Search

Hepatitis C-related fibrogenesis has been shown to involve complex interactions between peripheral and hepatic immune responses. Peripheral whole blood (PB) samples from patients with chronic hepatitis C (n = 36) were subjected to microarray analysis in order to identify gene expression patterns associated with immune pathways in PB and hepatic fibrosis. Distinct regulation of gene expression of inositol polyphosphate-5-phosphatase/145kDa (INPP5D or SHIP), a TLR2/TLR4-inhibitor, and heat shock protein 8/22 kDa (HSPB8), an endogenous TLR4-ligand, during fibrogenesis was identified and could be confirmed by quantitative reverse-transcription polymerase chain reaction. These results suggest a potential link between peripheral activity of the TLR4-pathway, peripheral SHIP-dependent immune regulation, and liver fibrosis. [ABSTRACT FROM AUTHOR]

The majority of chronic viral hepatitis cases are induced via infection with the hepatitis B virus (HBV), hepatitis C virus (HCV), or hepatitis D virus (HDV). These patients are at increased risk for progressive liver disease leading to cirrhosis as well as hepatocellular carcinoma (HCC). HBV infection is well controlled by the currently available nucleosides as well as nucleotides, and the development of cirrhosis can be prevented. Additionally, it has been shown that HBV-induced liver fibrosis can regress during successful antiviral treatment; however, a "functional cure", i.e., loss of HBsAg, is a rare event when these drugs are used. Therefore, novel therapeutic strategies are aiming at the selective suppression of HBsAg levels in combination with immunostimulation. The development of directly acting antivirals (DAAs) has revolutionized HCV therapy, as almost all patients can be cured via this treatment. Additionally, DAA therapy has few, if any, side effects, and is generally well tolerated by patients. HDV remains the most challenging type of chronic viral hepatitis. Although novel therapeutic options have recently been approved, response rates are still less favorable compared to HBV and HCV. This review discusses current and future options for the treatment of chronic HBV, HCV, and HDV infection.

The role of non-parenchymal liver cells as part of the hepatic, innate immune system in the defense against hepatotropic viruses is not well understood. Here, primary human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells were isolated from liver tissue obtained after tumor resections or liver transplantations. Cells were stimulated with Toll-like receptor 1-9 ligands for 6-24 h. Non-parenchymal liver cells expressed and secreted inflammatory cytokines (IL6, TNF and IL10). Toll-like receptor- and cell type-specific downstream signals included the phosphorylation of NF-κB, AKT, JNK, p38 and ERK1/2. However, only supernatants of TLR3-activated Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells contained type I and type III interferons and mediated an antiviral activity in the interferon-sensitive subgenomic hepatitis C virus replicon system. The antiviral effect could not be neutralized by antibodies against IFNA, IFNB nor IFNL, but could be abrogated using an interferon alpha receptor 2-specific neutralization. Interestingly, TLR3 responsiveness was enhanced in liver sinusoidal endothelial cells isolated from hepatitis C virus-positive donors, compared to uninfected controls. In conclusion, non-parenchymal liver cells are potent activators of the hepatic immune system by mediating inflammatory responses. Furthermore, liver sinusoidal endothelial cells were identified to be hyperresponsive to viral stimuli in chronic hepatitis C virus infection.

Nucleic acid polymers (NAPs) block the release of subviral particles from hepatocytes, a mechanism consistent with their antiviral activity against hepatitis B virus (HBV) in patients. Analysis of immunostimulatory properties of NAPs were conducted with several NAP species: REP 2006, the prototypic degenerate NAP [dN] 40 , containing TLR9-stimulatory CpG; REP 2055 a clinically active NAP with a sequence [dAdC] 20 devoid of CpG content; REP 2139 (also clinically active) and REP 2165 (REP 2055 analogues further rendered immunologically inactive by replacing cytidine with 5-methylcytidine and incorporating 2'-O methylation of riboses). These analyses revealed pro-inflammatory responses in human peripheral blood mononuclear cells with REP 2006 and with REP 2139 and REP 2165 only at high dose but displayed no significant antiviral activity. In primary isolated human hepatocytes and liver sinusoidal endothelial cells no significant inflammatory or antiviral responses were detected for any NAPs. In human Kupffer cells pro-inflammatory activity was observed with REP 2006 and REP 2055, whereas a weak but significant induction of interferon genes was only observed with REP 2006 at the highest concentration. We therefore hypothesize that the antiviral activity of NAPs optimized to treat HBV infection in patients cannot be explained by direct induction of innate antiviral responses.

Background & Aims: GALAD and BALAD-2 are statistical models for estimating the likelihood of the presence of hepatocellular carcinoma (HCC) in individual patients with chronic liver disease and the survival of patients with HCC, respectively. Both models use objective measures, particularly the serum markers α-fetoprotein (AFP), AFP-L3, and des-γ-carboxyprothrombin. We aimed to validate these models in an international cohort of patients with HCC and assess their clinical performance., Methods: We collected data on cancer diagnosis and outcomes of 6834 patients (2430 with HCC and 4404 with chronic liver disease) recruited from Germany, Japan, and Hong Kong. We also collected data from 229 patients with other hepatobiliary tract cancers (cholangiocarcinoma or pancreatic adenocarcinoma) and 92 healthy individuals (controls). For reference, the original UK cohort (on which the GALAD model initially was built and BALAD-2 was validated) was included in the analysis. We assessed the effects of tumor size and etiology on GALAD model performance, and its ability to correctly discriminate HCC from other hepatobiliary cancers. We assessed the performance of BALAD-2 in patients with different stages of HCC., Results: In all cohorts, the area under the receiver operating characteristic curve (AUROC), quantifying the ability of GALAD to discriminate patients with HCC from patients with chronic liver disease, was greater than 0.90-similar to the series on which the model originally was built (AUROC, 0.97). GALAD discriminated patients with HCC from those with other hepatobiliary cancers with an AUROC value of 0.95; values were slightly lower for patients with small unifocal HCCs, ranging from 0.85 to 0.95. Etiology and treatment of chronic viral hepatitis had no effect on the performance of this model. BALAD-2 analysis assigned patients with HCC to 4 distinct prognostic groups-overall and when patients were stratified according to disease stage., Conclusions: We validated the performance of the GALAD and BALAD-2 models for the diagnosis of HCC and predicting patient survival, respectively (based on levels of the serum markers AFP, AFP-L3, and des-γ-carboxyprothrombin), in an international cohort of almost 7000 patients. These systems might be used in HCC surveillance and determination of patient prognosis., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV.

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055. Ducks with persistent DHBV infection were treated with NAP 2055 to determine if the post-entry inhibitory activity exhibited by NAPs could provide a therapeutic effect against established DHBV infection in vivo. In all REP 2055-treated ducks, 28 days of treatment lead to initial rapid reductions in serum DHBsAg and DHBV DNA and increases in anti-DHBs antibodies. After treatment, 6/11 ducks experienced a sustained virologic response: DHBsAg and DHBV DNA remained at low or undetectable levels in the serum and no DHBsAg or DHBV core antigen positive hepatocytes and only trace amounts of DHBV total and covalently closed circular DNA (cccDNA) were detected in the liver at 9 or 16 weeks of follow-up. In the remaining 5/11 REP 2055-treated ducks, all markers of DHBV infection rapidly rebounded after treatment withdrawal: At 9 and 16 weeks of follow-up, levels of DHBsAg and DHBcAg and DHBV total and cccDNA in the liver had rebounded and matched levels observed in the control ducks treated with normal saline which remained persistently infected with DHBV. These data demonstrate that treatment with the NAP REP 2055 can lead to sustained control of persistent DHBV infection. These effects may be related to the unique ability of REP 2055 to block release of DHBsAg from infected hepatocytes.

Background & Aims: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen., Methods: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity., Results: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7) hepatocytes, 1.8 ± 0.5 × 10(6 )Kupffer cells, 4.3 ± 1.9 × 10(5) liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5) stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2%) and exhibited phagocytic activity, as determined with 1 μm latex beads. Endothelial cells were CD146(+) (97.8 ± 1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence., Conclusions: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

Selective internal radiation therapy (SIRT) using 90-yttrium is a local therapy for unresectable liver malignancies. Non-targeted 90-yttrium diversion via a patent hepatic falciform artery (HFA) is seen as risk for periprocedural complications. Therefore, this study aimed to evaluate the impact of a patent HFA on SIRT. 606 patients with SIRT between 2006 and 2012 were evaluated retrospectively. SIRT preparation was performed by digital subtraction angiography including (99m)Tc-HSAM administration and subsequent SPECT/CT. Patients with an angiographically patent HFA were analyzed for procedural consequences and complications. 19 of 606 patients (3%) with an angiographically patent HFA were identified. Only 11 of these 19 patients received 90-yttrium in the hepatic vessel bed containing the HFA. Initial coil embolization of the HFA succeeded only in three of 11 patients. Out of the eight remaining patients four had no abdominal wall (99m)Tc-HSAM accumulation. The other four patients presented with an abdominal wall (99m)Tc-HSAM accumulation, for those a reattempt of HFA embolization was performed or ice packs were administered on the abdominal wall during SIRT. In summary, all patients tolerated SIRT well. A patent HFA should not be considered a SIRT contraindication. In patients with abdominal wall (99m)Tc-HSAM accumulation HFA embolization or ice pack administration seems to prevent complications.

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Clinical studies have evaluated the safety and efficacy of radioembolization with yttrium-90 in patients with unresectable hepatocellular carcinoma (HCC). Citing literature published within the last 5 years, we review the clinical evidence of survival outcomes and safety of yttrium-90 treatment in patients with unresectable HCC. This paper is primarily focused on survival rates following the typical application of yttrium-90 in HCC treatment, and also includes time to progression and safety data. Also discussed are special indications and new developments related to yttrium-90 therapy in HCC, as well as patient selection and its correlation with successful treatment outcomes., Competing Interests: Financial & competing interests disclosure The author has provided consultancy services for BTG International Inc. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Gretchen Pezza, a compensated employee of BTG International Inc. provided editorial and writing assistance with this manuscript.

Unlabelled: We have previously shown that poly(I:C) activates murine hepatic cells to produce interferon (IFN) and suppresses hepatitis B virus (HBV) replication in vitro. Therefore, we addressed whether poly(I:C) is able to induce the clearance of HBV in vivo. The chronic HBV replication mouse model was established by the hydrodynamic injection (HI) of pAAV-HBV1.2 into the tail veins of wild-type and IFN-α/βR-, IFN-γ-, and CXCR3-deficient C57BL/6 mice. Fourteen days post-HI of pAAV-HBV1.2, mice were administered poly(I:C) by intraperitoneal injection, intramuscular injection, or HI. Only treatment of poly(I:C) by HI led to HBV clearance in wild-type C57BL/6 mice. Serum HBsAg disappeared within 40 days postinfection (dpi) in mice that received poly(I:C) by HI, and this was accompanied by the appearance of anti-HBs antibodies. HBV-specific T-cell and antibody responses were significantly enhanced by HI of poly(I:C). HBV replication intermediates and HBcAg-positive hepatocytes were eliminated in the liver. HI of poly(I:C) induced the production of IFNs in mice and enhanced the levels of cytokines, IFN-stimulated genes, and T-cell markers in the liver. Importantly, poly(I:C)-induced HBV clearance was impaired in IFN-α/βR-, IFN-γ-, and CXCR3-deficient mice, indicating that the induction of type I IFN and the stimulation and recruitment of T cells into the liver are essential for HBV clearance in this model. Taken together, the application of poly(I:C) by HI into the liver enhances innate and adaptive immune responses and leads to HBV clearance in an HBV mouse model, implicating the potential of intrahepatic Toll-like receptor 3 (TLR3) activation for the treatment of chronic hepatitis B patients., Importance: It has become well accepted that immunomodulation is a potentially useful approach to treat chronic viral infection. Recently, combinations of antiviral treatment and therapeutic vaccinations were evaluated for therapies of chronic hepatitis B virus (HBV) infection. Activation of the innate immune branch may also be important for viral control and contributes to HBV clearance. Our present study demonstrated that hepatic TLR3 activation led to clearance of hepatitis B virus in an HBV mouse model. For the first time, we showed that HBV clearance in this model is dependent not only on type I interferon (IFN) but also on type II IFN, indicating a coordinated action of innate and adaptive immune responses. T-cell recruitment appeared to be critical for the success of TLR3-mediated antiviral action. These findings implicate the potential of intrahepatic TLR3 activation for the treatment of chronic HBV infection., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Local ablative techniques such as selective internal radiation therapy (SIRT) have become the mainstay of treating hepatocellular carcinoma (HCC) in the bridging-to-transplant and palliative setting. We recently demonstrated that epithelial circulating tumor cells (CTCs) correlate to an unfavorable outcome. We wanted to scrutinize whether molecular markers detected in this specific CTC subgroup may also have clinical implications., Materials & Methods: Mononuclear cells and CTCs were isolated from peripheral blood samples using density gradient centrifugation followed by depletion of hematopoietic and enrichment of epithelial (EpCAM(+)) cells employing immunomagnetic beads. The mRNA expression of candidate markers was correlated with response to SIRT in 25 patients using quantitative real-time reverse-transcription PCR., Results: IGFBP1 mRNA expression levels were significantly correlated with time to progression in a Kaplan-Meier log rank test (p = 0.04; 0 vs 4 months) and receiver operating characteristic analysis demonstrated a potential use to predict patients with shortened time to progression (area under the curve: 0.8; 95% CI: 0.44-0.98; p = 0.03)., Conclusion: The EpCAM fraction of CTCs may be useful to detect novel molecular markers to individualize treatment decision in patients with HCC.

Objectives: The therapeutic application of small interfering RNAs (siRNAs) is limited by the induction of severe off-target effects, especially in the liver. Therefore, we assessed the potential of differently modified siRNAs to induce the hepatic innate immune system in vitro and in vivo., Methods: Primary isolated liver cells were transfected with siRNAs against apolipoprotein B1 (APOB1), luciferase (LUC) or galactosidase (GAL). For in vivo use, siRNAs were formulated in lipid nanoparticles (LNPs) and administered intravenously to C57BL/6 mice. Liver tissue was collected 6-48 h after injection and knock-down efficiency or immune responses were determined by quantitative reverse-transcription-linked PCR., Results: Unmodified GAL siRNA transiently induced the expression of TNF-α, IL-6, IL-10, IFN-β and IFN-sensitive gene 15 in vivo, whereas a formulation of 2'-O-methylated-LUC siRNA had no such effects. Formulation of unmodified APOB1-specific siRNA suppressed APOB1 mRNA levels by ~80% in the liver 48h after application. The results were paralleled in vitro, where transfection of liver cells with unmodified siRNAs, but not with chemically modified siRNAs, led to cell-type-specific induction of immune genes. These immune responses were not observed in MYD88-deficient mice or in chloroquine-treated cells in vitro., Conclusions: Our data indicate that siRNAs activate endosomal Toll-like receptors in different liver-derived cell types to various degrees, in vitro. LNP-formulated siRNA selectively leads to hepatic knock-down of target genes in vivo. Here, off-target immune responses are restricted to non-parenchymal liver cells. However, 2'-O-methyl modifications of siRNA largely avoid immune-stimulatory effects, which is a crucial prerequisite for the development of safe and efficient RNA-interference-based therapeutics.

We have previously identified 15 genes that are associated with the development of severe depressive side effects during the standard therapy with interferon alpha and ribavirin in the peripheral blood of hepatitis C virus infected patients. An enhanced expression of these genes was also found in the blood of psychiatric patients suffering severe depressive episode. Herein, we demonstrate that the same depression-related interferon-inducible genes (DRIIs) are also upregulated in post-mortem brains of suicidal individuals. Using cultured mouse hippocampal and prefrontal neurons we show that costimulation with murine IFN (mIFN) and the TLR3 agonist poly(I:C) promotes the expression of the described DRIIs, at the same time inducing pro-inflammatory cytokine expression through Stat1 and Stat3 activation, promoting neuronal apoptosis. Consequently, the upregulation of selective DRIIs, production of inflammatory cytokines and inhibition of neuronal plasticity may be involved in the pathogenesis of IFN-associated depression.

Aims: The current prognostic model to estimate the survival in hepatocellular carcinoma (HCC) patients treated with transarterial hepatic selective internal radiotherapy (SIRT) is not fully characterized. The aim of this study was to establish a new scoring model including assessment of both tumor responses and therapy-induced systemic changes in HCC patients to predict survival at an early time point post-SIRT., Methods and Materials: Between 2008 and 2012, 149 HCC patients treated with SIRT were included into this study. CT images and biomarkers in blood tested at one month post-SIRT were analyzed and correlated with clinical outcome. Tumor responses were assessed by RECIST 1.1, mRECIST, and Choi criteria. Kaplan-Meier methods were used to estimate survival curves. Cox regression was used in uni- and multivariable survival analyses and in the establishment of a prognostic model., Results: A multivariate proportional hazards model was created based on the tumor response, the number of tumor nodules, the score of the model for end stage liver disease (MELD), and the serum C-reactive protein levels which were independent predictors of survival in HCC patients at one month post-SIRT. This prognostic model accurately differentiated the outcome of patients with different risk scores in this cohort (P<0.001). The model also had the ability to assign a predicted survival probability for individual patients., Conclusions: A new model to predict survival of HCC patients mainly based on tumor responses and therapy-induced systemic changes provides reliable prognosis and accurately discriminates the survival at an early time point after SIRT in these patients.

In this study, Response Evaluation Criteria in Solid Tumors (RECIST), modified RECIST (mRECIST), Choi and modified Choi criteria were compared to determine which method is optimal for response evaluation in hepatocellular carcinoma (HCC) patients treated with transarterial radioembolization (TARE) with yttrium-90 microspheres. Responses were evaluated by RECIST, mRECIST, Choi and modified Choi criteria in 113 patients with HCC undergoing TARE. Results were compared at 12 weeks after therapy. Kaplan-Meier survival analyses and Cox regression were used to assess differences in time to progression (TTP) and overall survival (OS) between the responders and non-responders defined by each method. The results demonstrated that the responders and non-responders defined by mRECIST and Choi criteria successfully identified patients with a long TTP (400 and 280 days) or short TTP (188 and 166 days) (P=0.004 and 0.002, respectively). Neither RECIST nor modified Choi criteria discriminated between patients who had a short or long clinical benefit. Cox regression analysis revealed that Choi response was a prognostic factor of OS (P=0.004) and was associated with a 53% risk reduction. There was no significant association between survival and RECIST, mRECIST and modified Choi responses. In conclusion, tumor response according to Choi criteria may be helpful to define early HCC patients who benefit from TARE. RECIST, mRECIST and modified Choi appeared inferior.

Background and Aims: Circulating tumor cells (CTCs) have been proposed as a monitoring tool in patients with solid tumors. So far, automated approaches are challenged by the cellular heterogeneity of CTC, especially the epithelial-mesenchymal transition. Recently, Yu and colleagues showed that shifts in these cell populations correlated with response and progression, respectively, to chemotherapy in patients with breast cancer. In this study, we assessed which non-hematopoietic cell types were identifiable in the peripheral blood of hepatocellular carcinoma (HCC) patients and whether their distribution during treatment courses is associated with clinical characteristics., Methods: Subsequent to few enrichment steps, cell suspensions were spun onto glass slides and further characterized using multi-immunofluorescence staining. All non-hematopoietic cells were counted and individual cell profiles were analyzed per patient and treatment., Results: We detected a remarkable variation of cells with epithelial, mesenchymal, liver-specific, and mixed characteristics and different size ranges. The distribution of these subgroups varied significantly between different patient groups and was associated with therapeutic outcome. Kaplan-Meier log-rank test showed that a change in the ratio of epithelial to mesenchymal cells was associated with longer median time to progression (1 vs 15 months; P = .03; hazard ratio = 0.18; 95% confidence interval = 0.01-2.75)., Conclusions: Our data suggest that different CTC populations are identifiable in peripheral blood of HCC patients and, for the first time in HCC, that these individual cell type profiles may have distinct clinical implications. The further characterization and analysis of patients in this ongoing study seems to be warranted.

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.

Hepatitis C virus (HCV) is considered to have a causative role in B-cell lymphoproliferative diseases, including B-cell lymphomas, in chronic virus carriers. Previous data from in vitro HCV-infected B-cell lines and peripheral blood mononuclear cells from HCV-positive individuals suggested that HCV might have a direct mutagenic effect on B cells, inducing mutations in the tumor suppressor gene TP53 and the proto-oncogenes BCL6 and CTNNB1 (β-catenin). To clarify whether HCV indeed has a mutagenic effect on B cells in vivo, we analyzed naive and memory B cells from the peripheral blood of four chronic HCV carriers and intrahepatic B cells from the livers of two HCV-positive patients for mutations in the three reported target genes. However, no mutations were found in the TP53 and CTNNB1 genes. For BCL6, which is a physiological target of the somatic hypermutation process in germinal-center B cells, the mutation levels identified were not higher than those reported in the respective B-cell subsets in healthy individuals. Hence, we conclude that in chronic HCV carriers, the virus does not generally induce mutations in the cancer-related genes TP53, CTNNB1, and BCL6 in B cells. Based on these findings, new targets have to be investigated as potential mediators of HCV-associated B-cell lymphomagenesis.

Background: In chronic hepatitis C virus (HCV) infection, liver tissue pathology and HCV genotype are important determinants of clinical and/or treatment-related outcome. Although consistent epidemiological and/or molecular-biological clues derived from different studies on single virus-host interactions are meanwhile published, the in vivo transcriptional responses and cellular pathways affected in >1 key aspects of the disease or treatment process are far from being understood., Methods: Microarray analysis was performed in peripheral whole blood (PB) samples from 36 therapy-naïve HCV-infected patients with known liver histology. Linear regression analysis identified gene expression profiles significantly correlating (P < 0.015) with ≥1 out of 7 variables: sustained viral response (SVR), viral non-response (NR), end of treatment viral response (ETR), viral breakthrough (VB), HCV genotype (Gt. 1 vs. Gt. 2/3), stage of hepatic fibrosis [St. 0/1 vs. St. 2/3/4] and grade of hepatic inflammation (Gr. 0/1 vs. Gr. 2/3/4). Correlation values across all seven contrasts were considered for hierarchical clustering (HCL)., Results: A total of 1,697 genes showed ≥1 significant correlation results and genes involved in cell differentiation (183), immune response (53), and apoptosis (170) were leading fractions. HCL grouped the genes into six major clusters. Functional annotation analysis using DAVID (http://david.abcc.ncifcrf.gov) revealed that expression profiles that best linked these variables were highly enriched in cytokine/chemokine activity (Fisher-exact P < 0.0001) and specific biological module-centric algorithms finally led our focus on four out of fifty-three immune response genes: SMAD family member 3 (SMAD3), interleukin 1 receptor accessory protein (IL1RAP), tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), and chemokine 'C-C motif' receptor 5 (CCR5). Of those, TNFRSF1A and CCR5 showed significant correlation with two out of seven variables based on microarray and/or quantitative real-time polymerase chain reaction (qRT-PCR) data., Conclusion: We identified molecular targets of the innate and adaptive immune system and validated their transcriptional specificity in vivo suggesting significant involvement in two unique outcomes during HCV treatment.

Background: Though an important percentage of patients with chronic hepatitis C virus (HCV) undergoing interferon (IFN) therapy develop depressive symptoms, the role of the IFN system in the pathogenesis of depressive disorders is not well understood., Methods: 50 patients with HCV infection were treated with standard combination therapy (pegylated IFN-α2a/ribavirin). IFN-induced gene expression was analyzed to identify genes which are differentially regulated in patients with or without IFN-induced depression. For validation, PBMC from 22 psychiatric patients with a severe depressive episode (SDE) and 11 controls were cultivated in vitro with pegylated IFN-α2a and gene expression was analyzed., Results: IFN-induced depression in HCV patients was associated with selective upregulation of 15 genes, including 6 genes that were previously described to be relevant for major depressive disorders or neuronal development. In addition, increased endogenous IFN-production and selective hyper-responsiveness of these genes to IFN stimulation were observed in SDE patients., Conclusions: Our data suggest that selective hyper-responsiveness to exogenous (IFN therapy) or endogenous (depressive disorders) type I IFNs may lead to the development of depressive symptoms. These data could lead to the discovery of novel therapeutic approaches to treat IFN-induced and major depressive disorders.

Objective: Only little is known about the mechanisms of action of corticosteroids in the treatment of inflammatory liver diseases. As there is increasing evidence that stimulation of the innate immune system plays an important pathogenetic role in these conditions, we hypothesized that steroids may interfere with the activation of the Toll-like receptor (TLR) system of the liver., Methods: To test this hypothesis, murine non-parenchymal liver cells (Kupffer cells, liver sinusoidal endothelial cells) and primary hepatocytes were stimulated with TLR 1-9 ligands in the presence or absence of dexamethasone. Expression of pro- and anti-inflammatory cytokines was determined by quantitative reverse transcription-PCR or ELISA, respectively. Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) activation was assessed by western blot analysis., Results: TLR agonists induced the expression of pro- [tumor necrosis factor-α (TNF-α), IL-6, IL-1β, IFN-β] and anti-inflammatory cytokines [IL-10, transforming growth factor-β (TGF-β)], which was differentially modulated by steroid treatment. TNF-α and IL-6 expression was suppressed by dexamethasone, while IL-10 but not TGF-β was enhanced after TLR stimulation. IFN-β production induced by TLR 4 agonists but not TLR 3 agonists was inhibited by dexamethasone. TLR expression itself was down-regulated by steroid treatment in a cell type-specific manner. These effects were associated with suppression of the TLR-mediated activation of NF-κB., Conclusions: TLR signaling is modulated by corticosteroids in a cell type-specific fashion resulting in down-regulation of TLR expression, suppression of pro-inflammatory and up-regulation of anti-inflammatory cytokines. This represents an as yet unknown mechanism of action for corticosteroids that may at least in part explain their therapeutic effects in inflammatory liver diseases.

We quantified antibody responses to the hepatitis C virus (HCV) proteome that are associated with sustained virologic response (SVR) in human immunodeficiency virus (HIV)/HCV-coinfected patients treated with pegylated interferon and ribavirin. Analysis of pre- and posttreatment samples revealed significant decreases in the combined anti-core, anti-E1, and anti-NS4 HCV antibody titers in those with SVRs but not in those who experienced relapse or who did not respond. Furthermore, anti-HIV p24 antibody titers inversely correlated with treatment response. These results suggest that profiling anti-HCV antibody is useful for monitoring HCV therapy, especially in discriminating between those who experience relapse and those who have SVRs at 48 weeks.

Little is known of how the Toll-like receptor (TLR) system can modulate the function of non-parenchymal liver cells (NPC) as a major component of the innate and adaptive immune system of the liver. To investigate the diversification of TLR signalling pathways in NPC, we isolated Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC) from wild-type C57BL/6 mice and examined their responses to TLR1 to TLR9 agonists. The data show that KC respond to all TLR ligands by producing tumour necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6), to TLR3 and TLR4 ligands only by producing interferon-beta (IFN-beta), to TLR1 and TLR8 ligands by significantly up-regulating major histocompatibility complex (MHC) class II and costimulatory molecules, and to TLR1, -2, -4 and -6 ligands by inducing high levels of T-cell proliferation and IFN-gamma production in the mixed lymphocyte reaction (MLR). Similarly, LSEC respond to TLR1 to -4, -6, -8 and -9 ligands by producing TNF-alpha, to TLR3 and -4 ligands by producing IL-6, and to TLR3 ligands by producing IFN-beta. Interestingly, despite significant up-regulation of MHC class II and co-stimulatory molecules in response to TLR8 ligands, LSEC stimulated by TLR1, -2 or -6 could stimulate allogeneic T cells as assessed by MLR. By contrast, myeloid dendritic cells, used as positive control for classical antigen-presenting cells, respond to TLR1, -2, -4 and -9 ligands by both up-regulation of CD40 and activation of allogeneic T cells. In conclusion, NPC display a restricted TLR-mediated activation profile when compared with 'classical' antigen-presenting cells which may, at least in part, explain their tolerogenic function in the liver.

Our previous studies have shown that Toll-like receptor (TLR) ligands, Poly I:C and lipopolysaccharide (LPS), are able to activate non-parenchymal liver cells and trigger the production of interferon (IFN) to inhibit hepatitis B virus replication in vivo and in vitro. However, little is known about TLR-mediated cellular responses in primary hepatocytes. By the model of woodchuck hepatitis virus (WHV) infected primary woodchuck hepatocytes (PWHs), Poly I:C and LPS stimulation resulted in upregulation of cellular antiviral genes and relevant TLRs mRNA expression respectively. LPS stimulation led to a pronounced reduction of WHV replicative intermediates without a significant IFN induction. Poly I:C transfection resulted in the production of IFN and a highly increased expression of antiviral genes in PWHs and slight inhibitory effect on WHV replication. LPS could activate nuclear factor kappa B, MAPK and PI-3k/Akt pathways in PWHs. Further, inhibitors of MAPK-ERK and PI-3k/Akt pathways, but not that of IFN signalling pathway, were able to block the antiviral effect of LPS. These results indicate that IFN- independent pathways which activated by LPS are able to downregulate hepadnaviral replication in hepatocytes.

Aim: To investigate the molecular events involved in liver regeneration following subtotal hepatectomy (SH) as previous studies have largely focused on partial hepatectomy (PH)., Methods: Male Wistar rats were subjected to 70% PH or 90% SH, respectively, and sacrificed at different times after surgery. Untreated and sham-operated animals served as controls. Serum and liver samples were obtained to investigate liver function, apoptosis (TUNEL assay) and transcription factors (NF-kappaB, Stat3; ELISA) or cytokines (HGF, TNF-alpha, IL-6, TGF-alpha, TGF-beta; quantitative RT PCR) involved in liver regeneration., Results: Serum levels of ALT and AST in animals with 70% PH differed significantly from sham-operated and control animals. We found that the peak concentration 12 h after surgery returned to control levels 7 d after surgery. LDH was increased only at 12 h after 70% PH compared to sham. Bilirubin showed no differences between the sham and 70% resection. After PH, early NF-kappaB activation was detected 12 h after surgery (313.21 +/- 17.22 ng/mL), while there was no activation after SH (125.22 +/- 44.36 ng/mL) compared to controls (111.43 +/- 32.68 ng/mL) at this time point. In SH, however, NF-kappaB activation was delayed until 24 h (475.56 +/- 144.29 ng/mL). Stat3 activation was similar in both groups. These findings correlated with suppressed and delayed induction of regenerative genes after SH (i.e. TNF-alpha 24 h postoperatively: 2375 +/- 1220 in 70% and 88 +/- 31 in 90%; IL-6 12 h postoperatively: 2547 +/- 441 in 70% and 173 +/- 82 in 90%). TUNEL staining revealed elevated apoptosis rates in SH (0.44% at 24 h; 0.63% at 7 d) compared to PH (0.27% at 24 h; 0.15% at 7 d)., Conclusion: The molecular events involved in liver regeneration are significantly influenced by the extent of resection as SH leads to suppression and delay of liver regeneration compared to PH, which is associated with delayed activation of NF-kappaB and suppression of proregenerative cytokines.

Background and Aims: In extended liver resections, the preservation of vascular and biliary structures of the entire remnant liver is of paramount importance. The impact of venous outflow impairment and its consequences for liver regeneration and function are still a matter of debate., Materials and Methods: Rats (n = 75) were subjected to a 90% partial hepatectomy (PH), to a 70% liver resection with narrowing of the hepatic outflow of an additional 20% parenchyma (70%+ PH) or to an anatomic 70% PH. Postoperatively hepatocyte proliferation (Ki-67), liver function and survival were assessed. Gene expression analysis for markers of regeneration was determined by in-house complementary (DNA) arrays and quantitative real-time polymerase chain reaction (RT-PCR)., Results: Ninety percent PH led to a greater regenerative response as shown Ki-67 compared to animals with a 70%+PH (p < 0.05). However, liver function was equally impaired in both groups. Rats with 70% PH showed a greater proliferation index with less hepatic injury and better liver function. While mortality was 0% in the group of 70% PH, rats with 90% PH and 70+PH had a reduced survival of 75% (p < 0.05), Conclusion: Venous outflow obstruction leads to an impairment of liver regeneration and liver function. In cases with critically small liver remnants, restoration of an adequate venous outflow may be mandatory.

Aim: To characterize the IFN-response and its modulation by the antiviral compound lamivudine in HBV-transfected HepG2.2.15 cells., Methods: HepG2.2.15 and HepG2 cells were stimulated with various concentrations of IFN-alpha2a in the presence or absence of lamivudine. Then, total RNA was extracted and analysed by customised cDNA arrays and northern blot for interferon-inducible genes (ISGs). In addition, cellular proteins were extracted for EMSA and western blot. HBV replication was assessed by southern blot or ELISAs for HBsAg and HBeAg., Results: Two genes (MxA, Cig5) with completely abolished and 4 genes (IFITM1, -2, -3, and 6-16) with partially reduced IFN-responses were identified in HepG2.2.15 cells. In 2 genes (IFITM1, 6-16), the response to IFN-alpha could be restored by treatment with lamivudine. This effect could not be explained by a direct modulation of the Jak/Stat signalling pathway since EMSA and western blot experiments revealed no suppression of Stat1 activation and ISGF3 formation after stimulation with IFN-alpha in HepG2.2.15 compared to HepG2 cells., Conclusion: These results are consistent with the assumption that chronic hepatitis B may specifically modulate the cellular response to IFN by a selective blockage of some ISGs. Antiviral treatment with lamivudine may partially restore ISG expression by reducing HBV gene expression and replication.

We investigated the psychosocial effects of a right hepatectomy on donors for adult living donor liver transplantation (ALDLT). Questionnaires were sent to 66 actual donors, who had undergone ALDLT between August 1998 and September 2003, as well as to 139 potential donors, who had been examined as possible candidates for ALDLT; the latter had been excluded and had not undergone surgery. All actual donors reported full recovery within an average period of 14.41 (standard deviation = 8.86) weeks; all had returned to their preoperative employment. In preparation for ALDLT, they had received significantly more support from their families in the decision-making process than the potential donors had (t = 2.02; degree of freedom = 79; P = 0.047); they also felt better informed about donation than the potential donors (t = 2.04; df = 64; P = 0.045). Psychiatric problems occurred in 6 (14%) female donors in the perioperative period, mostly in connection with unrealistic outcome expectations. Donors with severe postoperative complications (n = 3) demonstrated higher scores of psychiatric symptoms (chi-square = 6.39; df = 2; P = 0.041). When we compared potential and actual donors, a significant difference in emotional quality of life was not demonstrated (t = 0.41; df = 76; P = 0.684), and it corresponded to that of the normative sample. For donors, perceived emotional quality of life did not depend on the course of recovery of the recipients. Six to 9 months after donation, potential donors reported a significantly higher physical quality of life than actual donors (t = 2.20; df = 56; P = 0.032). In conclusion, female donors, donors with their own major complications, or donors with unrealistic outcome expectations should be provided with adequate psychosocial care. With regard to the psychosocial outcome, ALDLT is a safe intervention for the donor.

We have used cDNA arrays to compare the activation of various cellular genes in response to infection with Sendai viruses (SeV) that contain specific mutations. Three groups of cellular genes activated by mutant SeV infection, but not by wild-type SeV, were identified in this way. While some of these genes are well known interferon (IFN)-stimulated genes, others, such as those for interleukin-6 (IL-6) and IL-8, are not directly induced by IFN. The gene for beta IFN (IFN-beta), which is critical for initiating an antiviral response, was also specifically activated in mutant SeV infections. The SeV-induced activation of IFN-beta was found to depend on IFN regulatory factor 3, and the activation of all three cellular genes was independent of IFN signaling. Mutations that disrupt four distinct elements in the SeV genome (the leader RNA, two regions of the C protein, and the V protein) all lead to enhanced levels of IFN-beta mRNA, and at least three of these viral genes also appear to be involved in preventing activation of IL-8. Our results suggest that SeV targets the inflammatory and adaptive immune responses as well as the IFN-induced intracellular antiviral state by using a multifaceted approach.

A sensitive, specific, reproducible, robust, and cost-effective customized cDNA array system based on established nylon membrane technology has been developed for convenient multisample expression profiling for several hundred genes of choice. The genes represented are easily adjusted (depending on the availability of corresponding cDNAs) and the method is accordingly readily applicable to a wide variety of systems. Here we have focused on the expression profiles for interferon-alpha2a, the most widely used interferon for the treatment of viral hepatitis and malignancies, in primary cells (peripheral blood mononuclear cells, T cells, and dendritic cells) and cell lines (Kit255, HT1080, HepG2, and HuH7). Of 150 genes studied, only six were consistently induced in all cell types and donors, whereas 74 genes were induced in at least one cell type. IRF-7 was identified as the only gene exclusively induced in the hematopoietic cells. No gene was exclusively induced in the nonhematopoietic cell lines. In T cells 12, and in dendritic cells, 25 genes were induced in all donors whereas 45 and 42 genes, respectively, were induced in at least one donor. The data suggest that signaling through IFN-alpha2 can be substantially modulated to yield significant cell-type and donor-specific qualitative and quantitative differences in gene expression in response to this cytokine under highly standardized conditions.

Signaling through Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) is central to the responses to the majority of cytokines and some growth factors, including the interferons (IFNs) and the IL-6 family of cytokines. The biological responses to stimulation through the widely distributed IL-6 and IFN-gamma receptors are, however, completely different. Remarkably, it is shown here that, in mouse embryo fibroblasts lacking STAT3, IL-6 mediates an IFN-gamma-like response including prolonged activation of STAT1, the induction of multiple IFN-gamma-inducible genes, the expression of class II MHC antigens, and an antiviral state. Normal cells exposed to IL-6 thus require a STAT3-dependent function(s) to down-regulate STAT1 activity and prevent an IFN-gamma-like response. The data encourage the view that the very disparate IFN-gamma and IL-6 JAK/receptor complexes mediate a common set of generic or "core" signals which are subject to STAT3-dependent modulation to provide IL-6 specificity. The switching of one cytokine response to one closely mimicking another as a result of the loss of a single signaling component has profound implications, for example, for the interpretation of the phenotypes of knockout mice and for the clinical use of inhibitors of signaling.

Objective: There is increasing evidence that coinfection of hepatitis C (HCV) with HIV is associated with accelerated progression of liver cirrhosis. The aim of this pilot study was to investigate toxicity and efficacy of interleukin-2 (IL-2) for treatment of affected patients., Design: Because low-dose, daily IL-2 therapy is well tolerated and can elevate CD4 cell counts and improve immune functions, patients were treated with 1-2 million units (MU) IL-2 subcutaneously daily., Methods: This pilot trial included 7 HIV/HCV-coinfected individuals. During therapy, clinical, virologic, and laboratory parameters were closely monitored., Results: All patients responded to IL-2 therapy with either improvement of either CD4 cell counts or liver function test results. In 2 patients, HCV-RNA in serum became negative 2 and 4 months, respectively, after cessation of therapy. HCV-RNA has remained undetectable in these 2 patients for 18 and 24 months, respectively. Therapy was well tolerated and no grade III or IV toxicities were observed., Conclusions: Low-dose, daily IL-2 therapy can improve both CD4 cell counts and liver function test results in patients with HIV/HCV coinfection and may in some cases lead to sustained suppression of viremia of HCV.

A tripartite receptor comprising the external region of the erythropoietin (Epo) receptor, the transmembrane and JAK-binding domains of the gp130 subunit of the interleukin-6 (IL-6) receptor, and a seven amino acid STAT1 recruitment motif (Y440) from the interferon (IFN)-gamma receptor, efficiently mediates an IFN-gamma-like response. An analogous completely foreign chimeric receptor in which the Y440 motif is replaced with the Y905 motif from gp130 also mediates an IFN-gamma-like response, but less efficiently. The IFNGR1 signal-transducing subunit of the IFN-gamma receptor is tyrosine phosphorylated through the chimeric receptors and the endogenous IL-6 and OSM receptors. Cross phosphorylation of IFNGR1 is not, however, required for the IFN-gamma-like response through the chimeric receptors, nor does it mediate an IFN-gamma-like response to IL-6 or OSM. The data argue strongly for modular JAK/STAT signalling and against any rigid structural organization for the "pathways" involved. They emphasize the likely high degree of overlap between the signals generated from disparate JAK-receptor complexes and show that relatively minor changes in such complexes can profoundly affect the response.

The combination of high and low density cDNA filter array technology potentially permits both the identification of subsets of induced genes and convenient and rapid multisample expression profiling of such subsets under a variety of conditions. The JAK/STAT1 pathway for IFN-gamma signaling in human cells has been well characterized, but the extent and importance of additional pathways remain to be established. Here, using high-density filter arrays of the RZPD UniGene set, we identified 18 novel IFN-gamma-inducible genes. Expression profiling was carried out using low-density arrays representing both novel and known IFN-gamma-inducible genes. Initial experiments failed to detect evidence for any novel non-JAK-dependent pathways in cells expressing a kinase-dead JAK2. The data, however, validated the potential of the combined methods in establishing rapid and convenient expression profiling of several hundred genes in response to any ligand of choice.

The pro-inflammatory cytokine interleukin (IL)-6 (refs. 1-5) can bind to cells lacking the IL-6 receptor (IL-6R) when it forms a complex with the soluble IL-6R (sIL-6R) (trans signaling). Here, we have assessed the contribution of this system to the increased resistance of mucosal T cells against apoptosis in Crohn disease (CD), a chronic inflammatory disease of the gastrointestinal tract. A neutralizing antibody against IL-6R suppressed established experimental colitis in various animal models of CD mediated by type 1 T-helper cells, by inducing apoptosis of lamina propria T cells. Similarly, specific neutralization of sIL-6R in vivo by a newly designed gp130-Fc fusion protein caused suppression of colitis activity and induction of apoptosis, indicating that sIL-6R prevents mucosal T-cell apoptosis. In patients with CD, mucosal T cells showed strong evidence for IL-6 trans signaling, with activation of signal transducer and activator of transcription 3, bcl-2 and bcl-xl. Blockade of IL-6 trans signaling caused T-cell apoptosis, indicating that the IL-6-sIL-6R system mediates the resistance of T cells to apoptosis in CD. These data indicate that a pathway of T-cell activation driven by IL-6-sIL-6R contributes to the perpetuation of chronic intestinal inflammation. Specific targeting of this pathway may be a promising new approach for the treatment of CD.

Background: Crohn's disease is a chronic inflammatory disease of the alimentary tract. Azathioprine is an effective agent in the management of chronic active Crohn's disease leading to long term remission of disease activity. Such treatment leads to limited efficacy or side effects in a small subset of patients., Aims: To compare efficacy and side effects of treatment with azathioprine plus corticosteroids versus mycophenolate mofetil (MMF) plus corticosteroids in patients with chronic active Crohn's disease., Methods: Seventy patients with chronic active Crohn's disease (Crohn's disease activity index (CDAI) greater than 150) were randomised for treatment with azathioprine/cortisone or MMF/cortisone. Corticosteroid dosage was tapered according to a standard protocol. Disease activity was monitored by clinical scores after one, two, three, and six months., Results: Treatment of patients with moderately active (CDAI 150-300) Crohn's disease with MMF/cortisone led to a significant reduction in clinical activity scores comparable to treatment with azathioprine/cortisone. Treatment of patients with highly active Crohn's disease (CDAI greater than 300) with MMF/cortisone caused significant suppression of clinical activity earlier than azathioprine/cortisone treatment. Treatment with MMF/cortisone was associated with few adverse effects., Conclusion: Treatment of chronic active Crohn's disease with MMF plus cortisone appears to be effective and well tolerated and should be considered in patients allergic to azathioprine or in whom azathioprine has failed.

The aim of this study was to examine the immunomodulating effects of rhIL-12 on the immune response induced by hepatitis B virus (HBV) antigens in clinical subgroups of patients with HBV infection. Peripheral blood mononuclear cells (PBMC) of 80 patients were stimulated with HBsAg, HBcAg, pre-S1Ag and tetanus toxoid in the absence or presence of IL-12 (0.01, 0.1 and 1 ng/ml). Stimulation by anti-CD3+ anti-CD28 and lipopolysaccharide (LPS) were used as controls. Proliferation and cytokine production were determined by 3H-thymidine uptake and ELISA after 72 h. After stimulation with HBV antigens only, production of tumour necrosis factor-alpha (TNF-alpha) or IL-10 was observed in all patients, while interferon-gamma (IFN-gamma) was detectable in only 27 patients. After costimulation with IL-12 and HBV antigens, however, large amounts of IFN-gamma were found in all patients, while HBV-induced IL-10 production remained mostly unchanged. When clinical subgroups including patients with compensated liver cirrhosis were compared, PBMC from patients with HBeAg+ hepatitis showed the lowest capacity to produce IFN-gamma after HBV antigen-positive IL-12. These data suggest that the ability of IL-12 to enhance IFN-gamma production against HBV antigens is correlated with the presence of HBeAg and is not impaired in patients with advanced liver disease. In addition, IL-12 and IL-10 production by antigen-presenting cells may be a critical factor that determines the efficacy of the immune response against the hepatitis B virus.

Immunosuppressive therapy with methotrexate (MTX) has been established as effective treatment for patients with rheumatoid arthritis. To analyse the therapeutic potential and mechanisms of action of MTX, we determined serum cytokine levels and cytokine production by splenic T cells and macrophages in untreated and MTX-treated mice. Furthermore, we assessed the role of MTX in a murine model of experimental arthritis induced by collagen type II (CIA). MTX reduced spontaneous and IL-15-induced tumour necrosis factor (TNF) production by splenic T cells but not by macrophages from healthy mice in vitro in a dose-dependent manner. In contrast, interferon-gamma (IFN-gamma) production was less strikingly reduced and IL-4 production was virtually unaffected. In addition, treatment of healthy mice with MTX in vivo led to reduced TNF serum levels and diminished TNF production by splenic T cells and macrophages. Intraperitoneal administration of MTX prior to the onset of arthritis completely prevented clinical and pathological signs of CIA. This was associated with a striking reduction of TNF production by spleen cells from MTX-treated mice. The role of TNF in MTX-mediated effects on cytokine production was further underlined by the finding that MTX effects on IFN-gamma production were augmented in TNF-transgenic mice but abrogated in mice in which the TNF-alpha gene had been inactivated by homologous recombination. Thus, MTX specifically modulates spontaneous and IL-15-induced TNF-alpha production in mice and prevents experimental murine CIA. These data suggest that TNF production by T cells is an important target of MTX and may serve as a basis to understand and further analyse MTX-mediated mechanisms of immunosuppression in patients with RA.

Objectives: To investigate the influence of the Th1 and Th2 lymphokines interleukins (IL)-4 and IL-13, interferon gamma (IFN gamma), and several monokines on the adhesion of mononuclear cells to synovial fibroblasts and intercellular adhesion molecule-1 (ICAM-1) expression and cytokine production of synovial fibroblasts in patients with osteoarthritis., Methods: Synovial fibroblasts were isolated from patients with osteoarthritis and stimulated with IL-1 beta, IL-4, IL-6, IL-10, IL-12, IL-13, tumour necrosis factor alpha (TNF alpha), and IFN gamma. Subsequently, we determined the production of IL-1 alpha, IL-1 beta, IL-6, IL-10, IL-12, IFN alpha and TNF alpha, and the expression of ICAM-1 lymphocyte function associated antigen 3 (LFA-3), BB7, and major histocompatibility complex class II molecules on these cells. Furthermore, the adhesion of freshly isolated mononuclear cells from the peripheral blood was tested using a colourimetric cell-cell adhesion assay., Results: Only production of IL-6 and the expression of ICAM-1 were observed. IL-1 beta and TNF alpha were the most potent stimulatory mediators of both cytokine production and ICAM-1 expression. IL-4 and IL-13 had differential effects as they upregulated cytokine production but downregulated IFN gamma induced ICAM-1 expression. In functional adhesion assays, TNF alpha, IL-1 alpha and, to a lesser extent, IFN gamma led to increased adhesion of mononuclear cells, whereas IL-4 and IL-13 had no effect., Conclusions: Our data indicate that Th1 and Th2 lymphokines can modulate the function (cytokine production and expression of adhesion molecules) of synovial fibroblasts.

In patients with chronic renal failure alterations in monokine production are a common feature. Their clinical relevance has not yet been proven. We show here a correlation between an overproduction of interleukin-(IL)-6 and tumor necrosis factor alpha (TNF alpha) upon stimulation with LPS by mononuclear cells in vitro and the clinical grade of immunodeficiency found in these patients. Higher levels of IL-6 and TNF alpha were correlated with an immunocompromized state, that is, non-responsiveness to hepatitis B vaccination, whereas patients with a better immune competence showed the same levels of these cytokines as healthy controls. Only the patients with a good immune function showed a high secretion of IL-10. The feedback mechanism of IL-10 for reducing monokine synthesis seems to be intact in these patients. Thus the secretion of IL-10 might be regarded as a compensatory mechanism which controls monokine induction by chronic renal failure and hemodialysis treatment. Immunocompromized patients who are unresponsive to hepatitis B vaccination seem to be unable to enhance IL-10 synthesis for control of monokine overproduction. This results in higher levels of IL-6 and TNF alpha that might be involved in the pathogenesis of reduced immune defense.

Although the pathogenesis of psoriasis vulgaris is still unknown, several characteristics point to an immunologically mediated process. Epidermal psoriatic lesions are characterized by a hyperproliferation of keratinocytes and an infiltration of T lymphocytes and granulocytes. Because the former may be mediated in part by lymphokines secreted by T cells, we have focused our interest on the in vivo and in vitro cytokine secretion patterns of T lymphocytes from psoriatic lesions. In five patients T lymphocytes were obtained from epidermal specimens. The cells were propagated with lectin and irradiated feeder cells and subsequently cloned by limiting dilution. The resulting T-cell clones were phenotypically and functionally characterized. Our data show that the majority of T-cell clones were CD4+ (74%), whereas only 25% were CD8+ and 1% were CD4-/CD8-. Also, we have further investigated the cytokine secretion pattern of T-cell lines or CD4+ T-cell clones, respectively. All cells tested produced interferon-gamma whereas only a minority secreted interleukin (IL)-4. Moreover, these cells produced high amounts of IL-2 but only little or no IL-10 or tumor necrosis factor-alpha. To correlate these data with the in vivo situation, biopsies from psoriatic lesions of five patients were investigated for the presence of the mRNA of IL-4, IL-10, and interferon-gamma using the polymerase chain reaction. In these biopsies only the mRNA for the Th1 cytokine interferon-gamma but not for the Th2 cytokines IL-4 and IL-10 could be detected. Identical experiments were performed to test the in vivo cytokine production of synovial fluid mononuclear cells of two patients with arthropathia psoriatica. Again, only the mRNA for interferon-gamma but not IL-4 could be detected. This indicates that T cells involved in psoriasis exhibit a Th1-like cytokine secretion profile.