Identification of High-Risk Aberrant Crypt Foci and Mucin-Depleted Foci in the Human Colon With Study of Colon Cancer Stem Cell Markers.

BACKGROUND: During colonoscopic screening, only macroscopic lesions will be identified, and these are usually the result of multiple genetic abnormalities. Magnification endoscopic detection of aberrant crypt foci (ACF), long before they acquire complex genetic abnormalities, is promising. However, the features of high-risk ACF-like lesions need to be identified. MATERIALS AND METHODS: In the present cross-sectional study, grossly visible normal mucosal flaps were shaved from 152 colectomies, including 96 colorectal cancer (CRC) cases and 56 controls (22 control specimens with disease with malignant potential and 34 without malignant potential). Methylene and Alcian blue stains were performed directly on the unfixed mucosal flaps to identify ACF and mucin-depleted foci (MDF). Detailed topographic analyses, with immunohistochemical staining for ß-catenin and cancer stem cell (CSC) markers (CD44, CD24, and CD166) were performed. RESULTS: ACF, MDF, and ß-catenin-accumulated crypts were detected more in specimens with adjacent CRC. The left colon had ACF with a larger diameter and greater crypt multiplicity, density, and gyriform pit pattern and were considered the high-risk ACF group. MDF, more commonly associated with dysplasia, is also a marker of possible carcinogenesis. The CD44 CSC marker was significantly upregulated in ACF specimens compared with normal controls. Our 3-tier ACF-only pit pattern classification system showed better linearity with mucosal dysplasia than did the 6-tier Kudo classification. CONCLUSION: High-risk ACF, when detected during chromoendoscopic screening, should be followed up. CSCs might play an important role in pathogenesis. Larger studies and genotypic risk stratification for definite identification of high-risk ACF are needed.

Patients with mild enteropathy have apoptotic injury of enterocytes similar to that in advanced enteropathy in celiac disease.

BACKGROUND: Severity of villous atrophy in celiac disease (CeD) is the cumulative effect of enterocyte loss and cell regeneration. Gluten-free diet has been shown to benefit even in patients having a positive anti-tissue transglutaminase (tTG) antibody titre and mild enteropathy. AIM: We explored the balance between mucosal apoptotic enterocyte loss and cell regeneration in mild and advanced enteropathies. METHODS: Duodenal biopsies from patients with mild enteropathy (Marsh grade 0 and 1) (n=26), advanced enteropathy (Marsh grade ≥2) (n=41) and control biopsies (n=12) were subjected to immunohistochemical staining for end-apoptotic markers (M30, H2AX); markers of cell death (perforin, annexin V); and cell proliferation (Ki67). Composite H-scores based on the intensity and distribution of markers were compared. RESULTS: End-apoptotic markers and marker of cell death (perforin) were significantly up-regulated in both mild and advanced enteropathies, in comparison to controls; without any difference between mild and advanced enteropathies. Ki67 labelling index was significantly higher in crypts of mild enteropathy, in comparison to controls, suggesting maintained regenerative activity in the former. CONCLUSIONS: Even in patients with mild enteropathy, the rate of apoptosis is similar to those with advanced enteropathy. These findings suggest the necessity of reviewing the existing practice of not treating patients with mild enteropathy.

The G1 phase E3 ubiquitin ligase TRUSS that gets deregulated in human cancers is a novel substrate of the S-phase E3 ubiquitin ligase Skp2.

E3 ubiquitin ligases have been implicated in the ubiquitination and proteasome-mediated degradation of several key regulators of cell cycle. Owing to their pleotropic behavior, E3 ubiquitin ligases are tightly regulated both at transcriptional and post-translational levels. The E3 ubiquitin ligase TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein) which negatively regulates c-Myc, are found down-regulated in most human cancer cell lines. However, the mechanism of regulation of intracellular levels of TRUSS remains elusive. Here we show that TRUSS is expressed majorly during the G1 phase of cell cycle and its level starts to decline with the expression of S-phase specific E3 ligase Skp2. Enforced expression of Skp2 led to a marked increase in the ubiquitination of TRUSS after its phosphorylation by GSK3ß and followed by rapid proteolytic degradation. Our co-immunoprecipitation studies suggested a direct interaction between Skp2 and TRUSS through the LRR motif of Skp2. Interestingly, the human tumor samples that exhibited elevated expression of Skp2, showed relatively poor expression of TRUSS. Further, enforced expression of HBx, the oncoprotein of Hepatitis B virus which is known to stabilize c-Myc and enhance its oncogenic potential, led to the intracellular accumulation of TRUSS as well as c-Myc. Apparently, HBx also interacted with TRUSS which negatively impacted the TRUSS-c-Myc and TRUSS-Skp2 interactions leading to stabilization of TRUSS. Thus, the present study suggests that TRUSS is a novel substrate of E3 ligase Skp2 and that disruption of TRUSS-Skp2 interaction by viral oncoproteins could lead to pathophysiological sequelae.

Mechanism of villous atrophy in celiac disease: role of apoptosis and epithelial regeneration.

CONTEXT: The data on status of apoptosis in patients with celiac disease are conflicting. Furthermore, complex interaction between intrinsic and common apoptotic pathways, apoptotic inhibitors, and epithelial cell proliferation is largely unclear for patients with celiac disease. OBJECTIVES: To determine the role of apoptosis and epithelial cell regeneration in celiac disease. DESIGN: Twenty-five treatment-naïve patients with celiac disease and 6 patients with functional dyspepsia, as controls, were included and duodenal biopsy specimens from all were subjected to immunohistochemistry with markers of intrinsic apoptotic pathway (AIF, H2AX, p53), common pathway (CC3, M30), apoptotic inhibitors (XIAP, Bcl2), and epithelial proliferation (Ki-67). Apoptotic and proliferation indices were calculated. RESULTS: Expression of end-apoptotic products, that is, H2AX in the cell nuclei (P = .01) and M30 in the cell cytoplasm (P < .01), was significantly upregulated in celiac disease in comparison to controls. Cleaved caspase-3 was also upregulated in villous cytoplasm in celiac disease. Apoptotic inhibitor Bcl2 was significantly down-regulated in celiac disease in comparison to controls. In addition, Ki-67 proliferation index was upregulated both in the crypts and villous mucosal epithelium in comparison to the crypts of the controls. CONCLUSIONS: Treatment-naïve patients with celiac disease have significantly higher level of apoptosis that involves both the common and intrinsic apoptotic pathways. Increased apoptosis and unequaled cell regeneration in crypts probably results in villous atrophy. Down-regulation of apoptotic inhibitors in treatment-naïve celiac disease imparts an additional pro-apoptotic effect.

Hepatitis e: molecular virology and pathogenesis.

Hepatitis E virus is a single, positive-sense, capped and poly A tailed RNA virus classified under the family Hepeviridae. Enteric transmission, acute self-limiting hepatitis, frequent epidemic and sporadic occurrence, high mortality in affected pregnants are hallmarks of hepatitis E infection. Lack of an efficient culture system and resulting reductionist approaches for the study of replication and pathogenesis of HEV made it to be a less understood agent. Early studies on animal models, sub-genomic expression of open reading frames (ORF) and infectious cDNA clones have helped in elucidating the genome organization, important stages in HEV replication and pathogenesis. The genome contains three ORF's and three untranslated regions (UTR). The 5' distal ORF, ORF1 is translated by host ribosomes in a cap dependent manner to form the non-structural polyprotein including the viral replicase. HEV replicates via a negative-sense RNA intermediate which helps in the formation of the positive-sense genomic RNA and a single bi-cistronic sub-genomic RNA. The 3' distal ORF's including the major structural protein pORF2 and the multifunctional host interacting protein pORF3 are translated from the sub-genomic RNA. Pathogenesis in HEV infections is not well articulated, and remains a concern due to the many aspects like host dependent and genotype specific variations. Animal HEV, zoonosis, chronicity in immunosuppressed patients, and rapid decompensation in affected chronic liver diseased patients warrants detailed investigation of the underlying pathogenesis. Recent advances about structure, entry, egress and functional characterization of ORF1 domains has furthered our understanding about HEV. This article is an effort to review our present understanding about molecular biology and pathogenesis of HEV.

Association of pro-inflammatory cytokines, adipokines & oxidative stress with insulin resistance & non-alcoholic fatty liver disease.

BACKGROUND & OBJECTIVES: The cytokines, adipokines, and oxidative stress have been implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD); however, such data remain scarce in India. The present study evaluated pro-inflammatory cytokines, adipokines, and markers of oxidative stress in patients with non-alcoholic fatty liver disease (NAFLD), and their association with degree of adiposity, insulin resistance and markers of disease severity. METHODS: The present prospective cross-sectional pilot study included 79 subjects; 34 NAFLD, 22 chronic hepatitis B (CH-B) and 23 healthy controls (HC). The parameters studied were adiponectin, leptin, tumour necrosis factor α (TNFα), interleukin-1 and 6 (IL-1, IL-6), and systemic markers of oxidative stress. RESULTS: The mean body mass index (kg/m 2 ) in NAFLD patients, CHB, and HC were 26.4±3.7, 21.3±2.3, and 22.3±2.7, respectively. The median serum levels of all pro-inflammatory cytokines were significantly higher (P<0.001) in NAFLD compared to control groups. Compared to HC, levels of adiponectin and leptin were significantly (P<0.05, P<0.01) reduced in both NAFLD and CHB. IL-6 showed marked and selective increase only in NAFLD patients. The levels of IL-6 were significantly (P<0.02) higher in NAFLD patients with advanced histology grade and correlated with IR (r=0.42, P=0.02). In a sub-group, markers of oxidative stress were significantly higher, and that of antioxidant potential were significantly lower among NAFLD patients compared to control subjects. INTERPRETATION & CONCLUSIONS: Patients with NAFLD revealed significantly elevated levels of pro-inflammatory cytokines, increased oxidative stress, and a significant association of IL-6 with IR and advanced histopathology.

Effect of addition of short course of prednisolone to gluten-free diet on mucosal epithelial cell regeneration and apoptosis in celiac disease: a pilot randomized controlled trial.

BACKGROUND: Identification of adjuvant treatment is necessary for rapid and effective treatment in patients with celiac disease. In a pilot randomized controlled trial, the effect of prednisolone on enterocyte apoptosis and regeneration in celiac disease was investigated. PATIENTS AND METHODS: Thirty-three treatment-naïve patients with celiac disease were randomized to either gluten-free diet (GFD, n = 17) or GFD + prednisolone (1 mg/kg for 4 weeks, n = 16). Duodenal biopsies were taken at baseline and at 4 and 8 weeks posttreatment. Six patients with functional dyspepsia were recruited as controls. All these biopsies were stained for markers of intrinsic apoptotic pathway (AIF, H2AX, p53), common apoptotic pathway (CC3, M30), apoptotic inhibitors (XIAP, Bcl2), and epithelial proliferation (Ki-67). Apoptotic (AI) and proliferation indices (PI) were compared. RESULTS: At baseline duodenal biopsies, the end apoptotic products H2AX and M30 were significantly increased. In comparison with those treated with GFD alone, after 4 weeks of GFD + prednisolone treatment, some markers of both intrinsic and common apoptotic pathways showed rapid decline. After prednisolone withdrawal, there was overexpression of H2AX, CC3, and p53 in the latter group. In comparison with those treated with only GFD, patients treated with prednisolone showed suppression of mucosal PI, which started rising again after withdrawal of prednisolone. CONCLUSIONS: Apoptosis takes place in mucosal epithelium in celiac disease. Addition of short course of prednisolone suppresses apoptosis rapidly. However, it also suppresses epithelial regeneration; hence, if used, it should be withdrawn after an initial short course.

Comparative tight junction protein expressions in colonic Crohn's disease, ulcerative colitis, and tuberculosis: a new perspective.

We intended to see the pattern of TJ protein expression along with ultrastructural changes in colonic biopsies from patients with Crohn's disease (CD), ulcerative colitis (UC), and tuberculosis (cTB). Colonic biopsies from 11 patients with active CD and ten patients each with active UC and untreated cTB were taken along with biopsies from six patients with irritable bowel syndrome as controls. These were evaluated for expression pattern of key TJ proteins which included claudin-2 as TJ pore-forming protein, claudin-4 as pore-sealing protein, ZO-1 as scaffold protein, and occludin as TJ protein related to cell migration and polarity. Claudin-2 expression was upregulated along the whole length of intercellular junction (ICJ) in biopsies from patients with active CD and UC in comparison to the biopsies from cTB patients and controls, where its expression was limited to the uppermost part of ICJ. There was reduced expression of ZO-1 in UC, CD, and cTB. On transmission electron microscopic examination, the pentalaminar structure of TJs was destroyed in patients with CD and UC but no significant change was seen in those with cTB and in controls. The expression of claudin-2 was distinctly different in active CD and UC in comparison to its expression pattern in patients with cTB and in controls. The redistribution of claudin-2 expression was in accordance with the TJ ultrastructural changes in patients with UC, CD, and cTB. Altered claudin-2 expression, along with destroyed TJs, may result in loss of selective permeability in patients with UC and CD.

Clinical profile, etiology and therapeutic outcome in 324 hepatocellular carcinoma patients at a tertiary care center in India.

OBJECTIVE: To study the profile and outcome of therapy for hepatocellular carcinoma (HCC) in India. METHODS: Data analysis of HCC patients enrolled in liver clinic between 1990 and 2005. RESULTS: We registered 324 HCC patients [males 284 (88%), mean age 52.4 +/- 13.1 years]. The etiology of HCC was: hepatitis B virus 165 (51%), hepatitis C virus 38 (12%), alcohol 20 (6%), combined 31 (10%) and unknown 70 (21%). Serum alpha-fetoprotein was >400 ng in 36%, portal vein invasion was seen in 40% and distant metastases in 13%. Therapy was offered to 141 (43.5%) patients, but survival data was available in only 130 (93%) of them. Treatment given and median survival time was as follows: surgical resection, 19 months (n = 14); transarterial chemoembolization, 11 months (n = 23); transarterial rhenium therapy, 26 months (n = 7); radiofrequency ablation, 24 months (n = 4); acetic acid ablation, 13 months (n = 17); oral chemotherapy, 26 months (n = 33), and combination therapy, 26 months (n = 32). Vascular invasion, Okuda staging and therapy were independent factors associated with survival. Treated patients had longer median survival compared to untreated ones (16 months vs. 7 months, p < 0.05). CONCLUSIONS: Hepatitis B infection is the predominant cause of HCC in India. Serum alpha-fetoprotein was diagnostic in only one third of our patients. Most patients present late, when curative therapies are not possible. Treated patients had better survival than untreated ones.

HBV genotypes in India: do they influence disease severity?

AIMS: Association of HBV genotypes (especially A and D) with severity of liver disease is controversial. We studied the influence of HBV genotypes on liver disease severity among Indian patients. METHODS: We selected 247 HBV infected patients (42 acute hepatitis, 87 carriers, 44 chronic hepatitis B [CHB], 35 liver cirrhosis [LC] and 40 hepatocellular carcinoma [HCC]). Genotyping of stored sera was performed using genotype-specific enzyme-linked immunosorbent assay (ELISA) and restriction fragment length polymorphism (RFLP). The distribution of genotypes in disease states of differing clinical, histological and biochemical severity were compared. RESULTS: The most common genotype was D (162/237, 68.3%), followed by A (61, 25.7%) and C (14, 5.9%). The distribution of HBV genotypes between patients with acute hepatitis and CHB (carriers + CHB + LC + HCC), or between carriers and disease states (CHB + LC + HCC), or between mild chronic infection (carriers + CHB) and complications of chronic HBV infection (LC + HCC) was similar. Eighty-seven patients had liver biopsy; the median histological activity index (HAI) and fibrosis stage at baseline were similar between genotype groups (four [1-9] genotype A [n = 28]), three (2-4) genotype C (n = 4) and four (1-10) genotype D (n = 55); P = 0.33 for HAI score; (0.5 [0-6] genotype A, 0.5 [0-4] genotype C and 1 [0-6] genotype D; P = 0.92 for fibrosis stage). The response to therapy was similar between the genotypes. CONCLUSION: Clinical, histological severity and therapeutic responses are similar among patients with HBV genotypes A and D.