The Relationship between Helicobacter pylori and Beta-2 Microglobulin in Humans

H. pylori is related to various gastrointestinal diseases. β 2 Microglobulin (β 2M) is an intrinsic element of major histocompatibility complex (MHC I). Serum β 2M level may increase in inflammatory states. The aim of current study is to evaluate the relationship between β 2M and H. pylori bearing CagA strains. Methods. H. pylori status was determined by histopathology of samples taken from stomach. CagA status and β 2M level were measured from blood samples of patients. Eradication therapy was administered to the patients with H. pylori infection. β 2 Microglobulin levels were measured before and after treatment. Results. 35 (29.2%) H. pylori(−) patients and 85 (70.8%) H. pylori (+) patients were included in the study. There were 52 (43.3%) patients with CagA negative and 33 (27.5%) patients with CagA positive H. pylori infection. The mean serum β 2M level was 1.83 mg/L in H. pylori (−) group, 1.76 mg/L in H. pylori (+) CagA (−) group, and 1.93 mg/L in H. pylori and CagA (+) group (P > 0.05). Serum β 2M levels (1.82 versus 1.64 mg/L P < 0.05) were decreased after eradication. Conclusion. H. pylori and CagA status did not affect β 2M level. Relationship between low grade systematic inflammation and H. pylori should be investigated to find out new predictors for diseases associated with inflammation.


Introduction
Discovery of Helicobacter pylori (H. pylori) changed the management and treatment of many gastrointestinal diseases. Depending on the geographic location, H. pylori is an inhabitant of half of the world population's stomach. H. pylori plays a crucial role in the development of gastrointestinal diseases such as chronic gastritis, gastric ulcer and duodenal ulcer mucosa associated lymphoid tissue lymphoma (MAL-TOMA), and gastric adenocarcinoma [1,2].
Despite its high prevalence, most of H. pylori infected subjects are asymptomatic. However some of them do not remain in gastritis stage and progress to gastric ulcer, duodenal ulcer, or gastric cancer [3]. Factors that contribute to disease progression have been recognised in the past decade. Cytotoxin-associated antigen (CagA) is one of the examples of H. pylori virulence factors that are encoded by genes of H. pylori "pathogenicity island" [4]. CagA is associated with significant inflammation in gastric mucosa. 2 -microglobulin ( 2 M) is the light chain domain of major histocompatibility complex (MHC I) [5]. It plays a crucial role in antigen presentation by chaperoning the assembly of the complex [6,7]. 2 M is synthesized by all nucleated cells but is released form lymphocytes mainly so it can be a marker for inflammation [8]. 2 M is bound noncovalently, so it can be detected as free monomer in serum, urine, and cerebrospinal fluid. After degradation of MHC I molecules, heavy chain remains in the cell but 2 M is released into circulation. Therefore, 2 M serum level is associated with cell turnover and renal function. Impaired glomerular function and increased cell turnover raise 2 M levels. Elevated serum 2 M level is poor prognosis of multiple myeloma, lymphomas, and leukemias [9][10][11].
We hypothesized that CagA positive H. pylori infection can be linked with increased levels of 2 M. Relationship between 2 M and H. pylori has been evaluated in the past decades [12][13][14] but CagA positivity in H. pylori infection and its association with 2 M have not been evaluated.
The purpose of this study was to evaluate the relationship between 2 M and CagA status of H. pylori infection. We also aimed to investigate the effect of eradication on 2 M levels.

Material and Methods
The patients with dyspepsia who were admitted to the gastroenterology outpatient clinic between June 2012 and December 2012 were evaluated. Patients between 18 and 65 years of age who were referred to upper endoscopy from outpatient clinic were enrolled in the study. Patients who used antibiotics or nonsteroidal anti-inflammatory drugs for any reason in the past two weeks were excluded from the study. Local ethical committee approved the study protocol and informed consent was obtained from each enrolled patient.
Also all patients were evaluated for prior chronic diseases. Routine hemogram tests and biochemical parameters which include alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), total and direct bilirubin, urea, creatine, and serum electrolytes were performed for all participants. Patients with abnormal values and patients with coronary artery disease, liver failure, kidney failure, cerebrovascular disease, and malignant disease which may contribute to serum levels of 2 M were also excluded from the study.
All patients who fulfilled the criteria underwent upper gastrointestinal tract endoscopy and patients who were taken biopsies from antrum and corpus were considered eligible for assessment. Other patients for whom biopsy material could not be obtained were also excluded from the study. We also excluded patients with gastric cancer and gastric and duodenal ulcer determined in endoscopy. Samples from antrum and corpus were transported in formalin contained solutions and were embedded in paraffin blocks. Fourmicrometer-thick sections from each specimen were stained with hematoxylin-eosin for histopathologic evaluation and with Giemsa for H. pylori evaluation. Not only diagnosis of H. pylori infection but also assessment of intensity of H. pylori, inflammation, activation, atrophy, and intestinal metaplasia according to Sydney classification has been determined from the stained specimens. Pathologists who were blinded to patients reported the histopathological assessments as normal (0), mild (1), moderate (2), and severe (3) for each parameter of Sydney classification.
Fasting blood samples were obtained by the venipuncture of the large antecubital veins of the studied patients without stasis, after a 12-hour fast for measured 2 M and H. pylori CagA status.
Comparison of serum levels of 2 M before and after the eradication therapy was another aim of the current study. Participants who were diagnosed with H. pylori infection were administered H. pylori eradication therapy for 14 days.
Blood samples of patients who were negative for ureabreath test were collected. The samples were then centrifuged immediately; the plasma was separated and stored at −80 ∘ C. In order to avoid variation, all samples were studied on the same day and using the same kit. Commercial kit (DIA.PRO Diagnostic Bioprobes Srl, Milano, Italy) was used for analysis of H. pylori CagA status according to the manufacturer's instructions. 2 M analysis was performed by using commercial kit (DiaSorin, Saluggia, Italy) according to the manufacturer's instructions.
SPSS version 15.0 statistical software was used for statistical analyses. Descriptive results were reported as mean ± standard deviation and median with minimum and maximum values. Results were analysed for normal distribution by using Kolmogorov-Smirnov. Student's -test, Mann Whitney , and Kruskal Wallis test were used where appropriate. Wilcoxon test was used to determine the comparison between serum levels of 2 M before and after the eradication therapy. Correlation between serum levels of 2 M and parameters of Sydney classification was determined by Spearman's correlation test.
value <0.05 was accepted as statistically significant.

Results
A total of 120 patients were included in the study. There There was no statistically significant difference between H. pylori positive and negative (36.7 versus 37.2 resp.; > 0.05) or CagA positive and negative ( > 0.05) subjects with regards to age and gender.
There was no significant difference in mean serum 2 M levels (1.82 mg/L versus 1.83 mg/L resp.; > 0.05) between patients with and without H. pylori infection. There was also no significant difference in mean serum 2 M levels between H. pylori negative patients group, H. pylori positive-CagA negative group, and H. pylori positive-CagA positive group (1.83 mg/L versus 1.76 mg/L versus 1.93 mg/L, > 0.05 resp.) (Figure 1).
Sydney classification parameters were evaluated in biopsy specimens which were conducted from antrum and corpus. The highest value of these parameters was used in correlation analysis of H. pylori intensity, acute and chronic inflammation, atrophy, intestinal metaplasia, and serum 2 M level. There was no significant correlation between Sydney classification parameters and serum 2 M levels ( > 0.05) ( Table 1).
Eighty-five patients who were H. pylori positive were administered H. pylori eradication therapy. Ten patients were

Discussion
The current study showed no association with H. pylori infection and serum 2 M level. Although H. pylori infection causes inflammation of gastric mucosa, it has no effect on serum 2 M levels. H. pylori infection with CagA strains did not have any effect on 2 M level. Although patients with CagA positive H. pylori infection had the highest serum 2 M levels, it was not statistically significant. Severity of gastric inflammation did not correlate with serum 2 M; however, serum 2 M levels were significantly decreased after H. pylori eradication.
H. pylori infection leads to accumulation of neutrophils and lymphocytes in the gastric mucosa. This accumulation progresses into chronic inflammation because H. pylori cannot be eradicated from mucosa by immune system. This local inflammation may trigger a systematic response in the host. H. pylori is suggested to be related with low grade inflammation [15]. This low grade inflammation regarding H. pylori infection is associated with diabetes mellitus and atherosclerosis [16,17]. CagA is one of the most virulent factors of H. pylori. CagA positive H. pylori causes more serious gastric mucosa damage. H. pylori infection with CagA strains was associated with coronary artery diseases and cerebrovascular disease related with low grade inflammation more than CagA negative H. pylori infection [18,19].
As serum 2 M level reflects the activated lymphocytes, it is also considered as a marker of inflammation. There is growing evidence regarding the relationship between 2 M and diseases associated with systemic inflammation. One study determined that elevated 2 M levels reflect the severity of Crohn disease [20]. 2 M levels were found to be elevated and correlated with disease severity in peripheral artery disease [21]. It is considered that hypoxia shed 2 M from cell membranes. Also 2 M level predicts cardiovascular mortality and all-cause mortality in general population, in population with renal impairment, and in elderly population [22][23][24]. Considering the association of 2 M and inflammatory states, we hypothesized that 2 M may increase in H. pylori infection, especially with CagA positive strains.
Previous studies have reported the same results on the relationship of serum 2 M level and H. pylori infection. Our study had reiterated these previous results. First study in the literature was from pre-H. pylori era. In this study, serum 2 M levels were measured in patients with malign and benign diseases of stomach. Elevated levels were determined in both groups of diseases but patients with gastric adenocarcinomas and gastric ulcer had elevated serum 2 M levels more frequent than patients with gastritis and duodenal ulcer. But the difference was not statistically significant and H. pylori was not discovered yet when this study was published [12].
Two studies have reported that serum 2 M levels have no relationship with H. pylori infection. Also correlation between serum 2 M levels and H. pylori severity and intensity according to Sydney classification cannot be found. Akay et al. compared serum 2 M levels of 30 patients with H. pylori and 22 patients without H. pylori infection. They failed to find a statistically significant difference in the mean of serum 2 M levels between two groups. H. pylori intensity had increasing effect on serum 2 M levels but it was not statistically significant [13].
Dincer et al. have conducted a study that aimed to find a correlation between serum 2 M levels and parameters of Sydney classification for three regions of stomach (incisura angularis, antrum, and corpus). They were not able to find a correlation, but although it was not significant, biopsies from corpus showed that moderate and severe inflammation caused elevated serum 2 M levels compared to mild inflammation [14].
While the previous reports did not determine difference in the mean of serum 2 M levels, tissue concentrations of 2 M did differ. In the same study, Conz et al. showed that 19 of 30 patients had subendothelial 2 M but none of the control group had subendothelial 2 M. Also one study conducted on uremic dialysis patients showed accumulation of 2 M in gastric mucosa with H. pylori infection. Patients without H. pylori infection did not show this finding [25].
Our study had some limitations. Although the present study had the highest number of patients among the previous studies, the patient number was low. Tissue concentrations of 2 M were not measured. These limitations may be a reason for the nonsignificant results.

Conclusion
To the best of our knowledge, this is the first report that investigated the association between 2 M, CagA, and eradication therapy. Although there is no association, this possible relationship should be investigated by further studies with more patients. It is important to find reliable biomarkers for systemic inflammation and possible associations with chronic infections like H. pylori because diseases associated with systemic inflammations' severity and extension can be predicted with these biomarkers.