P. Bedossa

Increased Infiltration of Macrophages in Omental Adipose Tissue Is Associated With Marked Hepatic Lesions in Morbid Human Obesity

In human obesity, white adipose tissue (WAT) is enriched in macrophages. How macrophage infiltration in WAT contributes to the complications of obesity is unknown. This study tested the hypothesis that recruitment of macrophages in omental WAT is associated with hepatic damage in obese patients. Paired biopsies of subcutaneous and omental WAT and a liver biopsy were collected during gastric surgery in 46 obese women and 9 obese men (BMI 47.9 ± 0.93 kg/m2). The number of HAM56+ macrophages in WAT was quantified microscopically, and correlations with clinical and biological parameters and histological liver pathology were investigated. There were twice as many macrophages in omental as in subcutaneous WAT (P < 0.0001). After adjustment for age, omental WAT macrophage infiltration was correlated to fasting glucose and insulin, quantitative insulin sensitivity check index, triglycerides, aspartate aminotransferase (AST), and γ-glutamyltranspeptidase. We propose an easy equation to estimate the amount of macrophages in omental WAT. Increased macrophage accumulation specifically in omental WAT was associated with hepatic fibroinflammatory lesions (P = 0.01). The best predictive model for the severity of hepatic damage includes adiponectinemia, AST, and omental WAT macrophages. These data suggest that the presence of macrophages in omental WAT participates in the cellular mechanisms favoring hepatic fibroinflammatory lesions in obese patients.

Adipose tissue transcriptomic signature highlights the pathological relevance of extracellular matrix in human obesity

BackgroundInvestigations performed in mice and humans have acknowledged obesity as a low-grade inflammatory disease. Several molecular mechanisms have been convincingly shown to be involved in activating inflammatory processes and altering cell composition in white adipose tissue (WAT). However, the overall importance of these alterations, and their long-term impact on the metabolic functions of the WAT and on its morphology, remain unclear.ResultsHere, we analyzed the transcriptomic signature of the subcutaneous WAT in obese human subjects, in stable weight conditions and after weight loss following bariatric surgery. An original integrative functional genomics approach was applied to quantify relations between relevant structural and functional themes annotating differentially expressed genes in order to construct a comprehensive map of transcriptional interactions defining the obese WAT. These analyses highlighted a significant up-regulation of genes and biological themes related to extracellular matrix (ECM) constituents, including members of the integrin family, and suggested that these elements could play a major mediating role in a chain of interactions that connect local inflammatory phenomena to the alteration of WAT metabolic functions in obese subjects. Tissue and cellular investigations, driven by the analysis of transcriptional interactions, revealed an increased amount of interstitial fibrosis in obese WAT, associated with an infiltration of different types of inflammatory cells, and suggest that phenotypic alterations of human pre-adipocytes, induced by a pro-inflammatory environment, may lead to an excessive synthesis of ECM components.ConclusionThis study opens new perspectives in understanding the biology of human WAT and its pathologic changes indicative of tissue deterioration associated with the development of obesity.

Identification of a new marker of hepatocellular carcinoma by serum protein profiling of patients with chronic liver diseases

Surface‐enhanced laser desorption ionization time‐of‐flight mass spectrometry (SELDI‐TOF MS) is a proteomic technique that enables the profiling of proteins present in any biological material studied. We used this approach to identify new biomarkers of hepatocellular carcinoma (HCC) in the sera of patients with cirrhosis. Sera from 82 patients with cirrhosis, either without (n = 38) or with (n = 44) HCC, were analyzed by SELDI‐TOF MS, and the results of the two groups were compared. The most efficient protein peaks leading to discrimination of patients with HCC were selected (receiver operative characteristic curves). The highest‐scoring peak combination was established in a first group of serum samples (multinomial regression) and was tested in an independent group. The protein corresponding to the highest discrimination was purified and characterized further. The intensity of 30 protein peaks significantly differed between cirrhotic patients with and without HCC. An algorithm including the six highest‐scoring peaks allowed correct classification (presence or absence of HCC) of 92.5% of patients in the test sample set and 90% in the validation sample set. The highest discriminating peak (8,900 Da) was purified further and was characterized as the C‐terminal part of the V10 fragment of vitronectin. An in vitro study suggested that the increase of the 8,900‐Da fragment in the serum of patients with HCC may proceed from the cleavage of native vitronectin with metalloproteases, a family of enzymes whose activity is enhanced in HCC. In conclusion, global protein profiling is an efficient approach that enabled us to identify a catalytic fragment of vitronectin as a new serum marker of HCC in patients with chronic liver diseases. (HEPATOLOGY 2005;41:40–47.)

Liver extracellular matrix in health and disease

Liver fibrosis is the hallmark of every chronic liver disease. It is also the major factor of morbidity and mortality due to the development of cirrhosis and its complications including hepatocellular carcinoma. But even at the beginning of the process of liver fibrosis and due to the strategic position of the extracellular matrix at the interface between blood flow and epithelial compartment, any quantitative or qualitative modification of extracellular matrix will rapidly affect structure and function of the liver. The development of several animal models of liver fibrosis as well as isolation and cultivation of hepatic stellate cells, the major fibrogenic cell type in the liver, led to the gathering of recent knowledge on the mechanism of liver fibrosis. Activation of hepatic stellate cells is a key event in this process and many details on this finely tuned mechanism are now available. In addition to these experimental data, experience from chronic hepatitis C now allows the development of new concepts and perspectives such as liver fibrosis regression and antifibrotic therapies. Copyright

Efficacy of peginterferon alpha-2b in chronic hepatitis delta: relevance of quantitative RT-PCR for follow-up.

Hepatitis delta virus (HDV) can cause severe acute and chronic liver disease in patients infected by hepatitis B virus. Interferon alpha at high doses, although poorly efficient, is the only treatment reported to provide some benefit in chronic hepatitis delta. Pegylated interferon alpha (PEG‐IFN) has not yet been evaluated. Treatment is usually monitored by the qualitative detection of HDV‐RNA in serum. In this study, safety and efficacy of PEG‐IFN were assessed in chronic hepatitis delta, and serum HDV‐RNA kinetics were determined using quantitative RT‐PCR. Fourteen patients with chronic hepatitis delta received subcutaneous PEG‐IFN alpha‐2b during 12 months (1.5 μg/kg per week). Serum HDV‐RNA was quantified at initiation and during the course of therapy, and during the posttreatment follow‐up period, which ranged from 6 to 42 months (median 16 months). PEG‐IFN alpha‐2b was well tolerated, inducing no serious adverse effect. Sustained biochemical response was obtained in 8 patients (57%). At the end of treatment, 8 patients (57%) had achieved virological response (undetectable HDV‐RNA). Sustained virological response throughout the posttreatment follow‐up period was observed in 6 patients (43%). HDV‐RNA kinetics were predictive of the response: after 3 months of PEG‐IFN, HDV‐RNA levels were significantly lower in the responders than in the nonresponders group (P = .018). After 6 months of therapy, a negative HDV‐RNA was predictive of sustained response (P = .021). In conclusion, this preliminary study indicates that PEG‐IFN alpha‐2b is safe and efficient for treatment of chronic hepatitis delta. The follow‐up of HDV‐RNA levels during therapy, which allows the differentiation of various profiles of virological responses, improves treatment monitoring. (HEPATOLOGY 2006;44:728–735.)

Evidence for a role of nonalcoholic steatohepatitis in hepatitis C: A prospective study

Although steatosis is a common histological feature in chronic hepatitis C (CHC), nonalcoholic steatohepatitis (NASH) has not yet been clearly characterized in this context. The aim of this prospective study was to investigate the characteristics of patients with NASH and CHC. Biopsies were categorized as CHC alone (178 patients [57%]), CHC+steatosis (94 patients [34%]), or CHC+NASH (24 patients [9%]). Patients with CHC+NASH had significantly higher AST and triglyceride levels and lower high‐density lipoprotein (HDL) cholesterol or total cholesterol than patients with CHC+steatosis. They also showed more steatosis and higher METAVIR fibrosis stage than patients with CHC+steatosis. Genotype 3 was more frequent in patients with CHC+NASH than in patients with CHC+steatosis or CHC alone. Patients with genotype 3 and CHC+NASH were similar to those with CHC+steatosis or with CHC alone according to triglyceride or the homeostasis model for assessment of insulin resistance (HOMA‐IR), whereas in patients with genotype 1, HOMA‐IR and triglyceride increased progressively from CHC alone to CHC+steatosis to CHC+NASH. In multivariate analysis, triglyceride and HDL cholesterol were predictors of NASH in patients with genotype 1, whereas in patients with genotype 3, AST was the only predictor. Conclusion: Patients with CHC+NASH differ significantly from those with CHC+steatosis and CHC alone in terms of biological and metabolic parameters and more advanced histopathological lesions. NASH is more common in genotype 3 and is not associated with metabolic dysfunctions in this subgroup, suggesting that NASH may complicate steatosis in CHC irrespective of etiology of steatosis. (HEPATOLOGY 2007.)

Molecular Profiling of Hepatocellular Carcinomas (HCC) Using a Large-Scale Real-Time RT-PCR Approach: Determination of a Molecular Diagnostic Index

The aim of this study was to develop and validate a molecular index for the diagnosis of hepatocellular carcinoma (HCC) based on genes whose specificity and level of expression are the most discriminating for the diagnosis of HCC. The level of expression of 219 genes was assessed with a real-time reverse transcription-polymerase chain reaction approach in a training set of samples including normal livers (15), cirrhosis (12), and HCC (16). The most informative genes were selected for the molecular index. This index was prospectively validated in a new set of 40 samples (testing set) and in a set of 45 cirrhotic macronodules. 44 out of the 219 genes were differentially expressed in HCC. 13 out of these 44 genes were finally selected for the molecular index according to their diagnostic performance and after exclusion of most redundant genes. Using this index, 42 out of 43 samples of the training set and 39 out of the 40 samples of the testing set were correctly ranked as HCC or not HCC (normal liver or cirrhosis). The index also enabled correct ranking of 44 out of 45 cirrhotic macronodules into 2 groups: benign (including macroregenerative and dysplastic macronodules) and malignant macronodules. This molecular diagnostic index is an efficient tool both for identification of overt HCC as well as minute lesions (cirrhotic macronodules). It might be useful to correctly diagnose borderline lesion or small well-differentiated hepatocellular carcinomas whose diagnosis is often difficult on a histopathological basis.

Expression of the RNA component of human telomerase (hTR) in prostate cancer, prostatic intraepithelial neoplasia, and normal prostate tissue

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected by a polymerase chain reaction (PCR)‐based assay (TRAP) in many immortal cell lines and various cancers, including prostate cancers. To investigate the role of telomerase in prostate cancer at the cellular level, the expression of one of the ribonucleoprotein complexes, the RNA component of human telomerase (hTR), was studied in normal, preneoplastic, and cancerous prostate tissues using a non‐radioactive in situ hybridization procedure. Nine human prostates resected at the time of radical prostatectomy were studied. In each case, archival paraffin‐embedded samples from normal tissue, prostatic intraepithelial neoplasia (PIN) lesions, the putative precancerous lesion, and prostate carcinomas were selected for in situ hybridization. hTR mRNA expression was detected in carcinomatous glands of seven out of the nine cancers (75 per cent). Furthermore, in seven out of the eight cases showing PIN lesions, the epithelial cells of PIN foci also expressed hTR mRNA. By contrast, in normal tissue, epithelial cells were negative, whereas hTR mRNA expression was detected in the basal cells. The detection of hTR mRNA in PIN lesions clearly strengthens the link between PIN and carcinomatous glands and suggests that telomerase expression occurs early in prostate carcinogenesis. Furthermore, this study confirms previous experimental data suggesting that the basal cell layer is the stem cell compartment in prostate. Copyright

The role of replicative senescence in chronic allograft nephropathy.

Abstract Strong evidence suggests that replicative senescence is involved in vivo because senescent cells have been detected in human tissues associated with physiological and pathological aging processes. Chronic allograft nephropathy (CAN) appears to be a major determinant of long-term survival in kidney transplantation. Several mechanisms are potentially involved; the aim of this study was to assess the impact of replicative senescence in CAN. Replicative senescent cells were detected on renal tissue cryosection using expression of a specific marker, senescence-associated β-galactosidase (SA-β-Gal) at pH 6. A total of 80 frozen renal samples (67 cases of CAN and 13 controls) were studied. To validate this marker, we measured in situ telomere length in cells expressing or not expressing SA-β-Gal using a validated quantitative fluorescence in situ hybridization technique. The presence of senescent cells was correlated with clinicopathologic data. Telomere length was significantly lower in cells expressing SA-β-Gal than in cells that did not. Replicative senescence was present in 45 out of 67 (67%) biopsy specimens and was significantly associated with the severity of CAN. No correlation with the notion of a previous episode of acute tubular necrosis, acute rejection, extrarenal epuration, duration of cold ischemia, and the delay between transplantation and biopsy was observed. However, the age of the donor, but not that of the recipient, was correlated with the occurrence of senescent cells. These results suggest that replicative senescence is a mechanism that might be involved in the development of CAN. The age of the donor appears to be the major determinant factor in replicative senescence.

Diagnostic value of serum protein profiling by SELDI-TOF ProteinChip compared with a biochemical marker, FibroTest, for the diagnosis of advanced fibrosis in patients with chronic hepatitis C

FibroTest has been validated for the diagnosis of liver fibrosis in patients with chronic hepatitis C.