Inducible nitric oxide synthase attenuates chronic colitis in human histocompatibility antigen HLA-B27/human beta2 microglobulin transgenic rats.

Rats transgenic for HLA-B27/human beta2-microglobulin develop a spontaneous multisystem inflammatory disorder that closely mimics human spondyloarthropathies. Prominent features of this disorder are gut inflammation that predominates in the colon, and arthritis. Several mediators such as IFN-gamma, IL-1beta, TNF-alpha, and inducible nitric oxide synthase (iNOS) have been found increased in the inflamed colonic mucosa. In the colon of HLA-B27 transgenic rats, iNOS is predominantly expressed by epithelial cells, and iNOS transcripts are detected in the hip cartilage of those rats, but not in nontransgenic littermates. The role of iNOS in this disorder was evaluated by administering the corticosteroid dexamethasone, or the NOS inhibitor L-N6-(1-iminoethyl)lysine (L-NIL) to HLA-B27 transgenic rats with established disease. Treatment with dexamethasone attenuated some aspects of gut inflammation, although it had no effect on iNOS expression. In contrast, treatment with L-NIL effectively inhibited iNOS activity, and resulted in an increase in colitis. Cytokine transcripts in the colon were modified by these treatments: IFN-gamma and IL-1beta were decreased after dexamethasone treatment, whereas administration of L-NIL resulted in decreased IFN-gamma, and TNF-alpha. A trend towards increased IL-1b expression was observed which could have contributed to the L-NIL pro-inflammatory effect. These results suggest that iNOS exerts a protective effect on colitis, in the inflammatory disorder of HLA-B27 transgenic rats.

A case of Enterocytozoon bieneusi infection in an HIV-negative renal transplant recipient.

Reported here is a case of microsporidiosis that occurred in an HIV-negative renal transplant recipient. The patient developed protracted diarrhea 18 months following transplant surgery. Many spores of Enterocytozoon bieneusi were detected in stool smears using a modified trichrome staining method. Identification was confirmed using the polymerase chain reaction. Histological examination of duodenal biopsies revealed numerous spores in the cytoplasm of enterocytes. Tacrolimus and steroid regimens were decreased, treatment with mycophenolate mofetil was discontinued, and the patient was given albendazole and metronidazole for 2 weeks. The diarrhea resolved after 15 days of treatment; 2 months later the patient had recovered completely. A more systematic search for microsporidia using specific staining procedures should be performed in transplant recipients who develop severe diarrhea.

MRI of intralesional hemolysis in focal nodular hyperplasia of the liver.

A case of Kasabach-Merritt syndrome caused by focal nodular hyperplasia of the liver is presented with atypical magnetic resonance findings due to intratumoral hemosiderin deposition. The high sensitivity of magnetic resonance imaging for iron served to identify the site of hemolysis in this patient with Kasabach-Merritt syndrome.

Nuclear accumulation of mutated beta-catenin in hepatocellular carcinoma is associated with increased cell proliferation.

Inappropriate activation of the Wnt pathway resulting from beta-catenin gene alterations has recently been implicated in the development of hepatocellular carcinoma (HCC). To explore the in vivo effects of mutated beta-catenin, HCC specimens from 32 patients carrying one or several tumors were screened for somatic mutations in exon 3 of the beta-catenin gene, and the expression and subcellular localization of beta-catenin was studied by immunohistochemistry. Missense mutations or interstitial deletions in beta-catenin exon 3 were detected in 12 of 35 (34%) HCC samples. After immunostaining, most tumors exhibited increased membranous and/or cytoplasmic expression of beta-catenin compared with adjacent nontumoral liver. Strong nuclear accumulation of beta-catenin was observed either focally or uniformly in 15 of 35 (43%) tumor specimens, but not in cirrhotic nodules or dysplastic liver cells in adjacent liver. Aberrant nuclear expression of beta-catenin was significantly associated with the presence of mutations in the beta-catenin gene (P < 0.005). Moreover, nuclear beta-catenin staining correlated significantly with increased Ki-67 proliferative index in tumor (P < 0.001) and seemed to be associated with poor outcome in patients with HCC. In conclusion, our data indicate that activation of the Wnt/beta-catenin pathway in HCC results mainly from somatic mutations in the beta-catenin gene and may promote tumor progression by stimulating tumor cell proliferation.

Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions.

Matrix metalloproteinase-2 (MMP-2) is involved in extracellular matrix remodeling. It is secreted as a proenzyme and activated by membrane type-MMPs (MT-MMP), such as MT1-MMP. In liver fibrosis, MMP-2 is highly expressed in myofibroblasts and may have a profibrogenic role. The mechanisms of its activation in the liver are still unclear. The aim of this work was to show that pro-MMP-2 is efficiently activated in human fibrotic liver and to investigate the role of cell-matrix interactions in this process. Liver specimens obtained from patients with active cirrhosis were compared to normal liver specimens. Human hepatic myofibroblasts were cultured either on plastic, fibronectin, laminin, or on collagen I gels. MMP-2 activity was visualized by gelatin zymography. MMP-2 active form (59 kd) was detected in active cirrhosis but not in normal liver. Myofibroblasts cultured on plastic, fibronectin, or laminin predominantly expressed inactive pro-MMP-2 (66 kd). In contrast, myofibroblasts cultured on collagen I markedly activated the enzyme. Similar results were obtained using membrane fractions from cells previously cultured on collagen or plastic. Activation was inhibited by the tissue inhibitor of metalloproteinases-2 but not by tissue inhibitor of metalloproteinases-1, implicating a MT-MMP-mediated process. Culture on collagen I up-regulated MT1-MMP protein detected by Western blotting, but decreased MT1-MMP mRNA. This study shows that MMP-2 is activated in fibrotic liver. It suggests that interactions between collagen I and myofibroblasts promote this process through a post-translational increase of MT1-MMP expression in these cells.

Biological effects of C-type natriuretic peptide in human myofibroblastic hepatic stellate cells.

During chronic liver diseases, hepatic stellate cells (HSC) acquire a myofibroblastic phenotype, proliferate, and synthetize fibrosis components. Myofibroblastic HSC (mHSC) also participate to the regulation of intrahepatic blood flow, because of their contractile properties. Here, we examined whether human mHSC express natriuretic peptide receptors (NPR). Only NPR-B mRNA was identified, which was functional as demonstrated in binding studies and by increased cGMP levels in response to C-type natriuretic peptide (CNP). CNP inhibited mHSC proliferation, an effect blocked by the protein kinase G inhibitor 8-(4 chlorophenylthio)-cGMP and by the NPR antagonist HS-142-1 and reproduced by analogs of cGMP. Growth inhibition was associated with a reduction of extracellular signal-regulated kinase and c-Jun N-terminal kinase and with a blockade of AP-1 DNA binding. CNP and cGMP analogs also blunted mHSC contraction elicited by thrombin, by suppressing calcium influx. The relaxing properties of CNP were mediated by a blockade of store-operated calcium channels, as demonstrated using a calcium-free/calcium readdition protocol. These results constitute the first evidence for a hepatic effect of CNP and identify mHSC as a target cell. Activation of NPR-B by CNP in human mHSC leads to inhibition of both growth and contraction. These data suggest that during chronic liver diseases, CNP may counteract both liver fibrogenesis and associated portal hypertension.

Myofibroblasts and hepatocellular carcinoma: an in vivo and in vitro study.

BACKGROUND/AIMS: The number of perisinusoidal myofibroblasts has been shown to be increased in hepatocellular carcinoma, as compared to cirrhosis. This increase might suggest a cooperative relationship between tumour cells and myofibroblasts. To assess this relationship, we undertook: (a) an immunohistochemical study to confirm the existence of an increased number of perisinusoidal myofibroblasts in human hepatocellular carcinoma, as compared to cirrhosis with or without liver cell dysplasia, (b) an in vitro study testing the role of normal or tumoral human hepatocytes in myofibroblast proliferation. METHODS: Forty explanted cirrhotic livers, including 14 with hepatocellular carcinoma and 24 with liver cell dysplasia, were studied. Myofibroblasts were detected by immunohistochemistry using an antibody directed against alpha-smooth muscle actin. Hepatic myofibroblasts in culture were obtained by outgrowth from human liver explants. RESULTS: There was a progressive increase in the number of perisinusoidal myofibroblasts, from cirrhotic nodules without dysplasia to liver cell dysplasia and hepatocellular carcinoma. Conditioned medium from isolated normal human hepatocytes had only minor mitogenic effects on myofibroblasts, as assessed by measuring DNA synthesis and cell growth. In contrast, conditioned medium from a human hepatoma cell line (HepG2 cells) markedly stimulated the proliferation of human myofibroblasts. This mitogenic activity was stored in HepG2 cells and secreted in the extracellular medium rather than being simply released following cell lysis. CONCLUSIONS: These results suggest that the increased number of myofibroblasts in hepatocellular carcinoma might be due to a paracrine mechanism involving soluble mitogenic factor(s) secreted by tumour cells.

A histopathological study of the effects of 6-month versus 12-month interferon alpha-2b therapy in chronic hepatitis C.

BACKGROUND/AIMS: Interferon therapy has been shown to have beneficial effects in chronic hepatitis C, but the optimal duration of treatment has not been clearly defined. The aims of this study were: (a) to perform a detailed histological comparison of the effects of a 6-month and a 12-month treatment using the Knodell score as well as a recently proposed grid of analysis, (b) to determine possible histological predictive factors of response to therapy, and (c) to attempt to relate histological and biochemical modifications. METHODS: Liver biopsies obtained before and 18 months after beginning of treatment were therefore compared in 26 patients treated for 6 months, and in 34 patients treated for 12 months. RESULTS: Six months of treatment induced a significant decrease in periportal (p = 0.02) and intralobular (p = 0.004) hepatocyte necrosis. The same items were improved in the 12-month-treated patients but, in addition, portal inflammation (p = 0.01), bile duct lesions (p = 0.03), lymphoid aggregates (p = 0.002) and fibrosis (p = 0.008) were also improved, according to the Knodell score. Low scores for fibrosis, steatosis and cholangiolar proliferation on the pretreatment liver biopsy could be considered predictive factors for alanine aminotransferase normalization at 6 months. There was no relationship between biochemical response and modification of fibrosis. CONCLUSION: Our results suggest that: (a) a decrease in fibrosis might be detected only after a 12-month interferon treatment, and (b) initial fibrosis, cholangiolar proliferation and steatosis are predictive of a lack of biochemical response. The absence of a relation between biochemical response and evolution of fibrosis implies that the evaluation of treatments in chronic hepatitis C should always include a detailed histopathological study.

A randomized trial comparing colchicine and ursodeoxycholic acid combination to ursodeoxycholic acid in primary biliary cirrhosis. UDCA-PBC Study Group.

The efficacy of colchicine combined with ursodeoxycholic acid (UDCA) and UDCA alone in the treatment of patients with nonadvanced primary biliary cirrhosis (PBC) was evaluated in a 2-year controlled study. Seventy-four patients with PBC who had been treated previously with UDCA (at least 8 months) but still had abnormal liver test results, especially elevated alkaline phosphatase activity, were randomized to be administered colchicine (1 mg/d, 5 days per week) (n = 37) or a placebo (n = 37). In addition, the patients were treated with UDCA (13-15 mg x kg(-1) x day(-1)). The patients underwent clinical examination and liver tests every 6 months and upper endoscopy and liver biopsy at entry and at 2 years. Procollagen type III aminoterminal peptide (PIIINP), hyaluronic acid, and sulfobromophthalein (BSP) elimination kinetics were determined at entry and after 2 years. After 2 years of treatment, relative to UDCA, colchicine combined with UDCA did not significantly improve symptoms, laboratory findings (serum bilirubin level, alkaline phosphatase and alanine transaminase [ALT] activities, immunoglobulin [Ig] M level), serum markers of fibrosis, or histological features, except lobular inflammation. Colchicine did tend to slightly reduce the progression of esophageal varices; however, the difference was not significant. BSP elimination kinetics (45-minute retention percentage) was significantly improved when treated with colchicine. During the 2-year study, the only clinical complications were variceal bleeding in one patient administered colchicine and two administered the placebo. Two patients died from nonliver causes. One severe adverse effect (peripheral neuromyopathy) was observed in a colchicine-treated patient. In conclusion, this study suggests that colchicine appears to provide a slight advantage relative to UDCA alone in patients with nonadvanced PBC.

Identification of intracellular stages of Cyclospora species by light microscopy of thick sections using hematoxylin.

Cyclospora sp is a recently identified coccidia responsible for enteric infection in humans. Most reports have failed to detect this parasite in intestinal biopsies by light microscopy, although the different stages have been ultrastructurally described in jejunum enterocytes. Very recently, some investigators have reported the detection by light microscopy of parasitophorous vacuoles in intestinal biopsies; however, only transmission electron microscopy (TEM) could clearly identify the parasitic stages. To improve the histological diagnosis without using TEM, we have tested different staining methods in biopsies obtained from a patient infected by the human immunodeficiency virus who was shedding Cyclospora oocysts. Hematoxylin stain alone for 15 minutes on 5 micrometer-thick sections of duodenal biopsies was found to be the most efficient method for observing different stages of the parasite. In particular, the banana-shaped merozoites were visualized and appeared very similar to the human coccidia Isopora belli. This simple technique may be useful in diagnosing Cyclospora infection.

Hepatocellular adenoma: color Doppler US and pathologic correlations.

PURPOSE: To describe the color Doppler ultrasound (US) features of hepatocellular adenoma (HA) and correlate these findings with pathologic findings, with special emphasis on the blood vessels. MATERIALS AND METHODS: Color Doppler US was prospectively performed in eight patients with histologic proof of HA. Eleven lesions were studied. RESULTS: In seven lesions, color Doppler US demonstrated central color flow. In six of these lesions, pulse-wave Doppler US demonstrated a continuous and flat venous spectrum with frequency shifts of 0.20-0.60 kHz (mean, 0.37 kHz), which corresponded to pathologic findings of intratumoral veins 1-5 mm in diameter. The other lesion with central color flow demonstrated a triphasic venous waveform with 1.20-kHz frequency shift, which corresponded pathologically to a central vein 10 mm in diameter. In these seven lesions, color Doppler examination demonstrated both venous and arterial peritumoral flow. CONCLUSION: Color Doppler US enables detection of intratumoral veins associated with peritumoral veins and arteries in patients with HA; these findings correlate well with pathologic data. These results might help in the differential diagnosis of HA and focal nodular hyperplasia.

Bronchoalveolar lavage eosinophilia associated with Pneumocystis carinii pneumonitis in AIDS patients. Comparative study with non-AIDS patients.

Lower pulmonary tract cell populations collected by bronchoalveolar lavages (BAL) were evaluated in three groups of immunocompromised patients: HIV infected patients with Pneumocystis carinii (PC) pneumonitis (n = 22), or pneumonitis not related to PC (n = 29), and non-HIV-infected, immunocompromised patients with a PC pneumonitis (n = 18). In AIDS patients with PC pneumonitis, the cell populations were 59.3 +/- 4.5 percent alveolar macrophages (AM), 19.6 +/- 2.5 percent lymphocytes, 14.6 +/- 4.4 percent polymorphonuclear cells (PMN), and 10.3 +/- 3.6 percent eosinophils. In HIV-infected patients without PC pneumonitis, they were 76.5 +/- 3.3 percent AM, 13 +/- 2.1 percent lymphocytes, 9.2 +/- 0.3 percent PMN, and 0.6 +/- 0.2 percent eosinophils, and in non-HIV-infected, immunocompromised patients with PC pneumonitis, they were 43.9 +/- 5.7 percent AM, 30.2 +/- 4.3 percent lymphocytes, 20.4 +/- 4.7 percent PMN, and 0.9 +/- 0.4 percent eosinophils. The most striking finding was a marked BAL eosinophilia in AIDS patients with PC pneumonitis. The significance of this particular cellular pulmonary response to PC is not clear, and its consequences on the lung structures and/or PC require evaluation.