The sterilizing activity of the regimen used to treat multidrug resistant tuberculosis (MDR TB) has not been studied in a mouse model.Mycobacterium tuberculosis (TB) strain H37Rv, treated with second-line drug combinations with or without the diarylquinoline TMC207, and then followed without treatment for 3 more months to determine relapse rates (modified Cornell model).Swiss mice were intravenously inoculated with 6 log10 of Bactericidal efficacy was assessed by quantitative lung colony-forming unit (CFU) counts. Sterilizing efficacy was assessed by measuring bacteriological relapse rates 3 months after the end of treatment.The relapse rate observed after 12 months treatment with the WHO recommended MDR TB regimen  was equivalent to the relapse rate observed after 6 months treatment with the recommended drug susceptible TB regimen . When TMC207 was added to this MDR TB regimen, the treatment duration needed to reach the same relapse rate dropped to 6 months. A similar relapse rate was also obtained with a 6-month completely oral regimen including TMC207, moxifloxacin and pyrazinamide but excluding both amikacin and ethionamide.In this murine model the duration of the WHO MDR TB treatment could be reduced to 12 months instead of the recommended 18–24 months. The inclusion of TMC207 in the WHO MDR TB treatment regimen has the potential to further shorten the treatment duration and at the same time to simplify treatment by eliminating the need to include an injectable aminoglycoside. As the leading cause of death from curable infectious diseases worldwide, tuberculosis (TB) is a serious global health issue Mycobacterium tuberculosis (M.tb) is very low (0.06 µg/ml). In mice intravenously (IV) inoculated with M.tb, TMC207 combined with first-line anti-TB drugs shortened the treatment duration from 6 to 4 months TMC207 (R207910) is a diarylquinoline with a new mechanism of action (inhibition of ATP synthase), and is in clinical development for treatment of both drug susceptible (DS) and MDR TB. Its minimal inhibitory concentration (MIC) against The laboratory has been approved on July 27th, 2004 to carry out animal experiments. Nicolas Veziris who carried the animal experiments has the following license number: 75-1531. We followed the animal experiment guidelines of the Faculté de Médecine Pierre-et-Marie Curie.TMC207 was provided by Johnson & Johnson , while the other compounds were purchased from the following manufactures: isoniazid (H) from Laphal , rifampin (R), pyrazinamide (Z) from Aventis , moxifloxacin (M) from Bayer , amikacin from Bristol-Myers Squibb , and ethionamide from Sigma (France).Mycobacterium bovis BCG and was further diluted with normal saline to obtain a 0.2 mg/ml suspension for mouse inoculation.The H37Rv strain of M.tb was grown on Löwenstein-Jensen medium (LJ slants). Colonies were subcultured in Dubos broth  for 7 days at 37°C. The turbidity of resulting suspension was adjusted with normal saline to match that of standard 1 mg/ml suspension of 6 colony forming units (CFU) of M.tb H37Rv.Four hundred seventy female four-week-old Swiss mice were purchased from the Janvier Breeding Center . They were intravenously infected in the tail vein with 0.5 ml of bacterial suspension containing approximately 1.1×10Mice were randomized into ten groups . NegativTreatment began 19 days after intravenous infection (D0) i.e when a large bacterial population had developed. All groups were treated 5 days per week. Drug suspensions were prepared weekly and kept at 4°C except for TMC207 which was prepared monthly in 20% hydroxypropyl-B-cyclodextrin and kept at 4°C. Amikacin was given by subcutaneous injection at 150 mg/kg as previously described To assess the extent of M.tb infection and to provide baseline values before initiation of chemotherapy, 10 untreated control mice were sacrificed at day 1 and 20 at day 19 after infection respectively (i.e. day −18 and day 0 in relation to the initiation of treatment). Treated mice were sacrificed either at the end of treatment to assess the bactericidal activity or 3 months after the end of treatment to assess the relapse rate (sterilizing activity) .The severity of infection and the effectiveness of the treatments were assessed by survival rate, spleen weight, gross lung lesions  and lung CFU counts.The number of CFU in the lungs was determined by plating homogenized lung suspensions in triplicate on Lowenstein-Jensen (LJ) slants, either diluted for untreated control mice or undiluted for treated mice. In the latter case the whole suspension (3.5 ml) was plated on 15 LJ slants. The CFU count was assessed after 6 weeks of incubation at 37°C Mean spleen weights were compared using the Student's t test. Proportion of positive mice 3 months after the end of treatment were compared using the Fisher's exact test (FET) to compare relapse rates since samples were small (less than 30 mice).Fify-two out of 440 mice died during the 15 months experiment. All of the 10 untreated mice died between 18 and 94 days after infection. Deaths among treated mice during the first month were assumed to be due to tuberculosis as mice appeared to be severely ill and had continuous weight loss after becoming infected whereas death occurring after the first month were due to gavage or injection accidents . In group 2, 2 mice out of 39 died because of gavage accidents after the first month, in group 3, 2 mice out of 30 died (1 during first month and 1 afterwards), in group 4, 18 mice out of 90 died (6 during the first month and 11 because of gavage or injection accident afterwards), in group 5, 6 mice out of 90 died (5 during first month and one because of a gavage accident), in group 6, 4 mice out of 30 died (3 during first month and one because of gavage or injection accident), in group 7, no mice died, in group 8, 2 mice out of 30 died in the first month, in group 9, 3 mice died (2 during first month and one because of gavage accident) and in group 10, 6 mice out of 30 died (2 during the first month and 4 afterwards).The day after infection (D-18), the mean spleen weight was 115±30 mg, while 18 days later, at the start of treatment (D0), the mean spleen weight had increased to reach 608±144 mg. During treatment, mean spleen weights decreased significantly in all the groups (p<0.005), compared to the mean spleen weight on D0 (results not shown).The number of gross lung lesions increased from 0 the day after inoculation to ++18 days later at the start of treatment. After three and four months of treatment, the number of lesions decreased progressively. There was no difference in gross lung lesions among treated groups (results not shown).The day after infection, the mean lung CFU count was 5.6 log10. On D0, the mean lung CFU had increased to 7.1 log 10.All mice were culture negative at the end of treatment in all treatment groups except 1 mouse in the JZM group after 4 months (3 colonies), 1 mouse in the AEtMZ group after 9 month (2 colonies) and 1 mouse in the AEtMZ group after 12 months (1 colony).The proportion of mice that relapsed (i.e. became culture positive) 3 months after the end of treatment was determined for each combination .In the group receiving 2 months of RHZ followed by 4 months of RH, 3 out of 28 mice (11%) relapsed with a mean 1.8 log CFU. In the group receiving 2 months of JRZ followed by 2 months of JR, 3 out of 19 mice (16%) relapsed with a mean 2.5 log10 CFU. Among mice treated with the MDR WHO regimen, i.e. 2 months of AEtMZ followed by 4, 7 or 10 months of EtM, the relapse rates were 11/19 (58%) with a mean 2.4 log10 CFU, 8/16 (50%) with a mean 2.0 log10 CFU and 4/18 (22%) with a mean 0.9 log10 CFU. In the group receiving 2 months of JZM followed by 2, 4 or 7 months of JM, relapse rates were 8/20 (40%) with a mean 1.7 log10 CFU, 2/19 (11%) with a mean 0.3 log10 CFU and 5/20 (25%) with a mean 2.2 log10 CFU. Five out of 18 mice (28%) that received WHO MDR regimen combined with J, i.e. 2 months of JAEtMZ followed by 4 months of JEtM, relapsed with a mean 1.5 log10 CFU. Nine out of 20 mice (45%) that received the WHO MDR regimen in which J replaced M, i.e. 2 months of JAEtZ followed by 4 months of JEt, relapsed with a mean 2.3 log10 CFU. Ten out of 20 mice (50%) that received the WHO MDR regimen in which J replaced Z, i.e. 2 months of JAEtM followed by 4 months of JEtM, relapsed with a mean 1.8 log10 CFU. And finally, nine out of 20 mice (45%) that received the 6 months JM combination relapsed with a mean 1.8 log10 CFU and four out of 18 mice (22%) that received the 6 months JZ combination relapsed with a mean 0.9 log10 CFU.Compared to the standard WHO DS regimen (group 2), the relapse rate of the WHO MDR regimen  was significantly worse after 6 and 9 months (p = 0.0009 and 0.009 respectively) but not after 12 months (p = 1). When J was added to the WHO MDR regimen (group 6) the relapse after 6 months became equivalent to that of RHZ (p = 0.23). When M or Z were removed (groups 7 and 8) from this regimen the relapse rate increased again and became significantly worse compared to that of RHZ .Compared to the standard WHO regimen for DS TB , the relapse rate of regimen JZM was significantly worse after 4 months (p = 0.03) but was equivalent after 6 and 9 months . Even the regimen combining only J and Z (group 10) for 6 months achieved an acceptable relapse rate in this experiment.We have previously shown that the MDR TB regimen combining amikacin, moxifloxacin, ethionamide and pyrazinamide has the highest bactericidal activity in the mouse model, resulting in culture conversion after 9 months of treatment Several factors complicate the extrapolation of mouse data to the human situation. Firstly, the pathology of tuberculosis in mice is different from humans, the spread of the bacilli is haematogenous rather than bronchogenous, tuberculosis develops much faster than in humans (mice die in less than 2 months), and mice do not develop lung cavities Secondly, some dosages used here may not exactly mimic those used in humans. E.g. the 150 mg/kg dose of amikacin generates a Cmax in mice that is approximately four times higher than the one obtained in patients taking a 15 mg/kg dose. On the other hand, the intracellular penetration of aminoglycosides is known to be low and their efficacy is therefore expected to be better in patients with mainly extracellular TB replication compared to mice with mainly intracellularly replication The third limitation is the statistical power of the study. To achieve a power of 80% to detect superiority between regimens, mouse relapse studies with 20 animals per group require an absolute difference of 40% in relapse rates between regimens The last limitation is that MDR TB isolates are by definition resistant to isoniazid and rifampin, but usually have also acquired additional resistance mutations. In this context, the impact of resistance to pyrazinamide on minimal treatment durations may be a very important one. Acquired pyrazinamide resistance is very common in MDR TB patients, and the data summarized in The second aim of this study was to assess the contribution of the diarylquinoline TMC207 to the sterilizing activity of MDR regimens. TMC207 has been shown to improve both the bactericidal and the sterilizing activity of the first line regimen RHZ The slight increase in relapse rate obtained by extending treatment with JM from 6 to 9 months (groups 5B and 5C) is puzzling. Such increase could be due to the fact that too few mice were studied per group (as discussed above in the limitations of the study), to the development of resistance, or to the possibility that the combination of TMC207+M is not active against bacilli persisting after 6 months of treatment. The emergence of resistance was excluded by testing the sensitivity of strains isolated after 9 months. The combination of TMC207+M might indeed not be sufficient to kill all persistent bacilli, but is very efficient if Z is added, and not stopped after 2 months. Indeed, it was recently reported that the regimen JZM resulted in acceptable relapse rates after just 5 months, and complete bacteriological cure (0% relapses) after 6 months of treatment The control group receiving the DS TB regimen JRZ achieved a relapse rate equivalent to that of the 6 months standard WHO regimen RHZ after 4 months. This confirms an earlier study In conclusion, we showed that treatment of mice with a moxifloxacin+pyrazinamide based regimen, as recommended by WHO for the treatment of MDR TB, results in stable cure in 12 months. An entirely oral regimen combining TMC207, moxifloxacin and pyrazinamide could reduce the duration needed to produce stable cure to only 6 months. These treatment duration estimates need to confirmed in clinical trials. Preliminary results of an ongoing clinical trial with TMC207 against MDR TB are encouraging


Serological analysis revealed that compared to wild-type littermates (+/YHmgn5), mice with a targeted mutation in the HMGN5 gene (tm1/YHmgn5), had elevated serum albumin, non-HDL cholesterol, triglycerides, and alanine transaminase, suggesting mild hepatic abnormalities. Metabolomics analysis of liver extracts and urine revealed clear differences in metabolites between tm1/YHmgn5 and their +/YHmgn5 littermates. tm1/YHmgn5 mice had a significant increase in hepatic glutathione levels and decreased urinary concentrations of betaine, phenylacetylglycine, and creatine, all of which are metabolically related to the glutathione precursor glycine. Microarray and qPCR analysis revealed that expression of two genes affecting glutathione metabolism, glutathione peroxidase 6 (Gpx6) and hexokinase 1 (Hk1), was significantly decreased in tm1/YHmgn5 mouse liver tissue. Analysis of chromatin structure by DNase I digestion revealed alterations in the chromatin structure of these genes in the livers of tm1/YHmgn5 mice. Thus, functional loss of HMGN5 leads to changes in transcription of Gpx6 and Hk1 that alter glutathione metabolism.High mobility group nucleosome-binding protein 5 (HMGN5) is a chromatin architectural protein that binds specifically to nucleosomes and reduces the compaction of the chromatin fiber. The protein is present in most vertebrate tissues however the physiological function of this protein is unknown. To examine the function of HMGN5  High mobility group (HMGN) proteins are ubiquitously expressed in vertebrate cells and are known to affect both chromatin structure and the levels of post-translational modifications to histone tails; two important epigenetic processes involved in the regulation of gene expression tm1/tm1Hmgn3 mice develop glucose intolerance due to disruptions in insulin release tm1/tm1Hmgn1 mice are deficient in DNA repair and also display behavioral abnormalities Genome-wide analysis revealed that the HMGN1 variant binds preferentially to regulatory elements in the genome, such as DNase hypersensitive sites and gene promoters HMGN5 is the most recently discovered member of the HMGN family in vivo were determined through the use of a genetically engineered mouse that carries a targeted disruption in the nucleosome binding region of the protein. Evaluation of blood chemistries of these mice +/yHmgn5 and tm1/yHmgn5 littermates revealed alterations in the expression of glutathione peroxidase 6 (Gpx6) and hexokinase 1 (Hk1), two enzymes known to be involved in glutathione metabolism In this study, the biological consequences of the functional loss of HMGN5 tm1 denotes “targeted mutation #1”. The Hmgn5 gene is located on chromosome X therefore male Hmgn5y/tm1 do not contain an untargeted allele. The targeting vector for generating the conditional to Hmgn5 tm1/tm1 mice was constructed by a recombinogenic cloning strategy Hmgn5 gene was retrieved from the BAC clone into the targeting vector PL253 by recombination in the DY380 bacteria strain. The neo gene with the phosphoglycerate kinase 1 promoter (pGKneo) was employed as a positive selectable marker and the pGK-thymidine kinase cassette was used as a negative selectable marker loxP/Frt-flanked positive selectable marker and the loxP site for conditional deletion of the HMGN5 exons were inserted as described in Hmgn5 gene injected into C57BL/6 blastocysts generated chimeras that transmitted the mutated allele to progeny FLP-mice, and the genomic fragment containing exons II, III, and IV of the Hmgn5 gene was removed by crossing with EIIA-Cre mice. The mice containing the targeted allele were backcrossed into the C57BL/6 background for at least 5 generations. HNGN5 knockout mice were designated tm1/YHmgn5 and their wild-type littermates denoted a +/YHmgn5. Mice were bred in a specific, pathogen-free facility with food and water ad libitum.The nomenclature of the genetically altered mice confirms to the nomenclature recommended by the mouse genome nomenclature committee and is used by the Jackson laboratory. The superscript The immunofluorescence assay was performed as previously described ad libitum. Standard 12-h light/dark cycles were used. Wild type and tm1/YHmgn5 male mice were placed in metabolic cages  for 24 h to collect urine samples on three separate occasions to acclimatize mice, separated by at least 24 h in a traditional cage. At 10–12 weeks of age (n = 13), mice were killed by CO2 asphyxiation, and tissues were harvested and frozen in liquid N2. All animal studies were approved by the National Cancer Institute Animal Care and Use Committee.Food (NIH31 standard chow) and water was provided 18 column  using an ACQUITY UPLC system (Waters Corp.) with a gradient mobile phase comprising 0.1% formic acid and acetonitrile containing 0.1% formic acid. A 0.5 ml/min flow rate was maintained in a 10-min run. The eluent was introduced directly into a Waters Q-TOF Premier mass spectrometer by electrospray ionization operating in either positive (ESI+) or negative (ESI−) ionization mode. For mass spectrometry scanning, the data were acquired in the centroid mode from 50–850 m/z. To confirm the identity of markers, authentic standards were compared with urine samples for retention time and tandem mass spectrometry fragmentation pattern when collision energies ranging from 15–35 V were applied.Urine (1∶5 dilution) was collected and diluted with 62.5% acetonitrile containing 0.5 µM of the internal standard chlorpropamide and centrifuged at 18,000 g for 20 min at 4°C to remove precipitated protein and other particulates, and the supernatant was transferred to an autosampler vial. Liver tissue samples were homogenized in a solvent comprised of 50% acetonitrile and HPLC grade water containing 0.5 µM chlorpropamide as the internal standard. Following homogenization, liver samples were agitated on a shaking platform at 1000 rpm for 20 minutes at 30°C, centrifuged at 18,000 g for 20 min at 4°C, and the supernatant was transferred to an autosampler vial. Samples (5 µl/injection) were subjected to reverse-phase chromatography on a 50−× 2.1-mm ACQUITY 1.7 µm BEH Cm/z and retention time. For urine samples, the peak area corresponding to protonated creatinine  was used to normalize the peak areas of other ions in a sample. This procedure minimized differences in analyte concentrations that were due to variations in renal physiology. For liver metabolomics, peak areas were normalized according to tissue weight. Data from ESI+ and ESI− were combined to generate a data matrix suitable for downstream analysis.The mass chromatographic data were aligned using MarkerLynx software (Waters) to generate a data matrix consisting of peak areas corresponding to a unique Elemental compositions of ions were determined using the METLIN metabolite database established by the Scripps Center for Metabolomics corr values generated by the OPLS loadings scatter S-plot as well as scores group contribution analysis were used to determine those ions that contributed most to the separation between +/YHmgn5 and tm1/YHmgn5 mouse samples.Centroided and integrated chromatographic mass data from 50–850 m/z were processed by MarkerLynx (Waters) to generate a multivariate data matrix. Pareto-scaled MarkerLynx matrices including information on sample identity were analyzed by PCA and OPLS using SIMCA-P+ version 12.0.1 . Ptm1/YHmgn5 mice were diluted (20- to 500-fold) in 50% acetonitrile containing the internal standard chlorpropamide (0.5 µM) for reversed phase chromatography and alpha-aminopimelic acid (1.0 uM) for HILIC chromatography. The mass spectrometer was operated in MRM mode, and optimal transition energies for each metabolite were monitored using the following m/z transitions: Betaine 118 → 59+; Creatine: 132 → 165+; Creatinine: 114 → 86+; Glutathione: 308 → 179+; Pantothenic Acid: 220 → 116+; Phenylacetylglycine: 192 → 74 −. Each urine metabolite concentration is expressed as micromoles per millimole creatinine. Liver metabolites are expressed as micromoles per milligram tissue weight.Metabolite concentrations were determined using an ACQUITY UPLC system coupled to a Xevo-TQ triple quadrople mass spectrometer (Waters). Chromatography was as described for UPLC-ESI-QTOF-MS analysis for all metabolites with the exception of glutathione, which was performed using a HILIC method using 50×2.1 mm ACQUITY 1.7 um BEH Amide column Gapdh mRNA as the internal control, and statistical analyses were performed using the ΔCt values. Primer sequences for gene expression analysis are available on request.The data for total analysis of the transcriptome of livers from wild type and mutant mice is available in the GEO database under accession number GSE39062. For additional verification of selected genes, total RNA was prepared from frozen liver using Trizol reagent  and the RNeasy mini kit  was used for RNA cleanup. cDNA was synthesized from 500 ng total RNA using iScript cDNA synthesis kit . For qPCR analysis, primers crossed exon-exon junctions, and NCBI-BLAST searches confirmed sequence specificity. Fermentas Maxima SYBR Green PCR Master Mix  was used for analysis on an Applied Biosystems  Prism 7900HT system. Relative expression calculated by the ΔΔCt method using Nuclei from liver samples were isolated as previously described 2 digested with various amounts of DNase I  for 2 min at 37°C. Digested DNA was incubated overnight with proteinase K  at room temperature, followed by extraction with water-saturated ether. Purified DNA was amplified by qPCR using the ABI Prism 7900HT system. qPCR reactions were performed using SYBR Green master kit (ThermoFisher). The primer sequences used for amplifications are available on request. Two intergenic regions known to be insensitive to DNase I digestion were used as loading controls, and the Ct of the average of both intergenic regions was used as the normalization control (ΔCt). The mean Ct of both intergenic regions was subtracted from the Ct value for each amplicon (ΔΔCt), The values displayed represent the fold difference of DNA recovered relative to the undigested sample (2−ΔΔCt). For statistical analysis, two way analysis of variance (ANOVA) was used to determine differences between genotypes using the ΔCt values.For DNase I digestions, nuclei prepared as described above were washed in buffer A . Samples containing 50 µg genomic DNA were then diluted in buffer A containing 6 mM CaClHmgn5 gene, located on chromosome X, only the region corresponding to exons II, III, and IV which code for the nucleosomal binding domain of the protein +/yHmgn5 and tm1/YHmgn5 mice, reveal loss of the HMGN5 protein from the nuclei of the genetically altered mice , operating in both positive and negative ionization mode. The mass to charge (m/z) ratio and retention time and the abundance data generated, were subjected to principal components analysis (PCA) and orthogonal projection to latent structures data analysis (OPLS-DA).To determine the mechanism for the mild hepatic dysfunction suggested by the blood chemistry, metabolite changes in liver and urine samples from tm1/yHmgn5 mice from their +/yHmgn5 littermates  in hepatic glutathione and a significant decrease in the urinary phenylacetylglycine (p = 0.023), a glycine conjugate. In addition, both creatine and betaine, compounds which are synthesized from glycine, were significantly reduced in the tm1/YHmgn5 mice, by 42% and 52% respectively. Urinary pantothenic acid was also reduced compared to wild-type littermates; however, this difference narrowly missed statistical significance (p = 0.056). In summary, data from the metabolomics screening suggested a defect in glutathione utilization in tm1/YHmgn5 mice, prompting further investigation into the molecular mechanism.Quantification of metabolites was carried out by use of specific ion monitoring with standards and triple-quadrupole mass spectrometry . Hmgn5tm+/YHmgn5 and tm1/YHmgn5 mice, microarray analysis was carried out +/YHmgn5 and tm1/YHmgn5 revealed that loss of HMGN5 altered the expression of 97 genes. Significantly, two of the genes that were most affected were related to glutathione metabolism: glutathione peroxidase 6 (Gpx6), which was the gene with was most down regulated (2−4.08) and hexokinase 1 (Hk1) which was down regulated by more than 4 fold (2−2.32) in the liver of tm1/YHmgn5 mice. qPCR analysis of RNA obtained from the liver of a new cohort of mice validated the results of the array (Gpx6 is an isoform of a glutathione-dependent enzyme responsible for the neutralization of hydrogen peroxide as well as reducing lipid hydroperoxides Hk1 is an isoform of a pentose phosphate shunt pathway enzyme involved in the generation of NADPH and ultimately the regeneration of reduced glutathione To examine the alterations in gene expression that could lead to the changes in metabolites between the he array . Gpx6 isGpx6 or Hk1 chromatin. Nuclei isolated from the liver of +/Y and Hmgn5tm1/YHmgn5 mice were digested with DNaseI and the amount of undigested DNA in 3 distinct genomic regions of Gpx6 or Hk1 (Gxp6 and amplicons 2 and 3 in Hk1 (Given that HMGN5 can alter chromatin compaction 6 or Hk1 , was qua3 in Hk1 , suggest+/YHmgn5 mice from their mutant tm1/YHmgn5 littermates, while transcriptional analysis and DNase I digestion studies link these changes the altered chromatin structure and expression of Gpx6 and Hk1. Thus, the results provide insights into the biological function of this HMGN variant.This study revealed that loss of HMGN5, a nucleosome binding protein that affects chromatin structure and function, alters the metabolomic profile of liver and urine. Metabolomic analysis of both liver and urine clearly separated the wild-type tm1/YHmgn5 and +/YHmgn5 mice was an elevation in hepatic glutathione concentrations, approximately 41% higher when quantified using triple-quadrupole mass spectrometry. In urine, metabolomic analysis demonstrated that tm1/YHmgn5 mice had significantly decreased concentrations of betaine, creatine, and phenylacetylglycine, all of which are metabolites of the glutathione precursor glycine, a finding that is in agreement with previous results suggesting that glycine is a requisite for maximal glutathione synthesis The most prominent difference between the livers of tm1/yHmgn5 mice did not show any obvious abnormalities in any of these factors we suggest that the observed changes in glutathione levels are due to the altered expression of the Gpx6 and Hk1 genes involved in the utilization and synthesis of glutathione, respectively. Impairments in glutathione utilization by glutathione peroxidases such as GPX6 could account for the mild elevation in the reduced form of glutathione observed. Furthermore, reduced glutathione is regenerated from its oxidized form through an NADPH dependent reaction catalyzed by the enzyme glutathione reductase Hk1 expression could be a consequence of the elevation in glutathione, as sufficient glutathione production could diminish the need to generate NADPH via the pentose phosphate shunt.The level of hepatic glutathione is regulated by a variety of factors such as insulin tm1/YHmgn5 mouse could be due to altered hepatic glutathione metabolism.Deregulation of glutathione metabolism has been implicated in several diseases including liver dysfunction tm1/YHmgn5 mice. Taken together with the previous finding that  tm1/tm1Hmgn1 mice display an impaired ability to repair damaged DNA as well as elevated tumorigenesis tm1/tm1Hmgn3 mice are mildly diabetic In conclusion, this study demonstrates that functional loss of HMGN5 disrupts glutathione metabolism, most likely due to chromatin changes that lead to altered expression of genes encoding two enzymes involved in glutathione utilization and synthesis, resulting in differences in the metabolism of this important thiol. Disruption of glutathione metabolism could lead to the mild hepatic differences between wild type and


HEY1-NCOA2 fusion gene was recently detected in 10 of 15 investigated MCs. The fusion probably arises through intrachromosomal rearrangement of chromosome arm 8 q. We report a new case of MC showing a t as the sole karyotypic aberration. Through FISH and whole transcriptome sequencing analysis we found a novel fusion between the IRF2BP2 gene and the transcription factor CDX1 gene arising from the translocation. The IRF2BP2-CDX1 has not formerly been described in human neoplasia. In our hospital’s archives three more cases of MC were found, and we examined them looking for the supposedly more common HEY1-NCOA2 fusion, finding it in all three tumours but not in the case showing t and IRF2BP2-CDX1 gene fusion. This demonstrates that genetic heterogeneity exists in mesenchymal chondrosarcoma.Mesenchymal chondrosarcomas (MCs) account for 3–10% of primary chondrosarcomas. The cytogenetic literature includes only ten such tumours with karyotypic information and no specific aberrations have been identified. Using a purely molecular genetic approach a  The classification of sarcomas describes over 50 different histological subtypes Mesenchymal chondrosarcomas (MCs) are rare tumours that account for 3–10% of primary chondrosarcomas (HEY1) gene and the nuclear receptor coactivator 2 (NCOA2) gene was detected in 10 out of 15 analysed MCs (67%) According to the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer IRF2BP2) and the caudal type homeobox 1 (CDX1) gene. Based on the recent report by Wang et al (2012) HEY1-NCOA2 gene fusion, finding it in all three.We report the finding of a balanced t as the sole karyotypic abnormality in an MC. The translocation led to a new fusion between the interferon regulatory factor 2 binding protein 2 gene . However, a tumour in the left iliacus muscle was detected, 3 cm in largest diameter. Biopsies revealed a spindle cell tumour of uncertain malignant potential. The patient received chemotherapy for PCNSL according to Abrey et al. The Norwegian Radium Hospital (NRH) is the largest referral centre for Norwegian patients with bone and soft tissue tumours covering a population of 2.6 million. To identify additional patients with a diagnosis of MC, a database search was performed for cases with this disease. Three additional patients (patients 2–4) were identified  was retrieved from The Radium Hospital biobank  in 2008). Specific permission to perform RNA analysis/sequencing was obtained from patient 1 after approval by the Regional Ethics Committee for Medical and Health Research Ethics South-East (REC number: 2010/1389A). The entire study was also approved by the institutional review board at the Norwegian Radium Hospital. All data were analyzed anonymously.Fresh tissue from a representative area of the tumour (patient 1) was received and analysed as part of our diagnostic routine. The samples were disaggregated mechanically and enzymatically with collagenase II . The resulting cells were cultured and harvested using standard cytogenetic techniques http://genome.ucsc.edu/). The selected clones (see Fluorescence in situ hybridization (FISH) was performed using probes from bacterial artificial chromosomes (BACs). BACs and fosmid clones flanking and covering the breakpoint positions were selected using the Human Genome Browser at the University of California web site . DNA labelling was done in a nick translation reaction and the synthesized probes were hybridized to previously G-banded slides. All procedures were performed as previously described Representative samples of tumour tissue were frozen and stored at −80°C after surgery. DNA was isolated using Genomic-tip  to obtain pure high molecular weight DNA. RNA was extracted from tumour tissue using the Trizol reagent (Life Technologies) with a homogenizer . The RNA quality was evaluated using the Experion Automated Electrophoresis System . cDNA was synthesized using the iScript kit and random primers (Bio-Rad Laboratories). All procedures were done according to the manufacturers’ recommendations.Sequencing was performed according to the TruSeq paired-end RNA-sequencing protocols from Illumina for Solexa sequencing on a Genome Analyzer IIx with paired end module . 3.5 µg total RNA was used as starting material for library construction, using the TruSeq RNA Sample Preparation Kit v2 where the steps include poly-A mRNA isolation, fragmentation, and cDNA synthesis before adapters are ligated to the products and amplified to a final cDNA library. Shearing to about 150 bp fragments was achieved using divalent cations under elevated temperature. Approximately 58 million clusters were generated by the TruSeq PE Cluster Kit v2 on the Illumina cBot Cluster Generation System, and 76 base pairs were sequenced, from each side of the fragments, using reagents from the TruSeq SBS Kit v5 .http://genome.ucsc.edu/, accessed May 2012). UniGene clusters were downloaded from National Center for Biotechnology Information  to assist in locating potential gene fusions. Three spanning reads and two split reads were required to call sequence reads a gene fusion.The Illumina software pipeline was used for processing of image data into raw sequencing data (SCS 2.9 and Casava 1.8.2), and only sequence reads marked as “passed filtering” were used in the downstream data analysis. A total of 91 million reads were obtained. We utilized the fusion discovery software deFuse (version 0.4.3) HEY1- NCOA2 fusion were identical to the primers used by Wang et al www.ncbi.nlm.nih.gov/BLAST/). All cases were tested for expression of a zinc-finger gene suppressor of zeste 12 homolog (Drosophila) (SUZ12) to assess RNA quality.Primers used in PCR were designed with the FastPCR software PCR experiments on genomic DNA were performed using ∼100 ng DNA as template in 25 µl PCR reactions using TaKaRa LA Taq following the manufacturer’s recommendations for LD-PCR: 30 cycles of 98°C for 10 sec (denaturation) followed by 68°C for 15 min . PCR products were purified using GeneJET PCR purification kit  and sent for Sanger sequencing (GATC Biotech).The cytogenetic analysis of the only tumour (patient 1) from which we got a fresh sample revealed a balanced t as the sole abnormality in all cells analysed . AnalysiHEY1-NCOA2 fusion which was recently identified in 10 of 15 investigated MCs HEY1 exon 4 and NCOA2 exon 13 in cases 2–4, identical to the one previously described PDGFRβ) and the large intron 1 of CDX1.All cases  as well as the three archival tumours) were tested for the IRF2BP2-CDX1 transcript involving two coding regions yielded the highest split count value (number of split reads supporting the fusion) of all the predicted fusions and was predicted to be in-frame. We therefore chose to focus on this putative fusion. cDNA PCR experiments with specific primers were run to validate the IRF2BP2-CDX1 fusion. Two distinct bands were identified using the primer IRF2BP2-895F, located in exon 1, in combination with CDX1-771R, located in exon 3 , whereas on chromosome 5 it was in the large intron 1 of CDX1 (chr5∶149551799 bp).Next, we wanted to identify the genomic breakpoints of the 1-26970R  we managIRF2BP2 gene also by FISH, we hybridized BAC clones overlapping IRF2BP2 to metaphases obtained from the cultured cells , confirming that this fusion gene is common in MC. In a majority of the well-known translocation-related sarcomas such as myxoid liposarcoma and low-grade fibromyxoid sarcoma, more than one defining fusion variant has been detected. Often one fusion variant is more common than the others IRF2BP2-CDX1 could be such an additional fusion variant identified in a subset of MCs, but only analysis of larger series of tumours can determine the prevalence of the IRF2BP2-CDX1.MCs are rare, and at our institution only five patients (four included in this study) received this diagnosis during the last 25 years. We identified the HEY1-NCOA2-positive MCs all had tumour manifestations detected in bone, whereas the MC showing the t and IRF2BP2-CDX1 fusion originated from soft tissue. Although most common in bone, one-fifth to one-third of MCs do arise in soft tissue HEY1-NCOA2 was first described Of possible interest is the fact that the three Table S1BAC probes used for FISH experiments in case 1.(XLS)Click here for additional data file.Table S2List of fusions suggested by the deFuse algorithm.(XLS)Click here for additional data file.


ARMS2/HTRA1 genes at the 10q26 locus have been associated with risk of age-related macular degeneration (AMD), with the most significantly associated variants being A69S (rs10490924), del443ins54 (EU427539) and rs11200638. We wished to explore the association of the del443ins54 in two ethnically different populations from India and Australia.The The del443ins54 was screened in a large cohort of ~1500 subjects from these two populations by a combination of PCR-based agarose gel electrophoresis and validated by resequencing. Statistical analysis comprised the calculations of allele, genotype and haplotype frequencies along with their p values and corresponding odds ratios (OR), and 95% confidence intervals (95% CI) and measures of linkage disequilibrium (LD).−13; OR=2.80, 95%CI, 2.12–3.70) and Australian cohorts . These associations were similar to those previously identified for the A69S and the rs11200638 variant in these populations that also exhibited high degrees of LD (D’ of 0.87-0.99). A major risk haplotype of “T-indel-A”  and a protective haplotype of “G-wild type-G”  were identified in the Indian and Australian cohorts, respectively.The del443ins54 was significantly associated with AMD in both the Indian (p=1.74×10These data provide an independent replication of the association of del443ins54 variant in two different ethnicities, despite differences in allele and haplotype frequencies between them. High levels of LD in both populations limit further genetic dissection of this region in AMD. ARMS2 (rs10490924) and HTRA1 (rs11200638) in multiple populations worldwide [EU427539) that affects the stability of ARMS2 mRNA by the removal of a polyadenylation signal (443 bases) and insertion of a 54bp AU rich element in the 3′-UTR (del443ins54), has also been identified in the 3′ end of the ARMS2 gene as increasing risk of AMD by several fold in both Caucasian [−5 to p=8.4×10−34) in individuals of European origin [Age-related macular degeneration (AMD) is a complex multifactorial disease and a leading cause of irreversible blindness in the world . A majorARMS2 rs0490924 a1 rs1120038 in mulARMS2 rs490924 anrs10490924) and rs11200638 variants with AMD in both South Indian and Australian cohorts [rs11200638 variants with AMD susceptibility in two ethnically different cohorts from Southern India and Australia.We have previously reported a significant association of the A69S  and normal controls from cohorts in India (n=433) and Australia (n=1054). The detailed methods of clinical diagnosis along with the inclusion and exclusion criteria have been previously reported ,12. AmplPLINK software [rs11200638 variants and the estimated haplotype frequencies and linkage disequilibrium (LD) were assessed with the Haploview software (version 4.2) that uses the EM algorithm [Allele and genotype frequencies were determined by the gene counting method and estimates of Hardy–Weinberg equilibrium were assessed. Odds ratios (OR) and 95% confidence intervals (95%CI) were calculated to assess the risk conferred for each variant using the software . Haplotylgorithm .rs11200638 genetic variants. Each variant was in Hardy–Weinberg equilibrium in both the Indian and Australian cohorts (p>0.05). Allele frequencies for each of the risk variants  exhibited a relatively higher frequency (0.60–0.63) in the Indian cohort compared to the European cohorts (0.36–0.53) but lesser than that reported in Han Chinese (0.73–0.77) or the in the Japanese (0.86–0.88) populations  and Australian , cohorts (ARMS2) and the rs11200638 (HTRA1) variants in both the Indian and Australian cohorts, respectively  or Australian cohorts .Homozygosity of the indel and the other variants were strongly associated with an increased risk of AMD in both the Indian and Australian cohorts. Combined homozygosities at the A69S and the rs11200638 variants were remarkably high across this 10q26 region with relatively higher values in the Australian  compared to the Indian  cohorts  between the A69S, del443ins54 and  cohorts . Two maj cohorts . Differewo genes .ARMS2 del443ins54 variant in two cohorts, and to the best of our knowledge for the first time among South Indians with AMD. The strong association of the del443ins54 along with the A69S and rs11200638 variants in the Indian and Australian cohorts were consistent with that observed in other populations [These data provide an independent replication of the association of the ulations . HaplotyARMS2 and HTRA1 genes are in high LD in European populations and thus dissecting out the role of one gene over the other has proved difficult. The advantage of undertaking a comparative analysis of genetic variants in populations of differing ethnicities expands the genetic diversity available and may provide the opportunity of identifying a more defined but associated region for further study. The current study highlighted similar degrees of associations across these three variants despite a relatively lower LD between the A69S, del443ins54 and rs11200638 variants in the Indian compared to the Australian cohort. However, it did confirm the presence of stratification differences between ethnicities with the allele frequency of the Indel of del443ins54 in South Indians being higher at 0.63 in cases compared to that in European populations (0.36–0.53) but lower than other Asian populations (0.73–0.88). The allele frequency in cases is similar to that previously shown by us in the assessment of the A69S (0.63) and the rs11200638 (0.60) variants of the ARMS2 and HTRA1 genes respectively, in the South Indian cohort. Evidence of population stratification has also been observed in AMD studies of the protective CFHR3–1 deletion, with the highest frequencies of the deleted allele being present in African populations (16%–20%) compared to Asians (<2%) [The ns (<2%) ,16.ARMS2 and HTRA1 gene in AMD is still unclear but functional dissection of the effect of the rs11200638 promoter variant in the HTRA1 gene has revealed that this variant resides within a putative transcription binding site for the factors AP2α and SRF (serum response factor) [HTRA1 expression levels revealed consistently higher levels of expression with the AA genotype compared to the GG genotype [rs11200638 variant have revealed no functional effect on HTRA1 expression [The potential role of the  factor) . Initialgenotype . In contpression .ARMS2 del443ins54 indel found decreased ARMS2 expression and almost 3.0 fold increase in HTRA1 expression [ARMS2 risk del443ins54 results in decrease in mRNA transcription levels of the ARMS2 gene, a non risk associated variant (rs2736911) also leads to significantly reduced ARMS2 transcript levels suggesting that ARMS2 protein deficiency alone is unlikely to be pathogenic in AMD [ARMS2 in mitochondrial homeostasis has also been suggested and the biology concerning mitochondrial dysfunction and the effects on age supports this notion [ARMS2 in retinal epithelial ARPE-19 cells and COS7 transfected cells to the cytosol rather than the mitochondria suggesting that ARMS2 may not confer risk to AMD through the mitochondrial pathway [HTRA1 expression are equivocal and further investigations on the functional role of these variants are required.Analysis of the chromosome 10q26 risk haplotype inclusive of the pression . Interesc in AMD . A functARMS2 and HTRA1 gene in this region will require much larger patient cohorts than have currently been assessed, or through the identification of other ethnic populations which show relatively lower levels of LD over this 10q26 region.In conclusion, we provide convincing evidence for the association of the del443ins54 variant with AMD, despite differences in allele, genotype and haplotype frequencies and LD across the 10q26 region reflecting population stratification differences in two different ethnicities. AMD, a complex multi-factorial disease is associated with multiple genomic regions with varying magnitudes of effect and the relevance of genetic associations differ between populations. Further, elucidation of the genetic basis of this disease through the analysis of individuals from different ethnic groups has the potential to provide useful insights into the genetic diversity of risk and protective variants within a gene as well as their contributions to disease. Also, meaningful genetic dissection of the


MFD can be used to study formulations of drugs and active agents in oil, water and emulsions.Molecular Fragment Dynamics (MFD) is a mesoscopic simulation technique based on Dissipative Particle Dynamics (DPD). Whereas DPD beads in general may not necessarily be identified with chemical compounds at all the MFD variant uses specific molecules or molecular fragments as its basic 6E6, C10E6, C12E6 and C16E6 at the water-air interface are performed to study their nanoscale structures and surface properties. The simulations of the self-aggregation of the polyoxyethylene alkyl ether surfactants lead to equilibrium nanoscale structures and computationally determined surface tensions which are in agreement with experimental data for different surfactant concentrations [MFD simulations of the nonionic polyoxyethylene alkyl ether surfactants C


KRAS, CTNNB1, PIK3CA and FGFR2 have been identified in endometrial cancer. The aim of this study was to provide insight into the clinicopathological features associated with patterns of mutation in these genes, a necessary step in planning targeted therapies for endometrial cancer. 466 endometrioid endometrial tumors were tested for mutations in FGFR2, KRAS, CTNNB1, and PIK3CA. The relationships between mutation status, tumor microsatellite instability (MSI) and clinicopathological features including overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier survival analysis and Cox proportional hazard models. Mutations were identified in FGFR2 (48/466); KRAS (87/464); CTNNB1 (88/454) and PIK3CA (104/464). KRAS and FGFR2 mutations were significantly more common, and CTNNB1 mutations less common, in MSI positive tumors. KRAS and FGFR2 occurred in a near mutually exclusive pattern (p = 0.05) and, surprisingly, mutations in KRAS and CTNNB1 also occurred in a near mutually exclusive pattern (p = 0.0002). Multivariate analysis revealed that mutation in KRAS and FGFR2 showed a trend (p = 0.06) towards longer and shorter DFS, respectively. In the 386 patients with early stage disease (stage I and II), FGFR2 mutation was significantly associated with shorter DFS  and OS  and KRAS was associated with longer DFS . In conclusion, although KRAS and FGFR2 mutations share similar activation of the MAPK pathway, our data suggest very different roles in tumor biology. This has implications for the implementation of anti-FGFR or anti-MEK biologic therapies.Mutations in multiple oncogenes including  Endometrial cancer comprises about 4% of cancer in women globally, with higher incidence in developed countries. The American Cancer Society estimates endometrial cancer will be the fourth most common cancer diagnosed and the eighth leading cause of cancer deaths in women in 2010 Considerable effort has gone into developing systems to more effectively identify patients with endometrioid endometrial cancer that carry an elevated risk of recurrence so they can be targeted for adjuvant therapies . Those patients that present with extrauterine disease (stage III/IV) carry a high risk of recurrence and progression. The majority of patients (∼80%), however, present with tumors clinically confined to the uterus (stage I/II). In these early stage patients, multiple studies have shown that the risk of recurrence is associated with tumor grade, depth of myometrial invasion, occult extension into the cervix and tumor cell invasion of lymphatic vessels (lymphovascular space invasion: LVSI), where high grade is the most widely accepted adverse prognostic marker Since 1988, the International Federation of Gynecology and Obstetrics (FIGO) has recommended full systematic pelvic and para-aortic lymphadectomy as part of staging for endometrial cancer. A new 2009 FIGO staging system has recently been implemented where tumors with no evidence of myometrial invasion are combined with tumors that show invasion to less than 50% of the myometrium and grouped into stage 1A fibroblast growth factor receptor 2 (FGFR2) mutations in 18/115 (16%) endometrioid endometrial cancers FGFR2, CTNNB1, KRAS and PIK3CA mutation in a large, unselected cohort of endometrioid endometrial cancers and to determine the relationship between mutation status and clinicopathologic variables including outcome. Mutations in PTEN were not included in this analysis due to the increased cost associated with sequencing all 9 exons of this tumor suppressor gene. In addition, the high prevalence of PTEN aberration (70%) argued against a possible association with poor prognosis in this tumor type.FGFR2 has been shown to be activated in a number of cancers due to gene amplification All research subjects provided written consent to ongoing protocols 91-507 and 93-0828, approved by the Washington University's Human Research Protection Office continuing Review Committee. The work performed at TGen was determined to be exempt from IRB approval following review and receipt of a Verification of Protections for Human research subjects form signed by Dr Goodfellow and a copy of the blank consent form.Tumor specimens were prospectively collected at the time of hysterectomy (1991–2006) for patients treated by the Division of Gynecologic Oncology at Washington University School of Medicine/Barnes–Jewish Hospital. Surgical staging and tumor grade was assigned on the basis of FIGO 1988. Patients who had received preoperative radiation or chemotherapy were excluded from analysis. The prospectively collected clinical and pathologic information was stored in a computerized database. Following their initial treatment, these patients were routinely followed at 3-month intervals for the first 2 years and then at 6-month intervals for at least 3 years. Disease surveillance included physical examination and periodic pap smears. Diagnostic imaging and directed biopsies were performed as clinically indicated. Histological confirmation of all recurrences was performed. Follow-up data were abstracted from clinic charts, hospital records, and the Siteman Cancer Center/Barnes-Jewish Hospital's cancer registry.Patients for whom follow-up data were unavailable or who died perioperatively (within 30 days of hysterectomy) were excluded from the analyses. The study population comprised 466 patients with endometrioid endometrial cancer, 386 of which had disease confined to the uterus (stage I or II).Tissue specimens and blood were obtained at the time of surgery, snap frozen, and stored at −70°C. Tumors were evaluated to select tissues with >66% neoplastic cellularity for DNA preparations. DNA was isolated using proteinase K and phenol extraction or the DNeasy Tissue Kit . DNA was extracted from peripheral blood leukocytes or, when blood was not available, from uninvolved myometrium, as previously described FGFR2, exon 2 of KRAS, exon 3 of CTNNB1, and exons 9 and 20 of PIK3CA were tested for mutations by direct sequencing. PCR primers and conditions are available upon request FGFR2 and KRAS and CTNNB1. For PIK3CA, rare and novel mutations were confirmed to have arisen somatically and common tumor-associated mutations were confirmed in the majority of samples.Exons 7, 8, 10, 13 and 15 of MSI analysis is routinely performed for all tumors. The MSI status and methods used for the majority of the cases reported here have been previously described p-value of 0.05. Statistical analyses were performed using SAS , as well as the cmprsk R (http://biowww.dfci.harvard.edu/~gray) statistical packages for competing risk analysis.The relationship between gene mutation status and covariates was assessed using Fisher's exact test or Student's t-test as appropriate. Overall survival (OS) was defined as the time from date of surgery to death due to any cause. Survivors were censored at the date of last contact. Disease free survival (DFS) was defined as the time from surgery to recurrence or progression. Patients were excluded if they had died within 30 days of surgery. The Kaplan-Meier product limit method was used to estimate OS and DFS. Univariate and multivariate Cox proportional hazard models were fitted to assess the effects of the covariates on OS and DFS, and the proportional hazard assumptions were checked using scaled Schoenfeld residuals FGFR2 ; KRAS and PIK3CA ; and CTNNB1 . Mutation data for all four genes was obtained for 453 cases (97%).The mean age at diagnosis for the 466 cases analyzed was 63.7 years with a mean follow-up time of 70.2 months (0.7–176). The majority of patients presented with early-stage disease (386 or 83% stage I or II) . MutatioFGFR2 mutations in 48/466 (10.3%) tumors  was excluded from analyses because of uncertainty as to whether the sequence change was functionally significant. The most common mutations were S252W  and N550K . All together, 7 mutations affecting 6 codons  accounted for 90% of the mutations identified (We identified ) tumors , includientified . We idenKRAS in 87/464 (19%) samples, including 115 previously investigated cases We identified mutations at codons 12 and 13 in PIK3CA in a total of 104/464 (22%) cases  cases . The majCTNNB1 in 88/454 (19%) endometrioid tumors  compared to microsatellite stable (MSS) tumors  . Similarly, mutations in FGFR2, were significantly more common in MSI positive tumors  compared to MSS tumors  (p = 0.016). In contrast, mutations in CTNNB1 were significantly less common in MSI positive tumors  compared to MSS tumors . Mutations in PIK3CA were more common in MSI positive tumors  compared to MSS tumors , although this was not significant (p = 0.08). 158/466 (34%) of tumors were MSI positive. Mutations in FGFR2 and KRAS mutations would occur in a mutually exclusive pattern. Indeed, only 4/87 (5%) KRAS mutation-positive tumors carried a FGFR2 mutation , whereas 44/377 (12%) KRAS mutation negative tumors carried an FGFR2 mutation . To investigate whether the tumors carrying mutations in both FGFR2 and KRAS were polyclonal, DNA from a different portion of the tumor was extracted from archived paraffin tissue and in all four cases both mutations were confirmed.Based on our understanding of receptor tyrosine kinase-MAPK signaling, and our preliminary analysis of 115 endometrial tumors, we anticipated that KRAS and CTNNB1 demonstrated a similar pattern of mutual exclusivity and rarely occurred together. In the 453 tumors sequenced for both genes, 88 and 85 carried mutations in CTNNB1 and KRAS, respectively. Of those tumors with CTNNB1 mutations, only 5/88 (5.7%) carried KRAS mutations, whereas 80/365 (22%) of the CTNNB1-wildtype tumors carried a KRAS mutation . Given CTNNB1 mutations were significantly more common in MSS tumors, we looked for the relationship between KRAS and CTNNB1 mutations in both MSS and MSI tumors. This association was even stronger in those tumors that demonstrated microsatellite stability where 1/71 (1%) CTNNB1 mutation positive tumors carried a KRAS mutation, whereas 44/230 (19%) of the CTNNB1 wildtype tumors carried a KRAS mutation . In contrast, this association was not present in those tumors with MSI as 4/17 (24%) CTNNB1 mutation positive tumors carried an activating KRAS mutation whereas 36/135 (27%) of the CTNNB1 wildtype tumors carried a KRAS mutation.Perhaps the most surprising finding from this cohort is that mutations in FGFR2 and KRAS, and of CTNNB1 and KRAS, no such pattern was seen for FGFR2 and CTNNB1. Specifically 8/88 (9%) CTNNB1 mutation positive tumors carried an FGFR2 mutation, whereas 40/365 (11%) CTNNB1 wildtype tumors carried an FGFR2 mutation. Within the MSS cohort of tumors, 7/71 (10%) CTNNB1 mutation positive tumors carried an FGFR2 mutation whereas 17/230 (7%) of the CTNNB1 wildtype tumors carried an FGFR2 mutation.Surprisingly, given the near mutual exclusivity of FGFR2, KRAS, PIK3CA mutation and age at diagnosis. CTNNB1 mutations were, however, significantly more common in patients diagnosed before age 60  compared to those diagnosed after age 60  . We chose 60 as our age cutoff based on previous data indicating reduced survival in patients >60 FGFR2 mutations were more common in Caucasian/Asian cases  than African American patients , albeit this was not significant (p = 0.10). PIK3CA mutations were significantly more common in stage I/II tumors  compared to late stage tumors   ; grade 2, 25/149 (17%); grade 3, 4/62 (6%)  and FGFR2 mutations showed a trend towards an association with grade ; grade 2 17/152 (11%); grade 3, 2/65 (3%) (p = 0.10)  compared to lower grade tumors 84/392, (21%)  as were FGFR2 mutations ; grade 3, 2/65 (3%) .There was no association between ct test) . CTNNB1  = 0.10) . As wellMutation status for the four oncogenes investigated was not associated with overall survival (OS) in the total cohort of 466 cases. OS was associated with age >60 (p = 0.0002), advanced stage (III/IV) (p<0.0001), FIGO tumor grade 2 (p = 0.0014), FIGO grade 3, p<0.0001) and adjuvant therapy (p<0.0001) . MultivaKRAS mutation was associated with longer disease free survival (DFS)  whereas the mutation status of other genes was not significantly associated with DFS. As expected, DFS was associated with higher stage (III/IV) (p<0.0001), FIGO tumor grade 2 (p = 0.0019) and 3 (p<0.0001) and adjuvant therapy (p<0.0001) in univariate analysis. Multivariate analysis showed that the presence of a KRAS mutation remained significantly associated with longer DFS   although this finding was of marginal statistical significance (CTNNB1 and PIK3CA mutations had no effect on the multivariate model (data not shown). We did not include adjuvant therapy in the multivariate model as analysis indicated it was not independent of stage and grade.The presence of = 0.048) . When FGificance . When boificance . CTNNB1 FGFR2 mutation positivity and grade 2 differentiation showed a trend towards shorter OS . When FGFR2 mutation was analyzed taking into consideration the effects of known prognostic factors variables, it became more significantly associated with OS  , stage II (p = 0.007) and high tumor grade (FIGO grade 3) (p<0.0001) . Both FG= 0.025) .FGFR2 mutation (p = 0.019) were significantly associated with shorter disease free survival (DFS) (KRAS mutation showed a trend towards associating with longer DFS (HR = 0.26 95% CI 0.06–1.11 p = 0.067) whereas CTNNB1 and PIK3CA mutations were not associated with DFS. When each gene was analyzed alone in multivariate analysis of early stage cancers, FGFR2 mutation status remained a significant factor associated with reduced DFS  (KRAS was significantly associated with longer DFS (HR = 0.23 CI 0.05–0.97 p = 0.045). When both genes were included in the model, FGFR2 remained significant (HR = 3.03 CI 1.26–7.27 p = 0.013). Kaplan-Meier survival plots showing the relationship between FGFR2 mutation and DFS and OS in early stage cancers are presented in Univariate analysis revealed only high grade (p = 0.0005); stage II (p = 0.009); adjuvant therapy (p = 0.049) and the presence of an al (DFS) . KRAS mu= 0.008)  and KRASHere we show the patterns of mutations in four endometrial oncogenes in the largest cohort of endometrioid endometrial tumors reported to date (n = 466). Given the large number of tumors in this single institution Washington University School of Medicine cohort, novel insights have been revealed which have not been evident with smaller subsets of tumors or in some cases where disparate evidence had been reported in smaller panels of tumors CTNNB1 and KRAS mutations in this cohort. Although 5 tumors were identified with mutations in both genes the vast majority of tumors only carried mutations in either KRAS or CTNNB1 (p = 0.0002). This finding was not a reflection of an association with MSI positive and negative tumors because when we looked in only the MSS tumors, the association was even more significant. Only 1% CTNNB1 mutation positive tumors carried a KRAS mutation whereas 19% of the CTNNB1 wildtype tumors carried a KRAS mutation . In most other cancers, mutual exclusivity of gene activation is observed between two proteins that map to the same signaling pathway, which makes intuitive sense, as activation of the same pathway at two different nodes is redundant. Although KRAS and CTNNB1 have very distinct roles in the MAPK pathway and the Wnt/TCF signaling pathway respectively, recent data suggests novel points of pathway crosstalk in some cell types One finding that may have implications for understanding the biology underlying endometrial cancer is the hereto-unrecognized mutual exclusivity of PIK3CA are not associated with poor prognosis In contrast to a previous study, our data suggest that mutations in exon 20 of FGFR2 mutation rate was 10% (48/466). The 10% mutation rate for this large, unselected series is consistent with the mutation rate reported by Dutt et al.  FGFR2 mutations in endometrial cancers we oversampled for cases that had recurred and tumors with microsatellite instability FGFR2 mutation with both defective DNA repair and recurrence in the current unselected cohort.In this single institution series of endometrioid endometrial cancers, the overall A number of clinical and pathologic prognostic factors have been evaluated in the search for markers to more accurately predict risk of recurrence or death for patients with endometrial carcinoma. Past studies have suggested tumor markers p53, p16, estrogen receptor, progesterone receptor and HER2/neu may have clinical utility in endometrial cancer for predicting lymph node metastasis, prognosis and in directing treatment FGFR2 and KRAS have prognostic significance within the cohort of endometrioid endometrial cancers. Our data suggest that FGFR2 mutations occur more often in the well and moderately differentiated endometrioid tumors  compared to undifferentiated tumors and possibly identify the “bad actors” in an otherwise better prognosis histological subgroup. Recent data in an independent cohort of endometrial tumors reported a similar frequency of mutations across G1–G3 tumors FGFR2 with shorter DFS is more significant in the multivariate analyses where the association of high grade with poor prognosis is accounted for, compared to univariate analysis. These findings strongly suggest that the observed effect of FGFR2 is not simply due to the confounding effects of other known prognostic factors, and underscore the likely functional significance of this gene in determining survival.In this study we have identified that KRAS mutation is associated with longer DFS in the total cohort in both univariate and multivariate analysis. In the subset of early stage cases, KRAS mutation was significantly associated with longer DFS in multivariate analysis after adjusting for grade and stage. We can speculate that the pattern of mutual exclusivity of FGFR2 and KRAS suggests that the role of these two genes in endometrial cancer initiation is likely to be through activation of the MAPK signaling pathway. The fact that they have different and indeed opposing effects on disease free survival leads us to further speculate that activation of “non-MAPK” pathways downstream of FGFR2 is driving the association of this gene with poor prognosis.A novel finding of this present study is that FGFR2 mutation is an independent prognostic marker in patients with early stage endometrioid endometrial cancer suggests that FGFR2 mutation testing could ultimately prove useful in the management of endometrial cancer. Current National Comprehensive Cancer Network (NCCN) guidelines for endometrioid endometrial cancer confined to the uterus recommends more aggressive adjuvant therapy as tumor grade and tumor stage increases, and also where multiple adverse prognostic indicators are present, including lymphovascular space involvement. We envisage that the mutation status of FGFR2 could be used to inform clinical decision making in a similar way to a poorly differentiated histology. Specifically, the presence of an FGFR2 mutation and absence of a KRAS mutation would stratify a patient as having high-risk disease, resulting in a recommendation for more aggressive therapy  that the patient samples are from a single institution, 2) the frequency of recurrence in early stage endometrioid cases is relatively low in this unselected cohort and 3) we had low number of late stage G1 and G2 tumors in this cohort which may have contributed to lack of statistical significance for FGFR2 in the entire cohort. We are currently sequencing the four exons of FGFR2 containing almost all reported mutations in endometrial cancer samples collected as part of the multi-institutional GOG-210 clinical trial “Molecular Staging of Endometrial Cancer”. This cohort also allows the assessment of FGFR2 mutations on endometrial cancer specific survival as well as overall survival, given the extensive clinical annotation of these samples.FGFR2 mutation testing may identify patients whose tumors will be sensitive to FGFR inhibition FGFR2 mutations as an independent prognostic marker in early stage tumors and the eventual identification of an FGFR inhibitor with clinical activity in patients with metastatic endometrial cancer, holds the promise of utilizing anti-FGFR therapies in an adjuvant setting to reduce the risk of recurrence in patients diagnosed with FGFR2 mutation positive endometrial cancer.Preclinical data suggests that FGFR2 was associated with shorter disease free progression and this was significant in patients diagnosed with early stage disease. This finding has clinical significance in that FGFR2 mutation status could function as a starting point in developing a molecular prognostic risk assessment score that could be used to identify patients that may benefit from more aggressive adjuvant radiation and/or chemotherapy following an initial hysterectomy. In the longer term, anti-FGFR agents could be tested in patients with FGFR2 mutation positive tumors to evaluate whether these agents reduce the frequency of recurrence in the adjuvant setting, in addition to the metastatic setting where they are currently being evaluated. As KRAS mutations were associated with reduced recurrence risk in this cohort, our data would suggest that MEK inhibition may not be effective in an adjuvant setting to prevent recurrence.In conclusion, our mutation analysis of four oncogenes frequently mutated in the endometrioid histology of endometrial cancer revealed that mutated Figure S1Kaplan Meier curves for recurrence/progression free survival (A) and overall survival (B) by FGFR2 mutation status in patients with early stage endometrial cancer.(TIF)Click here for additional data file.Table S1Clinicopathological features of endometrial tumors with FGFR2 mutations.aNumbering relative to NM_022970.2 bNumbering relative to NP_075259.2 cThese mutations have been reported previously (8).(DOC)Click here for additional data file.Table S2KRAS Mutations in Endometrial Tumors.(DOC)Click here for additional data file.Table S3PIK3CA Mutations in Endometrial Tumors.#These mutations are novel and do not appear in Cosmic (May 2011).(DOC)Click here for additional data file.Table S4CTNNB1 Mutations in Endometrial Tumors.(DOC)Click here for additional data file.Table S5Frequency of MSI and mutations, according to FIGO stage.(DOC)Click here for additional data file.Table S6Frequency of MSI and mutations, according to tumor grade.(DOC)Click here for additional data file.


HMGA2 and PLAG1 in 14 benign and 23 malignant thyroid tumors. To address the question if PLAG1 can be activated by HMGA2, the expression of both genes was quantified in 32 uterine leiomyomas 17 of which exhibited an overexpression of HMGA2. All leiomyomas with HMGA2 overexpression also revealed an activation of PLAG1 in the absence of detectable chromosome 8 abnormalities affecting the PLAG1 locus. To further investigate if the overexpression of PLAG1 is inducible by HMGA2 alone, HMGA2 was transiently overexpressed in MCF-7 cells. An increased PLAG1 expression was observed 24 and 48 h after transfection. Likewise, stimulation of HMGA2 by FGF1 in adipose tissue-derived stem cells led to a simultaneous increase of PLAG1 mRNA. Altogether, these data suggest that HMGA2 is an upstream activator of PLAG1. Accordingly, this may explain the formation of tumors as similar as lipomas and lipoblastomas resulting from an activation of either of both genes by chromosomal rearrangements.In pleomorphic adenomas of the salivary glands (PASG) recurrent chromosomal rearrangements affecting either 8q12 or 12q14∼15 lead to an overexpression of the genes of the genuine transcription factor PLAG1 or the architectural transcription factor HMGA2, respectively. Both genes are also affected by recurrent chromosomal rearrangements in benign adipocytic tumors as e. g. lipomas and lipoblastomas. Herein, we observed a strong correlation between the expression of  HMGA2 is located within the region 12q14∼15 which is frequently affected by chromosomal alterations PLAG1 (Pleomorphic adenoma gene 1) mapping to 8q12 encodes a genuine transcription factor encompassing seven zinc finger domains and a carboxyterminal transactivation domain. PLAG1 is developmentally regulated and highly expressed in certain fetal tissues PLAG1 plays a key role in the development of lipoblastomas IGF-II) promoter and to stimulate its activity The DNA-binding protein high mobility group AT-hook 2 (HMGA2) and the zinc finger protein PLAG1 share a common role in the molecular pathogenesis of certain benign tumors, e. g. of the salivary glands and of adipose tissue. Pleomorphic adenomas are benign tumors of myoepithelial origin most often located in the parotid glands. Based on the existence of clonal chromosomal aberrations cytogenetic subtypes of pleomorphic adenomas can be distinguished HMGA2 are a common finding in lipomas often as a t HMGA2 locus has been described as well HMGA2 has been found to allow a good discrimination between benign and malignant thyroid lesions HMGA2 ranked at the first position followed by Kallikrein 7 (KLK7), Mannose receptor, C type 2 (MRC2), Leucine-rich repeat kinase 2 (LRRK2), and PLAG1Similar but not identical to what is seen in pleomorphic adenomas both genes participate in the genesis of benign adipose tissue tumors. Chromosomal translocations affecting 12q14∼15 and targeting HMGA2 and PLAG1 in the molecular pathogenesis of salivary gland adenomas and adipose tissue tumors, we also quantified and compared the expression of HMGA2 and PLAG1 mRNA in thyroid adenomas as well as in papillary and follicular thyroid carcinomas. To further analyze the relationship between these two genes, we also quantified the PLAG1 expression in 32 uterine leiomyomas (UL) with as well as without 12q14 rearrangements. In addition, the PLAG1 expression was quantified in adipose tissue-derived stem cells (ADSCs) upon a stimulation of HMGA2 by FGF1. Furthermore, the MCF-7 breast cancer cell line, which has previously been used as a model in transfection experiments aiming at the functions of HMG proteins PLAG1 can be transcriptionally activated by HMGA2.Because of the apparent relationship of Formalin-fixed, paraffin-embedded tissue samples of 37 thyroid tumors were classified histologically. Fourteen cases were classified as follicular adenomas (FA), eleven tumors were diagnosed as papillary carcinomas (PTC), and four of them were follicular variants (FV PTC). The remaining twelve tumors were follicular thyroid carcinomas (FTC). Cryopreserved tissue samples of 32 uterine leiomyomas that have been analyzed cytogenetically In case of thyroid tumors, RNA was isolated from six 5 µm sections of FFPE tissues with the RNeasy FFPE kit . RNA from cryopreserved leiomyoma tissues as well as from cultivated cells (ADSCs and MCF-7 cell line) was isolated with the RNeasy Mini kit (Qiagen). Of each sample, 250 ng RNA was used for reverse transcription with M-MLV reverse transcriptase and random hexamers  according to the manufacturer’s instructions.Quantitative real-time RT-PCR was performed with the TaqMan Universal PCR Master Mix on a 7300 Real-Time PCR System (Applied Biosystems) as described elsewhere HMGA2 expression was performed with the HMGA2-specific TaqMan assay Hs00171569_m1 . For the detection of the endogenous control HPRT1, primers 5′-GGC AGT ATA ATC CAA AGA TGG TCA A-3′ and 5′-GTC TGG CTT ATA TCC AAC ACT TCG T-3′ were used in combination with the HPRT1-specific hydrolysis probe 6FAM-CAA GCT TGC TGG TGA AAA GGA CCC C-TAMRA. The PLAG1 expression was quantified with the TaqMan assay Hs00231236_m1 (Applied Biosystems). Reaction conditions were as follows: 2 min at 50°C and 10 min at 95°C followed by 50 cycles of 15 sec at 95°C and 1 min at 60°C.The relative quantification of the 5 cells/9.6-cm dish. After 24 hours the serum concentration was reduced to 1%. Another 24 hours later the medium was replaced by serum-free medium supplemented with 25 ng/ml of human recombinant FGF1 . Twelve, 24, and 72 hours after growth factor addition, cells were harvested and total RNA was extracted. As controls cells were cultured in medium 199 supplemented with 1% fetal bovine serum (FBS) without FGF1.Human adipose tissue-derived stem cells were obtained and treated as described previously Plasmid pCR3.1 containing the sequence coding for human wild type HMGA2 as well as the empty vector serving as a control . The day before transfection, 150,000 cells were seeded in 6-well plates  in 2 ml medium 199 supplemented with 20% FBS and allowed to attach for 24 h. Transfection complexes were prepared in a total volume of 500 µl in serum free medium 199 without antibiotics, 2.5 µg of the respective plasmid DNA and 7 µl Lipofectamine LTX transfection reagent (Life Technologies). A mock control treated with transfection reagent only as well as a non-treated control were included for each incubation period. During the formation of transfection complexes (25 min at room temperature), old growth medium was replaced by 2 ml fresh medium. Transfection complexes were then pipetted drop-wise to the cells. After incubation for 24 h or 48 h, cells were harvested in 350 µl RLT buffer (Qiagen) containing β-mercaptoethanol for RNA isolation using the RNeasy Mini Kit and Qiacube (Qiagen). As a positive control, the expression of IGF2BP2 (synonym: IMP2), which is known to be regulated by HMGA2 The cell line MCF-7 (breast cancer) was maintained in medium 199 (Life Technologies) supplemented with 20% FBS in an incubator at 37°C and 5% COHMGA2 and PLAG1 expression levels, Spearman’s rank correlation coefficient was calculated using the data of all 37 thyroid tumor samples analyzed. In addition, Pearson’s correlation coefficient was calculated using the relative expression values as well as the ΔCT values. In case of uterine leiomyomas, the expression levels of both HMGA2 and PLAG1 were classified as high or low based on the ΔCT values . The agreement between the HMGA2 and the PLAG1 classes was then quantified and tested by the kappa coefficient of agreement.As a measure of association between the relative This study was conducted in accordance with the Declaration of Helsinki HMGA2 and PLAG1 was quantified in 37 thyroid tumors. An adenoma with low expression of both genes was chosen as calibrator for the relative quantification. The highest relative HMGA2 expression observed in 14 follicular adenomas was 17.4, whereas the expression levels in four FV PTC ranged between 23.9 and 156.9. Even higher expression levels ranging between 128.2 and 1207.5 were observed in seven PTC . In contrast, PLAG1 was expressed at levels between 13.9 and 55.9 in six follicular carcinomas overexpressing HMGA2. No overexpression of PLAG1 (0.9) was detected in only one FTC with high HMGA2 expression.The relative expression of 14 cases  and 2. IHMGA2 and PLAG1 expression for all 37 tumor samples and resulted in a high value  indicating that the expression of both genes is strongly correlated in thyroid tumors. For reasons of comparison, Pearson’s correlation coefficient was calculated and also indicates a strong correlation when using the ΔCT values . Due to the exponential transformation, the linear relationship is less pronounced on the basis of relative gene expression values .Thus, as to the expression level of both genes a strong variation was noted even within the group of malignant tumors. If an alternative overexpression of either gene represents different pathways of tumorigenesis one would expect no or even an inverse correlation. On the other hand a correlation would indicate their involvement in just one pathway. Spearman’s correlation coefficient thus was calculated as a measure of association between the HMGA2 in uterine leiomyomas. Therefore, UL (n = 32) were chosen to study a presumable effect of HMGA2 on the PLAG1 expression. One myoma with a normal 46,XX karyotype was chosen as calibrator for the qRT-PCR data obtained for both genes.Recurrent rearrangements affecting the chromosomal band 12q14 are known to cause an overexpression of HMGA2 locus according to FISH HMGA2 was detected in 16 cases. In the remaining case without elevated HMGA2 expression despite a cytogenetically visible t (no. 503) FISH revealed no rearrangement of the HMGA2 locus Of 17 UL with known 12q14 rearrangement including case 646 without cytogenetically visible breakpoint but rearranged HMGA2 expression. A hidden rearrangement of HMGA2 was suspected but no evidence for such a rearrangement was obtained by FISH (case 520).Only 1/15 UL without cytogenetically detectable 12q14 aberration also showed an elevated PLAG1 was clearly elevated in all 17 UL which overexpress HMGA2. In 15 UL with low HMGA2 expression, elevated levels of PLAG1 were detected in only two cases . Similarly a high and significant degree of agreement was found between the low/high expression classification of samples obtained by both parameters , see also The expression of HMGA2 in ADSCs, FGF1 was chosen because it is a known inducer of HMGA2HMGA2 mRNA level 24 h after the addition of FGF1. Simultaneously, the PLAG1 expression increased by 2-fold  as well as after transfection with the empty vector. Cells transfected with the vector containing the HMGA2 insert, however, showed an approximately 2.8-fold increase in PLAG1 expression as compared to mock transfected cells 24 h as well as 48 h after transfection , both genes are expressed at higher levels than in follicular adenomas. Follicular carcinomas with high HMGA2 locus and the HMGA2 protein expression has been shown in uterine leiomyomas HMGA2 and PLAG1 is detectable on the mRNA as well as on the protein level HMGA2 and PLAG1 mRNA expression described herein is expected to reflect a correlation at the protein level as well.A correlation between chromosomal rearrangements affecting the PLAG1 in pleomorphic adenomas of the salivary glands occurs also in tumors with 12q14∼15 abnormalities lacking 8q12 aberrations PLAG1. In the same study, the PLAG1 expression was investigated in three UL, and two cases were also found to overexpress PLAG1, but no cytogenetic data were available for these three tumors.Besides the typical rearrangements involving chromosomal band 8q12 including the most frequent t, an activation of PLAG1 activation in tumorigenesis other than gene rearrangements. The results presented herein point to HMGA2 as an upstream regulator of PLAG1 and are additionally confirmed by the correlation between the expressions of both genes in uterine leiomyomas. An activation of HMGA2 in UL by 12q14 aberrations is well known PLAG1 and HMGA2 simultaneously. Of the 15 UL showing low HMGA2 levels, 13 also showed low levels of PLAG1. The two remaining cases showed an elevated PLAG1 expression despite a low HMGA2 mRNA expression, thus pointing to mechanisms other than HMGA2 upregulation being responsible for PLAG1 activation. In pleomorphic adenomas of the salivary gland cryptic, intrachromosomal 8q rearrangements have been observed leading to a fusion of PLAG1 with CHCHD7 or TCEA1PLAG1 by promoter swapping. Similar events that escape detection by conventional cytogenetics may have caused the upregulation of PLAG1 observed in two UL without visible rearrangements affecting 8q12. In all 17 UL with elevated HMGA2 levels a concomitant overexpression of PLAG1 was noted. The fact that increased HMGA2 levels were always linked to elevated PLAG1 levels suggests HMGA2 as an activator of PLAG1. Because no chromosomal rearrangements affecting 8q12 were present in these 17 UL, we assumed that elevated HMGA2 levels caused by 12q14 abnormalities trigger the activation of PLAG1. In turn, HMGA2 may exert its stimulating effect on the growth of UL with 12q14 aberrations PLAG1, which was shown to possess transactivating capacity IGF-II.These findings suggest alternative mechanisms of HMGA2 is accompanied by increased PLAG1 expression levels Click here for additional data file.


The differences that exist between the CD4 count strata at which certain opportunistic infections occur in sub Saharan Africa suggest that targeting prophylaxis at HIV patients in this part of the world based on data generated from developed countries may be one of the reasons why there is high HIV-related morbidity in Africa.The aim of this study was to investigate the changing incidence of some AIDS defining illnesses in Northern and Southern parts of Nigeria, relating them to different CD4 count strata.In this study, sputum samples of 234 HIV positive patients were analyzed for Mycobacterium tuberculosis and Pneumocystis jiroveci while 202 stool samples of HIV patients were analyzed for the presence of opportunistic intestinal parasites. We considered five CD4 strata . Incidence of the various opportunistic infections and their occurrence within each CD4 strata were estimated using simple statistical method. CD4 count of each patient was estimated using the Becton Dickenson FACSCount system.The prevalence of Mycobacterium tuberculosis, Pneumocystis jirovecii, Cryptosporidium parvum, Isospora belli and Cyclospora spp were 10.3%, 41.9%, 30.8%, 24.2% and 4.4% respectively. All the opportunistic infections occurred at higher rates in CD4 counts less than 200cells/ul (p < 0.0001) except for tuberculosis which occurred highest at CD4 counts >200cells/ul (16 out of the 24 positive sputum smears were recovered in patients with CD4 counts >200 cells/ul). Despite pronounced immunosuppression, P. jiroveci was not detected in sputum samples of 8% patients with CD4 count <200 cells/ul.Opportunistic parasites occurred almost exclusively at CD4 count <200 cells/ul. However, 6.4% of these parasites were isolated in patients with CD4 >200 cells/ul. Cryptosporidium parvum (30.8%) was the most frequently encountered opportunistic parasite, followed by Isospora belli (24.2%) and Cyclospora specie (4.4%).Although HIV related opportunistic infections are often reported to occur exclusively at CD4 count <200 cells/ul in patients; the result of this study shows that disparities exist and so, the possibility of opportunistic pathogens must remain in the differential diagnosis of infections in HIV patients independent of absolute CD4 count.


Asthma is a serious global health problem. Global prevalence of asthma ranges from 1% to 18% of population in different countries.In India, prevalence of asthma is 3% of the population. Major depressive disorder is the most common mood disorder often found to be higher among people with chronic health conditions like asthma. Presence of depression may lead to increased severity of asthma making it an uncontrolled asthma.Our objective was to see prevalence of depression among asthma patients and effect of asthma control on severity of depression.All patients who met the inclusion criteria evaluated with Goldberg’s The General Health Questionnaire (GHQ 28), Bengali Version of Beck Depression Inventory (BDI) and Holmes & Rahe’s Life Event Scale. Severity of asthma and level of asthma control determined as per GINA guidelines. Follow up was done after 3 months and patients were again evaluated with Goldberg’s The General Health Questionnaire (GHQ 28), Bengali Version of Beck Depression Inventory (BDI) and Holmes & Rahe’s Life Event Scale.Among 100 patients 35 (35%) had normal BDI score, mild mood disturbance was found in 23 (23%), borderline clinical depression in 12 (12%), moderate depression in 20 (20%), severe depression in 9 (9%), extreme depression in 1 (1%) patients. Follow up done at 3 months showed 68 patients had controlled and 32 patients had partially controlled asthma. Follow up evaluation with BDI showed 37 (37%) patients had normal score. Mild mood disturbance was found in 24 (24%), borderline clinical depression in 16 (16%), moderate depression in 14 (14%), severe depression in 8 (8%), extreme depression in 1 (1%) patients.There is no significant correlation between severity of asthma and severity of depression .There is also no correlation of asthma control on severity of depression .Depression is highly prevalent among asthma patients. There is no significant correlation between severity of asthma and severity of depression. There is also no correlation of asthma control on severity of depression.


Tropomyosin has been reported to be responsible for the cross-reactivity between shrimp and dust mites and the measurement of tropomyosin specific IgE was found superior to shrimp extract for the predicting of shrimp allergic reaction. A total of 55 dust mite allergic patients were recruited to analyse the cross-reactivity between shrimp and mites . Tropomyosin specific IgE for shrimps , Anisakis ( rAnis 3 ), house dust mites ( rDer p10 ) and german cockroach ( nBla g7 ) were measured using Phadia diagnotic test. Two recombinant allergens ( rTyr p10 for Tyrophagus putresstienciae and rPer a 7 for American cockroach ) were used to investigate the cross-reactivity . Basophil histamine release (BHR) assay was used to evaluate its biological activities. The results showed that there were 13 patients  sensitive to tropomyosin. The allergen specific tropomyosin were further analysed, there were 69% (9/13) sensitive to rAnis 3, 62%(8/13) sensitive to rDer p10, 54%(7/13) sensitive to nBla g7 and nPen m1. Most patients were sensitive to at least two different species of tropomyosin. Immuno-Dot study showed that there were 8/13 sensitive to rPer a7 and 7/13 sensitive to rTyr p10. Despite the percent inhibition of BHR were similar between Der p and shrimp stimulation the relationship between the tropomyosin sensitive of all species allergens were not correlated.In conclusion, tropomyosin specific IgE can be detected in 23.6% (13/55) of Der p sensitive patients and the percentage of shrimp specific tropomyosin allergy was higher in AD than BA. The poor relationship of the specific IgE between the shrimp specific tropomyosin with other species of allergen indicated that the allergenic component of torpomyosin from all allergen should be generated and used for the definite diagnosis of food allergy.