Renata Auricchio

Randomized Feeding Intervention in Infants at High Risk for Celiac Disease

BACKGROUND A window of opportunity has been suggested for reducing the risk of celiac disease by introducing gluten to infants at 4 to 6 months of age. METHODS We performed a multicenter, randomized, double-blind, placebo-controlled dietary-intervention study involving 944 children who were positive for HLA-DQ2 or HLA-DQ8 and had at least one first-degree relative with celiac disease. From 16 to 24 weeks of age, 475 participants received 100 mg of immunologically active gluten daily, and 469 received placebo. Anti-transglutaminase type 2 and antigliadin antibodies were periodically measured. The primary outcome was the frequency of biopsy-confirmed celiac disease at 3 years of age. RESULTS Celiac disease was confirmed by means of biopsies in 77 children. To avoid underestimation of the frequency of celiac disease, 3 additional children who received a diagnosis of celiac disease according to the 2012 European Society for Pediatric Gastroenterology, Hepatology, and Nutrition diagnostic criteria (without having undergone biopsies) were included in the analyses (80 children; median age, 2.8 years; 59% were girls). The cumulative incidence of celiac disease among patients 3 years of age was 5.2% (95% confidence interval [CI], 3.6 to 6.8), with similar rates in the gluten group and the placebo group (5.9% [95% CI, 3.7 to 8.1] and 4.5% [95% CI, 2.5 to 6.5], respectively; hazard ratio in the gluten group, 1.23; 95% CI, 0.79 to 1.91). Rates of elevated levels of anti-transglutaminase type 2 and antigliadin antibodies were also similar in the two study groups (7.0% [95% CI, 4.7 to 9.4] in the gluten group and 5.7% [95% CI, 3.5 to 7.9] in the placebo group; hazard ratio, 1.14; 95% CI, 0.76 to 1.73). Breast-feeding, regardless of whether it was exclusive or whether it was ongoing during gluten introduction, did not significantly influence the development of celiac disease or the effect of the intervention. CONCLUSIONS As compared with placebo, the introduction of small quantities of gluten at 16 to 24 weeks of age did not reduce the risk of celiac disease by 3 years of age in this group of high-risk children. (Funded by the European Commission and others; PreventCD Current Controlled Trials number, ISRCTN74582487.).

Effective Detection of Human Leukocyte Antigen Risk Alleles in Celiac Disease Using Tag Single Nucleotide Polymorphisms

Background The HLA genes, located in the MHC region on chromosome 6p21.3, play an important role in many autoimmune disorders, such as celiac disease (CD), type 1 diabetes (T1D), rheumatoid arthritis, multiple sclerosis, psoriasis and others. Known HLA variants that confer risk to CD, for example, include DQA1*05/DQB1*02 (DQ2.5) and DQA1*03/DQB1*0302 (DQ8). To diagnose the majority of CD patients and to study disease susceptibility and progression, typing these strongly associated HLA risk factors is of utmost importance. However, current genotyping methods for HLA risk factors involve many reactions, and are complicated and expensive. We sought a simple experimental approach using tagging SNPs that predict the CD-associated HLA risk factors. Methodology Our tagging approach exploits linkage disequilibrium between single nucleotide polymorphism (SNPs) and the CD-associated HLA risk factors DQ2.5 and DQ8 that indicate direct risk, and DQA1*0201/DQB1*0202 (DQ2.2) and DQA1*0505/DQB1*0301 (DQ7) that attribute to the risk of DQ2.5 to CD. To evaluate the predictive power of this approach, we performed an empirical comparison of the predicted DQ types, based on these six tag SNPs, with those executed with current validated laboratory typing methods of the HLA-DQA1 and -DQB1 genes in three large cohorts. The results were validated in three European celiac populations. Conclusion Using this method, only six SNPs were needed to predict the risk types carried by >95% of CD patients. We determined that for this tagging approach the sensitivity was >0.991, specificity >0.996 and the predictive value >0.948. Our results show that this tag SNP method is very accurate and provides an excellent basis for population screening for CD. This method is broadly applicable in European populations.

Natural History of Potential Celiac Disease in Children

BACKGROUND & AIMS The presence of celiac disease-associated autoantibodies (antiendomysium and antitissue transglutaminase [anti-TG2]) with normal jejunal mucosa indicate potential celiac disease. We performed a prospective, 3-year cohort study to determine the natural history of potential celiac disease in children. METHODS The study included 106 children with potential celiac disease, based on serology analysis and normal duodenal architecture. All but 2 carried the HLA-DQ2 and/or DQ8 haplotype. In all children, every 6 months, growth, nutritional parameters, celiac disease serology, and autoimmunity were investigated. In biopsies, γδ intraepithelial-, CD3-, and lamina propria CD25-positive cells were counted; duodenal deposits of anti-TG2 immunoglobulin A were detected. Biopsy analysis was repeated after 2 years on patients with persistent positive serology and/or symptoms. RESULTS Celiac disease was detected primarily in first-degree relatives and patients with autoimmune disorders (40.6%). A gluten-free diet was prescribed to 20/106 patients because of symptoms, which were relieved in only 11. Eighty-nine of the 106 patients entered the follow-up study, with normal daily consumption of gluten. During the follow-up antibodies disappeared in 14.6% and fluctuated in 32.6%. Villous atrophy was observed in 12/39 patients (30.8%) who underwent a repeat biopsy. CONCLUSIONS Most children with potential celiac disease remain healthy. After 3 years, approximately 33% of patients develop villous atrophy. Intestinal deposits of anti-TG2 IgA identify children at risk for villous atrophy.

Systematic review: early infant feeding and the prevention of coeliac disease

PREVENTCD, Prevent Coeliac Disease, is an international project investigating the hypothesis of possible induction of tolerance to gluten in genetically predisposed children through introducing small quantities of gluten during the period of breastfeeding.

Safety for Patients With Celiac Disease of Baked Goods Made of Wheat Flour Hydrolyzed During Food Processing

BACKGROUND & AIMS Celiac disease (CD) is characterized by an inflammatory response to wheat gluten, rye, and barley proteins. Fermentation of wheat flour with sourdough lactobacilli and fungal proteases decreases the concentration of gluten. We evaluated the safety of daily administration of baked goods made from this hydrolyzed form of wheat flour to patients with CD. METHODS Patients were randomly assigned to consumption of 200 g per day of natural flour baked goods (NFBG) (80,127 ppm gluten; n = 6), extensively hydrolyzed flour baked goods (S1BG) (2480 ppm residual gluten; n = 2), or fully hydrolyzed baked goods (S2BG) (8 ppm residual gluten; n = 5) for 60 days. RESULTS Two of the 6 patients who consumed NFBG discontinued the challenge because of symptoms; all had increased levels of anti-tissue transglutaminase (tTG) antibodies and small bowel deterioration. The 2 patients who ate the S1BG goods had no clinical complaints but developed subtotal atrophy. The 5 patients who ate the S2BG had no clinical complaints; their levels of anti-tTG antibodies did not increase, and their Marsh grades of small intestinal mucosa did not change. CONCLUSIONS A 60-day diet of baked goods made from hydrolyzed wheat flour, manufactured with sourdough lactobacilli and fungal proteases, was not toxic to patients with CD. A combined analysis of serologic, morphometric, and immunohistochemical parameters is the most accurate method to assess new therapies for this disorder.

Filamin A Is Mutated in X-Linked Chronic Idiopathic Intestinal Pseudo-Obstruction with Central Nervous System Involvement

We have previously reported that an X-linked recessive form of chronic idiopathic intestinal pseudo-obstruction (CIIPX) maps to Xq28. To select candidate genes for the disease, we analyzed the expression in murine fetal brain and intestine of 56 genes from the critical region. We selected and sequenced seven genes and found that one affected male from a large CIIPX-affected kindred bears a 2-bp deletion in exon 2 of the FLNA gene that is present at the heterozygous state in the carrier females of the family. The frameshift mutation is located between two close methionines at the filamin N terminus and is predicted to produce a protein truncated shortly after the first predicted methionine. Loss-of-function FLNA mutations have been associated with X-linked dominant nodular ventricular heterotopia (PVNH), a central nervous system (CNS) migration defect that presents with seizures in females and lethality in males. Notably, the affected male bearing the FLNA deletion had signs of CNS involvement and potentially has PVNH. To understand how the severe frameshift mutation we found can explain the CIIPX phenotype and its X-linked recessive inheritance, we transiently expressed both the wild- type and mutant filamin in cell culture and found that filamin translation can start from either of the two initial methionines in these conditions. Therefore, translation of a normal shorter filamin can occur in vitro from the second methionine downstream of the 2-bp insertion we found. We confirmed this, demonstrating that the filamin protein is present in the patients lymphoblastoid cell line that shows abnormal cytoskeletal actin organization compared with normal lymphoblasts. We conclude that the filamin N terminal region between the initial two methionines is crucial for proper enteric neuron development.

Immunoglobulin A anti-tissue transglutaminase antibody deposits in the small intestinal mucosa of children with no villous atrophy.

Objectives: Anti-tissue transglutaminase (anti-TG2) immunoglobulin A (IgA) autoantibodies are detectable in the serum of most patients with untreated celiac disease (CD). Their deposits in the intestine of patients with CD with severe enteropathy are considered specific for this condition. The histological spectrum of CD includes cases with normal villous architecture. The aim of this study was to look for anti-TG2 IgA deposits in the intestine of children with normal villous architecture and to relate them with other markers of gluten sensitivity. Patients and Methods: A total of 57 children with normal duodenal villous architecture and markers of gluten sensitivity were considered. Of those, 39 showed positive serum anti-endomysium antibodies and/or high levels of anti-TG2 antibodies (group 1), and 18 were seronegative with only a greater density of γδ intraepithelial lymphocytes (group 2). Thirty-four children with no markers of gluten sensitivity and a normal mucosa represented the control group (group 3). The duodenal sections of all patients were investigated for deposited anti-TG2 IgA by double immunofluorescence. Human lymphocyte antigen molecular typing was performed. Results: In group 1 and in group 2, 33 of 39 children (85%) and 12 of 18 children (66%) showed subepithelial anti-TG2 IgA intestinal deposits, respectively. Only in 3 of 34 (8.8%) children with no markers of gluten sensitivity were anti-TG2 IgA deposits noted. Conclusions: A subgroup of children with no serum CD-associated autoantibodies, but greater density of γδ intraepithelial lymphocytes, shows a clear anti-TG2 IgA deposition in the duodenal mucosa. These children must be investigated further for possible gluten sensitivity.

The preventCD study design: towards new strategies for the prevention of coeliac disease

Background PreventCD (www.preventcd.com) is a European multicentre study, which studies the influence of infant nutrition, and that of genetic, immunologic and environmental factors, on the risk of developing coeliac disease (CD). The hypothesis is that it is possible to induce tolerance to gluten by introducing small quantities of gluten to infants, preferably while they are still being breast-fed, and that this might also reduce the risk for related autoimmune disorders. Aim To describe the design of this ongoing European CD research project. Methods PreventCD encompasses two study designs and two study populations: (i) a European multicentre study: a prospective, double-blind, randomized dietary-intervention study among infants from families with high risk of CD, and (ii) a Swedish population-based CD screening study among 12-year-olds from the general population, divided into two birth cohorts that differ with respect to infant feeding practices. Discussion PreventCD is expected to elucidate some of the genetic and immunological mechanisms involved in the process of immune intolerance.

In NIH-3T3 Fibroblasts, Insulin Receptor Interaction with Specific Protein Kinase C Isoforms Controls Receptor Intracellular Routing*

Insulin increased protein kinase C (PKC) activity by 2-fold in both membrane preparations and insulin receptor (IR) antibody precipitates from NIH-3T3 cells expressing human IRs (3T3hIR). PKC-α, -δ, and -ζ were barely detectable in IR antibody precipitates of unstimulated cells, while increasing by 7-, 3.5-, and 3-fold, respectively, after insulin addition. Preexposure of 3T3hIR cells to staurosporine reduced insulin-induced receptor coprecipitation with PKC-α, -δ, and -ζ by 3-, 4-, and 10-fold, respectively, accompanied by a 1.5-fold decrease in insulin degradation and a similar increase in insulin retroendocytosis. Selective depletion of cellular PKC-α and -δ, by 24 h of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure, reduced insulin degradation by 3-fold and similarly increased insulin retroendocytosis, with no change in PKC-ζ. In lysates of NIH-3T3 cells expressing the R1152Q/K1153A IRs (3T3Mut), insulin-induced coprecipitation of PKC-α, -δ, and -ζ with the IR was reduced by 10-, 7-, and 3-fold, respectively. Similar to the 3T3hIR cells chronically exposed to TPA, untreated 3T3Mut featured a 3-fold decrease in insulin degradation, with a 3-fold increase in intact insulin retroendocytosis. Thus, in NIH-3T3 cells, insulin elicits receptor interaction with multiple PKC isoforms. Interaction of PKC-α and/or -δ with the IR appears to control its intracellular routing.

Gluten sensitivity in a subset of children with insulin dependent diabetes mellitus

Abstract Objectives The association between celiac disease and insulin dependent diabetes mellitus (IDDM) is well established. Rectal gluten challenge has been used in patients with celiac disease and in first degree relatives as a tool to assess the mucosal immune response to gluten. The aim of this study was to assess the mucosal immune response to gluten in IDDM children by rectal gluten challenge. Methods Rectal biopsy specimens were obtained from 19 children with IDDM before and 6 h after rectal challenge with 2 g of a peptic tryptic digest of gliadin. A total of 16 treated celiac patients and 10 control subjects were also investigated. Epithelium and lamina propria CD3+ and γδ+ lymphocytes were counted with reference to a standard reference area of muscolaris mucosae (104 μm2). Results After a local instillation of gliadin, a significant (>mean + 1 SD) percentage increment of lamina propria and epithelium CD3+ and of lamina propria and epithelium γδ+ lymphocytes was observed in five IDDM children, as compared to 11 and 13 celiac patients and one and two controls, respectively. A discriminant analysis allowed correct classification of 100% of patients with celiac disease and controls. The same analysis classified four of 19 IDDM children in the group of celiac patients. The positivity was associated with normal serology (antigliadin antibody, antiendomysial antibody, and antitissue transglutaminase antibodies) and a morphologically normal jejunal mucosa. All four patients had HLA-DQ alleles associated with celiac disease. Conclusions Approximately 20% of IDDM children react to rectal instillation of gliadin. Long term follow-up is necessary to establish whether these subjects are at increased risk for developing celiac disease.