Carmen Calvo

HLA-DQ2-Negative Celiac Disease in Finland and Spain

Genetic susceptibility to celiac disease (CD) is strongly associated with DQA1*0501 and DQB1*02 (= DQ2). To study whether CD patients without DQ2 share other MHC class II or TNF alleles, we screened DQ2-negative patients in Finland and Spain. Twelve of 84 (14%) Finnish patients and 13 of 189 (6%) Spanish patients were negative for DQ2. We observed that all but two of altogether 25 DQ2-negative patients had the DR4 DQ8 haplotype, or either DQA1*0501 or DQB1*02 alone. Also, all but three were positive for DRB4*01. The only patients without any of these alleles were both positive for DR 13. There was a clear difference between Finland and Spain: Ten (83%) of the 12 Finnish DQ2-negative patients but only five (38%) of the 13 Spanish patients had DRB1*03, DQA1*03, DQB1*0302 (= DQ8) alleles. Of the Spanish patients, eight (62%) had DQB1*02 without DQA1*0501 and three (23%) had DQA1*0501 without DQB1*02. None of the TNF, TAP, or DPB1 alleles was found to be significantly associated with CD. Our results indicate that in addition to the DQ2 heterodimer, the other major risk alleles for CD are DR4 DQ8, and either DQA1*0501 or DQB1*02 alone. Patients without these alleles appear to be very rare, only two (0.7%) were identified in altogether 253 patients tested.

Influence of Environmental and Genetic Factors Linked to Celiac Disease Risk on Infant Gut Colonization by Bacteroides Species

ABSTRACT Celiac disease (CD) is an immune-mediated enteropathy involving genetic and environmental factors whose interaction might influence disease risk. The aim of this study was to determine the effects of milk-feeding practices and the HLA-DQ genotype on intestinal colonization of Bacteroides species in infants at risk of CD development. This study included 75 full-term newborns with at least one first-degree relative suffering from CD. Infants were classified according to milk-feeding practice (breast-feeding or formula feeding) and HLA-DQ genotype (high or low genetic risk). Stools were analyzed at 7 days, 1 month, and 4 months by PCR and denaturing gradient gel electrophoresis (DGGE). The Bacteroides species diversity index was higher in formula-fed infants than in breast-fed infants. Breast-fed infants showed a higher prevalence of Bacteroides uniformis at 1 and 4 months of age, while formula-fed infants had a higher prevalence of B. intestinalis at all sampling times, of B. caccae at 7 days and 4 months, and of B. plebeius at 4 months. Infants with high genetic risk showed a higher prevalence of B. vulgatus, while those with low genetic risk showed a higher prevalence of B. ovatus, B. plebeius, and B. uniformis. Among breast-fed infants, the prevalence of B. uniformis was higher in those with low genetic risk than in those with high genetic risk. Among formula-fed infants, the prevalence of B. ovatus and B. plebeius was increased in those with low genetic risk, while the prevalence of B. vulgatus was higher in those with high genetic risk. The results indicate that both the type of milk feeding and the HLA-DQ genotype influence the colonization process of Bacteroides species, and possibly the disease risk.

Interleukin 18 maintains a long-standing inflammation in coeliac disease patients

Dietary gluten induces an early response in the intestine of coeliac disease patients (CD), within a few hours, and this is driven by high levels of proinflammatory cytokines, including IFNγ and IL‐15, as has been thoroughly shown by gluten stimulation of biopsy explants. Our aim was to identify the immune mediators involved in the long‐standing inflammation in untreated CD patients at diagnosis. mRNA and protein levels of TNFα, IL‐12(p35), IL‐12(p40), IL‐15, IL‐18 and IL‐23(p19) were quantified in biopsies from active CD patients, CD patients on a gluten‐free diet (GFD), healthy controls, and patients with non‐CD inflammation and mild histological changes in the intestine. Biopsies from CD patients on a GFD were also stimulated in vitro with gliadin, and protein expression of IL‐15 and IL‐18 was analysed. Levels of IL‐12 and IL‐23 mRNA are nearly absent, and TNFα levels remain unchanged among different groups. Both the active and inactive forms of IL‐18 protein have been found in all samples from active CD, and protein expression was only localized within the crypts. Levels of IL‐15 mRNA remain unchanged, and protein expression, localized within the lamina propria, is found in a small number of samples. In vitro stimulation with gluten induces the expression of IL‐15 and IL‐18. In active CD, the early response following gluten intake characterized by high IFNγ levels is driven by IL‐18, and probably IL‐15, and this alternates with periods of long‐standing inflammation with moderate IFNγ levels, maintained by IL‐18 alone.

TNFα and LTα gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

Abstract. TNFα and TNFβ, or linfotoxin (LTα), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFα and linfotoxin α (TNFβ) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA –308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position –308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.

Is it true that coeliacs do not digest gliadin? Degradation pattern of gliadin in coeliac disease small intestinal mucosa

Prolyl-endopeptidase supplementation has been proposed to favour gliadin degradation as an alternative treatment for coeliac disease (CD), although the real usefulness of this therapy in vivo is still under discussion.1 However, our data point to alternative treatments aiming to modify the intestinal microbiota in patients with CD by the use of probiotics and/or prebiotics. We propose that the induction of gliadin proteolysis in the human gut might not be the solution but the origin of CD. We have carried out gliadin zymograms using a complete protein solution from duodenal mucosa of patients with CD, both untreated patients with positive serology, genetics and duodenal inflammation (n = 20), and treated patients on a gluten-free diet (GFD) with negative serology and recovering mucosa (n = 9). We have also analysed 18 non-CD controls, without mucosal inflammation and negative serology. Figure 1 reveals for the first time to our knowledge, and in contrast to what would be expected, the existence of a specific gliadinase pattern in duodenal samples from patients with CD. This pattern was observed regardless of the phase …

IL6, IL10 and TGFB1 gene polymorphisms in coeliac disease: differences between DQ2 positive and negative patients

UNLABELLED Predisposition to coeliac disease (CD) might be partially due to an individual pattern of hyper-inflammatory biased immune response. One of these patterns of intense response may be linked to the haplotype carrying HLA-DQ2 alleles and TNF -308A allele. However, 10 % of CD patients do not express the DQ2 heterodimer and these do not usually carry the TNF -308A allele. A similar response might be achieved by genes codifying other cytokines. OBJECTIVES To study biallelic polymorphisms in genes codifying for TNFalpha, IL10, IL6 and TGFbeta1 in DQ2 negative CD patients and to compare the results with DQ2 positive patients and healthy controls, in order to establish whether any of these polymorphisms have a role in CD susceptibility. METHODS TNF -308 (G > A), IL-6 -174 (G > C) and TGFB1 codon 10 (+ 869, T > C) and codon 25 (+ 915, G > C) polymorphisms and IL-10 haplotype of polymorphisms in positions -1082 (G > A), -819 (C > T) and -592 (C > A) were typed by a SSP-PCR technique. RESULTS The distribution of allele frequencies for TNF -308 is different between DQ2 positive CD patients and controls and the same occurs for haplotype frequencies of the IL10 promoter (-1082, -819, -592): The frequencies of the TNF -308A allele (p = 0.027), TNF -308A carriers (p = 0.031) and of IL10GCC haplotype are increased (p = 0.013) in DQ2 positive CD patients. However, the IL6 -174 allele G is more frequent in DQ2 negative patients than in healthy controls (p = 0.018), DQ2 negative controls (p = 0,018), and DQ2 positive patients (p = 0.008). CONCLUSIONS DQ2 negative CD patients show an increased frequency of genotypes associated to IL6 high production. These were mainly allele G homozygous for the IL6 gene (-174) polymorphism. The IL6 -174GG genotype (homozygous) may be an additional risk marker for CD in DQ2 negative patients, representing an alternative susceptibility factor for CD when TNF -308A is negative.

Spectrum of CFTR mutations in the middle north of Spain and identification of a novel mutation (1341G→A)

We have analyzed 39 unrelated cystic fibrosis (CF) families by denaturing gradient gel electrophoresis (DGGE) and direct sequencing in order to determine the spectrum of CF mutations in our population. This approach has allowed us to detect 72 out of the 78 CF chromosomes (92.3%). The DF508 mutation was found to be present in 51/78 (65.4%) CF chromosomes, in accordance with the predicted Northwest‐Southeast gradient within the European population. Another 14 known mutations, and the novel 1341G→A mutation were identified. Nine out of fifteen non DF508 mutations were present in a single chromosome. The 1341G→A mutation, found in 2 unrelated patients, is a new mutation associated to severe phenotype, causing pancreatic insufficiency and chronic lung infections. Our data suggest a different distribution of non‐DF508 mutations in our population when compared with previous studies carried out in Spanish CF families. Six out of the 14 non‐F508 in our study were not present in a recent study carried out in 640 Spanish families with CF. These six mutations account for 29.6% non DF508 chromosomes in our sample. Hum Mutat 14:89, 1999.

Detection of Specific IgA Antibodies against a Novel Deamidated 8-Mer Gliadin Peptide in Blood Plasma Samples from Celiac Patients

We studied whether celiac disease (CD) patients produce antibodies against a novel gliadin peptide specifically generated in the duodenum of CD patients by a previously described pattern of CD-specific duodenal proteases. Fingerprinting and ion-trap mass spectrometry of CD-specific duodenal gliadin-degrading protease pattern revealed a new 8-mer gliadin-derived peptide. An ELISA against synthetic deamidated 8-mer peptides (DGP 8-mer) was used to study the presence of IgA anti-DGP 8-mer antibodies in plasma samples from 81 children (31 active CD patients (aCD), 17 CD patients on a gluten-free diet (GFD), 10 healthy controls (C) and 23 patients with other gastrointestinal pathology (GP)) and 101 adults (16 aCD, 12 GFD, 27 C and 46 GP-patients). Deamidation of the 8-mer peptide significantly increased the reactivity of the IgA antibodies from CD patients against the peptide. Significant IgA anti-DGP 8-mer antibodies levels were detected in 93.5% of aCD-, 11.8% of GFD- and 4.3% of GP-patients in children. In adults, antibodies were detected in 81.3% of aCD-patients and 8.3% of GFD-patients while were absent in 100% of C- and GP-patients. Duodenal CD-specific gliadin degrading proteases release an 8-mer gliadin peptide that once deamidated is an antigen for specific IgA antibodies in CD patients which may provide a new accurate diagnostic tool in CD.

Usefulness of antiendomysial antibodies as a serological marker in coeliac children

The new diagnostic criteria of coeliac disease (CD) give more importance to serological markers. Immunoglobulin A antiendomysial antibodies (IgA‐EmA) were determined in 138 sera from 79 coeliac children and the antibody levels compared to IgG and IgA antigliadin antibodies (IgG‐AGA, IgA‐AGA) in the sera. The assessment was also carried out in 29 children with other gastrointestinal diseases, 29 with non‐gastrointestinal diseases and 35 healthy children. The IgA‐EmA had a 91.4% specificity and a 88.4% sensitivity for active CD. The corresponding figures were 89.8% and 64.4% for IgA‐AGA and 73.7% and 86.2% for IgG‐AGA, respectively. The results of IgA‐EmA determinations were concordant with the intestinal biopsy findings in 90% of cases, versus 80% for IgA‐AGA and 83% for IgG‐AGA. In most of the discordant cases the biopsy showed only minor changes, making the classification difficult. All patients with positive IgA2‐EmA also had positive IgA1 EmA antibodies. IgA‐EmA are an excellent serological marker of CD activity in children and they are useful to decrease the number of intestinal biopsies which are needed to confirm the diagnosis in coeliac patients.

Increased prevalence of pathogenic bacteria in the gut microbiota of infants at risk of developing celiac disease: The PROFICEL study

ABSTRACT Celiac disease (CD) is an immune-mediated enteropathy involving genetic and environmental factors, whose interaction influences disease risk. The intestinal microbiota, including viruses and bacteria, could play a role in the pathological process leading to gluten intolerance. In this study, we investigated the prevalence of pathogens in the intestinal microbiota of infants at familial risk of developing CD. We included 127 full-term newborns with at least one first-degree relative with CD. Infants were classified according to milk-feeding practice (breastfeeding or formula feeding) and HLA-DQ genotype (low, intermediate or high genetic risk). The prevalence of pathogenic bacteria and viruses was assessed in the faeces of the infants at 7 days, 1 month and 4 months of age. The prevalence of Clostridium perfringens was higher in formula-fed infants than in breast-fed over the study period, and that of C. difficile at 4 months. Among breastfed infants, a higher prevalence of enterotoxigenic E. coli (ETEC) was found in infants with the highest genetic risk compared either to those with a low or intermediate risk. Among formula-fed infants, a higher prevalence of ETEC was also found in infants with a high genetic risk compared to those of intermediate risk. Our results show that specific factors, such as formula feeding and the HLA-DQ2 genotype, previously linked to a higher risk of developing CD, influence the presence of pathogenic bacteria differently in the intestinal microbiota in early life. Further studies are warranted to establish whether these associations are related to CD onset later in life.