Antibody Array 22 – Irritable Bowel / SIBO ScreenTM

Cyrex’s Array 22 – Irritable Bowel / SIBO Screen assesses IgG, IgA and IgM antibodies to Bacterial Cytotoxins and to Cytoskeletal Proteins. Cytotoxins are lethal xenobiotics released by commensal coliform bacteria. Bacterial cytotoxins can destroy epithelial cells of the intestinal barrier. Therefore, Cyrex also assesses antibodies to cytoskeletal proteins. Once this epithelial cell damage occurs, bacterial cytotoxins can freely enter circulation. Like systemic lipopolysaccharides (Array 2 – Intestinal Antigenic Permeability Screen), when bacterial cytotoxins enter the blood stream, they produce chronic inflammation and can open the blood brain barrier (Array 20 – Blood-Brain Barrier Permeability Screen).

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder. Despite this, as many as about 75% of affected individuals do not seek medical care.4 This is partially due to the lack of sensitive and specific evaluation strategies that has resulted in frustration for both patients and healthcare providers.

A large portion of IBS occurs exclusively after infection with bacteria such as E. coli, Samonella, Shigella and Campylobacter jejuni.1 2 All these organisms produce in common cytolethal distending toxins, which have a major role in the disruption of both tight junction and cytoskeletal proteins such as vinculin, talin and α-actinin, thus weakening the epithelial cell structures. Dysregulated immunity, low-grade inflammation and altered gastrointestinal (GI) permeability, the presence of mood disorders, fatigue, fibromyalgia and migraine found in up to 70% of patients add complexity to the accurately diagnosing this disorder.5

Currently, diagnosis is made by exclusion of disorders that mimic IBS and by focusing on IBS common symptoms. Furthermore, it is common for patients with small intestinal bacterial overgrowth (SIBO) have symptomatologies that overlap with IBS.6 SIBO is defined as the presence in the small intestine of excessive gut bacteria that originates from the colon.

In IBS and SIBO patients, the goals of lab testing are to determine the cause and to establish the diagnosis as early as possible in order to initiate successful treatment regimens.

One such lab test that was established based on animal models and in human subjects with IBS/SIBO is the measurement of antibodies against bacterial cytotoxins and cytoskeletal proteins in human blood.7 8 This is based on the scientific fact that bacteria from the colon move up to the upper gut, and their concentrations become much higher than 105/mL. By producing cytotoxins, these bacteria affect the delicate environment of the small intestine, and then gain entry into the cell, where, by binding to the cellular DNA, they induce apoptosis (cell death).

They then find their way to the submucosa, regional lymph nodes, and into the circulation. Immune response against the bacterial cytotoxins results in the production of IgA, IgM and IgG antibodies against them.

Due to the antigenic similarity between E. coli, Salmonella, Shigella toxins, Campylobacter jejuni and human cytoskeletal proteins such as vinculin and talin, these antibodies cross-react with the cytoskeletal proteins, causing depolymerization of and release of these proteins from the epithelial cells, with subsequent antibody production against the cytoskeletal proteins vinculin, talin, and actinin.

These IgA, IgM and IgG antibodies against bacterial cytotoxins and cytoskeletal proteins are detected in the blood with high accuracy and reproducibility. Thus, bacterial over-growth can be an instigator of extra- intestinal autoimmunity.

See Figure 1.

Figure 1. The Production of Antibodies Due to Bacterial CDT and Breakdown of Intestinal Cells. CDT released by E. coli (1) undergoes endocytosis (2), attacks cytoskeletal proteins (3), then enters the submucosa (4). CDT-assisted breakdown of the tight junctions results in the release of bacterial cytotoxins and cytoskeletal proteins, and the penetration of food antigens. Antibody production against these molecules can result in immunological activity within the gut and beyond.

SIBO is seen as a quantitative change in luminal bacteria of the small intestine disrupts digestion and absorption.

According to Bohm et al, SIBO can result from the failure of the gastric acid barrier, failure of small bowel clearance, small bowel anatomic alteration, local and systemic immune deficiency and systemic disease associations.12

Once coliform bacteria begin to thrive in the small intestine, a series of events can occur, which leads to SIBO. See Figure 2.

Figure 2. The Pathogenesis of SIBO. If coliform gram negative bacteria take up residence in the small intestine, the bacteria can ferment ingested carbohydrates, which increases gas production within the intestine, which results in bloating and flatulence, which then contributes to abdominal pain and/or discomfort.

Bacteria and the fermentation of carbohydrates produce toxic by-products that can further alter mucosa, gut immunity and damage the intestinal barrier. When the intestinal barrier is damaged, the body is at risk for autoimmunity. The mechanism of bacterial cytotoxin-induced autoimmunity is described below.

Pimentel et al. have shown the sensitivity and specificity of assessing antibodies to bacterial cytotoxins8 and to cytoskeletal protein,7 in patients with D-IBS and in patients with D-IBS with SIBO. This combination of antigen assessments provides clinically useful information and can help differentiate D- IBS from IBD patients with chronic diarrhea.8 Furthermore, Array 22 identifies, altered gut microbial populations, SIBO, intestinal cell damage, and even possible Celiac disease.

As described above, altered gut microbiome can occur due to a variety of reasons.

Once the gram- negative bacteria, thrive in their new environment, they may use their cytolethal distending toxins (CDTs) to infiltrate gut epithelial cells. CDTs have three subunits A, B and C designated as CdtA, CdtB, CdtC respectively.

CdtA and CdtC bind to the epithelium cell wall, CdtB then enters the cell where it acts on the cell’s nucleus to disrupt cell cycle and cause apoptosis (cell death).

The programmed death of epithelial cell results in a broken intestinal barrier allowing for the translocation of bacterial toxins such as CDTs and lipopolysaccharides (Array 2) from the gut into circulation.

Systemic bacterial products contribute to systemic inflammation.

Cytoskeletal proteins from intestinal epithelial cells are also found in other body barriers.

This production of antibodies against gut cytoskeletal proteins due to broken intestinal barrier may contribute to autoimmunity against the cytoskeletal proteins.

Figure 3. IBS/SIBO Mechanisms of Extra-Intestinal Autoimmunity. Something so small as bacteria can have far-reaching effects. When the intestinal microbiome is altered, cytotoxins from gram negative bacteria are released. These cytotoxins assist the bacteria in the invasion of epithelial cells where they attack the cell’s DNA. This causes a breakdown in the intestinal barrier, releasing bacterial cytotoxins and other immunogens into the bloodstream. The systemic inflammation due to these immunogens may play a role in autoimmune reactivity in the body.

Mechanisms of extra-intestinal autoimmunity due to irritable bowels with or without SIBO may include a breakdown of the intestinal barrier followed by systemic inflammation or antibody cross-reactivity.

See Figure 3. These three scenarios are known to play a role in autoimmunity.

Furthermore, the survival of intestinal cells with DNA damage by CdtB may promote genomic instability, which can lead to gastrointestinal tumor initiation and/or progression.13

Some gram negative, diarrhea-causing bacteria, such as Shigella14, and Rickettsia,15 also employ molecular mimicry in order to bind to vinculin as an entry point into the host cell. Shigella injects invasion proteins into the epithelial cell which reorganizes the actomyosin network.

Actomyosin calls vinculin to the cell interior wall where Shigella can access it. Shigella has engineered two vinculin binding sites that mimic those on talin. Vinculin is fooled, binds to Shigella and thus draws the bacterium into the cell.

CLINICAL ASPECTS OF ARRAY 22

Array 22 comprises both an environmental trigger (Bacterial Cytotoxins) and an indicator of a broken intestinal barrier (Cytoskeletal Proteins). Cyrex’s Array 22 – Irritable Bowel / SIBO Screen assesses IgG, IgA and IgM antibodies to Bacterial Cytotoxins and to Cytoskeletal Proteins. Cytotoxins are lethal xenobiotics released by commensal coliform bacteria. Bacterial cytotoxins can destroy epithelial cells of the intestinal barrier. Therefore, Cyrex also assesses antibodies to cytoskeletal proteins. Once this epithelial cell damage occurs, bacterial cytotoxins can freely enter circulation. Like systemic lipopolysaccharides (Array 2 – Intestinal Antigenic Permeability Screen), when bacterial cytotoxins enter the blood stream, they produce chronic inflammation and can open the blood brain barrier (Array 20 – Blood-Brain Barrier Permeability Screen).

With Array 22, one can identify a trigger of irritable bowels and the specific intestinal barrier damage they cause.

Environmental Exposures

The development of bowel disorders may be influenced by the genes a person inherits together with the way the person’s immune system responds to certain environmental triggers, such as stress, infectious agents and toxic chemicals. It is important to consider, 9 12 40

Dietary risk factor

• High sugar intake
• High salt intake
• Low protein intake

Medical history

• H.pylori infection
• Proton pump inhibitor
• Hypochlorhydria
• Vagotomy
• Gastrectomy
• Gastric by-pass
• Medications (opiates, tricyclic antidepressants, anticholinergics)

Connective tissue diseases

• Resection of ileocecal valve
• Duodenal and jejunal diverticulosis

Mucosal and/or systemic immune deficiency

• Celiac disease
• Cirrhosis
• Chronic pancreatitis

Stress

• Childhood exposure to victimization (verbal, physical, sexual abuse)
• Loss of a parent at a young age
• Learned illness behavior
• Depression
• Anxiety

Array 22 can be used to:

• Identify the overgrowth of large intestinal bacteria in the small intestine and the release of bacterial cytotoxin.
• Evaluate a breach of intestinal barrier by bacterial cytotoxins and their entry into circulation.
• Assist in setting guidelines for treatment of IBS/SIBO and reduced risk of igniting the autoimmune process.

Array 22 is recommended for patients who:

• Have irritable bowels.
• Exhibit symptoms of malabsorption, including weight loss, anemia or fatty stools.
• Have associated conditions such as fatigue, reflux, skin disorders, obesity or food intolerances.
• Pimental et al. from the Gut Motility Program, Division of Gastroenterology, Cedars-Sinai Medical Center in Los Angeles, California, have conducted extensive research on bacterial

REFERENCES

1. Gwee K-A, Leong Y-L, Graham C, et al. The role of psychological and biological factors in postinfective gut dysfunction . Gut, 1999; 44:400-406.
2. Marshall JK, Thabane M, Garg AM, et al. Incidence and epidemiology of irritable bowel syndrome after a large waterborne outbreak of bacterial dysentery . Gastroenterology, 2006; 131:445-450.
3. Saha L. Irritable bowel syndrome: pathogenesis, diagnosis, treatment, and evidence-based medicine . W J Gastroenterol, 2014; 20(22):6759-6773.
4. Hungin AP, Chang L, Locke GR, et al. Irritable bowel syndrome in the United States: prevalence, symptom patterns and impact. Aliment Pharmacol Ther, 2005; 21(11):1365-1375.
5. Lacy BE, Weiser K, De Lee R. The treatment of irritable bowel syndrome. Therap Adv Gastroenterol, 2009; 2(4):221-238.
6. Ghoshal UC and Srivastava D. Irritable bowel syndrome and small intestinal bacterial overgrowth: Meaningful association or unnecessary hype. World J Gastroenterol, 2014; 20(10):2482-2491.
7. Pimentel M, Morales W, Pokkunuri V, et al. Autoimmunity links vinculin to the pathophysiology of chronic functional bowel changes following Campylobacter jejuni infection in a rat model. Dig Dis Sci, 2015; 60(5):1195-1205.