Alejandra Claudia Cherñavsky

Confirmation of an association between rs6822844 at the IL2-IL21 region and multiple autoimmune diseases: Evidence of a general susceptibility locus

OBJECTIVE Autoimmune diseases often have susceptibility genes in common, indicating similar molecular mechanisms. Increasing evidence suggests that rs6822844 at the IL2-IL21 region is strongly associated with multiple autoimmune diseases in individuals of European descent. This study was undertaken to attempt to replicate the association between rs6822844 and 6 different immune-mediated diseases in non-European populations, and to perform disease-specific and overall meta-analyses using data from previously published studies. METHODS We evaluated case-control associations between rs6822844 and celiac disease (CD) in subjects from Argentina; rheumatoid arthritis (RA), type 1 diabetes mellitus (DM), primary Sjögrens syndrome (SS), and systemic lupus erythematosus (SLE) in subjects from Colombia; and Behçets disease (BD) in subjects from Turkey. Allele and gene distributions were compared between cases and controls. Meta-analyses were performed using data from the present study and previous studies. RESULTS We detected significant associations of rs6822844 with SLE (P = 0.008), type 1 DM (P = 0.014), RA (P = 0.019), and primary SS (P = 0.033) but not with BD (P = 0.34) or CD (P = 0.98). We identified little evidence of population differentiation (F(ST) = 0.01) within cases and controls from Argentina and Colombia, suggesting that association was not influenced by population substructure. Disease-specific meta-analysis indicated significant association for RA (P(meta) = 3.61 x 10(-6)), inflammatory bowel disease (IBD; Crohns disease and ulcerative colitis) (P(meta) = 3.48 x 10(-12)), type 1 DM (P(meta) = 5.33 x 10(-5)), and CD (P(meta) = 5.30 x 10(-3)). Overall meta-analysis across all autoimmune diseases reinforced association with rs6822844 (23 data sets; P(meta) = 2.61 x 10(-25), odds ratio 0.73 [95% confidence interval 0.69-0.78]). CONCLUSION Our results indicate that there is an association between rs6822844 and multiple autoimmune diseases in non-European populations. Meta-analysis results strongly reinforce this robust association across multiple autoimmune diseases in both European-derived and non-European populations.

Exploratory, randomized, double-blind, placebo-controlled study on the effects of Bifidobacterium infantis natren life start strain super strain in active celiac disease.

Background/Aims: The aim of this exploratory trial was to establish if the probiotic Bifidobacterium natren life start (NLS) strain strain may affect the clinical course and pathophysiological features of patients with untreated celiac disease (CD). Positive findings would be helpful in directing future studies. Methods: Twenty-two adult patients having 2 positives CD-specific tests were enrolled. Patients were randomized to receive 2 capsules before meals for 3 weeks of either Bifidobacterium infantis natren life start strain super strain (Lifestart 2) (2×109 colony-forming units per capsule) (n=12) or placebo (n=10), whereas they also consumed at least 12 g of gluten/day. A biopsy at the end of the trial confirmed CD in all cases. The primary outcome was intestinal permeability changes. Secondary endpoints were changes in symptoms and the Gastrointestinal Symptom Rating Scale, and in immunologic indicators of inflammation. Results: The abnormal baseline intestinal permeability was not significantly affected by either treatment. In contrast to patients on placebo, those randomized to B. infantis experienced a significant improvement in Gastrointestinal Symptom Rating Scale (P=0.0035 for indigestion; P=0.0483 for constipation; P=0.0586 for reflux). Final/baseline IgA tTG and IgA DGP antibody concentration ratios were lower in the B. infantis arm (P=0.055 for IgA tTG and P=0.181 for IgA DGP). Final serum macrophage inflammatory protein-1&bgr; increased significantly (P<0.04) only in patients receiving B. infantis. The administration of B. infantis was safe. Conclusions: The study suggests that B. infantis may alleviate symptoms in untreated CD. The probiotic produced some immunologic changes but did not modify abnormal intestinal permeability. Further studies are necessary to confirm and/or expand these observations.

Azathioprine in refractory sprue: results from a prospective, open-label study

OBJECTIVE:Refractory sprue is a rare and severe malabsorptive disorder that mimics celiac disease but is refractory to a gluten-free diet and is without initial evidence of overt lymphoma. Treatment is largely empiric and often ineffective, with steroids and immunosuppression being the mainstream therapeutic options. The aim of this study was to evaluate prospectively the effect of azathioprine on a group of patients diagnosed with refractory sprue.METHODS:We studied seven consecutive patients (five women and two men) with a well-defined diagnosis of refractory sprue and a lack of response to oral or parenteral steroids. At diagnosis, five patients had endoscopic evidence of ulcerative jejunitis, and five underwent exploratory laparotomy for exclusion of malignancies. The characteristic monoclonal TCRγ gene rearrangement was shown in five of six patients studied. Patients were treated for a mean of 11 months (range 8–12 months), and clinical, biochemical, molecular, and histological parameters were reassessed at the end of the trial. The study was a prospective, open-label, non-placebo-controlled study using azathioprine (2 mg/kg/day) plus oral prednisone (1 mg/kg/day). A gluten-free diet (n = 7) as well as enteral (n = 6) and parenteral nutrition (n = 5) were administered during the trial.RESULTS:After treatment, five patients had a complete clinical remission, and biochemical and nutritional parameters were significantly improved. Steroids were tapered after the onset of azathioprine, and no patient was on steroids at the end of the trial. Intestinal histology improved significantly in all cases (normal histology in three cases and minor infiltration in the lamina propria in two). Two patients did not respond to treatment at any time and died in months 10 and 9, of an irreversible ventricular fibrillation and sepsis, respectively. No overt lymphoma was demonstrated during the follow-up.CONCLUSIONS:The present study confirms earlier anecdotal reports on the efficacy of azathioprine in refractory sprue, with clear clinical and histological improvement shown in most patients. However, monoclonality persisted after treatment. We consider that a larger number of patients should be evaluated before a definitive recommendation is adopted for use of this drug in refractory sprue.

Non-synonymous variant (Gly307Ser) in CD226 is associated with susceptibility to multiple autoimmune diseases

OBJECTIVES Recently, a non-synonymous (Gly307Ser) variant, rs763361, in the CD226 gene was shown to be associated with multiple autoimmune diseases (ADs) in European Caucasian populations. However, shared autoimmunity with CD226 has not been evaluated in non-European populations. The aim of the present study is to assess the association of this single nucleotide polymorphism (SNP) with ADs in non-European populations. METHODS To replicate this association in non-European populations, we evaluated case-control association between rs763361 and coeliac disease (CED) samples from Argentina; SLE, RA, type-1 diabetes (T1D) and primary SS (pSS) from Colombia; and SLE samples from China and Japan. We genotyped rs763361 and evaluated its genetic association with multiple ADs, using chi(2)-test. For each association, odds ratio (OR) and 95% CI were calculated. RESULTS We show that rs763361 is significantly associated with Argentinean CED (P = 0.0009, OR = 1.60). We also observed a trend of possible association with Chinese SLE (P = 0.01, OR = 1.19), RA (P = 0.047, OR = 1.25), SLE (P = 0.0899, OR = 1.24) and pSS (P = 0.09, OR = 1.33) in Colombians. Meta-analyses for SLE (using our three populations) and T1D (our population and three published populations) yielded significant association with rs763361, P = 0.009 (OR = 1.16) and P = 1.1.46 x 10(-9) (OR = 1.14), respectively. CONCLUSIONS Our results demonstrate that the coding variant rs763361 in CD226 gene is associated with multiple ADs in non-European populations.

Bone-specific antibodies in sera from patients with celiac disease: characterization and implications in osteoporosis.

Osteopenia and osteoporosis are well-known complications detected in celiac disease patients with still obscure pathogenesis. In the present study we investigated the presence of circulating anti-bone autoantibodies in patients with celiac disease and explored their role in the associated bone disease. We evaluated serum samples from 33 patients at the time of diagnosis and from 20 of them after treatment. Sera from patients with inflammatory bowel disease (n = 9), nonceliac osteoporotic (n = 18), and healthy individuals (n = 10) were used as controls. The presence of IgA specific anti-bone antibodies was first investigated using indirect immunofluorescence on cryosections of fetal rat tibia (20-day pregnancy). Furthermore, samples were homogenized and total tissue extracts were subjected to Western blot analysis to confirm immunoreactivity. At diagnosis, sera from 51.5% (17/33) of celiac patients had antibodies that recognized antigenic structures in chondrocytes and the extracellular matrix along mature cartilage, bone interface, and perichondrium of fetal rat bone. Among controls, only two osteoporotic patients showed very low titles of anti-bone autoantibodies. The immunostaining was localized in areas where an active mineralization process occurred and was similar to the distribution of the native bone tissue transglutaminase. The frequency of patients with positive baseline titers of anti-bone antibodies diminished significantly after treatment (P = 0.048). Western blot assays confirmed the presence of autoantibodies in sera from patients with a positive immunofluorescence staining. Autoantibodies recognized a major protein band on tissue extracts with a molecular weight of 77–80 kDa, which could be displaced when sera were preadsorbed with human recombinant tissue transglutaminase. We provide original evidence that patients with celiac disease have IgA-type circulating autoantibodies against intra- and extracellular structures of fetal rat tibia. Our findings suggest that these antibodies recognize bone tissue transglutaminase as the autoantigen, and based on the localization of the immunoreactivity we speculate that they might have an active role in the pathophysiology of celiac disease-associated bone complications.

Altered Phenotype and Functionality of Circulating Immune Cells Characterize Adult Patients with Nonalcoholic Steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a chronic inflammatory liver disease associated with insulin resistance and its metabolic consequences. Leukocyte mobilization, intrahepatic activation, and an exacerbated production of reactive oxygen species (ROS) and cytokines contribute to the development of NASH. Though alterations in peripheral blood (PB) T cell proportions and functionality remain unidentified, they might play a main role in NASH progression. We have compared the phenotype and Th1/Th2 commitment of peripheral immune cell reservoirs in adult patients and controls as well as the ability of neutrophils and monocytes to handle an ex vivo challenge. Also, we correlated those parameters with the main histological characteristics in NASH. Compared with controls, patients showed increased numbers of CD4+ cells and both CD4+ and CD8+ CD45RO subsets together with a higher frequency of IFN-γ-producing CD4+ and CD8+ T cells. We also found a decreased number of CD4+ and CD8+ CD45RA subsets. The distinctive production of IFN-γ highlights the significance of the observed skewed frequencies of PB T cells. Whereas ROS production by monocytes from NASH patients did not differ from controls, circulating neutrophils displayed a particularly higher phorbol myristate acetate-induced production of ROS. A negative correlation between oxidative burst and fibrosis grade was observed. This study reveals the presence of a characteristic profile of peripheral immune cells in NASH. We also discuss the probable influence of obesity on some of our present findings.

Reduced Dietary Omega-6 to Omega-3 Fatty Acid Ratio and 12/15-Lipoxygenase Deficiency Are Protective against Chronic High Fat Diet-Induced Steatohepatitis

Obesity is associated with metabolic perturbations including liver and adipose tissue inflammation, insulin resistance, and type 2 diabetes. Omega-6 fatty acids (ω6) promote and omega-3 fatty acids (ω3) reduce inflammation as they can be metabolized to pro- and anti-inflammatory eicosanoids, respectively. 12/15-lipoxygenase (12/15-LO) enzymatically produces some of these metabolites and is induced by high fat (HF) diet. We investigated the effects of altering dietary ω6/ω3 ratio and 12/15-LO deficiency on HF diet-induced tissue inflammation and insulin resistance. We examined how these conditions affect circulating concentrations of oxidized metabolites of ω6 arachidonic and linoleic acids and innate and adaptive immune system activity in the liver. For 15 weeks, wild-type (WT) mice were fed either a soybean oil-enriched HF diet with high dietary ω6/ω3 ratio (11∶1, HFH), similar to Western-style diet, or a fat Kcal-matched, fish oil-enriched HF diet with a low dietary ω6/ω3 ratio of 2.7∶1 (HFL). Importantly, the total saturated, monounsaturated and polyunsaturated fat content was matched in the two HF diets, which is unlike most published fish oil studies in mice. Despite modestly increased food intake, WT mice fed HFL were protected from HFH-diet induced steatohepatitis, evidenced by decreased hepatic mRNA expression of pro-inflammatory genes and genes involved in lymphocyte homing, and reduced deposition of hepatic triglyceride. Furthermore, oxidized metabolites of ω6 arachidonic acid were decreased in the plasma of WT HFL compared to WT HFH-fed mice. 12/15-LO knockout (KO) mice were also protected from HFH-induced fatty liver and elevated mRNA markers of inflammation and lymphocyte homing. 12/15-LOKO mice were protected from HFH-induced insulin resistance but reducing dietary ω6/ω3 ratio in WT mice did not ameliorate insulin resistance or adipose tissue inflammation. In conclusion, lowering dietary ω6/ω3 ratio in HF diet significantly reduces steatohepatitis.

The Role of Innate Cells Is Coupled to a Th1-Polarized Immune Response in Pediatric Nonalcoholic Steatohepatitis

BackgroundNonalcoholic steatohepatitis (NASH) is a chronic inflammatory liver disease influenced by risk factors for the metabolic syndrome. In adult patients, NASH is associated with an altered phenotype and functionality of peripheral immune cells, the recruitment of leukocytes and intrahepatic activation, and an exacerbated production of reactive oxygen species (ROS) and cytokines. It remains unclear if the previously described differences between pediatric and adult nonalcoholic fatty liver diseases also reflect differences in their pathogenesis.AimsWe aimed to investigate the phenotype and functionality of circulating immune cells and the potential contribution of liver infiltrating leukocytes to the immunological imbalance in pediatric NASH.ResultsBy a real-time PCR-based analysis of cytokines and immunohistochemical staining of liver biopsies, we demonstrated that the hepatic microenvironment is dominated by interferon-gamma (IFN-γ) but not interleukin-4 and is infiltrated by a higher number of CD8+ cells in pediatric NASH. The number of infiltrating neutrophils positively correlated with ROS generation by peripheral polymorphonuclear cells. By a flow cytometric analysis of peripheral blood lymphocytes, a distinctive increase in CD8+ CD45RO and CD8+ CD45RA subpopulations and an increased production of IFN-γ by CD4+ and CD8+ cells were shown. The production of ROS following PMA stimulation was augmented in circulating neutrophils but not in monocytes.ConclusionIn sum, the distinctive phenotype and functionality of infiltrating and circulating cells suggest that the role of innate cells is coupled to a Th1-polarized immune response in pediatric NASH.

Liver infiltrating mononuclear cells in children with type 1 autoimmune hepatitis

Objective: To investigate infiltrating cells in the liver of children with type 1 autoimmune hepatitis (AH-1). Methods: liver biopsies from 24 untreated AH-1 patients (14 children, 10 adults), five patients with hepatitis C virus related chronic hepatitis (HCV), and 10 control liver specimens (CL) were processed for immunohistochemical cell characterisation. Results: Two different cell distribution patterns were detected in the liver of patients with AH-1: (1) CD4+ and CD20+ cells were found in the central areas of the portal tracts (portal distribution); (2) CD8+ cells were observed at the periphery of the portal space (periportal distribution). Some cell subsets, like CD56, CD57, Fas-L, and Bak, showed a non-defined distribution pattern. The presence of two well defined patterns of cell distribution was not observed in HCV and CL (CD4+, CD20+, and CD8+ cells were uniformly distributed in the portal space). In AH-1 and CL, the NK markers CD56 and CD57 were found scattered throughout the liver parenchyma. However, in HCV biopsies, CD56+ cells were also clearly increased in both the portal and the periportal areas. Biopsies of AH-1 and HCV patients showed a uniform distribution of Fas-L and Bak in the portal and periportal areas, with Bak staining also detected in the hepatic parenchyma. Conclusions: Despite clinical and genetic differences, there was a similar distribution of liver infiltrating mononuclear cells in children and adults with AH-1. These results raise the possibility of reclassifying cryptogenic chronic hepatitis by immunohistochemical analysis of infiltrating liver cells.

Evidences of the Involvement of Bak, a member of the Bcl-2 Family of Proteins, in Active Coeliac Disease

Apoptosis of enterocytes is a feature that characterises the development of lesions in coeliac disease (CD). However, the intracellular pathways that lead to apoptosis of enterocytes have not been completely clarified. Bak is a member of the Bcl-2 family of proteins that acts as an endogenous promoter of apoptosis in normal enterocytes. However, its role in coeliac lesions has not been explored. We used small intestinal mucosa from patients with CD to evaluate the differential expression of members of the Bcl-2 family of proteins. Gene expression of Bak was analysed by RT-PCR of biopsies from 14 patients with untreated CD and from 19 controls without CD. In these samples, we also investigated the localisation of the Bak protein by immunohistochemistry and its apoptotic activity. In patients with untreated CD there was a 2.3-fold higher expression of Bak mRNA ( p =0.026), without significant differences in the expression of related genes bax or bcl-2. The higher expression of interferon gamma (IFN n ) ( p =0.036) and the higher number of apoptotic cells identified by the TUNEL method ( p =0.032) confirmed the proapoptotic status in the intestinal mucosa of CD patients. We found a significant positive correlation ( p <0.0001) between the expression of IFN n and Bak mRNA in patients with untreated CD. The expression of Bak protein was higher in patients with CD, and the immunoreactivity was almost restricted to the epithelium. We found that Bak mRNA and its protein were overexpressed in the intestinal lesions of CD patients and that IFN n confers increased susceptibility for enterocytes to undergo apoptosis via upregulation of Bak.