Anti-vinculin antibodies as a marker of irritable bowel syndrome-diarrhea in Egyptian patients
Essam Biomy a Manal Sabry a, Naglaa Raafat AbdRaboh b
a Department of Internal Medicine, Faculty of Medicine, Ain Shams University, Cairo, Egypt
b Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Abstract:
Background: Circulating anti-vinculin antibodies have been used as a marker for irritable bowel syndrome IBS- predominant diarrhea (IBS-D) and for differentiating it from inflammatory bowel disease. There is no available data regarding their use in the diagnosis and follow up of IBS-D patients in the Middle East. We aim to evaluate the diagnostic value of anti-vinculin antibodies in Egyptian patients of IBS-D. Subjects and methods: The current study was carried out over nine months on ninety individuals, divided into two groups: (Group-1) included seventy patients with diarrhea predominant IBS (IBS-D), (Group-2) included twenty healthy volunteers as a normal control group. All subjects have been subjected to colonoscopic examination, in addition to measurement of serum levels of selected inflammatory markers and anti-vinculin antibodies. Results: the serum level of anti-vinculin antibodies was significantly higher in IBS-D patients’ group as compared to the control group. Moreover, there was a highly significant difference between the two groups in some inflammatory markers (erythrocyte sedimentation rate, C-reactive protein, and total leucocyte count). Conclusion: the findings of the present work strongly suggest the potential use of anti-vinculin antibodies as a diagnostic tool in Egyptian patients with post infectious-IBS.
Introduction
A very common functional gastrointestinal disorder that has a serious impact on every day quality of life is irritable bowel syndrome (IBS) (El-Salhy et al., 2012). It is not a life threatening disease, but it may be reflected on economic level of society, and impair the work productivity (Peery et al., 12).
IBS affects a substantial proportion of the population, The prevalence estimates of IBS within the community is usually range between 10% and 25% with rates vary significantly between countries and depend on diagnostic criteria used (Canavan et al., 2014). Meta-analysis shows a pooled estimate of international IBS prevalence of 11.2% (95% confidence interval 9.8–12.8) (Lovell and Ford, 2012), with variation by geographic region; the lowest occurring in South Asia (7.0%) and the highest in South America (21.0%). A study by Abdul Majeed Ahmed 2011, included 117 Egyptian individuals have shown higher prevalence of IBS in women (41.1%) more than men (21.0%), with overall prevalence of 34.2% in the studied sample.

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IBS is a functional disorder as there is no anatomical, physiological or biochemical defects, and hence there is no specific laboratory, histopathological, radiological or endoscopic tests for its diagnosis (Karantanos et al., 2010). Patients with IBS are diagnosed according to Rome-III criteria, including abdominal pain and discomfort lasting at least three days a month in the last three months, associated with two or more of following criteria: improvement with defecation, altered frequency of stools or altered consistency of stools. IBS is divided into three subtypes according to the predominant bowel pattern: diarrhea-predominant (IBS-D), constipation-predominant (IBS-C), and alternating diarrhea and constipation patterns (IBS-M) (Longstreth et al., 2006). Rome criteria IV released in 2016 had some differences from Rome III criteria of IBS diagnosis. The three different patterns are the same as previously mentioned, but the bouts of abdominal pain and alteration of frequency and form of stool should be at least one day per week over three months (Schmulson and Drossman, 2017).
Based on emerging animal models, about 10% of individuals who had acute gastroenteritis develop long-lasting IBS-D symptoms, referred to as post-infectious IBS (PI-IBS) which may be linked to changes in the gut microbiome (Saha et al., 2014). It was proved that the development of PI-IBS does not appear to be specifically linked to a particular types of bacteria (Thabane ; Marshall, 2009). The toxicity of the infecting bacteria and the severity of the initial illness, as well as the longer period of illness are considerable risk factors for the development of PI-IBS (Spiller ; Garsed, 2009).
In rats, Campylobacter jejuni infection induces a phenotype that resembles human PI-IBS, and leads to significant variation in small bowel microbial flora. (Morales et al., 2011). Progression to an IBS-like phenotype was predicted by the presence of a bacterial toxin called cytolethal distending toxin B (CdtB). In rats exposed to these toxins, levels of circulating antibodies to CdtB were associated with altered gut microbial population and reduction in interstitial cells of Cajal (Morales et al., 2011). Anti-CdtB antibodies were found to cross react with the host cell adhesion protein, vinculin via molecular mimicry. In addition, levels of circulating antibodies to CdtB and vinculin correlated with the levels of small intestinal bacterial overgrowth (SIBO) in these animals (Pimentel et al., 2014). Anti-CdtB antibodies and anti-vinculin antibodies have been reported to distinguish between IBS-D and inflammatory bowel disease (IBD) (Pimentel et al., 2015).
As a biomarker of IBS-D, anti-vinculin antibodies were not fully tested in the Middle East, we aim in the present study to evaluate the serum level of anti-vinculin antibodies in comparison to the routine inflammatory and biochemical findings in Egyptian patients with IBS-D.

Subjects and methods:
Ninety subjects were enrolled in this cross sectional study, divided into two groups: Seventy patients with diarrhea predominant IBS (IBS-D), based on the presence of ROME III criteria (Drossman and Dumitrascu, 2006), (patients’ group) with mean age ± SD of 30.2 ±7.45 years. In addition to, twenty healthy volunteers were included as a control group, with mean age ± SD of 30.2 ± 6.22 years, over the period January 2017, and March 2018.
All patients and healthy controls were recruited from Ain Shams University Outpatient Clinic and Colonoscopy Unit after approval of the ethical committees of the University and Hospital, an informed consent was obtained from the included subjects in writing.
All healthy controls were screened for previous history of gastrointestinal diseases and active symptoms at the time of study. Exclusion criteria included: the presence of diabetes mellitus, human immunodeficiency virus (HIV), neoplasms and autoimmune disease, pancreatic or biliary disease, positive colonoscopic examination; drug abusers, and recent taking of antibiotics (during the past one month).
Recruited participants were subjected to history taking stressing on GI symptoms to diagnose IBS-D patients depending on ROME III criteria questionnaire, and to exclude these symptoms in the control group. General and local physical examinations were performed in both groups. Stool analysis, abdominal ultrasound examination; followed by colonscopy for patients’ group to exclude patients with abnormal findings.
Venous blood samples were collected from all subjects with subsequent laboratory hematological assessment, including complete blood count (CBC), erythrocyte sedimentation rate (ESR) and C reactive protein (CRP). The collected blood samples were centrifuged at 3500 rpm for 15 minutes at 4 ? and then stored frozen at -80°C until the time of assay.
Biochemical measurement of the serum levels of liver enzymes, albumin, bilirubin, creatinine and random blood sugar (RBS) were performed using commercial assay kits according to the recommendation of manufacturers (Diagnostic system, Holzheim, Germany).

Plasma level of anti-vinculin:
Enzyme linked immunosorbent assay (ELISA) was performed using full length human vinculin protein (Novoprotein scientific, USA) as an antigen at a concentration of 1.2 ?g/ml. the antigen was immobilized overnight at 4°C onto high-binding 96-well plates in 100mM borate buffered saline with pH 8.2 (sigma Aldrich). The wells were blocked using 3% bovine serum albumin for one hour at room temperature, then the wells were incubated with a 1:32 dilution of plasma for 1 hour at room temperature. Anti-vinculin antibodies (R and D systems) were used as positive control. This was followed by one hour incubation with horseradish peroxidase conjugated secondary antibodies (Millipore – Merch). Three time wash with 0.05% phosphate buffered saline and Tween 20 (pH 7.4) separated each incubation. Tetramethylbenzidine substrate solution (BioRad) was used for detection using microtitration plate reader (stat Fax-1200, Awareness Technology, Inc. FL 34990, USA). The optical densities (OD) were read at 370nm, the values of OD were used for the data analysis. OD values were used for the data analysis (Pimentel et al 2015)

Statistical analysis:
Results of the different parameters were statistically analyzed by appropriate statistical methods using SPSS program version 24. Descriptive data were expressed as mean + standard deviation of the mean (SD) for quantitative values and as a percentage for qualitative data. Independent t-test is used to assess the statistical significance of differences between mean values in the two studied groups. Bivariate Pearson’s correlation test was carried out to study the correlations between the optical density of plasma level of anti-vinculin antibody and different parameters. Statistical significance was considered when P-value is ? 0.05.

RESULTS
In the present study 90 individuals have been recruited and divided into two age and sex matched groups. The patient’s group (group-1) included 70 subjects 25 males (35.7 %) and 45 females (64.3 %), with age range 18 to 45 years, mean age value of 30.2 + 7.5. The control group included 20 individuals, 9 males (45.0 %) and 11 females (55.0 %), with age range 19 to 39 years, with a mean value of 30.2 + 6.2. The details of biochemical and hematological parameters measured in the study sample are shown in table 1 and the only statistically significant difference between the two studied groups was detected in the total leucocytic count which was higher in patient’s group.

Table 1: Clinical and laboratory data of the study participants:
Parameters
IBS group
(n =70)
Mean + SD Control group
(n = 20)
Mean + SD Level of significance (p)
Gender
Male (%)
Female (%)
25 (35.7 %)
45 (64.3 %)
9 (45.0 %)
11 (55.0 %)
Age (years) 30.2 + 7.5 30.2 + 6.2 0.81
Serum creatinine (mg/dl) 0.96 + 0.17 0.95 + 0.16 0.82
Random blood sugar (mg/dl) 139.5 + 24.7 148.1 + 19.8 0.15
ALT ( IU/L) 25.9 + 10.9 22.4 + 9.5 0.19
AST (IU/L) 23.9 + 9.3 24.4 + 10.3 0.84
Serum albumin (g/dl) 4.1 + 0.4 4.2 + 0.3 0.30
Serum bilirubin (mg/dl) 0.8 + 0.2 0.7 + 0.1 0.08
Hb% (g/dl ) 13.1 + 1.0 13.3 + 0.9 0.42
TLC (103/mm3) 6.8 + 2.5 5.2 + 0.1

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