Michelle Pietzak

Zonulin Upregulation Is Associated With Increased Gut Permeability in Subjects With Type 1 Diabetes and Their Relatives

Zonulin, a protein that modulates intestinal permeability, is upregulated in several autoimmune diseases and is involved in the pathogenesis of autoimmune diabetes in the BB/Wor animal model of the disease. To verify the association between serum zonulin levels and in vivo intestinal permeability in patients with type 1 diabetes, both parameters were investigated in different stages of the autoimmune process. Forty-two percent (141 of 339) of the patients had abnormal serum zonulin levels, as compared with age-matched control subjects. The increased zonulin levels correlated with increased intestinal permeability in vivo and changes in claudin-1, claudin-2, and myosin IXB genes expression, while no changes were detected in ZO1 and occludin genes expression. When tested in serum samples collected during the pre–type 1 diabetes phase, elevated serum zonulin was detected in 70% of subjects and preceded by 3.5 ± 0.9 years the onset of the disease in those patients who went on to develop type 1 diabetes. Combined, these results suggest that zonulin upregulation is associated with increased intestinal permeability in a subgroup of type 1 diabetic patients. Zonulin upregulation seems to precede the onset of the disease, providing a possible link between increased intestinal permeability, environmental exposure to non–self antigens, and the development of autoimmunity in genetically susceptible individuals.

Celiac Disease, Wheat Allergy, and Gluten Sensitivity: When Gluten Free Is Not a Fad

As the gluten-free diet (GFD) gains in popularity with the general public, health practitioners are beginning to question its real health benefits. For those patients with celiac disease (CD), the GFD is considered medical nutrition therapy, as well as the only proven treatment that results in improvements in symptomatology and small bowel histology. Those with wheat allergy also benefit from the GFD, although these patients often do not need to restrict rye, barley, and oats from their diet. Gluten sensitivity is a controversial subject, where patients who have neither CD nor wheat allergy have varying degrees of symptomatic improvement on the GFD. Conditions in this category include dermatitis herpetiformis (DH), irritable bowel syndrome (IBS), and neurologic diseases such as gluten-sensitive ataxia and autism. It is important for patients and healthcare practitioners to understand the differences between these conditions, even though they may all respond to a GFD. Patients with CD can experience comorbid nutrition deficiencies and are at higher risk for the development of cancers and other autoimmune conditions. Those with wheat allergy and gluten sensitivity are thought not to be at higher risk for these complications. Defining the symptoms and biochemical markers for gluten-sensitive conditions is an important area for future investigations, and high-quality, large-scale randomized trials are needed to prove the true benefits of the GFD in this evolving field.

Stratifying Risk for Celiac Disease in a Large At-Risk United States Population by Using HLA Alleles

BACKGROUND & AIMS Susceptibility to celiac disease (CD) is related to HLA-DQ2 and DQ8 alleles and the heterodimers they encode. The objective of this study was to stratify risk for CD on the basis of HLA-DQ genotype. METHODS DNA from 10,191 subjects who are at risk for CD was analyzed for HLA-DQ haplotypes. Individuals with CD were identified as those who tested positive for anti-endomysial immunoglobulin A (EMA+) in an immunofluorescence assay. RESULTS Samples homozygous for DQ2.5 (HLA-DQA1 05-DQB1 02) or DQ2.2/DQ2.5 (HLA-DQA1 05-DQB1 02 and HLA-DQA1 0201-DQB1 02) comprised 5.38% of the total; 28.28% of these were EMA+ (95% confidence interval [CI], 24.55-32.26). Of the samples that were DQ2.5 heterozygous (HLA-DQA1 05-DQB1 02); 9.09% were EMA+ (95% CI, 7.82-10.51). Among samples in which HLA-DQ8 (HLA-DQA1 03-DQB1 0302) was detected, 8.42% of homozygotes (95% CI, 3.71-15.92) and 2.11% of heterozygotes (95% CI, 1.43-3.00) were EMA+. Samples with DQ2.2/DQ8 or DQ2.5/DQ8 comprised 5.08% of the total, and 11.78% of these were EMA+ (95% CI, 9.13-14.87). HLA-DQ2 and HLA-DQ8 were absent in 4283 samples (42.03% of the total); 0.16% of these samples were EMA+ (95% CI, 0.07-0.34). CONCLUSIONS High-resolution, sequence-specific oligonucleotide probe typing with 35 DQA1-specific and 37 DQB1-specific probes of DNA from more than 10,000 subjects was used to stratify risk of CD in an at-risk U.S. population. DQ2 homozygosity (DQ2.5/DQ2.2+2.5) increased risk for CD, estimated by the rate of EMA positivity, compared with the entire sample population and other DQ genotypes. These data suggest a quantitative relationship between the type/proportion of DQ heterodimers and the risk of CD and identify potential immunotherapeutic targets.

Bacterial colonization of the neonatal gut.

The indigenous microflora of the human gastrointestinal tract are not merely innocent bystanders, but rather play a critical role in normal physiology as well as in the modulation of systemic disease states. At birth, the human gastrointestinal tract is sterile, but within hours is colonized by ingested environmental and maternal coliforms and streptococci (1–4). It is well known that animals raised in a germ-free environment lack intestinal bacterial flora (5). Evidence that colonization occurs via the oral route comes from studies of newborn infants with intestinal obstruction, which reveal a sterile distal bowel with colon-like flora proximal to the obstruction (6). Rates and patterns of neonatal colonization are influenced by the route of delivery, hygiene of the neonatal environment, maternal bacterial flora and diet (7). By one week of age, healthy full-term infants delivered vaginally are colonized by anaerobic bacteria, predominantly Bacteroides (8). However, infants delivered by Caesarian section exhibit delayed colonization by anaerobes, and are colonized by non-E. coli Enterobacteriaceae (1). Remarkably, the flora of infants delivered via C-section resembles that of the surrounding hospital environment more than that of their mothers! Formula and breast-fed infants have very different early intestinal bacterial flora (9). The predominant bacteria of breast-fed infants are lactobacilli and bifidobacteria, which may outnumber Enterobacteriaceae by 1000-fold (10,11). The stools of formula fed infants contain predominantly Enterobacteriaceae. Of interest, the flora of the formula-fed infant develops more quickly than that of the breast-fed infant (12). This may be in part due to the iron in infant formulas, which fosters the growth of a more complex flora (13). Shortly before and after weaning, the composition of the microflora shifts to a more adult pattern, with fewer E. coli and Clostridium spp, and more Bacteroides and gram-positive anaerobic cocci (14,15). The human gastrointestinal tract is the home to more than 400 species of aerobic and anaerobic bacteria, residing in specific ‘niches‘ throughout the gut. Because of peristalsis and the antimicrobial effects of gastric acid, the microflora of the stomach (with the exception of H. pylori), is composed mostly of gram-positives at a concentration of <10 per gram of intraluminal fluid (16,17). The ileum is a ‘microbiologic transition zone‘ from the sparse jejunal aerobic flora to the dense anaerobic bacteria of the colon, where gram-negatives predominate at 10 to 10 organisms per gram of luminal content (18). In this respect, humans are more prokaryotic than eukaryotic! The impact of this internal environment on the development of chronic gastrointestinal and systemic inflammatory diseases is only now being defined and appreciated. The maturation and integrity of the neonatal immune system is dependent upon exposure to microbial antigens. In germ free animals, bacterial colonization causes inflammation and increased cellularity of the lamina propria (19–22). The normal gut microflora may also protect the infant from enteric pathogens by competing with pathogens for nutrients, by degrading their toxins, and by interfering with their adherence and growth (4,5,23–26). Normal flora also secrete antibacterial substances which protect against pathogens (27,28). In this issue of JPGN, Bailey et al. propose that bacterial colonization of the gut might be impacted by stress in the pregnant mother (29). Stress is presumed to suppress normal mucosal immunity and enhance the growth of enteric pathogens by enhancing the secretion of stressinduced neurohormones (30–35). Previous work by Bailey and Coe (36), also in infant rhesus monkeys, suggests that the stress produced by the disruption of the maternal-infant bond may increase vulnerability to disease. The stools of infant monkeys separated from their mothers contained significantly fewer bacteria, especially lactobacilli. Behavioral and cortisol responses to separation were correlated with the microflora and the Address correspondence and reprint requests to Dr. Michelle Pietzak, Childrens Hospital Los Angeles, Gastroenterology, Mailstop #78, 4650 Sunset Boulevard, Los Angeles, CA 91207 (e-mail: mpietzak@chla.usc.edu). Journal of Pediatric Gastroenterology and Nutrition 38:389–391

Celiac Disease: Going Against the Grains

Celiac disease (CD) occurs in genetically predisposed individuals who demonstrate a permanent intolerance to gluten, found in wheat, barley, and rye. This leads to the development of an autoimmune enteropathy, resulting in the malabsorption of critical vitamins, minerals, and calories. Signs and symptoms of the disease classically include diarrhea, short stature, iron-deficient anemia, and chronic abdominal pain. However, patients may present with another autoimmune disease (such as type I diabetes) or a family history of the disease, with no gastrointestinal symptoms. Serum antibodies can be used to screen for CD. However, the keys to confirming the diagnosis remain a small intestinal biopsy and the patients clinical response to a gluten-free diet. Clinicians in the United States must maintain a high index of suspicion for CD, because it is significantly underdiagnosed in this country.

Chapter 11 – Immunologic Reactions to Wheat: Celiac Disease, Wheat Allergy and Gluten Sensitivity

Although wheat is a staple in the human diet, many would argue that humans are not evolved to digest it properly. Immune reactions to the foreign proteins in wheat can result in a variety of symptoms. In celiac disease, genetically predisposed patients react to the gliadin fraction of wheat gluten, resulting in both intestinal and extra-intestinal manifestations. In wheat allergy, an IgE-mediated response can result in gastrointestinal, skin, and respiratory symptomatology. Gluten sensitivity has an evolving definition, but is thought to occur when patients who have neither celiac disease nor wheat allergy have varying degrees of symptomatic improvement upon dietary gluten withdrawal. Conditions in this category include dermatitis herpetiformis, irritable bowel syndrome, and neurologic conditions such as autism and gluten-sensitive ataxia. It is important for patients and healthcare practitioners to understand the differences between these conditions, even though they may all respond to similar dietary interventions, because of their potential health complications.Abstract Although wheat is a staple in the human diet, many would argue that humans are not evolved to digest it properly. Immune reactions to the foreign proteins in wheat can result in a variety of symptoms. In celiac disease, genetically predisposed patients react to the gliadin fraction of wheat gluten, resulting in both intestinal and extra-intestinal manifestations. In wheat allergy, an IgE-mediated response can result in gastrointestinal, skin, and respiratory symptomatology. Gluten sensitivity has an evolving definition, but is thought to occur when patients who have neither celiac disease nor wheat allergy have varying degrees of symptomatic improvement upon dietary gluten withdrawal. Conditions in this category include dermatitis herpetiformis, irritable bowel syndrome, and neurologic conditions such as autism and gluten-sensitive ataxia. It is important for patients and healthcare practitioners to understand the differences between these conditions, even though they may all respond to similar dietary interventions, because of their potential health complications.

Dietary Supplements in Celiac Disease

Celiac disease (CD) is an immune-mediated disorder that occurs in genetically predisposed individuals who ingest gluten. Gluten is a dietary protein found in wheat, barley, and rye. In the susceptible person, this gluten-containing diet can lead to the development of an autoimmune enteropathy, causing malabsorption of carbohydrates, proteins, fats, and critical vitamins and minerals. Deficiencies of key nutrients in celiac disease occur due to malabsorption, poor dietary intake, and lack of fortification of gluten-free foodstuff. This chapter reviews the data regarding common dietary supplements that may enhance a healthy gluten-free lifestyle, including iron, B-complex vitamins, fat-soluble vitamins, trace metals and minerals, fiber, and probiotics. Special attention is given to dietary supplements that promote bone health, pregnancy, and fertility, and alleviate symptoms in refractory celiac disease (RCD). Clinicians must maintain a high index of suspicion for these nutritional deficiencies, as they may present with a myriad of symptoms that closely mimic other disorders. If diagnosed early, the nutritional complications of long-standing celiac disease can be completely avoided by strict adherence to the gluten-free diet and appropriate dietary supplements.

A 9-Year-Old Girl With Poor Weight Gain and Postprandial Vomiting

Question: A 9-year-old girl presented with epigastric pain, postprandial vomiting, anorexia, and poor weight gain for 6 months. On examination, she was less than the fifth percentile for height, weight, and body mass index. An esophagogastroduodenoscopy (EGD) report from 3 months before arrival to our hospital revealed erythematous gastric mucosa with ulcerations and edema of the pyloric canal. She was treated for Helicobacter pylori and cytomegalovirus (CMV) without improvement. Laboratory results showed the following: White blood cell count 4.4 10/L (27.3% neutrophils, 64% lymphocytes, 6.6% monocytes, 1.4% eosinophils, 0.7% basophils), hemoglobin 9.5 g/dL, mean corpuscular volume 82 fL, red cell distribution width 24.2%, platelet count 556 10/L, total protein 4.7 g/dL, and albumin 2.5 g/dL. Review of records showed negative anti-neutrophil cytoplasmic antibodies, anti-Saccharomyces cerevisiae antibodies (ASCA), and tissue transglutaminase immunoglobulin (Ig)G and IgA. Serum total IgA levels were normal. Upper gastrointestinal series showed thickened gastric folds and narrowing of the pyloric canal (Figure A). Repeat EGD showed erythematous and friable mucosa, superficial ulcerations, and nodularity (Figure B–E). Friability, ulceration, and scarring of the prepyloric area and pylorus were appreciated; therefore, the duodenum was not able to be visualized. Colonoscopy was unremarkable. Histologic sections of the gastric biopsy specimens are shown in Figures F–I. What is the diagnosis? Look on page 905 for the answer and see the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI.

Yellow flank discoloration and abdominal distension in an infant.

Division of Gastroenterology and NutritionQuestion: A7-month-old infant pre-sented to the hospitalwith vomiting andabdominal distensionfor 3 days. On exami-nation, the baby waswell developed andalert. Abdomen wasnontender and dis-tended with hypo-active bowel sounds.Faint, localized yellowdiscoloration wasnoted over the bilat-eral flanks (Figure A).Laboratory results revealed the following: white blood cell count, 11.2 K/cumm (29.6% neutrophils, 58.4% lympho-cytes, 11.0% monocytes, 0.5% eosinophils, 0.5% basophils); hemoglobin, 10.0 g/dL; platelet count, 333 K/cumm; alkalinephosphatase, 83 U/L; total protein, 4.3 g/dL; albumin, 2.9 g/dL; aspartate aminotransferase, 33 U/L; alanine amino-transferase, 14 U/L; total bilirubin, 1.5 U/L; direct bilirubin, 1.0 U/L; amylase, 103; and lipase, 30. Urine analysis wasicototest positive. Fecal occult blood test was guaiac negative. Plain abdominal radiograph showed dilated small and largebowel suggestive of partial obstruction versus ileus (Figure B). Abdominal ultrasonography was ordered.What is the diagnosis?Look on page 000 for the answer and see the Gastroenterology web site (www.gastrojournal.org) for moreinformation on submitting your favorite image to Clinical Challenges and Images in GI.

Comparative study between American and Italian families of Celiac disease patients based on HLA profiles

Abstract Objective: To identify the HLA profiles of American celiac patients and their relatives and to compare the HLA profile of Italian and American families. Celiac disease (CD) is an autoimmune disorder triggered by gluten ingestion in genetically predisposed subjects. HLA class II alleles DQA1*0501/DQB1*02 and DQB1*0302 (encoding DQ2 and DQ8 haplotype respectively) represent the genetic markers for CD. Methods: Fourteen American celiac families (14 CD patients and 38 first degree relatives, including both parents) and 14 Italian celiac families (14 CD patients and 45 first degree relatives, including both parents) were included in this study. DNA was extracted from whole blood samples. Using the Eu-DQ ® Kit (Eurospital, Trieste—Italy), we performed the HLA typing here described. This Kit is composed of two multiplex PCR reactions, one containing DQα1*0501–DQβ1*0302 primers and Beta globin primers as internal control. The second one contains DQβ1*02 and Beta globin primers as internal control. The amplicons obtained were revealed on 2% agarose gel that is stained using Ethidium Bromide. Results: The prevalence of DQ2 alleles among American celiac patients was 64.3% as compared to 85.7% found among the Italian celiac patients (Table 1) Table 1 . American Controls Italian Controls N subjects screened 52 50 59 50 CD patients 14 14 Relatives of CD pts 38 45 Celiac DQ2 9 (64.3%) 12 (85.7%) Celiac DQ8 3 (21.4%) 2 (14.3%) Celiac DQ2/DQ8 2 (14.3%) 0 (0%) . Both the DQ8 alleles and the DQ2/DQ8 heterodimer were more frequent among the American CD subjects than the Italian patients. Interestingly, a similar difference between American and Italian subjects was also observed among the first degree relatives screened (Table 1) . Conclusions: American CD patients and their first degree relatives have a higher prevalence of DQ8 alleles, either alone or in combination with DQ2, as compared to Italian CD families.