Heterodimerization of cholecystokinin 1 and cholecystokinin 2 receptors in gallbladder cancer: a new mechanism for carcinogenesis.

PURPOSE: Cholecystokinin is present in abundance in gallbladder tissue and mediates function through two structurally related receptors, CCK1R and CCK2R. Heterodimerization of these receptors is known to impact cell growth in vitro. However, the significance of these heterodimers in gallbladder carcinogenesis is relatively unknown. METHODS: Therefore, we evaluated the expression and the dimerization status of CCK1 and CCK2 receptors in human gallbladder carcinoma cell line (GBC-SD) and resected gallbladder tissue from normal (n = 10), cholelithiasis (n = 25) and gallbladder cancer (n = 25) by immunofluorescence/immunohistochemistry and western blot. The dimerization status of CCK1R and CCK2R was evaluated by co-immunoprecipitation. To understand the effect of heterodimerization of these receptors on growth-related signaling pathways, the expression of p-AKT, rictor, raptor and p-ERK was evaluated by western blot. RESULTS: We demonstrated the expression and heterodimerization of CCK1 and CCK2 receptor in GBC-SD gall bladder carcinoma cell line. Knockdown of CCK1R and CCK2R in the cell line led to significant reduction in p-AKT (P = 0.005; P = 0.0001) and rictor (P < 0.001; P < 0.001) levels. In tissue samples, significantly higher expression of CCK1R and CCK2R was observed in gallbladder cancer when compared to other groups both by immunohistochemistry (P = 0.008 and P = 0.013) and western blot (P = 0.009 and P = 0.003). An increase in heterodimer formation of CCK1R with CCK2R was observed in gallbladder cancer when compared to normal and cholelithiasis tissues. No significant difference in the expression of p-AKT and p-ERK was observed between the three groups. CONCLUSION: Our results provide the first evidence of heterodimerization of CCK1R and CCK2R in gallbladder tissue, and its association with development of gallbladder cancer. This finding has potential clinical and therapeutic significance.

Characterization of microfibers emission from textile washing from a domestic environment.

Microfibers and microplastics are widely recognized emerging pollutants, which have the potential to cause an Eco-toxicological effect. Cellulosic and synthetic fibers are being released almost equally to the environment. Synthetic fibers released were non-biodegradable resulting in a significant negative impact on the environment. In the present study, four different households using fully automated washing machines (2 top-load, 2 front-load) were studied in a domestic laundry environment under real conditions. Laundry effluents were collected and contaminants analysis was carried out. The results estimated that the average emission rate of the four households was 7,453,635 MF/7Kg (FL, H1), 7,375,500 MFs/6Kg (FL, H2), 10,692,255 MFs/7Kg (TL, H3) and 7,589,017 MFs/6.2Kg (TL, H4). Synthetic fiber's emission rate was only about 19 %, and the average length range of microfibers released was found to be in the range of ≤5 µm (48.64 %), and the least amount of emission was found in the >500 µm range (11.49 %).

Expression of Antimicrobial Peptides and Cytokines in Human Omentum Following Abdominal Surgery.

Introduction Omentum can secrete out biological agents like different growth factors, cytokines, and antimicrobial peptides. The aim of our study was to determine the expression of antimicrobial peptides and cytokines in human omentum tissue and its response to intra-abdominal infection. Methodology Omentum tissue was obtained from 60 patients: control (n=20) and cases (n=40). mRNA expression of antimicrobial peptides (LL-37, HBD-1, HBD-2, HNP1-3) and cytokines (TNF- α, IL-8, IL-10, IL1ß) was evaluated using Real-Time PCR. Protein quantification was done by Immunoblotting and ELISA. Results Significantly higher expression of antimicrobial peptides (LL-37, HBD-1, HBD-2, HNP1-3) and cytokines (TNF- α, IL-8, IL-10, IL1ß) was observed in cases as compared to control at both the transcriptional and translational level (p<0.0001). Conclusion Omentum governs a population of antimicrobial peptides with potent immunologic functions. The expression of antimicrobial peptides and cytokines is inducible and increases with the severity of infection. Omentum is thus an immunologically active and adaptable organ but its complete regulatory mechanism is still elusive.

Antibacterial spectrum of human omentum and differential expression of beta defensins.

BACKGROUND: Human ß defensins (hBD1 and hBD2) are cationic, cysteine-rich peptides and form an integral part of the mammalian innate immune system. hBD1 is constitutively expressed in epithelial cells, whereas hBD2 increases in response to bacterial infection. Human omentum is known for its anti-inflammatory properties and also possesses an antibacterial activity of its own. We hypothesized that antimicrobial peptides, ß defensins, may govern host defense mechanism in the microbe-rich environment of the peritoneal cavity. Therefore, we analyzed the expression of hBD1 and hBD2 in omentum tissue in vivo and also studied the antibacterial activity of omentum against common pathogens. METHODOLOGY: Omentum tissues were obtained from 30 patients (15 cases and 15 controls). Real-time polymerase chain reaction (PCR) was used to evaluate the mRNA expression of hBD1 and hBD2. Protein quantification was done using Western blotting technique. Antibacterial susceptibility was performed to check the antibacterial activity of omentum. RESULT: Significantly higher expression of hBD2 was observed in cases compared to controls at both the transcriptional and translational levels. In comparison with an array of antibiotics, activated omentum also showed antibacterial property even at lower concentration of its extract. CONCLUSION: Omentum directly responds to bacterial infection, which may be due to differential expression of hBD1 and hBD2 in human omental tissue. These peptides (hBD1 and hBD2) may be an ideal candidate for novel antibiotic class with a broad-spectrum activity.

A let-7 microRNA binding site polymorphism in the KRAS 3'UTR is associated with increased risk and reduced survival for gallbladder cancer in North Indian population.

PURPOSE: Gallbladder cancer is a lethal malignancy of hepato-biliary system with high incidence in North India, especially along gangetic plain. The let-7 microRNAs play a key role in regulating KRAS expression and a polymorphism in 3' untranslated region (rs61764370, T/G) of KRAS leads to its higher expression. This polymorphism is known to be associated with increased risk and prognosis of various cancers but its association with gallbladder cancer has not been evaluated. To address this research question, we evaluated whether rs61764370 variant is associated with gallbladder cancer susceptibility and clinical outcomes. METHODS: In present case-control study, we enrolled 541 patients with gallbladder malignancy and 307 controls. Genomic DNA was obtained from peripheral blood and genotyping was performed using Taqman allelic discrimination assay. RESULTS: Heterozygous (TG) individuals are at a significant higher risk for GBC as compared with wild genotype (TT) (p = 0.007, odds ratio = 2.56, 95 % CI 1.27-5.18). At allelic level, allele G has significant higher risk for GBC as compared with T allele (p = 0.008, odds ratio = 2.5, 95 % CI 1.25-5.01). Survival analysis reveals decrease in overall survival for heterozygous genotype (p < 0.0001, hazard ratio = 3.42, 95 % CI 1.21-4.20). Also, significant decrease in overall survival was observed for patient carrying allele G (p < 0.0001, HR = 2.89, 95 % CI 1.21-4.20) as compared with allele C. CONCLUSIONS: We conclude that KRAS rs61764370 polymorphism is significantly associated with risk and prognosis of gallbladder malignancy in this endemic belt.

Polymorphism and Expression Profile of Cholecystokinin Type A Receptor in Relation to Gallstone Disease Susceptibility.

In the present study, we investigated expression pattern of Cholecystokinin type A receptor (CCKAR) in relation to its commonly studied polymorphism (rs1800857, T/C) in gallstone disease (GSD) patients and controls. A total of 502 subjects (272 GSD and 230 controls) were enrolled, and genotyping was performed by evaluating restriction fragments of PstI digested DNA. For analyzing expression pattern of CCKAR in relation to polymorphism, gallbladder tissue samples from 80 subjects (GSD-55; control-25) were studied. Expression of CCKAR mRNA was evaluated by reverse transcriptase-PCR and confirmed using real-time PCR. Protein expression was evaluated by enzyme-linked immunosorbent assay. We observed significantly (p < 0.0001) lower expression of CCKAR mRNA and protein in GSD tissues as compared with control. Significantly higher frequency of A1/A1 genotype (C/T transition) (p = 0.0005) was observed for GSD as compared with control. Expression of CCKAR protein was found to be significantly lower (p < 0.0001) in A1/A1 genotype as compared with other genotypes for GSD patients. Perhaps, this is the first report providing evidence of alteration in CCKAR expression in relation to its polymorphism elucidating the molecular pathway of the disease. Additional investigations with lager sample size are needed to confirm these findings.