PI3K/Akt/mTOR signaling & its regulator tumour suppressor genes PTEN & LKB1 in human uterine leiomyomas.

Background & objectives: Despite their high occurrence and associated significant level of morbidity manifesting as spectrum of clinical symptoms, the pathogenesis of uterine leiomyomas (ULs) remains unclear. We investigated expression profile of tumour suppressor genes PTEN (phosphatase and tensin homolog deleted on chromosome ten) and LKB1 (liver kinase B1), and key signaling components of P13K (phosphatidylinositol 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in leiomyomas and adjacent normal myometrium in women of reproductive age, to explore the possibility of targeting this pathway for future therapeutic implications. Methods: Real time PCR (qPCR) was used to quantify relative gene expression levels of PTEN, Akt1, Akt2, mTOR, LKB1 and VEGFA (vascular endothelial growth factor A) in leiomyoma as compared to adjacent normal myometrium. Immunohistochemistry was subsequently performed to analyze expression of PTEN, phospho-Akt, phospho-mTOR, phospho-S6, LKB1 and VEGFA in leiomyoma and adjacent normal myometrium. Results: Significant upregulation of PTEN (2.52 fold; P=0.03) and LKB1 (3.93 fold; P=0.01), and downregulation of VEGFA (2.95 fold; P=0.01) genes were observed in leiomyoma as compared to normal myometrium. Transcript levels of Akt1, Akt2 and mTOR did not vary significantly between leiomyoma and myometrium. An increased immunoexpression of PTEN (P=0.015) and LKB1 (P<0.001) and decreased expression of VEGFA (P=0.01) was observed in leiomyoma as compared to myometrium. Immunostaining for activated (phosphorylated) Akt, mTOR and S6 was absent or low in majority of leiomyoma and myometrium. Interpretation & conclusions: Upregulation of PTEN and LKB1 in concert with negative or low levels of activated Akt, mTOR and S6 indicates that PI3K/Akt/mTOR pathway may not play a significant role in pathogenesis of leiomyoma.

Performance characteristics of bisulfite conversion and SYBR green based quantitative PCR for DNA methylation analysis.

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes play vital role in development of severe disease like cancer. Many techniques used for assessment of DNA methylation, bisulfite treatment followed by methylation specific polymerase reaction (MSP) are one of them, which introduce conversion of unmethylated cytosine into uracil. The significant level of bisulfite treated DNA degradation results in the failure of methylation detection. Therefore, this step is to be properly controlled to avoid the degradation of DNA. In the present study, an attempt has been made to access the incubation time of DNA with bisulfate treatment at three time points i.e. 2.5, 4 and 16 hrs to get complete conversion of cytosine to uracil. Currently, the experiments were undertaken using oral cancer tissue, with varying incubation time of bisulfite treatment and 2 representative genes viz MGMT and p16 were selected for the quantitative assessment of methylation by real time PCR. Both genes are frequently methylated at promoter region in carcinogenesis. The short term incubation for 4hrs indicated better real time threshold value for p16 and MGMT gene methylation (Ct 25.55, 27.25) and unmethylation (Ct 18.82, 25.84) in tissue whereas it was 28.16, 37.35 and 21.98, 26.19 in blood sample, respectively as compared to other incubation time which shows less degradation of full length DNA.

Molecular and phenotypic expression of decorin as modulator of angiogenesis in human potentially malignant oral lesions and oral squamous cell carcinomas.

Background: Decorin is an extracellular matrix, multifunctional small proteoglycan molecule in tumor stroma that has been shown to be modulator of angiogenesis. No clinical data is available so far on decorin expression and survival outcome of oral cancer. Aim: The aim of the present study was to examine molecular and phenotypic expression of two angiogenesis modulators viz. decorin and vascular endothelial growth factor-A (VEGF-A) in human potentially malignant oral lesions (PMOLs) and oral squamous cell carcinomas (OSCC) in relation to clinico-pathological variables and survival outcome. Materials and Methods: Tissue biopsies were obtained from 72 PMOLs, 108 OSCC and 52 healthy controls. The PMOLs included cases of leukoplakias and oral submucous fi brosis. Immunohistochemistry was performed using antibodies against decorin, VEGF-A and CD-31. Messenger-ribonucleic acid (mRNA) expression was analyzed by using real-time polymerase chain reaction. Results: Cytoplasmic staining of decorin was observed in the basal layer of epithelium in 53 (73.61%) cases of PMOLs and in peritumoral stroma in 55 (50.92%) cases of OSCC. None of the cases showed nuclear expression of decorin. Decorin expression both at phenotypic and molecular level was found to be down-regulated from PMOLs to OSCC. Lymph node metastasis and reduced decorin expression independently correlated with overall survival in OSCC. VEGF-A expression had no signifi cant impact on survival outcome. Conclusion: Micro vessel density and VEGF-A expression were signifi cantly associated with reduced decorin expression in tumor stroma suggesting, decorin as angiogenic modulator in OSCC. Down-regulation of decorin expression and the presence of lymph node metastasis were adverse factor independently affecting overall survival in OSCC.

Body mass index and per capita income influence duodenal ulcer healing and H. pylori eradication whilst dietary factors play no part.

BACKGROUND: The role of dietary and sociodemographic factors in the healing of duodenal ulcer following H. pylori eradication remains undefined. AIM: To assess the role of diet, sociodemography and body mass index in the healing of duodenal ulcer and eradication of H. pylori. METHODS: A cross-sectional study consisting of 67 consecutive duodenal ulcer patients was undertaken. Sociodemographic factors studied included age, sex, occupation, educational status, religion, type of family, number of family members, per capita income and residence (urban vs. rural). Personal habits studied included alcohol consumption and smoking. Regular dietary intake over a two-month period was assessed using the food frequency questionnaire. All patients had documented H. pylori infection at the time of inclusion and received standard triple eradication therapy. Follow-up endoscopy and testing for H. pylori were done 4 weeks after completion of eradication therapy. RESULTS: The mean age of the 67 patients (60 male, 7 female) was 39.9+/-13.6 years. Healing of duodenal ulcer was documented in 51 patients. H. pylori infection was successfully eradicated in 31 patients but not in the other 36. There was no difference between the groups (Group A1: H. pylori eradicated, Group B1: H. pylori not eradicated) with regard to dietary and socio-demographic variables, except for BMI, which was significantly higher in patients in whom H. pylori had been eradicated. Per capita income was significantly higher in Group A2 (healed duodenal ulcer) as compared to Group B2 (duodenal ulcer not healed) while there was no difference in dietary and socio-demographic variables in these two groups. CONCLUSION: We found that higher body mass index and higher per capita income were associated with successful H. pylori eradication and duodenal ulcer healing, respectively, and that diet had no role to play in either. Further epidemiological studies from different parts of India and studies that control for Helicobacter pylori are required to establish predictive factors.

Abdominal tuberculosis.

Tuberculosis can involve any part of the gastrointestinal tract and is the sixth most frequent site of extrapulmonary involvement. Both the incidence and severity of abdominal tuberculosis are expected to increase with increasing incidence of HIV infection. Tuberculosis bacteria reach the gastrointestinal tract via haematogenous spread, ingestion of infected sputum, or direct spread from infected contiguous lymph nodes and fallopian tubes. The gross pathology is characterized by transverse ulcers, fibrosis, thickening and stricturing of the bowel wall, enlarged and matted mesenteric lymph nodes, omental thickening, and peritoneal tubercles. Peritoneal tuberculosis occurs in three forms : wet type with ascitis, dry type with adhesions, and fibrotic type with omental thickening and loculated ascites. The most common site of involvement of the gastrointestinal tuberculosis is the ileocaecal region. Ileocaecal and small bowel tuberculosis presents with a palpable mass in the right lower quadrant and/or complications of obstruction, perforation or malabsorption especially in the presence of stricture. Rare clinical presentations include dysphagia, odynophagia and a mid oesophageal ulcer due to oesophageal tuberculosis, dyspepsia and gastric outlet obstruction due to gastroduodenal tuberculosis, lower abdominal pain and haematochezia due to colonic tuberculosis, and annular rectal stricture and multiple perianal fistulae due to rectal and anal involvement. Chest X-rays show evidence of concomitant pulmonary lesions in less than 25 per cent of cases. Useful modalities for investigating a suspected case include small bowel barium meal, barium enema, ultrasonography, computed tomographic scan and colonoscopy. Ascitic fluid examination reveals straw coloured fluid with high protein, serum ascitis albumin gradient less than 1.1 g/dl, predominantly lymphocytic cells, and adenosine deaminase levels above 36 U/l. Laparoscopy is a very useful investigation in doubtful cases. Management is with conventional antitubercular therapy for at least 6 months. The recommended surgical procedures today are conservative and a period of preoperative drug therapy is controversial.