Anti-idiotypic antibody specific to GAD65 autoantibody prevents type 1 diabetes in the NOD mouse

Overt autoantibodies to the smaller isoform of glutamate decarboxylase (GAD65Ab) are a characteristic in patients with Type 1 diabetes (T1D). Anti-idiotypic antibodies (anti-Id) directed to GAD65Ab effectively prevent the binding of GAD65 to GAD65Ab in healthy individuals. Levels of GAD65Ab-specific anti-Id are significantly lower in patients with T1D, leading to overt GAD65Ab in these patients. To determine the possible protective role of GAD65Ab-specific anti-Id in T1D pathogenesis, we developed the monoclonal anti-Id MAb 8E6G4 specifically targeting human monoclonal GAD65Ab b96.11. MAb 8E6G4 was demonstrated as a specific anti-Id directed to the antigen binding site of b96.11. MAb 8E6G4 recognized human antibodies in sera from healthy individuals, T2D patients, and T1D patients as established by ELISA. We confirmed these MAb 8E6G4-bound human antibodies to contain GAD65Ab by testing the eluted antibodies for binding to GAD65 in radioligand binding assays. These findings confirm that GAD65Ab are present in sera of individuals, who test GAD65Ab-negative in conventional detection assays. To test our hypothesis that GAD65Ab-specific anti-Id have an immune modulatory role in T1D, we injected young Non Obese Diabetic (NOD) mice with MAb 8E6G4. The animals were carefully monitored for development of T1D for 40 weeks. Infiltration of pancreatic islets by mononuclear cells (insulitis) was determined to establish the extent of an autoimmune attack on the pancreatic islets. Administration of MAb 8E6G4 significantly reduced the cumulative incidence rate of T1D and delayed the time of onset. Insulitis was significantly less severe in animals that received MAb 8E6G4 as compared to control animals. These results support our hypothesis that anti-Id specific to GAD65Ab have a protective role in T1D.     

Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein

We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.     

Age-Related Changes in the Global DNA Methylation Profile of Leukocytes Are Linked to Nutrition but Are Not Associated with the MTHFR C677T Genotype or to Functional Capacities

Global DNA methylation of peripheral blood leukocytes has been recently proposed as a potential biomarker for disease risk. However, the amplitude of the changes in DNA methylation associated with normal aging and the impacts of environmental changes on this variation are still unclear. In this context, we evaluated the association of global DNA methylation with nutritional habits, tobacco smoking, body mass index (BMI), clinical laboratory parameters, polymorphism C677T MTHFR, functional cognition and the daily practice of physical activity in a cancer-free older population. Leukocyte global DNA methylation from 126 older individuals was quantified using a high-throughput ELISA-based method. Global DNA hypomethylation was observed in older individuals when compared to a younger population (p = 0.0469), confirming changes in DNA methylation in the aging process. Furthermore, the methylation profile of elders was correlated with the daily ingestion of carbohydrates (p = 0.0494), lipids (p = 0.0494), vitamin B6 (p = 0.0421), magnesium (p = 0.0302), and also to the serum levels of total protein (p = 0.0004), alpha 2 globulin (p = 0.0013) and albumin (p = 0.0015). No statistically significant difference was observed when global DNA methylation were stratified according to C677T MTHFR genotypes (p = 0.7200), BMI (p = 0.1170), smoking habit (p = 0.4382), physical activity in daily life (p = 0.8492), scored cognitive function (p = 0.7229) or depression state (p = 0.8301). Our data indicate that age-related variations in the global DNA methylation profile of leukocytes might be modulated by the daily intake of carbohydrates, lipids, vitamin B6, and magnesium and be associated with serum protein levels, however it is independent of C677T MTHFR genotype and not correlated with BMI, smoking habit, cognitive function or the routine physical activities.     

Newly diagnosed latent autoimmune diabetes in adults (LADA) is associated with low level glutamate decarboxylase (GAD65) and IA-2 autoantibodies. Diabetes Incidence Study in Sweden (DISS).

A quantitative assay with microSepharose was used to determine GAD65Ab and IA-2Ab levels in 771 population-based patients diagnosed with diabetes mellitus at 15 to 34 years of age, and in 828 matched controls. Among the patients, 587 (76%) were classified with type I, 108 (14%) with type II, and 76 (10%) with unclassifiable diabetes. The levels above normal demonstrated a prevalence of GAD65Ab in 66% of type I diabetes, 50% of type II diabetes and 54% of unclassifiable patients and for IA-2Ab in 40%, 17% and 21%, respectively. Among the autoantibody-positive sera, the LADA patients had a lower GAD65Ab index (median 0.19, p < 0.0001) and IA-2Ab index (median 0.28, p < 0.0001) than the type I patients (median 0.37 and 0.66). Patients with unclassifiable diabetes had a GAD65Ab (median 0.43) or IA-2Ab (median 0.63) index which was not different from the type I diabetes patients. Our data demonstrate that young adult new-onset LADA patients have low level GAD65Ab and IA-2Ab. The low-level autoantibodies may signify a less aggressive beta-cell autoimmunity, which may explain why these patients are often classified with type II or non-insulin-dependent diabetes.     

Circulating Protein Fragments of Cartilage and Connective Tissue Degradation Are Diagnostic and Prognostic Markers of Rheumatoid Arthritis and Ankylosing Spondylitis

Inflammation driven connective tissue turnover is key in rheumatic diseases, such as ankylosing spondylitis (AS). Few biomarkers are available for measuring disease prognosis or the efficacy of interventions applied in these tissue-related conditions. Type II collagen is the primary structural protein of cartilage and type III collagen of connective tissues, and obvious targets for the collagenalytic, which increase during tissue inflammation. The objective of the study was to investigate the diagnostic and prognostic utility of cartilage, C2M, and synovial, C3M, turnover biomarkers in AS. Serum samples were retrieved from patients suffering from AS (n = 103), RA (n = 47) and healthy controls (n = 56). AS progressors were defined as having new vertebral syndesmophytes or more that 3 unit change in mSASSS over a two-year period. Type II collagen degradation markers in serum were measured by the C2M ELISA, and type III collagen degradation by the C3M ELISA. Logistic regression and dichotomized decision tree were used to analyze the prognostic value of the markers individually or in combination. Both C2M and C3M levels were significantly higher in RA patients than in healthy controls (p<0.0001). Diagnostic utility was analyzed by ROC and areas under the curve (AUCs) were 72% and 89% for C2M and C3M, respectively. Both C2M and C3M, were significantly higher in serum samples from AS patient than from healthy controls (p<0.0001). The AUCs of C2M and C3M, respectively, were 70% and 81% for AS. A combination of C2M and C3M, dichotomized according to best cut-offs for individual markers, could correctly identify 80% of the progressors and 61% of the non-progressors. The present study is the first to show that specific biomarkers of cartilage and connective tissue degradation facilitate both diagnosis and prediction of progression of RA and AS.     

Association of Single Nucleotide Polymorphisms in the Lens Epithelium-Derived Growth Factor (LEDGF/p75) with HIV-1 Infection Outcomes in Brazilian HIV-1+ Individuals

The lens epithelium-derived growth factor p75 (LEDGF/p75), coded by the PSIP1 gene, is an important host co-factor that interacts with HIV-1 integrase to target integration of viral cDNA into active genes. The aim of this study was to investigate the association of SNPs in the PSIP1 gene with disease outcome in HIV-1 infected patients. We performed a genetic association study in a cohort of 171 HIV-1 seropositive Brazilian individuals classified as rapid progressors (RP, n = 69), typical progressors (TP, n = 79) and long-term nonprogressors (LTNP, n = 23). The exonic SNP rs61744944 and 9 tag SNPs were genotyped. A group of 192 healthy subjects was analyzed to determine the frequency of SNPs and haplotypes in the general population. Linkage disequilibrium (LD) analyses indicated that the SNPs analyzed were not in high LD (r²<0.8). Logistic regression models suggested that patients carrying the T allele rs61744944 (472L) were more likely to develop a LTNP phenotype (OR = 4.98; p = 0.05) as compared to TP group. The same trend was observed when LTNPs were compared to the RP group (OR = 3.26). Results of haplotype analyses reinforced this association, since the OR values obtained for the haplotype carrying allele T at rs61744944 also reflected an association with LTNP status (OR = 6.05; p = 0.08 and OR = 3.44; p = 0.12 for comparisons to TP and RP, respectively). The rare missense variations Ile436Ser and Thr473Ile were not identified in the patients enrolled in this study. Gene expression analyses showed lower LEDGF/p75 mRNA levels in peripheral blood mononuclear cells obtained from HIV-1 infected individuals. However, these levels were not influenced by any of the SNPs investigated. In spite of the limited number of LTNPs, these data suggest that the PSIP1 gene could be associated with the outcome of HIV-1 infection. Further analyses of this gene may guide the identification of causative variants to help predict disease course.     

An analysis of the cross-reactivity of autoantibodies to GAD65 and GAD67 in diabetes

Background

Autoantibodies to GAD65 (anti-GAD65) are present in the sera of 70–80% of patients with type 1 diabetes (T1D), but antibodies to the structurally similar 67 kDa isoform GAD67 are rare. Antibodies to GAD67 may represent a cross-reactive population of anti-GAD65, but this has not been formally tested.

Methodology/Principal Findings

In this study we examined the frequency, levels and affinity of anti-GAD67 in diabetes sera that contained anti-GAD65, and compared the specificity of GAD65 and GAD67 reactivity. Anti-GAD65 and anti-GAD67 were measured by radioimmunoprecipitation (RIP) using ¹²⁵I labeled recombinant GAD65 and GAD67. For each antibody population, the specificity of the binding was measured by incubation with 100-fold excess of unlabeled GAD in homologous and heterologous inhibition assays, and the affinity of binding with GAD65 and GAD67 was measured in selected sera. Sera were also tested for reactivity to GAD65 and GAD67 by immunoblotting. Of the 85 sera that contained antibodies to GAD65, 28 contained anti–GAD67 measured by RIP. Inhibition with unlabeled GAD65 substantially or completely reduced antibody reactivity with both ¹²⁵I GAD65 and with ¹²⁵I GAD67. In contrast, unlabeled GAD67 reduced autoantibody reactivity with ¹²⁵I GAD67 but not with ¹²⁵I GAD65. Both populations of antibodies were of high affinity (>10¹⁰ l/mol).

Conclusions

Our findings show that autoantibodies to GAD67 represent a minor population of anti-GAD65 that are reactive with a cross-reactive epitope found also on GAD67. Experimental results confirm that GAD65 is the major autoantigen in T1D, and that GAD67 per se has very low immunogenicity. We discuss our findings in light of the known similarities between the structures of the GAD isoforms, in particular the location of a minor cross-reactive epitope that could be induced by epitope spreading.     

Analysis of GAD65 autoantibodies in Stiff-Person syndrome patients

Autoantibodies to the 65-kDa isoform of glutamate decarboxylase GAD65 (GAD65Ab) are strong candidates for a pathological role in Stiff-Person syndrome (SPS). We have analyzed the binding specificity of the GAD65Ab in serum and cerebrospinal fluid (CSF) of 12 patients with SPS by competitive displacement studies with GAD65-specific rFab-derived from a number of human and mouse mAbs specific for different determinants on the Ag. We demonstrate considerable differences in the epitope specificity when comparing paired serum and CSF samples, suggesting local stimulation of B cells in the CSF compartment of these patients. Moreover, these autoantibodies strongly inhibit the enzymatic activity of GAD65, thus blocking the formation of the neurotransmitter gamma-aminobutyric acid. The capacity of the sera to inhibit the enzymatic activity of GAD65 correlated with their binding to a conformational C-terminal Ab epitope. Investigation of the inhibitory mechanism revealed that the inhibition could not be overcome by high concentrations of glutamate or the cofactor pyridoxal phosphate, suggesting a noncompetitive inhibitory mechanism. Finally, we identified a linear epitope on amino acids residues 4-22 of GAD65 that was recognized solely by autoantibodies from patients with SPS but not by serum from type 1 diabetes patients. A mAb (N-GAD65 mAb) recognizing this N-terminal epitope was successfully humanized to enhance its potential therapeutic value by reducing its overall immunogenicity.     

Mitochondrial DNA (mtDNA) Haplogroups Influence the Progression of Knee Osteoarthritis. Data from the Osteoarthritis Initiative (OAI)

Objective

To evaluate the influence of the mtDNA haplogroups on knee osteoarthritis progression in Osteoarthritis Initiative (OAI) participants through longitudinal data from radiographs and magnetic resonance imaging (MRI).

Methods

Four-year knee osteoarthritis progression was analyzed as increase in Kellgren and Lawrence (KL) grade, in addition to increase in OARSI atlas grade for joint space narrowing (JSN), osteophytes and subchondral sclerosis in the tibia medial compartment of 891 Caucasian individuals from the progression subcohort. The influence of the haplogroups on the rate of structural progression was also assessed as the four-year change in minimum joint space width (mJSW in millimetres) in both knees of (n = 216) patients with baseline unilateral medial-tibiofemoral JSN. Quantitative cartilage measures from longitudinal MRI data were those related to cartilage thickness and volume with a 24 month follow-up period (n = 381).

Results

During the four-year follow-up period, knee OA patients with the haplogroup T showed the lowest increase in KL grade (Hazard Risk [HR] = 0.499; 95% Confidence Interval [CI]: 0.261–0.819; p<0.05) as well as the lowest cumulative probability of progression for JSN (HR = 0.547; 95% CI: 0.280–0.900; p<0.05), osteophytes (HR = 0.573; 95% CI: 0.304–0.893; p<0.05) and subchondral sclerosis (HR = 0.549; 95% CI: 0.295–0.884; p<0.05). They also showed the lowest decline in mJSW (standardized response means (SRM) = −0.39; p = 0.037) in those knees without baseline medial JSN (no-JSN knees). Normalized cartilage volume loss was significantly lower in patients carrying the haplogroup T at medial tibia femoral (SRM = −0.33; p = 0.023) and central medial femoral (SRM = −0.27; p = 0.031) compartments. Cartilage thickness loss was significantly lower in carriers of haplogroup T at central medial tibia-femoral (SRM = −0.42; p = 0.011), medial tibia femoral (SRM = −0.32; p = 0.018), medial tibia anterior (SRM = +0.31; p = 0.013) and central medial femoral (SRM = −0.19; p = 0.013) compartments.

Conclusions

Mitochondrial genome seems to play a role in the progression of knee osteoarthritis. mtDNA variation could improve identification of patients predisposed to faster or severe progression of the disease.     

Insulin-Like Growth Factor 1 Receptor (IGF1R) Expression and Survival in Operable Squamous-Cell Laryngeal Cancer

Introduction

Prognosis of patients with operable laryngeal cancer is highly variable and therefore potent prognostic biomarkers are warranted. The insulin-like growth factor receptor (IGFR) signaling pathway plays a critical role in laryngeal carcinogenesis and progression.

Patients and Methods

We identified all patients with localized TNM stage I–III laryngeal cancer managed with potentially curative surgery between 1985 and 2008. Immunohistochemical (IHC) expression of IGF1R-alpha, IGF1R-beta and IGF2R was evaluated using the immunoreactive score (IRS) and mRNA levels of important effectors of the IGFR pathway were assessed, including IGF1R, IGF-binding protein 3 (IGFBP3), suppressor of cytokine signaling 2 (SOCS2) and members of the MAP-kinase (MAP2K1, MAPK9) and phosphatidyl-inositol-3 kinase (PIK3CA, PIK3R1) families. Cox-regression models were applied to assess the predictive value of biomarkers on disease-free survival (DFS) and overall survival (OS).

Results

Among 289 eligible patients, 95.2% were current or ex smokers, 75.4% were alcohol abusers, 15.6% had node-positive disease and 32.2% had received post-operative irradiation. After a median follow-up of 74.5 months, median DFS was 94.5 months and median OS was 106.3 months. Using the median IRS as the pre-defined cut-off, patients whose tumors had increased IGF1R-alpha cytoplasm or membrane expression experienced marginally shorter DFS and significantly shorter OS compared to those whose tumors had low IGF1R-alpha expression (91.1 vs 106.2 months, p = 0.0538 and 100.3 vs 118.6 months, p = 0.0157, respectively). Increased mRNA levels of MAPK9 were associated with prolonged DFS (p = 0.0655) and OS (p = 0.0344). In multivariate analysis, IGF1R-alpha overexpression was associated with a 46.6% increase in the probability for relapse (p = 0.0374). Independent predictors for poor OS included node-positive disease (HR = 2.569, p<0.0001), subglottic/transglottic localization (HR = 1.756, p = 0.0438) and IGF1R-alpha protein overexpression (HR = 1.475, p = 0.0504).

Conclusion

IGF1R-alpha protein overexpression may serve as an independent predictor of relapse and survival in operable laryngeal cancer. Prospective evaluation of the IGF1R-alpha prognostic utility is warranted.     

Host HLA B*Allele-Associated Multi-Clade Gag T-Cell Recognition Correlates with Slow HIV-1 Disease Progression in Antiretroviral Therapy-Na?ve Ugandans

Background

Some HIV infected individuals remain asymptomatic for protracted periods of time in the absence of antiretroviral therapy (ART). Virological control, CD4 T cell loss and HIV-specific responses are some of the key interrelated determinants of HIV-1 disease progression. In this study, possible interactions between viral load, CD4 T cell slopes, host genetics and HIV-specific IFN-γ responses were evaluated in chronically HIV-1-infected adults.

Methodolology/Principal Findings

Multilevel regression modeling was used to stratify clade A or D HIV-infected individuals into disease progression groups based on CD4 T cell slopes. ELISpot assays were used to quantify the frequency and magnitude of HIV-induced IFN-γ responses in 7 defined rapid progressors (RPs) and 14 defined slow progressors (SPs) at a single time point. HLA typing was performed using reference strand conformational analysis (RSCA). Although neither the breadth nor the magnitude of the proteome-wide HIV-specific IFN-γ response correlated with viral load, slow disease progression was associated with over-representation of host immunogenetic protective HLA B* alleles (10 of 14 SPs compared to 0 of 7; p = 0.004, Fisher''s Exact) especially B*57 and B*5801, multiclade Gag T-cell targeting (71%, 10 of 14 SPs compared to 14%, 1 of 7 RPs); p = 0.029, Fisher''s Exact test and evident virological control (3.65 compared to 5.46 log10 copies/mL in SPs and RPs respectively); p<0.001, unpaired student''s t-test

Conclusions

These data are consistent with others that associated protection from HIV disease with inherent host HLA B allele-mediated ability to induce broader Gag T-cell targeting coupled with apparent virological control. These immunogenetic features of Gag-specific immune response which could influence disease progression may provide useful insight in future HIV vaccine design.     

Clinical Outcomes for Gastric Cancer following Adjuvant Chemoradiation Utilizing Intensity Modulated versus Three-Dimensional Conformal Radiotherapy

Purpose/Objective(s)

To determine if intensity modulated radiation therapy (IMRT) in the post-operative setting for gastric cancer was associated with reduced toxicity compared to 3D conformal radiation therapy (3DCRT).

Materials/Methods

This retrospective study includes 24 patients with stage IB-IIIB gastric cancer consecutively treated from 2001–2010. All underwent surgery followed by adjuvant chemoradiation. Concurrent chemotherapy consisted of 5-FU/leucovorin (n = 21), epirubicin/cisplatin/5FU (n = 1), or none (n = 2). IMRT was utilized in 12 patients and 3DCRT in 12 patients. For both groups, the target volume included the tumor bed, anastomosis, gastric stump, and regional lymphatics.

Results

Median follow-up for the entire cohort was 19 months (range 0.4–8.5 years), and 49 months (0.5–8.5 years) in surviving patients. The 3DCRT group received a median dose of 45 Gy, and the IMRT group received a median dose of 50.4 Gy (p = 0.0004). For the entire cohort, 3-year overall survival (OS) was 40% and 3-year disease free survival (DFS) was 41%. OS and DFS did not differ significantly between the groups. Acute toxicity was similar. Between 3DCRT and IMRT groups, during radiotherapy, median weight lost (3.2 vs. 3.3 kg, respectively; p = 0.47) and median percent weight loss were similar (5.0% vs. 4.3%, respectively; p = 0.43). Acute grade 2 toxicity was experienced by 8 patients receiving 3DCRT and 11 receiving IMRT (p = 0.32); acute grade 3 toxicity occurred in 1 patient receiving 3DCRT and none receiving IMRT (p = 1.0). No patients in either cohort experienced late grade 3 toxicity, including renal or gastrointestinal toxicity. At last follow up, the median increase in creatinine was 0.1 mg/dL in the IMRT group and 0.1 mg/dL in the 3DCRT group (p = 0.78).

Conclusion

This study demonstrates that adjuvant chemoradiation for gastric cancer with IMRT to 50.4 Gy was well-tolerated and compared similarly in toxicity with 3DCRT to 45 Gy.     

Docetaxel,Cisplatin and Fluorouracil (DCF) Regimen Compared with Non-Taxane-Containing Palliative Chemotherapy for Gastric Carcinoma: A Systematic Review and Meta-Analysis

Background

Gastric carcinoma (GC) is one of the highest cancer-mortality diseases with a high incidence rate in Asia. For surgically unfit but medically fit patients, palliative chemotherapy is the main treatment. The chemotherapy regimen of docetaxel, cisplatin and 5-fluorouracil (DCF) has been used to treat the advanced stage or metastatic GC. It is necessary to compare effectiveness and toxicities of DCF regimen with non-taxane-containing palliative chemotherapy for GC.

Methods

PubMed, EmBase, Cochrane Central Register of Controlled Trials and China National Knowledge Infrastructure databases were searched to select relative randomized controlled trials (RCTs) comparing DCF to non-taxane-containing chemotherapy for patients with palliatively resected, unresectable, recurrent or metastatic GC. Primary outcome measures were 1-year and 2-year overall survival (OS) rates. Secondary outcome measures were median survival time (MST), median time to progression (TTP), response rate and toxicities.

Results

Twelve RCTs were eligible and 1089 patients were analyzed totally (549 in DCF and 540 in control). DCF regimen increased partial response rate (38.8% vs 27.9%, p = 0.0003) and reduced progressive disease rate (18.9% vs 33.3%, p = 0.0005) compared to control regimen. Significant improvement of 2-year OS rate was found in DCF regimen (RR = 2.03, p = 0.006), but not of 1-year OS rate (RR = 1.22, p = 0.08). MST was significantly prolonged by DCF regimen (p = 0.039), but not median TTP (p = 0.054). Both 1-year OS rate and median TTP had a trend of prolongation by DCF regimen. Chemotherapy-related mortality was comparable (RR = 1.23, p = 0.49) in both regimens. In grade I-IV toxicities, DCF regimen showed a major raise of febrile neutropenia (RR = 2.33, p<0.0001) and minor raises of leucopenia (RR = 1.25, p<0.00001), neutropenia (RR = 1.19, p<0.00001), and diarrhea (RR = 1.59, p<0.00001), while in other toxicities there were no significant differences.

Conclusion

DCF regimen has better response than non-taxane containing regimen and could potentially improve the survival outcomes. The chemotherapy-related toxicity of DCF regimen is acceptable to some extent.     

Pre-miR-146a (rs2910164 G>C) Single Nucleotide Polymorphism Is Genetically and Functionally Associated with Leprosy

Mycobacterium leprae infects macrophages and Schwann cells inducing a gene expression program to facilitate its replication and progression to disease. MicroRNAs (miRNAs) are key regulators of gene expression and could be involved during the infection. To address the genetic influence of miRNAs in leprosy, we enrolled 1,098 individuals and conducted a case-control analysis in order to study four miRNAs genes containing single nucleotide polymorphism (miRSNP). We tested miRSNP-125a (rs12975333 G>T), miRSNP-223 (rs34952329 *>T), miRSNP-196a-2 (rs11614913 C>T) and miRSNP-146a (rs2910164 G>C). Amongst them, miRSNP-146a was the unique gene associated with risk to leprosy per se (GC OR = 1.44, p = 0.04; CC OR = 2.18, p = 0.0091). We replicated this finding showing that the C-allele was over-transmitted (p = 0.003) using a transmission-disequilibrium test. A functional analysis revealed that live M. leprae (MOI 100∶1) was able to induce miR-146a expression in THP-1 (p<0.05). Furthermore, pure neural leprosy biopsies expressed augmented levels of that miRNA as compared to biopsy samples from neuropathies not related with leprosy (p = 0.001). Interestingly, carriers of the risk variant (C-allele) produce higher levels of mature miR-146a in nerves (p = 0.04). From skin biopsies, although we observed augmented levels of miR-146a, we were not able to correlate it with a particular clinical form or neither host genotype. MiR-146a is known to modulate TNF levels, thus we assessed TNF expression (nerve biopsies) and released by peripheral blood mononuclear cells infected with BCG Moreau. In both cases lower TNF levels correlates with subjects carrying the risk C-allele, (p = 0.0453 and p = 0.0352; respectively), which is consistent with an immunomodulatory role of this miRNA in leprosy.     

Interaction between PNPLA3 I148M Variant and Age at Infection in Determining Fibrosis Progression in Chronic Hepatitis C

Background and Aims

The PNPLA3 I148M sequence variant favors hepatic lipid accumulation and confers susceptibility to hepatic fibrosis and hepatocellular carcinoma. The aim of this study was to estimate the effect size of homozygosity for the PNPLA3 I148M variant (148M/M) on the fibrosis progression rate (FPR) and the interaction with age at infection in chronic hepatitis C (CHC).

Methods

FPR was estimated in a prospective cohort of 247 CHC patients without alcohol intake and diabetes, with careful estimation of age at infection and determination of fibrosis stage by Ishak score.

Results

Older age at infection was the strongest determinant of FPR (p<0.0001). PNPLA3 148M/M was associated with faster FPR in individuals infected at older age (above the median, 21 years; −0.64±0.2, n = 8 vs. −0.95±0.3, n = 166 log₁₀ FPR respectively; p = 0.001; confirmed for lower age thresholds, p<0.05), but not in those infected at younger age (p = ns). The negative impact of PNPLA3 148M/M on fibrosis progression was more marked in subjects at risk of altered hepatic lipid metabolism (those with grade 2–3 steatosis, genotype 3, and overweight; p<0.05). At multivariate analysis, PNPLA3 148M/M was associated with FPR (incremental effect 0.08±0.03 log₁₀ fibrosis unit per year; p = 0.022), independently of several confounders, and there was a significant interaction between 148M/M and older age at infection (p = 0.025). The association between 148M/M and FPR remained significant even after adjustment for steatosis severity (p = 0.032).

Conclusions

We observed an interaction between homozygosity for the PNPLA3 I148M variant and age at infection in determining fibrosis progression in CHC patients.     

Heart Failure Induces Significant Changes in Nuclear Pore Complex of Human Cardiomyocytes

Aims

The objectives of this study were to analyse the effect of heart failure (HF) on several proteins of nuclear pore complex (NPC) and their relationship with the human ventricular function.

Methods and Results

A total of 88 human heart samples from ischemic (ICM, n = 52) and dilated (DCM, n = 36) patients undergoing heart transplant and control donors (CNT, n = 9) were analyzed by Western blot. Subcellular distribution of nucleoporins was analysed by fluorescence and immunocytochemistry. When we compared protein levels according to etiology, ICM showed significant higher levels of NDC1 (65%, p<0.0001), Nup160 (88%, p<0.0001) and Nup153 (137%, p = 0.004) than those of the CNT levels. Furthermore, DCM group showed significant differences for NDC1 (41%, p<0.0001), Nup160 (65%, p<0.0001), Nup153 (155%, p = 0.006) and Nup93 (88%, p<0.0001) compared with CNT. However, Nup155 and translocated promoter region (TPR) did not show significant differences in their levels in any etiology. Regarding the distribution of these proteins in cell nucleus, only NDC1 showed differences in HF. In addition, in the pathological group we obtained good relationship between the ventricular function parameters (LVEDD and LVESD) and Nup160 (r = −0382, p = 0.004; r = −0.290, p = 0.033; respectively).

Conclusions

This study shows alterations in specific proteins (NDC1, Nup160, Nup153 and Nup93) that compose NPC in ischaemic and dilated human heart. These changes, related to ventricular function, could be accompanied by alterations in the nucleocytoplasmic transport. Therefore, our findings may be the basis for a new approach to HF management.     

Estimating Cognitive Reserve in Healthy Adults Using the Cognitive Reserve Scale

The concept of cognitive reserve emerged from observed disparities between brain pathology and clinical symptoms. It may explain better neuropsychological performance in healthy individuals. The objectives of this study were to measure reserve in healthy subjects using a new Cognitive Reserve Scale (CRS), analyze the internal consistency of the CRS, and analyze validity evidence. A total of 117 healthy individuals were divided into two groups: 87 adults (aged 18–64 years) and 30 elderly adults (≥65 years). All subjects completed the CRS and a comprehensive neuropsychological battery. The internal consistency of the scale was satisfactory (α = 0.77). No significant differences were observed between genders (t = 0.51, p = 0.611), and age was corrected by averaging the CRS score. The study of validity evidence showed that education affected the CRS (t = −2.98, p = 0.004, partial h² = 0.07) and there was no significant relationship between the CRS and IQ (r = 0.09, p = 0.33). Occupational attainment and the CRS were not related (F_2,116 = 0.11, p = 0.898). In line with previous studies on reserve, heterogeneity was observed in the analyses of relationships between the CRS and cognitive performance. There were significant relationships between CRS score and the Verbal Learning Spanish–Complutense Test last trial (r = 0.24, p = 0.009), sum (r = 0.32, p = 0.000), short-term (r = 0.29, p = 0.002) and long-term memory (r = 0.22, p = 0.018), Matrix Reasoning subtest (r = 0.20, p = 0.027) and Block Design subtest (r = 0.20, p = 0.029). No other neuropsychological variables correlated with the CRS (p>0.05). The CRS is a reliable instrument that reflects the frequency of participation in brain-stimulating activities across the lifetime. The associations between the CRS and education and neuropsychological performance support validity evidence.     

Influence of HLA Class I Haplotypes on HIV-1 Seroconversion and Disease Progression in Pumwani Sex Worker Cohort

We examined the effect of HLA class I haplotypes on HIV-1 seroconversion and disease progression in the Pumwani sex worker cohort. This study included 595 HIV-1 positive patients and 176 HIV negative individuals. HLA-A, -B, and -C were typed to 4-digit resolution using sequence-based typing method. HLA class I haplotype frequencies were estimated using PyPop 32-0.6.0. The influence of haplotypes on time to seroconversion and CD4+ T cell decline to <200 cells/mm³ were analyzed by Kaplan-Meier analysis using SPSS 13.0. Before corrections for multiple comparisons, three 2-loci haplotypes were significantly associated with faster seroconversion, including A*23∶01-C*02∶02 (p = 0.014, log rank(LR) = 6.06, false-discovery rate (FDR) = 0.056), B*42∶01-C*17∶01 (p = 0.01, LR = 6.60, FDR = 0.08) and B*07∶02-C*07∶02 (p = 0.013, LR = 6.14, FDR = 0.069). Two A*74∶01 containing haplotypes, A*74∶01-B*15∶03 (p = 0.047, LR = 3.942, FDR = 0.068) and A*74∶01-B*15∶03-C*02∶02 (p = 0.045, LR = 4.01, FDR = 0.072) and B*14∶02-C*08∶02 (p = 0.021, LR = 5.36, FDR = 0.056) were associated with slower disease progression. Five haplotypes, including A*30∶02-B*45∶01 (p = 0.0008, LR = 11.183, FDR = 0.013), A*30∶02-C*16∶01 (p = 0.015, LR = 5.97, FDR = 0.048), B*53∶01-C*04∶01 (p = 0.010, LR = 6.61, FDR = 0.08), B*15∶10-C*03∶04 (p = 0.031, LR = 4.65, FDR = 0.062), and B*58∶01-C*03∶02 (p = 0.037, LR = 4.35, FDR = 0.066) were associated with faster progression to AIDS. After FDR corrections, only the associations of A*30∶02-B*45∶01 and A*30∶02-C*16∶01 with faster disease progression remained significant. Cox regression and deconstructed Kaplan-Meier survival analysis showed that the associations of haplotypes of A*23∶01-C*02∶02, B*07∶02-C*07∶02, A*74∶01-B*15∶03, A*74∶01-B*15∶03-C*02∶02, B*14∶02-C*08∶02 and B*58∶01-C*03∶02 with differential seroconversion or disease progression are due to the dominant effect of a single allele within the haplotypes. The true haplotype effect was observed with A*30∶02-B*45∶01, A*30∶02-C*16∶02, B*53∶01-C*04∶01 B*15∶10-C*03∶04, and B*42∶01-C*17∶01. In these cases, the presence of both alleles accelerated the disease progression or seroconversion than any of the single allele within the haplotypes. Our study showed that the true effects of HLA class I haplotypes on HIV seroconversion and disease progression exist and the associations of HLA class I haplotype can also be due to the dominant effect of a single allele within the haplotype.     

Genetic Variation in the Interleukin-28B Gene Is Associated with Spontaneous Clearance and Progression of Hepatitis C Virus in Moroccan Patients

Background

Genetic variation in the IL28B gene has been strongly associated with treatment outcomes, spontaneous clearance and progression of the hepatitis C virus infection (HCV). The aim of the present study was to investigate the role of polymorphisms at this locus with progression and outcome of HCV infection in a Moroccan population.

Methods

We analyzed a cohort of 438 individuals among them 232 patients with persistent HCV infection, of whom 115 patients had mild chronic hepatitis and 117 had advanced liver disease (cirrhosis and hepatocellular carcinoma), 68 individuals who had naturally cleared HCV and 138 healthy subjects. The IL28B SNPs rs12979860 and rs8099917 were genotyped using a TaqMan 5′ allelic discrimination assay.

Results

The protective rs12979860-C and rs8099917-T alleles were more common in subjects with spontaneous clearance (77.9% vs 55.2%; p = 0.00001 and 95.6% vs 83.2%; p = 0.0025, respectively). Individuals with clearance were 4.69 (95% CI, 1.99–11.07) times more likely to have the C/C genotype for rs12979860 polymorphism (p = 0.0017) and 3.55 (95% CI, 0.19–66.89) times more likely to have the T/T genotype at rs8099917. Patients with advanced liver disease carried the rs12979860-T/T genotype more frequently than patients with mild chronic hepatitis C (OR = 1.89; 95% CI, 0.99–3.61; p = 0.0532) and this risk was even more pronounced when we compared them with healthy controls (OR = 4.27; 95% CI, 2.08–8.76; p = 0.0005). The rs8099917-G allele was also associated with advanced liver disease (OR = 2.34; 95% CI, 1.40–3.93; p = 0.0100).

Conclusions

In the Moroccan population, polymorphisms near the IL28B gene play a role both in spontaneous clearance and progression of HCV infection.