Synergistic infection of Helicobacter pylori with IL-Iβ Gene Polymorphism Among the Liver Transplant Recipients

Background and Objective: Patients receiving liver transplantation are more likely to develop a wide range of infectious complications such as Helicobacter pylori ( H. pylori ). This study aims to assess the prevalence of H. pylori in individuals receiving liver transplants through the lymph nodes. Moreover, to investigate the relationship between the polymorphisms in the IL-1β gene and the H. pylori


Introduction
Liver diseases, both acute and chronic, are significant global sources of morbidity and mortality.The most prevalent non-neoplastic cause of mortality among gastrointestinal tract diseases currently is liver cirrhosis (1,2).The "decompensated phase" of cirrhosis, which is more advanced and marked by the emergence of ascites, portal hypertension gastrointestinal hemorrhage, encephalopathy, and jaundice, ranges from the relatively stable and usually asymptomatic "compensated phase" (3).The most severe acute decompensation of cirrhosis is likely acute-onchronic liver failure (ACLF), which is defined as acute decompensation of chronic liver disease accompanied by (multiple) organ failures, despite developments in pathophysiology and the best available therapy (4).Liver transplantation (LT) is the most effective and fundamental form of treatment for patients who have unfavorable side effects from internal medicine and artificial livers (5).
One of the most significant clinical issues in patients with decompensated liver cirrhosis, particularly in those who are hospitalized, is bacterial infections, which are linked to high rates of mortality and morbidity (6,7).About 20-35% of patients in certain series and 20-60% of patients in other series of cirrhotic patients have infections at the time of admission or acquire them while they are hospitalized (8).On the other hand, even though dissemination through the lymphatic system is occasionally proposed as a possible channel that aids in deeper microbial invasion, it is often regarded as being unimportant due to ineffective tissue absorption and efficient lymph node filtering (9).The study did not include patients who had a history of chronic diseases like diabetes, kidney problems, or hematologic disorders.

Samples collection
Slices of the 4 µm thick, paraffin-embedded lymph node tissue blocks were inserted in sterile 1.5 ml centrifuge tubes after being treated in formalin.A distinct microtome knife was used for each sample to avoid cross-contamination.Fresh venous blood samples (10 mL) were taken from all patients after an overnight fast.Five mL were taken without any anticoagulants for serum testing, 2.5 mL were taken with EDTA for complete blood count (CBC), and 2.5 mL were taken with citrate for coagulation profile analysis.Following that, the serum samples were separated into aliquots and stored at 20°C pending further analysis.The samples were then taken right away to the lab for examination.

Serologic Laboratory analysis
All serum samples used in this study were subjected to a variety of laboratory tests, including those for the liver (alanine aminotransferase (ALT), aspartate aminotransferase (AST), total and direct bilirubin, and albumin), kidneys (urea, creatinine, ureic aid, Creatinine clearance, GFR, and urine creatinine), blood sugar profile (fasting blood sugar (FBS), postprandial blood sugar (PPBS), and glycated hemoglobin (HbA1C)], lipid profile

Genotyping of IL-1β Polymorphisms
Restriction fragment length polymorphisms in the polymerase chain reaction were used to genotype the IL-1β T-31C and C3954T SNPs (PCR-RFLP).

Effect of CagA gene on biochemical profiles
Fifteen

Association between H.pylori infection and tumor markers
Table 4 illustrates the levels of AFP, CEA, and CA19.9 were performed to analyze the difference in tumor marker levels between the H. pylori (+) and H. pylori (−) groups, Also, the association with CagA status.The levels of AFP in subjects in the H. pylori infection group were significantly lower than those in the H. pylori (−) group, while CEA was significantly higher than those in the H. pylori (−) group.Subjects showed no significant differences in the levels of the CA19.9 between two groups (P>0.05).On the other hand, subjects with CagA (+) and Cag A (-) showed no significant differences in the levels of the studied tumor markers (P>0.05).

Association between IL-1β gene Polymorphisms and H.pylori infection
The distribution of IL-1β genotypes and alleles (+3954 C/T and -31 T/C) is summarized in Table 5.
According to the analysis of IL-1β (+3954 C/T), TT genotype was significantly lower in the uninfected group than infected (OR= 0.124, CI=0.027-0.580,P=0.008), while the CT genotype was higher in infected than uninfected group, but insignificantly (P<0.05).In addition, infected patients' CC genotype and C allele frequencies were slightly higher than those of H. pylori-infected patients (OR=1.242,1.391 respectively).Concerning IL-1β (-31 T/C), the analysis demonstrated no statistical significance in the distribution of all genotypes or alleles between the infected and non-infected groups.However, in case of CagA positive showed a significantly low frequency of the C allele, which might be a protective from infection with CagA positive strain (OR= 0.268 CI= 0.086-0.831,P=0.022).

Haplotype analysis of IL-1β C3954T and T-31C
SNPs is shown in Table 6.Among all examined haplotypes, the TT haplotype was significantly lower in infected than uninfected 26% vs. 47.5%, while CagA positive showed an insignificant increase in the TT haplotype 37.5% vs. 16.7%(P >0.05).
As a result, lymphatics are frequently vaguely and sparsely mentioned in the literature on bacterial infections.Instead, the perspectives on invasion are dominated by studies on direct microbial disruption of epithelial and vascular endothelial cell layers (10,11).Cirrhotic patients experience a steady, gradual rise in capillary filtration, which is predominantly brought on by an increase in hydrostatic pressure.This helps to increase the amount of lymph produced, which leads to compensatory lymphatic responses such as an increase in the number and size of lymphatic vessels, which helps the interstitial fluid drain more effectively (12).Increased lymphatic flow restricts edema and reduces tissue dendritic cell retention when pathogens like Helicobacter pylori are present, whereas lymphostasis can result in persistent inflammation (13).Helicobacter pylori (H.pylori), a gram-negative, spiral-shaped opportunistic infection, is frequently linked to gastritis, peptic ulcers, and other gastrointestinal disorders (14).Around 50% of the people in developed countries were thought to have H. Pylori infections, whereas 90% of those in underdeveloped nations appeared to have this bacterium (15).Researchers from all over the world are interested in the part that H. pylori plays in the development of liver conditions including liver cirrhosis.A curious finding is that H. pylori has also been linked to the etiology of hepatic encephalopathy, a common complication that affects between 30 and 70% of people with liver cirrhosis (16,17).In addition to causing localized inflammation due to its cytopathic effect, H. pylori infection also leads to a generalized rise in the number of pro-inflammatory cytokines (18,19).Two key single nucleotide polymorphisms (SNPs) were detected in the IL-1β gene (IL-1β T-31C and IL-1β C3954T), where they were associated with H. pylori infection (20,21).This study aims to assess the prevalence of extra gastric translocation of H. pylori in the enlarged lymph nodes of seropositive ascites patients with decompensated end-stage liver disease.Moreover, to investigate the relationship between the polymorphisms in the IL-1β gene (IL-1β T-31C and IL-1β C3954T) and the H. pylori infection.Patients and Methods Study design and Patient enrollment Between April 2019 and May 2021, patients were recruited from the liver transplantation program of (NHTMRI) at the National Hepatology and Tropical Medicine Research Institute and participated in this retrospective cross-section pilot study.We prospectively enrolled 43 patients, and data on the patient's clinical, radiological, demographic, hematological, and biochemical findings were assessed both at the start of the study and throughout the follow-up periods.This study included patients with liver cirrhosis who were above the age of 20.

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cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and verylow-density lipoprotein (VLDL)], as well as enzymes amylase, lipase, lactate dehydrogenase (LDH), and homocysteine, tested using an automated Cobas c 111 analyzer by Roche Diagnostics.Modular Cobas 6000 C501 systems were used to test the iron profile (iron, Ferritin, Transferrin, and Total Iron-Binding Capacity, or TIBC), Thyroid profile [TSH, T3, T4] tested by Anylab F1 Thyroid Machine and electrolytes [Sodium (Na), Potassium (K), Total and ionized calcium] tested on ST-200 Plus Electrolyte.On a Cobas t 511 coagulation analyzer, the coagulation profile (prothrombin time (PT), INR, fibrinogen, AT III, protein C, and protein S) of all citrated plasma samples was evaluated.Additionally, the CBC of the EDTA blood samples was performed using the Sysmex Xn-550 Cell Counter and blood types.By using the Cobas 6000 (e 601 module), RT-PCR for HCV RNA and HBV DNA, and the hepatitis B surface antigen (HBsAg), anti-HCV antibodies were identified.Serum levels assessment of AFP, CEA, and CA 19-According to the manufacturer's instructions, the ARCHITECT i2000sr automated immunoassay analyzer (Abbott Diagnostics, Abbott Park, IL, USA) was used to measure the serum levels of the markers Alpha-fetoprotein (AFP), Carcinoembryonic antigen (CEA), and CA19-9.DNA Extraction Using Thermo Scientific TM K0171 following the manufacturer's instructions, DNA was extracted from Formalin-fixed paraffin-embedded (FFPE) enlarged lymph node tissues by deparaffinization, rehydration, and homogenization as described before by Yun et al., 2014 (22).Until usage, all extracted DNA was kept in a -20°C freezer.Using NanoDropTM 2000/2000c (Thermo Fisher Scientific, Waltham, MA, USA), DNA from tissues was examined for purity and concentration.Detection of H. pylori Nested PCR (n-PCR) for H. pylori targeting the UreA gene was performed initially for infection screening, then to confirm H. pylori species by detecting the virulence gene (CagA).When the amplified PCR products were electrophoresed on a 1.5% agarose gel, they revealed a 200-bp fragment of the UreA and a 550-bp fragment of the cagA.The amplified DNA was stained with ethidium bromide and examined on a gel while being compared to a 100bp DNA ladder (Fermentas, Thermo Fisher Scientific Inc.).

Figure 1 :
Figure 1: Immunohistochemical staining with anti-Helicobacter polyclonal antibodies of a node showing granular cytoplasmic brown staining in the paracortical area (arrow) by (original magnification X400).

Table 1
lists the PCR conditions and utilized primer.

Table 1 :
The sequences of reverse and forward primers and PCR conditions

Table 2 .
When biochemical parameters and H. pylori infection were analyzed, albumin, Na, decreased significantly in the infected group (P<0.05).Figure1presents the tissue histological features in the presence of H. pylori.

Table 2 :
Comparison of sociodemographic and clinical parameters according to H.pylori infection *Significant difference.

Table 3 :
Comparison of sociodemographic and clinical parameters according to CagA *Significant difference.

Table 4 :
Association between H.pylori infection and tumor markers

Table 5 :
Genotype distribution and allelic frequency of IL-1β (+3954 C/T and -31 T/C) in H.pylori infected and non-infected subjects.

Table 6 :
Analysis of IL-1β haplotypes of the examined polymorphisms In comparison to cagA-negative patients, H. pylori CagA-positive individuals displayed significantly decreased levels of albumin, urinary creatinine, HB, platelets, total calcium, iron, and AT III.These findings support the findings of Caliskan et al. (2014) who suggested that infection with H. pylori may help to minimize proteinuria (36).According to Xu et al. (2017), the individuals they evaluated had lower hemoglobin levels and a greater correlation between anemia and H. Pylori infection (37).

Lee et al. (2020), and Kishore et al., (2021) found
in infected than uninfected (26% vs. 47.5%) and the genotypes (CC and CT) were higher in infected than uninfected patients, which might increase the risk of H. pylori infection but insignificantly.In the haplotype analysis in the case of the CagA positive strain, the TT haplotype was present in 37.5% of the CagA positive group compared to 16.7% of the Cag A negative group, which might indicate that the CT haplotype is a risk for infection with virulent strain (OR=2.250,CI 0.877-5.775,P=0.092).To the best of our knowledge, it is the first time to study the interaction results of the study are in line with those of Lu et al. (2018) who found a link between H. pylori infection and uric acid (40).Ndebi et al. (2018) confirm a potential link between H. pylori infection, lipid profile, and uric acid (41), and Milutin et al., (2014) the TT genotype was higher in H. pylori-positive subjects compared to the control ones (25).From haplotype analysis, the present study showed that in four haplotypes (CC, CT, TC, and TT), the frequency of the TT haplotype was significantly lower interference role of IL-1β with H. pylori infection in LT patients.