CIRCULATING CONCENTRATIONS OF IL-2, IL-6, TNF-ALPHA AND ENDOTOXIN IN PATIENTS OF CARCINOMA GALL BLADDER

Introduction: Gallbladder Cancer represents the most common and aggressive type among the biliary tree cancers (BTCs). Carcinoma Gallbladder has an unusual geographic distribution. The highest incidence of carcinoma of gallbladder is in Chileans, American Indians, and in parts of North India. Objective: The present study was designed to characterise the circulating concentrations of endotoxin and different inflammatory mediators in patients with Carcinoma Gall bladder. Methods and Material: One hundred twenty eight Carcinoma Gall bladder patients were included in the study. Enzyme-linked immunosorbent assay was used for determining the circulating concentrations of Interleukin-2 (IL-2), Interleukin -6 (IL-6), Tumor Necrosis Factor-α (TNF-α) and Endotoxins in human serum by LAL Method. A cross sectional trial was performed Results: The mean age of the patients was 50.86±12.6 years. The mean duration of jaundice was since last 74.91±50.0 days. Fifty five percent patients had cholelithiasis association with Carcinoma Gall bladder. The mean Body Mass Index (BMI) of Carcinoma Gall bladder patients were 18.33±2.8. The mean circulating concentrations of IL-2 were 33.31±56.6 pg/ml, IL-6 were 167.16±216.4 pg/ml, TNF-α were 56.00±56.7 pg/ml and endotoxin were 0.66±0.2 EU/ml. Conclusions: Mean circulating concentrations of IL-2 and TNF-α were lower and mean circulating concentrations of IL-6 were higher than the previous studies. The mean circulating concentrations of endotoxin were within the normal limits. Which concludes that cytokines may participate in gall bladder carcinogenesis and the study provides evidence for a strong link between chronic inflammation and Carcinoma Gall bladder.


Introduction:
Carcinoma of the gallbladder is the most common malignant tumour of the biliary tract and high incidence is observed in Chile, Japan, Latin America and Northern India. The prevalence of carcinoma of the gallbladder is highly variable across the world. 1,2 The Risk factors of carcinoma of gall bladder are: Gender, approximately twice more common in women than men, mostly between the ages of 50 and 60.Early diagnosis is not generally possible. 3 The prognosis of Carcinoma Gall bladder is poor due to unsatisfactory treatment. 4 Obstructive jaundice causes increased exposure to endotoxin by lack of bile in the gut lumen and diminished clearance of endotoxin by Kupffer cells. 5 The present study was designed to characterize the circulating concentrations of endotoxin and different inflammatory mediators in patients with CAGB.

Materials and Methods:
Five millilitre blood samples were obtained from the patients. Serum was centrifuged and then stored at −20°C until testing. Biochemical tests were performed and a questionnaire was used to obtain the general information and biochemical test results.

Selection and Description of Participants:
One hundred and twenty eight Carcinoma Gall bladder patients were included in the study. The patients were taken from the outpatient department and were between 20-80 years of age. Patients were eligible for inclusion in the study when they presented with obstructive jaundice with bilirubin concentrations less than five mg%.  Fig.1).The mean body mass index of patients in the present study was (18.33±2.8) and majority of the patients were malnourished ( Table 3).The mean Functional status of the patients was above 60% (Table 3), which suggests that the patients required some help with daily work, were caring for self, could take care of most personal requirements but were not capable of normal activity or work. The mean hemoglobin level in the study was 10.69±1.8 g/dl i.e. the patients were anaemic and the mean leukocyte count was found to be 7880.08±2559.7 ( Table 3). The mean S. Bil. (Serum Bilirubin) level (total) was 15.93±7.0 mmol/L, mean S. Bil. level (direct) was 11.60±4.9 mmol/L, mean SGPT (Serum glutamic pyruvic transaminase), SGOT (Serum glutamic oxaloacetic transaminase) levels were 103.86±80.9 IU/L and 110.53±67.6 IU/L and the mean S.Alp. (Serum Alkaline Phosphatase)level was 1586.97±1103.6 IU/L ( Table 3). The levels of IL-2 in the present study were 33.31±56.6 pg/ml (Table 3). IL-6 levels were 167.16±216.4 pg/ml. The endotoxin level in the observed 128 Carcinoma Gall bladder patients was found to be 0.66±0.2 EU/ml and the TNF-α level was 56.00±56.7 pg/ml ( Table 3).None of the patients were positive for HIV, HbSAg or HCV.

Discussion:
The aim of this study was to determine the circulating concentrations of IL-2, IL-6, TNF-α and Endotoxin in Patients of Carcinoma Gall bladder. Early carcinoma gallbladder has no specific clinical presentation and preoperative diagnosis is rarely possible. Most of these patients are asymptomatic while a few present with clinical features suggestive of benign disease such as right upper abdominal pain interspersed with occasional attack of nausea and vomiting. 6 Cancer of the gallbladder is an aggressive malignancy that occurs predominantly in elderly people. More than 75% of patients with this malignancy are older than 65 years. 7 Studies from Pakistan have shown that the age of occurrence is younger than this. 4 The mean duration of jaundice in the present study was 74.91±50.0 days prior to presentation ,whereas in previous studies the average duration of jaundice was 1.15 months i.e.45-46 days. 8 In another study, patients had symptoms for an average duration of 3.8 months prior to presentation. 9 The female to male ratio in the study was 11:3 (73 female: 55 male), in previous studies it was found to be 2:1 (57 female: 29 male). 2 A high incidence among females suggests a role of female hormones in the aetiology of the disease and the majority of females were in 5th and 6th decade of life. 1,4 GBC usually arises in a background of cholelithiasis cholecystitis. 10 11 The symptoms of carcinoma of the gall bladder mimic benign gall bladder disease until persistent pain due to invasion of the surrounding structure gives a clue to the correct diagnosis. Anorexia, weight loss and jaundice are common but occur late in the disease. Hepatomegaly (often nodular) and a palpable gall bladder lump indicate an advanced stage of the disease, 12 which in the present study was found in 67(52.34%) patients (Fig.-2). In a study by Burgess et al, out of 29 patients eighteen patients (62%) presented with jaundice, 11 patients (38%) had a palpable mass, only eight (28%) had abdominal pain on diagnosis. 13 The most common symptoms in the study by Pandey et al was pain in the abdomen (82.8%), followed by anorexia (41.4%) and significant weight loss (39.4%). An abdominal mass was present in about three-fourths of the cases. 9 Nausea/vomiting were present in 69(53.91%) patients in the present study (Fig. 2), whereas in a study by Ballinger et al five of 19 (26-3%) patients complained of mild nausea and constipation was present in two patients. 14 In the present study, Constipation was present in 96 patients (Fig. 2). Ultrsonography examination of the abdomen was carried out in all the patients and it showed the presence of gallstones in 69 (69.7%) cases. A mass in the gallbladder was evident in 93 patients (93.9%); it was associated with a calculus in 63.6% patients. Sixty-seven patients (67.7%) had a sonographic evidence of spread of disease beyond the gallbladder. The diagnosis was confirmed preoperatively in 73 patients who presented with a palpable abdominal lump; intraoperatively hepatic involvement was seen in 53 (76.8%) patients. 9 In the present study, mass in gall bladder was present in 71 (55.47%) cases (Fig. 3). Short periods of biliary obstruction rarely induce malnutrition but prolonged obstruction of the bile duct with low bile acid secretion may affect fat absorption. It has also been suggested that the presence of endotoxins and liver dysfunction may lead to anorexia. The high incidence of malnutrition in obstructive jaundice-related neoplasia may be caused by the tumor per se and by the anorexic properties of tumor necrosis factor-a (TNFa). 15 Present study supports the study by Rai et al. in which, 43% of GBC patients were malnourished with low body mass index. 16 Majority of the patients in present study were anemic (Table 3). In previous studies, Anemia was present in 86 (86.9%) patients. 9 The hemoglobin levels in case and control groups in the study by Rai et al were 10.87 g/dl (+/-1.81 SD) and 11.62 g/dl (+/-1.89 SD), respectively (P < 0.001). 16 The bilirubin levels both total and direct, SGPT, SGOT and alkaline phosphatase were quite high in the present study (Table 3). Total bilirubin concentration was less than 5 mg/dl only in 3.1% of the patients and the level increased to above 16 mg/dl in 31.4% of them. Increased serum SGOT and SGPT levels were found in 72.2% and 71.8% of the patients and a high level of serum alkaline phosphatase was found in 74.8%. 11 IL-2 is a cytokine normally produced by the body during an immune response and IL-6 is a pro-inflammatory cytokine secreted by T-cells and macrophages to stimulate immune response to trauma, especially burns or other tissue damage leading to inflammation. Aggarwal et al. suggested that IL-2, IL-6, TNF are responsible for Autocrine growth. 17 In the present study, the IL-2 and IL-6 levels were 33.31±56 and 167.16±216.4 pg/ml (  19 In other study by Kimmings et al the IL-6 levels were 4.2 (0.9) pg/ml and TNF -α levels were 21.7 (3.1) pg/ml. 5 TNF, a kind of cytotoxic protein, is produced by monocytes and macro phagocytes because of production of bacterial endotoxins, tissue injury or tumor cells. Endotoxin is a strong substance to stimulate the production of TNF. Obstructive jaundice causes increased exposure to endotoxin by two different mechanisms. Firstly, the lack of bile in the gut lumen is thought to be responsible for an increase in translocation of endotoxins through the intestinal mucosa. Secondly, biliary obstruction causes a reduction in liver reticuloendothelial system function leading to a diminished clearance of endotoxin by Kupffer cells. 5 In a study by Yang et al. the endotoxin levels in Carcinoma Gall bladder patients was 0.70+0.25 EU/ml and the TNF-α levels were 2.43+0.57 pg/ml. 20 The endotoxin levels were lower in the present study but the TNF-α levels comparatively were very high (Table 3). Inflammatory interleukins have been linked with tumorigenesis, which suggests that inflammation is associated with cancer development.
Interleukins mediate different steps in the pathway leading to tumorigenesis. The mean circulating concentrations of IL-2 and TNF-α were lower than previous studies. The mean circulating concentrations of IL-6 were higher than the previous studies and the mean circulating concentrations of endotoxin were within the normal limits. Since the TNF-a is dysregulated, it can mediate cancer and also can act as an endogenous tumor promoter. This concludes that cytokines may participate in gallbladder carcinogenesis and the study provides evidence for a strong link between chronic inflammation and Carcinoma Gall bladder.