Trinidad Caballero

Automatic quantification of liver fibrosis: design and validation of a new image analysis method: comparison with semi-quantitative indexes of fibrosis

BACKGROUND/AIMS Liver fibrosis is one of the most important and characteristic histologic alterations in progressive and chronic liver diseases. Thus, in both clinical and experimental practice, it is fundamental to have a reliable and objective method for its precise quantification. Several semi-quantitative scoring systems have been described. All are time-consuming and produce partially subjective fibrosis evaluations that are not very precise. This paper describes the design and validation of an original image analysis-based application, FibroQuant, for automatically and rapidly quantifying perisinusoidal, perivenular and portal-periportal and septal fibrosis and portal-periportal and septal morphology in liver histologic specimens. METHODS The implemented image-processing algorithms automatically segment interstitial fibrosis areas, while extraction of portal-periportal and septal region is carried out with an automatic algorithm and a simple interactive step. For validation, all automatically extracted areas were also manually segmented and quantified. RESULTS Statistical analysis showed significant intra- and interoperator variability in manual segmentation of all areas. Automatic quantifications did not significantly differ from mean manual evaluations of the same areas. Comparison of our image analysis quantifications with staging histologic evaluations of liver fibrosis showed significant correlations (Spearmans, 0.72<r<0.83; p<0.0001) and that the latter are based more on the distribution patterns than on the quantity of fibrosis. CONCLUSIONS FibroQuant is a sensitive, precise, objective and reproducible method of fibrosis quantification, which complements semi-quantitative histologic evaluation systems. This novel tool could be of special value in clinical trials and for improving the prognosis and follow-up among patients with fibrosis-inducing hepatic diseases.

Liver fibrosis assessment with semiquantitative indexes and image analysis quantification in sustained-responder and non-responder interferon-treated patients with chronic hepatitis C

BACKGROUND/AIMS The effect of interferon on the reduction of liver fibrosis is controversial. We aimed to compare semiquantitative methods with a quantitative digital image analysis system to assess liver fibrosis in biopsies from patients with chronic hepatitis and different responses to interferon. METHODS We studied 98 liver biopsies with chronic hepatitis C before and after recombinant interferon alfa-2 treatment, using conventional histological assessment, grading of histological activity, scoring/staging of fibrosis (Knodell and Scheuer), and quantification of fibrosis with image analysis (FibroQuant). RESULTS Sustained-responders to interferon showed a significant reduction in histological lesions and in their Knodell and Scheuer activity indexes. The semiquantitative systems showed no reduction in fibrosis. The FibroQuant application showed a significant reduction in porto-periportal and septal areas among sustained-responders (P < 0.001) and non-responders (P < 0.05), and in porto-periportal and septal fibrosis areas only in sustained-responders (P < 0.001), whereas the percentage of fibrosis increased in non-responders (P < 0.001). CONCLUSIONS The Scheuer system is useful for the daily evaluation of fibrosis, but the FibroQuant application provides more objective data on the anti-fibrogenic effects of interferon, which include a reduction in the porto-periportal area in sustained-responders and non-responders, accompanied by a reduction in the area of fibrosis only when the viral replication has ceased.

Inhibition of poly adenosine diphosphate‐ribose polymerase decreases hepatocellular carcinoma growth by modulation of tumor‐related gene expression

Hepatocellular carcinoma (HCC) is associated with a poor prognosis due to a lack of effective treatment options. In HCC a significant role is played by DNA damage and the inflammatory response. Poly (ADP‐ribose) polymerase‐1 (PARP‐1) is an important protein that regulates both these mechanisms. The objective of this study was to examine the effect of pharmacology PARP‐1 inhibition on the reduction of tumor volume of HCC xenograft and on the hepatocarcinogenesis induced by diethyl‐nitrosamine (DEN). Pharmacologic PARP‐1 inhibition with DPQ greatly reduces tumor xenograft volume with regard to a nontreated xenograft (394 mm3 versus 2,942 mm3, P < 0.05). This observation was paralleled by reductions in xenograft mitosis (P = 0.02) and tumor vasculogenesis (P = 0.007, confirmed by in vitro angiogenesis study), as well as by an increase in the number of apoptotic cells in DPQ‐treated mice (P = 0.04). A substantial difference in key tumor‐related gene expression (transformed 3T3 cell double minute 2 [MDM2], FLT1 [vascular endothelial growth factor receptor‐1, VEGFR1], epidermal growth factor receptor [EPAS1]/hypoxia‐inducible factor 2 [HIF2A], EGLN1 [PHD2], epidermal growth factor receptor [EGFR], MYC, JUND, SPP1 [OPN], hepatocyte growth factor [HGF]) was found between the control tumor xenografts and the PARP inhibitor‐treated xenografts (data confirmed in HCC cell lines using PARP inhibitors and PARP‐1 small interfering RNA [siRNA]). Furthermore, the results obtained in mice treated with DEN to induce hepatocarcinogenesis showed, after treatment with a PARP inhibitor (DPQ), a significant reduction both in preneoplastic foci and in the expression of preneoplastic markers and proinflammatory genes (Gstm3, Vegf, Spp1 [Opn], IL6, IL1b, and Tnf), bromodeoxyuridine incorporation, and NF‐κB activation in the initial steps of carcinogenesis (P < 0.05). Conclusion: This study shows that PARP inhibition is capable of controlling HCC growth and preventing tumor vasculogenesis by regulating the activation of different genes involved in tumor progression. (HEPATOLOGY 2010;51:255–266.)

Fibrolamellár hepatocellular carcinoma. An immunohistochemical and ultrastructural study

Two cases of fibrolamellar carcinoma of the liver are reported in young female patients of 12 and 21 years of age. Small amounts of perinuclear alpha‐fetoprotein were found, unrelated to hyaline globules, as well as alpha1‐antitrypsin in a periglobular fashion in isolated cells. Ferritin was present in a large number of cells. Ultrastructurally, the well differentiated nature of the neoplasm was substantiated by previously unreported findings such as intercellular lumina analogous to bile canaliculi and peroxisome‐like bodies containing a central crystalloid. Filamentous material resembling Mallorys type of hyaline was also found. We conclude that both immunohistochemical and ultrastructural features reflect a high degree of differentiation.

Hepatic Expression of Adiponectin Receptors Increases with Non-alcoholic Fatty Liver Disease Progression in Morbid Obesity in Correlation with Glutathione Peroxidase 1

BackgroundThe prevalence of non-alcoholic fatty liver disease (NAFLD) in obesity is very high. The role of adiponectin receptors in NAFLD progression remains still unclear. We speculate that changes in the hepatic expression levels of the two adiponectin receptors may be associated with the expression of oxidative stress-related genes.MethodsWe studied 60 morbidly obese patients with NAFLD, who underwent liver biopsy at the time of bariatric surgery. We measured the hepatic messenger-RNA concentration of adiponectin receptors (ADIPOR1 and ADIPOR2), glutathione peroxidase 1 (GPx1), glutathione reductase (GRd) and inducible oxide nitric synthase. Additionally, biochemical parameters and oxidative stress markers were determined in blood samples. According to the Kleiner score, the patients were divided into two groups: group 1 (25 patients without steatohepatitis) and group 2 (25 patients with probable steatohepatitis and ten patients with steatohepatitis).ResultsThe messenger-RNA concentration of all genes analysed in the study was higher among the patients in group 2. However, no differences in blood oxidative stress markers were observed. Strong correlations were found among the expression levels of ADIPOR1, ADIPOR2 and GPx1. The multivariate analysis showed that the only independent variable associated with NAFLD progression was the increase in GPx1 expression levels.ConclusionsNAFLD progression in morbid obesity is associated with increase in hepatic adiponectin receptor and oxidative stress-related genes. The linear correlations suggest that ADIPOR1, ADIPOR2 and GPx1 share key molecular factors in the regulation of the genetic expressions.

Intraepithelial and lamina propria leucocyte subsets in inflammatory bowel disease: an immunohistochemical study of colon and rectal biopsy specimens.

AIMS--To gain new insights into the pathogenesis and differential diagnosis of ulcerative colitis and colonic Crohns disease. METHODS--Immunohistochemistry for different leucocyte subsets was performed in biopsy specimens of the sigmoid colon and rectum from 55 patients with inflammatory bowel disease and 11 healthy controls. RESULTS--Colonic biopsy specimens from patients with active ulcerative colitis had significantly higher numbers of CD45+ and CD3+ leucocytes compared with those from patients with inactive disease, and higher numbers of total leucocytes and macrophages than those from patients with Crohns disease. Rectal biopsy specimens from patients with Crohns disease had greater numbers of intraepithelial leucocytes (CD45, CD3 and CD8 cells) than specimens from patients with active or inactive ulcerative colitis, or from healthy controls. CONCLUSIONS--Because of the phenotypic differences in the inflammatory infiltrate in the mucosa from the sigmoid colon and the rectum, the segment of the intestine to be biopsied should be specified. Assessment of the leucocytic component of the intraepithelial infiltrate in rectal biopsy specimens was more useful than examination of colonic biopsy specimens in the differential diagnosis of ulcerative colitis and Crohns disease.

PARP inhibition attenuates histopathological lesion in ischemia/reperfusion renal mouse model after cold prolonged ischemia

We test the hypothesis that PARP inhibition can decrease acute tubular necrosis (ATN) and other renal lesions related to prolonged cold ischemia/reperfusion (IR) in kidneys preserved at 4°C in University of Wisconsin (UW) solution. Material and Methods. We used 30 male Parp1+/+ wild-type and 15 male Parp10/0 knockout C57BL/6 mice. Fifteen of these wild-type mice were pretreated with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) at a concentration of 15 mg/kg body weight, used as PARP inhibitor. Subgroups of mice were established (A: IR 45 min/6 h; B: IR + 48 h in UW solution; and C: IR + 48 h in UW solution plus DPQ). We processed samples for morphological, immunohistochemical, ultrastructural, and western-blotting studies. Results. Prolonged cold ischemia time in UW solution increased PARP-1 expression and kidney injury. Preconditioning with PARP inhibitor DPQ plus DPQ supplementation in UW solution decreased PARP-1 nuclear expression in renal tubules and renal damage. Parp10/0 knockout mice were more resistant to IR-induced renal lesion. In conclusion, PARP inhibition attenuates ATN and other IR-related renal lesions in mouse kidneys under prolonged cold storage in UW solution. If confirmed, these data suggest that pharmacological manipulation of PARP activity may have salutary effects in cold-stored organs at transplantation.

Nonalcoholic Steatohepatitis in a Patient with Ataxia-Telangiectasia

Ataxia-telangiectasia (A-T) is a rare disease characterized by neurodegenerative alterations, telangiectasia, primary immunodeficiency, extreme sensitivity to radiation, and susceptibility to neoplasms. A-T patients have inactivation of ataxia-telangiectasia-mutated (ATM) protein, which controls DNA double-strand break repair and is involved in oxidative stress response, among other functions; dysfunctional control of reactive oxygen species may be responsible for many of the clinical manifestations of this disease. To the best of our knowledge, hepatic lesions of steatohepatitis have not previously been reported in A-T patients. The present study reports the case of a 22-year-old man diagnosed with A-T at the age of 6 years who was referred to our Digestive Disease Unit with a three-year history of hyperlipidemia and liver test alterations. Core liver biopsy showed similar lesions to those observed in nonalcoholic steatohepatitis. Immunohistochemical staining disclosed the absence of ATM protein in hepatocyte nuclei. We suggest that the liver injury may be mainly attributable to the oxidative stress associated with ATM protein deficiency, although other factors may have made a contribution. We propose the inclusion of A-T among the causes of nonalcoholic steatohepatitis, which may respond to antioxidant therapy.

Estudio de la coinfección por el virus de la hepatitis G en la hepatitis crónica C. Respuesta al tratamiento con interferón alfa

BACKGROUND: It is thought that the cytopathic effect of HGV is not important. Nevertheless, the cytopathic effect on liver is less known in the cases of co-infection with HCV. The aim was to study the prevalence of co-infection in patients with chronic hepatitis C (CHC) and to analyse the clinical-epidemiological and histological data and the interferon (IFN) response. PATIENTS AND METHODS: We included 180 patients with CHC and the HGV-RNA was determined. RESULTS: The prevalence of co-infection was 12.2% (n = 22). No statistical differences were observed between the non co-infected and co-infected groups with regard to the age, sex, mechanism of transmission and alcohol abuse. Also, there were no differences in the hepatic biochemical, no organ-specific antibodies, histological lesions and Knodell index. The HCV biochemical response (BR) and virological response (VR) after 6 months post-IFN were the same in both groups (HGV negative: BR = 29%, VR = 12%; HGV positive: BR = 22%, VR = 18%). HGV was determined after 6 months posttreatment in the co-infected group (first cycle of IFN, n = 22; second cycle of IFN, n = 9): 12 (55%) were HGV-RNA negative and 5 (23%) HCV-RNA negative, (p = 0.021). When we compared the BR vs VR in this group, there were 12 HGV-RNA negative but only two had BR (NS). On the contrary, the BR was related to HCV-RNA negative (p = 0.023). CONCLUSION: The prevalence of HGV co-infection is important in our area (12.8%). The HGV does not increase the pathogenicity of HCV and does not change the IFN response, although the HGV is more IFN sensible than HCV. The determination of HGV is not necessary in patients with HCV.

Pseudomyxoma peritonei et ovarii associated with sequential ovarian and appendicular tumors and acromegaly

Pseudomyxoma peritonei associated with highly differentiated mucinous, intestinal-type ovarian tumor is reported in a 46-year-old acromegalic patient. Five years after its discovery a second operation revealed a mucinous cystadenoma of the appendix. The coexistence of pseudomyxoma peritonei with ovarian and appendicular tumors is rare, raising questions about the primary was origin of the peritoneal tumor. In this case the primary was ovarian while the appendicular tumor occurred later. Since acromegaly is associated with a high risk for the development of colorectal tumors, it could also have elicited a similar response in the intestinal-type ovarian tumor and the appendicular neoplasm found in this patient. The role of appendectomy in the surgical treatment and staging of ovarian tumors is stressed.