Whether information sources influence health protective behaviours during influenza pandemics or other emerging infectious disease epidemics is uncertain.Data from cross-sectional telephone interviews of 1,001 Hong Kong adults in June, 2009 were tested against theory and data-derived hypothesized associations between trust in  information, understanding, self-efficacy, perceived susceptibility and worry, and hand hygiene and social distancing using Structural Equation Modelling with multigroup comparisons.Trust in formal (government/media) information about influenza was associated with greater reported understanding of A/H1N1 cause (β = 0.36) and A/H1N1 prevention self-efficacy (β = 0.25), which in turn were associated with more hand hygiene . Trust in informal  information was negatively associated with perceived personal A/H1N1 susceptibility (β = −0.21), which was negatively associated with perceived self-efficacy (β = −0.42) but positively associated with influenza worry (β = 0.44). Trust in informal information was positively associated with influenza worry (β = 0.16) which was in turn associated with greater social distancing (β = 0.36). Multigroup comparisons showed gender differences regarding paths from trust in formal information to understanding of A/H1N1 cause, trust in informal information to understanding of A/H1N1 cause, and understanding of A/H1N1 cause to perceived self-efficacy.Trust in government/media information was more strongly associated with greater self-efficacy and handwashing, whereas trust in informal information was strongly associated with perceived health threat and avoidance behaviour. Risk communication should consider the effect of gender differences. Pandemic influenza A/H1N1 has a clinical profile similar to seasonal influenza, despite initially appearing more severe Predictors of population uptake of health protective behaviours in RID epidemics have begun to be studied Ethics approval was obtained from the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. For this telephone interview, written informed consent was waived by the IRB but verbal consent was required from all the respondents and agreement to participate in the interview was taken as further consent. Before the interview began, a brief introduction about the study aims and interview contents was given and then respondents were asked whether the interview could start. If approval was received this was recorded and the interview performed. If not, respondents were thanked and the call was terminated.More than 98% of Hong Kong households have landline telephones and all local calls are free. Random-digit dialled telephone numbers and within-household random-sampling grids (Kish grids) are a cost-effective way to survey highly representative random population samples. Kish grids are matrices containing random numbers for different sized households that facilitate random selection of individuals within households and help minimize sampling bias. The number of eligible household residents, “n”, is determined by asking the person of first contact in the household. The Kish grid provides a randomly generated number “k” between 1 and “n” which is used by the interviewer. Ordering by age and starting from the oldest eligible member in the household, the k'th member is then invited to participate in the survey. Different grid values are used for each household. As part of a series of surveys to monitor A/H1N1 epidemic activity, a commercial polling organization administered the questionnaire using this telephone-survey methodology, targeting 1,000–1,500 participants on each occasion, a sample size calculated to give an estimate of A/H1N1 health protective behaviours with a precision of ±3%. The survey with the largest sample was selected for this analysis. Sampling was performed during the evening to minimize exclusion of young working adults.rd–25th June, 2009, two weeks after the first community transmission had been identified in Hong Kong.Data on attitudes, knowledge, situational awareness, risk perception and preventive behaviours  were colExisting theoretical frameworks of behaviour change have been adapted to predict health-related behaviour-change for chronic, non-communicable diseases Reporting delays, uncertainty and other biases affect publicly available information on the characteristics of newly-emergent communicable diseases, such as A/H1N1 lay knowledge of infection-related risks can be limited. The resulting uncertainty about disease severity and transmissibility at the epidemic onset extends to the utility and timing of adopting preventive measures. Information cues to individuals about initiating protective action must therefore be synthesized from various sources. Perceived information reliability or trustworthiness influences decisions to utilize any given information source Perceived risk is influenced by several stimulus characteristics, including unfamiliarity, invisibility, dreadfulness and inequity Because data were collected using telephone interviews we had to adapt measures to suit a brief format in order to avoid people hanging up mid-way or providing invalid answers to hurry the interview, a problem encountered with this data collection method. We therefore used parsimonious measure to minimize assessment fatigue and low response rates which threaten representativeness.Trust in government/media  information: We asked about respondents' agreement with three statements . ResponsTrust in interpersonal  information: Respondents' agreement with two statements . ResponsUnderstanding cause of A/H1N1 (“I understand how Swine flu is caused”) and self-efficacy (confidence in one's ability to act in a way that achieves desired future outcomes) for A/H1N1 prevention (“I am confident that I can protect myself against Swine flu”): Each was assessed using responses on 5-point scales of agreement with these two single item statements .Perceived personal susceptibility: Two items, one assessing absolute susceptibility (perceived absolute probability of developing A/H1N1) and another assessing relative susceptibility (perceived probability of developing A/H1N1 relative to peers) formed a latent variable for perceived personal susceptibility . The CroWorry about contracting H1N1. Respondents were asked to indicate their level of worry over the past one week about contracting influenza A/H1N1. Responses were 5-point scales of worry ranged from “never thought about it” to “extremely worried” .Hand hygiene. Respondents were asked to indicate frequencies of use of four hand hygiene practices over the three days prior to interview: hand washing after sneezing, coughing and touching nose; hand washing after returning home, use of liquid soap for hand washing, and hand washing after touching common objects. Responses were on a 4-point scale of frequency: 1 “never”, 2 “sometimes”, 3 “usually” and 4 “always”. Cronbach's α was 0.62 .Social distancing behaviours: a. Social Avoidance. Respondents were asked to indicate if they had adopted any of four avoidance behaviours due to influenza A/H1N1 in the past 7 days: avoiding eating out, avoiding using public transport, avoiding going to crowded places, and rescheduling travel plans Responses were coded as 1 “yes” and 0 “no”. Cronbach's α was 0.61 .We first compared the demographic structure of the sample against that of the general population derived from the Hong Kong government General Household Survey to identify any sample differences.Our model proposes that trust in formal (government and media sources) and informal (from other people) information affects RID epidemic health protective behaviours, the former by informing about generic risk and response characteristics for dealing with a potential threat (causes and protective responses), the latter about threat imminence, severity and response effectiveness (seeing how others behave). We refer to the product of these combined processes as situational awareness, and propose that rather than driving behaviour directly information acts through altering the cognitive/affective domain of situational awareness. Thus the model is predicated on several premises: that understanding of the disease and perceived personal susceptibility influence self-efficacy Structural Equation Modeling (SEM) is a method for simulating and testing multiple and interrelated causal relationships simultaneously in statistical data, making it suitable for theory development and testing In our hypothesized model, trust in formal information, trust in informal information, perceived personal susceptibility, hand washing and social distancing behaviours were entered as latent (inferred) variables while other constructs were entered as observable (directly measured) variables because they were assessed with only one item. Two different health protective behaviors, hand washing and social distancing, were entered as the HPB outcomes because we hypothesized that different influences may act on each of these. We assumed that the “disturbances” of the two health behavior outcomes were correlated. Disturbance represents the unexplained variances of the latent variables predicted by the specified independent variables Adequacy of the measurement models was tested before testing the full structural model. To test the full structural model, all constructs  were ent2 = 147.69, p<0.001), and 89% of male compared to 80% of female respondents, obtained at least secondary education . Since the numbers of tertiary educated respondents and primary (elementary) educated respondents were too small to produce stable models, we limited stratification to gender and age only and acknowledge that this also incorporates indefinable education and income effects.Responses are likely to differ by sociodemographic factors Consequently, we used a multi-group SEM to assess the invariance of the model  across gFinally, to help interpret these multigroup SEM comparisons, we performed a post-hoc examination of the model variable means for different gender and age groups and tested differences using the Mann-Whitney test, which tests differences between two groups on ordinal scales of measurements.A total of 1,001/1,449 (69.1% response rate) Hong Kong adults successfully completed the interview. The characteristics of the sample were compared against the Hong Kong 2006 by-census population data Both formal and informal information trust were correlated with all situational awareness variables except worry about contracting A/H1N1 (“Worry”), while formal information trust was also independent of perceived personal susceptibility (“Susceptibility”). In turn, understanding of H1N1 cause (“Understanding”) and Perceived self-efficacy (“Self-efficacy”) were significantly associated with hand washing while Worry and Susceptibility were significantly associated with social distancing .The SEM model fitted well to the data with CFI = 0.977, TLI = 0.969 and RMSEA = 0.026. Standardized coefficients indicated two primary features in the model; the first one linking Formal information and hand hygiene and a second linking Informal information and Social distancing . Paths wThe second feature of the model is reflected in a different set of paths associating informal information trust with social distancing. Trust in informal information sources was inversely associated with Susceptibility (β = −0.21), which was associated positively with Worry (β = 0.44), and inversely with Self-efficacy (β = −0.42). However, more confidence in informal information sources was associated with more Worry (β = 0.16) and finally, only Worry was associated with social distancing (β = 0.36). Trust in informal information was independent of Understanding and Self-efficacy.The only remaining notable feature of the model was a strong inverse association (β = −0.42) between Susceptibility and Self-efficacy. This suggests some interaction between these two variables that could strongly influence both sets of paths mentioned so far.Overall, the model explained 11.3% of the variance in hand hygiene and 16.1% of the variance in social distancing behaviors.2 for “DIFFTEST” = 29.30, df = 19, p = 0.061). However, three sets of associations differed significantly between females and males: those between Formal information trust and Understanding, from Informal information trust to Understanding, and from Understanding to Self-efficacy. These paths were set free and estimated separately in female and male. The model with these paths freely estimated fit well to the data with CFI = 0.978, TLI = 0.976 and RMSEA = 0.023, and did not differ significantly from the least constrained model . Across gender, both the least constrained model and the most constrained models fit the data well with CFI>0.970, TLI≥0.970, RMSEA = 0.025. The most constrained model did not differ significantly from the least constrained model  than males (β = 0.25); the association between Informal information trust and Understanding was weakly positive in males (β = 0.12) but weakly negative in females (β = −0.14), and; the association between Understanding and Self-efficacy was positive (β = 0.12) in males but non-significant in females (β = −0.01).2 for “DIFFTEST” = 15.85, df = 19, p = 0.667). No path was found to be significantly different between the two age groups.Across the two age groups, both the least constrained model and the most constrained model fit well to the data with CFI>0.960, TLI≥0.950, RMSEA≤0.030. The most constrained model did not differ significantly from the least constrained model  information, situational awareness variables  and different types of health protective behaviours  for influenza protection. The model suggested that two different sets of influences relate trust in information to hand hygiene, and to social distancing respectively. The strongest associations observed were between Susceptibility and Self-efficacy (negative), Susceptibility and Worry (positive), Trust in Formal information and Understanding (positive), Trust in Formal information and Self-efficacy (positive), and Trust in Informal information and Susceptibility (negative), and Worry and social distancing (positive).Neither age nor gender contributed significant variation to the association between Trust in Formal information and Self-efficacy, and Self-efficacy and hand hygiene. These findings are consistent with other studies showing self-efficacy is enhanced by procedural information The association between Trust in Formal information and Understanding of influenza cause differed by gender but not age, with females showing a stronger association. Men tend to have poorer health knowledge than women Trust in Informal information seems to be associated with less perceived susceptibility to health threat. This may reflect rational processes or cognitive bias. Trusting social cues involves comparison and conformity influences, and can enhance optimistic bias (the tendency to view oneself less likely to experience negative events but more likely to experience positive events) in personal risk estimates Susceptibility was strongly associated with both Self-efficacy (negatively) and Worry (positively). Neither Worry nor Susceptibility varied significantly by gender or age group. This is plausible and theoretically consistent The model tested explained only a modest proportion of the variance in adoption of HBPs, suggesting that there are significant theoretical gaps that remain to be filled. These await further research.Social distancing is unassociated with formal HPB messages, suggesting potential susceptibility to a “herd-like” response in this Chinese community, particularly if confidence in formal (government or doctors) information is low. Voerten and colleagues describe such a pattern of response in the early stages of SARS Self-efficacy in preventing A/H1N1 influences hand hygiene but has little influence on social distancing. Formal health education messages that focus on enhancing the public's sense of their ability to protect themselves by adopting hygiene practices would seem to be the most effective to improve hand hygiene, but where the practice is already established, high levels of trust in these messages are not likely to significantly increase hand hygiene.This study is limited in being cross-sectional and relying on hypothesized modeling to infer causality. This is potentially error-prone and can only be confirmed by specific longitudinal tests of the hypotheses proposed above. There are potential limitations related to measurement imposed by the need to be parsimonious in questioning due to use of telephone interviews. Where this is not done refusal rates would have been unacceptably high Many factors influence RID protective behaviour. This study has examined a very limited number of these. Confidence in formal information such as health education messages is associated with greater compliance to recommended preventive measures for influenza A/H1N1 A simple version of our findings can be found it the supporting file .Table S1(0.05 MB DOC)Click here for additional data file.Table S2(0.03 MB DOC)Click here for additional data file.Table S3(0.04 MB DOC)Click here for additional data file.Table S4(0.04 MB DOC)Click here for additional data file.Text S1This is a simple version of our study findings for nonspecialists.(0.03 MB DOC)Click here for additional data file.


Finally, the staining pattern of protein tyrosine nitration, assessed by immunohistochemistry, parallelled that of NOS1 expression in normal renal parenchyma and benign tumours, supporting the concept that protein nitration was accounted for by NOS1 activity. In contrast, in malignant tumours, protein tyrosine nitration was accounted for by the production of reactive nitrogen oxide species by the inflammatory infiltrate. Altogether, these findings argue for a pattern of NO signalling similar in normal kidney and benign renal tumours, whereas it is completely different in malignant renal tumours.In normal human kidney, NOS1 and soluble guanylate cyclase (sGC) are expressed in tubular epithelial cells, suggesting a physiological autocrine NO signalling pathway. Therefore, we investigated both NOS1 and sGC expressions in benign and malignant renal tumours. In addition, we examined the pattern of protein tyrosine nitration in normal and tumour tissue. NOS1 expression and activity were found to be downregulated, correlating with the tumour grade, as shown by immunohistochemistry, quantitative RT–PCR analysis, and histochemical detection of the NADPH-diaphorase activity of nitric oxide synthases (NOS). These results show that the autocrine NO signalling pathway is maintained in benign tumours and lost in malignant tumours. In contrast, sGC expression was maintained in renal tumours whatever the tumour type, a finding showing that tumour cells remain sensitive to the bioregulatory role of exogeneous NO We used red blood cells in order to distinguish protein tyrosine nitration resulting either from an intracellular production of ONOOTumour tissue and normal tissue at distance from the tumour were collected from 32 patients undergoing nephrectomy for oncocytoma (six cases), chromophobe renal cell carcinoma (RCC) (four cases) and clear cell RCC . For each case, samples of tumour and normal tissue were snap frozen and stored in liquid nitrogen, and also fixed in formalin and embedded in paraffin. These samples were collected according to the guidelines of the French Ethics Committee for Research on Human Tissues.μm formalin-fixed paraffin sections of different renal tumours with the polyclonal N-terminus NOS1, the sCG and the 3-nitrotyrosine antibodies, and on 5 μm acetone-fixed cryostat sections for staining with the monoclonal C-terminus NOS1, the NOS2 and the NOS3 antibodies. The specificity and conditions of use of primary antibodies directed to NOS isoforms are listed in Table 1M solution, pH 7.4) prior to incubation on tissue sections. Immunohistochemical staining was assessed by two independent observers  including a pathologist (KR).Immunohistochemistry was performed on 5 μm cryostat sections were fixed for 15 min in a freshly made paraformaldehyde solution (4% w v−1 in PBS) and then washed four times in PBS. The NADPH-diaphorase (NADPH-d) activity of NOS, which is paraformaldehyde-resistant, was demonstrated by enzymatic reduction of nitroblue tetrazolium in the presence of NADPH, as previously described  and 0.4 mg ml−1 NBT  for 30 min at 37°C, washed in PBS, dehydrated and mounted without any counterstaining. NADPH was omitted in negative controls.In total, 5 μg) was denaturated at 72°C for 3 min and then reverse transcribed for 60 min at 42°C in a 20 μl reaction volume  containing 0.5 μg of random hexamers , dNTPs (1 mM each), RNasin (50 U) and RnaseH− MMLV reverse transcriptase (200 U).Total RNA was extracted from four cases of Führman III RCC and normal renal parenchyma from the same patients using the GenElute Mammalian Total RNA kit  and the Fast Prep cell disrupter . RNA . PCR amplifications were performed using Titanium Taq DNA polymerase . The reaction mixture contained 2 μl of the supplied 10 × Titanium Taq PCR buffer (containing magnesium chloride), 2 μl of a 1/1000 dilution of SYBR Green I , 1 μl of each primer (0.4 μM each), 0.4 μl of Titanium Taq DNA polymerase, 0.5 μl of dNTPs (10 mM each) and PCR-grade water to a volume of 15 μl. Microtubes (0.2 ml) were loaded with 15 μl of this master mix and 5 μl of the template (cDNA diluted 1/50) and the run was initiated. The cycling conditions were as follows: denaturation for 5 min at 95°C; amplification for 35 cycles, with denaturation for 5 s at 95°C, annealing for 10 s at 63°C and extension for 20 s at 72°C. To exclude primer-dimer artifacts, fluorescence was not measured at the end of the extension step, but a separate detection step was added (15 s) at a temperature (88°C) above the melting point of primer-dimers and below the melting point of the specific PCR product (92°C).The amplification conditions of the NOS1 and Primers NOS1 sense (5′-TCT CCT CCT ACT CTG ACT CC-3′) and NOS1 antisense (5′-TTG TGG ACA TTG GAT AGA CC-3′) were designed from the sequence of the human NOS1 cDNA (accession number NM-000620). They were selected for binding to separate exons  to avoid false positive results arising from amplification of contaminating genomic DNA.After completion of the cycling process, samples were subjected to a temperature ramp from 63 to 99°C, with continuous fluorescence monitoring for melting curve analysis. For each amplification, apart from primer-dimers, a single narrow peak was obtained at the expected melting temperature (92°C), indicating specific amplification without significant by-products.vs the corresponding Ct (threshold cycle) values. The correlation coefficient of these curves was always greater than 0.99. The amount of NOS1 transcript was calculated from these standard curves using the RotorGene software. A relative unit of 100 was assigned for the standard cDNA diluted 1/500. Samples were tested in triplicate and the average values were used for quantification.An external standard curve was generated with serial five-fold dilutions of cDNA  prepared from normal kidney RNA. The reference curve was constructed by plotting the relative amounts of these dilutions β-actin cDNA was determined, as described for NOS1 with primers 5′-CCT TCC TGG GCA TGG AGT CCT G-3′ (actin sense) and 5′-GGA GCA ATG ATC TTG ATC TTC-3′ (actin antisense). The ratio between the relative amount of NOS1 and β-actin was then calculated to compensate for variations in quantity or quality of starting mRNA as well as for differences in reverse transcriptase efficiency.For each sample, the relative amount of human We assessed the expression of NOS1 in tumour samples by using an antibody directed to the C-terminus part of NOS1 on cryostat sections. To confirm the staining pattern, we used another antibody directed to the N-terminus part of NOS1, on paraffin sections of a few cases of each type of tumour. The overall staining pattern was found identical with both antibodies .− or other reactive nitrogen species   , it was We assessed the pattern of protein nitration by immunohistochemistry in three cases of normal parenchyma and in three cases of each tumour type. In normal renal parenchyma the overall staining pattern was identical to the NOS1 expression pattern  the autocrine signalling pathway of NO previously observed in normal human tubular epithelial cells (− and long-range action of other reactive nitrogen species produced by inflammatory cells. The most well-known mechanism of protein nitration is the ONOO− pathway, resulting from the reaction of equimolar NO• and O2−, a phenomenon restricted to an organelle or cellular compartment such as mitochondria (− chemistry limited to close proximity of its source. In addition, we can rule out the involvement of long-range acting nitrogen species since red blood cells are devoid of any staining in normal renal tissue and in oncocytoma. In malignant tumours containing an important inflammatory infiltrate, the interpretation of protein nitration pattern is more complex. Indeed, other nitrating species known to have a long range of action are produced by inflammatory cells and result from the oxidation of circulating nitrite NO2− into NO2•, a reaction catalysed by myeloperoxidase or other haeme proteins in the presence of H2O2 (− transfer from HOCl to NO2− generates NO2Cl, a nitrating agent (The third major finding of this study concerns the pattern of protein tyrosine nitration, which is identical in normal kidney and in benign renal tumours, but completely different in malignant tumours. Nitration is a covalent modification of the tyrosine residues of some proteins with activating or inhibitory activities that could be involved in physiological conditions as well as in pathological states such as inflammatory processes and tumours (chondria . Our obs of H2O2 . In addiIn conclusion, our study helped decipher the pattern of NO signalling in renal tumours of different grades compared with that of normal renal parenchyma. The bioregulatory activity of NO is similar in benign tumours and in normal kidney, whereas there is a shift of NO signalling in high-grade malignant tumours, with a loss of endogeneous NO synthesis, but the maintenance of sGC, the receptor for exogeneous NO. The demonstration of nitrated proteins in renal tumours implies that exogeneous NO, especially reactive nitrogen species produced by inflammatory cells, have an impact on cancer cells, whose biological significance remain to be determined.


M.tuberculosis from children.MODS is a novel liquid culture based technique that has been shown to be effective and rapid for early diagnosis of tuberculosis (TB). We evaluated the MODS assay for diagnosis of TB in children in Viet Nam. 217 consecutive samples including sputum (n = 132), gastric fluid (n = 50), CSF (n = 32) and pleural fluid (n = 3) collected from 96 children with suspected TB, were tested by smear, MODS and MGIT. When test results were aggregated by patient, the sensitivity and specificity of smear, MGIT and MODS against “clinical diagnosis” (confirmed and probable groups) as the gold standard were 28.2% and 100%, 42.3% and 100%, 39.7% and 94.4%, respectively. The sensitivity of MGIT and MODS was not significantly different in this analysis (P = 0.5), but MGIT was more sensitive than MODS when analysed on the sample level using a marginal model (P = 0.03). The median time to detection of MODS and MGIT were 8 days and 13 days, respectively, and the time to detection was significantly shorter for MODS in samples where both tests were positive (P<0.001). An analysis of time-dependent sensitivity showed that the detection rates were significantly higher for MODS than for MGIT by day 7 or day 14 (P<0.001 and P = 0.04), respectively. MODS is a rapid and sensitive alternative method for the isolation of  It is estimated that 9.27 million new tuberculosis (TB) cases occurred worldwide in 2000, of which approximately 11% were in children Pham Ngoc Thach Hospital in Ho Chi Minh City Viet Nam admits approximately 200 children with suspected TB each year. In 2007, only 14.9% (59/395) of treated pediatric TB cases had microbiological confirmation. The majority of confirmed cases had pulmonary TB. Confirmation of extrapulmonary TB is even more challenging in children. This is consistent with reports from other settings The confirmation of TB is based on bacterial diagnosis. Sputum microscopy for TB diagnosis is widely used, rapid and straightforward but insensitive because of difficulty in obtaining adequate quality and volume of sputum samples from children. Lowenstein-Jensen (LJ) culture has been considered the gold standard for the diagnosis of TB for almost a hundred years, but it has a median time to positivity of four weeks and 70%–75% sensitivity against clinically defined probable adult cases. Commercial automated liquid culture methods including Mycobacterial Growth Indicator Tube (BATEC MGIT 960 - Becton Dickinson USA) are widely used in developed country settings for routine TB diagnosis. This method has a sensitivity of approximately 75% against clinically defined probable cases. Although such automated systems are faster than LJ culture, the median detection time is still approximately 14 days and the cost is relatively high, restricting their utility in high-burden settingstuberculosis; the growth in liquid medium is considerably faster than that on solid medium, and the distinctive morphology of M.tuberculosis in liquid culture as cording, strings or tangles. Recent evaluations have shown it to be an economical and rapid, sensitive and specific method for M.tuberculosis detection making it ideal for use globally but particularly in low income countries Recently, a new method known as Microscopic Observation Drug Susceptibility Assay – MODS - has been evaluated for the diagnosis of TB in children at the Instituto de Salud del Niño of Lima, Peru All children ≤16 years of age with clinical suspicion of tuberculosis presenting to the paediatric ward at Pham Ngoc Thach Hospital, Ho Chi Minh City, Viet Nam from May 2008 to December 2008 were enrolled into the study. Any patient already receiving TB therapy for more than seven days was excluded from the study. Data on socioeconomic and demographic features, TB history, TB contact history, HIV status and presenting clinical features were prospectively collected on a standard case report form (CRF). Samples were collected as per routine care as deemed appropriate by the treating physician. No additional samples were collected as part of this study. All specimen types from suspected cases were included in the study except blood. The definition of TB was based on microbiological confirmation by either smear or MGIT, intention to treat, treatment management and outcome. Tuberculosis was defined as “confirmed TB” if the patient had clinical symptoms consistent with TB and either smear or MGIT was positive in any sample. A positive MODS was not considered as part of the ‘confirmed TB’ group definition because this was the test under evaluation.The patient was defined as “probable TB” on ‘intention to treat’: if patients had clinical symptoms consistent with TB Patients were defined as “TB unlikely” if they recovered without TB treatment, had TB treatment but deteriorated or received an alternative diagnosis and treatment. It was impossible to ‘rule-out’ TB in these patients because clinical deterioration may have been due to undetected drug-resistant TB although MDR rates are low in this population (<3%) The protocol was approved by the Institutional Review Board (IRB) at Pham Ngoc Thach Hospital and the Health Services of Ho Chi Minh City. Informed consent was not sought because the study was conducted on routine samples only and it did not involve any intervention, additional samples or change in patient management. This patient consent waiver was approved by the IRB of Pham Ngoc Thach Hospital in the protocol.All samples including sputum, cerebral spinal fluid (CSF), gastric fluid and pleural fluid were collected and transferred to the microbiology department on the same day (or the following day if they were collected after 4 pm). The samples were then submitted for smear, MGIT and MODS culture. The number of specimens per patient and the specimen type were decided by physicians.3Citrate 0.05, NALC (N-Acetyl-L-Cysteine) and Phosphate buffer (PO4 10X - 0.67 M). Phosphate buffer 1X, homogenization buffer and decontamination buffer (HDB) were then prepared from the kit for sample processing. In brief, 3–5 ml sample was added to 3–5 ml HDB contained in a 50 ml falcon tube. The tube was shaken slightly by automated shaker and left at room temperature for 20minutes. After that, 35–39 ml phosphate buffer 1X was added into the mixture. The mixture was shaken by hand and then centrifuged at 3000 g, 4°C for 30 minutes. The supernatant was then discarded and 0.5 ml pellet at the bottom was re-suspended with 2 ml distilled water. The deposit was then aliquoted into 3 parts for smear, MGIT culture and MODS.All samples, except for CSF, were homogenised and decontaminated by Sputaprep (NaOH-NALC 2%) manufactured by Nam Khoa Company-Viet Nam prior to testing. The kit contains Mucoprep . After discarding the supernatant, 2 ml pellet was used for smear, MGIT and MODS culture. Technicians responsible for each test were blinded to the other microbiological results and clinical data on the patient records.Two drops of pellet from each sample were put onto a slide for homogenous smear preparation. The smears were then stained by ZN method according to World Health Organisation (WHO) standard protocol Processed samples were subjected to MGIT culture following the protocol of Becton Dickinson. In brief, 0.1 ml PANTA, 0.5 ml OADC and 0.5 ml of each processed sample were added into a MGIT medium tube. The mixture was inversion mixed by hand and then inoculated and incubated at 37°C in the MGIT machine. Positive results were reported automatically by the MGIT system.et al.The MODS culture was conducted in a biosafety cabinet class I which was placed in a separated room from the sample processing room, smear preparation room and MGIT culture room. The MODS method was performed as described in Park All cultures positive by MODS or MGIT were subcultured on LJ media (Becton Dickinson) in duplicate and incubated at 37°C for several weeks. These isolates were then subjected to standard biochemical identification tests and DNA extracted and archived.DNA extraction was done using the CTAB method.Isolates were archived in 7H9 media supplemented with 20% glycerol (7H9A). One or two loops of isolates collected from LJ culture were transferred into a cryotube containing 7H9A medium. These tubes were stored at −20°C.Spoligotyping was done according to the standard international Spoligotyping protocol Spoligotyping was done for all cultures positive by MODS (n = 73). If MODS was contaminated while subculturing from MODS to LJ for DNA extraction (n = 2) or MODS was negative but MGIT positive (n = 8), cultures positive by MGIT were used for spoligotyping.Accuracy measures of the 3 tests were calculated for two different definitions of the ‘gold standard’ reference test: (1) microbiological confirmation (confirmed group) or (2) ‘clinical diagnosis’ . In addition, we analyzed data on a ‘per patient’ or a ‘per sample’ basis.For the ‘per patient’ analysis, the data was aggregated to provide one result per patient, i.e. the ‘per patient’ test was regarded as positive, if at least one sample yielded a positive test result. Reported confidence intervals for accuracy measures  were calculated according to the method of Pearson and Clopper. Comparisons of accuracies between tests were done using McNemar's test.In the ‘per sample’ analysis we used a binary marginal generalized linear regression (GLM) models with an identity link function for all analyses. These models are very flexible, allow for the inclusion of covariates and account for the fact that results of multiple samples from the same patient or test results of different tests on the same sample may be dependent.t if the respective test was positive overall and reached the positive value at most t days after sample collection. Time-dependent sensitivity curves were estimated with the Kaplan-Meier method and samples without a positive test result were formally regarded as censored on day “infinity”. Time-dependent sensitivities of MGIT and MODS by days 7 and 14, respectively, were compared using a marginal regression model as described above. In addition, the time to positive MGIT and MODS, respectively, was compared in samples were both tests reached positivity with the Cox proportional hazards regression model. Robust standards were used to adjust for possible dependence of multiple samples from the same patient or test results of different tests on the same sample.For the ‘per sample’ analysis, we also calculated time-dependent sensitivity curves for MGIT and MODS. A test result was considered as positive by time Comparison of demographic and clinical features of patients between TB diagnoses  was done with Fisher's exact test for categorical data and the Kruskal-Wallis test for continuous data.All reported confidence interval are two-sided 95% confidence intervals and p-values ≤0.05 were regarded as statistically significant. All analyses were done with Stata version 9  except graphs which were created with R version 2.90 .One hundred children suspected of TB were enrolled into the study. Data of 96 (96%) children were included in the analysis. Four patients were excluded from the study because of not meeting the inclusion criteria (older than 16 years of age) . A totalGeneral demographic characteristics of the study population are shown in Clinical symptoms of TB of this referral study population included cough (67.7%), fever (80.2%), weight loss (45.8%) and chest X-ray consistent with TB (75%) . Other sThe clinically diagnosed gold standard was defined as patients who satisfied characteristics of either “confirmed TB” group or “probable TB” group. 78 patients were diagnosed with TB by clinical features and/or microbiological confirmation and 18 patients were classified as TB unlikely.When analysed by patient, the sensitivity of smear, MGIT and MODS were 28.2%,  (n = 22/78), 42.3%,  (n = 33/78) and 39.7%,  (n = 31/78). MODS was more sensitive than smear (P = 0.011)  but comparable to MGIT . Specificity, positive predictive value (PPV) of smear and MGIT was 100% because it was part of the definition for confirmed TB, but all patients with a positive smear/MGIT had clinical symptoms consistent with TB and were considered to have TB by the treating clinician. Specificity and PPV of MODS were 94.4%,  (n = 17/18) and 96.8%, , (n = 31/32), respectively. Negative predictive value (NPV) of smear, MGIT and MODS was 24.3%,   and 26.6%,  (n = 17/64).When analysed by sample, the sensitivity of MODS, MGIT and smear was 43.8%  (n = 74/169), 48.5%  (n = 82/169) and 27.2%  (n = 46/169), respectively. MODS was more sensitive than smear  but less sensitive than MGIT . aureus infection.Of 35 patients with confirmed TB, 31 (88.6%) patients had at least one sample positive by MODS Figure 2When analysed by sample, MODS yielded a sensitivity of 81.3%  (n = 74/91), specificity of 99.2%  (n = 125/126), PPV of 98.6%  (n = 74/75) and NPV of 88.0%  (n = 125/142). Using MODS we recultured 8 of the samples which had been positive by MGIT but negative by MODS. These samples remained culture negative by the MODS technique.To investigate whether sample type had a strong impact on the sensitivity of the three methods, we investigated the number of each sample type collected from 78 clinically diagnosed TB patients and analyzed the sensitivity of these methods in terms of sputum sample (n = 101), gastric fluid (n = 35), CSF (n = 30) and pleural fluid (n = 1) . Our dat175 samples of all types from patients receiving TB treatment in this admission were collected on the day of TB treatment or within seven days of starting TB therapy. TB treatment for more than 1 day reduced the sensitivity of all 3 microbiological tests, but this difference did not reach significance;  for smear,  for MGIT and  for MODS .Time to detection was defined as the number of days from sample processing (day 1) to results available. Smear results were available one day after sample processing due to the high volume of samples in this laboratory. Median time to detection of MODS was faster than MGIT, 8 days (IQR: 6–12) vs 13 days (IQR: 9–19), respectively in samples positive by MODS or MGIT. In the 73 samples which were both MGIT and MODS positive, MODS was faster in 64 (88%) samples and the median (IQR) time difference was 3 (1 to 6) days in favor of MODS (p<0.001).“Time-dependent sensitivity” counted only samples positive by both MGIT and MODS which reached positivity by day 7 or 14, respectively. The time-dependent sensitivity against clinical gold standard was significantly higher for MODS compared to MGIT on both day 7 (18.34% vs. 7.10% P = <0.001), and on day 14  .Four CSF samples positive by MGIT had a median time to positive of 24.5 days (IQR: 23–25). One CSF sample was positive by MODS 6 days after inoculation.To evaluate if MODS is useful for detection of smear negative cases, we evaluated the sensitivity of MGIT and MODS among 56 smear negative TB cases (confirmed and probable) and 123 smear negative TB samples including either MODS or MGIT positive. The sensitivity of MGIT and MODS by patient were comparable, , which were 21.4% (n = 12/56) and 17.85 (n = 10/56), respectively. The sensitivities were 30.08%,  (n = 37/123) and 25.20% (n = 31/123) for MGIT and MODS, respectively, in terms of sample with P = 0.053, 95%CI for difference . The median time to positive of MODS for smear negative samples was considerably faster than MGIT, which was 12 days (IQR: 8–17 days) compared to 19 days (IQR: 15–22 days). In the 30 samples where both MGIT and MODS were positive, the median time difference was 6 days (IQR: 3–11days) in favor of MODS (p = 0.005). Therefore, MODS appeared to have an advantage over MGIT in terms of time to detection.To investigate whether time to positive of MGIT and MODS is related to smear positive grading, we analyzed the association of time to detection of MGIT and MODS with smear grade. 3 (IQR: 13–110) and 100% were lymphocytic (IQR: 100%–100%). CSF parameters were not significantly different among groups (data not shown). The CSF volume available for testing varied with a median value of 3 ml (IQR: 2.5 ml–5 ml). The median CSF volume of children in the younger age group (under five years of age) (n = 19) was 3 ml (IQR: 2 ml–3 ml) and it was 5 ml (IQR: 5 ml–5 ml) in the older group (over 12 years old) (n = 4).One CSF was collected from each patient with suspected TBM. Thirty-two out of ninety-six patients (33.4%) had a lumbar puncture performed to establish a diagnosis of TBM. Of these, 6 patients were classified as confirmed TBM , 24 patients were defined as probable TBM on ‘intention to treat’ basis as previously published criteria In terms of contamination with fungi, the original contamination rate of MGIT and MODS were 1.39% (n = 3/217) and 0.9% (n = 2/217), respectively. All MGIT contaminated samples were decontaminated again and reinoculated in MGIT medium. No contamination was observed after decontamination. Reprocessing for sample contaminated by MODS was not attempted because of low volume .37Rv) at the wells closest to the positive control well. In terms of cross-contamination with TB bacteria, one sample and four negative control wells were contaminated with positive control isolate  and as sensitive as MGIT (P = 0.5) on per patient analysis (n = 78),. However, on per sample analysis (n = 169), MODS was more sensitive than smear (P<0.001) but less sensitive than MGIT (P = 0.027). Larger sample size (169 samples compared to 78 patients) may account for this difference.With microbiological diagnosis, MODS diagnosed 88% of TB cases, similar to a report from Peru in 2006 in which MODS detected 87% of pediatric TB cases Pediatric TB samples are paucibacillary and it is therefore important to consider which sample types yield highest sensitivity from the limited sample volumes that are inevitably available from children. Our study found that sputum and gastric fluid were appropriate for use to diagnose pediatric TB, in agreement with other groups who have also found gastric fluid to improve confirmed TB rates in sputum-negative pediatric TB. 5 to 106 bacteria/ml are required for the machine to signal a positive result. In MODS culture, the result was read under an inverse microscope with 400× magnification. Therefore, a few bacteria in each well can be detected by MODS several days before MGIT. From this study, we also observed that the time to positive is inversely related to the number of bacilli in each sample or smear results. Since the sensitivity of MODS and MGIT were similar in smear negative samples and MODS has a faster time to positive than MGIT, MODS is a candidate method for routine use to diagnose smear negative pediatric TB.In patients where both tests MODS and MGIT reached positivity, the time to positive of MODS was shorter than MGIT with a median time difference of 3 days to 6 days. Technical issues affect the turn around time of each method. In MGIT culture, 1037Rv. Our results were similar to the results of a study conducted in 2006 which recorded a specificity of 99.6% due to cross-contamination between samples. M.tuberculosis by spoligotying and no sample with cross-sample contamination was detected. No mis-identification of Non-tuberculous mycobacteria (NTM) was recorded in this study but this needs further evaluation in populations with higher rates of NTM, such as patients coinfected with HIV.The specificity of MODS (98.4%) was slightly lower than smear (100%) and MGIT (100%) because we had one false positive sample due to cross-contamination with H37Rv) to a single sample and to negative control wells; all cross-contaminated wells were sited at the positions closest to the positive control wells. A high concentration of H37Rv is inoculated into positive control wells (>105 bacteria/100 ul), and therefore cross-contamination was more likely from the positive control than from paucibacillary samples.Contamination is a common problem with all liquid culture and MODS is not an exception; careful manipulation and rigorous aseptic technique must be maintained to minimize contamination. Contamination with fungi was rare at only 1.4%. We have not detected any cross-contamination between samples but we did have cross-contamination from the positive control isolate , inverted microscope and tissue culture plates. In addition, the cost of MODS is low  and so, MODS should be evaluated for application in provincial TB laboratories within national TB programs.Overall, MODS is a sensitive, quick and reliable culture test for use in the diagnosis of children with TB, allowing these vulnerable individuals to get onto TB treatment earlier. It is also cheaper than the alternative rapid culture or molecular methods currently available. Further studies should be done to evaluate the specificity, cross-contamination, and feasibility of MODS in National TB Programs.


The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8×10−57), CCL4L1 (p = 3.9×10−21), IL18 (p = 6.8×10−13), LPA (p = 4.4×10−10), GGT1 (p = 1.5×10−7), SHBG (p = 3.1×10−7), CRP (p = 6.4×10−6) and IL1RN (p = 7.3×10−6) genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA), variation in gene copy number (CCL4L1) and altered transcription (GGT1). We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha  levels (p = 6.8×10−40), but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These include the presence of strong genetic effects in cis locations. The identification of protein quantitative trait loci (pQTLs) may be a powerful complementary method of improving our understanding of disease pathways.There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts –  cis effects including common variants in or near the IL6R, CCL4, IL18, LPA, GGT1, SHBG, CRP and IL1RN genes, all associated with blood levels of their respective protein products. Mechanisms implicated included altered transcription (GGT1) but also rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA) and variation in gene copy number (CCL4). Blood levels of many of these proteins are correlated with human diseases and the identification of “pQTLs” may in turn help our understanding of disease.One of the central dogmas of molecular genetics is that DNA is transcribed to RNA which is translated to protein and alterations to proteins can influence human diseases. Genome-wide association studies have recently revealed many new DNA variants that influence human diseases. To complement these efforts, several genome-wide studies have established that DNA variation influences mRNA expression levels. Loci influencing mRNA levels have been termed “eQTLs”. In this study we have performed the first genome-wide association study of the third piece in this jigsaw – the role of DNA variation in relation to protein levels, or “pQTLs”. We analysed 42 proteins measured in blood fractions from the InCHIANTI study. We identified eight  The identification of gene variants that alter the risk of common diseases has proven difficult. Recent genome-wide association studies of disease cases and controls have improved this situation but have shown that, with a few exceptions, most genetic effects on common disease are likely to be small cis effects), whilst others are found elsewhere in the genome (trans effects). The identification of these effects on gene expression may help understand disease aetiology. However, these data are limited by the fact that they assess gene expression, usually from a single cell type, rather than protein levels, which are likely to be more directly implicated in disease processes One successful complementary approach to studying gene-disease associations is to study associations between genetic variation and gene expression. Several genome-wide studies have shown that genetic variation influences gene expression in vivo studies of serum and plasma levels of proteins. There are likely to be many factors that influence serum and plasma protein levels, only one of which is genetic DNA variation leading to differences in mRNA transcription and subsequent mRNA translation to protein. Other mechanisms could include epigenetic factors, stochastic factors, environmental factors influencing regulation of expression, rates of secretion into the blood from the site of synthesis, proteolysis and clearance, and post-translational modifications such as glycosylation.There are no genome-wide analyses of the role of human genetic variation on large numbers of proteins. One way of testing this, and a way that could be relevant to the understanding of human diseases, is In this study we tested the hypothesis that common genetic variation influences protein levels in a human population. We used 1200 European individuals from the population based InCHIANTI study2>0.8.We used data from 496,032 single nucleotide polymorphisms (SNPs) from across the autosomal genome with minor allele frequencies >1% and which had passed stringent quality control checks see . These Scis effects and trans effects. Cis effects were defined as those in the gene(s) coding for the protein or within 300 kb either side of that gene. This was based on a recent study of HapMap variation in relation to gene expression that showed that most cis expression effects occur within this distance of genes We separated our results into 2 0.96 and 0.91 for IL6R and CRP respectively) and are associated with 0.69 (95%CIs:0.62–0.77), and 0.20 (95%CIs:0.12–0.29) per allele standard deviation differences in their respective protein levels. The SNP in the sex-hormone binding globulin (SHBG) gene, rs6761, was associated with SHBG protein levels with a per-allele effect size of 0.21 (95%CIs:0.13–0.30) standard deviations. This association appeared to be independent of a previously reported variant, rs1799941 2 = 0.1) and both remain associated with SHBG levels in the InCHIANTI study when correcting for the presence of the other (p = 0.008 for rs6761 correcting for rs1799941 and p = 0.003 for rs1799941 correcting for rs6761). We therefore genotyped these two variants in an additional 4590 individuals from the WATTs (n = 546) and the The Northern Finland 1966 Birth Cohort  studies. Details of replication studies are given in −12) and meta-analysis of all three studies provided very strong evidence of association (p = 1.8×10−16). Conditional analyses using all three studies showed that the association was driven by rs1799941 (p = 1.6×10−13 correcting for rs6761) rather than rs6761 (p = 0.38 correcting for rs1799941).For three of the eight genes showing cis effects, the associations have been reported in other studies, as part of candidate gene approaches. Variants in or close to the interleukin 6 receptor (IL6R) and C-reactive protein (CRP) genes, are closely correlated with those previously reported −6). A SNP in the parathyroid hormone (PTH) gene region was associated with PTH levels . Full details of the best cis association for each of the 42 protein levels measured are shown in Five of the cis findings have not been reported in other studies, although we recently reported those in the interleukin18 (IL18)−9) and permutation testing. Specifically, we identified a polymorphism (rs505922) close to the ABO blood group gene, that was very strongly associated with serum TNF-alpha levels (p = 6.76×10−40) (2 = 0.82) with the three alleles that determine the A, B and O alleles of ABO blood group. Separate genotyping of an additional SNP allowed us to accurately recode individuals with their ABO blood group based on a two SNP haplotype (rs8176746 and rs8176719)  = 2.4×106×10−40) . Using 18176719) . Individ8176719)  with tho8176719) . There w8176719)  and no aSix of the nine associations relate to proteins correlated with inflammatory or metabolic based disease processes so we further tested the robustness of the associations in InCHIANTI when correcting for a number of further covariates, including presence of cardio-vascular disease, diabetes, smoking status and use of steroid anti-inflammatory or lipid-lowering drugs. We also additionally corrected for total protein levels. All associations remained with very similar effect sizes .−5) at r2 = 0.71. This suggests that the GGT1 association we have seen with protein levels is due to altered transcript levels. There was no evidence that SNPs near the other genes were associated with altered transcript levels (p>0.001), although data were not available from rs1799941.We next assessed the likely mechanisms of the cis effects. Positions of SNP-protein-level associations relative to genes are shown in cis SNP, rs1143627, associated with transcript levels at LOD = 6.1. However, there was no association between this signal and serum protein levels, based on a SNP, rs10169916, in very strong linkage disequilibrium with rs1143627 .We next looked more extensively at the publicly available mRNA data cis effects and these cis effects are often the strongest in the genome. Further studies are needed to investigate the one trans finding we identified with TNF-alpha using one assay but not others. We did not find evidence for a fourth feature highlighted by genetic studies of gene expression: we did not find any “multi-trans” effects, where gene variants are associated with levels of multiple proteins.Our study shows that the human genetics of serum and plasma protein levels share several features of the genetics of gene expression levels cis effects that did not reach our cut off for significance. The need to correct p values for the number of tests performed meant that our study was not well powered to detect cis effects less than ∼0.22 or ∼0.18 standard deviations per allele for minor allele frequencies 0.1 and 0.5 respectively (based on p = 0.0005). Known variants that did not reach our criteria included those in the FGB (fibrinogen beta chain) and CCL2 genes, known to alter levels of their protein products, fibrinogenFGB gene p = 0.051 and rs1024611 in CCL2 p = 0.02). Additional trans effects may also exist but the need to correct for both the genome-wide number of SNPs and number of phenotypes meant that our study would not have detected effects less than ∼0.30 standard deviations per allele (based on p = 2.4×10−9). Given that our Bonferroni-based statistical cut-offs are likely to be conservative we also calculated false discovery rates −7, ∼3.×10−7 and ∼1.0×10−6 respectively.It is likely that there are other cis findings, the mechanism is known – differential cleavage of bound to unbound receptor caused by an amino acid changing SNP (nsSNP) results in different levels of soluble IL6 receptor cis findings the associations may relate to copy number variants (CNVs). There are reports that different sized LPA proteins, caused by different numbers of kringle repeats, are likely to result in altered secretion rates from the liver into the blood stream CCL4L1 gene, although further studies are needed to assess the extent of linkage disequilibrium between the LPA and CCL4L1-region variants we have found and CNVs in these genes. For the remaining cis effects, we have found little correlation between SNPs altering gene expression levels in lymphocytes and protein levels, with the exception of the GGT1 finding. This is perhaps not surprising given the numerous processes that could influence protein levels and is consistent with the observation in yeast experiments that there is considerable variation in the correlation between expression levels and protein abundanceSHBG, and R1270S in LPA are present in dbSNP, and neither of these are strongly (r2<0.5) correlated with the most associated SNPs in our study (both are present in HapMap).For one of the eight The mechanism of the association between ABO blood group and TNF-alpha levels is not known and further work is needed to identify the source of the discrepancy between the associations when different assays are used. The poor correlation between the two TNF-alpha measurements in the same study suggests the two assays are measuring different parts or fractions of the multi-meric TNF-alpha molecule, which can exist in transmembrane form, as a freely circulating protein, or as bound to soluble TNF receptors. Alternatively the association may be caused by cross-reactivity with ABO antigens. If shown in other studies to have a physiological effect the association of ABO blood group with TNF-alpha levels could help the understanding of the mechanisms behind the associations between blood group O and a reduced risk of thrombotic related diseasesAn important implication of our findings is that they may help dissect the causal direction of the associations between protein levels and correlated traits. Serum and plasma concentrations of many proteins change with disease status, ranging from metabolic and cardiovascular diseases to inflammatory and infectious states. Often it is not known whether altered levels of proteins are involved in disease aetiology or are simply a result of the disease processThe InCHIANTI study is a population based sample that includes 298 individuals of <65 age and 1155 individuals of age ≥65 years. The study design and protocol have been described in detail previously Venipuncture was performed in the morning after a 12-hour fast. Summary details of mean trait values and the numbers of individuals those measures were available in are given in www.HapMap.org). Experiments were performed as per the manufacturers instructions using 750 ng of genomic DNA extracted from whole blood. After processing chips were scanned on Illumina BeadStation scanners. All data were analyzed in BeadStudio , genotype calls were made using the standard cluster files provided by Illumina. Samples were initially assessed for genotype success rate (>98%) and concordance of reported and genotype gender. Nine samples were removed from further analysis due to gender mismatch. Eighty seven samples failed the cut off genotype success rate of 98%; forty eight of these samples were re-purified and successfully genotyped, thus in total 48 samples were removed from further analysis. Manual checking of genotype clusters was performed for all SNPs listed in Genome-wide genotyping was performed using the Illumina Infinium HumanHap550 genotyping chip (ver1 and ver3 chips were used). This product assays >555,000 unique SNPs derived primarily from stages I and II of the International Haplotype Map Project (http://pngu.mgh.harvard.edu/purcell/plink/index.shtml) LD-based SNP pruning function (using parameters –indep-pairwise 200 10 0.1)) that included InCHIANTI and HapMap CEU, JPT+CHB and YRI samples (http://www.HapMap.org). Only SNPs with a MAF >10% in HapMap were used in the analysis. This revealed that all individuals were of European ancestry  were also excluded from the analyses. We calculated how well SNPs passing the QC criteria covered common variation in the genome by identifying all European HapMap proxies at r2 ≥0.8 for minor allele frequencies (MAF) of 5% and 1%, and then comparing this number to the HapMap count of all autosomal SNPs ≥ the MAF.We only used SNPs that were called in >98% of samples and had minor allele frequencies in our sample of >1%. SNPs deviating appreciably from the expected population distribution (Hardy Weinberg Equilibrium p<1×10A SNP (rs1799941) previously reported to be associated with SHBG levels was not present on the Illumina chip or in HapMap. We therefore genotyped this separately using Taqman probes (Applied Biosystems).Many of the proteins were not normally distributed and so we performed appropriate statistical transformations. Where a simple log transformation was not appropriate, we used the STATA version 9 “ladder” command, which searches a subset of the ladder of powers to attempt to detect a simple transformation. Where such simple transformations were not appropriate (i.e. where the distributions were heavily skewed) we considered the STATA “lnskew0” command which performs a log transformation after adding a constant, thus creating a zero-skewness logged variable. Where this transformation was still not appropriate we considered the STATA “bcskew0” command which performs a box-cox power transformation to approximate normality. For proteins identified as significant using these transformations we further tested the robustness of the results by performing a probit transformation: we ranked all individuals for each trait and assigned Z scores corresponding to percentiles in a normal distribution.For eight proteins there were a small percentage of individuals who had levels below the assay detection limits. In each case there were less than 13 (1%) individuals with levels below detectable limits, except for Macrophage inflammatory protein beta, for which there were 77 individuals below the detectable limits. The values for these individuals for these traits were coded as zero. For a ninth protein, TNF-alpha, there were seven individuals who had levels above the assay detection limits and the values for these individuals were coded at the maximum detectable value 39.4 pgml-1. Non-parametric analyses using quantile regression in Stata v9.0, for MIP-beta and TNF-alpha showed that the highly significant associations observed with these two markers were not affected by the inclusion of individuals with levels out of the assay range.For six proteins  there were >8% of individuals that had levels below the detectable limits. For these we dichotomized traits at the median, or if there was more then 50% below detectable limits, at this point. There was no transformation which made LipoproteinA normally distributed but 14 mg/dl is used as a standard clinical cut off point for high levels and so was used to dichotomise the variable.For each autosomal SNP for each of the 36 proteins with levels as quantitative traits, we performed linear regression using PLINK software with age and sex as covariates. This means we tested just one genetic model, an additive model with one degree of freedom. This model tests if the trait alters by equal amounts with each additional allele across the three genotypes. For the six markers dichotomized into high and low values we also performed a single per allele test across genotypes using PLINK (Cochran-Armitage 1df test for trend).http://pngu.mgh.harvard.edu/purcell/plink/index.shtml but briefly each permutation randomly swaps phenotype values between individuals to provide a new dataset sampled under the null hypothesis, but which preserves any correlation between genotypes. The program then compares each observed test statistic against the maximum of all permuted statistics  for each single replicate. For the trans effect we performed 10,000 permutations across the entire genome and for the cis effects we performed 100,000 permutations across the region (“region-wide”) containing the gene and 300 kb each side. This approach meant that permutation tests were not corrected for relatedness but given the relatively small inflation factors for each trait and the fact that the largest permutation p value in cis) and rs1880887 with alkaline phosphatase levels (trans) but these associations did not remain after either permutation (p>0.05) or non-parametric tests (p>0.05 after multiplication by number of SNPs).To assess empirical significance of SNPs reaching significance after Bonferroni correction, we used the maxT function in PLINK. Full details are available at To assess false discovery rates we calculated the equivalent q statistic as implemented in the “Qvalue” software The 3 major ABO blood groups are determined by SNPs in the ABO geneSummary details of replication studies are given in http://www.ebi.ac.uk/swissprot/): SHBG - PO4278, TNFa - PO1375, IL-6sR - P08887, MIPb - P13236, IL18 - Q14116, LPA - P08519, GGT1 - P19440, CRP - P02741, IL1RA - P18510. Accession numbers for genes are taken from Ensembl (http://www.ensembl.org/index.html): ABO - ENSG00000175164, IL6R - ENSG00000160712, CCL4L2 - ENSG00000129277, IL18 - ENSG00000150782, LPA - ENSG00000198670, GGT1 - ENSG00000100031, SHBG - ENSG00000129214, CRP - ENSG00000132693, IL1RN - ENSG00000136689.Accession numbers for proteins are taken from Swissprot  which is the difference between the first and third quartile values (Q3–Q1). The upper value is the largest data value that is less than or equal to the third quartile plus 1.5 X IQR and the lower adjacent value is the smallest data value that is greater than or equal to the first quartile minus 1.5 X IQR. Values exceeding the upper and lower adjacent values are called outside values and are displayed as markers. a) Cis effects b) Trans effect.Plots represent box-plots except for (0.04 MB DOC)Click here for additional data file.Figure S2A) Map of ABO gene from UCSC genome browser, May04, showing positions of the Illumina panel genotyped SNPs (rs8176746 and rs505922) and the functional O blood group polymorphism (rs8176719). b) Linkage disequilibrium (r2) between the 3 SNPs after the O deletion was typed separately in the InCHIANTI samples. c) The four haplotypes formed by the three SNPs shows how rs505922 splits the A blood group allele haplotype. d) Haplotypes formed by rs8176746 and rs8176719  and how they define ABO phenotype.(0.13 MB DOC)Click here for additional data file.Figure S3Comparison of TNFA results in InCHIANTI. A) Correlations between transformed TNFA levels (log transformation) measured using an ELISA method  and a LINCOplex method, (Luminex (HADK2-61K-B). B) Correlations between each of the two transformed TNFA measures and three other key proteins, IL6 levels, high sensitivity C reactive protein levels and albumin levels. C) i)–ii)Histograms of raw TNFA measures, iii)–iv)associations with ABO blood group shown as box plots; and v) associations of R&D systems method with ABO blood group showing association is strongest in the one third of individuals with highest TNFA levels.(0.09 MB DOC)Click here for additional data file.Figure S4InCHIANTI and HapMap samples plotted for the first two principal components obtained from multidimensional scaling of a matrix of "identity by state" genotypes. All InCHIANTI samples cluster tightly around the European HapMap samples. INCH = InCHIANTI samples, CEU, JPT+CHB and YRI = European, combined Japanese and Han Chinese and Yoruban samples from HapMap, respectively.(0.04 MB DOC)Click here for additional data file.Table S1Summary details of participants and mean traits. Abbreviations for proteins are included if they are used elsewhere.(0.10 MB DOC)Click here for additional data file.Table S2Details of SHBG and TNF-alpha replication studies.(0.03 MB DOC)Click here for additional data file.Table S3Cis results for 42 proteins. Details of only the most strongly associated cis SNP for each gene are given, except for the eight reaching significance in which case details of all SNPs in the cis region <0.001 are also given. 3b Trans results. Details of the most strongly associated SNP in the genome wide scan, excluding the gene coding for the protein, plus 600kb of flanking sequence. For TNF-alpha details of all SNPs in the ABO region <0.001 are also given.Supplementary Table 3a and 3b Full details of trans and cis effects for 42 proteins. For the nine regions reaching overall significance we include all SNPs in that region that cross the significance threshold. IL = interleukin. 3a (0.42 MB DOC)Click here for additional data file.Table S4Associations of the eight cis and one trans finding in InCHIANTI using different covariates and exclusion criteria. MAF = Minor allele frequency. GC P = p values from (0.04 MB DOC)Click here for additional data file.Table S5Positions of the eight genes with significant cis effects based on Jan 07, NCBI 35, dbSNP125, HapMap phase II data release 21a, in relation to the region covered by all HapMap SNPs tagged at r2>0.2 by the most significant cis effect SNP.(0.04 MB DOC)Click here for additional data file.


An early step in the carcinogenesis of hereditary non-polyposis colorectal cancer (HNPCC) and some sporadic colorectal cancers (CRCs) is the acquisition of a ‘mutator phenotype’ resulting from defects in DNA mismatch repair (MMR) genes, which normally maintain genomic stability. This mutator phenotype causes an approximately 100–1000-fold increase in base substitutions and small insertion/deletion mutations thereby driving carcinogenesis. It also causes genome-wide microsatellite instability (MSI) due to the inability to repair mutations within these small, hard to replicate, repetitive DNA elements. In contrast, less is known about the role of mutator phenotypes in microsatellite stable (MSS) CRC. In this report, we have measured the mutation rates in 11 MSS CRC cell lines to obtain an estimate of the prevalence of mutator phenotypes in MSS carcinogenesis. Of the 11 cell lines, three of them (27%) possess spontaneous hypoxanthine phosphoribosyltransferase mutation rates approximately 10–100-fold above background. When challenged with alkylating and oxidising agents, the degree of survival and apoptotic responses are different, indicating that these cell lines may represent more than one mutator phenotype. These data demonstrate that a significant portion of MSS CRC cell lines has increased mutation rates and that this may play a role in MSS CRC carcinogenesis. Since the low spontaneous mutation rate in normal cells is difficult to reconcile with the relatively large number of mutations observed in tumours , Loeb an4 patients possess inherited defects in nucleotide excision repair (NER) and a predilection to skin cancer, and the mutation rate is significantly elevated in their cells when exposed to UV light UV light  and provN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2). They are at least in part independent of the recently reported MutY mutations (We hypothesised that mutator phenotypes might occur commonly in microsatellite stable (MSS) CRC carcinogenesis and to test this, we screened a panel of MSS CRC cell lines. To test this hypothesis, we employed the classic selectable mutation marker for human studies, hypoxanthine phosphoribosyltransferase (HPRT). We report here that three out of 11  MSS CRC cell lines tested possess elevated HPRT mutation rates. The MSS CRC cell lines with elevated mutation rates demonstrate unique patterns of sensitivity to the cytotoxic chemicals The MSS CRC cell lines were either isolated and generously provided by Dr James KV Willson  or purchased from the ATCC. Other than there being microsatellite stable, there were no other criteria for selecting these cell lines. One cell line was excluded because it had a low cloning efficiency.μg ml−1 6TG to test for mutations. Plates were fed every 2 weeks and wells scored positive for growth by phase microscopy after 6 weeks. This was repeated until approximately 30 million clonogenic units had been analysed  as published previously  for CRC (μl buffered solution with 20 U of RNasin (Promega), 0.5 μg of Oligo (dT)12–18 primer , and 20 mM of dNTPs. After the incubation, 5 μM of M13 tailed forward sense primer S-27 (−27 to −12 relative to the AUG) 5′-GTAAAACGACGGCCAG-TCAGCCCGCGCGCCGGC-3′, 5 μM of M13 tailed reverse primer AS661 (661-557) 5′-CAGGAAACAGCTATGAC-TCAACTTGAACTCTC-3′ and 1 μl of template DNA were added to the Taq PCR Master Mix  and incubated for 10 min at 94°C. The cDNA was amplified for a total of 30 cycles with each cycle consisting of 1 min at 94°C, 1 min at 60°C and 3 min at 72°C. Final extension of the PCR amplified product was for 10 min at 72°C. PCR amplified samples were sequenced using M13 primers and Big Dye cycle sequencing on an ABI3700 sequencer .To confirm the presence of mutations, RNA from 6TG resistant colonies was isolated using the RNAgents Total RNA Isolation System . The isolated RNA (approximately 500 ng) was reverse-transcribed using 5 U AMV reverse transcriptase  in a 20 μM MNNG (Sigma) for 45 min at 37°C or with 0–300 μM H2O2 (Sigma) for 60 min at 37°C. After treatment, the cells were washed once with MEM2+media (−1 (200 000 for Vaco8) in six-well plates. The cells were grown for 5 days (for H2O2) or 6 days (for MNNG), subcultured and counted using trypan blue exclusion with a haemocytometer. Experiments were performed three times, each in triplicate.For the cell viability assay, exponentially growing cells were trypsinised and washed twice in serum-free minimal essential medium (SF-MEM). Washed cells were suspended in SF-MEM and treated with 0–5 μM MNNG or 300 μM H2O2 as described above, and monitoring the cells at various time points after treatment. Apoptosis was determined as described (μg ml−1 DAPI (Sigma) for 10 min, followed by 60 μg ml−1 propidium iodide (Sigma) and fluorescence microscopy. In all, 200 cells were counted and the percent of cells that demonstrated apoptotic bodies calculated.Apoptosis was assessed after treating cells with either_5 −8 mutations per locus per cell division . Furthermore, the mutation frequency of one of them (Vaco411) appears to be higher than the other two. Limited sequencing of the hprt cDNA (hprt were indeed present (manuscript in preparation). The ages of the patients when the tumours were diagnosed were 32, 56 and 81 years old for Vaco411, Vaco8 and SW948, respectively.We measured the spontaneous HPRT mutation rate in 11 MSS CRC cell lines , while SW948 and Vaco8 were both intermediate, demonstrating more resistance than both SW480 and Vaco411 but less than HCT116 .A consistent feature of cells with elevated mutation rates due to defective MMR has been the tolerance to alkylating agents such as MNNG (2O2). 2O2 treatment (41 and 42% of cells remaining respectively after 300 μM H2O2) than the other three cell lines.We have previously demonstrated that two of the three mutators have elevated levels of the mutagenic base 8-oxoG . The majority  however, showed normal baseline rates.In MMR-deficient CRC, the spontaneous mutation rates are generally elevated at least 100–1000-fold above the baseline normal rate. In contrast, the increased HPRT mutation rates we report in this current study in the MSS CRC cell lines are approximately 10–100-fold increased relative to control levels in nonmutator MSS CRC cells and accordingly are designated as ‘intermediate’ elevated levels. It is noteworthy that intermediate mutation rates are present in other diseases such as Bloom's syndrome   defective in mutY possess elevated mutation rates approximately 30-fold above the background rate  mouse cells contain increased levels of oxidative DNA damage  There is a mutator phenotype but it was not detected. This seems unlikely since the HPRT assay detects such a wide range of mutations  did not possess elevated utations  (though utations . (3) Finet al, 1997; in situ hybridisation, it reports for changes in chromosome number whereas the HPRT assay is not expected to detect such changes. However, because CIN (production of aneuploidy) occurs at substantial rates, it can easily produce the second hit in tumour suppressor genes through loss of heterozygosity, and is therefore probably appropriate to consider CIN a mutator phenotype  feature of malignancy, because it explains how malignant cells acquire many of the critical features that distinguish them from their normal counterparts (e.g. ability to metastasise and acquire resistance to chemotherapeutics). One possible explanation is that most of the cell lines without a functional HPRT mutator phenotype manifest the chromosome instability (CIN) phenotype  of MSS CRCs exist with elevated mutation rates that are likely independent of one another. This suggests that mutator phenotypes may also play a role in MSS CRC carcinogenesis.


A defect in hypothalamic-pituitary-adrenal (HPA) axis function has been suggested to contribute to susceptibility to rheumatoid arthritis (RA).To investigate polymorphisms of the glucocorticoid receptor (GR) gene and determine any associations with RA.Three GR polymorphisms that tag 95% of all haplotypes across the GR gene were genotyped. These are an intron B Bcl1 polymorphism, a ttg insertion/deletion within intron F (rs2307674) and the single nucleotide polymorphism (SNP) lying in the 3′ untranslated region of exon 9b (rs6198). The dye terminator-based SNaPshot method or size resolution by capillary electrophoresis was performed. The study population comprised 198 UK Caucasian RA cases and 393 ethnically matched controls.No significant single point or haplotypic associations were found for GR polymorphisms with RA susceptibility. Furthermore, no evidence for GR polymorphisms with aspects of RA severity was seen.In this study of the most comprehensive coverage of GR polymorphisms with RA, no significant contributing role for GR polymorphisms with RA was found. Studies from both animals and humans suggest that a defect in the neuroendocrine system may be important in the disease rheumatoid arthritis (RA). Inappropriately normal plasma cortisol and a blunted response of plasma cortisol to surgical stress have been shown for RA patients.Gcs exert their effect by binding to the intracellular receptor, the glucocorticoid receptor alpha (GRα). The human GR gene  locus 5q31) is 10 exons in length and exons 1-9α are transcribed into GRα mRNA, which is translated into a functional receptor (GRα). Alternative splicing of the primary transcript results in an mRNA containing exons 1-9β, which gives rise to the GRβ. Unlike GRα, GRβ does not bind hormone and is transcriptionally inactive.q31 is 107et al. described an A to G polymorphism at position 3736 in exon 9b (now rs6198).9Derijk now rs619.8 This pet al. originally detailed the genotype and allele frequencies of the rs6198 polymorphism in a very small sample size of 30 RA cases compared with 24 controls and found carriage of the mutant allele to be associated with significant increased risk of RA.Derijk Blood samples were obtained with informed written consent. Ethical approval was obtained from Multicentre Research Ethics Committee (MREC) (99/8/84) and the University of Manchester committee on the ethics of research on human beings (8/92/(i)).All patients and controls were of UK Caucasoid origin.One hundred and ninety-eight RA cases from the Arthritis Research Campaign National Repository for families with RA were studied. Only one affected case per family was selected, at random, for investigation. All RA cases had disease that met the American College of Rheumatology 1987 criteria, as modified for genetic studies.DNA was available for 393 control subjects. These were individuals recruited from blood donors and from the records of general practitioners.4 buffer (Bioline), 1 µl of dNTPs , 0·3 µl MgCl2  and 0·2 µl of Taq polymerase . The primers and probes used were (5′ to 3′): for the intron B Bcl1 SNP: forward primer CTA TTC TTC AAA CTG AAT CTT CTG, reverse primer CAA CAC GTA TAT CTA CAT TTA GAA C, and probe GAC ACC AAT TCC TCT CTT AAA GAG ATT; for the rs6198 SNP: forward primer AGT GTC TTT TTA CCT ACG C, reverse primer ATG TTT CTC CAT ATT TGG C, and probe TGT GGT TTG GTA ATA CCA GAA CAG CAA ATT TAA A.SNP genotyping was by the dye terminator-based SNaPshot method . In brief, polymerase chain reactions  contained 10 ng of genomic DNA, 0·4 µl of each primer (25 pmol/µl), 1 µl of 10 × NHThe presence or absence of a ttg insert (rs2307674) was determined using capillary electrophoresis for size resolution, relative to a ROX 450 internal size standard, on an ABI Prism 310 DNA Genetic Analyser (Applied Biosystems), using a fluorescent dye (HEX)-labelled forward primer. The results were analysed using Genescan analysis and Genotyper 3·6 software (Applied Biosystems). The rs2307674 forward primer was ACC TCA AGT GAT CCA CCC, and the reverse primer GTA CAT GGT TAT ACT CAT ATA TAA C.The RA cases and a proportion of the controls had been previously genotyped for HLA-DRB1 alleles and shared epitope (SE) status assigned.2-test  and correction for multiple testing was performed by Bonferroni's method. Haplotype analysis was carried out using the expectation-maximization algorithm implemented in HelixTree . Stratification analysis was performed to investigate the effects of gender, disease severity, as assessed by the presence of erosive disease, age at disease onset , and carriage of SE alleles .Associations between the GR polymorphisms and RA were analysed using the χP = 0·05).The study had > 85% power to detect an odds ratio (OR) of 1·8 or greater with RA susceptibility (r2 = 1) was seen for the polymorphisms studied in both the case and control groups.No deviation from Hardy-Weinberg equilibrium was seen for any polymorphism in the cases or the controls. Strong linkage disequilibrium (LD) (No association with any GR polymorphism was found with the RA cases when considered as a whole group . Similarn = 3) rendered this observation nonsignificant (Pcorr = 0·15).On stratified analysis, a weak positive association with an excess of rs6198 AA (wild-type) homozygotes was seen when the SE negative cases were compared with SE negative controls . FurtherNo other single or haplotypic association of GR polymorphisms and no other parameters of RA severity were found.et al.Inappropriately low endogenous cortisol production may be a contributing factor to the onset and progression of RA. How this arises has not been ascertained.No evidence for genetic association with GR polymorphisms and RA susceptibility was observed. This is in agreement with the findings of a Korean study of RA, in which the 149 RA cases and controls were compared and no association with the intron B Bcl1 polymorphism was found with the RA group as a whole, or on stratification for disease severity.In summary, we have looked at three changes across the GR gene that capture 95% of all commonly occurring haplotypes, but find no evidence for GR polymorphisms contributing a substantial genetic effect, either as single point or as haplotypic associations, to risk of RA. We conclude that GR polymorphisms do not confer significant risk of RA.


However, the biochemical stability of the DGC and the functional performance of −/−adbn muscle have not been characterized. Here we show that the biochemical association between dystrophin and β-dystroglycan is compromised in −/−adbn skeletal muscle, suggesting that α-dystrobrevin plays a structural role in stabilizing the DGC. However, despite muscle cell death and DGC destabilization, costamere organization and physiological performance is normal in −/−adbn skeletal muscle. Our results demonstrate that myofiber degeneration alone does not cause functional deficits and suggests that more complex pathological factors contribute to the development of muscle weakness in muscular dystrophy.α-Dystrobrevin is a component of the dystrophin-glycoprotein complex (DGC) and is thought to have both structural and signaling roles in skeletal muscle. Mice deficient for α-dystrobrevin ( In particular, whether the absence of α-dystrobrevin leads to DGC instability or causes deficits in muscle function remain to be assessed.α-Dystrobrevin is a dystrophin-related protein that binds directly to dystrophin, syntrophin, and the sarcoglycan complex in vivo and ex vivo physiological performance of −/−adbn muscle. Our results suggest that the interaction between dystrophin and β-dystroglycan is compromised by the loss of α-dystrobrevin, implicating a role for α-dystrobrevin in stabilizing the DGC. However, muscle function was not compromised in −/−adbn mice, which demonstrates that substantial muscle cell necrosis can occur without adverse effect on physiological performance.To further investigate the consequences of α-dystrobrevin deficiency, we examined the biochemical stability of the DGC and also measured the +/−adbn and −/−adbn mice  muscles from adbn+/− and adbn−/− mice were determined at 2 and 7 months of age (−/−adbn muscle did not show an increased force drop after four eccentric contractions (ECCs). Since work done to stretch the muscle is the best predictor of the magnitude of contraction-induced injury adbn+/− and adbn−/− muscle. In addition, we did not see an increase in Procion orange infiltration of −/−adbn myofibers compared to control after eccentric contractions (data not shown), indicating that sarcolemmal stability is not affected in −/−adbn muscle.To investigate the functional consequences of DGC instability  and muscs of age . At both1) and the average of the top five responses (WBT1–5) did not differ between adbn+/− and adbn−/− animals at either 2 or 7 months of age , defined as the forward pulling tension exerted by an individual mouse in response to a tail pinch stimulus s of age . To eval+/− mice . Togetheadbn−/− mice exhibit skeletal and cardiac muscle cell necrosis adbn−/− mouse has been considered an animal model for muscular dystrophy. However, as muscular dystrophy is characterized by both muscle cell death and muscle weakness, we examined the functional performance of −/−adbn muscle. Surprisingly, adbn−/− muscle was indistinguishable from control muscle using both in vivo and ex vivo analyses. These results were unanticipated, as −/−adbn mice exhibit an elevated presence of centrally-nucleated fibers similar to other mouse models of muscular dystrophy Since the initial observation that adbn−/− mice and other DGC-associated muscular dystrophy mouse models may help to explain the lack of a functional deficit in adbn−/− mice. Unlike muscle from dystrophin-, sarcoglycan- and dystroglycan-deficient animals −/−adbn muscle does not exhibit significant fibrosis, mononuclear cell infiltration or increased membrane damage. Therefore, fibrosis, inflammation and membrane fragility may be factors contributing to the force deficits observed in dystrophic animal models. In fact, replacement of muscle fibers with less elastic fibrotic connective tissue has previously been suggested to contribute to decreased force production Some fundamental differences in the histological observations between −/−adbn mice, Warner et al. mdx mice exhibit muscle cell necrosis in the absence of sarcolemmal damage. These results indicate that sarcolemmal instability is not the sole cause of cell death. In the absence of sarcolemmal fragility, it has been hypothesized that cell death in adbn−/− mice results from aberrant signaling. Grady et al. adbn−/− mice adbn−/− mice, which is similar to the histology of δ-sarcoglycan null mice in which vascular function is perturbed adbn−/− mice since nNOS-deficient mice are not detectably dystrophic adbn−/− mice may provide insight into the molecular and cellular requirements for muscle cell death in other animal models of muscular dystrophy.While the cause of muscle cell death in DGC-related muscular dystrophies is unclear, a leading hypothesis is that damage to the sarcolemma during muscle contraction causes a loss of calcium homeostasis, leading to activation of proteases and eventual cell death −/−adbn mice have suggested that all other DGC components are expressed at normal levels and localize properly to the sarcolemma −/−adbn skeletal muscle extracts, but reduced by 70% in DGC-enriched fractions from −/−adbn muscle. This result suggests that the association between dystrophin and the glycoprotein complex is compromised in the absence of α-dystrobrevin. Although the destabilization of the DGC may contribute to the muscle cell necrosis in −/−adbn mice, it appears that the degree of dystrophin destabilization is insufficient to render the membrane susceptible to damage, cause costamere disorganization or alter the functional performance of the skeletal muscle. Furthermore, the upregulation of the cytoskeletal protein syncoilin While it has previously been proposed that α-dystrobrevin plays a structural role in the DGC adbn−/− mice exhibit defects in the maturation and maintenance of the postsynaptic membrane at the NMJ as the result of increased mobility and turnover of acetylcholine receptors (AChRs) adbn−/− mice provides a possible explanation for the increased AChR mobility. While acetylcholinesterase (AChE) mobility is also increased in adbn−/− mice adbn−/− mice. In support of this idea, mutations in the Caenorhabditis elegans dystrobrevin-like gene cause hypercontractility, which leads to muscle degeneration when on a sensitized background In addition to the sarcolemma, α-dystrobrevin is also enriched at the neuromuscular junction (NMJ). Previous work has shown that adbn−/− mouse has provided insight into the pathological factors that contribute to the development of muscular dystrophy. The observation that muscle cell necrosis and functional deficits are separable phenotypes forces us to reconsider the traditional view that muscle cell death is a causative factor for muscle weakness in muscle diseases. Furthermore, the adbn−/− mouse suggests that membrane fragility is not the sole contributing factor for muscle cell death in DGC-related muscular dystrophies. Further studies are needed to compare the cellular and molecular differences between the adbn−/− mouse and other DGC-related mouse models in order to help dissect the mechanisms underlying the development of muscular dystrophies.Study of the −/−adbn mice was generously provided by Dr. R. Mark Grady . To generate +/−adbn mice for controls, we outcrossed an −/−adbn male (SV129J/ C57Bl/6J mixed background) to an SV129J wild-type female to obtain +/−adbn mice. Subsequent mating between +/−adbn and −/−adbn mice provided −/−adbn and littermate control +/−adbn animals. Genotypic analyses were performed by standard PCR methods.A breeding pair of −/−adbn and +/−adbn 7 month-old mice, quickly frozen in liquid nitrogen-cooled isopentane and mounted in O.C.T medium . 10 µm cryosections were cut on a Leica CM3050 cryostat, air-dried and stained with hematoxylin and eosin-phloxine. Images were obtained on a Zeiss Axiovert 25 microscope fitted with a Leica DFC300 FX camera using Image Pro Plus 5.1 software.For light microscopy, muscles were dissected from 3) and incubated with a 1∶100 dilution of Rabbit 2 polyclonal antibody to dystrophin Costamere images were obtained according to previously described methods DC Protein Assay Kit II . To enrich for the DGC, pulverized muscle was solubilized in 10 volumes/g of 50 mM Tris-HCl, 0.5 M NaCl, 1% digitonin and a cocktail of protease inhibitors for 1 hour at 4°C with gentle mixing. After high-speed centrifugation, the supernatant was applied to a WGA-Sepharose column  pre-equilibrated with wash buffer  for 2 hours at 4°C to enrich for dystroglycan and associated proteins. The column was washed and bound proteins eluted with Laemmli sample buffer (3% SDS and 1% β-mercaptoethanol) at 100°C. Dystrophin and β-dystroglycan immunoreactivity in SDS muscle solubilates  and WGA elutes were measured by quantitative western blot analysis using the monoclonal antibodies NCL-DYS1 to dystrophin and NCL-b-DG to β-dystroglycan . Secondary antibodies were IRDye 800- or IRDye680-conjugated goat anti-mouse IgG and the fluorescent signals were detected and quantified using the Odyssey Infrared Imaging System . Relative levels of dystrophin from WGA elutes were determined by normalizing dystrophin signals to the corresponding β-dystroglycan signal. Statistical significance at p<0.05 was determined using a one-sample t-test.Skeletal muscle was dissected, snap-frozen in liquid nitrogen, and pulverized in a liquid nitrogen-cooled mortar and pestle. Total muscle homogenates were prepared by solubilizing pulverized muscle in 1% SDS, 5 mM EGTA, and protease inhibitors for 2 minutes at 100°C. The supernatant was collected after brief centrifugation and its protein concentration determined using the Bio-Rad 2+-Ringer's solution continuously gassed with 95% O2/5% CO2 to maintain a pH of 7.6 at 30°C. Platinum plate electrodes were positioned on either side of the muscle and stimulation induced by a single pulse lasting 200 microseconds. The muscle was adjusted to the optimal length (0L) at which maximal twitch force was achieved. Muscle was then subjected to an ECC regimen which consisted of five maximal tetanic stimulations at a frequency of 150 Hertz. Each stimulation was carried out over 700 milliseconds; over the final 200 milliseconds the muscle was lengthened at a velocity of 0.5 0L/second resulting in a total stretch of 10% 0L. A five-minute recovery time was allowed between measurements. The average force produced during a stretch was calculated by integrating the force-time curve and dividing this value by the duration of the stretch. Work was calculated by multiplying the average force produced during a stretch by the length of the displacement and normalized to muscle mass. Upon completion of the ECC protocol, muscle was weighed and subsequently bathed in 0.1% Procion orange, washed, and frozen for cryosectioning. Cross sectional area of each muscle was calculated according to Brooks and Faulkner 0L, the EDL fiber-to-muscle length constant 0.44, and 1.06 grams/centimeter3, the density of mammalian skeletal muscle All force measurements were obtained as previously described The WBT protocol was carried out as previously described +/−adbn and −/−adbn mice at each time point were assessed by using a Student's two-tailed t-test for independent samples to determine significance at p<0.05.Maximal exercise performance was tested on a Columbus Instruments treadmill with an uphill grade of 15°. Mice were acclimated to the treadmill by running at a speed of 10 meters per minute for 5 minutes, three times a week, for two weeks. To determine maximal exercise performance mice were run on the treadmill for 5 minutes, at a speed of 10 meters per minute, followed by a 1 meter per minute increase in speed every minute until exhaustion. Mice were considered exhausted when they refused to stay off a shock bar for at least 5 seconds. Maximal exercise capacity was determined as the average duration of two trials separated by two days. Differences between