Jiri Nevoral

Celiac disease, gluten-free diet, and oats

Oats in a gluten-free diet increase the diets nutritional value, but their use remains controversial. Contamination with prolamins of other cereals is frequent, and some clinical and experimental studies support the view that a subgroup of celiac patients may be intolerant to pure oats. Thus, this issue is more complex than previously suggested. In order to produce oats that are safe for all celiac patients, the following topics should be addressed: selection of oat cultivars with low avenin content, research on such recombinant varieties of oats, development of assay methods to detect avenins in oat products, guidelines for the agricultural processing of oats and the manufacture of oat products, as well as guidelines for following up with celiac patients who consume oats.

Long-term follow-up of Wilson Disease: natural history, treatment, mutations analysis and phenotypic correlation

Background and aims: Wilson disease (WD) is an inherited disorder of copper metabolism. When treated, the outcome can be excellent, although the long‐term survival has yet to be well documented. The aim of this study was to describe the long‐term outcome of a cohort of patients with WD and to assess those factors affecting the phenotypic manifestation of WD.

Changes of orexin A plasma levels in girls with anorexia nervosa during eight weeks of realimentation.

OBJECTIVE Orexin A (OXA) is a hypothalamic neuropeptide involved in regulation of food intake and nutritional status. There are multiple disturbances of neuropeptide signaling described in girls with anorexia nervosa (AN), but OXA levels have not been addressed in this population to date. Therefore, we analyzed OXA levels of AN girls in this study. METHOD OXA (radioimmunoassay/RIA/method), leptin, insulinlike growth factor-1 (IGF-1), and insulinlike growth factor-1 binding protein-3 (IGFBP-3) levels were measured before and after 8 weeks of realimentation in 36 girls with AN and in 14 healthy controls (control group: CG). RESULTS Average weight increased significantly in AN during the study (p < .0001), while plasma levels of OXA decreased (before realimentation: 56.2 ± 2.4 pg/ml; after realimentation: 47.5 ± 1.4 pg/ml; p = .0025). OXA levels before realimentation differed from levels in the CG (47.15 ± 2.6 pg/ml, p = .034), but not afterward. We did not find any correlation between OXA and age, height, weight, BMI; or IGF-1, IGFBP-3, and leptin levels. DISCUSSION OXA levels in untreated AN patients differ significantly from healthy subjects and decrease during realimentation. These findings indicate that OXA may be involved in the nutritional regulation of malnourished children and adolescents.

Variants of CARD15, TNFA and PTPN22 and susceptibility to Crohn’s disease in the Czech population: high frequency of the CARD15 1007fs

Crohns disease (CD) has been shown to be associated with the variants in the CARD15 gene as well as in other genes involved in the immune response. The frequencies of the variants profoundly differ among populations and so does the associated risk. We examined the associations of variants in the CARD15, TNFA and PTPN22 genes with pediatric-onset and adult-onset CD in the Czech population. Genotype, phenotype and allelic frequencies were compared between 345 patients with CD (136 pediatric-onset and 209 adult-onset patients) and 501 unrelated healthy controls. At least one minor allele of the CARD15 gene was carried by 46% patients and only 21% control subjects (OR = 3.2, 95% CI 2.4-4.4). In a multiple logistic regression model, the strongest association with CD was found for the 1007fs variant (OR = 4.6, 95% CI 3.0-7.0), followed by p.G908R (OR = 2.9, 95% CI 1.5-5.7) and p.R702W (OR = 1.7, 95% CI 1.0-2.9), while no independent association was found for the remaining variants in the CARD15 gene (p.268S, p.955I and p.289S), for the p.R620W variant in the PTPN22 gene or for the g.-308G>A variant in the TNFA gene. The age at CD onset was strongly modified by positivity for the 1007fs allele: it was present in 42% pediatric-onset and only 25% adult-onset patients. In conclusion, we report a high frequency of the minor allele of the CARD15 1007fs polymorphism in the Czech population and a strong effect of this allele on the age at disease onset.

The CTLA4 variants may interact with the IL23R- and NOD2-conferred risk in development of Crohn's disease

BackgroundThe CTLA4 (cytotoxic T-lymphocyte antigen 4) gene is associated with several immunopathologic diseases and because of its important immuno-regulatory role it may be considered also a plausible candidate for a genetic association with inflammatory bowel diseases. Previously published studies found no association of CTLA4 with Crohns disease itself, but some indicated an association with its subphenotypes. The aim of this study was to assess the association in the Czech population, using a set of markers shown to associate with other diseases.MethodsSix polymorphisms within the CTLA4 region were investigated in 333 patients with Crohns disease and 482 unrelated healthy controls, all Caucasians of Czech origin. The genotypes of the SNPs were determined using the TaqMan SNP genotyping assays. Haplotypes were reconstructed using an expectation-maximization algorithm, and their association with the condition was assessed using log-linear modeling. Then, potential interactions were tested between the CTLA4 variants and other genetic factors known to confer the disease susceptibility.ResultsNo crude associations with Crohns disease were found for the tested CTLA4 variants under the log-additive or dominant models. However, when stratified for the genetic risk conferred by the variants in the NOD2 (the p.Leu1007fsX1008, rs5743293) or the IL23R (p.R381Q, rs11209026), a significant negative association emerged for the minor alleles of CTLA4 CT60 (rs3087243), JO31 (rs11571302), JO27-1 (rs11571297) polymorphisms. This negative association with CTLA4 was apparent only in the strata defined by presence minor alleles at the NOD2 rs5743293 (here the CTLA4 CT60 A coffered an OR = 0.43, 95%CI 0.19 - 0.95 for the presence of CT60 A), or IL23R rs11209026 (here the OR for presence of CT60 A was 0.23, 95%CI 0.07 - 0.71). We observed this effect also for the haplotype consisting of minor alleles of the three tightly linked CTLA4 markers. Furthermore, this haplotype was associated with the younger age at diagnosis (OR 1.52, 95%CI 1.09 - 2.11, p = 0.014).ConclusionsA protective effect of a CTLA4 haplotype was unmasked after stratification for the risk variants in the NOD2 and IL23R genes, and may point towards the biological relevance of the molecule in the pathogenesis of the disease.

Disease activity is an important factor for indeterminate interferon-γ release assay results in children with inflammatory bowel disease.

Background: Interferon-&ggr; release assay (IGRA) is widely used for screening of latent tuberculosis (TB) before and during biological therapy (BT). An indeterminate result of IGRA represents a limitation in the management of inflammatory bowel disease (IBD). Data on factors influencing IGRA results are scarce in children. The aim of the study was to identify factors influencing IGRA results in children with IBD. Methods: Seventy-two children with IBD (59 Crohn disease, 11 ulcerative colitis, 2 IBD-unclassified) indicated for BT were tested for TB infection (history, TB skin test, chest radiograph, IGRA; QuantiFERON—TB Gold in tube [QFT]) and consecutively retested using QFT in 1-year intervals. Results: We recorded 165 results of QFT (3% positive, 87% negative, and 10% indeterminate results). During follow-up we identified 4 conversions of negative QFT to positivity (3%) and 4 reversions (4%). Patients with indeterminate results of QFT had significantly lower actual weight-for-height z score (P = 0.022), higher platelet count (P = 0.00017), and lower levels of serum albumin (P = 0.015) compared with patients with positive or negative QFT. Indeterminate QFT was associated with corticosteroid treatment, BT, and disease activity, but not with treatment by immunomodulators. In a subanalysis of patients with Crohn disease alone, Pediatric Crohns Disease Activity Index was identified as single independent risk factor for indeterminate results (P = 0.00037). Conclusions: Although corticosteroid treatment is traditionally considered to be the main risk factor for indeterminate results of IGRA, the disease activity of IBD has even more profound effects on the results.

Comparison of intestinal microflora in healthy infants and infants with allergic colitis.

Twenty-eight exclusively breast-fed healthy infants and 16 infants also exclusively breast-fed with allergic colitis (aged 85 ± 60 and 98 ± 58 d, respectively) were screened for differences in fecal flora. Bifidobacteria were detected in 23 healthy infants and only in 4 fecal samples of infants with allergic colitis. All bifidobacteria-free infants possessed Gram-positive regular rods as a major group of their fecal flora. These bacteria were identified as clostridia using genus-specific FISH probe. Infants with allergy colitis possessed significantly lower counts of bifidobacteria and total anaerobes and significantly higher counts of clostridia in their feces. In healthy infants, Bifidobacterium longum was the most frequently found species (54.5% of the samples), followed by B. adolescentis (20.0), B. breve (18.2), B. bifidum (16.4), B. dentium (10.9) and B. pseudocatenulatum (1.80). Bifidobacterial isolates from two babies with allergic colitis were identified as B. longum, one child from patients group contained species B. dentium and one baby B. adolescentis. Our results suggest that there are significantly lower counts of bifidobacteria in infants with allergic colitis than in healthy infants.

Two independent genetic factors responsible for the associations of the IBD5 locus with Crohn's disease in the Czech population

Background: The role of the IBD5 locus in development of Crohns disease (CD) has not been clarified. In the Czech population we examined its genetic association using variants of the SLC22A4 (rs1050152), SLC22A5 (rs2631367), two single nucleotide polymorphisms (SNPs) shown to be associated with CD in genome‐wide studies (rs6596075 and rs2188962), and four SNPs previously shown to tag the haplotype blocks 4, 7, 9, 10 of the IBD5 locus (IGR2063b_1, IGR2230a_1, IGR100Xa_1, IGR3236a_1). Methods: The genotype, phenotype, and allelic frequencies were compared between 469 unrelated patients with CD (177 pediatric‐onset, 292 adult‐onset) and 470 unrelated healthy controls, all Caucasians of Czech ancestry. Results: The most significant difference between patients and controls was found for the SNP rs6596075 (odds ratio [OR] = 0.70 for the G allele; 95% CI 0.52‐0.94) in the dominant model and SNP IGR2063b_1 (OR = 1.38 for the G allele; 95% CI 1.14‐1.67) in the log‐additive model. We found a strong linkage disequilibrium across the IBD5 locus except rs6596075. The haplotype consisting of minor alleles of all tested SNPs except rs6596075 was carried by 31% patients and 23% control subjects (OR = 1.35, 95% CI 1.06‐1.72). The association of variants in SLC22A4 and SLC22A5 was dependent on this risk haplotype, while the strong association of the rs6596075 was seemingly independent. In the analysis of subphenotypes we found only an association of the penetrating disease with rs6596075 (OR = 2.13; 95% CI 1.31‐3.47). Conclusions: Our study confirms the importance of IBD5 in determining CD susceptibility, and demonstrates that two independent genetic factors may be responsible for the association observed within this locus. (Inflamm Bowel Dis 2010)

Association of IL23R p.381Gln and ATG16L1 p.197Ala with Crohn disease in the Czech population.

Objectives: An association of variants in the genes encoding the interleukin 23 receptor (IL23R, p.Arg381Gln, rs11209026), and the autophagy-related gene 16-like 1 (ATG16L1, p.Ala197Thr, rs2241880) with Crohn disease (CD) was identified by whole genome association studies, and subsequently confirmed by other works. The aim of this study was to assess this association in the Czech population. Subjects and Methods: In a case-control study 333 patients with CD (137 paediatric and 196 adult-onset) and 499 unrelated healthy controls were genotyped using TaqMan SNP assays. Results: The IL23R p.381Gln allele was protective against CD in the Czech population (allelic frequency 3.2% in patients vs 5.5% in control subjects; OR 0.56, 95% CI 0.33–0.93, P = 0.02). ATG16L1 p.197Ala allele conferred increased risk of CD (allelic frequency 60% in patients vs 51% in controls; OR 1.25, 95% CI 1.02–1.52, P = 0.03). There was no appreciable difference in the effect of the associated alleles across the strata of CARD15-conferred risk. The IL23R and ATG16L1 variants did not influence the age at diagnosis, and in the genotype-phenotype analysis, the only detected association was a weak one between IL23R p.381Gln and involvement of the upper gastrointestinal tract (uncorrected P = 0.031). Conclusions: We confirmed the role of IL23R and ATG16L1 in the CD susceptibility in the Czech population, and found a weak protective effect of IL23R p.381Gln against upper gastrointestinal tract involvement.

Intestinal microbiota in exclusively breast-fed infants with blood-streaked stools

Intestinal microbiota in exclusively breast-fed infants with blood-streaked stools and in healthy exclusively breast-fed babies was compared. Total anaerobes, bifidobacteria, lactobacilli, coliform bacteria, enterococci and clostridia were quantified by cultivation methods in feces of 17 full-term exclusively breastfed patients (aged 16.3 ± 7.4 weeks) with blood-streaked stools and in the control group of 22 healthy fullterm exclusively breast-fed infants (13.7 ± 6.4 weeks). Specific fluorescence in situ hybridization kits for Bifidobacterium spp. were used for the quantitative detection of bifidobacteria in samples. Control samples had significantly (p < 0.05) higher counts of total anaerobes. Bifidobacteria were not detected in patients’ samples in 65 % and in controls in 36 % (p < 0.01). Bifidobacteria counts were also significantly higher in the control group (p < 0.01). Furthermore, clostridia strains were detected only in feces from bifidobacteria-negative infants reaching counts >8 log CFU/g. Lactobacilli were not detected in 65 % patients and in 45 % control samples. However, this difference was not significant as well as the difference in lactobacilli counts. Eosinophilia was observed in 35 % of patients, low IgA concentration in 71 % and also low IgG concentration in 71 %. pANCA positivity was found in 53 % of patients. In conclusion a significant low proportion of bifidobacterial microbiota in patients with blood-streaked stools was shown in comparison with controls.