. EM9 strain with pBAD vector alone (ECHP1007) on M63 with IPTG; ECHP1007 on M63 with arabinose (0.5% (W/V)); ECHP1007 on M63 without IPTG and arabinose. C. EM9 strain caring pBAD map vector (ECHP1005) on M63 with arabinose (0.5% (W/V)); ECHP1005 on M63 with IPTG; ECHP1005 on M63 without IPTG; ECHP1005 on M63 with arabinose (0.02% (W/V)); ECHP1005 on M63 with arabinose (0.1% (W/V)). D. EM9 strain caring pBAD yflG vector (ECHP1006) on M63 with arabinose (0.5% (W/V)); ECHP1006 on M63 with IPTG; ECHP1006 on M63 without IPTG; ECHP1006 on M63 with arabinose (0.1% (W/V)); ECHP1006 on M63 with arabinose (0.02% (W/V)).
Figure 6 Expression of map and yflG lacZ transcriptional fusions. β-galactosidase expression of map and yflG lacZ transcriptional fusion during the growth curve of strains BSHP7042 and BFS4611 in minimal medium. OD600 (○) and β-galactosidase activity (●) were measured for strain BSHP7042 with map lacZ transcription fusion. OD600 (□) and β-galactosidase activity (■) were measured for strain BFS4611 with yflG lacZ transcription fusion.
Figure 7 Identification of the transcription start point of the yflG promoter. Primer extension and sequencing reaction were performed with the same primer. The size of the extended product is compared to a DNA-sequencing ladder of the corresponding region. An arrow indicates the +1 site. Predicted -10 and -35 regions are in bold and boxed; the experimental transcription start site is in bold; the translational start site is underlined.
Figure 8 Expression of yflG lacZ transcriptional fusions in yvoA disrupted strain. A. gene organization around yflG and yvoA. The arrows indicate the direction of gene transcription; B. β-galactosidase expression yflG lacZ transcriptional fusion in yvoA disrupted strain BSIP8004 and yvoA wild type strain BFS4611 grown in minimal medium. OD600 (○) and β-galactosidase activity (●) were measured for strain BSIP8004. OD600 (□) and β-galactosidase activity (■) were measured for strain BSIP8004 and strain BFS4611.
Table 1 Reaction rate of MAP_Bs and YflG_Bs with different peptides as the substrates
Specific Activity (U/mg)
Peptide (4 mM) MAP_Bs YflG_Bs
MGMM 2.6 ± 0.3 232.0 ± 8.2
MAS 2.0 ± 0.1 41.0 ± 5.5
MG 1.2 ± 0.1 1.3 ± 0.1
Three measurements for each of the proteins with different substrates were made.
Table 2 MAP activity in crude cell extracts of E. coli and B. subtilis
Specific Activity (U/mg)
Peptide (4 mM) E. coli (BL5) B. subtilis wild type (168) B. subtilis yflG mutant (BFS4611)
MGMM 1.5 ± 0.2 1.5 ± 0.1 1.5 ± 0.1
MAS 1.1 ± 0.2 1.1 ± 0.1 1.1 ± 0.2
MG 0.6 ± 0.1 0.8 ± 0.2 0.4 ± 0.1
Cells in stationary growth phase of each of the strains were collected and sonically disrupted. Supernatants of each of the cell debris were used to measure MAP activity. Totally, three times of measurements were carried out.
Table 3 Distribution of def and map genes in selected genomes
Numbers in Each Genome
Genome Name
def
map
EMBL ID
Streptomyces coelicolor 4 3 AL645882
Streptomyces avermitilis 3 4 BA000030
Nocardia farcinica IFM 10152 3 3 AP006618
Shewanella oneidensis MR-1 3 1 AE014299
Silicibacter pomeroyi DSS-3 3 1 CP000031
Bacillus cereus ZK 2 3 CP000001
Bacillus thuringiensis serovar konkukian 2 3 AE017355
Bacillus anthracis 'Ames Ancestor' 2 3 AE017334
Bacillus anthracis Sterne 2 3 AE017225
Bacillus cereus ATCC 10987 2 3 AE017194
Bacillus anthracis Ames 2 3 AE016879
Bacillus cereus ATCC 14579 2 3 AE016877
Leifsonia xyli subxyli CTCB07 2 3 AE016822
Synechocystis PCC 6803 1 3 BA000022
Bacillus clausii KSM-K16 1 3 AP006627
Table 4 Bacterial strains and plasmids used or created in this study
Strains Genotype Source or Reference
Escherichia coli
XL1-Blue K12 supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lac- F'[proAB+ lacIq lacZΔM15 Tn10(tetR)] Laboratory collection
TG1 K12 supE hsdΔ5 thi Δ (lac-proAB) F'[traD36 proA+ proB+ lacIq lacZΔM15] Laboratory collection
BL5(DE3) F- Invitrogen
EM9 PT7-map (on chromosome) Chiron Culture Collection Centre
ECIP8003 F- pIPP8003 This work
ECIP8004 F- pIPP8004 This work
ECHP1005 EM9 pHPP1017 This work
ECHP1006 EM9 pHPP1018 This work
ECHP1007 EM9 pBAD This work
Bacillus subtilis
168 trpC2 Laboratory collection
BFS4611 trpC2 yflG::lacZ Functional analysis projecta
BFS817 trpC2 yvoA::lacZ Functional analysis projecta
BSHP7042 trpC2 map-lacZ This work
BSHP7045 trpC2 pDG148-map This work
BSHP7046 trpC2 map::lacZ pDG148-map This work
BSHP7037 trpC2 map::lacZ pDG148-yflG This work
BSHP7038 trpC2 pDG148-yflG This work
BSHP7043 trpC2 yflH::lacZ This work
BSIP8001 trpC2 amyE::gfp-map This work
BSIP8002 trpC2 amyE::gfp-yflG This work
BSIP8003 trpC2 yvoA::[(lacZ-ery)::pEC23(Kan)] This work
BSIP8004 trpC2 yflG::lacZ yvoA::[(lacZ-ery)::pEC23(Kan)] This work
Plasmids Description Source or Reference
pET24b(+) expression vector, KanR Novagen
pBAD-abg genetic rescue vector, AmpR [43]
pDG148 expression vector, AmpR KanR [44]
pJM783 cloning vector, CmR [45]
pSG1729 N-terminal GFP fusion vector, AmpR SpecR [42]
pEC23 integrative plasmid, KanR M. Simon and P. Stragier, unpublished
pIPP8003 pET24b map This work
pIPP8004 pET24b yflG This work
pHPP1017 pBAD map This work
pHPP1018 pBAD yflG This work
pHPP7017 pJM783 map-lacZ This work
pHPP7018 pJM783 yflH::lacZ This work
pHPP7021 pDG148 map This work
pHPP7022 pDG148 yflG This work
pHPP7025 pJM783 map::lacZ This work
pIPP8001 pSG1729 map This work
pIPP8002 pSG1729 yflG This work
a This strain has been constructed during the frame of the project for the functional characterization of the genome of B. subtilis in Japan [46].
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Moszer I Jones LM Moreira S Fabry C Danchin A SubtiList: the reference database for the Bacillus subtilis genome Nucleic Acids Res 2002 30 62 65 11752255 10.1093/nar/30.1.62
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
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Tatusov RL Koonin EV Lipman DJ A Genomic Perspective on Protein Families Science 1997 278 631 637 9381173 10.1126/science.278.5338.631
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BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-5-581621265310.1186/1471-2180-5-58Research ArticleNon-classical protein secretion in bacteria Bendtsen Jannick D [email protected] Lars [email protected]øll Anders [email protected] Søren [email protected] Center for Biological Sequence Analysis, BioCentrum-DTU, Building 208, Technical University of Denmark, DK-2800 Lyngby, Denmark2005 7 10 2005 5 58 58 21 6 2005 7 10 2005 Copyright © 2005 Bendtsen 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 present an overview of bacterial non-classical secretion and a prediction method for identification of proteins following signal peptide independent secretion pathways. We have compiled a list of proteins found extracellularly despite the absence of a signal peptide. Some of these proteins also have known roles in the cytoplasm, which means they could be so-called "moon-lightning" proteins having more than one function.
Results
A thorough literature search was conducted to compile a list of currently known bacterial non-classically secreted proteins. Pattern finding methods were applied to the sequences in order to identify putative signal sequences or motifs responsible for their secretion. We have found no signal or motif characteristic to any majority of the proteins in the compiled list of non-classically secreted proteins, and conclude that these proteins, indeed, seem to be secreted in a novel fashion. However, we also show that the apparently non-classically secreted proteins are still distinguished from cellular proteins by properties such as amino acid composition, secondary structure and disordered regions. Specifically, prediction of disorder reveals that bacterial secretory proteins are more structurally disordered than their cytoplasmic counterparts. Finally, artificial neural networks were used to construct protein feature based methods for identification of non-classically secreted proteins in both Gram-positive and Gram-negative bacteria.
Conclusion
We present a publicly available prediction method capable of discriminating between this group of proteins and other proteins, thus allowing for the identification of novel non-classically secreted proteins. We suggest candidates for non-classically secreted proteins in Escherichia coli and Bacillus subtilis. The prediction method is available online.
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Background
The secretion of proteins across biological membranes is in most cases mediated by translocation machinery recognising a specific sequence tag or motif in the protein to be secreted. In bacteria, the classical tripartite structured Sec signal peptide governs most of the targeting to the secretion pathway. In addition to this Sec-dependent secretion, various other secretion pathways have been discovered, which work in a Sec-independent fashion. Most predominant is the twin-arginine translocation (Tat) secretion pathway where a twin-arginine consensus motif is located within the signal peptide itself [1,2]. While the Sec- and Tat-dependent secretion pathways translocate proteins across only the inner membrane in Gram-negative bacteria, additional translocation machinery components are found in the outer membrane of this group of organisms. The N-terminal signal peptide plays a central role in these secretory systems as the tag signalling secretion.
Surprisingly, some bacterial proteins have been found to be secreted without any apparent signal peptide. This phenomenon, termed non-classical secretion, was identified in eukaryotes approximately 15 years ago, when interleukin 1β and thioredoxin were found to be secreted despite being devoid of any identifiable signal peptide [3-5].
Some proteins, which have been found to display a function in the cytoplasm, have also been shown to actively participate in biological processes in the extracellular environment [6]. This does not imply that the function they uphold in the extracellular environment is identical to that in the cytoplasmic environment. Such proteins, which display two unrelated functions, have been named "moonlighting" proteins [7,8].
The detection of non-classically secreted proteins in the extracellular environment could obviously be attributed to cell lysis during experimental handling. However, some of the proteins have been detected extracellularly by different groups in several bacterial species supporting the argument that they are, indeed, exported from the intact cell.
Non-classically secreted proteins can be identified through inactivation of Sec-dependent secretion by mutation or chemical treatment. Hirose et al. used SecA mutants to disrupt the translocation machinery, thereby identifying several non-classically secreted proteins in B. subtilis [9]. Under such conditions, secretion must occur in a Sec independent manner.
It is currently unknown whether secretion by non-classical means occurs at a specifically localised membrane microdomain as seen for secretion of SpeB in Streptococcus pyrogenes [10]. Indeed, the mechanism or mechanisms responsible for non-classical secretion are unknown.
Examples of non-classical secretion in bacteria
The first published study of non-classical secretion in bacteria reports the secretion of glutamine synthetase (GlnA) in the human pathogen Mycobacterium tuberculosis – one of the most important bacterial pathogens studied and responsible for millions of fatalities each year [11,12]. GlnA has been shown to be localised solely to the cytoplasm of the non-pathogen Mycobacterium smegmatis (although this difference need not be related to the pathogenicity of M. tuberculosis). A recombinant GlnA from M. tuberculosis expressed in M. smegmatis is also secreted, indicating that the signal for export is contained within the protein sequence [12].
For many years it has been known that M. tuberculosis secretes antigenic proteins without apparent signal peptides. ESAT-6 (early secretory antigenic target) is a small 6 kDa protein secreted by a novel secretion mechanism, the underlying details of which are still unknown. Another protein belonging to the same family, the small 10 kDa protein CFP-10, has subsequently been found to be secreted in spite of not possessing a signal peptide either (reviewed in [13]). The RD1 gene cluster in M. tuberculosis seems to encode the secretory system responsible for the secretion of the small antigenic proteins [14,15].
Unfortunately, the field has not yet agreed on a name for the new secretion system, although Stanley et al. designate the secretion system Snm for secretion in mycobacteria [14]. Snm1 (Rv3870), Snm2 (Rv3871) and Snm4 (Rv3877) mutants are defective in secretion of ESAT-6 (EsxA) and CFP-10 (EsxB) and attenuated in virulence. The Snm1 and Snm2 proteins are part of a subfamily of ATPases containing an AAA domain, which is associated with chaperone-like functions. All three proteins may constitute parts of the translocation machinery of the Snm system [14]. ESAT-6, CFP-10 and homologous proteins are reported to share a WXG motif as identified by PSI-BLAST [16]. Whether the WXG motif alone is sufficient for secretion is unknown. In Staphylococcus aureus, the ESAT-6 homologs (EsxA and EsxB), have been found to be secreted in a similar fashion [17].
Superoxide dismutase (SodA) is a protein regularly found in the cytoplasm of M. tuberculosis. It has also been reported to be secreted in the same organism, but does not contain a signal peptide [18]. Again as with GlnA, SodA is not secreted in the non-pathogenic mycobacterium M. smegmatis [18,19]. In M. tuberculosis, a protein required for superoxide dismutase secretion was identified and named SecA2 [20]; deletion of the SecA2 gene abolishes virulence of M. tuberculosis in mice. This suggests that this new secretory pathway plays a role in the export of virulence factors in Gram-positive bacteria [20].
SecA2-dependent secretion has also been demonstrated in another Gram-positive pathogen, Listeria monocytogenes [21]. In this study, ten proteins without a classical Sec-signal peptide were found to be secreted, and the authors furthermore examined whether these proteins were translocated to the cell wall or secreted to the extracellular medium. In addition to these ten proteins, seven proteins with signal peptides were found to be secreted in a SecA2-dependent manner. The SecA2 pathway thus seems to be involved in both signal peptide dependent and non-classical secretion. It has been speculated that the SecA2 secretion pathway could be analogous to Type III secretion of virulence factors commonly found in pathogenic Gram-negative bacteria. All the non-classically secreted proteins detected in L. monocytogenes clearly have a cytoplasmic functional role whereas the precise extracellular functionality of the proteins remains unknown.
Another example is the staphylococcal nuclease from M. smegmatis. It contains a signal peptide and is secreted but secretion also occurs when the signal peptide is removed by mutation [22]. Experiments indicated that the release of the staphylococcal nuclease to the extracellular environment was not due to cell lysis. Recchi et al. were not able to characterise components of the secretion apparatus, but since no functional signal peptide was present, secretion must have taken place in a non-classical fashion [22].
ClyA, a pore-forming protein displaying a cytotoxic effect in mammalian cells, does not carry an N-terminal signal peptide but is nevertheless released from E. coli via vesicles that pinch off from the outer membrane. The secretion of ClyA is independent of the five known secretion pathways (Type I-V) in Gram-negative bacteria, thus bacterial "membrane blebbing" could be a novel form of secretion in bacteria [23,24].
One of the most comprehensively studied Gram-positive bacteria, B. subtilis, is also capable of secreting proteins via one or more non-classical pathways. Various studies have tried to identify the entire proteome of B. subtilis by use of 2D-electrophoresis, mass-spectrometry and prediction methods [9,25,26], including proteins localised to the extracellular environment. Due to differences in experimental setup and laboratory conditions, the various proteomic studies in B. subtilis do not agree on its extracellular proteome. Recently, a review on protein secretion in B. subtilis suggested that signal peptide independent protein secretion in bacteria is perhaps more common than previously thought [27]. This review lists 24 proteins found in the extracellular environment without having classical Sec signal peptides. This list of extracellular proteins was compiled from studies having different experimental setups as different cellular conditions were investigated [9,25,28].
Through a structure-function analysis of the Foldase protein (PrsA), Vitikainen et al. discovered a number of seemingly non-classically secreted proteins in B. subtilis, although this was not their initial aim [28]. As PrsA is an essential chaperone in B. subtilis involved in post-translational folding of exported proteins, mutations in prsA might lead to unpredictable alterations in protein secretion and overall stability of the cell. As mentioned by the authors themselves, the modifications cause significant cell lysis making it difficult to assess the degree of true non-classical secretion in this study.
Proteins involved in carbohydrate metabolism (Eno, PdhB, PdhD and CitH) were identified as being extracellular by Vitikainen et al. [28], although none of these proteins have a known extracellular function. Proteins involved in metabolism of amino acids, RocA and RocF, were initially found by Antelman et al. [25] to be non-classically secreted, but only RocF was later identified by Vitikainen et al. [28].
The motility and chemotaxis protein Hag was initially identified by Hirose et al. [9] but later also FlgK and FliD were found to be localised extracellularly [25]. Each of these three proteins is known to possess extracellular functions. The detoxification proteins KatA and SodA were initially identified extracellularly being secreted from a SecA B. subtilis deletion strain [9]. Later, also YceD was found extracellularly in stationary-phase B. subtilis [25]. In Legionella pneumophila, it has been shown that the ortholog of one of these detoxification proteins, KatA, is critical for stationary-phase redox reactions in the periplasm [29].
Although the extracellular function of KatA in B. subtilis is unknown, KatA could have a similar role in stationary-phase B. subtilis. The elongation factor Ef-G and the protein folding chaperone GroEL has been identified in the extracellular environment both by Antelmann et al. [25] and by Vitikainen et al. [28].
These two proteins were not found in the SecA deletion study by Hirose et al. and their potential extracellular function is unknown [9]. Although GroEL has a cytoplasmic function, the GroEL homolog HspB has been shown to be actively secreted in stationary-phase Helicobacter pylori [30]. The phage related proteins XepA, XkgG, XkdK, XkdM and XlyA were all identified by Antelmann et al. [25] while XdkG was identified in [9]. CwlC is a protein involved in the metabolism of the cell wall and was identified extracellularly [25].
B. subtilis provides a good example of why inconsistencies in the reports of the extracellular proteome of a specific organism makes it difficult to produce a list of validated non-classically secreted proteins. The discrepancies can be attributed to experimental conditions as the extracellular proteome of any organism will vary depending on the state of the cell and the nature of its environment. Cell lysis and protein degradation are other sources of errors that are difficult to entirely account for in all assays.
Characterising non-classically secreted proteins
Little is known about the dynamical aspects of the non-classical secretion apparatus and whether the above mentioned cases are secreted through one or more different secretion systems. The only common theme to the phenomenon is the extracellular presence of these proteins despite the lack of a recognisable signal peptide or other conserved motifs.
Proteins entering the non-classical secretion pathway cannot be correctly identified using prediction methods such as PSORT [31] or SignalP [32]. Due to the lack of any apparent sequence motif in the N-terminal region of the sequence, it is doubtful whether secretion depends on this part of the protein in the way it does for other secretion pathways. Neither can non-classically secreted proteins be identified by their sequence homology to either known classically secreted proteins or to cytoplasmic proteins, as non-classically secreted proteins often seem to have a cytoplasmic function as well as an extracellular functional role.
Prediction methods for functional classification have been developed that allows for classification of proteins based on many sequence-derived features. For example, the method has been applied to predict protein functional categories [33] and to determine whether or not a protein is cell-cycle regulated [34]. Classification is based on a neural network evaluation of calculated and predicted protein sequence features such as post-translational modifications, secondary structure, isoelectric point and sequence length [33-36]. The idea behind such a classification scheme is that proteins can be described by their feature characteristics instead of by their sequence. Typically, some features are shared among proteins with similar function or subcellular localisation, despite the fact that they display no overall sequence similarity. Each protein sequence is assigned a feature-profile, and a neural network algorithm is trained to classify the proteins based on their feature-profiles. To avoid situations where data is unavailable, all features are either calculated or predicted directly from the protein sequence, but experimentally determined features can, in principle, be used as well.
We have previously applied this method with success to the problem of predicting non-classical secretion in mammals [36] and have now extended this to bacteria as described here. To better cover the diversity of bacteria, we have developed two different methods: one trained on and suited for proteins from Gram-positive bacteria and one for proteins from Gram-negative bacteria. Both methods are available from our website [37].
We show that, indeed, bacterial non-classically secreted proteins can be described in terms of sequence-derived features and use that property to propose additional secreted proteins in E. coli and B. subtilis.
Results and discussion
We have performed an exhaustive literature search and compiled a list of apparently non-classically secreted proteins (Table 1). Due to the sensitivity of protein detection techniques, it obviously cannot be excluded that some of the proteins detected in the extracellular environment originate from cell lysis, as we discuss further below.
Only one of the publications make a distinction between cell surface localised proteins and proteins dispersed in the extracellular medium [21]. Thus, only in these cases are we able to distinguish between cell surface localised proteins and other extracellular proteins (Table 1).
No simple sequence motifs in proteins undergoing non-classical secretion
No simple sequence motifs have been found that target all the known examples of non-classical secretion to the extracellular environment. Pallen et al. found a short WXG sequence motif in the ESAT-6 family of proteins by use of a PSI-BLAST approach [16]. However, we were not able to identify this as a common sequence motif in all proteins known to be localised extracellularly (data not shown). We searched for a common sequence motif in the 22 non-classically secreted proteins in B. subtilis using both a Gibbs-sampling approach [38] and the TEIRESIAS Pattern Discovery Algorithm [39], but found no conserved motifs.
As an alternative strategy, we attempted to identify the non-classically secreted proteins by means of their specific biological and chemical properties or characteristics. We have done this with success for mammalian proteins resulting in the prediction method SecretomeP [36]. The SecretomeP method for mammalian proteins is based on the fact that secreted proteins share certain features regardless of the mechanism by which they are secreted. A combination of such features can be used to distinguish them from non-secreted proteins.
We calculated or predicted approximately seventy different features for each sequence and tested each of them for discriminatory value. Subsequently, those contributing most strongly to the predictive performance were combined in a neural network approach as described previously [33,36]. Due to the relatively small number of known non-classically secreted proteins, we used as positive training examples Sec-dependent secreted proteins (with the signal peptide removed) and validated that this approach will identify non-classically secreted proteins correctly as it did for mammalian proteins [36].
Features characterising non-classically secreted proteins
Many sequence-derived features will be characteristic to proteins undergoing secretion as well as to cytoplasmic proteins. We therefore searched for combinations of features with discriminatory value. Using an iterative scheme, in which features were tested individually and in combination, we eventually obtained a set of features together that had optimal discriminatory power. Two different prediction methods were trained; one for Gram-positive bacteria and one for Gram-negative bacteria.
When used independently, the predictive performance of the six protein features selected by the iterative scheme ranges from very poor (for example based on threonine contents, which on its own obtains a correlation coefficient below 0.4) to fairly good (for example based on the amino acid composition network yielding a correlation coefficient just above 0.7). However, combining the features in a single network increases the performance and the robustness of the method considerably. When the network is provided with information about the different protein features simultaneously, it is capable of correctly classifying a protein as either secreted or non-secreted approximately 9 out of 10 times regardless of the pathway of secretion (for qualitative performance and evaluation, see below).
The SecretomeP method is also capable of discriminating cytoplasmic proteins from classical secretory proteins (carrying classical Sec signal peptides). We tested the method on the dataset from SignalP 3.0, which is a widely used prediction method for identification of classically secreted proteins carrying signal peptides [32]. 87.6% of the Gram-negative positive data set (secreted proteins) for SignalP 3.0 received a SecretomeP score above 0.5. 96.4% of the negative data set (cytoplasmic proteins) received a SecretomeP score below 0.5. 89.5% of the Gram-positive SignalP positive data set received a SecretomeP score above 0.5 and 94.7% of the corresponding negative SignalP data set received a SecretomeP score below 0.5 using the Gram-positive prediction method. This indicates slightly better identification of cytoplasmic proteins over secreted proteins for both bacterial methods. While these results are far from those obtained using SignalP 3.0, they nevertheless demonstrate the power of a feature-based approach. The correlation coefficient for the Gram-positive and Gram-negative feature-based method were 0.85 and 0.87, respectively. As mentioned above, Tat substrates constitute yet another class of secretory proteins. In contrast to the Sec-dependent secretion pathway, the Tat-dependent secretion pathway is capable of translocating fully folded proteins [1]. Tat substrates have signal peptides with a tripartite structure much like Sec signal peptides, and a prediction method for Tat substrates was recently published [40]. We tested the SecretomeP method on Tat substrates, and conclude from the results (not shown) that the method is unsuitable for the prediction of this class of secretory proteins. The folded conformation of Tat substrates during translocation may constrain these proteins with regards to the chemical and structural properties that we evaluate and interpret in SecretomeP.
An interesting observation regards the difference in output scores from DisEMBL [41] of secreted and cytoplasmic proteins. Secretory proteins show a greater degree of disorder for both Gram-negative and Gram-positive bacteria (Figure 1). Correspondingly, DisEMBL was chosen by the iterative scheme as an important feature for both prediction methods. This difference in disorder is an interesting observation, which could perhaps be attributed to a difference in the functional range of secreted proteins to that of cytoplasmic proteins.
Table 2a lists the features selected for the predictor for Gram-positive proteins. For prediction of Gram-negative non-classically secreted proteins, arginine contents, DisEMBL [41], instability index [42] and a specially designed amino acid composition network (see Materials & Methods) were selected as features (see Table 2b). Several amino acid composition based features were selected for both predictors confirming previous results, which have demonstrated the importance of amino acid composition in relation to this problem [31,32,43-46].
Others have published prediction methods for the subcellular localisation of proteins, but these were based solely on amino acid composition [44,47]. Furthermore, neither of the methods were developed with the aim of discovering non-classically secreted proteins, and neither of them seem to be publicly available (June 2005). Mammalian secretory proteins can also be classified to a certain extent based on amino acid composition [36].
Besides amino acid composition based features, structural features improve classification performance. Both PSIPRED (secondary structure prediction) and TMHMM (transmembrane helix prediction) were selected by the neural networks for their discriminatory value (see Methods section) when identifying secreted proteins from Gram-positive bacteria.
Prediction results for known non-classically secreted proteins
Although the human pathogens M. tuberculosis and M. smegmatis are both from the phylum Actinobacteria, we have grouped them with the firmicutes as they stain positive in Gram staining. The tuberculosis causing M. tuberculosis has been studied intensively and a few proteins have been found to be secreted through a truly Sec independent pathway as described above.
The first case of non-classical secretion described in bacteria was a glutamine synthetase [12]. This protein [Swiss-Prot:POA590] was correctly predicted as secreted by our method. We tested a known cytoplasmic glutamine synthetase from B. subtilis [Swiss-Prot:P12425], which received a correct negative prediction with a score of 0.109. The localisation of glutamine synthetase to the cytoplasm in B. subtilis has previously been demonstrated [26].
Both the early secretory antigenic target proteins from M. tuberculosis, ESAT-6 and CFP-10, are classified as being secreted using the SecretomeP method. They obtain high scores of 0.557 and 0.813, respectively. The superoxide dismutase (SodA) is secreted via non-classical means in M. tuberculosis, whereas the homolog in M. smegmatis is not. However, a score indicating secretion of SodA from M. tuberculosis was not obtained.
Most of the reported examples of non-classical secretion in Gram-positive bacteria originate from B. subtilis (Table 1). The B. subtilis proteins with known extracellular functions (CwlC, FlgK, FliD, XepA, XkdG, XkdK, XkdM, XlyA) [27] receive high scores when evaluated by the prediction method except for two (Hag & XkdG).
The catalase KatA from B. subtilis was shown to be secreted independently of the Sec-secretion apparatus [9] under normal cellular conditions, but also during prolonged starvation [25]. KatA correctly receives a score of 0.759 in B. subtilis using the SecretomeP prediction method. In Legionella pneumophila, it has been shown that KatA is critical for stationary-phase redox reactions in the periplasm [29], correspondingly, L. pneumophila KatA scores 0.935.
The E. coli cytotoxin ClyA [Swiss-Prot:P77335] is the only reported example of non-classical protein secretion in Proteobacteria. The prevalence in other phyla prompted us to train a prediction method for Gram-negative bacteria as well. As for Gram-positive bacteria, we have determined a combination of discriminative features, which allows for correct classification of secretory and cytoplasmic proteins.
Despite being able to test on only one true example of non-classical secretion from this group of organisms, we expect performance values on the order of those obtained for the prediction method for Gram-positive bacteria. Since the performance on the independent examples from Gram-positive bacteria meets our expectations from the cross-validated test sets, we have no reason to believe this will not be the case for the Gram-negative prediction method. However, the sole example, cytotoxin ClyA, receives a somewhat low prediction score of 0.225.
A number of mammalian sequences are known to be non-classically secreted [48]. We searched the proteomes of B. subtilis and E. coli, but most of the mammalian proteins have no close bacterial homologs.
An exception is the human thioredoxin family protein [Swiss-Prot:P10599], for which both bacterial strains have reasonably close homologs with approximately 30% shared amino acid residues. However, none of the bacterial homologs are predicted to be secreted using the SecretomeP prediction method.
PSORTb version 2 [49] classified correctly as 'extracellular' five of the proteins with known extracellular function, while the remaining were classified either as 'cytoplasmic' or 'unknown'. However, PSORTb was not developed with the aim of identifying non-classically secreted proteins.
Secretion, lysis, or leak?
At least one of studies that report known cytoplasmic proteins in the extracellular environment suggests lysis as result of experimental handling to be the cause [28]. Furthermore, it has been observed that B. subtilis is capable of causing lysis to surrounding cells in order to postpone sporulation [50].
Several of the B. subtilis proteins reported to be detected extracellularly are very abundant in the cytoplasm [26]. This observation leads to the speculation that very abundant cytoplasmic proteins leak to the extracellular milieu. These proteins all receive low scores from the SecretomeP 2.0 method (Table 1). Nonetheless, several proteins are repeatedly detected in the extracellular environment – even in different species. We believe that these proteins could be secreted proteins even though the secretion system is unknown and that system could, indeed, be several different systems.
Cross species comparison
Prompted by the seemingly different localisation pattern of the superoxide dismutase (SodA) in different organisms, we have examined a few other proteins for the same property. Submitting homologous proteins from different bacterial species to the method revealed mostly similar secretion patterns. As seen from Figure 2, KatA is predicted to be secreted in four of the bacterial species investigated. To our knowledge, it is currently unknown whether KatA in M. tuberculosis has an extracellular function as observed for L. pneumophila.
The chaperone GroEL has been shown to have a "moonlighting" function in L. monocytogenes [21]. Whether this is an ubiquitous property of the protein is difficult to assess as all examined orthologs receive similarly low prediction scores. This could support the notion that GroEL is predominantly cytoplasmic. Finally, the glutamine synthetase (GlnA) which has a known extracellular role in M. tuberculosis [11,12], is predicted to have a cytoplasmic role in all the other bacterial species inspected here.
The Gram-negative pathogen Pseudomonas aeruginosa was recently submitted to a thorough computational and experimental analysis to determine its secretome. It was shown that 19.4% of the proteome is secreted [51]. Most of the secreted proteins were carrying a cleavable signal peptide. Using our method, a similar proportion (13.4%) of the Pseudomonas aeruginosa proteome obtains a score above 0.5 thus indicating secretion.
We inspected the proteomes of E. coli and B. subtilis for proteins entering a non-classical secretory pathway using SecretomeP. After having removed sequences with a predicted signal peptide using SignalP 3.0 [32], the 100 highest scoring proteins from both organisms were investigated. Lists from both bacterial strains contained many proteins with possible extracellular functions. For E. coli, we found that 51 of the proteins were hypothetical or with unknown function. Six were membrane-associated and eight were annotated as being related to flagellar function. The remaining 35 had other annotations.
For the Gram-positive bacterium B. subtilis, 56 of the 100 high scoring proteins had no functional annotation. Five sequences were annotated as membrane associated and four were involved in antibiotic resistance. The remaining 35 of the potentially non-classically secreted proteins in B. subtilis had other annotations. These lists are available as supplementary from our website [37].
Experimental verification of the predicted extracellular localisation of these proteins is obviously needed. As mentioned, non-classically secreted proteins have previously been observed to localise extracellularly only occasionally and have known cytoplasmic functions, thereby displaying functional roles in both environments. This means that designing an experiment, which matches the conditions required for the predicted proteins to migrate across the cellular membrane is not necessarily an easy task.
Conclusion
We have compiled a list of bacterial proteins observed in the extracellular medium despite their apparent lack of a signal peptide. The list is based on an exhaustive literature search and we believe it to be almost complete. Non-classical secretion occurs in several different bacterial species and for a diverse group of proteins.
Furthermore, we present a novel method for prediction of non-classically secreted proteins in Gram-positive and Gram-negative bacteria. With high confidence based on a number of protein features, the method classifies as secreted most of the proteins reported to have an extracellular function. Coincidentally, most of the proteins which obtain low scores with our method have no currently known extracellular function. In summary, non-classical protein secretion is clearly supported by overwhelming evidence, and although the route(s) of export are still unclear, we are able to predict some of these proteins based on features which they share with classically secreted proteins. Other routes of export may allow a set of protein properties different from those of classically secreted proteins. This could explain the low scores some of the proteins reported to be non-classically secreted obtain with our method.
Methods
Generation of data sets
Ideally, our positive data set should consist of a large number of proteins secreted via non-classical pathways. Unfortunately, it was not possible to obtain a sufficiently large data set as only a small number of proteins undergoing non-classical secretion are known. Since we are looking for features shared among extracellular proteins, the mechanism by which a protein is secreted should not be important. We therefore used for training the large number of proteins known to be secreted via the classical Sec-dependent secretion mediated mechanism. All sequence data was extracted from Swiss-Prot release 44.0. Two individual training sets were created for Firmicutes and Proteobacteria, respectively.
A set of 690 extracellular proteins from Firmicutes (Gram-positive) and a set of 2185 extracellular proteins from Proteobacteria (Gram-negative) were extracted from the Swiss-Prot database based on annotations in the feature table (FT) and comments line (CC) [52]. Partial sequences were excluded from the data set. As we wanted to train a predictor that works in the absence of signal peptides, the signal peptide part of each sequence was removed according to the Swiss-Prot annotation. These lists of secreted proteins formed our positive data sets. Negative training sets were constructed by extracting 1084 proteins for Firmicutes and 2098 proteins for Proteobacteria from Swiss-Prot, which were annotated as localised to the cytoplasm. After redundancy reduction of the data sets based on a structural similarity criteria [53], 152 and 350 extracellular sequences were left in the positive data sets for Firmicutes and Proteobacteria, respectively. In the negative data sets, 140 and 334 sequences remained for Firmicutes and Proteobacteria, respectively. For Gram-positive bacteria (Firmicutes and Actinobacteria) a set of non-classically secreted proteins was retrieved from Swiss-Prot based on literature searches (see Table 1).
All data sets used are available as supplementary information from our website [37].
For identification of putative non-classically secreted proteins in E. coli and B. subtilis, we used the following accession numbers to extract the annotated and translated proteomes: [Genbank:NC_000913] for E. coli and [Genbank:NC_000964] for B. subtilis.
Neural network architecture and feature integration
The construction of a non-classical secretion predictor based on protein features followed the scheme from [33,36]. Briefly, the procedure included: 1) Calculating and assigning the protein features for each protein sequence, 2) Encoding features for processing by a neural network, 3) Training neural networks using three-fold cross validation and various combinations of features, and 4) Determining the combination of features yielding the best performance based on correlation coefficient.
An extra feature predictor was constructed prior to network training. This feature was based on amino acid composition alone (inspired by Reinhardt and Hubbard [44]) and aimed at distilling into a single score information about the specific contents of all amino acids, thereby keeping the input dimensionality in feature space low. Care was taken to prevent hidden optimisation from taking place by ensuring that this feature followed the cross-validation scheme.
The prediction methods assigns a score to each protein between 0 and 1, where a score above 0.5 is considered indicative of secretion. 0.5 was chosen as the cut-off value for discrimination as this was the value used during training.
Authors' contributions
JDB carried out sequence retrieval and drafted the manuscript. LK performed pattern finding, neural network training (with AF) and assisted with the drafting of the manuscript. AF was responsible for the amino acid composition network and the figure layout. SB provided general inputs and improvements to the manuscript.
Acknowledgements
This work was supported by grants from the Danish National Research Foundation, the Danish Natural Science Research Council, and the Danish Center for Scientific Computing.
Figures and Tables
Figure 1 Bacterial secreted proteins are more disordered in structure than cytoplasmic proteins. The predicted number of coils (average per residue) by DisEMBL is higher for secreted proteins than for cytoplasmic ones. The tendency also holds for the two other measures of disorder predicted by DisEMBL (not shown).
Figure 2 Prediction on different proteins in different organisms. Four proteins from five bacterial species. Scores above 0.5 indicate predicted secretion of that particular protein. For details, please refer to the text.
Figure 3 Putative non-classical secretory proteins. The top 100 scoring proteins are grouped based on annotation. Several groups have obvious relations to extracellular functions. For proteins with no annotation (grouped in 'Unknown'), our prediction method suggests an extracellular role. Proteins grouped in 'Other' may have an extracellular function, although this is not apparent in the current annotation.
Table 1 Non-classical secretory proteins in bacteria. The table lists proteins known to be localised extracellularly in Gram-positive bacteria. Many of the proteins are found localised to the cell surface, where as others are found in the surrounding media. Only one study distinguish between cell surface and extracellular localisation of the proteins [21]. ClyA is the only protein from Gram-negative bacteria reported to be non-classically secreted. Proteins listed above the horizontal line have known extracellular functions (see text for details). Abbreviations: Ex – Extracellular, Cs – Cell surface, BS – B. subtilis, MT – M. tuberculosis, LM – L. monocytogenes, EC – E. coli. aCytoplasmic abundance of proteins (% of total protein in the cell) in B. subtilis ('-' no data available) [26].
%a Protein Score Function or similarity Species Location Reference
- FliD 0.845 Flagellar hook-associated protein 2 BS Ex [25]
- EsxB 0.813 CFP-10 MT Ex [13]
- FlgK 0.795 Flagellar hook-associated protein 1 BS Ex [25]
- XkdK 0.733 PBSX prophage gene BS Ex [25]
- XlyA 0.684 Amidase (PBSX prophage lysin) BS Ex, Cs [25,54]
- CwlC 0.634 N-Acetylmuramoyl-L-alanine amidase BS Ex, Cs [25]
- EsxA 0.557 ESAT-6, 6 kDa early secretory antigenic target MT Ex [17]
- XepA 0.545 PBSX prophage lytic exoenzyme BS Ex [25]
- XkdM 0.544 PBSX prophage gene BS Ex [25]
- GlnA 0.539 Glutamine synthetase 1 MT Ex [11,12]
- ClyA 0.225 Cytotoxic protein EC Ex [23,24]
1.27 Hag 0.218 Flagellin protein BS Cs [9,25]
1.07 SodA 0.209 Superoxide dismutase [Fe] M. tuberculosis BS, MT, LM Ex [6,9,18,28]
- XkdG 0.090 PBSX prophage gene BS Ex [9]
- ManA 0.832 Phosphomannose isomerase LM Cs [21]
- KatA 0.759 Vegetative catalase 1 BS Ex [9,25,29]
- SodA 0.701 Superoxide dismutase BS Ex [9,28]
- YceD 0.551 Similar to tellurium resistance protein BS Ex [25]
- DnaK 0.375 Heat shock protein LM Cs [6,21]
- PdhC 0.152 Pyruvate dehydrogenase (E2 subunit) LM Ex, Cs [6,21]
0.71 PdhA 0.126 Pyruvate dehydrogenase (El α subunit) BS Ex [28]
1.20 CitH 0.118 Malate dehydrogenase BS Ex [25,28]
1.20 Gap 0.118 Glyceraldehyde-3-phosphate dehydrogenase BS, LM Ex, Cs [6,9]
1.23 Eno 0.108 Enolase BS, LM Cs [6,21,25,28]
- RpoB 0.100 RNA polymerase β subunit LM Ex, Cs [21]
- RocF 0.082 Arginase BS Ex [25,28]
5.17 EF-Tu 0.075 Elongation factor Tu LM Cs [6,21]
- RS9 0.073 Ribosomal protein S9 LM Cs [21]
- RocA 0.070 Pyrroline-5 carboxylate dehydrogenase BS Ex, Cs [25]
1.91 Ef-G 0.070 Elongation factor G BS, LM Ex, Cs [6,25,28]
- RpoC 0.059 RNA polymerase β' subunit LM Ex, Cs [21]
0.76 PdhD 0.052 Pyruvate dehydrogenase (E3 subunit) BS, LM Ex [6,25,28]
- RL19 0.050 Ribosomal protein L19 LM Cs [21]
0.57 PdhB 0.047 Pyruvate dehydrogenase (El β subunit) BS Ex [25,28]
1.30 GroEL 0.035 Class I heat shock protein (chaperonin) BS Ex, Cs [21,28]
Table 2 Protein features found to be discriminative for identification of non-classically secreted Gram-positive (a) and Gram-negative (b) bacterial proteins
Feature Program Reference
Threonine contents
Composition see Materials & Methods
Transmembrane helices TMHMM 2.0 [55]
Gravy ExPASy, ProtParam [56]
Protein disorder DisEMBL [41]
Secondary structure PSIPRED [57]
Feature Program Reference
Arginine contents
Composition see Materials & Methods
Instability index ExPASy, ProtParam
Protein disorder DisEMBL [41]
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BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-5-611622568010.1186/1471-2180-5-61Research ArticleComparison between Gram stain and culture for the characterization of vaginal microflora: Definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora Verhelst Rita [email protected] Hans [email protected] Geert [email protected] Gerda [email protected] Simaey Leen [email protected] Ganck Catharine [email protected] Backer Ellen [email protected] Marleen [email protected] Mario [email protected] Department Clinical Chemistry, Microbiology & Immunology, Ghent University Hospital, UGent, Ghent, Belgium2 Department of Obstetrics & Gynaecology, Ghent University Hospital, UGent, Ghent, Belgium2005 14 10 2005 5 61 61 25 1 2005 14 10 2005 Copyright © 2005 Verhelst 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 microbiological diagnosis of bacterial vaginosis is usually made using Nugent's criteria, a useful but rather laborious scoring system based on counting bacterial cell types on Gram stained slides of vaginal smears. Ison and Hay have simplified the score system to three categories and added a fourth category for microflora with a predominance of the Streptococcus cell type. Because in the Nugent system several cell types are not taken into account for a final score, we carried out a detailed assessment of the composition of the vaginal microflora in relation to standard Gram stain in order the improve the diagnostic value of the Gram stain. To this purpose we compared Gram stain based categorization of vaginal smears with i) species specific PCR for the detection of Gardnerella vaginalis and Atopobium vaginae and with ii) tDNA-PCR for the identification of most cultivable species.
Results
A total of 515 samples were obtained from 197 pregnant women, of which 403 (78.3%) were categorized as grade I microflora, 46 (8.9%) as grade II, 22 (4.3%) as grade III and 8 (1.6%) as grade IV, according to the criteria of Ison and Hay. Another 36 samples (7.0%) were assigned to the new category 'grade I-like', because of the presence of diphtheroid bacilli cell types. We found that 52.7% of the grade I-like samples contained Bifidobacterium spp. while L. crispatus was present in only 2.8% of the samples and G. vaginalis and A. vaginae were virtually absent; in addition, the species diversity of this category was similar to that of grade II specimens.
Based on the presence of different Lactobacillus cell types, grade I specimens were further characterized as grade Ia (40.2%), grade Iab (14.9%) and grade Ib (44.9%). We found that this classification was supported by the finding that L. crispatus was cultured from respectively 87.0% and 76.7% of grade Ia and Iab specimens while this species was present in only 13.3% of grade Ib specimens, a category in which L. gasseri and L. iners were predominant.
Conclusion
Further refinement of Gram stain based grading of vaginal smears is possible by distinguishing additional classes within grade I smears (Ia, Iab and Ib) and by adding a separate category, designated grade I-like. A strong correlation was found between grade Ia and the presence of L. crispatus and between grade I-like and the presence of bifidobacteria. This refinement of Gram stain based scoring of vaginal smears may be helpful to improve the interpretation of the clinical data in future studies, such as the understanding of response to treatment and recurrence of bacterial vaginosis in some women, and the relationship between bacterial vaginosis and preterm birth.
==== Body
Background
Currently the criteria as defined by Nugent et al. [1] are considered as the standard procedure to score vaginal smears by Gram stain [2]. This method scores the smears in a standardized manner by quantification of some of the cell types present – designated as Lactobacillus, Gardnerella vaginalis, Bacteroides and Mobiluncus 'morphotypes'. However, the Nugent scoring system conflates women with potentially very different vaginal microflora in a single category [3]. Since the method requires considerable time and skill, simpler versions have been described which assess the categories in a more qualitative manner [4-6]. Recent developments in our knowledge of the vaginal microflora – including the observation of different Lactobacillus species producing different amounts of hydrogen peroxide [7-9] with a potential effect on pregnancy outcome [10,11] urge to refine the Gram stain criteria in an effort to increase the agreement between Gram stain and the true composition of the vaginal microflora. In addition, a strong association of the metronidazole resistant fastidious anaerobic coccobacillus Atopobium vaginae with bacterial vaginosis [12-14] might have important implications in the pathophysiology of bacterial vaginosis related preterm labour and birth. The more accurate allocation of subjects according to vaginal microflora status, as assessed by Gram stain, may enhance the validity of studies on the etiology of bacterial vaginosis, and help to better understand response to treatment and recurrence in some women, as well as its relation to preterm birth.
Here we report our findings obtained by studying a total of 515 vaginal samples by Gram stain, by DNA-based techniques – like cloning and sequencing of amplified 16S rRNA-genes [13-15] and species specific PCR [12,15-18] – which make it possible to detect fastidious bacteria like A. vaginae [13,16,17] and by culture in combination with tDNA-PCR [20,21], which allows the rapid identification of large numbers of cultured isolates, including isolates from different Lactobacillus species [22]. Based on these findings, we propose refined criteria to categorize the status of the microflora of vaginal smears.
Results
We studied the composition of the vaginal microflora of 515 vaginal swabs from a prospective cohort of 197 unselected pregnant women at three time points during pregnancy using i) Gram stain based grading according to modified Ison & Hay criteria [6] – which will be further denoted here as the criteria of Claeys, ii) culture in combination with molecular identification of cultured organisms by tDNA-PCR and iii) species specific PCR for G. vaginalis and A. vaginae.
Detailed observation of the Gram stained vaginal smears in combination with specific PCR and tDNA-PCR based identification of cultured isolates led to subdivision of grade I samples and the recognition of a separate category, designated grade I-like: Grade I specimens were characterized as grade Ia when only Lactobacillus crispatus cell types (plump, mostly short rods) were present (Figure 1a – 1b), as grade Ib when only other Lactobacillus cell types were present (smaller or more elongated and less stained than in Ia smears)(Figure 1c – 1d) and as grade Iab when both L. crispatus and other lactobacilli were present (Figure 1e – 1f). Furthermore a number of samples were designated as grade I-like because of the presence of Gram positive rods, either quite small and short or otherwise irregularly shaped with clubbing, curved edges and irregular staining and often arranged like Chinese letters ('diphtheroid cell types') (Figure 1g – 1h). To corroborate that grade I-like samples indeed represent a separate class, cloning was carried out for two samples that had been categorized as grade I-like. For completeness, figures 1i – 1j represent grade II vaginal smears and figures 1k – 1l represent grade III vaginal smears.
Comparison between Gram stain and culture
Using the criteria of Claeys, 162 vaginal smears were scored as grade Ia, 181 as grade Ib, 60 as grade Iab, 36 as grade I-like, 46 as grade II, 22 as grade III and eight as grade IV (Table 1).
We cultured 1108 isolates anaerobically out of the 515 vaginal swabs and identified these with tDNA-PCR. A total of 136 isolates remained unidentified, since no corresponding tDNA-PCR fingerprint could be found in the database or because no amplification was obtained. A total of 72 species were identified, of which 17 belonged to the genus Lactobacillus and six to the genus Bifidobacterium (Table 1). The most common species recovered from grade Ia, Ib and Iab specimens were lactobacilli. L. crispatus (87.0%) and L. jensenii (22.2%) were the most abundant bacteria in grade Ia samples, whereas L. gasseri (32.0%) and L. iners (39.8%) were the most frequently present species in grade Ib specimens. Grade I-like specimens were found to contain mostly bifidobacteria (54.9%) and L. gasseri (52.8), while L. crispatus was almost absent (2.8%). In 19.8% of grade I-like specimens bifidobacteria were present while lactobacilli were absent. Bifidobacteria were more frequent in grade I-like samples than in other samples (χ2 = 120.6, p < 0.001, Table 2).
L. crispatus was present in 87.0% of grade Ia, 76.7% of grade Iab and 37.5% of grade IV samples but in less than 13.3% in all other grades. L. crispatus was the only Lactobacillus species that was linked to a single grade, namely grade Ia (χ2 = 186.3, p < 0.001), while the other lactobacilli were more evenly distributed over all samples (Table 3, 4, 5, 6). L. jensenii was the second most abundant species in grade Ia (22.2%), but was also frequent in most other grades, for example in 47.8% of grade II. L. vaginalis, the third most abundant species in grade Ia (9.3%) was absent from grade III and present in less than 20% of all other grades. L. gasseri and L. iners were more abundant in grade Ib (32.0 and 39.8%), grade I-like (52.8 and 19.4%), grade II (54.3 and 26.1%) and grade III (9.1 and 31.8%) than in grade Ia (6.8 and 3.7%).
The most characteristic cultured organisms in grade II and grade III specimens were G. vaginalis (respectively 21.7% and 72.7%) (χ2 = 120.6, p < 0.001, Table 7), Actinomyces neuii (respectively 6.5% and 9.1%), Aerococcus christensenii (respectively 4.3% and 22.7%), A. vaginae (respectively 4.3% and 13.6%), Finegoldia magna (respectively 2.2% and 9.1%) and Varibaculum cambriense (respectively 2.2% and 13.6%). These were virtually absent from grade I and grade IV, although G. vaginalis was present in approximately 2.0% of grade I samples. L. jensenii (47.8%) and L. gasseri (54.3%) were the most common lactobacilli in grade II specimens. Furthermore, whereas L. crispatus and L. vaginalis were never cultured from grade III specimens, L. iners (31.8%) was the lactobacillus mostly present in grade III. Mobiluncus curtisii and Peptostreptococcus sp. were cultured from grade III specimens only (both 4.5%). Dialister sp. (22.7%) and Prevotella spp. (22.6%) were frequently cultured from grade III specimens and only sporadically from other specimens.
The average number of species cultured per sample ranged from 1.5 for grade Ia specimens to 3.6 for grade III specimens (Table 8). Overall, the species diversity of the grade I-like category was higher (0.83) than that of the grade I subcategories (0.17, 0.21 and 0.30 for grades Ia, Ib, and Iab respectively) and comparable to that of the grade II category (0.76). The grade III category had the highest species diversity (1.50) (Table 8).
Comparison between Gram stain and species specific PCR for Gardnerella vaginalis and Atopobium vaginae
The series of 515 vaginal samples were analyzed by PCR with 16S rRNA gene based primers specific for A. vaginae and 16S–3S spacer primers specific for G. vaginalis.
After amplification with the ato167f A. vaginae primer set, respectively 14.7% of grade I, 8.3% of grade I-like, 28.3% of grade II, 86.4% of grade III and 12.5% of grade IV samples showed an amplicon. The percentage of positive samples for G. vaginalis specific PCR was respectively 28.9%, 19.4%, 47.8%, 86.4% and 12.5%.
The simultaneous presence of A. vaginae and G. vaginalis in a vaginal swab specimen had an accuracy of 90% [95% CI: 86–92%], a sensitivity of 82% [95% CI: 59–94%], a specificity of 90% [95% CI: 87–92%], a positive predictive value of 26% [95% CI: 17–39%] and a negative predictive value of 99% [95% CI: 98–100%] in assessing bacterial vaginosis (defined as a grade III smear).
Comparison between culture and cloning of grade I-like samples
Cloning of two grade I-like samples from trimesters 1 and 2 of the same patient, revealed the presence of Bifidobacterium breve (respectively 33.1 and 53.5%), Lactobacillus delbrueckii (64.8 and 13.3%) and L. gasseri (2.1 and 33.1%) clones. This was in agreement with the culture results which revealed the presence of B. breve in both trimesters, L. delbrueckii only in the first and L. gasseri only in the second.
In general, grade I-like samples were found by culture to contain more frequently Bifidobacterium (19/36 samples) and more different Bifidobacterium species (6) than samples from all other categories. Of the Bifidobacterium species, B. breve was most clearly associated with grade I-like, grade II and grade III.
Discussion
The importance of correct diagnosis of bacterial vaginosis and of more detailed characterization of the vaginal microflora
Although not causing a vaginal inflammatory response, bacterial vaginosis is considered to be the most common cause of vaginitis in pregnant and non-pregnant women and prevalences between 4.9% and 36.0% have been reported from European and American studies [23]. Several studies suggest the possibility that bacterial vaginosis increases the risk of acquiring HIV [24,25] and that the bacterial flora associated with bacterial vaginosis increases genital-tract HIV shedding [26]. A recent meta-analysis by Leitich et al. [27] established an odds ratio of 8 for preterm birth in association with bacterial vaginosis during early pregnancy. Spontaneous preterm birth occurs in 7–11% of pregnancies but accounts for three quarters of perinatal morbidity and mortality and for half of long term neurological impairment in children [28,29].
Bacterial vaginosis is characterized by the replacement of the normal vaginal microflora of lactobacilli by Gardnerella vaginalis and anaerobic organisms. Recently, different groups showed that the strict anaerobe Atopobium vaginae is another organism that is strongly associated with bacterial vaginosis [12,13,16,17]. The association between A. vaginae and bacterial vaginosis might help explain why some women suffer from recurrent bacterial vaginosis. For example, a recent study pointed to great in vitro efficacy of metronidazole, since this antibiotic inhibited growth of 99% of the vaginal isolates from bacterial vaginosis samples [30], but most likely overlooked the fastidious metronidazole resistant A. vaginae, shown in this study to be present in 86.4% of bacterial vaginosis samples when detected with species specific PCR.
Given the possibility that certain not yet characterized subgroups within the presumably heterogenic clinical entity of women with bacterial vaginosis could identify a group at higher risk for preterm birth than women with bacterial vaginosis as a whole and that adequate treatment of women from this higher risk group may allow for more targeted preterm birth prevention, better understanding of the composition and dynamics of the vaginal microflora and accurate diagnosis of bacterial vaginosis are warranted. Also, our data indicate that refined characterization of vaginal microflora may be necessary for more accurate interpretation of the results of clinical studies. For example, thus far Atopobium vaginae has been overlooked in clinical studies and furthermore, the fact that different Lactobacillus species may confer different strengths of colonisation resistance [10,11] has not been taken into account, partly because most laboratories lack the access to rapid and accurate methods for the identification of lactobacilli to the species level. In other words, several studies concerning the relation between the status of the vaginal microflora and different gynecologic and obstetric diseases and their treatments thus far may have reached biased conclusions due to insufficiently precise characterization of the microflora.
Criteria for microbiological categorization of vaginal microflora status
Spiegel et al. [31] defined a scoring system based on some of the bacterial cell types that can be seen in Gram stained smears of vaginal secretion. This was later refined by Nugent et al. [1], who provided a scoring system that evaluates the changes in vaginal microflora, from the normal condition to bacterial vaginosis status, as a continuum. Although the Nugent criteria have gained wide acceptance for the evaluation of the condition of the vaginal microflora [2,32], further refinement is warranted for several reasons. First, no definite criteria have been described to distinguish the Lactobacillus cell types from the Gardnerella and Bacteroides-Prevotella cell types. In practice and in our experience, 'morphotypes' are often difficult to assign to one of these groups. Also, some genera and species that are clearly associated with bacterial vaginosis, like Peptostreptococcus spp. [32] and A. vaginae [12,17,13] are not included in the Nugent score. Furthermore, Forsum et al. [2] found major discrepancies in scoring when the lactobacillary cell types were few in number and Larsson et al. [33] reported several problems in the interpretation of smears. For example, using the Nugent criteria, the presence of different Lactobacillus cell types in smears from patients with bacterial vaginosis can lead to assignation to grade II, whereas patients without bacterial vaginosis but with smears with more than 300–500 pleomorphic Lactobacillus cells may be regarded as containing G. vaginalis, also because some of these cells are very small. Additionally, the Nugent scoring system conflates women with potentially very different vaginal microflora in a single category [3].
In this study, the clinical microbiologist (GC) could not grade some of the smears due to the presence of cell types not scored in the system developed by Nugent [1] and classified these samples as grade I-like. Further detailed observation lead to the splitting up of grade I samples into subcategories designated grade Ia, grade Ib and grade Iab. After blind grading of the vaginal smears into grades Ia, Ib, Iab, I-like, II, III and IV, this classification was compared with the culture results and with species specific PCRs.
Grade Ia and Iab: Agreement with the presence of L. crispatus
From this comparison it became obvious that it is possible to recognize the presence of L. crispatus by means of Gram stain, since this species was cultured in 81.9% of the grade Ia samples and 76.7% of the grade Iab samples. Nevertheless, L. crispatus was not cultured from 21 of the 162 grade Ia samples. This may be explained by the fact that L. crispatus is not as easily cultured as other lactobacilli. Indeed, L. crispatus colonies were quite often observed as satellites of other bacteria and in some cases no growth at all was observed in samples with numerous L. crispatus-like lactobacilli on Gram stain. Using non culture dependent t-RFLP-analysis (data not presented) the Ia samples negative for L. crispatus culture were tested and 16 were positive for L. crispatus, bringing the agreement between Gram stain grading as grade Ia and the presence of L. crispatus to 96.9%. Similarly, when taking into account t-RFLP-analysis results, the agreement between categorization as grade Iab and t-RFLP-analysis positive for L. crispatus was 92.9% whereas L. crispatus was detected by t-RFLP-analysis only in 27.3%, 20.0%, 22.5% and 0% for grades Ib, I-like, II and III, respectively. These results indicate that – for a trained microbiologist – it is possible to recognize L. crispatus bacteria upon cell morphology, a finding that is of importance since this species is clearly associated with healthy microflora, and possibly better ensures stable healthy microflora than other lactobacilli [9]. Samples were scored as grade Ib when no L. crispatus cell types were observed, but other Lactobacillus cell types were predominant. The agreement with culture results was high: only 13.3% contained L. crispatus upon culture, whereas L. gasseri, L. iners, and L. jensenii were present in respectively 32.0, 39.8, and 24.3% of the grade Ib samples. These were clearly grade I samples since bacterial vaginosis-associated organisms were mostly absent.
The colonisation resistance conferred differs between Lactobacillus species
Overall the frequency of isolation of all Lactobacillus species together was comparable for the different grades in our population, since lactobacilli were cultured from 96.9% of grade Ia, 94.5% of grade Ib, 96.7% of grade Iab, 78.9% of grade I-like, 93.5% of grade II, 59.1% of grade III and 62.5% of grade IV samples. This is in agreement with previous reports [32,34]. However, we observed a clear difference with regard to the Lactobacillus species frequency distribution for the different grades. While L. crispatus, known as a strongly H2O2-producing species [7,8], was cultured from 87.0% of grade Ia specimens, it was absent in grade III specimens and only present in 2.8% of grade I-like specimens. In contrast, L. iners, reported as a weakly H2O2-producing species [7,8], was present in only 3.7% of Ia specimens but in 39.8% of grade Ib and 31.8% of grade III specimens. Whether it is the hydrogen peroxide production by L. crispatus that confers colonisation resistance remains a matter of debate, since a correlation between the presence of hydrogen peroxide production and the type of vaginal microflora was found by some [35], though not by others [36]. Possibly other species specific characteristics, present in L. crispatus, but absent in species like L. gasseri and L. iners, confer colonisation resistance. It has also been hypothesized that the onset of perturbation leading to bacterial vaginosis may be due to competition between Lactobacillus species [36], a situation possibly reflected by grade Iab specimens.
Grade I-likes: a separate category of vaginal microflora status
A number of samples were initially difficult to score because the predominant cell types could not be categorized as Lactobacillus, Gardnerella, Bacteroides-Prevotella or Mobiluncus cell types. These samples were considered as belonging to a separate category because of the presence of Gram positive rods, either quite small and short or otherwise irregularly shaped with clubbing, curved edges and irregular staining and often arranged like Chinese letters ('diphtheroid cell types'). Since it is likely that most microbiologists would score this cell type as 'Lactobacillus-like' and that therefore it would be scored in most cases as grade I, we designated it as 'grade I-like'. Culture and species specific PCR confirmed that indeed these samples represent a separate kind of vaginal microflora. This is reflected by the increased species diversity of 0.83, which is much higher than that for grades Ia, Ib and Iab (0.15–0.30) and which is comparable to that of grade II (0.76), but even more so by the virtual absence of L. crispatus (cultured from only one of 36 samples) as well as of G. vaginalis and A. vaginae (cultured from respectively 1 and 0 samples) and the presence of Bifidobacterium spp. in 19 of 36 samples, a much higher prevalence than in samples from all other grades. This was confirmed by cloning of two grade I-like samples, which contained only L. delbrueckii, L. gasseri and B. breve.
Rosenstein et al. [34] mentioned a category of vaginal smears with aberrant morphology, which they designated as grade I revertants. At first sight, their category shows resemblance with the category we describe here as I-like, because of low numbers of G. vaginalis and increased numbers of bifidobacteria, but on the other hand they reported even more bifidobacteria in their grade II and grade III samples and they designated this category as grade I revertants because the vaginal microflora of all 41 women with such smears reverted to grade I, which was not the case in our study (data to be presented elsewhere).
Importantly, since Gram stain based categorizing can result in the interpretation of grade I-like samples as genuine grade I samples (whereof their designation), this class of samples may jeopardize – and probably has done so in the past – the interpretation of the results of clinical studies.
Grade II: a microbiologically intermediate stage between healthy microflora and bacterial vaginosis
Our results confirm that grade II samples represent a microbiologically clearly intermediate status between grade I and III. L. crispatus is still present in 10.9% of the samples (compared to 59.0% of grade I and 0% of grade III samples), whereas the number of samples with L. gasseri (54.3%) is increased compared to grade I (21.3%) and grade III (9.1%). Species diversity of grade II is intermediate between that for grade I and grade III and species typically associated with bacterial vaginosis, like A. neuii, A. christensenii, A. vaginae, B. ureolyticus, F. magna, G. vaginalis, Peptoniphilus sp. and V. cambriense, are present, but again in a lower number of samples than in grade III specimens.
Grade III: Characterization of bacterial vaginosis -related organisms
The following species are generally considered as bacterial vaginosis related anaerobe organisms: Anaerococcus (Peptostreptococcus) tetradius, A. (Peptostreptococcus) vaginalis, Atopobium vaginae, Bacteroides ureolyticus, Finegoldia (Peptostreptococcus) magna, G. vaginalis, Gemella (Peptostreptococcus) morbillorum, Mobiluncus curtisii, Mycoplasma hominis, Peptoniphilus sp., Peptostreptococcus sp., Prevotella bivia, Prevotella ruminicola and Prevotella sp. [37,38]. Using tDNA-PCR we were able to identify 87.8% of the cultured isolates to the species level and found our results to be largely in agreement, but in addition we cultured Actinomyces neuii, Aerococcus christensenii, Dialister sp. and Varibaculum cambriense, whereas Mobiluncus spp., Mycoplasma hominis and Ureaplasma urealyticum were not or only sporadically cultured from grade II and grade III specimens. The absence of the latter species in our study can be explained by the fact that we did not use the specific culture methods for these fastidious organisms.
In this study we confirmed the strong association, as established previously [12,13,17], between A. vaginae and bacterial vaginosis.
Conclusion
In summary, our characterization of the vaginal microflora by means of detailed Gram stain interpretation and by culture in combination with genotypic identification helps to refine our understanding of normal and disturbed vaginal microflora. We showed that L. crispatus can be recognized as such on Gram stain, we established the existence of a separate additional category, characterized by the absence of L. crispatus and the abundance of bifidobacteria and we confirmed the association of Atopobium vaginae with bacterial vaginosis.
These data have implications for the basic understanding of the vaginal microflora and bacterial vaginosis; in addition, they may add to the value of Gram smear based diagnosis of bacterial vaginosis because of better defined Gram stain criteria.
Methods
Study population and sample collection
A total of 515 vaginal swabs were collected by sampling 197 pregnant women attending our out-patient clinic, each at three time points during pregnancy (respectively 197, 171 and 147 first, second and third trimester samples were collected). The swabs were obtained during the first, second and third pregnancy trimester, at mean gestational ages of 9.1 +/- 3.2 weeks, 20.4 +/- 2.3 weeks and 32.2 +/- 1.7 weeks, respectively.
Sampling was carried out by insertion of three sterile cotton swabs into the vaginal vault, after placement of a non-lubricated speculum. The swabs were rotated against the vaginal wall at the midportion of the vault and were carefully removed to prevent contamination with microflora of the vulva and introitus. The first swab was used to prepare a smear on a glass slide for the purpose of grading according to the criteria of Claeys (this study). The second swab was returned to a sterile tube (Copan, Brescia, Italy), for the purpose of DNA-extraction (dry swab). The third swab was placed into Amies transport medium (Nuova Aptaca, Canelli, Italy) for anaerobic culture. The unstained smear and both swabs were sent to the microbiology laboratory and were processed within 4 hours.
Grading of slides
Smears were dried, Gram stained (Mirastainer, Merck-Belgolabo, Overijse, Belgium) and examined under oil immersion at a magnification of 1000. Gram stained smears from vaginal swabs were all scored by one clinical microbiologist (GC) according to Ison & Hay criteria [5,6]: samples were categorized as grade I when only Lactobacillus cell types (large Gram positive rods) were present, as grade II (intermediate) when both Lactobacillus and Gardnerella or Bacteroides-Prevotella cell types were present, as grade III (bacterial vaginosis) when Lactobacillus cell types were absent and only Gardnerella,Bacteroides-Prevotella or Mobiluncus cell types were present and as grade IV when Gram positive cocci were predominantly present. Further subdivision of grade I samples into categories Ia, Iab and Ib and the description of a separate category, designated grade I-like, is presented in the Results section.
Culture and identification of cultured isolates by tDNA-PCR
For 515 specimens collected from 197 women, the swab on Amies transport medium was streaked onto Schaedler agar enriched with 5% sheep blood, vitamin K, hemin and sodium pyruvate (Becton Dickinson, Franklin Lakes, NJ) and incubated anaerobically at 37°C upon arrival at the microbiology laboratory. After 4 days of incubation, all the isolates with different colony morphology were selected for identification. DNA was extracted by simple alkaline lysis: one colony was suspended in 20 μl of 0.25% sodium dodecyl sulfate-0.05 N NaOH, heated at 95°C for 15 min and diluted with 180 μl of distilled water. tDNA-PCR and capillary electrophoresis were carried out as described previously [20,22]. The species to which each isolate belonged was determined by comparing the tDNA-PCR fingerprint obtained from each isolate with a library of tDNA-PCR fingerprints obtained from reference strains, using an in-house software program [20]. The library of tDNA-PCR fingerprints is available at our website [39] and the software can be obtained upon request.
DNA extraction of vaginal swab samples
For DNA extraction from the dry vaginal swabs, the QIAamp DNA mini kit (Qiagen, Hilden, Germany) was used according to the manufacturer's recommendations, with minor modifications, as described previously [13]. DNA-extracts were stored at -20°C and were used for the purpose of species specific PCR and cloning experiments.
Species specific PCR for Gardnerella vaginalis
G. vaginalis species specific primers as designed by Zariffard et al. (GZ) [19] were used. Briefly, a 20 μl PCR mixture contained respectively 0.05 and 0.4 μM primers, 10 μl of Promega master mix (Promega, Madison, WI), 2 μl of Qiagen DNA-extract of the samples and distilled water. Thermal cycling with GZ primers consisted of an initial denaturation of 10 min at 94°C, followed by 50 cycles of 5 s at 94°C, 45 s at 55°C and 45 s at 72°C, and a final extension of 10 min at 72°C. Five microliter of the amplified product was visualized on a 2% agarose gel.
Species specific PCR for Atopobium vaginae
A primer set that allowed amplification of the 16S rRNA gene of A. vaginae and that lacked homology with non-target bacteria was used as described earlier [13]. Briefly, a 10 μl PCR mixture contained 0.2 μM each of the primers ato167f (5' GCGAATATGGGAAAGCTCCG) and ato587r (5' GAGCGGATAGGGGTTGAGC), 5 μl of Promega master mix (Promega, Madison, WI), 1 μl of Qiagen DNA-extract of the samples and distilled water. Thermal cycling consisted of an initial denaturation of 5 min at 94°C, followed by three cycles of 1 min at 94°C, 2 min at 58°C and 1 min at 72°C, followed by 35 cycles of 20 sec at 94°C, 1 min at 58°C and 1 min 72°C, with a final extension of 10 min at 72°C, and cooling to 10°C. Five microliter of the amplified product was visualized on a 2% agarose gel. The primers amplified a DNA-fragment of 420 base pairs from A. vaginae and showed no cross reactivity to other organisms, including A. rimae and A. parvulum (data not presented).
Cloning of amplified mixtures of 16S rDNA
Cloning and sequencing was carried out largely as described previously [13]. However, to increase the amplification efficiency of the 16S rRNA-genes of G. vaginalis and bifidobacteria the following mixture of primers (0.1 μM each) was used for the initial amplification of the samples prior to cloning: primers 10 f (5' AGTTTGATCCTGGCTCAG), 534r (5' ATTACCGCGGCTGCTGG) and GV10f (5' GGTTCGATTCTGGCTCAG).
Statistical analysis
The Simpson's Diversity Index was calculated as D = 1-∑ (n/N)2 where n is the number of isolates of a particular species and N is the total number of isolates. Chi square analyses were carried out using the statistical software package SPSS v.11.0.
Authors' contributions
RV, GC, GV, EDB and MV participated in the development of the study design, the analysis of the study samples, the collection, analysis and interpretation of the data, and in the writing of the report. HV and MT participated in the development of the study design, the collection of the study samples, the collection, analysis and interpretation of the data, and in the writing of the report. LVS and CDG participated in the analysis of the study samples. All authors read and approved the final manuscript.
Acknowledgements
This work was supported through a research grant by the Marguerite-Marie Delacroix Foundation, the Bijzonder Onderzoeksfonds of the University of Gent (UGent) and the Fund for Scientific Research Flanders (Belgium).
We thank the Culture Collection of the University of Göteborg, Sweden for kindly providing Atopobium vaginae isolates.
Figures and Tables
Figure 1 Microscopic image (100 ×) of Gram-stained vaginal smears illustrating the different categories of vaginal microflora described:. a, b: grade Ia, i.e. mainly Lactobacillus crispatus cell types, plump quite homogeneous lactobacilli. c, d: grade Ib, i.e. non-L. crispatus cell types, long or short, thin lactobacilli. e, f: grade Iab, i.e. containing mixtures of L. crispatus and non-L. crispatus cell types. g, h: grade I-like, i.e. irregular-shaped Gram positive rods. i, j: grade II, i.e. mixture of Lactobacillus cell types and bacterial vaginosis-associated bacteria (Gardnerella, Bacteroides-Prevotella and Mobiluncus cell types). k, l: grade III, i.e. bacterial vaginosis.
Table 1 Detailed composition of the vaginal microflora of 515 vaginal swab samples, as determined by culture and tDNA-PCR based identification
Species Vaginal smears graded by Gram stain
Grade Ia Ib Iab I-like II III IV Total
Number of samples 162 181 60 36 46 22 8 515
Lactobacillus spp.
Lactobacillus crispatus 87.0a 13.3 76.7 2.8 10.9 37.5 42.7
Lactobacillus jensenii 22.2 24.3 43.3 13.9 47.8 18.2 12.5 26.8
Lactobacillus gasseri 6.8 32.0 25.0 52.8 54.3 9.1 25.0 25.6
Lactobacillus iners 3.7 39.8 8.3 19.4 26.1 31.8 21.2
Lactobacillus vaginalis 9.3 12.7 15.0 11.1 6.5 20.0 1.7
Lactobacillus casei 1.1 1.7 2.8 2.2 4.5 12.5 1.4
Lactobacillus coleohominis 1.2 5.5 5.0 2.2 3.1
Lactobacillus delbrueckii 0.6 5.6 2.2 0.8
Lactobacillus fermentum 0.6 1.1 3.3 4.5 1.2
Lactobacillus kalixensis 0.6 0.2
Lactobacillus mucosae 4.3 0.4
Lactobacillus nagelii 2.2 0.2
Lactobacillus oris 4.3 0.4
Lactobacillus pontis 94% 0.6 0.2
Lactobacillus reuteri 1.7 1.7 5.6 1.2
Lactobacillus rhamnosus 0.6 8.3 4.3 4.5 12.5 1.6
Lactobacillus salivarius 0.5 0.2
Bifidobacterium spp.
Bifidobacterium biavatii 0.6 5.6 12.5 0.8
Bifidobacterium bifidum 0.6 2.8 4.5 0.6
Bifidobacterium breve 0.6 25.0 10.9 9.1 3.3
Bifidobacterium dentium 0.6 8.3 4.5 1.0
Bifidobacterium longum 0.6 0.6 5.6 4.5 1.0
Bifidobacterium sp. 0.6 5.6 0.6
Bacterial vaginosis-related anaerobe organisms
Actinomyces neuii 6.5 9.1 1.0
Aerococcus christensenii 4.3 22.7 1.4
Anaerococcus tetradiusb 2.2 0.2
Anaerococcus vaginalisb 2.8 0.2
Atopobium vaginae 0.6 4.3 13.6 1.2
Bacteroides ureolyticus 0.6 2.8 2.2 9.1 1.0
Dialister sp. 22.7 12.5 1.2
Finegoldia magnab 0.6 0.6 2.2 9.1 1.0
Gardnerella vaginalis 1.2 2.8 1.7 2.8 21.7 72.7 6.8
Gemella morbilloriumb 2.2 0.2
Mobiluncus curtisii 4.5 0.2
Mycoplasma hominis 4.3 0.4
Peptoniphilus sp.b 3.1 1.7 2.8 6.5 9.1 2.7
Peptostreptococcus sp. 4.5 0.2
Prevotella bivia 0.6 2.8 13.6 1.0
Prevotella ruminicola 4.5 0.2
Prevotella sp. 4.5 0.2
Varibaculum cambriense 2.2 13.6 0.8
Other species
Actinomyces europaeus 96% 0.6 0.2
Actinomyces urogenitalis 4.5 0.2
Arcanobacterium bernardiae 0.6 0.2
Arthrobacter albus 4.3 0.4
Atopobium parvulum 4.5 0.2
Clostridium colicanis 3.3 0.4
Clostridium sp. 4.5 0.2
Corynebacterium amycolatum 2.8 0.2
Corynebacterium coyleae 0.6 0.2
Corynebacterium pseudogenitalium 0.6 0.2
Corynebacterium sp. 0.6 4.3 0.6
Enterococcus faecalis 2.5 4.4 3.3 2.2 4.5 3.1
Escherichia coli 0.6 3.3 2.8 9.1 1.2
Haemophilus influenzae 2.2 0.2
Helcococcus sp. 4.5 0.2
Pediococcus pentosaceus 2.8 4.5 0.4
Propionibacterium acnes 1.9 0.6
Propionibacterium avidium 2.8 0.2
Serratia sp. 0.6 0.2
Staphylococcus aureus 0.6 0.6 2.2 0.6
Staphylococcus capitis 0.6 0.2
Staphylococcus epidermidis 1.2 5.5 1.7 5.6 4.3 3.3
Staphylococcus haemolyticus 1.7 2.2 0.4
Staphylococcus hominis 1.1 2.8 2.2 0.8
Streptococcus agalactiae 1.9 4.4 1.7 11.1 4.5 75.0 4.5
Streptococcus anginosus group 3.1 3.9 1.7 5.6 4.3 9.1 3.7
Streptococcus gallolyticus 1.7 0.2
Streptococcus mitis 2.8 2.2 0.4
Streptococcus salivarius 0.6 1.7 0.8
Veilllonella atypica 1.2 0.6 2.8 0.8
Veillonella sp. 0.6 1.1 2.2 0.8
a Numbers represent percentage of samples from which the species was cultured.
b Formerly known as Peptostreptococcus.
Table 2 Presence of Bifidobacterium spp. in grade I like samples versus other samples.
Bifidobacterium spp. Grade I-like Other grades Total
Cultured 19 18 37
Not cultured 17 461 478
Total 36 479 515
Table 3 The presence of Lactobacillus species in grade Ia and grade Ib samples.
Grade Ia Grade Ib Total
Total 162 181 343
L. crispatus 141 24 165
L. jensenii 36 44 80
L. gasseri 11 58 69
L. iners 6 72 78
Table 4 Presence of L. gasseri or L. iners in grade II and grade III samples versus presence in other samples.
L. gasseri or L. iners Grades II and III Other grades Total
Cultured 41 184 225
Not cultured 27 263 290
Total 68 447 515
Table 5 Number of samples with lactobacilli in grade Ia versus the other grades.
Species Grade Ia Other grades Total
L. crispatus
185 35 220
L. jensenii
36 102 138
L. gasseri
11 123 132
L. iners
6 103 109
Table 6 Number of samples with lactobacilli in grade I versus the other grades.
Species Grades I a + Iab + Ib Other grades Total
L. crispatus
211 9 220
L. jensenii
106 32 138
L. gasseri
84 48 132
L. iners
83 26 109
Table 7 Presence of G. vaginalis in grade II and grade III samples versus presence in other samples.
G. vaginalis
Grades II and III Other grades Total
Cultured 26 9 35
Not cultured 42 438 480
Total 68 447 515
Table 8 Numbers of species cultured per patient and species diversity indices
Grade Number of species cultured Diversity
Average Range Number of Species/Number of Samples (Index) Simpson's Diversity Index
Ia 1.5 1–6 27/162 (0.17) 0.6
Ib 1.7 1–7 38/181 (0.21) 0.9
Iab 2.0 1–7 18/60 (0.30) 0.8
I-like 2.3 1–8 30/36 (0.83) 0.9
II 2.7 1–6 35/46 (0.76) 0.9
III 3.6 2–8 33/22 (1.50) 0.9
IV 2.0 1–3 5/8 (0.63) 0.8
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Ferris MJ Masztal A Martin DH Use of Species-Directed 16S rRNA Gene PCR Primers for Detection of Atopobium vaginae in Patients with Bacterial Vaginosis J Clin Microbiol 2004 42 5892 4 15583334 10.1128/JCM.42.12.5892-5894.2004
Obata-Yasuoka M Ba-Thein W Hamada H Hayashi H A multiplex polymerase chain reaction-based diagnostic method for bacterial vaginosis Obstet Gynecol 2002 100 759 764 12383546 10.1016/S0029-7844(02)02201-9
Zariffard MR Saifuddin M Sha BE Spear GT Detection of bacterial vaginosis-related organisms by real-time PCR for lactobacilli, Gardnerella vaginalis and Mycoplasma hominis FEMS Immunol Med Microbiol 2002 34 277 281 12443827 10.1016/S0928-8244(02)00397-8
Baele M Baele P Vaneechoutte M Storms V Butaye P Devriese LA Verschraegen G Gillis M Haesebrouck F Application of tDNA-PCR for the identification of enterococci J Clin Microbiol 2000 38 4201 4207 11060090
Welsh J McClelland M Genomic fingerprints produced by PCR with consensus tRNA gene primers Nucleic Acids Res 1991 19 861 866 2017367
Baele M Vaneechoutte M Verhelst R Vancanneyt M Devriese LA Haesebrouck F Identification of Lactobacillus species using tDNA-PCR J Microbiol Methods 2002 50 263 271 12031576 10.1016/S0167-7012(02)00035-0
Morris M Nicoll A Simms I Wilson J Catchpole M Bacterial vaginosis: a public health review BJOG 2001 108 439 50 11368127 10.1016/S0306-5456(00)00124-8
Hashemi FB Ghassemi M Roebuck KA Spear GT Activation of human immunodeficiency virus type 1 expression by Gardnerella vaginalis J Infect Dis 1999 179 924 30 10068588 10.1086/314674
Sewankambo N Gray RH Wawer MJ Paxton L McNaim D Wabwire-Mangen F Serwadda D Li C Kiwanuka N Hillier SL Rabe L Gaydos CA Quinn TC Konde-Lule J HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis Lancet 1997 23 546 50 10.1016/S0140-6736(97)01063-5
Sha BE Zariffard MR Wang QJ Chen HY Bremer J Cohen MH Spear GT Female genital-tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Mycoplasma hominis J Infect Dis 2005 191 25 32 15592999 10.1086/426394
Leitich H Bodner-Adler B Brunbauer M Kaider A Egarter C Husslein P Bacterial vaginosis as a risk factor for preterm delivery: a meta-analysis Am J Obstet Gynecol 2003 189 139 47 12861153 10.1067/mob.2003.339
Peters KD Kochanek KD Murphy SL Deaths: final data for 1996 Natl Vital Stat Rep 1998 47 1 100 9824931
Paneth NS The problem of low birth weight Future Child 1995 5 19 34 7633862
Beigi RH Austin MN Meyn LA Krohn MA Hillier SL Antimicrobial resistance associated with the treatment of bacterial vaginosis Am J Obstet Gynecol 2004 191 1124 9 15507930 10.1016/j.ajog.2004.05.033
Spiegel CA Amsel R Holmes KK Diagnosis of bacterial vaginosis by direct gram stain of vaginal fluid J Clin Microbiol 1983 18 170 7 6193137
Delaney ML Onderdonk AB Nugent score related to vaginal culture in pregnant women Obstet Gynecol 2001 98 79 84 11430961 10.1016/S0029-7844(01)01402-8
Larsson PG Carlsson B Fahraeus L Jakobsson T Forsum U Diagnosis of bacterial vaginosis: need for validation of microscopic image area used for scoring bacterial morphotypes Sex Transm Infect 2004 80 63 7 14755040 10.1136/sti.2003.006106
Rosenstein IJ Morgan DJ Sheehan M Lamont RF Taylor-Robinson D Bacterial vaginosis in pregnancy: distribution of bacterial species in different gram-stain categories of the vaginal flora J Med Microbiol 1996 45 120 6 8683547
Jones SA A study of the prevalence of hydrogen peroxide generating Lactobacilli inbacterial vaginosis: the determination of H2O2 concentrations generated, in vitro, by isolated strains and the levels found in vaginal secretions of women with and without infection J Obstet Gynaecol 1998 18 63 7 15512007 10.1080/01443619868325
Rosenstein Rosenstein IJ Fontaine EA Morgan DJ Sheehan M Lamont RF Taylor-Robinson D Relationship between hydrogen peroxide-producing strains of lactobacilli and vaginosis-associated bacterial species in pregnant women Eur J Clin Microbiol Infect Dis 1997 16 517 22 9272387 10.1007/BF01708235
Hill GB The microbiology of bacterial vaginosis Am J Obstet Gynecol 1993 169 450 4 8357043
Thorsen P Jensen IP Jeune B Ebbesen N Arpi M Bremmelgaard A Moller BR Few microorganisms associated with bacterial vaginosis may constitute the pathologic core: a population-based microbiologic study among 3596 pregnant women Am J Obstet Gynecol 1998 178 580 7 9539529
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BMC Med Inform Decis MakBMC Medical Informatics and Decision Making1472-6947BioMed Central London 1472-6947-5-331622130810.1186/1472-6947-5-33Research ArticleAn adaptive prediction and detection algorithm for multistream syndromic surveillance Najmi Amir-Homayoon [email protected] Steve F [email protected] National Security Technology Department, The Johns Hopkins University Applied Physics Laboratory, Laurel, MD 20723-6099, USA2005 12 10 2005 5 33 33 25 2 2005 12 10 2005 Copyright © 2005 Najmi and Magruder; licensee BioMed Central Ltd.2005Najmi and Magruder; 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
Surveillance of Over-the-Counter pharmaceutical (OTC) sales as a potential early indicator of developing public health conditions, in particular in cases of interest to biosurvellance, has been suggested in the literature. This paper is a continuation of a previous study in which we formulated the problem of estimating clinical data from OTC sales in terms of optimal LMS linear and Finite Impulse Response (FIR) filters. In this paper we extend our results to predict clinical data multiple steps ahead using OTC sales as well as the clinical data itself.
Methods
The OTC data are grouped into a few categories and we predict the clinical data using a multichannel filter that encompasses all the past OTC categories as well as the past clinical data itself. The prediction is performed using FIR (Finite Impulse Response) filters and the recursive least squares method in order to adapt rapidly to nonstationary behaviour. In addition, we inject simulated events in both clinical and OTC data streams to evaluate the predictions by computing the Receiver Operating Characteristic curves of a threshold detector based on predicted outputs.
Results
We present all prediction results showing the effectiveness of the combined filtering operation. In addition, we compute and present the performance of a detector using the prediction output.
Conclusion
Multichannel adaptive FIR least squares filtering provides a viable method of predicting public health conditions, as represented by clinical data, from OTC sales, and/or the clinical data. The potential value to a biosurveillance system cannot, however, be determined without studying this approach in the presence of transient events (nonstationary events of relatively short duration and fast rise times). Our simulated events superimposed on actual OTC and clinical data allow us to provide an upper bound on that potential value under some restricted conditions. Based on our ROC curves we argue that a biosurveillance system can provide early warning of an impending clinical event using ancillary data streams (such as OTC) with established correlations with the clinical data, and a prediction method that can react to nonstationary events sufficiently fast. Whether OTC (or other data streams yet to be identified) provide the best source of predicting clinical data is still an open question. We present a framework and an example to show how to measure the effectiveness of predictions, and compute an upper bound on this performance for the Recursive Least Squares method when the following two conditions are met: (1) an event of sufficient strength exists in both data streams, without distortion, and (2) it occurs in the OTC (or other ancillary streams) earlier than in the clinical data.
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Background
Surveillance of Over-the-Counter pharmaceutical (OTC) sales as a potential early indicator of developing public health conditions, in particular in cases of interest to biosurvellance, has been suggested in the literature [1]. Sales of over-the-counter pharmaceuticals (OTCs) offer several advantages as possible early indicators of public health. They are very widely used [2], and reliable and detailed electronic records of their sales exist.
Another possible advantage is the timeliness of OTC sales relative to other observable events that might occur when the public health is threatened. This is a particularly difficult aspect since it requires the identification of specific events in all the data streams before a judgment can be reached as to the correlations and the timeliness of those events.
We have, in a previous article [3], provided evidence that when judiciously grouped, the OTC data show time-dependent correlations with clinical data, and that the present days values of the latter can be estimated well from the present and past values of the former using a set of linear filters hj[m], where the subscript j refers to the particular OTC product group (multiple groups are used) and the index m refers to the time step. If we denote the clinical data time series on day number n by y[n], and the OTC time series on the same day number n by xj[n], (the index j denotes the OTC product group), then the estimation problem discussed in our previous paper refers to using today's and past days' OTC data to estimate today's clinical data, in the sense that the estimated quantity is . The linear filters hj[m] are assumed to have a span of M points (days).
This estimate is to be compared with the actual value of the clinical data today. The "prediction" problem, the subject of the present paper, refers to an attempt to estimate future values of the clinical data using today's and past days' values of the OTC channels, i.e. the predicted quantity is now , k > 0. In the parlance of linear filter theory, the data set whose prediction is desired (the dependent variable) is termed the primary data channel. All other data sets (distinct from the primary channel) that are used to make the predictions are known as reference channels, otherwise known as independent variables. When the primary data set (the dependent variable) is used to predict its own values, then the primary channel is also the reference channel (the independent variable).
We present a prediction method based on an adaptive recursive least squares filter. In addition, we compare these predictions, which we term auto predictions, with similar predictions that use the same method applied to the clinical data alone without referencing any OTC channels. It is our contention that when the auto prediction results (i.e. when using the clinical data in the past to predict its future values) are equally (in the sense of minimum squared error) effective as or better than those predictions based solely on OTC streams, in all time intervals, then it is highly probable that no event of interest to biosurveillance actually exists in the clinical data. This is based on the fundamental premises of linear optimal predictors that a nonstationary and relatively short duration event superimposed on an otherwise stationary and predictable background cannot be predicted from the stationary background data alone. We argue that the best performance comparison, in the context of a biosurveillance system whose objective is to detect an outbreak early, among all method/data stream combinations is tied closely to the existence of such events. Lacking any real specific events of sufficient signal strength, we perform a study based on simulated events in order to compute an upper bound on the indicated performances. We emphasize that the system whose performance we are investigating here is a predict and detect system, in the sense that it uses historical clinical and other ancillary data streams in order to predict clinical data many days into the future. The detection performance is then based on a study of probabilities of true detections versus the probabilities of false alarms.
The meaning of an upper bound on the detection performance in this context is in the following sense. Given a data stream yt that includes an event of short duration then the detection performance of a specific prediction method, is related to the quantity where is the predicted value of the data stream when an event exists and yt is the value of the data stream in the absence of the event. This predicted value could be based on the data stream itself, or it could be based on a combination of the data stream and several other correlated data sets. In a real-time situation one might perform detections based on the quantity , where is a prediction of the data stream in the absence of the signal, because the actual quantity yt is not available when the predictions are made at t - Δt. We contend that an upper bound on detection performance is obtained when we use the "actual background" yt instead of the "predicted background" .
Methods
Data grouping and recursive least squares prediction
JHU/APL is currently collecting large quantities of daily OTC sales data. We receive sales records of 622 different products under the general category of cold remedies from a single vendor, with similar numbers from other vendors. Many of these products are used to treat very similar conditions. Product sales from some of these product groups are known to be good indicators of the corresponding clinical data. For instance, chest rub sales are highly correlated with the count of physician diagnosis of acute bronchitis or acute bronchiolitis [4].
The OTC products of interest were grouped based on a combination of the syndromes the product is intended to alleviate, the physical description of the product (e. g. a pill, a powder, a lip balm, etc.) and the age/sex group the product is targeted for. There were 15 syndrome groups, 15 physical types, and 4 target age/sex groups (mostly age, but 4 of the products were designated as intended especially for women). Some combinations contained no products, but there were a total of 92 combinations that did, so there was still some need for aggregation of these groups. The aggregation procedure has been reported elsewhere [5]. The groups we eventually used are shown in Table 1. The clinical data are counts of outpatient encounters, based on physicians diagnoses (according to the International Classification of Diseases, Ninth Revision (ICD9) standards [6]) reported in insurance claims that fall within a particular set of acute respiratory conditions (see table 1). Encounters were included only for patients 12 years old and older. The encounters are further restricted to include only patients living in the National Capitol Region (NCR), which includes the District of Columbia, Baltimore, suburban portions of Maryland and the Washington suburbs in Virginia. The encounters are time-tagged according to the day of occurrence.
Table 1 OTC Adult Medication Product Groups
Product Group Name
ALLERGY
BRONCHIAL
COLD-ALLERGY
COUGH
FLU
POWDER
SINUS
THROAT
Here, we consider the clinical data, the dependent variable, as the primary data channel (in the parlance of adaptive filter theory) whose values are to be predicted. The OTC product groups (the independent variables) are then used to predict the daily clinical data in the following manner. Today's and several past days' OTC data are combined to make a future clinical data prediction, which is then compared to the actual value of that day's clinical data when it becomes available, and the error is used to update the filter coefficients in such a way as to minimize the square of the error. For simplicity and to illustrate the method we consider the estimation problem in which there is only one reference channel whose value at each time n is denoted by x[n] (note that the subscript j denoting the particular product group is now missing since we are using an example with only 1 product group). The latter is used to estimate the present value y[n] of the primary channel (the dependent variable – office visit data). The estimation equations, once put into the recursive form are then easily generalized to the prediction problem. A linear estimate of the primary channel in terms of a single reference channel is given by , where we have assumed a filter of length M. The last equation can be written as a vector dot product , where h= [h[0], h[1],..., h[M -1]]T, x[n] = [x[n], x[n -1],..., x[n - (M - 1)]]T, and the superscript T denotes the transposition operation (the transpose of a row vector is a column vector). A linear predictor of the primary channel (the dependent variable) at k steps ahead is then given by .
Clearly we could perform a similar prediction process when we use the clinical data by itself instead of the OTC data streams, as well as simply including the clinical data as an "additional" reference data stream. Let P denote the number of days ahead to predict, M denote the number of linear filter coefficients, and N denote the number of OTC data channels. Then the filter vector hwill have M × N elements when we predict the clinical data using the OTC channels, and it will have M elements when we use the clinical data to predict itself, and (M + 1) × N elements when we combine the OTC channels and the clinical data to predict the clinical data. The covariance/correlation matrix of the reference channels will then be a square matrix of the appropriate dimension in each case. The filter application and updates are recursive. Denoting the clinical data (the dependent variable) on day n by y[n] and the reference data (the independent variables) by xj, the Recursive Least Squares P – days ahead prediction equation is [7] Where L denotes the number of reference data streams and it is equal to N when only OTC data are used, or 1 if the clinical data is used to predict itself, or N + 1 if both clinical and OTC channels are used to predict the clinical data. This step is repeated as many times as required in order to obtain the predicted values . The recursion equations and the new method of minimum multiple look error feedback is described in Additional file 1. We should point out that when we use the clinical data alone (i.e. for self prediction) then the prediction equation is of the form , where the subscript j could be left out since only one filter is used.
Our simulated signal was constructed by combining the following assumptions about an event of interest that can be reasonably expected to arise if a biological attack were to occur. The event is the result of a deterministic multiplicative signal s(t), in the sense that if y(t) denotes the clinical data in the absence of the signal, the presence of the signal will lead to the following clinical data y(t){1+s(t)}. The corresponding event has a sharp rise of about 5–10 days from a minimum of 0 to a maximum value that shows the corresponding percent increase of clinical data at the peak of the outbreak, above the normal "background" number. We consider the rise time of about a week to be a reasonable assumption based on observations of infectious disease characteristics [8]. In addition, we assume that the event has a fall off period of about twice that of the rise time. This signal can be easily modeled by a function of the form . The log-normal shape is based on observations as reported by Sartwell [8]. Figure 1 shows a specific example matching the requirements of an event described above.
Figure 1 Simulated multiplicative event.
The simulation consists of applying this deterministic signal with a given maximum value to the OTC data, on any given day, and applying the same deterministic signal to the clinical data with a time delay. Then we compute predictions of the clinical data once using OTC data only, and a second time using the clinical data itself. In both cases we use the predicted clinical data for detection and the detector output is , where denotes the predicted value of the clinical data on day t. The predictions are performed once with no signal present (to compute false alarms), and once with signal present to compute true detections, and for all day numbers 100 through 550. A range of thresholds are used to calculate the total number of detections and the total number of false alarms. These numbers are then averaged over the total number of days to give the probability of detection pd and the probability of false alarm pfa, both of which are functions of threshold. The Receiver Operating Characteristic (ROC) curve is then obtained by eliminating the threshold variable and plotting pd as a function of pfa. This curve provides the most concise form of evaluating the performance of any detection system [9]. The best performance is, by definition, a horizontal line pd = 1, while the worst is the line pd = 0, for all values of pfa. The 45° line represents the performance of a hypothetical detector that decided on the presence of a true signal by tossing a fair coin, i.e. equal probabilities of detection vs false alarm.
Simulation parameters are as follows. The signal maximum strength takes on values 10%, 100% and 200% (percentage increases refer to the background counts). We have chosen 2 signal lag times of 5 and 10 days (lag times refer to the lag between the application of the maximum signal strength to the OTC channels and the office visit count channel). The predictor uses a filter length of 5 days and we try 2 sets of predictions: 5 days ahead and 10 days ahead.
Results and discussion
Figure 2 shows the actual clinical data, and figure 3 shows all the OTC channels, for the period 11/1/2001 through 5/14/2003 consisting of 560 days. Figure 4 shows the 5-days ahead prediction results using only the OTC channels. Figure 5 shows a similar output when the office visits data are added in as another reference channel. Figure 6 shows the output when only the office visits data are used to predict their own future values, i.e. OTC channels are not used here. All results use a 5 point filter, i.e. for each OTC product group j the corresponding filter is hj[m] and 0 ≤ m ≤ 4. The effective memory of each filter is set at approximately 1 month.
Figure 2 Office visit data [clinical data] between 11/1/2001 and 5/14/2003.
Figure 3 OTC sales data for the same period.
Figure 4 5-days ahead predictions of office visit data using OTC sales data, versus actual data.
Figure 5 5-days ahead predictions of office visit data using OTC sales data and office visit data, versus actual data.
Figure 6 5-days ahead predictions of office visit data using OTC sales data and office visit data, versus actual data.
A simple measure of the effectiveness of the predictor performance (not the detector performance) is a plot of the mean versus standard deviation of the difference between the actual and the predicted values (prediction error vector). Figure 7 shows the means versus standard deviations for all 3 cases and for 5-days ahead as well as 10-days ahead predictions. The prediction error vector was computed between days 100 and 550, to allow for filter initialization in the beginning; we could have started making predictions as early as 50 days from the beginning since the effective memory of the filter is set at 30 days, but chose to begin making predictions at day 100 to be absolutely safe. These prediction results are quite encouraging and show significant correlations between OTC and office visit data, in the sense that the predictions are quite close to the actual values. What is perhaps more surprising, is the fact that using office visit data for self prediction apparently has the lowest error. Although these errors are computed over the entire time section, there are no time intervals over which the self prediction results are worse than those when the OTC are used to predict the clinical data.
Figure 7 Performance characteristics of 5-days and 10-days ahead predictions, using OTC sales data alone, OTC data plus office visit data, and office visit data alone.
Our interpretation is that this particular office visit data has sufficiently strong autocorrelations at long lags that allows for a better prediction when compared to the predictions made using the cross correlations. We emphasize that one cannot draw any conclusions as to the best combination of method/data for prediction from these results when in fact no identifiable and significant events of interest exist in the present data set. For instance, one cannot state that OTC data can be safely ignored in the prediction problem in favour of using the clinical data itself. In order to illustrate this point and to place an upper bound on detection performance of a biosurveillance system that relies on predicting the clinical data from OTC and/or clinical data, we have performed an analysis based on simulated events superimposed on the present data sets.
Figure 8 shows the ROC curves (pd vs pfa) for a 5-day lag and 5 days ahead prediction for all three signal maximum amplitudes. The dotted lines represent the auto-predictions made using only the clinical data. The solid lines show the predictions using the OTC data. The thickness of the lines in each case represents the signal maximum amplitude. Based on this figure alone, we can summarize these results as follows. Given the assumptions in this simulation, the auto-predictions do not appear to perform well in a predict-ahead and detect surveillance system. For instance, even at signal maximum amplitude of 200%, for a = 0.2, corresponding to a false alarm rate of once every 5 days, the probability of detection when using auto-predictions is barely above 60%, whereas if one uses the OTC predictions that probability is 100%. If a signal maximum amplitude is lowered to 100%, the auto-prediction probability of detection is down to 40%, and that of OTC predictions is still a very healthy 97%. These results continue to hold so long as the lag value is larger than or equal to the number of prediction days, as can be seen clearly in figures 9 and 10. Figure 11 shows a dramatic fall in performance when the latter condition is not satisfied, i.e. when the number of prediction days exceeds the lag number. Clearly, if we try to predict "too many" days ahead (irrespective of the lag number), the detection results worsen considerably. The present data set predictions appeared to hold to about 12 days ahead.
Figure 8 ROC curves of auto-predictions (office visit data alone to predict itself), and OTC predictions (OTC data to predict office visit data), for 3 signal strengths, using a simulated actual lag of 5 days between OTC and office visit data and a 5-days ahead predictor.
Figure 9 ROC curves of auto-predictions (office visit data alone to predict itself), and OTC predictions (OTC data to predict office visit data), for 3 signal strengths, using a simulated actual lag of 10 days between OTC and office visit data and a 5-days ahead predictor.
Figure 10 ROC curves of auto-predictions (office visit data alone to predict itself), and OTC predictions (OTC data to predict office visit data), for 3 signal strengths, using a simulated actual lag of 10 days between OTC and office visit data and a 10-days ahead predictor.
Figure 11 ROC curves of auto-predictions (office visit data alone to predict itself), and OTC predictions (OTC data to predict office visit data), for 3 signal strengths, using a simulated actual lag of 5 days between OTC and office visit data and a 10-days ahead predictor.
Our choice of the synthetic signal requires further discussion. Since we are interested in placing upper bounds on the performance of a multi-stream syndromic surveillance system that uses a prediction method to detect an outbreak we decided to concentrate on a type of epidemic curve that has a reasonably fast rise time and a slower fall off. We chose the 1-week rise time and 2-weeks fall off because they are reasonable numbers in the context of early detection of most biological attack scenarios. It so happens that these numbers appear to fit the observations by Sartwell and so we used a log-normal shape. It turns out the results are quite insensitive to the analytic form of the signal, for instance, we could have used a "triangle" signal with the same rise-time characteristics and reached similar conclusions.
Finally we should discuss our results in view of the fact that we applied the same multiplicative signal in all data streams without distortion. A complete simulation study of the performance of a multi-stream syndromic surveillance system that uses a prediction method to detect an outbreak would include all possible signal distortions (including amplitude reduction, but also changes in the shape of the signal), and all reasonable time delays; this is a huge task and well beyond the scope of this publication. What we have attempted here is obtaining an upper bound on this performance by varying the time delay and the maximum amplitude of the signal but keeping the signal undistorted. Any distortion of the signal would clearly degrade the ROC curves. In the absence of a general theory of infectious disease evolution and the uncertainties associated with the impact of an infectious disease outbreak upon all the data streams in a multi-stream syndromic surveillance system we have found performance upper bounds on the limited number of cases we have studied, in conjunction with the prediction algorithm presented here.
Conclusion
Based on our simulation results we can state the following broad conclusions regarding a multistream syndromic surveillance system that operates by predicting the clinical data several days in advance and issuing early warnings if the predicted values exceed a given thershold. This predict-and-detect system must include ancillary data streams (such as OTC) with established correlations with the clinical data, and a prediction method that can react to nonstationary events sufficiently fast. Any predictions of the clinical data using only the clinical data, i.e. relying on self-correlations of the clinical data rather than cross-correlations with other data streams such as OTC data, can be an effective estimate of the background conditions. Whether OTC (or other data streams yet to be identified) can provide the best source of predicting clinical data is still an open question. The system must also include a prediction algorithm that can react sufficiently fast to nonstationary changes. The Recursive Least Squares Minimum Distance Error algorithm presented here seems to satisfy this condition. Finally, we have no way of knowing the likelihood that events of interest will always be present in both the clinical data and the ancillary streams, without significant distortion, and with reasonable time lags. But if any event satisfies these conditions, we have provided the framework for a system that has an excellent chance of detecting it in advance.
Abbreviations
OTC: over the counter (medications).
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
The idea of predicting clinical data using OTC, and the algorithm using recursive least squares with feedback of minimum error among multiple looks, and the associated computer programmes were developed by A. H. Najmi. The OTC data were grouped from among a much larger set into a small set used in this study, via a maximum likelihood method developed by S. F. Magruder.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional File 1
Recursive Least Squares Prediction with minimum distance multiple look error feedback.
Click here for file
Acknowledgements
This research is sponsored by the Defense Advanced Research Projects Agency and managed under Naval Sea Systems Command (NAVSEA) contract N00024-98-D-8124. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied of the Defense Advanced Research Projects Agency, NAVSEA, or the United States Government.
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Anna Goldenberg PNAS Early statistical detection of Anthrax outbreaks by tracking over the counter medication sales 2002 99 5237 5240
Self-care in the New Millennium, report by Roper Starch Worldwide, Inc, prepared for the Consumer Healthcare Products Association, Roper-Starch 2001
Najmi AH Magruder SF BMC Medical Informatics and Decision Making Estimation of Hospital Emergency Room Data Using OTC Pharmaceutical Sales and Least Mean Square Filters 2004 4 5
Magruder SF Johns Hopkins University Applied Physics Laboratory Technical Digest Evaluation of OTC pharmaceutical sales as a possible early warning indicator of public health 2003 24
Magruder SF Happel-Lewis S Najmi AH Florio AE Special Issue on the Proceedings of the National Syndromic Surveillance Conference Progress in Understanding and Using Over-the-Counter Pharmaceuticals for Syndromic Surveillance of Public Health, Morbidity and Mortality Weekly Report 2004 53 S117 122
See for example Diagnostic Coding Essentials 2001 Ingenix Publishing Group, Salt Lake City, Utah
Mathematical methods and Algorithms for Signal Processing 2000 Moon and Stirling, Prentice Hall
The Incubation Period of Poliomyelitis 1952 42 Philip Sartwell, American Journal of Public Health 1403 1408
Detection of Signals in Noise 1995 McDonough and Whalen, Academic Press
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BMC Med Inform Decis MakBMC Medical Informatics and Decision Making1472-6947BioMed Central London 1472-6947-5-341622568610.1186/1472-6947-5-34Research ArticleInformation resource preferences by general pediatricians in office settings: a qualitative study Kim George R [email protected] Edward L [email protected] Harold P [email protected] Division of Health Sciences Informatics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA2 Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA2005 14 10 2005 5 34 34 22 3 2005 14 10 2005 Copyright © 2005 Kim et al; licensee BioMed Central Ltd.2005Kim 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
Information needs and resource preferences of office-based general pediatricians have not been well characterized.
Methods
Data collected from a sample of twenty office-based urban/suburban general pediatricians consisted of: (a) a demographic survey about participants' practice and computer use, (b) semi-structured interviews on their use of different types of information resources and (c) semi-structured interviews on perceptions of information needs and resource preferences in response to clinical vignettes representing cases in Genetics and Infectious Diseases. Content analysis of interviews provided participants' perceived use of resources and their perceived questions and preferred resources in response to vignettes.
Results
Participants' average time in practice was 15.4 years (2–28 years). All had in-office online access.
Participants identified specialist/generalist colleagues, general/specialty pediatric texts, drug formularies, federal government/professional organization Websites and medical portals (when available) as preferred information sources. They did not identify decision-making texts, evidence-based reviews, journal abstracts, medical librarians or consumer health information for routine office use.
In response to clinical vignettes in Genetics and Infectious Diseases, participants identified Question Types about patient-specific (diagnosis, history and findings) and general medical (diagnostic, therapeutic and referral guidelines) information. They identified specialists and specialty textbooks, history and physical examination, colleagues and general pediatric textbooks, and federal and professional organizational Websites as information sources. Participants with access to portals identified them as information resources in lieu of texts.
For Genetics vignettes, participants identified questions about prenatal history, disease etiology and treatment guidelines. For Genetics vignettes, they identified patient history, specialists, general pediatric texts, Web search engines and colleagues as information sources. For Infectious Diseases (ID) vignettes, participants identified questions about patients' clinical status at presentation and questions about disease classification, diagnosis/therapy/referral guidelines and sources of patient education. For ID vignettes, they identified history, laboratory results, colleagues, specialists and personal experience as information sources.
Conclusion
Content analysis of office-based general pediatricians' responses to clinical vignettes provided a qualitative description of their perceptions of information needs and preferences for information resource for cases in Genetics and Infectious Diseases. This approach may provide complementary information for discovering practitioner's information needs and resource preferences in different contexts.
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Background
Information behaviors of pediatricians have been examined in different clinical environments: in general patient care at an academic medical center [1,2], in neonatal intensive care [3] and in hospital on-call care [4], and as part of a subset of practitioners caring for patients in a broader age range [5]. Collectively, these studies demonstrate distinctions in information resource preferences by pediatric practitioners in different clinical environments. A systematic review of information seeking behaviors of physicians in general [6] found wide variation of information resource preferences, distributed among colleagues, text sources and electronic databases, from which the authors inferred a need for further categorization of information needs and resources.
Published studies on pediatricians' information use have focused on hospital-based practitioners and house staff, although most pediatricians practice in office settings, away from medical libraries, librarians and hospital-based specialists [7]. Office-based practitioners have perspectives distinct from hospital-based practitioners that may influence their information needs and resource preferences. Among these perspectives may be practical constraints, such as the need to transport (rather than manage) unstable patients or the cost and space needed to maintain onsite repositories of information.
In contrast to these constraints has been the increasing adoption of information and communication technologies (ICT) into practice [8]. In addition to patient management tools such as electronic medical records (EMRs), electronic prescribing and secure messaging, pediatric practitioners have a growing number of traditional and new electronic knowledge resources available by subscription and for free, from medical center libraries [9,10], government agencies [11], professional organizations [12] and medical publishers. With increasing availability of telemedicine services [13] and marketing of inpatient specialty services as "product" [14], the information environment of the academic medical center is expanding to include community physicians' offices.
Domain competencies for US pediatricians have traditionally been defined by formal training curricula and board certification [15], but information management competencies linked to professional certification maintenance for continuous professional development (CPD) [16] and evidence-based practice improvement [17] are evolving. The day-to-day management of knowledge and incorporation of evidence into care have not been well defined [18] and are part of an informal practitioner curriculum [19], based on personal experience, availability and reputations of resources and local organizational cultures [20] that may influence what is and is not used.
This pilot study explores information needs and resource preferences of (a) a characterized group of practitioners (general pediatricians) within (b) a characterized clinical workflow (office-based practice) for (c) problems in characterized domains (genetics and infectious diseases) using short clinical vignettes. Vignettes [21] have been used to teach and evaluate problem-solving skills and to elicit practitioners' information needs [22]. Their advantages include cost effectiveness and case-mix control for new or rare conditions. Genetics and Infectious Diseases were chosen as two representative domains in which pediatricians receive formal training and clinical experience.
In this study, we explore several questions:
• Can vignettes be used to elicit information needs and resource preferences from a group of practitioners with common attributes, clinical environments and patient problems?
• What are information needs and resource preferences of office-based general pediatricians, and are there commonalities with pediatricians in other settings?
Methods
Design
We conducted semi-structured interviews of twenty office-based general pediatricians to elicit their: (a) demographic and computer use data, (b) verbalized uses of specified information resources and (c) "think aloud" perceptions of questions and identification of information resource preferences in response to short vignettes representing clinical cases in genetics and infectious diseases. This study was approved by the appropriate institutional review board.
Setting
Participants were interviewed individually by telephone by one of the authors (GRK).
Subjects
A sample of twenty general pediatricians was recruited from urban/suburban office-based practices responding to general calls for subjects through two professional pediatric group newsletters (Winter-Spring 2004), through hospital pediatric department and joint community practice affiliations and through direct telephone/e-mail solicitation. Participants were enrolled by telephone.
Vignette creation
Sixteen short vignettes [See Additional file 1] representing hypothetical patients with problems in Genetics (8) or Infectious Diseases (8) were created to provide a variety of cases reflecting different combinations of familiarity, acuity and information problem (diagnosis or therapy) to general pediatricians. Domain experts in general pediatrics reviewed all vignettes (GRK, ELB, HPL) for presentation.
Data collection
The vignettes were randomized into non-repeating sequential groups of four vignettes and presented to participants by telephone in a standardized fashion. Participants' verbal responses were recorded and transcribed immediately post-interview for content analysis. Interviews lasted approximately 30 minutes each. One participant was replaced after it was discovered post-interview that the audio recorder had malfunctioned.
For each of the twenty participants, after a standard introduction to the study and consent, data was collected in a continuous three-part fashion:
1. In response to each of four sequential, randomized vignettes, the participant was asked to state perceptions of the questions presented by the vignette and how s/he would approach the problems in a "think-aloud" fashion [23], indicating information resources that s/he would consult in managing or approach the vignette. For each information resource indicated, the participant was asked what s/he would do if the resource consulted did not have the answer, until the problem was "resolved".
2. In response to a list of medical and pediatric information resources, the participant was asked to state the types of questions, problems or information needs for which s/he would use each resource type.
3. Information about demographics (practice type, years in practice, computer and Internet availability) was elicited.
Data analysis
Demographic information was tabulated for descriptive analysis [see Results: Demographics].
Indicated use of specific information resource types and their relative frequencies of indicated use (using the participant-resource pair as the unit of analysis) were tabulated for descriptive analysis [see Additional file 2 and Results: Information resources survey].
Transcribed responses to vignettes were coded consecutively according to the constant comparison method using open coding [24] to identify questions and information resources using the participant-vignette pair as the unit of analysis. Content analysis of interview transcripts was performed (GRK, HPL) using a combination of open-source software (AnSWR [25] from the Centers for Disease Control and Prevention) and desktop relational database/spreadsheet tools. Discrepancies were resolved through discussion (GRK, ELB).
Codes representing questions perceived from vignettes were sorted into nine categories forming two broad categories of Question Types about patient-specific and general medical information [see Additional file 3]. Question Types involving general medical knowledge were mapped to the closest entity from a published taxonomy of Generic Questions [26]. Codes representing information resource preferences were sorted into Information Resource Types describing patient-specific and general medical information sources [see Additional file 4]. Relative frequencies of perceived Question Types [see Additional file 5] and identified Information Resource preferences for all vignettes and for vignettes according to domain (genetics or infectious diseases) [see Additional file 6] were tabulated [see Results: Responses to vignettes].
Validation
Validation of the results was by member-checking. A summary of the results [see Results] was presented to all participants with a 90% return. All returns concurred with the summary.
Results
Demographics
Participants and work environments
Twenty pediatricians (11 male, 9 female) in urban/suburban pediatric practice participated. Average time in practice was 15.4 yrs (range 2–28 yrs, median 17 years (male mean 17.5 yrs, median 19 yrs; female mean 12.7 yrs, median 16 yrs; t-test NS)). Twelve participants classified their practices as private practice, 4 as managed care and 4 as free clinic/federally qualified health center. All 20 classified their clinical work as general pediatrics, with 7 adding adolescent medicine, 2 adding adult medicine and 1 adding developmental pediatrics. Thirteen worked in private offices, 4 in multiple specialty settings, 2 in multi-site clinics and 1 in an expanded urgent care (with limited well child care).
Information resources and computer availability
All participants shared office computers with Internet access in clinical areas. All had billing functions performed by separate computer systems. Twelve had access to a private networked computer, 10 used handheld computers, 6 had wireless access, 4 had mobile computers and 4 used electronic prescribing. Three used an EMR, 4 had access to an EMR that provided read-only capability. Three used computers in consultation rooms. Two had computers for other projects (research and patient education). Twelve had the capability to communicate with their patients by e-mail, but only 2 did so more than very rarely.
Information resources survey
Frequently and rarely identified resources
The most frequently identified resources were specialists, generalist colleagues and general pediatric texts, followed by drug formularies and specialty textbooks. Professional organization and federal health Websites (in particular the American Academy of Pediatrics and the Centers for Disease Control and Prevention) were identified as sources of guidelines and other information. When commercial medical portals were available (usually through institutional affiliation), they were frequently identified as a consulted information resource.
Resources rarely or not identified as sources of information in the office included: algorithmic/flow-chart decision-making texts, evidence-based medical reviews, medical journal abstracts, medical librarians or consumer health information. Several participants identified evidence-based medicine resources for specific problems or for self-education.
Identified uses of resources
Pediatric handbooks (such as the Harriet Lane Handbook [27]) were identified for drug dosing, cardiology information (electrocardiogram interpretation, blood pressure/pulse normal values) and disease-specific protocols. General pediatric texts were identified as first resources for resolving differential diagnosis and management questions, for general review and for self-education. Specialty texts were identified for domain-specific questions, with infectious disease (AAP Red Book [28]), genetics (Smith's Recognizable Patterns of Human Malformation [29]) and dermatology resources identified most frequently. Formularies were identified for questions on drug dosages, adverse reactions, packaging information, and for general information about drugs with which a participant had little or no experience. Guidelines and organizational policy statements, in particular those from the American Academy of Pediatrics [30], were identified for questions on disease management, for continuing education and for reference in policy-making.
Generalist colleagues were identified for confirmation of findings and for discussion of diagnostic and management decisions. Specialists were identified as information resources in anticipation of referral. Contact with generalist colleagues was usually face-to-face (in the office) while contact with specialists was via telephone and with a person or institution with which the participant had prior interaction or contact through an established referral network.
The CDC Travel Health Website [31] was the most frequently identified online resource for current immunization and travel health information. The CDC Website [11] was also identified for general immunization information. The American Academy of Pediatrics general Website [12] was identified for policy statements, current professional news and consumer health information. Commercial medical portals, when available, were identified in lieu of print textbooks.
Responses to vignettes
Frequently perceived question types
Patient-specific question types
Collectively, patient-specific Question Types regarding details about history, findings (symptoms, signs, test results), diagnosis, and treatment plans were the most frequently perceived, followed by Question Types regarding current patient state (stability, general appearance/condition), social support and patient/family understanding of a condition. Participants also perceived pragmatic questions about legal and insurance issues regarding minors in non-custodial care.
Patient-specific questions about maternal, prenatal and birth history were perceived more frequently in response to Genetics vignettes and questions about patients' appearance/condition were perceived more frequently in Infectious Disease vignettes.
General medical question types
Collectively, general medical Question Types regarding etiology, and treatment/diagnosis guidelines for a condition were the most frequently perceived.
General medical questions about disease etiology occurred more frequently in response to Genetics vignettes and questions about diagnosis/referral criteria and disease classification were perceived more frequently in Infectious Disease vignettes.
Most general medical Question Types posed by participants had correspondences to Generic Medical Questions in the taxonomy developed by Ely et al [26], however, there were two for which there were no explicit matches:
• "What are the classifications of a disease?" (to decide on an approach to management):
• "...what type of sickle cell [the patient] has, if he has SS [a type of sickle cell disease] or one of the more favorable ones..."
• "...what type of school failure, is it in one area, speech?"
• "What is available treatment?" (to locate new and uncommon resources):
• "...what treatments are available, what's experimental? If it exists, where is it provided? Would [the patient] qualify for participation in a study?"
• "What is the latest therapy available for patients with a disease X?"
Frequently identified information resources
Collectively, specialists were the most frequently identified Information Resource Type. They were also the most frequently identified last resource when an answer could not otherwise be found.
Patient-specific resources
The most frequently identified patient-specific resources were patient history and examination, followed records from and communication with previous providers and institutions (inpatient hospital or nursery), which were similar for both Genetics and Infectious Diseases vignettes.
General medical resources
After specialists, the most frequently identified general medical resources were specialty and general pediatric texts and colleagues (pediatric generalists), although participants with access to medical portals indicated them as resources in lieu of texts. Participants also identified Web search engines, guidelines from professional organizations (American Academy of Pediatrics) and local institutions (hospitals, social services, health departments, schools).
For Genetics vignettes, genetic specialists, dysmorphology texts/atlases and genetic databases (Online Mendelian Inheritance in Man (OMIM) [32]) were more frequently identified than for Infectious Diseases vignettes, for which specialists in infectious disease and other domains, infectious disease texts and guidelines (in particular the AAP Red Book), federal and local government resources (CDC Website and local health departments, schools and social services) and their own experience and training were more frequently identified.
Discussion
Emerging model
The information process of physicians in patient care has been described as iterations of inquiries/interventions (history, examination, diagnostic testing, treatment) and responses to updates of a patient's clinical status and/or a practitioner's information state [33]. Parallel to this process is the process by which decisions are made about further inquiries/interventions, for which the practitioner may need to consult external resources [34].
Practitioners anticipate information needs by organizing and maintaining "personal information collections, defined as subsets of... information...that individuals [build] conceptually and physically over time" [35], including "sources and channels...that can be located again easily." These preferences follow a principle of "least effort" [36], in which selection of resources (tools) are optimized to meet needs (jobs) in terms of the effort and cost to maintain and use them.
Characterization of such collections and their use by practitioners may be helpful on several fronts. First, it can help plan placement of resources to promote their effective use. It has recently been suggested that the way to promote evidence-based medical practice is to promote information management [37]. Second, it may help to determine practitioner training needs (knowing what questions to ask and what resources to use) in problems of new awareness. A recent systematic review [38] suggests that the longer physicians are practice, the greater their need for quality improvement interventions. Third, it may help practitioners develop and maintain situational awareness (SA) of information as it relates to patient care, practice improvement and professional development. SA of clinical information has been described within an intensive care unit (ICU) [39], but not within physician offices. Lastly, it may help determine factors that influence practitioners to use specific resource types.
Participants' information needs
In response to vignettes, patient-specific questions focused on patient history, examination and management. General medical questions focused on etiology, diagnostic/therapeutic management and guidelines. For Genetics vignettes, participants perceived questions about disease etiology more frequently than for Infectious Disease vignettes, for which they perceived questions about diagnostic and referral guidelines, disease classification and sources of patient education materials.
In comparison to an analysis of patient care questions asked at an academic medical center (AMC), the office-based (OB) participants perceived a higher relative frequency of questions about diagnosis and no questions about drugs. Reasons to explain this may include: 1) the OB group used hypothetical patients whereas the AMC-derived group used actual patients, 2) the OB group used only outpatient settings (where patient diagnoses are usually not known prior to the encounter) whereas more than half (58.8%) of the AMC-derived questions were generated from inpatient settings (where patients' admitting diagnoses are known), 3) the OB group used hypothetical encounters (where details of therapy may not be specified), whereas the AMC-derived group used actual patient cases and 4) the vignettes elicited questions about hypothetical cases in genetics and infectious diseases, whereas the AMC-derived questions came from patients from a number of different domains.
Question taxonomies
As noted in the AMC study of pediatricians' questions, the participants' questions fell within a few Generic Questions from the taxonomy derived from ethnographic observation of family practitioners. Interestingly, participants perceived two Question Types that did not have specific matches within the taxonomy: questions about disease classifications and about sources of the best and latest care for a disease. One suggestion that has been made is that pediatricians may have different information needs from family practitioners [1].
To express information needs of a group of practitioners, a question taxonomy should: a) match their perspectives (Example: parenteral nutrition and intravenous fluids are largely inpatient, not outpatient objects), the inventory (of objects/relationships) relevant to that perspective (Example: nutrition and immunizations may be therapy, but are not drugs) and the granularity (of knowledge) needed (Example: parenteral nutrition, infant formula and breastfeeding are distinct and important forms of nutrition) to express concepts and information needs easily and clearly [40]. The Generic Question taxonomy, while important, may not sufficiently express the practical information needs of pediatricians.
Participants' information resources
Within the context of Genetics and Infectious Diseases (as represented in these vignettes), participants identified a relatively small, but common set of defined resource types (general and specialty pediatric texts, generalist colleagues). They named a few specific, trusted online resources, although some were comfortable with "open" Web searching. They identified trusted and established sources of specialty care (frequently through a medical center), reachable by phone.
In comparison to AMC pediatricians, office-based general pediatricians have similar resource preferences, but also identified community resources, including the patient's family and social environment (school, etc.). Participants identified specific Websites (CDC, AAP) and medical portals when available (through academic affiliations) as trusted sources. In contrast to AMC-defined roles of "faculty" and "resident," participants identified in-office colleagues as informal consultants and specialists (by phone) as formal consultants. Specialists were frequently identified as "last" resource if an answer could not otherwise be found.
The use of vignettes
The described use of vignettes can generate a description of information resource use, but it does not provide information on what users actually do. It also does not provide information on barriers to pursuing answers to questions. It is not intended to supplant other methods of discovering information needs (such as surveys, interviews and ethnographic observations), but as a way of exploring information needs and resource preferences in a flexible and low-cost fashion. It may be useful in "fine-tuning" exploration of information needs and resource preferences:
• Vignettes about rare or new conditions may be used to identify practitioners' unrecognized information needs and/or to guide the development and deployment of new information resources to fulfill needs
• Vignettes about new morbidities may be used to identify the unrecognized practitioner information needs and/or to guide the development of practitioner education and awareness programs
In addition to the qualitative frequency analyses described in this paper, exploratory factor analysis of the results may be used to discover categories of resources that may help determine their selection by practitioners in different circumstances (using a Hierarchy of Abstraction Model [41]).
Conclusion
Using content analysis of semi-structured responses to short clinical vignettes in the domains of genetics and infectious diseases, we have explored information needs and resource preferences of office-based general pediatricians. This approach yielded descriptive information that demonstrates commonalities and distinctions with other studies on the information needs of general pediatricians.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
GRK participated in the design of the study and obtaining IRB approval, developed the vignettes and questionnaire, organized agreement on attribute assignments, recruited participants, interviewed the participants, coded transcripts (simple, axial and selective), and drafted the manuscript. ELB participated in the design of the study and obtaining IRB approval, tested the vignettes and reviewed the questionnaire and reviewed the manuscript. HPL participated in the design of the study and obtaining IRB approval, helped in the development of vignettes and questionnaire, participated in agreement on attribute assignments, participated in coding of transcripts and 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:
Supplementary Material
Additional file 1
Vignettes used in semi-structured interviews Sixteen vignettes listed by domain (Genetics (8), Infectious Diseases(8))
Click here for file
Additional file 2
Information resource survey Relative frequency of resource use identified by participants. Cited uses of specific resources
Click here for file
Additional file 3
Question types perceived in vignettes Patient-specific and general medical question types perceived by participants in response to vignettes with closest match of general medical question types to Generic Question taxonomy [26]
Click here for file
Additional file 4
Information resource preferences identified in vignettes Patient-specific and general medical information resources identified by participants in response to vignettes
Click here for file
Additional file 5
Frequency of perceived question types in vignettes Patient-specific and general medical question types perceived by participants in response to vignettes according to frequency of report
Click here for file
Additional file 6
Frequency of identified information resource preferences in vignettes Information resources identified by participants in response to vignettes according to frequency of report (for all vignettes and for vignettes by domain)
Click here for file
Acknowledgements
This study was funded by NLM Training Grant T15-LM07452-01. We acknowledge aid in recruitment from the American Academy of Pediatrics Steering Committee on Clinical Information Technology (SCOCIT) and the Maryland Chapter, the Johns Hopkins Community Physicians and the Howard University Department of Pediatrics. We also acknowledge the pediatricians who donated time and expertise as participants in this project.
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BMC NeurolBMC Neurology1471-2377BioMed Central London 1471-2377-5-181620737610.1186/1471-2377-5-18Research ArticlePrevalence of abnormal findings on brain magnetic resonance (MR) examinations in adult participants of brain docking Tsushima Yoshito [email protected] Ayako [email protected] Keigo [email protected] Department of Radiology, Motojima General Hospital, Ohta, 373-0033 Japan2 Department of Diagnostic Radiology and Nuclear Medicine Gunma University Hospital, Maebashi, Japan2005 5 10 2005 5 18 18 6 4 2005 5 10 2005 Copyright © 2005 Tsushima et al; licensee BioMed Central Ltd.2005Tsushima 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 determine the prevalence of abnormal findings on brain magnetic resonance (MR) examinations in adult participants of brain docking in order to assess its usefulness.
Methods
We analyzed screening brain MR examinations for 1113 adults (age, 52.6+/-8.5 years; range, 22–84; 761 male and 352 female) performed during 6-year period from April 1998 to March 2004. All participants voluntarily sought a brain MR examination at their own expense. All subjects were studied using the same 1.0-T MR scanner, on axial T1-weighted spin echo (SE) images, proton-density-weighted and T2-weighted fast SE images, and intracranial MR angiography (MRA). All abnormal findings were classified into three basic categories: (1) findings with no referral necessary; (2) findings not requiring further evaluation, but which needed to be reported to the referring physician; (3) findings requiring further evaluation.
Results
Participants with abnormal MR findings requiring further evaluation accounted for 1.3 %, but five of seven suspected intracranial aneurysms were not confirmed by other imaging modalities (false positive). No malignant tumors or other life-threatening pathology was detected, and only three participants (0.27 %) with abnormalities underwent surgical treatment. No participant groups were identified from our data as being high risk for MR abnormal findings requiring further evaluation.
Conclusion
Brain-docking participants had a variety of abnormalities on brain MR examinations, but only a small percentage of these findings required further evaluation. The usefulness of the brain docking with MRI and MRA has yet to be proven, and at this time we cannot approve this screening procedure.
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Background
"Brain docking", a method of screening for brain disease, has become popular in Japan in recent years. This unique Japanese practice, which may be performed as a part of an annual medical check-up, usually consists of brain magnetic resonance (MR) imaging and MR angiography (MRA) in addition to routine physical and laboratory examinations. It has been believed that brain docking may be beneficial for early diagnosis of some brain disorders, since it is well established that unexpected abnormalities are sometimes detected on brain MR examinations, usually in the setting of an investigation for a reason unrelated to the abnoramlity [1-4].
A screening examination is an examination on individuals who are at risk for a particular disease or condition, but who lack any signs or symptoms of the disease or condition, to determine if the disease or condition is present. It should be done only when clinical studies have demonstrated that screening examinations may do more good than harm [5]. The extraordinary spatial resolution of MR imaging seems to promise earlier intracranial disease detection and improved patient outcomes. However, to our knowledge, there has not been any scientific evidence to demonstrate that a screening brain MR examination may provide more benefit than harm to people being screened. Screening studies may raise issues regarding false-positive findings, overdiagnosis, and unnecessary additional medical examinations when the result is falsely interpreted as abnormal. Cost-effectiveness should also be determined.
In the current study, we analyzed the results of screening brain MR examinations in order to assess its usefulness. We also attempted to identify participant groups which are high risk for intracranial abnormalities on brain MR examinations, as limiting the screened population to those who are high risk for target disease would be better for cost effectiveness.
Methods
Participants and clinical data
We included 1113 consecutive adult participants (age, 52.6+/-8.5 years; range, 22–84; 761 male and 352 female) on whom brain MR examinations were performed during 6-year period from April 1998 to March 2004 as a part of an annual medical check-up in our hospital. All participants voluntarily sought a brain MR examination at their own expense. They usually believed that they were neurologically healthy at the time of the medical check-up, although some participants had a past history of brain infarction, bleeding, trauma or tumor. Some of them had recurrent headache or vertigo/dizziness, but they did not feel that immediate medical advice was required.
All participants underwent a clinical interview, physical and neurological examinations by a well-experienced neurologist. The presence of recurrent or chronic headache and vertigo/dizziness was always questioned by the neurologist. Since vertigo and dizziness were not always clearly differentiated, these two symptoms were combined in our analyses. Participants' demographic data consisted of age and sex. The body-mass-index (BMI), calculated as weight/height2 (kg/m2), was used as the index of relative weight. Self-reported data on cigarette smoking were used to classify subjects as nonsmokers, current smokers of 1 to 20 cigarettes per day (moderate smokers), and those smoking more than 20 cigarettes per day (heavy smokers). Regular alcohol consumption was recorded as grams of average absolute ethanol per day, and categorized as follows; nondrinkers, low alcohol intake (< 60 g/day; moderate drinkers), and high alcohol intakes (≥ 60 g/day; heavy drinkers). Individuals were considered to have systemic hypertension if their blood pressure readings had repeatedly exceeded 140 mmHg systolic or 90 mmHg diastolic, if they were currently taking antihypertensive therapy, or if they had a past medical history of systemic hypertension. Diabetes mellitus was diagnosed by a fasting serum glucose concentration of > 140 mg/dl or under current treatment for diabetes. High serum cholesterol level was diagnosed by a fasting serum total cholesterol level of > 250 mg/dl, or having a past medical history of high serum cholesterol level.
Brain stroke was defined as a history of physician-diagnosed symptomatic infarction and hemorrhage. Transient ischemic attack (TIA) was not considered a brain stroke. Cardiac diseases were defined as a history of congestive heart failure, myocardial infarction, angina pectoris, left ventricular hypertrophy, or atrial fibrillation; or electrocardiographic evidence of past myocardial infarction, left ventricular hypertrophy, or atrial fibrillation.
MR imaging and interpretation
All participants were studied on the same 1.0-T MR scanner (Magnex ®, Shimadzu, Kyoto, Japan), including axial T1-weighted spin echo (SE; TR = 450 ms, TE = 15 ms), and proton-density-weighted and T2-weighted fast SE (FSE; TR = 4000 ms, TE = 20 and 100 ms, echo train length = 8) images. The matrix was 256 * 192 and section thickness was 5 mm with a gap of 2.5 mm for all sequences. MR angiography (MRA) was also performed using time-of-flight (TOF) technique (TR = 40 ms, TE = 9 ms, flip angle = 20°, field of view = 200 mm; slice thickness = 1.0 mm; volume thickness = 54 mm; matrix = 256 * 174; number of acquisitions = 1; acquisition time = 6 minutes 15 seconds), and 16 projections of the MRA of the circle of Willis were created by a maximum-intensity projection (MIP) algorithm around the head-to-foot axis and right-to-left axis. Contrast-enhanced T1-weighted images were not obtained in any cases.
All MR images were interpreted by a board-certified diagnostic radiologist (YT) with 17-years of experience as a general radiologist, and all MR abnormalities were classified into three basic categories: (1) findings with no referral necessary; (2) findings not requiring further evaluation, but which needed to be reported to the referring physician; (3) findings requiring further evaluation. A well-experienced neurosurgeon also separately interpreted all images. When there was disagreement in image interpretation and neutral consensus could not be reached, the radiologist made the final decision. For MRA image interpretation, only the MIP images were evaluated, and the source images were not used.
White-matter signal abnormalities on MR images were considered present if visible as high intensity on proton-density and T2-weighted images, without prominent low intensity on T1-weighted images. According to Fazekas scale [6], periventricular hyperintensity (PVH) was graded as 0 = absence, 1 = caps or pencil-thin lining, 2 = smooth halo, and 3 = irregular PVH extending into the deep white matter. Separate deep white matter high intensities (DWMH) were rated as 0 = absent, 1 = punctuate foci, 2 = beginning confluence of foci, and 3 = large confluent areas. Both PVH and DWMH were considered abnormal when the grades were 2 or 3 [6], and classified as a finding not requiring further evaluation, but which needed to be reported to the referring physician.
When an intracranial aneurysm was suspected on MRA, a three-dimensional computed tomography (3D-CT) examination with contrast material or digital subtraction angiography (DSA) was recommended to confirm the diagnosis. 3D-CT was performed using a single-detector helical CT scanner (HiSpeed ®, GE-Yokogawa, Tokyo, Japan) with an intravenous bolus injection (2.5 ml/sec) of contrast material (iopamidol, Iopamiron 370 ®, Nihon Schering, Osaka, Japan; 80 ml), and surface rendering (SR) images were constructed from 40 or 50 axial images (thickness = 1.0 mm, pitch = 1.0) using an Advantage Workstation ® (Version 3.1; GE-Yokogawa, Tokyo, Japan).
In our hospital, no approval of the ethics committee was necessary for this kind of a retrospective study. The Declaration of Helsinki principles was followed.
Statistical analyses
The data were expressed by mean +/- standard deviation (SD). For statistical analyses, Student t test and Fisher exact test were used. P values less than 0.05 were considered significant. We set the screening cost for MR examination at US$200, although we know that the cost is much higher in US and EU countries.
Results
Nine of 1113 participants had a past history of intracranial disease or trauma (two with cerebral bleedings, two with infarctions, two with brain contusions, one subdural hematoma [postoperative], one acoustic neurinoma [postoperative] and one intracranial aneurysm [operated]). Of 1113 participants, 939 (84.4 %) were categorized was having no abnormal findings or findings with no referral necessary. Abnormal MR findings were demonstrated in 15.6 % of the participants and were classified as follows: 159 (14.3 %) with findings not requiring further evaluation, but needed to be reported to the referring physician; 15 (1.3 %) with findings requiring further evaluation (Table 1,2). No malignant tumors or other life-threatening pathology were detected.
Table 1 Findings requiring further evaluation (15 participants, 1.3%).
MR diagnosis No. of cases
Aneurysm, confirmed 1
Aneurysm, unconfirmed 6
Arachinoid cyst in the quadrigerminal plate cistern 1
Pituitary adenoma 3
Meningioma 1
Epidermoid tumor 1
Superficial siderosis 1
Major vessel stenosis 1
Total 15
Table 2 Findings not requiring further evaluation, but needing to be reported to the referring physician (159 participants, 14.3%).
MR diagnosis No. of cases
White-matter signal abnormalities 113
Lacunar infarction 31
Old lobar or cerebellar infarction 13
Venous malformation 6
Arachinoid cyst in the middle cranial fossa 4
Old bleeding 4
Old traumatic lesion 2
Cerebral atrophy 2
Total 175
On MRA, seven intracranial aneurysms and one middle cerebral artery stenosis in eight participants were suspected (Table 1), and all were categorized as having findings requiring further evaluation. Of these eight participants, one aneurysm of the anterior communicating artery (8 mm) and a middle cerebral artery stenosis were confirmed by 3D-CT. Five presumed aneurysms, all of which were measured less than 5 mm in diameter on MRA, were not confirmed on 3D-CT (n = 2) or DSA (n = 3), thus we considered that the MRA findings of these five participants were to be false positives. These false positive findings were suspected to be due to the low image quality of the MRA images. One remaining participant did not wish to confirm the diagnosis of aneurysm on other imaging modalities.
In our screened population, three participants (0.27%) with abnormalities (one aneurysm, one pituitary adenoma and one epidermoid tumor) underwent surgical treatment.
Demographic and clinical data of the participants were summarized in Table 3. There were no statistically significant differences between the participants with and without abnormal MR findings requiring further evaluation.
The cost for a screening brain MR examination was approximately US$200 in Japan, thus the estimated cost for the identification of one participant with a finding requiring further evaluation (1.3 %) was US$ 14840. When the cases with unconfirmed intracranial aneurysms were excluded, there were nine participants who had findings requiring further evaluation, and the cost for the identification of one participant with a finding requiring further evaluation (0.8 %) rose to US$ 24733.
Table 3 Demographic and clinical data of the participants with and without abnormal MRI findings requiring further evaluation.
Variables Findings requiring further evaluation (n = 15) No abnormality or findings not requiring further evaluation (n = 1098)
Age range Total Age range Total
34–59 y.o. (n = 11) 60–84 y.o. (n = 4) 34–59 y.o. (n = 910) 60–84 y.o. (n = 188)
Age (y.o.) 54.3 +/- 10.0 52.5 +/- 8.5
(range) (34–75) (22–84)*
Sex, male:female 9:2 1:3 10:5 622:288 129:59 751:347§
BMI (kg/m-2) 24.5 +/- 4.9 23.7 +/- 0.7 24.3 +/- 4.1 23.6 +/- 3.7 23.5 +/- 3.0 23.6 +/- 3.7
(range) (19.5–35.6) (22.7–24.4) (19.5–35.6) (13.8–59.0) (13.8–33.5) (13.8–59.0)*
Hypertension 3 1 4 (26.7%) 204 (22.4%) 59 (31.3%) 263 (24.0%)§
Diabetes mellitus 1 0 1 (6.7%) 94 (10.3%) 37 (19.7%) 131 (11.9%)§
Hyperlipidemia 6 1 7 (46.7%) 279 (30.7%) 74 (39.4%) 353 (32.0%)§
Cardiac disease 0 0 0 (0.0%) 21 (2.3%) 15 (8.0%) 36 (3.3%)§
Heavy smoking 1 0 1 (6.7%) 114 (12.5%) 16 (8.5%) 130 (11.8%)§
Heavy drinker 0 0 0 (0.0%) 40 (4.4%) 2 (1.1%) 42 (3.8%)§
Headache 1 0 1 (6.7%) 125 (13.7%) 9 (4.8%) 134 (12.2%)§
Vertigo/Dizziness 4 0 4 (26.7%) 111 (12.2%) 24 (12.8%) 135 (12.3%)§
There were no statistically significant differences between the participants with and without abnormal MR findings requiring further evaluation (*, Student t test; §, Fisher exact test). No statistics were done on the breakdowns (age range) of each group.
Discussion
In the current study, a variety of abnormal findings was discovered on screening brain MR examinations, but most of them were not serious. The prevalence of the abnormal findings requiring further evaluation in our study was only 1.3 %, and no malignant tumors or other life-threatening pathology was detected in any participants.
Some studies have attempted to determine the prevalence of abnormal findings in screening brain MR examinations, although most of these prior reports have focused on some specific abnormalities, such as silent infarctions [7], brain tumors [8] and unruptured intracranial aneurysms [9]. For instance, Onizuka et al. [8] reviewed screening brain MR examinations of 4000 individuals (24–85 years; mean, 56.0) without neurological signs or symptoms. They have focused on the prevalence of brain tumors, and found 11 incidental brain tumors (0.28 %). Some studies (Table 4) reported the prevalence of various incidental findings on MR examinations in neurologically healthy individuals in the setting of an investigation for other reasons [1-4]. Katzmen et al. [2] enrolled 1000 volunteers (3–83 years; mean, 30.6) who participated as control subjects for various research protocols, and reported the prevalence of 1.1 % of routine referral and 0 % of immediate referral necessary. Kim et al. [3] retrospectively reviewed 225 MR examinations performed for various research purposes in neurologically healthy children (1 month -18 years; mean 11.2 years), and reported a single lesion (< 1 %) requiring urgent referral. The prevalence of abnormal findings requiring further evaluation in our study (1.3 %) was close to the results previously reported in the adult population [1,2,4]. A very small percentage of participants (three participants; 0.27%) with abnormal findings required surgery in our study, and Yue et al. [1] also reported a similar low prevalence for abnormalities requiring surgery (0.25%).
Table 4 The studies evaluating the frequency of clinically important abnormalities on head MRI in neurologically normal participants of research protocols.
Study & Year No. of participants
(male/female) Mean age (range) MRI results
No referral* Routine referral Urgent referral Immediate referral
Yue NC, et al. (1997) (1) 3672 > 65 3608 (98.3 %) 64 (1.7 %)
Katzman GL, et al. (1999) (2) 1000 (546/454) 30.6 (3–83) 971 (97.1 %) 18 (1.8 %) 11 (1.1 %) 0 (0 %)
Kim BS, et al. (2002) (3) 225 (100/125) 11.2 (0–18) 206 (91.6 %) 17 (8 %) 1 (< 1 %) 0 (0 %)
Illes J, et al. (2004) (14) 151 (82/69) 47.1 (18–90) 141 (93.4 %) 7 (4.6 %) 3 (2.0 %) 0 (0 %)
SD: standard deviation.
* Including the participants without any abnormal findings.
It was quite difficult to determine the cost effectiveness of brain docking, since a variety of intracranial diseases were discovered. It would be better to calculate quality-adjusted life-years (QALYs) gained by this screening procedure but this was also difficult for the same reason. From our study, however, since no malignant tumors or other life-threatening diseases (except for an aneurysm) were discovered in any participants, the cost-effectiveness of this screening procedure would largely depend on the prevalence of intracranial aneurysms. However, the wisdom of searching for and treating small asymptomatic intracranial aneurysms has been widely questioned.
Yoshimoto et al. [10] reported that screening asymptomatic populations to identify and treat unruptured aneurysms would not be cost-effective assuming the incidence of unruptured aneurysm of 3.0 % in 50-year-old subjects and annual rupture rates of 0.02–0.005. In the current study, the prevalence of a possible intracranial aneurysm was only 0.62 % (seven of 1113 participants), and only one aneurysm was surgically treated, thus based on their study it was less likely that the screening brain MR examination was cost-effective in our population. Baba et al. [11] also studied the cost-effectiveness of screening for asymptomatic unruptured intracranial aneurysms and concluded that such a mass screening was not cost-effective unless the diagnostic accuracy of MRA was considerably increased or the annual rate of subarachinoid hemorrhage due to unruptured aneurysms was high (0.01 to 0.02 per year). The diagnostic accuracy of intracranial aneurysms affects the usefulness of screening MRA. It has been reported that MRA can depict intracranial aneurysms 5 mm or larger with good accuracy [12]. However, MRA is less useful for the identification of smaller aneurysms, and the accuracy in the detection of intracranial aneurysms depends on the field strength, i.e. high field strength confers higher accuracy [13]. The development of both hardware and software of the MR unit also contributes to the improvement in the accuracy of the detection of intracranial aneurysms. The field strength of our MR unit used in this study was 1.0 T and both hardware and software were relatively old, so many small aneurysms may have escaped a correct diagnosis on the MRA in the current study. For a better understanding of the false positive aneurysms it is better to make comparisons to newer MR technologies, although it is quite difficult to discuss this issue only from our current data. Further studies using newer MR equipments are encouraged, and we are now trying to evaluate MRA images in the brain docking in our hospital using new 1.5-T MR equipment.
Not only MRA but also brain MRI may miss some conditions, and a diagnosis of normal might be inaccurate. However, it was less likely that the field strength of the MR unit and the quality of hardware and software would significantly affect the accuracy of detecting clinically important intracranial abnormalities on MRI, since findings less conspicuous than those detected in our study were unlikely to be clinically important. The lack of clinical follow-up, which may result in the underestimation of false negative findings, was also a shortcoming of our study. Currently, the false negative rate of brain docking is unknown.
Another important concern was that the diagnoses of uncertain or unimportant findings may require additional invasive testing or elaborate follow-up, which may place the persons at risk for unexpected health consequences and lead them to unwarranted health expenditures, and will result in increased patient anxiety [14]. In fact in the current study, five of seven presumed intracranial aneurysms were not confirmed by other imaging modalities.
From the view point of cost-effectiveness, it is better to limit the screened population who are at high risk for the target diseases. However, we were not able to identify any groups which were high risk for having intracranial MR abnormalities requiring further evaluation.
Conclusion
Brain-docking participants had a variety of abnormalities on brain MR examinations, but the percentage of these findings requiring further evaluation was small. The usefulness of the brain docking with MRI and MRA has yet to be proven, and at this time we cannot approve this screening procedure.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YT participated in the design of the study and performed the statistical analysis. KE and ATT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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Yue NC Longstreth WT JrElster AD Jungreis CA O'Leary DH Poirier VC Clinically serious abnormalities found incidentally at MR imaging of the brain: data from the Cardiovascular Health Study Radiology 1997 202 41 46 8988190
Katzman GL Dagher AP Patronas NJ Incidental findings on brain magnetic resonance imaging from 1000 asymptomatic volunteers JAMA 1999 282 36 39 10404909 10.1001/jama.282.1.36
Kim BS Illes J Kaplan RT Reiss A Atlas SW Incidental findings on pediatric MR images of the brain AJNR Am J Neuroradiol 2002 23 1674 1677 12427622
Illes J Rosen AC Huang L Goldstein RA Raffin TA Swan G Atlas SW Ethical consideration of incidental findings on adult brain MRI in research Neurology 2004 62 888 890 15037687
Hillman BJ CT screening: who benefits and who pays Radiology 1993 228 26 28 12832572
Fazekas F Chawluk JB Alavi A Hurtig HI Zimmerman RA MR signal abnormalities at 1.5 T in Alzheimer's dementia and normal aging AJR 1987 149 351 356 3496763
Kobayashi S Okada K Koide H Bokura H Yamaguchi S Subcortical silent brain infarction as a risk factor for clinical stroke Stroke 1997 28 1932 1939 9341698
Onizuka M Suyama K Shibayama A Hiura T Horie N Miyazaki H Asymptomatic brain tumor detected at brain check-up Neurol Med Chir (Tokyo) 2001 41 431 435 11593969 10.2176/nmc.41.431
Nakagawa T Hashi K The incidence and treatment of asymptomatic, unruptured cerebral aneurysms J Neurosurg 1994 80 217 223 8283259
Yoshimoto Y Wakai S Cost-effectiveness analysis of screening for asymptomatic, unruptured intracranial aneurysms: a mathematical model Stroke 1999 30 1621 1627 10436111
Baba Y Takahashi M Korogi Y Cost-effectiveness of screening unruptured cerebral aneurysms in Japan Eur Radiol 2000 10 S362 S365 11001447
Korogi Y Takahashi M Mabuchi N Miki H Fujiwara S Horikawa Y Nakagawa T O'Uchi T Watabe T Shiga H Intracranial aneurysms: diagnostic accuracy of three-dimensional, Fourier transform, time-of-flight MR angiography Radiology 1994 193 181 186 8090889
Korogi Y Takahashi M Mabuchi N Watabe T Shiokawa Y Shiga H O'Uchi T Nakagawa T Miki H Horikawa Y Fujiwara S Furuse M MR angiography of intracranial aneurysms: a comparison of 0.5 T and 1.5 T Comput Med Imag Graph 1997 21 111 116 10.1016/S0895-6111(96)00064-X
Illes J Fan E Koenig BA Raffin TA Kann D Atlas SW Self-referred whole-body CT imaging: current implications for health care consumers Radiology 2003 228 346 351 12893896
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BMC Pregnancy ChildbirthBMC Pregnancy and Childbirth1471-2393BioMed Central London 1471-2393-5-131622567810.1186/1471-2393-5-13Research ArticleLonger postpartum hospitalization options – who stays, who leaves, what changes? Watt Susan [email protected] Wendy [email protected] Paul [email protected] School of Social Work, McMaster University, Hamilton, Ontario, Canada2 School of Nursing, McMaster University, Hamilton, Ontario, Canada3 Department of Clinical Epidemiology & Biostatistics, McMaster University, Hamilton and Senior Research Associate, St. Joseph's Health System Research Network, Brantford, Ontario, Canada2005 14 10 2005 5 13 13 11 2 2005 14 10 2005 Copyright © 2005 Watt et al; licensee BioMed Central Ltd.2005Watt 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 examines the practice implications of a policy initiative, namely, offering women in Ontario Canada up to a 60-hour postpartum in-hospital stay following an uncomplicated vaginal delivery. This change was initiated out of concern for the effects of 'early' discharge on the health of mothers and their infants. We examined who was offered and who accepted extended stays, to determine what factors were associated with the offer and acceptance of this option, and the impact that these decisions had on post-discharge health status and service utilization of mothers and infants.
Methods
The data reported here came from two related studies of health outcomes and service utilization of mothers and infants. Data were collected from newly delivered mothers who had uncomplicated vaginal deliveries. Questionnaires prior to discharge and structured telephone interviews at 4-weeks post discharge were used to collect data before and after policy implementation. Qualitative data were collected using focus groups with hospital and community-based health care managers and providers at each site. For both studies, samples were drawn from the same five purposefully selected hospitals. Further analysis compared postpartum health outcomes and post discharge service utilization of women and infants before and after the practice change.
Results
Average length of stay (LOS) increased marginally. There was a significant reduction in stays of <24 hours. The offer of up to a 60-hour LOS was dependent upon the hospital site, having a family physician, and maternal ethnicity. Acceptance of a 60-hour LOS was more likely if the baby had a post-delivery medical problem, it was the woman's first live birth, the mother identified two or more unmet learning needs in hospital, or the mother was unsure about her own readiness for discharge. Mother and infant health status in the first 4 weeks after discharge were unchanged following introduction of the extended stay option. Infant service use also was unchanged but rate of maternal readmission to hospital increased and mothers' use of community physicians and emergency rooms decreased.
Conclusion
This research demonstrates that this policy change was selectively implemented depending upon both institutional and maternal factors. LOS marginally increased overall with a significant decrease in <24-hour stays. Neither health outcomes nor service utilization changed for infants. Women's health outcomes remained unchanged but service utilization patterns changed.
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Background
In this paper, we examine changes in postpartum length of stay (LOS) in five Ontario hospitals following a provincial policy initiative that intended that women be offered the option of up to a 60-hour postpartum stay in hospital after an uncomplicated vaginal birth. In 1999, the Ontario Ministry of Health and Long-Term Care made the decision that, as part of the Healthy Babies Healthy Children program, hospitals should provide women with the option of up to 60 hours of in-hospital postpartum care [1]. The change was initiated out of concern for the impact of 'early' discharge on mother and infant health. The examination of postpartum LOS is based on two research studies, The Ontario Mother and Infant Survey (1998–2000) and The Ontario Mother and Infant Survey II (2001–2004).
In exploring the implementation of this initiative, we examined who was offered and who accepted a 60-hour stay. We compared groups of women who were offered/not offered and accepted/did not accept extended stays to determine what factors were associated with the offer and acceptance of this option. Additionally, we examined the impact of this change on the post-discharge health outcomes and service utilization of mothers and infants.
Postpartum hospital length of stays
In the 20th century, there was a shift to delivering the majority of infants in hospitals [2,3]. For example, in the United States, the most frequent reason for hospitalization of females is pregnancy [4]. In Canada, the second most prevalent reason for hospitalization of females is pregnancy and childbirth (24% of all women hospitalised) [5]. However, while hospital delivery has become the norm, a search of the literature revealed that the length of the subsequent postpartum hospitalization has been the subject of opinion, conjecture and change.
One constant of postpartum in-hospital LOS over the last century has been the extension and contraction of the time considered appropriate. Variously, LOS has ranged from 14-day lying-in periods [3] to 'drive-through' deliveries [6-8] with only several hours of postpartum in-hospital care.
Over the past 20 years, international literature about postpartum LOS reveals vast practice differences among stakeholders. For example, in reviewing the postpartum discharge practices of eight international clinical trials, Brown, Small, Faber, Krastev, and Davis identified that there was a wide range of definitions of what constituted an 'early discharge' [9]. In 2002, Britton, Baker, Spino and Bernstein reported that, in the United States, 59% of paediatricians surveyed believed that for healthy newborns, a 37 to 48 hour post-delivery hospital stay was optimal and that LOS was a clinical decision that should be determined by the infant's physician [10].
Changing health policies
The policies of insurers rather than clinicians largely have driven postpartum LOS practices. For example, in the United States in the 1990's, private insurers refused to pay for postpartum hospital stays beyond 12–24 hours, forcing physicians and hospitals to discharge mothers and infants as quickly as possible [2]. Similarly, Brown et al reported that, in Australia, postpartum LOS has decreased significantly in the last 12 years [9]. However, "while the proportion of women with private health coverage who stay in hospital for ≥5 days has fallen, there continues to be only a small number of women in this category who leave hospital before day three (<2% in 1999)" [[9], p. 5].
The literature also describes what happens when institutions attempt to structurally standardize postpartum LOS. As Declercq and Simms make clear, in the United States, the legislative imposition of a minimum 48 hour LOS guarantee was meant to contravene the highly criticized, fiscally driven actions of insurers and the 'drive through' postpartum practices of many medical centres [6].
In the United States, Lichtenstein, Brumfield, Cliver, Chapman, Lenze and Davis observed that "the rules of early discharge, while framed by state and federal laws, were bureaucratically organized to meet hospital priorities" [11]. Lichtenstein et al also explored who was offered and who accepted the offer of an extended stay. They argued that, "early discharge was normative" [[11], p 89] and determined that sociodemographic factors, (i.e., age, education, marital status, urban or rural homes) combined with institutional norms, "produce differential outcomes in the length of postpartum care" [[11], p 89].
On the other hand, single events rather than systematic change, can trigger changes in LOS. In Ontario, the death of an infant after 'early' release, combined with pronouncements by the Canadian Paediatric Society, spurred a policy guarantee of a 60-hour LOS [12,13].
Changing health practices
The literature about postpartum LOS also revealed differences of opinion among stakeholders regarding the outcomes of 'early' discharge. Usually, the debate has focussed on health of the infant. U.S. paediatricians who took part in the study by Britton et al expressed concern that, because of shortened postpartum LOS, "they had cared for infants who were discharged early and experienced adverse outcomes related to the short stay" [[10], p 53]. D'Amour, Goulet, Labadie, Bernier, and Pineault reported that nurses surveyed in Canada were concerned about release in under 72 hours because "It is often at the third day following birth, when the mother and the baby have returned home, that the main health problems arise" [[14], p 398] and that "shorter stays in hospital often result in parents not having enough time to receive or fully integrate hospital information about care for both the baby and the mother" [[14], p 398]. On the other hand, many articles reported no adverse outcomes because of early postpartum release [9,15,16] and that the relationship between a shorter stay and newborn infant adverse health outcomes, including readmission, are inconsistent [19-27].
Methods
Data collection for The Ontario Mother and Infant Survey (TOMIS) occurred between November 1998 and June 1999 and was staggered across sites. The Hospital Stay and Postpartum Home Visiting Program extension to the Health Babies Healthy Children program was added in November 1999. TOMIS II data collection was started in September 2001, with initiation staggered across sites, and was completed in March 2002.
Data for TOMIS I and TOMIS II were collected using quantitative cross-sectional surveys completed at discharge (self administered questionnaire) and approximately 4-weeks post-discharge (structured telephone interview). The survey methods and instruments used for TOMIS II paralleled those used in TOMIS, which allowed for an appropriate comparison of data at two points in time. The same study sites, sample size, eligibility criteria, recruitment strategy, and instruments were used for the two surveys [16]. Five purposefully selected Ontario hospitals provided respondents who constituted a cross-section of mothers and newborn infants with diverse socio-economic characteristics and access to varying health and social services. The characteristics of the hospitals were as follows:
Site 1 Southern, suburban, teaching hospital, metropolitan catchment area, 3900 annual births
Site 2 Central east regional centre, urban & rural catchment areas, 1500 annual births
Site 3 Central south regional centre, urban & rural catchment areas, 4500 annual births
Site 4 Southern, urban, teaching, metropolitan catchment area, 2700 annual births
Site 5 Central north regional centre, urban & rural catchment areas, 2000 annual births.
Participants for both studies included the first 250 eligible, consenting women from each site, totalling 1,250 participants in each study. This sample size was determined to be large enough to allow for the examination of many variables together, and was in keeping with the generally accepted guideline of 30 subjects per variable [28]. Women were eligible if they (a) had given birth vaginally to a single live infant, (b) were being discharged from hospital at the same time as their infant, (c) were assuming care of their infant at the time of discharge, and (d) were competent to give consent to participate. Women were excluded if they (a) had an infant who required admission to a neonatal intensive care or special care nursery for more than 24 hours or (b) were unable to communicate in one of the study languages – English, French, Chinese, and Spanish. In previous studies it had been determined that this way of recruiting subjects produced samples at each site that reflected the populations known to be served by each hospital thereby permitting generalizability of the results to this healthy group of women and their newborn children [16]. Each study hospital continued to utilize its own postpartum care protocols throughout the recruitment period. Full descriptions of the methodology have been published [16]. The ethics review committees of McMaster University and each of the hospitals involved in the study granted ethical approval.
Descriptive statistics were computed by site for all variables measured, including frequency counts and percentages, or means and standard deviations (as appropriate). T-tests, chi-square tests or, when appropriate, Fisher's exact tests were used to determine differences between sites or differences between TOMIS and TOMIS II data. Chi-square tests were used to identify variables associated with being offered the option of a 60-hour stay in hospital following delivery and variables associated with acceptance of a 60-hour length of stay. The decision about which variables to include in these bivariate analyses were made a priori based on the literature, available data, and clinical judgement. Unadjusted odds ratios, corresponding 95% confidence intervals, and p-values are reported for these associations. Only variables that were found to be statistically significant in the bivariate analyses or were judged to be clinically relevant were included in the logistic regression. Multiple logistic regression analysis was used to identify the best predictors of being offered the option of a 60-hour stay as well as to identify the best predictors of acceptance of a 60-hour stay. The final results are reported as adjusted odds ratios (OR) and 95% confidence intervals. The goodness of fit of the logistic regression model was assessed using the rho-squared statistic [29]. A rho-square value between 0.20 and 0.40 suggests a very good fit of the model. A probability level of <0.05 was used to determine statistical significance. SPSS was used for all statistical computations.
Results
Study participants
Of the 1250 women recruited for TOMIS II, 890 (61.2% to 82.8% per site) participated in the follow-up telephone interview at 4-weeks post discharge. Table 1 provides a profile of the women and infants who took part in the survey. The profiles of the samples generally reflect the census profiles of women aged 15 to 35 in the tracts from which each hospital normally draws its patient population. In keeping with ethical standards of research practice in hospitals, data on non-consenting women were not collected
Table 1 Characteristics of TOMIS II study participantsa
Characteristic Site 1 (n = 250) Site 2 (n = 250) Site 3 (n = 250) Site 4 (n = 250) Site 5 (n = 250)
Maternal age in years (mean ± SD)b 31.7 ± 4.9 28.8 ± 5.1 29.3 ± 5.2 29.7 ± 5.7 27.0 ± 5.1
Gestation in weeks (mean ± SD) 39.5 ± 1.4 39.7 ± 1.4 39.7 ± 1.4 39.4 ± 1.7 39.4 ± 1.3
Birth weight in grams (mean ± SD)b 3344 ± 452 3525 ± 516 3564 ± 485 3404 ± 682 3517 ± 557
% % % % %
First live birth 43.4 42.0 40.4 46.0 44.0
Martial status c
Married 88.8 71.3 79.9 78.3 59.3
Common-law/living with partner 6.0 21.9 14.5 12.3 27.8
Never married/separated/widowed/divorced 5.2 6.9 5.6 9.4 12.9
Family income c,d
<$20,000 12.1 14.7 7.4 28.5 23.8
$20,000 to $39,999 18.2 20.7 13.0 18.4 19.7
$40,000 to $59,999 17.3 29.7 23.4 16.7 18.8
$60,000 to $79,999 16.0 17.2 22.1 13.6 17.0
≥$80,000 36.4 17.7 34.2 22.8 20.6
Born in Canada c 37.6 93.6 81.1 34.1 96.8
Self-reported ethnicity c
Canadian 26.9 94.3 79.2 37.0 93.6
Other than Canadian 73.1d 5.7 20.8 63.0f 6.4
Language spoken at home c
English/French 55.2 99.6 86.0 63.9 99.6
Other than English/French 44.8e 0.4 14.0 36.1f 0.4
Highest level of education c
Less than high school 4.5 9.7 11.6 17.1 13.4
High school 9.7 13.3 14.1 20.8 10.2
Some community college/technical school 5.3 14.5 10.4 8.6 13.4
Completed community college/technical school 19.8 33.5 24.1 17.6 29.7
Some university 10.1 5.6 9.6 6.9 5.3
University 50.6 23.4 30.1 29.0 28.0
a originally published in Sword, W., Watt, S., Krueger, P. (2004). Implementation, Uptake, and Impact of a Provincial Postpartum Program, Canadian Journal of Nursing Research, 36, 60–82
b ANOVA indicated a statistically significant difference across sites (p < 0.05)
c Chi-square test indicated a statistically significant difference across sites (p < 0.05)
d 8.4% of the total sample did not report family income
e 26.9% of the total sample at Site 1 "Chinese"; 15.5% "Jewish"; 23.6% spoke Chinese at home
f 11.9% of the total sample at Site 4 "South Asian"; no predominant language "Other than English/French"
There were no statistically significant differences in any of these variables between those women who completed the telephone interview and those who did not. This finding suggests that subjects lost to follow-up were similar in terms of sociodemographic characteristics to those who participated in the interview. Statistically significant differences were found between sites for all of the variables except mean length of gestation and first live birth (which are less likely to vary by site given the inclusion criteria), thereby reflecting the diversity in the sample we had intended to achieve with the selection of study sites. When TOMIS II participants were compared to TOMIS participants, there were minimal differences in sociodemographic characteristics reported in Table 1.
Implementation of an extended lengths of stay
There were wide and statistically significant differences (p < 0.05) in implementation of the 60-hour stay option across sites, with between 11.7% and 81.2% of women reportedly having been offered an extended hospital stay (Site 1 – 11.7%; Site 2 – 41.9%; Site 3 – 81.2%; Site 4 – 39.9%; Site 5 – 52.3%). Of those women who were offered a 60-hour stay, between 21.1% and 39.4% accepted (Site 1 – 21.1%; Site 2 – 39.4%; Site 3 – 30.4%, Site 4 – 31.3%; Site 5 – 21.3%). No statistically significant difference was found across sites for these acceptance rates.
Implementation challenges in offering a 60-hour stay identified by focus group participants included a limited capacity due to recent downsizing and reorganization of catchment areas for obstetrical units. These factors were compounded by a recent increase in the number of deliveries at some of the sites. At sites where physical capacity was an issue, care providers acknowledged that they did not routinely offer an extended stay but rather made clinical judgments in determining an appropriate LOS for each woman admitted to their unit.
Those women who accepted a longer stay had a variety of reasons for doing so, including their own health (31.7%), the health of their baby (39.8%), and breastfeeding difficulties (20.2%). Those women who declined the 60-hour stay offer identified wanting to go home as the major reason for turning it down (39.5%). Additionally, this group identified readiness for discharge (25.0%), dissatisfaction with their hospital accommodation or care (16.0%), and other children at home (10.2%) as reasons for declining a longer hospital stay.
Satisfaction with postpartum LOS increased significantly following the change in policy and the resultant increase in LOS. Satisfaction increased from 74 to 89% (p < 0.01). Although not statistically significant, women who were not satisfied preferred longer stays (77.3% and 69.2%; p = 0.220). While women who preferred a shorter postpartum stay increased from 21% to 28%, it is interesting to note that of those offered a 60-hour stay, 96.1% were satisfied with their postpartum LOS whether or not they made use of the offer. Conversely, 80% of the women who were not offered a 60-hour stay were dissatisfied with their LOS.
Offer of an extended length of stay
Based on the literature and the findings of TOMIS, a number of variables were examined to determine what factors might be associated with being offered up to a 60-hour postpartum LOS. Five variables were found to be significantly associated – hospital site, having a family physician, self-defined ethnicity of mother as Canadian, English or French language spoken at home, and being born in Canada (Table 2).
Table 2 Variables associated with being offered the option of a 60-hour stay in hospital following delivery a (n = 1230)
Offered 60 Hr LOS
Variables Yes (%) No (%) P value b Unadjusted Odds Ratio 95% Confidence Interval
Information Collected From Mother Prior to Discharge From Hospital
Site (n = 1230):
1 19 (7.7) 227 (92.3) 1.00 -
4 65 (27.2) 174 (72.8) 4.46 (2.58, 7.72)
2 89 (35.9) 159 (64.1) 6.69 (3.92, 11.42)
5 127 (51.0) 122 (49.0) 12.44 (7.32, 21.13)
3 216 (87.1) 32 (12.9) <0.001 80.64 (44.3, 146.6)
Age of mother (n = 1228)
20 to 39 468 (41.7) 653 (58.3) 1.00 -
<20 or >39 47 (43.9) 60 (56.1) 0.663 1.09 (0.73, 1.63)
First live birth (n = 1226):
No 286 (40.9) 413 (59.1) 1.00 -
Yes 228 (43.3) 299 (56.7) 0.409 1.10 (0.88, 1.38)
Has a family physician (n = 1226):
No 17 (26.6) 47 (73.4) 1.00 -
Yes 497 (42.8) 665 (57.2) 0.011 2.07 (1.17, 3.64)
Baby had medical problems since birth (n = 1229):
No 442 (41.1) 634 (58.9) 1.00 -
Yes 74 (48.4) 79 (51.6) 0.087 1.34 (0.96, 1.88)
Number of concernsc prior to discharge (n = 1230):
Two or more 203 (39.3) 314 (60.7) 1.00 -
One or fewer 313 (43.9) 400 (56.1) 0.104 1.21 (0.96, 1.52)
Mother had medical problems since birth (n = 1229):
No 478 (41.5) 674 (58.5) 1.00 -
Yes 38 (49.4) 39 (50.6) 0.176 1.37 (0.87, 2.18)
Mother has other children (n = 1224):
Yes 293 (40.8) 426 (59.2) 1.00 -
No 219 (43.4) 286 (56.6) 0.361 1.11 (0.88, 1.40)
Language spoken most often at home (n = 1230):
Other 55 (23.8) 176 (76.2) 1.00 -
English or French 461 (46.1) 538 (53.9) <0.001 2.74 (1.98, 3.80)
Ethnic or cultural group (n = 1216):
Other 95 (23.6) 307 (76.4) 1.00 -
Canadian 419 (51.5) 395 (48.5) <0.001 3.42 (2.62, 4.48)
Place of birth (n = 1229):
Other 99 (26.1) 280 (73.9) 1.00 -
Canada 417 (49.1) 433 (50.9) <0.001 2.72 (2.09, 3.56)
Marital status (n = 1223):
Partnered 470 (41.8) 655 (58.2) 1.00 -
No Partner 45 (45.9) 53 (54.1) 0.426 1.18 (0.78, 1.79)
Total income before taxes and deductions of all household members (n = 1134):
Less than $20,000 70 (36.3) 123 (63.7) 1.00 -
$20,000 or more 406 (43.1) 535 (56.9) 0.078 1.33 (0.97, 1.84)
Highest level of education (n = 1221):
Completed high school or less 125 (41.4) 177 (58.6) 1.00 -
Education beyond high school 387 (42.1) 532 (57.9) 0.826 1.03 (0.79, 1.34)
Mother feels that help and support at home will meet both her and baby's needs (n = 1220):
Other response 215 (39.7) 326 (60.3) 1.00 -
Definitely yes 299 (44.0) 380 (56.0) 0.131 1.19 (0.95, 1.50)
Mother feels she and baby are ready to be discharged (n = 1227):
Definitely/Probably no/Don't know 65 (36.1) 115 (63.9) 1.00 -
Definitely/Probably yes 450 (43.0) 597 (57.0) 0.085 1.33 (0.96, 1.85)
a Exact question asked was, "Were you offered the option of a 60-hour stay in hospital after your delivery? Yes/No".
b Chi-square test
c Concerns included: breast-feeding; bottle-feeding; infant care and behaviour; signs of illness in infant; physical changes and care of yourself; sexual changes and intercourse; emotional changes in yourself.
The final logistic regression model found that the first three factors were the most important predictors of being offered a 60-hour postpartum LOS (Table 3). The hospital in which a woman delivered remained the single most important predictor.
Table 3 Final logistic regression model of the most important predictors of being offered a 60-hour stay in hospital following delivery (n = 1212a)
Predictors Adjusted Odds Ratio b 95% Confidence Interval
Site
1 1.00 -
4 7.37 (4.66, 11.68)
2 13.22 (8.29, 21.09)
5 14.84 (9.13, 24.14)
3 33.68 (33.68, 115.3)
Mother has a family physician:
No 1.00 -
Yes 2.45 (1.28, 4.69)
Ethnic or cultural group:
Other 1.00 -
Canadian 1.84 (1.26, 2.70)
Final Logistic Regression Model Statistics: Rho-square = 0.25 (a pseudo R2, values between 0.2 and 0.4 suggest a very good fit)
Hosmer and Lemeshow Goodness-of-fit test = 0.98 (values greater than 0.25 indicate good fit) 74.3% correctly classified
a Eighteen of the 1230 (1.5%) mothers had missing values for one or more of the variables included in the final model.
b Odds ratios for categorical variables represent comparisons with the referent group (OR = 1.00) after adjustment for all other variables in the model.
Acceptance of an extended length of stay
The ten variables found to be associated with the acceptance of the offer are the following:
1. first live birth;
2. the baby having medical problems since birth;
3. the mother having two or more concerns prior to discharge;
4. the mother having medical problems since birth;
5. the mother being unsure that help and support at home will meet both her and the baby's needs;
6. the mother being not sure that she and the baby are ready for discharge;
7. the mother having 2 or more unmet learning needs while in hospital;
8. the mother's rating of her own health as only good/fair/poor since having the baby;
9. the mother having a low affective social support score; and,
10. the mother not being able to tell when her baby is sick (See Additional file 1).
Four of these variables emerged as the best predictors of acceptance – unmet learning needs in hospital, infant medical problems, first live birth, and mother's perception of her own discharge readiness (Table 4).
Table 4 Final logistic regression model of the most important predictors of accepting an offer for a 60-hour stay in hospital following delivery (n = 379)
Predictors Adjusted Odds Ratio a 95% Confidence Interval
Number of unmet identified learning needs while in hospital:
Less than 2 1.00 -
2 or more 1.98 (1.27, 3.10)
Baby had medical problems since birth b:
No 1.00 -
Yes 2.82 (1.49, 5.34)
First live birth b:
No 1.00 -
Yes 1.86 (1.20, 2.89)
Mother feels she and baby are ready to be discharged b:
Definitely yes 1.00 -
Other response 1.84 (1.19, 2.86)
Final Logistic Regression Model Statistics: Rho-square = 0.10 (a pseudo R2, values between 0.2 and 0.4 suggest a very good fit)
Hosmer and Lemeshow Goodness-of-fit test = 0.36 (values greater than 0.25 indicate good fit) 64.9% correctly classified
a Odds ratios for categorical variables represent comparisons with the referent group (OR = 1.00) after adjustment for all other variables in the model.
b Information collected from mother prior to discharge from hospital
Health outcomes and post-discharge service utilization
The question then arose, "If postpartum LOS has increased, what is the impact of this change on the health status of the mother and infant, and on their service utilization following discharge?" The health status of both the mother and her infant, as reported by the mother, was unchanged from TOMIS. In TOMIS 88.4% and in TOMIS II 90.7% of women reported their own health as excellent, very good, or good (p = 0.12). Infant health was reported in both studies as excellent, very good, and good by 98.0% of mothers (p = 0.33).
Similarly, service utilization in terms of use of community physicians and emergency rooms and hospital readmissions for infants was unchanged (Table 5). Women, however, experienced more hospital readmissions but fewer had contacts with community physicians and emergency rooms (Table 5).
Table 5 Service utilization of mother and infant
Service Utilization TOMIS n = 875 TOMIS II n = 890 p-value
No. % No. %
Mother
Community physician a 670 76.6 640 71.9 0.03
ER 59 7.1 29 3.3 0.001
Readmission 8 0.9 35 4.0 <0.001
Infant
Community physician b 826 94.4 857 96.3 0.076
ER 70 8.0 66 7.4 0.71
Readmission c 35 4.0 40 4.5 0.69
a includes family physicians, gynaecologists, and walk-in clinics; excludes exclusive ER or midwife contact
b includes family physicians, paediatricians, and walk-in clinics; excludes exclusive ER or midwife contact
c excludes day admissions for circumcisions
Discussion
The results demonstrate that implementation of this policy was dependent upon the specific hospital's approach to postpartum care and what the decision makers in each hospital viewed as an appropriate LOS. The attitudes of providers, namely, physicians and nurses, and the perception of administrators about the adequacy of their facilities to accommodate what they believed would be a significant uptake of the offer of an extended stay played a role in determining whether or not the practice was implemented in each of the sites. It was clear that providers did not perceive the 60-hour stay policy as a practice requirement. Yet the policy appears to have increased the sensitivity of providers to the possible problems associated with a short hospital stay. Hence, stays of <24 hours decreased. It would appear that a new norm developed for a 24 to 48 hour postpartum LOS for uncomplicated vaginal deliveries.
Although providers had been quite concerned about short stays, women were, overall, very satisfied with their LOS whether or not they accepted an extended stay. The anticipated increased demand for extended stays did not materialize. Rather, for a variety of reasons, many women appeared to prefer to leave hospital as soon as they and their infants were able to do so and did not suffer negative health consequences in the following 4 weeks.
The two features of the social location of a mother that would most likely lead to being offered a 60-hour postpartum stay, self-identified ethnicity as Canadian and having a family physician, suggests that women who were offered an extended stay were not marginalized and were likely to have social supports. The women who often are deemed by health professionals as being most at risk for poor outcomes with early discharge, the young, the poor, and the unsupported, did not appear to be targeted by this selective policy implementation.
Uptake of the offer of a 60-hour postpartum stay had much more to do with a woman's own sense of readiness to leave hospital with her infant than with structural factors. A first time mother was more likely to have concerns about leaving hospital and to see the hospital as a "safe haven". She was not obligated to leave hospital by the need to mother other children. Since cost was not a factor at the level of the individual in a system of universal health insurance, these mothers could wait until they believed they were ready to go home.
The question then was asked of whether a lengthening of hospital stay, in the circumstances defined in this study, made any difference in outcomes. Evidence from this study showed no change in the self-evaluated health status of the mother or in her evaluation of her infant's health at 4 weeks post discharge. Similarly, no change was found in infant service utilization of community physicians, emergency rooms, or hospital readmission. Maternal service utilization decreased in relation to the use of community physicians and emergency rooms but readmission increased overall, ranging from 1% to 4% with statistically significant increases at two of the five sites. These findings suggest that, at least in the short term, increasing postpartum LOS increased hospital postpartum care costs without producing substantial health improvement or reduction in costs of service use following discharge.
Conclusion
The findings of TOMIS and TOMIS II suggest that if practice is to be changed, policy directives, while necessary, may be insufficient incentives. Compelling clinical evidence is required to change practice. Clinicians allocate resources on the basis of their clinical judgement rather than policy guidelines.
Healthy women with healthy babies, when given options, seem to make choices about how long to stay in hospital and choose to stay for the shortest possible period based on their perceptions of their own health status and that of their infant. Flexibility rather than strict rules is needed if both clinical judgement and a woman's preference are to be considered in individual LOS decisions.
Neither health outcomes nor economies in service utilization were found in our study to provide justification for an extended postpartum stay for healthy women and infants. Patient satisfaction may be the most important factor to consider. Women are more satisfied with their hospital experiences when they are offered the option of a stay of up to 60 hours whether or not they stay for this period. Not surprisingly, the ability to exercise some degree of control over one's own care continues to be an important issue in patient satisfaction and probably a factor in a woman's decision about how long to stay in hospital. Therefore, finding optimal lengths of stay for healthy women following an uncomplicated vaginal delivery is appropriately less a matter of policy and more an issue of good clinical judgement on the part of both women and their health care providers.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SW had a major role in designing the study and writing the proposal, co-supervised all aspects of study implementation, participated in data analysis, and was the lead writer of this manuscript.
WS had a major role in designing the study and writing the proposal, co-supervised all aspects of study implementation, participated in data analysis, contributed to the manuscript, and provided editorial comments.
PK contributed to the study design, implementation, analysis and interpretation, as well as the writing of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional file 1
Table – Variables associated with acceptance of a 60-hour length of stay. This table provides the P-value, Unadjusted Odds Ratio, and Confidence Intervals for the variables associated with the acceptance of the offer of a 60-hour postpartum length of stay in hospital.
Click here for file
Acknowledgements
The Canadian Health Services Research Foundation funded the Ontario Mother and Infant Survey (TOMIS). The Canadian Institutes of Health Research funded TOMIS II.
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-1031621265710.1186/1471-2458-5-103Research ArticleWork ethics and general work attitudes in adolescents are related to quality of life, sense of coherence and subjective health – a Swedish questionnaire study Axelsson Lars [email protected] Ingemar [email protected]åkansson Anders [email protected] Göran [email protected] Department of Health Sciences, Kristianstad University, S-291 88 Kristianstad, Sweden2 Department of Clinical Sciences, Malmö, Family Medicine, Lund University, S-205 02 Malmö, Sweden2005 7 10 2005 5 103 103 26 4 2005 7 10 2005 Copyright © 2005 Axelsson et al; licensee BioMed Central Ltd.2005Axelsson 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
Working life is an important arena in most people's lives, and the working line concept is important for the development of welfare in a society. For young people, the period before permanent establishment in working life has become longer during the last two decades. Knowledge about attitudes towards work can help us to understand young people's transition to the labour market. Adolescents are the future workforce, so it seems especially important to notice their attitudes towards work, including attitudes towards the welfare system. The aim of this study was to describe and analyse upper secondary school students' work attitudes, and to explore factors related to these attitudes.
Methods
The sample consisted of 606 upper secondary school students. They all received a questionnaire including questions about quality of life (QOL), sense of coherence (SOC), subjective health and attitudes towards work. The response rate was 91%. A factor analysis established two dimensions of work attitudes. Multivariate analyses were carried out by means of logistic regression models.
Results
Work ethics (WE) and general work attitudes (GWA) were found to be two separate dimensions of attitudes towards work. Concerning WE the picture was similar regardless of gender or study programme. Males in theoretical programmes appeared to have more unfavourable GWA than others. Multivariate analyses revealed that good QOL, high SOC and good health were significantly related to positive WE, and high SOC was positively related to GWA. Being female was positively connected to WE and GWA, while studying on a practical programme was positively related to GWA only. Among those who received good parental support, GWA seemed more favourable.
Conclusion
Assuming that attitudes towards work are important to the working line concept, this study points out positive factors of importance for the future welfare of the society. Individual factors such as female gender, good QOL, high SOC and good health as well as support from both parents, positive experience of school and work contacts related positively to attitudes towards work. Further planning and supportive work have to take these factors into account.
==== Body
Background
The increasing number of people being outside the labour market in most European countries is a huge problem for both the societies and the individuals involved. Working life is an important arena in most people's lives, and also for a well-functioning society, so it is important to focus on people's attitudes towards work.
There are new demands and expectations of both the labour market and the workforce [1], and young people do not fully comply with these demands of improved flexibility [2]. According to a previous study, young people find it more acceptable to take advantage of social benefits compared to old people [3]. The structure of the labour market and working life is continuously changing, a development that has been especially obvious in recent decades. The working field has changed from a focus on industrial production towards a focus on information and media and improved educational skills [4]. Concerning young people, the period before permanent establishment in working life has become longer during the last two decades [5].
Working life is of fundamental importance for the development of welfare in a society [6]. An essential part of the welfare system is the working line concept [7], which has different meanings in different countries. The fundamental meaning of the concept is that everybody should earn his or her own living through work if possible. When needed, support to manage work should be given before public financial support is offered. Those who are unable to work should be financially compensated by the social welfare system. In Sweden, a debate is in progress about a general decline in ethics in the use of the social welfare system. This is probably related to huge restructuring of the public welfare system and increased individualisation among members of society [8].
The welfare system in Sweden, as in other parts of Scandinavia, is quite generous compared to many other countries, but the development of welfare also depends on the working line concept, which seems to bolster the welfare in a society. Living in generous welfare state like those in Scandinavia improves the experience of intrinsic work values rather than affecting work ethics negatively [9]. In Sweden, the social welfare compensation system is related to the loss of income through unemployment, disease and parental leave. However, the working line concept in Sweden has become weaker in its capability to motivate those on sick leave and in unemployment to re-enter working life [6].
Because important parts of the welfare system, and thereby equity between people in society, rely on the existence of a functional working line concept, it seems urgent to maintain it. Knowledge about attitudes towards work can help us to understand young people's transition into the labour market. As adolescents will constitute the forthcoming manpower, it seems especially important to notice their attitudes towards work, working life and the welfare system. In this respect, it has been highlighted that there is an extensive literature on personality and work, but the research on individual differences in attitudes and motivations to work seem to be rare [10].
Bearing in mind the great importance of the working line concept for a society's welfare, the aim of this study was to describe and analyse upper secondary school students' work attitudes, and to explore factors related to these attitudes.
Methods
The Swedish upper secondary school consists of 16 different study programmes. The two theoretical programmes are either natural sciences or social sciences/humanities. The other 14 programmes are practical, training pupils for occupations, e.g. mechanics, building workers, industrial workers, electricians, assistant nurses or hotel and restaurant staff. Approximately 98% of all teenagers in Sweden attend upper secondary school, which lasts for three years.
This study was performed in Kristianstad municipality in the south of Sweden with approximately 75,000 inhabitants and with six upper secondary schools. About 1100 students were studying in their last year in those schools and about half of them were invited to participate in the study.
In January and February 2004, a questionnaire was distributed to 606 students (median age 18 years). In order to obtain a manageable and representative sample concerning sex and study programme, all students in three of the schools and all students in the theoretical programmes in another school were invited to participate in the study. In this study, students from 13 of the 16 ordinary programmes participated.
The questionnaire included questions about immigration, parental employment status, social support, subjective health and quality of life (QOL), satisfaction with aspects of life, sense of coherence (SOC), and different aspects of attitudes towards life and work. First, interviews, as a basis for the questions were performed with eleven students in practical and theoretical programmes. The questionnaire was developed, then tested in a pilot study among 14 students in practical and theoretical programmes, and adjusted before use.
The students answered the questionnaire anonymously in school. Students not present at the survey day received an envelope with the questionnaire when they were back in school again. Their main teacher collected the envelopes and returned them by post to the investigator. The final response rate was 91%, which means that 551 students responded to the questionnaire (Table 1).
Table 1 Number of students and percentages of dropout in theoretical and practical programmes. Males and females.
Theoretical programmes Practical programmes
Males Females Males Females Total
Number of students in the sample 103 128 245 130 606
Number of respondents 99 118 220 110 547a
Number of dropouts (%) 4 (4) 10 (8) 25 (10) 20 (15) 59 (9)
Note: aFour respondents had not declared sex.
Measurements and definitions
By using factor analysis, a set of answers to seven questions concerning attitudes towards work was analysed. Two dimensions of attitudes towards work appeared. The first four statements in Table 2 describe the attitudes towards parts of the social welfare system, i.e. unemployment and sick leave, and are labelled as work ethics (WE). The last three statements describe the general work attitudes (GWA) as a context, i.e. the importance of having a good life, having fun, and having spare time in relation to having a job.
Table 2 Results from the factor analysis of seven items from the questionnaire.
WE (Factor 1) GWA (Factor 2)
Eigenvalue = 2.87 Eigenvalue = 1.09
Factor loadings Factor loadings
It is important that we all get a job, otherwise society will not function -0.70 <0.10
It is OK to be unemployed even when there is a job available 0.78 0.18
It is OK to be reported sick even when not sick 0.60 0.28
It is unnecessary to work because the state provides compensation to the unemployed 0.63 0.10
It is possible to have a good life even without having a job 0.38 0.64
It is more important to have fun during life than to have a job 0.23 0.80
Spare time is more important for a good life than it is to have a job <0.10 0.85
The response alternatives to each item were on a five-graded scale ranging from "completely agree" to "completely disagree". In order to perform logistic regression models, the dependent variables WE and GWA had to be dichotomised. A dichotomous WE variable was then constructed. Based on the logical distribution of the answers, those 69% who partly disagreed or completely disagreed with all four statements (the answers to the first statement were inverted before the WE variable was created) were designated as those who have positive WE. Those 31% who did not dismiss all the statements were designated as those having negative WE.
Another approach was used with the GWA factor because of the skewed distribution of answers. Those who partly disagreed or completely disagreed with all the three statements in the GWA variable were as few as 16%, so a GWA index ranging from 3–15 was made. The index was dichotomised as close as possible to the median; value 10–15 (high index) and value 3–9 (low index). High index means that the respondents disagreed rather than agreed with the statements and low index that they agreed rather than disagreed with the statements. The internal consistency of the index established by Cronbach's α was 0.71.
QOL, as we measured it, refers to the individuals' evaluation of their life contents, i.e. their global QOL. This definition is in accordance with definitions in earlier studies of QOL [11,12]. Global QOL is measured here by the answer to the question "How do you feel about your life as a whole just now?" This question had five response categories, scored from "very good" to "very bad". The item used in the questionnaire to measure QOL have been used previously [13]. The question has been chosen after pilot studies as an item giving meaningful results, and has been followed by interviews that confirm the results. This indicates that the questions asked have high validity when used in order to shed light on a person's global QOL [13].
Sense of coherence (SOC) was measured by Antonovsky's [14] Orientation to Life Questionnaire, short form (SOC-13) which includes 13 items with seven response alternatives each. This index ranged from 13–91 and was divided into four groups by quartiles. The internal consistency of the SOC index established by Cronbach's α was 0.85 in this study.
Subjective health was measured by the answers to the question: "How would you describe your overall health status at present?" This question had five response categories, scored from "very good" to "very bad". This question is in accordance with questions about self-rated health status in other studies i.e. [15].
As far as the other independent variables are concerned, the scales and the method for categorising the variables can be seen in Table 3.
Table 3 Independent variables in the logistic regression models.
Variable Type of data a Categorised
Sex N: Man/woman Man/woman
Study programme N: Theoretical/practical Theoretical programmes/practical programmes
Work experiences during the whole upper secondary school period O: Very positive (1) to very negative (5) Very positive (1)/rather positive (2)/neither positive nor negative (3)/negative (4–5)
Contentedness with the upper secondary school period O: Very content (1) to very discontented (5) Content (1–2)/neither content nor discontented (3)/discontented (4–5)
Spare time O: Very content (1) to very discontented (5) Very content (1)/not very content (2–5)
Financial situation O: Very content (1) to very discontented (5) Content (1–2)/not content (3–5)
Parental support measured by two variables: support from father and support from mother O: Very good support (1) to no support (5) and not relevant (6) Very good support from both parents/very good support from one of them/not very good support from either of them (a combination of both variables established the three categories above)
Support from a friend O: Very good (1) to no support (5) and not relevant (6) Very good (1)/not very good support (2–6)
Possibilities to make own decisions about the future O: Very good (1) to very bad (5) Very good (1)/not very good (2–5)
QOL O: Very good (1) to very bad (5) Good (1–2)/not very good (3–5)
SOC index Num: 13–91 points Group 1 (25–50)/group 2 (51–59)/group 3 (60–68)/group 4 (69–91)
Subjective health O: Very good (1) to very bad (5) Good (1–2)/not very good (3–5)
a N = Nominal; O = Ordinal; Num = Numerical.
Statistical analyses
The significance of bivariate relations between variables was tested by chi-squared test. When the groups were small or the expected frequencies were low, Fisher's exact test was used for the comparison. The significance of differences between means was tested by a one-way Anova test. Correlation was established by Spearman's rank order correlation coefficient.
The factor analysis was done by means of principal component analysis. For factor extraction the varimax method for orthogonal rotation was used and eigenvalue >1 was set as a criterion. As can be seen from Table 2, variables with practically significant (>0.5) varimax loadings [16] were used to establish logical units (WE and GWA).
Multivariate analyses were carried out as logistic regression models (method: enter). The WE variable and the dichotomised GWA index were used as dependent variables. Variables included in the model were those with a significant (p < 0.20) relation to the dependent variable and with low correlation (rs2 < 0.20) to each other. As QOL, SOC and subjective health were highly correlated, they were analysed in separate models (Tables 6 and 7).
Table 6 Positive odds ratios (POR) and 95% confidence intervals (CI) for variables related to positive work ethics (WE), in three different models using QOL, SOC and subjective health as explanatory variables (n = 540).
QOL included SOC included Subjective health included
POR CI POR CI POR CI
Sex: female 1.60 1.06–2.44 1.88 1.21–2.93 1.65 1.08–2.51
Study programme: practical 1.22 0.81–1.84 1.23 0.80–1.89 1.17 0.78–1.77
Work experiences:
negative 1.00 1.00 1.00
neither/nor 1.11 0.46–2.72 1.04 0.42–2.56 1.16 0.48–2.82
rather positive 2.05 0.88–4.81 1.76 0.75–4.16 2.18 0.94–5.09
very positive 3.60 1.46–8.85 3.13 1.26–7.80 3.89 1.59–9.50
Spare time: very content 0.95 0.60–1.51 0.95 0.61–1.50 1.06 0.67–1.68
Support from a friend: very good 0.88 0.58–1.34 0.82 0.54–1.26 0.87 0.58–1.32
Possibilities to make own decisions about the future: very good 1.48 0.93–2.36 1.46 0.92–2.32 1.63 1.03–2.56
QOL:
not good 1.00
rather good 1.98 1.24–3.15
very good 3.01 1.58–5.75
SOC:
group 1, 25–50 1.00
group 2, 51–59 1.73 1.02–2.94
group 3, 60–68 2.24 1.26–3.96
group 4, 69–91 4.25 2.21–8.16
Subjective health:
not good 1.00
good 1.76 1.12–2.78
very good 2.06 1.12–3.79
Notes: Figures in bold when POR significant.
Variables not showing a bivariate relation (p < 0.20) to WE and therefore not included in the logistic regression model were: immigration, parental employment status, contentedness with the upper secondary school period, support from a relative, partner, other persons, number of social contacts outside home, close contact with persons outside home, parental support, self-confidence, contentedness with the personal financial situation, relations to friends, feeling lonesome, length of work experience, plans for a working or studying career.
Table 7 Positive odds ratios (POR) and 95% confidence intervals (CI) for variables related to high general work attitude (GWA) index, in three different models using QOL, SOC and subjective health as explanatory variables (n = 514–525).
QOL included SOC included Subjective health included
POR CI POR CI POR CI
Sex: female 2.38 1.61–3.51 2.77 1.84–4.16 2.40 1.62–3.54
Study programme: practical 1.81 1.23–2.66 1.81 1.22–2.69 1.80 1.22–2.65
Spare time: very content 1.06 0.71–1.60 0.95 0.63–1.44 1.07 0.71–1.60
Financial situation: content 1.41 0.96–2.08 1.27 0.85–1.90 1.43 0.97–2.10
Support from a friend: very good 0.74 0.50–1.10 0.69 0.46–1.04 0.74 0.50–1.10
Contentedness with the upper secondary school period:
discontent 1.00 1.00 1.00
neither/nor 1.73 0.81–3.72 1.78 0.83–3.81 1.71 0.79–3.68
content 2.09 1.08–4.07 1.93 1.00–3.72 2.09 1.08–4.06
Parental support:
not very good 1.00 1.00 1.00
very good support from father or mother 1.55 0.94–2.54 1.49 0.90–2.47 1.55 0.95–2.54
very good support from both parents 1.66 1.04–2.65 1.57 0.98–2.54 1.66 1.04–2.65
QOL: good 1.19 0.73–1.94
SOC: high 1.92 1.28–2.90
Subjective health:
good 1.19 0.75–1.88
Notes: Figures in bold when POR significant.
Variables not showing a bivariate relation (p < 0.20) to GWA index and therefore not included in the logistic regression model were: immigration, parental employment status, close contact with persons outside home, support from a relative, support from other persons, relations to friends, number of social contacts outside home, support from partner, self-confidence, feeling lonesome, possibilities to make own decisions, length of work experience, work experiences, plans of a working or studying career.
Independent variables were first analysed as categorical, and when not significantly related to the dependent variable they were presented as dichotomous instead. They were dichotomised as close as possible to the median value in order to give a neutral split, free from subjective influence (Table 3). The goodness of fit for the logistic regression models used was established by Hosmer and Lemeshow's [17] goodness of fit test, i.e. a not significant goodness of fit test, and high agreement was found between observed and expected frequencies in the two by ten tables derived from these tests.
The results of the logistic regression models were expressed as odds ratios (OR) with 95% confidence interval (CI). The odds ratios were calculated in an ordinary way, but in line with the salutogenetic approach the positive and negative outcome in the dependent variable, as well as in the explanatory variables, were changed and expressed as positive odds ratios (POR) [18].
The significance level was set to 0.05. For the statistical procedures SPSS for Windows version 9.0 and EPI 5 (Epi Info) were used.
The study has been approved by the Ethics Committee at the Faculty of Medicine, University of Lund (LU 669-03).
Results
The factor analysis established work ethics (WE) and general work attitudes (GWA) as two separate dimensions of attitudes towards work, and the correlation between those two components was rs = 0.32. As can be seen from Table 4, there were no differences in the four statements that constituted WE according to sex and study programme. Almost all of the students disagreed with the statements concerning sick leave and unemployment benefits. Concerning GWA, there were significant differences in the three statements principally because fewer males, especially in theoretical programmes, had disagreed with the statements. These differences can also be seen according to the GWA index (Table 5).
Table 4 Agreement/disagreement with the statements about work ethics (WE) and general work attitudes (GWA) and % in relation to gender and type of programme.
Theoretical programmes Practical programmes
Males Females Males Females p-valuea
WE n = 97–99 n = 115–118 n = 216–219 n = 108–110
Agree that
It is important that we all get a job otherwise the society doesn't work 80 85 89 89 0.115
Disagree that
It is OK to be unemployed even when there is a job available 87 95 93 96 0.066
It is OK to be reported sick even when not sick 95 98 94 97 0.387
It is unnecessary to work because the state provides compensation to the unemployed 96 98 97 97 0.775
GWA
Disagree that
It is possible to have a good life even without having a job 57 75 72 81 0.001
It is more important to have fun during life than to have a job 60 73 73 85 0.001
Spare time is more important for a good life than it is to have a job 53 77 68 77 0.000
Notes: χ2 analyses when comparing distributions of all four groups.
a p-values in bold when significant.
Table 5 Work ethics (WE), general work attitudes (GWA), QOL, SOC and subjective health according to study programmes and sex.
Theoretical programmes Practical programmes
Males Females Males Females p-valuea
(n = 95–99) (n = 114–118) (n = 213–220) (n = 107–110)
Positive WE % 60 72 69 76 0.062
High GWA index % 30 56 47 63 0.000
Good QOL % 75 77 81 71 0.214
Good subjective health % 71 70 81 66 0.008
SOC: Mean 60.2 58.7 61.4 55.9 0.003
Notes: χ2 analyses when comparing distributions of all four groups of WE, GWA-index, QOL and subjective health and a one-way Anova test for the comparison of all four groups of SOC.
a p-values in bold when significant.
According to our definition of WE, 69% of the students reported positive WE. The highest percentages of students with good subjective health and the highest SOC scores were found among the males in practical programmes (Table 5).
The logistic regression model that includes QOL as an explanatory variable (Table 6) shows that positive WE was related to being female (POR = 1.60), having very positive work experiences (POR = 3.60), and having rather or very good QOL (POR = 1.98 and 3.01, respectively). SOC and subjective health correlated highly with each other and with QOL (rs2 > 0.20), and were therefore not included in the same logistic regression model. When additional logistic regression models were tested with SOC or subjective health as explanatory variables instead of QOL, both SOC and subjective health were significantly related to WE. Principally the same variables, with almost the same figures of POR, were significantly related to WE when QOL, SOC or subjective health were used as explanatory variables (Table 6).
In another logistic regression model including QOL as an explanatory variable (Table 7), it is shown that high GWA index was related to being female (POR = 2.38), studying on a practical programme (POR = 1.81), being content with the upper secondary school period (POR = 2.09), and having very good parental support (POR = 1.66). In contrast to QOL and subjective health, SOC was significantly related to GWA. Otherwise using SOC, subjective health or QOL as explanatory variables gave similar results in relation to GWA.
Discussion
Our study presents data on attitudes towards work among adolescents. Two independent factors, work ethic (WE) and a general work attitude (GWA) appeared as important.
According to our results, having negative WE and low GWA index include negative attitude towards work as an action of solidarity with society. A majority among the students reported positive WE. Focusing on factors that are related to positive WE and GWA might open ways to moderate young people's outlook concerning work in relation to the welfare system.
It can be assumed that negative WE and a low GWA index also mirror negative attitudes towards the working line concept. But the societal norm, on which the working line concept relies, is that as many as possible should earn their living through work if possible.
Consequently, it is important that citizens do not use benefits derived from the social welfare system except when necessary. Therefore, support to people, making them enter working life or encouraging them to stay in working life, is important. Support in this can be built up by knowledge about factors that are related to attitudes towards work. Our results in this study highlight some variables that are significantly related to attitudes towards work, in terms of WE and GWA.
First, having positive WE was related to good QOL, high SOC and good subjective health, and having high GWA index was related to high SOC. However, the result showed that the figures for POR when SOC or subjective health were used as explanatory variables in the models were principally the same as when QOL was used in the model. Antonovsky's [14] SOC concept has affinities with other salutogenetic concepts, e.g. self-efficacy [19] and hardiness [20]. Antonovsky [14] suggested that the stronger the SOC world outlook of a person is, the more likely he or she is to cope successfully with life stressors. He also suggested that SOC contributes to good health as an individual with high SOC will perceive stressors to be less stressful and the negative consequences of a stressful life would therefore be less. Recently it has been suggested that SOC has a unique relation to health and that the concept of SOC also refers to an active self-esteem structure and self-determination [21]. Based on our results, we suggest that QOL and subjective health, although measured as single items, may be markers of a similar concept to SOC, which is related to WE and GWA as well.
WE and GWA represent different dimensions of attitudes towards work, but the results derived from the logistic regression models also suggest similarities in the outcomes concerning these variables i.e. in gender and work or school experiences. Females reported a more positive WE and a higher GWA index than males did. One explanation from the different attitudes can be found in males' and females' different work values. It has been postulated that a radical change in attitudes and values concerning work is in progress [22]. "Postmateralistic" values such as the importance of good quality of life, high self-expression, belonging and intellectual satisfaction, are ranked higher, especially by females, than materialistic values such as economic growth, law and order and security. It has also been shown that females ranked altruism as a work value [23], and endorsed work ethics more than males did [10]. Our results show that males expressed a more positive attitude towards being voluntarily unemployed and to improper use of the social welfare system, and they gave less priority to work as something important for a good life compared to females.
In the present study, WE relates to former work experiences and GWA relates to former school experiences. For many students, work experiences occur during the upper secondary school period. It is also common that young people work part-time in the paid labour market in the evenings, and/or in the summer while still full-time students [24]. If these experiences of work are mainly positive it seems reasonable that they influence WE positively.
It has previously been shown that the quality of work experiences during high school has significant consequences for well-being among young people[25]. On the one hand, work can be beneficial for youth development because work can limit depressive affect. On the other hand, employment including early work stressors can cause harmful consequences to adolescents' mental well being [25]. It seems important that schools work to obtain high-quality practical placements for the students and that employers are aware of the connection between WE and work experiences. In order to moderate the experiences of work to give positive events, schools and employers can support adolescents in balancing their commitments in both school and work.
GWA was not related to former work experiences, but was related to whether the upper secondary school period is a positive event or not. In Sweden, 97% of adolescents apply to upper secondary school in their final year of compulsory school, although one of five lacks an upper secondary school education at the age of 20 [26]. The most common reason for leaving after compulsory school or for dropping out of upper secondary school was school fatigue. Our results show that those who were less content with the school period also had low GWA index. Therefore, school experiences, i.e. school fatigue, should be recognised, as they can influence willingness to complete school and thereby secondarily affect the possibilities to enter working life as well.
Parental support is an important part of social support, and in this study parental support was related to GWA, but not WE. Social support has been described as a moderator of life stress and as a potent isolator against stressful conditions in life [27]. It has also been shown that young people perceive family/friends as the primary providers of social support [28]. Concerning unemployed young people, it has recently been shown that parental support decreases the risk of having mental health problems [29]. It has also been suggested that fathers are important in supporting young people to enter working life [26]. Our result is complementary to that; very good support from both parents relates to the attitude that work is important for a good life, an attitude that can be assumed to increase the chances of entering working life.
According to our results, those who follow a practical study programme are more likely than those in theoretical programmes to have high GWA index. One explanation might be that those in practical programmes give more priority to working life, i.e. they are concentrated on getting a job when school is finished, as they have recently chosen and accomplished a work-related education. It can also be assumed that those in theoretical programmes, to a larger extent than those in practical programmes, give priority to further studies. It has been shown that students in work-related courses pay less importance to work goals than students in theoretical programmes do [30]. In the present study, high GWA index is related to studying on a practical programme. Therefore, it seems possible that those students pay more attention to working life than those in theoretical programmes do.
Methodological considerations
The advantage of this study is that 606 students were invited to participate and most of them (91%) completed the study. A non-response analysis showed no significant differences in dropout according to sex or study programme. Thus, our results might be generalised to other students in similar settings. The result of the factor analysis makes sense. The appropriateness of the analyses was 0.80 when established by Kaiser-Meyer-Olkin measure of sampling adequacy. The factor analysis logically combined items, with practically significant factor loadings, that established work ethics and general work attitudes as two important components in students' attitudes towards work. The study design was cross-sectional, which limits the possibilities to draw causal conclusions. Therefore we refer to relations between variables instead of emphasising the concept determinants. Another limitation of the results is the possibility of bias related to the self-report. The questions asked should have limited recall problems but some statements of WE may give socially acceptable answers. In that way the figure of 69% reporting a positive WE could be some overestimation. However, the procedure with strictly anonymous responses may partly compensate for such bias. The ethical attitude could also be influenced by personality traits not covered in our study. The findings suggest that interpretations of the students' attitudes towards work, working life and the welfare system and related variables might be a basis for further studies on this field.
Conclusion
Factors related to attitudes towards work are of importance for increased understanding of adolescents' transition to working life. Several factors appeared as positively related to either of the two dimensions of work attitudes found in this study: female gender, good QOL, high SOC, support from both parents, school experiences and work contacts. It is important that schools as well as employers help students to balance their study commitments and work as it might improve experiences of school and work. Knowledge about these factors might increase adolescents' possibilities to enter working life. However, further studies of this kind that explore attitudes towards work among adolescents are necessary for an understanding of young adults' transition to working life.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
LA participated in conceiving the study, carrying out the study and had the main responsibility for writing the manuscript.
IA participated in conceiving the study and writing the manuscript.
AH participated in conceiving the study and writing the manuscript.
GE participated in conceiving the study and writing the manuscript.
All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-1041621266610.1186/1471-2458-5-104Research ArticleDiffering mental health practice among general practitioners, private psychiatrists and public psychiatrists Younès N [email protected] MC [email protected] B [email protected] V [email protected] MP [email protected] I [email protected] Academic Unit of Psychiatry, Centre Hospitalier de Versailles, 177 Rue de Versailles 78157 Le Chesnay Cedex. France2 National Institute of Health and Medical Research (INSERM-U669), Hôpital Cochin, AP-HP, Paris, France3 Mental Health Foundation, MGEN, Paris, France4 Direction of Medical Policy, Assistance Publique – Hôpitaux de Paris, Paris2005 7 10 2005 5 104 104 19 5 2005 7 10 2005 Copyright © 2005 Younès et al; licensee BioMed Central Ltd.2005Younès 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
Providing care for mental health problems concerns General Practitioners (GPs), Private Psychiatrists (PrPs) and Public Psychiatrists (PuPs). As patient distribution and patterns of practice among these professionals are not well known, a survey was planned prior to a re-organisation of mental health services in an area close to Paris
Methods
All GPs (n = 492), PrPs (n = 82) and PuPs (n = 78) in the South-Yvelines area in France were informed of the implementation of a local mental health program. Practitioners interested in taking part were invited to include prospectively all patients with mental health problem they saw over an 8-day period and to complete a 6-month retrospective questionnaire on their mental health practice. 180 GPs (36.6%), 45 PrPs (54.9%) and 63 PuPs (84.0%) responded.
Results
GPs and PrPs were very similar but very different from PuPs for the proportion of patients with anxious or depressive disorders (70% v. 65% v. 38%, p < .001), psychotic disorders (5% v. 7% v. 30%, p < .001), previous psychiatric hospitalization (22% v. 26 v. 61%, p < .001) and receiving disability allowance (16% v. 18% v. 52%, p < .001). GPs had fewer patients with long-standing psychiatric disorders than PrPs and PuPs (52%, 64% v. 63%, p < .001). Time-lapse between consultations was longest for GPs, intermediate for PuPs and shortest for PrPs (36 days v. 26 v. 18, p < .001). Access to care had been delayed longer for Psychiatrists (PrPs, PuPs) than for GPs (61% v. 53% v. 25%, p < .001). GPs and PuPs frequently felt a need for collaboration for their patients, PrPs rarely (42% v. 61%. v. 10%, p < .001).
Satisfaction with mental health practice was low for all categories of physicians (42.6% encountered difficulties hospitalizing patients and 61.4% had patients they would prefer not to cater for). GPs more often reported unsatisfactory relationships with mental health professionals than did PrPs and PuPs (54% v. 15% v. 8%, p < .001).
Conclusion
GP patients with mental health problems are very similar to patients of private psychiatrists; there is a lack of the collaboration felt to be necessary, because of psychiatrists' workload, and because GPs have specific needs in this respect. The "Yvelines-Sud Mental Health Network" has been created to enhance collaboration.
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Background
In developed countries, mental health problems, especially anxious and depressive disorders, are frequent and a leading cause of disability in terms of cost to the individual and society [1-6]. Since they are potentially remediable when adequately treated at an early stage, they represent a major public health challenge [7,8].
Mental Health care concerns the entire health system. First of all, there are general practitioners (GPs) who play a pivotal role, as first line and as the main health professional consulted [1,8-10]. Since primary care is known to be insufficient on its own, access to mental health professionals (psychiatrists, psychologists) needs to be improved to enhance mental health care overall [11-16].
In France up till now patients were free to consult GPs, psychiatrists in private practice in the community (PrPs) or psychiatrists working in the public sector (PuPs). There were 60 815 GPs in France in 1996, and 11 816 PrPs and PuPs in 1997 [17]. Patient distribution, patterns of practice and job satisfaction among these professionals are not well known.
In a pilot area ("Yvelines Sud" area, South-West of Paris), prior to a reorganization of mental health care, a survey was conducted among local physicians involved in mental health care. First General Practitioners' opinions on their practice in mental health and their collaboration with mental health professionals were studied [18]. Then the aim was to gain a better understanding of the overall organization of mental health care. The present article compares general practitioners (GPs), private psychiatrists (PrPs) and public psychiatrists (PuPs) according to their mental health patient population, their mental health practice and their job satisfaction.
Methods
Population
The 492 GPs, the 82 PrPs and the 75 PuPs in the area of "South Yvelines" (600 000 inhabitants) were approached by post in spring 2000 and informed of the local mental health program. They were asked if they were willing to recruit for the survey, with a postage-paid reply envelope if they agreed to take part. 180 GPs (response rate of 36.6%), 45 PrPs (54.9%) and 63 PuPs (84.0%) were included. The global response rate is 44.4%.
Data collected
The mental health professionals responded to two questionnaires requiring approximately 30 minutes to complete:
1. A prospective patient questionnaire completed by the physician.
GPs were asked to include prospectively over an 8-day period all consulting patients over 15 years old for whom a Mental Health Problem was "the main current problem", distinguishing between new patients and those already in follow-up. They were also asked to give the overall number of consultations during the same period. 1519 patients with mental health problems were enrolled by GPs, representing 15.0 % of the overall number of consultations. On average participating GPs saw 8 patients with mental health problems (range 0–35).
PrPs and PuPs were asked to complete the questionnaire in a prospective manner for the first 30 consulting patients, older than 15, also distinguishing new patients from the others. They included 606 new patients and 1645 patients already known to them.
2. A 6-month retrospective practitioner questionnaire measuring the physicians' opinions on their practice in general and on their mental health practice.
Statistical analyses
Analyses were performed with SAS 8.2 Software. Three groups were considered: GPs, PrPs and PuPs. Descriptive and comparative analyses were carried out on physician demographics, patient profiles, mental health practice and job satisfaction. As appropriate, the chi-square test was used for categorical variables and ANOVA tests for continuous variables. A 5% p level of significance was chosen.
Results
Characteristics of respondent physicians (table 1)
Table 1 Description of respondent physicians (N = 288)
General Practitioners
N = 180 Private psychiatrists
N = 45 Public psychiatrists
N = 63
Demographics (%)
Age class
25 – 35 years old 9.0 0.0 23.8
36 – 55 years old 84.5 88.9 73.0
56 years old and more 6.2 11.1 3.2
Gender: female 30.6 46.7 37.7
Duration of professional activity
Less than 5 years 15.0 13.3 47.6
From 5 to 10 years 18.9 28.9 27.0
More than 10 years 66.1 57.8 25.4
Professional activity (% of time spent)
Consultations 76.6 80.2 34.7
Hospitalization 0.0 0.0 31.3
Emergencies 0.0 0.0 8.7
Paper work 10.0 6.3 11.0
Exchanges with colleagues 5.0 3.9 9.9
Further medical education 8.4 9.6 4.4
Respondent physicians were predominantly experienced providers, male and between 36 and 54 years old. PuPs were on average younger than the others (some being residents). Professional activity consisted mostly in clinical activity: consultations for private physicians and more diverse activities for public psychiatrists (also involved in hospitalisation and emergencies). More minor activities were paper work, further education and exchanges with colleagues. 95.6% of PrPs and 65.1% of PuPs reported practising structured psychotherapies (mainly psychoanalysis).
Comparison of mental health patients of GPs, private and public psychiatrists (table 2)
Table 2 Comparison of Patients with mental health problems, already known, seen during one week, for General Practitioners, Private and Public Psychiatrists (N = 2724)
Patients of General Practitioners
N = 1079 Patients of Private Psychiatrists
N = 1130 Patients of Public Psychiatrists
N = 515 Chi2 test
Demographics (%)
Age class <0.0001
15 – 25 years old 5.8 9.7 15.7
26 – 65 years old 78.9 86.9 75.8
66 years old and more 15.3 3.4 8.5
Gender: female 68.9 74.7 52.4 <0.0001
Current professional activity 60.1 69.7 44.4 <0.0001
Living alone 25.7 30.0 28.8 ns
Main mental health problem (%) <0.0001
Anxiety and mood disorders 70.4 65.3 38.4
Psychotic disorders 4.6 6.9 30.2
Alcohol and Substance misuse 6.9 1.3 8.0
Other 18.1 28.3 22.6
MHP severity (%)
Past psychiatric hospitalization 21.8 26.1 61.3 <0.0001
National disability allowance 16.2 18.9 51.8 <0.0001
MHP chronicity (%)
MHP duration more than 3 years 52.2 62.9 63.8 <0.0001
The GP and PrP patients with mental health problems already known to the practitioner were very similar for gender and employment rate. PuP patients were younger, more often male and non-working than GP and PrP patients. There are respectively five times and three times more patients aged 65 or more among GP patients than among PrP and PuP patients.
GPs and PrPs were very similar for percentages of patients diagnosed as anxious or depressed (67.8%) and for percentages of psychotic patients (5.8%). Psychotics patients were much more numerous and anxious or depressed patients much less numerous among PuP patients than among community physician patients. Alcohol and drug misuse were more often treated by GPs and PuPs (7.5%) than by PrPs (1%).
GP and PrP patient percentages did not differ for previous psychiatric hospitalization and national disability allowance. PuPs had patients with more severe characteristics for these variables than GPs and PrPs. Psychiatrists (PrPs and PuPs) had more patients with long term psychiatric disorders than did GPs.
Comparison of mental health practice between GPs, private and public psychiatrists (table 3)
Table 3 Comparison of Mental Health Practice concerning patients seen during one week for General Practitioners, Private and Public Psychiatrists.
Patients of General Practitioners Patients of private psychiatrists Patients of public psychiatrists Chi2 test or ANOVA
New patients (N = 603) (Percentage of MHP patients) N = 439 (28.9%) N = 73 (6.1%) N = 91 (15.1%)
Patient recruitment (%) <0.0001
Patient 88.4 61.6 42.8
Family 14.1 23.3 17.6
GPs 0.0 26.0 24.2
Psychiatrist 0.0 6.8 19.8
Percentage of patients who consulted too late according to the professional (%) 25.2 60.7 52.6 <0.0001
Care project = management by the professional (%) 70.2 89.1 88.6 <0.0001
Wish for collaboration with another physician (%) 42.3 9.6 61.1 <0.0001
Patients already known (N= 2724) (Percentage of MHP patients) N = 1079 (71.1%) N = 1130 (93.9%) N = 515 (84.9%)
Mean days from last consultation (sd) 36.4 (34.9) 17.6 (16.4) 25.7 (18.5) <0.0001
Collaboration with other professionals (%) 26.3 29.6 53.4 <0.0001
Type of care (%)
Pharmacological treatment - 18.9 51.5
Psychotherapy - 52.9 16.8
Both - 28.2 31.8
The proportion of new patients among consultants was the highest for GPs, intermediate for PuPs and the lowest for PrPs. Patient recruitment differed: GPs had no patients referred by another physician, while a quarter of psychiatrists' patients were referred by GPs. GPs had fewer new patients for whom they considered that access to mental health care had occurred late. They more often actually sought collaboration for care provision, and much more often stated they would like to have some form of collaboration.
For patients already known to the practitioners, time-lapse between consultations was the longest for GPs, intermediate for PuPs and the shortest for PrPs. Collaboration with another professional less often occurred for community physician patients than for PuP patients. Among psychiatrists, different patterns of care were noted: PrPs were more likely to use psychotherapy than PuPs, and conversely PuPs more often used pharmacological treatments.
Comparison of job satisfaction of GPs, private and public psychiatrists (table 4)
Table 4 Comparison of job satisfaction of General Practitioners, Private and Public Psychiatrists.
General Practitioners
N = 180 Private psychiatrists
N = 45 Public psychiatrists
N = 63 Chi2 test
MHP practice satisfaction (%)
Having MHP patients that the practitioner would prefer not to cater for 64.2 55.6 64.4 ns
Having difficulties hospitalizing MHP patients (always/often) 46.9 40.0 41.0 ns
Unsatisfactory or very unsatisfactory relationships with
... GPs 19.0 33.3 29.7 0.05
... private psychiatrists 49.1 11.3 27.1 <0.0001
... public psychiatrists 29.9 29.7 18.8 <0.0001
... colleagues in general 16.2 23.5 10.5 ns
Relationships with mental health professionals are worse than with other health professionals 53.7 15.4 8.1 <0.0001
Having insufficient or very insufficient scope for taking on new patients (workload) 39.7 93.4 77.1 <0.0001
Scope for entrusting part of care to another professional insufficient or very insufficient 46.3 73.2 80.7 <0.0001
General practice satisfaction (%)
- clinical activities
Independence is essential or important 98.9 100.0 95.2 ns
Exchanges with colleagues are essential or important 99.4 88.8 98.5 ns
Possibility for being replaced insufficient or very insufficient 89.9 86.3 73.3 0.008
- other activities
Income is unsatisfactory or very unsatisfactory 44.8 56.8 57.7 ns
Administrative duties are demanding or very demanding 91.6 77.8 64.0 <0.0001
Time for further medical education is unsatisfactory or very unsatisfactory 65.7 57.8 77.4 ns
Time for reading medical journals is unsatisfactory or very unsatisfactory 64.4 66.7 78.1 ns
Opportunities for writing medical articles are unsatisfactory or very unsatisfactory 84.1 85.7 80.7 ns
Opportunities for being involved in research and evaluation studies are unsatisfactory or very unsatisfactory 98.3 87.1 68.4 ns
GPs, PrPs and PuPs did not differ according to their general practice satisfaction, except for scope for finding replacements and administrative paperwork: more private physicians (88.1% and 84.7%) complained about these issues than PuPs (73.3% and 64%).
Satisfaction with mental health practice was low for all three categories of physicians: 42.6% encountered difficulties hospitalizing patients and 61.4% had patients they would prefer not to cater for. GPs, PrPs and PuPs however differed according to their mental health practice satisfaction. Psychiatrists (PuPs and especially PrPs) experienced more difficulties in taking on new patients because of workload (88.1%), and in entrusting part of their care to another professional (84.7%) than did GPs. Workload was lower for GPs than for PrPs and PuPs.
Regarding physicians' opinions on their relationships with colleagues, the most frequent unsatisfactory rating was for relationships between GPs and PrPs (for both). The best relationships were among private psychiatrists. GPs more often reported unsatisfactory relationships with mental health professionals than did PrPs and PuPs.
Discussion
This survey was undertaken to obtain better insight into how practice in mental health is distributed among medical professionals in a French area, prior to re-organisation of mental health services. To our knowledge no other survey has been addressed exhaustively to all physicians involved in mental health care in a particular geographical area. This limits scope for comparisons with other work.
Limitations
The first limitation is the moderate response rate, reflecting differing interest for the mental health program according to the professional group. GPs may feel less concerned than psychiatrists for different reasons. First, GPs in France, as first line professionals, are contacted by numerous care networks (asthma, diabetes etc) which could take up a lot of their time, even if they are interested in mental health care. Second, GPs may present an interest variable: respondents were probably more involved in mental health care in their ordinary practice than non-respondents. Among psychiatrists, public psychiatrists (PuPs) seemed more concerned than private psychiatrists (PrPs) possibly because they are more concerned about public health issues. Their response rate is comparable to that obtained by studies among Australian or Finnish public psychiatrists [19,20].
The second limitation is that the results are based on reports from the professionals, and particularly in the case of GPs, on their reporting of mental health patients that they themselves identified as having mental health problems. This means of assessment could involve a recruitment bias with a selection of particular patients. However the survey did not intend to assess the prevalence of psychiatric disorders in practice, or needs for mental health treatment, already studied [8,21-24]. The study option was to compare how physicians perceived their usual mental health activity, and how satisfied they were with it, prior to the mental health care reorganization, the aim being to adapt the mental health program to these particular attitudes.
Mental health patient distribution among professionals
This is the first survey studying mental health patient distribution with a recruitment via the professionals, and comparing GPs, PuPs and PrPs. In particular very few studies have explored PrP practice. In Ontario, Canada, a community survey has shown the influence of certain demographic variables on distribution of patients with mental health problems (age, marital status) but not the influence of severity variables (which were only approximately determined)[21]. In the United States, a large, nationally representative sample of patient visits showed that men, African Americans, other non-white persons, and patients under 15, between 65 and 74, and 75 and over, made proportionally more visits to primary care physicians than to psychiatrists[25]. Severity has been shown to influence the specialist/generalist division of responsibility for patients with mental disorders : specialists were resorted to for patients with psychotic, affective, and schizophrenic disorders, whereas general medical practitioners were more likely treat neurotic disorders in which symptoms of anxiety and depression predominated[24]. Finally in Michigan, USA, a study compared criteria-defined MDD patients of GPs (resorting to primary care) and psychiatrists (outpatients of a university department of psychiatry). Depressed patients consulting a psychiatric practitioner were reported as more severely depressed, more likely to be male, more highly educated and younger. Depressed primary care patients were less likely to have received prior treatment for depression and less likely to present past and current psychiatric comorbidity. The authors concluded that depressed patients encountered in routine primary care are substantially different from those seen in psychiatric settings[22]. The results of the present study confirm the difference between patients with mental health problems encountered in primary care and those encountered in public psychiatric setting (where patients are younger, more often male and more severe). But the difference is smaller between primary care and private psychiatric settings, where patients were in fact more similar than different on demographics, diagnosis and severity criteria. It confirms that GPs had to cater for patients with severe mental health problems. The biggest difference between GP patients and psychiatrist patients was the chronic nature of the mental health problem for the latter, which raises the issues of early help-seeking behaviors in relation to specialist care[26].
Mental health practice
An important result of the survey lies to the unequal access to mental health care for patients in the light of the first professional consulted: GP, PrP or PuP. Patients with mental health problems seemed fairly similar between primary care and private psychiatric settings. It can be supposed that the first professional consulted is determined by social and educational levels. Whatever the professional category of the practitioner first consulted, these professionals catered for their patients on their own. Thus, the care provided was different. PrPs tended to see their patients more often than did GPs. PrPs were likely to practise psychotherapies while GPs provided other forms of care, without structured psychotherapies.
Regarding mental health practice, PuPs were radically different from both GPs and PrPs: they used more pharmacological treatment and they more often shared practice (team work is more frequent in hospitals); this is coherent with the fact that their particular patients with mental health problems were more frequently psychotic and their condition more severe.
Mental health practice seemed a burden to all professionals (GPs, PrPs and PuPs). Physicians, and especially psychiatrists, were overworked and had difficulty providing the care they considered suitable (hospitalization for instance). This is a problem for all physicians, and not only for PrPs, as shown in Australia where the lack of beds was their most frequent reason for dissatisfaction[19].
The survey showed another aspect that is important for the efficiency of the whole care system: the poor relationships with physicians of other professional categories. GPs, who, as we have shown, manage patients with severe mental health problems but see their patients less often than do PrPs, expressed dissatisfaction with their relationships with psychiatrists. They were particularly dissatisfied with their relationships with PrPs, possibly because they felt closer to them (both are private) so that they may have more expectations in terms of relationships and collaboration with them. GPs desired some form of collaboration for their new patients much more frequently than PrPs. This result evidencing poor relationships among physicians is important because infrequent and unsatisfactory links between primary care and specialist health care are a reason for concern in several countries. It raises the whole issue of help-seeking behaviors [26-29]. However this survey shows that it may be that psychiatrists, overworked and working in isolation, cannot find time or scope for more collaboration with GPs, unless there is a complete reorganization of the mental health system.
The results on job satisfaction among these professionals has revealed a moderate to poor level of satisfaction. All physicians (GPs and psychiatrists) complained about insufficient time for further education and above all, for writing medical articles and for research. Private professionals complained about administrative demands. Time pressure and paperwork have already been shown as frequently reported factors in stress and job dissatisfaction among Australian GPs[30], insufficient participation in research was reported among Canadian psychiatrists[31] and finally, administrative demands were noted among Australian psychiatrists[19]. The present survey did not study litigation and compensation issues, shown to be the most frequent reason for dissatisfaction for private psychiatrists in previous studies in other countries. In France, litigation is still relatively rare. Insufficient time for further education is confirmed by results on time allocation. The main apportionment of waking time is roughly similar when compared with previous studies: first clinical activities then further education and paper work for all professionals, even if this survey did not preclude biased recall of retrospective agendas, as did the survey using a hand-held computer[32]. The present results revealed that physicians without an academic inscription had less time for education and research than other European general physicians in academic departments, American residents or American psychiatrists [32-35].
Finally professionals attached great importance to their clinical independence as well as to scope for collaboration. Physicians' ability to obtain outpatient and inpatient services they required has been shown to be the most consistent and powerful predictor of changes in levels of practice satisfaction over time in an American nationally representative sample of primary care physicians and specialist physicians (including psychiatrists)[36]. This suggests that reorganization of mental health care needs to take account of professionals' dual need for independence and collaboration.
Conclusion
The present results confirm the need to implement more collaborative practices among practitioners involved in mental health, not in the form of the classic referral to specialists as the major therapeutic option, but in the form of emphasis on collaborative relationships with mental health specialists. Results from this survey have been integrated into the "South Yvelines Mental Health Network" created in June 2001, by promoting this type of collaborative relationships in the area (workshops, educational interventions, targeted collaborative actions ...). It was organized along the lines of the "individualized stepped care" proposed by Von Korff and colleagues[37,38]. For example, patients who pose problem for their primary care physician will benefit from prompt public psychiatric consultations, or brief interventions in support of primary care management without transferring the responsibility to specialist care. Only if necessary, will the transfer to specialist care by private or publics psychiatrists be organized. Further evaluations of the impact of the South Yvelines Mental Health Network are in completion.
List of abbreviations
GPs (General Practitioners). PrPs (Private Psychiatrists). PuPs (Public Psychiatrists).
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
Study concept and design: Gasquet, Kovess, Hardy-Bayle. Acquisition of data, study supervision : Chaillet. Analysis and interpretation: Younès, Gasquet. Drafting of the manuscript : Younès. Statistical expertise : Younès, Falissard. Critical revision : Gasquet, Younès, Falissard, Hardy-Bayle
Pre-publication history
The pre-publication history for this paper can be accessed here:
Supplementary Material
Additional File 1
Mental Health Practice Questionnaire. The questionnaire is divided into two parts : a retrospective questionnaire on professional activity and a prospective patient questionnaire.
Click here for file
Additional File 2
Questionnaire sur les Pratiques en Santé Mentale. French version of the Mental Health Practice Questionnaire.
Click here for file
Acknowledgements
All the professionals of the Réseau Santé Mentale Yvelines Sud. This survey was made possible through funding from local hospitals and by an unrestricted grant from Eli Lilly and Company, France.
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-1061621611710.1186/1471-2458-5-106Research ArticleUnfavourable birth outcomes of the Roma women in the Czech Republic and the potential explanations: a population-based study Bobak Martin [email protected] Jan [email protected] Ivo [email protected] Radim J [email protected] International Centre for Health and Society, Department of Epidemiology and Public Health, University College London, 1–19 Torrington Place, London WC1E 6BT, UK2 Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, Videnska 1083, Prague 4, Czech Republic2005 10 10 2005 5 106 106 11 7 2005 10 10 2005 Copyright © 2005 Bobak et al; licensee BioMed Central Ltd.2005Bobak 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
Data on the health status of the Roma people in Central and Eastern Europe are sparse and the reasons for their poor health are not clear. The objective of this study was to quantify the differences in birth outcomes between Roma and non-Roma mothers in the Czech Republic and to investigate the potential causes of such differences.
Method
A population-based study recruited 8938 non-Roma and 1388 Roma hospitalised singleton births that occurred in two Czech districts (Teplice and Prachatice) between 1995 and 2004. During their stay in hospital, mothers completed a questionnaire on their demographic and socioeconomic characteristics and maternal smoking and alcohol consumption. Data on maternal height and weight and on infants' birth weight and gestational age were taken from hospital records.
Results
Birth weight and gestational age of Roma infants was 373 (SE 15) g and 0.92 (0.05) weeks, respectively, lower than in non-Roma infants. Controlling for demographic, socioeconomic and behavioural factors reduced these differences to 133 (18) g and 0.57 (0.06) weeks, respectively (all p-values < 0.001). In terms of binary outcomes, the Roma vs. non-Roma odds ratios were 4.5 (95% CI 3.7–5.4) for low birth weight (< 2500 g), 2.8 (2.2–3.4) for preterm birth (< 37 weeks of gestation), and 2.9 (2.5–3.4) for intrauterine grown retardation (<10th percentile of birth weight for gestational age); controlling for all covariates reduced these odds ratios to 1.7 (1.3–2.2), 1.5 (1.1–2.0) and 1.3 (1.0–1.6), respectively. Maternal education made the largest contribution to the ethnic differences; the role of health behaviours was relatively modest.
Conclusion
There are striking differences in birth outcomes between Roma and non-Roma mothers. The causes of these differences are complex but largely socioeconomic.
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Background
The Roma people (Romanies, Gypsies, of northern Indian origin) are the most important ethnic minority in Central and Eastern Europe. It has been known for a long time that the socioeconomic conditions of most Roma people in Central and Eastern Europe are worse than those of the general population, and it has been suspected that their health follows a similar pattern. However, the main conclusion of two recent reviews was that there is a striking lack of information about the Roma people [1,2]; even the estimates of the size of their population vary widely [1], and data on their health are sparse and incomplete [1,2].
Available data suggest that the health of the Roma people is poor [1,2]. Similarly to ethnic differences in other countries [3], the contribution of different factors, such as culture, genes, behaviours or socioeconomic status, remains unclear. One particular area of great interest is reproductive health and birth outcomes. Data from Bulgaria, Hungary, Czech Republic and Slovakia indicate that abortions, low birth weight and premature birth are more common in Roma than non-Roma populations [4-9]. Given the problems with obtaining funding for such research and the difficulties with recruiting the Roma people into epidemiological studies, virtually all studies so far were of small size and most were conducted in non-representative population samples.
In this paper, we analysed a large population based study of births in two Czech communities. The study was originally designed to investigate the effect of air pollution on birth outcomes [10] but it also collected data on ethnicity and health behaviours known to be associated reproductive outcomes. Preliminary results from the first year of the study indicated that ethnicity was an important determinant of birth weight, gestational age and foetal growth [5]. Here we report the analyses of 10-year birth series. The paper has two objectives. First, to quantify the differences between Roma and non-Roma (i.e. European origin) infants in birth weight, gestational age and intrauterine growth. The second objective was to estimate the contribution of demographic variables, socioeconomic disadvantage and health behaviours to the ethnic differences.
Methods
Setting and subjects
As mentioned above, the study was set up to examine the effects of air pollution [10]. It was conducted in two Czech districts: Teplice (North Bohemia, high levels of air pollution) and Prachatice (South Bohemia, clean air). All singleton births from April 1995 through 2004 to women with at least 1 year of residence in the district were included in the study; exclusions due to short-term residence were very rare and cannot not bias the study. Virtually all deliveries in these districts during the study period were hospitalised, and women were enrolled during their stay in hospital. Given the almost universal hospitalisation of deliveries and the low refusal rate (less than 5% in each district), the births recruited into the study are representative for the two communities. Multiple births were excluded.
Measurements
During hospitalisation, women completed a self-administered questionnaire with a help of a specially trained nurse. Ethnicity was based on maternal self-report; the dataset contained the following options: Asian; European; Roma, and "other".
Mother reported their highest attained education (primary or less; apprenticeship; secondary; university), marital status (married; cohabiting; single; divorced/widowed), smoking (cigarettes per day) and consumption of alcohol (drinks per week) before and during pregnancy. Data on mothers' height (in cm), pre-pregnancy weight (in kg), birth weight (in grams) and gestational age (in weeks) were taken from hospital records. The study attempted to collect data on fathers but we did not use these data because of the high rate of missing values (> 60% of values in variables related to father were missing).
Statistical analysis
Because the numbers of Asian and "other" infants were too small for meaningful analyses (84 and 62 births, respectively), the final dataset was restricted to European and Roma babies. The statistical analysis was conducted in several steps. First, socioeconomic and life style variables were tabulated by ethnicity. Second, we compared crude differences between the two ethnic groups in the following birth outcomes: birth weight and gestational age measured as continuous variables, and low birth weight (< 2500 g), preterm birth (gestational age < 37 weeks), and intrauterine growth retardation (IUGR, < 10th gender-specific percentile of birth weight for gestational age) defined as binary variables. Finally, we examined the extent to which the differences between Roma and non-Roma birth outcomes can be explained by available covariates. The statistical models (for both binary and continuous outcomes) were built as follows. First, crude effects of ethnicity were estimated. Second, the ethnic differences were adjusted separately for: (i) demographic characteristics of the infant (gender and district of birth); (ii) maternal demographic characteristics (maternal age and number of pregnancies); (iii) maternal social characteristics (education and marital status); (iv) maternal size (height and weight); and (v) maternal behaviours (smoking and alcohol consumption). Finally, all these covariates were entered into one model. All analyses were conducted using the STATA statistical software (Stata Corporation, College Station, Texas, USA).
Results
The final dataset consisted of 10,326 singleton births, of whom 1388 (13%) occurred among Roma women. The distribution of socioeconomic characteristics and behaviours is shown in table 1. The high proportion of Roma infants in Teplice reflects the ethnic composition of the two districts. Roma mothers (and infants) had much less favourable profile in most characteristics, except of body mass index and alcohol consumption. Roma infants had considerably lower birth weight, somewhat shorter gestation, and much higher rate of IUGR (table 2).
Table 1 Descriptive characteristics of the infants/mothers.
Non-Roma (n = 8938) Roma (n = 1388) p-value
District
Teplice 77% 94% < 0.001
Prachatice 22% 6%
Male sex (%) 51% 51% 0.995
Number of pregnancies (%)
1 37% 27% < 0.001
2 30% 22%
3+ 33% 51%
Maternal age group (%)
< 19 7% 21% < 0.001
20–24 38% 38%
25–29 34% 24%
30–34 15% 10%
35+ 5% 6
Marital status (%)
Married 72% 29% < 0.001
Cohabiting 11% 26%
Single 12% 41%
Divorced/widowed 5% 4%
Education (%)
Primary or less 15% 81% < 0.001
Apprenticeship 46% 17%
Secondary 33% 2%
University 6% 0%
Maternal height, cm, mean (SD) 166.2 (6.4) 159.4 (7.0) < 0.001
Maternal weight, kg, mean (SD) 62.7 (12.2) 56.7 (11.5) < 0.001
Maternal body mass index, kg/m2, mean (SD) 22.6 (4.0) 22.4 (4.3) 0.01
Maternal smoking (%)
Ever 42% 78% < 0.001
Before pregnancy 35% 73% < 0.001
During 1st trimester 25% 67% < 0.001
During 2nd trimester 18% 65% < 0.001
During 3rd trimester 16% 63% < 0.001
Maternal alcohol consumption, any (%)
Before pregnancy 48% 31% < 0.001
During pregnancy 22% 21% 0.491
Table 2 Birth outcomes in the Roma and non-Roma infants.
Non-Roma (n = 8938) Roma (n = 1388) p-value
Birth weight, g, mean (SD) 33442 (483) 2970 (522) < 0.001
Gestational age, weeks, mean (SD) 39.6 (1.5) 38.7 (2.0) < 0.001
Low birth weight (< 2500 g) (%) 3.6% 14.1% < 0.001
Preterm birth (< 37 weeks) (%) 3.9% 9.9% < 0.001
IUGR (< 10th percentile)(%) 8.9% 22.2% < 0.001
Table 3 shows the extent to which different characteristics explain differences between Roma and non-Roma infants in birth weight and gestational age. In crude analyses, Roma infants were 373 g lighter at birth and their gestational age was 0.92 weeks shorter than of non-Roma babies. Gender, district, maternal age and number of pregnancies contributed only marginally to these differences. In contrast, maternal education "explained" more than one third of the difference in birth weight and more than one fifth of the difference in gestational age (the role of marital status was only marginal). Maternal size, and to a lesser extent smoking, also accounted for some of the ethnic differences. After controlling for all covariates available, the ethnic differences in birth weight were reduced by almost two thirds (from 373 g to 133 g); for gestational age, the reduction was smaller, about 40% (from 0.92 weeks to 0.57 weeks).
Table 3 Explaining ethnic differences in birth weight (g) and gestational age (weeks): results of linear regression (differences between Roma and non-Roma infants, standard errors and p-values).
Adjusted for Birth weight Gestational age
Diff. (SE) p-value Diff. (SE) p-value
Crude -373 (14) < 0.001 -0.92 (0.05) < 0.001
Infants' demographic characteristics (gender and district) -363 (14) < 0.001 -0.89 (0.05) < 0.001
Mothers' demographic characteristics (maternal age, number of pregnancies) -354 (15) < 0.001 -0.87 (0.05) < 0.001
Mothers' social characteristics (education, marital status) -237 (17) < 0.001 -0.73 (0.06) < 0.001
Maternal height and weight -260 (15) < 0.001 -0.80 (0.05) < 0.001
Maternal smoking and alcohol consumption -292 (16) < 0.001 -0.81 (0.05) < 0.001
All covariates listed above -133 (18) < 0.001 -0.57 (0.06) < 0.001
Results of logistic regression for binary outcomes were similar (table 4). For low birth weight, the crude odds ratio of 4.5 was reduced to 1.7 in the full model; for preterm birth, the odds ratio 2.8 fell to 1.5 after controlling for all covariates; and for IUGR, the covariates explained most of the excess in Roma infants. For all outcomes, education made the largest contribution to the reduction of the crude odds ratios.
Table 4 Explaining ethnic differences in low birth weight, preterm birth and IUGR: results of logistic regression (odds ratios for Roma vs non-Roma, 95% confidence intervals and p-values).
Adjusted for: Low birth weight Preterm birth IUGR
OR 95% CI p-value OR 95% CI p-value OR 95% CI p-value
Crude 4.5 (3.7–5.4) < 0.001 2.8 (2.2–3.4) < 0.001 2.9 (2.5–3.4) < 0.001
Infants' demographic characteristics
(gender and district) 4.3 (3.5–5.2) < 0.001 2.5 (2.1–3.1) < 0.001 2.9 (2.5–3.3) < 0.001
Mothers' demographic characteristics
(age, number of pregnancies) 3.9 (3.2–4.8) < 0.001 2.4 (1.9–3.0) < 0.001 2.8 (2.4–3.3) < 0.001
Mothers' social characteristics
(education, marital status) 2.6 (2.0–3.3) < 0.001 2.0 (1.5–2.6) < 0.001 1.8 (1.5–2.1) < 0.001
Maternal height and weight 2.9 (2.4–3.6) < 0.001 2.2 (1.8–2.8) < 0.001 2.0 (1.7–2.3) < 0.001
Maternal smoking and
alcohol consumption 3.3 (2.6–4.1) < 0.001 2.1 (1.7–2.7) < 0.001 2.2 (1.9–2.6) < 0.001
All covariates
listed above 1.7 (1.3–2.2) < 0.001 1.5 (1.1–2.0) 0.010 1.3 (1.0–1.6) 0.029
In additional analyses, we found no evidence for an interaction between ethnicity and calendar year: the differences between Roma and non-Roma groups remained constant over the study period (not shown in table). There was also no interaction between ethnicity and district; despite the difference in the size of the Roma population between the two districts, the differences between Roma and non-Roma groups were similar in Teplice and in Prachatice (not shown in table).
Discussion
This study in a representative community sample of births found striking ethnic differences in all birth outcomes studied and in most socio-demographic characteristics. The covariates available in the study, particularly maternal education, explained a considerable part of these differences.
Several limitations of the study should be considered when interpreting the results. First, more detailed data would provide a more complete picture of the family environment. It is likely that if information on economic and material conditions (e.g. income, housing, household amenities) would contribute further to the explanation of the ethnic differences. Data on husbands would also help but, as explained above, the proportion of missing data on fathers was too high to be used in the analyses. Data on household assets would also be valuable to improve the assessment of families' socioeconomic status. Similarly, data on factors that could mediate the link between ethnicity, socioeconomic characteristics and birth outcomes, such as nutrition, would be also useful. Finally, ethnic differentials in birth outcomes may be further exacerbated by unmeasured area-level characteristics [11].
Second, some of the measurements might have been inaccurate. In particular, the definition of ethnicity is a difficult issue. Some misclassification is likely; some Roma mothers may have identified themselves as non-Roma. However, self-reported ethnicity, used in this study, has been suggested as the preferred method [3], and alternative methods are not ideal either. In addition, mothers may have misreported some of the information (e.g. smoking or alcohol consumption). Birth outcome variables were taken from medical records; while in a few cases these variables may have been recorded inaccurately by the hospital personnel, the validity of such information in this study is high [12]. In addition, assuming that such misclassification was random, it would tend to underestimate the associations studied, rather than lead to spurious findings.
Finally, the number of Roma births was much smaller than that of non-Roma births, and the unbalanced structure of the sample might have reduced the statistical power. However, given the overall size of the study, the statistical power was sufficient to demonstrate, in Roma vs. non-Roma comparisons, an odds ratio of 1.20 or higher at 95% confidence level. The probability of the beta error is therefore low.
In the research on ethnic differences in health in general, an important questions has been debated for some time, namely what are the reasons for such differences [3]. For different health outcomes, the proposed explanations range from genetic to socioeconomic factors [13-17] but addressing this questions has been hampered by limitations of available data [3]. Our results shed new light at one specific type of ethnic differences in health – the poor birth outcomes of Roma mothers. It has been generally perceived that the poor health of Roma people is largely due to their unhealthy behaviours, such as smoking and drinking. Our results suggest that the explanation is more complex. Smoking before and during pregnancy was considerably more common in Roma women but it statistically explained a relatively modest part of the excess of poor birth outcomes of Roma mothers. Alcohol consumption, on the other hand, was not more common in Roma mothers, and cannot therefore be implicated in their poor pregnancy outcomes.
Maternal education made by far the largest single contribution to explaining the poor birth outcomes in Roma mothers. This is not surprising, because there were huge differences in educational attainment between Roma and non-Roma mothers in this study (table 1), and because maternal education has been previously shown to be the key determinant of low birth weight, preterm birth, intrauterine growth and infant mortality in the Czech population [18,19]. In this study, the crude differences in mean birth weight between infants born to mothers with primary and university education was 322 g. It is therefore entirely plausible that education plays an essential part in the differences between the two ethnicities, not least because maternal education is a good proxy for a variety of measures of deprivation. Marital status was also strongly associated with birth outcomes (e.g. the crude differences in birth weight between married and single mothers was 232 g) but its contribution to the ethnic differences was smaller than that of education.
Maternal size, particularly height, partly reflects socioeconomic conditions in earlier life [20]. Reproductive history, indicated by the number of pregnancies, is also associated with social status. It is therefore likely that the ethnic differences in birth outcome in the Czech Republic are to a considerable extent determined by socioeconomic factors. This is consistent with the conclusions of a recent review of the literature on the ethnic differences in health in United States and Britain [3]. It would be interesting to explore whether factors such as nutrition or use, access to and quality of antenatal care can help further clarify the pathways linking ethnicity, socioeconomic circumstances and health.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MB analysed the data and drafted the paper. JD and RJS designed and coordinated the study and commented on the drafts of the paper. IS contributed to the data collection and data management and commented on the drafts of the paper.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This study was supported by the Ministry of Environment of the Czech Republic (grants No. VaV/340/2/00 and VaV/740/5/03), and by the Academy of Sciences of the Czech Republic (1QS500390506). MB's work is supported by the Wellcome Trust and the MarArthur Foundation.
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Nazroo JY The structuring of ethnic inequalities in health: economic position, racial discrimination, and racism Am J Public Health 2003 93 277 284 12554585
Semerdjieva M Mateva N Dimitrov I Sexual culture of gypsy population Folia Med (Plovdiv ) 1998 40 72 75 10205999
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Joubert K Size at birth and some sociodemographic factors in gypsies in Hungary J Biosoc Sci 1991 23 39 47 1999445
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Rimarova K Ostro A Bernasovska K Holecyova G Aghova L Reproductive indicators of Roma mothers: cross-sectional study Living conditions and health 2004 Bratislava, Public Health Office of the Slovak Republic 110 114
Sram RJ Benes I Binkova B Dejmek J Horstman D Kotesovec F Otto D Perreault SD Rubes J Selevan SG Skalik I Stevens RK Lewtas J Teplice program - the impact of air pollution on human health Environ Health Perspect 1996 104 (Suppl. 4) 699 714 8879999
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Gupta S de Belder A Hughes LO Avoiding premature coronary deaths in Asians in Britain: spend now on prevention or pay later for treatment BMJ 1995 311 1035 1036 7580644
Stewart JA Dundas R Howard RS Rudd AG Wolfe CD Ethnic differences in incidence of stroke: prospective study with stroke register BMJ 1999 318 967 971 10195965
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Sheldon TA Parker H Race and ethnicity in health research J Public Health Med 1992 14 104 110 1515192
Koupilova I Vagero D Leon DA Pikhart H Prikazsky V Holcik J Bobak M Social variation in size at birth and preterm delivery in the Czech Republic and Sweden, 1989-1991 Paediatr Perinatal Epidemiol 1998 12 7 24 10.1111/j.1365-3016.1998.00075.x
Koupilova I Bobak M Holcik J Pikhart H Leon DA Increasing social variation in birth outcomes in the Czech Republic after 1989 Am J Public Health 1998 88 1343 1347 9736874
Kuh D Wadsworth M Parental height: childhood environment and subsequent adult height in a national birth cohort Int J Epidemiol 1989 18 663 668 2807671
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-1081621910410.1186/1471-2458-5-108Research ArticleAssessing immunization data quality from routine reports in Mozambique Mavimbe João C [email protected] Jørn [email protected] Gunnar [email protected] Faculty of Medicine, Eduardo Mondlane University, Maputo, Mozambique2 Faculty of Mathematics and Natural Sciences, Department of Informatics, University of Oslo, Norway3 Faculty of Medicine, Department of General Practice and Community Medicine, Section of International Health, University of Oslo, Norway2005 11 10 2005 5 108 108 3 5 2005 11 10 2005 Copyright © 2005 Mavimbe et al; licensee BioMed Central Ltd.2005Mavimbe 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
Worldwide immunization coverage shows an increase in the past years but the validity of the official reports for measuring change over time has been questioned. Facing this problem, donor supported initiatives like the Global Alliance for Vaccine and Immunizations, have been putting a lot of effort into assessing the quality of data used, since accurate immunization information is essential for the Expanded Program on Immunization managers to track and improve program performance. The present article, discusses the practices on record keeping, reporting and the support mechanism to ensure data quality in Mozambique.
Methods
A process evaluation study was carried out in Mozambique in one district (Cuamba) in Niassa Province, between January and March 2003. The study was based on semi-structured interviews, participant observation and review of the data collection materials.
Results
Differences were found for all vaccine types when comparing facility reports with the tally sheets. The same applies when comparing facility reports with district reports. The study also showed that a routine practice during supervision visits was data quality assessment for the outpatient services but none related to data consistency between the tally sheets and the facility report. For the Expanded Program on Immunization, supervisors concentrated more on the consistency checks between data in the facility reports and the number of vaccines received during the same period. Meetings were based on criticism, for example, why health workers did not reach the target. Nothing in terms of data quality was addressed nor validation rules.
Conclusion
In this paper we have argued that the quality of data, and consequently of the information system, must be seen in a broader perspective not focusing only on technicalities (data collection tools and the reporting system) but also on support mechanisms. Implications of a poor data quality system will be reflected in the efficiency of health services facing increased demands, with stagnant or decreasing resources.
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Background
Immunization is an important means of controlling diseases and has been considered as the most cost effective health intervention [1]. Immunization is provided in most countries through the Expanded Program on Immunization (EPI) and as a part of the primary health care approach. Different approaches are used to enhance immunization coverage such as fixed vaccination posts, outreach services and national immunization days [2].
Worldwide immunization coverage shows an increase in the past years but the validity of the official reports for measuring change over time has been questioned [3,4]. For example, the World Health Organization (WHO) experts showed in a previous report that there was a tendency to overstate the number of fully immunized children against vaccine preventable diseases [3,5].
Facing this problem, donor supported initiatives like the Global Alliance for Vaccine and Immunizations (GAVI) have been putting a lot of effort into assessing the quality of data used, since accurate immunization information is essential for EPI managers to track and improve program performance [5].
Several studies have reported inconsistencies in data reporting as well as poor support mechanisms to ensure data quality at the district level [6-8]. For example a study done in Nepal found that data obtained from the facility registers were lower than the data reported at the district level; showing a tendency of over-reporting to the higher levels [9]. Other studies showed that errors in reporting were due to lack of supervision and feedback from the superior levels as well as inadequate incentives to health workers [6,7].
EPI in Mozambique started in 1979 following the national immunization campaign against smallpox. The EPI targets seven vaccine preventable diseases in children less than two years of age plus neonatal tetanus through immunization of pregnant women. These are Bacillus Calmette Guerin (BCG), Diphtheria, Pertussis, Tetanus and Hepatitis B (DTP+HepB), Polio and measles vaccines. The national coverage estimates for 2003 were 87% for BCG, 70% for the third dose of Polio vaccine, 72% for DTP+HepB and 77% for measles vaccine [10].
From its inception, the EPI has undergone changes along the years which led to the development of new data collection tools. The most fundamental change was full integration into primary health care services.
As with other health programs in Mozambique, EPI is hampered by chronic shortages of resources and difficult logistics due to the large geographical area, poor communications and infrastructure. This lack of resources – especially human resources – forced the Ministry of Health to adopt strategies that require inadequately trained personnel located in the most remote health facilities to cope with the challenges of general information management and routine personnel shortage [11].
With the present article, we try to answer the following research questions:
1. What are the current processes of record keeping and reporting within the EPI?
2. What are the existing support mechanisms to ensure data quality on EPI at the district level?
3. How can data quality be improved on EPI at the district level?
These questions were addressed as part of an ongoing research initiative of the Health Information System Program (HISP), which is a joint collaborative of the University of Oslo in Norway, the Eduardo Mondlane University (UEM) in Mozambique, the University of Western Cape in South Africa, and the Ministries of Health of Mozambique and South Africa. This project involved a combination of semi-structured interviews, participant observation, and review of data collection materials. The HISP program focuses on improving information to support decision making in primary health care.
Methods
Study scope
A process evaluation study was carried out in Mozambique in one district (Cuamba) in Niassa Province, between January and March 2003. Cuamba has an estimated population of 180,000 habitants with approximately 7200 children less than one year of age [12]. The district health infrastructure is composed of 13 health facilities of which 12 provide primary health care services, and one is a rural hospital. Cuamba at the time of the study had two medical doctors and thus most facilities were managed by paramedical staff [13].
Selection of research site
Niassa province was chosen because the provincial directorate had identified constraining factors related to the functionality of the EPI; for example, the appearance of epidemics (like measles) in areas reporting high coverage rates [14-16]. Niassa is also among the pilot sites for HISP in Mozambique and Cuamba was selected because it constitutes a strategic point for HISP implementation as it is the referral district for the southern region of the Niassa province; Cuamba also has a relation with the Medical Faculty of the UEM, being a training site for last year medical students.
The district provides vaccination in 7 health facilities. For our study, all health facilities providing vaccination in Cuamba were selected. Since the outreach services are organized by the district headquarters and thus do not have to compete with other services and priorities like in a health facility, data from this activity has been excluded from our study.
Sources of information
Both qualitative and quantitative approaches have been used. The qualitative materials were gathered through semi-structured interviews with open questions directed to 7 health workers from the facility level. Health workers were asked questions regarding the flow of information, the support mechanism (the quality of supervision visits, feedback and supplies) and the interaction between them and the District Health Management Team (DHMT) called NEP. Participant observation was used for the meeting held at the district level and to understand work practices at the facility level.
Quantitative materials were collected from three different sources: tally sheets, facility reports and district reports corresponding to the whole year of 2002. The assessed immunizations were BCG; the third dose of DTP+HepB; and measles vaccine in children less than one year of age.
Information and data flows in EPI
In Mozambique, each district has an organization structure with different directorates, including the district health directorate. The district health directorate is composed of several health facilities which provide health services to the population and the DHMT that is responsible for the health management at the district level. The health facilities provide mother and child services, outpatient services and (for some facilities) the EPI services.
The official national immunization coverage figures are based on routine data produced at the facility level and by the outreach services. The outreach services are sustained by district level authorities.
At the first contact between a child and the health facility, the activity is recorded in the immunization tally sheet, called the A01 form for BCG, DTP+HepB (1, 2 and 3), Polio (birth, 1, 2 and 3), measles, and the A02 form for the tetanus vaccine. These constitute the basic data sources of the immunization status of the child population in the area (Figure 1).
Figure 1 Information flow of the EPI in Mozambique.
On a monthly basis, the A01 and A02 registers are collated in a facility form (A03). The outreach services follow the same procedure as a facility, filling out a tally sheet and later the A03 form. The data are aggregated at the district level into a district form (A04) which does not discriminate information from various facilities.
The district form is sent to the provincial level on a monthly basis where the data are entered in a computer database and on a quarterly basis sent to the national level using a floppy disk (Planning Department) or in a paper format (Community Health Department).
Data analysis
Data from the interviews were analyzed according to three different themes: information and data flows; work practices; and the existing support mechanisms. Participant observation data was used as a narrative description of the data analysis process during the quarterly meeting held at the district level. Some interviewee responses are given; these are based on verbatim notes, which are quoted in italics and have been translated from Portuguese to English.
Quantitative data (tally sheets, facility reports and district reports) were entered in a computer database using as interface Epidata ver. 3.0 (The EpiData Association) and later converted into SPSS version 11 (SPSS Inc.). All sets of data were based on the same target population from three levels: tally sheets, facility reports and district reports.
These three sets of data were analyzed by descriptive statistics. Frequencies and cross-tabulations were computed between the different sources of data. Later the data was compared and the consistency between them assessed. Significant differences were defined as those with p < 0.05.
Since district reports represent aggregated data, data from the outreach services were then subtracted from the district reports for comparative purposes. Due to the existing similarities in the distribution per facility, vaccine type and months, only BCG vaccine was used to illustrate the distribution per facility and per month. For the sake of anonymity the names of the health facilities were represented as A, B, C, D, E, F and G.
Study limitations
Certain limitations of the study should be borne in mind. First of all, the findings may not be entirely applicable to other areas in Mozambique. Secondly, the sample is small and may not be entirely representative of the overall EPI program in Mozambique.
Results
Vaccination data at the entry point
In each facility one health worker is responsible for the EPI. It is usually the mid-wife who also holds multiple health care program responsibilities, for example, maternal health care. After vaccinating the child, the health workers take the tally sheet and register the vaccinated children.
Analyzing the data at this level, differences were found for all vaccine types when comparing facility report with the tally sheets (Table 1). Most of the facility reports showed higher values compared with the tally sheets. For example, for the BCG, the values ranged from 224% higher to 7% lower and the average was 44% higher whereas for DTP+HepB, they ranged from 474% higher to 19% lower with an average of 95% higher. For measles they ranged from 268% higher to 8% lower and the average was 72% higher for facility reports when compared with the tally sheets.
Table 1 Distribution of vaccines in Cuamba during 2002: differences between tally sheets and facility reports per facility
Number of vaccines per health facility
A B C D E F G
BCG
Register 89 3497 108 402 588 634 165
PHC Report 288 3537 108 448 549 655 294
Difference (%) 223.6% 1.14% 0.00% 11.44% -6.63% 3.31% 78.18%
DPT3
Register 19 2282 58 449 356 221 72
PHC Report 109 2282 62 479 288 248 206
Difference (%) 473.7% 0.0% 6.9% 6.7% -19.1% 12.2% 186.1%
Polio 3
Register 19 2294 58 449 370 230 64
PHC Report 109 2294 68 479 334 240 147
Difference (%) 473.7% 0.0% 17.2% 6.7% -9.7% 4.3% 129.7%
Measles
Register 47 1849 56 412 386 303 42
PHC Report 173 1849 60 442 356 317 135
Difference (%) 268.1% 0.0% 7.1% 7.3% -7.8% 4.6% 221.4%
The facility analysis showed that the data increase was mainly in facilities A and facility G for all vaccine types. Conversely, facility E showed decreased values in all vaccine types. The findings showed that most of the data generated in facility B were concordant but showed an increase of 2% for BCG.
The distribution per vaccine and per month (Table 2) showed that, for facility A, a set of "zeros" was found in April and June to December. This was due to a non existent tally sheets at the facility level. A Similar situation was found in facility G for the period of April until July, September and October. For facility C, it was found that EPI services were not done from January to July due to a sick leave of the midwife. For this same facility, the study showed that there were filled tally sheets but nothing was reported to the district level during August for two vaccine types.
Table 2 Distribution of BCG vaccine in Cuamba during 2002: differences between tally sheets and facility reports per facility and per month (Cuamba, 2002)
Facility A Facility B Facility C Facility D Facility E Facility F Facility G
TS FR TS FR TS FR TS FR TS FR TS FR TS FR
Jan 18 18 251 251 0 0 35 35 32 32 47 47 0 0
Feb 18 18 246 246 0 0 33 33 64 64 16 16 0 0
Mar 19 19 282 282 0 0 41 40 29 29 15 15 0 0
Apr 0 19 268 308 0 0 25 25 30 30 39 39 0 28
May 34 34 233 233 0 0 41 41 90 90 23 23 0 50
Jun 0 22 246 246 0 0 30 30 38 38 36 36 0 20
Jul 0 51 336 336 0 0 44 44 109 86 80 80 0 20
Aug 0 9 344 344 12 0 27 27 51 51 33 33 86 0
Sep 0 29 346 346 40 52 36 47 70 70 65 86 0 51
Oct 0 19 340 340 31 31 0 36 55 25 118 118 0 46
Nov 0 34 240 240 25 25 38 38 9 9 78 78 37 37
Dec 0 16 365 365 0 0 52 52 11 25 84 84 42 42
TS: Tally sheets FR: Facility Reports
The data analysis also showed that there were different values in the tally sheets when compared with the facility reports characterized by increased values during September for all vaccines types in facility C as well as for facility F.
The data at the district level
At the district level, each month, the health facilities send the facility form (A03) which is aggregated together with the outreach campaigns to make up the district average (A04). This aggregation is the responsibility of the District EPI Coordinator who, after compiling the data, sends it to the District Director to get the corresponding signature. For our study, data from the outreach campaigns are excluded.
Our study showed that numbers of all vaccines types were different in three sets of data when comparing tally sheets, regarding facility registers and district reports (Table 3). The vaccines reported by the health facilities showed an increase averaging 7% for all vaccine types compared to the tally sheets. Differences were also seen between the numbers of vaccines from the facility reports when compared with the district reports. These changes were 0.4% for BCG and 2.8% for measles, whereas the DPT+HepB showed a decrease of 0.5%.
Table 3 Comparison of different data sources for the EPI (Cuamba, 2002)
BCG DPT+HepB Measles
Sources Value Difference (%) Value Difference (%) Value Difference (%)
Tally Sheets (A01) 5483 - 3457 - 3095 -
Facility Reports (A03) 5879 7.2% 3674 6.3% 3332 7.7%
District Reports (A04) 5903 0.4% 3656 -0.5% 3428 2.9%
The support mechanism
All health workers received supervision visits at intervals of less than 3 months. Supervision visits were supported by an American NGO (Medical Care Development International) which provided vehicles, fuel, and allowances. All health workers considered the supervision visits as good and as a way to increase their skills in the provision of care as well as in data analysis.
A routine practice during supervision visits was data quality assessment for the outpatient services but none related to data consistency between the tally sheets and the facility report. For the EPI, supervisors concentrated more on the consistency checks between data in the facility reports and the number of vaccines received during the same period. It was also said that supervisors were looking for miscalculations in the rows and columns of the facility report. Feedback mechanism was rare. When they existed it was based on criticism, for example "...why aren't you reaching the target ...".
All health workers reported a lack of archiving files, punchers and staples to organize the raw data. Health workers' interaction with the district management team was characterized as "...they want a good performance, so we provide them good data..." therefore "...they are not coming after us.." but " ...they are not providing enough supplies nor incentives to do so ...for example, to get a stapler, or a file to store the tally sheets at the facility, it is extremely difficult, ...to get fuel for the motorbike is also a very difficult process...". It was also said that "... if we don't provide good data showing that we are achieving the target, we are threatened not to receive the salary....".
Errors are also related to miscalculations despite the use of a calculator. Some health workers said "... the boss is always asking for good quality data but these calculators have very small buttons and screen, so the numbers are difficult both to type and to see...".
Meetings took place at the district level in a monthly and quarterly basis to discuss achievements and to produce the quarterly report that has to be sent to the provincial level. Data completeness was discussed but very little about data validation. In these meetings they used a standardized spreadsheet where coverage, targets and the fulfillment index were discussed for each facility, and for each health program.
The meeting was based on criticism, for example, why health workers did not reach the target. Nothing in terms of data quality was addressed nor validation rules. For example, during one quarterly meeting where one health facility reached immunization coverage rates for BCG of 335%, for the first quarter in 2003, the health worker said that he was vaccinating the neighboring district population since they were using his services in cases of sickness. No further discussion was raised on this matter.
Discussion
Data collection begins with the first contact between patient and health care provider. Counts of individual vaccinations are then forwarded to the next administrative level (district) where they are aggregated as a district average. Data that are recorded accurately at the facility level should correspond to data reported at the district level.
Our study showed that there were data differences between facility and district reports suggesting that there are errors in the reporting system at the facility level. These errors caused increased values for two vaccine types when comparing district reports with facility reports.
Our findings from a district perspective might not look important due to the correct data provided by facility B, which performs most of the immunizations activities in the district. On the other hand, with a facility analysis, it could be seen that facilities are over-reporting for all vaccines types and the average over-reporting ranges from 44% for BCG to 95% for DPT+HepB.
It is also important to note that analysis per facility and per month showed that the facilities reported immunized children even in the months were no children were recorded in the immunization tally sheets. This is mainly due to the way district manager's approach health workers that "did not perform well", for example, threats of salary cut.
Other abnormalities were vaccinations performed but with nothing reported to the district level. In some cases, the health worker attributed to the lack of adequate archiving materials to the absence of some tally sheets showing that the existing support mechanism is still inadequate to support the recording, storing and reporting practices.
In general, at the facility level, there is a tendency to show a "good performance" as seen by the example of the health worker that was vaccinating people at the neighboring district. This situation means that there is poor capacity to understand and to establish clear catchment areas per facility.
The notion of vaccination target and the absolute need to achieve them, since it constitutes the basis for good performance, leads to an emphasis on imperatively reaching fixed targets with success rather than effects of prevention or public health. Consequently, health workers experienced the pressure to immunize eligible children as a burden or even over reporting.
This rigidity and "discipline" imposed by the managers for target achievement may be understood as an indication of what Streefland calls, metaphorically, "a military organization model" which so often emerges in vaccination programs [17]. Further errors in the facility reporting might be added due to lack of motivation of the health personnel, lack of feedback, no concern for quality information and no cross-checking mechanism.
The system in general, as it is designed, invites "data cooking" as well as lack of interest in supporting practices such as record keeping and data use. For managers the major concern is to achieve the target; therefore, the information system is seen as an "upward system" and not as a system that may support their own work.
A common perception is that to improve accuracy and timeliness of data, redesigning the forms and data collection procedures constitutes the main solution [18]. Using this approach, implementing a register book at the facility level to ensure record keeping, could be a suggestion. However, we believe that the most important aspect is to relate information needs to interventions with focus on how information generated could be used and influence local decisions [17,18].
Some experiences, for example in Kyrgyzstan and in South Africa, showed improved data quality by giving health workers the basic skills to monitor their own work, leading to a sense of ownership of the generated information [19,20].
Different approaches can be used to improve the support mechanisms, for example, increasing the quality of the supervision visits regarding the quality of data from the tally sheet, as well as providing adequate feedback mechanism to the producers of data at the remote sites.
On the other hand, supervision visits could include a more comprehensive data analysis on EPI. It could be used as a way to do in-job training on basic concepts and monitoring indicators, a strategy used in some countries producing satisfactory results [19]. The "eyeballing" approach (a quick look at the forms), the 3C's approach (completeness, correctness and consistency) could also be promoted as a first step towards data quality improvement, and could be an essential part of health workers at the remote sites.
Conclusion
In this paper we have argued that the quality of data, and consequently of the information system, must be seen in a broader perspective not focusing only on technicalities (data collection tools and the reporting system) but also on support mechanisms. Implications of poor data quality system will be reflected in the efficiency of health services facing increased demands, with stagnant or decreasing resources.
Within the existing study limitations, we believe that the present paper constitutes an important contribution applicable to the EPI in Mozambique and other country settings where poor data quality constitutes a bottle neck for good information systems as well as good decision making. It uses both quantitative and qualitative methods to highlight problems in a complex system where lack of resources is a constant problem.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
João Carlos de Timóteo Mavimbe participated in all phases of the study from data collection to the preparation of the manuscript and is corresponding author. Jørn Braa and Gunnar Bjune have participated on the interpretation of results and the critical revision of the paper for substantial intellectual content. 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 all the authorities in Niassa Province (Dr. Ussene Isse) for the support given and openness to discuss the findings. We also like to thank Prof. Sundeep Sahay, Joystna Sahay, Jose Nhampossa, Kristoffer Fossum and Bonang Nkoane all from the University of Oslo, for suggestions and comments on the final draft. We wish to extent our gratitude to health workers at the most remote sites, which spared some time to participate in the study, despite their pressing work demand.
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-1101622568310.1186/1471-2458-5-110Research ArticleImproving rates of pneumococcal vaccination on discharge from a tertiary center medical teaching unit: A prospective intervention Thomas Chandra M [email protected] Andrea [email protected] Carla [email protected] Norman RC [email protected] Department of Medicine, University of Calgary, Calgary, Canada2005 14 10 2005 5 110 110 20 3 2005 14 10 2005 Copyright © 2005 Thomas et al; licensee BioMed Central Ltd.2005Thomas 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
Pneumococcal disease causes significant morbidity and mortality in at-risk individuals, and is complicated by emerging antibiotic resistance. An effective, safe and cost-effective vaccine is available, but despite this many patients who would benefit from pneumococcal vaccination remain unvaccinated. The purpose of this study was to determine the rates of missed opportunities to provide pneumococcal vaccination to patients being discharged from a tertiary center medical teaching unit and to determine if a nurse coordinator-based intervention would increase rates of pneumococcal vaccination prior to discharge home.
Methods
We conducted a prospective, controlled study in the setting of a Medical Teaching Unit at a tertiary care centre to assess the impact of a nurse coordinator based intervention on the rates of vaccination of eligible patients on discharge home. The rates of vaccination during an eight-week usual-care period (February 20 to April 16, 2002) and an eight-week intervention period (April 22 to June 16, 2002) were compared.
Results
Prior to the intervention none of thirty-eight eligible patients were vaccinated prior to discharge home from the Medical Teaching Unit. After the intervention 27 (54%) of fifty eligible patients were vaccinated prior to discharge.
Conclusion
There are significant missed opportunities to provide pneumococcal vaccination to inpatients who are discharged home from a medical unit. Using a patient care coordinator we were able to significantly improve the rates of vaccination on discharge.
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Background
Invasive infection by streptococcus pneumoniae is an important cause of morbidity and mortality, particularly in individuals older than 65 years of age, and those with chronic health conditions. The incidence of invasive pneumococcal disease almost triples in those over 65. Despite appropriate antibiotic therapy and supportive care there is a high case fatality rate in pneumococcal bacteremia of 15–20% in adults overall with highest rates in those over 65 and those with chronic medical conditions. The case fatality rate has been reported as 12.1% for individuals aged 16 to 64 with indications for vaccination compared to 5.4% to individuals of the same age with no indications [1].
Previously there was uniform susceptibility to penicillins. The National Centre for Streptococcus in Edmonton, Alberta now has reported 10.2% of isolates have diminished susceptibility to penicillin [2]. Macrolide resistance among invasive S. pneumoniae isolates also dramatically increasing [3].
Given the high morbidity and mortality of pneumococcal disease, and increasing antibiotic resistance, the focus is shifting to primary prevention. The 23-valent pneumococcal vaccine is between 50–80% effective against invasive pneumococcal disease in older and high-risk individuals [4]. The vaccine is also cost-effective in high-risk individuals [5,6].
The rates of vaccination against pneumococcus are well below targets and studies have shown significant missed opportunities for vaccination of at-risk individuals [7]. The Health Canada target vaccination rate is 80% of patients who meet the Canadian Immunization Guidelines criteria for pneumococcal vaccination [8]. However, only 42% of those over 65 and 15% of those eligible persons younger than 65 have received pneumococcal vaccination [8]. Studies have shown that 36% to 70% of patients admitted to hospital with invasive pneumococcal infection had been inpatients in the five years prior to admission [9,10]. Brull et al demonstrated that pneumococcal vaccine is the most commonly overlooked preventive health intervention among medial patients who are discharged from a tertiary care hospital [11]. Another study showed that a computerized reminder system in a teaching hospital resulted in a pneumococcal vaccination rate of 35.8% of eligible patients compared to 0.8% in the control group [12]. Many hospitals do not have specialized computer systems such as that described in this study, therefore the generalizability of the study is limited.
Given the evidence that there was room for improvement in the delivery of pneumococcal vaccination to at-risk individuals discharged from hospital, we embarked on a study to determine if a nurse-coordinator-based intervention would increase the rate of pneumococcal vaccination of eligible patients discharged home from an inpatient medical teaching unit.
Methods
Our study was conducted on the medical teaching unit (MTU) at Foothills Medical Centre, a tertiary referral centre in Calgary, Canada with over 700 inpatient beds. The MTU had an average patient census of 20 to 30 with a median length of stay of 7 days. Primary medical care was provided by 3 to 4 clinical clerks and 4 to 5 junior residents under the supervision of 2 senior residents and an attending physician. The team also had a dedicated clinical pharmacist and a care coordinator. The care coordinator is a registered nurse who routinely meets with patients on the day of admission to describe the structure of the MTU, liaises with the patient's outpatient primary care and specialist physicians and coordinates appropriate resources and follow-up for the patient on discharge from hospital.
Patients were considered to be eligible for our study if they met Canadian Immunization Guidelines criteria to receive pneumococcal vaccination, these criteria are described in Appendix 1 [8]. Patients who died, were transferred to other services or had been vaccinated prior to admission were excluded. Patients transferred to other services included those transferred to critical care services and surgical services for surgical intervention. Medically stable patients for which a protracted hospitalization was anticipated were transferred to a non-teaching service prior to discharge home or to long term care facilities.
The first admission was used for patients admitted multiple times to the medical teaching unit. Patients admitted during the usual-care period were eligible to be included in the intervention period if they continued to meet eligibility criteria.
Previous vaccination status was determined by asking the patient and by reviewing the Calgary Public Health vaccination database, which includes information on all pneumococcal vaccinations given in the Calgary Health Region since 1997.
At the outset of the study a multidisciplinary team comprised of internal medicine residents, attending internists, an MTU Care Coordinator, a pharmacist, a nurse manager and a representative from public health was assembled. The team noted that there was no process in place to ensure that eligible patients would be vaccinated on discharge. They embarked on the task of developing a flowchart that would represent a rational process that, if followed, would result in vaccination of patients who were discharged home from the MTU.
The final process is diagrammed in Figure 1.
Figure 1 The study algorithm for vaccinating patients discharged during the usual-care and intervention periods.
The final study consisted of a pre and post intervention where an assessment of pneumococcal vaccine provision on discharge home took place during a usual care period and then an intervention period where the process described in figure 1 and outlined below in the 'Intervention Period' was implemented.
Usual-care period
The usual care period consisted of an eight-week period where the medical students and residents on the MTU were provided with a standardized form outlining eligibility for pneumococcal vaccination. These team members were responsible for assessment of patient eligibility, determining vaccination status and, if appropriate, ordering a dose of vaccine. However, no advice on how best to carry out these tasks was provided. The MTU Care Coordinator determined vaccine eligibility and gathered information on previous vaccination status and whether or not a vaccine dose was ordered at the time a patient was discharged from hospital. This information was not provided to the medical students or residents. The usual care period extended from February 20 to April 16, 2002.
Intervention period
The intervention period involved rolling out the process described above and outlined in Figure 1. The first step in the process included a formalized method of identification of eligible patients. A standardized eligibility form was developed and a consistent individual, the MTU care coordinator, was given the responsibility of ensuring that the form was completed. The completed eligibility form was faxed to the public health office where the database was examined and the vaccination status of the patients admitted to the MTU was reported to the MTU care coordinator. Patients who were eligible to receive the pneumococcal vaccine but had not been previously vaccinated were provided with educational materials on the vaccine by the MTU care coordinator who would, in concert with the floor nurses, housestaff and the attending internist, answer any patient questions on the vaccine. The patient was then offered the vaccine and, if they agreed, it was ordered by the housestaff on the team. The MTU care coordinator oversaw this process and would ensure that the responsible housestaff ordered the vaccine. Nursing staff administered the vaccine as ordered and completed a vaccination form that was sent to the pharmacy. The pharmacy would forward these forms on to public health so that the vaccination database could be updated. All of the individuals and procedures described above were present prior to the intervention but were not previously linked together by a responsible individual – the care coordinator.
A nursing inservice was held on April 17, 2002 to inform staff of the initiative, the background issues and to review vaccine administration and charting. At this time the vaccination rates from the initial 8 week assessment were posted for nursing staff and residents. On April 22, 2002 a computerized message appeared on terminals throughout the Foothills Medical Centre reminding staff to vaccinate eligible patients against pneumococcus on discharge. From April 22 to June 16, 2002 the process described above was implemented.
Statistical analysis
The overall characteristics of patients admitted to the MTU and those for the usual-care and the intervention period were described using means or medians for continuous variables and proportions for categorical variables. Differences between the groups were assessed using Student's t-test. The vaccination rates during the study period were compared using the Chi-square test. All analyses were carried out using Stata Version 5 [13].
Results
The final study population consisted of 38 individuals in the usual-care group and 50 individuals in the intervention group (Figure 2). There were 229 individuals admitted to the MTU during the two eight-week study periods (108 in the usual-care period and 121 during the intervention period). Eight individuals had multiple admissions. Of the patients admitted to the MTU 80.8% (n = 186) were eligible to receive pneumococcal vaccine. Ninety-one (48.9%) of patients were eligible because they were aged greater than 65 years. For patients less than 65 years of age the most common reason for eligibility was liver disease 45.3% (n = 43). Fifty-one (27.4%) of the individuals who met criteria for vaccination had been vaccinated prior to admission, 15.7% of the patients less than 65 years of age and 39.6% of the greater than 65 years age group, and were, therefore excluded from the final study population. Sixty-five percent of eligible patients who were not previously vaccinated were discharged home leaving a final study population of 88 patients.
Figure 2 The flow of patients in the intervention and usual care periods of the study.
The characteristics of the final study population by intervention group are described in Table 1. The study population was similar prior to and during the intervention with no significant difference between the two groups with respect to age, length of stay or sex. The primary study outcome was vaccination rate on discharge home. Fifty-four percent (n = 27) of patients in the intervention group were vaccinated prior to discharge home compared to none of the patients in the usual-care group. The vaccination rate for individuals greater than 65 years of age tended to be higher than the younger group (64.7% versus 48.5%). However, this difference was not statistically significant (p = 0.276). Individuals vaccinated on discharge also had a longer median length of stay (5.5 versus 10 days) but the interquartile ranges for these two values overlap (i.e. the difference was not statistically significant).
Table 1 Characteristics of study population
Characteristic Usual-care (n = 38) Intervention (n = 50)
Age (mean with 95% CI) 56.1 (50.9–61.2) 54.7 (49.7–59.6)
<65 years of age % (n) 63.2% (24) 66% (33)
Reason for vaccine eligibility if age < 65 years*
Splenectomy 1.5% (1) 3.0% (1)
Cardiovascular disease 15.4% (10) 6.1% (2)
Pulmonary disease 13.9% (9) 6.1% (2)
Liver disease (includes alcoholism) 30.8% (20) 51.5% (17)
Renal disease 4.6% (3) 9.1% (3)
Diabetes mellitus 16.9% (11) 24.2% (8)
Immunosuppressed 6.2% (4) 24.2% (8)
Length of stay (median with IQ range) 7 (3–14) days 7 (4–11) days
Female (%) 52.6% (20) 38.0% (19)
*percentages add up to more than 100 because individuals are present in more than one category
To determine if the study had a broader impact on prescribing of pneumococcal vaccines in the Foothills Hospital, we examined ordering of pneumococcal vaccine outside of the MTU setting. In addition to the 27 patients vaccinated on the MTU, 73 patients were vaccinated on other services. Vaccine use was assessed in the same time periods as the study in the year prior to and after the study. There was an increase from 13 vaccinations in 2001 to 219 in 2003 while the Foothills hospital bed capacity increased by only 36 beds (April 2001 761 beds, April 2002 766 beds and April 2003 797 beds). This information is presented in Figure 3.
Figure 3 Pneumococcal vaccination use in Foothills Hospital*. * The number of vaccinations administered in the Foothills Hospital prior to and following the study. The time periods were chosen to correspond with the study periods in 2002 – usual-care period from Feb. 20 to April 16 and the intervention period from April 22 to June 16. The bar graph shows an increase in administration of pneumococcal vaccinations after the intervention and indicating there were significant effects outside of the unit where the intervention occurred and that the effect was sustained after the study was terminated.
Discussion
We demonstrated that there are significant missed opportunities to provide pneumococcal vaccination to eligible patients discharged home from a medical teaching unit service. Further, we demonstrated that by a collaborative, multidisciplinary effort and with the involvement of a nurse coordinator, we were able to assess 100% of patients for eligibility and reach a vaccination rate of 54% [14,15]. Additionally, one year after the intervention the use of pneumococcal vaccine remains increased compared to prior to this study. The successful intervention involved developing a sustainable process for vaccinating patients on discharge by reorganizing existing resources.
The vaccination rate in this study was superior to that which is currently achieved on an outpatient basis. Furthermore, it was also superior to the improvement that was shown in a recent study assessing the impact of a highly specialized computerized reminder system that found a rate of 35.8% after their intervention suggesting that a multimodality intervention is superior to ensure vaccination on discharge [12]. Other interventions that utilized computer-generated admissions list and pre-printed order forms for the vaccine and a dedicated nurse vaccine manager did not manage to achieve rates of vaccination as high as that which was reached in our study. We think the superior results achieved in our study stem from the systematic process developed using integral team members to ensure vaccination on discharge and there being a single responsible individual being accountable.
Based on the known efficacy of the vaccine, and its cost effectiveness in high risk populations, we would anticipate that this intervention will diminish invasive pneumococcal disease, hospitalizations and health care costs in the future.
One limitation in this study was vaccine supply. The hospital pharmacy had anticipated an increase in vaccine utilization. However, the degree of increased vaccine ordering far exceeded that which was anticipated and there was a one week time period when patients were unable to receive vaccination prior to discharge due to lack of availability. A further limitation was that occasionally patients were discharged and left hospital prior to receiving an ordered vaccination. We were unable to quantify the number of theses cases. The continuity of a nurse coordinator in the setting of a MTU where housestaff are constantly rotating was pivotal, and this approach will be most applicable on services where there is a nurse care coordinator. A strength of our study was the presence of a population-based database of vaccination. This database allowed the intervention to taget unvaccinated patients. Without the database 27% of patients entering the hospital could have been revaccinated.
A meta-analysis of interventions that increase adult immunization supports organizational change as the most effective means of improving preventive care measures, surpassing financial incentives, patient reminders, patient education and feedback [16]. We found also that organizational change, with the involvement of a nurse coordinator to do specific prevention activities was effective. With the flux of staff on the MTU the long-term sustainability of this intervention remains to be proven.
Conclusion
There are significant missed opportunities to provide pneumococcal vaccination to hospital inpatients who are at high risk from pneumococcal pneumonia. The use of a patient care coordinator and minor system changes significantly improved the rates of vaccination on discharge.
List of abbreviations
MTU: Medical teaching Unit
Appendix
Guidelines for use of pneumococcal vaccination [7].
• All persons ≥65 years of age
• All persons >2 years with the following: asplenia, splenic dysfunction or sickle cell disease
• All persons >2 years with the following chronic conditions: chronic cardiorespiratory disease (except asthma), cirrhosis, alcoholism, chronic renal disease, nephritic syndrome, diabetes mellitus, chronic CSF leak, HIV infection and other conditions associated with immunosuppression (Hodgkins disease, lymphoma, multiple myeloma, induced immunosuppression for organ transplantation
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
CT, AL and CC collectively assisted in designing, conducting the research and revising the manucsript. CT conducted the statistical analysis and drafted the manuscript. NC supervised the research project and assisted in all aspects of the trial, and revising the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
We would like to acknowledge the following individuals for their assistance in completing this project: Colleen Johnston RN, MTU care coordinator, Duane Bates BSc Pharm, Afsheen Remtulla RN, Barb Kathol RN, Jill Gay CCHRA (C) and the nursing staff on the patient care units associated with the Medical Teaching Unit. The project was part of a quality assurance program on the Medical Teaching Units.
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Robinson KA Baughman W Rothrock G Barrett NL Pass M Lexau C Damaske B Stefonek K Barnes B Patterson J Zell ER Schuchat A Whitney CG Epidemiology of invasive Streptococcus pneumoniae infections in the United States, 1995–1998: Opportunities for prevention in the conjugate vaccine era JAMA 2001 285 1729 35 11277827 10.1001/jama.285.13.1729
Preventing pneumococcal disease: a Canadian consensus conference. 16–18 February 1998 Can Commun Dis Rep 1999 25 25 35 10079579
Hyde TB Gay K Stephens DC Vugia DJ Pass M Johnson S Barrett N Schaffner W Cieslak PR Maupin PS Zell ER Jorgensen JH Facklam RR Whitney CG Macrolide resistance among invasive Streptococcus pneumoniae isolates JAMA 2001 286 1857 62 11597287 10.1001/jama.286.15.1857
Fedson DS The clinical effectiveness of pneumococcal vaccination: a brief review Vaccine 1999 17 S85 90 10471188 10.1016/S0264-410X(99)00113-9
Sisk JE Moskowitz AJ Whang W Lin DL Fedson DS McBean AM Plouffe JF Cetron MS Butler JC Cost-effectiveness of vaccination against pneumococcal bacteremia among elderly people JAMA 1997 278 1333 9 9343464 10.1001/jama.278.16.1333
Sisk JE Whang W Butler JC Sneller V-P Whitney CG Cost-effectiveness of vaccination against invasive pneumococcal disease among people 50 through 64 years of age: role of comorbid conditions and race Ann Intern Med 2003 138 960 8 12809452
Squires SG Macey JF Tam T Progress towards Canadian target coverage rates for influenza and pneumococcal immunizations Can Commun Dis Rep 2001 27 90 1 11396048
Canadian Immunization Guide 1998 5 Health Canada: Ottawa
Fedson DS Baldwin JA Previous hospital care as a risk factor for pneumonia. Implications for immunization with pneumococcal vaccine JAMA 1982 248 1989 95 7120627 10.1001/jama.248.16.1989
Fedson DS Chiarello LA Previous hospital care and pneumococcal bacteremia. Importance for pneumococcal immunization Arch Intern Med 1983 143 885 9 6679231 10.1001/archinte.143.5.885
Brull R Ghali WA Quan H Missed opportunities for prevention in general internal medicine CMAJ 1999 160 1137 40 10234343
Dexter PR Perkins S Overhage JM Maharry K Kohler RB McDonald CJ A computerized reminder system to increase the use of preventive care for hospitalized patients N Engl J Med 2001 345 965 70 11575289 10.1056/NEJMsa010181
Stata Corporation, Stata 1996 Stata Corporation: College Station, Texas
Landis S Scarbrough ML Using a vaccine manager to enhance in-hospital vaccine administration J Fam Pract 1995 41 364 9 7561710
Nowalk MP Middleton DB Zimmerman RK Hess MM Skledar SJ Jacobs MA Increasing pneumococcal vaccination rates among hospitalized patients Infect Control Hosp Epidemiol 2003 24 526 31 12887242
Stone EG Morton SC Hulscher ME Maglione MA Roth EA Grimshaw JM Mittman BS Rubenstein LV Rubenstein LZ Shekelle PG Interventions that increase use of adult immunization and cancer screening services: a meta-analysis Ann Intern Med 2002 136 641 51 11992299
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BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-5-1121623231610.1186/1471-2458-5-112Research ArticleDiet induced weight loss accelerates onset of negative alliesthesia in obese women Frankham Patrick [email protected] Caroline [email protected] Michel [email protected] Département d'Anatomie et Physiologie, Faculté de Médecine, Université Laval, Québec, CA2 Groupe Interdisciplinaire de Recherche en Obésité (GIRO), Université Laval, Québec, CA2005 18 10 2005 5 112 112 15 2 2005 18 10 2005 Copyright © 2005 Frankham et al; licensee BioMed Central Ltd.2005Frankham 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 physiological and behavioral responses to hypocaloric diet are to increase energy intake to defend a steady body weight. We utilized the method of "negative alliesthesia" for measuring the hedonic reponse to sweet stimulus before (Initial session) and 3 months after entering a weight loss program. The negative alliesthesia test is known by physiologists but few clinical data exist. It is based on the observation that repeated pleasant gustatory stimuli turn into unpleasantness in the process of alliesthesia. At first visit participants repeatedly ingested sweet stimuli until they found them unpleasant and rated quantitatively on a linear analogue scale their hedonic experience. This procedure was repeated every 3 min until participants felt displeasure to end the session. The same protocol was followed after three months of following a weight loss diet. Dieting energy intake was from 1400 – 2000 kcal/d for 8 wk. Energy composition was 50% carb:25% prot: 25% lipid. After 8 wk caloric intake increased by 50 kcal/wk, to reach daily intake of 1800 – 2400 kcal/d. Energy composition was 50% carb:22% prot: 27% lipid. We report results on the effect of slow weight loss on negative alliesthesia in ten obese female participants enrolled in a commercial diet program based on Canada's Food Guide (Mincavi®).
Results
Results showed that diet lowered the mean BMI (Initial session 36.8 +/- 1.8 vs. 3 mo 34.9 +/- 1.8 kg/m2). At 3 mo the onset of negative alliesthesia, time to abandon experimental session, was shortened (Initial session 33 vs. 3 mo 24 min). The same trend was observed in the time to reach indifference (Initial session 21.9 +/- 3.8 vs. 3 mo 16.2 +/-2.4 min). There was no observed difference in maximum (Initial session +79.5 +/- 11.7; 3 mo +94.5 +/- 9.9 mm) and minimum (Initial session -90.0 +/- 14.4; 3 mo -106 +/- 11.1 mm) hedonic rating.
Conclusion
Earlier onset of negative alliesthesia, as seen in our participants, is not consistent with previous hedonic studies that showed delayed or absent negative alliesthesia in participants when below their initial body weight. Therefore, it is hypothesized that the accelerated onset of negative alliesthesia observed in our obese participants after weight loss is suggestive of a lowered body weight set-point. Factors inherent to the weight loss diet studied here, such as mild energetic restriction, lowered palatability, and diet composition, may have played a role in this experimental outcome.
==== Body
Background
Energy balance is dependent upon a constant equilibrium between the energy intake and energy expenditure. The former is controlled by "appetite" through an intermediate phase termed "satiation" and its termination via "satiety" [1-4]. The role of satiety in controlling food intake begins once food has interacted with the receptors on the tongue and nose. The bolus of food follows the oropharyngeal duct to the stomach and duodenum, onward to the jejuno-ileum and colon to be expelled once digested. Along the transit, satiety signals arise from visceral afferent nerve fibers through the gastro-intestinal tract itself and the blood. At every step there is a cascade of neuromodulators that are postulated to participate, in part or entirely, in satiety (bombesin, CCK, PYY, ghrelin, leptin, insulin, glucocorticoids) [5-9]. Maladjustment in any component may favor increase daily intake and thus contribute to an upward drift in body weight and eventually obesity.
Some authors attribute the rising prevalence of obesity [10-13] simply to ever increasing caloric intake [14] and decreased physical activity while others have implicated over- or under-responses of satiating hormones or maladjusted satiety cues [15,16]. Regardless of the underlying etiology of obesity, this pathology is further complicated by such biobehavioral factors as dietary variety, portion size, snacking, cafeteria foods, increased palatability, and low cost of calorie dense foods [14].
In the 50's, Keys' experiment of semi starvation and refeeding in healthy normal-weight men [17] has provided data supporting the existence of autoregulatory feedback signals linking the state of depletion of fat stores to compensatory mechanisms operating via both food intake and regulatory thermogenesis [18-20]. In the following years, a number of authors have defended the hypothesis that body weight is regulated and that there exists a ponderostat [21-45]. According to that point of view, the major signal responsible for the overall stability of body weight is the set-point. That point of view entails that obesity results from a rise in body weight set-point, an analog signal that is the aim the system tends to achieve. If this were not the case, all defense mechanisms would operate to counter the rise of body weight.
Others have argued against the set-point theory and have instead substituted the term settling point [46,47]. According Davis & Wirtshafter [46] the absence of a set-point would then mean the absence of regulation per se. The same point of view against a set-point also existed in temperature regulation. For Webb [48,49] the system defends heat rather than temperature. Such a concept of regulatory biological systems is actually equal to an absence of any regulation and systems operating as simple steady states. With temperature as well as with body weight, the fact that responses (shivering, sweating, alliesthesia, thermogenesis, hoarding etc.) oppose changes to temperature and to body weight minimize the significance of the settling point hypothesis.
In previous studies, our laboratory has found that negative alliesthesia occurred when normal weight subjects were maintained on a ad libitum bland monotonous diet [50]. These subjects also experienced significant weight loss. In addition, when maintained on a bland diet normal weight subjects' lose weight without reporting a sensation of chronic hunger, a pattern understood as a lowering of the body weight set-point [50]. Inversely, when obese subjects lower their body weight while maintained on a caloric restriction diet it has been shown that satiety was decreased or suppressed [51]. The only difference between normal and obese patients is that the response takes place at a higher body weight in the latter [51].
The gustatory pleasure evoked by a sweet stimulus in subjects maintained under varying weight conditions reveals valuable information about the internal state of the biological system [32,51-53]. The decrease of initial pleasure after repeated ingestion of a sweet stimulus demonstrates an important physiological mechanism termed "negative alliesthesia".
The relationship between obese individuals' alliesthesia and their body weight has scarcely been studied in the clinical setting. The aim of the present clinical study was to compare the onset of negative alliesthesia in obese subjects before- and three months after following a weight loss diet.
Methods
Study participants
Ten female participants from the greater Quebec City Region were recruited from the Minçavi weight loss program. All participants were at least 18 yr of age and had stable body weight at the time of joining the program. Stable body weight was defined as not having gained or lost more than 2 kg in the past month. Patient demographics are outlined in Table 1. The protocols were described in detail to the participants, without any use of the word set-point, and without mentioning the aim of the study.
Table 1 Participant's demographics and descriptive data analyses. Obesity Class I, II & III according to World Health Organization Classification (World Health Organization, Geneva, 1998). Where Class I = BMI 30.0 to 34.9, Class II = BMI 35.0 to 39.9 and Class III BMI > 40.0. At first visit participants chose either Cantin® caramel candy 7-g or Ensure® Vanilla 7-ml as stimulus for determination of hedonic rating. The stimulus was retained for following visits. At first visit (Initial session) participants were asked to subjectively indicate the age at which they considered themselves to have become obese. Our study participants were all female, the age at perceived onset of obesity correlated with first childbearing in all participants.
Participants Mean (± S.E.) Demographics
Sex (n)
Female 10
Age 31.5 ± 3.2
Body Mass Index †
Initial BMI 36.8 ± 1,8
3-Month Diet BMI 34.9 ± 1,8
Loss (%) 5.2 ± 0.2
Initial Body Weight (kg) 94.7 ± 3.0
3-Month Body Weight (kg) 89.9 ± 3.0
Loss (%) 5.2 ± 0.2
Obesity Class (BMI)
I (30.0 to 34.9) 3
II (35.0 to 39.9) 4
III (>40.0) 3
Stimulus chosen
Liquid (Ensure® liquid diet) 3
Solid (Cantin® caramel candy) 7
Age of Perceived Onset of Important Body Weight ‡ 24 ± 2 yr
†: The body mass index is the weight in kilograms divided by the square of the height in meters (kg/m2).
‡: This variable is defined as that time when participants were aware that they were "heavier" than "average" individuals. This response was correlated with childbearing in all participants.
Participants were instructed to fast overnight and arrive at our laboratory early the following morning. At each visit, only one participant was taken at a time. Participants completed a general health questionnaire and body weights were recorded before initiating the experimental sessions.
Inclusion and exclusion criteria
Diabetics (Type I and II) were excluded from participating in the study. Smokers were also excluded because previous experimental results with the same hedonic method used here have shown that transient nicotine can lower the set-point for body weight [54], a result that was confirmed in rats [15].
Ethics, informed consent and compensation
The study protocol was approved by University Ethics Committee and each participant signed an informed consent before study initiation. Participants received forty dollars per visit as a compensation for their participation in the study. The monetary amount given is believed not to have influenced participant's ratings.
Minçavi® commercial weight loss program
Minçavi® (phonetically: "thin for life" in French), is a commercial weight loss program, based on Canada's Food Guide recommendations. Upon joining the program, participants, mostly women, receive a recipe book and are explained the importance of eating 3 meals/day and choosing foods from the four major food groups (grain products, fruits/vegetables, milk/dairy products, meat/meat alternate). The proposed recipes are based on inexpensive, readily available whole grain products, vegetables, fruits, lean meats, and low-fat dairy products. Group leaders teach participants how to prepare a variety of nutritious, well balanced and easy to cook meals. Group leaders are people who have lost excess body weight and have been maintaining a healthy body weight for at least two years by following the very program they promote. Participants are free to decide for themselves how much weight they want to lose. However, the group leaders establish the milestones in accordance to a recommended 5% loss in body weight.
Depending on their BMI, sex, age and physical activity, participants are assigned one of the four diet plans that range from 1400 to 2000 kcal/d (5852 to 8360 kJ/d) in the weight loss phase. During that phase, it is estimated that 50% of the energy is derived from carbohydrates, 25% from protein, and 25% from lipids. When participants reach the goal weight, they enter the "weight maintenance phase" and are taught to increase their caloric intake by 50 kcal /wk, in a minimum of 8 wk, to eventually reach a daily intake of 1800 to 2400 kcal/d (7524 to 10 032 kJ/day) depending on energy needs. During this second phase, diet composition changes slightly, with a decrease in protein and an increase in lipid content (carbohydrates = 50%, protein = 22%, lipids = 27%).
In groups of 50 to 100, participants are instructed on how to record everything they consume in a daily journal designed for that purpose and they are invited to share them with their group leader every week. Weigh-in sessions followed by a 30 to 45 min lecture on various topics (e.g., weight loss, nutrition, motivation) and recipe sampling; take place on a weekly basis. In addition, the commercial program provides support from a dietician through telephone line and Internet.
Sweet stimuli
At first visit, participants were allowed to choose between one of two sweet stimuli that would be presented to them over the experiment. Participants were explained that the stimuli would be ingested repeatedly over the session and retained for the next visit. Stimuli were either Cantin® caramels (7-g) to be chewed or Ensure® Vanilla (7-ml) to be drunk. Two different stimuli were offered, without being tasted, to offer an alternative to those participants who might not like caramel, or would be unable to chew due to dental prosthesis.
Hedonic ratings
Participants ingested one sweet stimulus, and after 15 s were asked to report their hedonic rating on a linear analogue scale. The entire stimulus was masticated (caramel) and swallowed, or drunk (Ensure®). Participants did not rinse after ingesting the stimuli. This procedure was repeated every 3 min until participants felt displeasure and decided to end the experimental session. Participants were instructed that they would have to decide by themselves when they chose to end the session out of displeasure (disgust for the stimulus: negative alliesthesia). Participants were instructed to indicate with a felt pen on a washable board the hedonicity aroused by the stimulus. They were to use an analogue scale with (0) in the middle for an indifferent sensation, a positive (+) for a pleasant sensation, and (-) for a displeasurable sensation. The distance (mm) from zero (0) would indicate the intensity of pleasure or displeasure. After the participant wrote her mark on the scale, the distance from zero (indifference) was measured, then the mark was erased. The repeated ratings were then plotted against time. Subjects performed the first alliesthesia test before starting the diet and the second one, three months after entering the program, while still on the weight loss diet.
Data analysis
The percent change in body weight at three months from initial session was calculated after the second session. This value was determined by dividing the difference in body weight between experimental sessions by initial session body weight. Value was expressed as a percent. Endpoints measured at each session were:
i. Body Mass Index (BMI). Determined by dividing participant's body weight (kg) / participant's height squared (m2).
ii. Time to reach zero rating for the hedonicity of taste sensation. This variable is defined as the time taken by participant to reach indifference after ingesting sweet stimuli every 3 min. This variable is interpreted as indicative of satiation.
iii. Time to abandon experimental session. This variable is interpreted as full satiety through negative alliesthesia since the participant was free to withdraw whenever she chose.
iv. Amplitude of hedonic rating. The highest (maximal) hedonic rating was the most pleasurable rating reported by participants after ingesting sweet stimulus. In opposition, the lowest (minimal) hedonic rating was the most displeasurable rating reported by participants after ingesting a sweet stimulus.
v. To compare rate of alliesthesia, the Kaplan Meier's method was used as for survival rate.
Results
After following the Minçavi® weight loss program for three months, mean group body weight was significantly lower (Initial session 94.7 +/- 3.0; 3 mo 89.9 +/- 3.0 kg; Student's paired t = 9.90; p < 0.0001; two tailed; Table 1). Maximal weight loss observed in our participants was 6.8 kg and minimal was 0.9 kg (Figure 1). Expressed as BMI, the weight loss program caused a significant decrease after three months (Initial session 36.8 +/- 1.8; 3 mo 34.9 +/- 1.8 kg/m2; Student's paired t = 10.4; p < 0.0001; two tailed). Body weight loss at 3 mo was 5.2 % ± 0.2 (Table 1)
Figure 1 Individual body weight loss in 10 obese participants at Initial session and 3 mo following a commercial regimen (Minçavi®). Mean group body weight loss showed a statistically significant decrease (Initial session 94.7 +/- 3.0; 3 mo 89.9 +/- 3.0 kg; Student's paired t = 9.90; p < 0.0001; two tailed). The range of body weight loss was from 0.9 kg to 6.8 kg for all participants who followed the Minçavi® diet for three months. One participant (ID. S-MAV-01) was invited to return for a follow-up visit after 6 mo on diet. Her body weight showed a continued decrease of 0.9 kg at 3 mo and 8.6 kg at 6 mo.
When patients repeatedly ingested sweet stimuli and reported their hedonic rating, the entire cohort of fasted participants started with reports of pleasurable sensation and gave positive hedonic rating. Over the course of the session the positive rating fell to zero or became negative, indicating negative alliesthesia. A representative alliesthesia kinetic from one participant before and while on the Minçavi® diet is shown in Figure 2. The mean time to reach zero rating, or indifference, revealed a statistically significant earlier delay after dieting (Initial session 21.9 +/- 3.8; 3 mo 16.2 +/- 2.4 min; Student's paired t = 2.48; p = 0.0351; two tailed, Figure 3). The solid stimulus was chosen by 7/10 of participants, over the liquid stimulus. Regardless of the solid or liquid chosen, participants still yielded identical kinetic profiles for alliesthesia before and after weight loss.
Figure 2 A representative satiation kinetic from one obese participant (Class I) at Initial session and 3 mo following a commercial regimen (Minçavi®). The curve shows that time to reach zero rating significantly decreased from 21 min at Initial session to 12 min at 3 mo on diet. The figure also shows that the overall time to abandon the experimental session, negative alliesthesia, was significantly decreased from 36 min at Initial session to 21 min at 3 mo on diet. From this satiation kinetic, the initial rating of pleasure (Initial session 125 mm) remained the same (3 mo 127 mm). The amplitude of rating (minimal and maximal hedonicity) was also similar from Initial session to 3 mo.
Figure 3 Mean (+/- SE) time to reach zero rating or indifference. The overall time to reach zero rating was significantly decreased after dieting (Initial session 21.9 +/- 3.8; 3 mo 16.2 +/- 2.4 min; Student's paired t = 2.48; p = 0.0351; two tailed).
In order to follow the time course of alliesthesia in our experimental groups, we created survival curve using the product limit method of Kaplan-Meier. This procedure would cancel the fact that not all participants followed the same time course and stayed for some duration. No event times thus were considered missing or censored. The median time to achieve negative alliesthesia was significantly increased after diet (Log-rank chi-square = 4.312, df = 1, p = 0.0378; Figure 4). Diet lowered the median time to achieve negative alliesthesia (Initial session 33 vs. 3 mo 24 min). There was no difference in maximal and minimal median hedonic rating (maximal Initial session +79.5 +/- 11.7 mm; 3 mo +94.5 +/- 9.9 mm; Wilcoxon = 14.5; p = 0.1934 or minimal Initial session -90.0 +/- 14.4 mm; 3 mo -106 +/- 11.1 mm Wilcoxon = 22.0; p = 0.6250). The absence of detecting a significant difference in this endpoint reveals that participant's hedonic rating for sweet stimulus was unchanged after diet.
Figure 4 Kaplan-Meier curve was created to determine the time to achieve an event: satiety. Diet showed a significant increase in the satiety rate at 3 mo vs. Initial session (Log-rank chi-square = 4.312, df = 1, p = 0.0378). The median time to achieve satiety was lower after diet (24 min vs. 33 min).
Discussion
Obese participants enrolled in the present study all lost weight by following the Minçavi® weight loss program. Although the overall group weight loss was statistically significant, the clinical relevance of such a loss for any individual participant (range 0.9 – 6.8 kg) cannot be ascertained. However, for obese people even moderate weight loss is thought to be beneficial if not physiologically at least psychologically[55,56]. All but one participant met the objective of a 5% decrease in BMI purported by the Minçavi® program (Figure 1).
Historical data from a controlled clinical trial in morbidly obese participants awaiting bariatric surgery and weight-matched un-operated controls utilizing the same experimental approach over a longer period revealed a similar alliesthesia kinetic [57]. Curve parameters from the present diet study suggest that negative alliesthesia as seen in our participants is comparable to those reported in morbidly obese patients (Historical controls initial session: 25.5 min, vs. Historical controls 3 month: 30 min).
The pleasure aroused by sweet stimuli faded earlier at the three month mark than at the initial session. The relationship of pleasure with intake is obvious as a stimulus that turns toward unpleasantness should be rejected; such negative alliesthesia is suggestive of a satiation process. Earlier onset of negative alliesthesia was observed in each participant who had lost weight. A weak negative correlation was observed when we tested the relationship between amount of weight loss from diet to time to achieve negative alliesthesia (Pearson r = -0.53). Correlation data did however suggest the use of greater patients in future studies utilizing the method. At first visit, all participants yielded a response kinetic, that could be interpreted as three distinct phases, namely; appetite, satiation, and satiety. Because participants all arrived at our laboratory after an overnight fast, their initial rating of hedonicity was positive at every session. Each participant transitioned through to what could be referred to as a "satiation phase", indicative of a zero or indifferent hedonicity rating. The final phase, suggestive of satiety, was the abandonment due to significant displeasure and disgust for the sweet stimulus. Thus, our obese participants displayed a negative alliesthesia pattern similar to that observed in normal weight subjects. It is likely that changes in alliesthesia observed here were linked to that specific diet and that upon returning to their previous dietary habits, subjects would probably return to their baseline response.
Previous experiments have focused mainly on shift in taste preferences in obese and lean individuals after dieting [53,58]. These investigations yielded equivocal results, and assessed the changes in taste correlated with lowered body weight. Our participants did not modify their initial ratings although their weights were lowered from dieting. Such a result would not support a changed taste preference related to body weight in fasted subjects. Cummings, Weigle, Frayo, Breen, Ma, Dellinger, & Purnell, [59] studied the effects of diet-induced weight loss on plasma ghrelin levels. Their subjects showed a significant 17% decrease in BMI after six months. Although the population studied was similar to ours, their experimental approach to weight loss was more drastic than the Minçavi® diet. Subjects from the Cummings study were initially on a three-month liquid diet (1000 kcal/d) and transitioned to a solid diet for the remainder of the study.
Conclusion
An earlier onset of negative alliesthesia was obvious at three months when participants had lost weight and were still on the Minçavi® diet. In the light of previous work in animals and in humans cited earlier, these findings could be interpreted has a lowering of body weight set-point. Dietary factors may have played a role in this phenomenon. Since Minçavi® promotes a diet that is lower in fat and in added sugar, it is likely that the participants have found it to be less palatable than their usual diet. Earlier work has shown that a decrease in palatability could lower one's body weight set-point [50]. Some studies also suggest that diets that are relatively high in protein [60-62] and that have a low glycemic index [63-67] such as the Minçavi® diet, may promote satiety and weight loss. It is possible such factors, when present in the diet, contribute to lower one's set-point.
Maintaining a lowered set-point, by consuming a sensible diet that promotes satiety and gradual weight loss, may be the key for long-term success, as the body strives to maintain a body weight close to that set-point by reducing food intake and enhancing energy expenditure. Further studies are needed to better understand alliesthesia in obese individuals and its link to body weight set-point.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
PF carried out the experimental alliesthesia sessions, and data analyses. MC & CG conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Figure 5 A Kaplan-Meier curve was created using historical control data obtained from morbidly obese patients (BMI > 50) awaiting bariatric surgery. Subjects did not receive any diet intervention. Participants all received the same test for negative alliesthesia described in the methods section. Both groups showed a similar satiety kinetic for sweet stimulus (Log-rank chi-square = 0.045, df = 1, p = 0.8311, NS). The median time to achieve satiety in both groups were comparable (Initial session 25.5 min vs. 3 month 30 min). These results suggest the test is reproducible in a clinical setting for patients.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
This work was supported by the Conseil de la Recherche en Sciences Naturelles et en Génie (NSERC) Canada. The authors wish to thank Ms. Danielle Dubois, Dtp. and Minçavi® group leaders for their helpful assistance.
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BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-5-341622130010.1186/1471-244X-5-34Case ReportChronic koro-like symptoms – two case reports Kar Nilamadhab [email protected] Wolverhampton Primary Care Trust, Corner House Resource Centre, 300, Dunstall Road, Wolverhampton, WV6 0NZ, United Kingdom2005 12 10 2005 5 34 34 2 6 2005 12 10 2005 Copyright © 2005 Kar; licensee BioMed Central Ltd.2005Kar; 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
Koro is a culture bound syndrome, which has been reported usually from Asian countries. It has been described as an acute, brief lasting illness, which often occurs in epidemics. There is no description in literature of a chronic form of this syndrome.
Case presentation
Two sporadic cases with koro-like symptoms from East India are presented where the illness had a chronic course with durations spanning more than ten years. In contrast to acute, good prognosis, psycho-education responsive form that is usually seen in epidemics; the chronic form, appeared to be associated with greater morbidity and poorer response to interventions.
Conclusion
There is a possibility of a chronic form of koro syndrome.
==== Body
Background
Koro is considered to be a cultural-bound syndrome, characterised by the belief of retraction of the genitals into the abdomen and is associated with anxiety symptoms [1-3]. It has been commonly reported from India and other Southern Asian countries. It often manifests as epidemics [4,5] though sporadic cases are also noted [6]. The syndrome is described to be short lasting. There is no description in literature of a chronic form of this syndrome. Two patients with koro-like symptoms from Orissa in East India are presented here, whose symptoms continued for prolonged duration of more than ten years, highlighting the possibility of a chronic form of this syndrome.
Case presentation
Case: 1
A 30-year-old unmarried, male, clerk presented with the persistent fear that his penis was shrinking gradually for last 12 years. He stated that due to the shrinkage he remained weak, anxious and sleepless. He continuously brooded over it. He got panicky whenever he saw his penis, as he felt that it is shrinking back, and remained perturbed for days together after that. Due to this he consciously avoided to see or contact his penis, even in bathroom. He explained that it was due to his nocturnal emissions and a couple of masturbatory acts in middle adolescence. He was extremely religious and attended religious institutions (like that of Satsang and Brahmakumaris). He considered penis as sacred and its gradual shrinkage as an omen of his ultimate destruction and death. He believed that this was due to sinful (anti-religious) incidences of nocturnal emission and masturbatory acts. He had decided not to marry in order to avoid any possible retraction. He remained preoccupied with these thoughts, avoided company, and interaction with females. He requested for medicines that would stop further retraction.
He had been given anxiolytics, sedatives, antidepressants and anti-psychotics at various point of time by many practitioners and had been counselled. His symptoms persisted with intermittent exacerbations without much relief.
Unlike obsession his thoughts were never resisted. His belief had influenced his behaviour and way of life, was shakeable, and appeared more like overvalued ideas. His mood state was predominantly anxious with occasional panic attacks associated with somatic equivalents. He did not have any other major psychiatric syndrome.
Case: 2
A 41-year-old unmarried, unemployed male from a business family, presented with the complaints of gradual retraction of penis and scrotum into the abdomen. He had frequent panic attacks feeling that the end had come. The symptoms had persisted more than 15 years with a waxing and waning course. During exacerbations he spent most of his time measuring the penis by a scale and pulling it in order to bring it out of abdomen. He tied a string around it and attached it to a hook above to prevent its shrinkage during night. There was a history of excessive sexual indulgence in the early twenties with prostitutes and he had suffered repeatedly from sexually transmitted diseases characterised by discharges (probably gonorrhoea) and ulcers (probably syphilis). He was adequately treated for that. He did not have remorse or guilt over those happenings. He continued visiting prostitutes occasionally and described no inadequacy in sex. He engaged his partner in fellatio in order to bring the penis out. He refused marriage, as he feared that his life was at stake due to this shrinkage as he had had many experiences of nearing death due to that. He did not have regular work and was mostly dependent on the family.
He had been treated with anxiolytics and occasionally with antipsychotic medications with minimal benefit. He had received psycho-education. He frequently engaged in doctor shopping and tried to see doctors before the appointment day expressing concern over his symptoms. He often used medicines from other fields of medicine (homeopathy, Ayurveda, unani) to prevent the retraction.
Conclusion
The presenting symptoms of these two patients, characterised by excessive anxiety and belief of shrinkage of genitalia, are similar to those found in koro patients. Age of onset of the koro symptoms in the index patients are comparable to that (20–40 years) observed in the epidemics [5]. Factors such as extramarital intercourse, venereal disease and scrotal filaria were found to be significantly commoner in koro patients [7]. One of the index patients had veneral diseases and had contact with prostitutes. Significant premorbid or sexual psychopathologies were absent in most cases in epidemics [5], so also in the index cases. The symptoms of these patients were not secondary to any other major psychiatric disorder.
The findings in these index cases are supported by the view that individual koro patients exhibit a symptomatology indicative of major psychiatric conditions (i.e. psychosis or affective disorder), and appear unrelated to collective episodes which involve social, cultural, cognitive and physiological factors in the diffusion of koro-related beliefs [6]. A sporadic case of koro was reported to be associated with psychotic depression [8].
However compared to koro patients reported in literature, the index cases had few differences considering duration of illness, continued presence of significant psychiatric morbidity characterised by koro-like symptoms which affected their life significantly, and non-response to interventions. Multiple episodes have been described in some patients in koro epidemics but with only minor residual symptoms [5], however in the index patients the symptoms have been chronic. Majority of the individuals affected in koro epidemic in India were reported from the lower socio-economic strata and were poorly educated, which were not the case with the index patients. Epidemics of koro were known to be contained or benefited by mass education programmes [9]. In the index patients the symptoms of koro did not respond in spite of various interventions, which included individual sessions of psycho-education.
While the issues concerning phenomenology, diagnosis and nosology of koro are still being discussed [5,6,10], it is apparent that koro, which presents both sporadically and in epidemics as an acute anxiety state, may also have a chronic form. In contrast to acute, good prognosis, psycho-education responsive form that is usually seen in epidemics; the chronic form, appears to be sporadic with greater morbidity, and with poorer response to interventions.
The underlying dynamics resulting in chronicity of symptoms of koro are not clear. It is possible, as observed before [11], that many psychiatric symptoms that share a similar 'surface grammar' differ in their 'deep grammar' or structure. Though the index cases suggest that koro can be chronic, with a poorer prognosis; further information regarding this presentation and factors behind it are needed.
Competing interests
The author(s) declares that he has no competing interests.
Authors' contributions
NK examined the patients, treated, followed up, conceptualised, did literature survey and wrote the paper.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Written consent was obtained from the patients for publication of the patients' details. Department of Psychiatry, Mental Health Institute, Cuttack, India and Quality of Life Research and Development Foundation supported the study in part.
==== Refs
Kar N Kar N, Kar GC Cultural variations in sexual practices Comprehensive Textbook of Sexual Medicine 2005 New Delhi: Jaypee 121 136
Chowdhury AN Dysmorphic penis image perception: the root of koro vulnerability. A longitudinal study Acta Psychiatr Scand 1989 80 518 20 2688365
Chowdhury AN Glans penis perception of koro patients Acta Psychiatr Scand 1993 87 355 7 8517176
Nandi DN Banerjee G Saha H Boral GC Epidemic koro in West Bengal, India Int J Soc Psychiatry 1983 29 265 8 6642919
Sachdev PS Koro epidemic in North-East India Aust N Z J Psychiatry 1985 19 433 8 3869009
Bartholomew RE The social psychology of 'epidemic' koro Int J Soc Psychiatry 1994 40 46 60 8005778
Chowdhury AN Biomedical potential for symptom choice in koro Int J Soc Psychiatry 1989 35 329 32 2628376
Westermeyer J A case of koro in a refugee family: association with depression and folie a deux J Clin Psychiatry 1989 50 181 3 2715141
Dutta D Koro epidemic in Assam Br J Psychiatry 1983 143 309 10 6626846
Cheng ST A critical review of Chinese koro Cult Med Psychiatry 1996 20 67 82 8740959 10.1007/BF00118751
Rejon Altable C Rodriguez Urrutia A Koro-misidentification syndrome in schizophrenia? A plea for clinical psychopathology Psychopathology 2004 37 249 52 15353891 10.1159/000080721
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BMC Psychiatry. 2005 Oct 12; 5:34
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BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-5-371622568910.1186/1471-244X-5-37Research ArticleA survey of participants in two internet support groups for people with hair-pulling Bruwer Belinda R [email protected] Dan J [email protected] MRC Unit on Anxiety Disorders, University of Stellenbosch, Cape Town, South Africa2 Department of Psychiatry, University of Cape Town, Cape Town, South Africa2005 14 10 2005 5 37 37 18 3 2005 14 10 2005 Copyright © 2005 Bruwer and Stein; licensee BioMed Central Ltd.2005Bruwer and Stein; 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 substantial number of patients suffering from psychological problems or psychiatric disorders have turned to internet support groups for help. This paper reports on the perceived effectiveness of trichotillomania (TTM) internet support groups for people suffering from hair-pulling.
Methods
A questionnaire was sent via e-mail to all subscribers of two mailing lists devoted to TTM, each of which takes a somewhat different approach to the condition. The questionnaire addressed the possible benefits and problems associated with belonging to a TTM virtual support group.
Results
Subscribers had similar demographic features as clinical samples of trichotillomania patients. Subscribers to both internet lists found them helpful in terms of feeling supported and in obtaining information. The different approaches to TTM on the two lists were associated with differences in treatments attempted by participants.
Conclusion
Internet support groups can potentially contribute to increasing awareness about and knowledge of psychiatric disorders such as TTM, as well as to their management. Nevertheless, additional effort is required to ensure that subscribers are able to make informed, evidence-based decisions.
==== Body
Background
A substantial number of people suffering from psychological problems or psychiatric disorders have turned to internet based groups for information and support. Such groups have potential strengths, as well as possible limitations [1]. Previous work has, for example, indicated that virtual groups can be experienced as supportive, but there is also evidence that the quality of medical information on internet websites has limitations [2,3].
In working with people with obsessive-compulsive and spectrum disorders, we regularly encourage patients to seek information from standard websites such as those hosted by the Obsessive-Compulsive Foundation and the Trichotillomania Learning Center. We have previously reported that people with obsessive-compulsive disorder (OCD) can experience virtual OCD groups as both supportive and informative [3].
Although trichotillomania is increasingly recognized to be a prevalent disorder, there are relatively few resources available to those who suffer from this condition [4]. We were interested in the experience that people with trichotillomania (TTM) have of virtual TTM groups. We were intrigued to note that two internet mailing lists (the TTM Telemailer group and the Trichees Yahoogroup) appeared to have somewhat different approaches to this condition, and therefore chose to survey participants in both. The list-owner of the TTM Telemailer has formulated the "John Kender Diet", while the Trichees Yahoogroup seemed to focus on a range of other treatments.
The "John Kender Diet" is based on a hypothesis that people inclined to TTM have a natural biochemistry irreversibly inclined towards certain allergic reactions. The hypothesis is that people with TTM are allergic to yeast (Malessizia sp.) found normally in the skin and gut, and reacts with itching and irritation followed by hair-pulling. Thus, there are "bad" foods and "good" foods and every TTM sufferer should determine his or her optimal diet. "Bad" foods include sugar, egg yolk, peanuts, legumes, butter, shellfish, organ meats, caffeine, nutrasweet, raisins, grapes, watermelon and seeds. "Good" foods include allicins (onions, garlic), tannins, yogurt and tropical fruits [5]. There are no published studies of the efficacy of this diet in trichotillomania.
On the Trichees Yahoogroup listserv, a range of other treatments are discussed, including a "Fish Oil Diet". While essential fatty acids found in fish oil have recently been found effective in controlled trials of a number of different psychiatric disorders [6,7], there are no studies of their efficacy in trichotillomania. In contrast, a number of different medications and psychotherapy interventions have been studied for trichotillomania in randomized controlled trials. Although there are few treatment guidelines for this condition, there is growing evidence for the efficacy of habit reversal, and some patients may benefit from serotonergic medications [4].
Methods
Two mailing internet lists known to the authors were chosen for the study. The TTM Telemailer was started in March 1995 by John R Kender and had 470 members at the time of the study. The Trichees Yahoogroup was started in October 1998 and had 540 members at the time of the study. Although list-owners do not formally moderate the discussions, they do participate in the discussion, and so are able to influence views of participants.
The project was approved by the Institutional Review Board of the University of Stellenbosch Medical School. A questionnaire was drafted to obtain basic demographic details of subscribers to the mailing lists, and to elicit the perceived benefits and problems of participating in the lists. The questionnaire was informed by a previous survey of participants in an OCD mailing list [3]. With the consent of the list-owners, the questionnaire was sent twice over a three-month period to all list-serve members. Subscribers voluntarily chose whether or not to respond to the questionnaires via e-mail.
A total of 85 questionnaires were received, 58 from the TTM Telemailer (return rate 12.34%), and 27 from the Trichees Yahoogroup (return rate 5%). Four questionnaires were excluded due to incomplete information. Data were tabulated using Microsoft Excel and compared with t-tests or chi-square as appropriate (Table 1&2). At the end of the questionnaire, a section was left for participants to provide any additional comments; the authors read all comments and a representative selection of these is included in the appendix.
Table 1 Data from two internet support groups (percentages)
Telemailer Trichees chi-square p-values
Gender distribution
Females 91.2 100 0.985 ns
Males 8.8 0
Interest in TTM
I have TTM 85.9 87.5 3.647 ns
My child has TTM 8.7 8.3
Significant other has TTM 1.8 0
My friend has TTM 1.8 0
My child and I have TTM 1.8 0
Significant other and I have TTM 0 4.2
Mental health professional 0 0
Diagnosis and treatment of TTM
Professionally diagnosed 61.4 70.8 3.635 ns
Not professionally diagnosed 38.6 29.2
Treated previously, not presently 38.6 41.7
Treated previously and presently 22.8 37.5
Never been professionally treated 38.6 20.8
Means of discovering support group
Surfing the internet 68.5 91.7 5.402 ns
Support group members 7 0
Psychologist 3.5 0
Allied mental health professional 1.8 0
Friend 1.8 0
Family member/ significant other 1.8 0
Additional resources 15.6 8.3
Length of time belonging to group
More than 12 months 63.1 8.3 29.951 <0.001
6 – 12 months 12.3 20.8
3 – 6 months 8.8 33.3
1 – 3 months 12.3 8.3
1 week to 1 month 3.5 12.5
Less than 1 week 0 16.7
Frequency read and/or respond to messages
More than once a day 8.8 8.3 4.676 ns
Once a day 26.3 41.7
Less than once a day 15.8 20.8
Weekly 19.3 25
Monthly 14 0
Time spend making use of support group
Less than one hour per week 45.6 16.7 6.182 ns
1 – 2 hours per week 31.6 50
2 – 5 hours per week 15.8 25
More than 5 hours per week 7 8.3
Seeking professional help
Support group was very helpful 10.5 15.5 6.548 ns
Only somewhat helpful 26.3 16.7
Somewhat helpful 8.8 8.3
Unhelpful 26.3 8.3
Already sought professional help 28.1 54.2
The John Kender Diet
Attempted, with good results 33.3 0 18.195 <0.001
Attempted, without good results 19.3 12.5
Did not attempt the diet 45.6 66.7
Never heard about the diet 1.8 20.8
The Fish Oil Diet
Attempted, with good results 0 10 4.600 ns
Attempted, without good results 9.1 40
Did not attempt the diet 81.8 40
Never heard about the diet 9.1 10
Mislead by information from support group
Yes 12.3 4.2 2.480 ns
Maybe 8.8 4.2
I don't think so 54.4 70.8
No 24.6 20.8
Helpfulness to family and friends
Support group was helpful 40.4 33.3 2.004 ns
Support group was unhelpful 19.3 25
Did not share TTM 31.6 37.5
Table 2 Perceived helpfulness: Data from two internet support groups (percentages)
Telemailer Trichees chi-square p -values
Learning about symptoms of TTM
Helpful 89.5 95.8 0.247 ns
Unhelpful 10.5 4.2
Learning about causes of TTM
Helpful 77.2 79.2 20.009 ns
Unhelpful 22.8 20.8
Learning about treatment of TTM
Helpful 82.4 91.6 0.523 ns
Unhelpful 17.6 8.4
Tips to decrease hair-pulling
Helpful 87.7 79.2 2.977 ns
Unhelpful 8.8 20.8
Not applicable (I don't have TTM) 3.5 0
Feeling supported
Helpful 87.7 87.5 2.612 ns
Unhelpful 12.3 8.3
Not applicable 0 4.2
Realizing that you're not alone with TTM
Helpful 98.2 91.6 2.849 ns
Unhelpful 1.8 4.2
Not applicable 0 4.2
Giving a name to the hair-pulling problem
Helpful 77.2 75 0.409 ns
Unhelpful 21.1 20.8
Not applicable 1.7 4.2
Learning about cognitive- behavioral therapy
Helpful 57.9 58.3 0.044 ns
Unhelpful 42.1 41.7
Learning about John Kender Diet
Helpful 94.8 50 19.534 <0.001
Unhelpful 5.2 50
Learning about Fish Oil Diet
Helpful 90.9 70 0.439 ns
Unhelpful 9.1 30
Results
Membership and participation
Both groups had similar demographic features: most members were females with hair-pulling who had discovered the support group by surfing the internet, the mean age of subscribers was 32.4 (range 16 – 54), and mean age of onset of TTM was 12.5 (0.5 – 34). A large number of participants (62.5%) were not receiving professional help.
There was a significant difference between the groups in how long people had been members (χ2 = 29.951, p < 0.001); more than 60% of respondents had been members of the TTM Telemailer for longer than 12 months, while more than half of the Trichees Yahoogroup respondents had subscribed within the past 3 to 12 months. Conversely, although most respondents read and or responded to messages posted on the support group once a day, participants in the Trichees Yahoogroup spent significantly more time logged on (Table 1).
Perceived helpfulness and informativeness
Participants in both lists found these helpful in terms of feeling supported, learning about the symptoms, causes and treatments for TTM, as well as tips on ways to decrease hair-pulling (see Appendix for a range of participant views). The majority of subscribers to both groups believe that they had not been misled by information provided on the support group. More than a third found that the support group had been of help to family and friends. In contrast, a third of participants had not discussed their TTM with family or friends (Table 1).
Participants in both lists found them somewhat helpful in terms of learning about cognitive behavioral therapy and the Fish Oil Diet. The TTM Telemailer was experienced as very helpful in learning about the John Kender Diet, and more participants in this group had tried this diet and found it helpful (Table 1 and 2).
Discussion
The main findings of this study were that 1) subscribers to both internet lists found them helpful in terms of feeling supported and in obtaining information, and 2) the different approaches to TTM on the two lists were associated with differences in treatments attempted by participants.
Respondents noted in their comments that a great deal of the information provided was useful, as it came from people dealing with the same problem. They also found it useful to interact frequently with someone who had experienced their problem. The internet support groups were helpful in terms of learning about the symptoms and treatment of TTM, tips on how to decrease hair pulling, and in giving a name to their hair-pulling problem. Only a small number of members reported in their comments that their symptoms increased when reading the messages posted on the support group.
Consistent with this positive experience the majority of subscribers were members for more than a year, read and/or responded to messages posted by members once a day or weekly, and spent approximately one to two hours per week making use of the support group. It is notable that many sufferers had not sought professional help, had discontinued treatment, or had not discussed symptoms with family or friends. Indeed, subjects reported having received inadequate information and advice from professionals.
Differences in the apparent content of the two support groups did seem to impact on attempted treatments. The TTM Telemailer subscribers found the support group very helpful in terms of learning about the John Kender Diet, while both support group subscribers found the groups somewhat helpful in learning about the Fish Oil Diet. This may indicate the influence the list-owner has regarding information provided and topics under discussion, as well as attitudes of members towards certain forms of treatments.
Most subscribers had not followed either of these diets or had not achieved good results with them. Nevertheless, people did not feel misled by information, and they continued to participate in the groups. Indeed, members of TTM Telemailer had been subscribers for a longer period than those to Trichees Yahoogroup, suggesting that the former had a longer lasting influence, or alternatively, that people interested in particular nutrition-based interventions were more likely to find this list and to remain subscribers.
Perceived problems related to the support groups included the tediousness of reading and/or responding to large numbers of messages, the abrupt loss of discussion threads when a subscriber suddenly quit the mailing list, the deviation of the discussion away from problems related to TTM, or disagreements with the other subscribers or the list owner's viewpoint. In both groups, English is the language used and this might be difficult for people with English as their second or third language. Another problem mentioned was that members can have different expectations from the support groups, i.e. as a group providing support, as a forum to discuss issues, or as a place to gain information about TTM and recent research.
There are several limitations to the data presented here. First, the sample was a convenience sample; it represents participants who chose not only to continue to participate in the list, but also to answer the questionnaire. Indeed, there was a low response rate, although this might be an underestimation given that not all of the participants in the groups were in fact active members at the time of the study. Second, the survey was not comprised of standardized rating scales, and the extent to which self-report data collected is reliable or valid is unknown. For example, while it seems safe to assume that many participants in the groups had hair pulling, we are unable to confirm that a diagnosis of trichotillomania was in fact present. Possible bias in the survey is compounded by the cross-sectional rather than longitudinal design; there may well be a disjunction between perceived increase or decrease in hair pulling during participation in the survey, and actual changes in symptoms. Participants in the groups have some level of computer literacy, knowledge about how and where to search for the internet support groups, as well as access to a computer and the internet; and the data cannot necessarily be extrapolated to other groups of people with trichotillomania. Nevertheless, membership of the support groups was similar to that described in the clinical literature on hair pulling, i.e. mostly young females [8].
Conclusion
In conclusion, although there are important limitations to the study, the data here suggest that there may well be a place for internet virtual groups in the management of some people with trichotillomania. Internet lists may also be of value to family, friends and 'significant others' of people with TTM. Mental health professionals may find that participation in internet support groups provides insight into various conditions. Although participants do not feel misled, there is not a focus on evidence-based advice in these TTM lists, and patients should be advised in detail about this issue before being referred to an internet support group. Conversely, subscribers to internet lists should be educated about the potential value of professional evaluation and evidence-based treatment. A systematic evaluation of the effectiveness of internet support groups in reducing hair pulling and distress, and increasing quality of life, is needed.
Appendix: Comments by subscribers
"This group has helped me to be pull-free for more than 1 month – that is the longest I've ever been pull-free, before this I couldn't even go for one day without pulling."
"The amount of research and knowledge of the participants, as well as practical experiences, has been invaluable in understanding and helping me with trich. The support is amazing!!"
"The only problem is that it's so time consuming to read all the messages and then respond to all the applicable ones..."
"Sometimes I think that talking about it or reading about TTM a lot actually makes me want to pull more..."
"I am involved with someone with TTM... it helps for me to understand what they are all going through and learn from their experiences."
"It is very useful for information on recent research... I live in Australia, and there are very few support groups here, so it's good to be in contact with people all over the world..."
"Over the past seven years, I have found helping the list members very satisfying. I've met electronically, and then sometimes in person, some great people, too."
"The group support has also helped me to confront my feelings about this disorder... and use some of the knowledge I've gained to enlighten family members... and talk about it with friends."
"With partial use of the Diet and a few dealing mechanisms I have been able to stop pulling for 5 months now. I thank this mailer."
"It helps me to understand it better so then it's easier to explain to my friends. Also gave me the confidence to go to a doctor."
"As of today I am 123 days absolutely pull free from scalp hair and 7–8 days pull free from my eyelashes and I have stopped pulling my eyebrows. I might not have come so far if it weren't for this support group."
"The support groups that I belong to have helped me to realize that professional help is VERY limited. The best help you can get is from each other and people like John Kender..."
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DS contributed to the concept and design of the study. BB carried out the study, conducted an initial analysis and drafted the manuscript. DS revised the analysis and manuscript. Both authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
The authors wish to acknowledge the trichotillomania list owners, John R Kender and Julie Jaarsma-Holdom for their support and for permission to do this study, and would like to thank the subscribers to these lists for participating in this study and for sharing their experiences.
==== Refs
Edejer TT Disseminating health information in developing countries: the role of the internet BMJ 2000 321 797 800 11009519 10.1136/bmj.321.7264.797
Kunst H Groot D Latthe PM Latthe M Khan KS Accuracy of information on apparently credible websites: survey of five common health topics BMJ 2000 7337 581 582
Stein DJ Psychiatry on the internet: Survey of an OCD mailing list Psychiatric Bulletin 1997 21 95 98
O' Sullivan RL Keuthen NJ Christenson GA Mansueto CS Stein DJ Swedo SE Trichotillomania: Behavioral symptom or clinical syndrome? American Journal of Psychiatry 1997 10 1442 1449
Amanda's trichotillomania guide
Ranjekar PK Hinge A Hegde MV Ghate M Kale A Sitasawad S Wagh UV Debsikdar VB Mahadik SP Decreased antioxidant enzymes and membrane essential polyunsaturated fatty acids in schizophrenic and bipolar mood disorder patients Psychiatry Res 2003 121 109 22 14656446 10.1016/S0165-1781(03)00220-8
Arvindakshan M Ghate M Ranjekar PK Evans DR Mahadik SP Supplementation with a combination of omega-3 fatty acids and antioxidants (vitamin E and C) improves outcome of schizophrenia Schizophr Res 2003 62 195 204 12837515 10.1016/S0920-9964(02)00284-0
Ko SM Under-diagnosed psychiatric syndrome I: Trichotillomania Ann Acad Med Singapore 1999 2 279 81 10497682
Stemberger RM Thomas AM Mansueto CS Carter JG Personal toll of trichotillomania: Behavioral and interpersonal sequelae J Anxiety Disord 2000 1 97 104 10770238 10.1016/S0887-6185(99)00028-6
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BMC Psychiatry. 2005 Oct 14; 5:37
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BMC SurgBMC Surgery1471-2482BioMed Central London 1471-2482-5-201622130210.1186/1471-2482-5-20Technical AdvanceLaparoscopic repair of high rectovaginal fistula: Is it technically feasible? Kumaran Saravanan S [email protected] Chinnusamy [email protected] Alfie J [email protected] Ramakrishnan [email protected] Murugayyan [email protected] Department of Gastrointestinal Surgery, Gem Hospital, Coimbatore, India2 Department of Advanced Laparoscopic and Gastrointestinal Surgery, Gem Hospital, Coimbatore, India3 Department of Advanced Laparoscopic Surgery, Gem Hospital, Coimbatore, India4 Department of Endogynec Surgery, Gem Hospital, Coimbatore, India2005 12 10 2005 5 20 20 30 5 2005 12 10 2005 Copyright © 2005 Kumaran et al; licensee BioMed Central Ltd.2005Kumaran 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
Rectovaginal fistula (RVF) is an epithelium-lined communication between the rectum and vagina. Most RVFs are acquired, the most common cause being obstetric trauma. Most of the high RVFs are repaired by conventional open surgery. Laparoscopic repair of RVF is rare and so far only one report is available in the literature.
Methods
We present a case of high RVF repaired by laparoscopy. 56-year-old female who had a high RVF following laparoscopic assisted vaginal hysterectomy was successfully operated laparoscopically. Here we describe the operative technique and briefly review the literature.
Results
The postoperative period of the patient was uneventful and after a follow up of 6 months no recurrence was found.
Conclusion
Laparoscopic repair of high RVF is feasible in selected patients but would require proper identification of tissue planes and good laparoscopic suturing technique.
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Background
Rectovaginal fistula (RVF) is an epithelium-lined communication between the rectum and vagina. Most RVFs are acquired, although congenital abnormalities do exist. Acquired RVFs can occur due to various causes. The most common acquired cause is obstetric. RVFs can be classified into low and high variety. Although perineal approach is the preferred one for low variety, high fistulas are best approached transabdominally. High RVFs are most commonly approached by a conventional open technique. We describe laparoscopic technique of repairing a case of high RVF. So far there is only one report in literature mentioning primary closure of RVF by laparoscopic technique [1].
Methods
We are describing our technique of laparoscopic management of high RVF in a 56 year old lady who developed RVF following laparoscopic assisted vaginal hysterectomy. She was admitted with complaints of passing flatus and feces per vagina for 11 months. Laparoscopic assisted vaginal hysterectomy with bilateral salphingo-oopherectomy was done 18 months back for leiomyoma uterus. She started passing flatus and feces per vagina 4 weeks after the surgery. Clinically the patient was obese (BMI-32). Patient had no features of sphincter disturbance. Per vaginal examination revealed a small area of induration high in the vault. The vault was healthy. Routine blood investigations were normal. USG abdomen was normal. Instillation of methylene blue into the rectum with a vaginal tampon confirmed the fistula. After thorough bowel preparation, DVT prophylaxis and prophylactic antibiotics patient was taken up for surgery.
The patient was placed in modified lithotomy position. The abdomen was prepared and draped. Cleansing of the vaginal lumen with an antiseptic solution (Povidone-iodine) was done. A Foley catheter was inserted into the urinary bladder. The surgery was performed under general anesthesia. Team position is diagrammatically represented in Figure 1A. Pneumoperitoneum was created with Veress needle. 10 mm ports were placed at the umbilicus for camera; in the right lower midclavicular line for right working hand and in the right upper midclavicular line for rectal retraction. 5 mm ports were placed in the left lower midclavicular line for left working hand and in the left iliac fossa for bladder retraction (Figure 2A).
Figure 1 Schematic picture of the surgical procedure. A – Team positions. B – Picture showing RVF. C – RVF Dissected. D – Rectal and vaginal defects sutured separately.
Figure 2 Port Positions and Intraoperative pictures. A – Port positions. B – Fistulous tract exposed. C – Fistulous opening on the vaginal side. D – Completion of repair.
There were adhesions between the rectum and the bladder peritoneum which was dissected using scissors. On retraction of the rectum, the dense fibrosis in the region of the fistula was identified which was divided with sharp scissors (Figure 2B). There was a 5 mm fistula between the vault of the vagina and the middle third rectum (Figure 2C). The fistulous tract was opened and thorough visualization of the fistula on the rectal and vaginal side was done. Resection of the fibrous fistulous tract was done. Mobilization of the rectum 2 cms distal to the fistula was also done.
The rent on the rectal side was closed in 2 layers with 2-0 Vicryl by intracorporeal suturing. The vaginal side of the rent was also approximated with 2-0 Vicryl in single continuous layer (Figure 2D). An omental patch was placed and sutured in situ between the repaired rectum and vagina. Thorough wash was given. Drain was placed in the pelvis through the right flank. Proximal colostomy was not performed.
Results
The postoperative period was uneventful. The patient was started on oral fluids on the 3rd postoperative day. The drain was removed on 5th postoperative day and discharged on the same day. On follow up (6 Months), the patient had no specific complaints. There was no recurrence which was confirmed by performing vaginography.
Discussion
Most RVFs are acquired. Acquired fistulas can occur due to various causes. These include trauma (including operative, obstetric, and traumatic injuries), infection, inflammatory bowel disease (IBD), carcinoma and radiation. The vaginal passage of gas and stool can cause physical symptoms due to inflammation and irritation. Patients may also suffer from significant psychosocial and sexual dysfunction.
In general, obstetric trauma is the most common cause of RVFs, occurring in up to 88% of published series [2-5]. But this is not the case at some institutions due to their referral pattern. For example, in a series from the Mayo Clinic, only 11% of their rectovaginal fistulas were secondary to obstetric injuries whereas 24% were due to inflammatory bowel disease [6]. Surgical trauma is another etiology for a RVF. Both anorectal and vaginal operations present a risk. Abdominal surgeries like hysterectomies, low anterior resections and ileo-anal anastomosis also carries the risk of developing a RVF. The fistula may result from a direct injury during the surgery or from infection or anastomotic leak postoperatively.
RVFs can be classified into low and high varieties. Low RVF is between the lower third of the rectum and the lower half of the vagina. A high fistula is between the middle third of the rectum and the posterior vaginal fornix. Small-sized fistulas are less than 0.5 cm in diameter, medium-sized fistulas are 0.5–2.5 cms, and large-sized fistulas exceed 2.5 cms.
A thorough history and physical examination are necessary to help identify the etiology of the fistula, as well as to assess its location, any ongoing inflammation, and whether a sphincter defect is present or not. Continence evaluation is of paramount importance in RVFs following perineal lacerations. All this information is essential prior to surgical treatment. Procto-sigmoidoscopy should be done. The fistulous opening may be seen as a small dimple or pit and occasionally can be gently probed for confirmation. Flexible endoscopy (sigmoidoscopy or colonoscopy) is used to fully evaluate the possibility of IBD. Evaluation of established RVFs with endo-rectal ultrasound and trans-vaginal ultrasound examination is more important if the patient complains of incontinence or if the underlying cause is obstetric trauma. Yee LF et al found that non-contrast endoanal ultrasound was not useful in imaging RVF and did not recommend this as a diagnostic or screening tool for the identification of a RVF [7]. High fistulas may not be readily apparent on physical examination or vaginal inspection and may even be missed by endoscopy. Methylene blue enema with a vaginal tampon in place, looking for staining on the tampon is used to confirm the diagnosis. Vaginography with a water soluble contrast medium has a reported sensitivity of 79% to 100% [8-10]. CT and MRI also play a role in the diagnosis and evaluation of the RVF as they may give insight into the underlying cause of the fistula.
The management of RVF depends on size, location, cause, anal sphincter function and overall health status of the patient. It also depends on the skill and judgment of the surgeon. Treatment of established RVFs should be surgical. The treatment of RVF must be tailored to the individual fistula. Transabdominal approach is the standard surgical approach for high fistulas. Different surgical techniques have been described. These include fistula division and closure with or without bowel resection and use of local flaps, such as the bulbocavernosus flap and a variety of muscle and musculocutaneous flaps for repair of large defects. Diversion colostomy is preferred to safeguard the anastomosis.
Perineal approach is the choice in the management of low RVFs. These include transvaginal, transperineal, transanal or conversion of the fistula into a complete perineal laceration with subsequent repair. The transanal endorectal advancement flap is the most popular technique of repairing simple low RVFs by colorectal surgeons while the gynecologists prefer the transvaginal approach. The perineal approach is not preferable because of the damage to the perineal body but the exposure is excellent and is indicated in failed cases following transanal and transvaginal approach.
Laparoscopic management of the RVFs is still in its infancy. Some surgeons have performed laparoscopic assisted procedures. Schwenk W et al reported a case of RVF for which they had performed a laparoscopic resection of the sigmoid colon with the fistulous tract and intracorporeal colorectal anastomosis [11]. Pelosi et al performed laparoscopic upper rectovaginal mobilization to facilitate the transvaginal repair of recurrent RVF [12]. Total laparoscopic repair is still rare, because of the complexity of the procedure. Nezhat CH reports correction of two cases of RVF by laparoscopy [1]. Our case is probably the first reported case of RVF following laparoscopic assisted vaginal hysterectomy, which has been repaired by a total laparoscopic technique.
We feel that simple high RVF can be repaired laparoscopically after proper assessment of the patient and the fistula. As in our case, there was no need for a diversion colostomy in cases where the fistulous tract is completely excised and a proper suturing of the defect is done. Good preparation of the bowel is essential to avoid any fecal contamination of the operative area. Adequate laparoscopic experience with proper identification of the tissue planes and the fistulous tract and meticulous surgical technique is required to accomplish a total laparoscopic repair. Laparoscopic repair of RVF has all the advantages of a minimal access surgery. It is associated with minimal wound complications, less postoperative pain and early recovery.
Conclusion
Laparoscopic repair of RVFs can be demanding and would require proper identification of tissue planes and adequate experience in advanced laparoscopic procedures. We conclude that laparoscopic resection of simple high RVFs with primary intracorporeal closure is feasible and should be considered in selected cases as an alternative to open surgery. Safety and long term results of laparoscopic repair of RVF need to be confirmed by further studies.
Abbreviations
RVF: Rectovaginal Fistula
IBD: Inflammatory bowel disease
BMI: Body mass index
DVT: Deep vein thrombosis
CT: Computerized tomography
MRI: Magnetic resonance imaging
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SSK: Acquisition of data and preparing the manuscript.
CP: Management of the case, critical evaluation and overall supervision.
AJK: Preparing the manuscript and critical evaluation.
RP: Collection of the data and drafting the manuscript.
MN: Management of the case and final approval
Pre-publication history
The pre-publication history for this paper can be accessed here:
Acknowledgements
Written consent was taken from the patient for publishing her clinical details and operative photographs.
==== Refs
Nezhat CH Bastidas JA Pennington E Nezhat FR Raga F Nezhat CR Laparoscopic treatment of type IV rectovaginal fistula Am Assoc Gynecol Laparosc 1998 5 297 9
Belt RL JrBelt RL Repair of anorectal vaginal fistula utilizing segmental advancement of the internal sphincter muscle Dis Colon Rectum 1969 12 99 104 5774855
Hibbard LT Surgical management of rectovaginal fistulas and complete perineal tears Am J Obstet Gynecol 1978 130 139 41 619652
Lowry AC Thorson AG Rothenberger DA Repair of simple rectovaginal fistula. Influence of previous repairs Dis Colon Rectum 1988 31 676 8 3168676
Russell TR Gallagher DM Low rectovaginal fistulas approach and treatment M J Surg 1997 134 13 8
Lescher TC Pratt JH Vaginal repair of the simple rectovaginal fistula Surg Gynecol Obstet 1967 124 1317 21 6024913
Yee LF Birnbaum EH Read TE Kodner IJ Fleshman JW Use of endoanal ultrasound in patients with rectovaginal fistulas Dis Colon Rectum 1999 42 1057 64 10458131
Arnold MW Aguilar PS Stewart WRC Vaginography: an easy and safe technique for diagnosis of colovaginal fistulas Dis Colon Rectum 1990 33 344 5 2323285 10.1007/BF02055482
Bird D Taylor D Lee P Vaginography: the investigation of choice for vaginal fistulae? Aust N Z J Surg 1993 63 894 6 8216069
Giordano P Drew PJ Taylor D Vaginography – investigation of choice for clinically suspected vaginal fistulas Dis Colon Rectum 1996 39 568 72 8620810 10.1007/BF02058713
Schwenk W Bohm B Grundel K Muller J Laparoscopic resection of high rectovaginal fistula with intracorporeal colorectal anastomosis and omentoplasty Surg Endosc 1997 11 147 9 9069147 10.1007/s004649900318
Pelosi MA 3rdPelosi MA Transvaginal repair of recurrent rectovaginal fistula with laparoscopic-assisted rectovaginal mobilization J Laparoendosc Adv Surg Tech A 1997 7 379 83 9449089
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BMC Surg. 2005 Oct 12; 5:20
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BMC SurgBMC Surgery1471-2482BioMed Central London 1471-2482-5-211622570010.1186/1471-2482-5-21Research ArticleIs there a relationship between weather conditions and aortic dissection? Repanos Costa [email protected] Neil K [email protected] ENT Department, Derriford Hospital, Plymouth, United Kingdom2 ENT Department, Torbay Hospital, Torquay, United Kingdom3 Blackpool Victoria Hospital, Blackpool, United Kingdom2005 15 10 2005 5 21 21 3 12 2004 15 10 2005 Copyright © 2005 Repanos and Chadha; licensee BioMed Central Ltd.2005Repanos and Chadha; 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
Bleeding and rupture of blood vessels has been correlated with weather conditions in the past. This is the first study in the world literature with the aim of investigating the relationship between atmospheric pressure and temperature with the presentation of aortic dissection.
Methods
The dates of all emergency aortic dissection repairs from 1996–2002 in a regional cardiothoracic unit at Blackpool Victoria Hospital were obtained. Hourly temperature and pressure data from a regional weather station for this time period was supplied by the Meteorological Office. The mean and standard deviation of hourly temperature and pressure data for that month were compared to the mean and standard deviation of the data 24 and 48 hours prior to the aortic dissection.
Results
26 patients were found to have been operated on during the time period studied. There was no statistically significant correlation between temperature or atmospheric pressure readings, and the incidence of aortic dissection, using a Bonferonni-corrected significance p-value of 0.005
Conclusion
This study is the first to examine the relationship between atmospheric pressure, temperature and dissecting thoracic aorta. No statistically significant relationship was demonstrable.
==== Body
Background
Aortic dissection occurs when there is a defect in the intimal layer of the aorta. This causes blood to track through the aortic layers, creating a false lumen between the intima and the overlying adventitia, tending to obliterate the blood supply to branches along its route. The dissection may spread distally to involve the renal, spinal and iliac arteries, or proximally to involve the head and neck vessels, coronary vessels, or aortic root. Once the dissection has occurred it can rupture into the main lumen itself, in which case the patient may survive for some time, or rupture externally leading to haemorrhage and potentially a rapid death.
The known risk factors for aortic dissection are male sex, hypertension, atherosclerosis, diabetes, hyperlipidemia, smoking, syphilis, Marfan's disease, and Ehlers-Danlos disease. In a large series of necropsies of acute aortic dissections more than 40% of patients with proximal dissection died immediately, the rate of death ranged between 1 and 3% per hour. Within 24 hours 70%, within 1 week 94% and within 5 weeks 100% of people with proximal aortic dissection died [1]. After surgical treatment of proximal aortic dissection the survival rate is approximately 70% after 3 years [2].
The aetiology of an acute aortic dissection remains poorly understood. It is thought that the moment of dissection may be precipitated by higher than normal blood pressures, and it has been postulated that variablilty of atmospheric pressure may add to this. There is also a seasonal variation in blood pressure [3,4] and it has been shown that there is a seasonal increase in the rate of non-traumatic aortic dissections in the winter [5,6].
Previous work has observed an increase in ruptured abdominal aortic aneurysms (AAA) in winter [7], and a correlation with atmospheric pressure [8]. Other vascular disruptions, including subarachnoid aneurysm rupture and spontaneous cervical artery dissection, have also been demonstrated to correlate with atmospheric pressure changes [9] or seasonal variations [10].
There have been no previous studies specifically investigating the relationship between weather conditions and the incidence of thoracic aortic dissection. Considering the similarity in seasonal incidence between AAA rupture and aortic dissection, we aim to explore whether there is an association between atmospheric pressure, temperature, and the presentation of aortic dissection.
Methods
Hospital records were used to identify all emergency aortic dissection repairs between 1996 and 2002 in a regional cardiothoracic unit at Blackpool Victoria Hospital. Data was available from the cardiothoracic minimum dataset, and cases were confirmed by reference to the hospital notes. Ethical approval was obtained to use this data. Temperature and pressure recordings from a nearby regional weather station were obtained for this time period from the United Kingdom Meteorological Office. Blackpool cardiothoracic unit receives referrals from northern Lancashire, and Crosby is the weather station closest to the centre of the unit's catchment area.
The meteorological data used was hourly temperature measurements and hourly atmospheric pressure measurement for a full month centred on the occurrence of each aortic dissection.
Using the meteorological data, the following temperature indicators were calculated (Table 1):
Table 1 Temperature indicators
1 Mean temperature for the whole month of the operation
2 Mean temperature 48 hours before operation date
3 Mean temperature 24 hours before operation date
4 Mean daily temperature range for the whole month of the operation
5 Mean daily temperature range 48 hours before operation date
6 Mean daily temperature range 24 hours before operation date
The following pressure indicators were also calculated (Table 2):
Table 2 Pressure indicators
7 Mean pressure for the whole month of the operation
8 Mean pressure 48 hours before operation date
9 Mean pressure 24 hours before operation date
10 Mean daily pressure range for the whole month of the operation
11 Mean daily pressure range 48 hours before operation date
12 Mean daily pressure range 24 hours before operation date
13 Standard deviation of pressure for the whole month
14 Standard deviation of pressure 48 hours before operation date
15 Standard deviation of pressure 24 hours before operation date
Indicators were then paired for comparison using each set of monthly data against the corresponding data from 48 hours, and then 24 hours before the operation. The null hypothesis was that there were no differences within each pair of indicators. These hypotheses were tested using paired t-tests and Wilcoxen signed rank tests (depending on the distribution of the data).
The Bonferroni correction is a statistical adjustment to allow for multiple comparisons. A Bonferroni correction was applied by dividing the p-values by the number of outcomes being tested. In this study we have made 10 comparisons between indicators, and have therefore used α = 0.005 for hypothesis testing. The null hypothesis was therefore rejected for p values ≤ 0.005, and the probability of making a Type I error therefore 0.05.
Results
Data for 26 consecutive operated dissecting aortic aneurysms were obtained for the study time period. Results for the temperature indicators and atmospheric pressure indicators including comparisons between pairs are shown in Table 3 and Table 4 respectively.
Table 3 Temperature indicator values and comparison of results.
Indicator A Mean of A (°C) n = 26 Indicator B Mean of B (°C) n = 26 Indicator A vs. Indicator B p-value
Paired t-test (t-value)
Mean temperature for whole month 11.36 Mean temperature 48 hours before 10.60 -1.7243 0.097
Mean temperature for whole month 11.50 Mean temperature 24 hours before 10.91 1.4658 0.156
Temperature range for whole month 5.94 Temperature range 48 hours before 5.54 0.9972 0.329
Temperature range for whole month 5.82 Temperature range 24 hours before 5.62 0.4275 0.673
Table 4 Pressure indicator values and comparison of results.
Indicator A Mean of A (millibars) n = 26 Indicator B Mean of B (millibars) n = 26 Indicator A vs. Indicator B p-value
Paired t-test (t-value) Wilcoxon signed rank test (z-value)
Mean pressure for whole month 1011.32 Mean pressure 48 hours before 1009.39 N/A 0.724 0.469
Mean pressure for whole month 1011.32 Mean pressure 24 hours before 1009.88 N/A 0.444 0.657
Pressure range for the whole month 6.99 Pressure range 48 hours before 8.51 N/A -1.943 0.052
Pressure range for whole month 6.99 Pressure range 24 hours before 8.40 -1.9275 N/A 0.065
Standard deviation of pressure for whole month 2.22 Standard deviation of pressure 48 hours before 2.73 N/A -2.283 0.022
Standard deviation of pressure for whole month 2.22 Standard deviation of pressure 24 hours before 2.64 N/A -1.905 0.057
Table 3 and 4 compare both mean temperature and pressure over the month of the event to the mean temperature and pressures respectively over the preceding 24 and 48 hours.
The distribution of dissections through the year is represented in Figure 1.
Figure 1 Seasonal variation of aortic dissections (1996–2002).
Figure 1 depicts the spread of dissections by the month in which they occurred. The numbers are too small to make any comment about seasonality.
Discussion
Aortic dissection is a condition that presents infrequently to hospitals and is sometimes initially misdiagnosed. Increasingly as hospitals and sub-speciality departments merge to form larger institutions, they will cover a greater population, and the incidence in hospitals will apparently rise.
In our study the most significant result (p = 0.022) showed a trend towards the standard deviation of pressure in the 48 hour period prior to the dissection, being greater than the standard deviation of pressure for that whole month (2.73 vs. 2.22 millibars). This did not reach statistical significance however, after the Bonferroni correction has been applied.
The pressure range was calculated by subtracting the lowest from the highest reading of the day. The mean pressure range for the whole month (6.99 millibars) had a trend towards being lower than the mean 48 hours before dissection (8.51 millibars, p = 0.047) and 24 hours before dissection (8.40 millibars, p = 0.066). This was not statistically significant.
The incidence of aortic dissection had no seasonal or monthly correlation. This is demonstrated by the bar chart (Figure 1). Other studies have demonstrated a seasonal increase in the rate of non-traumatic aortic dissections in the winter [5,6]. The same seasonal variation has been noted in several studies for ruptured abdominal aortic aneurysms (AAA) with an increase in incidence in the winter [7,11,12]. One study found a statistically significant correlation with rupture of AAA and mean atmospheric pressure [8] and the only other published studies found no correlation between the incidence of ruptured AAA and barometric pressure or humidity [7,13]. There have also been studies examining the incidence of other vascular disruptions with changing season and weather conditions. In one study a modest correlation was found between high atmospheric pressure, daily change in pressure and the risk of rupture of subarachnoid aneurysms [9]. Another study demonstrated seasonal variation in spontaneous cervical artery dissection [10].
Previous work has shown that the only physiological parameter positively associated with atmospheric pressure is arterial blood gas concentrations [14]. However the exact mechanism by which reduced arterial oxygen tension may cause rupture of an aneurysm of dissection remains unknown. The onset of labour is the only other medical change thought to be associated with falling atmospheric pressure [15]. Though this possible association may not seem immediately useful, it is known that pregnant women have a higher incidence of spontaneous aortic dissection [16].
The analysis in our study involved using accurate meteorological data and operation dates from a regional cardiothoracic centre covering a large geographical area. Despite careful methodology there are potential errors that must be remembered. The weather station, although centrally placed over the catchment area, may not have reflected the patients' movements over the region in that time period and does not account for weather variations over an area 50 miles in diameter.
There is also likely to be variability in the timing of presentation. Some patients may have begun aortic dissection a long time prior to presenting to hospital and a tamponading effect may have slowed the progression of collapse. We have attempted to account for this by looking at weather data in both the 24 and 48 hour time periods prior to the day of operation. It may be that a sudden change in the indicator being examined between 48 and 24 hours prior to dissection may be related to the onset of dissection.
Only patients who had surgery were included in this study and there are undoubtedly others who either did not survive long enough to have surgery or who were managed without surgery. It is presumed that the inclusion of these patients would not have changed the trends obtained in relation to weather conditions. In practice this study may not have any immediate applications to alter clinical practice, but serves to further elucidate the complex aetiology of a rare and sometimes lethal condition.
Conclusion
This study is the first to examine the relationship between atmospheric pressure, temperature and dissecting thoracic aorta, and was unable to demonstrate any statistically significant relationship.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
CR and NC drafted the manuscript. CR conceived the study. Statistical analysis was performed by ED of the statistics department (see acknowledgements). CR and NC 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:
Acknowledgements
We would like to thank the Blackpool Victoria Hospital Cardiothoracic Unit for the use of their hospital data, the Meteorological Office for the use of their temperature and atmospheric pressure data, and Emma Dark of North Bristol Healthcare Trust, for her help with statistical analysis.
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Goodwin J Pearce VR Taylor RS Read KLQ Powers SJ Seasonal cold and circadian changes in blood pressure and physical activity in young and elderly people Age and Ageing 2001 30 311 317 11509309 10.1093/ageing/30.4.311
Minami J Kawano Y Ishimitsu T Yoshimi H Takishita S Seasonal variations in office, home, and 24 h ambulatory blood pressure in patients with essential hypertension J Hyperten 1996 14 1421 1425
Manfredini R Portaluppi F Salmi R Zamboni P La Cecilia O Kuwornu Afi H Regoli F Bigoni M Gallerani M Seasonal variation in the occurrence of non-traumatic rupture of thoracic aorta Am J Emerg Med 1999 17 672 4 10597086 10.1016/S0735-6757(99)90156-0
Upshur RE Mamdani MM Knight K Are there seasonal patterns to ruptured aortic aneurysms and dissections of the aorta? Eur J Vasc Endovasc Surg 2000 20 173 6 10942690 10.1053/ejvs.2000.1139
Sterpetti AV Cavallari N Allegrucci P Agosta F Cavallaro A Seasonal variation in the incidence of ruptured abdominal aortic aneurysm J R Coll Surg Edinb 1995 40 14 5 7738888
Bown MJ McCarthy MJ Bell PR Sayers RD Low atmospheric pressure is associated with rupture of abdominal aortic aneurysms Eur J Vasc Endovasc Surg 2003 25 68 71 12525814 10.1053/ejvs.2002.1798
Buxton N Liu C Dasic D Moody P Hope DT Relationship of aneurysmal subarachnoid haemorrhage to changes in atmospheric pressure: results of a prospective study J Neurosurg 2001 95 391 392 11565858
Schievink WI Wijdicks EF Kuiper JD Seasonal pattern of spontaneous cervical artery dissection J Neurosurg 1998 89 101 3 9647179
Ballaro A Cortina-Borja M Collin J A seasonal variation in the incidence of ruptured abdominal aortic aneurysms Eur J Vasc Endovasc Surg 1998 15 429 31 9633499 10.1016/S1078-5884(98)80205-0
Varty K Reid A Jagger C Bell PR Vascular emergencies: what's in season? Cardiovasc Surg 1995 3 409 11 7582996 10.1016/0967-2109(95)94160-X
Kurtoglu M Yanar H Aksoy M Seasonality in the incidence of abdominal aortic aneurysm ruptures: a review of eight years Ulus Travma Derg 2004 10 39 41 14752685
Burnett RW Itano M An interlaboratory study of blood-gas analysis: dependence of pO2 and pCO2 results on atmospheric pressure Clin Chem 1989 35 1779 17981 2503269
Noller KL Resseguie LJ Voss V The effect of changes in atmospheric pressure on the occurrence of the spontaneous onset of labour in term pregnancies Am J Obstet Gynecol 1996 174 1192 1197 8623846
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BMC Vet ResBMC Veterinary Research1746-6148BioMed Central London 1746-6148-1-41621611610.1186/1746-6148-1-4Research ArticleMolecular characterisation of the caprine (Capra hircus) lymphocyte function-associated antigen-1 alpha subunit-encoding cDNA Fett Thomas [email protected] Laurent LM [email protected] Etienne A [email protected] Daniel JM [email protected] Pathology Department, Faculty of Veterinary Medicine, University of Liege, 4000 Liege, Belgium2005 10 10 2005 1 4 4 4 7 2005 10 10 2005 Copyright © 2005 Fett et al; licensee BioMed Central Ltd.2005Fett 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
Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alpha L beta 2) is required for many cellular adhesive interactions during the immune response.
Results
The Capra hircus CD11a-encoding cDNA was sequenced and compared with its human, murine, rat, bovine and ovine counterparts. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues.
Conclusion
Therefore, along with the caprine CD18-encoding cDNA, which has been available for a few months, the sequence data revealed here will allow the Capra hircus LFA-1 expression in vitro as a tool to explore the specificities of inflammation in the caprine species.
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Background
Lymphocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) is a member of the β2-integrin subfamily of cell surface receptors. Integrins consist of a 120 to 180 kDa α subunit (CD11a in this case) and a 90 to 110 kDa β subunit that are noncovalently associated single-pass transmembrane proteins [1]. The bulk of each integrin subunit is extracellular, where it typically functions as a receptor for extracellular matrix molecules or as a counterreceptor for surface proteins of apposed cells [2]. The heterodimer CD11a/CD18 is expressed on all leukocytes and mediates high affinity adhesion to a variety of cell types that express one or more of the β2-integrins ligands, intercellular adhesion molecules (ICAM-1 to -5) [3-5]. The adhesion process mediated is a critical step of a wide range of immunological activities, including cytolysis of target cells, cross-interaction and cross-stimulation between lymphocytes, phagocytosis of complement-coated targets, neutrophils clearance from inflamed tissue, and the regulation of leukocyte traffic between the bloodstream and tissues [6-9]. As the relevance of the goat model for studying leukocyte traffic, diapedesis and pathologic tissue infiltration is well established in such important areas as mastitis [10-13] or lentivirus-associated diseases [14-16], increasing our knowledge about caprine β2 integrins is of great importance to offer new possibilities for research and to provide additional insights into those fields. Along with the caprine CD18-encoding cDNA, which is available for a few months [17], the sequence data provided here will allow the Capra hircus β2-integrin CD11a/CD18 expression in vitro as a tool to examine the specificities of inflammation in the caprine species.
Methods
RNA isolation
Total RNA from phorbol myristate acetate (PMA)-stimulated (25 ng/ml for 15 min) caprine (Boer breed) peripheral blood mononuclear cells (PBMC) was extracted with TRIzol (Invitrogen, USA) as described by the manufacturer. The PBMCs were obtained by density gradient centrifugation with Ficoll-Paque Plus (Amersham, USA) and maintained in RPMI 1640 supplemented with 10% foetal bovine serum (Gibco BRL, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in a 5% CO2 atmosphere.
Amplification of cDNA ends
We used SMART RACE technology (Clontech Laboratories Inc., USA) to obtain caprine CD11a (CaCD11a) 5'- and 3'-ends and RT-PCR to amplify full-length CaCD11a CDS. For first-strand cDNA synthesis, and according to the sequence of bovine CD11a available [GenBank: AY267467], gene-specific primers were designed which were expected to give non overlapping ~1 kb RACE products: a sense primer for the 3'-RACE PCR : 5'-TGCAATGTRAGCTCTCCCATCTTC-3' (corresponding to nt 2572 to nt 2595) and an antisense primer for the 5'-RACE PCR : 5'-CCGGCCTCCTCTCTGCTCCCCATAG-3' (nt 1470 to nt 1446). Reverse transcription and polymerase chain reactions (PCR) were carried out according to the instruction manual of the SMART RACE cDNA Amplification Kit. The 5'- and 3'-RACE products were gel-purified using the S.N.A.P.™ Gel Purification Kit (Invitrogen, USA), TA-cloned into pCRII-TOPO (Invitrogen, USA) and seeded on kanamycin IPTG plates. Miniprep were obtained from colonies grown in 5 ml LB-kanamycin broth and the clones were sequenced on the ABI-3730 Genetic Analyzer using the Big Dye terminator chemistry (Applied Biosystems, USA).
Molecular cloning of full-length cDNA
Total RNA from PMA-stimulated PBMCs was reverse transcribed using Improm II (Promega, USA). The full-length cDNA was then generated by long distance PCR using Platinum Taq DNA polymerase High Fidelity (Invitrogen, USA) with primers designed from the distal ends of both 5'- and 3'-RACE products : 5'-GTCGCCAGTAAATCCCAAGA-3' (sense, within the 5'-UTR) and 5'-GCACCTCAATCTCCACCACT-3' (antisense, 3'UTR). The procedures recommended by the manufacturer were followed, with these cycling parameters : 5 min at 94°C, then 35 cycles including (i) 30 s at 94°C, (ii) 30 s at 58°C and (iii) 3 min 30 s at 68°C, followed by a final extension at 68°C for 5 min. Resulting PCR products were then processed for sequencing as aforementioned for the RACE products. The CD11a cDNA sequence was deduced from sequences obtained from nine independent clones. Sequence data have been deposited at GenBank under accession No. AY773018 and AY773019.
Bioinformatics
Primers design was performed with Netprimer [18] and Primer 3 [19]. Nucleotidic sequence and similarity analyses were carried out using respectively Chromas v.2.21 [20] and BLAST programs [21]. Alignment of amino acids sequences were drawn by GeneDoc v.2.6.002 [22] following the BLOSUM62 matrix. SignalP v.2.0.b2 [23] and NetNGlyc v.1.0 [24] provided peptide signal and N-glycosylation sites prediction, respectively.
Results & discussion
Characterisation of CaCD11a-encoding cDNA and deduced aa sequence
Two alleles have been identified for the CaCD11a cDNA. The sequence contains ~4200 bp with an ORF of 3498 [Genbank: AY773019] or 3495 bp [Genbank: AY773018] depending on the allele that codes for 1165 or 1164 aa followed by ~600 bp in the 3'-UTR (Fig. 1). The mature CaCD11a contains a 23-aa putative leader peptide, an extracellular domain of 1061 or 1062 residues (24-1084/1085), a single hydrophobic transmembrane region of 24 residues (1085/1086–1108/1109) and a cytoplasmic tail of 57 residues (Fig. 1). Nine N-linked putative glycosylation sites (Asn-X-Thr/Ser) are present in the extracellular domain (Fig. 2). The mature protein contains 19 cysteine residues among which one is located into the cytoplasmic tail (Fig. 2). The extracellular domain also contains an inserted (I) domain of 172 amino acids (residues 153–324) quite similar to those found in all the leukocyte integrin α subunits sequenced to date and located between the β sheets 2 and 3 of a seven bladed β-propeller region [25]. The I-domain is homologous with repeated domains found in von Willebrand factor and cartilage matrix protein [1] and can be expressed as an isolated domain. Its three-dimensional structure consists of a five-stranded parallel β-sheet core surrounded on both faces by α-helices, with a short antiparallel strand occurring on one edge of this sheet [26]. The I-domain contains a metal ion-dependent adhesion site (MIDAS) [27] (residues 159–163, 228, 261) (Fig. 2) and an I-domain allosteric site (IDAS) that plays a functional role in ICAM-1 binding [28-30]. Three repeats with a divalent cation binding motif are found at amino acid residues 465–473, 527–535 and 587–595 (Fig. 2). All the conserved cysteines and all but one N-glycosylation sites are found outside the I region and divalent cation binding motifs (Fig. 2), consistent with the hypothesis that these regions may undergo conformational changes important in ligand binding [31,32].
Figure 1 The nucleotide and deduced amino acid sequences of Capra hircus CD11a cDNA. The putative leader peptide and transmembrane region are underlined. Nine independent clones were sequenced in both directions. Sequence data have been deposited at GenBank under accession No. AY773018 and AY773019 (shown here), respectively without and with Gln-743 (#).
Figure 2 Comparison of the caprine (Ca-), bovine (Bo-), human (Hu-), murine (Mu-) and rat (Ra-) α subunits amino acid sequences. The letters in the top row identify the constitutive blocks: putative signal peptide (s), extracellular domain (e), transmembrane region (t), cytoplasmic tail (c), I-domain (i), MIDAS motif (m) and divalent cation binding motifs (d). Black, dark grey and light grey columns represent identity among 6, 5 and 4 species, respectively. Cysteine residues (#) and potential N-glycosylation sites (*) are marked at the bottom of the sequences. The important Glu-332 residue (+) and the Gln-743 residue which is absent in the smaller allele (=) are identified.
General comparison among species
Overall, the general organization of caprine, bovine [33], ovine [34], human [32], murine [35] and rat [GenBank: NW_047562] CD11a proteins is quite similar (Fig. 2). Comparison between mature CaCD11a sequence and its bovine, ovine, human, murine and rat counterparts shows overall 94%, 98%, 77%, 68% and 55% identity, respectively, with the highest identity for the MIDAS, the cation binding motifs and the transmembrane region and the lowest identity for the cytoplasmic tail (Table 1). The high conservation of the MIDAS and the putative cation binding motifs is consistent with an involvement of these regions in the functional activity of LFA-1 α subunit, as suggested by the requirement of Mg2+ and Ca2+ for CD11a/CD18-dependent cellular interactions [31] or binding to purified ICAM-1 [36,37]. The transmembrane region shows also a high degree of conservation that could be explained by (i) physicochemical, and (ii) functional constraints. Indeed, (i) residues lying in the membrane have to possess rather hydrophobic character to allow liposolubility, which is confirmed by the presence of many leucine residues (figure 2) and (ii) bi-directional integrin signalling (inside-out and outside-in) is accomplished by transmission of information across the plasma membrane [38]. By contrast, the low conservation of the cytoplasmic tail suggests that it is not required to guarantee adequate functioning of LFA-1. This is in agreement with the observation that truncation of the LFA-1 α subunit cytoplasmic domain has no effect on binding to ICAM-1, whereas binding is markedly diminished by β subunit cytoplasmic domain truncation [39]. Residue Glu-332 that is located in the linker following the I-domain and that is known to be critical for communication to the β2 I-like domain, rolling, integrin extension and activation by Mn2+ of firm adhesion [8] is strictly conserved.
Table 1 Between-species percent identities of CD11a constitutive blocks. Ca, Bo, Ov, Hu, Mu and Ra: caprine, bovine, ovine, human, murine and rat CD11a, respectively; MIDAS: metal-ion dependent adhesion site.
Block Ca vs. Bo (%) Ca vs. Ov (%) Ca vs. Hu (%) Ca vs. Mu (%) Ca vs. Ra (%)
Overall 94 98 77 68 55
Putative signal peptide 86 95 60 41 4
Extracellular domain 94 98 78 69 60
Transmembrane region 95 100 95 79 87
Cytoplasmic tail 92 94 58 50 47
I-domain 95 99 86 72 75
MIDAS 100 100 100 85 85
Putative cation binding motif 1 100 100 88 66 55
Putative cation binding motif 2 100 100 77 88 88
Putative cation binding motif 3 100 100 88 77 88
Every cysteine residue in the caprine extracellular portion of mature CD11a is present at the same location in bovine, ovine, human, murine and rat CD11a, which is consistent with a role in maintaining the global structure of the protein whereas two cysteine residues (positions 1009 and 1048) are absent from caprine CD11a and therefore do not seem to be indispensable. The mouse version distinguishes by an additional cysteine residue at position 199 (mouse numbering) within the extracellular portion. Of nine potential Asn-glycosylation sites in caprine CD11a, the ones present at amino acids 185, 667, 723 and 859 are strictly conserved, one is only absent from murine and rat CD11a (residue 894), without predictable consequences on a functional point of view.
Interestingly, as in sheep [34] and human [GenBank: NM_002209 and AY892236], an allelic variant with a triplet insertion resulting in an additional Glu744 in the extracellular domain was consistently identified, which suggests an allelic polymorphism that might be biologically relevant. Studies of genomic sequences will permit to know if this addition represents two alleles or not.
Finally, one has to note that the lowest between-species percent identities are observed with the rat CD11a sequence which has been derived from an annotated genomic sequence. Cloning and characterisation of rat CD11a from rat PBMCs would probably give a higher identity.
Conclusion
This study reports for the first time the isolation and sequencing of the caprine LFA-1 α subunit (CD11a) cDNA, and demonstrates that, despite some focal differences, it shares all the main characteristics of its known mammalian homologues. Along with the caprine CD18-encoding cDNA which is now available [17], the sequence data provided here will allow the successful expression of caprine LFA-1 in vitro as a tool to examine the specificities of inflammation in the caprine species.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TF carried out cloning and sequencing, participated in the sequence alignment and to the draft of the manuscript. LZ participated in the sequence comparison and to the draft of the manuscript. EB participated in the design of the study. DD conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This study is supported by the Belgian federal services for public health and security of the food chain and environment, grant S-6107. The authors are grateful to Prof M. Georges for giving free access to all the facilities of the laboratory of molecular genetics and to Prof. J.-F. Beckers for the caprine PBMCs.
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BMC Vet ResBMC Veterinary Research1746-6148BioMed Central London 1746-6148-1-61622568410.1186/1746-6148-1-6Research ArticleMetoclopramide-induced central nervous system depression in the chicken Al-Zubaidy Muna HI [email protected] Fouad K [email protected] Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Mosul, PO Box 11136, Mosul, Iraq2005 14 10 2005 1 6 6 1 6 2005 14 10 2005 Copyright © 2005 Al-Zubaidy and Mohammad; licensee BioMed Central Ltd.2005Al-Zubaidy and Mohammad; 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
Metoclopramide is a dopamine D2-receptor antagonist used as an antiemetic and gastroprokinetic agent in man and animals. The drug causes sedation as a side effect in man. Such a sedative action of metoclopramide has not been documented in the chicken as the drug is not used clinically in this species. The present study examines the central nervous system depressant effects of metoclopramide in 7–14 days old broiler chicks.
Results
Injection of metoclopramide at 50, 100 and 200 mg/kg, subcutaneously (s.c.) induced sedation in the chicks in a dose dependent manner. The chicks manifested, within 3.6–19 minutes of metoclopramide injection, signs of sedation characterized by drooping of the head and wings, closed eyelids, reduced motility and decreased distress calls. The duration of sedation ranged between 37.2 to 163.4 minutes. Metoclopramide at 100 and 200 mg/kg induced, within 12.2 and 6.2 minutes, sleep (loss of righting reflex) for 43.8 and 158.6 minutes, respectively. The median effective doses of metoclopramide for induction of sedation and sleep in the chicks were 11 and 53 mg/kg, s.c., respectively. Lower doses of metoclopramide (5 and 10 mg/kg, s.c.) significantly decreased the open-field activity of the chicks and increased the durations of their tonic immobility. All treated-chicks recovered from the central nervous system depressant effect of metoclopramide without any observable adverse effects.
Conclusion
The data suggest that metoclopramide induces central nervous system depression in chicks, and the drug could have potential clinical applications as a sedative-hypnotic agent in avian species not intended for human consumptions.
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Background
Metoclopramide (methoxychloroprocainamide) is a dopamine D2-receptor antagonist used as an antiemetic and gastroprokinetic agent in man [1] as well as in dogs [2-4] and cats [3-5]. The drug also has serotonergic effects [6] and indirect parasympathomimetic activity [7,8]. Metoclopramide has been used experimentally in pigeons as an antiemetic agent at 10, 20 and 40 mg/kg, body weight [9]. Antidopaminergic drugs such as phenothiazine tranquilizers are known to induce a state of sedation in different animal species, including the chicken [3,4,10]. Metoclopramide treatment has been reported to cause sedation in man [1,11,12]. Such a sedative action of metoclopramide, though a side effect, has not been documented in the chicken as the drug is not used clinically in this species. In this communication, we present the central nervous system (CNS) depressant activity of metoclopramide in 7–14 days old broiler chicks. This age group of the chicken is suitable for examining sedative effects of CNS depressants [13-15].
Results
Injection of metoclopramide at 50, 100 and 200 mg/kg, subcutaneously (s.c.) induced sedation in the chicks in a dose dependent manner. The chicks manifested within 3.6–19 minutes of metoclopramide injection (Table 1) signs of sedation characterized by drooping of the head and wings, closed eyelids, reduced motility and decreased distress calls. The duration of sedation ranged between 37.2 to 163.4 minutes and it was also dose dependent (Table 1). Metoclopramide at 100 and 200 mg/kg induced, within 12.2 and 6.2 minutes, sleep (loss of righting reflex) in the chicks for 43.8 and 158.6 minutes, respectively (Table 1).
Table 1 Metoclopramide -induced sedation and sleep (loss of righting reflex) in chicks
Metoclopramide (mg/kg, subcutaneously) Latency to onset of sedation (minute) Latency to onset of sleep (minute) Duration of sedation (minute) Duration of sleep (minute) Recovery time (minute)
50 19.0 ± 2.2 none 37.2 ± 7.6 none 43.4 ± 7.8
100 8.8 ± 2.5* 12.2 ± 2.2 66.4 ± 5.5* 43.8 ± 3.5 74.6 ± 6.4*
200 3.6 ± 0.5* 6.2 ± 0.5† 163.4 ± 17.4*† 158.6 ± 23.3*† 168.4 ± 16.6*†
Values are mean ± SE of 5 chicks/group.
* Significantly different from the 50 mg/kg treatment group, P < 0.05.
† Significantly different from the 100 mg/kg treatment group, P < 0.05.
A control group of chicks was also treated with physiological saline solution (5 ml/kg, subcutaneously).
The median effective doses (ED50s) of metoclopramide for the induction of sedation and sleep in the chicks, as determined by the up-and-down method, were 11 and 53 mg/kg, s.c., respectively (Table 2). Metoclopramide at lower doses (5 and 10 mg/kg, s.c.) also caused CNS depression in chicks; the drug significantly decreased the open field activity of the chicks and significantly increased the durations of their tonic immobility (Table 3). All chicks recovered smoothly from metoclopramide-induced CNS depression and none of them suffered from adverse effects or died during the study.
Table 2 Determination of median effective doses (ED50) of metoclopramide for induction of sedation and sleep (loss of righting reflex) in chicks by the up-and-down method*
Variable Result
Sedation
ED50 11 mg/kg, subcutaneously (s.c.)
Range of the doses used 25-5 = 20 mg/kg, s.c.
Initial dose 25 mg/kg, s.c.
Last dose 10 mg/kg, s.c.
Number of chicks used 8 (XXXXOOXO)
Increase or decrease in the dose 5 mg/kg, s.c.
Range of latency to induce sedation 20-5 = 15 minutes
Range of duration of sedation 44-7 = 37 minutes
Sleep
ED50 53 mg/kg, s.c.
Range of the doses used 100-50 = 50 mg/kg, s.c.
Initial dose 100 mg/kg, s.c.
Last dose 60 mg/kg, s.c.
Number of chicks used 9 (XXXXXOXOX)
Increase or decrease in the dose 10 mg/kg, s.c.
Range of latency to induce sleep 12-2 = 10 minutes
Range of duration of sleep 67-11 = 56 minutes
*Dixon [18].
X = sedation or sleep; O = no sedation or sleep
Table 3 Effect of metoclopramide on open-field activity and tonic immobility test in chicks
Metoclopramide (mg/kg, subcutaneously) Squares crossed/5 minutes Duration of immobility (second)
0 (saline-control) 3.5 ± 1.4 10.5 ± 2.7
5 1.0 ± 1.0* 54.9 ± 18.1
10 0.1 ± 0.1* 130.6 ± 26.3*†
Open-field activity was measured 20 min after drug administration. Tonic immobility test was done immediately after the open-field test.
Values are mean ± SE of 8 chicks/group.
* Significantly different from the respective control values, P < 0.05.
† Significantly different from the 5 mg/kg treatment group, P < 0.05.
Discussion
Sedation has been reported clinically in man treated with regular therapeutic (or higher) doses of metoclopramide [1,11,12]. This effect has not been quantitatively reported in animals, especially in avian species. In the present report, metoclopramide -induced CNS depression in the chicks was concluded depending on the signs of sedation and sleep described in avian species [13-15]. The durations of sedation and sleep were also quantitatively reported (Table 1). The ED50s of metoclopramide for the induction of sedation and sleep were calculated for the first time in chicks by the up-and down method. Open-field activity and tonic immobility tests presented additional evidences for the CNS depressant activity of metoclopramide in the chicks. Little information are available on the pharmacological profile of metoclopramide in birds. The drug affects gastrointestinal motility in hispaniolan parrots [16] and prevents reserpine-induced emesis in pigeons [9]. Further, metoclopramide was found to increase gastrointestinal tract motility and inhibit plasma cholinesterase activity in chicks [17]. These effects are similar to those found in other animal species [2-5,8] and further suggest that the sedative activity of metoclopramide in the chicken should be potentially expected.
Metoclopramide depression is usually considered a side effect in man [1,11,12] and possibly in animals. Sedation could then be an additional pharmacological (side) effect of this drug to be expected in avian species. Further exploration of the potential research and possibly therapeutic applications of this drug is needed as a sedative agent in avian species not intended for human consumption, as the drug is not approved for use in food producing animals. The chicks in the present study were successfully used as a model to show the CNS depressant action of metoclopramide. Chicks were reported to be a suitable animal model for examining the CNS depressant action of drugs [13-15]. The sedative-hypnotic doses (50, 100 and 200 mg/kg, s.c.) of metoclopramide used in the present study are higher than the therapeutic ones used in dogs and cats [2-5]. However, the doses (especially the ED50s) of metoclopramide used in the present study are close to its antiemetic ones (10, 20 and 40 mg/kg, body weight, orally) used in pigeons treated with reserpine [9]. Using the open-field activity test, metoclopramide-induced CNS depression could be detected in the present study even at a dose as low as 5 mg/kg, s.c. (Table 3). Further, species differences in the magnitude of response to metoclopramide doses or its quality should be expected between mammals and birds or even among various avian species. Such a species difference has been reported with sedatives like xylazine [4,10,15].
Overall, the findings of the present study indicate the sedative and hypnotic (CNS depressant) effects of metoclopramide in chicks. These effects could be attributed to the antidopaminergic action of metoclopramide at the level of the CNS [1]. Centrally acting antidopaminergic drugs such as phenothiazine derivatives and butyrophenones are known to induce CNS depression in different animals as well as in avian species [3,4,10].
Conclusion
The data suggest that metoclopramide induces CNS depression in chicks, and the drug could have potential research and clinical applications as a sedative-hypnotic agent in avian species not intended for human consumption.
Methods
Unsexed, 7–14 days old, broiler chicks (body weight 52–95 g) were used. They were maintained in batches of 20–30 chicks at a time in a room at a temperature of 30–34°C-controlled by electric heaters with constant lighting. Litter consisted of wood shavings. Water and feed were given ad libitum. The chicks were treated s.c. with physiological saline solution at 5 ml/kg body weight (control group) or with metoclopramide HCl (Yuhan Corp., South Korea) at 50, 100 and 200 mg/kg body weight. Metoclopramide was dissolved in physiological saline solution, and the volume of administration was at 5 ml/kg body weight. The site of s.c. injection was on either lateral side of the chest. Care was taken so that leakage did not occur during or after the drug administration from the site of injection. The choice of metoclopramide doses was based on preliminary experiments in chicks in which doses more than 20 mg/kg, s.c. induced signs of sedation characterized by drooping of the head, closed eyelids, reduced motility or immotility, and decreased distress calls as well as recumbeny. After the injection of metoclopramide the chicks were monitored for the onset of sedation (drooping of the head) and sleep (loss of righting reflex after placing the chick on one side). The durations of sedation and sleep as well as recovery times were recorded too. Recovery time was the time from the onset of sedation until the chick moved freely. The ED50s of metoclopramide for the induction of sedation or sleep in the chicks were determined by the up-and-down method [18].
Further, the CNS depressant action of metoclopramide at lower doses (5 and 10 mg/kg) was also monitored by examining the open field activity [13,19] of the chicks and then subjecting them to the tonic immobility test [20]. In this experiment the chicks were treated s.c. with either physiological saline solution (control) at 5 ml/kg, or with metoclopramide at 5 and 10 mg/5 ml saline/kg body weight. Twenty minutes after the injection, each chick was placed alone on the center of the arena of an open field box (90 × 60 × 50 cm); the arena was divided into 24 equal squares and 50 g of wheat grains were scattered on the surface [13]. Open field activity was monitored by counting, within 5 minutes, the number of squares entered by both feet. After the open field activity test, each chick was subjected to tonic immobility test [20] by holding the chick in both hands and placing it on a wooden table for 15 seconds, then the hands were withdrawn and the chick was timed to upright itself and standing.
All experiments complied with regulations addressing animal use, and proper attention has been given to ethical consideration towards the chicks used in the present study. The data were statistically analyzed by one way analysis of variance followed by the least significant difference test [21]. Non-parametric data (open-field activity) were subjected to Mann-Whitney-U-test [22]. The level of significance was at P < 0.05.
Authors' contributions
MHIA executed the experiments, shared in statistical analysis and shared in drafting the manuscript.
FKM conceptualized the study, designed the experiments, supervised drug administration and behavioral tests, shared in statistical analysis and drafted the manuscript.
The authors read and approved the manuscript.
Acknowledgements
The study was supported by the College of Veterinary Medicine, University of Mosul, Iraq.
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Qualie JM Hall J Study of oral high-dose metoclopramide in cytotoxic drug-induced nausea and vomiting Pharmacol J 1989 243 PR21
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Al-Baggou' BKH Al-Dewachi OS Said MOM Mohammad FK Behavioral performance, serum glucose level and differential leukocyte count in local domestic chicks treated with xylazine Iraqi J Vet Sci 1999 12 223 232
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Kuwahara MD Sparber SB Behavioral consequences of embryonic or early postnatal exposure to 1ℓ-α-noracetylmethadol (NLAAM) in the domestic chicken Neurobehav Toxicol Teratol 1982 4 323 329 7099352
Hennig CW Fazio JK Hughes CA Castaldi WR Spencer BD Duration of tonic immobility in chickens as a function of alpha adrenergic receptor stimulation and blockade Pharmacol Biochem Behav 1984 20 731 738 6146144 10.1016/0091-3057(84)90192-8
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Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-4-551619755710.1186/1475-925X-4-55ResearchSynchronization analysis of the uterine magnetic activity during contractions Ramon Ceon [email protected] Hubert [email protected] Pam [email protected] James D [email protected] Curtis [email protected] Hari [email protected] Department of Electrical Engineering, University of Washington, Seattle, WA, USA2 MEG Center, University of Tübingen. Tübingen, Germany3 Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA4 Graduate Institute of Technology, University of Arkansas at Little Rock, Little Rock, Arkansas, USA2005 1 10 2005 4 55 55 29 6 2005 1 10 2005 Copyright © 2005 Ramon et al; licensee BioMed Central Ltd.2005Ramon 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
Our objective was to quantify and compare the extent of synchronization of the spatial-temporal myometrial activity over the human uterus before and during a contraction using transabdominal magnetomyographic (MMG) recordings. Synchronization can be an important indicator for the quantification of uterine contractions.
Methods
The spatialtermporal myometrial activity recordings were performed using a 151-channel noninvasive magnetic sensor system called SARA. This device covers the entire pregnant abdomen and records the magnetic field corresponding to the electrical activity generated in the uterine myometrium. The data was collected at 250 samples/sec and was resampled with 25 samples/sec and then filtered in the band of 0.1–0.2 Hz to study the primary magnetic activity of the uterus related to contractions. The synchronization between a channel pair was computed. It was inferred from a statistical tendency to maintain a nearly constant phase difference over a given period of time even though the analytic phase of each channel may change markedly during that time frame. The analytic phase was computed after taking Hilbert transform of the magnetic field data. The process was applied on the pairs of magnetic field traces (240 sec length) with a stepping window of 20 sec duration which is long enough to cover two cycle of the lowest frequency of interest (0.1 Hz). The analysis was repeated by stepping the window at 10 sec intervals. The spatial patterns of the synchronization indices covering the anterior transabdominal area were computed. For this, regional coil-pairs were used. For a given coil, the coil pairs were constructed with the surrounding six coils. The synchronization indices were computed for each coil pair, averaged over the 21 coil-pairs and then assigned as the synchronization index to that particular coil. This procedure was tested on six pregnant subjects at the gestational age between 29 and 40 weeks admitted to the hospital for contractions. The RMS magnetic field for each coil was also computed.
Results
The results show that the spatial patterns of the synchronization indices change and follow the periodic pattern of the uterine contraction cycle. Spatial patterns of synchronization indices and the RMS magnetic fields show similarities in few window frames and also show large differences in few other windows. For six subjects, the average synchronization indices were: 0.346 ± 0.068 for the quiescent baseline period and 0.545 ± 0.022 at the peak of the contraction.
Discussion
These results show that synchronization indices and their spatial distributions depict uterine contractions and relaxations.
==== Body
Background
Phase synchronization analysis of the uterine electrical or magnetic activity could be used as a tool to quantify the uterine contractions and it may also help us to distinguish between true and false labor. The electrical and the associated magnetic activity of the uterus arise from the generation and transmission of the action potentials in the uterine muscle. It is also possible that the depolarization of the uterine muscle tissue is the result of chemical stimulation at the cellular level. These action potentials or tissue level depolarizations occur in groups and the related measured electrical or magnetic activity appears as a burst activity. The frequency of the action potential within a burst, the duration of the burst, and total number of simultaneously active cells are directly related to the frequency, amplitude, and duration of a uterine contraction[1,2]. The electrical activity, also called electromyography (EMG), of the uterus has been recorded earlier with internal and abdominal surface electrodes [3]. Recently the spontaneous magnetic activity, also called magnetomyography (MMG) of the uterus has also been recorded with a 151-channel SQUID biomagnetometer [2] at the University of Arkansas, Little Rock, USA. The magnetic coils were designed to completely accommodate the frontal area of the gravid abdomen and uterus.
The investigation of synchronization is based on the hypothesis that the uterus remains quiescent throughout most of the pregnancy and close to the time of delivery there is an increase in the electrical activity. It is also assumed that during real labor the uterine cells are tightly coupled and activate in a coordinated way. This will give rise to the increased synchronization of the electrical burst activity throughout the uterine wall leading to the uterine contractions. Here, the synchronization between a pair of channels was defined as a statistical tendency to maintain a nearly constant phase difference over a given period of time even though the analytic phase of each channel may change markedly during that time frame [4,5]. From the collected data set one could examine how the phase synchronization changes between the adjacent channels during a contraction cycle. Based on the resolution of the recording device, several channels should pick up this rhythmic activity.
Our preliminary results show for the first time that the spatial and temporal patterns of phase synchronization and averaged magnetic fields change significantly during a contraction cycle. This was investigated in six subjects exhibiting contractions. Subjects had gestational age between 29 and 40 weeks. Synchronization analysis can be used in further studies to determine the propagation of contractions in the uterine wall and also will help in determining the normal or abnormal contractions for clinical work.
Methods
Uterine magnetic field data collection
Procedures for collecting the uterine magnetic field data are described elsewhere [1,2]. A brief summary is given here. Transabdominal MMG recordings of six adult female subjects were collected with the 151-channel SARA (VSM MedTech, Ltd., Port Coquitlam, B.C., Canada) system. Here, the acronym SARA stands for SQUID Array for Reproductive Assessment. The sensors are arranged in a concave array that spans the maternal abdomen and the uterus. A photograph of the system during a routine examination is shown in figure 1 and a layout of the sensors is shown in figure 2. A photograph of the sensor coils is in the left side of the figure. A person sits in front of these coils during data collection. A schematic layout of the primary sensor coils is shown in the right side of the figure 2. In this plot, the radiological coordinates are used where right and left are switched. All 151 primary magnetic sensors are spaced approximately 3 cm apart over an area of about 850 cm2 [1]. The primary sensor coils are of 2 cm diameter. The mother simply sits and leans forward against the smooth surface of the array allowing the SQUID (Superconducting Quantum Interference Device) sensors to receive magnetic signals from the entire maternal abdomen [1,2].
Figure 1 SARA system for measurement of the magnetic activity of the uterine. (Courtesy of CTF Corporation.)
Figure 2 (Left) A frontal photograph of the arrangement of sensor coils. A person sits in front of these coils during data collection. (Right) A schematic layout of the primary sensor coils as seen in front of a subject.
The data of six subjects were collected with the above described SARA system. During these recordings, one of the sensor coils was not in operation due to high noise level. The position of this sensor is identified in figure 2. The magnetic signature at the missing sensor is estimated by averaging the data from the surrounding six sensors.
The subjects recruited in this group, presented themselves at the labor and delivery unit of the hospital complaining of contractions. They were not in active or latent phase of labor and no cervical changes were detected. The study was approved by the Institutional Review Board and a written consent was obtained from each subject. The gestation period of subjects ranged from 29 to 40 weeks. In general, recording sessions ranged from 8 to 12 minutes. For each subject, a continuous 4 minute long data set was selected for synchronization analysis containing at least two contractions. The subject indicated by a finger raise, measured by a light barrier start and end of a perceived contraction. The data was collected with a sampling rate of 250 samples/sec. The data was then downsampled at 25 samples/sec and filtered with a bandpass filter (0.1–0.2 Hz) for further analysis. The bandpass of 0.1–0.2 Hz was selected to extract the signal related to the primary contraction activity of the uterus [1,2]. A montage of 60 second long data set is shown in figure 3. One should note that a typical uterine contraction last 45 to 60 seconds [1] and, generally, MMG activity precedes the perceived contraction.
Figure 3 A montage of the 60 sec long uterine magnetic field data.
Data analysis
The synchronization and decoherence indices between two sensor channels were computed after taking Hilbert transform of the data. The synchronization between a pair of channels was inferred from a statistical tendency to maintain a nearly constant phase difference over a given period of time even though the analytic phase of each channel may change markedly during that time frame [4,5]. The Hilbert transform was applied on the pairs of magnetic field traces (240 second length) with a stepping window of 20 sec duration which is long enough to cover two cycles of the lowest frequency of interest (0.1 Hz). The analysis was repeated by stepping the window at 10 sec intervals.
A typical magnetic field of a channel and the stepping windows are given figure 4. This channel is identified by a circle in figure 3. The top trace shows the temporal pattern of the magnetic field over 240 sec duration. One can clearly see the peaks of the magnetic activity which are related to contraction peaks. Peak contraction activity is very noticeable near to 18, 108 and 175 seconds. There is also an irregular contraction activity between 60 to 100 seconds. The bottom trace is a magnified view with the 20-seconds stepping windows marked on it.
Figure 4 (Top) An example of the magnetic signal in one of the sensing coils, (bottom) magnified view of the signal with 20 second window frames marked on it. The step size for stepping the window is 10 sec. Peaks related to the uterine contractions are very noticeable at 18, 108 and 175 seconds. There is also an irregular contraction activity between 60 to 100 seconds.
The phase of the analytic signal shows a sawtooth pattern which is unwrapped to produce a cumulative linear phase of the signal. The phase differences between the two channels were computed by subtracting the phase of one channel from the other. This phase difference was then used to determine the synchronization and decoherence indices. The mathematical techniques for computing synchronization and decoherence indices are given in detail elsewhere [4-6]. We computed synchronization indices based on Shanon entropy function [5]. Phase locking, i.e., synchronization between the phases of two signals within a stepping window was given by Shanon entropy function, e(t), defined as:
where pi was the relative frequency of finding the phase difference modulus of 2π in the ith bin. The function e(t) varied between zero and its maximum value of emax = ln N. We used 100 bins (N = 100) for the phase difference in a 20 sec stepping window. This phase locking was normalized and called synchronization index, q(t), and is represented as:
The synchronization index, q(t), has a value of zero for uniform distribution of phase differences and a value of one for a spike, or delta, distribution of phase differences between two signals. The decoherence index is defined as the standard deviation of the phase difference between two channels and, in general, varied inversely with the synchronization index [4].
The spatial patterns of the synchronization indices covering the anterior transabdominal area were computed. For this, regional coil-pairs were used. For a given coil, the coil pairs were constructed with the surrounding six coils. This gives us 21 unique combinations of coil pairs to compute the synchronization and decoherence indices for a particular coil. For the coils at the edges, six nearest coils were also used. This provided 21 unique coil pairs for each coil at the edges. The spatial averaging of the synchronization indices was not uniform for the coils at the edges. It was slightly biased towards the inner coils. The spatial averaging of the synchronization indices was uniform for the coils away from the edges.
The synchronization indices were computed for each coil pair. These values were averaged over the 21 coil-pairs and then assigned as the synchronization indices to that particular coil. This analysis was performed for all 151 coils to make 2-D spatial plots of the synchronization indices covering the transabdominal area. The synchronization indices were referenced to a common average reference for plotting and comparative analysis. In the temporal direction, the above procedures were repeated for each 20-sec window.
The RMS (root mean square) magnetic field for each channel in the 20 sec windows is also computed and presented in figures 10 and 11.
Figure 10 Contour plots of the RMS magnetic field in 20 sec windows.
Figure 11 Spatially averaged synchronization indices and the RMS magnetic fields.
For a comparative analysis, the synchronization and decoherence indices were also computed for the randomly shuffled magnetic field data for each channel. It is anticipated that synchronization indices of the randomly shuffled data will be much lower than the unshuffled data. The random shuffling of the time-series data of a given sensor was done by random permutations. For a coil pair, the random shuffling was independently performed for both sensors. All data analysis was performed using MATLAB 7.1 (The Mathworks, Natick, MA., USA) software on a 2.8 GHz Windows Intel workstation.
Results
Two channel synchronization
An example of how the synchronization indices are computed for a pair of channels is given in figures 5, 6, 7, 8. For this we selected two sensor channels in the middle of the transabdominal area which contain moderate level of contraction and relaxation activity of the uterus. The uterine magnetic fields, analytic signal magnitudes and phases are shown in figure 5. For the sake of clarity, this analysis is shown only for the first 100 seconds even though the analysis was performed for the entire 240 sec long data sets. The top row of plots are the uterine magnetic fields of channel 1 and 2. All the left plots are for channel 1 and the right ones are for the channel 2. The middle row of plots are for the magnitude of the analytic signal which was obtained after taking Hilbert transform of the magnetic field data shown in the top row. Similarly, bottom row plots are the unwrapped phases of the analytic signal.
Figure 5 The uterine magnetic fields of two channels in the middle of the transabdomianl area. Also the magnitudes and phases of analytic signal obtained after taking Hilbert transform of the magnetic fields.
Figure 6 The analytic phases of two signals.
Figure 7 (Top) The phase difference between two signals, (middle) temporal derivative of the analytic phase of one signal, and (bottom) temporal derivative of the analytic phase of the other signal.
Figure 8 (Top) Synchronization index and (bottom) decoherence index of two signals. The mean value of the synchronization index of the randomly shuffled data is also shown in the top frame.
The cumulative analytic phase of the MMG data over a 240 sec period was obtained by unwrapping the phase of the analytic signal, a part of which is shown in figure 5. The analytic phases are plotted in figure 6. Both channels have almost a similar slope of approximately 0.96 rad/sec (or 0.15 Hz) over the 240 sec length of MMG data. The phase difference between two channels is plotted in figure 7, top plot. The middle and the bottom plots are for the phase derivatives (rad/sec) of the channel 1 and channel 2 phases, respectively. Over small sections of time windows (~2–4 sec), it can be observed that phase differences between two channels are constant, i.e., horizontal sections of the phase difference plots. In these short time windows, the two channels are in phase coherence, or synchronized. While in other places the phase difference is noticeably changing signifying that two channels are going in and out of phase. This becomes more visible when one looks at the derivatives of the phases of channel 1 and 2 given in the middle and in the bottom plot. As expected, derivative plots are almost flat lines except for squiggles and some well defined sharp spikes. At the spikes, the changes in the phase differences are very large and one would expect a high decoherence near to those spikes. The calculated synchronization and decoherence indices are shown in figure 8. The synchronization and decoherence indices are almost mirror images of each other. The synchronization indices show the maintained level of synchrony between two signals while the decoherence indices emphasize the episodic departure from the synchrony [4].
Spatial Synchronization Patterns
Spatial patterns of the synchronization indices and the RMS magnetic fields are given in figures 9 and 10 respectively. The window frames are identified in figure 3. All plots are scaled to the same scale which is given in window 20. The intensity scale is normalized in the range of zero (blue color) to 1.0 (red color). The red areas in the plots depict higher synchronization. Spatial patterns of the synchronization indices show some correlation with the RMS magnetic field patterns. On a window by window visual comparison, the ones that seem to match are 1, 2, 3, 4, 10, 11, 16, 17 and 20, while those that show poor correlation are 5, 6, 7, 8, 9, 12, 13, 14, 15, 18, and 19. Thus, one could conclude that there is some correlation between the synchronization index and the RMS magnetic field. These plots in figure 9 also show that during contraction larger areas of uterine have higher levels of synchronization as compared to the 20 sec window before and after the contraction. For example refer to the plots in windows 9, 10, 11 and 12. Here, windows 10 and 11 are on the contraction peak as shown in figure 4.
Figure 9 Contour plots of the synchronization indices during 20 sec stepping windows.
The minimum and maximum values of the synchronization indices and the RMS magnetic fields were computed for each window and then averaged over the 20 window values. The average for the minimum values of synchronization indices is 0.3 ± 0.06 and the average for the maximum values is 0.59 ± 0.06. Similarly, for the RMS magnetic fields, the average of the minimum values was 0.153 ± 0.05 pT and the average of the maximum values was 0.346 ± 0.03 pT. Here the minimum values refer to the windows where the uterine contraction activity is minimum or there is no activity. The maximum values should refer to the windows where there is significant uterine contraction activity.
Spatial averages of synchronization indices and the RMS magnetic fields for each window were computed and are given in figure 11. Both show a similar pattern, i.e., higher averaged values at the contraction peaks as compared to the lower values during baseline activity of the uterus. The range of the average synchronization indices was 0.3 to 0.6. The average synchronization indices of all window frames for the randomly shuffled data were also computed. Its value was 0.088 ± 0.04. Compared to this, the averaged synchronization indices in each frame are much higher (0.3–0.6) for the actual data without random shuffling.
Spatial patterns of the synchronization indices and the RMS magnetic fields of all six subjects were also computed. For each subject we identified the windows where the synchronization activity was high and spread in large areas. These windows were considered to contain large areas of uterine contraction. Similarly, windows were identified where the synchronization activity was low and, in general, relate to the relaxation period of the uterus. In a 4 minute long data set, we identified two window frames for the contraction activity and two for the relaxation activity. The averaged synchronization indices and the averaged RMS magnetic fields for these windows were computed and are given in table 1. The mean values of all six subjects are given at the bottom of the table. Synchronization indices and the RMS magnetic fields, both are higher during contraction period as compared to the relaxation period.
Table 1 Average of the of synchronization indices and RMS magnetic fields at the peak of the uterine contraction and at relaxation.
Synchronization Indices RMS Magnetic Fields
Subject No. Contraction Relaxation Contraction Relaxation
1 0.563 0.395 0.305 0.15
2 0.525 0.21 0.190 0.12
3. 0.56 0.35 0.3 0.2
4. 0.56 0.37 0.215 0.15
5. 0.51 0.37 0.28 0.135
6. 0.55 0.38 0.23 0.15
Mean 0.545 ± 0.022 0.346 ± 0.068 0.253 ± 0.048 0.151 ± 0.027
Discussion
Synchronization indices and their spatial distributions depict uterine contractions. The analysis of the two channel data shows that there are definite changes in the synchronization and decoherence indices over a given contraction cycle of 1–2 minute long data. Based on these observed changes, the spatial patterns of the synchronization changes over the transabdominal cavity were computed. These show that the spatial patterns of the synchronization activity and the RMS magnetic fields change during a contraction cycle. One can follow these changing patterns over two contraction cycles in a 240 sec long data set which we have used.
Here we have restricted our analysis to the primary frequency band of the uterine contraction, viz., 0.1–0.2 Hz which likely represents the plateau and repolarization phase of the action potentials. This is a very low frequency band which forced us to use a stepping window of 20 sec length for computing the synchronization indices. It is a long window which smears the uterine biological information over a 20 sec long window. For a more accurate analysis, closer to the real time-frame, a small window size and a small step size is needed. This would require that the uterine magnetic field data in a higher band of 0.3–1 Hz be used for the phase synchronization analysis. This would provide additional information to what we have reported here. If one wants to study the spikelike activity of the initial inward currents of the tissue, probably a higher frequency band (10–100 Hz) will be more appropriate for data analysis.
Spatial patterns shown in figures 9 and 10 do show similarities in few window frames and also show large differences in few other window frames. One would expect a significant correlation between each frame of synchronization indices of figure 9 with that of the RMS magnetic field shown in figure 10. However, there are reasons for these observed differences. The synchronization indices are averaged over 21 regional coil pairs while the RMS magnetic field is not averaged over the nearby coils. It is computed individually for each coil. This could create differences in the spatial patterns as observed in figure 9 and 10. At the peak of the uterine contraction, one would expect a high degree of phase synchronization and this will show up as a good correlation between the synchronization index and the magnetic field spatial plots. During the low-level contraction activity and also during the relaxation periods, uterine magnetic fields could still have a good amount of phase synchronization. This could be one of the main reasons for the observed differences in the corresponding windows of figures 9 and 10.
Magnetomyography offers a new, noninvasive technique for the evaluation of uterine signals. The synchronization index could be an indicator to track the spatial patterns of spread of uterine myometrial activity and thereby increasing our understanding of uterine contractions. In future studies, this application could contribute significantly to the development of methodologies for differentiating true and false labor and prediction of labor thus resulting in better management of pregnancies.
Acknowledgements
This was supported by the NIH grant number 5R33 EB00987.
==== Refs
Eswaran H Preissl H Wilson JD Murphy P Robinson SE Lowery CL First magnetomyographic recordings of uterine activity with spatialtemporal information with a 151-channel sensor array Am J Obstet Gynecology 2002 187 145 151 10.1067/mob.2002.123031
Eswaran H Preissl H Wilson JD Murphy P Lowery CL Prediction of labor in term and preterm pregnancies using non-invasive magnetomyographic recordings of uterine contractions Am J Obstet Gynecology 2004 190 1598 1602 discussion 1602–1603. 10.1016/j.ajog.2004.03.063
Garfield RE Chwalisz K Shi L Olson G Saade GR Instrumentation for the diagnosis of term and preterm labour: review J Perinat Med 1998 26 413 436 10224598
Freeman WJ Rogers LJ Fine Temporal Resolution of Analytic Phase Reveals Episodic Synchronization by State Transitions in Gamma EEGs J Neurophysiology 2002 87 937 945
Tass P Rosenblum MG Weule J Kurths J Pikovsky AS Volkmann J Schnitzler A Freund H-J Detection of n:m phase locking from noisy data: application to magnetoencephalography Physical Review Letters 1998 81 3291 3294 10.1103/PhysRevLett.81.3291
Freeman WJ Origin, structure, and role of background EEG activity. Part 2. Analytic phase Clin Neurophysiology 2004 115 2089 2107 10.1016/j.clinph.2004.02.028
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Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-4-581621909810.1186/1475-925X-4-58ResearchA coarse-to-fine approach to prostate boundary segmentation in ultrasound images Sahba Farhang [email protected] Hamid R [email protected] Magdy M [email protected] Medical Instrument Analysis and Machine Intelligence Group, University of Waterloo, Waterloo, Canada2 Department of Systems Design Engineering, 200 University Avenue West, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada3 Department of Electrical and Computer Engineering, 200 University Avenue West, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada2005 11 10 2005 4 58 58 4 8 2005 11 10 2005 Copyright © 2005 Sahba et al; licensee BioMed Central Ltd.2005Sahba 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 paper a novel method for prostate segmentation in transrectal ultrasound images is presented.
Methods
A segmentation procedure consisting of four main stages is proposed. In the first stage, a locally adaptive contrast enhancement method is used to generate a well-contrasted image. In the second stage, this enhanced image is thresholded to extract an area containing the prostate (or large portions of it). Morphological operators are then applied to obtain a point inside of this area. Afterwards, a Kalman estimator is employed to distinguish the boundary from irrelevant parts (usually caused by shadow) and generate a coarsely segmented version of the prostate. In the third stage, dilation and erosion operators are applied to extract outer and inner boundaries from the coarsely estimated version. Consequently, fuzzy membership functions describing regional and gray-level information are employed to selectively enhance the contrast within the prostate region. In the last stage, the prostate boundary is extracted using strong edges obtained from selectively enhanced image and information from the vicinity of the coarse estimation.
Results
A total average similarity of 98.76%(± 0.68) with gold standards was achieved.
Conclusion
The proposed approach represents a robust and accurate approach to prostate segmentation.
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1 Introduction
Prostate cancer is one of the most frequently diagnosed forms of cancer in the male population and the second cancer-related cause of death for this group [1,2]. Ultrasound imaging is a widely used technology for prostate biopsy. The accurate detection of the prostate boundary in ultrasound images is crucial for some clinical applications, such as the accurate placement of the needles during the biopsy, accurate prostate volume measurement from multiple frames, constructing anatomical models used in treatment planning and estimation of tumor border. These images are the result of reflection, refraction and deflection of ultrasound beams from different types of tissues with different acoustic impedance [3]. However, in ultrasound images the contrast is usually low and the boundaries between the prostate and background are fuzzy. Also speckle and weak edges make the ultrasound images inherently difficult to segment. Furthermore, the quality of the image depends on the type and particular settings of the machine. All these factors make the analysis of ultrasound images challenging. But it still remains an important image modality for clinical applications and an automatic segmentation of these images is highly desirable. This work is organized as follows: In section II existing literature on prostate segmentation is briefly reviewed. The motivation for this work is summarized at the end of section II. Section III introduces the proposed approach and describes its main components in detail. Section IV validates the performance of the method via visual inspection and some quantitative measures. Section V concludes the work.
2 Related work
Currently, the prostate boundaries are generally extracted from TRUS images [3]. As previously mentioned, in TRUS images of the prostate, the signal-to-noise ratio is very low. Therefore, traditional edge detectors fail to extract the correct boundaries. Consequently, many methods have been introduced to facilitate more accurate and automatic or semi-automatic segmentation of the prostate boundaries from the ultrasound images.
Knoll et al. [4] considered deformable contours for prostate segmentation in medical images for both initialization and modeling. They have proposed a method based on a one-dimensional dyadic wavelet transform as a multiscale contour parameterization technique to constrain the shape of the prostate model.
Richard et al. [5] presented an algorithm which segments a set of parallel 2D images of the prostate into prostate and non-prostate regions to form a 3D image of the prostate. This texture-based algorithm is a pixel classifier based on four texture energy measures associated with each pixel in the image. Clustering techniques are then used to label each pixel in the image with the label of the most probable class.
Arnink et al. [6] described a method for determination of the contour of the prostate in ultrasound images. They have used an edge detection technique based on nonlinear Laplace filtering. The method then combines the information about edge location and strength to construct an edge intensity image. Finally, edges representing a boundary are selected and linked to build the final outline.
Ladak et al. [7,19] proposed an algorithm for semi-automatic segmentation of the prostate from 2D ultrasound images. The algorithm uses model-based initialization and a discrete dynamic contour. First, the user must select four points around the prostate. Then the outline of the prostate is estimated using cubic interpolation functions and shape information. Finally, the estimated contour is deformed automatically to better fit the image. This semi-automatic algorithm can segment a wide range of prostate images, but at least four initial points must be selected manually by the user (radiologist).
Prater et al. [8] presented a method for segmentation of the prostate in transrectal ultrasound images based on feed-forward neural networks. This method segments images to prostate and non-prostate regions. Three neural network architectures have been proposed. These networks are trained using a small portion of a training image segmented by an expert and then applied to the entire training image.
Wang et al. [30] presented two methods for semiautomatic three-dimensional (3-D) prostate boundary segmentation using 2-D ultrasound images. The segmentation process is initiated by manually placing four points on the boundary of a selected slice. Then an initial prostate boundary is determined. It is refined using the Discrete Dynamic Contour until it fit the actual prostate boundary. The remaining slices are then segmented by iteratively propagating the result to another slices and implementing the refinement.
Hu et al. [31] proposed an algorithm for semiautomatic segmentation of the prostate from 3D ultrasound images. In this method the authors use model-based initialization and mesh refinement using deformable models. Six points are required to initialize the outline of the prostate using shape information. The initial outline is then automatically deformed to better fit the prostate boundary.
Chiu emphet al. [32] introduced a semi-automatic segmentation algorithm based on the dyadic wavelet transform and the discrete dynamic contours. In this method first a spline interpolation is used to determine the initial contour based on four user-defined initial points. Then the discrete dynamic contour refines the initial contour based on the approximate coefficients and the wavelet coefficients generated using the dyadic wavelet transform. A selection rule is used as well to choose the best contour based.
Abolmaesumi et al. [33] used a segmentation technique to extract prostate contours from Transrectal Ultrasound images. In this method an Sticks filter is used to reduce the speckle. The problem is then discretized by projecting equispaced radii from an arbitrary seed point inside the prostate cavity towards its boundary. Candidate edge points obtained along each radius include the measurement points and some false returns. This modelling approach is used for prostate contour extraction.
Ghanei et al. [9] proposed a three-dimensional deformable surface model for prostate segmentation based on a discrete structure which is made from a set of vertices in the 3D space as triangle facets. The model converges from using a weighted sum of the internal and external forces. The model is initialized manually from a few human-drawn polygons drawn on different slices.
Pathak et al. [10] proposed an algorithm for guided edge delineation which provides automatic prostate edge detection as a visual guide to the observer. It is followed by manual editing. The edge detection algorithm contains three stages. First, the sticks algorithm is used to enhance contrast and reduce speckle in the image. Second, the resulting image is smoothed using an anisotropic diffusion filter. Finally, some basic prior knowledge of the prostate, such as shape and echo pattern, is used to detect the most probable edges which indicate the prostate shape. In the last stage, the information is integrated by using a manual linking procedure on the detected edges.
Shen et al. [11] introduced a statistical shape model to segment the prostate in transrectal ultrasound images. First, a Gabor filter bank is used in both multiple scales and multiple orientations to characterize the prostate boundaries. The Gabor features are reconstructed to be invariant to the rotation of the ultrasound probe. Then, a hierarchical deformation strategy is used. The model focuses on the similarity of different Gabor features at different deformation stages using a multiresolution technique. The authors have also introduced an adaptive focus deformable model, which uses the concept of an attribute vector [12].
In another work Gong et al. [28] presented an approach based on deformable models. In this technique, model initialization and constraining model evolution are based on prior knowledge about the prostate shape. The prostate shape has been modeled using deformable superellipses.
Betrouni et al. [29] discussed a method for the automatic segmentation of trans-abdominal ultrasound images of the prostate. In this method a filter is used to enhance the contours without changing the information in the image. Adaptive morphological and median filtering are employed to detect the noise-containing regions and smooth these areas. Then a heuristic optimization algorithm begins to search for the contour initialized from a prostate model.
Generally, prostate segmentation methods have limitations when the image contains shadows with similar gray level and texture attached to the prostate, and/or missing boundary segments. In these cases the segmentation error may increase considerably. Another obstacle may be the lack of a sufficient number of training (gold) samples if a learning technique is employed. Algorithms based on active contours have been quite successfully implemented with the major drawback that they depend on user interaction to determine the seed points (initial snake).
Based on an analysis of the existing literature a new approach should ideally possess certain properties:
• User interaction (e.g. defining seed points) may not be always desirable due to its drawbacks such as time consumption, human error etc. The new technique, therefore, should require a minimum level of user interaction.
• Providing a large number of training samples in medical environments is generally difficult, specially if the samples are being prepared by an expert. Hence, sample-based learning should be avoided.
• The approach must be robust with respect to the presence of noise and shadow.
In this paper, by introducing a multi-stage, coarse-to-fine approach, we establish a straightforward algorithm which attempts to satisfy the above conditions as much as possible. In this method some input parameters must be adjusted for series of images (i.e. images captured with a certain equipment setting).
3 Proposed approach
In this section, we will present a new region- and grayscale-based approach to prostate segmentation in ultrasound images. The proposed approach contains four main stages (see Fig. 1):
Figure 1 Schematic diagram of the proposed approach.
• Pre-Processing: After smoothing, using a locally-adaptive contrast enhancement technique, a primary version of the image which has sufficient contrast across the image is produced (see section 3.1).
• Coarse Segmentation: The obtained image is thresholded and some morphological operators are applied until an isolated object related to the prostate is produced. The central point of this object can be considered as the central point of the prostate. Subsequently, using a Kalman estimator, a coarse estimation for the prostate boundary is obtained. This coarse estimation is not accurate, but we just use it as input for the next stage (see section 3.2).
• Selective Enhancement: Using coarse estimation, one eroded and one dilated version can be produced. These are used to establish regional fuzzy membership functions. By defining a fuzzy inference system based on the produced membership functions, a selective contrast enhancement can be obtained in the area of the prostate (see section 3.3).
• Prostate Segmentation: Finally, the algorithm finds potential boundary pieces and extracts the prostate boundary (see section 3.4).
In the following sections, each stage of the proposed approach will be described in detail.
3.1 Pre-processing
Transrectal ultrasound images are heavily corrupted with noise. Since we just need a rough estimation for the next stage, we can remove the noise using a median filter (7 × 7 or 9 × 9). It should be mentioned that this smoothed version is only used for the next step. For the final segmentation we use the "original" ultrasound image. Therefore, the median filter does not manipulate the edge information needed for the final segmentation. Fig. 2 shows the original TRUS and its smoothed version.
Figure 2 Top: Original image, Bottom: Smoothed image using a median filter 9 × 9.
In order to enhance the contrast of the image, a locally-adaptive method based on fuzzy sets has been used to create a primary version which has enough contrast [13,14,25].
In a fuzzy approach, an image can be presented as an array of fuzzy singletons (a membership function with only one supporting point). Each of these singletons has a value that denotes the membership of each pixel to a predefined image property. The properties can be defined globally for the whole image or locally for sub-images [13]. In the case of contrast enhancement using fuzzy techniques, some parameters of spatial domain, such as minimum gray-level gmin or maximum gray-level gmax, are needed. In a global method, finding those parameters is simple, but if we want to enhance the image in a locally approach, then we must calculate these parameters for each local neighborhood to adjust the membership function. Tizhoosh et al. [13] have presented a locally adaptive approach to find the parameters for some support points for an M × N-image and interpolate these values to obtain corresponding values for each pixel. It is clear that these interpolated parameters are not precise, but because the concept of fuzziness is incorporated, the input data have not to be exact [13,14]. In the method proposed in [13], first the image is divided into MS rows and NS. This leads to a matrix with the size of MS × NS. Each element in this matrix corresponds to a supporting point for the image. A window around each supporting point is considered to find the local information for that point. In a general case the size of window around each supporting point depends on the degree of homogeneity or edginess of local pixels in the corresponding sub-image. Small window sizes do not only increase the computational cost but also sometimes fail to capture correct information due to noise sensitivity. On the other hand, with large window sizes, values obtained by interpolated parameters lose their accuracy. In our application we use constant window sizes around each supporting point to find the local information. Just to avoid loss of information during the interpolation, the size of windows is chosen in a way that they have enough overlap with each other. Using the local windows we can extract local parameters, like gmin,local and gmax,local. Applying a 2-D interpolation function (e.g. linear or cubic), the membership function parameters can be calculated for the whole image [13].
To apply this method in our case we have used a maximum window size of 40 × 40 and a minimum window size of 20 × 20, both determined heuristically. The algorithm calculates a proper window size between these two values using fuzzy rules. Among methods introduced in [13] we have employed a fuzzy rule-based approach to enhance the contrast for the entire image [13-16]. The input membership functions used in this fuzzy rule-based approach are shown in Fig. 3.
Figure 3 Input membership function for locally adaptive contrast enhancement. It represents three terms for the fuzzy set "pixel intensity". Generally b is in the middle value of LocalMin and LocalMax, a is the middle values of LocalMin and b, and c is the middle value of LocalMax and b.
For the output membership function we have used three singleton values gS1 = 10, gS2 = 100 and gS3 = 255.
The result of applying the above method on the smoothed image followed by global thresholding on the enhanced image are shown in Fig. 4 and 5, respectively. As we can see in Fig. 4, applying the primary contrast enhancement makes a significant gray level difference between the dark areas, including prostate, and bright gaps around the prostate.
Figure 4 Primary high contrast version of the smoothed image from Fig. 2.
Figure 5 (a): The result of global thresholding on primary high contrast version in Fig. 4, (b): Filled version of the global thresholded image.
For global thresholding, all gray levels g less than a threshold value T are set to zero and those greater than this threshold are set to 255 such that a binary image with black and white values g' is obtained. Fig. 5(a) shows the result of applying global thresholding to the image in Fig. 4. The main advantage of applying the primary contrast enhancement is that the bright gaps produced around the prostate have distinctly large gray level intensities. Consequently, an image with a bimodal histogram is produced. T is chosen as a middle value between two peaks in the histogram and for a large category of ultrasound images, good performance of global thresholding can be expected.
3.2 Coarse estimation
This section demonstrates how a central point inside the prostate can be found. We will see that using this point and some other information how a Kalman estimator achieves a coarse estimation of the prostate boundary.
The binary image obtained after thresholding contains several black holes (Fig 5(a)). These holes can be filled such that a completely white region is archived (Fig 5(b)) [17]. Applying the primary contrast enhancement stage ensures the extraction of a white area corresponding to the prostate in the thresholded image. This area must be isolated from other white regions. One straightforward method uses the morphological opening [17]. This operator must be applied with a large structuring element to isolate the object corresponding to the prostate. Considering the geometrical location and the measurement of area, the isolated object with high likelihood to be part of the prostate can be distinguished. A point O inside the prostate is required to perform the next stage. This point can be simply obtained by finding the centroid point of this isolated object.
Now we want to find a coarse estimation of the prostate boundary. This can be solved using a Kalman filter [18] for tracing the edges. We can use some properties of this filter to extract the valid data from the thresholded image and remove the irrelevant parts. To implement such a system we interpret the problem of edge tracing as a dynamic tracking system. In this system the pixels located on the edge of an object of interest are used as the input (measurement data) for the tracking filter. Using such a system the Kalman filter can track a trajectory along the border of the object. Each new pixel along the border brings updated information for the current and future position. We assume each pixel along the border to be in a dynamic movement. For this movement we can consider the position and velocity as the variables which describe the state of the system. Using a Kalman filter we can estimate the position which is actually the border of the object of interest. In fact we use a tracking system to follow the border. In a 2D image we have two variables for position and two variables for velocity. Because of the shape of the prostate it is more efficient to represent the state variables in the form of relative polar coordinates. We can use the internal point O to define such state variables and consider the following state vector:
where r is the distance between the internal point O(xc, yc) and pixels (xp, yp) located on the border of the prostate and θ is the angle between the vertical axis and . Hence, the following equations can be considered for r, θ, and :
where , are the radial and angular velocity, respectively. Using the above state vector we represent the sequential pixels on the border of the object of interest (corresponding to the movement of vehicle on a path) by radial and angular position and velocity. Kalman filter considers a discrete dynamic model contains state and measurement equations:
xk = Ak-1xk-1 + Bk-1Wk-1, (6)
Zk = Hkxk + Vk, (7)
where x is the state vector in equation 1. Other components are defined as follows:
where Wk ~ N(0,Qk) and Vk ~ N(0, Rk) are the process and measurement noise, respectively. T is the interval which represents the changes in the state and measurement equations. The value of T does not affect the final result and therefore we can choose it as T = l for simplicity. R and Q are diagonal matrices. The values in these matrixes are the measurement and process noise covariance, respectively. In the area that border is changing smoothly the measurement data is placed inside the association gate and the value of measurement noise R should be small. In the case when there is no data inside the association gate we cannot be ensure about the validity of measurement data. Therefore the value of measurement noise R should be large. The large and small values for the measurement noise depend on the quality of image and can be adjusted between 5 to 100. The value of process noise in matrix Q simulates the small variation around the estimated point. For process noise we can consider a value between 4 to 8. In the discrete dynamic equations the accelerations in the radius and angle (the changes of radial and angular velocities) is modeled as a zero-mean, white, Gaussian noise W. Also the measurement data Zk which is the location of boundary pixels in the form of r and θ is assumed to be a noisy version of the actual position of the boundary. Kalman filtering is done using prediction and update steps. The prediction equations include
xk|k-1 = Ak-1xk-1|k-1, (11)
whereas update equations can be given as follows
Kk-1 = Pk|k-1Hk-1S-1, (14)
xk-1|k-1 = xk-1|k-2 + Kk-1[Zk-1 - Hk-1xk-1|k-2], (15)
where P is error covariance matrix, K is kalman gain and S is innovation covariance matrix. For calculation of the coarse version, the Kalman filter receives its initial data from the nearest point placed on the vertical axis and on the top of the internal point O. Then it starts the estimation using the data located on the border of the object in the thresholded image. In each sequential iteration, the points along the prostate border can be used as measured data and the Kalman filter estimates the next r and θ. These predicted values determine a pixel as the next pixel on the border. Also it predicts and for the next iteration. When we go to the next iteration the new pixel on the border is the new measured data for the filter. This data is compared to the predicted position from the previous iteration. If there is sufficient correlation between them the measured data is incorporated to update the filter state, otherwise the prediction point is considered as measured point and after updating the filter starts the next iteration. To measure the correlation we implement an association process between the predicted and measured data. For this association process we use a gate, the so-called "association gate" around the predicted pixel. Only the pixels located on the border and inside of this gate are considered as valid measured data for updating the filter. For good performance, the association gate must be adaptive. This means that the size of this gate must be varied based on the covariance of the Kalman filter. The gate must maximize the presence of valid data and minimize invalid data. To find such a gate, the probability ratio function, L, at the kth iteration must be maximized [18]:
where p1(k) and p0(k) are the probabilities of the presence of valid data and invalid data in the gate, respectively. This prompts us to check the value of a statistical distance d with respect to a threshold D as follows [18]:
d2 ≤ D, (17)
with
d2 = lTP-1l, (18)
where l is the vector of geometrical distance between the predicted point and an arbitrary point l = (l1, l2), and P is the covariance matrix of the Kalman filter. In two dimensional problems, equation 18 is reduced to the following form:
where a, b, c and e are the values of covariance matrix. This is an equation for the points inside of an ellipse. The value of D is generally a constant between 1 to 10. In the case when there is no data inside the association gate we need to capture the data. Therefore, the value of D should gradually increase. Fig. 6 illustrates this local polar coordinate schema and elliptical gating.
Figure 6 local polar coordinate schema and elliptical gating for extracting the coarse estimation.
When the border reaches a shadow, or a missing boundary segment is encountered, then there is an abrupt change in the pixel path. These sharp changes are considered as new paths for tracking process. After a few iterations Kalman filter detects that such cases do not belong to the true path because the data do not correlate with the followed path. The filter adaptively enlarges the association gate until it again captures the true data on the border which have enough correlation with the prediction. Using the association technique the data belonging to shadows and missing segments on the prostate border are detected and eliminated. During the process, Kalman estimator follows the prostate border variations in a coarse manner and irrelevant parts are isolated. Fig. 7 shows this coarse estimation for the prostate.
Figure 7 Isolated object corresponding to the prostate and its center.
In the most parts, the gray level difference produced by primary contrast enhancement makes the prostate border distinguishable after thresholding. Applying the Kalman estimator ensures that the prostate is isolated from any irrelevant parts and a coarse estimation is achieved. An interesting result is that if the quality of ultrasound images are good enough there is no irrelevant parts attached to the prostate and the boundary can be visible after primary contrast enhancement and thresholding. In these cases we do not need to employ the Kalman filter and coarse estimation may even be used as the final segmented image.
The quality of this coarse estimation directly depends on the quality of the ultrasound image in terms of original contrast, the presence and intensity of shadow, the noise and so on. For all tested images the extracted coarse estimation had sufficient accuracy to be passed to subsequent stages.
3.3 Selective enhancement
In this section a new regional approach is introduced to increase the prostate contrast. This will amplify the strength of the outer prostate edges.
The previous section contained some techniques to find a coarse estimation. Using the boundary generated by the coarse estimation, two contours can be obtained such that the true boundary is ideally located between them. In our approach, these are called inner and outer contours. For extracting the inner contour, an erosion operator with a disk-shaped structuring element can be employed on the coarse estimation. With the same structuring element and using a dilation operator, the outer contour can be obtained as well. These two contours are employed as points of departure to define a membership function.
This membership function determines to what degree does a pixel belong to the prostate. Fig. 8 illustrates the membership function μlocation based on pixel position. Because the boundary of the coarse estimation is extracted from the object corresponding to the prostate, it can be assumed that the true edges of the prostate are located around this boundary. If the pixel is located outside of the outer contour, the membership value is 0, if it is inside of the inner contour, the membership value is 1. For the pixels in between, the nearest distance from the inner and outer contours is determined and the value of membership is calculated. Because the pixels located between the inside contour and the coarse estimation boundary most likely belong to the prostate, the membership function has more emphasis on this interval.
Figure 8 the membership function μlocation based on pixel position. In this Figure and dinsideand doutside are the shortest euclidian distance to the outside and inside contours respectively.
Similarly, this function has less emphasis for the pixels located between the coarse estimation boundary and the outer contour because the pixels located around the outer contour most likely do not belong to the prostate. In fact, the membership value μ depends on the following equation:
where α = 6, β = 0.5 (heuristically selected parameters for the sigmoidal function) and the value of d is calculated as follows
where dinside and doutside are the shortest euclidian distance of arbitrary points to the outer and inner contours, respectively. For the establishment of a fuzzy inference system, we need to define appropriate fuzzy rules [13,14]. The area between the two contours is important for the extraction of the prostate boundary. Since we assume that there is generally a dark to light transition from the inside to outside of the prostate, any algorithm improving the strength of edges in this area is useful. A straightforward method is to make the dark and gray pixels darker and bright pixels brighter. This can increase the strength of the edges around the prostate boundary. In addition, as previously discussed, we want to enhance the contrast just for the area within the prostate. Therefore, simple rules can be defined as follows:
• IF the pixel does not belong to the prostate, THEN leave it unchanged
• IF the pixel belongs to the prostate AND is dark, THEN make it darker
• IF the pixel belongs to the prostate AND is gray, THEN make it dark
• IF the pixel belongs to the prostate AND is bright, THEN make it brighter
The last rule is mainly designed to enhance the brighter boundary pixels (bright pixels within the prostate are not relevant at this stage). The membership function for input gray levels is shown in Fig. 9. In this figure T4 is the brightest gray level in the image. T1, T2 and T3 can be calculated based on local information, but for simplicity we have used the values 25, 50 and 80, respectively. For the output membership function, fuzzy singletons with the values GS1= 1, GS2 = 64 and GS3= 255 are defined empirically.
Figure 9 Membership functions for input gray level values.
Using these rules we enhance the contrast of the image not only based on gray level values but also based on the location of each pixel. The result is shown in Fig. 10. It clearly demonstrates that the result is an image with higher contrast in the prostate area.
Figure 10 Top: Original image. Bottom: Enhanced Image using proposed fuzzy inference.
3.4 Final segmentation
In the previous section we achieved an image with a highly contrasted prostate area and almost no changes in other regions such that strong edges were created in the boundaries of the prostate. Also we know that the true boundary is located between the inner and outer contours and somewhere most likely close to the coarse estimation. Therefore, we must first detect the edges located between these two contours. For this purpose, a Canny edge detector is used [21]. Fig. 11 shows the result for the area between two contours. This image contains many potential boundary pieces. Among these pieces, those with higher likelihood of being a true edge should be considered. We start from a point above the internal point O, traverse along the coarse boundary and consider small pieces (3 to 5 pixels) on this boundary, sequentially. For each edge segment on the coarse boundary there are several edge pieces. We use three criteria to extract the final edges:
Figure 11 The result of applying Canny edge detection on the enhanced image (Fig. 10). Solid line is the coarse estimation outline.
1. The average distance of N candidate pixels in the vicinity of the coarse boundary – we can calculate the vicinity distance by the following equation:
where di is the distance of ith pixel with respect to the boundary of the coarse estimation.
2. The value of the gradient – In the preceding section, we enhanced the contrast of the prostate area. This increases the local gradient values of the pixels located on the true boundary pieces. The local gradient value in 3 × 3 neighborhoods is determined as:
where Gx and Gy are the gradient values in x and y directions, respectively.
3. The angle of edge pieces with respect to the coarse boundary – we can calculate the absolute value of angle between the coarse boundary piece and edge piece. This value can vary from 0 to π/2. Zero radians reflects the most and π/2 the least compatibility.
After these criteria are considered in the presented priority order, the final boundaries must be extracted from potential pieces. Using the above criteria we must extract the edge pieces which have the greatest likelihood of being a true edge. Among all pieces, a piece with minimum distance, maximum gradient, or minimum angle with respect to the boundary of the coarse estimation should be chosen. If we consider the border of the coarse version where there was no shadow and missing boundary segment, the information of the coarse version is more reliable. Therefore, in these parts our criteria for choosing an edge piece is the distance of that piece with respect to the coarse version. If the distances are equal, then the angles of pieces are considered. If the angles are equal as well, then the gradient values are compared. But in the parts where the Kalman filter has estimated the border of the coarse estimation the strengths of the edge pieces (using gradient values) are the only criteria for the selection. Subsequently, the algorithm goes directly to the next piece and continues the procedure until the complete prostate outline is achieved. For the coarse boundary pieces, if there is no edge piece around them we can continue the edge extracted in the previous coarse boundary piece so that it has the minimum angle with respect to the coarse estimation. A straightforward way to fill the gaps between adjacent pieces is to fill them by straight lines. Finally, we can again apply a Kalman filter to smooth these edges and make a consistent outline for the prostate to achieve the final result. This smoothing filter is just like what we used to find the coarse estimation except that it uses all final data on the prostate border.
A sample result of employing the proposed approach on the extracted edge is shown in Fig. 12. This is the final result for the prostate segmentation. This figure shows the outline obtained manually by a radiologist (solid line). Visually, the difference between the two contours is negligible. To evaluate the algorithm for a low quality case, we have applied the proposed method in the image shown in Fig. 13. As we can see, the segmentation in the strongly shadowed areas have some error but in the other areas, the result is almost the same as manual segmentation.
Figure 12 Automatic and manual boundaries are shown on the original images. Solid lines are manually segmented images and dash lines are the result of proposed algorithm.
Figure 13 A low quality TRUS image and the result of automatic and manual boundaries shown on the original images. Solid lines are manually segmented images and dash lines are the result of proposed algorithm.
4 Experiments and results
Other algorithms mentioned in section 2 have used different numbers of ultrasound images (from 8 [11] to 90 [7,19]) to validate their approaches. They have compared the algorithm-based segmentations with the manual segmentations (as a gold standard). We have selected the images to include regular and difficult cases and examined 42 different TRUS images. The images were noisy, with low contrast and shadow effects. The results of the proposed method have been evaluated by comparing the algorithm-based segmentation and the manual segmentation (gold standards).
As it was mentioned throughout the paper some parameters need to be adjusted in this algorithm. But for a set of similar TRUS images (i.e. images captured with a certain machine setting), most of them do not require any change. In the conducted experiments, the following parameter configurations have been used:
• The size of the median filter used for smoothing was 7 × 7.
• The structuring element used in the opening procedure was a disk with diameter 15.
For quantitative evaluation, we have used the following error measures to validate the performance of our segmentation compared to the manual segmentation by a radiologist for those images [22]:
Distance δ = Average Euclidean distance (in pixels) between the algorithm-based segmentation and the manual segmentation. For each pixel the distance is defined as the shortest Euclidean distance between that pixel and the pixels located on the other contour.
Area EA = Area error (%) = 100.,
where SMan is the area of the manual segmentation and SAlg is the area of the algorithm-based segmentation.
Similarity In addition, we have used a similarity measure, η, based on the misclassification rate as a more general criterion in image segmentation [23,24]:
where B0 and F0 denote the background and foreground of the original image (manually segmented), BT and FT denote the background and foreground area pixel in the result image, and |.| is the set cardinality.
Table I summarizes the results along with the average, m, and standard deviation, σ, for the three quantitative measures for all 42 test images.
Table 1 Quantitative evaluation of proposed approach in comparison with manual segmentation (gold standard). The average m and deviation σ for three performance measures are calculated for 42 test images and their corresponding gold standards.
Number δ EA η
m 3.67 5.62 98.76
σ 1.08 2.98 0.68
Considering the quality of the images in terms of the shadow effect, the quantitative results are promising. From the results, it can be seen that in the areas that there is no shadow the proposed method is able to deliver very accurate results. In the areas, in which strong shadows cover the prostate the result of the algorithm is still acceptable. Of course, in these areas error is increased because the gray level and the texture of the shadow is very similar to those of the prostate. Qualitatively, the difference between the algorithm-based segmentations and the manual segmentations is not considerable. The proposed approach, implemented in MatLab, needs (in average) less than 10s to segment the prostate using a 2.8 GHz Pentium IV. However, this time measurement is based on experimental setup. It can be expected that a considerable speedup can still be achieved if the algorithm is implemented and optimized in realtime platforms such as C++.
5 Conclusion
A novel approach to prostate segmentation containing a coarse estimation and a new selective fuzzy contrast enhancement model has been presented in this paper. Because of the characteristics of the ultrasound images, we first smooth the original image using two filters. This smoothed image is enhanced using a locally-adaptive contrast technique. The output image has large gaps with high intensity around the prostate. Using global thresholding and morphological operators, an isolated object containing the prostate was obtained. The center of this object was considered as an internal point of the prostate. A Kalman estimator with the polar coordinates was implemented to find a coarse estimation of the prostate border. Using erosion and dilation of this estimation, inner and outer contours were obtained. A fuzzy inference system based on these regional contours and spatial gray level information was designed to selectively enhance the contrast of original image within the prostate region. The output of this fuzzy enhancement system provided an image with high contrast and strong edges on the prostate borders. Finally, the edges between inner and outer contours were extracted. In order to correctly recognize the prostate boundaries, potential boundary pieces were marked and based on pixels gradients, the vicinity and angle relative to the coarse estimation boundary, the final segmentation was achieved. The proposed approach has been examined for typical TRUS images. In comparison with manually segmented images, the experimental results show that our approach can segment the prostate boundary accurately. A total average similarity of 98.76%(± 0.68) with the gold standards was achieved. The test images contain not only regular but also noisy, low-contrasted and shadowy cases. In this approach we have designed a straightforward and fast algorithm with minimum level of user interaction. It also does not need training samples for implementation. More samples containing the manual prostate segmentation by radiologists are desired in order to verify the segmentation accuracy more reliably. In addition, by using an adaptive approach for noise reduction, we can improve the image quality, especially the prostate edges. This can give us better results in subsequent stages. In addition, developing of a similar technique for 3D prostate segmentation can be a subject for further work.
6 Authors' contributions
Farhang Sahba developed the main parts of the approach. Hamid R. Tizhoosh provided guidance and was involved in the theoretical aspects of algorithm development. Magdy M. Salama provided guidance. All authors read and approved the final manuscript.
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Cardiovasc DiabetolCardiovascular Diabetology1475-2840BioMed Central London 1475-2840-4-151619755610.1186/1475-2840-4-15Original InvestigationCircadian rhythm of autonomic activity in non diabetic offsprings of type 2 diabetic patients Fiorentini A [email protected] A [email protected] A [email protected] P [email protected] L [email protected] III Clinica Medica, Department of Clinical Medicine, University "La Sapienza", Rome, Italy2 Medicina Interna E, Department of Clinical Medicine, University "La Sapienza", Rome, Italy2005 1 10 2005 4 15 15 1 8 2005 1 10 2005 Copyright © 2005 Fiorentini et al; licensee BioMed Central Ltd.2005Fiorentini 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 aim of the present study was to evaluate, by heart rate variability (HRV) with 24-hours ECG Holter (HRV), the circadian autonomic activity in offspring of type 2 diabetic subjects and the relation with insulin-resistance. METHODS: 50 Caucasian offsprings of type 2 diabetic subjects were divided in two groups: insulin-resistant offsprings (IR) and non insulin-resistant offsprings (NIR). Autonomic nervous activity was studied by HRV. Time domain and spectral analysis (low frequency, LF, and high frequency, HF, provide markers of sympathetic and parasympathetic modulation when assessed in normalized units) were evaluated. RESULTS. Time domain showed a reduction of total SDNN in IR (p < 0.001) and NIR (p 0.047) versus controls. Spectral analysis showed a total and night LF higher in IR and NIR than in control group (all p < 0.001). CONCLUSION. In frequency domain, the analysis of sympathetic (LF) and parasympathetic (HF) component evidenced an association between the offspring of type 2 diabetic subjects and a sympathetic overactivity. A global reduction and alteration of circadian rhythm of autonomic activity are present in offspring of type 2 diabetic patients with and without insulin resistance. The data of our study suggested that an autonomic impairment is associated with the familiarity for type 2 diabetes independently to insulin resistance and that an impairment of autonomic system activity could precede the insulin resistance.
heart rate variabilitycircadian rhythminsulin resistance
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Background
Sympathetic and parasympathetic components of neurovegetative system regulate cardiac activity. Spectral analysis of heart rate variability (HRV) is a non invasive metodic used to assess cardiac autonomic activity. The autonomic activities can be assessed in HRV by the relative distribution (evaluated in normalized units) of low frequency (LF), as an index of sympathetic modulation and of high frequency (HF), as an index of parasympathetic modulation. The analysis of HRV has been used for evaluate the autonomic cardiac activity in numerous pathophisyologic conditions [1-9]: an impaired heart rate variation is a marker of autonomic neuropathy [10,11] as in diabetic subjects [12-16].
Several studies have showed a sympathetic overactivity also in non diabetic insulin resistant group [17-19]. However, little is known about the association between the familiarity for type 2 diabetes mellitus and the autonomic activity. An increased LF/ HF ratio (low frequency/high frequency ratio) has identified in only insulin resistant offsprings of type 2 diabetic subjects [20]. De Angelis et al [21] has showed this condition at rest but not under stimulated conditions. Laitinen et al [22] has identified a sympathetic overactivity during acute hyperinsulinemia both in insulin resistant and non insulin resistant offsprings of type 2 diabetic subjects.
The aim of the present study was to evaluate, by heart rate variability with 24-hours ECG Holter registration, the autonomic activity and circadian autonomic rhythm in offspring of type 2 diabetic subjects and to evaluate the possible impact of sympathetic activity on insulin resistance. We tested the hypothesis that in non insulin resistant offsprings of type 2 diabetic subjects an alteration of circadian rhythm of autonomic activity are present and that the impairment of autonomic system activity could precede the insulin resistance.
Subjects and methods
70 consecutive caucasian offsprings of type 2 diabetic subjects, admitted to our department, were screened. In all subjects, after an overnight fast, oral glucose tolerance tests (OGTTs) was performed: samples blood for glucose and plasma insulin were collected before and 2 h after a glucose load consisting of 75 g glucose anhydrate in 300 ml of water ingested over the course of 5 min. Also, fasting plasma insulin was measured to evaluate the insulin-resistance by the homeostasis model assessment-index (HOMA-I). Among them, 50 caucasian subjects (age: 47.71 ± 9.96 years, 32 men and 18 women), with normal OGTTs, were admitted in this study and were divided in two groups: offsprings with insulin resistance and offsprings without insulin resistance:
Subjects with hypertension [23], diabetes mellitus, impaired fasting glycemia, impaired glucose tolerance [24], obesity, dyslipidemia, cardiac arrhythmias, microalbuminuria and with drug treatment or diseases that could potentially disturbs carbohydrate metabolism (glucocorticoids, furosemide, beta-blockers, etc.) and cardiac autonomic activity (beta-blockers, anti-arrhythmics, ACE-inhibitors) were excluded.
The control group consisted of 25 sex and age matched healthy non insulin resistant subjects with normal OGTTs and without familiarity for type 2 diabetes mellitus.
Height, weight and body circumferences were measured on all subjects. Body mass index (BMI, kg/m2) was calculated as weight divided by height squared. Waist-to-hip ratio (WHR) was defined as waist circumference divided by hip circumference.
Informed consent was obtained from all participants; all the investigations were performed in accordance with the participants of the Declaration of Helsinki.
Insulin resistance
The insulin-resistance was evaluated by the homeostasis model assessment index (HOMA-I) [25-28]. The HOMA-I was calculated by the formula: fasting plasma glucose (mmol/L) x fasting plasma insulin (μU/ml)/ 22,5, as described by Matthews and coworkers [29]. Insulin-resistance was defined as the third and fourth quartiles of HOMA-I.
The index subject were subdivide into two groups based on HOMA-I: 1) group of insulin-resistant offsprings (IR); 2) group of non insulin-resistant offsprings (NIR).
HRV assessment
Autonomic nervous activity was evaluated by heart rate variability (HRV) analysis during 24-hour ECG recording. All Holter recordings were performed using a three-channel recorder. Autonomic nervous activity was analysed following the recommendations of the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology [30]. Spectral estimates of R-R interval were obtained from stationary regions free of ectopic beats and technical artefacts. The standard deviation of normal-to-normal RR intervals (SDNN) (ms) and the square root of the mean of the sum of the squares of differences between adjacent NN intervals (RMS-SD), correlated with parasympathetic system, were calculated and were divided in two periods: night (0 am – 6 am) and day (7 am – 9 pm). Fast Fourier Transform was used to obtain power spectral estimates of HRV. Total power in the frequency range (0 – 0.40 Hz) was divided into low frequency (LF: 0.04 – 0.15 Hz, modulated by sympathetic system) and high frequency (HF: 0.15 – 0.40 Hz, modulated by parasympathetic system). The power of LF and HF components was considered in normalized units (n.u.). Subjects were analysed for 24 hours, at 10 minutes interval. Artificial data and arrhythmic were excluded. The day was divided in four periods: night (0 am – 6 am), morning (7 am – 12 am), afternoon (1 pm – 6 pm), evening (7 pm – 11 pm). Data analyses were performed with software Del Mar Avionics Accuplus 363, Irvine California, USA.
Statistical analysis
All analysis were done with SPSS 12.0 (SPSS Inc., Chicago, IL, USA) for Windows XP. Data are presented as means + SD. For data with multiple time points, variables were analysed by the general linear model ANOVA and simple regression analyses were carried out by standard techniques 95% confidence intervals (CT) were calculated for regression coefficient. Means values were considered significant at p < 0.05.
Results
Clinical characteristics
The groups had not statistically significant difference in age, sex and anthropometrics parameters (i.e. BMI, waist and hip circumference, waist to hip ratio, table 1).
Table 1 Clinical characteristics for each study-group of offsprings and controls.
IR NIR Controls p Value
Sex (M/F) 16/9 18/7 16/9
Age (years) 49,12 ± 6,68 49,80 ± 6,53 53,69 ± 3,72 0,190
BMI (kg/m2) 25.41 ± 4.67 25.50 ± 3.69 25.09 ± 2.23 0,120
Glycemia '0 (mg/dl) 93,375 ± 11,06 99,66 ± 1,30 94,00 ± 6,15 0,095
Glycemia '120 (mg/dl) 97,62 ± 17,45 90,00 ± 26,60 92,66 ± 18,22 0,095
Fasting plasma insulin (uU/ml) 17,95 ± 6,35 7,66 ± 0,50 7,16 ± 0,21 0,000
HOMA index 4,41 ± 0,80 1.83 ± 0,12 1,45 ± 0,25
SP (mmHg) 123,20 ± 3,70 122,60± 6,35 120.20 ± 7.09 0,013
DP(mmHg) 80,00 ± 3,16 79,80± 3,56 78.80 ± 4.90 0,014
HR (bpm) 74.75 ± 4.57 70.25 ± 4.11 68.50 ± 5.92 0,011
SP: systolic pressure, DP: diastolic pressure, HR: heart rate, BMI: body mass index, HOMA-I: the homeostasis model assessment-index, IR: insulin resistant offsprings of type 2 diabetic patients, NIR: non insulin resistant offsprings of type 2 diabetic patients.
Heart rate variability
Tables 2 and 3 show the means of autonomic function measures for each group.
Table 2 Means (95%CI) RR intervals in the different time periods for each offspring group and controls.
R-R (msec) IR NIR Controls
Night (0–6 am) 895,39 (821,61–969,17) 873,36 (781,09–965,64) 1126,75 (1060,44–1193,05)
morning (7–12 am) 792,44 (741,58–843,29) 752,26 (719,55–784,98) 870,29 (805,43–935,15)
afternoon (1–6 pm) 690,28 (658,75–721,82) 689,16 (638,25–740,07) 749,54 (615,63–883,46)
evening (7–11 pm) 734,37705,02–763,72) 596,87 (360,36–833,39) 910,82 (808,78–1012,87)
Value are means (95% CI), IR: insulin resistant offsprings of type 2 diabetic patients, NIR: non insulin resistant offsprings of type 2 diabetic patients, RR: normal-to-normal R-R interval (ms)
Table 3 Autonomic function measures for each offspring group and controls.
IR NIR Controls P value
SDNN (msec) 86.10 ± 35.96 119.70 ± 28.22 131.90 ± 29.52 0,000
SDNN day (msec) 105.21 ± 28.37 128.77 ± 30.60 120.29 ± 26.97 0,062
SDNN night (msec) 118.99 ± 46.16 112.40 ± 7.54 119.57 ± 38.25 0,822
RMS-SD (msec) 43.15 ± 20.60 42.63 ± 9.18 36.75 ± 10.59 0,331
RMS-SD day (msec) 41.58 ± 18.64 34.77 ± 5.75 31.37 ± 9.07 0,050
RMS-SD night (msec) 47.47 ± 28.80 55.70 ± 18.46 47.47 ± 10.60 0,485
LF u.n. 70.88 ± 6.20 67.59 ± 4.46 55.46 ± 7.49 0,000
HF u.n. 23.63 ± 6.67 27.09 ± 3.74 37.65 ± 7.53 0,000
LF/HF 3.34 ± 1.37 3.72 ± 0.72 2.44 ± 1.09 0,004
LF night u.n. 3.34 ± 1.37 3.72 ± 0.72 2.44 ± 1.09 0,000
HF night u.n. 25.72 ± 8.19 36.53 ± 3.17 58.62 ± 6.59 0,000
LF/HF night 3.06 ± 1.49 2.21 ± 0.54 0.79 ± 0.48 0,000
LF u.n. morning 73.45 ± 9.61 69.04 ± 7.41 69.54 ± 8.36 0,298
HF u.n. morning 22.12 ± 7.17 24.30 ± 8.52 23.05 ± 7.13 0,765
LF/HF morning 4.15 ± 2.80 5.69 ± 3.11 3.70 ± 1.72 0,122
LF u.n. afternoon 70.59 ± 10.62 70.06 ± 4.26 70.09 ± 6.62 0,979
HF u.n. afternoon 23.21 ± 10.25 21.42 ± 3.79 22.30 ± 5.09 0,794
LF/HF afternoon 4.65 ± 1.71 3.74 ± 0.46 3.67 ± 1.30 0,647
LF u.n. evening 71.90 ± 10.70 71.90 ± 10.70 56.75 ± 7.75 0,000
HF u.n. evening 23.52 ± 6.28 24.28 ± 9.08 35.75 ± 9.14 0,001
LF/HF evening 3.30 ± 1.00 4.12 ± 2.21 2.00 ± 0.88 0,009
Value are means ± SD, IR: insulin resistant offsprings of type 2 diabetic patients, NIR: non insulin resistant offsprings of type 2 diabetic patients, SDNN: standard deviation of all sinus rhytm RR intervals, RMS SD: the square root of the mean of the sum of the squares of differencies between adjacent NN intervals, LF: low frequence, HF: high frequence, hours of Holter registration: night time = 0–6 am, morning = 7–12 am, afternoon = 1–6 pm, evening = 7–11 pm. P Value is between groups.
Time domain
Time domain analysis of HRV showed a reduction statistically significant of total SDNN in IR (p < 0.001) and NIR (p 0.047) groups (IR: 86.10 ± 35.96 ms, NIR: 119.70 ± 28.22 ms) versus control group (131.90 ± 29.52 ms) (fig. 1). Total SDNN were reduced in IR group when compared with (NIR) NIR group (p 0.003). These results showed a total activity reduction of autonomic system in insulin resistant and non insulin resistant offsprings of type 2 diabetic patients. The autonomic activity reduction is major in IR group than NIR group.
Figure 1 SDNN value in offsprings of type 2 diabetic patients and controls. SDNN: standard deviation of all sinus rhythm RR intervals, IR: insulin resistant offsprings of type 2 diabetic patients, NIR: non insulin resistant offsprings of type 2 diabetic patients. #p:0.003IR vs NIR; *p:0.047 controls vs NIR and † p:0.001 controls vs IR
RMS-SD in night time not increased in IR group. Therefore insulin-resistance was associated with alteration of circadian rhythm of parasympathetic component (at the night).
Frequency domain
Frequency domain analysis of HRV showed a total LF higher (all p < 0.001) in IR (70.88 ± 6.2 n.u.) and NIR (67.59 ± 4.46 n.u.) groups than in control group (55.46 ± 7.49 n.u.). No difference was noticed between IR and NIR groups (p: 0.157). HF were lower in IR (23.63 ± 6.67 n.u) and NIR (27.09 ± 3.74 n.u.) than in control group (37.65 ± 7.53) (all p < 0.001), while not statistically significant difference were between the groups of offsprings type 2 diabetic patients, p: 0.127. LF/HF total is higher in IR (3.34 ± 1.37) and NIR (3.72 ± 0.72) groups versus control group (2.44 ± 1.09), respectively p 0.016 and p 0.002.
Night (0 am – 6 am)
LF value are higher in IR (67.80 ± 9.70 n.u.) and NIR (59.60 ± 1.75 n.u.) groups than in control groups (35.21 ± 5.97) (all p < 0.001) and are higher in IR group when compared with NIR group, p 0.001). HF are lower in IR (25.72 ± 8.19 n.u.) and NIR group (36.53 ± 3.17) than in control groups (58.62 ± 6.59) (all p < 0.001 vs controls).
Morning (7 am – 12 am)
LF and HF had not statistically difference between IR (LF: 73.45 ± 9.61 n.u.; HF n.u.: 22.12 ± 7.17 n.u.), NIR (LF: 69.04 ± 7.41 n.u.; HF: 24.30 ± 8.52 n.u) and control groups (LF: 69.54 ± 8.36 n.u.; HF: 23.05 ± 7.13 n.u.).
Afternoon (1 pm – 6 pm)
LF, HF and LF/HF had not statistically difference between IR (LF: 70.59 ± 10.62 n.u.; HF: 23.21 ± 10.25 n.u.; LF/HF: 4.65 ± 1.71), NIR (LF: 70.06 ± 4.26 n.u.; HF: 21.42 ± 3.79 n.u.; LF/HF: 3.74 ± 0.46) and control groups (LF: 70.09 ± 6.62 n.u.; HF: 22.30 ± 5.09 n.u.; LF/HF: 3.67 ± 1.30).
Evening (7 pm – 11 pm)
LF were higher in IR (71.90 ± 10.70 n.u.) and NIR (71.90 ± 10.70 n.u.) groups than control group (56.75 ± 7.75 n.u.) (all p < 0.001). HF were lower in IR (23.52 ± 6.28 n.u.) and NIR (24.28 ± 9.08 n.u.) groups than control group (35.75 ± 9.14 n.u.), respectively p: 0.001 and p: 0.002. LF/HF is higher in IR (3.30 ± 1.00) and NIR (4.12 ± 2.21) groups than control group (2.00 ± 0.88) (respectively p: 0.04 and p: 0.002).
Discussion
The data of our study suggested that an autonomic impairment is associated with the familiarity for type 2 diabetes independently of insulin resistance. In frequency domain, the analysis of sympathetic (LF) and parasympathetic (HF) component and the symphatovagal balance (LF/HF) evidenced an association between the familiarity and a sympathetic overactivity, especially in nocturnal period, demonstrated by increase of LF (figure 2 and figure 3) and LF/HF ratio (figure 4). This autonomic impairment is major in insulin resistant offsprings than non insulin resistant offsprings of type 2 diabetic patients. Moreover, our study had demonstrated, in time domain analysis of HRV, a significant reduction of the total autonomic system activity in both groups, expressed by progressive decrease of SDNN value from NIR to IR groups. These results indicated that the familiarity of type 2 diabetes mellitus is related to a global reduction of autonomic nervous system and that the dysautonomia increases if offsprings are insulin resistant.
Figure 2 LF value in offsprings of type 2 diabetic patients and control. IR: insulin resistant offsprings of type 2 diabetic patients, NIR: non insulin resistant offsprings of type 2 diabetic patients, LF: low frequency, hours of Holter registration: 0–6 am= night time, 7–12 am = morning. * p < 0.001 NIR and IR vs controls, #: p:0.001 IR vs NIR; & p < 0.001 controlsvs NIR and IR.
Figure 3 Circadian variation of LF in IR, NIR and in control group. Gray circles: group of non insulin resistant offsprings of type 2 diabetic patients; black squares: group of insulin resistant offsprings of type 2 diabetic patients; white triangles: control group; IR: insulin resistant offsprings of type 2 diabetic patients, NIR: non insulin resistant offsprings of type 2 diabetic patients, LF: low frequency.
Figure 4 LF/HF value in offsprings of type 2 diabetic patients and control. IR: insulin resistant offsprings of type 2 diabetic patients, NIR: non insulin resistant offsprings of type 2 diabetic patients, LF/HF: low frequency/high frequency, hours of Holter registration: 0–6 am= night time, 7–12 am = morning. *p.0.016 controls vs IR; †p:0.002 control vs NIR; # p:0.015 IR vs NIR; £ p:0.0001 controls vs NIR and IR; ç p:0.043 controls vs NIR.
In summary, a global reduction and alteration of circadian rhythm of autonomic activity are present in offspring of type 2 diabetic patients, without and with insulin resistance (figure 2, 3).
A long term observation can answer the question whether autonomic abnormality precede the occurence of insuline resistence and play a role in the complex pathogenesis of the insulin resistance and type 2 diabetes mellitus. Others studies occurs to explain a possible pathogenetic role of autonomic dysfunction in the development of insulin resistance and type 2 diabetes mellitus.
Limitations
A limitation in this study is the use of the HOMA index as a conventional indicator of insulin resistance. The best method for assessment of insulin resistance is the glucose clamp technique, however, the HOMA model has proved be a robust clinical and epidemiological tool in descriptions of the pathophysiology of diabetes, already quoted in > 500 publications, it has become one of standard tools in the armamentarium of the clinical physiologist [23].
Other study are necessary to determine the mechanism whereby insulin-resistance may be related to autonomic dysfunction.
List of abbreviations
BMI: body mass index; DM: type 2 diabetes mellitus; HF: high frequency; HOMA-I: the homeostasis model assessment-index; HRV: heart rate variability; LF: low frequency; OGTTs: oral glucose tolerance tests; RMS-SD: the square root of the mean of the sum of the squares of differences between adjacent NN intervals; SDNN: The standard deviation of normal-to-normal RR intervals; WHR: waist-to-hip ratio.
Competing interests
The author(s) declare that they have no competing interests.
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Cerebrospinal Fluid ResCerebrospinal Fluid Research1743-8454BioMed Central London 1743-8454-2-61617429310.1186/1743-8454-2-6ReviewIntegration of the subarachnoid space and lymphatics: Is it time to embrace a new concept of cerebrospinal fluid absorption? Koh Lena [email protected] Andrei [email protected] Miles [email protected] Neuroscience Program, Department of Laboratory Medicine and Pathobiology, Sunnybrook and Women's College Health Sciences Centre, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5, Canada2005 20 9 2005 2 6 6 22 6 2005 20 9 2005 Copyright © 2005 Koh et al; licensee BioMed Central Ltd.2005Koh 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.
In most tissues and organs, the lymphatic circulation is responsible for the removal of interstitial protein and fluid but the parenchyma of the brain and spinal cord is devoid of lymphatic vessels. On the other hand, the literature is filled with qualitative and quantitative evidence supporting a lymphatic function in cerebrospinal fluid (CSF) absorption. The experimental data seems to warrant a re-examination of CSF dynamics and consideration of a new conceptual foundation on which to base our understanding of disorders of the CSF system. The objective of this paper is to review the key studies pertaining to the role of the lymphatic system in CSF absorption.
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Review
Evidence for cranial CSF-lymphatic connections
The classical textbook theory assumes that the projections of the arachnoid membrane into the cranial venous sinuses represent the primary sites for CSF clearance and when absorption through these sites is blocked, this leads to disorders of the CSF system. However, this view is increasingly being contested [1-5].
Apart from one study in which Prineas [6] reported what appeared to be lymphatic vessels within the brain parenchyma of individuals with neurological disorders, it is now accepted that lymphatic vessels do not exist in the brain and spinal cord [7]. However, the literature is filled with reports of a physiological relationship between the CSF and extracranial lymph compartments.
We first learn of an affiliation between CSF and lymph through studies conducted over 100 years ago. Schwalbe demonstrated a connection between the subarachnoid space and the cervical lymphatic system in dogs and rabbits with the use of Berlin blue [8]. A similar experiment performed in dogs revealed CSF tracer in the submaxillary and cervical lymph nodes [9]. From these early studies, numerous reports solidified a link between CSF and lymph (Table 1). In general terms, these studies indicated that various tracers injected into the CSF or brain parenchyma made their way into lymphatic vessels external to the cranium and into a variety of lymph nodes in the head and neck. The cribriform plate appeared to be central to this clearance. The various tracers seemed to move from the subarachnoid space through the cribriform foramina associated with the olfactory nerves (fila olfactoria). The tracer was then observed in the lymphatic vessels in the submucosa of the olfactory and respiratory epithelium. Based on unpublished data using 20% fluorescein isothiocyanate-dextran, we found that these vessels become visible within 10 minutes after administration into the cisterna magna in sheep. As can be seen in Table 1, the association between CSF and lymph has been established in mice, rats, rabbits, cats, guinea pigs, pigs, sheep, dogs, monkeys and humans. It seems safe to assume that CSF-nasal lymphatic absorption is a characteristic feature of mammalian systems.
Table 1 Summary of important experiments illustrating a link between CSF and the lymphatic system
Site of Recovery
First Author Date Species Tracer Site of
Injection Retropharyngeal Cervical
lymph nodes Olfactory Nerves Nasal Lymphatics Nasal Interstitium Spinal Nerves
Schwalbe [8] 1869 dog, rabbit Berlin blue CSF +
Quincke [9] 1872 dog mercuric sulfide CSF +
Key & Retzius [86] 1875 human Richardson's blue CSF + + + +
Goldmann [87] 1913 dog, rabbit trypan blue CSF + +
Weed [88] 1914 cat ferrocyanide-iron solution CSF + + + +
Mortensen & Sullivan [23] 1933 dog brominized oil or thorotrast CSF +
Faber [89] 1937 rabbit X-ray medium CSF + + + +
Yoffey & Drinker [90] 1939 rabbit, monkey India ink CSF + + + +
Brierley & Field [39] 1948 rabbit India ink CSF + + +
Brierley [91] 1950 rabbit India ink CSF +
Courtice & Simmonds [31] 1951 rabbit blue dye plasma CSF + + +
Simmonds [92] 1952 rabbit, cat blood CSF +
Schurr [24] 1953 dog pantopaque CSF + + + +
Woollam [93] 1953 neonatal rat colloidal carbon CSF +
Bowsher [94] 1957 cat S35 labeled protein CSF +
Svane-Knudsen [95] 1958 guinea pig iron solution CSF + +
Di Chiro [96] 1972 dog RISAb CSF + +
Potts [26] 1972 dog radiopaque mixture CSF +
Bradbury [53] 1980 sheep RISAb CSF +
Bradbury & Cole [54] 1980 cat, rabbit RISAb CSF +
Hasuo [33] 1981 dog, cat India ink; 99 mTc-DTPA CSF +
McComb [62] 1982 rabbit RISAb with dextran or dye CSF + +
Bradbury & Westrop [55] 1983 rabbit RISAb CSF + +
Pile-Spellman [97] 1984 cat, rabbit radiolabelled colloid CSF + +
Love & Leslie [63] 1984 cat artificial CSF & dextran CSF +
McComb [27] 1984 cat RISAb & Elliott's B CSF + +
Szentistvanyi [98] 1984 rat HPc & or Evan's blue albumin PARa + +
Gomez [99] 1985 rabbit HPc & Evan's blue CSF + + +
Erlich [100] 1986 rabbit ferritin CSF + +
Leeds [101] 1989 dog Ringer's lactate or blue dye CSF +
Harling-Berg [81] 1989 rat human serum albumin CSF +
Wang & Casley-Smith [50] 1989 rat India ink PARa +
McComb & Hyman [56] 1990 monkey RISAb CSF + +
Yamada [102] 1991 rabbit RISAb PARa +
Tsay [103] 1992 rabbit saline CSF +
Zhang [12] 1992 rat India ink CSF / PARa + +
Kida [11] 1993 rat India ink CSF + +/- + +/-
Kida [14] 1994 rat India ink PARa + +/- + +/-
Botel [69] 1994 cat, rat, dog, monkey X-ray medium CSF + + +
Brinker [30] 1994 cat, dog, monkey dye, dextran, X-ray medium CSF + + + +
Hunter [104] 1995 rabbit nanoparticulate contrast CSF / PARa +
Slusarczyk [105] 1996 rat India ink CSF +
Boulton [106] 1996 sheep RISAb CSF + +
Miura [40] 1998 monkey carbon particles CSF + +
Boulton [58] 1999 rat RISAb CSF +
Silver [65] 1999 sheep artificial CSF CSF +
Bozanovic-Sosic [41] 2001 sheep RISAb CSF +
Zakharov [16] 2003 neonatal sheep Microfil CSF + + + +/- +
Vega & Jonakait [107] 2004 rat India ink CSF +
Zakharov [17] 2004 neonatal sheep Microfil CSF + + + +/-
Johnston [15] 2004 sheep, pig, rabbit, rat mouse, monkey, human Microfil CSF + + + +/-
Johnston [108] 2005 monkey Microfil CSF + + + +/-
a parenchyma
b radioisotope iodinated serum albumin
c horseradish peroxidase
Nature of anatomical connections between CSF and lymphatic system
The cellular parameters associated with the physiological 'coupling' of the CSF and extracranial lymph compartments will no doubt have an influential role in determining how CSF is absorbed and by inference, also create potential pathological targets for obstruction and interference with CSF absorption. The most basic issue is whether CSF convects first into an intervening interstitial compartment (the submucosal interstitium associated with the olfactory and respiratory epithelium) or whether there is some form of direct connection between the CSF and nasal lymphatics. Table 1 highlights support for both of these propositions.
Jackson and colleagues have postulated two possible mechanisms for CSF uptake into lymphatics [10]. The first is the "open cuff model" in which the perineural sheath cells disappear distal to the cribriform plate, allowing CSF to dissipate into the interstitial space where it is absorbed by the initial lymphatics in the olfactory and respiratory submucosa. The "closed cuff model" depicts the perineural space as a cul de sac. In this case, lymphatic vessels may fuse with the perineural cells and in some way get direct access to CSF that has convected along the olfactory nerve. However, more recent data suggests a third possibility.
In rats, CSF may move directly from the subarachnoid space into submucosal lymphatics that emerge at the level of the cribriform plate [11-14]. This concept is supported by recent studies with Microfil, a silicon rubber injection compound. When Microfil was infused into the subarachnoid compartment of mice, rats, rabbits, sheep, pigs, monkeys and humans, it entered an extensive lymphatic network adjacent to the extracranial surface of the cribriform plate [4,15-17]. Lymphatics filled with Microfil were especially conspicuous around the olfactory nerves close to the point of exit from the cribriform plate (Figs. 1A–C). Microfil was also observed in the afferent lymphatic vessels entering into the retropharyngeal nodes (Fig. 1D). While some Microfil was scattered throughout the nasal submucosal tissues in some preparations, this pattern was the exception rather than the rule. It is possible that the high pressures required to infuse the Microfil in the post-mortem state could have ruptured the lymphatic vessels occasionally. Additionally, it was clear that the longer the period between death and infusion of the contrast agent, the greater the chance of Microfil being observed within the nasal interstitial space due to tissue deterioration.
Figure 1 Anatomical relationships between cerebrospinal fluid and lymphatic vessels. A – Illustration of cribriform plate and lymphatic vessels in the rat. In this example, yellow Microfil has been injected into the cisterna magna. An extensive network of lymphatics filled with yellow Microfil can be observed in the olfactory submucosa. Black arrows-cribriform plate; OB – olfactory bulb. B – Lymphatics filled with yellow Microfil (injected into the cisterna magna) in the ethmoid turbinates of the pig. C – Lymphatics filled with yellow Microfil (injected into the cisterna magna) in the ethmoid turbinates of the sheep. Blood vessels (red) can be seen interspersed between the lymphatic networks. D – Lymphatics filled with yellow Microfil (injected into the cisterna magna) converge on several lymph nodes. In this example, prenodal lymphatic vessels can be observed converging onto one of the retropharyngeal nodes in sheep. E – When Evans blue dye is injected into the spinal subarachnoid space in sheep, it enters the epidural tissues around the spinal cord. F – Lymphatic vessels filled with Evans blue dye (injected into the spinal subarachnoid space) can be observed draining to the intercostal lymph nodes in sheep.
It is of interest to note that some contrast agents can be taken up into lymphatic vessels readily after injection into the interstitial space post-mortem. Evans blue dye is an example. However, this does not seem to be true of Microfil. This silastic material was developed to outline vascular networks after injection into a vessel lumen. It is relatively viscous and is unlikely to be taken up readily from an interstitial compartment. In the Microfil studies [17], the authors failed to visualize lymphatic vessels following subcutaneous injection of Microfil. The material accumulated at the depot site but did not enter the initial lymphatics post-mortem. This implied that a direct connection had to exist between the CSF and lymph compartments to facilitate uptake into lymphatic vessels.
Histological investigation in the sheep Microfil studies showed that the lymphatic vessels fused to the sheaths of the olfactory nerves within the submucosa proximal to the cribriform plate [17]. Upon closer examination, lymphatics filled with Microfil appeared to form a collar around olfactory nerves close to the extracranial portion of the cribriform plate (Fig. 2).
Figure 2 Anatomical connections between the olfactory nerve and extracranial lymphatic vessels. In schematic (A) the lymphatics are connected directly with the CSF space. In A1, the lymphatic vessels form a collar around the emerging olfactory nerve root with the lymphatic endothelium fusing to the perineural sheath of the nerve and the periosteum or dura associated with the cribriform plate. In effect this lymphatic collar provides a 'seal' that ensures that little or no CSF enters the submucosal interstitium. In A2, the lymphatics join with the cribriform plate and nerve as above but in this scenario, a collar of CSF follows the nerve some distance into the submucosa. This CSF collar is delimited by the lymphatic vessel. As in the scenario outlined in A1, no CSF is permitted to enter the interstitium. In (B), the lymphatics are not connected directly with the olfactory nerves or cribriform plate but are interspersed throughout the olfactory submucosa. In this proposal, CSF must convect first into the interstitium of the submucosa from which it is absorbed into blind ending lymphatic vessels. (C) Uptake of Microfil by lymphatic vessels adjacent to cribriform plate. This histological section was stained with hematoxylin and eosin. In this example, yellow Microfil was infused into the CSF space (appears dark brown in section) and blue Microfil was injected into the arterial circulation. Distended lymphatic vessels containing Microfil are especially prominent in the area surrounding the olfactory nerve roots as they emerge from the cribriform plate (red arrows). Lymphatics are also observed fused to the olfactory nerves at discrete locations away from the cribriform plate (yellow arrows). Microfil is not observed free within the interstitium of the submucosa. Regarding the relationship between cranial CSF and lymph, examples such as this would appear to support the schema illustrated in A. BV – blood vessels.
Some studies have reported the existence of a perineural sheath around the olfactory nerves composed of flattened cells [18]. Whether this layer is oriented sparsely around the nerve [19] or represents a more substantial connective tissue sheath [20] is open to debate. More recently, the olfactory ensheathing cells have been identified. In mammals, these cells appear to be responsible for the regeneration of unmyelinated olfactory axons throughout life [21] and have been observed along the nerves from the olfactory mucosa to the olfactory bulbs [22]. Whatever the nature of the outer cell layer, it is evident that the lymphatic endothelial cells fuse to this tissue [17].
Direct connections between CSF and lymph would appear to make sense from a theoretical perspective. One might imagine that CSF leaks would be very common if CSF convected routinely into the nasal submucosal interstitium since the fluid would be separated from the air spaces only by a layer of olfactory or respiratory epithelium. The need for effective CSF clearance under a wide variety of intracranial pressures and the requirement to protect the brain from air-borne infection would seem to be best met by a CSF absorption system that limits CSF access to well-defined lymphatic drainage pathways rather than permit random CSF dispersion throughout the extracellular spaces of the olfactory and respiratory submucosa.
Other CSF-lymphatic connections
The most important lymphatic CSF absorption pathway is no doubt the olfactory route leading to cervical lymphatic vessels but there are other nerves that may conduct CSF extracranially. Even though the bulk of evidence favors the olfactory nerves as facilitating CSF-lymph connections, tracers injected into the CSF system appear to exit the cranium along almost all of the cranial nerves including the trigeminal, acoustic [7], hypoglossal and vagus nerves [16].
Injection of tracer into the subarachnoid space resulted in the appearance of tracer in the optic nerve [16,23-31]. Although the eye does not appear to contain lymphatics, one report noted edema of the eye in cats following resection of the cervical lymph nodes and vessels [32]. Hasuo and colleagues proposed CSF drainage from the subarachnoid space of the optic nerve through arachnoid granulations into the orbital connective tissue from which lymphatics were believed to transfer the fluid to the cervical lymph nodes [33].
One possible location for lymphatic CSF absorption that has been ignored generally is the dura itself. In rats, lymphatics exist around the wall of the sagittal sinus, in the areas of the confluence of sinuses in proximity to the mesothelial cells of the subdural spaces and close to the vasculature of the dural tissues [34]. Lymphatic vessels have also been observed in the dura of the base of the skull of dogs [35]. 125I-albumin injected into the subdural space in rabbits was observed to enter plasma [36] and it seems likely that dural lymphatics contributed to this clearance. In the studies by Killer et al, India ink injected into the subarachnoid space of the optic nerve penetrated the arachnoid and entered the interstitial compartment and lymphatics in the dura of the nerve [37]. There is however, at least one theoretical objection to a possible role for dural lymphatics in CSF drainage. The cellular architecture and the presence of tight junctions between arachnoid cells are believed to contribute to the blood-brain/CSF barrier [38]. Without this barrier function, the extravasated fluid and solutes from the permeable dural capillaries would enter the dural interstitium and possibly gain access to CSF. However, for any dural CSF absorption to occur, presumably CSF would have to pass through the supposed barrier provided by the arachnoid membrane to enter dural tissues.
Lymphatics also appear to play a role in spinal CSF absorption. India ink infused into the ventricles or cisterna magna of rabbits has been found around emerging spinal nerve roots. The tracer passed from the subarachnoid space cul de sac into lymphatic vessels and nodes of the cervical and lumbosacral region [39]. Similarly, an accumulation of carbon particles was found in the lumbar para-aortic lymph nodes in rats following infusion of India ink into the cisterna magna [11]. In monkeys, lymphatic vessels have been observed in spinal epidural tissues [40]. Unlike the situation with olfactory nerves, there is no evidence for direct spinal CSF-lymph connections. It is clear that CSF from the spinal subarachnoid compartment must first pass into the epidural tissues from which absorption takes place into blind ending lymphatic vessels (Figs. 1E, F).
From a quantitative perspective, the drainage of CSF from the spinal cord subarachnoid space plays a role in total volumetric CSF absorption. Studies performed in sheep showed that the relative proportion of CSF absorption by the spinal compartment represents approximately 25% of total CSF clearance [41].
Relationship between parenchymal interstitial fluid and the lymphatic circulation
The CNS has a complex extracellular space that connects with the internal (ventricular) and external (subarachnoidal) CSF through the ependymal layer and pia-mater and the Virchow-Robin spaces [42]. The Virchow-Robin spaces are extensions of the subarachnoid space (also termed perivascular spaces) that penetrate with blood vessels into the brain. Fluid within this space appears to be continuous with CSF and the parenchymal interstitial liquid [43]. Other studies have also shown a direct anatomical connection between the perivascular space of intracerebral arteries and the perivascular space of arteries in the subarachnoid space in humans and rats [43,44]. The studies of Cserr et al [45-47] support the concept of bulk drainage of interstitial fluid from its formation at the capillary-glial complex and its movement through the perivascular and subependymal regions into the ventricular system and subarachnoid space. Following injection of radioactive albumin into the caudate nucleus of rabbits, about 50% of the tracer cleared from the brain was accounted for by passage to lymph [48]. Additionally, Kida performed a series of studies in rats demonstrating a direct drainage of interstitial fluid and CSF into the deep cervical lymph nodes [14]. Therefore, at least a portion of parenchymal interstitial fluid drains ultimately into lymphatic vessels.
Földi developed the concept of parenchymal interstitial fluid draining into extracranial lymphatics located in the adventitia of the internal carotid artery [49]. Wang and colleagues observed that a carbon tracer injected into the cerebral hemispheres drained extracranially along the adventitia of internal carotid arteries and vertebral arteries of rats [50]. These adventitial spaces were considered to be prelymphatic, as subsequent tracer was found in the deep cervical lymph nodes.
Quantitative evidence for volumetric CSF absorption into cervical lymphatics
It is difficult to quantify volumetric CSF absorption by lymphatics due to the complexity of the anatomical pathways involved. Some investigators have simply taken CSF tracer recovery in lymph nodes as a reflection of lymphatic function. For example, Marmarou's group measured a very low recovery in the cervical, retropharyngeal, parotid, and mandibular lymph nodes in cats 8 h after infusion of radioactive albumin into the brain [51]. However, at a given point in time, the amount of lymph within a node is very small and represents only a miniscule fraction of the mass of tracer that would have traversed the node over a given period. A more appropriate approach is to collect lymph from the cervical lymphatic vessels. For example, Boulton et al collected lymph from sheep cervical lymphatic vessels overtime after administration of a radioactive tracer into the cisterna magna, and found that there were measurable amounts of tracer in the lymph 1 h after injection (the rate of lymphatic CSF absorption peaked at 1.86 ml/hr, 3 h after injection) [52].
Courtice and Simmonds were among the first to quantify the absorption of a CSF dye into plasma and cervical lymph [31]. They found that on average, 4.7% of the total amount of dye injected into the CSF space was recovered in cervical lymph of cats during the 3.5–4.5 h duration of the experiment. In sheep, Bradbury and colleagues monitored cervical lymph flow for over 24 h after a single injection or continuous intraventricular infusion of I125-albumin. Approximately 32% of CSF was recovered in the cervical lymphatics of sheep [53]. Similar experiments were performed in the cat and rabbit (6–8 h duration), and tracer recoveries were 13% and 39%, respectively [54]. When the cribriform plate was sealed intracranially in the rabbit (with kaolin injection or with removal of the olfactory bulbs followed by application of cyanoacrylate glue to the plate), recoveries in cervical lymph dropped by approximately 90% [55]. In primates, a recovery of between 30–50% of I125-albumin was observed in extracranial tissue spaces and lymphatics after continuous infusion into the lateral ventricles [56].
While these studies hinted at an important role of lymphatics in CSF absorption, it was difficult to envision how protein recoveries translated into volumetric data. Additionally, a crucial element in designing an approach to quantify the lymphatic contribution to CSF absorption is the ability to correct the recovery data for errors introduced by filtration of the CSF tracer. In other words, presuming that arachnoid villi and granulations transport CSF into the plasma, the CSF in the plasma will eventually filter into the lymphatic compartment. Without correction, the cannulated lymphatic vessels might receive CSF tracer not only from the CSF compartment directly but also from re-circulated plasma tracer. This would result in an overestimation of the lymphatic contribution to CSF drainage. Similarly, the non-lymphatic contribution to CSF clearance would be underestimated if the loss of CSF tracer due to the normal filtration of proteins from the vasculature were not taken into consideration. Indeed, one study showed that the loss of tracer from sheep plasma was over 5%/h [52].
To correct the tracer recovery data for filtration errors and to permit the estimation of volumetric data from protein tracer approaches, a three-compartment mathematical model was developed and applied to sheep data. The data suggested that 40–48% of all CSF removed from the cranial compartment in adult sheep was cleared by lymphatics [52]. Additionally, plasma recoveries of a CSF tracer dropped by approximately 50% in sheep [57] and rats [58] when the cervical lymphatics were diverted or obliterated, further supporting the view that the cervical lymphatic vessels are responsible for about one-half of total CSF clearance.
While protein tracer studies have played an important role in focusing attention on lymphatic CSF absorption, perhaps the most striking data have been obtained from studies in which the cribriform plate has been sealed in sheep. In this procedure the nasal mucosa, olfactory nerves and all soft tissue on the extracranial surface of the cribriform plate were scraped away with a curette and the bone surface sealed with either bone wax or tissue glue. Sheep were challenged with constant flow or constant pressure infusions of artificial CSF into the CSF compartment before and after the extracranial side of the cribriform plate was sealed. The rate of CSF absorption was reduced significantly by this blockage and remarkably, the data suggested that the majority (> 80%) of cranial CSF absorption occurred through the cribriform plate at opening CSF pressures in adult [59] and in newborn animals [60]. When radioactive CSF protein tracers were injected into the CSF compartment of fetal sheep, the highest concentrations were measured in lymph collected from the cervical lymphatics compared with samples obtained from the thoracic duct or plasma [61]. These data suggest that lymphatics have an important role in CSF absorption before birth as well.
Relationship between intracranial pressure and lymphatic CSF absorption
McComb and colleagues noted that an increase in intracranial pressure (ICP) in rabbits and cats resulted in greater levels of a CSF radioactive tracer in the optic nerve, olfactory bulbs, episcleral tissue, and deep cervical lymph nodes [27,62]. Hasuo observed that cervical lymph flow in dogs and cats increased 2–5 fold when ICP was raised to 30–70 cm H2O [33]. A temporary increase in cervical lymph flow has been observed in cats during cisternal infusions [63]. Protein concentrations declined during the experimental period due presumably to the increased amount of CSF draining via the lymph vessels.
In sheep, cervical lymphatic pressures and flow rates were closely related to ICP [64]. Silver and colleagues measured the cervical lymphatic pressure and lymph flow rates under incremental changes in ICP (10–70 cm H2O). At baseline CSF pressures, about 10% of the lymph in sheep cervical lymphatic vessels had its origins as CSF. As ICP was elevated, the proportion increased. At 70 cm H2O ICP, cervical lymph flow rates were 4 fold higher compared to baseline conditions and nearly 80% of the lymph in these ducts was estimated to originate in the CSF compartment [65].
Implications of blockage of lymphatic CSF absorption
Edema of the brain, elevation of ICP, EEG anomalies and behavioural alterations have been demonstrated after chronic ligation of the cervical lymphatic vessels of dogs [49,66]. Similarly, removal of cervical nodes and ligation of cervical lymphatic vessels in rabbits led to cellular changes in the brain including necrotic neurons, and a dense infiltration of phagocytes [67]. Ligation of the cervical lymphatics result in edema of the brain and increased concentration of protein in cats and rabbits [32,68]. Botel and colleagues obstructed the retropharyngeal lymph nodes and vessels in cats by coagulation [69]. This group observed that CSF outflow resistance doubled, but ICP remained the same compared to control animals.
In recent studies, baseline ICP was elevated after the cribriform plate was obstructed on the nasal side [70]. Mean, diastolic, and systolic ICPs doubled when CSF absorption through the cribriform plate was prevented. An important element of the experimental design was the separation of the cranial and spinal subarachnoid compartments. With this approach, cranial CSF absorption could be assessed without the added complexities of compensatory CSF drainage mechanisms associated with the spinal cord. Therefore, with a major absorption site negated, the ability of the host to balance CSF production was impaired. In order to establish a new equilibrium condition, much higher ICPs were required.
Following bolus infusions of saline into the CSF compartment of adult sheep, obstruction of CSF absorption through the cribriform plate increased the peak ICP after infusion and augmented the time required for ICP to return to baseline [71]. Moreover, analysis of the data indicated that CSF outflow resistance was elevated significantly. Cribriform plate obstruction reduced cranial CSF absorption in adult [59] and neonatal sheep [60]. For a given ICP, CSF clearance was reduced substantially after sealing the cribriform plate. It was evident that much higher CSF pressures were required to maintain a given CSF absorption rate when CSF access to lymphatic vessels in the nasal submucosa was prevented. Additionally, obstruction of the cribriform plate also increased the concentration of the radioactive tracer in the superior sagittal sinus [3].
Are disorders of the CSF system associated with impaired lymphatic CSF absorption?
Very little information is available on this subject but there are some interesting observations that may impact on this issue. Surgical procedures in humans that ablate the olfactory nerves do not seem to be associated regularly with any discernible problems with CSF circulation. It is plausible that CSF might be diverted to the spinal subarachnoid space (and thence, into lymphatics associated with the spinal epidural tissues) to compensate for the obstruction to absorption at the cribriform plate. Nonetheless, it is noteworthy that a study showed that 8% of patients developed hydrocephalus in the immediate postoperative period of cranial base surgery, with half of these patients also exhibiting CSF leaks [72].
Lack of development of the olfactory bulbs in humans [73] and mice [74] has also been associated with hydrocephalus. It is not clear whether the olfactory neurons are absent or defective in these examples but, if this is the case, the important lymphatic connections in the vicinity of the cribriform plate may not exist. In this regard, cranial skeletal anomalies have been associated with CSF disorders. The forkhead transcription factor Foxc1 mouse mutant demonstrates hydrocephalus and other defects [75]. The skeletal defects in the head are extensive with many bones being distorted or absent including those associated with the base of the skull [76]. Additionally, the nasal septum (within which a repository of lymphatics exists with known connections to the CSF compartment) is reduced in size. These alterations might affect the architecture of the cribriform plate and reduce the number of lymphatics that have access to CSF. These animals also exhibit extensive edema [76]. While no reason for the edema has yet been proposed it is of interest to note that targeted disruptions of the related Foxc2 gene are associated with abnormal development of lymphatic vessels [77].
The time taken for India ink to move from the CSF into the cervical lymph nodes was increased relative to controls in a model of TGFβ1 induced hydrocephalus in the mouse [78]. This suggests that the cribriform-lymphatic connection is disrupted in these animals. When bismuth (Bi) subnitrate was injected into the peritoneal cavity of mice the animals developed hydrocephalus [79]. High concentrations of Bi were present in the olfactory bulb and hypothalamus. Additionally, high Bi-levels were associated with diffusion from fenestrated blood vessels of the circumventricular organs and olfactory epithelium. Whether bismuth toxicity elicits some pathological process at the level of the olfactory-lymph connections has never been determined but seems worth investigating. Further study in these animal models may help to elucidate whether impaired lymphatic CSF absorption is linked to disorders of the CSF system.
A lymphatic-CSF relationship would also seem to have immunological implications. For example, a humoral immune response in mice was generated mainly by the deep cervical lymph nodes after injection of sheep red blood cells into various intracerebral sites [80]. In rats, infusion of human serum albumin into the cranial CSF [81] or administration of ovalbumin into the spinal subarachnoid space [82] led to antibody production by the cervical lymph nodes. Antibody titers in the peripheral circulation were reduced when cervical lymphatics were obliterated [81].
After the induction of experimental autoimmune encephalomyelitis in rats, a severe immune response was generated, resulting in cerebral lesions [83]. Removal of the deep and superficial cervical lymph nodes following induction of autoimmune encephalomyelitis reduced the severity of the pathology significantly. Therefore, the cervical lymph nodes may act to prime immune cells to target the brain. Some investigators have speculated that lymphatic drainage of brain antigens could conceivably contribute to the pathogenesis of Alzheimer's disease and multiple sclerosis [84].
Conclusion
The tenets that form the basis of our understanding of CSF absorption do not appear to have received critical appraisal in recent years. The arachnoid projections into the cranial venous sinuses are believed to represent the primary sites for CSF absorption and current views on the pathophysiology of the CSF system have often focused on impaired CSF clearance through these elements [85]. However, this concept may be in need of revision. The possibility that CSF may drain into extracranial lymphatic vessels in significant volumes has been generally ignored even though an association between CSF and lymph has been known for over 100 years. CSF mainly flows along the extensions of the subarachnoid compartment associated primarily with olfactory nerves, convects through the cribriform plate and is absorbed ultimately by lymphatics in the nasal submucosa. It seems to be an appropriate time to create a new conceptual foundation on which to base our understanding of CSF parameters. Attention directed to lymphatic CSF absorption may reveal new insights into the cause of CSF disorders and provide novel targets for therapeutic intervention.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
LK had the primary responsibility of writing and organizing the review. AZ and MJ contributed ideas and helped in the preparation of the manuscript. All authors read and approved the final manuscript.
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Harm Reduct JHarm Reduction Journal1477-7517BioMed Central London 1477-7517-2-191621265510.1186/1477-7517-2-19Brief ReportNeedle and syringe sharing practices of injecting drug users participating in an outreach HIV prevention program in Tehran, Iran: A cross-sectional study Vazirian Mohsen [email protected] Bijan [email protected] Saman [email protected] Masako [email protected] Masahiro [email protected] Ravari Shahrzad [email protected] Mohammad Mehdi [email protected] Substance Abuse Prevention and Treatment Office, Ministry of Health and Medical Education, Iran2 Iranian National Center for Addiction Studies (INCAS), Tehran University of Medical Sciences, Tehran, Iran3 Persepolis Society, Tehran, Iran4 Department of Global Health and Socio-epidemiology, Kyoto University School of Public Health, Kyoto, Japan5 Center for Disease Management, Ministry of Health and Medical Education, Iran2005 7 10 2005 2 19 19 29 6 2005 7 10 2005 Copyright © 2005 Vazirian et al; licensee BioMed Central Ltd.2005Vazirian 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.
HIV infection rates have reached epidemic proportions amongst injecting drug users (IDUs) in Iran. Although a number of community-based interventions have being implemented in the country, there is little information on the risk behaviors of IDU participants in these programs. This cross-sectional report aimed to compare the risk behaviors of injecting drug users with differential exposure rates to an HIV outreach program in Tehran, Iran. Results indicated that shared use of needle/syringe in the past month was significantly lower among IDUs who received estimated ≥ 7 syringes per week than those who did not [adjusted odds ratio (OR) = 14.36, 95% confidence interval (CI) 2.30–89.56]. While the effectiveness of this outreach program needs further evaluation through a longitudinal investigation, our preliminary findings suggest that the outreach program in Tehran may have been beneficial in reducing direct sharing among those who received more than several needles/syringes from the program.
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Findings
Injecting drug use, to date, has been reported as the main route of HIV transmission in Iran [1,2] and recent figures show that the prevalence of HIV infection has reached to high levels among IDUs in the capital, Tehran [3]. In a series of attempts to prevent HIV transmission among IDUs, an outreach program for HIV prevention was supported by the United Nations Office on Drug and Crimes (UNODC) and the Ministry of Health of Iran in 2003 [4]. As a part of this outreach program, distribution and exchange of needles and syringes have been facilitated by a non-governmental organization (NGO) named Persepolis Society in its single (at that time) drop-in centre and through outreach activities in a neighborhood in Tehran where many drug users live and street-based drug sales have been going on. While methadone maintenance therapy and referrals for detoxification were available at/through the drop-in centre, IDUs who did not want to participate in substitute therapy or detoxification were provided with a package containing 4 syringes/needles, 4 extra needles, water vials, filters, alcohol pads, and 2 condoms at each visit. However, IDUs were provided with large numbers of needles/syringes and condoms if they requested so. Cookers were also offered, but infrequently.
In October 2004, one year after the establishment of the outreach program, a convenience sample of drug users (consisting 213 IDUs and 85 non-IDUs) was recruited at the drop-in centre, and at parks and streets in the area. Active drug users were approached by an ex-user staff member of the NGO for recruitment and were then interviewed by a male researcher not affiliated with the Persepolis Society. In the questionnaire-based interview, participants were asked about their demographics and HIV risk characteristics, as well as their contact with the outreach program, estimated length of contact, and the total number of syringes they received from the program.
Of 131 male injecting drug users who ever received free needles/syringes from the program, data of the 105 male IDUs with a complete set of variables for multivariate analysis were considered for this report and then categorized based on the rate of exposure to the program. The sub-sample was divided into two groups of IDUs who received few needle/syringes (< 7 syringes per week) (37/105 = 35%) and those who received estimated ≥ 7 syringes per week during their participation in the program (68/105 = 65%). While adjusted for demographics and basic drug use characteristics, a logistic regression analysis was performed to assess the association between having received ≥ 7 syringes per week from the program and the related behavioral variables (Table). The research protocol was approved by the Ethical Committee of the Iranian National Center for Addiction Studies at Tehran University of Medical Sciences in Iran and by the Committee for Research on Human Subjects at Kyoto University in Japan.
Table 1 Association between having received ≥ 7 syringes/week from the outreach program and related behavioral characteristics
Characteristics Total No. (%) Received < 7 syringes per week from the program Received ≥ 7 syringes per week from the program Crude odds ratio (95% CI) P valuea Adjusted odds ratio (95% CI) P value
Overall 105 37 68 - - - -
Did not share needle/syringe during last month 90 (85.7) 24 (64.9) 66 (97.1) 17.87 (3.75–85.08) 0.000 14.36 (2.30–89.56) 0.004
Did not share a cooker at last injection 37 (35.2) 11 (29.7) 26 (38.2) 1.46 (0.62–3.45) 0.383 0.65 (0.20–2.08) 0.468
Used condom at last sex/never had sex 40 (38.1) 14 (37.8) 26 (38.2) 1.01 (0.44–2.32) 0.968 0.67 (0.22–2.06) 0.488
Know HIV can be transmitted through shared needle/syringe 101 (96.2) 36 (97.3) 65 (95.6) 0.60 (0.06–6.00) 1.000b 1.98 (0.14–26.43) 0.605
Ever tested for HIV 63 (60.0) 22 (59.5) 41 (60.3) 1.03 (0.45–2.34) 0.934 0.62 (0.20–1.96) 0.424
Variables shown in this table are controlled for age, ethnicity, marital status, educational levels, recruitment site, length of residency in the area, length of lifetime drug injecting, size of drug use network, and frequency of daily drug injecting.
aP values based on chi-square test of proportions unless otherwise specified.
bTwo-tailed Fisher exact test. OR, odds ratio; CI, confidence interval.
The median age of IDUs in this sub-sample was 32.0 years and 67% had educational levels of junior high school or more. Up to 61% were jobless at the time of interview and 52% have ever been married. Among socio-demographic characteristics, only being recruited from the drop-in centre and having lived for 6 years or more in that area were associated with having received ≥ 7 syringes per week from the program in the bivariate analysis.
While 18.9% (7/37) of those who received few needles/syringes from the program reported having injected using a shared needle or syringe at last injection, none of the 68 IDUs who received ≥ 7 syringes per week from the program shared a needle/syringe at last injection (P = 0.000) (not in the Table). Also, shared use of needles/syringes in the past month was significantly lower among those IDUs who received ≥ 7 syringes per week than those who did not (adjusted OR = 14.36, 95% CI, 2.30–89.56; P value = 0.004). However, there was no difference between two groups in the use of a shared cooker at last injection, condom use during last sex, levels of HIV/AIDS knowledge, and a history of previous HIV testing (Table).
Effectiveness of needle/syringe programs in reducing needle and syringe sharing among IDUs has been shown in many countries [5-9]. Our findings show that those IDUs who received many syringes from the program reported less sharing of needles/syringes in the past month than those who received few syringes. While it is possible that like many other observations [5], the distribution/exchange of free needles/syringes resulted in the lower sharing of needles/syringes among IDUs, a rival hypothesis should also be considered: those IDUs who shared needles/syringes less frequently in the past month may have already had higher risk perception and actively sought and received more syringes from the program. In either situation, the needle and syringe program in Tehran can be considered beneficial in that it either resulted in less sharing of needles and syringes, or at least provided easy access to clean needles/syringes for IDUs whether or not any behavior change occurred.
Cooker sharing that has been less targeted in the program was at high rates among both groups without any significant difference. Because of the importance of cooker sharing in the transmission of blood-borne infections [10,11], the need to address the barriers to distributing cookers has been identified by this study, and efficient ways to prevent cooker sharing are being examined.
This study had several limitations. Though the association between having received many needles/syringes from the program and low rates of sharing needles/syringes in the past month is quite strong, because of the cross-sectional design of this study, it is not possible to draw a conclusion on the direction of this association. In addition, our comparison might have been affected by the selection bias due to convenience sampling. Though yet to be confirmed in studies with more robust design, the above preliminary findings suggest that the HIV outreach program in Tehran may have been beneficial in reducing needle/syringe sharing but not in reducing cooker sharing among IDU participants.
List of abbreviations
AIDS = Acquired immunodeficiency syndrome
HIV = human immunodeficiency virus
IDU = injecting drug user
INCAS = Iranian National Center for Addiction Studies
NGO = non-governmental organization
UNODC = United Nations office on Drug and Crimes
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MV and BN coordinated the study and collected the data in the field. SZ was responsible for the statistical analysis and writing the paper. MOK and SMR provided assistance in analyzing and interpreting the data. MK and MMG secured funding and supervised the collaborative socio-epidemiologic study between Japan and Iran. All authors read and approved the final manuscript.
Acknowledgements
We greatly acknowledge the participants of this study and also thank J Koerner for her comments and assistance in preparing the manuscript and H Tajdar, M Borumand, and M Rafiee for their research assistance. This research was financed by the Department of Global Health and Socio-epidemiology, Kyoto University School of Public Health in Japan and by a grant from the Ministry of Health and Medical Education in Iran.
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UNAIDS/UNICEF/WHO Epidemiological Fact Sheets on HIV/AIDS and Sexually Transmitted Infections- Islamic Republic of Iran, 2004 Update 2004 Geneva
Center for Disease Management, Ministry of Health and Medical Education AIDS/HIV Surveillance Report (December 2004) 2004 Tehran, Iran
Zamani S Kihara M Gouya MM Vazirian M Ono-Kihara M Razzaghi EM Ichikawa S Prevalence of and factors associated with HIV-1 infection among drug users visiting treatment centers in Tehran, Iran AIDS 2005 19 709 716 15821397 10.1097/01.aids.0000166094.24069.72
Vazirian M Review of drug demand reduction programs in Iran: Advices for development and strategic planning Social Welfare Quarterly (in Persian) 2003 9 145 201
World Health Organization Evidence for Action: Effectiveness of Community-Based Outreach in Preventing HIV/AIDS among Injecting Drug Users 2004 Geneva
Watters JK Estilo MJ Clark GL Lorvick J Syringe and needle exchange as HIV/AIDS prevention for injection drug use JAMA 1994 271 115 20 8264065 10.1001/jama.271.2.115
Donoghoe MC Stimson GV Dolan K Alldritt L Changes in HIV risk behavior in clients of syringe-exchange schemes in England and Wales AIDS 1989 3 267 272 2504230
Peak A Rana S Maharjan SH Jolley D Crofts N Declining risk for HIV among injecting drug users in Kathmandu, Nepal: the impact of a harm-reduction programme AIDS 1995 9 1067 1070 8527080
Bluthenthal RN Kral AH Gee L Erringer EA Edlin BR The effect of syringe exchange use on high-risk injection drug users: a cohort study AIDS 2000 14 605 611 10780722 10.1097/00002030-200003310-00015
Crofts N Caruana S Bowden S Kerger M Minimising harm from hepatitis C virus needs better strategies British Medical Journal 2000 321 899 11021890
Grund JP Friedman SR Stern LS Jose B Neaigus A Curtis R Des Jarlais DC Syringe-mediated drug sharing among injecting drug users: patterns, social context and implications for transmission of blood-borne pathogens Soc Sci Med 1996 42 691 703 8685737 10.1016/0277-9536(95)00193-X
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Int J Behav Nutr Phys ActThe International Journal of Behavioral Nutrition and Physical Activity1479-5868BioMed Central London 1479-5868-2-141620971610.1186/1479-5868-2-14ResearchSchool lunch and snacking patterns among high school students: Associations with school food environment and policies Neumark-Sztainer Dianne [email protected] Simone A [email protected] Peter J [email protected] Mary [email protected] Jayne A [email protected] Division of Epidemiology and Community Health, University of Minnesota, Minneapolis, Minnesota, USA2 School of Nursing, University of Minnesota, Minneapolis, Minnesota, USA2005 6 10 2005 2 14 14 8 3 2005 6 10 2005 Copyright © 2005 Neumark-Sztainer et al; licensee BioMed Central Ltd.2005Neumark-Sztainer 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.
Objectives
This study examined associations between high school students' lunch patterns and vending machine purchases and the school food environment and policies.
Methods
A randomly selected sample of 1088 high school students from 20 schools completed surveys about their lunch practices and vending machine purchases. School food policies were assessed by principal and food director surveys. The number of vending machines and their hours of operation were assessed by trained research staff.
Results
Students at schools with open campus policies during lunchtime were significantly more likely to eat lunch at a fast food restaurant than students at schools with closed campus policies (0.7 days/week vs. 0.2 days/week, p < .001). Student snack food purchases at school were significantly associated with the number of snack machines at schools (p < .001) and policies about the types of food that can be sold. In schools with policies, students reported making snack food purchases an average of 0.5 ± 1.1 days/week as compared to an average of 0.9 ± 1.3 days/week in schools without policies (p < .001). In schools in which soft drink machines were turned off during lunch time, students purchased soft drinks from vending machines 1.4 ± 1.6 days/week as compared to 1.9 ± 1.8 days/week in schools in which soft drink machines were turned on during lunch (p = .040).
Conclusion
School food policies that decrease access to foods high in fats and sugars are associated with less frequent purchase of these items in school among high school students. Schools should examine their food-related policies and decrease access to foods that are low in nutrients and high in fats and sugars.
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Background
Research studies have clearly shown that adolescents' dietary intakes are not consistent with national recommendations. Areas of concern include high intakes of saturated fat, total fat, and soft drinks, and low intakes of fruits, vegetables, fiber, and calcium-rich foods [1-3]. These dietary patterns are of concern because of their potential for increasing risk for developing obesity, heart disease, osteoporosis, dental caries, and various types of cancer [4].
Adolescent eating patterns are influenced by factors proximal to the adolescent such as individual food preferences [5], family meal patterns [6], and parental role modeling [7]. However, it is increasingly becoming clearer that adolescent eating patterns are also influenced by more distal factors such as media messages and social norms [8,9]. Since adolescents spend a large amount of time in school, an important question – is to what extent does the school food environment influence adolescent eating patterns [10]. In Bronfenbrenner's ecological model, which shows concentric spheres of influences on the individual ranging from proximal factors (i.e., individual characteristics) to distal factors (i.e. social norms and public policies), the school lies in the middle [11].
Although there are federal regulations regarding the types of foods that can be served in the United States Department of Agriculture (USDA) reimbursable school meals [12,13], few regulations are in place for alternative foods, such as those served a la carte in the cafeteria or in snack bars, and in vending machines [14]. A study of 55 high schools revealed that school environments do not always foster healthful eating practices consistent with national dietary guidelines [15]. In a statewide survey of food policies in 336 Minnesota high schools, two-thirds of the principals indicated that only healthy food choices should be provided to students at school, yet only one-third reported that their school had an overall policy about nutrition and food. Even fewer reported the presence of specific policies about the types of foods and beverages sold in vending machines, school stores, or at school functions [16]. School vending machines were prevalent and 77% of the principals reported that their school or district had a contract with a soft drink company. Vending machine hours were limited in some way in 81% of the schools, but only about a third of the schools limited the vending machine hours to before and after school only or after all lunch periods were completed. While it is important to respect adolescents increasing autonomy and decision-making skills, research clearly shows that food availability is one of the strongest correlates of food choices in adolescents [17,18]. Schools provide a setting in which it is possible to increase the availability and attractiveness of a range of healthy food options from which students can make choices, and restrict the availability of foods that are low in nutrients and high in fats and sugars.
The few studies that have examined associations between school food environment and student eating patterns suggest that the school food environment has a significant impact on food choices. Cullen and colleagues [19] found that fourth grade students attending elementary schools without a la carte food items consumed more fruits, juices, and vegetables than fifth graders who attended a middle school with a la carte food line (or snack bar). Kubik et al found that among seventh-grade students in 16 schools, having a school a la carte program was associated with lower intakes of fruits and vegetables and higher intakes of calories from total and saturated fats [20]. They also found that the number of snack vending machines was associated with lower fruit intakes, suggesting that students may be choosing alternative snack foods from the vending machines rather than fruit. However, policies were not examined regarding the types of foods sold in vending machines and their hours of operation and the impact of such policies on vending machine purchases.
This study expands on the limited, but growing, body of literature that explores the role of schools in influencing the dietary practices of youth. Specifically, our study objectives are: 1) to describe school lunch practices and vending machine purchases in a large sample of high school students; and 2) to examine associations between eating patterns of high school students and school food environment and policies.
Methods
Study population and study design
The study population included 1088 high school students from 20 high schools in the Minneapolis/St. Paul metropolitan area in Minnesota. These schools were participating in TACOS (Trying Alternative Cafeteria Options in Schools), a two-year, group-randomized, school-based nutrition intervention trial [21,22]. Participating schools were predominantly suburban, and ranged in enrollment from 812–3157 students (median = 1713). The study population was nearly equally divided on gender (47.0% male, 53.0% female). All students were in 9th–12th grade (18.0%, 26.2%, 30.0%, and 25.8% in 9th, 10th, 11th, and 12th grade, respectively). Race/ethnicity breakdown was as follows: 84.3% White, 4.6% Asian American, 2.5% Hispanic, 2.4% Black, and 6.2% American Indian/other). Nine percent of the students were eligible for free or reduced school lunch.
Data on adolescent school lunch patterns and vending machine practices were collected at baseline, prior to the beginning of the first year of the TACOS intervention with surveys that were mailed to the homes of a random sample of 75 students from each of the 20 participating schools. The University of Minnesota's Institutional Review Board Human Subjects Committee and the research review committees of the participating school districts approved all study protocols. A parental passive consent letter was included with the survey, as part of the cover letter; if the parent agreed to have their child participate they were asked to give the survey to their child. Students received ten dollars for completing the survey. The response rate for survey completion was 75%. Data on school food policies were collected with surveys that were mailed to principals and food service directors at each of the 20 participating schools at the end of the first intervention year. In one school, neither principal nor food service director responded, while in another school the principal did not respond but the food service director did, resulting in data from 19 schools from either the principal or the food service director. Questions on these surveys assessed school food-related policies and practices during the previous school year. The survey instrument was developed, based on previously published surveys about the school food environment [14-16,23-25]. Data on vending machine availability and hours of operation were collected – through site visits by trained research staff. Vending machines were included if they were in locations that were accessible to students (e.g., lunchroom, hallways, student locker areas, gymnasiums, commons area, etc., but not in faculty lounge areas).
Description of measures
Adolescent Eating Patterns
School lunch patterns among adolescents were assessed with the following questions on the student survey: "During a normal school week, how may days per week do you...1) Get lunch in the school cafeteria main lunch line? 2) Get lunch in one of the school cafeteria a la carte or snack bar lines? 3) Bring lunch from home? 4) Get lunch off campus at a fast food restaurant? 5) Get lunch off campus at a convenience store?" Vending machine practices were assessed with two similar questions: "During a normal school week, how many days per week do you...1) Get food from a school snack/food vending machine?; and 2) Get soft drinks from a school vending machine?" Response categories were days per week (range: 0–5).
School food-related policies and food environment
School food-related policies about open/closed campus during lunchtime and the types of food served in vending machines were assessed with the principal survey [22]. Data from the food service director survey were used in the analysis in the few instances in which there were missing data from the principal, or the principal gave the response: "don't know." The existence of a closed campus policy during lunchtime was assessed with the question: "Is the high school campus open or closed for lunch periods?" The existence of policies about type of food sold in vending machines was assessed with the question: "Are there any school policies about what is sold in the school vending machines (yes/no/don't know)?"
Policies regarding hours of operation of vending machines during lunchtime were determined for snack and soft drink vending machines through direct observations by research staff [22]. Direct observations by research staff were also used to assess the number of vending machines including number of snack vending machines, number of soft drink vending machines, and number of other vending machines. Snack vending machines were defined as those that were non-refrigerated, and sold items including candy bars, candy, chips, pretzels, pastry, and gum. Soft drink machines were defined as those that sold primarily soft drinks, but could include one or more sleeves of water, juice, juice drinks, or sports drinks. However, if more than half of the machine columns were filled with drinks other than soft drinks, the machine was recorded under "other" vending machines (e.g., fruit juice, juice drinks, water, or sports drinks).
Data analysis
School food-related policies and measures of school environment (e.g., the number of vending machines) are school-level data implying that all students in a school are under the same policy. Adolescent eating patterns are individual-level data. In descriptive analyses of student eating patterns we present individual level means and standard deviations. For analyses of the association of school policies and student eating patterns, we used mixed models (SAS Release 8.2, proc MIXED) specifying the school as nested in the policy, that is, each student in a school is under the same policy. Standard errors for differences between categories of a policy are inflated, degrees of freedom are two fewer than the number of schools for which policy data is available.
Results
School lunch patterns and vending machine practices among students
Students more frequently ate meals from the main lunch line (Mean = 2.4 days/week) than any of the other options, although they also ate frequently from the a la carte line (M = 1.8 days/week) (Table 1). On average, students brought lunch from home once a week (M = 0.9 days/week). Lunch purchases from off-campus fast food restaurants and convenience stores were less frequent, although standard deviations were large, indicating variation across students. On average, students purchased snacks from vending machines nearly once a week (M = 0.9 days/week). Students purchased soft drinks from vending machines 1.6 days/week; nearly two-thirds (61.5%) of the students reported purchasing soft drinks at least one day/week.
Table 1 Lunch patterns andvending machine practices at school or during school hours in past school week (five days)
Total N† Mean days/week Days/week (percentage reporting)
Mean SD 0 1–2 3–4 5
Lunch patterns:
Regular school lunch 1080 2.4 1.9 29.2 21.8 27.3 21.7
A la carte lunch 1077 1.8 1.7 31.6 36.2 22.0 10.2
Bring lunch 1076 0.9 1.7 71.9 9.8 7.5 10.8
Fast food restaurant 1075 0.4 1.0 81.6 13.0 3.8 1.6
Convenience store 1067 0.1 0.6 92.5 5.5 1.5 0.5
Vending machine practices:
Vending snacks 1081 0.9 1.3 56.8 31.2 9.2 2.8
Vending soft drinks 1081 1.6 1.7 38.5 34.0 17.5 10.0
† Summary statistics are at the individual student level.
School lunch patterns and vending machines were examined across gender and grade level. Boys ate meals from the main lunch line more frequently than girls (M = 2.8 ± 2.0 vs. 2.0 ± 1.8 days/week; p < .001) and brought lunch from home less frequently than girls (M = 0.7 ± 1.6 vs. 1.1 ± 1.8 days/week, p < .001). Boys purchased soft drinks from vending machines on more days/week than girls (M = 1.8 ± 1.8 vs. 1.4 ± 1.6; p < .001). There were no gender differences in a la carte, fast food, and convenience store lunch purchases or in snack food vending machine purchases across gender (data not shown).
A la carte, fast food restaurant, and convenience store lunch practices differed across grade in school. A la carte food purchases were most frequent among students in the 9th and 10th grades and declined among students in the 11th and 12th grades (M = 2.0 ± 1.8 2.1 ± 1.7, 1.7 ± 1.6, and 1.5 ± 1.6 days/week in 9th, 10th, 11th, and 12th graders, respectively; p < .001). In contrast, eating lunch at a fast food restaurant was much more frequent among youth in 11th and 12th grades (M = 0.1 ± 0.6, 0.2 ± 0.6, 0.4 ± 1.0, and 0.7 ± 1.3 days/week in 9th, 10th, 11th, and 12th graders, respectively; p < .001). Getting lunch from a convenience store was not common, but youth in upper grades reported getting lunch more frequently from convenience stores than youth in lower grades (M = 0.1 ± 0.5, 0.1 ± 0.5, 0.2 ± 0.5, and 0.3 ± 0.8 days/week in 9th, 10th, 11th, and 12th graders, respectively; p < .001). Eating from the regular lunch line, bringing lunch from home, and snack and soft drink vending machine purchases did not differ across grade in school (data not shown).
School food environment and policies
Variables assessing the school food environment and policies of potential relevance to students' lunch patterns and vending machine practices were examined (Table 2). About two-thirds of the schools had a closed campus policy during lunchtime (68.4% n = 13 schools). With regard to vending machines, only three (15.8%) of the schools had policies regarding the types of food that could be sold in vending machines. The mean number of snack vending machines in each school was 2.7; four schools had none, six schools had 1–2, six schools had 3–4, and four schools had 5–7 snack vending machines. The mean number of soft drink vending machines in each school was 5.3; three schools had 1–2, seven schools had 3–4, six schools had 5–7, and four schools had 8+ soft drink vending machines. Out of the 16 schools that had snack vending machines, 25% (n = 4), had them closed during lunchtime. Out of the 20 schools that had soft drink machines, 55% (n = 11) had them closed during lunchtime.
Table 2 School food policies and environment in 20 high schools
School policies: Yes No Percent with policy
Closed campus policy during lunch 13† 6† 68%
Policies about food sold in vending machines 3† 16† 16%
Vending machines closed during lunch
Snack machines 4 12‡ 25%
Soft-drink machines 11 9 55%
Number of vending machines/school: Mean SD
Snack Machines 2.7 2.5
Soft-drink machines 5.3 2.7
Other type of machine offerings 5.1 2.4
† One (1) school missing
‡ Four (4) schools had no snack vending machines
Open/closed campus policy during lunchtime and student lunch practices
As shown in Table 3, students at schools with open campus policies during lunchtime were significantly more likely to eat lunch at a fast food restaurant (0.7 days/week vs. 0.2 days/week) or a convenience store (0.3 days/week vs. 0.1 days/week) than students at schools with closed campus policies. There were no significant differences for eating from the main lunch line, eating a la carte foods, or bringing lunch from home between students at school with open campus policies vs. closed campus policies.
Table 3 Student lunch patterns (days/week) by school policies regarding open/closed policy during lunch hours
Closed campus (N = 13 schools) Open campus (N = 6 schools)
Mean (SD) Mean (SD) p-value
Regular lunch line 2.5 (2.0) 2.0 (1.8) .156
Ala carte lunch 1.8 (1.7) 1.6 (1.6) .493
Bring lunch 0.9 (1.7) 1.1 (1.8) .344
Fast food restaurant 0.2 (0.8) 0.7 (1.3) <.001
Convenience store 0.1 (0.4) 0.3 (0.8) <.001
School policies/availability of vending machines and student vending machine practices
Having a school policy about the types of foods sold in vending machines was significantly inversely associated with frequency of student snack food purchases from vending machines (Table 4). In schools with policies, students reported making snack food purchases an average of 0.5 days/week as compared to an average of 0.9 days/week in schools without policies. Similar nonsignificant trends were found for soft drink purchases.
Table 4 Student vending machine (VM) practices by policies regarding food sold in VM, policies regarding hours of operation of VM, and number of VM at school
Policy about food sold in VM VM closed during lunch† Number of VM†
Yes No Yes No 0 1–2 3–4 6+ 8+
M (SD) M (SD) p-value M (SD) M (SD) p-value M (SD) M (SD) M (SD) M (SD) M (SD) p-value
Student snack food VM purchases (days/week) 0.5 (1.1) 0.9 (1.3) <.001 0.9 (1.2) 1.0 (1.3) .477 0.4 (1.0) 0.8 (1.2) 1.1 (1.4) 1.1 (1.4) <.001
Student soft drink VM purchases (days/week) 1.4 (1.7) 1.6 (1.7) .110 1.4 (1.6) 1.9 (1.8) .043 1.3 (1.6) 1.7 (1.7) 1.4 (1.6) 1.9 (1.8) .173
† Done for policies regarding school snack and soft drink policies in accordance with whether student or soft drink
VM purchases are being examined.
Associations between vending machine availability at schools and student snack food and soft drink vending purchases were also examined (Table 4). Student snack food purchases from vending machines were significantly more frequent among students from schools with a greater number of snack food vending machines. Policies regarding hours of operation of snack food machines were not associated with snack food purchases in the 16 schools that had snack machines. In contrast, student soft drink purchases from vending machines were not significantly associated with the number of soft drink vending machines, but were significantly lower in schools in which machines were turned off during lunchtime.
Discussion
This study examined associations between school food policies and student lunch practices and vending machine purchases. Study findings have implications for schools and suggest steps that schools could take to encourage healthier eating practices among students. A closed campus policy during the lunch hour was associated with fewer lunch purchases from fast food restaurants and convenience stores by students. The existence of school policies regarding the types of foods that can be sold in vending machines was associated with fewer student snack food purchases from vending machines. Student snack food purchases from vending machines was also associated with the number of snack food vending machines at school. Finally, limited hours of operation of soft drink vending machines was associated with fewer student purchases of soft drinks from vending machines. While previous studies have found associations between the food school environment (e.g, number of vending machines, availability of a la carte foods) and student eating behaviors [19,20], to the best of our knowledge, this is the first study that has examined and observed associations between school food policies and student eating behaviors.
It is encouraging to note that students most frequently reported eating the regular school lunch, which is regulated in terms of nutritional standards. However, the students also reported frequent consumption of a la carte foods for lunch, which have only minimal regulations in terms of nutrition. Previous analyses have shown that the most commonly available a la carte foods within schools tend to be high in energy and low in nutrients [22,25,26]. The high intake of a la carte foods points to a need for ensuring that healthy foods that are appealing to high school students and reasonably priced, are offered as a la carte choices, and that access to food high in fat and sugar is limited. The finding that high school students bring lunch from home an average of about once a week suggests that interventions aimed at improving the dietary intake of adolescents might also include ideas for healthy brown bag alternatives.
Differences in school lunch and vending machine practices across grade in school suggest that as students enter the upper grades, they are less likely to eat a la carte foods within the school and are more likely to make food purchases outside the school premises at fast food restaurants and convenience stores. These findings suggest that avenues for "alternative" and probably less healthful and more costly food options change as youth get older and have more independence due to having cars and being allowed the freedom to leave school during the day. School-based interventions need to take into account the different eating patterns of younger and older students. Interventions also need take consider factors likely to be influencing school eating practices such as the proximity of different food outlets to the school campus.
Strengths and limitations of the study need to be taken into account in interpreting the findings. A major strength of this study, which contributes to the utility of the findings, was that data on food policies and the school food environment were collected from multiple sources including principals, food service directors, and observations by trained research staff. Since data were collected from 20 schools, which differed from each other in terms of school size, populations served, and school food environments and some generalizations of the findings are possible. However, the 20 schools that participated in the study were from one Midwestern state in the United States and served a population with low representation of minorities and adolescents from lower socio-economic levels. Thus, caution should be taken in making generalizations to other areas and populations. The wording of questions on the student survey also limited some of the conclusions that we were able to draw from the data. For example, for vending machine purchases, questions asked about days per week that students made purchases. The item did not assess number of items purchased per day so could not capture students making numerous purchases in a single day. Finally, additional questions on student eating practices (e.g., fast food intake) after school hours and total dietary intake would have also been informative.
Further research is needed to explore the impact of changing the school food environment and policies on student eating practices both during, and after, school hours. For example, if schools implement closed campus policies during lunchtime, will students make healthier food choices at school or eat more of the less healthful a la carte choices? And will they eat more or less frequently at fast food restaurants after school? If vending machines in schools offered fewer high fat and high sugar foods and more healthful options, what would students choose to drink at school and after school? Further research is also needed to replicate findings from this study in different school populations and assess different types of food policies.
Conclusion
In conclusion, school food policies that decrease access to foods high in fats and sugars are associated with less frequent purchase of these items among high school students. Based upon these findings, it is recommended that schools examine their food-related policies and consider policies to decrease access to foods and beverages that are low in nutrients and high in fats and sugars. Strategies suggested by our data include having closed campus policies during the lunch hour, having policies regarding the types of food that can be sold in vending machines (e.g., more healthful options), keeping soft drink machines turned off during the lunch hour, and limiting the number of snack food vending machines. Schools should also consider strategies for making healthier alternatives more accessible and attractive to students in terms of appearance, taste, and cost. Clearly factors other than eating practices at school are associated with the overall quality of dietary intake and health outcomes of youth; nevertheless, since 35–40% of calories are consumed at school [23,24], eating practices at school are likely to be making a significant contribution. As educational institutions, schools have a crucial role to play in providing youth with healthy eating opportunities.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DNS wrote the manuscript and worked with the analyst to develop an analysis plan and incorporated input from all other authors on the manuscript. SAF developed and directed the overall study. PJH contributed to the study design and conducted the data analysis. MS contributed to the study design and implementation. JAF contributed to study implementation and oversaw evaluation activities.
Acknowledgements
This research was supported by NIH R18 HL61305 with supplemental funding from the Centers for Disease Control and Prevention.
David Crawford, PhD, served as guest editor for this manuscript.
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Neumark-Sztainer D Story M Hannan PJ Croll J Overweight status and eating patterns among adolescents: Where do youth stand in comparison to the Healthy People 2010 Objectives? Am J Public Health 2002 92 844 851 11988458
French SA Story M Neumark-Sztainer D Fulkerson JA Hannan P Fast food restaurant use among adolescents: Associations with nutrient intake, food choices, behavioral and psychosocial variables International Journal of Obesity 2001 25 1823 1833 11781764 10.1038/sj.ijo.0801820
Munoz KA Krebs-Smith SM Ballard-Barbash R Cleveland LE Food intakes of US children and adolescents compared with recommendations [published erratum appears in Pediatrics 1998 May;101(5):952-3] Pediatrics 1997 100 323 329 9282700 10.1542/peds.100.3.323
Story M McAnarney ER, Kreipe RE, Orr DE and Comerci DG Nutritional requirements during adolescence Textbook of Adolescent Medicine 1992 Philadelphia, WB Saunders 75 84
Woodward DR Boon JA Cumming FJ Ball PJ Williams HM Hornsby H Adolescents' reported usage of selected foods in relation to their perceptions and social norms for those foods Appetite 1996 27 109 117 8937616 10.1006/appe.1996.0039
Neumark-Sztainer D Hannan PJ Story M Croll J Perry C Family meal patterns: Associations with sociodemographic characteristics and improved dietary intake among adolescents Journal of the American Dietetic Association 2003 103 317 322 12616252 10.1053/jada.2003.50048
Fisher J Mitchell D Smiciklas-Wright H Birch L Maternal milk consumption predicts the tradeoff between milk and soft drinks in young girls' diets J Nutr 2001 131 246 250 11160541
Story M Neumark-Sztainer D French S Individual and environmental influences on adolescent eating behaviors Journal of the American Dietetic Association 2002 102 S40 51 11902388 10.1016/S0002-8223(02)90421-9
French SA Story M Jeffrey RW Environmental influences on eating and physical activity Annual Review of Public Health 2001 22 309 335 11274524 10.1146/annurev.publhealth.22.1.309
Wechsler H Devereaux RS Davis M Collins J Using the school environment to promote physical activity and healthy eating Preventive Medicine 2000 31 S121 S137 10.1006/pmed.2000.0649
Bronfenbrenner M Ecology of the family as a context for human development: Research perspectives Developmental Psychology 1986 22 723 742 10.1037/0012-1649.22.6.723
U.S. Department of Agriculture National school lunch program and school breakfast program: Nutrition objectives for school meals Federal Register 1994 59 30218 30251
U.S. Department of Agriculture National school lunch program and school breakfast program: Compliance with the Dietary Guidelines for Americans and food-based menu systems Food and Consumer Service 1995
Wechsler H Brener ND Kuester S Miller C Food service and foods and beverages available at school: Results from the School Health Policies and Programs Study 2000 Journal of School Health 2000 71 313 324 11586874
Story M Hayes M Kalina B Availability of foods in high schools: Is there cause for concern? Journal of the American Dietetic Association 1996 96 123 126 8557936 10.1016/S0002-8223(96)00039-9
French SA Story M Fulkerson JA School food policies and practices: A state-wide survey of secondary school principals Journal of the American Dietetic Association 2002 102 1785 1789 12487541 10.1016/S0002-8223(02)90382-2
Neumark-Sztainer D Story M Perry C Casey MA Factors influencing food choices of adolescents: Findings from focus-group discussions with adolescents Journal of the American Dietetic Association 1999 99 929 937 10450307 10.1016/S0002-8223(99)00222-9
Neumark-Sztainer D Wall M Perry C Story M Correlates of fruit and vegetable intake among adolescents. Findings from Project EAT Prev Med 2003 37 198 208 12914825 10.1016/S0091-7435(03)00114-2
Cullen KW Eagan J Baranowski T Owens E de Moor C Effect of a la carte and snack bar foods at school on children's lunchtime intake of fruits and vegetables Journal of the American Dietetic Association 2000 100 1482 1486 11138440 10.1016/S0002-8223(00)00414-4
Kubik MY Lytle LA Hannan PJ Perry CL Story M The association of the school food environment with dietary behaviors of young adolescents American Journal of Public Health 2003 93 1168 1173 12835204
French SA Story M Fulkerson JA Hannan P An environmental intervention to promote lower-fat food choices in secondary schools: Outcomes from the TACOS study American Journal of Public Health 2004 94 1507 1512 15333303
French SA Story M Fulkerson JA Gerlach AF Food environment in secondary schools: A la carte, vending machines, and food policies and practices American Journal of Public Health 2003 93 1161 1167 12835203
Fox MK Crepinsek MK Connor P Battaglia M School nutrition dietary assessment study II: Summary of findings 2001 Alexandria, VA, U.S. Department of Agriculture, Food and Nutrition Service, Office of Analysis, Nutrition and Evaluation
Dwyer J The School Nutrition Dietary Assessment Study American Journal of Clinical Nutrition 1995 61 173S 177S 7832161
Harnack L Snyder P Story M Holliday R Lytle L Neumark-Sztainer D Availability of a la carte food items in junior and senior high schools: A needs assessment Journal of the American Dietetic Association 2000 100 701 703 10863576 10.1016/S0002-8223(00)00204-2
Zive MM Elder JP Prochaska JJ Conway TL Pelletier RL Marshall S Sallis JF Sources of dietary fat in middle schools Preventive Medicine 2002 35 376 382 12453715 10.1006/pmed.2002.1089
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Int Semin Surg OncolInternational seminars in surgical oncology : ISSO1477-7800BioMed Central London 1477-7800-2-211623616110.1186/1477-7800-2-21ResearchExploring strategies to prevent post-lobectomy space: transient diaphragmatic paralysis using Botulinum Toxin Type A (BTX-A) Kaya Seyda Ors [email protected] Habip [email protected] Hakan Rıza [email protected]Özcan Ali Vefa [email protected] İbrahim [email protected] Burhan [email protected] Koray [email protected] Pamukkale University Medical School, Department of Thoracic Surgery, Denizli, Turkey2 Pamukkale University Medical School, Department of Anaesthesiology, Denizli, Turkey3 Pamukkale University Medical School, Department of Cardiovascular Surgery, Denizli, Turkey4 Pamukkale University Medical School, Department of General Surgery, Denizli, Turkey2005 19 10 2005 2 21 21 2 3 2005 19 10 2005 Copyright © 2005 Kaya et al; licensee BioMed Central Ltd.2005Kaya 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.
Objective
Various techniques to reduce air space after pulmonary lobectomy especially for lung cancer have been an important concern in thoracic surgical practice. The aim of this study was to assess the effectiveness of Botulinum toxin A (BTX-A) injection into the diaphragm to reduce air space after right lower pulmonary lobectomy in an animal model.
Methods
Twelve male New Zealand rabbits were randomly allocated into two groups. All animals underwent right lower lobectomy. Then, normal saline of 0,1 ml and 10 units of 0,1 ml Botulinum toxin type A were injected into the muscular part of the right hemidiaphragm in control (n = 6) and BTX-A groups (n = 6) respectively. Residual air space and diaphragmatic elevation were evaluated with chest X-ray pre- and postoperatively. Diaphragmatic elevation was measured as a distance in millimetre from the line connecting the 10th ribs to the midpoint of the right hemidiaphragm.
Results
The mean diaphragmatic elevation in BTX-A and control groups were 7.0 ± 2.5 and 1.3 ± 1.2 millimetres respectively. Diaphragmatic elevations were significantly higher in BTX-A group (p = 0.0035).
Conclusion
Intraoperative Botulinum toxin type A injection may reduce postlobectomy spaces effectively via hemidiaphragmatic paralysis in rabbits. Further studies are needed to validate the safe use of Botulinum toxin type A in human beings.
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Introduction
Lung resection alone is the most effective therapy for the patients with non-small cell lung carcinoma limited to the lung without distant metastasis. Complete resection is the goal of all operations for lung cancer. Every patient with locoregional lung cancer should be approached as a potential candidate for resection. For patients with adequate lung function, the current standard cancer resections include lobectomy, bronchoplastic lobectomy, bilobectomy and pneumonectomy, based on the extent of disease. The appropriate operation depends on the clinical and surgical stage of the tumor. Lobectomy is the ideal operation for resection of a lung cancer confined to the parenchyma of a single lobe[1,2].
After a lobectomy, the pleural space is drained routinely. Approximately 10% of persistent spaces become complicated with empyema or bronchopleural fistula). Lung lobectomies especially in the presence of fibrotic parenchymal disorders often leave a pleural space, and the remaining lobe or lobes may not be sufficient to fill the ipsilateral hemithorax. Residual air space and prolonged air leak after pulmonary lobectomies can potentially cause serious complications such as bronchopleural fistula and empyema, requiring longer hospital stay and increased health costs. Although it is not universally accepted among thoracic surgeons, reducing postlobectomy space could theoretically be useful in preventing the above-mentioned complications. In this respect, several manoeuvres can be used to attempt to decrease the size of the hemithorax including pleural tents, muscle flaps, pneumoperitoneum and phrenic nerve manipulation [1-4].
Botulinum toxin type A (BTX-A) is an extremely potent neurotoxin that interacts selectively with cholinergic neurons to inhibit the presynaptic release of the neurotransmitter acetylcholine [5]. BTX-A is currently used for cosmetic and therapeutic goals for years, but to our knowledge, has not been reported for the treatment of postlobectomy spaces in literature so far. The aim of our present study was to evaluate whether BTX-A can be effective for hemidiaphragmatic paralysis to prevent postlobectomy space in an animal model.
Materials and methods
The protocol was approved by the Institutional Animal Care and Use Committee, and all animals were housed in the facilities of the Medical Faculty of Pamukkale University.
Animals
This study was carried out on twelve male New Zealand rabbits weighing between 1.5–2.0 kg. The animals were housed in wire bottom cages at 21–24°C room temperature with 12-hour light dark cycle. All animals were fed on standard laboratory diet and water but received only water for 12 h before surgery.
After an overnight fast, the rabbits were anesthetized by an intramuscular injection of ketamine, 35 mg/kg and xylazine 5 mg/kg. After cardiac monitorization and endotracheal intubation, ventilation was maintained artificially (SAR-830 Rodent ventilator, Geneq Inc., Montreal, Canada).
Surgical procedure
The right chest wall was shaved, and the animal placed in a left side down position. A skin incision of 3–4 centimetre in length was made on the right anterolateral chest wall under aseptic conditions. The muscles in the 6th intercostal space were bluntly dissected to expose the right thoracic cavity. Right lower lobectomy was performed by ligation of bronchial artery, vein and bronchi with 2/0 silk suture. Then, animals were randomly allocated to two groups. Following right lower lobectomy, the animals in Group 1 (n = 6) served as controls, and 0,1 ml of 0.09% NaCI was injected into the medial and lateral muscular part of the right hemidiaphragm. The animals in Group 2 (n = 6) were injected with 10 U (0, 1 ml) BTX-A (BOTOX®, Allergan Pharmaceutical Ltd., Ireland) into the same region as in Group 1. All injections were made using a 26-gauge needle attached to a sterile 1 ml syringe. All animals had a 10F chest tube placed along the diaphragm and for the following two hours aspirated intermittently and chest tube was removed after having negative pressure. Both groups received single dose prophylactic antibiotic, and received diclofenac sodium for postoperative analgesia for 3 days.
Postoperative period
After the surgery, the rabbits were closely monitored for clinical evidence of pain (vocalisation, tachypnea, and restlessness) for seven days. Chest radiographs were taken for evaluation of diaphragmatic elevation, residual air space and complications. X-rays were taken during in inspiration. Since the onset of paralytic effect of Botulinum toxin type A begins in 2nd day, diaphragmatic elevation was evaluated radiographically in four consecutive antero-posterior-chest radiographs (preoperatively, postoperatively, 3rd and 7th day). All the roentgenograms were taken at a 90 cm distance from the cassette while animals were in erect position. Diaphragmatic elevation was measured as a distance in millimetre from the line connecting the 10th ribs to the midpoint of the right hemidiaphram. Reversal of paralysis was observed by fluoroscopic examination of diaphragmatic movements.
Statistical analysis
The results are expressed as mean and ± standard deviation (SD). Differences among the groups were evaluated using Mann-Whitney U test. A P value of less than 0.05 was considered significant.
Results
There were no respiratory distress, prolonged air leak and any other complications requiring treatment in any group. Two animals in BTX-A group had about less than 10% increase in respiratory rate at the 3rd day and normalised at the 4th day (From about 70 to 80 breaths per minutes). There was no changes in dietary patterns, and neither vocalisation nor agitation in any animal.
Immediate postoperative and 3rd day roentgenograms revealed no complications such as hemothorax, mediastinal shift and atelectasis. Minimal residual air space in the base of right hemidiaphragm was detected in 3rd day roentgenograms of two rabbits in BTX-A and three rabbits in control groups. There was no residual air space In BTX-A group but was still remaining in 2 rabbits of control group in 7th day radiographs.
The mean preoperative and postoperative 7th day measurements of diaphragmatic heights are shown in Table 1 for both groups. The mean right hemidiaphragmatic elevations were 7.0 ± 2.5 in BTX-A (Figure 1) and 1.3 ± 1.2 in control (Figure 2) groups. Diaphragmatic elevations were significantly different (p = 0.0035) between groups. Fluoroscopic examination of diaphragmatic movements revealed that paralyses reversed in 8–12 weeks.
Table 1 Diaphragmatic heights, and diaphragmatic elevation differences in both groups (mean ± SD).
Control group BTX-A group P value
Preop diaphragmatic heights (mm) 23.8 ± 5.4 24.8 ± 5.7 NS
Postop 7th day diaphragmatic heights (mm) 25.2 ± 4.4 31.8 ± 4.3 0.045
Elevation difference (mm) 1.3 ± 1.2* 7.0 ± 2.4 0.0035
Figure 1 Postoperative 7th day chest radiograph of a rabbit of BTX-A group.
Figure 2 Postoperative 7th day chest radiograph of a rabbit of control group.
Discussion
Some of the major factors delaying early hospital discharge following pulmonary lobectomy for lung cancer can be secondary to the adverse consequences of postlobectomy space. With increasing concern for quality of medical care, length of hospital stay, and cost of health care delivery, it has become the responsibility of the surgeon to control all factors that lead to a prolonged hospitalization.
Although several techniques have been used for prevention of postlobectomy space, optimal methods have not been described. Among these methods diaphragmatic elevation techniques have been used [1,2]. Phrenic nerve paralysis by crushing or local anaesthetic injection are some of the applied techniques for this purpose. Complication of permanent paralysis by crushing or ineffectiveness of short term paralysis by local anaesthetic injection reduces the usefulness of diaphragmatic elevation method. In general, diaphragmatic paralysis leads up to 20% loss of in pulmonary functions in adults with fully expanded normal lungs, in which elevated diaphragm will compress the expanded lung [6-8]. However when a patient had a lobectomy, diaphragmatic paralysis will not collapse an expanded lung, but instead obliterates a dead space.
In several rabbit studies, botulinum toxin A has been used as 5 to 10 units for relaxation of different muscles [9,10]. In the study of Aoki, the safety margin of BTX-A was determined as 13.9 ± 1.7 U/kg in mice [11]. As the duration of act of BTX is dose dependent, 10 U of BTX injection per animal was preferred into the diaphragm, which was safe and provided sufficient paralysis [12].
Botulinum toxin type A has an average clinical onset of action approximately 12 to 72 hours after injection, with a peak effect at one week. Then, plateau effect continues for 1 to 2 months [13]. For this reason, we took radiographs at the 3rd and 7th day for the evaluation of diaphragmatic elevation. We detected significant diaphragmatic elevation in the BTX-A group at day 7. Fluoroscopic examination of diaphragmatic movements revealed that paralysis reversed in 8–12 weeks. Our results are generally in concordance with these studies.
In this study, we showed that injection of BTX into the diaphragm can provide effective elevation for the prevention of postlobectomy space in an animal model.
Further studies are now required to evaluate its effectiveness in the clinical setting, particularly after pulmonary resection for lung cancer.
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Martini N Ginsberg RJ Pearson FG, Cooper JD, Deslauriers J, Ginsberg RJ, Hiebert CA, Patterson GA, Urschel HC Lobectomy Thoracic Surgery 2002 New York: Churchill Livingstone 981 90
Shields TW Ponn RB Shields TW, LoCicero III J, Ponn RB Complications of pulmonary resection General Thoracic Surgery 2000 Philadelphia: Lippincott Williams and Wilkins 481 505
Okur E Kir A Halezeroglu S Alpay AL Atasalihi A Pleural tenting following upper lobectomies or bilobectomies of the lung to prevent residual air space and prolonged air leak Eur J Cardiothorac Surg 2001 20 1012 5 11675190 10.1016/S1010-7940(01)00947-2
Cerfolio RJ Holman WL Katholi CR Pneumoperitoneum after concomitant resection of the right middle and lower lobes (bilobectomy) Ann Thorac Surg 2000 70 942 6 discussion 946–7 11016338 10.1016/S0003-4975(00)01675-1
Graham HK Aoki KR Autti-Ramo I Boyd RN Delgado MR Gaebler-Spira DJ Gormley ME Guyer BM Heinen F Holton AF Matthews D Molenaers G Motta F Garcia Ruiz PJ Wissel J Recommendations for the use of botulinum toxin type A in the management of cerebral palsy Gait Posture 2000 11 67 79 10664488 10.1016/S0966-6362(99)00054-5
Shields TW Shields TW, LoCicero III J, Ponn RB Diaphragmatic function, diaphragmatic paralysis and eventration of the Diaphragm General Thoracic Surgery 2000 Philadelphia: Lippincott Williams and Wilkins 617 36
Urmey WF McDonald M Hemidiaphragmatic paresis during interscalene brachial plexus block: effects on pulmonary function and chest wall mechanics Anesth Analg 1992 74 352 7 1539813
Fell SC Surgical anatomy of the diaphragm and the phrenic nerve Chest Surg Clin N Am 1998 8 281 94 9619305
Ohtsuki H Hasebe S Okano M Furuse T Morphological changes in the orbital surface layer muscle of the rabbit eye produced by botulinum toxin Ophthalmologica 1998 212 53 60 9438587 10.1159/000027261
Kim HS Hwang JH Jeong ST Lee YT Lee PK Suh YL Shim JS Effect of muscle activity and botulinum toxin dilution volume on muscle paralysis Dev Med Child Neurol 2003 45 200 6 12613778 10.1017/S0012162203000380
Aoki KR Botulinum neurotoxin serotypes A and B preparations have different safety margins in preclinical models of muscle weakening efficacy and systemic safety Toxicon 2002 40 923 8 12076646 10.1016/S0041-0101(02)00086-7
Aoki KR Guyer B Botulinum toxin type A and other botulinum toxin serotypes: a comparative review of biochemical and pharmacological actions Eur J Neurol 2001 8 21 9 11851731 10.1046/j.1468-1331.2001.00035.x
Tilton AH Injectable neuromuscular blockade in the treatment of spasticity and movement disorders J Child Neurol 2003 18 S50 66 13677571
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Int Semin Surg Oncol. 2005 Oct 19; 2:21
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Lipids Health DisLipids in Health and Disease1476-511XBioMed Central London 1476-511X-4-221620215810.1186/1476-511X-4-22ResearchThe antihyperlipidemic activities of 4(3H) quinazolinone and two halogenated derivatives in rats Refaie Fawzia M [email protected] Amr Y [email protected] Soad M Abdel [email protected] Aida M [email protected] Mona A [email protected] Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt2 Department of Organic Chemistry, Faculty of Science, Al-Azhar University, Cairo, Egypt3 Department of Clinical Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt2005 4 10 2005 4 22 22 21 9 2005 4 10 2005 Copyright © 2005 Refaie et al; licensee BioMed Central Ltd.2005Refaie 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.
In the present study, the effects of subchronic treatments (4 weeks) of hypercholesterolemic (single) and diabetic-hypercholesterolemic (combined) rats with 4 (3H) quinazolinone and 2 halogenated derivatives (6, 8-dibromo-2-methy-4 (3H) quinazolinone and 6-iodo-2-methyl-4(3H) quinazolinone) at a sublethal dose level (2 mg/Kg) on cholesterol metabolism were investigated. Bezafibrate, a hypolipidemic drug was used as a reference compound for data comparison. Treatment of rats with single and combined hypercholesterolemia with quinazolinone compounds gave rise to highly significant reductions in serum total cholesterol and cholesterol ester levels, whereas serum triacylglycerol level was significantly reduced only after treatment with halogen-substituted quinazolinones in single hyper-cholesterolemia, compared to the control group. The effects of different quinazolinones and bezafibrate on reduction of serum LDL-C level were comparable in single hypercholesterolemia but significantly different in combined hypercholesterolemia. Results obtained from this study suggest that the antihyperlipidemic effect of quinazolinone compounds was brought about by inhibition of dietary cholesterol absorption and / or intestinal ACAT activity.
QuinazolinoneHalogenated quinazolinonesBezafibratehypercholesterolemiaDiabetes mellitusTriglyceridesCholesterollipoproteinsRat
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Introduction
Cardiovascular diseases remain by far the number one cause of death for both men and women of all ethnic backgrounds. Although many causative factors of these diseases are recognized (smoking, high blood pressure, genetic background, diabetes mellitus and obesity) high serum LDL-C and elevated total cholesterol levels are the most prevalent indicators for susceptibility to atherosclerotic heart disease [1,2]. Atherosclerosis is a disorder of the arterial wall characterized by accumulation of cholesterol ester in cells derived from the monocyte-macrophage line, smooth muscle cell proliferation and fibrosis, and results in narrowing the blood vessel [3]. An association of dietary cholesterol with cardiac and cerebral vascular diseases is based on several lines of evidence, including studies in animal models and epidemiological data in humans [4].
Dyslipidemia, hallmarked by low plasma HDL-C and high LDL-C and triacylglycerol levels, is common in patients with diabetes mellitus. These lipoprotein abnormalities are held to be responsible for considerable cardiovascular disease-related morbidity and mortality [5]. The risk for cardiovascular disease is increased approximately 2 to 4 fold in patients with diabetes mellitus compared with non-diabetic controls [6].
There are many classes of lipid lowering agents available, these drugs have different mechanisms of action and variable efficacy depending on the lipid profile of an individual. In spite of their lipid-lowering effect, these drugs have many side effects. Thus, research is still pursuing to find out novel agents that are more effective and safe. The 4(3H)-quinazolinone derivatives (a derivative of the parent compound quinazoline) have been shown as a group of compounds of broad medical interest. Quinazolinones are reported to exhibit antibacterial [7], antiviral [8], anticancer [9], antihypertensive [10] and anti-inflammatory activities [11]. The antihyperlipidemic and antihypercholesterolemic activities of quinazolinone derivatives are reported and the activities of the tested compounds were almost equal to that of β-sitosterol (a plant sterol of hypolipidemic activity) [1,12].
The present work aims at investigating the antihyperlipidemic activities of 4 (3H) quinazolinone and two halogenated derivatives (6, 8-dibromo- and 6-iodo-2-methyl-4(3H) quinazolinone) in hypercholesterolemic and/or diabetic rats. Bezafibrate was adopted in this study as a reference hypolipidemic drug for data comparison.
Materials and methods
Chemicals
4(3H)-quinazolinone was synthesized by stirring a mixture of formamide and anthranilic acid at 120–125°C for 4 h. [7]. The two halogenated derivatives; 6, 8-dibromo-2-methyl-4(3H)-quinazolinone and 6-iodo-2-methyl-4(3H)-quinazolinone were synthesized as previously described [13]. The crude compounds were filtered, washed several times with hot water then recrystallized from ethanol. The structures of the synthesized compounds were verified by IR and mass spectral analyses.
Animals
A total of 130 adult male Swiss albino rats weighing 150–180 g were used throughout this study. The animals were housed in steel mesh cages (4/cage) and maintained for a week-acclimatization period on a commercial pellet diet, which was finely ground before being administered to animals.
Preparation and Administration of Chemical Compounds
One tablet (200 mg) of the bezafibrate (trivial name: Bezalip, GlaxoWellcome, UK) was finely ground and dissolved in 50 ml of distilled water to prepare a stock solution of 4 mg/ml. The solution was prepared weekly and stored in a well stoppered bottle at 4°C. Bezafibrate was daily administered to rats by a stainless-steel gavage needle at a dose level of 18 mg/kg body weight, which is equivalent to the human dose calculated from the conversion table of Paget and Barnes [14].
A weight of 50 mg quinazolinone and its halogenated derivatives was dissolved in 50 ml of 10% (v/v) polyethylene glycol to prepare stock solutions (1 mg/ml). The solutions were prepared weekly and stored in a well-stoppered bottle at 4°C. The quinazolinone compounds were orally administered to animals at a sublethal dose level of 2 mg/kg body weight. The LD50 value of quinazolinone was previously reported to be 320 mg/kg body weight on i.p. administration in rats [15]. The toxicities of dibromo-and iodoquinazolinone derivatives were very low as they did not cause any mortality of rats in excess of 800 mg/kg body weight.
Preparation of High Cholesterol Diet
The high cholesterol diet was prepared as previously described [16]. 1% cholesterol (w/w) and 0.2% sodium cholate (w/w) were well mixed with the finely-ground commercial diet.
Induction of Diabetes Mellitus
Induction of diabetes mellitus in rats was carried out by a single i.p. injection of freshly prepared alloxan monohydrate (Sigma, USA) solution at a concentration of 120 mg/kg body weight after a fast of 12 h. [17]. After 3 days of diabetes mellitus induction rats were put on the high cholesterol diet for 4 weeks.
Study Design
Rats were allocated into 4 main reference groups (I-IV) as follows: Group I. Normal Controls (NC) comprised 8 normal rats fed a normal diet and left intact without any treatment, Group II. Vehicle Controls consisted of 8 normal rats fed a normal diet and received daily an oral dose of 0.5 ml 10% polyethylene glycol, Group III. Hypercholesterolemia, which was subdivided into 5 subgroups (8 rats each): (a) Hypercholesterolemia Controls, in which rats fed a high cholesterol diet and left without any treatment and (b, c, d, e) subgroups, which consisted of hypercholesterolemic rats treated daily with a single oral dose of bezafibrate, 4 (3H) quinazolinone, 6, 8-dibromo or 6-iodo-2-methyl-4 (3H) quinazolinone, respectively, and Group IV. Combined Hypercholesterolemia (C), which was also subdivided into 5 subgroups (8 rats each): (a) Diabetes- Hypercholesterolemia controls, in which diabetic rats fed a high cholesterol diet and left without any treatment and (b, c, d, e) subgroups, in which diabetic rats fed a high cholesterol diet and treated daily with a single oral dose of bezafibrate, 4 (3H) quinazolinone, 6, 8-dibromo or 6-iodo-2-methyl-4 (3H) quinazolinone, respectively.
Treatment with the different compounds started on the same day of feeding high cholesterol diet and lasted for 4 weeks. Body weight of the animals in all groups was recorded weekly until the end of the experiment.
Blood Collection and Tissue Sampling
Blood samples were taken from the retro-orbital venous plexus under light ether anesthesia using a glass capillary tube after a fast of 12 h. and immediately centrifuged. Serum samples were aliquoted and stored at -20°C until lipid profile analysis, except for fasting glucose, LDL and HDL levels, which were determined on the same day without delay. Liver was excised, rinsed from blood in isotonic sterile saline, blotted dry and weighed. A weighed portion of fresh liver tissue was dropped into a test tube containing concentrated sulphuric acid for total lipid determination. The remainder of liver tissue was stored in physiological saline at -20°C until biochemical analysis.
Biochemical Assays
Fasting serum glucose level was determined by an enzymatic colorimetric method [18] using a commercial assay kit (Diamond Diagnostics, Egypt). Serum and hepatic total cholesterol and cholesterol ester levels were measured according to Zlatkis et al. [19]. Serum triacylglycerol level was assayed by the method of Jacobs and Van Denmark [20] (Diamond Diagnostics, Egypt). Serum and hepatic total lipid levels were determined as described by Knight et al. [21]. Serum HDL-C [22] and LDL-C levels [23] were also assessed (Diamond Diagnostics, Egypt and Quimica Clinica Aplicada, Spain, respectively). Serum VLDL-C level and the atherogenic index were determined by calculation [24,25]. Liver and kidney function tests (serum albumin, urea, creatinine, alanine and aspartate aminotransferases activity) were determined using commercial assay kits (Diamond Diagnostics, Egypt). The concentrations of triacylglycerol and malondialdehyde were also estimated in the whole liver homogenate (5%) [26,27].
Statistical Analysis
Data were expressed as means ± SD and the least significant difference (LSD) test for multiple comparisons between the different treated subgroups and along with their respective controls was applied [28].
Results
Fig. 1 shows a gradual increase in the percentage of total body weight gain in hypercholesterolemic rats treated with bezafibrate, quinazolinone and its halogenated derivatives throughout the experimental period. Non significant changes were recorded in the percentage of body weight gain in the treated subgroups at the 4th week point, except for the bezafibrate-treated subgroup, which showed a significant decrease compared to normal controls.
Figure 1 Changes in the percentage of body weight gain in hypercholesterolemic rats treated with the different modalities throughout the experimental period. *denotes statistical significance (p < 0.05) at the 4th week point compared to normal controls.
Fig. 2 demonstrates irregular changes in the percentage of body weight gain in diabetic-hypercholesterolemic rats treated with bezafibrate, quinazolinone and its halogenated derivatives culminated in a highly significant reduction at the end of the experiment, which reached 79.32, 74.92, 85.32 and 59.48% (P < 0.01), respectively, compared to normal controls.
Figure 2 Changes in the percentage of body weight gain in diabetic-hypercholesterolemic rats treated with the different modalities throughout the experimental period. *denotes statistical significance (p < 0.05) at the 4th week point compared to normal controls.
Table 1 illustrates non significant differences in relative liver weights in both non diabetic and diabetic-hypercholesterolemic groups treated with the different compounds.
Table 1 Effects of bezafibrate, quinazolinone and its halogenated derivatives on relative liver weights of rats with single and combined hypercholesterolemia compared to normal controls.
Single Hypercholesterolemia Combined Hypercholesterolemia
Groups Liver wt./b.wt. Groups Liver wt./b.wt.
N.C N.C
Mean ± SD 0.031 ± 0.005 Mean ± SD 0.031 ± 0.005
Range (0.024–0.039) Range (0.024–0.039)
HCD+B C+B
Mean ± SD 0.030 ± 0.003 Mean ± SD 0.037 ± 0.005
Range (0.025–0.032) Range (0.032–0.045)
Change% -11.76 Change% 5.71
HCD+Q C+Q
Mean ± SD 0.033 ± 0.003 Mean ± SD 0.030 ± 0.002
Range (0.029–0.037) Range (0.028–0.033)
Change% -2.94 Change% -14.29
HCD+BrQ C+BrQ
Mean ± SD 0.031 ± 0.003 Mean ± SD 0.029 ± 0.003
Range (0.028–0.035) Range (0.025–0.036)
Change% -8.82 Change% -17.14
HCD+IQ C+IQ
Mean ± SD 0.030 ± 0.002 Mean ± SD 0.030 ± 0.001
Range (0.027–0.032) Range (0.027–0.031)
Change% -11.76 Change% -14.29
Table 2 shows that treatment of hypercholesterolemic rats with bezafibrate, quinazolinone, dibromo- and iodo-derivatives caused a significant reduction in fasting serum glucose level, which reached 11.63, 29.78, 22.47 and 29.40%, respectively, compared to the control group. Multiple comparison analysis showed that quinazolinone and its halogenated derivatives produced a more pronounced antihyperglycemic effect than bezafibrate. Bezafibrate and the three quinazolinone compounds gave rise to a comparable decrease of 18.12, 27.73, 31.66 and 32.31%, respectively, in serum total lipids concentration. A parallel reduction in serum triacylglycerol level was noticed in bezafibrate (26.65%), dibromo- (16.83%) and iodoquinazolinone (15.74%) subgroups, compared to the control group. Treatment with bezafibrate, quinazolinone and its dibromo-derivative caused a comparable reduction in serum total cholesterol and cholesterol ester levels, which reached 28.78 & 37.7, 39.93 & 47.77 and 41.41 & 46.85%, respectively. Iodoquinazolinone showed a more notable decrease of 43.83 and 50.78%, respectively. Serum free cholesterol and HDL-C levels were insignificantly changed in all treatment subgroups. In contrast, significantly parallel reductions in serum LDL-C level and atherogenic index were recorded in all treated subgroups, and reached 38.56 & 41.29, 55.03 & 56.73, 55.65 & 49.91 and 54.44 & 37.70% in bezafibrate, quinazolinone, dibromo- and iodoquinazolinone, respectively. Serum VLDL-C level was significantly reduced in bezafibrate (26.63%), dibromo- (16.83%) and iodoquinazolinone (15.72%) subgroups, compared to controls. Multiple comparison analysis showed non significant changes in serum VLDL-C level among the different treatment subgroups.
Table 2 Statistical significance of fasting serum glucose level and lipid profile in hypercholesterolemic rats treated with bezafibrate, quinazolinone and halogenated derivatives, compared to control group.
Parameters HCD HCD+B HCD+Q HCD+BrQ HCD+IQ
Glucose(mg/dl)
Mean ± SD 123.57 ± 11.99a 109.20 ± 14.31b 86.77 ± 9.87c 95.80 ± 8.28c 87.24 ± 5.32c
Range (105.11–135.02) (82.69–125.22) (77.45–106.22) (86.36–111.10) (77.54–93.81)
Change% - -11.63 -29.78 -22.47 -29.40
Total lipids(g/L)
Mean ± SD 4.58 ± 0.67a 3.75 ± 0.68b 3.31 ± 0.95b 3.13 ± 0.43b 3.10 ± 0.78b
Range (3.62–5.67) (2.82–4.79) (2.02–4.54) (2.40–3.90) (2.17–4.72)
Change% - -18.12 -27.73 -31.66 -32.31
T.G.(mg/dl)
Mean ± SD 94.46 ± 14.23a 69.29 ± 11.04b 87.05 ± 9.18ac 78.56 ± 12.79bc 79.59 ± 15.94bc
Range (73.33–108.89) (54.40–86.65) (75.56–102.22) (66.67–98.50) (52.44–100.89)
Change% - -26.65 -7.84 -16.83 -15.74
T.Cholesterol(mg/dl)
Mean ± SD 159.06 ± 26.04a 113.28 ± 17.52b 95.55 ± 18.11bc 93.19 ± 14.29bc 89.35 ± 22.37c
Range (119.35–87.50) (95.93–139.06) (75.63–121.41) (65.63–104.69) (70.31–123.44)
Change% - -28.78 -39.93 -41.41 -43.83
F.Cholesterol(mg/dl)
Mean ± SD 36.66 ± 7.14a 37.88 ± 8.94a 32.12 ± 8.15a 29.45 ± 4.11a 30.14 ± 6.39a
Range (25.76–45.20) (29.59–52.36) (21.14–46.18) (22.11–33.82) (25.04–40.33)
Change% - 3.33 -12.38 -19.67 -17.79
E.Cholesterol(mg/dl)
Mean ± SD 122.40 ± 19.10a 76.25 ± 8.89b 63.93 ± 10.02bc 65.05 ± 7.96bc 60.25 ± 15.29c
Range (91.85–142.30) (66.01–89.04) (50.54–76.01) (54.00–74.33) (43.32–83.11)
Change% - -37.7 -47.77 -46.85 -50.78
HDL-C (mg/dl)
Mean ± SD 24.27 ± 3.23 a 27.73 ± 5.48a 28.33 ± 6.15a 25.68 ± 6.34a 21.14 ± 8.93a
Range (19.75–28.80) (18.30–35.15) (20.36–36.00) (18.92–33.32) (14.44–37.84)
Change% - 14.26 16.73 5.81 -12.90
LDL-C (mg/dl)
Mean ± SD 116.09 ± 26.74a 71.32 ± 17.78 b 52.20 ± 15.23b 51.49 ± 10.43 b 52.89 ± 19.31b
Range (74.82–141.75) (49.96–89.66) (32.83–76.84) (29.54–62.17) (26.84–79.28)
Change% - -38.56 -55.03 -55.65 -54.44
VLDL-C (mg/dl)
Mean ± SD 18.89 ± 2.85a 13.86 ± 2.21b 17.41 ± 1.84ac 15.71 ± 2.56bc 15.92 ± 3.19bc
Range (14.67–21.78) (10.88–17.33) (15.11–20.44) (13.33–19.70) (10.49–20.18)
Change% - -26.63 -7.83 -16.83 -15.72
A.I.
Mean ± SD 5.57 ± 0.87a 3.27 ± 1.30b 2.41 ± 0.42b 2.79 ± 1.02b 3.47 ± 0.91b
Range (4.22–7.21) (1.80–6.11) (1.94–3.14) (1.67–4.52) (2.26–4.70)
Change% - -41.29 -56.73 -49.91 -37.70
For each parameter similar characters denote insignificance between groups. The mean difference is significant at p < 0.05.
Table 3 illustrates that treatment of diabetic-hypercholesterolemic rats with bezafibrate caused significant reductions in fasting serum glucose and total lipid levels (17.36 and 23.84%, respectively), while more pronounced reductions were recorded after treatment with quinazolinone (67.48 & 48.32%), dibromo- (66.09 & 49.76%) and iodo- derivatives (67.39 & 47.52%), compared to the control group. Significant decreases were recorded in serum levels of total cholesterol and cholesterol ester levels after treatment with quinazolinone (43.66 & 62.33%), dibromo- (49.86 & 55.34%) and iodo-derivative (49.12 & 56.74%, respectively), whereas bezafibrate caused a less notable decrease of serum cholesterol ester level (19.93%) and did not affect total cholesterol level. None of the foregoing treatments significantly affected serum triacylglycerol, HDL-C and VLDL-C levels. Treatment of diabetic-hypercholesterolemic rats with quinazolinone, dibromo- and iodo- derivatives produced significant decreases in serum LDL-C level (71.74, 65.69 and 60.74%, respectively) and atherogenic index (50.24, 69.54 and 51.99%). Bezafibrate treatment caused a slight but significant reduction in the atherogenic index (28.39%), while serum LDL-C level remains unchanged.
Table 3 Statistical significance of serum fasting glucose level and lipid profile in diabetic-hypercholesterolemic rats treated with bezafibrate, quinazolinone and halogenated derivatives.
Parameters C C+B C+Q C+BrQ C+IQ
Glucose(mg/dl)
Mean ± SD 210.34 ± 16.56a 173.82 ± 26.86b 68.40 ± 5.22c 71.32 ± 8.42c 68.60 ± 5.66c
Range (189.24–230.00) (140.00–218.00) (61.65–79.47) (60.55–85.00) (60.76–75.36)
Change% - -17.36 -67.48 -66.09 -67.39
Total lipids(g/L)
Mean ± SD 6.25 ± 2.29a 4.76 ± 0.85b 3.23 ± 1.08c 3.14 ± 0.63c 3.28 ± 0.77c
Range (2.32–9.51) (3.76–5.96) (2.02–4.64) (2.02–4.15) (2.20–4.34)
Change% - -23.84 -48.32 -49.76 -47.52
T.G(mg/dl)
Mean ± SD 110.42 ± 18.75a 91.91 ± 15.64a 109.51 ± 31.98a 99.00 ± 15.06a 100.64 ± 10.33a
Range (88.14–133.33) (70.00–111.11) (66.67–165.00) (73.33–121.14) (86.67–117.11)
Change% - -16.76 -0.82 -10.34 -9.72
T.Cholesterol(mg/dl)
Mean ± SD 176.28 ± 42.14a 152.76 ± 36.36a 99.31 ± 22.80b 88.38 ± 26.64b 89.69 ± 10.02b
Range (127.34–224.38) (106.25–199.22) (74.22–126.56) (65.63–134.38) (73.44–104.22)
Change% - -13.34 -43.66 -49.86 -49.12
F.Cholesterol(mg/dl)
Mean ± SD 39.19 ± 11.02ab 44.37 ± 9.44a 30.31 ± 6.98b 30.03 ± 6.73b 31.06 ± 5.43b
Range (25.62–51.89) (28.62–53.98) (20.16–40.98) (23.09–41.30) (26.02–41.63)
Change% - 13.22 -22.66 -23.37 -20.75
E.Cholesterol(mg/dl)
Mean ± SD 135.37 ± 31.41a 108.39 ± 31.2b 69.00 ± 18.80c 60.46 ± 16.43c 58.56 ± 6.96c
Range (101.72–170.75) (77.63–145.24) (49.18–96.97) (48.40–88.08) (47.42–65.67)
Change% - -19.93 -62.33 -55.34 -56.74
HDL-C(mg/dl)
Mean ± SD 24.76 ± 3.62a 25.88 ± 2.74a 24.87 ± 4.71a 26.71 ± 2.58a 22.50 ± 1.77a
Range (18.00–27.94) (21.13–29.60) (19.83–31.52) (22.40–30.33) (19.80–24.32)
Change% - 4.52 0.44 7.88 -9.13
LDL-C(mg/dl)
Mean ± SD 115.29 ± 34.06a 84.56 ± 43.44a 32.58 ± 18.85b 39.56 ± 26.65b 45.26 ± 10.16b
Range (79.73–168.69) (44.05–137.56) (19.51–64.94) (16.24–82.42) (34.31–63.03)
Change% - -26.65 -71.74 -65.69 -60.74
VLDL-C(mg/dl)
Mean ± SD 22.08 ± 3.75a 18.38 ± 3.13a 22.52 ± 6.28a 19.80 ± 3.01a 20.13 ± 2.07a
Range (17.63–26.67) (14.00–22.22) (13.33–33.00) (14.67–24.23) (17.33–23.42)
Change% - -1.67 1.99 -10.33 -8.83
A.I.
Mean ± SD 6.27 ± 2.06a 4.49 ± 1.53b 3.12 ± 1.20b 1.91 ± 1.27b 3.01 ± 0.57b
Range (3.56–9.80) (2.59–6.91) (1.65–4.82) (0.99–4.33) (2.21–3.79)
Change% - -28.39 -50.24 -69.54 -51.99
For each parameter similar characters denote insignificance between groups.
The mean difference is significant at p < 0.05.
Table 4 demonstrates that treatment of hypercholesterolemic rats with bezafibrate, quinazolinone, dibromo- and iodo- derivatives caused a significant parallel reduction in hepatic total lipids concentration (20.11, 30.80, 25.01 and 25.21%, respectively) associated with a significant comparable elevation in hepatic malondialdehyde concentration (74.95, 63.11, 49.84 and 60.81%), compared to the control group. Hepatic triacylglycerol concentration was significantly decreased in bezafibrate (43.03%), dibromo- (25.59%) and iodoquinazolinone (23.70%) subgroups, while it remains unchanged in quinazolinone subgroup, compared to the control group. A significant elevation in hepatic free cholesterol and, in contrast a significant decrease of hepatic cholesterol ester concentrations were recorded in quinazolinone (120.34 & 50.67%, respectively), dibromo-(66.21 & 32.09%) and iodoquinazolinone (63.28 & 31.06%) subgroups. The LSD test revealed that halogenated quinazolinones exerted a more pronounced effect on cholesterol fractions than quinazolinone. Bezafibrate produced no significant effect on hepatic cholesterol fractions. Also, hepatic total cholesterol concentration was not significantly changed in all treatment subgroups.
Table 4 Statistical significance of hepatic lipid profile and malondialdehyde concentration in hypercholesterolemic rats treated with bezafibrate, quinazolinone and halogenated derivatives, compared to control group
Parameters HCD HCD+B HCD+Q HCD+BrQ HCD+IQ
Total lipids
Mean ± SD 75.30 ± 7.86a 60.16 ± 4.18b 52.11 ± 13.07b 56.47 ± 9.50b 56.32 ± 12.94b
Range (68.24–88.24) (54.90–66.27) (36.86–74.90) (41.57–68.24) (41.57–76.08)
Change% - -20.11 -30.80 -25.01 -25.21
Triacylglycerol
Mean ± SD 10.55 ± 2.66a 6.01 ± 1.39b 9.83 ± 2.17a 7.85 ± 2.75b 8.05 ± 1.35b
Range (7.09–14.91) (4.09–7.83) (7.30–13.91) (4.39–11.74) (6.65–10.50)
Change% - -43.03 -6.82 -25.59 -23.70
Total cholesterol
Mean ± SD 25.18 ± 2.77ab 25.64 ± 1.48a 22.71 ± 2.13b 22.80 ± 2.01b 22.83 ± 2.13b
Range (20.58–28.46) (23.98–28.32) (19.11–25.85) (20.00–25.39) (19.12–25.61)
Change% - 1.83 -9.81 -9.45 -9.33
Free cholesterol
Mean ± SD 5.80 ± 1.70a 5.23 ± 1.32a 12.78 ± 2.46b 9.64 ± 1.30c 9.47 ± 1.50c
Range (3.37–8.13) (3.85–6.83) (9.52–17.33) (7.68–11.45) (7.22–11.51)
Change% - -9.83 120.34 66.21 63.28
Ester cholesterol
Mean ± SD 19.38 ± 1.67a 20.42 ± 1.00a 9.56 ± 1.56b 13.16 ± 1.30c 13.36 ± 1.20c
Range (16.99–21.76) (19.35–22.47) (7.57–11.43) (11.11–14.51) (11.67–15.56)
Change% - 5.37 -50.67 -32.09 -31.06
MDA(nmol/g)
Mean ± SD 131.31 ± 15.10a 229.73 ± 4.76b 214.18 ± 23.85b 196.75 ± 26.44b 211.16 ± 14.54b
Range (113.33–155.42) (223.53–238.43) (172.42–246.01) (161.57–229.28) (188.37–230.33)
Change% - 74.95 63.11 49.84 60.81
All parameters are expressed in mg/g tissue unless otherwise stated.
For each parameter similar characters denote insignificance between groups.
The mean difference is significant at p < 0.05.
Table 5 shows that hepatic total lipids, triacylglycerol, total cholesterol and malondialdehyde concentrations were not significantly changed in diabetic-hypercholesterolemic rats treated with quinazolinone and its halogenated derivatives. A significant parallel elevation in hepatic free cholesterol and, in contrast a significant parallel decrease of hepatic cholesterol ester concentrations were recorded in quinazolinone (65.53 & 32.62%, respectively), dibromo-(68.94 & 30.48%) and iodoquinazolinone subgroups (67.44 & 25.59%). Bezafibrate treatment had no significant effect on all of the foregoing parameters.
Table 5 Statistical significance of hepatic lipid profile and malondialdehyde concentration in diabetic-hypercholesterolemic rats treated with bezafibrate, quinazolinone and halogenated derivatives
Parameters C C+B C+Q C+BrQ C+IQ
Total lipids
Mean ± SD 74.76 ± 7.34a 65.00 ± 7.37a 71.37 ± 6.01a 65.93 ± 11.54a 66.12 ± 9.47a
Range (65.88–84.71) (54.90–76.47) (62.16–77.65) (45.49–81.57) (48.92–78.43)
Change% - -13.06 -4.53 -11.81 -11.56
Triacylglycerol
Mean ± SD 12.10 ± 3.37a 9.90 ± 1.08a 11.90 ± 4.38a 12.03 ± 5.07a 11.80 ± 4.82a
Range (8.87–17.30) (8.38–11.30) (7.48–16.61) (6.09–18.78) (6.23–17.30)
Change% - -18.18 -1.65 -0.58 -2.48
Total cholesterol
Mean ± SD 26.53 ± 4.93a 26.58 ± 3.37a 23.87 ± 3.58a 25.62 ± 2.94a 26.57 ± 2.88a
Range (20.05–34.15) (21.14–31.71) (18.79–28.86) (23.58–32.52) (22.08–30.08)
Change% - 0.19 -10.03 -3.43 0.15
Free cholesterol
Mean ± SD 7.34 ± 2.45a 7.49 ± 1.93a 12.15 ± 2.42b 12.40 ± 3.17b 12.29 ± 2.59b
Range (4.04–10.41) (5.69–11.32) (8.29–16.03) (8.85–18.31) (8.98–14.80)
Change% - 2.04 65.53 68.94 67.44
Cholesterol ester
Mean ± SD 19.19 ± 2.61a 19.19 ± 1.95a 12.93 ± 2.78b 13.34 ± 2.77b 14.28 ± 3.82b
Range (15.31–23.74) (16.26–21.93) (9.25–16.69) (10.36–18.21) (9.57–19.02)
Change% - 0 -32.62 -30.48 -25.59
MDA(nmol/g)
Mean ± SD 235.18 ± 16.32a 238.29 ± 18.23a 250.98 ± 12.06a 250.23 ± 13.03a 244.19 ± 13.45a
Range (210.72–256.47) (218.99–269.02) (234.51–267.97) (237.65–276.24) (221.17–257.91)
Change% - 1.32 6.72 6.40 3.83
All parameters are expressed in mg/g tissue unless otherwise stated.
For each parameter similar characters denote insignificance between groups.
The mean difference is significant at p < 0.05.
Discussion
Although the literature implies a few evidence about the antihyperlipidemic activity of some quinazolinone derivatives, yet intensive research in quinazolinone chemistry is still in progress as indicated by the continuous flow of newly appearing derivatives. In the present study, we have demonstrated the effects of quinazolinone and two halogenated derivatives on the lipid profile in single and combined hypercholesterolemia in rats. Alloxan was used to induce diabetes mellitus in rats and, surprisingly treatment of single and combined hypercholesterolemia groups with quinazolinone compounds for 4 weeks significantly decreased the fasting serum glucose level, compared to bezafibrate and their respective control groups (Tables 2 &3). Although the antihyperglycemic activity of these compounds was not intentionally studied, this effect could be attributed to being cyclic amidine compounds. Metformin, a well known hypoglycemic drug, which acts as an inhibitor of hepatic glucose production, possesses guanidine and amidine functionalities in its molecular structure. Another class of compounds, triarylimidazoles, which has also an amidine moiety in a cyclic structure, displayed a significant glucagon antagonistic property [29]. The antihyperglycemic activity of the quinazolinone compounds is worth to be adequately investigated.
The quinazolinone compounds, being water insoluble, were dissolved in 10% polyethylene glycol as a solvent and thus a vehicle control group was established to evaluate its effect on all studied serum and liver tissue parameters. Statistical analysis showed that polyethylene glycol had no significant effect on the body weight of animals, as well as all serum and liver tissue parameters (data not shown). Accordingly, statistical significance was referred to the normal control or the reference groups.
In order to assess the toxic side effects of the administered dose levels of quinazolinone compounds, the change percent of body weight gain at each week point with respect to the initial body weight was recorded. In comparison to normal controls at the 4th week point, hypercholesterolemic rats treated with quinazolinone compounds exhibited non significant changes in the percentage of body weight gain, except for bezafibrate subgroup, which showed a significant reduction (P < 0.01) (Fig. 1). Diabetic-hypercholesterolemic rats manifested a significant decrease in the percentage of body weight gain (P < 0.01) in the different treatment modalities (Fig. 2). This clearly indicates that quinazolinone compounds had no significant effect on body weight, and that the reduction in the body weight gain of diabetic- hypercholesterolemic animals was mainly due to diabetes mellitus. Sellei et al. [30] reported that treatment with a therapeutic dose of any compound on the body weight loss must not exceed 10%. Also, Bissery et al. [31] stated that a drug dosage that produces a loss in body weight of 20% is considered as excessively toxic. Feron et al. [32] documented that in subchronic toxicity experiments, the weights of the major organs of the body may serve as a useful index of toxicity. However, decreased absolute weights in treated animals may be merely a reflection of lower body weight, thus calculation of organ weight to whole body weight ratio justifies the usefulness of the data obtained. In the present study, relative liver weights were recorded, as liver is the main site of drug activation and detoxification. Non significant differences in relative liver weights were observed in single and combined hypercholesterolemia subgroups (Table 1). Also, non significant alterations were recorded in serum levels of aminotransferases activity (ALT&AST) and albumin, as well as urea and creatinine concentrations, compared to normal controls (data not shown), which indicates the safety of the quinazolinone compounds at the adopted dose levels.
A comparable reduction in serum and hepatic triacylglycerol levels is reported in hypercholesterolemic rats treated with bezafibrate, dibromo- and iodoquinazolinone subgroups, compared to their reference groups (Tables 2 &4), which demonstrates that halogens substitution in the quinazolinone nucleus confered a triacylglycerol-lowering effect to the parent quinazolinone compound. However, this observation was not noticed in combined hypercholesterolemia (Tables 3 &5) and may be interpreted as simply due to increased fat mobilization as a result of insulin deficiency manifested by the significant reduction in the body weight of the animals at decapitation (Fig. 2).
The cholesterol-lowering effect of quinazolinone and its dibromo-and iodo-derivatives is represented by comparable reductions in serum total cholesterol and cholesterol ester levels in single and combined hypercholesterolemia (Tables 2 &3), which might be due to inhibition of dietary cholesterol absorption and/or its esterification. Since two enzymes are involved in these two processes; pancreatic cholesterol esterase [33] and intestinal acyl CoA-cholesterol acyl transferase enzyme (ACAT) [34], thus it could be suggested that quinazolinones inhibit one or both enzymes activity. The assumption that quinazolinones inhibit ACAT activity is confirmed by the significant decrease of hepatic cholesterol ester concentration and, in contrast the significant increase of hepatic free cholesterol concentration (Tables 4 &5). The remarkable elevation in hepatic MDA concentration in single hypercholesterolemia after treatment with quinazolinones, compared to their reference group (Table 4) may be explained due to increased fatty acids pool and consequently its peroxidation as a result of ACAT inhibition by quinazolinone compounds (as previously mentioned). Another suggestion for the cholesterol-lowering effect of the quinazolinones is the interaction between quinazolinones and cholesterol, which inhibits its entry into the enterocytes, thus not providing the required cholesterol pool for esterification.
Parallel results affirmed [16] a significant reduction in serum total cholesterol and unchanged hepatic total cholesterol concentrations in high cholesterol-fed rats treated with 1 mg/Kg quinazoline derivative (4-amino-2-(4-(bicycle (2, 2, 2) oct-2-ene-5-carbonyl)-1-piperazinyl)-6, 7-dimethoxy-quinazoline). The authors ascribed their findings due to direct inhibition of cholesterol absorption or due to increased biliary excretion of sterol and/or bile acids and the block of cholesterol movement from the liver to the blood.
Also, it is turned out that halogens substitution of quinazolinone has a negative effect on inhibition of hepatic cholesterol esterification displayed by the less pronounced reduction in hepatic free cholesterol and, in contrast the more notable elevation in hepatic cholesterol ester concentrations, compared to quinazolinone (Table 4). Surprisingly, this phenomenon was not observed in case of combined hypercholesterolemia (Table 5). In comparison to bezafibrate, iodoquinazolinone was found to produce a significant more pronounced reduction in serum total cholesterol and cholesterol ester levels. In case of combined hypercholesterolemia, treatment with quinazolinone and its dibromo- and iodo-derivatives produced a significantly favorable effect on serum and hepatic cholesterol profiles than bezafibrate (Tables 3 &5).
In regard to serum lipoproteins profile, the 3 quinazolinones reduced more or less equally serum LDL-C level, whereas serum VLDL-C level was highly reduced by dibromo- and iodoquinazolinone, compared to their reference group. The quinazolinone compounds did not affect significantly serum HDL-C level (Table 2). Taken together, these findings furtherly confirm the inhibitory effect of quinazolinones on cholesterol esterification, as it is well known that LDL particles have a high affinity to bind to cholesterol ester, while it is free cholesterol in case of HDL [35], and that halogens substitution of the quinazolinone increases its triacylglycerol-lowering activity. Consistent with our results, Seki et al. [16] referred the decrease of serum total cholesterol level in quinazoline derivative-treated rats to reduction in serum VLDL-C and LDL-C rather than HDL-C levels.
In combined hypercholesterolemia subgroups serum HDL-C and VLDL-C levels were not significantly affected by any of the treatments, whereas serum LDL-C level was significantly decreased by quinazolinones treatment, compared to either bezafibrate or their reference group (Table 3). It is interesting to remark that the more notable reduction in serum LDL-C level in combined than single hypercholesterolemia subgroups might be a sequela of enhanced lipid peroxidation in the former, which converted LDL to modified LDL, including glycosylated, oxidized and small dense LDL particles [36]. Increased liver malondialdehyde (MDA) concentration in the combined hypercholesterolemia subgroups confirms this explanation (Tables 4 &5). However, the non significant changes in hepatic MDA concentration in the different diabetic-hypercholesterolemic subgroups, compared to their reference group, may be due to enhanced lipolysis in diabetes mellitus that exceeded the effects of quinazolinone compounds against LDL peroxidation, which was displayed in non diabetic animals.
Our results are in line with those of Hsun et al. [37], who stated that quinazoline derivatives reduce significantly the level of serum LDL-C with respect to the cholesterol reference group. Previous studies documented that serum LDL-C reduction is due to inhibition of hepatic apoB secretion, which is the main apolipoprotein in LDL particles that is recognized by the LDL receptor, and that cholesterol ester availability may be important in regulating apo B secretion. Carr et al. [38,39] affirmed that when ACAT is highly inhibited in African green monkey, both apo B and cholesterol ester secretion are reduced, but when ACAT is moderately inhibited cholesterol ester secretion may be reduced without affecting apo B secretion.
Data presented in Tables (2 &3) demonstrate a significant reduction in the atherogenic index (AI) in the quinazolinones-treated subgroups of non diabetic and diabetic hypercholesterolemic rats, compared to their respective reference groups. Non significant differences in the atherogenic index were noticed between the quinazolinones and the bezafibrate. The reduced atherogenic index in both treatments was mainly due to the decrease in serum total cholesterol level rather than elevation in serum HDL-C level.
Conclusion
1-Subchronic treatments of non diabetic and diabetic-hypercholesterolemic rats with 4(3H)-quinazolinone and its 6, 8-dibromo and 6-iododerivatives (4 weeks) have no significant toxic side effects at the adopted sublethal dose levels (2 mg/Kg).
2-Quinazolinone and its halogenated derivatives possess potential antihyperlipidemic activity in single and combined hypercholesterolemia demonstrated by the dramatic reduction in serum total cholesterol and cholesterol ester levels, and most importantly LDL-C, which is a major risk factor for coronary and cerebral vascular atherosclerosis. The mode of action of quinazolinone compounds is possibly through inhibition of dietary cholesterol absorption and intestinal ACAT activity.
3-Halogen derivatization of quinazolinone confers a triacylglycerol-lowering activity to the parent compound in case of single hypercholesterolemia only.
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Mol CancerMolecular Cancer1476-4598BioMed Central London 1476-4598-4-371620971210.1186/1476-4598-4-37ResearchActivation of focal adhesion kinase enhances the adhesion and invasion of pancreatic cancer cells via extracellular signal-regulated kinase-1/2 signaling pathway activation Sawai Hirozumi [email protected] Yuji [email protected] Hitoshi [email protected] Yoichi [email protected] Hiroki [email protected] Hiromitsu [email protected] Tadao [email protected] Department of Gastroenterological Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 4678601, Japan2005 6 10 2005 4 37 37 12 9 2005 6 10 2005 Copyright © 2005 Sawai et al; licensee BioMed Central Ltd.2005Sawai 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
Interaction with integrin and focal adhesion kinase (FAK) regulates the cancer cell adhesion and invasion into extracellular matrix (ECM). In addition, phosphorylation of FAK correlates with the increase of cell motility and invasion. Adhesion and spreading of cancer cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK. In this study, we investigated the mechanism of activation of FAK and its downstream extracellular signal-regulated kinase (ERK)-1/2 signaling following stimulation by interleukin (IL)-1α and adhesion to ECM with subsequent enhancement of pancreatic cancer cell adhesion and invasion.
Results
In immunoblotting analysis, all three pancreatic cancer cell lines (AsPC-1, BxPC-3, and Capan-2) expressed the protein of FAK and β1 integrin. Enhancement of FAK protein association with β1 integrin when cells were plated on Coll IV was more increased by stimulation with IL-1α. Preincubation with anti-β1 integrin antibody and FAK siRNA transfection inhibited the association of FAK with β1 integrin of pancreatic cancer cells. FAK phosphorylation was observed by adhesion to Coll IV, furthermore, stronger FAK phosphorylation was observed by stimulation with IL-1α of pancreatic cancer cells adhered to Coll IV in time-dependent manner. Genistein, a tyrosine kinase inhibitor, markedly inhibited the FAK phosphorylation. IL-1α stimulation and Coll IV adhesion enhanced the activation of Ras, as evidenced by the increased Ras-GTP levels in pancreatic cancer cells. Activation of Ras correlated with the phosphorylation of ERK. While not statistical affecting the apoptosis of pancreatic cancer cells, IL-1α-induced adhesion and invasion on Coll IV were inhibited with FAK gene silencing by siRNA, β1 integrin blocking, and inhibition of FAK phosphorylation. PD98059, a MEK inhibitor, also inhibited IL-1α-induced enhancement of adhesion and invasion in pancreatic cancer cells.
Conclusion
Our results demonstrated that activation of FAK is involved with the aggressive capability in pancreatic cancer through Ras/ERK signaling pathway. Based on our results, we suggest that the modification of IL-1, FAK, and integrins functions might be a novel therapeutic approach to aggressive spread of pancreatic cancer.
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Background
Integrin binding to extracellular matrix (ECM) protein or integrin crosslinking increases tyrosine phosphorylation of focal adhesion kinase (FAK) [1,2]. FAK is a tyrosine kinase considered a central molecule in integrin-mediated signaling, and it is involved in cellular motility and protection against apoptosis [3-7]. The carboxyl-terminal tyrosine residue (tyr397) of FAK, constitutes a major site of phosphorylation, appears important for the tyrosine phosphorylation of focal complex associated proteins, and creates a high-affinity binding site recognized by the SH-2 domain of the Src family [8,9]. In vitro, the N-terminal domain of FAK binds directly to peptides corresponding to the cytoplasmic domain of integrin β subunits [2,10]. In addition, overexpression and phosphorylation of FAK correlates with the increase of cell motility and invasion [4,5,11,12]. Adhesion and spreading of cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK [3,4,7]. Furthermore, suppression of adhesion induced tyrosine phosphorylation of FAK may interrupt cancer cell-ECM interactions and affect the invasive and metastatic potential of cancer cells. Based on these results, considerable evidence points to a critical role of FAK participating in cancer cell-ECM interactions.
The integrin family ECM receptors are key mediators of cell proliferation and cell survival. Integrin-mediated cell adhesion is required for cell motility and also affects cell proliferation and invasion in many systems [13-15]. We previously proved that enhancement of α6β1-integrin expression by interleukin (IL)-1α acting through IL-1 receptor type I (IL-1RI) plays an important role in metastatic and invasive behaviors of pancreatic cancer, and that strong expression of α6 integrin in cancerous tissues significantly correlated with poor prognosis of pancreatic cancer patients [15,16]. β1 integrin is also reported to play an important role in invasiveness and metastasis formation of cancer cells [17-19].
Integrin-ECM interactions also activate signaling cascades such as extracellular signal-regulated kinase-1/2 (ERK1/2), mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3-K), and Akt [2-5,20-23]. Especially, the downstream targets of Ras signaling pathway are ERK1/2, which have been found to be regulated by activation of FAK with respect to different matrix components [3,21,24]. Therefore, integrin binding to the ECM creates and activates a bipartite kinase complex and transduces external stimuli from the ECM to the nucleus.
In this study, we investigated the mechanism of activation of FAK and its downstream ERK1/2 signaling following adhesion to ECM. Our results suggest that activation of FAK enhances the adhesive and invasive capabilities of pancreatic cancer cells through Ras/ERK signaling pathway.
Results
Expression of FAK and β1 integrin in pancreatic cancer cells
In immunoblotting analysis, all three pancreatic cancer cell lines also expressed FAK and β1 integrin (Fig. 1A). In this study, we transfected all three pancreatic cancer cells with siRNA that specifically targets FAK. Downregulation of FAK protein expression by siRNA was confirmed by immunoblotting. Transfection of siRNA resulted in a near total loss of FAK expression (Fig. 1B).
Figure 1 Expression of FAK and β1 integrin in pancreatic cancer cells. (A) FAK and β1 integrin protein expression in pancreatic cancer cell lines was determined in whole cell lysates by Western blotting analysis. Fifty micrograms of total cell lysates was separated on 10 % SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were probed with antibodies against FAK and β1 integrin. The β-actin Western blot served as a loading control. (B) Knockdown of FAK expression by siRNA was confirmed by immunoblotting in all three pancreatic cancer cells. Re-probing with an anti-β-actin antibody served as a control.
Interaction between FAK and β1 integrin
In order to determine if FAK interacts with β1 integrin subunit, β1 integrin was immunoprecipitated from cell lysates of AsPC-1, BxPC-3, and Capan-2 cells and Western blotted using anti-FAK antibody. As shown Fig. 2, more FAK protein associated with β1 integrin when cells were plated on Coll IV. Stimulation with recombinant human IL-1α (rIL-1α) of pancreatic cancer cells on Coll IV furthermore enhanced the association of FAK with β1 integrin. Preincubation with anti-β1 integrin antibody and FAK siRNA inhibited the association of FAK with β1 integrin of pancreatic cancer cells (Fig. 2).
Figure 2 FAK and β1 integrin subunit interaction in AsPC-1, BxPC-3, and Capan-2 cells. After incubating on Coll IV with 10 ng/ml IL-1α or 0.5 μg/ml anti-β1 integrin antibodies for 24 h, cells were added (2 × 105cells/well) to each well and incubated at 37°C and 5% CO2 for 30 min. After removing unattached cells, total cell lysates were immunoprecipitated with antibodies against β1 integrin subunit. Samples were resolved in 10 % SDS-PAGE gel under nonreducing conditions and transferred to polyvinylidene difluoride membranes. Membranes were then probed with anti-FAK or anti-β1 integrin antibodies. Samples from cells incubated on 3 % BSA were served as control.
Phosphorylation of FAK was enhanced by Coll IV adhesion and IL-1α stimulation
FAK activity was examined by FAK phosphorylation and total tyrosine phosphorylation in AsPC-1, BxPC-3, and Capan-2 cells. The lysates of pancreatic cancer cells were analyzed by immunoprecipitation with antibodies to FAK followed by Western blotting with an antibody which is specific for anti-phosphotyrosine (4G10). FAK activation was observed within 15 min of adhesion to Coll IV and remained high for 60 min. (Fig. 3). Stronger FAK phosphorylation was observed by stimulation with rIL-1α of pancreatic cancer cells adhered to Coll IV in time-dependent manner (Fig. 3). In contrast, incubation with Genistein, a tyrosine kinase inhibitor, markedly inhibited the FAK phosphorylation.
Figure 3 Phosphorylation of FAK in AsPC-1, BxPC-3, and Capan-2 cells. After incubating on Coll IV with 10 ng/ml IL-1α and/or 60 μM Genistein for 24 h, cells were added (2 × 105 cells/well) to each well and incubated at 37°C and 5 % CO2 for 15, 30, or 60 min. After removing unattached cells, cells were collected from each time point and lysed by lysis buffer and immunoprecipated with FAK antibody as described in Methods and materials. Effect of IL-1α and Genistein on Coll IV mediated phosphorylation of FAK was demonstrated. IP, immunoprecipitation; WB, Western blot.
Effect of FAK gene silencing and β1 integrin blocking on apoptosis of pancreatic cancer cells
After treated/untreated with FAK siRNA or control siRNA, pancreatic cancer cells were incubated with/without anti-β1 integrin antibody for 24 h, and then terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay was performed to investigate whether knockdown of FAK expression and β1 integrin blocking had any effects on pancreatic cancer apoptosis. Both FAK siRNA transfection and incubation with β1 integrin antibody induced a slight increase in the apoptotic fraction of calls in normal culture conditions, however, there was no statistical difference among these treatments (Table 1).
Table 1 Effect of FAK siRNA and β1 integrin blocking on apoptosis of pancreatic cancer cells
Apoptotic fraction (%)
Cell line Untreated Anti-β1 integrin antibody FAK siRNA Control siRNA
AsPC-1 1.37 ± 0.33 1.89 ± 0.51 2.20 ± 0.66 1.44 ± 0.54
BxPC-3 1.99 ± 0.31 2.38 ± 0.51 2.56 ± 0.53 2.02 ± 0.4
Capan-2 2.44 ± 0.30 2.59 ± 0.37 3.01 ± 0.52 2.50 ± 0.43
AsPC-1, BxPC-3, and Capan-2 cells were treated with anti-β1 integrin antibody, FAK siRNA, or control siRNA and incubated for 24 h, and then detection of apoptosis was performed by TUNEL assay and flow cytometry. Statistical significance was tested by one-way analysis of variance and post hoc test (Turkey Kramer multiple comparisons). All data are expressed as mean ± s.d. There was no significant difference among these treatments.
Involvement of FAK with adhesive and invasive capabilities of pancreatic cancer cells
We investigated whether the inhibition of FAK had any effects on adhesive and invasive response in pancreatic cancer cells. Knockdown of FAK expression with siRNA inhibited IL-1α-induced adhesion and invasion (Fig. 4A, 4B). The inhibitory antibodies against β1 integrin subunit treatment similarly inhibited the IL-1α-induced enhancement of adhesion and invasion in all three pancreatic cancer cells. Genistein and PD98059 (a MEK inhibitor) also inhibited these enhancements of adhesion and invasion by IL-1α stimulation in pancreatic cancer cells. While not statistical affecting cellular apoptosis, the basal adhesive and invasive capabilities of these cells were also suppressed by siRNA, anti-β1 integrin antibody, Genistein, and PD98059 (Fig. 4A, 4B). DMSO vehicle had no effect on adhesion and invasion assays. These data suggest that FAK regulation and β1 integrin subunit may have critical roles in adhesive and invasive capabilities of pancreatic cancer cells.
Figure 4 Involvement of FAK phosphorylation and integrin signaling with adhesion and invasion of pancreatic cancer cells. (A) AsPC-1, BxPC-3, and Capan-2 cells were incubated with 10 ng/ml IL-1α, with 10 ng/ml IL-1α and 60 μM Genistein, with 10 ng/ml rIL-1α and 25 μM PD98059, or with 10 ng/ml rIL-1α and equivalent amounts of DMSO vehicle for 24 h. FAK siRNA transfected cells were cultured with 10 ng/ml IL-1α for 24 h. After incubating for 30 min with/without anti-β1 antibody, cell adhesion assay was performed at 37°C for 30 min. Statistical significance was tested by one-way analysis of variance and post hoc test (Turkey Kramer multiple comparisons). The p-values indicate statistical significance between data in controls and each treatment. Bars indicate the s.d.*: p < 0.05. (B) After incubation for 30 min with/without antibody against β1 integrin, AsPC-1, BxPC-3, and Capan-2 cells were cultured with 10 ng/ml IL-1α, with 10 ng/ml IL-1α and 60 μM Genistein, with 10 ng/ml rIL-1α and 25 μM PD98059, or with 10 ng/ml rIL-1α and equivalent amounts of DMSO vehicle for 24 h in the inner chamber coated with Coll IV. FAK siRNA transfected cells were cultured with 10 ng/ml IL-1α for 24 h in the inner chamber. Statistical significance was tested by one-way analysis of variance and post hoc test (Turkey Kramer multiple comparisons). The p-values indicate statistical significance between data in controls and each treatment. Bars indicate the s.d. *: p < 0.05.
Activation of Ras and ERK pathway after IL-1α stimulation and Coll IV adhesion
We examined the activation of Ras/ERK pathway, a downstream target of FAK in pancreatic cancer cells, following adhesion of cells to Coll IV for 15, 30, or 60 min. IL-1α stimulation and Coll IV adhesion enhanced the activation of Ras, as evidenced by the increased Ras-GTP levels in three pancreatic cancer cell lines. Activation of Ras correlated with the phosphorylation of ERK. In contrast, IL-1α stimulation and Coll IV adhesion did not induce the Akt phosphorylation (data not shown). These results indicate that IL-1α and Coll IV adhesion may induce activation of ERK through a Ras-dependent pathway as a downstream of FAK activation (Fig. 5A). To evaluate whether FAK and β1 integrin affect IL-1α-induced activation of Ras and ERK, pancreatic cancer cells were transfected with FAK siRNA or treated with anti-β1 integrin antibody for 30 min before being exposed to rIL-1α for 30 min on Coll IV. Inhibition FAK expression and β1 integrin function inhibited the activation of Ras and phosphorylation of ERK in three pancreatic cancer cell lines (Fig. 5B). These results suggest that expression of FAK and β1-integrin has an important role in regulating IL-1α-induced activation of signaling pathways. Detection of total ERK 1/2 levels served as a loading control.
Figure 5 Involvement of FAK and β1 integrin subunit with the activation of Ras and ERK pathway in pancreatic cancer cells. Examination of Ras and downstream ERK activation was performed as described in Materials and Methods. Cell lysates were prepared according to the instructions provided in the Ras Activation Assay Kit, and affinity precipitation of GTP-bound Ras was performed using GST-tagged Raf-RBD. Levels of pull-downed Ras (Ras-GTP) were determined by anti-Ras immunoblotting.(A) AsPC-1, BxPC-3, and Capan-2 cells were serum starved for 24 h and then attached to Coll IV with 10 ng/ml IL-1α for 15, 30, or 60 min. The time-dependent Ras activation and ERK1/2 phosphorylation by adhesion to Coll IV was demonstrated. Detection of total ERK 1/2 levels served as a loading control.(B) AsPC-1, BxPC-3, and Capan-2 cells were serum starved for 24 h and then attached to Coll IV with 10 ng/ml IL-1α in the presence or absence of inhibitory antibodies against β1 integrin subunit for 30 min. FAK siRNA transfected pancreatic cancer cells were attached to Coll IV with 10 ng/ml IL-1α. Effects of IL-1α, FAK gene silencing, and β1 integrin blocking on the activation of Ras/ERK signaling pathway in pancreatic cancer cells were demonstrated. Detection of total ERK 1/2 levels served as a loading control.
Discussion
In this report, we demonstrate that FAK plays a critical role in adhesive behavior of pancreatic cancer cells via activating the Ras/ERK signaling pathways. FAK protein association with β1 integrin was increased when cells were attached on Coll IV and further increase was observed by stimulating with IL-1α. Knockdown of FAK expression with siRNA inhibited IL-1α-induced Ras/ERK activation with subsequent inhibition IL-1α-induced adhesion and invasion of pancreatic cancer cells on Coll IV while not statistical affecting cellular apoptosis.
The activation of FAK following cell adhesion to Coll IV and integrins engagement has been reported for many different cell types [3,4,7,25-27]. Recent reports have strongly implicated the FAK phosphorylation of lung cancer cells in adhesion to Coll IV [7]. In this study, the phosphorylation of FAK in pancreatic cancer cells was detectable with Coll IV adhesion in time-dependent manner. Furthermore, stronger FAK phosphorylation was observed by stimulation with IL-1α of pancreatic cancer cells adhered to Coll IV. The tyrosine phosphorylation of FAK was suppressed by Genistein, which has been reported as a potent inhibitor of several tyrosine kinases. It can be suggested that Genistein inhibits the activities of protein tyrosine kinases (PTKs), which are active in the upstream of FAK signaling pathway. Considering the effect of Genistein on FAK phosphorylation, it can be suggested that the induction of FAK phosphorylation in pancreatic cancer cells may proceed primarily through the inhibition of a specific protein tyrosine phosphatase. For instance, a dual-specific phosphatase, PTEN, has been shown to be associated with FAK and capable of FAK dephosphorylation [28-30]. It would be interesting to determine whether PTEN is associated with FAK in cancer cells after adhesion to Coll IV.
There is accumulating evidence that supports an important role of ERK pathway in promoting cell proliferation and invasion of cancer cell [4,31-35]. It has been reported in many cell lines that integrin dependent activation of MAPK requires FAK [24,36,37]. Coll IV-dependent activation of ERK1/2 in intestinal epithelial cells also requires FAK [3]. MAPK has been reported to regulate cell migration through the enhancement of myosin light chain phosphorylation [38]. Thus, the requirement for FAK activity in the efficient activation of MAPK could also be related to the effects of FAK on cell motility [39,40]. In addition, previous studies have indicated that loss of integrin signaling, such as FAK gene silencing and blocking of integrin signaling, can induce cellular apoptosis[14,20,41-44]. In contrast, these suppress of integrin signaling is reported to decrease the invasive behavior but not cellular apoptosis and proliferation in some cancer cells[2,45]. In this study, the similarity in the time course over which FAK and ERK1/2 were activated in response to IL-1α and adhesion to Coll IV of pancreatic cancer cells were observed. Both of knockdown of FAK expression with siRNA and blocking of β1 integrin inhibited IL-1α-induced Ras/ERK activation with subsequent inhibition IL-1α-induced adhesion and invasion of pancreatic cancer cells on Coll IV while not statistical affecting cellular apoptosis. Furthermore, we demonstrated that a MEK inhibitor PD98059 inhibited IL-1α-induced enhancement of adhesion and invasion in pancreatic cancer cells. Our results reveal that the relationship between FAK phosphorylation and the activation of its downstream Ras/ERK signaling pathway has an important role in the adhesive and invasive capabilities of pancreatic cancer cells. It has also been reported that IL-1 plays an important role in tumor invasion and angiogenesis [46-48]. Furthermore, recent reports demonstrated the involvement of IL-1 with the ERK1/2 signaling pathway activation [49-51]. Our results in this work are supported with these reports.
Integrin mediated signaling to ERK1/2 is dependent on the integrity of the actin cytoskeleton [52,53]. The disruption of cytoskeleton integrity completely inhibited fibronectin stimulated FAK tyrosine phosphorylation and ERK1/2 signaling. Also, in some cancer cells, the integrity of the cytoskeleton structure is required for ECM generated signals to ERK1/2. β1 integrin is reported to play an important role in adhesion and invasion of cancer cells [17-19]. And several intracellular signals have been suggested to mediate effects of IL-1, including activation of MAPK. Activation of MAPK by IL-1 subsequently induces the activator protein-1 and nuclear factor-κB DNA-binding activity, which promotes expression of the genes involved in cell survival, proliferation, and angiogenesis [54,55]. We also previously reported that the enhancement of α6β1-integrin expression by IL-1α acting through IL-1RI plays a critical role in adhesive and invasive behaviors in pancreatic cancer, and proved that the strong expression of the α6 integrin subunit in pancreatic cancer tissue significantly correlated with the poor prognosis and the presence of hepatic metastases in pancreatic cancer patients [15,16]. In this study, we investigated whether β1 integrin is physically associated with FAK or the activation of β1 integrin mediated signaling is sufficient to activate FAK in pancreatic cancer cells. We demonstrated that the enhancement of adhesion and invasion to Coll IV in pancreatic cancer cells is dependent on the presence of β1 integrin. Furthermore, we proved that FAK phosphorylation correlated with the activation of its downstream Ras/ERK signaling pathway. Our data indicates that the Coll IV-induced phosphorylation of FAK correlated with physical association of FAK with β1 integrin with subsequent the activation of ERK1/2 signaling pathways in pancreatic cancer cells.
Conclusion
We demonstrated that IL-1α stimulation and cell adhesion to Coll IV enhanced the FAK protein association with β1 integrin and FAK phosphorylation. And these enhancements correlated with the activation of Ras/ERK signaling pathways in pancreatic cancer cells. IL-1α-induced activation of these signaling pathways can be inhibited by knockdown of FAK expression with siRNA, consistent with the inhibition of adhesive and invasive capabilities of pancreatic cancer cells. Based on these results, we suggest that the modification of IL-1, FAK, and integrins functions might be a novel therapeutic approach to aggressive spread of pancreatic cancer.
Methods
Cell culture
The human pancreatic cancer cell lines, AsPC-1, BxPC-3 and Capan-2, were provided from the American Type Culture Collection (Rockville, MD, USA). The AsPC-1 and BxPC-3 cells were maintained in RPMI 1640 (Gibco BRL, Eggenstein, Germany) supplemented with 10 % fetal calf serum (FCS). Capan-2 cells were maintained in Dulbecco modified Eagle medium (Gibco BRL) with high glucose and 10 % fetal calf serum. All cells were incubated at 37°C in a humidified atmosphere of 5 % CO2 in air.
Reagents and Antibodies
rIL-1α was provided by Diaclone (Besançon, France). Coll IV (human) and Genistein were purchased from SIGMA (Saint Louis, MO, USA). PD98059 was purchased from New England Biolabs Inc (Beverly, MA, USA). The monoclonal antibodies used included anti-β1 (P5D2) from Chemicon International Inc. (Temecula, CA, USA); anti-phosphotyrosine (4G10) and anti-FAK (4.47) from Upstate Biotechnology Inc. (Lake Placid, NY, USA). The polyclonal anti-phospho-Akt (587F11), anti-phospho-ERK 1/2 and anti-ERK 1/2 antibodies were from Cell Signaling Technology (Beverly, MA, USA).
Western blot analysis
Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 % Triton X-100, 0.1 % SDS, 0.1 % Nonidet P-40, 2 mM PMSF, 1 mM vanadate, 5 μg/ml Trasylol, 10 μM Pepstatin A and 10 μM leupeptin). Protein concentrations were determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). The amounts of samples were 50 μg per each lane. Lysates were separated by 10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes (Immobilon PVDF; Nihon Millipore Ltd., Tokyo, Japan) and immunoblotted with each antibody. β-actin Western blots were served as controls.
Immunoprecipitation
Pancreatic cancer cells lysates were centrifuged at 14,000 rpm at 4°C for 15 min and the supernatants (750 μg of protein) were used for immunoprecipitation with either anti-β1 integrin (P5D2) or anti-FAK (4.47) antibody at 4°C overnight. After incubation for 1 h with antibody, either protein A- or protein G-sepharose was added to the lysates and incubated overnight at 4°C. The bead bound complexes were pelleted, washed several times with lysis buffer in PBS and boiled with SDS sample buffer for 5 min before loading on 10 % SDS-PAGE. For Western blot analysis, the proteins were transferred to polyvinylidene difluoride membranes after SDS-PAGE Specific binding was detected with the enhanced chemiluminescence system (ECL; Amersham Life Science Ltd., Buckinghamshire, United Kingdom).
RNA interference (siRNA)
Pancreatic cancer cells were transfected with siRNA for FAK and with control nonspecific siRNA using FAK siRNA/siAb™ Assay Kits (Upstate Biotechnology Inc.) according to the manufacture's instruction. Briefly, cells were grown in 35 mm dishes and overlaid with the transfection mixture containing siRNA at a concentration of 200 pmol/well. After 4 h incubation, complete medium with 10 % FCS was added and cells were incubated for another 48 h.
TUNEL assay
Detection of apoptosis was performed by TUNEL assay. After incubating for 24 h with/without anti-β1 integrin antibody, pancreatic cancer cells were collected by centrifugation, fixed in 4 % paraformaldehyde (pH 7.4) and then stained and analyzed for apoptosis using an In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics GmbH, Penzburg, Germany). Fixed cells were permeabilized using a mixture containing terminal deoxynucleotidyltransferase and fluorescein-dUTP at 37°C for 60 min. Flow cytometric analysis using a FACS scan (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA) was done to quantitate apoptosis[56].
Cell adhesion assay
Adhesion assay was performed as described previously with some modifications [15]. 24-well plates were coated either with Coll IV (5.0 μg/cm2) or 3 % bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Briefly, after incubating for 24 h with/without rIL-1α (10 ng/ml), Genistein (60 μM), PD98059 (25 μM), or equivalent amounts of DMSO vehicle, cells were added (2 × 105 cells/well) to each well and incubated at 37°C and 5 % CO2 for 15, 30, or 60 min. After removing unattached cells, the number of adherent cells was counted directly by light microscopy. Before the stimulating experiments with IL-1α were attempted, a concentration of 10 ng/ml was determined to be the lowest effective concentration for stimulating experiments (data not shown). In some experiments, 0.5 μg/ml anti-β1 integrin antibodies were added to cells for 30 min prior to seed to the well. Before the blocking experiments were attempted, a concentration of 0.5 μg/ml was determined to be the lowest effective concentration for blocking experiments (data not shown). Experiments were performed in triplicate and repeated at least three times.
Cell invasion Assay
In vitro invasion assays were performed as described previously with some modifications [15]. Polycarbonate filters (6.3-mm diameter, 8- μm pore size) of cell culture inserts (BD Biosciences Discovery Labware, Franklin Lakes, NJ, USA) were coated with 5.0 μg/cm2 Coll IV. Cancer cells were added (1 × 105 cells/well) to the inner chamber of a cell culture insert and incubated at 37°C for 24 h, either with 10 ng/ml rIL-1α, with 10 ng/ml rIL-1α and 60 μM Genistein, with 10 ng/ml rIL-1α and 0.5 μg/ml of anti-β1 integrin antibody, with 10 ng/ml rIL-1α and 25 μM PD98059, or with 10 ng/ml rIL-1α and equivalent amounts of DMSO vehicle. To quantitate invasion, the filters were fixed in 70 % ethanol for 30 min and stained with Giemsa. Cells were removed from the upper surface of the filters by rubbing gently with a cotton-tipped applicator. Cells that had invaded through the membrane were counted in five random microscope fields of the lower filter surface.
Ras activation assay
Ras activation state was determined using the Ras Activation Assay Kit provided from Upstate. Briefly, after serum starved for 24 h, pancreatic cancer cells were incubated on the 35-mm well coated with Coll IV (5.0 μg/cm2) in serum-free medium with/without rIL-1α (10 ng/ml) for 15, 30, or 60 min. Then, cells were harvested and lysed in lysis buffer (100 mM HEPES, pH 7.5, 200 mM NaCl, 1 % Nonidet P-40, 10 mM MgCl2, 5 mM EDTA and 10 % glycerol), and supernatant prepared by centrifugation for 5 min at 4°C at 14,000 g. Ras-GTP from various treated lysates was "pulled down" using the GST fusion protein corresponding to human Ras binding domain of Raf-1 bound to agarose. The presence of Ras-GTP was detected by Western blotting using anti-Ras antibody (Upstate). In some experiments, 0.5 μg/ml anti-β1 integrin antibodies were added to cells for 30 min prior to seed to the well.
Statistical analysis
Statistical comparisons were made using the Student's t test for paired observations or by one-way ANOVA with a post hoc test for multiple comparisons. Statistical significance was indicated by p < 0.05. Data are presented as mean ± standard deviation (s.d.). Each experiment was repeated 3 times and was carried out in triplicate.
Abbreviations
ECM, extracellular matrix; FAK, focal adhesion kinase; Coll IV, collagen type IV; IL, interleukin; IL-1RI, IL-1 receptor type I; ERK, extracellular signal-regulated kinase; MAPK, mitogen activated protein kinase; PI3-K, phosphatidylinositol 3-kinase; TUNEL, deoxynucleotidyl transferase-mediated nick end labeling; PTK, protein tyrosine kinase; FCS, fetal calf serum; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PBS, phosphate-buffered saline.
Authors' contributions
HS carried out the Western blots, immunoprecipitations, and the investigation of Ras activity in addition to the drafting of the manuscript. YO and HF contribute the adhesion and invasion assays and statistical analyses. YM and TH performed the cell culture, adhesion assay, and the literature search. HT designed the experiments and contributed to the writing of the manuscript. TM conceived the project and aided in experimental design. All authors read and approved the final manuscript.
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Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-3-491618535810.1186/1477-7827-3-49ResearchSelection of gonadotrophin surge attenuating factor phage antibodies by bioassay Sorsa-Leslie Tarja [email protected] Helen D [email protected] William J [email protected] Paul A [email protected] The Department of Obstetrics & Gynaecology, University of Aberdeen, Aberdeen, AB25 2ZD, UK2 The Department of Molecular & Cell Biology, IMS, University of Aberdeen, Aberdeen, AB25 2ZD, UK3 Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki, POB 63 (Haartmaninkatu 8), 00014 Helsinki, Finland4 The Division of Basic Medical Sciences and Division of Clinical Developmental Sciences, St. George's, University of London, London, SW17 0RE, UK2005 26 9 2005 3 49 49 12 8 2005 26 9 2005 Copyright © 2005 Sorsa-Leslie et al; licensee BioMed Central Ltd.2005Sorsa-Leslie 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 aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF).
Methods
A synthetic single-chain antibody (Tomlinson J) phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs) forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4–7). The candidate GnSAF protein bands and spots were then excised for peptide mass mapping.
Results
Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants.
Conclusion
This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the study of uncharacterised proteins that defy conventional approaches. In addition, we conclude that these data support suggestions that GnSAF may be structurally related to serum albumin or very tightly bound to serum albumin.
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Background
Phage display has proven to be a powerful tool for selecting proteins and peptides with specific binding properties from vast numbers of variants. Phage display is based on the simple fact that if gene fragments encoding polypeptides are fused to bacteriophage M13 coat protein genes, the protein products of these fusion genes are displayed on the surface of the filamentous phage. Studies had shown that functional antibody fragments can be expressed in the periplasmic space of E. coli [1,2]. In the case of antibodies, antibody fragments are displayed on the surface with the antigen-binding domains exposed to the outside environment. These phage-bearing particles can be bio-panned against immobilised antigen and those that bind can be eluted and used to infect a new E. coli population. This process can be repeated several times using lower concentrations of antigen each time, thus leading to significant enrichment of high affinity antigen-binding phage. The surface antibody fragments can then be sub-cloned into E. coli vectors that produce soluble antibodies which can be manipulated in the same way as any other recombinant protein.
Although five putative gonadotrophin surge-attenuating factor (GnSAF) amino acid sequences have been published [3-6], they have no significant homology, were derived from proteins between 12–69 kDa in mass, and have not been conclusively confirmed as GnSAF [7]. The purification of GnSAF using conventional chromatographic methods has been fraught with problems, including low concentrations of GnSAF protein in biological fluids despite high bioactivity, co-elution of GnSAF with serum albumin and the interference from large number of proteins in follicular fluid, serum and cell-conditioned medium [6,8,9]. These difficulties have hampered advances in the field. The production of specific GnSAF antibodies by conventional means has not been successful. The rat pAb reported by [6] reflects this problem, having good affinity for GnSAF bioactivity, but co-purifying inadequate amounts of protein, which are also contaminated with too many other proteins, to allow the production of homogenous GnSAF preparations.
Since conventional protein purification and polyclonal antibody strategies have failed to yield conclusive GnSAF candidate molecules, a strategy of combining phage display with our well-established rat pituitary cell bioassay for GnSAF [6,7] was developed. The rationale for this is as follows: Firstly, phage display will produce a range of antibodies to proteins even in the absence of prior purification of the proteins. Secondly, the GnSAF bioassay can be as easily used to detect the absence of GnSAF bioactivity as its presence. Thus, the bioassay could be utilised to pick out phage-derived antibodies that recognised bioactive GnSAF. Once a GnSAF-specific antibody was identified, it could be utilised in an immunopurification strategy to isolate the GnSAF molecule. The current study was therefore devised specifically to utilise the novel combination of phage display and bioassay in order to attempt to identify human ovarian GnSAF.
The rationale for the degree of effort that has been invested into GnSAF research revolves around its potential role in the negative regulation of pituitary responsiveness to GnRH [7]. GnSAF probably coordinates the LH signal with ovarian steroidogenesis and follicular development and, as such, would have considerable therapeutic and diagnostic potential for women and commercially important species.
Methods
GnSAF Bioactive Material and Bioassay
GnSAF bioassay
A critical part of this study was the GnSAF bioassay which was used to test and screen phage display library products for recognition of GnSAF bioactivity. However, the exact method employed to test these phage products varied according the stage of the study. The basic bioassay, which involves the addition of aqueous preparations for testing in a dose-response-design is described below. In the relevant sections detailing the production and testing of the phage display library products, the different approaches used to generate these aqueous preparations are outlined.
Adult female Sprague-Dawley rats (10–14 weeks old) were maintained under a constant 12-h light: 12-h dark, 22°C environment with ad libitum access to food and water. For each cell culture, 15 rats, selected at random during the estrous cycle, were killed by stunning and cervical dislocation. Dispersion and culture of the pituitary cells in serum-free defined medium (SFDM) was carried out as described [6,10]. Bioassays were carried out in quadruplicate wells: 200 μl of fresh SFDM was added, together with the treatments made up to 25 μl with SFDM. All the culture plates contained at least 12 control wells receiving SFDM only. After 24 h incubation with the test substances, the medium was collected and stored at -20°C for subsequent measurement of basal FSH as an index of inhibin bioactivity. The wells were then treated with 0.1 μM GnRH (Fertagyl: Intervet UK Ltd., Cambridge, UK) in 50 μl of SFDM. In all dishes 8 wells previously exposed to SFDM received GnRH alone while 4 wells previously exposed to SFDM received 50 μl of SFDM instead of the 50 μl of GnRH challenge. These acted as controls for the magnitude of the GnRH response. Cultures were terminated after 4 h incubation by collecting the media which was stored at -20°C for subsequent measurement of GnRH-induced LH as an index of GnSAF bioactivity (specifically reduced GnRH-induced LH, but not basal FSH secretion). The QC hFF preparations were added to each bioassay at 0, 1, 5 & 25 μl/well, in at least four wells/dose/separate culture, to act as a GnSAF quality control. Bioassays in which the QC hFF caused <30% suppression of GnRH-induced LH secretion, or in which the control GnRH response constituted <50% increase in LH, were repeated and the data discarded. Concentrations of gonadotrophins in cell-conditioned media from rat anterior pituitary cell cultures were determined using homologous rat time-resolved fluoro-immunoassay (DELFIA) for (a) FSH: with sensitivity and intra-assay and inter-assay C.V. values of 0.6 ng FSH/ml (NIDDK-rFSH-RP-2) using NIDDK-anti-rFSH-S11 and 7.1% and 11.2% respectively; (b) LH: with sensitivity and intra-assay and inter-assay C.V. values of 0.2 ng LH/ml (NIDDK-rLH-RP3) using NIDDK-anti-rLH-S11 and 5.4% and 7.9% respectively.
Human follicular fluid as a source of GnSAF bioactivity
All protocols employing human subjects were given Joint Ethical Committee Approval at Aberdeen and patients all gave informed consent. Follicular fluid (hFF) was aspirated from follicles ≤18 mm in diameter from 40 women undergoing routine IVF in Aberdeen and pooled and desalted as previously described [8]. Subsequently, 500 μl aliquots of the hFF pool were stored at -20°C and used as a GnSAF bioactivity quality control (QC), producing a 40–60% reduction in GnRH-induced LH at 50 μl/well, in all bioassays performed as part of the present study, as previously described [8].
Granulosa/luteal cell-conditioned medium (G/LCM) as a source of GnSAF bioactivity with reduced serum protein contamination
Granulosa/luteal cells (G/LC) were recovered from hFF obtained from women undergoing IVF and cultured as described by [6]. In the present study 2,500 ml of G/LC-conditioned medium (G/LCM) was processed, subjected to Dyematrex Blue A Dye reduction of serum albumin and tested to confirm GnSAF bioactivity as described [6].
Phage Display Library Use to Generate GnSAF Bioactivity-Specific Antibodies
Plasmids and bacterial strains
The single-chain antibody expression vector pIMS147 was derived from the pHELP1 vector [11-14]. This vector produces soluble single-chain antibody (scAb) fragments fused via the 3' end of the variable domain with the human Cκ (HuCκ) domain. This vector also contains six-histidine tag for purification by immobilised metal ion chelate affinity chromatography (IMAC). The intact immunoglobulin (IgG) molecules were obtained by using vectors VHE, VKExpress (a kind gift from A. Bradbury, Los Alamos USA) and pLNOH (a kind gift from L. Norderhaug, Oslo Norway). The modification was made by cloning the pLNOH IgG3 into VHE and adding a HindIII site.
The phage display of scAbs
500 μl of glycerol stock of the Tomlinson J Library was inoculated into 500 ml of 2 × TY broth supplemented with 1% glucose and 100 μg ampicillin/ml, and incubated, with shaking, at 37°C to an OD600 of 0.4 (1–2 h). KM13 helper phage [15] was added to 50 ml of the culture and the mixture incubated at 37°C, without shaking, for 30 min. Infected E. coli cells were pelleted, resuspended in 100 ml of 2 × TY with 0.1% glucose, 100 μg ampicillin/ml and 50 μg kanamycin/ml, and further incubated overnight, with shaking, at 30°C. Phage particles were precipitated with 20 ml polyethylene glycol in 2.5 M NaCl (20% w/v) as described previously [16]. Immunotubes (Greiner Labortechnik, Gloucestershire, UK) were coated by incubation overnight at 4°C with 4 ml of crude GnSAF preparation (10 μg total protein/ml of albumin-depleted G/LCM) in PBS (Oxoid Ltd, Hampshire, UK), washed with PBS, and blocked with 2% skimmed milk in PBS at room temperature (RT) for 2 h. Phagemid particles (approximately 1 × 1012) were added in 4 ml of 2% skimmed milk in PBS, and incubated at RT for 60 min on a rotating turntable, and a further 60 min without rotation. The tubes were washed 20 times with PBS containing 0.1% Tween 20 (Sigma-Aldrich Co. Ltd., Poole, Dorset, UK), and the bound phage eluted with 500 μl of trypsin-PBS (1 mg/ml) for 10 min, with rotation, at RT. Half of the eluted phage (0.25 ml) were infected into 1.75 ml of exponential phase TG1 cell culture suspension in 2 × TY broth, plated on TYE agar containing 1% glucose and 100 μg ampicillin/ml and incubated overnight at 37°C. 7 μl of 2 × TY broth containing 1% glucose, 100 μg ampicillin/ml was added to the plate and the bacteria scraped until loose. 50 μl of this cell suspension was then used to infect 50 ml of 2 × TY (1% glucose, 100 μg ampicillin/ml), incubated, with shaking, at 37°C to an OD600 of 0.4 and subsequently 1 ml of scraped bacteria was stored with 15% glycerol at -80°C. Selected phage were then rescued from the culture as described above. Selection (bio-panning) was repeated a further three times with reducing total protein concentrations of the immobilised GnSAF (albumin-depleted, concentrated, G/LCM) preparation (2nd and 3rd rounds 2.5 μg total protein/ml, 4th round 1.25 μg total protein/ml).
Affinity selection and initial screening of anti-GnSAF antibodies
After the fourth round of bio-panning, 300 individual clones were chosen randomly. The 10 clones with the highest affinity to GnSAF, but not skimmed milk or BSA were pre-selected using an affinity enzyme-linked immunosorbent assay (ELISA) and further selection was made by sequencing them to identify complementarity-determining regions (CDRs). Final selection was based on the scAb affinity for GnSAF as determined using duplicate rat pituitary monolayer bioassays described above. The bioassay was used in the first instance to test for pooled phage clones and subsequently for individual clones which had been subcloned into a soluble vector. As detailed in Fig. 1., pooled or individual scAbs or antibodies were immobilised on dishes pre-coated with secondary antibodies and then hFF or G/LCM or culture medium alone were added. Antibodies or scAbs with affinity for GnSAF bioactivity removed GnSAF molecules from the medium. When this medium was then added to the GnSAF bioassay these GnSAF-recognising scAbs or antibodies could be detected by the reduction in GnSAF bioactivity as revealed by less marked inhibition of GnRH-induced LH release from the rat pituitary cells relative to control preparations such as hFF incubated with medium only.
Figure 1 Diagram showing the strategy to identify GnSAF-recognising scAbs. (1) 96-well dishes were coated with secondary antibodies that would and would not bind the scAbs/IgGs and then (2) incubated with candidate anti-GnSAF and control scAbs/IgGs. When (3) GnSAF-containing QC hFF or G/LCM were added, immobilised GnSAF-recognising scAbs/IgGs bound some or all the GnSAF so that when the medium was (4) removed from the wells and added (5) to our rat pituitary monolayer bioassay, GnSAF bioactivity (6) would be reduced. Where the sequence of secondary and primary antibodies did not include the appropriate coating antibody and GnSAF-recognising scAb/IgG, GnSAF bioactivity would not be significantly reduced.
Expression and purification of bacterial single-chain antibody fragments
A single colony of TG1 cells containing the scAb vector (pIMS147) was grown as previously described [12] and then induced with isopropyl-β-D-thiogalactosidase (IPTG) at a final concentration of 1 mM. Induction was for 4 h at 25°C, after which the periplasmic contents were released to growth media using fractionation buffer (200 mM Tris-HCl, 20% (w/v) sucrose, 1 mM EDTA, 0.5 mg lysozyme/ml, pH 7.5) [12]. The periplasmic fraction containing scAb was removed and filtered through a 0.45 μm filter (Sartorius Ltd, Epsom, Surrey, UK).
Purification of single-chain antibody fragments
The expressed scAbs were purified with Ni2+-charged fast-flow sepharose, IMAC (Amersham Biosciences UK Ltd, Little Chalfont, Bucks, UK) [14] using the hexa-histidine tail as a binding target. A Ni2+-charged fast-flow sepharose resin column was prepared according to the manufacturer's instructions and 2 ml of crude periplasmic fraction used to re-suspend the resin. This suspension was allowed to bind for at least 1 h at 4°C and then passed through the column 3–4 times, each time returning the suspension to the same tube. The sepharose column was washed four times with 14 ml of 100 mM NaCl in PBS, four times with 14 ml of 10 mM imidazole in PBS followed by100 mM NaCl in PBS, pH 7.9, then twice with 14 ml ice-cold PBS and allowed to settle. The column was drained and proteins eluted with 250 mM imidazole in PBS. The eluates were collected and the elution step repeated a further 3 times. Following dialysis against PBS to remove the imidazole, the eluates were assayed for expression product by capture ELISA. The eluates were placed in a Slide-A-Lyzer dialysis cassette (molecular weight cut-off 10 kDa, Pierce and Warriner Ltd, Chester, Cheshire, UK) and dialyzed against 2 litres of PBS overnight at 4°C with constant stirring, after which time the PBS was changed and dialysis was continued for a further 3 h.
ELISA for determination of intact IgG produced by CHO-K1 cells
96-well flat bottomed Immulon® 4 ELISA plates (Dynex Technologies, Ashford, Middlesex, UK) were coated either overnight at 4°C or for 1–2 h at RT with 100 μl of antibody against heavy chain (anti-human IgG, Sigma) diluted 1:1000 in PBS. The plates were washed 3 times with 200 μl of 0.05% (v/v) Tween 20 in PBS (PBS-T) and then blocked for 2 h at RT or overnight at 4°C with 200 μl of 2% skimmed milk in PBS. After washing, as described above, with PBS-T, 100 μl of the neat culture media containing IgGs was added in duplicate to all wells. The plates were incubated for 2 h at RT or 1 h at 37°C and then washed 3 times with 200 μl of PBS-T. After this, the plates were incubated for 1 h at RT with 100 μl of the anti-light chain antibody (Sigma) conjugated with horseradish peroxidase (HRP) diluted 1:1000 in PBS. A standard curve was produced by a doubling dilution series of a known concentration of purified human IgG (Sigma, 4.8 mg/ml stock). Wells containing PBS alone were included as assay blanks. After the final incubation, the plates were washed 5 times with 200 μl of PBS-T and then developed with 100 μl of 0.1 mg/ml 3,3', 5,5' tetramethylbenzidine dihydrochloride (TMB/ml) and 0.2 μl/ml of 30% (v/v) hydrogen peroxide in 0.05 mol/l citrate phosphate buffer, pH 5.0. When optimal signal intensity was obtained, the reaction was stopped by the addition of 50 μl of 1 M H2SO4. The absorbance at 450 nm was read and corrected for absorbance at 405 nm to account for non-specific absorbance by the ELISA plate on an IEMS MF plate-reader (Labsystems Affinity Sensors, Cambridge, UK). The plate-reader was controlled by Genesis software (version 2.20) for Windows™. This ELISA was repeated using concentrated, fragment-free, IgG media and a standard curve was calculated using the known IgG standard to determine the concentrations of intact IgG.
ScAb-derived mammalian IgGs
CHO-K1 cells were used to express scAb-derived antibodies as intact human IgGs. The cells were grown in tissue culture flasks with surface area s of 10 cm2, 25 cm2 or 75 cm2, or in 96-well plates. The cultures were maintained with HAMS-F12 medium (Gibco BRL, Life Technologies Ltd., Paisley, UK) supplemented with 2 mM L-glutamine, 10% (v/v) FCS, penicillin 100 U/ml and streptomycin 100 μg/ml at 37°C in an atmosphere of 5% (v/v) CO2 incubator. The cells were passaged when the cultures became confluent (85–95% confluency) by splitting the cells and adding fresh media. CDRs from soluble expression plasmids (pIMS147) for two different GnSAF-recognising scAbs were amplified by PCR. Two unique cloning sites: PstI/BstEII for the heavy chain and SacI/XhoI for the light chain were then introduced. PCR products were separately sub-cloned into mammalian expression vectors for heavy and light chains (VHExpress and VKE).
Expression and purification of intact IgG molecules
The transfection of IgG vectors into cultured CHO-K1 cells was performed with Lipofectamine™ PLUS reagent (Gibco BRL, Life Technologies) according to manufacturer's instructions. The cells were cultured in 6-well plates, until 80% confluent, before proceeding to co-transfections. 16 μg of DNA diluted in 600 μl of serum-free Optimem media (Gibco BRL, Life Technologies) per plasmid construct (heavy and light chains) was precomplexed with 36 μl of PLUS Reagent and mixed, followed by incubation at RT for 15 min. 24 μl of Lipofectamine Reagent was diluted into 600 μl of medium (serum-free Optimem) in a second tube and mixed. Pre-complexed DNA was combined and diluted in Lipofectamine Reagent, mixed and incubated for 15 min at room temperature. Fresh serum-free Optimem media (800 μl/well) was added to the cells while the complexes were forming. DNA-PLUS-Lipofectamine Reagent complexes were added (200 μl/well) to each well, mixed into the medium gently, and incubated at 37°C at 5% CO2. After 3 h incubation the total volume of media was increased to 2 ml/well and the serum concentration was raised to 10%. After 24 h of transfection the media was replaced with fresh media and incubated for a further 3–4 days before collection and purification. The media, confirmed to contain the intact IgG, was concentrated 10-fold. IgG fragments were then removed by filtration through 100 kDa filter (Centricon Plus-80 PBHK Biomax, Amicon, Millipore Ltd., Watford, Hertfordshire, UK), followed by chromatographic purification with rProtein L™-Agarose (ACTIgen Ltd., Olso, Norway) according to manufacturer's instructions. An ELISA utilising antibodies against both heavy and light chains was used to monitor production and concentration.
Determination of scAb/IgG affinity for GnSAF bioactivity
(a) Binding of GnSAF bioactivity
To determine whether scAbs or scAb-derived IgGs would bind GnSAF bioactivity, in-vitro bioactivity-binding experiments were carried out as shown in Fig 1. In the case of the phage form of the scFvs, 96-well flat-bottomed Immulon 4 ELISA plates were coated (duplicates) either overnight at 4°C or for 2 h at RT with 200 ng of anti-M-13 antibody/well (Amersham Biosciences UK Ltd). For soluble scAb fragments in plasmid pIMS147 and for intact IgGs, produced by CHO-K1 cells, the plates were coated with 100 μl/well (1:1000 dilution) of goat anti-human kappa light chains (bound and free) antibodies (Sigma). Wells were washed three times with PBS, 0.1% Tween (200 μl/well), blocked with 2% (w/v) skimmed milk or 3% BSA in PBS for 2 h at RT or overnight at 4°C. The plates were washed with PBS-T (200 μl/well) four times, 10–50 μl per well of scAb preparation (1–10 μg) in PBS was added and the plates were then incubated for an hour at 37°C or 2 h at RT. Plates were washed again four times with 200 μl/well of PBS-T and twice with PBS before incubation with GnSAF-containing and controls preparations. The source for GnSAF for these bioassays was either QC hFF or G/LCM with GnSAF bioactivity. The GnSAF preparation was added (250 μl/well) to the immobilised scAbs/IgGs and incubated for 2 h on a rotating platform at 37°C. The fluid was recovered, duplicates combined, centrifuged at 20,000 × g for 10 min and the supernatant bioassayed at 0, 1, 5, 25 μl/well using duplicate rat pituitary cell monolayer bioassays.
(b) Blocking of GnSAF bioactivity
To determine whether scAbs or scAb-derived IgGs would block the GnSAF bioactivity, in-vitro bioactivity-blocking experiments were carried out as follows. A 1:1 ratio of QC hFF and scAb/IgG in PBS, or 2 μg of purified IgG with 250 μl of QC hFF, were incubated in duplicate for 2 h at 37°C on a rotating platform. The fluid was recovered, duplicates combined, centrifuged at 20,000 × g for 10 min and the supernatant bioassayed at 0, 1, 5, 25 μl/well using duplicate rat pituitary cell monolayer bioassays.
Immobilisation of ScAbs and IgGs with immunopurification supports
Coupling and use of scAb/IgG with CnBr-activated sepharose 4B
Cyanogen bromide activated sepharose 4 B (Pharmacia, Biotech Ltd., Knowhill, Milton Keynes, UK) was swollen for 15 min in 1 mM HCl (0.2 g/sample) and washed on a 3G sintered glass filter with the same solution (200 ml/g of dry powder). The washing solution was added in several aliquots and the supernatant was aspirated off by vacuum between successive additions. The swollen gel was then washed once with 5 ml of coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3) followed by suction of the excess buffer. When the beads were dry they were added to a mixture of coupling buffer combined with scAb or IgG (1–10 μg in PBS) and rotated in an end-over-end mixer for 1–2 h at RT or overnight at 4°C. The tubes were centrifuged at 500 × g for 10 min and washed 5 times with 1 ml of coupling buffer. The remaining active groups were blocked by 1 M ethanolamine, pH 8.0 or 0.2 M glycine, pH 8.0 for 2 h at RT or overnight at 4°C. To remove the excess of uncoupled ligand, the adsorbent was washed alternatively with high and low pH Sodium acetate buffer (0.1 M NaAc, 1 M NaCl, pH 8.0 and pH 4.0) for 5 times. The coupled sepharose was stored in PBS at 4°C unless used immediately.
Initially, 100 μl of coupled CnBr beads were combined with 100 μl of partially purified GnSAF (2 μg of protein from albumin-depleted G/LCM), equilibrated to pH 7.4 and end-over-end rotated for 2 h at RT or at 4°C overnight. The beads were centrifuged at 500 × g for 5 min and the buffer was aspirated = unbound fraction. One gel volume (200 μl) of washing buffer 1 (20 mM PBS, pH 7.2) was added, mixed gently and centrifuged for 5 min at 500 × g and eluates collected. This wash step was repeated 3 times and eluates were pooled together with the unbound fraction. The immuno-immobilised proteins (= bound fraction) were eluted by one of two methods. The first method used 300 μl (3 gel volumes) of 0.1 M glycine, pH 2.8 and neutralised immediately after centrifugation (5 min 500 × g) with 120 μl 1 M Tris-HCl, pH 13. The second method used 200 μl of 2 M NaI for repeated elution steps. The eluted fractions were then combined. Proteins from a final glycine elution step were not combined with the NaI-eluted bound fractions. Both bound fractions were desalted with HiTrap desalting columns (Amersham, Pharmacia Biotech Ltd.) or microspin G-25 (Amersham, Pharmacia Biotech Ltd.) before proceeding to GnSAF bioassay at 0, 1, 5, 25 μl/well doses in two separate rat pituitary cell monolayer bioassays. Once the recovery of GnSAF bioactivity was confirmed by bioassay, this process was scaled up to immobilise 120 ng of scAb-derived IgG and perform 15 consecutive immunopurifications of GnSAF from G/LCM. The bound proteins were pooled between each immunopurification. The recovered protein was double-desalted, checked for GnSAF and inhibin bioactivities, freeze-dried and reconstituted in 200 μl of 2-D lysis buffer for gel electrophoresis.
Coupling and use of rpAb with Dynabeads
Two ml of rat polyclonal anti-GnSAF antiserum [6] was processed using a 5 ml HiTrap Q column according to manufacturer's instructions for the purification of rat IgG and the partially purified rat IgG desalted into 0.1 M PBS. The IgG was coupled to 300 μl of anti-rat IgG magnetic Dynabeads (Dynal Biotech Ltd, Bromborough, Wilts, UK) and then cross-linked, to enhance the stability of the immobilisation. The authors had previously (unpublished observations) determined that using 2 M NaI to elute protein bound by immobilised antibodies enabled the antibody to maintain active binding of antigen for between 10 and 15 cycles of immunopurification of G/LCM, although recovery was reduced marginally with each successive elution. Fifteen cycles of GnSAF immunopurification were then carried out. For each cycle, a fresh aliquot of 500 μl of concentrated G/LCM was incubated with the immobilised rpAb for 15 min at RT on an orbital mixer. This was designed to ensure that the binding capacity of the immobilised rpAb was exceeded every time in order to maximise the amount of GnSAF protein recovered. Unbound material was removed after each cycle by drawing the beads to the bottom of the tube magnetically and removing the medium. The beads were then washed twice with 0.2 M phosphate buffer for 3 min and the bound proteins eluted by incubation with 500 μl of 2 M NaI for 15 min at RT. The bound proteins were pooled and between each immunopurification the beads were washed twice with 0.2 M phosphate buffer for 3 min each. The recovered protein was double-desalted, checked for GnSAF and inhibin bioactivities, freeze-dried and reconstituted in 200 μl of 2-D lysis buffer (0.01 M Tris-HCl, pH 7.4, 1 mM EDTA, 8 M Urea, 0.05 M DTT, 10% (v/v) glycerol 5% (v/v) NP40, 6% (w/v) pH 3–10 Resolyte, BDH Merck Ltd., Lutterworth, Leics, UK) for gel electrophoresis.
Other Methods
1-D SDS-Page and Western Blots
SDS-PAGE was performed using Bio-Rad Mini-Protean II™ gel apparatus and SDS-polyacrylamide system supplied by Life Technologies Ltd. After separation by SDS-PAGE, the protein was electro-blotted onto a polyvinylidene fluoride (PVDF) membrane (Immobilon®-P, Millipore Ltd.) or onto nitrocellulose membrane (Hybond-C extra, Amersham International plc, UK). For antibody fragments containing the HuCκ domain, HRP-conjugated goat anti-human kappa light chain (bound and free) antibodies (Sigma) were used and for whole IgG antibodies, either HRP-conjugated goat anti-human kappa light chains (bound and free) antibody or goat anti-human IgG (whole molecule) antibody with HRP conjugate (Sigma) were used, as appropriate. The blots were developed using the ECL Western blotting detection kit (Amersham Pharmacia biotech UK Ltd.) according to manufacturer's instructions before exposing the blot onto x-ray film and developed in a Kodak M35 X-OMAT processor (Kodak Ltd., Liverpool, UK). The scAb-derived IgG and rpAb immunopurified proteins in the bound and unbound fractions were separated by 1-D SDS-PAGE.
2-D gel electrophoresis
Proteins immunopurified using the immobilised scAb-derived IgG and rpAb and reconstituted in lysis buffer were analysed by 2-Dimensional gel electrophoresis (2-D gels) gels using a small format gel system [17] with immobilised pH gradient (IPG) gels for the first dimension separation. The proteins were separated in the first dimension using 7 cm, pH 4–7 IPG gel strips (Amersham-Pharmacia Biotech Ltd.). The dehydrated IPG strips were re-hydrated overnight in the IPG re-swelling buffer containing the immunopurified proteins [18]. Following their re-hydration the IPG gel strips were electrophoresed on a Multiphor II apparatus (Amersham Biotech UK Ltd.) as described in [19]. Proteins were located by staining with colloidal Coomassie brilliant blue G250.
Mass spectroscopic peptide mass mapping
The protein spots and bands were excised from the gels, washed, in-gel reduced, S-alkylated, and in-gel digested with sequencing-grade modified trypsin (Promega Madison, WI, USA) as described elsewhere [20,21]. An aliquot of the peptide extract produced by in-gel cleavage was passed through a GELoader tip which contained a small volume of POROS R2 sorbent (PerSeptive Biosystems, Framingham, MA, USA) as described [21]. The adsorbed peptides were washed extensively and then eluted in 0.5 μl of a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma) in 50% acetonitrile/5% formic acid. The mass spectra were acquired on a PerSeptive Biosystems Voyager-DE STR MALDI-TOF mass spectrometer operating in the reflectron-delayed extraction mode. Spectra were internally calibrated using trypsin auto-digestion products. Both the MASCOT and the MS-Fit database searching programs were used to search the NCBi protein database, with the masses of the tryptic peptides, to identify the proteins.
Statistical analysis
The in-vitro pituitary cell responses are expressed as percentages of the relevant control gonadotrophin concentrations secreted from blank control wells on the same culture dishes. These controls were either wells exposed to SFDM alone (basal secretion) or wells exposed to SFDM + 0.1 μM GnRH. The differences between treatment groups and dose-responses were assessed using two-way analysis of variance (ANOVA). Differences between treatments and controls were tested by Dunnet's Post Hoc Test and between treatments by the Bonferroni-Dunn Post Hoc Test. The analyses were performed using the Statview 5 programme (Abacus Concepts Inc., Berkley, CA, USA). All results are presented as means ± SEM.
Sequence of the study
The methods described above were combined in the following sequence: 1) production and partial purification of GnSAF bioactivity, 2) bio-panning of the Tomlinson Library, 3) identification of 300 clones by simple ELISA, 3) identification of 8 GnSAF-binding clones from the original 300 clones by bioassay, 4) subcloning of the 8 clones into soluble form, 5) selection of 3 scAbs with high affinity for GnSAF by bioassay, 6) expression of the 3 scAbs as intact human IgGs in CHO-K1 cells, 7) selection of the mammalian IgG with the highest affinity for GnSAF by bioassay, 8) immobilisation of rpAb (6) and phage-derived mammalian IgG, 9) validation of phage-derived mammalian IgG binding and blocking of GnSAF by bioassay, 10) immunopurification of GnSAF using immobilised rpAb and phage-derived mammalian IgG, 11) Western blot, 1-D, 2-D and mass spectroscopic peptide mass mapping of immunopurified GnSAF, 12) further candidate GnSAF molecules identified.
Results
Production of GnSAF from granulosa/luteal cells and depletion of serum proteins
BSA-free M199 conditioned by granulosa/luteal cells (G/LCM) contained significant quantities of GnSAF bioactivity with a dose of 25 μl/well reducing GnRH-induced LH secretion to <50% of control (p < 0.001). When serum albumin was depleted using Dyematrex Blue A affinity chromatography, the GnSAF activity remained in the unbound fraction, reducing GnRH-induced LH secretion to as little as 38 ± 3% of control (Fig. 2a). In contrast, the bound, serum albumin, fraction had no significant effect on GnRH-induced LH secretion (Fig. 2a).
Figure 2 Initial screening of pooled scAb populations. The Dyematrex Blue A process left GnSAF bioactivity in the unbound fraction (a) and this was used to pan the phage display library. Neither anti-M13 (closed circles) nor SFDM (open circles) affected GnSAF bioactivity levels in QC hFF (b). ScAbs recognising the Dyematrex Blue A bound fraction and a non-GnSAF protein (V peptide) also had no affinity for GnSAF bioactivity (c, e) while those recognising the unbound Dyematrex Blue A fraction significantly reduced GnSAF bioactivity in QC hFF. Data represent the mean of quadruplicate determinations from 2 different rat pituitary cell culture bioassays. The shaded horizontal bar (a) indicates the mean ± SE range for control GnRH-induced LH secretion while the shaded dose-response curves (b-e) indicates the mean ± SE range for QC hFF incubated with culture medium (b, open circles). The significance value (by ANOVA) indicates the significance of reduction of GnSAF bioactivity.
Selection of anti-GnSAF phage antibodies and their characterisation
The enrichment of phage antibodies from the Tomlinson J Library prior to affinity selection, and from each subsequent bio-pannning step, was indicated by the increase in the number of phage infections after each round of panning (Table I). The strategy was that selection stringency was increased by lowering the amount of antigen used to coat the immunotubes. After the 4th round of bio-panning, 300 individual clones were selected randomly to produce single chain variable fragment (scFv)-phage particles. These were then individually analysed for their ability to specifically bind to crudely purified GnSAF preparation, rather than to blocking agent, by monoclonal phage ELISA. The monoclonal binding ELISA was repeated for 32 positive clones with highest affinity for the GnSAF bioactive preparations and an additional control of human serum as a binding target to eliminate any cross-reactions was added, as was the candidate GnSAF internal peptide, EPQVYVHAPC [6], for hapten finding. Since there are large numbers of proteins in serum, the degree of cross-reactivity was marked. The ELISA was repeated and the ten clones showing the strongest affinity for GnSAF bioactive preparations were selected to be sequenced and then subcloned into pIMS147 to produce phage-free antibodies. Sequencing resulted in 8 unique phage clones which were pooled and tested for recognition of GnSAF bioactivity using our in-vitro rat monolayer bioassay. After sub-cloning the eight clones were induced to produce soluble scFv and the binding ELISA was repeated. This confirmed that clones had retained their ability to preferentially bind GnSAF but not milk or BSA. These clones, when immobilised in 96-well plates, reduced GnSAF bioactivity in-vitro (data not shown, similar to data in Fig. 2.).
Table 1 Augmentation of phage infection rate with each round of panning against albumin-depleted G/LCM containing GnSAF bioactivity.
Panning cycle number Amount of antigen used (μg ml-1) Phage infection titre (pfu ml-1)
1 10.00 7.0 × 106
2 2.50 8.0 × 107
3 2.50 1.0 × 108
4 1.25 4.7 × 1012
Expression and purification of ScAbs
Unfortunately, precipitation of the scAbs was repeatedly observed after dialysing purified scAbs against PBS overnight. This could be due to aggregation or improper folding of the scAbs. To determine the concentration of scAb by HuCκ capture ELISA, the samples were boiled for 10 min to avoid a possible aggregation effect before addition to the ELISA plate. This enabled the overall number of scAbs in the purified preparation, but not the amount of functional scAbs, to be determined.
Recognition of GnSAF by scAb antibodies
The pooled 8 clones were investigated for the ability to immunocapture GnSAF from QC hFF. The QC hFF incubated with culture medium alone, or with anti-M13 in the absence of the scAbs (Fig. 2b), or with the negative control a clone derived from phage library panned against a control peptide derived from a bacterial protein (V peptide, Fig. 2e), reduced GnRH-induced LH secretion to 37 ± 10%, 60 ± 2% and 55 ± 7% of control (p < 0.001 vs control). After incubation with the scAb against the non-bioactive, bound (rich in serum albumin), fraction of G/LCM following Dyematrex Blue A purification (Fig. 2c), the QC hFF retained its GnSAF bioactivity, reducing GnRH-induced LH secretion to 48 ± 7% of control, (p < 0.001 vs control). In contrast, after incubation with the scAb selected against the bioactive, unbound, fraction of G/LCM following Dyematrex Blue A purification (Fig 2d), the QC hFF had little GnSAF activity remaining (GnRH-induced LH secretion remained at 82 ± 5% of control, p > 0.05). When the clones were used to coat 96-well plates separately, 6 (Fig. 3c,d,e,f,j) had no significant effect on the GnSAF bioactivity seen in QC hFF (GnRH-induced LH secretion reduced to 37 ± 11% of control, p < 0.001, Fig. 3a) or in QC hFF incubated in V peptide-coated wells as a negative control (Fig. 3b). In contrast, GnSAF bioactivity was almost totally removed by the scAbs 3-c4c and 3-c4b and greatly reduced by the scAb 2-g3 (GnRH-induced LH secretion only reduced to 81 ± 12%, 91 ± 7% and 70 ± 10% of control respectively, Fig. 3g,h,i).
Figure 3 Representative example of bioassay testing of scAb/IgG affinity for GnSAF bioactivity. In this case antibody recognition of GnSAF bioactivity in QC hFF was tested compared with GnSAF bioactivity in QC hFF alone (a) or incubated with the non-GnSAF scAb, V peptide (b). Of the 8 individual scAbs tested, 3 showed high affinity for GnSAF, highly significantly reducing GnSAF bioactivity in QC hFF (g, h, i). Data represent the mean of quadruplicate determinations from 2 different rat pituitary cell culture bioassays. The shaded dose-response curves (a-j) indicates the mean ± SE range for QC hFF incubated with culture medium (a, open circles). The significance value (by ANOVA) indicates the significance of reduction of GnSAF bioactivity.
Binding and blocking experiments with IgG forms of scAbs
Based on the bioassay data above, full length (150 kDa) immunoglobulins were produced for the scAb clones 3-c4c and 3-c4b which showed the greatest affinity for GnSAF. This was done by co-transfecting the heavy and light chain vectors (containing the scAb CDR regions) into mammalian cells CHO-K1. Transient transfections were optimised, but the transfection efficiency was far better for the light chain vector and therefore the overall intact IgG yield was relatively low. The purified IgGs were used for blocking and binding experiments. While the clone 3-c4c-derived IgG had lost its functional activity, 3-c4b-derived IgG significantly altered the hFF dose-response curves, demonstrating continued affinity for GnSAF (data not shown, dose-response changes very similar to Fig. 3h). In bioactivity-binding experiments, GnSAF bioactivity was reduced 4.6-fold compared to control IgG (transient transfections expressing an unrelated IgG recognising the pesticide hapten atrazine) while in bioactivity-blocking experiments, GnSAF bioactivity was reduced 3.4-fold compared to control (data not shown, calculated from changes in ED50 in dose-response curves).
Immunopurification of GnSAF bioactivity
Following small-scale experiments to ensure that the scAb-derived IgG and rpAb continued to recognise GnSAF bioactivity once immobilised, all remaining stocks of rpAb and 3-c4b-derived IgG were immobilised and used to immunopurify the maximum possible quantity of GnSAF bioactivity. These data are shown in Figs. 4 &5. An extensive panel of controls were used and these established that elution with 2 M NaI did not interfere with recovered GnSAF bioactivity (Fig. 4a). Two molar NaI processed in the same way as eluted protein also had no effect on GnRH-induced LH secretion (data not shown). The supports used for immunopurification did not bind GnSAF bioactivity (Fig. 4b) or inhibin bioactivity (Fig. 4e) in a non-specific manner. In contrast, there was marked recovery of GnSAF bioactivity (as defined by the suppression of GnRH-induced LH secretion: Fig. 4c), but not inhibin bioactivity (as defined by the suppression of basal FSH secretion: Fig. 4f), by eluted protein recognised by the immobilised rpAb and scAb-derived IgG. The recovery of GnSAF bioactivity in the unbound fraction (Fig 4d) was expected because in each cycle of immunopurification the immobilised antibodies were overloaded with GnSAF bioactivity to ensure maximal purification. When visualised by 2-D gel electrophoresis both the scAb-derived IgG (Fig. 5a) and rpAb (Fig. 5c) had produced a spread of proteins between 60 and 70 kDa, pI 5.5–6.0, the expected range for GnSAF bioactivity. Only single bands, at 74 and 69 kDa were visualised by Coomassie blue staining of 1-D gels (Fig. 5b,d). Western blotting with the scAb-derived IgG (Fig. 5e) compared with Western blot with the secondary antibody only (Fig. 5f) demonstrated specific recognition of a unique protein band at 66 kDa (Fig. 5e, lane 3).
Figure 4 Immunopurification of GnSAF from granulosa/luteal cell-conditioned medium (G/LCM) using immobilised phage-derived monoclonal and rat polyclonal antibodies. (A) GnSAF bioactivity in G/LCM treated with (open circles) and without (closed circles) 2 M NaI elution buffer. GnSAF (b) and inhibin (e) bioactivities in quality control human follicular fluid (QC hFF, closed circles) but not in proteins non-specifically bound by the immunopurification media without immobilised antibody (non-specific binding: NSB, open circles). GnSAF (c) but not inhibin (f) bioactivity detected in G/LCM immunopurified using immobilised rat polyclonal (closed circles) or immobilised phage-derived antibody (3-c4b, open circles). GnSAF bioactivity remaining in excess protein not bound by immobilised phage-derived antibody (3-c4b, d). Data is from triplicate cell cultures, results are mean ± SE. The shaded dose-response curves (a-j) indicates the mean ± SE range for hFF incubated with culture medium (a, closed circles). The effects of unpurified G/LCM on basal FSH are not shown because of the high concentration of recombinant human FSH content which interferes with the rat FSH immunoassay.
Figure 5 GnSAF immunopurified from G/LCM. Immunopurification using phage-derived antibody immobilised on protein-L agarose (a,b) or rat polyclonal antibody immobilised on anti-rat IgG-coated magnetic beads (c,d) was pooled after 15 consecutive loading and elution cycles with 2 M NaI. Proteins detected by Coomassie blue staining of (a,c) 2-D (immunopurified protein only, numbers match those in Table II) and (b, d) 1-D (Unbound = flow-through, Bound = immunopurified) gels were investigated by mass spectroscopic peptide mass mapping. Western blot of the 3-c4b-immunopurified G/LCM (e,f) with either 3-c4b and secondary antibody (e) or secondary antibody only (f) show the elution of a specifically recognised 60–70 kDa protein band (arrow) by 2 M NaI, with no further protein eluted with glycine washes.
Candidate GnSAF molecules resulting from scab-derived IgG and rpAb immunopurification of G/LCM
The 1-D gel bands and the labelled spots in 2-D gels shown in Fig. 5 were then subjected to mass spectroscopic peptide mass mapping and the principal findings are shown in Table II. The main candidate GnSAF molecule identified was serum albumin, its precursor and variant forms. Once the human serum albumin peak was removed from the data for the 1-D rpAb-immunopurified protein, one further candidate molecule at a similar pI and kDa was revealed. The five spots visible in the 2-D gel of this material also produced positive matches with serum albumin precursor, two hypothetical proteins and other tissue-specific proteins. The keratin 10 is epidermal and therefore likely to have been due to contamination. While there was insufficient material to obtain positive identification from the IgG-immunopurified protein, the two 2-D spots again showed positive matches with serum albumin precursor and variant forms.
Table 2 Candidate molecules identified (NCBInr) from proteins immunopurified from G/LCM using immobilised phage-derived IgG and rat pAb.
Immuno-purification Ab Spot/band Database Protein MOWSE Score Accession Number
PI kDa
rpAb 1-D 66 kDa band 5.9 69.2 Serum albumin precursor 224,400 P02768
Excl albumin peaks 9.08 86.8 SAD1 kinase 767 Q8TDC3
2-D #1 6.0 56.8 PRO2619 372,800 11493459
5.9 69.2 Serum albumin precursor 228,770 6013427
#2 5.8 72.8 Solute carrier family 39, member 12 1,855 22749433
#3 5.0 57.2 Keratin 10 6,260 88041
#5 6.0 56.8 PRO2619 102,500 11493459
5.9 69.2 Serum albumin precursor 78,270 6013427
#6 6.0 56.8 PRO2619 2,067,000 11493459
5.9 69.2 Serum albumin precursor 1,302,000 6013427
Derived IgG 1-D 74 kDa band Insufficient protein
2-D #4 5.9 69.2 Serum albumin precursor 2.483e+11 6013427
#7 5.9 69.4 Hypothetical protein 6.366e+11 51476390
5.9 69.2 Serum albumin precursor 2.276e+11 6013427
Discussion
We have successfully created anti-GnSAF antibodies with the help of phage display technology. The antigen used was partially purified GnSAF and the bio-panning was combined in a novel manner with our GnSAF rat monolayer bioassay as the selection step. This is an alteration to the normal phage display experiment where the antigen is preselected prior to phage display. It has not been possible to obtain specific GnSAF antibodies by conventional means, because the GnSAF has not yet been definitely isolated. This is highlighted by the presence of several non-GnSAF antibodies in our previously described rat anti-GnSAF serum [6]. The process of bio-panning the Tomlinson J Library against crudely purified GnSAF was successful. The increase in the number of eluted phage particles after each round of panning (Table I) caused an enrichment of clones recognised by the immobilised antigen which was further confirmed by increased signal of binding ELISAs for polyclonal phage preparations (data not shown) and recognition for GnSAF bioactivity.
After purification of the scAbs some protein aggregates or misfoldings were observed and this made it impossible to accurately quantify scAbs, also possibly reducing the number of functional scAbs in the purified preparations. The same observation has previously been made with regard to the Tomlinson J library [22]. In order to overcome this precipitation problem, the scAbs were denatured by boiling for 10 min before their addition to ELISA plates. However, this can only provide the overall number of light chains present (the secondary antibody being anti-HuCκ), not the number of functional scAbs. When producing scAb-derived IgGs there was no aggregation observed and accurate quantification was carried out without boiling. The coating of plastic immunoplates with antibodies relied on the immunoabsorbent nature of plates. There is always a chance that the 3-dimentional structure of antibody might be assembled in such a manner that antigen-binding site was covered or sterically hindered. When the antibody was in the phage form, an anti-M13 was used as coating agent before adding the scFv to ensure that the antigen-binding site in of the scFv was left free to be in contact with GnSAF in solution.
There have been previous reports of panning antibody libraries against "unknown" targets. For instance in one study, a mixture of proteins separated by 2-D gels was blotted onto PVDF membrane and an antibody phage display library was panned against individual spots. This process produced specific monoclonal antibodies against protein spots of interest [23]. However, this strategy produces antibodies against denatured proteins and they will not necessarily work in biological fluids where protein is in its tertiary structure. In our approach on the other hand, the protein target was in its native structure when selected by rat monolayer bioassay and thus the antibodies created this way should be functional in biological environments, for example in bioassays, and physiological systems.
The candidate GnSAF proteins identified in this study are interesting in light of a recent publication [24] suggesting that GnSAF is a C-terminal component of the human serum albumin subdomain IIIB. The main positive identification in our study was serum albumin precursor and this suggests that GnSAF could be a modified form of serum albumin. Clearly the major serum albumin forms do not have GnSAF bioactivity since the bound fraction from the Dyematrex Blue A purification step, which consists largely of serum albumin, does not have GnSAF bioactivity (Fig. 2). Some evidence of possible endocrine effects of serum albumin were published in the early 1990's, including [25], although further studies suggested that any biological activity depended upon the albumin fatty acid content rather than effects on gonadotrophin actions [26]. Nevertheless, the fact that our synthetic antibodies clearly recognised GnSAF bioactivity and also appeared to immunopurify serum albumin supports the suggestion that GnSAF may be formed by post-translational modification of serum albumin [24] or may be a much smaller molecule very tightly bound to serum albumin. What is clear however, for instance by the separation of GnSAF activity and the majority of serum albumin in bioactive preparations, is that the mature serum albumin molecule does not have GnSAF bioactivity Scaling up the production of the GnSAF-recognising IgG and immunopurifying a much larger quantity of GnSAF bioactivity than in the present study, followed by depletion of serum albumin, would be the best way of confirming or disproving this possibility. Such an approach would allow the extensive validation studies required to confirm whether a specific candidate protein is in fact GnSAF.
Although most studies indicate a 60–70 kDa size for GnSAF bioactivity [7], previously published candidates [3-6,27] are not necessarily mutually exclusive. None of the previously published GnSAF candidate sequences [3-6,27] have significant homology with the candidate proteins presented in Table 2. The only conclusion that can be drawn from this at present is that these candidates are possibly not the GnSAF molecule. [4,24] present good evidence that the serum albumin IIIB domain might be GnSAF, particularly a 12 kDa C-terminus component, possibly post-translationally modified. This is supported by our findings of serum albumin, its precursor molecule and variant PRO2619 in the immunopurified GnSAF bioactive G/LCM fractions. However, the peptide mass matches in our study are scattered across the serum albumin molecule and not limited to the amino acids after 490 of the human serum albumin molecule. Clearly this is less supportive of the findings of [4,24] and more indicative of the serum albumin being present as a contaminant or GnSAF transporter. The possible identification of SAD1 kinase once the serum albumin peaks had been excluded from the peptide mass search of the 66 kDa band of rpAb-immuniopurified G/LCM, is interesting. However, there is nothing in the limited literaure about this protein to suggest that it might be GnSAF [28]. The solute carrier molecule is a ion transporter involved in maintaining intracellular zinc concentrations [29] and therefore highly unlikely to be GnSAF. In contrast, the hypothetical protein matched to spot 7 appears to be another variant of serum albumin precursor.
Although none of the candidate GnSAF identifications were conclusive, the present study has provided the gene sequences to allow transfection to produce antibodies that recognise GnSAF bioactivity or the occupied GnSAF transporter. Scaling-up the production of the scAb-derived IgG should be linked together with antibody-based depeltion of non-GnSAF bioactive albumin to further reduce the number of proteins present in bioactive fractions. Such a strategy should ultimately lead to the final characterisation of this molecule.
A key technique in the present study was the rat pituitary cell bioassay. The use of such bioassays in the study of GnSAF was extensively reviewed by [27]. The Fowler and Danforth groups (reviewed in [7,27]) in particular undertook studies to ensure that GnSAF bioactivity as determined by bioassay was not due to steroid hormones, inhibins, activin, follistatin or some endogenous pituitary effect. Extensive in-vivo studies of GnSAF in women by the Messinis group (reviewed in [7,27]) also show that GnSAF requires the ovary to be present, supporting the use of G/LCM in the present study The latest such data is evidence that post-menopausal women have a more rapid response to exogenous oestradiol in terms of sensitisation of the pituitary to GnRH than would be expected [30], confirming the effect of an ovarian compound in antagonising GnRH. Nevertheless, the possibility that there are multiple proteins in G/LCM that suppress GnRH-induced LH must be considered. This point is unlikely, but will only be disproven once GnSAF is finally identified. The use of G/LCM greatly reduces the possibility of non-specific effects since no substrate for the production of steroid hormones was provided, no serum or serum albumin was used in the culture medium and the biological activity of the material has been extensively investigated [6], with similar findings seen in other species [31].
Conclusion
We have used a novel strategy by utilising a rat monolayer bioassay in bio-panning procedure for the rapid isolation of recombinant antibody fragments from a naïve synthetic phage display library. scAbs modified to human IgG forms with the same block GnSAF bioactivity and immobilise GnSAF from biological fluids. Furthermore, we have used scAb-derived IgG to immunopurify and then Western blot a band of size 66 kDa, pI 5.5–6.0 which matches size and isoelectric behaviour suggested for GnSAF in most previous studies [7]. The repeated identification of serum albumin, precursor and variant, supports suggestions that GnSAF may be a post-translationally modified form of serum albumin, although not the mature serum albumin molecule itself. Alternatively, these identifications may be due to contamination and also suggests that GnSAF may be tightly bound to, and transported by, serum albumin. The fact that MS fingerprinting showed peptide matches across the whole serum albumin amino acid sequence introduces uncertainty as to whether GnSAF is actually part of the serum albumin domin IIIB [4,24], and supports the conclusion that GnSAF is more likely to be a separate molecule that is bound by serum albumin. Finally, this study has demonstrated how phage display and alternative screening strategies may be combined to improve protein identification strategies.
Authors' contributions
TSL carried out the phage and antibody panning and development and drafted the manuscript.
WJH designed the phage strategy and helped draft the manuscript.
HDM helped design the study and draft the manuscript.
PAF designed the bioassay strategy, carried out the bioassays and helped draft the manuscript.
All authors read and approved the final manuscript.
Acknowledgements
We are grateful to Mrs M Fraser, Mrs P Cunningham, Mrs Elaine Durward and Ms F MacGregor for their expert technical assistance. We thank the staff at the Biological Services Unit (University of Aberdeen) for maintaining the rats used in this study and Dr AF Parlow at NIDDK's National Hormone and Pituitary Program (Torrance, California, USA) and SAPU (Carluke Hospital, Scotland) for hormone assay materials. We thank Dr P Cash, Mrs E Argo, Mrs E Stewart and Mr I Davidson of the Aberdeen Proteome Facility for the invaluable assistance and patience. We are grateful to the BBSRC for their financial support of the project in two grants to PAF, HDM, WJ Harris which included TSL's salary. The BBSRC had no role in study design, collection, analysis and interpretation of the data.
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Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-3-541620216910.1186/1477-7827-3-54ReviewThe role of aromatase inhibitors in ameliorating deleterious effects of ovarian stimulation on outcome of infertility treatment Mitwally Mohamed FM [email protected] Robert F [email protected] Michael P [email protected] Division of Reproductive Endocrinology & Infertility, Department of Obstetrics & Gynecology, Wayne State University, Detroit, Michigan, USA2 Reproductive Sciences Division, Department of Obstetrics & Gynecology, University of Toronto, Toronto, Ontario, Canada2005 4 10 2005 3 54 54 7 7 2005 4 10 2005 Copyright © 2005 Mitwally et al; licensee BioMed Central Ltd.2005Mitwally 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.
Clinical utilization of ovulation stimulation to facilitate the ability of a couple to conceive has not only provided a valuable therapeutic approach, but has also yielded extensive information on the physiology of ovarian follicular recruitment, endometrial receptivity and early embryo competency. One of the consequences of the use of fertility enhancing agents for ovarian stimulation has been the creation of a hyperestrogenic state, which may influence each of these parameters. Use of aromatase inhibitors reduces hyperestrogenism inevitably attained during ovarian stimulation. In addition, the adjunct use of aromatase inhibitors during ovarian stimulation reduces amount of gonadotropins required for optimum stimulation. The unique approach of reducing hyperestrogenism, as well as lowering amount of gonadotropins without affecting the number of mature ovarian follicles is an exciting strategy that could result in improvement in the treatment outcome by ameliorating the deleterious effects of the ovarian stimulation on follicular development, endometrial receptivity, as well as oocyte and embryo quality.
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PART ONE
1 Introduction
Current epidemiological evidence suggests that 15% of couples will experience infertility. Background prevalence rates now appear to be reasonably stable, but there is evidence of an increase in the rate of referrals for medical help [1,2]. Farley and Belsey, 1988 [3], have reported estimates of the prevalence (percentage) of primary infertility by region and country. They estimated 6% for North America, 5.4% for Europe, 3% for the Middle East, 10.1% for Africa, 4.8% for Asia and Oceania, 3.1% for Latin America and 6.5% for the Caribbean. The American Society for Reproductive Medicine (ASRM) estimates that 5 million American heterosexual couples report difficulties in achieving a viable pregnancy, of which 1.3 million seek advice for the problem [4].
2 Ovarian stimulation and assisted reproduction for infertility management
After correcting the abnormalities detected during the diagnostic workup, ovulation induction is usually performed either for treatment of anovulation/oligo-ovulation, or empirically in regularly ovulating women. This approach results in a pregnancy rate of around 8%–15% per cycle depending on the agents used for ovulation induction and the characteristics of the couple, such as the woman's age and the presence or absence of a male factor. Couples who do not become pregnant with ovulation induction alone then undergo more sophisticated treatment modalities including intrauterine insemination (IUI) and in-vitro fertilization and embryo transfer (IVF-ET) as a treatment of last resort [5].
Since the birth of Louise Brown in 1978, IVF-ET has become the therapeutic mainstay for female infertility. It has become generally accepted as therapy for a wide array of fertility problems, and has been accompanied by the rapid expansion of IVF-ET clinics worldwide resulting in more than 1% of babies being conceived by IVF-ET in western countries [6].
2.1 Ovarian stimulation for assisted reproduction
In most assisted reproduction programs, gonadotropins are used alone or in combination to stimulate the growth and maturation of multiple follicles. This is essential because of the need to recruit a greater number of follicles, which provides the opportunity for retrieval of a large number of oocytes. This would improve the chance for fertilization of multiple oocytes and thereby allow an increased number of embryos for transfer in order to give acceptable success rates. Recent advances in the understanding of ovarian stimulation, the techniques of oocyte retrieval, the handling of gametes, the methods of assisted fertilization and improved conditions of culture media have steadily increased the fertilization rate. Fertilization rates of 60–70% can now be expected when conventional insemination, or even higher when intracytoplasmic sperm injection (ICSI) are carried out. However, there has not been a corresponding increase in implantation rates, which have remained steady at overall rates around 10%–15% [6].
2.2 Low implantation rates with assisted reproduction
Throughout the last five decades, a progressive series of revolutionary techniques have been developed to overcome infertility, starting with the successful fertilization of human oocytes in vitro [7] and followed nearly 10 years later by the birth of the first IVF-ET baby [8]. Several other new developments in assisted reproduction have emerged, including cryopreservation and storage of embryos for later transfer [9], fertilization of oocytes with a single injected spermatozoon to alleviate severe male infertility i.e. ICSI [10] and diagnosis of genetic defects from preimplantation embryos prior to intrauterine transfer [11]. However, although IVF-ET is now a standard, well-established treatment for infertility, success rates remain relatively low, with only about 33% of cycles resulting in pregnancy [12]. This is believed to be due to the low implantation rate that has not significantly increased as fertilization rates [13]. Efforts are being made to improve implantation rates after IVF-ET by improving culture conditions, optimizing gamete quality and developing new techniques of selecting viable embryos for transfer without significant success. For this reason, multiple embryos are generally transferred to improve pregnancy rates, but this has resulted in an unacceptably high rate of multiple-gestation pregnancies [14].
Although governed by multiple interactive events, embryo implantation depends mainly on the quality of embryos and the status of uterine receptivity. During the last two decades, several developments in controlled ovarian hyperstimulation [COH], fertilization, and embryo culture techniques have led to an optimization in the number and quality of embryos available for ET. In contrast, uterine receptivity has failed to benefit from parallel improvements, and its disarrangement is likely to represent an important cause of the sub-optimal embryo implantation rates observed in IVF-ET [15].
2.3 Poor outcome of infertility treatment associated with ovarian stimulation
In the following section we review in brief both animal and human evidence for the unfavorable outcome including impaired implantation and increased adverse outcomes in pregnancies achieved following ovarian stimulation when compared with spontaneous pregnancies.
2.3.1 Animal data
Increased pre- and post-implantation embryonic loss has been reported in mammals [16-21] including rats [16,18], mice [17-19], murine [20] and hamsters [21], in association with ovarian hyperstimulation. These effects have been attributed to ovarian stimulation using standard doses of gonadotropins. At higher doses of gonadotropins, studies have found increased frequencies of oocyte aneuploidy, embryo mortality, fetal growth retardation and congenital abnormalities [22,23].
The poor outcome after ovarian stimulation has been attributed to adverse effects on the maternal side and or the gametes and embryo side. On the maternal side: inadequate uterine synchrony or receptivity has been reported. On the gamete and embryo side, ovarian stimulation has been found associated with chromosomal defects in the oocyte leading to increased lethality during the preimplantation stages [22,23]. However, because species-specific variations in implantation strategies exist, these differences preclude the formulation of a unifying theme for the molecular basis of this event.
2.3.2 Human data
Many studies have found higher pregnancy rates in donor oocyte recipients than patients undergoing standard IVF-ET [24,25]. The higher success rates could be attributed to better quality oocytes from younger donors. However, in centers using a shared oocyte system, where the donor keeps half of the oocytes for herself, significantly higher pregnancy and implantation rates were found in the recipients [26].
Another evidence for adverse effects of ovarian stimulation on implantation is the higher implantation rate associated with IVF-ET in the natural or un-stimulated cycle. Although most studies were associated with a rather high proportion of cancelled cycles [25–75%] and a low clinical pregnancy rate per started cycle [range 0–23%], higher implantation rates have been reported (up to 30%) [27].
Adverse Obstetrical Outcome after ovarian stimulation
Induction of ovulation has been shown to raise the risk of miscarriage when compared with spontaneous pregnancies [28]. This was true even after controlling for advanced age, a known significant risk factor for miscarriage. Higher risks as compared with natural pregnancies are reported in pregnancies after IVF-ET, primarily owing to growth retardation and pre-term birth. Although this can be explained by the high multiple pregnancy rates in IVF-ET pregnancies [29], an increased rate of small for gestational age and pre-term birth children is reported in singleton IVF-ET pregnancies as compared with natural singleton pregnancies after adjustment for potential biases [30-32]. Moreover, twin pregnancies after assisted reproduction have a higher rate of perinatal mortality and lower birth weight as result of a higher rate of premature parturition when compared to spontaneously occurring twins [33,34]. In addition, it was reported that women who conceived multiple gestations through assisted reproductive technologies have a 2.1-fold higher risk of preeclampsia than those who conceive spontaneously [35]. Pregnancies associated with severe ovarian hyperstimulation syndrome have been found to be complicated with increased miscarriage rates [36,37]
While ovarian stimulation has been suggested to contribute, at least in part, for the adverse obstetric outcome other studies have shown that infertility itself is a factor that leads to increased obstetric risks and that sub-fertility is a predictor for low birth weight. Some believe that it is the cause of infertility itself, rather than the use of ovarian stimulation, that is the reason behind poor obstetric outcome after infertility treatment [38,39].
3 Postulated mechanisms behind adverse effects of ovarian stimulation
Several causes and targets have been suggested to explain the poor outcome associated with ovarian stimulation (Table 1). These include (1) supraphysiological levels of estrogen, and other steroids and peptides, attained during ovarian hyperstimulation, (2) the use of exogenous gonadotropins as well as other medications applied during ovarian stimulation such as Gonadotropin releasing hormone (GnRH) analogues (agonists and antagonists), human chorionic gonadotropin (hCG) and clomiphene citrate (3) Other possible undetermined factors. These factors are believed to act through their effects on (i) the endometrium, (ii) the developing oocyte, (iii) the developing embryo, (iv) the ovaries and corpus luteum, (v) the pituitary gland, and (vi) possibly on other targets such as fallopian tubes, the coagulation system, as well as the early developing placenta.
Table 1 Postulated mechanisms behind less favorable treatment outcome (reduced implantation rate and increased adverse obstetric outcome) after ovarian hyperstimulation
Causes: Targets
1-Supraphysiological estrogen levels attained during ovarian stimulation
2-Medications used during ovarian stimulation:
•Clomiphene citrate
•Gonadotropins
•GnRH analogues: agonists & antagonists
•HCG used to trigger ovulation
3-Other probable causes: abnormal levels of other hormones, peptides and local autocrine mediators associated with ovarian stimulation 1-Endometrium
2-Developing oocyte
3-Sperm
4-Developing embryo
5-Maternal endocrine system: ovaries (corpus luteum), pituitary gland and hypothalamus.
6-Other probable targets e.g.
•Fallopian tubes
•Early developing placenta
•Leptin-mediated effects
•Coagulation system
3.1 Effect of supraphysiological levels of estrogen
It is believed that the supraphysiological levels of estrogen, attained during ovarian stimulation, may explain, at least in part, for the adverse effects of ovarian stimulation on the outcome of infertility treatment [40-42]. Different mechanisms have been suggested for the deleterious effects of the supraphysiological levels of estrogen attained during ovarian stimulation Table 2.
Table 2 Deleterious effects of the supraphysiological estrogen levels attained during ovarian stimulation:
1-Effect on the endometrium: most of the available evidence
a: Dys-synchronization of the implantation window.
b: Abnormal Temporal expression of the endometrial pinopodes.
c: Defective endometrial estrogen and progesterone receptors.
d: Impaired endometrial blood flow
e: Abnormal endometrial integrins expression
2-Effect on the developing oocyte
a: Effect on chromosomal and cytogenetic integrity of the oocyte
b: Effect on the mitochondrial function
3-Effect on the sperm causing possible premature acrosome reaction and deactivation
4-Effect on the developing embryo and blastocyst hatching
5-Effect on the ovaries and pituitary (defective corpus luteum function and luteal phase):
a: Defective LH secretion:
- Abnormal LH surge
- Abnormal LH tonic pulse
b: Defective corpus luteum function
6-Other probable targets:
I-Leptin
II-Coagulation system: III-Fallopian tubes
• a: Effect on the ductal environment and ductal fluid
• b: Effect on the ductal transfer and motility
IV-Early developing placenta
Considering all the patients together, significant decreases in pregnancy and implantation rates were observed when estradiol (E2) concentrations were > 2500 pg/ml [>9000 pmol/L.] compared with patients having lower E2 concentrations. High serum E2 concentrations on the day of hCG injection in high and normal responder patients, regardless of the number of oocytes retrieved and the serum progesterone concentration were found to be detrimental to uterine receptivity [42]. Later, it has been shown that a significant reduction in the implantation and pregnancy rates occurred in almost all women with a higher serum E2 concentration of ~5–6000 pg/ml [19–22,000 pmol/L] [43,44]. Recently, we have presented data showing that high E2 levels are associated with less favorable treatment outcome in women undergoing controlled ovarian hyperstimulation and IVF-ET [45-51]. We studied the effect of E2 by looking at the area under the curve (AUC) for E2 levels along the stimulation cycle [45-51]. We believe this is more accurate than looking at a single measurement of E2 e.g. one day of hCG administration [46]. In addition to studying the AUC for E2 levels [45,46], we looked at the E2 production per mature follicle and per gonadotropin dose administered during COH [48] and the effect of age on these parameters [47]. We found significant correlation between these parameters and the outcome of IVF-ET treatment showing that high E2 levels are associated with lower clinical pregnancy and implantation rates [45-49] in addition to increased adverse obstetric outcomes including higher miscarriage rate [49] and lower birth weight [50]. Successful IVF-ET treatment cycles were associated with lower AUC-E2 compared to unsuccessful cycles at the same patients [49].
3.1.1 Effect of supraphysiological estrogen levels on the endometrium
One of the causes for the reduced implantation rates associated with ovarian stimulation may be an impairment of endometrial receptivity, due to high concentrations of sex steroids. This suggestion is supported by higher implantation rates in hormonal replacement treatment cycles after ovum donation, as opposed to the standard IVF-ET cycles as explained earlier.
Effect on Implantation Window
It is generally believed that the embryo-uterine interactions leading to implantation can only succeed when embryonic development is synchronized with the preparation of the endometrium to the receptive state. Typically, this means that the embryos have reached the blastocyst stage and that the endometrium has undergone certain hormone-dependent changes during a specific time window in the preimplantation phase that prepare it to be receptive to the developing blastocyst [52].
The concept of endometrial receptivity introduced by Psychoyos [53,54], has been shown to last only for several hours, thereby determining a narrow nidation window. The concept of an "implantation window" or "receptive endometrium" was initially established in rodents. In the rat, the fertilized embryo reaches the uterus on day 4 after fertilization, and implantation occurs in the afternoon of day 5 [53,54]. In humans, the ovum is fertilized in the fallopian tube, arrives in the uterine cavity around day 17 (day 14 is taken as day of ovulation of a 28-day cycle), and remains there as a free-floating embryo until about day 19; implantation then occurs between days 19 to 22 [55,56]. However, the precise timing and molecular basis of the receptive window in the human remain undefined. Unfortunately, endometrial receptivity knowledge in the human is limited due to the obvious experimental drawbacks and the lack of specific criteria to define a receptive endometrium.
In the literature, there is controversy regarding the effect of ovarian stimulation on endometrial development. Most of the investigators have reported adverse effects of high estrogen levels on endometrial development but there was no consensus on the actual effect. Some have shown endometrial advancement [57-61] while others showed endometrial retardation [62,63]. However, all studies confirm direct deleterious effects on the endometrial development that jeopardize the chance of implantation due to the lack of synchronization between the development of the endometrium and the early embryo development. Implantation failure has been suggested to result from the disparity in maturation between the endometrial stroma and the epithelium observed in histology. Since a paracrine communication between the epithelium and the stroma may be important at the beginning of implantation, this disparity could compromise uterine receptivity or early trophoblastic invasion [64,65].
The effect of high E2 concentrations (> 20,000 pmol/L) was found to be associated with gland-stromal dys-synchrony, which indicated a deficient secretory transformation of the endometrium that represents a sub-optimal endometrial environment for implantation. This finding substantiates clinical observation of significantly lower pregnancy rates in IVF-ET cycles of women with high E2 concentrations. In these patients, there was a marked stromal edema associated with a significantly greater number of stromal vessels, which suggested advanced stromal maturation [44].
In these studies, definitions of supraphysiological hormonal concentrations were variable. Also, the timing of the biopsies and the drug regimens used for ovarian stimulation were different. This, in addition to the variation in endometrial response to different E2 concentrations, may explain the disagreements reported in the literature [66]. Moreover, both the premature progesterone serum elevation, which occurs in 30% of stimulated IVF-ET cycles before hCG administration, and the advanced post-ovulatory rise in progesterone serum concentrations are believed to be responsible for the advanced endometrial development associated with ovarian stimulation [67,68]. However, other studies failed to confirm these observations [69,70].
Effect on endometrial pinopodes
At the time of implantation, the apical membranes of the epithelial cells lining the uterine cavity develop large and smooth membrane projections, named pinocytes due to their pinocytotic function [71]. Their development is progesterone-dependent with strict correlation with the implantation window in the rodents [72]. Similar structures have been seen in the human endometrium [73]. The number of pinopodes was found to have a strong correlation with implantation after embryo transfer [74,75].
Hormonal treatment has been shown to be associated with changes in the timing of pinopode formation. During ovarian stimulation with clomiphene citrate followed by human menopausal gonadotropins (hMG)/hCG, fully developed pinopodes were found 2 to 3 days earlier [76]. In contrast, with E2 and progesterone treatment, fully developed pinopodes were found to be about two days later. Pinopodes were found to form as early as 4 days after hCG administration [59].
Nikas et al. [61], studied the temporal expression of pinopodes as a specific marker for receptivity in IVF-ET cycles induced by gonadotropins, compared to women with regular menstrual periods and proven fertility who served as controls. They did not find ovarian stimulation to affect endometrial pinopode formation in terms of quantity and life span. Instead, the cycle days when pinopodes formed were specific to the individual, being on average 1–2 days earlier in cycles with ovarian stimulation than in natural cycles. These changes in pinopode expression may reflect shifts in the window of receptivity, resulting in ovo-endometrial asynchrony and limiting implantation success in IVF-ET [61]. In IVF-ET, embryonic development is probably delayed while the uterus is advanced, resulting in an early closure of the nidation window, before the embryo eventually reaches a stage capable of initiating implantation. These findings support the theory that reduced implantation rates in IVF-ET cycles could result from impaired or premature endometrial maturation.
Effect on estrogen and progesterone receptors in the endometrium
A lack of estrogen receptors (ER) has been reported [77] during ovarian stimulation cycles that rendered the endometrium functionally hypoestrogenic or hypoprogestogenic. Also, it has been reported that the expression of progesterone receptors (PR) in the endometrium was decreased in the early part of luteal phase after ovarian stimulation [78]. This premature PR decrease was consistent with an early high progesterone level [79]. Papanikolaou et al. [80], investigated prospectively the effect of multi-follicular ovarian stimulation for IVF on the late follicular phase endometrium histology and the expression of ER and PR. Endometrial biopsies were taken in a natural cycle on the day of the onset of the surge of the LH, and in a subsequent stimulation cycle on the day of hCG administration for final oocyte maturation. Histological examination of biopsies both in natural and stimulated cycles showed no secretory changes. However, in stimulated cycles, PR expression was significantly up-regulated compared to natural cycles in both glands (1.67 versus 1.34, P < 0.05) and stroma (1.98 versus 1.62, P < 0.05), whereas ER was down-regulated in glands (1.15 versus 1.43, P < 0.05). In IVF cycles, the progesterone measurements, although within normal values (range 0.8–1.4 microg/l), were significantly higher than in natural cycles (0.99 vs 0.63 microg/l, respectively, P = 0.008). An ongoing pregnancy rate of 37.5% was achieved in the stimulated cycles. The authors concluded that although the current study found no early secretory transformation in stimulated endometria before hCG administration, the ER and PR expression in these endometria was similar to the one described during the first days of the luteal phase in natural cycles. Supraphysiological concentrations of estradiol and subtle progesterone rises in the late follicular phase might be responsible for this modulated steroid receptor profile. They added that this phenomenon indicated accentuated maturation of the endometrium in IVF cycles from the pre-ovulatory phase onwards. [80].
Ovarian stimulation with GnRH-agonist/hMG was found to induce precocious secretory endometrial transformation around the time of oocyte retrieval. Compared to natural cycles, there was an imbalance between endometrial steroid receptor content, proliferation index, and maturation in the peri- and postovulatory phases of stimulated cycles. The lower ER in stimulated cycles on the day of oocyte pick up as compared to the natural cycle controls on the day of ovulation was mainly observed in the stroma, but failed to reach a significant difference in the glands. As for PR, the staining intensity of the stromal ER in the stimulated cycles was higher than that of luteal phase day 2 of a natural cycle. These findings suggest a relative imbalance in ER and PR content of the endometrium in stimulated cycles compared to their natural cycle counterparts. [79].
In conclusion, excessive ovarian response was suggested to lead to insufficient secretory transformation of the endometrium, as well as discordant glandular and stromal development at a time that coincides with the period of maximum uterine receptivity.
Effect on endometrial vasculature and endometrial blood flow
Angiogenesis has a critical role in female reproductive physiology. Growth of the endometrium and placentation is also accompanied by extensive angiogenesis. Thus, an actively maintained blood supply is an essential requirement for reproductive functions, including normal implantation [81]. Endometrial vasculature has been shown to play a prominent role in the early endometrial response to the implanting blastocyst, and vascular changes may contribute to uterine receptivity [82].
The introduction of transvaginal Doppler ultrasound makes the measurement of uterine artery blood flow possible, and at one time it was hoped that uterine arterial resistance changes might reflect uterine receptivity [83]. Applebaum first introduced the concept of evaluating uterine receptivity by a uterine score including the endometrial blood flow [84]. Different studies have demonstrated significant changes in the Doppler indices of uterine and ovarian vessels during ovarian stimulation and spontaneous cycles [85,86]. Basir et al., found evidence of impaired endometrial blood flow in association with significantly high estrogen levels in high responders to ovarian stimulation [87].
Although pregnancy outcome tended to be poor in patients with higher mean uterine arterial impedance indices, the predictive value of using a specific resistance index (RI) or pulsatility index [PI] variable in assessing endometrial receptivity seems to be limited [88]. One of the explanations is that the major uterine compartment is the myometrium and not the endometrium, and thus most of the blood passing through the uterine arteries never reaches the endometrium. A more logical approach would be to evaluate the vascularization around the endometrium directly in an attempt to assess endometrial receptivity.
Histological studies have confirmed that the sub-endometrial halo surrounding the endometrium represents the innermost layer of the myometrium, and compared with the outer myometrium, it consists of a distinct compartment of more tightly packed muscle cells with increased vascularity. Studies have shown that interactions between the junctional zone and the endometrium may play an important role in the implantation process [89,90] and endometrial-sub-endometrial blood flow distribution pattern assessed by transvaginal color Doppler before ET was found to correlate with the implantation and pregnancy rate after IVF-ET [91]. With the absence of sub-endometrial blood flow, even in the presence of other favorable parameters, no conception was achieved. By using a similar approach, Salle et al., calculated a uterine score in the secretory phase of the menstrual cycle preceding IVF-ET [92]. Immunocytochemistry study revealed that the sub-endometrial myometrium, also called the junctional zone myometrium or archimyometrium, exhibits a cyclic pattern of estrogen and PR expression that parallels that of the endometrium [93]. Moreover, the responsiveness of the junctional zone has been shown to be associated with implantation success during IVF-ET treatment [90].
Many investigators have also noted the correlation of junctional zone contractions with pregnancy outcome in both natural [94] and assisted reproduction cycles [95]. Less-active junctional zone contractility is associated with higher pregnancy rates
Kupesic et al. compared the 2-D and 3-D ultrasonographic scoring systems by combining parameters including endometrial thickness, volume, echogenicity, and sub-endometrial blood flow. They found the two systems had similar efficiencies in predicting pregnancy outcome of IVF-ET procedures [96]. Several investigators noticed that when the endometrial and sub-endometrial flow parameters were combined, significant differences were found between pregnant and nonpregnant patients [97-99]. In contrast, there was no significant difference if attention was only focused on intraendometrial or sub-endometrial blood flow [100]. These results imply that the endometrial/sub-endometrial area must be considered as a whole in evaluating endometrial perfusion.
One hallmark of implantation is increased vascular permeability at the implantation site. Vasoactive agents, including histamine, platelet-activating factor, vascular endothelial growth factor, and eicosanoids, have been studied during implantation [101,102]. Vaginal E2 administration improves endometrial proliferation and uterine perfusion, presumably because of combined local and systemic effects, but may interfere with P-induced uterine relaxation [103].
In a study by Basir et al., [104] the investigators compared the hemodynamic parameters of the utero-ovarian vasculature and the endometrial spiral arteries of women who showed a moderate response with women whose E2 concentrations were in excess of 20,000 pmol/l after ovarian stimulation. Despite low uterine PI and RI, the endometrial blood flow in high responders appears to be impaired. The authors concluded that this might contribute to the decline in implantation efficiency noted in high responders. The decreased endometrial blood flow despite the increased blood flow in the uterine arteries may indicate a shunt of blood flow from the endometrium into the myometrium.
The authors [104] found low pulsatility index and resistance index of the ovarian arteries indicating neovascularization and increased capillary permeability in the ovarian tissue of high responders. The authors suggested that the blood flow might be directed through the utero-ovarian collaterals to the ovaries. However, because the sample size in this study was small (19 patients with E2 > 20,000 pmol/L), further larger prospective studies are required to confirm the effect of excessively high concentrations of serum E2 on endometrial blood flow.
Moreover, the increase in hormonal concentrations in the peripheral plasma leads to a decrease in peripheral vascular resistance [105] and a decreased contractility of the uterine muscles. This results in relaxation and opening up of the small uterine vascular channels, which may also cause an increase in the capillary permeability. In a study on the endometrial morphological changes at high concentrations of E2, a significantly greater number of vessels and endometrial edema in women who responded excessively to ovarian stimulation was demonstrated. Therefore, it was postulated that the blood flow through these minute endometrial vessels may be very slow and the weak Doppler flow signals arising from them could not be picked up by the color Doppler despite low uterine PI and RI. The increase in capillary permeability and dilatation leads to extravasation of fluid from the intercellular to extracellular compartments, and hence endometrial edema [106]. In another study the investigators suggested that the blood flow per capillary might actually be reduced during edema [107].
Successful implantation and continuing development of implanted embryo depends on a complex series of cellular and molecular events between the blastocyst and the endometrium [108]. The decline in blood flow could therefore impede the exchange of essential nutrients, bioactive molecules and reactive compounds that are vital for implantation with absent endometrial and intra-endometrial vascularization appeared to be a useful predictor of failure of implantation in IVF-ET cycles [109].
Whether fertilization occurs in vivo or in vitro, most human embryos will not develop through gestation [110] with a very high proportion of developmental failure during the preimplantation stages associated with chromosomal defects of oocyte origin [111]. Van Blerkom suggested that the dissolved oxygen content of pre-ovulatory follicular fluid and the developmental competence of the corresponding oocyte were related [112]. A developmentally significant association between the chromosomal normality of the human oocyte and the level of intra-follicular oxygen and peri-follicular vascularity was reported suggesting that hypoxic intra-follicular conditions that result from the failure of an appropriate microvasculature to develop around the growing or pre-ovulatory follicle(s) could be a proximate cause of the maternal-age-related increase in the incidence of trisomic conditions [112-114].
Effect on Integrins
Integrins are a family of cell adhesion molecules. Previous work has shown the expression of integrins in the endometrium changes during the menstrual cycle [115]. Three integrins in particular (1β1, 4β1 and vβ3) are thought to play a vital role in implantation as all are expressed during the 'implantation window'. The β3 and β1 integrins have also been shown to be reduced in infertile patients using flow cytometry [116]. Aberrant patterns of integrin expression have also been associated with certain diagnoses in infertile patients, including luteal phase defects, endometriosis, hydrosalpinx and unexplained infertility [117].
The exact role of integrins remains controversial and results have not been duplicated in all studies. Creus and co-workers showed no difference in integrin expression in patients who became spontaneously pregnant compared with those that do not [118]. Thomas et al., demonstrated that integrin expression seems to be reduced in the glandular epithelium in the endometrium after ovulation induction, irrespective of the dating. The authors concluded that there might be an ideal E2 level that should be reached during IVF-ET treatment as low estrogen levels might reduce the yield of oocytes, but high levels might impair the receptivity of the endometrium reducing integrin expression and leading to lower implantation rates [119].
3.1.2 Effect of supraphysiological estrogen levels on the developing oocyte (follicular and oocyte development)
As the contributor of the bulk of the cytoplasm to the zygote and half of its nuclear DNA, the key importance of the oocyte is unquestionable. The principle, that embryogenesis is rooted in oogenesis, has been understood for nearly a century, and is now becoming better understood at the molecular level. During follicular growth, the oocyte is in close contact with granulosa cells through gap junctions and is therefore under the influence of the follicular environment. Since oocyte maturation is such a long process, any adverse events occurring during this period can damage vital structural molecules or whole organelles, resulting in oocytes with reduced developmental competence. These oocytes may in turn give rise to preimplantation embryos with a compromised viability, and hence, a reduced implantation potential [120]. The ER gene is expressed in the human cumulus-oocyte complexes and oocytes but not in granulosa/cumulus cells which might suggest a lack of receptor-mediated autocrine effect of estrogen during folliculogenesis. Conversely, estrogen secreted by granulosa/cumulus cells, may exert a paracrine effect to influence oocyte maturation and fertilization competence directly [121]. It was reported that retrieval of >10 oocytes during IVF-ET cycles was correlated with oocytes of lower quality, as manifested by a decrease in the fertilization rate [122].
Effect on the chromosomal structure and cytogenetics of the oocytes
Studies with animal systems have indicated that a single cycle of ovarian stimulation can have adverse effects on oocyte competence during early development, and may well have downstream effects on the normality of fetal growth and development. Whether developmental defects could result from chromosomal malsegregation during ovarian stimulation-induced meiotic maturation has been examined in the mouse system. Earlier studies compared oocytes obtained after spontaneous or hormonally induced ovulation and found no increase in the incidence of non-disjunction or oocyte aneuploidy after ovarian stimulation [123]. In contrast, cytogenetic analysis of pronuclear stage mouse eggs after single cycle of ovarian stimulation showed chromosomal aberrations largely confined to the female pronucleus, indicating developmental compromise prior to fertilization [124]. Sengoku and Dukelow found comparable frequencies of aneuploidy for cleavage-stage hamster embryos produced in natural and pregnant mare serum gonadotropins (PMSG)-stimulated cycles, indicating that peri-implantation mortality may have an epigenetic origin [125].
In IVF-ET programs, the high rate of oocyte recovery and successful fertilization in vitro contrasts with a relatively high rate of conception failures, as even the most advanced IVF-ET clinics can provide a 33% success rate per cycle at best [12], despite multiple embryo transfers. The possible reason for this conception failure is the high frequency of lethal chromosomal abnormalities that prevents embryonic development beyond the pre- and post-implantation stages. The high frequency of spontaneous abortions also indicates that the proportion of embryos with genetic defects is significant. Cytogenetic analysis of the early stages of cleavage indicates that chromosomal aberrations may be found in 35–44% of pre-implantation embryos produced in vitro [126]. It has to be emphasized that the majority of pre-implantation embryos carry numerical chromosomal defects. However, the high incidence of chromosomal anomalies is presumably biased by the fact that only embryos with a poor morphological score were analyzed (discarded embryos). It nevertheless indicates that the low implantation rates of human pre-implantation embryos in IVF-ET programs are the consequence of natural selection [127]. Aneuploidy is the most common abnormality found in normally developing embryos following ovarian stimulation and IVF-ET [128], but other aberrations were also revealed. Polyploidy and multinucleation were frequently described in arrested embryos
Estrogen is well known to induce chromosomal and cytogenetic damage. The natural hormone E2 has clearly been shown to induce alterations in chromosome number such as losses or gains of whole chromosomes [129,130], chromosome translocations [131,132], and gene amplifications [133,134]. In addition, there is preliminary evidence of estrogen-induced gene mutations and gene deletions [135,136].
The synthetic estrogen, diethylstilbestrol (DES) causes a severe yet reversible deterioration of meiotic spindle microtubule organization during maturation of the mouse oocytes [137]. High doses of E2 were found to induce numerical chromosome changes (both chromosome gains and losses) similar to the reported observations with the synthetic estrogen, diethylstilbestrol [129]. Estrogen is also known to cause direct DNA damage via its catecholestrogen metabolites [138,139]. There are several types of free radical-mediated DNA damage, which are induced by estrogens and/or their metabolites [140-142].
As explained earlier, supraphysiological estrogen levels associated with ovarian stimulation impair the uterovarian blood flow resulting in follicular hypoxia. Gaulden proposed that follicular hypoxia might have a potent adverse influence on spindle organization and the normality of chromosomal segregation in the human oocyte [143].
Effect on the mitochondrial function of the oocytes and embryos
Van Blerkom has suggested that the developmental competence of mouse and human early embryos is related to the metabolic capacity of the mitochondria. It is thought that mitochondrial replication does not begin until after implantation, and that paternal contribution is minimal. Therefore the preimplantation embryo is completely reliant on maternally inherited mitochondria in the oocyte. Deletions and mutations in oocyte mitochondrial DNA may lead to mitochondrial dysfunction, influencing energy production and apoptosis in oocytes and early embryos, resulting in aberrant chromosomal segregation or developmental arrest [144].
The classical model of E2 action has been described to be mediated by cytoplasmic/nuclear partitioning receptor proteins that stimulate gene transcription upon binding to specific DNA sequences [145]. However, there are increasing functional evidences for extra nuclear/cytoplasmic localization of steroid hormone receptors. Several studies showing rapid non-genomic actions of steroids have led to speculate about the existence of cell-surface resident receptor forms [146-148]. Reports have documented the presence of estrogen binding proteins localized at the plasma membrane [149,150]. Independently, the known direct effects of various steroids on mitochondrial gene transcription support the idea of receptor attachment to the mitochondrial genome [151]. It was also identified early that estrogen specific binding sites were associated with mitochondrial and microsomal structures [152].
The mitochondrial-enriched subfraction represented an important source of E2 binding, where the steroid was recognized in a stereospecific and high affinity manner. The existence of mitochondrial and membrane estrogen binding sites correlated with the presence of ER but mainly with ER proteins. Using macromolecular E2 derivatives in Ligand Blot studies, both mitochondrial and membrane estrogen binding proteins were found in the uterus, and the ovary. This differential cellular partitioning of ER and forms may contribute to the known diversity of E2 effects in target organs [153].
Recently, in myocardial cell model, it was reported that at physiological concentrations, which do not inhibit mitochondrial functions, estrogens can protect heart mitochondria from the loss of cytochrome c induced by high calcium, and this might be one of the possible mechanisms by which estrogens preserve myocardial cell viability after ischemia/reperfusion [154]. High concentrations of estrogens (50–100 M) have been found to have a damaging effect on mitochondrial functions by strongly inhibiting mitochondrial respiration and membrane potential presumably due to decreased activity of the respiratory chain. The inhibition of the respiratory chain may be due to non-specific binding of estrogens to hydrophobic regions of the mitochondrial membranes, which may change protein/lipid interactions, disturb electron transport through the inner mitochondrial membrane and reduce membrane potential [155].
We postulate that supraphysiological levels of estrogen attained during ovarian stimulation may affect the mitochondrial function of the developing oocyte and embryo. This could be one of the mechanisms behind impaired developmental capacity of the oocytes and embryos obtained after ovarian stimulation.
3.1.3 Effect of supraphysiological estrogen levels on the spermatozoa and sperm/oocyte interaction
Sperm are exposed to estrogens within the male tract, and P450 aromatase has also been identified in human spermatozoa [156]. This raises the possibility that the spermatozoa can provide a continuing local source of estrogens in the epididymis, as well as while in the female tract on its journey to fertilize the oocyte. Inside the female genital tract, the spermatozoa would also be exposed to estrogens, particularly in tubal fluid following follicle rupture and when in close vicinity to released oocytes [157].
Estrogen receptors in the spermatozoa
Non-genomic effects of estrogens have also been reported in several cell types including the spermatozoa [158]. Immunohistochemical detection of ER has been reported for human spermatozoa, with distribution in both the head and flagellum [159].
Possible effect of estrogen on the spermatozoa function
After ejaculation, in vitro, the spermatozoa are initially unable to fertilize [160]. The spermatozoa acquire the capacity to fertilize, after a certain period of time that is species-specific when exposed to an appropriate environment either in vivo or in vitro. This process is called 'sperm capacitation' [161]. Capacitated spermatozoa are able to express hyperactivated motility, to undergo the acrosome reaction, and to fertilize an oocyte [160]. Estrogens are believed to have a possible effect on these events (capacitation and acrosome reaction), which would have important consequences on fertility in vivo.
Recently, Adeoya-Osiguwa provided evidence that E2 and environmental estrogens can significantly stimulate mammalian sperm capacitation, acrosome reactions. They found that in uncapacitated cells, E2 at 00.001 μmol/l, significantly stimulated capacitation and acrosome reactions while in capacitated cells, E2 had no effect. The authors concluded that whether these responses have effects on fertility in vivo remains to be determined, along with the mechanisms of action involved [157]. It is pertinent to mention here that the average E2 concentrations in follicular fluid from mature oocyte are in the micromolar range [162,163].
The regulation of capacitation is very important in mammalian fertilization as evidence suggests that once capacitation has been initiated it will usually continue unchecked, frequently resulting in the spermatozoa undergoing spontaneous acrosome reactions and thus becoming non-fertilizing [164].
Adeoya-Osiguwa argued that E2 and the environmental estrogens appear primarily to stimulate the spermatozoa, accelerating the rate of capacitation and then promoting 'over-capacitation' in at least some of the cells, resulting in the acrosome reaction [157]. Since already acrosome-reacted spermatozoa are non-fertilizing [160], similar responses occurring in vivo could reduce the number of potentially fertilizing cells and so have an undesirable effect on fertility. As capacitation and fertilization occur in the female reproductive tract, it is likely that any effects of environmental estrogens on sperm function would be more pronounced in the female, but effects on mature spermatozoa awaiting ejaculation cannot be ruled out.
3.1.4 Effect of supraphysiological estrogen levels on the embryo
Mouse and human embryos, when cultured in vitro, undergo a delay in development compared with those grown in vivo. This delay can be caused by suboptimal culture conditions, but possible influences of ovarian stimulation cannot be excluded [165]. In order to determine if implantation failure associated with ovarian stimulation was due to abnormalities in the blastocysts or the endometrium, decidualization studies and embryo transfers to pseudopregnant recipients were performed [166,167]. The uterus of a large proportion of superovulated animals was unable to undergo decidualization in time, whereas embryo transfers to pseudopregnant females resulted in normally developing fetuses, which indicated that hormonally treated oocytes themselves were not affected. Some studies found that the E2 concentrations in the fresh cycle were not related to the success of frozen-thawed embryo transfer cycles indicating that embryo quality seemed unaffected by the high estrogen levels [168,169]. However, there is increasing evidence suggesting that ovarian stimulation and the associated high estrogen levels are detrimental on the embryo and associated with a decrease in the fertilization rate. When compared with blastocysts derived from naturally cycling mice, blastocysts that developed in vivo in superovulated mice were found to have fewer microvilli on their surface [170], a reduced [35S]-methionine uptake [171], and a lower cell number and mitotic index [172]. A reduced cell number and a two-fold decrease in viability post-transfer of embryos from gonadotropins-stimulated hamster females, was also observed [21]. Furthermore, it has been reported that the proportion of abnormal preimplantation embryos increases after superovulation, and that blastocysts have a smaller trophoblastic outgrowth in vitro [173]. Moreover, in mice as well as in humans, there is evidence for steroids being regulators of gene expression in the embryo and endometrium, and that embryo morphology and rate of development – both of which reflect embryo quality – have a genetic basis. Also, ovulation induction therapy has been found to be associated with an increased rate of mosaicism in the embryos, which fail to implant [52].
It has been proposed that high E2 levels after COH impair endometrial receptivity because oocyte quality, fertilization rate, and embryo cleavage (until day 2) were normal in patients with a high response [174] and the quality of embryos and the implantation rate seemed normal in subsequent frozen-thawed embryo transfer [43]. However, high E2 levels were found to be deleterious to embryo adhesion in vitro, mainly because they have a direct toxic effect on the embryo that may occur at the cleavage stage [175].
Mouse and human embryos, when cultured in vitro, undergo a delay in development compared with those grown in vivo. This delay can be caused by suboptimal culture conditions, but possible influences of ovarian stimulation cannot be excluded. In the mice, preimplantation embryonic development in vitro and in vivo was found to be negatively influenced by the ovarian stimulation itself, and results in an impaired blastocyst formation and fetal growth retardation at day 14 of gestation. The authors suggested that a similar negative effect of ovarian stimulation on oocyte and embryo quality seems likely in IVF-ET which might explain in part for the delay in embryonic development after IVF-ET, and for the low birth weight often observed after assisted reproductive technologies [165].
Effect of estrogen on blastocyst hatching
The successful hatching of the embryos is thought to be a key event in the implantation process. One reason for the low implantation rate that has been suggested is the limited ability of blastocysts to hatch from the zona pellucida. Suboptimal culture conditions might induce the hardening of zona pellucida, which could limit the hatching ability of embryos [176]. To help embryos hatching from their zona pellucida during blastocyst expansion, different types of assisted hatching have been developed, including mechanical partial zona dissection or zona drilling, chemical zona drilling with acidic Tyrode's solution, and the laser dissection technique [177]. However, there is controversy about the benefit of assisted hatching on the improvement of the implantation rate and pregnancy rate. Some agreed as to the benefit of assisted hatching in women with advanced age, in women with repeated IVF-ET failure [178].
The degree of zona pellucida thickness variation of the transferred embryos has been found to exhibit a strong correlation with clinical pregnancy outcome following IVF-ET treatment and to be important for embryo selection during clinical transfers [179,180]. Zona pellucida thickness variation and character were found to correlate with implantation. Implantation rates were found to range from 10% for embryos with uniform thickness to 29% with thin or irregular zona pellucida [181,182]. These reports suggested that patients transferred with embryos with thinner zonae had a better chance of successful implantation and pregnancy as compared to those transferred with embryos having thicker zonae. Other reports have proposed zona pellucida thickness variation as a reliable marker for selecting thawed as well as fresh human embryos for transfers [183,184]. Cohen [185] conducted a retrospective analysis of zona pellucida thickness of transferred embryos through video recordings and concluded that the variations in zona pellucida thickness rather than the zona pellucida thickness per se of the transferred embryos was a stronger predictor of the IVF-ET.
A significant linear relationship was reported to exist between the mean zona pellucida thickness of each patient and the maximum E2 level and an increasing one with the hMG dose. The authors found that the zona pellucida thickness was basically an individual feature that influenced the fertilization rate [186].
3.1.5 Effect of supraphysiological estrogen levels on the ovaries [corpus luteum], pituitary, and hypothalamus
Abnormalities in the luteal phase have been shown in virtually all the stimulation protocols used in ovarian stimulation, on the hormonal, as well as on the endometrial level. All three aspects of a defective luteal phase, that is, a shortened luteal phase and/or low mid-luteal serum progesterone concentrations and/or abnormal endometrial histology, have been regularly observed in IVF-ET cycles. For that reason, luteal-phase supplementation with hCG or progesterone increases pregnancy rates, and its necessity has been well established, at least in GnRH-agonist cycles [187].
In the 'pre-agonists era', Edwards and Steptoe were the first to postulate luteal phase inadequacy resulting from ovarian stimulation as a cause of failure of IVF-ET cycles. With the introduction of the GnRH-agonist, used in ovarian stimulation cycles to avoid premature luteinizing hormone [LH] surge, luteal phase inadequacy was reported. A meta-analysis of different clinical trials demonstrated beneficial effects of luteal support when ovarian stimulation was carried out with human menopausal gonadotropins [hMG] in association with GnRHa [187].
Recently, GnRH antagonists have become available for clinical use. Whether GnRH antagonists induce down-regulation of pituitary GnRH receptors is still a subject of investigation [188]. It has been demonstrated that chronic administration of the GnRH antagonist Cetrorelix in rats causes an important down-regulation of pituitary GnRH receptors [189].
Direct effect on corpus luteum
Different estradiol receprors have been detected in human corpus luteum, indicating that estrogen might be a local regulator of corpus luteum function [190]. In the pre-agonist era, the alteration of the Estradiol/progesterone (E2/P) ratio was considered a main cause of luteal-phase inadequacy, possibly through the luteolytic action of E2 [5]
Estrogen is thought to exert a direct luteolytic action in primates as exogenous administration of E2 reduces progesterone concentrations during the luteal phase [191], probably via inhibiting the enzyme 3 beta-hydroxysteroid dehydrogenase, which is mandatory for progesterone synthesis [192]. Moreover, although the exact mechanism has not yet been established, estrogen may play a role in the regulation of proteins involved in the process of luteal-cell apoptosis [193].
Normal corpus luteum function is dependent on the proper function of the pituitary gland and hypothalamus. Adequate luteinizing hormone [LH] surge during ovulation and continuous tonic LH pulses during the luteal phase are necessary for the proper development of the corpus luteum. Corpus luteum dysfunction due to the effect of ovarian stimulation on the pituitary gland and hypothalamus will be discussed later.
Effect on the pituitary gland and hypothalamus
Normal corpus luteum function requires optimal follicular development in the follicular phase, especially follicle stimulating hormone (FSH) stimulation, adequate luteinizing hormone (LH) surge during ovulation and continuous tonic LH pulses during the luteal phase. In turn, the normal luteal phase is characterized by an optimal hormonal environment and adequate endometrial secretory transformation. As many factors contribute to a normal corpus luteum function, any alteration might exert a deleterious effect on the final target, the endometrium, leading to embryo/endometrial asynchrony [5]. Luteal phase insufficiency due to corpus luteum defects and LH suppression is known to be associated with failure to achieve and maintain pregnancy [194].
Effect on LH secretion
The lifespan and steroidogenic capacity of the human corpus luteum is dependent on continuous tonic luteinizing hormone secretion as well as healthy adequate gonadotropins surge. Feedback mechanisms from ovarian steroids and GnRH pulses regulate LH secretion during the luteal phase, but a number of autocrine and paracrine factors within the ovary might also play a role in controlling corpus luteum function [5].
Effect on LH surge
It is obvious that during COH for ART, the prevention of endogenous LH surge is mandatory to avoid the occurrence of premature LH surge as well as for timing of oocyte retrieval.
However, during ovarian stimulation cycles in which no pituitary suppression is used, there are data that suggest a defective endogenous gonadotropins surge. The gonadotropins surge is an event crucial for final oocyte maturation, ovulation, and subsequently for corpus luteum function. The duration of the LH peak seems to be more important than its amplitude for the induction of ovulation [195]. Ovulation induction by hCG is not physiological; the absence of an FSH surge, and the long duration of LH activity associated with hCG action, would contribute to some of the luteal phase abnormalities [196].
Messinis demonstrated that an attenuated LH surge is obtained in normally cycling women during superovulation induction with sequential clomiphene/hMG treatment. The peak values and the duration of the LH surge to have significant negative correlations with the plasma E2 levels, the number of follicles, and the total follicular fluid volume aspirated at laparoscopy. This suggests that during superovulation induction for IVF-ET, the endogenous LH surge is attenuated by factors, which are related to the degree of ovarian hyperstimulation [197].
As shown above, the induction of superovulation in women with human gonadotropins may result in blockage of the endogenous LH surge, but the reasons for this are not known. A high number of small follicles have been suggested to have a suppressive effect on both tonic and mid-cycle gonadotropins secretion [198].
Effect of abnormal LH surge on nuclear maturation of the egg
A timely LH surge of adequate amplitude and sufficient duration is important to bring about rapid and complex cellular differentiation, resulting in cascades of tightly coupled biochemical events, which initiate oocyte maturation, ovulation, and corpus luteum formation [199]. It is known that a midcycle LH surge of sufficiently high amplitude and duration is important for both nuclear and cytoplasmic maturation, which ensure the normal fertilization and developing potential of oocytes [199]. For PMSG-hyperstimulated rats, higher doses of hCG are required to completely ovulate the expanded cohort of preovulatory follicles [200].
Effect on LH tonic pulse during the luteal phase
Low luteal LH levels have been described after human menopausal gonadotropins treatment6 and after GnRH-agonist treatment or after GnRH-antagonist treatment. These low, almost undetectable, luteal LH levels may not be able to support corpus luteum. As a result, a shortened luteal phase and low mid-luteal progesterone concentrations have been described in cycles stimulated with the association either of a GnRH agonist or a GnRH antagonist [195,196].
Supraphysiological progesterone serum concentrations may also interfere with the pituitary's luteinizing hormone secretion by disturbing the feedback control mechanisms and may result in a reduction of the LH serum levels [195,196]. Progesterone modulates LH secretion during the luteal phase by influencing the LH pulse amplitude and pituitary release of LH [201]. A longer exposure to progesterone or the combined action of estrogen and progesterone decreases LH release [302]. As ovarian stimulation results in supraphysiological steroid serum concentrations, these high steroid levels may adversely affect LH secretion via a long-loop feedback mechanism. In turn, disturbed LH secretion may induce a luteal-phase defect with premature luteolysis, low progesterone levels and shortened luteal phase. It might therefore be hypothesized that deviation from the normal hormonal environment could be a prevalent effect of ovarian stimulation in the luteal phase; despite the use of different stimulation protocols [7]. This might be a possible explanation of the observation that in natural cycle, luteal-phase length was normal after GnRH-antagonist treatment [203]. However, in GnRH-antagonist cycles and after minimal ovarian stimulation, luteal-phase length was normal despite an abnormal endocrine profile [204].
3.1.6 Other probable effects of supraphysiological estrogen levels
There are other less defined probable mechanisms through which supraphysiological estrogen levels may cause adverse effects on the outcome of infertility treatment.
(A) Leptin-mediated effect
Recently, an important role for the leptin, the secretory product of adipocytes, in reproductive medicine has emerged and it is interesting to discuss in brief a possible link between leptin, induction of ovulation and aromatase inhibitors.
Soon after its discovery in the early 1990s, it was recognized that leptin played a significant role in reproduction, providing a critical link between metabolic state and fertility. It now appears that leptin may have an important role in both normal ovarian physiology and pathophysiology. Research has revealed that a minimum level of leptin stimulation is required for maintenance of fertility in animals and humans. Conversely, elevated leptin levels may impair fertility [205].
Leptin is a secretory product of adipocytes that correlates significantly with the body mass index with increased levels in obese women [206]. It can influence reproduction through central (on GnRH neural system) and peripheral (on the ovary directly) mechanisms [205]. A positive correlation has been found between leptin and BMI, as well as between leptin and testosterone in women with polycystic ovarian syndrome (PCOS) [207].
Numerous studies have shown that circulating leptin concentrations rise in parallel with E2 during ovarian stimulation [208-213]. These changes are not likely to be a direct action of FSH because FSH decreases as E2 and leptin rise during natural cycles, and high FSH levels experienced during ovarian stimulation simulate endogenous levels in postmenopausal women, who have lower serum leptin concentrations than premenopausal women [205]. Bützow [206] found that the larger the increase in serum leptin concentrations during FSH stimulation, the poorer the ovarian response in terms of number of follicles and retrieved oocytes. Moreover, higher serum leptin levels were found in oligo- and amenorrheic women who failed to respond to clomiphene therapy [214].
The effect of body weight on outcomes of assisted reproduction has been investigated. Fedorcsak [215] reported that among patients who conceived, overweight patients (BMI >25) had fewer oocytes retrieved, a higher miscarriage rate, and lower live birth rate. In a much larger retrospective study that included 8822 embryo transfer cycles, the cumulative pregnancy rate progressively decreased as BMI increased from <25 to >35 [216].
Higher follicular fluid leptin concentrations correlated with lower intrafollicular oxygen concentration [pO2], [217] a condition that negatively impacts oocyte developmental competence [218] with a direct evidence of a relationship between leptin levels and ART outcome reported by Mantzoros [219] who found significantly lower follicular fluid leptin concentrations in women who became pregnant within three cycles of IVF-ET or gamete intrafallopian transfer (GIFT). More recently, a significant negative correlation between non-fasting serum leptin levels measured at the beginning of FSH stimulation and pregnancy success in women undergoing first attempt IVF-ET cycles was reported [220]. Moreover, as leptin receptors are expressed in the human endometrium, [221] a role for leptin in endometrial receptivity cannot be excluded. These results imply that elevated leptin may be a key factor in obesity-related fertility problems, and conversely that elevated leptin may negatively impact fertility independently of body mass. Fewer good embryos and lower implantation rate suggest that elevated leptin impairs oocyte developmental competence and/or early cleavage stage embryo development, possibly via direct actions on the follicle [205].
E2 has been reported to increase leptin mRNA expression. In human adipose tissue culture, E2 stimulated leptin secretion in women but not in men [222]. In women, E2 was found to increase ob mRNA expression and leptin release. Moreover, in adipose tissue of women, the estrogen precursors; testosterone and dehydroepiandrosterone also induced an increase in leptin secretion, an effect that was prevented by the aromatase inhibitor letrozole. Moreover, the stimulatory effect of E2 observed in women was antagonized by the antiestrogen ICI182780 [223].
(B) Coagulation system
Estrogen has been pointed out as a pre-thrombotic factor. It has long been established that both pregnancy and oral contraceptive use have resulted in an increased level of many coagulation parameters. Generally, this has been thought to be a result of the estrogen component. Although it has been well established that long-term exposure to exogenous contraceptive steroids can have a promoting influence on the potential for thrombosis in women, it is less clear what role high levels of endogenous steroids might play. Undefined coagulation abnormalities were reported after hMG- and hCG-induced hyperstimulation of the ovary in several women [224-227]. Kim et al., [225] noted a large increase in fibrinogen after hMG treatment, accompanied by "significant increases" in the prothrombin time. However, the statistically significant activation of clotting factors occurring during controlled ovarian hyperstimulation is usually not accompanied by clinically significant coagulation disorders that may be explained by either of the following: (1) the increased levels of clotting activity were still "within normal limits"; (2) none of the patients had any conditions known to predispose to coagulopathies (history of coagulopathies, phlebitis, damaged or compromised endothelial cells, etc.); or (3) a combination of the two [226]. However, such subtle coagulation disturbances may exert an adverse effect on endometrial receptivity and the early development of the placenta by affecting the microcirculation.
Two case reports were published of activated protein C-resistant women who suffered a thrombotic event during IVF-ET treatment [227,228]. Curvers [229] reported that the only coagulation parameter that changed considerably during IVF-ET treatment was the activated protein C (APC). In their study, the authors observed that hyperstimulation, i.e. high estrogen levels, induce APC resistance, and that under these conditions both the absolute values and the changes in the APC and the estrogen levels (hyperstimulation-baseline) correlate significantly. Prior to that study, it was reported that high estrogen levels were not associated with APC resistance [230,231]. However, the APC resistance test used in these studies, which is based on quantification of the effect of APC on the clotting of plasma initiated via the intrinsic coagulation pathway, is not very sensitive to changes in sex hormones [232]. On the other hand, Curvers et al. [232], used an assay that quantifies down-regulation of extrinsic coagulation by APC that is particularly sensitive to hormonal changes in women [232].
We think it is possible that the reported effect of ovarian stimulation-associated supraphysiological levels of estrogen on the coagulation system, though being modest, may contribute at least partially to the reduced implantation rate observed after assisted reproduction. This could be due to an impact on the microcirculation in the endometrium that could affect the early stages of the implantation as well as the early development of the placenta. However, more studies are needed to support this hypothesis.
(C) Tubes and tubal transfer
Effect on the oviductal environment
Oviducts are biologically active, providing an environment that sustains and enhances fertilization during early embryonic development as the embryo travels toward the uterine cavity. Following superovulation, the fluid from the oviduct seems to impair embryo development. Furthermore, a stimulated oviductal environment has also been shown to have a negative influence on the implantation capacity of mouse embryos [233].
Superovulation in the mouse was described as a model for intra-uterine growth retardation
Superovulation is associated with a slower preimplantation embryo development, a later and impaired implantation and a prolonged gestation [234]. This suggests that the oviductal milieu rather than the embryo quality are responsible for the adverse effects observed after superovulation. The stimulated oviductal environment impairs the developmental capacity of embryos in comparison with untreated pseudopregnant females. In-vitro culture is also suboptimal but better than the stimulated oviductal environment. However, a detrimental effect of hormonal stimulation upon the oviductal environment has not yet been demonstrated in the human. A possible potential negative effect, however, is not contradicted by observations reporting a higher pregnancy rate after ovarian stimulation and IUI compared to insemination alone [235]. In the human, there are observations that GIFT results in higher pregnancy rates in comparison with IVF-ET [236,237]. It is difficult to interpret these data since the embryos were exposed longer to the possible deleterious stimulated oviductal environment-using GIFT whereas in IVF-ET, possible "suboptimal" culture conditions as opposed to in-vivo conditions were used. Since the introduction of sequential culture media, incorporating amino acids, vitamins and growth factors, the in-vitro culture of embryos has been improved, resulting in implantation rates of >50% per transferred blastocyst [238]. Confirmation of these data in a trial would result in higher pregnancy rates than ever reported for GIFT [239].
Effect on the oviductal embryo transfer
Akira [240] has found that ovarian stimulation was associated with accelerated oviductal embryo transport. The authors have concluded that increased implantation failure in superovulated rats may result from the accelerated embryo transport resulting from elevated E2/P ratio. Accelerated oocyte/embryo transfer has been postulated to be the mechanism behind which high doses of estrogen work as postcoital emergency contraception as explained later.
Post-coital contraception with estrogen
The achievement of very high levels of estrogen by administering exogenous estrogen has been suggested for postcoital contraception as early as the 1960s, when high-dose estrogen was identified as a highly effective emergency contraceptive [241].
Greenwald [242] compared the response of the rabbit, rat, mouse, hamster, and guinea pig, to a single post-coital injection of E2 cyclopentylpropionate and showed that post-coital treatment with estrogens caused either tube locking of embryos or accelerated transport to the uterus. Although other effects were also detected, the alteration in oviductal transport accounted for the contraceptive effect. Embryos that entered the uterus prematurely were expelled whereas whose sojourn through the oviduct was prolonged, degenerated.
Other investigators found that a single injection of E2 was given at different times after coitus revealed that a wide range of effectiveness can be achieved and suggested different mechanisms can account for the contraceptive effect when the same steroid is given at different times post-coitus [243,244]. Some believed the main target was the endometrium where they observed stromal edema, hemorrhage, and loss of decidua, all of which was considered unsuitable for implantation. Administering high dose of estrogen in the preovulatory phase was found to depress endometrial growth and angiogenesis through a negative influence on the vascular endothelial growth factor [245]. This suggested that estrogens might interfere with endometrial receptivity even if given before ovulation.
Other mechanisms for the contraceptive effect of post-coital estrogen were found to operate in monkeys. If given in the follicular phase, so as to advance the preovulatory increment in plasma estrogen, they evoke a premature LH surge that does not trigger ovulation and the formation of a functional corpus luteum, and the spontaneous LH surge is delayed or suppressed [246]. ER present in the granulosa cells of antral and preovulatory follicles and in luteal cells [247] allow for a diversity of effects in the ovary. In rhesus monkey, supraphysiological doses of estrogen given in the mid or late follicular phase induce atresia or luteinization without rupture of the dominant follicle, reduce the viability of granulosa cells, reduce the synthesis of E2 and progesterone and are detrimental to the oocyte [248].
Whatever the mechanism of action of high dose of estrogen for emergency contraception is, success in preventing pregnancy provides another evidence of the toxic effect of the supraphysiological estrogen levels fertilization, implantation and early development of the embryo which result in failure of achieving pregnancy.
[D] Effect on other factors involved in decidualization and early developing placenta
Paracrine factors
The importance of paracrine factors in mediating the cellular and biochemical changes involved in embryo implantation has been recognized. Many growth factors and cytokines, such as inhibins and activins, whose expression is generally limited to developmental and pathological states, are produced by actively remodeling endometrial cells, and play crucial roles in regulating endometrial cell function. Example of these factors includes the inhibin and activin family in the paracrine regulation of endometrial receptivity, decidualization and implantation. Estrogen is known to play an important role in regulation of these factors as discussed by Jones [249].
Role of Calcitonin
The peptide hormone calcitonin is currently being evaluated as a potential marker of the fertile human endometrium. In ovariectomized animals, it has been shown that administration of estrogen together with progesterone inhibits progesterone-mediated calcitonin gene induction.29 Such antagonistic interactions between estrogen and progesterone pathways have been documented previously in breast and uterine cells. It has been proposed that these phenomena reflect transcriptional cross talk occurring between estrogen and PR co-expressed in the same target tissue. A complex interplay of the two ovarian hormones, progesterone and estrogen, in the uterine milieu is believed to be critical for optimal calcitonin gene expression [250,251].
3.2 Effect of the medications used for ovarian stimulation
3.2.1 Clomiphene citrate
In spite of the high ovulation rate with the use of clomiphene citrate (around 50–90%), the pregnancy rate is much lower (around 20–40%) [252-254]. Moreover, there is a higher than expected incidence of miscarriage in conception cycles following clomiphene citrate treatment [255]. Such discrepancy is believed to be due to the peripheral antiestrogenic effect of clomiphene citrate, particularly at the level of the cervical mucus [256,257] and endometrium, [258,259]. The persistence of the zu-isomer of clomiphene citrate in the body due to its long half-life (several weeks) and slow clearance adds to the accumulation of the antiestrogenic effects over subsequent cycles of administration [260,261]. There is also evidence of a direct harmful effect of a high concentration of clomiphene citrate and its isomers on fertilization, and on early mouse [262] and rabbit [263] embryo development. Such effects, however, were not confirmed in other studies [264,265]. There is still some controversy concerning a direct effect on the quality of oocytes associated with clomiphene citrate treatment [266,267]. Decreased uterine blood flow during the early luteal phase and the peri-implantation stage is another explanation for the poor outcome of clomiphene citrate treatment [268]. Other investigators have suggested the presence of other unrecognized infertility factors [269,270].
3.2.2 Gonadotropins
The existence of nongonadal gonadotropins (FSH, LH/hCG) receptors was first suggested by Ziecik [270] who conducted binding studies in the porcine uterus. Subsequently, nongonadal receptors were found in human tissues, including endometrium, myometrium, fallopian tube, umbilical cord, and brain, by using a variety of techniques, including immunohistochemistry; Northern, Western, and ligand blotting; and in situ hybridization [271-274]. This would suggest a possible direct action of the gonadotropins on the uterus and involvement in the endometrial development, implantation and establishment of pregnancy [275].
High concentrations of exogenous gonadotropins used for hyperstimulation of folliculogenesis were shown to be detrimental to oocyte and embryo development in many animal species. This is believed to be due to the associated supraphysiological E2 levels and other possible undetermined factors associated with ovarian stimulation. However, a direct effect of gonadotropins cannot be ruled out. It has been reported that early embryo loss due to superovulation could be rescued by an injection of goat antiserum against PMSG [276].
Effect of gonadotropins on LH surge
It has been suggested that during ovarian stimulation, the supraphysiological FSH levels that persist into the late follicular phase, thereby overriding selection of the single dominant follicle of the natural cycle, secretion of an ovarian factor(s) blocks estrogen-induced LH surges [277]. Accumulated evidence has indicated that the ovaries produce another non-steroidal substance, named "gonadotropins surge-attenuating factor" (GnSAF), which may play a role in the control of the midcycle luteinizing hormone surge in women [278-281]. Although GnSAF activity is present during superovulation induction, it is still unclear whether this factor plays a physiological role during the normal menstrual cycle. Treatment with FSH initially attenuated the response of LH to GnRH via the production of GnSAF from the ovaries, while around the midfollicular phase, the rising concentrations of E2 were able to overcome the attenuating effect of GnSAF and increase pituitary sensitivity to GnRH. The increased pituitary sensitivity in the midfollicular phase of the FSH-treated cycles, however, was not further enhanced in the late follicular phase despite the continuous rise in E2 values [282]. It is suggested that eventually GnSAF was able to overcome the sensitizing effect of E2
Effect of gonadotropins on chromosomal development
A dose-response relationship between the PMSG dose and the incidence of polyploidy in the CD-1 mouse has been reported with the level of polyploidy rising from 2.9% with 10 IU PMSG to 10.5% with 15 IU PMSG, in the zygot stage. Both a disturbance at maturation division and an error at fertilization were the cause of polyploidy [22]. Whether this is the direct effect of PMSG or the resultant ovarian stimulation is not known.
3.2.3 GnRH analogues
GnRH plays a pivotal role in the control of female reproduction and is secreted by hypothalamic neurons in a pulsatile way. It binds to specific receptors on pituitary gonadotrophs, which is followed by the secretion of the gonadotropins, LH and FSH, which regulate steroidogenesis and gametogenesis in the ovary [283].
GnRH analogues are able to suppress gonadotropins release and, subsequently, gonadal function. This is the basis for their clinical application in ovarian stimulation. Several agonistic or antagonistic GnRH analogues have been developed for this purpose [284].
Effect of GnRH analogues on ovarian steroidogenesis and corpus luteum function
GnRH receptors and GnRH receptor mRNA were found to be expressed in human granulosa-luteal cells GnRH receptors have been found in the luteinized human granulosa cells, and a possible direct effect of GnRH on ovarian steroidogenesis has been suggested [485,486].
Although there are many studies that investigated the possible direct effect of various GnRH agonists on ovarian steroidogenesis and hence the corpus luteum, there is no consensus of the effects in the human ovary. Also, there is little information concerning the direct effect of GnRH antagonists on ovarian steroidogenesis [487]. A few studies that looked at the effect of GnRH agonist treatments in vivo on in vitro steroidogenesis by human luteinized granulosa cells, found an impairment of progesterone production [287-290]. Minaretzis [291] have reported on the effect of the GnRH antagonist Nal-Glu compared with GnRH agonist leuprolide acetate treatment of patients undergoing controlled ovarian hyperstimulation. The progesterone production in human luteinized granulosa cells cultures was not different between the two groups. Both GnRH antagonists had no effect on basal or hCG-induced E2 or progesterone production by granulose lutein cells, independent of whether the cells were exposed to the compounds in vitro or in vivo. However, an in-vitro study demonstrated an inhibitory effect of GnRH antagonist on gonadal steroid secretion [292]. Pellicer and Miro who found that GnRH agonist exposure in vivo may affect human luteinized granulosa cells function in vitro. They compared the progesterone accumulation in human luteinized granulosa cells cultures between patients treated with clomiphene citrate/gonadotropins and GnRH agonist/gonadotropins. Granulosa cells obtained from patients treated with the GnRH agonist had lower progesterone production than cells isolated from women treated with clomiphene citrate/gonadotropins [293]. However, Minaretzis compared the human luteinized granulosa cells steroidogenesis in vitro from Nal-Glu and leuprolide acetate-treated women respectively. They reported that basal and gonadotropins-stimulated progesterone secretion was similar in the two treatment groups [291].
Weiss et al., found that in vivo treatment with triptorelin or the two GnRH antagonists, cetrorelix and ganirelix; did not have an effect on spontaneous or hCG-stimulated steroidogenesis. The authors also performed in vitro treatments with triptorelin, cetrorelix and ganirelix for up to 96 hours and did not find any effect of these treatments on basal or hCG-stimulated steroid production [287].
In all prior reports, which studied the effect of GnRH agonist treatment on ovarian steroidogenesis, only the effect of hCG on progesterone secretion by human luteinized granulose cells was investigated. Recently, we examined the effect of two known physiologic stimuli of ovarian steroidogenesis, hCG and insulin. We found that GnRH agonist treatment affected both the basal and hCG-stimulated progesterone production by human luteinized granulosa cells but the pattern of insulin-stimulated progesterone secretion was not affected. Because insulin and hCG may share common pathways beyond but not at the level of receptor activation, we hypothesize that in-vivo GnRH agonist might affect the expression and/or activation of LH receptors. In our study, we found that human luteinized granulosa cells from patients treated with GnRH antagonist responded to hCG in a fashion generally consistent with previously reported hCG-stimulated human luteinized granulosa cells progesterone production [294]. In addition, the basal progesterone production and absolute levels of insulin-stimulated progesterone production were both significantly higher than observed following GnRH agonist treatment. In contrast, human luteinized granulosa cells previously exposed in vivo to GnRH agonist had a blunted P response to hCG. Our results, therefore, support the hypothesis that GnRH agonists may have a direct negative effect on human ovarian steroidogenesis by the corpus luteum and suggest that luteal function may be less affected during IVF-ET cycles when GnRH antagonist is used. However, in another study, GnRH antagonist therapy in women undergoing ovarian stimulation was found to be associated with a significant effect on ovarian follicular steroidogenesis. The authors found the mean follicular fluid E2 concentration significantly lower in patients treated with GnRH antagonist than in those treated with GnRH agonist. However, no significant differences were found between groups in follicular fluid progesterone concentrations [295]. However, further studies are required to investigate the effect of GnRH agonist and antagonist on gonadotropins receptors and different enzymes involved in ovarian steroidogenesis by the corpus luteum.
Effect on LH secretion
Because pituitary LH secretion is dependent on GnRH stimulation and feedback mechanisms from ovarian steroids, any alteration may be deleterious. In cycles using GnRH agonists for ovarian stimulation, a significant drop in mid-luteal progesterone concentrations was observed, consistent with corpus luteum insufficiency. Long-term GnRH-agonist administration has been associated with a profound desensitization of the pituitary cells. In fact, studies on pituitary gonadotropins secretory capacity after GnRH-agonist treatment have indicated that this remains impaired for at least 14 days after the discontinuation of the GnRH-agonist and for the whole length of the luteal phase [296]. In a randomized trial, it was also demonstrated that, despite the early cessation of the GnRH agonist in the follicular phase, luteal-phase characteristics were abnormal [297].
GnRH-antagonist treatment has been shown to be effective in blocking the LH surge. GnRH antagonists bind competitively to pituitary GnRH receptors and cause an immediate inhibition of gonadotropins release. In contrast to GnRH agonists, it was suggested that treatment with GnRH antagonists might not adversely affect luteal LH secretion, since the pituitary maintains its responsiveness to the endogenous GnRH stimulus [298]. A normal luteal phase, in terms of duration and serum progesterone concentrations, was observed in natural cycles in which an antagonist was administered to prevent the LH surge [299]. However, data on the luteal phase from unsupplemented cycles after antagonist administration are limited as a result of the small number of patients involved and are rather controversial. Four out of six patients had either a shortened luteal phase or low progesterone concentrations in cycles stimulated with HMG and the GnRH antagonist Cetrorelix and receiving no luteal-phase supplementation. However, with or without luteal-phase supplementation, luteal LH levels were low, indicating that another mechanism might be involved [299,300].
3.2.4 Human chorionic gonadotropins
Supraphysiological steroid serum concentrations may interfere with LH secretion via long-loop feedback, but, additionally, the exogenously administered hCG might amplify LH secretion arrest via a second short-loop negative feedback. Although in the monkeys such a negative feedback exists [301] there is a debate in the literature about its existence in humans, with some of the studies supporting this hypothesis [302] and others not [303,304]. Nevertheless, findings from in-vitro studies further support this idea. GT1-7 neurons, which are morphologically and functionally similar to GnRH neurons, were found to contain LH/hCG receptors. In addition, exogenously administered hCG was found to decrease the expression of GnRH receptor gene in GT1-7 cells or GnRH secretion in immortalized GnRH neurons [305].
4 Measure to improve treatment outcome after ovarian stimulation and assisted reproduction
Even if the results of reproductive medicine have improved in terms of numbers of pregnancies, it is still striking that it is necessary to use stimulation which sometimes leads to hyperstimulation and multiple pregnancies, that embryo development in vitro is still limited, that implantation only occurs for 15–20% of transferred embryos and this ratio has not changed significantly along the last 25 years. We still need to improve techniques to gain pregnancy rates approaching 50% per embryo. During assisted reproduction technology interventions, controlled ovarian stimulation is used to obtain several oocytes in attempts to increase the likelihood of having at least one developmentally competent embryo available for transfer. However, current techniques for identifying the competent embryo[s] are by no means perfect. These limitations, coupled with pressures to maximize the chance of pregnancy, typically result in the transfer of multiple embryos. Not surprisingly, this practice has resulted in an unacceptably high rate of multiple pregnancies arising from IVF-ET. During the last few years, concerted efforts have focused on reducing these rates by restricting the number of embryos to transfer [306].
In order to fulfill this task, several approaches have been suggested to improve the outcome of treatment after ovarian stimulation and assisted reproduction. Table 3 summarizes the different approaches including the two main approaches tried so far i.e. optimizing ovarian stimulation protocols and improving embryology techniques. A third approach we suggest i.e. the use of aromatase inhibitors constitutes the third and most recent approach.
Table 3 Various approaches to improve treatment outcome after ovarian stimulation:
(1) Approaches involving ovarian stimulation protocols:
A: Reduce intensity of ovarian stimulation
- No stimulation: natural cycle
- Minimal stimulation
- Step-down protocol
B: The application of new medications
- Recombinant FSH
- Recombinant LH
- GnRH antagonists
C: The use of adjuvant medication
- Insulin sensitizers
- Corticosteroids
- Other medications
(2) Improving embryology techniques
- Improving embryo culture conditions, selection and extending embryo culture (blastocyst transfer)
- Preimplantation genetic diagnosis
- In vitro maturation
- Micromanipulation including: ICSI, assisted hatching, mitochondrial injection and cytoplasmic and nuclear transfer.
(3) The use of aromatase inhibitors for
- Improving implantation rates
- Reducing FSH dose required for ovarian stimulation:
- Other possible benefits:
* Improving response to FSH stimulation in poor responders
* Preventing premature endogenous gonadotropin surge
* Reducing the risk of severe ovarian hyperstimulation
We suggest a THIRD novel approach that we believe it may carry a hope for improving the outcome of treatment after ovarian stimulation and assisted reproduction. This approach involves the use of aromatase inhibitors.
4.1 Modified ovarian stimulation protocols
4.1.1 Reducing the intensity of ovarian stimulation protocols
Different approaches have been suggested to improve the treatment outcome during assisted reproduction by reducing the intensity of ovarian stimulation that would reduce the high estrogen levels [307]. These approaches include no-stimulation (natural cycle IVF-ET), minimal stimulation IVF-ET cycles, step-down protocols, FSH coasting and in vitro maturation.
However, all these measures are associated with the major drawback of affecting the main goal of ovarian stimulation, which is the achievement of a suitable number of embryos when a good number of oocytes retrieved.
Natural cycle IVF-ET
The first IVF-ET treatment in the literature was a natural cycle IVF-ET. Since then natural cycle IVF-ET has been largely replaced by IVF-ET with ovarian stimulation. Natural cycle IVF-ET has several advantages including a close to zero multiple pregnancy rate, and a zero risk of ovarian hyperstimulation syndrome as well as being less time consuming, physically and emotionally less demanding for patients, and cheaper than stimulated IVF-ET. However, it seems to be less effective. Unfortunately, good quality randomized controlled trials and formal cost-effectiveness analyses are lacking. Pelinck reviewed 20 selected studies that comprised a total of 1800 cycles of natural cycle IVF-ET, resulting in 819 embryo transfers (45.5% per cycle) and 129 ongoing pregnancies (7.2% per cycle and 15.8% per embryo transfer). The authors concluded that: in spite of being a low-risk, low-cost and patient-friendly procedure, high cancellation rates because of premature LH rise and premature ovulations hamper efficacy of natural cycle IVF-ET and that a randomized controlled trial comparing natural cycle IVF-ET with current standard treatment strategies is warranted. [27].
The cumulative live birth rates after four cycles of treatment was reported to be 32% which is comparable with the value of 34% for women having conventional IVF-ET treatment [308]. However, there is enough data on cumulative live birth rates following conventional IVF-ET, which is likely that current cumulative rates for IVF-ET will be higher than previously reported.
In the implantation rate per embryo after natural cycle IVF-ET was found quite acceptable up to 50%. Also, the fertilization rate was found to be 100% for the oocytes retrieved from the mature follicles (as reviewed by [27] Pelinck). We believe that the significantly better oocyte quality (100% fertilization rate) as well as better implantation rate (about 50%), reflect clearly the value of avoiding the deleterious effects of ovarian stimulation on the treatment outcome. Moreover, there is an important point to mention regarding the success rates of natural IVF-ET cycles, which is the bias in selecting patients who underwent natural IVF-ET cycles. Many studies included patients with unfavorable outcome such as poor responders, old age and repeated IVF-ET failures.
Minimal stimulation IVF-ET
Minimal stimulation for IVF-ET that is less expensive than full stimulation and minimizes monitoring and patient discomfort has been described for almost 10 years and found to be associated with acceptable pregnancy rates and was suggested as an attractive alternative to select patients undergoing IVF-ET [309]. This protocol involves the use of CC plus FSH alone or in conjunction with GnRH antagonist to prevent premature LH surge [310]. It is expected that the minimal-stimulation regimens produce fewer oocytes, fewer embryos available after fertilization, and fewer excess embryos available for cryopreservation. This leads to the main drawback of the minimal-stimulation regimen, which is the lower total reproductive potential (pregnancies resulting from fresh and frozen-thawed embryos resulting from a single stimulation cycle) [311].
FSH step-down and coasting protocols
Other measures have been tried by decreasing the FSH dose (step down protocol) to improve endometrial receptivity. With the use of a step-down regimen with FSH in high responders, uterine receptivity was believed to improve secondary to lowering E2 levels during the preimplantation period [312].
Coasting or withholding FSH injection for a period of time has been suggested in patients at substantial risk for the development of severe hyperstimulation syndrome. Coasting in a studied subset of IVF-ET patients did not adversely affect cycle outcome parameters when it was not prolonged [313].
4.1.2 Application of new medications for ovarian stimulation
The ongoing development of more effective and safer pharmacological compounds is driving fundamental changes in medicine [314]. Along the last decade several new medications have been developed for application in ovarian stimulation and assisted reproduction including medications prepared by the new recombinant technology [recombinant FSH and LH] and GnRH antagonists as well as other medications.
Recombinant FSH
The introduction of recombinant FSH into the clinical management of patients suffering from infertility appears to be associated with several treatment benefits when compared with urinary human menopausal gonadotropins
The fact that rFSH preparations have batch-to-batch consistency, are free from urinary protein contaminants and have the potential to be produced in limitless quantities is advantageous. The question whether newer, more pure FSH products are beneficial from the clinical perspective, has not been settled beyond reasonable doubt. The price of rFSH has been reported to be three times as high as the price of the former FSH preparations [315].
Although both FSH and LH are required for normal follicular growth and maturation, the precise role of LH is at present still uncertain. Since it was shown in the late 1980s that too high a concentration of LH might have a negative effect on fertilization and embryo quality, the idea arose that pure FSH preparations might be superior to hMG preparations for patients with endogenous LH production [316]. This hypothesis was tested in several clinical trials comparing uFSH with hMG with respect to pregnancy rates per IVF-ET treatment cycle. Statistical significance was not reached in any of these individual studies [316,317]. However, in 1995 a meta-analysis by Daya was published, which included eight studies and demonstrated a significant difference in favor of uFSH [316]. A few years later this finding was contradicted by a meta-analysis by Agrawal who argued that meta-analyses should take into account the different pituitary desensitization protocols used [318]. When pooling together 11 trials with the most commonly used GnRH agonist protocol [the long protocol], the overall odds ratio for comparing FSH and HMG was not significant. Although this meta-analysis was criticized on the issue of study selection bias, re-analysis following the inclusion and exclusion of selected studies did not change the overall results of the study [316,318,319].
The question whether rFSH also leads to more clinical pregnancies per IVF-ET cycle has been addressed by several clinical trials, none of which reached statistical significance. However, two meta-analyses pooling together the results of several trials did show significant treatment effects in favor of rFSH [320,321]. The interpretation of these meta-analyses is still debated, since they compare two types of rFSH (the alpha and beta variant), as well as different types of urinary FSH (uFSH and uFSH-HP, and in Out's meta-analysis one study with HMG was also included). In Daya's analysis, which received Cochrane status, an absolute increase in pregnancy rates of 3.7% (95% confidence interval [CI] 0.5–6.9%) was demonstrated comparing rFSH with uFSH/uFSH-HP.
Data on the third possible comparison, between rFSH and hMG, are scarce. The studies that have been conducted so far found no statistically significant differences with respect to ongoing pregnancy rates [322-324]. The evidence available at this time, comparing the different FSH containing gonadotropins preparations, concerns their usage in IVF-ET. However, almost half of all gonadotropins are used in other settings, such as IUI. Very little data are available comparing the preparations for this indication. With respect to adverse effects of exogenous FSH administration, no significant differences have been found between products. The main risk associated with the use of FSH containing gonadotropins products is the development of the ovarian hyperstimulation syndrome (OHSS). The incidence of OHSS does not differ between products [321-324].
Recombinant LH
hCG has been used as a surrogate LH surge because of the degree of homology between the two hormones. The major differences between the two hormones include the sequence of the β-subunit, the regulation of the secretion of the two hormones, and the pharmacokinetics of clearance of hCG as opposed to LH [325,326].
hCG has a slower plasma metabolic clearance, which consists of a rapid phase in the first 5–9 h following IM administration and a slower phase in the 1–1.3 days after administration. After 36 h, the calculated half-life of hCG was 2.32 days, as compared with LH, for which estimates have ranged from 1 h [326] to 3–5 h [327,328]. By day 10 after administration, less than 10% of the originally administered hCG was measurable [329]. However, the long serum half-life of hCG is likely to be an undesirable characteristic in clinical practice.
Until recently, there was no other biologic preparation that was as effective as hCG in triggering the final stage of ovulation. Recent developments using genetic engineering technologies and posttranscriptional biosynthesis have led to the production of rhLH, which has been available for use in clinical trials for several years [330]. The rhLH produced in vitro is purified and further formulated to yield a pharmaceutical preparation with very high specific immunoactivity and bioactivity. A crossover pharmacokinetic study has been performed in nonhuman primates to assess and compare pituitary, urinary, and rhLH [331]. After intravenous administration of pituitary and urinary-derived LH, and rhLH, mean concentration-time curves were parallel. The mean areas under the curve (AUC) for concentration by time after dose curves were similar, after correction for immunologic differences in dose. The mean clearance estimates and volume of distribution at steady state, distribution, and terminal half-lives were similar for all three types of LH. Furthermore, the results of studies in human volunteers show that rhLH and urinary-derived hLH have similar pharmacokinetic characteristics; both preparations presented an iv distribution half-life of 1.2 h and a terminal half-life of 10 h [332,333]. rhLH total body clearance was 2 L/h, with less than 5% of the dose being excreted by the kidneys. The steady state volume of distribution was approximately 8 L [332]. Furthermore, it has been demonstrated that co-administration of rhLH and rhFSH (follitropin α) does not modify their respective pharmacokinetic characteristics [332]. Finally, low doses of rhLH have been shown to promote ovarian steroidogenesis when follicular growth is induced with rhFSH in hypogonadotropic hypogonadal women [334].
The results show that a single dose of rhLH is effective in inducing final follicular maturation and early luteinization in IVF-ET and embryo transfer patients and is comparable with 5,000 IU u-hCG. A single dose of rhLH results in a highly significant reduction in OHSS compared with hCG. The dose of rhLH giving the highest efficacy to safety ratio was between 15,000 and 30,000 IU. This study has shown that the incidence of OHSS is significantly smaller when using a single dose of rhLH for induction of final maturation of follicles and oocytes in women treated for IVF-ET, compared with the use of 5,000 IU u-hCG or two doses of rhLH [335].
Chandrasekher [336] compared rhLH with u-hCG and pituitary hLH as an ovulatory stimulus in rhesus monkeys before IVF-ET. These investigators found that a single injection of 2,500 IU rhLH was as effective as 1,000 IU u-hCG in inducing oocyte maturation, oocyte fertilizability, and luteinization of granulosa cells. The data suggested that using a conversion factor of 2.5, a dose of 12,500 IU rhLH would be as effective as 5,000 IU u-hCG in humans. Another study (unpublished data) looked at the spontaneous LH surges in seven healthy female volunteers with normal menstrual cycles after a single injection of either 250 μg GnRH-agonist (buserelin) or 5,000 IU u-hCG administered when the dominant follicle reached a diameter of 17 mm. It was found that the mean Cmax of the spontaneous LH surge was 47 IU/L (95% CI, 28–65 IU/L) and the mean AUC was 1,019 IU h/L (95% CI, 718–1,320 IU h/L). Using rhLH pharmacokinetic characteristics defined in Phase I studies [332,333], it was calculated that, to obtain a Cmax and an AUC larger than the corresponding mean values observed in spontaneous surges in 95% of cases, single sc injections of between 14,000 IU and 25,000 IU rhLH should be used.
GnRH antagonists
GnRH antagonists do not induce an initial stimulation of gonadotropins release, but cause an immediate and rapid, reversible suppression of gonadotropins secretion. The principal mechanism of action of GnRH antagonists is competitive receptor occupancy of GnRH-r [337].
GnRH antagonists have been introduced clinically to prevent premature endogenous LH surge to replace the GnRH agonists that require time for a state of desensitization to be reached and, to start with, LH secretion actually increases (flare-up). With GnRH antagonists, immediate blockade of pituitary gonadotropins secretion when premature luteinization during IVF-ET stimulation is imminent seemed an obvious approach. Several studies of dose and treatment schedules have been done, [338,339] and two general approaches have emerged. The first is a single subcutaneous injection of a large dose on about the eighth day of stimulation with gonadotropins. The alternative is multiple (five or six daily) injections of a small dose from about day 6 of stimulation until the day that hCG for final oocyte maturation is given.
The next step in the development of GnRH antagonists use was to establish whether a GnRH antagonist is at least as effective as a GnRH agonist as the established reference medication testing the regimen of repeated antagonist injections [340,341] or the regimen of a single-dose regimen [342].
With repeated injections, treatment with gonadotropins was found to be shortened by 1–2 days with slightly fewer follicles at the time of hCG injection so the number of recovered oocytes tends to be lower. No significant difference was found with respect to percentages of metaphase II oocytes, fertilization rates, and number of good quality embryos [340,341].
Pregnancy rates were high in both groups in all four studies but in every one the rate was lower in the antagonist group. A meta-analysis of the five randomized trials, showed an overall significantly lower rate of pregnancy of 5%. This meta-analysis unfortunately included the study that compared a single-dose analogue regimen with different gonadotropins starting dose as an additional variable [342]. It has been suggested that the larger numbers of oocytes and embryos with agonists allow better selection [343], although the numbers of good quality embryos do not seem to be different. A direct adverse effect on the embryo cannot yet be ruled out but is not likely.
Antagonist blockade allows immediate reversal of pituitary gonadotropins secretion. This means that in IVF-ET ovulatory ripening can be triggered via endogenous gonadotropins by using a GnRH agonist [344] with the advantage of prevention of ovarian hyperstimulation syndrome, which is thought to result at least in part from the prolonged LH effect of hCG [345]. Finally, the use of GnRH antagonist blockade of premature luteinization can be used in IVF-ET with very low or without any hormonal stimulation, lower risk of overstimulation, and simplification of the procedure [346].
A possible disadvantage of an antagonist in IVF-ET is its narrow therapeutic range with the currently advised doses for repeated injections. Patient compliance needs to be high because there is a risk of premature LH secretion if an injection is missed [337].
Another disadvantage is the unpredictable timing of the start of the IVF-ET procedure, which begins with the administration of FSH on day 3 of the woman's cycle and so depends on how regular her menstruation is. This problem may be solved by pretreatment with an oral contraceptive [335].
4.1.3 Use of adjuvant medication during ovarian stimulation
Insulin sensitizers
The independent effect of insulin resistance on infertility treatment in PCOS is not well defined. Regardless of body weight, insulin-resistant PCOS women need higher gonadotropins doses during ovarian stimulation, and insulin resistance is also associated with a risk of multifollicular development and high cancellation rate [347]. Hyperinsulinemic PCOS women are more likely to produce lower quality oocytes that exhibit low fertilization rates after IVF-ET in association with lower implantation rates [348].
Improving insulin resistance with exercise, low-calorie diet and insulin-lowering drugs such as metformin, troglitazone and acarbose decrease insulin levels, correct the endocrine abnormalities induced by obesity and insulin resistance which may improve the outcome of infertility treatment [349-351]. This leads to the assumption that co-treatment with insulin-lowering drugs or weight reduction before and during down-regulation and ovarian stimulation.
Some studies did not find deleterious of insulin resistance on ovarian stimulation [352]. Metformin treatment has been reported to increase the number of mature oocytes retrieved from women with PCOS undergoing gonadotropins-stimulated IVF-ET and ICSI. A low dose of metformin of 500 mg twice daily was started on day 1 of the cycle prior to leuprolide acetate suppression, and continued to the day of the pregnancy test. Metformin treatment significantly increased the number of mature oocytes, fertilization rates, and number of embryos produced, but did not alter the total number of oocytes or peak E2 levels [353].
Later, the same authors reported that metformin therapy improves ovarian stimulation and IVF-ET outcomes in coasted patients with clomiphene-resistant PCOS [354]. No further published reports to date have examined this issue.
Corticosteroids
Successful use of corticosteroids in treatment of anovulatory infertility has been reported [355,356]. Later corticosteroids were used as adjuvant therapy together with clomiphene citrate or gonadotropins in ovulation induction. The theoretical basis for this application has not been fully elucidated, but it has been postulated that corticosteroid treatment could improve ovulation through reduced influence of the adrenal androgens on follicular development [357]. There have been reports on improved ovulation and pregnancy rates with adjuvant corticosteroid treatment in anovulatory women with elevated androgens or androgens within the higher range [358,359], as well as in normoandrogenic women [360]. Adding dexamethasone to gonadotropins in ovulation induction in women with normal serum concentrations of gonadotropins, androgens, and prolactin did not give an improved outcome in other studies [361].
Corticosteroids have also been used as adjuvant therapy in IVF-ET treatment. In 1986, Kemeter and Feichtinger [362] reported a significantly better pregnancy rate in a group of women with various infertility causes, except cycle abnormalities, undergoing IVF-ET with adjuvant prednisolone treatment, as compared to a group of women without adjuvant prednisolone. They stated that prednisolone would improve follicle maturation and thereby improve the pregnancy rate. In contrast, others [363] did not see any beneficial effects of adjuvant dexamethasone in a group of women with serum DHEA-S >2.5 μg/ml [6510 nmol/l] undergoing IVF-ET treatment, neither did Bider [361], in a group of women with tubal factor infertility after addition of dexamethasone.
In the above-mentioned study by Lobo [357], decreased serum concentrations of testosterone, unbound testosterone, and DHEA-S were noted after dexamethasone and clomiphene administration. In women who ovulated, testosterone and unbound testosterone increased again when clomiphene was added despite the continuation of dexamethasone. Decreased serum concentrations of DHEA-S and testosterone after ovarian stimulation with clomiphene citrate and human menopausal gonadotropins (HMG) and adjuvant prednisolone have been reported [362]. The other studies did not report data on androgen concentrations after glucocorticoid treatment. The present prospective, randomized, placebo-controlled study was performed to find out if adrenal suppression with prednisolone during ovarian stimulation before IVF-ET in a group of women with PCOS resulted in any changes in serum and follicular-fluid androgens. Clinical outcome variables, such as embryo quality, implantation rate, and clinical pregnancy rate were also noted.
Fridstrom [364] have reported that adjuvant glucocorticoids in ovarian stimulation before IVF-ET did not decrease the concentrations of adrenal androgens in serum and follicular fluid in PCOS. However, whether there were beneficial effects on ovum quality or implantation rate could not conclusively be determined in their study due to the small number of patients.
The addition of corticosteroids to ovarian stimulation protocols for assisted reproduction has been suggested in cases of recurrent pregnancy loss [365].
Progesterone antagonists: Receptor antagonist onapristone and mifeprisotone
Krusche found that after ovarian stimulation, the PR antagonist onapristone retarded endometrial transformation in the rabbit model. The authors concluded that since ovarian stimulation, used in human IVF-ET therapy, is frequently reported to cause an advancement of post-ovulatory endometrial development, a therapeutic application of PR antagonists to slow down such advanced endometrial transformation was suggested. Eventually, this modulation of advanced endometrial development may improve implantation rates [366].
4.2 Improving embryology techniques
Improving embryo techniques is a very important approach to improve the outcome of assisted reproduction treatment. This includes, improving culture conditions and extended culture till the blastocyst stage as well as improving the accuracy for selecting viable embryos for transfer. Another strategy employs pre-implantation genetic diagnosis (PGD). In addition there are other strategies to improve the embryo quality including oocyte donation and micromanipulation techniques (assisted hatching, and cytoplasmic and mitochondrial transfer) [367].
4.2.1 Improving embryo culture condition, selection and extended embryo culture (blastocyst transfer)
Over the past decade there has been considerable interest in optimizing culture media for supporting human embryos including reducing glucose concentrations [368], adding amino acids [369] and supplementing media with growth factors [370]. However, there have been no large prospective randomized studies on the effects of culture media on outcome after IVF-ET, apart from one, which reported that although glucose-free medium improved embryo quality, pregnancy rates were not increased [371]. All these improvements as well as the growing data on the composition of human female reproductive tract fluids and from embryo physiology studies has led to the proposition that extended embryo culture should take place till the blastocyst stage in more than a single medium formulation [372]. To this end, sequential media have been developed, tested extensively on animal models and subsequently used clinically [373].
One approach to increasing pregnancy rates is to improve the selection of viable embryos. Embryos are selected on the basis of morphology and rate of development, and the fastest developing embryos of the best morphology are selected for transfer. Although there is some correlation between morphology and blastocyst formation [374] and implantation [375], selection on the basis of morphology and rate of development remains an unsatisfactory and imprecise method of selecting viable embryos. It is impossible to identify visually with certainty, which embryos at early cleavage stages will subsequently arrest. One approach to improve the selection is the extended culture of the embryos to the blastocyst stage and transfer them on day 5 or 6, allowing the identification of developing embryos and the transfer of blastocysts, which are synchronous with the endometrium development, rather than cleavage stage embryos. It had been hoped also that, during extended culture, the chromosomally abnormal embryos so common at early cleavage stages [376] would arrest and fail to complete preimplantation development. However, although few human blastocysts have total chromosomal abnormality, a large number of blastocysts have various proportions of abnormal cells and are mosaic [377].
However, although many clinics have experienced increased success with extended culture, others have reported no benefit. So, it would be unwise to suggest that blastocyst culture and transfer represents a panacea for all clinics and all patients. On the contrary, transferring early stage embryos (zygot intrafallopian transfer) may help in repeated implantation failure rather than blastocyst transfer. [378].
4.2.2 Preimplantation genetic diagnosis
Preimplantation genetic diagnosis (PGD) allows the diagnosis of a genetic disorder in an embryo before its implantation in the uterus. PGD involves the removal of one or two blastomeres from an eight-cell stage embryo after IVF-ET, analysis of the blastomeres using fluorescent in situ-hybridization (FISH) or PCR, and identifying affected embryos. As only unaffected embryos are transferred back to the uterus after PGD, termination can be avoided. PGD is of benefit to couples at risk of passing on a genetic disease to their offspring as well as for couples with repeated unexplained IVF-ET failures. The transfer of unaffected embryos is believed to enhance the success rate of IVF-ET. Unfortunately, a large number of oocytes need to be retrieved for a successful PGD cycle, as not all will fertilize, cleave, undergo successful biopsy and be identified as suitable for transfer. Although most embryos (96%) survive biopsy, the pregnancy rates after transfer of biopsied embryos (16% per treatment cycle) [377] are less than those after conventional IVF-ET (23% per cycle) [379]. However, PGD is still in its early developmental stages [14].
4.2.3 In-Vitro Maturation of Oocytes
Immature oocytes can be aspirated from small follicles of > 2 mm in diameter. Resumption of meiosis in fully-grown oocytes, germinal vesicle breakdown, extrusion of the first polar body and acquisition of the ability to be fertilized may all occur during in vitro maturation (IVM). Although the first human birth after IVM was achieved 20 years ago [380], a few cases have been reported subsequently, including births after the aspiration and IVM of oocytes retrieved from women who have natural or partially stimulated cycles [381] and women with PCOS [382]. When oocytes are removed from small antral follicles and placed in culture, approximately 60% will have undergone nuclear maturation within 48 h. After exposure to spermatozoa, about 40% of oocytes will undergo normal fertilization, exhibiting two pronuclei and extruding the second polar body. Between 20 and 25% of fertilized oocytes will undergo cleavage [383]. However, pregnancy rates after the transfer of such embryos have been extremely low (1–2%) and most embryos arrest between the four- and eight-cell stages with only few encouraging results [384]. Several factors may influence the fertilization, cleavage, blastocyst and pregnancy rates achieved after IVM, including the hormonal environment in vivo or in vitro and the composition of the culture medium [385]. Further research in these areas will lead to greater understanding of oocyte maturation and should result in improved implantation and pregnancy rates [14].
4.2.4 Micromanipulation techniques
Intracytoplasmic sperm injection (ICSI)
The use of small numbers of spermatozoa during IVF-ET results in fertilization failure. In these cases, it is possible to inject a single spermatozoon into the ooplasm of a mature metaphase II oocyte, a procedure known as ICSI. The first successful pregnancies were in the early 1990s [10]. Since then, the technique has become a widely accepted treatment for couples with severe male factor infertility. Extending the indication of ICSI to include non-male factor infertility has been suggested to improve the fertilization and pregnancy rates. However, several studies have not supported the benefit of ICSI over conventional IVF-ET in improving the treatment outcome for non-male factor indications with growing consensus against applying ICSI for all cases undergoing IVF-ET [386-389].
Assisted hatching
To help embryos hatching from their zona pellucida during blastocyst expansion, different types of assisted hatching have been developed, including mechanical partial zona dissection or zona drilling, chemical zona drilling with acidic Tyrode's solution, and the laser technique [176]. However, there is controversy about the benefit of assisted hatching on the improvement of the implantation rate and pregnancy rate. Some investigators agreed as to the benefit of assisted hatching in women with advanced age, in women with repeated IVF-ET failure, and in the general population [177,178].
Mitochondrial injection and cytoplasm, and nuclear transfer
Van Blerkom suggested that the developmental competence of mouse and human early embryos is related to the metabolic capacity of the mitochondria [144]. Deletions and mutations in oocyte mtDNA may lead to mitochondrial dysfunction, influencing energy production and apoptosis in oocytes and early embryos, resulting in aberrant chromosomal segregation or developmental arrest [144]. In mice, cytoplasmic transfer from young to old oocytes improved older oocytes quality [390].
Cytoplasmic transfer from metaphase II donor oocytes to mature recipient oocytes from women with recurrent IVF-ET failure has been carried out in an attempt to restore normal growth in developmentally compromised oocytes and embryos [391].
Nuclear transfer has also been proposed for the treatment of mitochondrial disease [392] whereby a karyoblast containing metaphase II chromosomes from women with repeated failures of embryo development due to defective oocyte cytoplasm is fused to enucleated donor oocytes. The increased cost and complexity as well as the invasiveness of the procedure with the decreased implantation rates all make these procedures far from being applied routinely to improve the treatment outcome after assisted reproduction. Only selected cases might be expected to benefit from these newly developing techniques. It should be noted that currently in the United States, performance of cytoplasmic or nuclear transfer in humans can only be conducted following approval of the protocol by the FDA.
PART TWO
1. Aromatase inhibition to improve outcome of treatment after ovarian stimulation
1.1 Introduction
We hypothesize that aromatase inhibitors can be used to improve the treatment outcome after ovarian stimulation either alone, or in combination with IUI and assisted reproductive technology. The use of aromatase inhibitors during ovarian stimulation may have several benefits including: (1) The enhancement of implantation by lowering the supraphysiological levels of estrogen attained during ovarian hyperstimulation that is believed to adversely affect the development of the endometrium, oocytes and embryo, as well as other possible targets. (2) Reducing gonadotropins dose required for achievement of optimum ovarian stimulation. This would reduce possible deleterious direct effects of exogenous gonadotropins injection in addition to reducing the cost of treatment. (3) Other possible benefits such as improvement of ovarian response to FSH stimulation in poor responders, and prevention of premature endogenous gonadotropin surge, as well as lower risk of severe ovarian hyperstimulation syndrome.
The improvement in implantation, as well as the reduced cost of treatment by decreasing the gonadotropins dose required for ovarian hyperstimulation would encourage the policy of transferring one embryo to minimize the risk of multiple gestation. This would have a tremendous economic impact on the practice of assisted reproduction, as well as cost of health care for multiple gestation worldwide.
1.2 Outline of the use of aromatase inhibitors for ovarian stimulation
In the following section, we present brief outline on the aromatase enzyme, estrogen biosynthesis and the development of aromatase inhibitors followed by a summary of the available data concerning the use of aromatase inhibitors for ovarian stimulation.
The aromatase enzyme
Aromatase is a microsomal member of the cytochrome P450 hemoprotein-containing enzyme complex superfamily (P450 arom, the product of the CYP19 gene) that catalyzes the rate-limiting step in the production of estrogens, that is, the conversion of androstenedione and testosterone via three hydroxylation steps to estrone and E2 respectively [393]. Aromatase activity is present in many tissues, such as ovaries, brain, adipose tissue, muscle, liver, breast tissue, and in malignant breast tumors. The main sources of circulating estrogens are the ovaries in premenopausal women and adipose tissue in postmenopausal women [394,395].
The development of aromatase inhibitors
Aromatase is a good target for selective inhibition because estrogen production is a terminal step in the biosynthetic sequence. Several aromatase inhibitors have been utilized in clinical studies over the last 20 years. The most successful, third generation aromatase inhibitors are licensed for breast cancer treatment [396]. Aromatase inhibitors have been classified in a number of different ways, including first-, second-, and third-generation; steroidal and non-steroidal; reversible (ionic binding), and irreversible (suicide inhibitor, covalent binding) [397]. Table 4 lists the different classes of aromatase inhibitors.
Table 4 Different classes of aromatase inhibitors:
Generation Non-Steroidal Steroidal
First Generation Aminogltethimide
Second Generation Rogletimide Formestane
Fadrozole
Third Generation Anastrozole Exemestane
Letrozole
Vorozole
Table 5 The different degrees of whole body aromatase inhibition by the various aromatase inhibitors
Aromatase inhibitor Dose Mean percentage of total body aromatase inhibition
Aminoglutethimide in conjunction with hydrocortisone 1000 mg plus 40 mg of hydrocortisone (daily) 90.6
Formestane 250 mg (every 2 weeks, intramuscularly) 84.8
Exemestane 25 mg/day 97.9
Fadrozole 2 mg/day 82.4
Anastrozole 1 mg/day 97.3
Letrozole 2.5 mg/day >99.1
Vorozole 1 mg/day 93
Steroidal aromatase inhibitors are androstenedione analogues that act as a false substrate and bind irreversibly to the androgen-binding site of the enzyme [398]. Non-steroidal aromatase inhibitors exert their function through binding to the heme moiety of the cytochrome P450 enzyme [399]. The first of these inhibitors to be used clinically was aminoglutethimide, which induces a medical adrenalectomy by inhibiting many other enzymes involved in steroid biosynthesis [400]. Although aminoglutethimide is an effective hormonal agent in postmenopausal breast cancer, its use is complicated by the need for concurrent corticosteroid replacement, in addition to side effects like lethargy, rashes, nausea and fever that result in 8–15% of patients stopping treatment [401,402]. The lack of specificity and unfavorable toxicity profile of aminoglutethimide led to the search for more specific aromatase inhibitors. In addition, the above mentioned aromatase inhibitors were not able to completely inhibit aromatase activity in premenopausal patients.
Third generation aromatase inhibitors
The third-generation anti-aromatase agents commercially available include two non-steroidal preparations, anastrozole and letrozole, and a steroidal agent, exemestane. Anastrozole and letrozole are often referred to as aromatase inhibitors, whereas exemestane is called an aromatase inactivator [403,404]. Anastrozole, ZN 1033, (Arimidex®) and letrozole, CGS 20267, (Femara®) are selective aromatase inhibitors, available for clinical use in North America, Europe and other parts of the world for treatment of postmenopausal breast cancer. These triazole (antifungal) derivatives are reversible, competitive aromatase inhibitors, which are highly potent and selective. At doses of 1–5 mg/day, they inhibit estrogen levels by 97% to >99% resulting in estrogen concentrations below detection by most sensitive immunoassays. Table 5 shows the relative potencies of different aromatase inhibitors. Aromatase inhibitors are completely absorbed after oral administration, with mean terminal half-life (t1/2) of approximately 45 hours (range, 30–60 hours). They are cleared from the systemic circulation mainly by the liver. Gastrointestinal disturbances account for most of the adverse events, although these have seldom limited therapy. Other adverse effects are asthenia, hot flashes, headache, and back pain [406]. The average wholesale cost for a month's supply (thirty tablets) of aromatase inhibitors is about $150 to $250 [407]. Table 6 lists the clinical advantages of the third generation aromatase inhibitors as regards potential use for ovarian stimulation.
Table 6 Advantages of the third generation aromatase inhibitors:
• Extremely potent in inhibiting the aromatase enzyme
• Very specific in inhibiting the aromatase enzyme without significant inhibition of the other steroidogenesis enzymes
• Orally administered
• 100% bioavailability after oral administration
• Rapid clearance from the body (Short half-life, ~45 hours)
• No accumulation of the medications or their metabolites
• No significant active metabolites
• Well tolerated on daily administration for years
• Few side effects
• Very safe without significant contraindications
• Relatively inexpensive
Hypotheses behind the use of aromatase inhibitors for ovarian stimulation
In the late 1990s, we explored the hypothesis that it might be possible to mimic the action of clomiphene citrate, without depletion of ER, by administration of an aromatase inhibitor in the early part of the menstrual cycle. We hypothesized that the result of blocking estrogen production would be release of the hypothalamic/pituitary axis from estrogenic negative feedback, thereby increasing gonadotropin secretion and resulting in stimulation of ovarian follicular development. This first hypothesis is referred to as CENTRAL hypothesis. The selective non-steroidal aromatase inhibitors have a relatively short half-life (approximately 40 hours) compared to clomiphene citrate, and would be ideal for this purpose since they are eliminated from the body rapidly [408,409]. In addition, no adverse effects are expected on estrogen target tissues, since no ER down-regulation occurs as observed in clomiphene citrate treatment cycles.
We subsequently developed a second hypothesis, which was referred to as the PERIPHERAL hypothesis to explain another mechanism of action of the aromatase inhibitors in ovarian stimulation. We believe that aromatase inhibitors also act locally in the ovary to increase follicular sensitivity to FSH stimulation. This may result from the temporary accumulation of intraovarian androgens, since conversion of androgen substrate to estrogen is reversibly blocked by aromatase inhibition. There are data supporting a stimulatory role for androgens in early follicular growth in primates [410]. Testosterone was found to augment follicular FSH receptor expression in primates suggesting that androgens promote follicular growth and estrogen biosynthesis indirectly by amplifying FSH effects [411,412]. In addition, androgen accumulation in the follicle may stimulate insulin-like growth factor I (IGF-I), along with other endocrine and paracrine factors, which may synergize with FSH to promote folliculogenesis [413-416]. This hypothesis still waits for evidence to support it.
1.2.1 Use of aromatase inhibitors alone for ovarian stimulation
In the last few years, we have worked on the development of aromatase inhibitors for ovarian stimulation and infertility management and reported interesting success in different applications as follows:
Induction of ovulation in anovulatory women
Based on our CENTRAL hypothesis of using an aromatase inhibitor for induction of ovulation (as explained above), the success of an aromatase inhibitor in inducing ovulation in patients with PCOS was reported [417-420].
Augmentation of ovulation in ovulatory women
After demonstrating success in inducing ovulation in anovulatory women, we proceeded to test whether aromatase inhibition might enhance the release of endogenous gonadotropins enough to stimulate the development of multiple follicles in ovulatory women and result in augmentation of ovulation or even controlled ovarian hyperstimulation. The use of an aromatase inhibitor for augmenting ovulation in patients with ovulatory infertility was successful in women with unexplained infertility, endometriosis, and women undergoing therapeutic donor insemination, and in ovulating partners of infertile men [419,420].
Induction and augmentation of ovulation after clomiphene citrate failure
For over 40 years, clomiphene citrate has been the most commonly used treatment for the induction and augmentation of ovulation, accounting for about two thirds of the fertility drugs prescribed in the United States [421,422]. In spite of the high ovulation rate associated with the use of clomiphene citrate, the pregnancy rate is much lower than anticipated. This is particularly true when considering pregnancy rate per cycle after three cycles of CC treatment [421], with higher than expected incidence of miscarriage in conception cycles [255]. Such discrepancy is believed, as explained earlier, to be due to the peripheral antiestrogenic effect of clomiphene citrate particularly at the level of the cervical mucus and endometrium, and which manifest themselves even in the presence of gonadotropin treatment superovulation. The accumulation in the body of the isomers of clomiphene citrate due to the long half-life (several days to weeks) adds to the persistence of the antiestrogenic effect [261,262].
In order to improve the outcome of clomiphene citrate treatment, various approaches have been suggested to overcome the antiestrogenic effect including concomitant estrogen administration. Some investigators reported increased endometrial thickness and improved pregnancy rates with this approach [423-425] while others have reported no benefit [426] or even a deleterious effect of estrogen administration [427]. Another approach was to administer clomiphene citrate earlier during the menstrual cycle [428], to allow the anti-estrogenic effect to wear off earlier before the critical period of fertilization and implantation. A third approach has been to combine another selective ER modulator like tamoxifen, which has more estrogen agonistic effect on the endometrium with clomiphene citrate [429] or to use it as an alternative to clomiphene citrate [430]. However, none of these strategies have proved to be completely successful in avoiding the peripheral antiestrogenic effects of clomiphene citrate. In addition to a discrepancy between ovulation and pregnancy rates with clomiphene citrate treatment, 20% to 25% of anovulatory women are resistant to clomiphene citrate and fail to ovulate at doses up to 200 mg daily.
The success of aromatase inhibition in inducing and augmenting ovulation encouraged trying aromatase inhibitors in cases of clomiphene citrate failure that was found to be successful in achieving ovulation and pregnancy [419,420]. As explained above, the significantly shorter half-life of the third generation aromatase inhibitors compared to clomiphene citrate, allows rapid elimination from the body [408,409]. In addition, since no ER down-regulation occurs, any adverse effects on estrogen target tissues, as observed in clomiphene citrate treated cycles, is expected.
1.2.2 Adjunct use of aromatase inhibitors for ovarian stimulation
We investigated the idea of combining an aromatase inhibitor with FSH injections to reduce the dose of FSH required to achieve optimum controlled ovarian hyperstimulation, without adverse antiestrogenic effects [431,432]. A significant reduction in the FSH dose required (from 45% to 55% in women with PCOS and unexplained infertility) has been reported [433] without evidence of peripheral antiestrogenic effects [432,433].
Improving ovarian response to FSH stimulation in poor responders
Reducing FSH dose required for optimum controlled ovarian hyperstimulation encouraged us to explore the use of an aromatase inhibitor in conjunction with FSH to improve response to ovarian stimulation in poor responders. In a selected group of women who had a poor response to FSH stimulation in at least two prior treatment cycles, adding an aromatase inhibitor resulted in improvement in the response to FSH stimulation. All patients developed a significantly greater number of mature ovarian follicles and almost a third of the treatment cycles resulted in pregnancy. In addition, the dose of FSH required to achieve such optimum response was significantly less than the dose used in the prior cycles in which FSH was used alone [434,435].
Developing alternative regimens for administering aromatase inhibitors for ovarian stimulation
During all the above-mentioned studies, the aromatase inhibitor, letrozole, was administered orally as a daily dose of 2.5 mg from day 3 to day 7 of the menstrual cycle. Based on the pharmacokinetics of the new aromatase inhibitors (almost 100% bioavailability after oral administration, ~2 days half-life and no accumulation or significant metabolite accumulation), we thought of a potentially more convenient method of administering an aromatase inhibitor for ovulation induction. We hypothesized that significantly higher concentration of the aromatase inhibitor can be achieved early in the menstrual cycle with faster clearance later in the menstrual cycle with the administration of a high single dose of the aromatase inhibitor early in the menstrual cycle such as on day 3. A single dose regimen would satisfy two goals: first, achieving maximum estrogen suppression early in the menstrual cycle when it is desired, and second, to allow clearance of the aromatase inhibitor before the critical final stage of fertilization and embryogenesis, to maximize safety and avoid any possible undesirable effects of the aromatase inhibitors. Single dose administration of an aromatase inhibitor was found successful in inducing ovulation with ovulation and pregnancy rates comparable to the 5-day regimen [436].
Limitations of preliminary data on use of aromatase inhibitors for ovarian stimulation
In all our clinical trials described above, results were not obtained from randomized, prospective, placebo controlled studies, the optimum research design. However, due to the experimental nature of the use of the aromatase inhibitors for ovarian stimulation, which to our knowledge, has never been used before, we believed that the present observational trials were mandatory before proceeding into any definitive randomized studies.
The encouraging results of our data have led other investigators from different centers world-wide to study the use of aromatase inhibitors for ovarian stimulation and in general have reported findings similar to ours in terms of the success of aromatase inhibitors in infertility treatment [437-450].
In a randomized, prospective study, a superior uterine environment has been found in patients treated with an aromatase inhibitor compared with clomiphene citrate, reflecting the lack of the antiestrogenic effects with aromatase inhibitor treatment. Although a non-significant increase in pregnancy was observed in patients who received aromatase inhibitor treatment (16.7 versus 5.6% per patient, P = 0.55), this almost three-fold increase in pregnancy rate would have required about ten more patients per group to reach statistical significance. The superiority of a single dose administration of an aromatase inhibitor was also reported when compared to clomiphene citrate [439]. In other studies, the use of an aromatase inhibitor in conjunction with FSH was found to reduce the FSH dose [440] and improve response to ovarian stimulation in poor responders [441,442], confirming our previous reports.
In another prospective randomized trial, Biljan [438] studied two doses (2.5 and 5 mg per day) of the aromatase inhibitor, letrozole for ovarian stimulation in patients with unexplained infertility. They found patients treated with a higher dose of letrozole developed more follicles without a detrimental effect on endometrial development. The potential for different doses and regimens of administration of aromatase inhibitors for ovarian stimulation still requires a lot of study, but the use of aromatase inhibitors at high doses should be cautiously considered.
In all reports and most of studies of other investigators, the aromatase inhibitor, letrozole was the one used. However, anastrozole, another third generation aromatase inhibitor similar to letrozole, was used in other studies [443,444]. Due to the similar pharmacokinetics and pharmacodynamics, including similar potencies and specificity in inhibiting the aromatase enzyme, we believe there is likely to be no difference between the third generation aromatase inhibitors in their efficacy for ovarian stimulation. It does, however, need to be determined what dose of anastrozole is required to be equivalent to letrozole.
Future avenues for the use of aromatase inhibitors for infertility management
It seems that there are many interesting areas of research that need exploration as regards the development of aromatase inhibitors for infertility management. These directions for research would include: confirming the available preliminary data on the success of aromatase inhibition in induction and augmentation of ovulation, as well as reducing the dose of FSH needed for ovarian stimulation, improving response in poor responders, and finding the optimum regimen for administering aromatase inhibitors for infertility treatment
Moreover, the use of aromatase inhibitors for new applications including in-vitro maturation and prevention of severe ovarian hyperstimulation syndrome and endometriosis-related infertility are interesting future avenues for aromatase inhibitors potential use in infertility management.
In addition the use of aromatase inhibitors to improve the outcome of treatment after assisted reproduction as discussed earlier in this review is an exciting area of application.
1.3 Use of aromatase inhibitors to improve treatment outcome after ovarian stimulation and assisted reproduction
In addition to using aromatase inhibitors, ALONE, for ovarian stimulation, their use during assisted reproduction carries several potentials to improve the treatment outcome.
Improving implantation rates
We hypothesize that aromatase inhibition during assisted reproduction may improve the implantation rate mainly by reducing the estrogen levels attained during COH. In addition, two other mechanisms may apply including reducing the dose of FSH required for optimum COH as well as applying much simpler stimulation protocols that do not require the use of GnRH analogues, hence avoiding any possible direct deleterious effects of FSH and GnRH analogues on the endometrium.
1.3.1 Lowering supraphysiological estrogen levels
As discussed above, the undesirable effects of ovarian stimulation on the outcome of infertility treatment are believed to be due to the supraphysiological levels of estrogen irrespective to whatever mechanisms explain for that (various postulated mechanisms were discussed above). So, lowering estrogen levels may be associated with improved outcome of treatment in terms of improving the implantation and pregnancy rates in addition to lowering risk of sever ovarian hyperstimulation syndrome.
Reducing estrogen synthesis by aromatase inhibition seems to be an exciting idea to ameliorate the deleterious effects of the supraphysiological levels of estrogen on the endometrium, the developing oocyte and embryo as well as the luteal.
Until recently there was no suitable aromatase inhibitor that could be used clinically to reduce estrogen levels during ovarian stimulation. This is because the available aromatase inhibitors were not safe for clinical application during ovarian stimulation due to lack of specificity in inhibiting the aromatase enzyme without inhibiting other steroidogenesis enzymes (e.g. aminoglutethimide). The other aromatase inhibitors (steroidal androstenedione analogues) were irreversible in their effect on the aromatase enzyme besides being parentrally administered. Most important, these old aromatase inhibitors were not potent enough to inhibit the aromatase enzyme and lower estrogen levels in women of the reproductive age group. However, the third generation non-steroidal aromatase inhibitors group is very potent and specific in inhibiting the aromatase enzyme reversibly. These new aromatase inhibitors are orally administered with very high safety profile and well tolerability. Moreover, they are cheap with a relatively short half-life [~45 hours], and already approved for clinical use to reduce estrogen production in postmenopausal women with breast cancer.
They have not been used in women of the reproductive age group. However, we have found these aromatase inhibitors to be effective in inhibiting the aromatase enzyme and effectively decrease estrogen levels in women of the reproductive age group during their successful use for ovarian stimulation [417,418,431,432].
In our experience with the use of an aromatase inhibitor for ovarian stimulation, estrogen levels were significantly lower (especially E2 level/mature follicle) when compared with conventional stimulation protocols (clomiphene citrate, FSH and clomiphene citrate plus FSH). Such low E2 levels may be beneficial and explains at least partially the improved outcome of treatment in terms of achieving promising high pregnancy rates during aromatase inhibitor treatment.
It may not be only the supraphysiological estrogen levels, which explain totally for the reduced implantation rate during ovarian stimulation cycles. Local paracrine factors, or possibly other undetermined factors may be responsible for the reduced implantation rate during COH. In this case the aromatase inhibitors may not offer a complete solution to overcome such drawbacks of ovarian stimulation. However, the idea is exciting and seems to be promising and warrant trials because high estrogen levels may explain, at least in part, for the deleterious effects of ovarian stimulation on treatment outcome.
In ddition, we think that, reducing estrogen levels achieved during induction of ovulation by using aromatase inhibitors may prevent the significant increase in leptin concentrations avoiding its possible deleterious effects on the outcome of treatment as explained above. Because elucidation of leptin's specific role in reproductive function has been challenging, with conflicting results reported by various investigators, it is still quite early and highly speculative to hypothesize a link between aromatase inhibitors use and the role of leptin during induction of ovulation. However, the available strong data about a possible role of leptin in mediating reproductive disorders especially in obese women and the firm findings of a regulatory effect of estrogen on leptin production make this hypothesis exciting and interesting enough to warrant future investigation.
1.3.2 Reducing gonadotropins requirements for optimum ovarian stimulation
As mentioned earlier, the use of an aromatase inhibitor significantly reduces the dose of FSH required for optimum COH. In addition to the significant economic benefit, we believe that reducing the dose of FSH may improve the treatment outcome of ovarian stimulation by reducing any possible deleterious effect of the exogenously administered FSH on the endometrium, developing oocyte or other targets.
Other possible benefits
It is believed that the endogenous LH surge arises once serum estrogen levels surpass a set threshold for a certain period of time [451-453].
The supraphysiological estrogen levels attained during ovarian stimulation are believed to cause premature release of the endogenous LH surge resulting in cycle cancellation during assisted reproduction. For that reason, GnRH analogues have been the standard of practice during most of the stimulation protocols for assisted reproduction to prevent the occurrence of endogenous LH surge either by direct inhibition of LH surge (GnRH antagonists) or by down-regulation of the GnRH receptors at the pituitary levels resulting in pituitary desensitization and prevention of the release of endogenous LH [454,455].
Unfortunately, the use of GnRH analogues (which is a crucial part of the stimulation protocol to prevent cycle cancellation as explained above), is associated with several problems including increasing the dose of FSH required to achieve optimum COH (due to suppressing the endogenous gonadotropin secretion, and possible peripheral effect at the level of the ovaries), as well as the luteal phase defect due to a dysfunctional corpus luteum function secondary to persistent endogenous LH suppression as explained earlier. In addition, there is rising evidence of a possible direct deleterious effect of the GnRH analogues, especially the antagonist at the level of the endometrium [456].
The use of an aromatase inhibitor to reduce estrogen levels attained during COH may be effective in preventing the occurrence of premature ovarian surge. This would avoid the use of GnRH analogues during stimulation protocols for assisted reproduction which has several advantages including prevention of the possible deleterious effects of these agents as mentioned above in addition to reducing the cost of treatment as well allowing implementing much simpler stimulation protocols during assisted reproduction.
Aromatase inhibitors for endometriosis-related infertility
The expression of aromatase enzyme in endometriotic tissues with the significant role of locally produced estrogen in endometriosis progression [457] suggests a benefit of aromatase inhibitors in endometriosis-related infertility. The inhibition of local estrogen production in endometrial implants, and the lower peripheral estrogen levels associated with the use of aromatase inhibition for ovulation induction, is expected to protect, to some degree, against progression of endometriosis during infertility treatment.
1.4 Concerns regarding the use of aromatase inhibitors in infertility treatment
There are three main concerns that may arise regarding the use of the aromatase inhibitors to improve the outcome of treatment after ovarian stimulation and assisted reproduction. They include the possible deleterious effect of the low follicular estrogen milieu on the development of the oocytes, the possible direct effect of the aromatase inhibitors on the oocyte development, fertilization or embryogenesis, and the accumulation of the androgens that may result from inhibition of their conversion into estrogens.
1.4.1 Effect of low estrogen milieu on the developing oocyte
Palter et al. have reviewed the question whether an estrogen-free (or at least very low estrogen) intrafollicular environment is compatible with follicular development, ovulation, and corpus luteum formation [458]. The authors concluded that markedly reduced to nonexistent intrafollicular and circulating concentrations of estrogen are compatible with follicular "expansion", retrievable and fertilizable oocytes, as well as with cleavable and apparently transferable embryos. The authors drew their conclusion after discussing lessons from data in the literature including cases of deficiency of the 17-hydroxylase/17–20 lyase [459-466] and 3β-hydroxysteroid dehydrogenase [467,468] and the aromatase enzyme [469-473] as well as cases of severe hypogonadotropism [474-478]. In addition, data on the use of aromatase inhibitors in animals were reviewed [479-482].
Cases of enzyme deficiency leading to a status of very low estrogen levels
17 alpha-hydroxylase/17–20 lyase deficiency
17alpha-hydroxylase/17–20 lyase deficiency is one form of congenital adrenal hyperplasia, which is associated with marked impairment of glucocorticoid, androgen, and estrogen biosynthesis [459]. Women suffering from this enzyme deficiency suffer from hypergonadotropic hypogonadism and sexual infantilism. However, early reports noted the presence of many primary and secondary follicles in ovarian material [460] and many of them had bilateral multicystic ovaries at the time of laparotomy [461-463].
It is interesting that Rabinovici reported on a patient afflicted with virtually complete 17-hydroxylase/17–20 lyase deficiency that, despite castrate levels of estrogens, underwent an apparently successful induction of ovulation associated with progressive follicular expansion and oocyte retrieval, IVF-ET, and early embryonic cleavage followed suit [464].
3β-hydroxysteroid dehydrogenase [3β-HSD] deficiency
The deficiency of 3β-HSD is associated with markedly reduced levels of estrogen. However, the existence of normal ovulatory function in a woman with late-onset of a mild form of 3β-HSD has been reported [483].
In a group of cycling female Rhesus monkeys exposed to ovulation induction with hFSH and hFSH + hLH in the absence or presence of the 3β-HSD inhibitor trilostane given on days 1–8 of the menstrual cycle [467], apparently healthy oocytes were obtained by follicular aspiration 34 h after hCG administration. Importantly, treatment with the 3β-HSD inhibitor, trilostane, led to a reduction in serum E2 levels to 7% of that of control animals throughout the follicular phase. Despite this dramatic reduction in E2 levels, neither the total number of large antral follicles per animal (17 ± 1 vs. 18 ± 2) nor their size distribution differed significantly from 3β-HSD inhibitor-untreated controls. Furthermore, treatment with the 3β-HSD inhibitor did not alter the overall maturation pattern of collected oocytes (atretic, prophase I, metaphase I, or metaphase II). However, the authors found a reduction in the percentage of metaphase II oocytes that were successfully fertilized (15 vs. 65%). Moreover, metaphase oocytes that required more than 8 h to complete meiosis in vitro failed to fertilize in three of four animals receiving 3β-HSD inhibitor relative to controls (31%). These observations suggest that follicular development and the completion of meiosis may be unaffected by the low estrogen levels but that cytoplasmic oocyte maturation and/or function could be unfavorably affected [467].
Aromatase enzyme deficiency and the use of aromatase inhibitors
Obviously, aromatase (estrogen synthase) enzyme deficiency is associated with marked decrease or almost absence of estrogen production.
Extreme examples of complete aromatase deficiency due to mutations in the aromatase gene, CYP19 gene, in adult human females, however, were reported [469,470]. The affected patients suffered from ambiguous external genitalia, primary amenorrhea, sexual infantilism, and multicystic ovaries.
Morishima [471] reported on the aromatase deficiency in a 28-yr-old 46 XX proband followed since infancy. Null mutant mice for aromatase gene [ArKO] were generated [484], thereby affording the opportunity to examine the role of estrogen in the follicular development in the mouse ovary. Evaluation of the ovaries revealed the presence of many large follicles filled with granulosa cells and evidence of antrum formation, but no corpora lutea. The ovarian phenotype degenerated with age upon the appearance of hemorrhagic cystic follicles and the loss of secondary and antral follicles coincident with the infiltration of macrophages and with stromal hyperplasia [485-488]. Therefore, the ArKO females are infertile, due primarily to a complete lack of ovulation.
However, recently, Jones et al., reported that oocytes were harvested from the ovaries of 4- and 7-week old ArKO, wild type and heterozygote mice stimulated with 5 IU PMSG. The number of immature oocytes harvested from ArKO females did not differ from the number collected from wild type or heterozygote littermates of either age group. Oocyte in vitro maturation rates also did not differ between the three genotypes or two age groups, with almost 75% of the immature oocytes progressing to metaphase II. Chromatin staining confirmed the arrest of these oocytes at the second meiotic division with chromatin staining clearly present in the oocyte and polar body. Mature oocytes were inseminated and IVF-ET rates did not differ between the three genotypes or two age groups, with fertilization occurring in approximately 67% of oocytes. Fertilized oocytes were cultured to blastocysts. Again, blastocyst development rates did not differ between the groups, with approximately 65% of the zygotes developing into blastocysts. Blastocyst morphology was similar across all of the groups [489].
These results indicate that ArKO oocytes are competent to develop to at least the implantation stage. The authors concluded that estrogen might not be required for the production and maturation of developmentally competent oocytes. Rather, its role in folliculogenesis is probably via regulation of the hypophysial pituitary gonadal axis and thus gonadotropin secretion [489].
Regarding the effect of an estrogen-free/poor intrafollicular environment on gametogenic maturation, Palter et al. [458] concluded that the effect is a negative one. Their conclusion was based on a number of primate studies, which indicated that an estrogen-free/poor intrafollicular environment is associated with marked decrements in the rates of meiotic maturation and fertilization [467,490]. In addition data derived from rodent models suggested that further suggested a compromise in early embryonic development [491].
In cycling female rhesus monkeys, the aromatase inhibitor, 1,4,6-androstatrien-3, 17-dione (ATD) was used to inhibit estrogen production during gonadotropin ovarian stimulation [479]. Animals treated with ATD displayed a drastic reduction in serum 17β-E2 levels to 37% of that of controls within 8 h of ATD treatment and to 16% of control by the day of hCG injection. In turn, the circulating levels of androstenedione rose. Despite the drastic reduction in the circulating levels of E2 and the increase in the circulating levels of androgens, the overall number of large antral follicles [16 ± 3 for controls and 20 ± 3 for ATD-treated] and their size distribution (as assessed by ultrasonography) proved comparable for control and ATD-treated animals. Similarly, no difference was noted in the number of oocytes collected or in the proportion of oocytes reinitiating meiosis (MI at the time of collection). In contrast, ATD-treated animals displayed a marked increase (31 vs. 11%) in the proportion of prophase I oocytes. Moreover, ATD-treated oocytes displayed retarded in vivo completion of maturation to MII (4% vs. 26%). Interestingly, the latter retardation was not observed in vitro. Furthermore, two of the four ATD-treated animals yielded oocytes that were morphologically abnormal. Finally, oocytes from ATD-treated animals displayed significantly reduced rates of fertilization (9% vs. 25%) as compared with controls. However, the cleavage rate after successful fertilization was similar for ATD-treated vs. ATD-untreated controls. In vitro cultures of granulosa cells collected at the time of oocyte aspiration revealed equivalent 24-h progesterone production in treated and control animals. The authors concluded that these findings suggest that the acute reduction in E2 levels during the terminal stage of gonadotropin stimulation had little effect upon follicular recruitment and expansion but an apparent detrimental effect upon gametogenic function may, in fact, exist. However, we cannot extrapolate from this study that the findings are simply due to the acute reduction of estrogen production as it is important to realize that there was a concomitant significant rise in androstenedione. Moreover, the time of administering an aromatase inhibitor during the latter part of the follicular phase as well as the irreversible nature of aromatase inhibition constitute important differences from our model of using a REVERSIBLE aromatase inhibitor, TEMPORARILY, EARLY in the menstrual cycle that has a SHORT half-life.
Selvaraj [480,481] and Shetty [482] examine the effects of blocking estrogen biosynthesis during the follicular phase on follicular maturation in the adult female bonnet monkey. In their studies, they used one of the third-generation REVERSIBLE, aromatase inhibitors (CGS 16949A) starting EARLY on day 3 of the menstrual cycle. There were 53% and 70% reductions in the basal and surge levels of E2, respectively without obvious effect on follicular maturation, ovulation, and luteal function as assessed by serum hormone profiles as well as by laparotomy. Moreover, the concurrent administration of FSH and an aromatase inhibitor resulted in the suppression of the FSH-induced increase in the circulating levels of E2 (by 100%) with no effect noted on either the number of follicles developed or their size relative to control. In addition, granulosa and theca cells, removed on day 9 of the treatment cycle, were responsive to gonadotropins in vitro, disclosing no evidence of a deleterious effect on the cellular development and maturation of follicular cells.
Terry studied a possible role of high E2 levels in mediating the adverse effects of hyperstimulation with PMSG on early embryonic development in the rat [491]. They used the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA), to inhibit endogenous E2 production. The authors conducted three experiments. In the first, varying doses of 4-OHA were administered either concurrently with hCG to pro-estrus female rats hyperstimulated at early diestrus stage with 20 IU PMSG or alone into non-hyperstimulated pro-oestrus females. At high doses of 1000, 2000, or 5000 mg/rat, 4-OHA substantially improved the survival of embryos in hyperstimulated females with optimum protection at 2000 mg, while low doses of 100 and 500 mg/rat were ineffective. When administered alone, only the highest dose of 5000 mg/rat 4-OHA increased embryo count. In the second experiment, higher doses of PMSG were studied (30 or 40 IU), with or without 5000 mg/rat 4-OHA given at the time of hCG injection. PMSG proved to be more detrimental with increasing dose, and high doses of 4-OHA (5000 mg/rat) was needed to rescue embryos from death in the 30, but not 40, PMSG group. In the third experiment, the influence of the timing of 4-OHA treatment on its ability to improve the embryo count in hyperstimulated females was examined by introducing 4-OHA 24 h earlier, rather than at the time of hCG treatment. The results showed the importance of timing of 4-OHA administration, as 5000 mg/rat 4-OHA was able to restore embryo survival in the 40 PMSG hyperstimulated group only when it was administered 24 h before hCG injection. These results highlighted that 4-OHA, when administered at the appropriate time and dose, could reverse the negative effects of hyperstimulation from PMSG on early embryonic development. The authors concluded that this might be due to the suppression of estrogen production, thereby alleviating the supraphysiological level of E2, which is typically present in PMSG-treated females, which supports the hypothesis that excessive E2 is responsible for the negative effects of hyperstimulation with PMSG on early embryonic development.
The high success rates of ovulation and achievement of pregnancy in our reports on the use of aromatase inhibitor in ovarian stimulation, despite significantly lower estrogen levels could be due to several reasons. First: We used one of the third-generation "REVERSIBLE" aromatase inhibitors. Second: The aromatase inhibitor was administered EARLY in the follicular phase and for a limited period of time, which would allow the rapid clearance of the aromatase inhibitor from the body due to its SHORT half-life. Third: the reversible nature of the aromatase enzyme inhibition and the rising levels of FSH, which induce the expression of the aromatase enzyme, both would not allow for the estrogen levels to drop drastically below the low physiologic range. We believe that these low estrogen levels attained during the use of aromatase inhibitors for ovarian stimulation are compatible with healthy development of the oocytes, fertilization, embryo development, implantation and achievement of pregnancy. Fourth: the absence of significant rise in androgen levels would have prevented any possible unwanted effects on the terminal part of the oocyte maturation and ovulation.
1.4.2 Direct effect of the aromatase inhibitors on the developing oocyte, fertilization and embryogenesis
As explained above, the short half-life of the aromatase inhibitors and limiting the administration to the early part of the follicular phase would allow the rapid clearance of the medications from the body before the important stage of fertilization and embryogenesis. This in addition to the absence of accumulation of the aromatase inhibitors of any of their metabolites would make them safe for the ovarian stimulation. We have reported a preliminary data on the pregnancy outcome after the use of aromatase inhibitors for ovarian stimulation supporting the safety of using these medications for such indication [492].
Data have been accumulating regarding the absence of deleterious effects in association with aromatase inhibitor treatment on follicle/oocyte maturation and embryo development in mice [492,493]. Treatment with the aromatase inhibitor, anastrozole was associated with similar number of total follicles found per animal (30.4 follicles/animal in the control group and 27 follicles/animal in the anastrozole-treated group) as well as comparable rates of development of embryos, morulae, blastocysts, and hatching blastocysts between the two groups (P = 0.20, 0.10, 0.44, and 0.38, respectively) [493].
1.4.3 Effect of the accumulated androgens
As mentioned before, the rapid clearance of the aromatase inhibitors from the body due to their short half-life and the reversible nature of the aromatase enzyme inhibition as well as the rising levels of FSH, which induce the expression of the aromatase enzyme, all would not allow the clearance of any accumulating androgens by converting them to estrogen. The conversion of the androgens into estrogens (the step catalyzed by the aromatase enzyme) is a terminal step in the cascade of steroidogenesis. So, substrate (androgens) accumulation is not expected to be very significant, as other alternative earlier steps in the steroidogenesis pathways will work to clear out the accumulating androgens. For these reasons, pharmacokinetic studies on the new aromatase inhibitors reported the absence of significant androgen elevations or abnormal changes in other steroids in patients receiving aromatase inhibitors for breast cancer. In a subset of our patients who received an aromatase inhibitor for ovarian stimulation, we did not find significant change in the androgen levels while receiving the medication when compared to stimulation with gonadotropins alone (unpublished data).
Most recently we published data on the favorable pregnancy outcome after the use of letrozole for ovarian stimulation alone or in conjunction with gonadotropins [494]. This study looked at outcome of pregnancies achieved after ovarian stimulation with the aromatase inhibitor, letrozole, alone or in conjunction with gonadotropins. The study included a cohort of 394 pregnancy cycles achieved after letrozole and other ovarian stimulation treatments in a ddition to a control group of pregnancies spontaneously conceived without ovarian stimulation. These 394 pregnancy cycles were achieved as follows: 63 pregnancies with 2.5 mg of letrozole alone or with gonadotropins, 70 pregnancies with 5.0 mg of letrozole, 113 pregnancies with clomiphene alone or with gonadotropins, 110 pregnancies with gonadotropins alone, and 38 pregnancies achieved without ovarian stimulation. The study found pregnancies conceived after letrozole treatments were associated with similar miscarriage and ectopic pregnancy rates compared with all other groups. In addition, letrozole use was associated with a significantly lower rate of multiple gestation compared with clomiphene citrate. The findings of the stuy support the favorable pregnancy outcome and low multiple gestation rate in association with the use of aromatase inhibitors for ovarian stimulation [494].
5 Summary and conclusion
Ovarian stimulation particularly in conjunction with assisted reproductive technologies aims at stimulating the recruitment of several mature ovarian follicles that would enhance the chance of successful treatment by obtaining several embryos readily available for transfer into the uterus. It is obvious that supraphysiological levels of estrogen are inevitably attained during ovarian stimulation due to the significant contribution of estrogen production from each one of the several mature ovarian follicles. There is growing evidence that such supraphysiological estrogen levels are deleterious on the development of the endometrium, oocytes, embryos as well as other targets through different mechanisms. This is believed to explain, at least in part, the low pregnancy rates associated with assisted reproduction, mainly as a result of the persistently low implantation rates that have not improved impressively along almost three decades of assisted reproduction experience. This led several investigators to adopt the concept of minimal ovarian stimulation for assisted reproduction as an alternative approach to reduce the deleterious effects of the supraphysiological estrogen levels attained during routine controlled ovarian hyperstimulation. Unfortunately such approach, despite being logical, is associated with the major drawback of achieving fewer oocytes than desired, which, expectedly, reduces the chances of treatment success. Recently, we reported the success of the new generation aromatase inhibitors in ovarian stimulation. We found these agents to be effective in reducing the amount of estrogen production by mature ovarian follicles significantly. Furthermore, we found the use of these agents to be associated with improved ovarian response to stimulation by gonadotropins, resulting in significant reduction in the dose of gonadotropins required for controlled ovarian hyperstimulation. The novel idea of using aromatase inhibitors during ovarian stimulation for assisted reproduction combines the benefit of a significant reduction in estrogen production as well as gonadotropins dose, with the exiting advantage of achieving a good number of mature oocytes as a result of enhanced ovarian response to gonadotropins. This is expected to improve the various aspects of treatment outcomes after assisted reproduction including increased safety, reduced cost, as well as enhanced implantation rate. If confirmed in well-designed clinical trials, this would facilitate acceptance of the concept of single embryo transfer by infertile couples and practitioners, thereby reducing the epidemic of multiple births after assisted reproduction with its significant deleterious health and economical effects. This is particularly true in the light of recent data supporting the success of the approach of single embryo transfer [495-497].
Authors' contributions
MFMM has written the review article, RFC and MPD have reviewed the manuscript
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Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-3-541620216910.1186/1477-7827-3-54ReviewThe role of aromatase inhibitors in ameliorating deleterious effects of ovarian stimulation on outcome of infertility treatment Mitwally Mohamed FM [email protected] Robert F [email protected] Michael P [email protected] Division of Reproductive Endocrinology & Infertility, Department of Obstetrics & Gynecology, Wayne State University, Detroit, Michigan, USA2 Reproductive Sciences Division, Department of Obstetrics & Gynecology, University of Toronto, Toronto, Ontario, Canada2005 4 10 2005 3 54 54 7 7 2005 4 10 2005 Copyright © 2005 Mitwally et al; licensee BioMed Central Ltd.2005Mitwally 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.
Clinical utilization of ovulation stimulation to facilitate the ability of a couple to conceive has not only provided a valuable therapeutic approach, but has also yielded extensive information on the physiology of ovarian follicular recruitment, endometrial receptivity and early embryo competency. One of the consequences of the use of fertility enhancing agents for ovarian stimulation has been the creation of a hyperestrogenic state, which may influence each of these parameters. Use of aromatase inhibitors reduces hyperestrogenism inevitably attained during ovarian stimulation. In addition, the adjunct use of aromatase inhibitors during ovarian stimulation reduces amount of gonadotropins required for optimum stimulation. The unique approach of reducing hyperestrogenism, as well as lowering amount of gonadotropins without affecting the number of mature ovarian follicles is an exciting strategy that could result in improvement in the treatment outcome by ameliorating the deleterious effects of the ovarian stimulation on follicular development, endometrial receptivity, as well as oocyte and embryo quality.
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PART ONE
1 Introduction
Current epidemiological evidence suggests that 15% of couples will experience infertility. Background prevalence rates now appear to be reasonably stable, but there is evidence of an increase in the rate of referrals for medical help [1,2]. Farley and Belsey, 1988 [3], have reported estimates of the prevalence (percentage) of primary infertility by region and country. They estimated 6% for North America, 5.4% for Europe, 3% for the Middle East, 10.1% for Africa, 4.8% for Asia and Oceania, 3.1% for Latin America and 6.5% for the Caribbean. The American Society for Reproductive Medicine (ASRM) estimates that 5 million American heterosexual couples report difficulties in achieving a viable pregnancy, of which 1.3 million seek advice for the problem [4].
2 Ovarian stimulation and assisted reproduction for infertility management
After correcting the abnormalities detected during the diagnostic workup, ovulation induction is usually performed either for treatment of anovulation/oligo-ovulation, or empirically in regularly ovulating women. This approach results in a pregnancy rate of around 8%–15% per cycle depending on the agents used for ovulation induction and the characteristics of the couple, such as the woman's age and the presence or absence of a male factor. Couples who do not become pregnant with ovulation induction alone then undergo more sophisticated treatment modalities including intrauterine insemination (IUI) and in-vitro fertilization and embryo transfer (IVF-ET) as a treatment of last resort [5].
Since the birth of Louise Brown in 1978, IVF-ET has become the therapeutic mainstay for female infertility. It has become generally accepted as therapy for a wide array of fertility problems, and has been accompanied by the rapid expansion of IVF-ET clinics worldwide resulting in more than 1% of babies being conceived by IVF-ET in western countries [6].
2.1 Ovarian stimulation for assisted reproduction
In most assisted reproduction programs, gonadotropins are used alone or in combination to stimulate the growth and maturation of multiple follicles. This is essential because of the need to recruit a greater number of follicles, which provides the opportunity for retrieval of a large number of oocytes. This would improve the chance for fertilization of multiple oocytes and thereby allow an increased number of embryos for transfer in order to give acceptable success rates. Recent advances in the understanding of ovarian stimulation, the techniques of oocyte retrieval, the handling of gametes, the methods of assisted fertilization and improved conditions of culture media have steadily increased the fertilization rate. Fertilization rates of 60–70% can now be expected when conventional insemination, or even higher when intracytoplasmic sperm injection (ICSI) are carried out. However, there has not been a corresponding increase in implantation rates, which have remained steady at overall rates around 10%–15% [6].
2.2 Low implantation rates with assisted reproduction
Throughout the last five decades, a progressive series of revolutionary techniques have been developed to overcome infertility, starting with the successful fertilization of human oocytes in vitro [7] and followed nearly 10 years later by the birth of the first IVF-ET baby [8]. Several other new developments in assisted reproduction have emerged, including cryopreservation and storage of embryos for later transfer [9], fertilization of oocytes with a single injected spermatozoon to alleviate severe male infertility i.e. ICSI [10] and diagnosis of genetic defects from preimplantation embryos prior to intrauterine transfer [11]. However, although IVF-ET is now a standard, well-established treatment for infertility, success rates remain relatively low, with only about 33% of cycles resulting in pregnancy [12]. This is believed to be due to the low implantation rate that has not significantly increased as fertilization rates [13]. Efforts are being made to improve implantation rates after IVF-ET by improving culture conditions, optimizing gamete quality and developing new techniques of selecting viable embryos for transfer without significant success. For this reason, multiple embryos are generally transferred to improve pregnancy rates, but this has resulted in an unacceptably high rate of multiple-gestation pregnancies [14].
Although governed by multiple interactive events, embryo implantation depends mainly on the quality of embryos and the status of uterine receptivity. During the last two decades, several developments in controlled ovarian hyperstimulation [COH], fertilization, and embryo culture techniques have led to an optimization in the number and quality of embryos available for ET. In contrast, uterine receptivity has failed to benefit from parallel improvements, and its disarrangement is likely to represent an important cause of the sub-optimal embryo implantation rates observed in IVF-ET [15].
2.3 Poor outcome of infertility treatment associated with ovarian stimulation
In the following section we review in brief both animal and human evidence for the unfavorable outcome including impaired implantation and increased adverse outcomes in pregnancies achieved following ovarian stimulation when compared with spontaneous pregnancies.
2.3.1 Animal data
Increased pre- and post-implantation embryonic loss has been reported in mammals [16-21] including rats [16,18], mice [17-19], murine [20] and hamsters [21], in association with ovarian hyperstimulation. These effects have been attributed to ovarian stimulation using standard doses of gonadotropins. At higher doses of gonadotropins, studies have found increased frequencies of oocyte aneuploidy, embryo mortality, fetal growth retardation and congenital abnormalities [22,23].
The poor outcome after ovarian stimulation has been attributed to adverse effects on the maternal side and or the gametes and embryo side. On the maternal side: inadequate uterine synchrony or receptivity has been reported. On the gamete and embryo side, ovarian stimulation has been found associated with chromosomal defects in the oocyte leading to increased lethality during the preimplantation stages [22,23]. However, because species-specific variations in implantation strategies exist, these differences preclude the formulation of a unifying theme for the molecular basis of this event.
2.3.2 Human data
Many studies have found higher pregnancy rates in donor oocyte recipients than patients undergoing standard IVF-ET [24,25]. The higher success rates could be attributed to better quality oocytes from younger donors. However, in centers using a shared oocyte system, where the donor keeps half of the oocytes for herself, significantly higher pregnancy and implantation rates were found in the recipients [26].
Another evidence for adverse effects of ovarian stimulation on implantation is the higher implantation rate associated with IVF-ET in the natural or un-stimulated cycle. Although most studies were associated with a rather high proportion of cancelled cycles [25–75%] and a low clinical pregnancy rate per started cycle [range 0–23%], higher implantation rates have been reported (up to 30%) [27].
Adverse Obstetrical Outcome after ovarian stimulation
Induction of ovulation has been shown to raise the risk of miscarriage when compared with spontaneous pregnancies [28]. This was true even after controlling for advanced age, a known significant risk factor for miscarriage. Higher risks as compared with natural pregnancies are reported in pregnancies after IVF-ET, primarily owing to growth retardation and pre-term birth. Although this can be explained by the high multiple pregnancy rates in IVF-ET pregnancies [29], an increased rate of small for gestational age and pre-term birth children is reported in singleton IVF-ET pregnancies as compared with natural singleton pregnancies after adjustment for potential biases [30-32]. Moreover, twin pregnancies after assisted reproduction have a higher rate of perinatal mortality and lower birth weight as result of a higher rate of premature parturition when compared to spontaneously occurring twins [33,34]. In addition, it was reported that women who conceived multiple gestations through assisted reproductive technologies have a 2.1-fold higher risk of preeclampsia than those who conceive spontaneously [35]. Pregnancies associated with severe ovarian hyperstimulation syndrome have been found to be complicated with increased miscarriage rates [36,37]
While ovarian stimulation has been suggested to contribute, at least in part, for the adverse obstetric outcome other studies have shown that infertility itself is a factor that leads to increased obstetric risks and that sub-fertility is a predictor for low birth weight. Some believe that it is the cause of infertility itself, rather than the use of ovarian stimulation, that is the reason behind poor obstetric outcome after infertility treatment [38,39].
3 Postulated mechanisms behind adverse effects of ovarian stimulation
Several causes and targets have been suggested to explain the poor outcome associated with ovarian stimulation (Table 1). These include (1) supraphysiological levels of estrogen, and other steroids and peptides, attained during ovarian hyperstimulation, (2) the use of exogenous gonadotropins as well as other medications applied during ovarian stimulation such as Gonadotropin releasing hormone (GnRH) analogues (agonists and antagonists), human chorionic gonadotropin (hCG) and clomiphene citrate (3) Other possible undetermined factors. These factors are believed to act through their effects on (i) the endometrium, (ii) the developing oocyte, (iii) the developing embryo, (iv) the ovaries and corpus luteum, (v) the pituitary gland, and (vi) possibly on other targets such as fallopian tubes, the coagulation system, as well as the early developing placenta.
Table 1 Postulated mechanisms behind less favorable treatment outcome (reduced implantation rate and increased adverse obstetric outcome) after ovarian hyperstimulation
Causes: Targets
1-Supraphysiological estrogen levels attained during ovarian stimulation
2-Medications used during ovarian stimulation:
•Clomiphene citrate
•Gonadotropins
•GnRH analogues: agonists & antagonists
•HCG used to trigger ovulation
3-Other probable causes: abnormal levels of other hormones, peptides and local autocrine mediators associated with ovarian stimulation 1-Endometrium
2-Developing oocyte
3-Sperm
4-Developing embryo
5-Maternal endocrine system: ovaries (corpus luteum), pituitary gland and hypothalamus.
6-Other probable targets e.g.
•Fallopian tubes
•Early developing placenta
•Leptin-mediated effects
•Coagulation system
3.1 Effect of supraphysiological levels of estrogen
It is believed that the supraphysiological levels of estrogen, attained during ovarian stimulation, may explain, at least in part, for the adverse effects of ovarian stimulation on the outcome of infertility treatment [40-42]. Different mechanisms have been suggested for the deleterious effects of the supraphysiological levels of estrogen attained during ovarian stimulation Table 2.
Table 2 Deleterious effects of the supraphysiological estrogen levels attained during ovarian stimulation:
1-Effect on the endometrium: most of the available evidence
a: Dys-synchronization of the implantation window.
b: Abnormal Temporal expression of the endometrial pinopodes.
c: Defective endometrial estrogen and progesterone receptors.
d: Impaired endometrial blood flow
e: Abnormal endometrial integrins expression
2-Effect on the developing oocyte
a: Effect on chromosomal and cytogenetic integrity of the oocyte
b: Effect on the mitochondrial function
3-Effect on the sperm causing possible premature acrosome reaction and deactivation
4-Effect on the developing embryo and blastocyst hatching
5-Effect on the ovaries and pituitary (defective corpus luteum function and luteal phase):
a: Defective LH secretion:
- Abnormal LH surge
- Abnormal LH tonic pulse
b: Defective corpus luteum function
6-Other probable targets:
I-Leptin
II-Coagulation system: III-Fallopian tubes
• a: Effect on the ductal environment and ductal fluid
• b: Effect on the ductal transfer and motility
IV-Early developing placenta
Considering all the patients together, significant decreases in pregnancy and implantation rates were observed when estradiol (E2) concentrations were > 2500 pg/ml [>9000 pmol/L.] compared with patients having lower E2 concentrations. High serum E2 concentrations on the day of hCG injection in high and normal responder patients, regardless of the number of oocytes retrieved and the serum progesterone concentration were found to be detrimental to uterine receptivity [42]. Later, it has been shown that a significant reduction in the implantation and pregnancy rates occurred in almost all women with a higher serum E2 concentration of ~5–6000 pg/ml [19–22,000 pmol/L] [43,44]. Recently, we have presented data showing that high E2 levels are associated with less favorable treatment outcome in women undergoing controlled ovarian hyperstimulation and IVF-ET [45-51]. We studied the effect of E2 by looking at the area under the curve (AUC) for E2 levels along the stimulation cycle [45-51]. We believe this is more accurate than looking at a single measurement of E2 e.g. one day of hCG administration [46]. In addition to studying the AUC for E2 levels [45,46], we looked at the E2 production per mature follicle and per gonadotropin dose administered during COH [48] and the effect of age on these parameters [47]. We found significant correlation between these parameters and the outcome of IVF-ET treatment showing that high E2 levels are associated with lower clinical pregnancy and implantation rates [45-49] in addition to increased adverse obstetric outcomes including higher miscarriage rate [49] and lower birth weight [50]. Successful IVF-ET treatment cycles were associated with lower AUC-E2 compared to unsuccessful cycles at the same patients [49].
3.1.1 Effect of supraphysiological estrogen levels on the endometrium
One of the causes for the reduced implantation rates associated with ovarian stimulation may be an impairment of endometrial receptivity, due to high concentrations of sex steroids. This suggestion is supported by higher implantation rates in hormonal replacement treatment cycles after ovum donation, as opposed to the standard IVF-ET cycles as explained earlier.
Effect on Implantation Window
It is generally believed that the embryo-uterine interactions leading to implantation can only succeed when embryonic development is synchronized with the preparation of the endometrium to the receptive state. Typically, this means that the embryos have reached the blastocyst stage and that the endometrium has undergone certain hormone-dependent changes during a specific time window in the preimplantation phase that prepare it to be receptive to the developing blastocyst [52].
The concept of endometrial receptivity introduced by Psychoyos [53,54], has been shown to last only for several hours, thereby determining a narrow nidation window. The concept of an "implantation window" or "receptive endometrium" was initially established in rodents. In the rat, the fertilized embryo reaches the uterus on day 4 after fertilization, and implantation occurs in the afternoon of day 5 [53,54]. In humans, the ovum is fertilized in the fallopian tube, arrives in the uterine cavity around day 17 (day 14 is taken as day of ovulation of a 28-day cycle), and remains there as a free-floating embryo until about day 19; implantation then occurs between days 19 to 22 [55,56]. However, the precise timing and molecular basis of the receptive window in the human remain undefined. Unfortunately, endometrial receptivity knowledge in the human is limited due to the obvious experimental drawbacks and the lack of specific criteria to define a receptive endometrium.
In the literature, there is controversy regarding the effect of ovarian stimulation on endometrial development. Most of the investigators have reported adverse effects of high estrogen levels on endometrial development but there was no consensus on the actual effect. Some have shown endometrial advancement [57-61] while others showed endometrial retardation [62,63]. However, all studies confirm direct deleterious effects on the endometrial development that jeopardize the chance of implantation due to the lack of synchronization between the development of the endometrium and the early embryo development. Implantation failure has been suggested to result from the disparity in maturation between the endometrial stroma and the epithelium observed in histology. Since a paracrine communication between the epithelium and the stroma may be important at the beginning of implantation, this disparity could compromise uterine receptivity or early trophoblastic invasion [64,65].
The effect of high E2 concentrations (> 20,000 pmol/L) was found to be associated with gland-stromal dys-synchrony, which indicated a deficient secretory transformation of the endometrium that represents a sub-optimal endometrial environment for implantation. This finding substantiates clinical observation of significantly lower pregnancy rates in IVF-ET cycles of women with high E2 concentrations. In these patients, there was a marked stromal edema associated with a significantly greater number of stromal vessels, which suggested advanced stromal maturation [44].
In these studies, definitions of supraphysiological hormonal concentrations were variable. Also, the timing of the biopsies and the drug regimens used for ovarian stimulation were different. This, in addition to the variation in endometrial response to different E2 concentrations, may explain the disagreements reported in the literature [66]. Moreover, both the premature progesterone serum elevation, which occurs in 30% of stimulated IVF-ET cycles before hCG administration, and the advanced post-ovulatory rise in progesterone serum concentrations are believed to be responsible for the advanced endometrial development associated with ovarian stimulation [67,68]. However, other studies failed to confirm these observations [69,70].
Effect on endometrial pinopodes
At the time of implantation, the apical membranes of the epithelial cells lining the uterine cavity develop large and smooth membrane projections, named pinocytes due to their pinocytotic function [71]. Their development is progesterone-dependent with strict correlation with the implantation window in the rodents [72]. Similar structures have been seen in the human endometrium [73]. The number of pinopodes was found to have a strong correlation with implantation after embryo transfer [74,75].
Hormonal treatment has been shown to be associated with changes in the timing of pinopode formation. During ovarian stimulation with clomiphene citrate followed by human menopausal gonadotropins (hMG)/hCG, fully developed pinopodes were found 2 to 3 days earlier [76]. In contrast, with E2 and progesterone treatment, fully developed pinopodes were found to be about two days later. Pinopodes were found to form as early as 4 days after hCG administration [59].
Nikas et al. [61], studied the temporal expression of pinopodes as a specific marker for receptivity in IVF-ET cycles induced by gonadotropins, compared to women with regular menstrual periods and proven fertility who served as controls. They did not find ovarian stimulation to affect endometrial pinopode formation in terms of quantity and life span. Instead, the cycle days when pinopodes formed were specific to the individual, being on average 1–2 days earlier in cycles with ovarian stimulation than in natural cycles. These changes in pinopode expression may reflect shifts in the window of receptivity, resulting in ovo-endometrial asynchrony and limiting implantation success in IVF-ET [61]. In IVF-ET, embryonic development is probably delayed while the uterus is advanced, resulting in an early closure of the nidation window, before the embryo eventually reaches a stage capable of initiating implantation. These findings support the theory that reduced implantation rates in IVF-ET cycles could result from impaired or premature endometrial maturation.
Effect on estrogen and progesterone receptors in the endometrium
A lack of estrogen receptors (ER) has been reported [77] during ovarian stimulation cycles that rendered the endometrium functionally hypoestrogenic or hypoprogestogenic. Also, it has been reported that the expression of progesterone receptors (PR) in the endometrium was decreased in the early part of luteal phase after ovarian stimulation [78]. This premature PR decrease was consistent with an early high progesterone level [79]. Papanikolaou et al. [80], investigated prospectively the effect of multi-follicular ovarian stimulation for IVF on the late follicular phase endometrium histology and the expression of ER and PR. Endometrial biopsies were taken in a natural cycle on the day of the onset of the surge of the LH, and in a subsequent stimulation cycle on the day of hCG administration for final oocyte maturation. Histological examination of biopsies both in natural and stimulated cycles showed no secretory changes. However, in stimulated cycles, PR expression was significantly up-regulated compared to natural cycles in both glands (1.67 versus 1.34, P < 0.05) and stroma (1.98 versus 1.62, P < 0.05), whereas ER was down-regulated in glands (1.15 versus 1.43, P < 0.05). In IVF cycles, the progesterone measurements, although within normal values (range 0.8–1.4 microg/l), were significantly higher than in natural cycles (0.99 vs 0.63 microg/l, respectively, P = 0.008). An ongoing pregnancy rate of 37.5% was achieved in the stimulated cycles. The authors concluded that although the current study found no early secretory transformation in stimulated endometria before hCG administration, the ER and PR expression in these endometria was similar to the one described during the first days of the luteal phase in natural cycles. Supraphysiological concentrations of estradiol and subtle progesterone rises in the late follicular phase might be responsible for this modulated steroid receptor profile. They added that this phenomenon indicated accentuated maturation of the endometrium in IVF cycles from the pre-ovulatory phase onwards. [80].
Ovarian stimulation with GnRH-agonist/hMG was found to induce precocious secretory endometrial transformation around the time of oocyte retrieval. Compared to natural cycles, there was an imbalance between endometrial steroid receptor content, proliferation index, and maturation in the peri- and postovulatory phases of stimulated cycles. The lower ER in stimulated cycles on the day of oocyte pick up as compared to the natural cycle controls on the day of ovulation was mainly observed in the stroma, but failed to reach a significant difference in the glands. As for PR, the staining intensity of the stromal ER in the stimulated cycles was higher than that of luteal phase day 2 of a natural cycle. These findings suggest a relative imbalance in ER and PR content of the endometrium in stimulated cycles compared to their natural cycle counterparts. [79].
In conclusion, excessive ovarian response was suggested to lead to insufficient secretory transformation of the endometrium, as well as discordant glandular and stromal development at a time that coincides with the period of maximum uterine receptivity.
Effect on endometrial vasculature and endometrial blood flow
Angiogenesis has a critical role in female reproductive physiology. Growth of the endometrium and placentation is also accompanied by extensive angiogenesis. Thus, an actively maintained blood supply is an essential requirement for reproductive functions, including normal implantation [81]. Endometrial vasculature has been shown to play a prominent role in the early endometrial response to the implanting blastocyst, and vascular changes may contribute to uterine receptivity [82].
The introduction of transvaginal Doppler ultrasound makes the measurement of uterine artery blood flow possible, and at one time it was hoped that uterine arterial resistance changes might reflect uterine receptivity [83]. Applebaum first introduced the concept of evaluating uterine receptivity by a uterine score including the endometrial blood flow [84]. Different studies have demonstrated significant changes in the Doppler indices of uterine and ovarian vessels during ovarian stimulation and spontaneous cycles [85,86]. Basir et al., found evidence of impaired endometrial blood flow in association with significantly high estrogen levels in high responders to ovarian stimulation [87].
Although pregnancy outcome tended to be poor in patients with higher mean uterine arterial impedance indices, the predictive value of using a specific resistance index (RI) or pulsatility index [PI] variable in assessing endometrial receptivity seems to be limited [88]. One of the explanations is that the major uterine compartment is the myometrium and not the endometrium, and thus most of the blood passing through the uterine arteries never reaches the endometrium. A more logical approach would be to evaluate the vascularization around the endometrium directly in an attempt to assess endometrial receptivity.
Histological studies have confirmed that the sub-endometrial halo surrounding the endometrium represents the innermost layer of the myometrium, and compared with the outer myometrium, it consists of a distinct compartment of more tightly packed muscle cells with increased vascularity. Studies have shown that interactions between the junctional zone and the endometrium may play an important role in the implantation process [89,90] and endometrial-sub-endometrial blood flow distribution pattern assessed by transvaginal color Doppler before ET was found to correlate with the implantation and pregnancy rate after IVF-ET [91]. With the absence of sub-endometrial blood flow, even in the presence of other favorable parameters, no conception was achieved. By using a similar approach, Salle et al., calculated a uterine score in the secretory phase of the menstrual cycle preceding IVF-ET [92]. Immunocytochemistry study revealed that the sub-endometrial myometrium, also called the junctional zone myometrium or archimyometrium, exhibits a cyclic pattern of estrogen and PR expression that parallels that of the endometrium [93]. Moreover, the responsiveness of the junctional zone has been shown to be associated with implantation success during IVF-ET treatment [90].
Many investigators have also noted the correlation of junctional zone contractions with pregnancy outcome in both natural [94] and assisted reproduction cycles [95]. Less-active junctional zone contractility is associated with higher pregnancy rates
Kupesic et al. compared the 2-D and 3-D ultrasonographic scoring systems by combining parameters including endometrial thickness, volume, echogenicity, and sub-endometrial blood flow. They found the two systems had similar efficiencies in predicting pregnancy outcome of IVF-ET procedures [96]. Several investigators noticed that when the endometrial and sub-endometrial flow parameters were combined, significant differences were found between pregnant and nonpregnant patients [97-99]. In contrast, there was no significant difference if attention was only focused on intraendometrial or sub-endometrial blood flow [100]. These results imply that the endometrial/sub-endometrial area must be considered as a whole in evaluating endometrial perfusion.
One hallmark of implantation is increased vascular permeability at the implantation site. Vasoactive agents, including histamine, platelet-activating factor, vascular endothelial growth factor, and eicosanoids, have been studied during implantation [101,102]. Vaginal E2 administration improves endometrial proliferation and uterine perfusion, presumably because of combined local and systemic effects, but may interfere with P-induced uterine relaxation [103].
In a study by Basir et al., [104] the investigators compared the hemodynamic parameters of the utero-ovarian vasculature and the endometrial spiral arteries of women who showed a moderate response with women whose E2 concentrations were in excess of 20,000 pmol/l after ovarian stimulation. Despite low uterine PI and RI, the endometrial blood flow in high responders appears to be impaired. The authors concluded that this might contribute to the decline in implantation efficiency noted in high responders. The decreased endometrial blood flow despite the increased blood flow in the uterine arteries may indicate a shunt of blood flow from the endometrium into the myometrium.
The authors [104] found low pulsatility index and resistance index of the ovarian arteries indicating neovascularization and increased capillary permeability in the ovarian tissue of high responders. The authors suggested that the blood flow might be directed through the utero-ovarian collaterals to the ovaries. However, because the sample size in this study was small (19 patients with E2 > 20,000 pmol/L), further larger prospective studies are required to confirm the effect of excessively high concentrations of serum E2 on endometrial blood flow.
Moreover, the increase in hormonal concentrations in the peripheral plasma leads to a decrease in peripheral vascular resistance [105] and a decreased contractility of the uterine muscles. This results in relaxation and opening up of the small uterine vascular channels, which may also cause an increase in the capillary permeability. In a study on the endometrial morphological changes at high concentrations of E2, a significantly greater number of vessels and endometrial edema in women who responded excessively to ovarian stimulation was demonstrated. Therefore, it was postulated that the blood flow through these minute endometrial vessels may be very slow and the weak Doppler flow signals arising from them could not be picked up by the color Doppler despite low uterine PI and RI. The increase in capillary permeability and dilatation leads to extravasation of fluid from the intercellular to extracellular compartments, and hence endometrial edema [106]. In another study the investigators suggested that the blood flow per capillary might actually be reduced during edema [107].
Successful implantation and continuing development of implanted embryo depends on a complex series of cellular and molecular events between the blastocyst and the endometrium [108]. The decline in blood flow could therefore impede the exchange of essential nutrients, bioactive molecules and reactive compounds that are vital for implantation with absent endometrial and intra-endometrial vascularization appeared to be a useful predictor of failure of implantation in IVF-ET cycles [109].
Whether fertilization occurs in vivo or in vitro, most human embryos will not develop through gestation [110] with a very high proportion of developmental failure during the preimplantation stages associated with chromosomal defects of oocyte origin [111]. Van Blerkom suggested that the dissolved oxygen content of pre-ovulatory follicular fluid and the developmental competence of the corresponding oocyte were related [112]. A developmentally significant association between the chromosomal normality of the human oocyte and the level of intra-follicular oxygen and peri-follicular vascularity was reported suggesting that hypoxic intra-follicular conditions that result from the failure of an appropriate microvasculature to develop around the growing or pre-ovulatory follicle(s) could be a proximate cause of the maternal-age-related increase in the incidence of trisomic conditions [112-114].
Effect on Integrins
Integrins are a family of cell adhesion molecules. Previous work has shown the expression of integrins in the endometrium changes during the menstrual cycle [115]. Three integrins in particular (1β1, 4β1 and vβ3) are thought to play a vital role in implantation as all are expressed during the 'implantation window'. The β3 and β1 integrins have also been shown to be reduced in infertile patients using flow cytometry [116]. Aberrant patterns of integrin expression have also been associated with certain diagnoses in infertile patients, including luteal phase defects, endometriosis, hydrosalpinx and unexplained infertility [117].
The exact role of integrins remains controversial and results have not been duplicated in all studies. Creus and co-workers showed no difference in integrin expression in patients who became spontaneously pregnant compared with those that do not [118]. Thomas et al., demonstrated that integrin expression seems to be reduced in the glandular epithelium in the endometrium after ovulation induction, irrespective of the dating. The authors concluded that there might be an ideal E2 level that should be reached during IVF-ET treatment as low estrogen levels might reduce the yield of oocytes, but high levels might impair the receptivity of the endometrium reducing integrin expression and leading to lower implantation rates [119].
3.1.2 Effect of supraphysiological estrogen levels on the developing oocyte (follicular and oocyte development)
As the contributor of the bulk of the cytoplasm to the zygote and half of its nuclear DNA, the key importance of the oocyte is unquestionable. The principle, that embryogenesis is rooted in oogenesis, has been understood for nearly a century, and is now becoming better understood at the molecular level. During follicular growth, the oocyte is in close contact with granulosa cells through gap junctions and is therefore under the influence of the follicular environment. Since oocyte maturation is such a long process, any adverse events occurring during this period can damage vital structural molecules or whole organelles, resulting in oocytes with reduced developmental competence. These oocytes may in turn give rise to preimplantation embryos with a compromised viability, and hence, a reduced implantation potential [120]. The ER gene is expressed in the human cumulus-oocyte complexes and oocytes but not in granulosa/cumulus cells which might suggest a lack of receptor-mediated autocrine effect of estrogen during folliculogenesis. Conversely, estrogen secreted by granulosa/cumulus cells, may exert a paracrine effect to influence oocyte maturation and fertilization competence directly [121]. It was reported that retrieval of >10 oocytes during IVF-ET cycles was correlated with oocytes of lower quality, as manifested by a decrease in the fertilization rate [122].
Effect on the chromosomal structure and cytogenetics of the oocytes
Studies with animal systems have indicated that a single cycle of ovarian stimulation can have adverse effects on oocyte competence during early development, and may well have downstream effects on the normality of fetal growth and development. Whether developmental defects could result from chromosomal malsegregation during ovarian stimulation-induced meiotic maturation has been examined in the mouse system. Earlier studies compared oocytes obtained after spontaneous or hormonally induced ovulation and found no increase in the incidence of non-disjunction or oocyte aneuploidy after ovarian stimulation [123]. In contrast, cytogenetic analysis of pronuclear stage mouse eggs after single cycle of ovarian stimulation showed chromosomal aberrations largely confined to the female pronucleus, indicating developmental compromise prior to fertilization [124]. Sengoku and Dukelow found comparable frequencies of aneuploidy for cleavage-stage hamster embryos produced in natural and pregnant mare serum gonadotropins (PMSG)-stimulated cycles, indicating that peri-implantation mortality may have an epigenetic origin [125].
In IVF-ET programs, the high rate of oocyte recovery and successful fertilization in vitro contrasts with a relatively high rate of conception failures, as even the most advanced IVF-ET clinics can provide a 33% success rate per cycle at best [12], despite multiple embryo transfers. The possible reason for this conception failure is the high frequency of lethal chromosomal abnormalities that prevents embryonic development beyond the pre- and post-implantation stages. The high frequency of spontaneous abortions also indicates that the proportion of embryos with genetic defects is significant. Cytogenetic analysis of the early stages of cleavage indicates that chromosomal aberrations may be found in 35–44% of pre-implantation embryos produced in vitro [126]. It has to be emphasized that the majority of pre-implantation embryos carry numerical chromosomal defects. However, the high incidence of chromosomal anomalies is presumably biased by the fact that only embryos with a poor morphological score were analyzed (discarded embryos). It nevertheless indicates that the low implantation rates of human pre-implantation embryos in IVF-ET programs are the consequence of natural selection [127]. Aneuploidy is the most common abnormality found in normally developing embryos following ovarian stimulation and IVF-ET [128], but other aberrations were also revealed. Polyploidy and multinucleation were frequently described in arrested embryos
Estrogen is well known to induce chromosomal and cytogenetic damage. The natural hormone E2 has clearly been shown to induce alterations in chromosome number such as losses or gains of whole chromosomes [129,130], chromosome translocations [131,132], and gene amplifications [133,134]. In addition, there is preliminary evidence of estrogen-induced gene mutations and gene deletions [135,136].
The synthetic estrogen, diethylstilbestrol (DES) causes a severe yet reversible deterioration of meiotic spindle microtubule organization during maturation of the mouse oocytes [137]. High doses of E2 were found to induce numerical chromosome changes (both chromosome gains and losses) similar to the reported observations with the synthetic estrogen, diethylstilbestrol [129]. Estrogen is also known to cause direct DNA damage via its catecholestrogen metabolites [138,139]. There are several types of free radical-mediated DNA damage, which are induced by estrogens and/or their metabolites [140-142].
As explained earlier, supraphysiological estrogen levels associated with ovarian stimulation impair the uterovarian blood flow resulting in follicular hypoxia. Gaulden proposed that follicular hypoxia might have a potent adverse influence on spindle organization and the normality of chromosomal segregation in the human oocyte [143].
Effect on the mitochondrial function of the oocytes and embryos
Van Blerkom has suggested that the developmental competence of mouse and human early embryos is related to the metabolic capacity of the mitochondria. It is thought that mitochondrial replication does not begin until after implantation, and that paternal contribution is minimal. Therefore the preimplantation embryo is completely reliant on maternally inherited mitochondria in the oocyte. Deletions and mutations in oocyte mitochondrial DNA may lead to mitochondrial dysfunction, influencing energy production and apoptosis in oocytes and early embryos, resulting in aberrant chromosomal segregation or developmental arrest [144].
The classical model of E2 action has been described to be mediated by cytoplasmic/nuclear partitioning receptor proteins that stimulate gene transcription upon binding to specific DNA sequences [145]. However, there are increasing functional evidences for extra nuclear/cytoplasmic localization of steroid hormone receptors. Several studies showing rapid non-genomic actions of steroids have led to speculate about the existence of cell-surface resident receptor forms [146-148]. Reports have documented the presence of estrogen binding proteins localized at the plasma membrane [149,150]. Independently, the known direct effects of various steroids on mitochondrial gene transcription support the idea of receptor attachment to the mitochondrial genome [151]. It was also identified early that estrogen specific binding sites were associated with mitochondrial and microsomal structures [152].
The mitochondrial-enriched subfraction represented an important source of E2 binding, where the steroid was recognized in a stereospecific and high affinity manner. The existence of mitochondrial and membrane estrogen binding sites correlated with the presence of ER but mainly with ER proteins. Using macromolecular E2 derivatives in Ligand Blot studies, both mitochondrial and membrane estrogen binding proteins were found in the uterus, and the ovary. This differential cellular partitioning of ER and forms may contribute to the known diversity of E2 effects in target organs [153].
Recently, in myocardial cell model, it was reported that at physiological concentrations, which do not inhibit mitochondrial functions, estrogens can protect heart mitochondria from the loss of cytochrome c induced by high calcium, and this might be one of the possible mechanisms by which estrogens preserve myocardial cell viability after ischemia/reperfusion [154]. High concentrations of estrogens (50–100 M) have been found to have a damaging effect on mitochondrial functions by strongly inhibiting mitochondrial respiration and membrane potential presumably due to decreased activity of the respiratory chain. The inhibition of the respiratory chain may be due to non-specific binding of estrogens to hydrophobic regions of the mitochondrial membranes, which may change protein/lipid interactions, disturb electron transport through the inner mitochondrial membrane and reduce membrane potential [155].
We postulate that supraphysiological levels of estrogen attained during ovarian stimulation may affect the mitochondrial function of the developing oocyte and embryo. This could be one of the mechanisms behind impaired developmental capacity of the oocytes and embryos obtained after ovarian stimulation.
3.1.3 Effect of supraphysiological estrogen levels on the spermatozoa and sperm/oocyte interaction
Sperm are exposed to estrogens within the male tract, and P450 aromatase has also been identified in human spermatozoa [156]. This raises the possibility that the spermatozoa can provide a continuing local source of estrogens in the epididymis, as well as while in the female tract on its journey to fertilize the oocyte. Inside the female genital tract, the spermatozoa would also be exposed to estrogens, particularly in tubal fluid following follicle rupture and when in close vicinity to released oocytes [157].
Estrogen receptors in the spermatozoa
Non-genomic effects of estrogens have also been reported in several cell types including the spermatozoa [158]. Immunohistochemical detection of ER has been reported for human spermatozoa, with distribution in both the head and flagellum [159].
Possible effect of estrogen on the spermatozoa function
After ejaculation, in vitro, the spermatozoa are initially unable to fertilize [160]. The spermatozoa acquire the capacity to fertilize, after a certain period of time that is species-specific when exposed to an appropriate environment either in vivo or in vitro. This process is called 'sperm capacitation' [161]. Capacitated spermatozoa are able to express hyperactivated motility, to undergo the acrosome reaction, and to fertilize an oocyte [160]. Estrogens are believed to have a possible effect on these events (capacitation and acrosome reaction), which would have important consequences on fertility in vivo.
Recently, Adeoya-Osiguwa provided evidence that E2 and environmental estrogens can significantly stimulate mammalian sperm capacitation, acrosome reactions. They found that in uncapacitated cells, E2 at 00.001 μmol/l, significantly stimulated capacitation and acrosome reactions while in capacitated cells, E2 had no effect. The authors concluded that whether these responses have effects on fertility in vivo remains to be determined, along with the mechanisms of action involved [157]. It is pertinent to mention here that the average E2 concentrations in follicular fluid from mature oocyte are in the micromolar range [162,163].
The regulation of capacitation is very important in mammalian fertilization as evidence suggests that once capacitation has been initiated it will usually continue unchecked, frequently resulting in the spermatozoa undergoing spontaneous acrosome reactions and thus becoming non-fertilizing [164].
Adeoya-Osiguwa argued that E2 and the environmental estrogens appear primarily to stimulate the spermatozoa, accelerating the rate of capacitation and then promoting 'over-capacitation' in at least some of the cells, resulting in the acrosome reaction [157]. Since already acrosome-reacted spermatozoa are non-fertilizing [160], similar responses occurring in vivo could reduce the number of potentially fertilizing cells and so have an undesirable effect on fertility. As capacitation and fertilization occur in the female reproductive tract, it is likely that any effects of environmental estrogens on sperm function would be more pronounced in the female, but effects on mature spermatozoa awaiting ejaculation cannot be ruled out.
3.1.4 Effect of supraphysiological estrogen levels on the embryo
Mouse and human embryos, when cultured in vitro, undergo a delay in development compared with those grown in vivo. This delay can be caused by suboptimal culture conditions, but possible influences of ovarian stimulation cannot be excluded [165]. In order to determine if implantation failure associated with ovarian stimulation was due to abnormalities in the blastocysts or the endometrium, decidualization studies and embryo transfers to pseudopregnant recipients were performed [166,167]. The uterus of a large proportion of superovulated animals was unable to undergo decidualization in time, whereas embryo transfers to pseudopregnant females resulted in normally developing fetuses, which indicated that hormonally treated oocytes themselves were not affected. Some studies found that the E2 concentrations in the fresh cycle were not related to the success of frozen-thawed embryo transfer cycles indicating that embryo quality seemed unaffected by the high estrogen levels [168,169]. However, there is increasing evidence suggesting that ovarian stimulation and the associated high estrogen levels are detrimental on the embryo and associated with a decrease in the fertilization rate. When compared with blastocysts derived from naturally cycling mice, blastocysts that developed in vivo in superovulated mice were found to have fewer microvilli on their surface [170], a reduced [35S]-methionine uptake [171], and a lower cell number and mitotic index [172]. A reduced cell number and a two-fold decrease in viability post-transfer of embryos from gonadotropins-stimulated hamster females, was also observed [21]. Furthermore, it has been reported that the proportion of abnormal preimplantation embryos increases after superovulation, and that blastocysts have a smaller trophoblastic outgrowth in vitro [173]. Moreover, in mice as well as in humans, there is evidence for steroids being regulators of gene expression in the embryo and endometrium, and that embryo morphology and rate of development – both of which reflect embryo quality – have a genetic basis. Also, ovulation induction therapy has been found to be associated with an increased rate of mosaicism in the embryos, which fail to implant [52].
It has been proposed that high E2 levels after COH impair endometrial receptivity because oocyte quality, fertilization rate, and embryo cleavage (until day 2) were normal in patients with a high response [174] and the quality of embryos and the implantation rate seemed normal in subsequent frozen-thawed embryo transfer [43]. However, high E2 levels were found to be deleterious to embryo adhesion in vitro, mainly because they have a direct toxic effect on the embryo that may occur at the cleavage stage [175].
Mouse and human embryos, when cultured in vitro, undergo a delay in development compared with those grown in vivo. This delay can be caused by suboptimal culture conditions, but possible influences of ovarian stimulation cannot be excluded. In the mice, preimplantation embryonic development in vitro and in vivo was found to be negatively influenced by the ovarian stimulation itself, and results in an impaired blastocyst formation and fetal growth retardation at day 14 of gestation. The authors suggested that a similar negative effect of ovarian stimulation on oocyte and embryo quality seems likely in IVF-ET which might explain in part for the delay in embryonic development after IVF-ET, and for the low birth weight often observed after assisted reproductive technologies [165].
Effect of estrogen on blastocyst hatching
The successful hatching of the embryos is thought to be a key event in the implantation process. One reason for the low implantation rate that has been suggested is the limited ability of blastocysts to hatch from the zona pellucida. Suboptimal culture conditions might induce the hardening of zona pellucida, which could limit the hatching ability of embryos [176]. To help embryos hatching from their zona pellucida during blastocyst expansion, different types of assisted hatching have been developed, including mechanical partial zona dissection or zona drilling, chemical zona drilling with acidic Tyrode's solution, and the laser dissection technique [177]. However, there is controversy about the benefit of assisted hatching on the improvement of the implantation rate and pregnancy rate. Some agreed as to the benefit of assisted hatching in women with advanced age, in women with repeated IVF-ET failure [178].
The degree of zona pellucida thickness variation of the transferred embryos has been found to exhibit a strong correlation with clinical pregnancy outcome following IVF-ET treatment and to be important for embryo selection during clinical transfers [179,180]. Zona pellucida thickness variation and character were found to correlate with implantation. Implantation rates were found to range from 10% for embryos with uniform thickness to 29% with thin or irregular zona pellucida [181,182]. These reports suggested that patients transferred with embryos with thinner zonae had a better chance of successful implantation and pregnancy as compared to those transferred with embryos having thicker zonae. Other reports have proposed zona pellucida thickness variation as a reliable marker for selecting thawed as well as fresh human embryos for transfers [183,184]. Cohen [185] conducted a retrospective analysis of zona pellucida thickness of transferred embryos through video recordings and concluded that the variations in zona pellucida thickness rather than the zona pellucida thickness per se of the transferred embryos was a stronger predictor of the IVF-ET.
A significant linear relationship was reported to exist between the mean zona pellucida thickness of each patient and the maximum E2 level and an increasing one with the hMG dose. The authors found that the zona pellucida thickness was basically an individual feature that influenced the fertilization rate [186].
3.1.5 Effect of supraphysiological estrogen levels on the ovaries [corpus luteum], pituitary, and hypothalamus
Abnormalities in the luteal phase have been shown in virtually all the stimulation protocols used in ovarian stimulation, on the hormonal, as well as on the endometrial level. All three aspects of a defective luteal phase, that is, a shortened luteal phase and/or low mid-luteal serum progesterone concentrations and/or abnormal endometrial histology, have been regularly observed in IVF-ET cycles. For that reason, luteal-phase supplementation with hCG or progesterone increases pregnancy rates, and its necessity has been well established, at least in GnRH-agonist cycles [187].
In the 'pre-agonists era', Edwards and Steptoe were the first to postulate luteal phase inadequacy resulting from ovarian stimulation as a cause of failure of IVF-ET cycles. With the introduction of the GnRH-agonist, used in ovarian stimulation cycles to avoid premature luteinizing hormone [LH] surge, luteal phase inadequacy was reported. A meta-analysis of different clinical trials demonstrated beneficial effects of luteal support when ovarian stimulation was carried out with human menopausal gonadotropins [hMG] in association with GnRHa [187].
Recently, GnRH antagonists have become available for clinical use. Whether GnRH antagonists induce down-regulation of pituitary GnRH receptors is still a subject of investigation [188]. It has been demonstrated that chronic administration of the GnRH antagonist Cetrorelix in rats causes an important down-regulation of pituitary GnRH receptors [189].
Direct effect on corpus luteum
Different estradiol receprors have been detected in human corpus luteum, indicating that estrogen might be a local regulator of corpus luteum function [190]. In the pre-agonist era, the alteration of the Estradiol/progesterone (E2/P) ratio was considered a main cause of luteal-phase inadequacy, possibly through the luteolytic action of E2 [5]
Estrogen is thought to exert a direct luteolytic action in primates as exogenous administration of E2 reduces progesterone concentrations during the luteal phase [191], probably via inhibiting the enzyme 3 beta-hydroxysteroid dehydrogenase, which is mandatory for progesterone synthesis [192]. Moreover, although the exact mechanism has not yet been established, estrogen may play a role in the regulation of proteins involved in the process of luteal-cell apoptosis [193].
Normal corpus luteum function is dependent on the proper function of the pituitary gland and hypothalamus. Adequate luteinizing hormone [LH] surge during ovulation and continuous tonic LH pulses during the luteal phase are necessary for the proper development of the corpus luteum. Corpus luteum dysfunction due to the effect of ovarian stimulation on the pituitary gland and hypothalamus will be discussed later.
Effect on the pituitary gland and hypothalamus
Normal corpus luteum function requires optimal follicular development in the follicular phase, especially follicle stimulating hormone (FSH) stimulation, adequate luteinizing hormone (LH) surge during ovulation and continuous tonic LH pulses during the luteal phase. In turn, the normal luteal phase is characterized by an optimal hormonal environment and adequate endometrial secretory transformation. As many factors contribute to a normal corpus luteum function, any alteration might exert a deleterious effect on the final target, the endometrium, leading to embryo/endometrial asynchrony [5]. Luteal phase insufficiency due to corpus luteum defects and LH suppression is known to be associated with failure to achieve and maintain pregnancy [194].
Effect on LH secretion
The lifespan and steroidogenic capacity of the human corpus luteum is dependent on continuous tonic luteinizing hormone secretion as well as healthy adequate gonadotropins surge. Feedback mechanisms from ovarian steroids and GnRH pulses regulate LH secretion during the luteal phase, but a number of autocrine and paracrine factors within the ovary might also play a role in controlling corpus luteum function [5].
Effect on LH surge
It is obvious that during COH for ART, the prevention of endogenous LH surge is mandatory to avoid the occurrence of premature LH surge as well as for timing of oocyte retrieval.
However, during ovarian stimulation cycles in which no pituitary suppression is used, there are data that suggest a defective endogenous gonadotropins surge. The gonadotropins surge is an event crucial for final oocyte maturation, ovulation, and subsequently for corpus luteum function. The duration of the LH peak seems to be more important than its amplitude for the induction of ovulation [195]. Ovulation induction by hCG is not physiological; the absence of an FSH surge, and the long duration of LH activity associated with hCG action, would contribute to some of the luteal phase abnormalities [196].
Messinis demonstrated that an attenuated LH surge is obtained in normally cycling women during superovulation induction with sequential clomiphene/hMG treatment. The peak values and the duration of the LH surge to have significant negative correlations with the plasma E2 levels, the number of follicles, and the total follicular fluid volume aspirated at laparoscopy. This suggests that during superovulation induction for IVF-ET, the endogenous LH surge is attenuated by factors, which are related to the degree of ovarian hyperstimulation [197].
As shown above, the induction of superovulation in women with human gonadotropins may result in blockage of the endogenous LH surge, but the reasons for this are not known. A high number of small follicles have been suggested to have a suppressive effect on both tonic and mid-cycle gonadotropins secretion [198].
Effect of abnormal LH surge on nuclear maturation of the egg
A timely LH surge of adequate amplitude and sufficient duration is important to bring about rapid and complex cellular differentiation, resulting in cascades of tightly coupled biochemical events, which initiate oocyte maturation, ovulation, and corpus luteum formation [199]. It is known that a midcycle LH surge of sufficiently high amplitude and duration is important for both nuclear and cytoplasmic maturation, which ensure the normal fertilization and developing potential of oocytes [199]. For PMSG-hyperstimulated rats, higher doses of hCG are required to completely ovulate the expanded cohort of preovulatory follicles [200].
Effect on LH tonic pulse during the luteal phase
Low luteal LH levels have been described after human menopausal gonadotropins treatment6 and after GnRH-agonist treatment or after GnRH-antagonist treatment. These low, almost undetectable, luteal LH levels may not be able to support corpus luteum. As a result, a shortened luteal phase and low mid-luteal progesterone concentrations have been described in cycles stimulated with the association either of a GnRH agonist or a GnRH antagonist [195,196].
Supraphysiological progesterone serum concentrations may also interfere with the pituitary's luteinizing hormone secretion by disturbing the feedback control mechanisms and may result in a reduction of the LH serum levels [195,196]. Progesterone modulates LH secretion during the luteal phase by influencing the LH pulse amplitude and pituitary release of LH [201]. A longer exposure to progesterone or the combined action of estrogen and progesterone decreases LH release [302]. As ovarian stimulation results in supraphysiological steroid serum concentrations, these high steroid levels may adversely affect LH secretion via a long-loop feedback mechanism. In turn, disturbed LH secretion may induce a luteal-phase defect with premature luteolysis, low progesterone levels and shortened luteal phase. It might therefore be hypothesized that deviation from the normal hormonal environment could be a prevalent effect of ovarian stimulation in the luteal phase; despite the use of different stimulation protocols [7]. This might be a possible explanation of the observation that in natural cycle, luteal-phase length was normal after GnRH-antagonist treatment [203]. However, in GnRH-antagonist cycles and after minimal ovarian stimulation, luteal-phase length was normal despite an abnormal endocrine profile [204].
3.1.6 Other probable effects of supraphysiological estrogen levels
There are other less defined probable mechanisms through which supraphysiological estrogen levels may cause adverse effects on the outcome of infertility treatment.
(A) Leptin-mediated effect
Recently, an important role for the leptin, the secretory product of adipocytes, in reproductive medicine has emerged and it is interesting to discuss in brief a possible link between leptin, induction of ovulation and aromatase inhibitors.
Soon after its discovery in the early 1990s, it was recognized that leptin played a significant role in reproduction, providing a critical link between metabolic state and fertility. It now appears that leptin may have an important role in both normal ovarian physiology and pathophysiology. Research has revealed that a minimum level of leptin stimulation is required for maintenance of fertility in animals and humans. Conversely, elevated leptin levels may impair fertility [205].
Leptin is a secretory product of adipocytes that correlates significantly with the body mass index with increased levels in obese women [206]. It can influence reproduction through central (on GnRH neural system) and peripheral (on the ovary directly) mechanisms [205]. A positive correlation has been found between leptin and BMI, as well as between leptin and testosterone in women with polycystic ovarian syndrome (PCOS) [207].
Numerous studies have shown that circulating leptin concentrations rise in parallel with E2 during ovarian stimulation [208-213]. These changes are not likely to be a direct action of FSH because FSH decreases as E2 and leptin rise during natural cycles, and high FSH levels experienced during ovarian stimulation simulate endogenous levels in postmenopausal women, who have lower serum leptin concentrations than premenopausal women [205]. Bützow [206] found that the larger the increase in serum leptin concentrations during FSH stimulation, the poorer the ovarian response in terms of number of follicles and retrieved oocytes. Moreover, higher serum leptin levels were found in oligo- and amenorrheic women who failed to respond to clomiphene therapy [214].
The effect of body weight on outcomes of assisted reproduction has been investigated. Fedorcsak [215] reported that among patients who conceived, overweight patients (BMI >25) had fewer oocytes retrieved, a higher miscarriage rate, and lower live birth rate. In a much larger retrospective study that included 8822 embryo transfer cycles, the cumulative pregnancy rate progressively decreased as BMI increased from <25 to >35 [216].
Higher follicular fluid leptin concentrations correlated with lower intrafollicular oxygen concentration [pO2], [217] a condition that negatively impacts oocyte developmental competence [218] with a direct evidence of a relationship between leptin levels and ART outcome reported by Mantzoros [219] who found significantly lower follicular fluid leptin concentrations in women who became pregnant within three cycles of IVF-ET or gamete intrafallopian transfer (GIFT). More recently, a significant negative correlation between non-fasting serum leptin levels measured at the beginning of FSH stimulation and pregnancy success in women undergoing first attempt IVF-ET cycles was reported [220]. Moreover, as leptin receptors are expressed in the human endometrium, [221] a role for leptin in endometrial receptivity cannot be excluded. These results imply that elevated leptin may be a key factor in obesity-related fertility problems, and conversely that elevated leptin may negatively impact fertility independently of body mass. Fewer good embryos and lower implantation rate suggest that elevated leptin impairs oocyte developmental competence and/or early cleavage stage embryo development, possibly via direct actions on the follicle [205].
E2 has been reported to increase leptin mRNA expression. In human adipose tissue culture, E2 stimulated leptin secretion in women but not in men [222]. In women, E2 was found to increase ob mRNA expression and leptin release. Moreover, in adipose tissue of women, the estrogen precursors; testosterone and dehydroepiandrosterone also induced an increase in leptin secretion, an effect that was prevented by the aromatase inhibitor letrozole. Moreover, the stimulatory effect of E2 observed in women was antagonized by the antiestrogen ICI182780 [223].
(B) Coagulation system
Estrogen has been pointed out as a pre-thrombotic factor. It has long been established that both pregnancy and oral contraceptive use have resulted in an increased level of many coagulation parameters. Generally, this has been thought to be a result of the estrogen component. Although it has been well established that long-term exposure to exogenous contraceptive steroids can have a promoting influence on the potential for thrombosis in women, it is less clear what role high levels of endogenous steroids might play. Undefined coagulation abnormalities were reported after hMG- and hCG-induced hyperstimulation of the ovary in several women [224-227]. Kim et al., [225] noted a large increase in fibrinogen after hMG treatment, accompanied by "significant increases" in the prothrombin time. However, the statistically significant activation of clotting factors occurring during controlled ovarian hyperstimulation is usually not accompanied by clinically significant coagulation disorders that may be explained by either of the following: (1) the increased levels of clotting activity were still "within normal limits"; (2) none of the patients had any conditions known to predispose to coagulopathies (history of coagulopathies, phlebitis, damaged or compromised endothelial cells, etc.); or (3) a combination of the two [226]. However, such subtle coagulation disturbances may exert an adverse effect on endometrial receptivity and the early development of the placenta by affecting the microcirculation.
Two case reports were published of activated protein C-resistant women who suffered a thrombotic event during IVF-ET treatment [227,228]. Curvers [229] reported that the only coagulation parameter that changed considerably during IVF-ET treatment was the activated protein C (APC). In their study, the authors observed that hyperstimulation, i.e. high estrogen levels, induce APC resistance, and that under these conditions both the absolute values and the changes in the APC and the estrogen levels (hyperstimulation-baseline) correlate significantly. Prior to that study, it was reported that high estrogen levels were not associated with APC resistance [230,231]. However, the APC resistance test used in these studies, which is based on quantification of the effect of APC on the clotting of plasma initiated via the intrinsic coagulation pathway, is not very sensitive to changes in sex hormones [232]. On the other hand, Curvers et al. [232], used an assay that quantifies down-regulation of extrinsic coagulation by APC that is particularly sensitive to hormonal changes in women [232].
We think it is possible that the reported effect of ovarian stimulation-associated supraphysiological levels of estrogen on the coagulation system, though being modest, may contribute at least partially to the reduced implantation rate observed after assisted reproduction. This could be due to an impact on the microcirculation in the endometrium that could affect the early stages of the implantation as well as the early development of the placenta. However, more studies are needed to support this hypothesis.
(C) Tubes and tubal transfer
Effect on the oviductal environment
Oviducts are biologically active, providing an environment that sustains and enhances fertilization during early embryonic development as the embryo travels toward the uterine cavity. Following superovulation, the fluid from the oviduct seems to impair embryo development. Furthermore, a stimulated oviductal environment has also been shown to have a negative influence on the implantation capacity of mouse embryos [233].
Superovulation in the mouse was described as a model for intra-uterine growth retardation
Superovulation is associated with a slower preimplantation embryo development, a later and impaired implantation and a prolonged gestation [234]. This suggests that the oviductal milieu rather than the embryo quality are responsible for the adverse effects observed after superovulation. The stimulated oviductal environment impairs the developmental capacity of embryos in comparison with untreated pseudopregnant females. In-vitro culture is also suboptimal but better than the stimulated oviductal environment. However, a detrimental effect of hormonal stimulation upon the oviductal environment has not yet been demonstrated in the human. A possible potential negative effect, however, is not contradicted by observations reporting a higher pregnancy rate after ovarian stimulation and IUI compared to insemination alone [235]. In the human, there are observations that GIFT results in higher pregnancy rates in comparison with IVF-ET [236,237]. It is difficult to interpret these data since the embryos were exposed longer to the possible deleterious stimulated oviductal environment-using GIFT whereas in IVF-ET, possible "suboptimal" culture conditions as opposed to in-vivo conditions were used. Since the introduction of sequential culture media, incorporating amino acids, vitamins and growth factors, the in-vitro culture of embryos has been improved, resulting in implantation rates of >50% per transferred blastocyst [238]. Confirmation of these data in a trial would result in higher pregnancy rates than ever reported for GIFT [239].
Effect on the oviductal embryo transfer
Akira [240] has found that ovarian stimulation was associated with accelerated oviductal embryo transport. The authors have concluded that increased implantation failure in superovulated rats may result from the accelerated embryo transport resulting from elevated E2/P ratio. Accelerated oocyte/embryo transfer has been postulated to be the mechanism behind which high doses of estrogen work as postcoital emergency contraception as explained later.
Post-coital contraception with estrogen
The achievement of very high levels of estrogen by administering exogenous estrogen has been suggested for postcoital contraception as early as the 1960s, when high-dose estrogen was identified as a highly effective emergency contraceptive [241].
Greenwald [242] compared the response of the rabbit, rat, mouse, hamster, and guinea pig, to a single post-coital injection of E2 cyclopentylpropionate and showed that post-coital treatment with estrogens caused either tube locking of embryos or accelerated transport to the uterus. Although other effects were also detected, the alteration in oviductal transport accounted for the contraceptive effect. Embryos that entered the uterus prematurely were expelled whereas whose sojourn through the oviduct was prolonged, degenerated.
Other investigators found that a single injection of E2 was given at different times after coitus revealed that a wide range of effectiveness can be achieved and suggested different mechanisms can account for the contraceptive effect when the same steroid is given at different times post-coitus [243,244]. Some believed the main target was the endometrium where they observed stromal edema, hemorrhage, and loss of decidua, all of which was considered unsuitable for implantation. Administering high dose of estrogen in the preovulatory phase was found to depress endometrial growth and angiogenesis through a negative influence on the vascular endothelial growth factor [245]. This suggested that estrogens might interfere with endometrial receptivity even if given before ovulation.
Other mechanisms for the contraceptive effect of post-coital estrogen were found to operate in monkeys. If given in the follicular phase, so as to advance the preovulatory increment in plasma estrogen, they evoke a premature LH surge that does not trigger ovulation and the formation of a functional corpus luteum, and the spontaneous LH surge is delayed or suppressed [246]. ER present in the granulosa cells of antral and preovulatory follicles and in luteal cells [247] allow for a diversity of effects in the ovary. In rhesus monkey, supraphysiological doses of estrogen given in the mid or late follicular phase induce atresia or luteinization without rupture of the dominant follicle, reduce the viability of granulosa cells, reduce the synthesis of E2 and progesterone and are detrimental to the oocyte [248].
Whatever the mechanism of action of high dose of estrogen for emergency contraception is, success in preventing pregnancy provides another evidence of the toxic effect of the supraphysiological estrogen levels fertilization, implantation and early development of the embryo which result in failure of achieving pregnancy.
[D] Effect on other factors involved in decidualization and early developing placenta
Paracrine factors
The importance of paracrine factors in mediating the cellular and biochemical changes involved in embryo implantation has been recognized. Many growth factors and cytokines, such as inhibins and activins, whose expression is generally limited to developmental and pathological states, are produced by actively remodeling endometrial cells, and play crucial roles in regulating endometrial cell function. Example of these factors includes the inhibin and activin family in the paracrine regulation of endometrial receptivity, decidualization and implantation. Estrogen is known to play an important role in regulation of these factors as discussed by Jones [249].
Role of Calcitonin
The peptide hormone calcitonin is currently being evaluated as a potential marker of the fertile human endometrium. In ovariectomized animals, it has been shown that administration of estrogen together with progesterone inhibits progesterone-mediated calcitonin gene induction.29 Such antagonistic interactions between estrogen and progesterone pathways have been documented previously in breast and uterine cells. It has been proposed that these phenomena reflect transcriptional cross talk occurring between estrogen and PR co-expressed in the same target tissue. A complex interplay of the two ovarian hormones, progesterone and estrogen, in the uterine milieu is believed to be critical for optimal calcitonin gene expression [250,251].
3.2 Effect of the medications used for ovarian stimulation
3.2.1 Clomiphene citrate
In spite of the high ovulation rate with the use of clomiphene citrate (around 50–90%), the pregnancy rate is much lower (around 20–40%) [252-254]. Moreover, there is a higher than expected incidence of miscarriage in conception cycles following clomiphene citrate treatment [255]. Such discrepancy is believed to be due to the peripheral antiestrogenic effect of clomiphene citrate, particularly at the level of the cervical mucus [256,257] and endometrium, [258,259]. The persistence of the zu-isomer of clomiphene citrate in the body due to its long half-life (several weeks) and slow clearance adds to the accumulation of the antiestrogenic effects over subsequent cycles of administration [260,261]. There is also evidence of a direct harmful effect of a high concentration of clomiphene citrate and its isomers on fertilization, and on early mouse [262] and rabbit [263] embryo development. Such effects, however, were not confirmed in other studies [264,265]. There is still some controversy concerning a direct effect on the quality of oocytes associated with clomiphene citrate treatment [266,267]. Decreased uterine blood flow during the early luteal phase and the peri-implantation stage is another explanation for the poor outcome of clomiphene citrate treatment [268]. Other investigators have suggested the presence of other unrecognized infertility factors [269,270].
3.2.2 Gonadotropins
The existence of nongonadal gonadotropins (FSH, LH/hCG) receptors was first suggested by Ziecik [270] who conducted binding studies in the porcine uterus. Subsequently, nongonadal receptors were found in human tissues, including endometrium, myometrium, fallopian tube, umbilical cord, and brain, by using a variety of techniques, including immunohistochemistry; Northern, Western, and ligand blotting; and in situ hybridization [271-274]. This would suggest a possible direct action of the gonadotropins on the uterus and involvement in the endometrial development, implantation and establishment of pregnancy [275].
High concentrations of exogenous gonadotropins used for hyperstimulation of folliculogenesis were shown to be detrimental to oocyte and embryo development in many animal species. This is believed to be due to the associated supraphysiological E2 levels and other possible undetermined factors associated with ovarian stimulation. However, a direct effect of gonadotropins cannot be ruled out. It has been reported that early embryo loss due to superovulation could be rescued by an injection of goat antiserum against PMSG [276].
Effect of gonadotropins on LH surge
It has been suggested that during ovarian stimulation, the supraphysiological FSH levels that persist into the late follicular phase, thereby overriding selection of the single dominant follicle of the natural cycle, secretion of an ovarian factor(s) blocks estrogen-induced LH surges [277]. Accumulated evidence has indicated that the ovaries produce another non-steroidal substance, named "gonadotropins surge-attenuating factor" (GnSAF), which may play a role in the control of the midcycle luteinizing hormone surge in women [278-281]. Although GnSAF activity is present during superovulation induction, it is still unclear whether this factor plays a physiological role during the normal menstrual cycle. Treatment with FSH initially attenuated the response of LH to GnRH via the production of GnSAF from the ovaries, while around the midfollicular phase, the rising concentrations of E2 were able to overcome the attenuating effect of GnSAF and increase pituitary sensitivity to GnRH. The increased pituitary sensitivity in the midfollicular phase of the FSH-treated cycles, however, was not further enhanced in the late follicular phase despite the continuous rise in E2 values [282]. It is suggested that eventually GnSAF was able to overcome the sensitizing effect of E2
Effect of gonadotropins on chromosomal development
A dose-response relationship between the PMSG dose and the incidence of polyploidy in the CD-1 mouse has been reported with the level of polyploidy rising from 2.9% with 10 IU PMSG to 10.5% with 15 IU PMSG, in the zygot stage. Both a disturbance at maturation division and an error at fertilization were the cause of polyploidy [22]. Whether this is the direct effect of PMSG or the resultant ovarian stimulation is not known.
3.2.3 GnRH analogues
GnRH plays a pivotal role in the control of female reproduction and is secreted by hypothalamic neurons in a pulsatile way. It binds to specific receptors on pituitary gonadotrophs, which is followed by the secretion of the gonadotropins, LH and FSH, which regulate steroidogenesis and gametogenesis in the ovary [283].
GnRH analogues are able to suppress gonadotropins release and, subsequently, gonadal function. This is the basis for their clinical application in ovarian stimulation. Several agonistic or antagonistic GnRH analogues have been developed for this purpose [284].
Effect of GnRH analogues on ovarian steroidogenesis and corpus luteum function
GnRH receptors and GnRH receptor mRNA were found to be expressed in human granulosa-luteal cells GnRH receptors have been found in the luteinized human granulosa cells, and a possible direct effect of GnRH on ovarian steroidogenesis has been suggested [485,486].
Although there are many studies that investigated the possible direct effect of various GnRH agonists on ovarian steroidogenesis and hence the corpus luteum, there is no consensus of the effects in the human ovary. Also, there is little information concerning the direct effect of GnRH antagonists on ovarian steroidogenesis [487]. A few studies that looked at the effect of GnRH agonist treatments in vivo on in vitro steroidogenesis by human luteinized granulosa cells, found an impairment of progesterone production [287-290]. Minaretzis [291] have reported on the effect of the GnRH antagonist Nal-Glu compared with GnRH agonist leuprolide acetate treatment of patients undergoing controlled ovarian hyperstimulation. The progesterone production in human luteinized granulosa cells cultures was not different between the two groups. Both GnRH antagonists had no effect on basal or hCG-induced E2 or progesterone production by granulose lutein cells, independent of whether the cells were exposed to the compounds in vitro or in vivo. However, an in-vitro study demonstrated an inhibitory effect of GnRH antagonist on gonadal steroid secretion [292]. Pellicer and Miro who found that GnRH agonist exposure in vivo may affect human luteinized granulosa cells function in vitro. They compared the progesterone accumulation in human luteinized granulosa cells cultures between patients treated with clomiphene citrate/gonadotropins and GnRH agonist/gonadotropins. Granulosa cells obtained from patients treated with the GnRH agonist had lower progesterone production than cells isolated from women treated with clomiphene citrate/gonadotropins [293]. However, Minaretzis compared the human luteinized granulosa cells steroidogenesis in vitro from Nal-Glu and leuprolide acetate-treated women respectively. They reported that basal and gonadotropins-stimulated progesterone secretion was similar in the two treatment groups [291].
Weiss et al., found that in vivo treatment with triptorelin or the two GnRH antagonists, cetrorelix and ganirelix; did not have an effect on spontaneous or hCG-stimulated steroidogenesis. The authors also performed in vitro treatments with triptorelin, cetrorelix and ganirelix for up to 96 hours and did not find any effect of these treatments on basal or hCG-stimulated steroid production [287].
In all prior reports, which studied the effect of GnRH agonist treatment on ovarian steroidogenesis, only the effect of hCG on progesterone secretion by human luteinized granulose cells was investigated. Recently, we examined the effect of two known physiologic stimuli of ovarian steroidogenesis, hCG and insulin. We found that GnRH agonist treatment affected both the basal and hCG-stimulated progesterone production by human luteinized granulosa cells but the pattern of insulin-stimulated progesterone secretion was not affected. Because insulin and hCG may share common pathways beyond but not at the level of receptor activation, we hypothesize that in-vivo GnRH agonist might affect the expression and/or activation of LH receptors. In our study, we found that human luteinized granulosa cells from patients treated with GnRH antagonist responded to hCG in a fashion generally consistent with previously reported hCG-stimulated human luteinized granulosa cells progesterone production [294]. In addition, the basal progesterone production and absolute levels of insulin-stimulated progesterone production were both significantly higher than observed following GnRH agonist treatment. In contrast, human luteinized granulosa cells previously exposed in vivo to GnRH agonist had a blunted P response to hCG. Our results, therefore, support the hypothesis that GnRH agonists may have a direct negative effect on human ovarian steroidogenesis by the corpus luteum and suggest that luteal function may be less affected during IVF-ET cycles when GnRH antagonist is used. However, in another study, GnRH antagonist therapy in women undergoing ovarian stimulation was found to be associated with a significant effect on ovarian follicular steroidogenesis. The authors found the mean follicular fluid E2 concentration significantly lower in patients treated with GnRH antagonist than in those treated with GnRH agonist. However, no significant differences were found between groups in follicular fluid progesterone concentrations [295]. However, further studies are required to investigate the effect of GnRH agonist and antagonist on gonadotropins receptors and different enzymes involved in ovarian steroidogenesis by the corpus luteum.
Effect on LH secretion
Because pituitary LH secretion is dependent on GnRH stimulation and feedback mechanisms from ovarian steroids, any alteration may be deleterious. In cycles using GnRH agonists for ovarian stimulation, a significant drop in mid-luteal progesterone concentrations was observed, consistent with corpus luteum insufficiency. Long-term GnRH-agonist administration has been associated with a profound desensitization of the pituitary cells. In fact, studies on pituitary gonadotropins secretory capacity after GnRH-agonist treatment have indicated that this remains impaired for at least 14 days after the discontinuation of the GnRH-agonist and for the whole length of the luteal phase [296]. In a randomized trial, it was also demonstrated that, despite the early cessation of the GnRH agonist in the follicular phase, luteal-phase characteristics were abnormal [297].
GnRH-antagonist treatment has been shown to be effective in blocking the LH surge. GnRH antagonists bind competitively to pituitary GnRH receptors and cause an immediate inhibition of gonadotropins release. In contrast to GnRH agonists, it was suggested that treatment with GnRH antagonists might not adversely affect luteal LH secretion, since the pituitary maintains its responsiveness to the endogenous GnRH stimulus [298]. A normal luteal phase, in terms of duration and serum progesterone concentrations, was observed in natural cycles in which an antagonist was administered to prevent the LH surge [299]. However, data on the luteal phase from unsupplemented cycles after antagonist administration are limited as a result of the small number of patients involved and are rather controversial. Four out of six patients had either a shortened luteal phase or low progesterone concentrations in cycles stimulated with HMG and the GnRH antagonist Cetrorelix and receiving no luteal-phase supplementation. However, with or without luteal-phase supplementation, luteal LH levels were low, indicating that another mechanism might be involved [299,300].
3.2.4 Human chorionic gonadotropins
Supraphysiological steroid serum concentrations may interfere with LH secretion via long-loop feedback, but, additionally, the exogenously administered hCG might amplify LH secretion arrest via a second short-loop negative feedback. Although in the monkeys such a negative feedback exists [301] there is a debate in the literature about its existence in humans, with some of the studies supporting this hypothesis [302] and others not [303,304]. Nevertheless, findings from in-vitro studies further support this idea. GT1-7 neurons, which are morphologically and functionally similar to GnRH neurons, were found to contain LH/hCG receptors. In addition, exogenously administered hCG was found to decrease the expression of GnRH receptor gene in GT1-7 cells or GnRH secretion in immortalized GnRH neurons [305].
4 Measure to improve treatment outcome after ovarian stimulation and assisted reproduction
Even if the results of reproductive medicine have improved in terms of numbers of pregnancies, it is still striking that it is necessary to use stimulation which sometimes leads to hyperstimulation and multiple pregnancies, that embryo development in vitro is still limited, that implantation only occurs for 15–20% of transferred embryos and this ratio has not changed significantly along the last 25 years. We still need to improve techniques to gain pregnancy rates approaching 50% per embryo. During assisted reproduction technology interventions, controlled ovarian stimulation is used to obtain several oocytes in attempts to increase the likelihood of having at least one developmentally competent embryo available for transfer. However, current techniques for identifying the competent embryo[s] are by no means perfect. These limitations, coupled with pressures to maximize the chance of pregnancy, typically result in the transfer of multiple embryos. Not surprisingly, this practice has resulted in an unacceptably high rate of multiple pregnancies arising from IVF-ET. During the last few years, concerted efforts have focused on reducing these rates by restricting the number of embryos to transfer [306].
In order to fulfill this task, several approaches have been suggested to improve the outcome of treatment after ovarian stimulation and assisted reproduction. Table 3 summarizes the different approaches including the two main approaches tried so far i.e. optimizing ovarian stimulation protocols and improving embryology techniques. A third approach we suggest i.e. the use of aromatase inhibitors constitutes the third and most recent approach.
Table 3 Various approaches to improve treatment outcome after ovarian stimulation:
(1) Approaches involving ovarian stimulation protocols:
A: Reduce intensity of ovarian stimulation
- No stimulation: natural cycle
- Minimal stimulation
- Step-down protocol
B: The application of new medications
- Recombinant FSH
- Recombinant LH
- GnRH antagonists
C: The use of adjuvant medication
- Insulin sensitizers
- Corticosteroids
- Other medications
(2) Improving embryology techniques
- Improving embryo culture conditions, selection and extending embryo culture (blastocyst transfer)
- Preimplantation genetic diagnosis
- In vitro maturation
- Micromanipulation including: ICSI, assisted hatching, mitochondrial injection and cytoplasmic and nuclear transfer.
(3) The use of aromatase inhibitors for
- Improving implantation rates
- Reducing FSH dose required for ovarian stimulation:
- Other possible benefits:
* Improving response to FSH stimulation in poor responders
* Preventing premature endogenous gonadotropin surge
* Reducing the risk of severe ovarian hyperstimulation
We suggest a THIRD novel approach that we believe it may carry a hope for improving the outcome of treatment after ovarian stimulation and assisted reproduction. This approach involves the use of aromatase inhibitors.
4.1 Modified ovarian stimulation protocols
4.1.1 Reducing the intensity of ovarian stimulation protocols
Different approaches have been suggested to improve the treatment outcome during assisted reproduction by reducing the intensity of ovarian stimulation that would reduce the high estrogen levels [307]. These approaches include no-stimulation (natural cycle IVF-ET), minimal stimulation IVF-ET cycles, step-down protocols, FSH coasting and in vitro maturation.
However, all these measures are associated with the major drawback of affecting the main goal of ovarian stimulation, which is the achievement of a suitable number of embryos when a good number of oocytes retrieved.
Natural cycle IVF-ET
The first IVF-ET treatment in the literature was a natural cycle IVF-ET. Since then natural cycle IVF-ET has been largely replaced by IVF-ET with ovarian stimulation. Natural cycle IVF-ET has several advantages including a close to zero multiple pregnancy rate, and a zero risk of ovarian hyperstimulation syndrome as well as being less time consuming, physically and emotionally less demanding for patients, and cheaper than stimulated IVF-ET. However, it seems to be less effective. Unfortunately, good quality randomized controlled trials and formal cost-effectiveness analyses are lacking. Pelinck reviewed 20 selected studies that comprised a total of 1800 cycles of natural cycle IVF-ET, resulting in 819 embryo transfers (45.5% per cycle) and 129 ongoing pregnancies (7.2% per cycle and 15.8% per embryo transfer). The authors concluded that: in spite of being a low-risk, low-cost and patient-friendly procedure, high cancellation rates because of premature LH rise and premature ovulations hamper efficacy of natural cycle IVF-ET and that a randomized controlled trial comparing natural cycle IVF-ET with current standard treatment strategies is warranted. [27].
The cumulative live birth rates after four cycles of treatment was reported to be 32% which is comparable with the value of 34% for women having conventional IVF-ET treatment [308]. However, there is enough data on cumulative live birth rates following conventional IVF-ET, which is likely that current cumulative rates for IVF-ET will be higher than previously reported.
In the implantation rate per embryo after natural cycle IVF-ET was found quite acceptable up to 50%. Also, the fertilization rate was found to be 100% for the oocytes retrieved from the mature follicles (as reviewed by [27] Pelinck). We believe that the significantly better oocyte quality (100% fertilization rate) as well as better implantation rate (about 50%), reflect clearly the value of avoiding the deleterious effects of ovarian stimulation on the treatment outcome. Moreover, there is an important point to mention regarding the success rates of natural IVF-ET cycles, which is the bias in selecting patients who underwent natural IVF-ET cycles. Many studies included patients with unfavorable outcome such as poor responders, old age and repeated IVF-ET failures.
Minimal stimulation IVF-ET
Minimal stimulation for IVF-ET that is less expensive than full stimulation and minimizes monitoring and patient discomfort has been described for almost 10 years and found to be associated with acceptable pregnancy rates and was suggested as an attractive alternative to select patients undergoing IVF-ET [309]. This protocol involves the use of CC plus FSH alone or in conjunction with GnRH antagonist to prevent premature LH surge [310]. It is expected that the minimal-stimulation regimens produce fewer oocytes, fewer embryos available after fertilization, and fewer excess embryos available for cryopreservation. This leads to the main drawback of the minimal-stimulation regimen, which is the lower total reproductive potential (pregnancies resulting from fresh and frozen-thawed embryos resulting from a single stimulation cycle) [311].
FSH step-down and coasting protocols
Other measures have been tried by decreasing the FSH dose (step down protocol) to improve endometrial receptivity. With the use of a step-down regimen with FSH in high responders, uterine receptivity was believed to improve secondary to lowering E2 levels during the preimplantation period [312].
Coasting or withholding FSH injection for a period of time has been suggested in patients at substantial risk for the development of severe hyperstimulation syndrome. Coasting in a studied subset of IVF-ET patients did not adversely affect cycle outcome parameters when it was not prolonged [313].
4.1.2 Application of new medications for ovarian stimulation
The ongoing development of more effective and safer pharmacological compounds is driving fundamental changes in medicine [314]. Along the last decade several new medications have been developed for application in ovarian stimulation and assisted reproduction including medications prepared by the new recombinant technology [recombinant FSH and LH] and GnRH antagonists as well as other medications.
Recombinant FSH
The introduction of recombinant FSH into the clinical management of patients suffering from infertility appears to be associated with several treatment benefits when compared with urinary human menopausal gonadotropins
The fact that rFSH preparations have batch-to-batch consistency, are free from urinary protein contaminants and have the potential to be produced in limitless quantities is advantageous. The question whether newer, more pure FSH products are beneficial from the clinical perspective, has not been settled beyond reasonable doubt. The price of rFSH has been reported to be three times as high as the price of the former FSH preparations [315].
Although both FSH and LH are required for normal follicular growth and maturation, the precise role of LH is at present still uncertain. Since it was shown in the late 1980s that too high a concentration of LH might have a negative effect on fertilization and embryo quality, the idea arose that pure FSH preparations might be superior to hMG preparations for patients with endogenous LH production [316]. This hypothesis was tested in several clinical trials comparing uFSH with hMG with respect to pregnancy rates per IVF-ET treatment cycle. Statistical significance was not reached in any of these individual studies [316,317]. However, in 1995 a meta-analysis by Daya was published, which included eight studies and demonstrated a significant difference in favor of uFSH [316]. A few years later this finding was contradicted by a meta-analysis by Agrawal who argued that meta-analyses should take into account the different pituitary desensitization protocols used [318]. When pooling together 11 trials with the most commonly used GnRH agonist protocol [the long protocol], the overall odds ratio for comparing FSH and HMG was not significant. Although this meta-analysis was criticized on the issue of study selection bias, re-analysis following the inclusion and exclusion of selected studies did not change the overall results of the study [316,318,319].
The question whether rFSH also leads to more clinical pregnancies per IVF-ET cycle has been addressed by several clinical trials, none of which reached statistical significance. However, two meta-analyses pooling together the results of several trials did show significant treatment effects in favor of rFSH [320,321]. The interpretation of these meta-analyses is still debated, since they compare two types of rFSH (the alpha and beta variant), as well as different types of urinary FSH (uFSH and uFSH-HP, and in Out's meta-analysis one study with HMG was also included). In Daya's analysis, which received Cochrane status, an absolute increase in pregnancy rates of 3.7% (95% confidence interval [CI] 0.5–6.9%) was demonstrated comparing rFSH with uFSH/uFSH-HP.
Data on the third possible comparison, between rFSH and hMG, are scarce. The studies that have been conducted so far found no statistically significant differences with respect to ongoing pregnancy rates [322-324]. The evidence available at this time, comparing the different FSH containing gonadotropins preparations, concerns their usage in IVF-ET. However, almost half of all gonadotropins are used in other settings, such as IUI. Very little data are available comparing the preparations for this indication. With respect to adverse effects of exogenous FSH administration, no significant differences have been found between products. The main risk associated with the use of FSH containing gonadotropins products is the development of the ovarian hyperstimulation syndrome (OHSS). The incidence of OHSS does not differ between products [321-324].
Recombinant LH
hCG has been used as a surrogate LH surge because of the degree of homology between the two hormones. The major differences between the two hormones include the sequence of the β-subunit, the regulation of the secretion of the two hormones, and the pharmacokinetics of clearance of hCG as opposed to LH [325,326].
hCG has a slower plasma metabolic clearance, which consists of a rapid phase in the first 5–9 h following IM administration and a slower phase in the 1–1.3 days after administration. After 36 h, the calculated half-life of hCG was 2.32 days, as compared with LH, for which estimates have ranged from 1 h [326] to 3–5 h [327,328]. By day 10 after administration, less than 10% of the originally administered hCG was measurable [329]. However, the long serum half-life of hCG is likely to be an undesirable characteristic in clinical practice.
Until recently, there was no other biologic preparation that was as effective as hCG in triggering the final stage of ovulation. Recent developments using genetic engineering technologies and posttranscriptional biosynthesis have led to the production of rhLH, which has been available for use in clinical trials for several years [330]. The rhLH produced in vitro is purified and further formulated to yield a pharmaceutical preparation with very high specific immunoactivity and bioactivity. A crossover pharmacokinetic study has been performed in nonhuman primates to assess and compare pituitary, urinary, and rhLH [331]. After intravenous administration of pituitary and urinary-derived LH, and rhLH, mean concentration-time curves were parallel. The mean areas under the curve (AUC) for concentration by time after dose curves were similar, after correction for immunologic differences in dose. The mean clearance estimates and volume of distribution at steady state, distribution, and terminal half-lives were similar for all three types of LH. Furthermore, the results of studies in human volunteers show that rhLH and urinary-derived hLH have similar pharmacokinetic characteristics; both preparations presented an iv distribution half-life of 1.2 h and a terminal half-life of 10 h [332,333]. rhLH total body clearance was 2 L/h, with less than 5% of the dose being excreted by the kidneys. The steady state volume of distribution was approximately 8 L [332]. Furthermore, it has been demonstrated that co-administration of rhLH and rhFSH (follitropin α) does not modify their respective pharmacokinetic characteristics [332]. Finally, low doses of rhLH have been shown to promote ovarian steroidogenesis when follicular growth is induced with rhFSH in hypogonadotropic hypogonadal women [334].
The results show that a single dose of rhLH is effective in inducing final follicular maturation and early luteinization in IVF-ET and embryo transfer patients and is comparable with 5,000 IU u-hCG. A single dose of rhLH results in a highly significant reduction in OHSS compared with hCG. The dose of rhLH giving the highest efficacy to safety ratio was between 15,000 and 30,000 IU. This study has shown that the incidence of OHSS is significantly smaller when using a single dose of rhLH for induction of final maturation of follicles and oocytes in women treated for IVF-ET, compared with the use of 5,000 IU u-hCG or two doses of rhLH [335].
Chandrasekher [336] compared rhLH with u-hCG and pituitary hLH as an ovulatory stimulus in rhesus monkeys before IVF-ET. These investigators found that a single injection of 2,500 IU rhLH was as effective as 1,000 IU u-hCG in inducing oocyte maturation, oocyte fertilizability, and luteinization of granulosa cells. The data suggested that using a conversion factor of 2.5, a dose of 12,500 IU rhLH would be as effective as 5,000 IU u-hCG in humans. Another study (unpublished data) looked at the spontaneous LH surges in seven healthy female volunteers with normal menstrual cycles after a single injection of either 250 μg GnRH-agonist (buserelin) or 5,000 IU u-hCG administered when the dominant follicle reached a diameter of 17 mm. It was found that the mean Cmax of the spontaneous LH surge was 47 IU/L (95% CI, 28–65 IU/L) and the mean AUC was 1,019 IU h/L (95% CI, 718–1,320 IU h/L). Using rhLH pharmacokinetic characteristics defined in Phase I studies [332,333], it was calculated that, to obtain a Cmax and an AUC larger than the corresponding mean values observed in spontaneous surges in 95% of cases, single sc injections of between 14,000 IU and 25,000 IU rhLH should be used.
GnRH antagonists
GnRH antagonists do not induce an initial stimulation of gonadotropins release, but cause an immediate and rapid, reversible suppression of gonadotropins secretion. The principal mechanism of action of GnRH antagonists is competitive receptor occupancy of GnRH-r [337].
GnRH antagonists have been introduced clinically to prevent premature endogenous LH surge to replace the GnRH agonists that require time for a state of desensitization to be reached and, to start with, LH secretion actually increases (flare-up). With GnRH antagonists, immediate blockade of pituitary gonadotropins secretion when premature luteinization during IVF-ET stimulation is imminent seemed an obvious approach. Several studies of dose and treatment schedules have been done, [338,339] and two general approaches have emerged. The first is a single subcutaneous injection of a large dose on about the eighth day of stimulation with gonadotropins. The alternative is multiple (five or six daily) injections of a small dose from about day 6 of stimulation until the day that hCG for final oocyte maturation is given.
The next step in the development of GnRH antagonists use was to establish whether a GnRH antagonist is at least as effective as a GnRH agonist as the established reference medication testing the regimen of repeated antagonist injections [340,341] or the regimen of a single-dose regimen [342].
With repeated injections, treatment with gonadotropins was found to be shortened by 1–2 days with slightly fewer follicles at the time of hCG injection so the number of recovered oocytes tends to be lower. No significant difference was found with respect to percentages of metaphase II oocytes, fertilization rates, and number of good quality embryos [340,341].
Pregnancy rates were high in both groups in all four studies but in every one the rate was lower in the antagonist group. A meta-analysis of the five randomized trials, showed an overall significantly lower rate of pregnancy of 5%. This meta-analysis unfortunately included the study that compared a single-dose analogue regimen with different gonadotropins starting dose as an additional variable [342]. It has been suggested that the larger numbers of oocytes and embryos with agonists allow better selection [343], although the numbers of good quality embryos do not seem to be different. A direct adverse effect on the embryo cannot yet be ruled out but is not likely.
Antagonist blockade allows immediate reversal of pituitary gonadotropins secretion. This means that in IVF-ET ovulatory ripening can be triggered via endogenous gonadotropins by using a GnRH agonist [344] with the advantage of prevention of ovarian hyperstimulation syndrome, which is thought to result at least in part from the prolonged LH effect of hCG [345]. Finally, the use of GnRH antagonist blockade of premature luteinization can be used in IVF-ET with very low or without any hormonal stimulation, lower risk of overstimulation, and simplification of the procedure [346].
A possible disadvantage of an antagonist in IVF-ET is its narrow therapeutic range with the currently advised doses for repeated injections. Patient compliance needs to be high because there is a risk of premature LH secretion if an injection is missed [337].
Another disadvantage is the unpredictable timing of the start of the IVF-ET procedure, which begins with the administration of FSH on day 3 of the woman's cycle and so depends on how regular her menstruation is. This problem may be solved by pretreatment with an oral contraceptive [335].
4.1.3 Use of adjuvant medication during ovarian stimulation
Insulin sensitizers
The independent effect of insulin resistance on infertility treatment in PCOS is not well defined. Regardless of body weight, insulin-resistant PCOS women need higher gonadotropins doses during ovarian stimulation, and insulin resistance is also associated with a risk of multifollicular development and high cancellation rate [347]. Hyperinsulinemic PCOS women are more likely to produce lower quality oocytes that exhibit low fertilization rates after IVF-ET in association with lower implantation rates [348].
Improving insulin resistance with exercise, low-calorie diet and insulin-lowering drugs such as metformin, troglitazone and acarbose decrease insulin levels, correct the endocrine abnormalities induced by obesity and insulin resistance which may improve the outcome of infertility treatment [349-351]. This leads to the assumption that co-treatment with insulin-lowering drugs or weight reduction before and during down-regulation and ovarian stimulation.
Some studies did not find deleterious of insulin resistance on ovarian stimulation [352]. Metformin treatment has been reported to increase the number of mature oocytes retrieved from women with PCOS undergoing gonadotropins-stimulated IVF-ET and ICSI. A low dose of metformin of 500 mg twice daily was started on day 1 of the cycle prior to leuprolide acetate suppression, and continued to the day of the pregnancy test. Metformin treatment significantly increased the number of mature oocytes, fertilization rates, and number of embryos produced, but did not alter the total number of oocytes or peak E2 levels [353].
Later, the same authors reported that metformin therapy improves ovarian stimulation and IVF-ET outcomes in coasted patients with clomiphene-resistant PCOS [354]. No further published reports to date have examined this issue.
Corticosteroids
Successful use of corticosteroids in treatment of anovulatory infertility has been reported [355,356]. Later corticosteroids were used as adjuvant therapy together with clomiphene citrate or gonadotropins in ovulation induction. The theoretical basis for this application has not been fully elucidated, but it has been postulated that corticosteroid treatment could improve ovulation through reduced influence of the adrenal androgens on follicular development [357]. There have been reports on improved ovulation and pregnancy rates with adjuvant corticosteroid treatment in anovulatory women with elevated androgens or androgens within the higher range [358,359], as well as in normoandrogenic women [360]. Adding dexamethasone to gonadotropins in ovulation induction in women with normal serum concentrations of gonadotropins, androgens, and prolactin did not give an improved outcome in other studies [361].
Corticosteroids have also been used as adjuvant therapy in IVF-ET treatment. In 1986, Kemeter and Feichtinger [362] reported a significantly better pregnancy rate in a group of women with various infertility causes, except cycle abnormalities, undergoing IVF-ET with adjuvant prednisolone treatment, as compared to a group of women without adjuvant prednisolone. They stated that prednisolone would improve follicle maturation and thereby improve the pregnancy rate. In contrast, others [363] did not see any beneficial effects of adjuvant dexamethasone in a group of women with serum DHEA-S >2.5 μg/ml [6510 nmol/l] undergoing IVF-ET treatment, neither did Bider [361], in a group of women with tubal factor infertility after addition of dexamethasone.
In the above-mentioned study by Lobo [357], decreased serum concentrations of testosterone, unbound testosterone, and DHEA-S were noted after dexamethasone and clomiphene administration. In women who ovulated, testosterone and unbound testosterone increased again when clomiphene was added despite the continuation of dexamethasone. Decreased serum concentrations of DHEA-S and testosterone after ovarian stimulation with clomiphene citrate and human menopausal gonadotropins (HMG) and adjuvant prednisolone have been reported [362]. The other studies did not report data on androgen concentrations after glucocorticoid treatment. The present prospective, randomized, placebo-controlled study was performed to find out if adrenal suppression with prednisolone during ovarian stimulation before IVF-ET in a group of women with PCOS resulted in any changes in serum and follicular-fluid androgens. Clinical outcome variables, such as embryo quality, implantation rate, and clinical pregnancy rate were also noted.
Fridstrom [364] have reported that adjuvant glucocorticoids in ovarian stimulation before IVF-ET did not decrease the concentrations of adrenal androgens in serum and follicular fluid in PCOS. However, whether there were beneficial effects on ovum quality or implantation rate could not conclusively be determined in their study due to the small number of patients.
The addition of corticosteroids to ovarian stimulation protocols for assisted reproduction has been suggested in cases of recurrent pregnancy loss [365].
Progesterone antagonists: Receptor antagonist onapristone and mifeprisotone
Krusche found that after ovarian stimulation, the PR antagonist onapristone retarded endometrial transformation in the rabbit model. The authors concluded that since ovarian stimulation, used in human IVF-ET therapy, is frequently reported to cause an advancement of post-ovulatory endometrial development, a therapeutic application of PR antagonists to slow down such advanced endometrial transformation was suggested. Eventually, this modulation of advanced endometrial development may improve implantation rates [366].
4.2 Improving embryology techniques
Improving embryo techniques is a very important approach to improve the outcome of assisted reproduction treatment. This includes, improving culture conditions and extended culture till the blastocyst stage as well as improving the accuracy for selecting viable embryos for transfer. Another strategy employs pre-implantation genetic diagnosis (PGD). In addition there are other strategies to improve the embryo quality including oocyte donation and micromanipulation techniques (assisted hatching, and cytoplasmic and mitochondrial transfer) [367].
4.2.1 Improving embryo culture condition, selection and extended embryo culture (blastocyst transfer)
Over the past decade there has been considerable interest in optimizing culture media for supporting human embryos including reducing glucose concentrations [368], adding amino acids [369] and supplementing media with growth factors [370]. However, there have been no large prospective randomized studies on the effects of culture media on outcome after IVF-ET, apart from one, which reported that although glucose-free medium improved embryo quality, pregnancy rates were not increased [371]. All these improvements as well as the growing data on the composition of human female reproductive tract fluids and from embryo physiology studies has led to the proposition that extended embryo culture should take place till the blastocyst stage in more than a single medium formulation [372]. To this end, sequential media have been developed, tested extensively on animal models and subsequently used clinically [373].
One approach to increasing pregnancy rates is to improve the selection of viable embryos. Embryos are selected on the basis of morphology and rate of development, and the fastest developing embryos of the best morphology are selected for transfer. Although there is some correlation between morphology and blastocyst formation [374] and implantation [375], selection on the basis of morphology and rate of development remains an unsatisfactory and imprecise method of selecting viable embryos. It is impossible to identify visually with certainty, which embryos at early cleavage stages will subsequently arrest. One approach to improve the selection is the extended culture of the embryos to the blastocyst stage and transfer them on day 5 or 6, allowing the identification of developing embryos and the transfer of blastocysts, which are synchronous with the endometrium development, rather than cleavage stage embryos. It had been hoped also that, during extended culture, the chromosomally abnormal embryos so common at early cleavage stages [376] would arrest and fail to complete preimplantation development. However, although few human blastocysts have total chromosomal abnormality, a large number of blastocysts have various proportions of abnormal cells and are mosaic [377].
However, although many clinics have experienced increased success with extended culture, others have reported no benefit. So, it would be unwise to suggest that blastocyst culture and transfer represents a panacea for all clinics and all patients. On the contrary, transferring early stage embryos (zygot intrafallopian transfer) may help in repeated implantation failure rather than blastocyst transfer. [378].
4.2.2 Preimplantation genetic diagnosis
Preimplantation genetic diagnosis (PGD) allows the diagnosis of a genetic disorder in an embryo before its implantation in the uterus. PGD involves the removal of one or two blastomeres from an eight-cell stage embryo after IVF-ET, analysis of the blastomeres using fluorescent in situ-hybridization (FISH) or PCR, and identifying affected embryos. As only unaffected embryos are transferred back to the uterus after PGD, termination can be avoided. PGD is of benefit to couples at risk of passing on a genetic disease to their offspring as well as for couples with repeated unexplained IVF-ET failures. The transfer of unaffected embryos is believed to enhance the success rate of IVF-ET. Unfortunately, a large number of oocytes need to be retrieved for a successful PGD cycle, as not all will fertilize, cleave, undergo successful biopsy and be identified as suitable for transfer. Although most embryos (96%) survive biopsy, the pregnancy rates after transfer of biopsied embryos (16% per treatment cycle) [377] are less than those after conventional IVF-ET (23% per cycle) [379]. However, PGD is still in its early developmental stages [14].
4.2.3 In-Vitro Maturation of Oocytes
Immature oocytes can be aspirated from small follicles of > 2 mm in diameter. Resumption of meiosis in fully-grown oocytes, germinal vesicle breakdown, extrusion of the first polar body and acquisition of the ability to be fertilized may all occur during in vitro maturation (IVM). Although the first human birth after IVM was achieved 20 years ago [380], a few cases have been reported subsequently, including births after the aspiration and IVM of oocytes retrieved from women who have natural or partially stimulated cycles [381] and women with PCOS [382]. When oocytes are removed from small antral follicles and placed in culture, approximately 60% will have undergone nuclear maturation within 48 h. After exposure to spermatozoa, about 40% of oocytes will undergo normal fertilization, exhibiting two pronuclei and extruding the second polar body. Between 20 and 25% of fertilized oocytes will undergo cleavage [383]. However, pregnancy rates after the transfer of such embryos have been extremely low (1–2%) and most embryos arrest between the four- and eight-cell stages with only few encouraging results [384]. Several factors may influence the fertilization, cleavage, blastocyst and pregnancy rates achieved after IVM, including the hormonal environment in vivo or in vitro and the composition of the culture medium [385]. Further research in these areas will lead to greater understanding of oocyte maturation and should result in improved implantation and pregnancy rates [14].
4.2.4 Micromanipulation techniques
Intracytoplasmic sperm injection (ICSI)
The use of small numbers of spermatozoa during IVF-ET results in fertilization failure. In these cases, it is possible to inject a single spermatozoon into the ooplasm of a mature metaphase II oocyte, a procedure known as ICSI. The first successful pregnancies were in the early 1990s [10]. Since then, the technique has become a widely accepted treatment for couples with severe male factor infertility. Extending the indication of ICSI to include non-male factor infertility has been suggested to improve the fertilization and pregnancy rates. However, several studies have not supported the benefit of ICSI over conventional IVF-ET in improving the treatment outcome for non-male factor indications with growing consensus against applying ICSI for all cases undergoing IVF-ET [386-389].
Assisted hatching
To help embryos hatching from their zona pellucida during blastocyst expansion, different types of assisted hatching have been developed, including mechanical partial zona dissection or zona drilling, chemical zona drilling with acidic Tyrode's solution, and the laser technique [176]. However, there is controversy about the benefit of assisted hatching on the improvement of the implantation rate and pregnancy rate. Some investigators agreed as to the benefit of assisted hatching in women with advanced age, in women with repeated IVF-ET failure, and in the general population [177,178].
Mitochondrial injection and cytoplasm, and nuclear transfer
Van Blerkom suggested that the developmental competence of mouse and human early embryos is related to the metabolic capacity of the mitochondria [144]. Deletions and mutations in oocyte mtDNA may lead to mitochondrial dysfunction, influencing energy production and apoptosis in oocytes and early embryos, resulting in aberrant chromosomal segregation or developmental arrest [144]. In mice, cytoplasmic transfer from young to old oocytes improved older oocytes quality [390].
Cytoplasmic transfer from metaphase II donor oocytes to mature recipient oocytes from women with recurrent IVF-ET failure has been carried out in an attempt to restore normal growth in developmentally compromised oocytes and embryos [391].
Nuclear transfer has also been proposed for the treatment of mitochondrial disease [392] whereby a karyoblast containing metaphase II chromosomes from women with repeated failures of embryo development due to defective oocyte cytoplasm is fused to enucleated donor oocytes. The increased cost and complexity as well as the invasiveness of the procedure with the decreased implantation rates all make these procedures far from being applied routinely to improve the treatment outcome after assisted reproduction. Only selected cases might be expected to benefit from these newly developing techniques. It should be noted that currently in the United States, performance of cytoplasmic or nuclear transfer in humans can only be conducted following approval of the protocol by the FDA.
PART TWO
1. Aromatase inhibition to improve outcome of treatment after ovarian stimulation
1.1 Introduction
We hypothesize that aromatase inhibitors can be used to improve the treatment outcome after ovarian stimulation either alone, or in combination with IUI and assisted reproductive technology. The use of aromatase inhibitors during ovarian stimulation may have several benefits including: (1) The enhancement of implantation by lowering the supraphysiological levels of estrogen attained during ovarian hyperstimulation that is believed to adversely affect the development of the endometrium, oocytes and embryo, as well as other possible targets. (2) Reducing gonadotropins dose required for achievement of optimum ovarian stimulation. This would reduce possible deleterious direct effects of exogenous gonadotropins injection in addition to reducing the cost of treatment. (3) Other possible benefits such as improvement of ovarian response to FSH stimulation in poor responders, and prevention of premature endogenous gonadotropin surge, as well as lower risk of severe ovarian hyperstimulation syndrome.
The improvement in implantation, as well as the reduced cost of treatment by decreasing the gonadotropins dose required for ovarian hyperstimulation would encourage the policy of transferring one embryo to minimize the risk of multiple gestation. This would have a tremendous economic impact on the practice of assisted reproduction, as well as cost of health care for multiple gestation worldwide.
1.2 Outline of the use of aromatase inhibitors for ovarian stimulation
In the following section, we present brief outline on the aromatase enzyme, estrogen biosynthesis and the development of aromatase inhibitors followed by a summary of the available data concerning the use of aromatase inhibitors for ovarian stimulation.
The aromatase enzyme
Aromatase is a microsomal member of the cytochrome P450 hemoprotein-containing enzyme complex superfamily (P450 arom, the product of the CYP19 gene) that catalyzes the rate-limiting step in the production of estrogens, that is, the conversion of androstenedione and testosterone via three hydroxylation steps to estrone and E2 respectively [393]. Aromatase activity is present in many tissues, such as ovaries, brain, adipose tissue, muscle, liver, breast tissue, and in malignant breast tumors. The main sources of circulating estrogens are the ovaries in premenopausal women and adipose tissue in postmenopausal women [394,395].
The development of aromatase inhibitors
Aromatase is a good target for selective inhibition because estrogen production is a terminal step in the biosynthetic sequence. Several aromatase inhibitors have been utilized in clinical studies over the last 20 years. The most successful, third generation aromatase inhibitors are licensed for breast cancer treatment [396]. Aromatase inhibitors have been classified in a number of different ways, including first-, second-, and third-generation; steroidal and non-steroidal; reversible (ionic binding), and irreversible (suicide inhibitor, covalent binding) [397]. Table 4 lists the different classes of aromatase inhibitors.
Table 4 Different classes of aromatase inhibitors:
Generation Non-Steroidal Steroidal
First Generation Aminogltethimide
Second Generation Rogletimide Formestane
Fadrozole
Third Generation Anastrozole Exemestane
Letrozole
Vorozole
Table 5 The different degrees of whole body aromatase inhibition by the various aromatase inhibitors
Aromatase inhibitor Dose Mean percentage of total body aromatase inhibition
Aminoglutethimide in conjunction with hydrocortisone 1000 mg plus 40 mg of hydrocortisone (daily) 90.6
Formestane 250 mg (every 2 weeks, intramuscularly) 84.8
Exemestane 25 mg/day 97.9
Fadrozole 2 mg/day 82.4
Anastrozole 1 mg/day 97.3
Letrozole 2.5 mg/day >99.1
Vorozole 1 mg/day 93
Steroidal aromatase inhibitors are androstenedione analogues that act as a false substrate and bind irreversibly to the androgen-binding site of the enzyme [398]. Non-steroidal aromatase inhibitors exert their function through binding to the heme moiety of the cytochrome P450 enzyme [399]. The first of these inhibitors to be used clinically was aminoglutethimide, which induces a medical adrenalectomy by inhibiting many other enzymes involved in steroid biosynthesis [400]. Although aminoglutethimide is an effective hormonal agent in postmenopausal breast cancer, its use is complicated by the need for concurrent corticosteroid replacement, in addition to side effects like lethargy, rashes, nausea and fever that result in 8–15% of patients stopping treatment [401,402]. The lack of specificity and unfavorable toxicity profile of aminoglutethimide led to the search for more specific aromatase inhibitors. In addition, the above mentioned aromatase inhibitors were not able to completely inhibit aromatase activity in premenopausal patients.
Third generation aromatase inhibitors
The third-generation anti-aromatase agents commercially available include two non-steroidal preparations, anastrozole and letrozole, and a steroidal agent, exemestane. Anastrozole and letrozole are often referred to as aromatase inhibitors, whereas exemestane is called an aromatase inactivator [403,404]. Anastrozole, ZN 1033, (Arimidex®) and letrozole, CGS 20267, (Femara®) are selective aromatase inhibitors, available for clinical use in North America, Europe and other parts of the world for treatment of postmenopausal breast cancer. These triazole (antifungal) derivatives are reversible, competitive aromatase inhibitors, which are highly potent and selective. At doses of 1–5 mg/day, they inhibit estrogen levels by 97% to >99% resulting in estrogen concentrations below detection by most sensitive immunoassays. Table 5 shows the relative potencies of different aromatase inhibitors. Aromatase inhibitors are completely absorbed after oral administration, with mean terminal half-life (t1/2) of approximately 45 hours (range, 30–60 hours). They are cleared from the systemic circulation mainly by the liver. Gastrointestinal disturbances account for most of the adverse events, although these have seldom limited therapy. Other adverse effects are asthenia, hot flashes, headache, and back pain [406]. The average wholesale cost for a month's supply (thirty tablets) of aromatase inhibitors is about $150 to $250 [407]. Table 6 lists the clinical advantages of the third generation aromatase inhibitors as regards potential use for ovarian stimulation.
Table 6 Advantages of the third generation aromatase inhibitors:
• Extremely potent in inhibiting the aromatase enzyme
• Very specific in inhibiting the aromatase enzyme without significant inhibition of the other steroidogenesis enzymes
• Orally administered
• 100% bioavailability after oral administration
• Rapid clearance from the body (Short half-life, ~45 hours)
• No accumulation of the medications or their metabolites
• No significant active metabolites
• Well tolerated on daily administration for years
• Few side effects
• Very safe without significant contraindications
• Relatively inexpensive
Hypotheses behind the use of aromatase inhibitors for ovarian stimulation
In the late 1990s, we explored the hypothesis that it might be possible to mimic the action of clomiphene citrate, without depletion of ER, by administration of an aromatase inhibitor in the early part of the menstrual cycle. We hypothesized that the result of blocking estrogen production would be release of the hypothalamic/pituitary axis from estrogenic negative feedback, thereby increasing gonadotropin secretion and resulting in stimulation of ovarian follicular development. This first hypothesis is referred to as CENTRAL hypothesis. The selective non-steroidal aromatase inhibitors have a relatively short half-life (approximately 40 hours) compared to clomiphene citrate, and would be ideal for this purpose since they are eliminated from the body rapidly [408,409]. In addition, no adverse effects are expected on estrogen target tissues, since no ER down-regulation occurs as observed in clomiphene citrate treatment cycles.
We subsequently developed a second hypothesis, which was referred to as the PERIPHERAL hypothesis to explain another mechanism of action of the aromatase inhibitors in ovarian stimulation. We believe that aromatase inhibitors also act locally in the ovary to increase follicular sensitivity to FSH stimulation. This may result from the temporary accumulation of intraovarian androgens, since conversion of androgen substrate to estrogen is reversibly blocked by aromatase inhibition. There are data supporting a stimulatory role for androgens in early follicular growth in primates [410]. Testosterone was found to augment follicular FSH receptor expression in primates suggesting that androgens promote follicular growth and estrogen biosynthesis indirectly by amplifying FSH effects [411,412]. In addition, androgen accumulation in the follicle may stimulate insulin-like growth factor I (IGF-I), along with other endocrine and paracrine factors, which may synergize with FSH to promote folliculogenesis [413-416]. This hypothesis still waits for evidence to support it.
1.2.1 Use of aromatase inhibitors alone for ovarian stimulation
In the last few years, we have worked on the development of aromatase inhibitors for ovarian stimulation and infertility management and reported interesting success in different applications as follows:
Induction of ovulation in anovulatory women
Based on our CENTRAL hypothesis of using an aromatase inhibitor for induction of ovulation (as explained above), the success of an aromatase inhibitor in inducing ovulation in patients with PCOS was reported [417-420].
Augmentation of ovulation in ovulatory women
After demonstrating success in inducing ovulation in anovulatory women, we proceeded to test whether aromatase inhibition might enhance the release of endogenous gonadotropins enough to stimulate the development of multiple follicles in ovulatory women and result in augmentation of ovulation or even controlled ovarian hyperstimulation. The use of an aromatase inhibitor for augmenting ovulation in patients with ovulatory infertility was successful in women with unexplained infertility, endometriosis, and women undergoing therapeutic donor insemination, and in ovulating partners of infertile men [419,420].
Induction and augmentation of ovulation after clomiphene citrate failure
For over 40 years, clomiphene citrate has been the most commonly used treatment for the induction and augmentation of ovulation, accounting for about two thirds of the fertility drugs prescribed in the United States [421,422]. In spite of the high ovulation rate associated with the use of clomiphene citrate, the pregnancy rate is much lower than anticipated. This is particularly true when considering pregnancy rate per cycle after three cycles of CC treatment [421], with higher than expected incidence of miscarriage in conception cycles [255]. Such discrepancy is believed, as explained earlier, to be due to the peripheral antiestrogenic effect of clomiphene citrate particularly at the level of the cervical mucus and endometrium, and which manifest themselves even in the presence of gonadotropin treatment superovulation. The accumulation in the body of the isomers of clomiphene citrate due to the long half-life (several days to weeks) adds to the persistence of the antiestrogenic effect [261,262].
In order to improve the outcome of clomiphene citrate treatment, various approaches have been suggested to overcome the antiestrogenic effect including concomitant estrogen administration. Some investigators reported increased endometrial thickness and improved pregnancy rates with this approach [423-425] while others have reported no benefit [426] or even a deleterious effect of estrogen administration [427]. Another approach was to administer clomiphene citrate earlier during the menstrual cycle [428], to allow the anti-estrogenic effect to wear off earlier before the critical period of fertilization and implantation. A third approach has been to combine another selective ER modulator like tamoxifen, which has more estrogen agonistic effect on the endometrium with clomiphene citrate [429] or to use it as an alternative to clomiphene citrate [430]. However, none of these strategies have proved to be completely successful in avoiding the peripheral antiestrogenic effects of clomiphene citrate. In addition to a discrepancy between ovulation and pregnancy rates with clomiphene citrate treatment, 20% to 25% of anovulatory women are resistant to clomiphene citrate and fail to ovulate at doses up to 200 mg daily.
The success of aromatase inhibition in inducing and augmenting ovulation encouraged trying aromatase inhibitors in cases of clomiphene citrate failure that was found to be successful in achieving ovulation and pregnancy [419,420]. As explained above, the significantly shorter half-life of the third generation aromatase inhibitors compared to clomiphene citrate, allows rapid elimination from the body [408,409]. In addition, since no ER down-regulation occurs, any adverse effects on estrogen target tissues, as observed in clomiphene citrate treated cycles, is expected.
1.2.2 Adjunct use of aromatase inhibitors for ovarian stimulation
We investigated the idea of combining an aromatase inhibitor with FSH injections to reduce the dose of FSH required to achieve optimum controlled ovarian hyperstimulation, without adverse antiestrogenic effects [431,432]. A significant reduction in the FSH dose required (from 45% to 55% in women with PCOS and unexplained infertility) has been reported [433] without evidence of peripheral antiestrogenic effects [432,433].
Improving ovarian response to FSH stimulation in poor responders
Reducing FSH dose required for optimum controlled ovarian hyperstimulation encouraged us to explore the use of an aromatase inhibitor in conjunction with FSH to improve response to ovarian stimulation in poor responders. In a selected group of women who had a poor response to FSH stimulation in at least two prior treatment cycles, adding an aromatase inhibitor resulted in improvement in the response to FSH stimulation. All patients developed a significantly greater number of mature ovarian follicles and almost a third of the treatment cycles resulted in pregnancy. In addition, the dose of FSH required to achieve such optimum response was significantly less than the dose used in the prior cycles in which FSH was used alone [434,435].
Developing alternative regimens for administering aromatase inhibitors for ovarian stimulation
During all the above-mentioned studies, the aromatase inhibitor, letrozole, was administered orally as a daily dose of 2.5 mg from day 3 to day 7 of the menstrual cycle. Based on the pharmacokinetics of the new aromatase inhibitors (almost 100% bioavailability after oral administration, ~2 days half-life and no accumulation or significant metabolite accumulation), we thought of a potentially more convenient method of administering an aromatase inhibitor for ovulation induction. We hypothesized that significantly higher concentration of the aromatase inhibitor can be achieved early in the menstrual cycle with faster clearance later in the menstrual cycle with the administration of a high single dose of the aromatase inhibitor early in the menstrual cycle such as on day 3. A single dose regimen would satisfy two goals: first, achieving maximum estrogen suppression early in the menstrual cycle when it is desired, and second, to allow clearance of the aromatase inhibitor before the critical final stage of fertilization and embryogenesis, to maximize safety and avoid any possible undesirable effects of the aromatase inhibitors. Single dose administration of an aromatase inhibitor was found successful in inducing ovulation with ovulation and pregnancy rates comparable to the 5-day regimen [436].
Limitations of preliminary data on use of aromatase inhibitors for ovarian stimulation
In all our clinical trials described above, results were not obtained from randomized, prospective, placebo controlled studies, the optimum research design. However, due to the experimental nature of the use of the aromatase inhibitors for ovarian stimulation, which to our knowledge, has never been used before, we believed that the present observational trials were mandatory before proceeding into any definitive randomized studies.
The encouraging results of our data have led other investigators from different centers world-wide to study the use of aromatase inhibitors for ovarian stimulation and in general have reported findings similar to ours in terms of the success of aromatase inhibitors in infertility treatment [437-450].
In a randomized, prospective study, a superior uterine environment has been found in patients treated with an aromatase inhibitor compared with clomiphene citrate, reflecting the lack of the antiestrogenic effects with aromatase inhibitor treatment. Although a non-significant increase in pregnancy was observed in patients who received aromatase inhibitor treatment (16.7 versus 5.6% per patient, P = 0.55), this almost three-fold increase in pregnancy rate would have required about ten more patients per group to reach statistical significance. The superiority of a single dose administration of an aromatase inhibitor was also reported when compared to clomiphene citrate [439]. In other studies, the use of an aromatase inhibitor in conjunction with FSH was found to reduce the FSH dose [440] and improve response to ovarian stimulation in poor responders [441,442], confirming our previous reports.
In another prospective randomized trial, Biljan [438] studied two doses (2.5 and 5 mg per day) of the aromatase inhibitor, letrozole for ovarian stimulation in patients with unexplained infertility. They found patients treated with a higher dose of letrozole developed more follicles without a detrimental effect on endometrial development. The potential for different doses and regimens of administration of aromatase inhibitors for ovarian stimulation still requires a lot of study, but the use of aromatase inhibitors at high doses should be cautiously considered.
In all reports and most of studies of other investigators, the aromatase inhibitor, letrozole was the one used. However, anastrozole, another third generation aromatase inhibitor similar to letrozole, was used in other studies [443,444]. Due to the similar pharmacokinetics and pharmacodynamics, including similar potencies and specificity in inhibiting the aromatase enzyme, we believe there is likely to be no difference between the third generation aromatase inhibitors in their efficacy for ovarian stimulation. It does, however, need to be determined what dose of anastrozole is required to be equivalent to letrozole.
Future avenues for the use of aromatase inhibitors for infertility management
It seems that there are many interesting areas of research that need exploration as regards the development of aromatase inhibitors for infertility management. These directions for research would include: confirming the available preliminary data on the success of aromatase inhibition in induction and augmentation of ovulation, as well as reducing the dose of FSH needed for ovarian stimulation, improving response in poor responders, and finding the optimum regimen for administering aromatase inhibitors for infertility treatment
Moreover, the use of aromatase inhibitors for new applications including in-vitro maturation and prevention of severe ovarian hyperstimulation syndrome and endometriosis-related infertility are interesting future avenues for aromatase inhibitors potential use in infertility management.
In addition the use of aromatase inhibitors to improve the outcome of treatment after assisted reproduction as discussed earlier in this review is an exciting area of application.
1.3 Use of aromatase inhibitors to improve treatment outcome after ovarian stimulation and assisted reproduction
In addition to using aromatase inhibitors, ALONE, for ovarian stimulation, their use during assisted reproduction carries several potentials to improve the treatment outcome.
Improving implantation rates
We hypothesize that aromatase inhibition during assisted reproduction may improve the implantation rate mainly by reducing the estrogen levels attained during COH. In addition, two other mechanisms may apply including reducing the dose of FSH required for optimum COH as well as applying much simpler stimulation protocols that do not require the use of GnRH analogues, hence avoiding any possible direct deleterious effects of FSH and GnRH analogues on the endometrium.
1.3.1 Lowering supraphysiological estrogen levels
As discussed above, the undesirable effects of ovarian stimulation on the outcome of infertility treatment are believed to be due to the supraphysiological levels of estrogen irrespective to whatever mechanisms explain for that (various postulated mechanisms were discussed above). So, lowering estrogen levels may be associated with improved outcome of treatment in terms of improving the implantation and pregnancy rates in addition to lowering risk of sever ovarian hyperstimulation syndrome.
Reducing estrogen synthesis by aromatase inhibition seems to be an exciting idea to ameliorate the deleterious effects of the supraphysiological levels of estrogen on the endometrium, the developing oocyte and embryo as well as the luteal.
Until recently there was no suitable aromatase inhibitor that could be used clinically to reduce estrogen levels during ovarian stimulation. This is because the available aromatase inhibitors were not safe for clinical application during ovarian stimulation due to lack of specificity in inhibiting the aromatase enzyme without inhibiting other steroidogenesis enzymes (e.g. aminoglutethimide). The other aromatase inhibitors (steroidal androstenedione analogues) were irreversible in their effect on the aromatase enzyme besides being parentrally administered. Most important, these old aromatase inhibitors were not potent enough to inhibit the aromatase enzyme and lower estrogen levels in women of the reproductive age group. However, the third generation non-steroidal aromatase inhibitors group is very potent and specific in inhibiting the aromatase enzyme reversibly. These new aromatase inhibitors are orally administered with very high safety profile and well tolerability. Moreover, they are cheap with a relatively short half-life [~45 hours], and already approved for clinical use to reduce estrogen production in postmenopausal women with breast cancer.
They have not been used in women of the reproductive age group. However, we have found these aromatase inhibitors to be effective in inhibiting the aromatase enzyme and effectively decrease estrogen levels in women of the reproductive age group during their successful use for ovarian stimulation [417,418,431,432].
In our experience with the use of an aromatase inhibitor for ovarian stimulation, estrogen levels were significantly lower (especially E2 level/mature follicle) when compared with conventional stimulation protocols (clomiphene citrate, FSH and clomiphene citrate plus FSH). Such low E2 levels may be beneficial and explains at least partially the improved outcome of treatment in terms of achieving promising high pregnancy rates during aromatase inhibitor treatment.
It may not be only the supraphysiological estrogen levels, which explain totally for the reduced implantation rate during ovarian stimulation cycles. Local paracrine factors, or possibly other undetermined factors may be responsible for the reduced implantation rate during COH. In this case the aromatase inhibitors may not offer a complete solution to overcome such drawbacks of ovarian stimulation. However, the idea is exciting and seems to be promising and warrant trials because high estrogen levels may explain, at least in part, for the deleterious effects of ovarian stimulation on treatment outcome.
In ddition, we think that, reducing estrogen levels achieved during induction of ovulation by using aromatase inhibitors may prevent the significant increase in leptin concentrations avoiding its possible deleterious effects on the outcome of treatment as explained above. Because elucidation of leptin's specific role in reproductive function has been challenging, with conflicting results reported by various investigators, it is still quite early and highly speculative to hypothesize a link between aromatase inhibitors use and the role of leptin during induction of ovulation. However, the available strong data about a possible role of leptin in mediating reproductive disorders especially in obese women and the firm findings of a regulatory effect of estrogen on leptin production make this hypothesis exciting and interesting enough to warrant future investigation.
1.3.2 Reducing gonadotropins requirements for optimum ovarian stimulation
As mentioned earlier, the use of an aromatase inhibitor significantly reduces the dose of FSH required for optimum COH. In addition to the significant economic benefit, we believe that reducing the dose of FSH may improve the treatment outcome of ovarian stimulation by reducing any possible deleterious effect of the exogenously administered FSH on the endometrium, developing oocyte or other targets.
Other possible benefits
It is believed that the endogenous LH surge arises once serum estrogen levels surpass a set threshold for a certain period of time [451-453].
The supraphysiological estrogen levels attained during ovarian stimulation are believed to cause premature release of the endogenous LH surge resulting in cycle cancellation during assisted reproduction. For that reason, GnRH analogues have been the standard of practice during most of the stimulation protocols for assisted reproduction to prevent the occurrence of endogenous LH surge either by direct inhibition of LH surge (GnRH antagonists) or by down-regulation of the GnRH receptors at the pituitary levels resulting in pituitary desensitization and prevention of the release of endogenous LH [454,455].
Unfortunately, the use of GnRH analogues (which is a crucial part of the stimulation protocol to prevent cycle cancellation as explained above), is associated with several problems including increasing the dose of FSH required to achieve optimum COH (due to suppressing the endogenous gonadotropin secretion, and possible peripheral effect at the level of the ovaries), as well as the luteal phase defect due to a dysfunctional corpus luteum function secondary to persistent endogenous LH suppression as explained earlier. In addition, there is rising evidence of a possible direct deleterious effect of the GnRH analogues, especially the antagonist at the level of the endometrium [456].
The use of an aromatase inhibitor to reduce estrogen levels attained during COH may be effective in preventing the occurrence of premature ovarian surge. This would avoid the use of GnRH analogues during stimulation protocols for assisted reproduction which has several advantages including prevention of the possible deleterious effects of these agents as mentioned above in addition to reducing the cost of treatment as well allowing implementing much simpler stimulation protocols during assisted reproduction.
Aromatase inhibitors for endometriosis-related infertility
The expression of aromatase enzyme in endometriotic tissues with the significant role of locally produced estrogen in endometriosis progression [457] suggests a benefit of aromatase inhibitors in endometriosis-related infertility. The inhibition of local estrogen production in endometrial implants, and the lower peripheral estrogen levels associated with the use of aromatase inhibition for ovulation induction, is expected to protect, to some degree, against progression of endometriosis during infertility treatment.
1.4 Concerns regarding the use of aromatase inhibitors in infertility treatment
There are three main concerns that may arise regarding the use of the aromatase inhibitors to improve the outcome of treatment after ovarian stimulation and assisted reproduction. They include the possible deleterious effect of the low follicular estrogen milieu on the development of the oocytes, the possible direct effect of the aromatase inhibitors on the oocyte development, fertilization or embryogenesis, and the accumulation of the androgens that may result from inhibition of their conversion into estrogens.
1.4.1 Effect of low estrogen milieu on the developing oocyte
Palter et al. have reviewed the question whether an estrogen-free (or at least very low estrogen) intrafollicular environment is compatible with follicular development, ovulation, and corpus luteum formation [458]. The authors concluded that markedly reduced to nonexistent intrafollicular and circulating concentrations of estrogen are compatible with follicular "expansion", retrievable and fertilizable oocytes, as well as with cleavable and apparently transferable embryos. The authors drew their conclusion after discussing lessons from data in the literature including cases of deficiency of the 17-hydroxylase/17–20 lyase [459-466] and 3β-hydroxysteroid dehydrogenase [467,468] and the aromatase enzyme [469-473] as well as cases of severe hypogonadotropism [474-478]. In addition, data on the use of aromatase inhibitors in animals were reviewed [479-482].
Cases of enzyme deficiency leading to a status of very low estrogen levels
17 alpha-hydroxylase/17–20 lyase deficiency
17alpha-hydroxylase/17–20 lyase deficiency is one form of congenital adrenal hyperplasia, which is associated with marked impairment of glucocorticoid, androgen, and estrogen biosynthesis [459]. Women suffering from this enzyme deficiency suffer from hypergonadotropic hypogonadism and sexual infantilism. However, early reports noted the presence of many primary and secondary follicles in ovarian material [460] and many of them had bilateral multicystic ovaries at the time of laparotomy [461-463].
It is interesting that Rabinovici reported on a patient afflicted with virtually complete 17-hydroxylase/17–20 lyase deficiency that, despite castrate levels of estrogens, underwent an apparently successful induction of ovulation associated with progressive follicular expansion and oocyte retrieval, IVF-ET, and early embryonic cleavage followed suit [464].
3β-hydroxysteroid dehydrogenase [3β-HSD] deficiency
The deficiency of 3β-HSD is associated with markedly reduced levels of estrogen. However, the existence of normal ovulatory function in a woman with late-onset of a mild form of 3β-HSD has been reported [483].
In a group of cycling female Rhesus monkeys exposed to ovulation induction with hFSH and hFSH + hLH in the absence or presence of the 3β-HSD inhibitor trilostane given on days 1–8 of the menstrual cycle [467], apparently healthy oocytes were obtained by follicular aspiration 34 h after hCG administration. Importantly, treatment with the 3β-HSD inhibitor, trilostane, led to a reduction in serum E2 levels to 7% of that of control animals throughout the follicular phase. Despite this dramatic reduction in E2 levels, neither the total number of large antral follicles per animal (17 ± 1 vs. 18 ± 2) nor their size distribution differed significantly from 3β-HSD inhibitor-untreated controls. Furthermore, treatment with the 3β-HSD inhibitor did not alter the overall maturation pattern of collected oocytes (atretic, prophase I, metaphase I, or metaphase II). However, the authors found a reduction in the percentage of metaphase II oocytes that were successfully fertilized (15 vs. 65%). Moreover, metaphase oocytes that required more than 8 h to complete meiosis in vitro failed to fertilize in three of four animals receiving 3β-HSD inhibitor relative to controls (31%). These observations suggest that follicular development and the completion of meiosis may be unaffected by the low estrogen levels but that cytoplasmic oocyte maturation and/or function could be unfavorably affected [467].
Aromatase enzyme deficiency and the use of aromatase inhibitors
Obviously, aromatase (estrogen synthase) enzyme deficiency is associated with marked decrease or almost absence of estrogen production.
Extreme examples of complete aromatase deficiency due to mutations in the aromatase gene, CYP19 gene, in adult human females, however, were reported [469,470]. The affected patients suffered from ambiguous external genitalia, primary amenorrhea, sexual infantilism, and multicystic ovaries.
Morishima [471] reported on the aromatase deficiency in a 28-yr-old 46 XX proband followed since infancy. Null mutant mice for aromatase gene [ArKO] were generated [484], thereby affording the opportunity to examine the role of estrogen in the follicular development in the mouse ovary. Evaluation of the ovaries revealed the presence of many large follicles filled with granulosa cells and evidence of antrum formation, but no corpora lutea. The ovarian phenotype degenerated with age upon the appearance of hemorrhagic cystic follicles and the loss of secondary and antral follicles coincident with the infiltration of macrophages and with stromal hyperplasia [485-488]. Therefore, the ArKO females are infertile, due primarily to a complete lack of ovulation.
However, recently, Jones et al., reported that oocytes were harvested from the ovaries of 4- and 7-week old ArKO, wild type and heterozygote mice stimulated with 5 IU PMSG. The number of immature oocytes harvested from ArKO females did not differ from the number collected from wild type or heterozygote littermates of either age group. Oocyte in vitro maturation rates also did not differ between the three genotypes or two age groups, with almost 75% of the immature oocytes progressing to metaphase II. Chromatin staining confirmed the arrest of these oocytes at the second meiotic division with chromatin staining clearly present in the oocyte and polar body. Mature oocytes were inseminated and IVF-ET rates did not differ between the three genotypes or two age groups, with fertilization occurring in approximately 67% of oocytes. Fertilized oocytes were cultured to blastocysts. Again, blastocyst development rates did not differ between the groups, with approximately 65% of the zygotes developing into blastocysts. Blastocyst morphology was similar across all of the groups [489].
These results indicate that ArKO oocytes are competent to develop to at least the implantation stage. The authors concluded that estrogen might not be required for the production and maturation of developmentally competent oocytes. Rather, its role in folliculogenesis is probably via regulation of the hypophysial pituitary gonadal axis and thus gonadotropin secretion [489].
Regarding the effect of an estrogen-free/poor intrafollicular environment on gametogenic maturation, Palter et al. [458] concluded that the effect is a negative one. Their conclusion was based on a number of primate studies, which indicated that an estrogen-free/poor intrafollicular environment is associated with marked decrements in the rates of meiotic maturation and fertilization [467,490]. In addition data derived from rodent models suggested that further suggested a compromise in early embryonic development [491].
In cycling female rhesus monkeys, the aromatase inhibitor, 1,4,6-androstatrien-3, 17-dione (ATD) was used to inhibit estrogen production during gonadotropin ovarian stimulation [479]. Animals treated with ATD displayed a drastic reduction in serum 17β-E2 levels to 37% of that of controls within 8 h of ATD treatment and to 16% of control by the day of hCG injection. In turn, the circulating levels of androstenedione rose. Despite the drastic reduction in the circulating levels of E2 and the increase in the circulating levels of androgens, the overall number of large antral follicles [16 ± 3 for controls and 20 ± 3 for ATD-treated] and their size distribution (as assessed by ultrasonography) proved comparable for control and ATD-treated animals. Similarly, no difference was noted in the number of oocytes collected or in the proportion of oocytes reinitiating meiosis (MI at the time of collection). In contrast, ATD-treated animals displayed a marked increase (31 vs. 11%) in the proportion of prophase I oocytes. Moreover, ATD-treated oocytes displayed retarded in vivo completion of maturation to MII (4% vs. 26%). Interestingly, the latter retardation was not observed in vitro. Furthermore, two of the four ATD-treated animals yielded oocytes that were morphologically abnormal. Finally, oocytes from ATD-treated animals displayed significantly reduced rates of fertilization (9% vs. 25%) as compared with controls. However, the cleavage rate after successful fertilization was similar for ATD-treated vs. ATD-untreated controls. In vitro cultures of granulosa cells collected at the time of oocyte aspiration revealed equivalent 24-h progesterone production in treated and control animals. The authors concluded that these findings suggest that the acute reduction in E2 levels during the terminal stage of gonadotropin stimulation had little effect upon follicular recruitment and expansion but an apparent detrimental effect upon gametogenic function may, in fact, exist. However, we cannot extrapolate from this study that the findings are simply due to the acute reduction of estrogen production as it is important to realize that there was a concomitant significant rise in androstenedione. Moreover, the time of administering an aromatase inhibitor during the latter part of the follicular phase as well as the irreversible nature of aromatase inhibition constitute important differences from our model of using a REVERSIBLE aromatase inhibitor, TEMPORARILY, EARLY in the menstrual cycle that has a SHORT half-life.
Selvaraj [480,481] and Shetty [482] examine the effects of blocking estrogen biosynthesis during the follicular phase on follicular maturation in the adult female bonnet monkey. In their studies, they used one of the third-generation REVERSIBLE, aromatase inhibitors (CGS 16949A) starting EARLY on day 3 of the menstrual cycle. There were 53% and 70% reductions in the basal and surge levels of E2, respectively without obvious effect on follicular maturation, ovulation, and luteal function as assessed by serum hormone profiles as well as by laparotomy. Moreover, the concurrent administration of FSH and an aromatase inhibitor resulted in the suppression of the FSH-induced increase in the circulating levels of E2 (by 100%) with no effect noted on either the number of follicles developed or their size relative to control. In addition, granulosa and theca cells, removed on day 9 of the treatment cycle, were responsive to gonadotropins in vitro, disclosing no evidence of a deleterious effect on the cellular development and maturation of follicular cells.
Terry studied a possible role of high E2 levels in mediating the adverse effects of hyperstimulation with PMSG on early embryonic development in the rat [491]. They used the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA), to inhibit endogenous E2 production. The authors conducted three experiments. In the first, varying doses of 4-OHA were administered either concurrently with hCG to pro-estrus female rats hyperstimulated at early diestrus stage with 20 IU PMSG or alone into non-hyperstimulated pro-oestrus females. At high doses of 1000, 2000, or 5000 mg/rat, 4-OHA substantially improved the survival of embryos in hyperstimulated females with optimum protection at 2000 mg, while low doses of 100 and 500 mg/rat were ineffective. When administered alone, only the highest dose of 5000 mg/rat 4-OHA increased embryo count. In the second experiment, higher doses of PMSG were studied (30 or 40 IU), with or without 5000 mg/rat 4-OHA given at the time of hCG injection. PMSG proved to be more detrimental with increasing dose, and high doses of 4-OHA (5000 mg/rat) was needed to rescue embryos from death in the 30, but not 40, PMSG group. In the third experiment, the influence of the timing of 4-OHA treatment on its ability to improve the embryo count in hyperstimulated females was examined by introducing 4-OHA 24 h earlier, rather than at the time of hCG treatment. The results showed the importance of timing of 4-OHA administration, as 5000 mg/rat 4-OHA was able to restore embryo survival in the 40 PMSG hyperstimulated group only when it was administered 24 h before hCG injection. These results highlighted that 4-OHA, when administered at the appropriate time and dose, could reverse the negative effects of hyperstimulation from PMSG on early embryonic development. The authors concluded that this might be due to the suppression of estrogen production, thereby alleviating the supraphysiological level of E2, which is typically present in PMSG-treated females, which supports the hypothesis that excessive E2 is responsible for the negative effects of hyperstimulation with PMSG on early embryonic development.
The high success rates of ovulation and achievement of pregnancy in our reports on the use of aromatase inhibitor in ovarian stimulation, despite significantly lower estrogen levels could be due to several reasons. First: We used one of the third-generation "REVERSIBLE" aromatase inhibitors. Second: The aromatase inhibitor was administered EARLY in the follicular phase and for a limited period of time, which would allow the rapid clearance of the aromatase inhibitor from the body due to its SHORT half-life. Third: the reversible nature of the aromatase enzyme inhibition and the rising levels of FSH, which induce the expression of the aromatase enzyme, both would not allow for the estrogen levels to drop drastically below the low physiologic range. We believe that these low estrogen levels attained during the use of aromatase inhibitors for ovarian stimulation are compatible with healthy development of the oocytes, fertilization, embryo development, implantation and achievement of pregnancy. Fourth: the absence of significant rise in androgen levels would have prevented any possible unwanted effects on the terminal part of the oocyte maturation and ovulation.
1.4.2 Direct effect of the aromatase inhibitors on the developing oocyte, fertilization and embryogenesis
As explained above, the short half-life of the aromatase inhibitors and limiting the administration to the early part of the follicular phase would allow the rapid clearance of the medications from the body before the important stage of fertilization and embryogenesis. This in addition to the absence of accumulation of the aromatase inhibitors of any of their metabolites would make them safe for the ovarian stimulation. We have reported a preliminary data on the pregnancy outcome after the use of aromatase inhibitors for ovarian stimulation supporting the safety of using these medications for such indication [492].
Data have been accumulating regarding the absence of deleterious effects in association with aromatase inhibitor treatment on follicle/oocyte maturation and embryo development in mice [492,493]. Treatment with the aromatase inhibitor, anastrozole was associated with similar number of total follicles found per animal (30.4 follicles/animal in the control group and 27 follicles/animal in the anastrozole-treated group) as well as comparable rates of development of embryos, morulae, blastocysts, and hatching blastocysts between the two groups (P = 0.20, 0.10, 0.44, and 0.38, respectively) [493].
1.4.3 Effect of the accumulated androgens
As mentioned before, the rapid clearance of the aromatase inhibitors from the body due to their short half-life and the reversible nature of the aromatase enzyme inhibition as well as the rising levels of FSH, which induce the expression of the aromatase enzyme, all would not allow the clearance of any accumulating androgens by converting them to estrogen. The conversion of the androgens into estrogens (the step catalyzed by the aromatase enzyme) is a terminal step in the cascade of steroidogenesis. So, substrate (androgens) accumulation is not expected to be very significant, as other alternative earlier steps in the steroidogenesis pathways will work to clear out the accumulating androgens. For these reasons, pharmacokinetic studies on the new aromatase inhibitors reported the absence of significant androgen elevations or abnormal changes in other steroids in patients receiving aromatase inhibitors for breast cancer. In a subset of our patients who received an aromatase inhibitor for ovarian stimulation, we did not find significant change in the androgen levels while receiving the medication when compared to stimulation with gonadotropins alone (unpublished data).
Most recently we published data on the favorable pregnancy outcome after the use of letrozole for ovarian stimulation alone or in conjunction with gonadotropins [494]. This study looked at outcome of pregnancies achieved after ovarian stimulation with the aromatase inhibitor, letrozole, alone or in conjunction with gonadotropins. The study included a cohort of 394 pregnancy cycles achieved after letrozole and other ovarian stimulation treatments in a ddition to a control group of pregnancies spontaneously conceived without ovarian stimulation. These 394 pregnancy cycles were achieved as follows: 63 pregnancies with 2.5 mg of letrozole alone or with gonadotropins, 70 pregnancies with 5.0 mg of letrozole, 113 pregnancies with clomiphene alone or with gonadotropins, 110 pregnancies with gonadotropins alone, and 38 pregnancies achieved without ovarian stimulation. The study found pregnancies conceived after letrozole treatments were associated with similar miscarriage and ectopic pregnancy rates compared with all other groups. In addition, letrozole use was associated with a significantly lower rate of multiple gestation compared with clomiphene citrate. The findings of the stuy support the favorable pregnancy outcome and low multiple gestation rate in association with the use of aromatase inhibitors for ovarian stimulation [494].
5 Summary and conclusion
Ovarian stimulation particularly in conjunction with assisted reproductive technologies aims at stimulating the recruitment of several mature ovarian follicles that would enhance the chance of successful treatment by obtaining several embryos readily available for transfer into the uterus. It is obvious that supraphysiological levels of estrogen are inevitably attained during ovarian stimulation due to the significant contribution of estrogen production from each one of the several mature ovarian follicles. There is growing evidence that such supraphysiological estrogen levels are deleterious on the development of the endometrium, oocytes, embryos as well as other targets through different mechanisms. This is believed to explain, at least in part, the low pregnancy rates associated with assisted reproduction, mainly as a result of the persistently low implantation rates that have not improved impressively along almost three decades of assisted reproduction experience. This led several investigators to adopt the concept of minimal ovarian stimulation for assisted reproduction as an alternative approach to reduce the deleterious effects of the supraphysiological estrogen levels attained during routine controlled ovarian hyperstimulation. Unfortunately such approach, despite being logical, is associated with the major drawback of achieving fewer oocytes than desired, which, expectedly, reduces the chances of treatment success. Recently, we reported the success of the new generation aromatase inhibitors in ovarian stimulation. We found these agents to be effective in reducing the amount of estrogen production by mature ovarian follicles significantly. Furthermore, we found the use of these agents to be associated with improved ovarian response to stimulation by gonadotropins, resulting in significant reduction in the dose of gonadotropins required for controlled ovarian hyperstimulation. The novel idea of using aromatase inhibitors during ovarian stimulation for assisted reproduction combines the benefit of a significant reduction in estrogen production as well as gonadotropins dose, with the exiting advantage of achieving a good number of mature oocytes as a result of enhanced ovarian response to gonadotropins. This is expected to improve the various aspects of treatment outcomes after assisted reproduction including increased safety, reduced cost, as well as enhanced implantation rate. If confirmed in well-designed clinical trials, this would facilitate acceptance of the concept of single embryo transfer by infertile couples and practitioners, thereby reducing the epidemic of multiple births after assisted reproduction with its significant deleterious health and economical effects. This is particularly true in the light of recent data supporting the success of the approach of single embryo transfer [495-497].
Authors' contributions
MFMM has written the review article, RFC and MPD have reviewed the manuscript
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RetrovirologyRetrovirologyRetrovirology1742-4690BioMed Central 1742-4690-2-631623232010.1186/1742-4690-2-63ResearchDBR1 siRNA inhibition of HIV-1 replication Ye Ying [email protected] Leon Jessica [email protected] Noriko [email protected] Yathi [email protected] David [email protected] Department of Molecular Biology & Biochemistry, 2230 McGaugh Hall, University of California, Irvine, Irvine, CA 92697-3900, USA2005 18 10 2005 2 63 63 20 8 2005 18 10 2005 Copyright ©2005 Ye et al; licensee BioMed Central Ltd.2005Ye et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1) expression would specifically inhibit HIV-1 replication.
Results
We designed three short interfering RNA (siRNA) molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression.
Conclusion
These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.
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Background
Retrotransposons resemble retroviruses and contain long terminal repeats (LTR). They are mobile DNA elements that replicate via RNA intermediates. The Ty1 retroelement is among the best characterized of the retrotransposons of the yeast Saccharomyces cerevisiae [1-3]. Using a genetic screen aimed at identifying cellular factors involved in Ty1 transposition, Chapman and Boeke found that S. cerevisiae DBR1 mutants had lower Ty1 transposition frequency and were viable [4]. Dbr1 protein is a 2'-5' phosphodiesterase that cleaves intron RNA lariat branch points after splicing, facilitating ribonucleotide turnover [4,5]. DBR1 mutants produce mature mRNA, but accumulate intron lariats [3,4]. Mutations in DBR1 inhibit both Ty1 transposition and cDNA formation. Cheng and Menees provided evidence that Ty1 transcripts contain a 2'-5' branch characteristic of an RNA lariat, and demonstrated that the genomic RNA lariat is an unanticipated intermediate in the life cycle of Ty1 [2]. The location of this branch suggests that it may play a role during the formation of Ty1 cDNA by facilitating the transfer of nascent minus-strand of Ty1 cDNA from the upstream region of the Ty1 RNA template to the downstream region [2,3]. The similarity of Ty1 to animal retroviruses further suggests that the branch may be widely conserved among retroviruses as well as retroelements.
Human immunodeficiency virus (HIV) reverse transcription of the RNA genome into DNA is performed by the viral enzyme reverse transcriptase (RT). The primer for reverse transcription is a cellular tRNA. Retroviruses, long terminal repeat (LTR) retrotransposons, and long interspersed nucleotide element retrotransposons use cellular tRNAs to initiate cDNA synthesis [6-8]. Different tRNAs are used by different retroviruses and retrotransposons [9]. Early in the viral life cycle, the tRNA primes minus-strand strong-stop DNA synthesis, whereby the 5' end of the viral positive sense RNA genome is copied into minus-strand cDNA by RT, while the RNA template is degraded by the RNAse-H activity of RT. After minus-strand strong-stop DNA synthesis, a template shift from the 5' LTR to the 3' LTR is required to continue synthesis of the complete minus-strand cDNA, a requirement for converting the viral RNA genome into the proviral DNA genome. This "template switch" is required to synthesize the complete DNA genome, but its precise mechanism has never been identified. The recent work of Menees and colleagues showed that the 5' nucleotide (nt) of Ty1 RNA forms a 2'-5' bond with a nt at the beginning of R region in the 3' LTR of the same RNA, creating a lariat. The properties of the lariat suggest it forms by a novel mechanism and that branching and debranching may play roles in Ty1 reverse transcription at the minus-strand transfer step.
RNAi is a mechanism of gene silencing that has been widely used to study gene function in vitro [10-14]. RNAi occurs in cells via complex endogenous machinery that recognizes double stranded RNA, cleaves it into small fragments (19–21 nucleotides), and then uses those fragments as guides to specifically degrade RNA species displaying complementary sequence. The small fragments, called short interfering RNA (siRNA), can be introduced directly into mammalian cells to enter this pathway to induce the specific degradation of intracellular RNA. SiRNA-induced RNAi mediated degradation of DBR1 mRNA was used to study the role of DBR1 protein in HIV-1 replication.
Results
Transfection of GHOST cells with DBR1 siRNAs resulted in suppression of DBR1 mRNA
Three DBR1 siRNAs, targeted to DBR1 sequence positions 215 to 235, 436 to 456 and 971 to 991 were designed and inserted into pHyper (analogous to pSuper, obtained from Dr. V. Planelles of the University of Utah). For each DBR1 siRNA expression plasmid, two 64 nucleotide (nt) oligonucleotides were annealed to create 5' Bgl-II and 3' Hind III sites flanking a 21 nt target sequence in the DBR1 gene, a central 9 nt loop followed by a 21 nt antisense copy of the target sequence. The annealed 64 mers were cloned between the Bgl-II and Hind-III sites of the pHyper vector, as previously described for pSuper [15]. These three DBR1 siRNA plasmids were designated pHyper-D1, pHyper-D2 and pHyper-D3.
GHOST-R5X4 cells were transfected with various amounts of all three DBR1 siRNA expression plasmids combined in equal proportions (pHyper-D123) or control vector pHyper for forty-eight hours with Lipofectamine. To evaluate the effects of DBR1 siRNA, total RNA was extracted and real-time quantitative RT-PCR was performed with DBR1 specific primers. The DBR1 mRNA level was downregulated to a maximum of 80% following transfection with 12.8 μg/ml pHyper-D123 (Fig 1A). Next we examined the kinetics of DBR1 mRNA knockdown in GHOST-R5X4 cells transfected with each DBR1 siRNA plasmid separately (pHyper-D1, pHyper-D2, pHyper-D3) or together (pHyper-D123) compared to cells transfected with the control vector pHyper. We analyzed DBR1 message levels at various time points post-transfection by real-time quantitative RT-PCR. The data revealed that transfection of cells with any of the three DBR1 siRNA expression plasmids resulted in a reduction in the amount of DBR1 mRNA compared to cells transfected with the control vector pHyper (Fig 1B). This effect was maximal forty-eight hours post-transfection. Moreover, we found that the mixture of all three DBR1 siRNA expressing plasmids was the most effective and reduced the DBR1 mRNA level by more than 80%.
Figure 1 Suppression of DBR1 mRNA expression by DBR1 siRNA. (A) GHOST-R5X4 cells were transfected with various concentrations of DBR1 siRNA pHyper-D123 (0.8 μg/ml, 1.6 μg/ml, 3.2 μg/ml, 6.4 μg/ml, 12.8 μg/ml) or mock transfected. Forty-eight hours post transfection, RNA was isolated, treated with Dnase I and used as template in real-time quantitative RT-PCR. Each reaction included 300 ng RNA, 0.5 μM each gene-specific primer for DBR1 and GAPDH amplification. GAPDH was used as an internal control. (B) GHOST-R5X4 cells were transfected with 12.8 μg/ml DBR1 siRNA pHyper-D1, pHyper-D2, pHyper-D3, pHyper-D123 or control pHyper and cells were collected at the indicated time points. RNA was isolated, treated with Dnase I and analyzed by real-time quantitative RT-PCR. Results shown are from triplicate samples in a representative experiment. Error bars indicate standard deviations. Results that are significant by Student's t-test are indicated by asterisks (p < 0.05).
Knockdown of DBR1 or over expression of DBR1 is not toxic to cells
We used a commercial assay of cellular mitochondrial metabolism to determine whether DBR1 siRNA or DBR1 cDNA expression had adverse or cytotoxic effects. GHOST-R5X4 cells were transfected with pHyper-D123 and pHyper as a control or CDM9-DBR1 and CDM9 as a control. Forty-eight hours later, we assayed mitochondrial electron transport by its ability to reduce the tetrazolium salt, MTS, to produce formazan using a commercial kit. We did not detect significant cytotoxic effects following the addition of 3.2, 6.4, 12.8, 25.6, or 51.2 μg/ml of DBR1 siRNA pHyper-D123 and 1, 4, or 6 μg/ml of CDM9-DBR1 compared to the same concentration of each control plasmid (Fig 2).
Figure 2 Determination of cell viability. (A) GHOST-R5X4 cells were transfected with 3.2 μg/ml, 6.4 μg/ml, 12.8 μg/ml, 25.6 μg/ml or 51.2 μg/ml DBR1 siRNA pHyper-D123 (black bars) or the same concentrations of pHyper alone (white bars) as a control. (B) GHOST-R5X4 cells were transfected with 1 μg/ml, 4 μg/ml, 6 μg/ml CDM9-DBR1 (black bars) or the same concentrations of CDM9 alone (white bars) as a control. After forty-eight hours cellular viability was assayed with a commercial MTS assay kit (Promega). Results shown are from triplicate samples in a representative experiment. Error bars indicate standard deviations.
DBR1 knockdown inhibits HIV-1 replication in GHOST cells
To test whether DBR1 siRNA was capable of inhibiting HIV-1 replication, GHOST-R5X4 cells were transfected with pHyper-D1, pHyper-D2, pHyper-D3, pHyper-D123 or pHyper alone for forty-eight hours, then infected with R5-HIV-1 (JR-CSF). Twenty-four hours after HIV-1 infection, cell supernatants were collected to detect extracellular HIV-1 by ELISA for the viral capsid protein, p24. These data showed more than a 70% decrease in HIV-1 replication in GHOST-R5X4 cells expressing three DBR1 siRNA molecules compared to vector transfected cells (Fig 3). We also measured HIV-1 p24 in cell supernatants that were collected two and three days post-infection. Less suppression of viral replication was observed at these times, however, probably because the levels of DBR1 mRNA had returned to near normal (Fig. 1B and data not shown).
Figure 3 DBR1 siRNA inhibition of HIV-1 replication. GHOST-R5X4 cells were transfected with 12.8 μg/ml DBR1 siRNA pHyper-D1, pHyper-D2, pHyper-D3, pHyper-D123 or control pHyper. Forty-eight hours later, cells were infected with HIV-1 (JR-CSF) for twenty-four hours and p24 in supernatants was analyzed using a commercial HIV-1 p24 ELISA kit. All ELISA measurements were done in triplicate. Error bars indicate +/- standard deviation. Results marked with an asterisk are significant by Student's t-test (p < 0.05).
DBR1 over-expression counteracts the effect of DBR1 siRNA on HIV-1 replication
We next queried whether DBR1 over-expression would affect HIV-1 replication alone or when cotransfected with DBR1 siRNA. GHOST-R5X4 cells were transfected with a human DBR1 cDNA expressed by the plasmid vector, CDM9 at three concentrations or with vector alone. RNA was extracted forty-eight hours later and quantified by real-time quantitative RT-PCR with DBR1 specific primers (Fig. 4A). We observed a dose dependent increase in DBR1 mRNA expression as expected. Subsequently, GHOST-R5X4 cells were transfected with CDM9-DBR1, control vector CDM9, or DBR1 siRNA pHyper-D123, control vector pHyper or cotransfected with CDM9-DBR1 and DBR1 siRNA pHyper-D123, control vector CDM9 and pHyper, for forty-eight hours and then infected with HIV-1 (JR-CSF). To evaluate HIV-1 replication, the supernatants were analyzed twenty-four hours post infection for the presence of viral capsid, p24, with a commercial ELISA kit (Fig. 4B). We did not observe a significant effect of DBR1 overexpression on HIV-1 replication in GHOST-R5X4 cells. In contrast, the three DBR1 siRNA's significantly repressed HIV-1 replication and this inhibition could be reversed by cotransfection of DBR1 cDNA. This indicates that the DBR1 siRNA inhibited HIV-1 replication specifically, by lowering DBR1 expression.
Figure 4 DBR1 over-expression does not affect HIV-1 replication. (A) GHOST-R5X4 cells were transfected with various amounts of CDM9-DBR1 (1 μg/ml, 4 μg/ml, 6 μg/ml) or mock transfected for forty-eight hours. Total RNA was isolated and DBR1 expression was quantified by real-time RT-PCR. (B) GHOST-R5X4 cells were transfected with CDM9-DBR1 (black bar, CDM9-DBR1), control vector CDM9 (white bar, CDM9-DBR1) or DBR1 siRNA pHyper-D123 (black bar, DBR1 siRNA), control vector pHyper (white bar, DBR1 siRNA), or cotransfected CDM9-DBR1 and DBR1 siRNA pHyper-D123 (black bar, CDM9-DBR1 + DBR1 siRNA), control vector CDM9 and pHyper (white bar, CDM9-DBR1 + DBR1 siRNA) for forty-eight hours and then infected with HIV-1 (JR-CSF) for twenty-four hours. Supernatants were collected and p24 was assayed using a commercially available HIV-1 p24 ELISA kit. All ELISA measurements were done in triplicate. Error bars indicate standard deviations. The asterisk denotes p < 0.05 by Student's t-test.
DBR1 siRNA suppresses HIV-1 replication during reverse transcription
To more precisely determine the stage at which HIV-1 replication is inhibited by degradation of DBR1 mRNA, we transfected GHOST-R5X4 cells with DBR1 siRNA pHyper-D123 or control vector pHyper for forty-eight hours, then infected the cells with HIV-1, and harvested them twenty-four hours post-infection. The cells were lysed and DNA was isolated to evaluate the synthesis of HIV-1 cDNA by real-time quantitative PCR. The oligonucleotide primers M667 and AA55 specific for the R and U5 regions of the HIV-1 LTR respectively were used to detect early reverse transcription products, also known as strong-stop DNA, env primers Env1 and Env2 were used to detect intermediate reverse transcription products, while the LTR-R region and gag primers M667 and M661 were chosen to detect completely synthesized viral cDNA (Fig. 5A). Copies of HIV-1 were normalized against copies of β-globin to control for differences in cell number between samples. The results showed that early, products of reverse transcription were differentially affected by expression of DBR1 siRNA compared to intermediate and late reverse transcription products. The accumulation of HIV-1 strong-stop DNA was not affected by DBR1 siRNA expression (Fig. 5B), while the level of intermediate length and complete HIV-1 cDNA molecules was decreased similarly in the presence of DBR1 siRNA twenty-four hours post-infection (Fig. 5C,5D). This difference implies that the lariat debranching activity of DBR1 is needed after strong-stop DNA synthesis but prior to reverse transcription of the env gene or the completion of reverse transcription. To assay the specificity of inhibition of HIV-1 reverse transcription by DBR1 siRNA, we transfected GHOST-R5X4 cells with CDM9-DBR1, control vector CDM9 or with DBR1 siRNA pHyper-D123, control vector pHyper or cotransfected with CDM9-DBR1 and DBR1 siRNA pHyper-D123 or control vector CDM9 and pHyper for forty-eight hours and then infected with HIV-1 (JR-CSF). As shown in Fig 5, DBR1 over-expression did not significantly affect HIV-1 reverse transcription. Moreover, the effect of DBR1 siRNA expression on HIV-1 cDNA synthesis could be reversed by cotransfection of a DBR1 cDNA. This result indicates that the inhibition of HIV-1 reverse transcription by DBR1 siRNA was mediated specifically, by suppression of DBR1 expression.
Figure 5 DBR1 siRNA suppresses HIV-1 reverse transcription after minus-strand strong-stop DNA formation. (A) Location of primers. The oligonucleotide primers M667/AA55 specific for the R/U5 region of the LTR were used to detect early reverse transcripts (strong-stop DNA) (B) and env primers Env1/Env2 to were used detect intermediate reverse transcripts (C). LTR/gag primers M667/M661 were chosen to detect complete first-strand viral cDNA (D). In B, C and D, GHOST-R5X4 cells were transfected with DBR1 siRNA pHyper-D123 (black bars, siRNA), control vector pHyper (white bars, siRNA) or CDM9-DBR1 (black bars, cDNA), control vector CDM9 (white bars, cDNA) or co-transfected with CDM9-DBR1 and DBR1 siRNA pHyper-D123 (black bars, siRNA + cDNA), control vectors CDM9 and pHyper (white bars, siRNA + cDNA) for forty-eight hours, followed by infection with Dnase-treated HIV-1 (JR-CSF). Twenty-four hours later, the cells were lysed and DNA was isolated for real time quantitative PCR to evaluate the synthesis of HIV-1 cDNA. Copies of HIV-1 DNA were normalized against copies of β-globin. Error bars indicate standard deviations. Asterisks denote p < 0.05 by Student's t-test.
Discussion
SiRNAs are widely used for targeting and silencing genes by RNA interference. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells allows the use of this technology in research and perhaps eventually as a therapeutic tool. Inhibition of HIV-1 replication with RNAi has already been demonstrated with siRNAs against a variety of structural or regulatory genes including gag, pol, nef, vif, tat, env and rev [16-22]. These results demonstrate that siRNA directed to an HIV-1-specific gene could inhibit viral replication. In addition to targeting viral genes, many studies also investigated the efficacy of siRNAs in down regulating host cell molecules necessary for HIV-1 infection [23-28]. An advantage in targeting the interaction of cellular molecules with HIV-1 is that broad-spectrum efficacy against all clades of HIV-1 might be more readily achievable and the frequency of escape mutants might be lower. In this report, we have shown that siRNAs targeted to the host cellular gene DBR1 specifically decreased the amount of HIV-1 cDNA and capsid protein produced.
It has been previously found that the yeast gene DBR1 affects Ty1 transposition [1,4,29]. This gene encodes the RNA debranching enzyme, which hydrolyzes the 2'-5' bonds of intron RNA lariat branch points. A recent report suggested that the debranching enzyme influences Ty1 transposition because the Ty1 genomic RNA forms a lariat intermediate during cDNA synthesis [2]. This lariat intermediate was proposed to be formed by a 2'-5' phosphodiester bond between the 5' end of the genome and the first nucleotide in the 3' R region. Since retroviruses like HIV-1 and retroelements of Saccharomyces cerevisiae like Ty1 replicate via similar mechanisms and have similar LTR structures, we tested whether reduction in human DBR1 affected HIV-1 replication. We used three DBR1 siRNAs to specifically degrade DBR1 mRNA and measured the effects of DBR1 down regulation on HIV-1 replication.
We demonstrated that DBR1 siRNA expression reduced the level of DBR1 mRNA in a dose and time dependent manner and decreased the amount of virus produced from infected cells. This DBR1 siRNA-mediated inhibition of HIV-1 replication occurred after strong-stop DNA formation but prior to reverse transcription of the env gene or the completion of minus-strand synthesis. We found that DBR1 siRNA inhibited the formation of env gene cDNA and complete HIV-1 cDNA twenty-four hours post-infection (Fig. 5). In contrast, there was little effect on the synthesis of minus-strand strong-stop DNA. To determine if the lack of virus production and inhibition of reverse transcription were specifically due to DBR1 siRNA, we transfected DBR1 cDNA alone or with DBR1 siRNA and analyzed HIV replication. DBR1 over-expression alone had little effect on HIV-1 replication but it blocked the inhibitory effect of DBR1 siRNA when cotransfected. These results demonstrate the specific inhibition of HIV-1 reverse transcription by DBR1 siRNA after strong-stop DNA formation – perhaps during the minus-strand template switch. Nevertheless, we cannot exclude suppression of viral replication at other stages after strong-stop DNA formation.
In summary, our studies show that siRNA targeted to the human DBR1 gene specifically inhibited HIV-1 replication during reverse transcription. Although we do not know the role of the putative HIV-1 genomic RNA lariat in viral replication, it is likely that further investigation with DBR1 siRNA will provide insights into the mechanism of HIV-1 cDNA synthesis.
Conclusion
We have demonstrated that suppression of the host RNA lariat de-branching enzyme (DBR1) with siRNA specifically inhibits HIV-1 replication. Further studies showed DBR1 siRNA suppresses HIV-1 replication during reverse transcription. A recent report found that the minus-strand template switch in the yeast retroelement Ty1 is accomplished via an RNA lariat intermediate, which is formed by a 2'-5' phosphodiester bond between the 5' end and 3' LTR of the genomic RNA. HIV-1 is similar to retroelements like Ty1and our results suggest that HIV-1 may utilize a similar RNA lariat intermediate during minus-strand transfer. This finding may have profound implications for the development of new therapeutics for AIDS.
Methods
Construction of siRNA expression vectors
The mammalian expression vector, pHyper (analogous to pSuper) was obtained from Dr. Vicente Planelles of the University of Utah and used for expression of siRNA. For each siRNA expression plasmid, a pair of 64 nucleotide oligonucleotides were annealed to create a 5' Bgl-II and 3' Hind III site flanking a 21 nt target sequence in the DBR1 gene, a central 9 nt loop followed by a 21 nt antisense copy of the target sequence. Three pairs of cDNA oligonucleotides, targeting the human DBR1 gene at different locations were synthesized by MWG Biotech (Irvine, CA). Each pair of oligonucleotides was annealed at 90°C for 4 minutes, then at 70°C for 10 minutes, cooled to 37°C, and incubated for 20 minutes. The annealed dsDNA oligonucleotides were ligated into the pHyper vector between the Bgl-II and Hind III sites. These three 21 nt DBR1 sequences, D1, AACGAGGCGGATCTACGCTGC; D2, AAGGATCGGTGGAATCTCTGG; D3, AATGTGACTGGGCGCCTGTGG, were used to create three DBR1-siRNA plasmids, designated pHyper-D1, D2 and D3.
Cell culture, DNA transfection, viral stock preparation and infection
GHOST-R5X4 cells were cultured in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal bovine serum (FBS), 50 μg/ml gentamicin and 500 μg/ml of G418. Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS and 50 μg/ml gentamicin. GHOST-R5X4 cells were plated at 95% confluency for transfection and transfected using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were collected. Viral stocks were prepared by transfecting 293T cells (seeded at 9 × 106 cells per T75 flask) with 100 μg of πSV-JR-CSF plasmid coprecipitated with calcium phosphate. Two days post-transfection, culture supernatant was collected and frozen at -80°C until needed. HIV-1 virions in the supernatant were quantified using HIV-1 p24 ELISA kit (Perkin Elmer).
Cell viability assay
Cell viability assays were conducted with CellTiter 96 aqueous cell proliferation assay kit (Promega) according to the manufacturer's specifications. Briefly, cells were plated into flat-bottom 96-well plates at a density of 2 × 103 cells/well and allowed to attach overnight. Cells were then transfected with different concentrations of pHyper-DBR1 siRNA and pHyper as a control or CDM9-DBR1 and CDM9 as a control. After forty-eight hours of incubation, 20 μL of MTS reagent was added to each well, the plate was incubated for three hours at 37°C. A 490 nm absorbance value was determined using a Model-550 ELISA plate reader (Beckman Instruments Inc.). The percentage of viable cells was calculated as: (Abssample - Absblank)/(Abscontrol - Absblank) × 100.
RNA isolation and real-time quantitative RT-PCR
Total RNA was isolated using Trizol reagent (Life Technologies) according to the manufacturer's instructions. The amount of extracted RNA was quantified by measuring the absorbance at 260 nm. One microgram of RNA was treated with 1 unit Dnase I (Invitrogen) in a volume of 10 μl to remove contaminating DNA (37°C for 15 min, 75°C for 5 min). Three hundred ng of Dnase I treated RNA was reverse transcribed using a two-step reverse transcription kit (Applied Biosystems) in a final volume of 10 μl. Reverse transcription was performed for 60 min at 37°C. The total cDNA volume of 10 μl was frozen until real-time quantitative PCR was performed. After thawing for PCR experiments, the cDNA was diluted in 90 μl of distilled water and 2 μl of diluted cDNA was used for each PCR reaction. Real-time quantitative PCR was performed using ABI Prism 7700 sequence detection system, (PE Applied Biosystems) for amplification and detection. PCR conditions were as follows: initial denaturation at 95°C for 10 min then 40 rounds of cycling at 95°C for 15 sec and 60°C for 60 sec. Each PCR reaction in triplicate contained 15 μl SYBR green PCR master mix (Applied Biosystems), and 0.3 μM of each gene-specific primer for human DBR1 and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) in a 30 μl reaction volume. Forward and reverse primer sequences for amplifying DBR1 were GGAAACCATGAAGCCTCAAA (nt 247–266) and CCGATCCTTACACCTCGGTA (nts 444–425), respectively. Forward and reverse primer sequences for amplifying GAPDH were GGTGGTCTCCTCTGACTTCAA (nt 840–860) and GTTGCTGTAGCCAAATTCGTTGT (nt 966–944). A normalized DBR1 value was calculated by dividing the DBR1 copy number (determined from the appropriate standard curve) by the GAPDH (endogenous reference) copy number.
P24 ELISA
HIV-1 p24 ELISA was performed using a commercially available kit (Perkin Elmer) according to the manufacturer's instructions. For measuring p24 in the supernatants, 103 fold dilutions of the supernatants were used. All ELISA measurements were done in triplicate.
Quantitative real-time PCR
At each time point, 3.0 × 106 cells were lysed in 100 μl of 100 μg/ml proteinase K in 10 mM Tris-HCl, pH 8.0 at 56°C for 1 hour, followed by heat inactivation at 95°C for 10 min. Each PCR reaction contained 15 μl SYBR green PCR master mix (Applied Biosystems), 5 μl of cell lysate, 0.3 μM of each primer in a 30 μl reaction volume. HIV-1 specific primers AA55, CTGCTAGAGATTTTCCACACTGAC (nts 635–612) and M667, GGCTAACTAGGGAACCCACTG (nts 496–516) were used to detect minus strand strong stop. Env1, GGCAGGGATACTCACCCTTATCG (nts 8337–8360) and Env2, GGATTTCCCACCCCCTGCGTCCC (nts 8590–8566) were used to detect intermediate lengthe reverse transcription products. M661, CCTGCGTCGAGAGAGAGCTCCTCTGG (nts 695–672) and M667 were used to detect complete synthesis of first-strand cDNA. Copies of HIV-1 DNA were normalized against copies of β-globin using primers LA1 globin, ACACAACTGTGTTCACTAGC and LA2 globin, CAACTTCATCCACGTTCACC directed to β-globin [30].
DBR1 cDNA isolation and expression
A 1.7 kb cDNA fragment encoding human DBR1 was amplified by PCR from a human PHA-stimulated T cell cDNA library [31]. Amplification reactions were performed in a 25 μl mixture containing 0.625 Units FailSafe PCR Enzyme Mix (Epicentre), 500 nM forward (GAATTCGCCACCATGCGGGTGGCTGTGGCTGGCTGCTGCCACGG) and reverse (AACAAGTAAATCATCTTAAGCTGCATCG) primers in FailSafe PCR Premix-E buffer. The amplification reactions were carried out with the following program: one cycle of 2 min at 92°C, 35 cycles consisting of 30 sec at 95°C, 1 min at 55°C, 2 min at 72°C and a final extension step of 10 min at 72°C. PCR products were separated on 1% agarose gel pre-stained with ethidium bromide. A gel cleanup kit (Eppendorf) was used to purify and elute the PCR product DNA fragment for cloning and sequencing. The fragment was inserted between the Xho1 and Not1 sites of CDM9 and the sequence was found to be identical to the DBR1 sequence in the NCBI database. CDM9 is a derivative of CDM8 that contains a larger region of the human CMV immediate early region including the first intron and non-coding exon [32]. Various amounts of CDM9-DBR1 (1, 4 and 6 μg/ml) were used to transfect GHOST-R5X4 cells with Lipofectamine (Invitrogen) according to manufacturer's instructions, CDM9 alone was used as a control. Total cellular RNA was extracted and real-time quantitative RT-PCR was performed to quantify DBR1 expression.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YY carried out most of the experiments and drafted this manuscript. JDL performed most of the real-time PCR and analyzed the data. NY isolated the DBR1 cDNA and constructed some of the siRNA expression vectors. YN designed the DBR1 siRNAs and helped construct the siRNA expression vectors. DC conceived of the study, participated in its design and coordination and helped to revise the manuscript. All living authors read and approved the final manuscript.
Acknowledgements
We thank Dr. V. Planelles of the University of Utah for providing pHyper and for useful advice.
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Anderson J Banerjea A Planelles V Akkina R Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA AIDS Res Hum Retroviruses 2003 19 699 706 10.1089/088922203322280928 13678472
Anderson J Banerjea A Akkina R Bispecific short hairpin siRNA constructs targeted to CD4, CXCR4, and CCR5 confer HIV-1 resistance Oligonucleotides 2003 13 303 312 10.1089/154545703322616989 15000821
Butticaz C Ciuffi A Munoz M Thomas J Bridge A Pebernard S Iggo R Meylan P Telenti A Protection from HIV-1 infection of primary CD4 T cells by CCR5 silencing is effective for the full spectrum of CCR5 expression Antiviral Therapy 2003 8 373 377 14640383
Qin X An DS Chen ISY Baltimore D Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5 Proc Natl Acad Sci USA 2003 100 183 188 10.1073/pnas.232688199 12518064
Lauermann V Nam K Trambley J Boeke JD Plus-strand strong-stop DNA synthesis in retrotransposon Ty1 J Virol 1995 69 7845 7850 7494296
Zack JA Arrigo SJ Weitsman SR Go AS Haislip A Chen IS HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure Cell 1990 61 213 222 10.1016/0092-8674(90)90802-L 2331748
Camerini D James SP Stamenkovic I Seed B Leu-8/TQ1 is the human equivalent of the Mel-14 lymph node homing receptor Nature 1989 342 78 82 10.1038/342078a0 2509939
Seed B An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2 Nature 1987 329 840 842 10.1038/329840a0 3313052
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Retrovirology. 2005 Oct 18; 2:63
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==== Front
Respir ResRespiratory Research1465-99211465-993XBioMed Central London 1465-9921-6-1071616806210.1186/1465-9921-6-107ResearchTolerance to bronchodilation during treatment with long-acting beta-agonists, a randomised controlled trial Haney Sarah [email protected] Robert J [email protected] Freeman Hospital, Newcastle-upon-Tyne, UK2 Department of Respiratory Medicine, Waikato Hospital, Hamilton, New Zealand2005 16 9 2005 6 1 107 107 15 4 2005 16 9 2005 Copyright © 2005 Haney and Hancox; licensee BioMed Central Ltd.2005Haney and Hancox; 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
Regular use of beta-agonists leads to tolerance to their bronchodilator effects. This can be demonstrated by measuring the response to beta-agonist following bronchoconstriction using methacholine. However most studies have demonstrated tolerance after a period of beta-agonist withdrawal, which is not typical of their use in clinical practice. This study assessed tolerance to the bronchodilator action of salbutamol during ongoing treatment with long-acting beta-agonist.
Methods
Random-order, double-blind, placebo-controlled, crossover trial. After 1 week without beta-agonists, 13 asthmatic subjects inhaled formoterol 12 μg twice daily or matching placebo for 1 week. Eight hours after the first and last doses subjects inhaled methacholine to produce a 20% fall in FEV1. Salbutamol 100, 200 and 400 μg (cumulative dose) was then given at 5-minute intervals and FEV1 was measured 5 minutes after each dose. After a 1 week washout subjects crossed over to the other treatment. Unscheduled use of beta-agonists was not allowed during the study. The main outcome variable was the area under the salbutamol response curve.
Results
The analysis showed a significant time by treatment interaction indicating that the response to salbutamol fell during formoterol therapy compared to placebo. After 1 week of formoterol the area under the salbutamol response curve was 48% (95% confidence interval 28 to 68%) lower than placebo. This reduction in response remained significant when the analyses were adjusted for changes in the pre-challenge FEV1 and dose of methacholine given (p = 0.001).
Conclusion
The bronchodilator response to salbutamol is significantly reduced in patients taking formoterol. Clinically relevant tolerance to rescue beta-agonist treatment is likely to occur in patients treated with long-acting beta-agonists.
==== Body
Background
Long-acting beta-agonists are often added to inhaled corticosteroids to improve asthma control.[1] Despite this, most patients still need a short-acting beta-agonist for relief of breakthrough symptoms. The possibility that chronic long-acting beta-agonist use might adversely affect the acute response to short-acting beta-agonists is rarely considered.
It is well known that regular use of long-acting beta-agonists leads to tolerance to their bronchoprotective effects (their ability to prevent bronchoconstriction).[2,3] Studies looking for bronchodilator tolerance have had more variable results.[4,5] This has led to a widespread belief that clinically significant tolerance to bronchodilation does not occur.[6] However, several recent studies have clearly shown that bronchodilation tolerance does occur and becomes more apparent with increasing levels of bronchoconstriction. [7-11] This raises the possibility that beta-agonists will be less effective during acute severe asthma in patients using long-acting beta-agonists.
Much of the evidence for bronchodilator tolerance has come from studies using a 'challenge-rescue model' that measures the response to short-acting beta-agonists after bronchoconstriction has been induced with either methacholine or exercise.[7-11] Testing bronchodilation from a state of increased bronchomotor tone is thought to mimic patients' use of beta-agonists to relieve asthma symptoms. However, many of these studies have been performed after a period of withdrawal from maintenance beta-agonist [7,8,11] and the findings may not be relevant to patients who continue to use their long-acting beta-agonist twice daily. Other studies have been carried out at the time of peak effect of long-acting beta-agonist, when subjects are least likely to need additional bronchodilator and when results are confounded by differing baseline levels of bronchodilation and bronchial reactivity.[9,10]
The bronchodilator and bronchoprotective effects of long-acting beta-agonists peak within 1 hour of inhalation.[12] If they are taken twice daily as recommended then patients will be most vulnerable to bronchoconstriction and most likely to need their reliever inhalers 8–12 hours after inhalation. Tolerance to the bronchodilator effects of these inhalers at this time is therefore of the highest clinical relevance. Although tolerance to bronchoprotection is known to occur at this time,[3] changes in bronchodilation using this 'challenge-rescue'[10] model have not been studied in placebo-controlled trials.
This study was designed to assess tolerance to salbutamol bronchodilation 8 hours after regular formoterol treatment in a double-blind, randomised, placebo-controlled, cross-over trial.
Methods
Subjects had a physician diagnosis of asthma and a PD20 methacholine (provocative dose of methacholine required to produce a 20% fall in FEV1) <1.5 mg (<7.7 μmol). Those currently using long-acting beta-agonists were excluded. Those who had used oral corticosteroids in the previous 3 months or who had changed asthma treatment in the previous 6 weeks were excluded, as were pregnant and lactating women. All subjects provided written informed consent. Ethical approval for the study was granted by the Waikato Ethics Committee.
After a 1-week run-in period during which no beta-agonists were used, subjects were computer randomised to receive coded bottles of capsules containing formoterol 12 μg or matching placebo for use with the Foradil aerolizer device (Foradil, Novartis, Auckland, New Zealand). A methacholine challenge and salbutamol response was performed 8 hours after the first dose of study medication. The study medication was then taken twice daily for 1 week. A further challenge and response was performed 8 hours following the last dose. After a washout period of at least 1 week the study was repeated using the alternative medication. Use of additional beta-agonists was not allowed throughout the study. Ipratropium bromide (20 μg Atrovent metered dose inhaler, Boehringer Ingelheim, Auckland, New Zealand) was used for symptom relief.
Methacholine challenge was performed using a modified Yan technique.[13] Baseline FEV1 was the highest of 3 consistent measurements. Subjects then inhaled doubling doses of nebulised methacholine from 0.0073 mg to 3.728 mg from a dosimeter. FEV1 was measured 1 minute after each dose. Once the FEV1 had fallen by ≥20% from baseline, methacholine challenge was stopped. The PD20 (cumulative dose) was calculated by linear interpolation. Where the FEV1 fell <20% from baseline (2 subjects each on 1 occasion after formoterol) an arbitrary PD20 of 15 mg was assigned (twice the maximum cumulative dose given).
Salbutamol (Ventolin, GlaxoSmithKline, Auckland, New Zealand) 100 μg, 100 μg and 200 μg via metered dose inhaler and Volumatic spacer was given at 0, 5 and 10 minutes after challenge respectively. The FEV1 was measured 5 minutes after each dose of salbutamol, giving a total response time of 15 minutes.
The main outcome measurement was the area under the salbutamol response curve (AUC), expressed as a percentage of the methacholine-induced fall in FEV1. A secondary outcome was the final (15-minute) FEV1 after 400 μg salbutamol. Previous studies indicate that the pre-methacholine FEV1 and dose of methacholine used are significant covariates of the post-methacholine bronchodilator response [7] and analyses adjusted for these covariates using analysis of covariance. Methacholine doses were log-transformed for analysis to approximate a normal distribution. The test of the hypothesis used a general linear model with terms for subject, drug and time as well as the covariates of pre-methacholine FEV1 and log methacholine dose. Whether tolerance to bronchodilation occurred was assessed using the drug*time interaction term in the model. Post-hoc comparisons of the AUC and final FEV1 between placebo and formoterol treatments on day 1 and after 1 week of treatment used Tukey's exact method. Comparisons of pre-challenge FEV1 and PD20 methacholine use paired t tests. Analyses were performed using Minitab 13.2 and SPSS 10.0 for Windows. All available data were used in the main analysis. One subject did not attend for the second placebo challenge. Excluding this subject did not significantly alter the analysis.
Results
Eighteen subjects started the run-in period. Five subjects withdrew – one for personal reasons, one because he was prescribed oral steroids for acute gout, two for respiratory tract infections and one for an exacerbation of asthma. Thirteen subjects (5 male) completed the study. One subject did not attend for the challenge after 1 week of placebo therapy. Baseline data on the subjects are presented in table 1.
Table 1 Baseline data on subjects.
Subject Age (years) Sex Dose of inhaled steroids (mcg)* FEV1 at 1st placebo challenge (L) Percent predicted FEV1
1 34 Female 200 3.11 100
2 40 Female 400 2.60 89
3 26 Male 1000 3.52 85
4 23 Male 0 4.32 97
5 31 Female 0 2.41 86
6 36 Male 0 4.44 107
7 31 Female 0 3.09 98
8 19 Female 0 3.83 110
9 25 Male 0 3.93 87
10 23 Female 250 2.43 73
11 60 Female 400 1.73 73
12 24 Female 0 2.87 89
13 24 Male 500 4.18 90
* budesonide equivalent (beclometasone = budesonide = 2 × fluticasone)
Changes in baseline FEV1 and PD20
The pre-challenge FEV1 was higher during formoterol treatment than during placebo. This was of borderline significance after 1 week (table 2). The first dose of formoterol also increased the PD20 methacholine. After 1 week of formoterol this protection had decreased and the PD20 was not significantly different to placebo (table 2).
Table 2 Changes in FEV1, PD20 and fall in FEV1
Day 1 Day 8
Pre-methacholine FEV1(L) Mean and SD Placebo 3.26 (0.84) 3.24 (0.86)
Formoterol 3.44 (0.80) 3.36 (0.89)
Difference (95% CI) 0.18 (0.08, 0.28) 0.12 (-0.01. 0.24)
P = 0.002 P = 0.069
PD20(mg) Geometric mean and 95% CI for mean Placebo 0.10 (0.04, 0.24) 0.12 (0.04, 0.32)
Formoterol 0.45 (0.15, 1.35) 0.17 (0.05, 0.60)
Difference (doubling doses) (95% CI) 2.12 (0.88, 3.36) 0.51 (-0.49, 1.51)
P = 0.003 P = 0.286
Fall in FEV1 from baseline (%) Mean and SD Placebo 24.78 (4.74) 23.73 (4.54)
Formoterol 24.56 (5.24) 22.26 (4.34)
Difference (95% CI) -0.22 (-3.54, 3.10) -1.47 (-4.61, 1.66)
P = 0.886 P = 0.327
N = 13. As there is no data on subject 6 for day 8 placebo, data from day 1 have been used to calculate means.
Changes in salbutamol response
The area under the salbutamol response curve was lower during formoterol therapy compared to placebo. There was a significant time*treatment interaction indicating that the change in salbutamol response from day 1 to day 8 of formoterol treatment was different to the change in response from day 1 to day 8 of placebo (table 3). Comparisons between placebo and formoterol at each time point found that the reduction in AUC was not statistically significant after the first dose of formoterol, after adjusting the analysis for the increased dose of methacholine used. However, after 1 week of treatment the reduction was statistically significant even after adjusting for the dose of methacholine and the pre-challenge FEV1 (table 4). The mean difference in adjusted AUC between the day 1 and day 8 formoterol challenges was 58.1%.time (95% confidence interval 2.7, 113.4; p = 0.04).
Table 3 ANOVA table for AUC.
Source Degrees of freedom Seq sums of squares Adjusted mean squares F P
Baseline FEV1 1 2195 0 0 0.998
Log dose methacholine 1 88136 46295 22.13 0.000
Subject 12 83869 5368 2.57 0.016
Time 1 10109 5292 2.53 0.121
Treatment 1 24752 18182 8.69 0.006
Time*treatment 1 14283 14283 6.83 0.013
Error 33
Total 50
Table 4 Differences in area under the salbutamol response curve (AUC)
Day 1 Day 8
AUC as % of fall in FEV1 (%.time) mean and SD Placebo 196 (74) 205 (63)
Formoterol 120 (61) 107 (53)
Difference (least squares means, 95% confidence interval) 13.06 (-50.4, 76.53) 85.44 (31.78, 139.1)
P = 0.944 P = 0.001
Final FEV1(L) mean and SD Placebo 3.32 (0.84) 3.31 (0.89)
Formoterol 3.25 (0.22) 3.12 (0.84)
Difference (least squares means, 95% confidence interval) 0.06 (-0.18, 0.30) 0.26 (0.06, 0.46)
P = 0.909 P = 0.006
Differences in AUC by treatment and time using Tukey's exact method from covariate analysis (n = 13)
Despite the fact that pre-challenge FEV1 and post-methacholine FEV1 were higher during formoterol treatment, the FEV1 at the end of the salbutamol dose-response was lower on both formoterol days compared to placebo (see figure 1 for day 8). This difference was not statistically significant after 1 dose, but after 1 week of formoterol the mean difference was significant after allowing for covariates (table 4).
Figure 1 Response to salbutamol following methacholine challenge. Mean and standard error.
There was no effect of order of treatment on salbutamol response, PD20 or FEV1. Analysing AUC as absolute change in FEV1 instead of as a percentage of the fall in FEV1 gave similar results. In this study, baseline FEV1 was not a significant covariate in the analysis of AUC. Excluding this from the model did not materially alter the results of the analysis. There was no difference in the change in salbutamol response between those subjects taking and those not taking inhaled corticosteroids.
Discussion
This study has demonstrated a marked reduction in the bronchodilator response to salbutamol within the usual dosing interval of standard doses of formoterol (12 μg bd). After 1 week of formoterol therapy the area under the salbutamol response curve in the formoterol arm was nearly half that of placebo. This reduction in response was statistically significant after adjusting for the pre-challenge FEV1 and dose of methacholine used in the challenge
The area under the salbutamol response curve was also reduced after a single dose of formoterol. However, this was not statistically significant after adjusting for the dose of methacholine used in the challenge, which was higher after the first dose of formoterol. After 1 week of formoterol therapy the PD20 methacholine was not significantly different to placebo, indicating that tolerance to bronchoprotection had occurred. Hence at this time point there was little protection against bronchoconstriction and greatly reduced bronchodilation.
It is known that cellular tolerance to beta-agonists occurs very rapidly, within 8 hours in vitro, [14] so it is possible that 'tolerance' had occurred even prior to the challenge on day 1 of formoterol treatment. However, there may be other explanations for a reduced response to salbutamol during formoterol therapy, including the fact that beta2-receptors continue to be occupied by formoterol 8 hours after the last dose, leaving fewer receptors available to bind salbutamol. This could explain the reduction in the salbutamol response after the first dose of formoterol. However, the analysis showed a significant interaction between time and treatment, indicating that the bronchodilator response to salbutamol declined further during the formoterol treatment period. Lee et al [15] also found that the salbutamol response 1 hour after a single dose of salmeterol or formoterol was significantly lower after 1 week of treatment than after the first dose of long-acting beta-agonist. If receptor occupancy was the only explanation for the reduction in AUC we would expect to have found a similar reduction in AUC on the first and last day of formoterol treatment. Moreover, the reduction in salbutamol response after a week of formoterol treatment observed in this study was similar to a previous study in our laboratory, where the salbutamol response was measured 24 hours after the last dose of formoterol.[16] Receptor occupancy by formoterol would be much lower at this time, suggesting that the reduction in response is more likely to be due to receptor downregulation.
Regardless of the mechanism, we have shown that the bronchodilator response to salbutamol is reduced during long-acting beta-agonist treatment. Previous studies have been criticised for only analysing changes in FEV1 from baseline rather than actual post-bronchodilator levels of FEV1.[17] It is notable in this study that the final FEV1 after 400 μg of salbutamol was lower during formoterol treatment than during placebo, despite a higher pre-methacholine FEV1 (figure 1).
A recent meta-analysis of studies of regular beta-agonist therapy concluded that the bronchodilator response to subsequent beta-agonist is reduced.[18] However, most of the individual studies included in this analysis failed to show significant tolerance. In contrast, all of the published trials that have measured the bronchodilator response after methacholine challenge have shown significant tolerance.[7-9,11] The 'challenge-rescue model' appears to be more sensitive to changes in the response to bronchodilators. Bronchodilation is a 'closed-end scale'[19] with a maximal achievable level. The dose-response to beta-agonist bronchodilators is therefore dependent on the pre-bronchodilator FEV1.[20] Subjects with mild, stable asthma are often close to their maximum FEV1 and have little capacity for additional bronchodilation. Using methacholine to induce a controlled level of bronchoconstriction before testing bronchodilators ensures that there is capacity for bronchodilation and differences in responses can be shown more easily. Since asthmatics take bronchodilators to relieve symptoms caused by bronchoconstriction it seems logical to test bronchodilators from a state of bronchoconstriction.
This study is the first blinded, placebo-controlled trial to test for bronchodilator tolerance during the trough period of long-acting beta-agonist dosing using the 'challenge-rescue' model. A recent non-blinded trial compared the bronchodilator response to salbutamol after methacholine challenge between formoterol/budesonide or salmeterol/fluticasone to inhaled steroids alone, at 12 hours after inhalation.[21] This study found a similar degree of tolerance to the present study despite methodological differences including the use of salbutamol as-needed.
International guidelines recommend that long-acting beta-agonists are used only in those subjects already taking inhaled corticosteroids.[1] Not all of our subjects were taking such medication. However, this does not detract from the applicability of our study as it has been established that stable doses of inhaled corticosteroids do not alter the development of tolerance to beta-agonists.[7] Moreover, it is widely recognised that patients are often poorly compliant with their inhalers and it is likely that many patients will continue to use their long-acting beta-agonists without inhaled corticosteroids.
All but one of our subjects showed some decrease in the bronchodilator ability of salbutamol after 1 week of formoterol treatment, although the magnitude of this was variable (figure 2). The reasons for this variability are unknown. Lee et al found that tolerance occurred to the same extent in subjects who were homozygous for the Arg-16 or for the Gly-16 polymorphisms of the beta2-receptor.[15]
Figure 2 Individual data for AUC after 1 week of placebo and formoterol. AUC is expressed as a percentage of the fall in FEV1 induced by methacholine.
Conclusion
In conclusion, we have found that bronchodilator tolerance is present during the usual dosing interval of long-acting beta-agonists. Wraight found that tolerance to salbutamol bronchodilation increased with increasing levels of bronchoconstriction.[11] The level of bronchoconstriction induced in this study (20%) was mild compared to that likely to occur during a severe exacerbation of asthma. Patients using long-acting beta-agonists who experience exacerbations of asthma may have an inadequate response to beta-agonist relievers.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
RH conceived the trial, participated in its design, interpretation of results and helped to draft the manuscript. SH participated in the design of the study, acquired the data, performed the statistical analysis and helped to draft the manuscript. Both authors read and approved the final manuscript.
Acknowledgements
We would like to thank the study participants. We also thank Dr J McLachlan and the staff of the Waikato Hospital Respiratory Laboratory, Dr Graham Mills and the Waikato Respiratory Research Unit and Jan Cowan of the Respiratory Research Unit in the Dunedin School of Medicine.
==== Refs
British Thoracic society British guideline on the management of asthma Thorax 2003 58 suppl 1 1 83
Cheung D Timmers MC Zwinderman AH Bel EH Dijkman JH Sterk PJ Long-term effects of a long-acting β2-adrenoceptor agonist, salmeterol, on airway hyperresponsiveness in patients with mild asthma N Engl J Med 1992 327 1198 1203 1357550
Yates DH Sussman HS Shaw MJ Barnes PJ Chung KF Regular formoterol treatment in mild asthma. Effect on bronchial responsiveness during and after treatment Am J Respir Crit Care Med 1995 152 1170 4 7551366
Newnham DM McDevitt DG Lipworth BJ Bronchodilator subsensitivity after chronic dosing with eformeterol in patients with asthma Am J Med 1994 97 29 37 7913292 10.1016/0002-9343(94)90045-0
Aziz I Hall IP McFarlane LC Lipworth BJ β2-adrenoceptor regulation and bronchodilator sensitivity after regular treatment with formoterol in subjects with stable asthma J Allergy Clin Immunol 1998 101 337 41 9525449
Horiuchi T Castro M The pathobiologic implications for treatment. Old and new strategies in the treatment of chronic asthma Clin Chest Med 2000 21 381 95 10907595 10.1016/S0272-5231(05)70273-2
Hancox RJ Aldridge RE Cowan JO Flannery EM Herbison GP McLachlan CR Town GI Taylor DR Tolerance to beta-agonists during acute bronchoconstriction Eur Respir J 1999 14 283 7 10515402 10.1034/j.1399-3003.1999.14b08.x
Jones SL Cowan JO Flannery EM Hancox RJ Herbison GP Taylor DR Reversing acute bronchoconstriction in asthma: the effect of bronchodilator tolerance after treatment with formoterol Eur Respir J 2001 17 368 73 11405513 10.1183/09031936.01.17303680
Van der Woude HJ Winter TH Aalbers R Decreased bronchodilating effect of salbutamol in relieving methacholine induced moderate bronchoconstriction during high dose treatment with long acting β2 agonists Thorax 2001 56 529 535 11413351 10.1136/thorax.56.7.529
Storms WW Chervinsky P Ghannam A Bird S Hustad CM Edelman JM A comparison of the effects of oral montelukast and inhaled salmeterol on the response to rescue bronchodilation after challenge Respir Med 2004 98 1051 62 15526805 10.1016/j.rmed.2004.05.008
Wraight JM Hancox RJ Herbison GP Cowan JO Flannery EM Taylor DR Bronchodilator tolerance: the impact of increasing bronchoconstriction Eur Respir J 2003 21 810 5 12765426 10.1183/09031936.03.00067503
Rabe KF Jorres R Nowak D Behr N Magnussen H Comparison of the effects of salmeterol and formoterol on airway tone and responsiveness over 24 hours in bronchial asthma Am Rev Respir Dis 1993 147 1436 41 8503554
Yan K Salome C Woolcock AJ Rapid method for measurement of bronchial responsiveness Thorax 1983 38 760 5 6648855
Hadcock JR Wang H Malbon CC Agonist-induced destabilization of β-adrenergic receptor mRNA J Biol Chem 1989 264 19928 19934 2555338
Lee DKC Jackson CM Bates CE Lipworth BJ Cross tolerance to salbutamol occurs independently of β2 adrenoceptor genotype-16 in asthmatic patients receiving regular formoterol or salmeterol Thorax 2004 59 662 7 15282385 10.1136/thx.2003.019059
Haney S Hancox RJ Rapid onset of tolerance to beta-agonist bronchodilation Respir Med 2005 99 566 571 15823453 10.1016/j.rmed.2004.10.014
Barnes N Bronchodilator subsensitivity to salbutamol after salmeterol Lancet 1995 346 968 7564751 10.1016/S0140-6736(95)91589-3
Salpeter SR Ormiston TM Salpeter EE Meta-analysis: respiratory tolerance to regular β2-agonist use in patients with asthma Ann Intern Med 2004 140 802 813 15148067
Cockcroft DW Swystun VA Functional antagonism: tolerance produced by inhaled beta2 agonists Thorax 1996 51 1051 1056 8984693
Hendeles L Beaty R Ahrens R Stevens G Harman EM Response to inhaled albuterol during nocturnal asthma J Allergy Clin Immunol 2004 113 1058 62 15208585 10.1016/j.jaci.2004.03.046
Lee DKC Jackson CM Currie GP Cockburn WJ Lipworth BJ Comparison of combination inhalers vs corticosteroids alone in moderate persistent asthma Br J Clin Pharmacol 2003 56 494 500 14651722 10.1046/j.1365-2125.2003.01887.x
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Respir Res. 2005 Sep 16; 6(1):107
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==== Front
Respir ResRespiratory Research1465-99211465-993XBioMed Central London 1465-9921-6-1071616806210.1186/1465-9921-6-107ResearchTolerance to bronchodilation during treatment with long-acting beta-agonists, a randomised controlled trial Haney Sarah [email protected] Robert J [email protected] Freeman Hospital, Newcastle-upon-Tyne, UK2 Department of Respiratory Medicine, Waikato Hospital, Hamilton, New Zealand2005 16 9 2005 6 1 107 107 15 4 2005 16 9 2005 Copyright © 2005 Haney and Hancox; licensee BioMed Central Ltd.2005Haney and Hancox; 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
Regular use of beta-agonists leads to tolerance to their bronchodilator effects. This can be demonstrated by measuring the response to beta-agonist following bronchoconstriction using methacholine. However most studies have demonstrated tolerance after a period of beta-agonist withdrawal, which is not typical of their use in clinical practice. This study assessed tolerance to the bronchodilator action of salbutamol during ongoing treatment with long-acting beta-agonist.
Methods
Random-order, double-blind, placebo-controlled, crossover trial. After 1 week without beta-agonists, 13 asthmatic subjects inhaled formoterol 12 μg twice daily or matching placebo for 1 week. Eight hours after the first and last doses subjects inhaled methacholine to produce a 20% fall in FEV1. Salbutamol 100, 200 and 400 μg (cumulative dose) was then given at 5-minute intervals and FEV1 was measured 5 minutes after each dose. After a 1 week washout subjects crossed over to the other treatment. Unscheduled use of beta-agonists was not allowed during the study. The main outcome variable was the area under the salbutamol response curve.
Results
The analysis showed a significant time by treatment interaction indicating that the response to salbutamol fell during formoterol therapy compared to placebo. After 1 week of formoterol the area under the salbutamol response curve was 48% (95% confidence interval 28 to 68%) lower than placebo. This reduction in response remained significant when the analyses were adjusted for changes in the pre-challenge FEV1 and dose of methacholine given (p = 0.001).
Conclusion
The bronchodilator response to salbutamol is significantly reduced in patients taking formoterol. Clinically relevant tolerance to rescue beta-agonist treatment is likely to occur in patients treated with long-acting beta-agonists.
==== Body
Background
Long-acting beta-agonists are often added to inhaled corticosteroids to improve asthma control.[1] Despite this, most patients still need a short-acting beta-agonist for relief of breakthrough symptoms. The possibility that chronic long-acting beta-agonist use might adversely affect the acute response to short-acting beta-agonists is rarely considered.
It is well known that regular use of long-acting beta-agonists leads to tolerance to their bronchoprotective effects (their ability to prevent bronchoconstriction).[2,3] Studies looking for bronchodilator tolerance have had more variable results.[4,5] This has led to a widespread belief that clinically significant tolerance to bronchodilation does not occur.[6] However, several recent studies have clearly shown that bronchodilation tolerance does occur and becomes more apparent with increasing levels of bronchoconstriction. [7-11] This raises the possibility that beta-agonists will be less effective during acute severe asthma in patients using long-acting beta-agonists.
Much of the evidence for bronchodilator tolerance has come from studies using a 'challenge-rescue model' that measures the response to short-acting beta-agonists after bronchoconstriction has been induced with either methacholine or exercise.[7-11] Testing bronchodilation from a state of increased bronchomotor tone is thought to mimic patients' use of beta-agonists to relieve asthma symptoms. However, many of these studies have been performed after a period of withdrawal from maintenance beta-agonist [7,8,11] and the findings may not be relevant to patients who continue to use their long-acting beta-agonist twice daily. Other studies have been carried out at the time of peak effect of long-acting beta-agonist, when subjects are least likely to need additional bronchodilator and when results are confounded by differing baseline levels of bronchodilation and bronchial reactivity.[9,10]
The bronchodilator and bronchoprotective effects of long-acting beta-agonists peak within 1 hour of inhalation.[12] If they are taken twice daily as recommended then patients will be most vulnerable to bronchoconstriction and most likely to need their reliever inhalers 8–12 hours after inhalation. Tolerance to the bronchodilator effects of these inhalers at this time is therefore of the highest clinical relevance. Although tolerance to bronchoprotection is known to occur at this time,[3] changes in bronchodilation using this 'challenge-rescue'[10] model have not been studied in placebo-controlled trials.
This study was designed to assess tolerance to salbutamol bronchodilation 8 hours after regular formoterol treatment in a double-blind, randomised, placebo-controlled, cross-over trial.
Methods
Subjects had a physician diagnosis of asthma and a PD20 methacholine (provocative dose of methacholine required to produce a 20% fall in FEV1) <1.5 mg (<7.7 μmol). Those currently using long-acting beta-agonists were excluded. Those who had used oral corticosteroids in the previous 3 months or who had changed asthma treatment in the previous 6 weeks were excluded, as were pregnant and lactating women. All subjects provided written informed consent. Ethical approval for the study was granted by the Waikato Ethics Committee.
After a 1-week run-in period during which no beta-agonists were used, subjects were computer randomised to receive coded bottles of capsules containing formoterol 12 μg or matching placebo for use with the Foradil aerolizer device (Foradil, Novartis, Auckland, New Zealand). A methacholine challenge and salbutamol response was performed 8 hours after the first dose of study medication. The study medication was then taken twice daily for 1 week. A further challenge and response was performed 8 hours following the last dose. After a washout period of at least 1 week the study was repeated using the alternative medication. Use of additional beta-agonists was not allowed throughout the study. Ipratropium bromide (20 μg Atrovent metered dose inhaler, Boehringer Ingelheim, Auckland, New Zealand) was used for symptom relief.
Methacholine challenge was performed using a modified Yan technique.[13] Baseline FEV1 was the highest of 3 consistent measurements. Subjects then inhaled doubling doses of nebulised methacholine from 0.0073 mg to 3.728 mg from a dosimeter. FEV1 was measured 1 minute after each dose. Once the FEV1 had fallen by ≥20% from baseline, methacholine challenge was stopped. The PD20 (cumulative dose) was calculated by linear interpolation. Where the FEV1 fell <20% from baseline (2 subjects each on 1 occasion after formoterol) an arbitrary PD20 of 15 mg was assigned (twice the maximum cumulative dose given).
Salbutamol (Ventolin, GlaxoSmithKline, Auckland, New Zealand) 100 μg, 100 μg and 200 μg via metered dose inhaler and Volumatic spacer was given at 0, 5 and 10 minutes after challenge respectively. The FEV1 was measured 5 minutes after each dose of salbutamol, giving a total response time of 15 minutes.
The main outcome measurement was the area under the salbutamol response curve (AUC), expressed as a percentage of the methacholine-induced fall in FEV1. A secondary outcome was the final (15-minute) FEV1 after 400 μg salbutamol. Previous studies indicate that the pre-methacholine FEV1 and dose of methacholine used are significant covariates of the post-methacholine bronchodilator response [7] and analyses adjusted for these covariates using analysis of covariance. Methacholine doses were log-transformed for analysis to approximate a normal distribution. The test of the hypothesis used a general linear model with terms for subject, drug and time as well as the covariates of pre-methacholine FEV1 and log methacholine dose. Whether tolerance to bronchodilation occurred was assessed using the drug*time interaction term in the model. Post-hoc comparisons of the AUC and final FEV1 between placebo and formoterol treatments on day 1 and after 1 week of treatment used Tukey's exact method. Comparisons of pre-challenge FEV1 and PD20 methacholine use paired t tests. Analyses were performed using Minitab 13.2 and SPSS 10.0 for Windows. All available data were used in the main analysis. One subject did not attend for the second placebo challenge. Excluding this subject did not significantly alter the analysis.
Results
Eighteen subjects started the run-in period. Five subjects withdrew – one for personal reasons, one because he was prescribed oral steroids for acute gout, two for respiratory tract infections and one for an exacerbation of asthma. Thirteen subjects (5 male) completed the study. One subject did not attend for the challenge after 1 week of placebo therapy. Baseline data on the subjects are presented in table 1.
Table 1 Baseline data on subjects.
Subject Age (years) Sex Dose of inhaled steroids (mcg)* FEV1 at 1st placebo challenge (L) Percent predicted FEV1
1 34 Female 200 3.11 100
2 40 Female 400 2.60 89
3 26 Male 1000 3.52 85
4 23 Male 0 4.32 97
5 31 Female 0 2.41 86
6 36 Male 0 4.44 107
7 31 Female 0 3.09 98
8 19 Female 0 3.83 110
9 25 Male 0 3.93 87
10 23 Female 250 2.43 73
11 60 Female 400 1.73 73
12 24 Female 0 2.87 89
13 24 Male 500 4.18 90
* budesonide equivalent (beclometasone = budesonide = 2 × fluticasone)
Changes in baseline FEV1 and PD20
The pre-challenge FEV1 was higher during formoterol treatment than during placebo. This was of borderline significance after 1 week (table 2). The first dose of formoterol also increased the PD20 methacholine. After 1 week of formoterol this protection had decreased and the PD20 was not significantly different to placebo (table 2).
Table 2 Changes in FEV1, PD20 and fall in FEV1
Day 1 Day 8
Pre-methacholine FEV1(L) Mean and SD Placebo 3.26 (0.84) 3.24 (0.86)
Formoterol 3.44 (0.80) 3.36 (0.89)
Difference (95% CI) 0.18 (0.08, 0.28) 0.12 (-0.01. 0.24)
P = 0.002 P = 0.069
PD20(mg) Geometric mean and 95% CI for mean Placebo 0.10 (0.04, 0.24) 0.12 (0.04, 0.32)
Formoterol 0.45 (0.15, 1.35) 0.17 (0.05, 0.60)
Difference (doubling doses) (95% CI) 2.12 (0.88, 3.36) 0.51 (-0.49, 1.51)
P = 0.003 P = 0.286
Fall in FEV1 from baseline (%) Mean and SD Placebo 24.78 (4.74) 23.73 (4.54)
Formoterol 24.56 (5.24) 22.26 (4.34)
Difference (95% CI) -0.22 (-3.54, 3.10) -1.47 (-4.61, 1.66)
P = 0.886 P = 0.327
N = 13. As there is no data on subject 6 for day 8 placebo, data from day 1 have been used to calculate means.
Changes in salbutamol response
The area under the salbutamol response curve was lower during formoterol therapy compared to placebo. There was a significant time*treatment interaction indicating that the change in salbutamol response from day 1 to day 8 of formoterol treatment was different to the change in response from day 1 to day 8 of placebo (table 3). Comparisons between placebo and formoterol at each time point found that the reduction in AUC was not statistically significant after the first dose of formoterol, after adjusting the analysis for the increased dose of methacholine used. However, after 1 week of treatment the reduction was statistically significant even after adjusting for the dose of methacholine and the pre-challenge FEV1 (table 4). The mean difference in adjusted AUC between the day 1 and day 8 formoterol challenges was 58.1%.time (95% confidence interval 2.7, 113.4; p = 0.04).
Table 3 ANOVA table for AUC.
Source Degrees of freedom Seq sums of squares Adjusted mean squares F P
Baseline FEV1 1 2195 0 0 0.998
Log dose methacholine 1 88136 46295 22.13 0.000
Subject 12 83869 5368 2.57 0.016
Time 1 10109 5292 2.53 0.121
Treatment 1 24752 18182 8.69 0.006
Time*treatment 1 14283 14283 6.83 0.013
Error 33
Total 50
Table 4 Differences in area under the salbutamol response curve (AUC)
Day 1 Day 8
AUC as % of fall in FEV1 (%.time) mean and SD Placebo 196 (74) 205 (63)
Formoterol 120 (61) 107 (53)
Difference (least squares means, 95% confidence interval) 13.06 (-50.4, 76.53) 85.44 (31.78, 139.1)
P = 0.944 P = 0.001
Final FEV1(L) mean and SD Placebo 3.32 (0.84) 3.31 (0.89)
Formoterol 3.25 (0.22) 3.12 (0.84)
Difference (least squares means, 95% confidence interval) 0.06 (-0.18, 0.30) 0.26 (0.06, 0.46)
P = 0.909 P = 0.006
Differences in AUC by treatment and time using Tukey's exact method from covariate analysis (n = 13)
Despite the fact that pre-challenge FEV1 and post-methacholine FEV1 were higher during formoterol treatment, the FEV1 at the end of the salbutamol dose-response was lower on both formoterol days compared to placebo (see figure 1 for day 8). This difference was not statistically significant after 1 dose, but after 1 week of formoterol the mean difference was significant after allowing for covariates (table 4).
Figure 1 Response to salbutamol following methacholine challenge. Mean and standard error.
There was no effect of order of treatment on salbutamol response, PD20 or FEV1. Analysing AUC as absolute change in FEV1 instead of as a percentage of the fall in FEV1 gave similar results. In this study, baseline FEV1 was not a significant covariate in the analysis of AUC. Excluding this from the model did not materially alter the results of the analysis. There was no difference in the change in salbutamol response between those subjects taking and those not taking inhaled corticosteroids.
Discussion
This study has demonstrated a marked reduction in the bronchodilator response to salbutamol within the usual dosing interval of standard doses of formoterol (12 μg bd). After 1 week of formoterol therapy the area under the salbutamol response curve in the formoterol arm was nearly half that of placebo. This reduction in response was statistically significant after adjusting for the pre-challenge FEV1 and dose of methacholine used in the challenge
The area under the salbutamol response curve was also reduced after a single dose of formoterol. However, this was not statistically significant after adjusting for the dose of methacholine used in the challenge, which was higher after the first dose of formoterol. After 1 week of formoterol therapy the PD20 methacholine was not significantly different to placebo, indicating that tolerance to bronchoprotection had occurred. Hence at this time point there was little protection against bronchoconstriction and greatly reduced bronchodilation.
It is known that cellular tolerance to beta-agonists occurs very rapidly, within 8 hours in vitro, [14] so it is possible that 'tolerance' had occurred even prior to the challenge on day 1 of formoterol treatment. However, there may be other explanations for a reduced response to salbutamol during formoterol therapy, including the fact that beta2-receptors continue to be occupied by formoterol 8 hours after the last dose, leaving fewer receptors available to bind salbutamol. This could explain the reduction in the salbutamol response after the first dose of formoterol. However, the analysis showed a significant interaction between time and treatment, indicating that the bronchodilator response to salbutamol declined further during the formoterol treatment period. Lee et al [15] also found that the salbutamol response 1 hour after a single dose of salmeterol or formoterol was significantly lower after 1 week of treatment than after the first dose of long-acting beta-agonist. If receptor occupancy was the only explanation for the reduction in AUC we would expect to have found a similar reduction in AUC on the first and last day of formoterol treatment. Moreover, the reduction in salbutamol response after a week of formoterol treatment observed in this study was similar to a previous study in our laboratory, where the salbutamol response was measured 24 hours after the last dose of formoterol.[16] Receptor occupancy by formoterol would be much lower at this time, suggesting that the reduction in response is more likely to be due to receptor downregulation.
Regardless of the mechanism, we have shown that the bronchodilator response to salbutamol is reduced during long-acting beta-agonist treatment. Previous studies have been criticised for only analysing changes in FEV1 from baseline rather than actual post-bronchodilator levels of FEV1.[17] It is notable in this study that the final FEV1 after 400 μg of salbutamol was lower during formoterol treatment than during placebo, despite a higher pre-methacholine FEV1 (figure 1).
A recent meta-analysis of studies of regular beta-agonist therapy concluded that the bronchodilator response to subsequent beta-agonist is reduced.[18] However, most of the individual studies included in this analysis failed to show significant tolerance. In contrast, all of the published trials that have measured the bronchodilator response after methacholine challenge have shown significant tolerance.[7-9,11] The 'challenge-rescue model' appears to be more sensitive to changes in the response to bronchodilators. Bronchodilation is a 'closed-end scale'[19] with a maximal achievable level. The dose-response to beta-agonist bronchodilators is therefore dependent on the pre-bronchodilator FEV1.[20] Subjects with mild, stable asthma are often close to their maximum FEV1 and have little capacity for additional bronchodilation. Using methacholine to induce a controlled level of bronchoconstriction before testing bronchodilators ensures that there is capacity for bronchodilation and differences in responses can be shown more easily. Since asthmatics take bronchodilators to relieve symptoms caused by bronchoconstriction it seems logical to test bronchodilators from a state of bronchoconstriction.
This study is the first blinded, placebo-controlled trial to test for bronchodilator tolerance during the trough period of long-acting beta-agonist dosing using the 'challenge-rescue' model. A recent non-blinded trial compared the bronchodilator response to salbutamol after methacholine challenge between formoterol/budesonide or salmeterol/fluticasone to inhaled steroids alone, at 12 hours after inhalation.[21] This study found a similar degree of tolerance to the present study despite methodological differences including the use of salbutamol as-needed.
International guidelines recommend that long-acting beta-agonists are used only in those subjects already taking inhaled corticosteroids.[1] Not all of our subjects were taking such medication. However, this does not detract from the applicability of our study as it has been established that stable doses of inhaled corticosteroids do not alter the development of tolerance to beta-agonists.[7] Moreover, it is widely recognised that patients are often poorly compliant with their inhalers and it is likely that many patients will continue to use their long-acting beta-agonists without inhaled corticosteroids.
All but one of our subjects showed some decrease in the bronchodilator ability of salbutamol after 1 week of formoterol treatment, although the magnitude of this was variable (figure 2). The reasons for this variability are unknown. Lee et al found that tolerance occurred to the same extent in subjects who were homozygous for the Arg-16 or for the Gly-16 polymorphisms of the beta2-receptor.[15]
Figure 2 Individual data for AUC after 1 week of placebo and formoterol. AUC is expressed as a percentage of the fall in FEV1 induced by methacholine.
Conclusion
In conclusion, we have found that bronchodilator tolerance is present during the usual dosing interval of long-acting beta-agonists. Wraight found that tolerance to salbutamol bronchodilation increased with increasing levels of bronchoconstriction.[11] The level of bronchoconstriction induced in this study (20%) was mild compared to that likely to occur during a severe exacerbation of asthma. Patients using long-acting beta-agonists who experience exacerbations of asthma may have an inadequate response to beta-agonist relievers.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
RH conceived the trial, participated in its design, interpretation of results and helped to draft the manuscript. SH participated in the design of the study, acquired the data, performed the statistical analysis and helped to draft the manuscript. Both authors read and approved the final manuscript.
Acknowledgements
We would like to thank the study participants. We also thank Dr J McLachlan and the staff of the Waikato Hospital Respiratory Laboratory, Dr Graham Mills and the Waikato Respiratory Research Unit and Jan Cowan of the Respiratory Research Unit in the Dunedin School of Medicine.
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British Thoracic society British guideline on the management of asthma Thorax 2003 58 suppl 1 1 83
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Yates DH Sussman HS Shaw MJ Barnes PJ Chung KF Regular formoterol treatment in mild asthma. Effect on bronchial responsiveness during and after treatment Am J Respir Crit Care Med 1995 152 1170 4 7551366
Newnham DM McDevitt DG Lipworth BJ Bronchodilator subsensitivity after chronic dosing with eformeterol in patients with asthma Am J Med 1994 97 29 37 7913292 10.1016/0002-9343(94)90045-0
Aziz I Hall IP McFarlane LC Lipworth BJ β2-adrenoceptor regulation and bronchodilator sensitivity after regular treatment with formoterol in subjects with stable asthma J Allergy Clin Immunol 1998 101 337 41 9525449
Horiuchi T Castro M The pathobiologic implications for treatment. Old and new strategies in the treatment of chronic asthma Clin Chest Med 2000 21 381 95 10907595 10.1016/S0272-5231(05)70273-2
Hancox RJ Aldridge RE Cowan JO Flannery EM Herbison GP McLachlan CR Town GI Taylor DR Tolerance to beta-agonists during acute bronchoconstriction Eur Respir J 1999 14 283 7 10515402 10.1034/j.1399-3003.1999.14b08.x
Jones SL Cowan JO Flannery EM Hancox RJ Herbison GP Taylor DR Reversing acute bronchoconstriction in asthma: the effect of bronchodilator tolerance after treatment with formoterol Eur Respir J 2001 17 368 73 11405513 10.1183/09031936.01.17303680
Van der Woude HJ Winter TH Aalbers R Decreased bronchodilating effect of salbutamol in relieving methacholine induced moderate bronchoconstriction during high dose treatment with long acting β2 agonists Thorax 2001 56 529 535 11413351 10.1136/thorax.56.7.529
Storms WW Chervinsky P Ghannam A Bird S Hustad CM Edelman JM A comparison of the effects of oral montelukast and inhaled salmeterol on the response to rescue bronchodilation after challenge Respir Med 2004 98 1051 62 15526805 10.1016/j.rmed.2004.05.008
Wraight JM Hancox RJ Herbison GP Cowan JO Flannery EM Taylor DR Bronchodilator tolerance: the impact of increasing bronchoconstriction Eur Respir J 2003 21 810 5 12765426 10.1183/09031936.03.00067503
Rabe KF Jorres R Nowak D Behr N Magnussen H Comparison of the effects of salmeterol and formoterol on airway tone and responsiveness over 24 hours in bronchial asthma Am Rev Respir Dis 1993 147 1436 41 8503554
Yan K Salome C Woolcock AJ Rapid method for measurement of bronchial responsiveness Thorax 1983 38 760 5 6648855
Hadcock JR Wang H Malbon CC Agonist-induced destabilization of β-adrenergic receptor mRNA J Biol Chem 1989 264 19928 19934 2555338
Lee DKC Jackson CM Bates CE Lipworth BJ Cross tolerance to salbutamol occurs independently of β2 adrenoceptor genotype-16 in asthmatic patients receiving regular formoterol or salmeterol Thorax 2004 59 662 7 15282385 10.1136/thx.2003.019059
Haney S Hancox RJ Rapid onset of tolerance to beta-agonist bronchodilation Respir Med 2005 99 566 571 15823453 10.1016/j.rmed.2004.10.014
Barnes N Bronchodilator subsensitivity to salbutamol after salmeterol Lancet 1995 346 968 7564751 10.1016/S0140-6736(95)91589-3
Salpeter SR Ormiston TM Salpeter EE Meta-analysis: respiratory tolerance to regular β2-agonist use in patients with asthma Ann Intern Med 2004 140 802 813 15148067
Cockcroft DW Swystun VA Functional antagonism: tolerance produced by inhaled beta2 agonists Thorax 1996 51 1051 1056 8984693
Hendeles L Beaty R Ahrens R Stevens G Harman EM Response to inhaled albuterol during nocturnal asthma J Allergy Clin Immunol 2004 113 1058 62 15208585 10.1016/j.jaci.2004.03.046
Lee DKC Jackson CM Currie GP Cockburn WJ Lipworth BJ Comparison of combination inhalers vs corticosteroids alone in moderate persistent asthma Br J Clin Pharmacol 2003 56 494 500 14651722 10.1046/j.1365-2125.2003.01887.x
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Virol JVirology Journal1743-422XBioMed Central London 1743-422X-2-831621612310.1186/1743-422X-2-83MethodologyAn inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification Soliman Hatem [email protected] Mansour [email protected] Institute of Zoology, Fish Biology and Fish Diseases, Faculty of Veterinary Medicine, University of Munich, Germany2005 17 10 2005 2 83 83 27 5 2005 17 10 2005 Copyright © 2005 Soliman and El-Matbouli; licensee BioMed Central Ltd.2005Soliman and El-Matbouli; 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
Koi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment.
Results
A rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers – two inner primers, two outer primers and two loop primers – was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65°C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV.
Conclusion
This paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath.
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Background
Koi Herpesvirus (KHV) is a highly contagious viral disease which causes significant morbidity and mortality in common carp (Cyprinus carpio) and its ornamental domesticated form, koi carp [1]. Although the virus is currently regarded as a DNA-virus belonging to family Herpesviridae [1], some reports have disputed this classification and have renamed the virus as Carp Nephritis and Gill Necrosis Virus, CNGV [2]. More recently, reports based on morphology and genetics have demonstrated strong evidence that KHV is indeed a herpesvirus [3].
The international trade in live fish is arguably the most effective dispersal pathway of fish diseases through incidental movement of pathogenic organisms [4].With respect to koi, exhibitions and national and international trading have facilitated the rapid global spread of KHV. The disease struck koi population in the USA and Israel in 1998 and spread rapidly [5]; it has been reported in Germany [6], Korea [7,8], Indonesia [9], Japan [10], South Africa, and Thailand (unpublished data).
Clinical signs of KHV are often non-specific and mortality may occur rapidly. Discoloration and severe necrosis of the gills is the most consistent sign of infection, with disorientation and erratically swimming prior to death, which can occur within 24–48 hours after the onset of clinical signs [11,12]. KHV has caused considerable economic losses in both the koi and carp culture industries: to fish breeders, retailers and hobbyists impacted by the cumulative mortalities associated with outbreaks [4,2]. There is a clear need for a reliable, rapid diagnostic procedure for the detection of KHV infection.
Rapid virological diagnosis through isolation of the virus has proven difficult and time consuming. A far more efficient approach is nucleic acid amplification; one of the most valuable tools in virtually all life science fields [13]. One of the most widely used techniques is the polymerase chain reaction (PCR) which uses heat denaturation of double-stranded DNA products to promote the next round of DNA synthesis [14,15]. A widely used PCR assay for KHV was developed [16], and a second PCR assay for KHV has been described [12]. A real-time TaqMan PCR assay for KHV has also been developed to detect and quantify KHV DNA in infected tissues [17]. While these PCR techniques have significantly increased our ability to detect KHV infection in koi and common carp, their requirement for a high precision thermacycler has prevented their widespread use in private clinics, for example, as a routine diagnostic tool.
A novel nucleic acid amplification method, loop-mediated isothermal amplification (LAMP), has been developed that does not require a theramcycler. LAMP relies instead on autocycling strand displacement DNA synthesis by a Bst DNA polymerase, to amplify DNA with high specificity, efficiency, and speed under isothermal conditions [13,18,19]. LAMP requires two specially designed inner and two outer primers to improve specificity [20,21]; if two additional 'loop' primers are added, the reaction time can be halved [20]. The amplification products are stem-loop DNA structures with several inverted repeats of the target, and cauliflower-like structures comprising multiple loops [22]. In the present study, we used a LAMP technique for diagnosis of KHV, and evaluated its sensitivity, specificity, and applicability.
Results
Optimisation of the KHV LAMP reaction
The LAMP reaction was performed using purified KHV genomic DNA as a template to determine the optimal primer combination and duration of reaction. The amplicon was formed using either 4 or 6 primers. With 4 primers, a LAMP product was detected after 60 min at 65°C (Fig. 2) while with 6 primers the amplification product was detected as early as 30 min (Fig. 3). KHV DNA extracted either by kit or by boiling gave rise to a typical ladder pattern: many bands of different size up to the loading well as shown in Figures 2, 3 and 5. After addition of 1 μl of diluted SYBR Green I to the reaction tube, positive reactions (amplified products) turned green, whereas all negative controls remained orange, the starting colour of SYBR Green (Fig. 4). The optimal primer concentration is stated in Methods.
Figure 2 Agarose gel showing the effect of time on amplification of KHV DNA by LAMP assay, using four primers (FIP, BIP, F3, B3), carried out at 65°C for durations of 10–60 min. Lane mar = 100 bp DNA molecular weight standard, lane -ve = negative control. The LAMP assay detected KHV after 60 min.
Figure 3 Agarose gel showing the effect of time on amplification of KHV DNA by LAMP assay using six primers (FIP, BIP, F3, B3, loopF, loopB), carried out at 65°C for durations of 10–60 min. Lane mar = 100 bp DNA molecular weight standard, lane -veco = negative control. The LAMP assay detected KHV as early as 30 min.
Figure 4 Visual detection of KHV LAMP products using SYBR Green I stain. 1: negative LAMP reaction remained orange. 2: positive LAMP reaction turned green.
Figure 5 Agarose gel showing LAMP products of KHV DNA extracted by boiling. The reaction was carried out at 65°C using the 6 primer set. Lanes: mar = 100 bp molecular weight marker; 1 = KHV DNA extracted by boiling; 2 = negative fish tissue; 3 = negative control.
Specificity of the KHV LAMP primers and assay
Reaction products were detected only when KHV DNA was present, giving rise to a typical ladder-like pattern. There were no amplification products detected with Herpesvirus cyprini (CHV), channel catfish virus (CCV) or koi fish genomic DNA (Fig. 6).
Figure 6 Agarose gel illustrating the specificity of the designed primers to KHV DNA. The reaction was carried out at 65°C using the 6 primer set for 1 hr. Lanes: 1 = KHV DNA; 2 = Herpesvirus cyprini (CHV) DNA showing no amplification; 3 = channel catfish virus (CCV) showing no amplification; 4 = uninfected koi tissue; mar = 100 bp DNA molecular weight marker.
Sensitivity of the LAMP reaction in detection of KHV
The reaction was tested using 10-fold serial dilutions of KHV DNA from both purified viral DNA and from DNA extracted from positive clinical samples, and compared against results from the commonly used PCR assay. The detection limit of both the LAMP and PCR assays using purified KHV viral DNA was 10-7 (Fig. 7, 9). The detection limit of both assays was 10-5 for clinical samples (Fig. 8, 10). These were the limits for the KHV LAMP reaction under optimal conditions: using 6 primers at 65°C for 60 min. If the reaction was run for 30 min, the detection limit of the LAMP assay was 10-3 for the purified KHV viral DNA and 10-1 for clinical samples. Increasing the primer concentrations did not affect these detection limits (data not shown).
Figure 7 Agarose gel illustrating the sensitivity of the LAMP assay using 10-fold serial dilutions of purified KHV viral DNA. The amplification shows a ladder-like pattern, and detected purified KHV viral DNA down to a dilution of 107. Lanes: -1 = dilution of 10-1; -2 = 10-2 and so on; mar = 100 bp DNA molecular weight standard. -veco = negative control.
Figure 8 Agarose gel demonstrating the sensitivity of the LAMP assay using 10-fold serial dilutions of KHV DNA extracted from a clinical sample. The amplification shows a ladder-like pattern, and detected KHV DNA in a clinical sample at a dilution of 10-5. Lanes: 0 = undiluted KHV DNA; -1 = dilution of 10-1; -2 = 10-2 and so on; mar = 100 bp DNA molecular weight standard. -veco = negative control.
Figure 9 Agarose gel illustrating the sensitivity of the PCR assay using 10-fold serial dilutions of the purified KHV viral DNA. The PCR shows a 484 bp amplification product, and detected purified KHV viral DNA down to a dilution of 107. Lanes: 0 = undiluted KHV DNA; -1 = dilution of 10-1; -2 = 10-2 and so on; mar = 100 bp DNA molecular weight standard; -veco = negative control without target DNA.
Figure 10 Agarose gel showing the sensitivity of the PCR assay using 10-fold serial dilutions of the KHV DNA extracted from a clinical sample. The PCR reveals a 484 bp amplification product, and detected KHV in a clinical sample at a dilution of 10-5. Lanes: 0 = undiluted KHV DNA; -1 = dilution of 10-1; -2 = 10-2 and so on; mar = 100 bp DNA molecular weight standard; -ve = negative control without target DNA.
Applicability of the KHV LAMP reaction
50 clinical cases with suspected KHV infections were submitted to our laboratory and were investigated both with the LAMP assay and standard PCR. 37 out of 50 tested positive with both the PCR and LAMP; 13 were negative. No sample that was negative with the LAMP assay tested positive with the PCR, and vice versa.
Discussion
The most extensively used diagnostic methods for KHV are cell culture and PCR. These techniques, however, require a relatively long time to produce results or are not practical for commercial producers, retailers, and regulators because of the equipment and expertise needed to conduct the assays. Moreover, the Taq DNA polymerase used in the PCR assay is easily inactivated by tissue- and blood-derived inhibitors such as myoglobin, hem-blood protein complex and immunoglobulin G [25-28]. Loop-mediated isothermal amplification (LAMP) is a novel method that facilitates rapid nucleic acid amplification using only simple equipment [13]. In the first step of the LAMP reaction, Bst polymerase synthesises new DNA between the F3 and B3 primers; this is the same reaction as standard PCR and requires homology between the primers and the template DNA. In the next step, the newly synthesised strands are recognised by the inner primers FIP and BIP to start loop mediated autocycling amplification [29] to produce stem-loop DNA structures with several inverted repeats of the target and cauliflower-like structures with multiple loops [22]. Amplification is specific and rapid when template which includes sequences that the loop primers recognise is present [20]. To accelerate the LAMP reaction 6 primers were used instead of 4. The two additional primers hybridised to the stem-loops (except for those loops that had been hybridized by the inner primers) [20].
KHV DNA extraction was performed by boiling fish tissues in a buffer solution; a simple and rapid technique [31-33] AL buffer was used to inactivate DNase and to elute DNA from tissues. Immediately after boiling, both undiluted and diluted DNA samples were trialled as templates for the LAMP reaction. No amplification products were detected for the undiluted DNA; addition of 800 μl TE buffer was necessary to dilute reaction inhibitors which were present in the boiled solution [30]. The LAMP assay was sensitive enough to detect KHV DNA at this (1:4) dilution. A specific type of DNA polymerase was required for the LAMP reaction, Bst DNA polymerase, which has two distinct activities: linear target isothermal multimerisation and amplification, and cascade rolling-circle amplification [34]. The mechanism of loop mediated isothermal amplification is similar to cascade rolling circle amplification. Occasionally, a different LAMP amplification pattern appeared as a result of linear target isothermal multimerisation and amplification, as LAMP primers and target DNA seem to randomly multimerize [29]. Betaine was used in the LAMP reaction mixture to reduce base stacking [35-37] and to increase not only the overall rate of reaction but also target selectivity by significantly reducing amplification of irrelevant sequences [13]. Use of SYBR Green I for visual inspection of LAMP amplification products was a simple and superior technique, with no gel electrophoresis and staining with ethidium bromide required. Only 1 μl of diluted SYBR Green I added to the reaction mixture was enough to see a result: if the reaction mix turned from orange to green it was judged as positive. This visualisation technique is effective due to the high specificity and amplification efficiency of LAMP [22].
The detection limit of the KHV LAMP reaction was determined through amplification of 10-fold serial dilutions of both purified KHV viral DNA and DNA from positive clinical samples (containing both fish and KHV DNA). The LAMP reaction was performed at 65°C for 30 and 60 min, and compared with the results of the standard PCR assay. There was no difference between the detection limit of the LAMP reaction and the PCR reaction at 60 min: both were positive at 10-7 dilution of purified virus DNA, and at 10-5 from the clinical samples. However, at 30 min the LAMP detected down to only 10-3 dilution of viral DNA and 10-1 dilution DNA from clinical samples. Hence the optimal LAMP conditions were determined to be 65°C for 60 min to detect KHV virus down to a concentration of 0.1 pg. Although the LAMP reaction had equivalent sensitivity to the PCR test, it is considered superior because it is a simpler technique which can be carried out in most situations where a rapid diagnostic method is required: under field conditions, in private clinics, and at quarantine inspection stations. A water bath is the only equipment needed, and is used for both the DNA extraction and nucleic acid amplification.
Although the application of LAMP for the detection of KHV has been reported previously [38], these authors use only 4 primers which target the KHV tk gene. In the current study, 6 primers which recognise 8 distinct regions on the KHV DNA were used, thereby enhancing the specificity of the reaction and eliminating false positive results [20]. Also, DNA extraction by boiling prior to the LAMP test and visualisation of reaction products using SYBR Green I DNA stain were employed to reduce the time needed to perform the KHV test and to simplify the procedure.
In conclusion, the KHV LAMP reaction is a highly sensitive, rapid, and reliable method that can be used under field condition for diagnosis of the KHV infection.
Methods
DNA oligonucleotides
Six primers were designed from a KHV amplicon (Genbank Accession number AF411803), which recognise eight distinct regions of the target DNA. Forward inner primer (FIP) comprised the antisense sequence of F1 (23nt), a TTTT linker and a sense sequence of F2 (23nt): 5'- CAACAATGCTTCTTGTGATTACA-TTTT-GAACCCG AGGGGACTGCTCGCTT-3'. Backward inner primer (BIP) consisted of the sense sequence of B1 (23nt), a TTTT linker and the antisense sequence of B2 (23nt): 5'- CC GATGGAGTGAAACTGGAACTG-TTTT-CGTCATGCTCTCCGAGGCCAGCG-3'. The outer primers were F3 (19nt): 5'- GAGGAAGCGCAAAAAGAAC-3', and B3 (19nt): 5'- TTCAGTCTGTTCCTCAACC-3'. The loop primers were, loop F (20nt): 5'-ATTATTATAC AACAACAATA-3'; and loop B (20nt): 5'-TGAGCGTGGGGTCAAAGTT G-3'. (Fig. 1). Primers used in the PCR assay were constructed according to Gilad et al. (2002). Forward primer- KHV9/5F: 5'- GACGACGCCGGAGACCTTGTG-3', and reverse primer- KHV9/5R: 5'- CACAAGTTCAGTCTGTTCCTCAAC-3'. This primer set amplified a 484 bp segment of the KHV template.
Figure 1 Nucleotide sequence of the KHV amplicon (GenBank accession number AF411803) used for construction of the inner and outer primers. The primer sequences are indicated in bold letters. Inner primers FIP and BIP comprise sequences within the amplicon; FIP is the complementary sequence of F1 and F2, BIP is B1 plus the complementary sequence of B2.
DNA extraction
Gills, kidney, spleen, and brain were sampled from fish sent to our laboratory with suspected KHV infections. DNA extraction was performed using both a commercial kit and a tissue boiling method. For the QIAamp DNA mini kit (QIAGEN GmbH, Hilden Germany), one gram of each organ was ground thoroughly in liquid nitrogen using a mortar and pestle. 20 mg of tissue powder was placed in a 2 ml microfuge tube, 180 μl of lysis buffer and 20 μl of proteinase K were added, then incubated at 56°C in a water bath until the tissues were completely lysed (1–3 h). DNA extraction was then completed according to the manufacturer's instructions, with final elution of DNA in 100 μl elution buffer, and storage at -20°C.
The second method of DNA extraction was by boiling: 20 mg of each tissue were placed in 2 ml microfuge tubes with 200 μl AL buffer (QIAGEN GmbH, Hilden, Germany), and placed in boiling water for 15 min. 800 μl of Tris- EDTA buffer (TE: 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) was then added to the tube, mixed well, and centrifuged at 14,000 rpm for 3 min. The supernatant contained the DNA was used immediately in the KHV assays.
LAMP reaction
The 25 μl reaction mixture comprised: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 6 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Triton X-100, 1.6 M betaine, deoxynucleotide triphosphates 2.8 mM each, 1.6 μM each FIP and BIP, 0.8 μM each loop-F and loop-B, 0.2 μM each F3 and B3 primers, 8 U Bst DNA polymerase (New England BioLabs, GmbH, Frankfurt, Germany), 2 μl template DNA, distilled water to 25 μl. As a negative control, template DNA was omitted from the reaction. The mix was incubated at 65°C for 60 min and then heated at 80°C for 2 min to terminate the reaction.
Analysis of LAMP products
1 μl of 1:10 diluted SYBR Green I Nucleic acid gel stain, 10,000× concentration in DEMSO (Cambrex Bio Science, Rockland, Inc, ME USA) was added directly to the reaction tube and any colour change observed. The solution turned green if LAMP reaction products were present, otherwise it remained orange. Reaction products were also analysed by gel electrophoresis: 5 μl aliquots were analysed on a 2% agarose gel and subsequently stained with ethidium bromide; a DNA molecular weight marker, 100 bp DNA Ladder, (Cambrex Bio Science, Inc, Rockland, ME USA) was used to determine the size of the products.
PCR assay
Amplification was performed according to Gilad et al. (2002) in a standard reaction volume of 50 μl comprising 3 μl template DNA and 47 μl 1.1× ReaddyMix PCR Master mix: 75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 1.5 mM MgCl2, 0.01% Tween20, 0.2 mM each of dATP, dCTP, dGTP, dTTP, 1.25 U Taq DNA polymerase and red dye for electrophoresis (ABgene, Hamburg, Germany) and forward and reverse primers (20 pmol each). The reaction mixture was subjected to 39 amplification cycles under the following conditions: denaturation at 94°C for 1 min, annealing at 68°C for 1 min, extension at 72°C for 30s. The amplification cycles were preceded by a denaturation step at 94°C for 5 min and followed by an extended elongation step at 72°C for 7 min.
Detection of PCR products
products were analysed by electrophoresis on 1.5% agarose gels stained with ethidium bromide. 100 bp DNA Ladder (Cambrex Bio Science, Inc, Rockland, ME USA) was used to determine the size of the PCR products.
Optimisation of KHV LAMP reaction conditions
varying concentrations of the FIP, BIP, F3, B3, loop-F, loop-B primers were trialled, as well as use of only 4 primers (excluding loop-F and loop-B). Time of reaction was varied in 5 minute increments from 10–60 min to determine detection time of KHV genomic DNA.
Specificity of the KHV LAMP assay
The reaction was tested using DNA from Herpesvirus cyprini (CHV), channel catfish virus (CCV) and koi genomic DNA.
Sensitivity of the KHV LAMP reaction
The detection limits of the KHV LAMP assay were evaluated using 10-fold serial dilutions of purified KHV DNA and DNA extracted from positive clinical samples. The reaction was performed at 65°C for both 30 and 60 min, and compared with the PCR assay results.
Applicability of the KHV LAMP reaction
After the initial validation studies, the KHV LAMP reaction was used to test 50 suspected clinical cases submitted to our laboratory and the results compared with the PCR assay results of those 50 cases.
Author's contributions
ME conceived and supervised the study and drafted the manuscript. HS carried out all the experimental work and data acquisition.
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-3-691623232410.1186/1477-7819-3-69ResearchInjection of colorectal cancer cells in mesenteric and antimesenteric sides of the colon results in different patterns of metastatic diffusion: An experimental study in rats Boni Luigi [email protected] Angelo [email protected] Gianlorenzo [email protected] Francesca [email protected] Mario [email protected] Renzo [email protected] Department of Surgery, University of Insubria, Ospedale di Circolo e Fondazione Macchi, Varese, Italy2005 19 10 2005 3 69 69 6 9 2005 19 10 2005 Copyright © 2005 Boni et al; licensee BioMed Central Ltd.2005Boni 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 experimental study was designed to investigate the differences in pattern of local growth and diffusion of colorectal cancer cells injected into either mesenteric (M) or antimesenteric (AM) sides of the colon.
Methods
A total of 1 × 106 colonic adenocarcinoma cells (line DHD/K12-TRb) were injected into the cecal wall of BDIX syngeneic male rats at an M or AM site of the colon. At six weeks after injection, all animals were sacrificed and the presence or absence of tumor in the cecum as well as regional metastasis and peritoneal carcinomatosis were determined.
Results
Six weeks after injection, macroscopic tumor growth was observed in 27/37 (72%) animals in group M and 21/32 (65%) in group AM (P = 0.98). In group AM, diffuse peritoneal carcinomatosis was present in 19/21 rats (90.4%) versus 3/27 rats (11%) in group M; this difference was statistically significant (P = 0.025). Regional mesenteric lymph nodes were the only location in which tumor was detected in 23/27 rats (85%) in group M versus 2/21 (9.5%) in group AM; this difference too was statistically significant (P = 0.031)
Conclusion
The patterns of diffusion of tumors implanted in mesenteric and antimesenteric sites of the colon appear to be different, although the reason for this is not clear.
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Background
Colorectal cancer is the second leading cause of death from tumors in the world with a worldwide incidence of more than 1 million cases [1]. As for most malignant tumors, the degree of infiltration of the colonic wall (T stage) and presence of lymph node/ distant (N and M stage) metastases have been demonstrated to be strongly related to prognosis [2]. Identifying the pattern of cancer spread (local and distant) may be important in choosing the appropriate surgical and medical, strategies in relation to various factors, such as tumor location and site of growth.
Several experimental models have been developed to examine colorectal cancer kinetics, but most are based on heterotopic cell implantation (i.e. peritoneal, subcutaneous, etc.) [3,4] both in nude and syngeneic rats [5,6].
Other investigators have developed orthotopic models in which tumor implantation is performed directly into the wall of the colon of healthy rats, in order to study patterns of local growth and metastatic spread [7,8].
The present experimental study was designed to investigate the differences in pattern of local growth and diffusion of colorectal cancer cells injected into either mesenteric (M) or antimesenteric (AM) sides of the colon. Such differences may be of clinical importance, since metastases and diffusion could be influenced by tumor location.
Materials and methods
Animals
BDIX syngeneic male rats (Charles Rivers Italia – Lecco, Italy) with a mean weight of 290 ± 15 g were used for all experiments. They were kept in protective cages with controlled air in/outflow and allowed free access to food and water. They were divided into two groups, mesenteric (M) and antimesenteric (AM), of 40 rats each.
Tumor cell line
The colonic adenocarcinoma cell line DHD/K12-TRb (European Collection of Cell Culture, Salisbury, Wiltshire – UK) was used in all experiments. These cells were cloned [3] from a 1,2-dimethylhydrazine-induced colonic adenocarcinoma and maintained in monolayers using DMEM medium enriched with Ham's F10 (1:1, v/v; GIBCO, Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (GIBCO) and 0.005% gentamycin (GIBCO). The cells were divided every 72 hours after dispersion in 0.125% EDTA-trypsin.
Prior to injection, cells viability was confirmed by trypan-blue test, and was found to be greater than 95% in all cases.
Tumor implantation model and experimental procedure
Each animal was anaesthetized by intraperitoneal injection of 75 mg/kg of ketamine (Sigma-Aldrigh, Italy). A 2 cm midline laparotomy was performed and the cecum was exposed. Intraparietal injection (Fig. 1) of 1 × 106 cells in 0.25 ml of buffer solution was performed in a mesenteric (Group M) or antimesenteric (Group AM) side of the cecum (Figure 1).
Figure 1 Intraparietal injection of colonic cancer cells (mesenteric injection).
Extreme care was taken to avoid accidental spillage of cells into the abdominal cavity or tumor injection directly into the bowel lumen: the creation of a macroscopically visible blister in the bowel wall was required for confirmation of intra-parietal injection. The laparotomy was closed with reabsorbable 3/0 suture.
At six week after injection, all animals were sacrificed with a lethal dose of sodium thiopental (Sigma-Aldrigh, Italy) A wide xiphopubic incision was performed and the presence or absence of tumor in the cecum as well as regional metastasis (Figure 2) and peritoneal carcinomatosis (Figure 3) was recorded. Histopathological examination of the primary tumor and metastases was always performed to confirm the features of the specimen.
Figure 2 Mesenteric growth of implanted colonic cancer.
Figure 3 Peritoneal carcinomatosis after antimesenteric implantation of cancer cells.
Statistical analysis
Student's t-test was used to compare numbers of tumor lesions and the characteristics of diffusion, with P-values less than 0.05 considered significant.
Results
Three animals in group M and 8 in group AM died due to cardiorespiratory arrest during anesthesia or immediately after the procedure. Six weeks after tumor cell injection, macroscopic tumor growth was observed in 27/37 (72%) animals in group M and 21/32 (65%) in group AM; this difference was not significant (P = 0.98).
In group AM, diffuse peritoneal carcinomatosis (Figure 3) was present in 19/21 rats (90.4%) versus 3/27 cases (11%) in group M (Figure 4); this difference was statistically significant (P = 0.025).
Figure 4 Incidence of peritoneal carcinomatosis in groups M and AM (P = 0.025).
Regional mesenteric lymph nodes (Figure 2) (located near tumor growth) were the only manifestation of tumor diffusion in 23/27 rats (85%) in group M versus 2/21 (9.5%) in group AM (Figure 5); this difference was significant (P = 0.031). No liver or lung metastases were observed in either group. Histological confirmation of the presence of cancer cells was obtained in all cases.
Figure 5 Incidence of mesenteric growth in groups M and AM (P = 0.031).
Discussion
Study of the kinetics of local growth and metastatic diffusion of cancers is extremely important for increasing knowledge of the natural history of the tumors and for determination of appropriate surgical strategies and/or medical therapies.
Although experimental models are the best means for study of tumor kinetics, exposure of animals to carcinogenic agents results in excessive variability such as differences in differentiation, location and diffusion. On the contrary, implantation of cancer cells or solid tumor, directly into the site to be studied, it can reproduce a standard condition more suitable for this kind of studies [3-7].
Several experimental models of free cancer cell implantation into the wall of the colon have been described [3,4]. The animal model used in our experiments is well-established, since it has been fully demonstrated that DHD/K12/TRb colonic cancer cells are able to grow when injected subcutaneously, intraperitoneally and in the wall of the colon [3-5]. Nevertheless, some of these studies used nude animals, in which unusual immunological status may result in great difficulty in interpretation of results.
In our study, the incidence of tumor growth after implantation was almost 65%; similar results have been reported by other studies [4,7]. Incorrect parietal injection, low viability of tumor cells, intraluminar injection, or host immune response to cancer cells may be explanations of the 65% tumor growth incidence.
Garcia Almo et al, [7], injecting DHD/K12/TRb cells into the cecal wall of syngeneic rats, demonstrated progressive tumor growth from stage T1 to stage T4, as well as lymph node involvement and distant metastases, reproducing the same progressive development as occurs in human colon cancer. Nevertheless, with this kind of injection model, it seems to be extremely difficult to perform precise implantation into the mucosa and to enable progressive tumor growth corresponding to that observed clinically.
In order to avoid accidental spillage of tumor cells and induce true local growth, Balague et al, [8] developed a model in which solid tumors, derived from DHD/K12/TRb colonic cancer cells, were implanted into the wall of the colon. Nevertheless, variability in the number of viable cells contained in a single piece of implanted tumor may result in misinterpretation of results.
None of the above studies considered difference in tumor implantation side, mesenteric or antimesenteric, as affecting local growth and distant metastases.
This difference might be of clinical significance, since certain locations can result in more aggressive tumor behavior due to particular anatomical patterns of blood or lymphatic vessels. In our study, when injection of tumor cells was performed in the mesenteric site of the colon, cancer grew locally and spread to the regional mesenteric lymph nodes. On the other hand, with antimesenteric implantation, there was lymph node metastasis but a high incidence of peritoneal carcinomatosis.
Proximity to larger blood and lymphatic vessels of the mesenteric site of the colon could explain the tendency of implanted cells to spread to regional nodes. When cancer cells are injected into the antimesenteric wall, peritoneal diffusion appears to be promoted, probably due to direct contact of the tumor with loops of bowel and the peritoneum.
Another explanation may be related to differences in microvascular pattern of the wall of the colon. Preliminary results of an experimental vascular casting study that we are performing in collaboration with the Department of Human Anatomy, seem to support the theory that the antimesenteric side of the colon differs significantly from the mesenteric side in microvascular arterial density and distribution (unpublished data).
Since M and N stage are the most important prognostic factors in human colorectal cancer [2], differences in local and metastatic diffusion of cancers located on the mesenteric or antimesenteric sites of the colon may be of clinical importance [9].
Conclusion
The experimental model used in this study is able to induce local growth as well as peritoneal carcinomatosis in rats by parietal injection of malignant cells. The pattern of diffusion of tumors implanted at mesenteric and antimesenteric sites of the colon appear to differ, although the reason for this difference is unclear. Proximity of larger blood and lymphatic vessels or differences in vascular pattern in the wall of the colon between mesenteric and antimesenteric sites might account for these findings. Further clinical studies are needed to confirm the prognostic significance of the location of colorectal cancer in humans.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
LB: Clinical Lecturer in Surgery, Research leader he performed the experimental procedure and He wrote the experimental part of the manuscript
AB: Professor of Surgery, research project advisor, he supervised the experimental procedures and helped during the manuscript preparation
GD: Clinical Lecturer, assisted during the experimental procedure, he helped during the manuscript preparation (introduction and Discussion) and literature review
FR: Clinical Lecturer, She helped during the manuscript preparation especially in the statistical methods and discussion
MD: Staff surgeon, assisted during the experimental procedure
RD: Professor of Surgery, research project advisor, he helped during the manuscript preparation and final revision
All authors read and approved the final manuscript for publication.
Acknowledgements
Special thanks to Dr Fabrizio Cantore and Luisa Giavarini for their assistance during the experimental procedure.
The study was conducted as per the guidelines of research on animals of the Italian University and Research Ministry
The study was approved by the Italian University and Research Ministry committee
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Garcıa-Olmo D Garcıa-Rivas D Garcıa-Olmo DC Atienzar M Orthotopic implantation of colon carcinoma cells provides an experimental model in the rat that replicates the regional spreading pattern of human colorectal cancer Cancer Lett 1998 132 127 133 10397463 10.1016/S0304-3835(98)00174-8
Balague C Braumann C Fuhrer K Guski H Jacobi CA Validation of a new experimental model of colon cancer Surg Endosc 2001 15 833 836 11443462 10.1007/s004640090028
Benevento A Boni L Dionigi G Carcano G Capella C Capriata G Casula G Dettori G Dionigi R The mesenteric and antimesenteric location of colorectal cancer: the relationship with lymph nodes Surgeon 2004 2 214 220 15570829
|
16232324
|
PMC1266403
|
CC BY
|
2021-01-04 16:39:04
|
no
|
World J Surg Oncol. 2005 Oct 19; 3:69
|
utf-8
|
World J Surg Oncol
| 2,005 |
10.1186/1477-7819-3-69
|
oa_comm
|
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