Impact of Helicobacter pylori Eradication on Absolute Telomere Length in Gastric Mucosa

Patients and Methods: This study was conducted on (42) subjects divided into two groups, group I: included (17) H. pylori negative cases served as control group, group II: included (25) H. pylori positive patients. Absolute TLs was measured in base pairs (bp) in gastric mucosa and analyzed by real time polymerase chain reaction (RT-PCR) in all groups and reevaluated in H. pylori positive patients 4 weeks after eradication therapy.


INTRODUCTION
Helicobacter pylori (H.pylori) is a gram-negative, non-invasive, helixshaped, microaerophilic agent.It is the most common chronic bacterial infection in humans.H. pylori infection is strongly related with socioeconomic status and its prevalence is over 80% in developing countries and 20-50% in developed countries [1].H. pylori is the etiological agent of chronic gastritis, peptic ulcer, and has been documented to be linked with the development of gastric adenocarcinoma and lymphoma [2].Histological pre-neoplastic changes that might progress into gastric cancer are found in around 50% of people infected with H. pylori.Despite this fact, less than 2% of the patients develop gastric cancer [3].Although many factors may be related to H. pylori associated gastric carcinogenesis, the underlying molecular mechanisms are still unknown.The complications of chronic inflammation associated with H. pylori infection are believed to be related to the genetic features of the host, virulence factors of the bacteria strain, and environmental factors [4].Telomerase, located at the distal end of human chromosomes, comprises simple, repetitive and G-rich hexameric sequences (TTAGGG), and is vital for chromosomal stability and replication [5].Short telomeres are associated with cellular senescence and decreased tissue renewal capacity [6].Longer telomere length (TL) appears to prevent genomic instability and development of cancer in human aged cells by limiting the number of cell divisions.However, shortened telomeres impair immune function that might also increase cancer susceptibility [7].
Telomere length has been analyzed in many human cancers, and it has been found to be shorter in some tumors (e.g.colon cancer, gastric cancer and glioblastoma) and longer in others (e.g.chordoma) compared to normal tissue [8,9].In this study we aimed to assess the absolute (TLs) in gastric mucosa of H. pylori positive patients compared to H. pylori negative controls and to determine any changes in (TLs) by H. pylori eradication therapy.

Patients:
This cohort prospective study was carried out in the Hepatology, Gastroenterology and Infectious Diseases and Medical Microbiology and Immunology Departments, Faculty of Medicine, Benha University from January 2016 to July 2016.The study was approved by the local ethics committee of Benha University Hospitals and written consent was taken from each participant.Forty two adult patients were enrolled in this study were complaining of dyspeptic symptoms (defined as abdominal pain related to the meals).According to the results of upper GIT endoscopy, biopsy urease test and microbiological culture, selected patients were divided into 2 groups.Group I: (17) H. pylori negative patients with functional dyspepsia (according to Rome II criteria) [10] and they were considered as a control group.Group II: (25) H. pylori positive patients.Patients with chronic disease as liver cirrhosis, chronic renal failure, diabetes, hypertension, obstructive airway diseases, patients with history of non-steroidal anti-inflammatory drugs (NSAIDs), proton pump inhibitors (PPI) within previous month, heavy smokers and patients with cancers were excluded from the study.
All patients were subjected to full history taking, thorough clinical examination and routine laboratory investigation.
Upper GIT endoscopy: was done for all patients and controls by the same endoscopist after fasting at least 8 hours using video endoscope (OLYMPUS GIF TYPE Q180 2001629, Japan).Four quadrant biopsies were taken from antral mucosa within 5 cm of the pyloric opening for detection of H. pylori infection using urease test and microbiological culture and TL analysis.In positive H. pylori cases upper GIT endoscopy was repeated one month after H. pylori eradication therapy (a full dose PPI, Clarithromycin 500 mg and Metronidazole 400 mg, all twice daily for 2 weeks) and biopsy samples were again taken to determine the changes in telomere length after eradication therapy, gastric fragments were kept in thioglycolate broth (Difco Laboratories, Detroit, Mich.) at 4°C.Samples for TL analysis were sent to the laboratory in tubes with 0.9% NaCl.Then they were centrifuged at 3000g for 5 minutes, the supernatant was removed, and the tissue samples were stored at -80°C until TL was analyzed using (RT.PCR).

Identification of H. pylori
Urease test: Biopsies were placed in tubes containing christensen's 2% urea agar and examined within 24 h of incubation at 37ºC for urea hydrolysis.
Microbiological Culture: Fragments in thioglycolate broth (Difco Laboratories, Detroit, Mich.) were ground in a tissue homogenizer.Biopsies were rubbed onto Dent's agar plates using Columbia agar base supplemented with 7% human blood containing vancomycin, trimethoprim, cefsulodin and amphotercin B (Oxoid, Basingstoke, United Kingdom).Plates were incubated for 4-7 days in microaerophilic environment at 37°C.Bacteriological identification of H. pylori was done.Patients considered positive for H. pylori if direct urease and culture were positive or if culture alone was positive and considered negative if both tests were negative
HBG was used as a single copy gene needed for normalization.Necessary standards for absolute quantitation of β-globin expression were prepared using QuantiTect SYBR Green PCR Kit (Roche, Germany).
The telomere length assay was performed according to manufacturer's instructions with a Telo TAGGG Telomere Length Assay (Roche, Germany) kit.The product derived from the telomere PCR assay was normalized with the product obtained by the β-globin PCR assay and telomere length was measured in base pairs (bp).The primers used in amplifications were described in table1.
Cycling conditions (for both telomere and HBG amplicons) are: 10 min at 95°C, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min, followed by a meltingcurve analysis.
A plot of CT (the fractional cycle number at which the well's accumulating fluorescence crosses a set threshold that is several standard deviations above baseline fluorescence), versus log (amount of input target DNA) was generated by performing serial dilutions of the standards ranging between 10 -1 to 10 -6 dilution.This linear curve allows absolute quantitation of unknowns in the same PCR run.

Statistical analysis:
Collected data were analyzed using SPSS 16 (SpssInc, Chicago, ILL Company).Categorical data were presented as number and percentages while quantitative data were expressed as mean ± standard deviation, and range.Chi square test (X2) test was used to analyze categorical variables.Quantitative data were tested for normality using KolomogrovSmirnove test, using Wilcoxon test, Man Whitney U test and Spearman's correlation coefficient (rho) as proved to be non-parametric.P<0.05 was considered significant.

RESULTS
This cohort prospective study was conducted on 25 H. pylori-positive patients (16 males and 9 females) with mean age 39.4±8.12 years and 17 H.pylori-negative patients served as a control group (12 males and 5 females) with mean age 38.5±8.5 years.There was insignificant statistical difference between patients and controls as regard sex and age, (P value = 0.65 and 0.74 respectively) (Table 2).
The absolute TLs was shorter in H. pylori positive patients than control group (H.pylori negative patients) before treatment regimen (2326.4± 165.06 bp Vs 3046.7 ± 331.60 bp respectively) with highly statistically significant difference between both groups (P<0.001)(Table 3).
In Table (4) there was highly statistically significant difference between H. pylori positive patients before and after eradication therapy (P<0.001).The telomere length was significantly increased after eradication therapy (absolute TLs was 2326.4±165.06bp before treatment Vs 2842.3 ± 296.04 bp after treatment).
There was insignificant difference in absolute TLs in H. pylori positive patients after eradication therapy compared to control group (P = 0.053) (Table 5).
There was significant negative correlation between ages of patients and TLs before and after treatment (before treatment, r= -0.713, P<0.001, after treatment r= -0.846, P<0.001) (Figs. 1,2 respectively).While as regard the relation between sex and TLs there was no significant relation (before treatment TLs in males was 2335.5 ± 166.97 bp, in females it was 2310.1 ± 170.28 bp and P value = 0.51 and after eradication therapy TLs was in males 2881.6 ± 305.00 bp and in females it was 2772.3 ± 282.65 bp with P value = 0.55) (Table 6).
[11].Genomic DNA extraction: DNA was extracted using QIAamp DNA mini kit (Qiagen, Germany) according to the manufacturer instructions.The extracted DNA concentration was confirmed through measurement by NanoDrop 2000c Spectrophotometer.Readings were taken at wave lengths of 260 and 280 nm.Concentration of DNA samples were measured = 30 ng /µL at wave lengths 260 and 280 nm [12].Quantitative real time PCR: as described by Cawthon [13].

Table ( 2
): Comparison between studied groups regarding sex and age Comparison between H. pylori positive patients and control group as regard TLs before treatment *Telomere length, **Helicobacter pylori, ***MWU= Man Whitney U TestTable (4): TLs in H. pylori positive patients before and after eradication therapy *Telomere length, **Helicobacter pylori.

Table ( 5
): Comparison between H. pylori positive patients and control group as regard TLs after eradication therapy Aslan et al. [20] who found negative correlation but insignificant (r = 0.172, p>0.05), while Hou et al. [21] reported that telomeres were significantly shorter in association with aging (P<0.001), and the extent of telomere shortening may vary considerably among individuals within age groups, suggesting that environmental and lifestyle factors could play critical roles in the rate of telomere attrition.As regard relation between TLs and sex the present work found insignificant relation between TLs and gender before eradication therapy in patients group (2335.5 ± 166.97 bp in males and 2310.1 ± 170.28 bp in female with P value 0.51) this in agreement with Aslan et al. [21] as they found TLs before eradication was found (2721.3 ± 2003.44 bp in males, 2161.1 ± 1608.6 bp in females, p>0.05), on the same hand Hou et al. [21] reported insignificant relation between TLs and sex (P value = 0.09), when studying TLs in gastric cancer related to H. pylori positivity.CONCLUSION H. pylori positive patients have significantly shorter absolute TLs in their gastric mucosa compared to H. pylori negative controls.H. pylori eradication therapy increases the absolute TL in H. pylori infected patients and that can open new approach for consider effectiveness of H. pylori eradication therapy in increasing the telomere length one of preventable methods for gastric cancer.
Figure (1): Correlation between age and telomere length before treatment Figure (2): Correlation between age and telomere length after treatment Table (6): Relation between sex and telomere length