***NOTE: I wrote this paper in late 2013 for an audience of medical professionals, and I have been asked by other practitioners and patients to post it here to help clarify research and approach to gluten allergy. Research is always evolving, and I wanted to share both the literature available and the my belief in the importance of non-laboratory (elimination/challenge diet) assessment in some cases. To access information from or attend my monthly classes in Seattle and the greater Eastside, like my facebook page or follow me on twitter @DoctorGlynn. For more information on my practice or to make an appointment, visit the Naturopathic Clinic of Issaquah website or call (425)391-1080.
Celiac Disease & Gluten-Associated Conditions:
Using Laboratory Measures to Clarify Etiology and Determine Course of Treatment
The concept of gluten allergy has been around for many years, but only recently has the term become ubiquitous. More patients than ever are entering clinics with self-diagnoses of various reactions to gluten, leaving practitioners to decipher the intricacies of gluten-induced symptoms. Adding to the confusion is the assumption by many that gluten allergy and its autoimmune counterpart, celiac disease, are the same disease entity. This article will attempt to clarify this misconception in the exploration of terminology, pathophysiology, changing clinical picture, and differential diagnosis using laboratory medicine.
Appropriate Terminology for Gluten-Related Disorders
Historically, one of the greatest impediments to accurate assessment and treatment of gluten-induced symptoms was the lack of a standardized diagnostic criteria for food allergy in general. In 2010, the National Institute of Allergy and Infectious Diseases sought to remedy this situation, creating a schematic for the wide scope of adverse food reactions. Two main subcategories created by the expert panel are immune-mediated and non-immune mediated, the former encompassing food allergy and celiac disease and the latter encompassing food intolerances. Under these guidelines, any abnormal antibody response to gluten would be considered an allergy. However, the guidelines go on to assert that IgG antibodies should not be used to assess patients for food allergy. The use of IgA is implied in the schematic by the term “non-IgE mediated” but is never discussed as a marker of food allergy.
Immunoglobulins Produced in Response to Gluten Exposure
Some discourse still exists over whether non-IgE mediated reactions should be considered true allergy. IgE has long been the standard laboratory measurement for classical allergic response, its secretion in the body resulting in histamine release by mast cells and potential anaphylaxis. Skin-prick testing is the most common form of assessing IgE-induced allergy symptoms, although serum IgE assays can also be used in conjunction with other clinical and laboratory measures to identify allergy in children and adults.¹ IgE antibodies to gluten have been found in patients with atopic dermatitis and urticaria, and are beginning to be used in conjunction with skin prick testing to diagnose classic wheat allergy.
IgA is the first line of defense in mucosal immunity. It was discovered in the late 1960s that IgA-producing lymphocytes in the gastrointestinal system exist in twenty times greater quantity than those producing IgG. In a healthy gastrointestinal tract, enterocytes secrete IgA to inhibit colonization and invasion by various pathogens. IgA decreases antigen entry into the tissue space and activates lymphocytes; these cells then establish a common mucosal immunity by passage through the lymphatic system to other mucosal sites and subsequent secretion of antigen-specific IgA.  Because of this common mucosal immunity, salivary IgA offers a convenient way to screen for immune-mediated reaction to gluten in patients hesitant to complete a comprehensive elimination-challenge diet. It has been shown that total IgA can be elevated during states of inflammation such as inflammatory bowel disease, metabolic syndrome, and connective tissue diseases., IgA has also been shown to be depressed in family history and/or clinical presentation of atopy, making it important to screen for total IgA when running allergen-specific IgA food allergy panels. Currently, anti-TTG IgA is used as the primary antibody in the diagnosis of celiac disease.
In the greater medical community, IgG has yet to be established as a valid marker of food allergy. In fact, oral and sublingual allergy desensitization studies show that IgG antibody rises in conjunction with decreases in IgE mast cell reactivity and basophil responses. In line with this correlation, the European Academy of Allergy and Clinical Immunology (EAACI) asserts that IgG is more a marker of immunological tolerance than allergy. The National Institute of Allergy and Infectious Diseases lists allergen-specific IgG4 testing under the heading of ‘Non-Standardized and Unproven Procedures” in its 2010 Guidelines for the Diagnosis and Management of Food Allergy in the United States.¹
Despite the lack of wide-ranging acceptance for IgG-mediated food allergy, basic immunology tells us that in cases of mucosal endothelial cell destruction or when IgA is deficient, antigens from the lumen are complexed with IgG in the lamina propria.³ These immune complexes activate complement in a Type III hypersensitivity reaction and result in temporary movement of inflammatory mediators and IgG into the lumen between epithelial cells. Although the exact role of IgG in gluten allergy has yet to be elucidated in research, the immunoglobulin’s connection to enterocyte destruction and subsequent inflammation may explain why some patients’ symptoms resolve when elimination of IgG-positive foods takes place. For example, one study showed that when patients with irritable bowel syndrome (IBS) removed IgG-positive foods from their diets, they experienced relief of symptoms. Furthermore, IgG to deamidated gluten peptide (DGP) and IgG to tissue transglutaminase (TTG) are used in cases of IgA deficiency under the new guidelines for diagnosis of celiac disease.7 Patients with gastrointestinal symptoms and IgG-positive gluten assays are undoubtedly mounting an immune response. Whether IgG is acting in immune tolerance to gluten or as an indicator of allergy, however, may currently be clearer in clinical practice than in research.
Celiac Disease: Pathophysiology, Changing Clinical Picture and Diagnostic Criteria
In celiac disease, gluten intake leads to both 1) production of antibodies against TTG and 2) inflammatory cytokine release leading to enterocyte destruction. The process begins with gluten entering the tissue space of the small intestine through either paracellular of transcellular absorption. Gluten is then deamidated, forming DGP, or cross-linked to TTG, forming gluten-TTG. In the presence of HLA-DQ2 or HLA-DQ8 cell surface markers, DGP and gluten-TTG are presented to CD4+ Th1 cells by dendritic cells, initiating a Type IV hypersensitivity reaction. These CD4+ cells release IFN-gamma, which leads to the activation of the humoral immune response through the clonal expansion of B-cells. The resulting plasma cells produce IgA and IgG to gliadin and TTG. The tissue destruction component of this process is also perpetuated by IFN-gamma, which subsequently triggers lamina propria cells and fibroblasts to secrete matrix metalloproteinases. The metalloproteinases begin to degrade cellular matrix and basement membrane, while simultaneously enhancing the cytotoxicity of intraepithelial lymphocytes and NK cells. The latter facilitate apoptosis of enterocytes.
Celiac disease is therefore a mix of humoral and cellular immune responses, mediated by antibodies and various cytokines. The condition develops due to multiple factors, including genetic susceptibility, presence of antibodies to TTG and/or DGP, intestinal damage, and gluten as an environmental immunological trigger. HLA-DQ2 is positive in ninety-five percent of those with biopsy-confirmed celiac disease, and the remaining five percent have HLA-DQ8.7 These genes must be present in order for autoimmunity to develop, as they are essential to the process of generation of anti-TTG/anti-DGP antibodies and enterocyte destruction. While absence of these markers can be helpful in exclusion of celiac disease from a list of differential diagnoses, the presence of either is not diagnostic as they are common in individuals of Caucasian European descent.3 Positive HLA-DQ2 is found in approximately 25-30% of these individuals, making the assay useful for ― but not conclusive of ― diagnosis of celiac disease. It is clear that celiac disease is a very specific, genetically-influenced, autoimmune sequence of events within the umbrella of immune response to gluten, much like Hashimoto’s thyroiditis exists within the overarching diagnostic category of thyroid disease.
Along with our understanding of genetic factors, the clinical picture of celiac disease is changing. Celiac disease was originally considered a childhood condition, but the mean age of diagnosis as of 2010 was 45 years. The condition may also be more common than most practitioners realize, as about 1 in 133 people in the United States have the disease. In patients with a first-degree relative with celiac disease, prevalence increases to 1 in 22. Celiac disease was once considered an exclusively gastrointestinal disorder, but we now know the condition can manifest with extra-intestinal symptoms such as ataxia, peripheral neuropathy, skin eruptions, anemia, muscle weakness, and osteopenia. The disease also has associations with other autoimmune diagnoses, including but not limited to type I diabetes mellitus, idiopathic pulmonary hemosiderosis, systemic lupus erythmatosus, IgA nephropathy, polymyositis, and Sjögren’s syndrome.12
New diagnostic criteria from the American College of Gastroenterology (ACG) recommend anti-TTG IgA as the most sensitive and specific serologic marker for celiac disease. They also assert the significance of assessing total IgA in the diagnostic process. Separate diagnostic guidelines are laid out for IgA deficiency and include assays of anti-TTG IgG and anti-DGP IgG. In children younger than two years of age, anti-TTG IgG alone or in conjunction with anti-DGP IgG should be used due to high probability of insufficient total IgA. HLA-DQ2 and HLA-DQ8 genetic haplotypes continue to be recommended. Anti-gliadin antibodies are no longer endorsed in establishing the diagnosis of celiac disease; however, confirmatory endoscopy and biopsy of the duodenum are still required. It is now necessary that 1-2 of the requisite biopsies be in the region of the duodenal bulb in order to identify an additional 9-13% of celiac disease patients.7 A positive intestinal biopsy will reveal villous atrophy.3
Differential Diagnosis in Gluten-Sensitive Individuals
Because the presenting symptoms of gluten-related conditions can be complex, laboratory medicine can be an important too for differentiating between autoimmune, allergic and functional conditions. The following are some of the more common diagnoses and laboratory measures to consider when encountering a patient with gluten-induced symptoms:
Inflammatory Bowel Disease
Clinical characteristics of inflammatory bowel disease (IBD) are often similar to those in celiac disease and such functional bowel disorders as irritable bowel syndrome (IBS). The diseases comprising IBD ― Crohn’s disease and ulcerative colitis ― share the common symptoms of abdominal pain, diarrhea, fatigue, fever, weight loss, and possible blood in the stool. Endoscopy and colonoscopy with biopsy are the current standards of diagnosis for these conditions, but a fecal assay of calprotectin can serve as a relatively non-invasive way to distinguish patients urgently in need of biopsy from those with functional digestive issues. Calprotectin is a protein that is released from neutrophils during active inflammatory states, and has been correlated with degree of intestinal inflammation. Patients between flares of the disease with elevated fecal calprotectin have been shown to be at greater risk of relapse within one year. Moreover, fecal calprotectin may indicate even subclinical mucosal inflammation, and therefore may help identify when an increase in naturopathic or conventional treatment is necessary. It should be noted that gastrointestinal bleeding has not been associated with levels of calprotectin, so clinical signs and symptoms must continue to be monitored to determine severity of disease progression.
Eosinophilic esophagitis (EoE) is considered one disease within the spectrum of gluten-sensitive enteropathies. The clinical presentation of this condition can closely resemble that of celiac disease and includes abdominal pain, diarrhea, steatorrhea, and nausea and vomiting after meals. Weight loss is also common in adults and children. Eighty percent of patients with EoE will have symptoms of gastroesophageal reflux that do not respond to a two-month trial of proton pump inhibitors (PPI). An endoscopy would be indicated in these cases; a diagnosis of EoE would be made if biopsy revealed greater than or equal to 15 eosinophils per high power field.
Wheat Allergy and Non-Celiac Gluten Sensitivity
Some experts argue that celiac disease, wheat allergy, and gluten sensitivity are conditions characterized by three distinct immunological responses to gliadin protein with three separate histological and prognostic results. Wheat allergy is IgE-mediated and associated with allergic symptoms minutes to hours after exposure to gluten.2Non-celiac gluten sensitivity (NCGS) is a diagnosis of exclusion to consider in patients with gluten-induced symptoms that improve on a gluten free diet but lack genetic, immunologic, and endoscopic markers of celiac disease. Anti-gliadin IgA or IgG may be present in this condition. NCGS is not typically associated with intestinal damage and permeability, in contrast to the overt enterocyte destruction that occurs in celiac disease. The elevated fecal lactoferrin level and lactulose/mannitol ratio frequently seen in IBD and celiac disease are typically normal in NCGS.
Irritable Bowel Syndrome
Irritable bowel syndrome can manifest as reactivity to multiple foods, including non-celiac gluten sensitivity (NCGS).21 The diagnosis of IBS is currently considered one of exclusion, but does have its own specific Rome III diagnostic criteria. According to these guidelines, a patient must have recurrent abdominal pain or discomfort (an uncomfortable sensation not described as pain) for at least three days per month in the last three months. This abdominal pain or discomfort must be associated with two or more of the following characteristics: improvement with defecation, onset associated with a change in stool frequency, or onset associated with a change in form (appearance) of stool. Moreover, the criteria must have been fulfilled for the last three months with symptom onset at least six months prior to diagnosis. Laboratory measures to rule out autoimmune and inflammatory conditions may include fecal calprotectin, an iron panel to assess for anemia, food immunoglobulin testing, stool testing for parasites and intestinal bacterial overgrowth, celiac disease markers, and intestinal biopsy.
Gluten-Associated Disorders: Beyond the Gluten-Free Diet
Complaints of adverse physiologic reactions to gluten are becoming more common in medical offices. Many health professionals question whether this trend is due to an actual increase in incidence, an improvement in diagnostic methods, or simply a rise in awareness. Which of these is true remains to be clarified by research, but there is no doubt that our tools for identifying food allergies and furthering our understanding of the immune system are rapidly expanding.
With any food-related symptoms or diagnoses, the astute physician would recommend identification and ― at least temporary ― removal of offending foods from the diet. And while it is essential to determine if gluten is a problematic food protein for patients, we must take further steps in laboratory diagnosis to determine a patient’s exact immunological response to gluten in order to develop appropriate treatment plans and prevent further tissue destruction. The importance of identifying celiac disease is paramount, because if left untreated it may contribute to infertility, development of other related autoimmune disorders, and a higher incidence of certain cancers including lymphomas.12
We have yet to fully understand the implications of genetic susceptibility in autoimmune diseases, but it is known that specific HLA haplotypes are also associated with type I diabetes mellitus, multiple sclerosis, and Graves’ disease. Because of the potential for food to be antigenic, the impact of diet and genetics on autoimmune conditions can be pivotal in shifting the immune response. While gluten-free diets can alleviate symptoms, it is important that we continually review the literature and use of diagnostic testing, as this is an evolving discussion and recommendations are sure to change in the future. Researchers continue to discover immunologic and genetic etiologies of gluten-induced symptoms, leading to important branching points in treatment approach. Oral or sublingual immunotherapy, for example, may be a possibility in NCGS or IBS, while in celiac disease this therapy would be contraindicated due to the potential for autoimmune gastrointestinal and systemic sequelae.8Identifying the exact pathophysiology and category of immune response for each individual can aid not only in determining the necessary length and course of a gluten-free diet, but also in preventing comorbidities and improving autoimmune prognosis.
 National Institute of Allergy and Infectious Diseases. Guidelines for the diagnosis and management of food allergy in the United States. December 2010. Available at: http://www.jacionline.org/article/S0091-6749(10)01566-6/fulltext. Published Dec 2010. Accessed September 8, 2013.
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 Sandin A, Björkstén B, Böttcher M, et al. High salivary secretory IgA antibody levels are associated with less late-onset wheezing in IgE-sensitized infants. Pediatr Allergy Immunol 2011; 22:477-481.
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 Stapel S, Asero R, Ballmer-Weber B, et al. EAACI Task Force. Testing for IgG4 against foods is not recommended as a diagnostic tool: EAAC1 Task Force Report. Allergy 2008; 63(7):793-6.
 Atkinson W, Sheldon T, Shaath N, et al. Food elimination based on IgG antibodies in irritable bowel syndrome: a randomised controlled trial. Gut 2004; 53:1459–1464.
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 Fasano A, Berti I, Gerarduzzi T, et al. Prevalence of celiac disease in at-risk and not-at-risk groups in the United States. Archives of Internal Medicine 2003; 163(3):268-292.
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Burri E, Beglinger C, et al. Fecal calprotectin – a useful tool in the management of inflammatory bowel disease. Swiss Med Wkly 2012; 142:w13557.
 Dellon E, Gonsalves N, Hirano I. ACG clinical guideline: Evidence based approach to the diagnosis and management of esophageal eosinophilia and eosinophilic esophagitis. Am J Gastroenterol 2013; 108(5):679.
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