Effects of the Mammalian Target of Rapamycin Inhibitor Everolimus on Hepatitis C Virus Replication In Vitro and In Vivo.

BACKGROUND: The influence of immunosuppressants on hepatitis C virus (HCV) re-infection after liver transplantation, particularly mammalian target of rapamycin inhibitors, remains unclear. The aim of our study was to analyze the influence of everolimus (EVR) on HCV replication activity in the context of underlying molecular mechanisms, with focus on the pro-myelocytic leukemia protein (PML). METHODS: HCV viral load was recorded in 40 patients with post-transplant HCV re-infection before and 8 weeks after introduction of EVR. An HCV cell culture replicon system for genotype (GT) 1b, GT2b, and GT3a was used to compare the influence of EVR on HCV replication for the respective genotypes in vitro. Fluorescence-activated cell-sorting analysis was used to test for effects on cell proliferation. PML expression was silenced with the use of small hairpin RNA constructs, and PML expression was quantified by means of quantitative real-time polymerase chain reaction. RESULTS: In patients with HCV, the viral load of GT1a and GT1b was hardly affected by EVR, whereas the viral load was reduced in patients with GT2a (P ≤ .0001) or GT3 infection (P ≤ .05). In vitro EVR impairs HCV replication activity of GT2a and GT3a up to 60% (P ≤ .0005), whereas in GT1b cells, HCV replication activity is increased by 50% (P ≤ .005). Replicon cell viability was not impaired. HCV replication activity is impaired in the absence of PML, which can be reversed by overexpression of PML isoforms. Furthermore, in the absence of PML, the effect of EVR on HCV replication activity is nearly abrogated. CONCLUSIONS: The mammalian target of rapamycin inhibitor EVR influences HCV replication via PML. The herein presented results suggest a genotype-dependent benefit for an EVR-based immunosuppressive regimen in patients with GT2a or GT3 re-infection after liver transplantation.

Diagnostics and treatment of a severe humoral rejection after liver transplantation: donor-specific antibodies as a still underestimated cause of graft failure.

BACKGROUND: Donor-specific antibodies (DSAs) are increasingly being considered a cause of complications after liver transplant (LT). However, neither monitoring of DSAs nor the appropriate therapeutic procedures for humoral graft damage are yet standardized. Here we report a case of DSA-positive humoral rejection after LT that was successfully treated with plasmapheresis and immunoglobulins. METHODS: Human leukocyte antigen (HLA)-specific DSAs were detected by Luminex bead assay. Patient characteristics, laboratory values, and data about the patient's general condition were documented from April 2013 to June 2015. CASE REPORT: Eighteen months after LT, a 54-year-old man experienced severe hepatopathy with rapidly increasing transaminase activity and total bilirubin levels. Histologic findings were inconclusive, demonstrating chronic cholestasis and minimal positive staining for C4 d. However, an analysis for anti-HLA antibodies detected DSAs against HLA class II molecules with high mean fluorescence intensity. The patient underwent 8 courses of plasmapheresis, resulting in sustained amelioration of his condition and decreases in bilirubin levels and transaminase activity. CONCLUSION: De novo DSAs can be responsible for graft failure after LT. Thus, procedures aimed at detecting DSAs are recommended, and regular monitoring of DSAs after LT is important for individualized risk management. Plasmapheresis is an efficient therapeutic procedure for DSA-associated graft failure.

[Expert recommendations: Hepatitis C and transplantation]. / Empfehlungen zur antiviralen Therapie der chronischen Hepatitis C bei Patienten auf der Warteliste und nach Transplantation.

With the approval of new direct acting antiviral agents (DAA), therapeutic options for patients with chronic hepatitis C virus (HCV) infection are now generally available before and after liver transplantation (LT). Interferon-free DAA regimens are highly effective therapies and provide a good safety profile. However, the body of clinical evidence in this patient population is limited and the best treatment strategies for patients on the waiting list with (de)compensated cirrhosis and after LT are not well defined. The following recommendations for antiviral therapy in the context of LT are based on the currently available literature and clinical experience of experts in the field, and have been discussed in an expert meeting. The aim of this article is to guide clinicians in the decision making when treating patients before and after LT with DAAs.

[HCV reinfection after liver transplantation - management and first experiences with telaprevir-based triple therapy]. / HCV-Reinfektion nach Lebertransplantation. Management und erste Ergebnisse mit der Telaprevir-basierten Triple-Therapie.

BACKGROUND AND OBJECTIVE: The management of hepatitis C virus (HCV) recurrence after liver transplantation (LTx) is a major challenge in patient care. For patients with HCV GT1, treatment standard with pegylated interferon (PEG-IFN) and ribavirin (RBV) has been augmented in 2011 by first generation protease inhibitors (PI), telaprevir (TVR) and boceprevir (BOC). We report our first experiences with TVR-based triple therapy in patients with GT1-reinfection of the graft. PATIENTS AND METHOD: 13 patients with histologically proven HCV GT1-reinfection of the graft received 12 weeks of PEG-IFN/RBV/TVR followed by 12 weeks of PEG-IFN/ RBV only. During the triple therapy phase immune suppression was tightly monitored, and the patients were also closely monitored for side effects. RESULTS: The dosage of immunosuppressants had to be reduced significantly (TAC: 30-fold; CSA 3,5-fold). Stable levels were achieved by daily or over-daily dosing of a special size application of 0,1 mg tacrolimus (Tac) bid or a minimal dose of 10 mg cyclosporine (CSA) bid or qd, respectively. In all patients hematological side effects were observed, 65 % of which required RBV dose reduction, administration of erythropoietin or blood transfusions. Increase of kidney retention values requiring infusions occurred in 50 %. All side effects were reversible. There were no early discontinuations of therapy. An early viral response (EVR) with viral decline below limit of detection was noted at week 12 in 9/13 patients and at week 12 in further 3 patients. CONCLUSION: Our preliminary results show high EVR response rates of TVR-based triple therapy in LTx patients with HCV-GT1 re-infection.

Locoregional therapy for hepatocellular carcinoma: radioembolization with yttrium-90 microspheres.

Compared with other malignant tumours, hepatocellular carcinoma (HCC) exhibits particular characteristics regarding its supplying vessels and tumour biology. If a potentially curative surgical approach, such as resection or liver transplantation, is due to technical or prognostical reasons no option, these characteristics are a fundamental prerequisite for the possibility to effectively treat this tumour by local ablation methods. Microsphere and particle technology with selective transport of tumoricidal substances or radiation represents a new generation of therapeutics in interventional oncology. With the intrahepatic application of radioactive microspheres via the hepatic artery (radioembolization) local ablation can be performed even of diffuse and multifocal liver tumours, which hitherto, could only be approached with systemic therapy. The present standard for radioembolization, is the use of yttrium-90 glass or resin microspheres. The indications, technique and current results of radioembolization with yttrium-90 microspheres for the treatment of HCC are discussed in this review.

Hepatitis viruses: live and let die.

Viral hepatitis is a diffuse inflammatory reaction of the liver caused by hepatotropic viruses. Among the hepatitis viruses, only hepatitis B virus and hepatitis C virus are able to persist in the host and cause chronic hepatitis. In the course of persistent infection, inflammation forms the pathogenetic basis of chronic hepatitis that can lead to nodular fibrosis, which can progress to cirrhosis and, eventually, hepatocellular carcinoma (HCC). Of the different antiviral defense systems employed by the host, apoptosis significantly contributes to the prevention of viral replication, dissemination, and persistence. Pathomorphologic studies have shown acidophilic bodies and hepatocyte dropout, features that are compatible with apoptosis. The number of hepatocytes showing features of apoptosis in patients with chronic hepatitis B and C was found to be higher than in healthy subjects, indicating that apoptosis is involved in the pathogenesis of these diseases. There are various data suggesting that hepatitis B and C viral proteins may modulate apoptosis. Vice versa, mechanisms of apoptosis inhibition might represent central survival strategies employed by the virus which, in the end, may contribute to HCC development. While the expression and retention of viral proteins in hepatocytes may influence the severity and progression of liver disease, the mechanisms of liver injury in viral hepatitis are defined to be due not only to the direct cytopathic effects of viruses, but also to the host immune response to viral proteins expressed by infected hepatocytes. However, the exact role of these observations in relation to pathogenesis remains to be established. The mechanism and systems are complex. This report aims to provide an overview and intends to cite only a small number of pertinent references.

Dexamethasone desensitizes hepatocellular and colorectal tumours toward cytotoxic therapy.

The glucocorticoid dexamethasone is frequently used as co-treatment in cytotoxic cancer therapy, e.g. to prevent nausea, to protect normal tissue or for other reasons. While the potent pro-apoptotic properties and the supportive effects of glucocorticoids to tumour therapy in lymphoid cells are well studied, the impact to cytotoxic treatment of colorectal and hepatocellular carcinoma is unknown. We tested apoptosis-induction, viability, tumour growth and protein expression using 8 established cell lines, 18 surgical specimen and a xenograft on nude mice. In the presence of dexamethasone we found strong inhibition of apoptosis in response to 5-FU, cisplatin, gemcitabine or gamma-irradiation, enhanced viability and tumour growth of colorectal and hepatocellular carcinomas. No correlation with age, gender, histology, TNM, the p53 status and induction of therapy resistance by dexamethasone co-treatment could be detected. These data show that glucocorticoid-induced resistance occurs not occasionally but is common in colorectal and hepatocellular carcinomas implicating that the use of glucocorticoids may be harmful for cancer patients.

Overexpression and functional characterization of kinin receptors reveal subtype-specific phosphorylation.

G protein-coupled receptors such as the receptors for bradykinin are present in low copy numbers in most natural cells. To overcome the problems associated with the analysis of these receptors at the protein level, we used highly efficient expression systems such as the baculovirus/insect cell system. However, the structural and functional statuses of recombinant receptors have often remained elusive. We have expressed the two types of human kinin receptors, B1 and B2, in Sf9 cells. Both receptors are found on the surface of infected cells where they display the same pharmacological profiles as their cognate receptors of native cells. The functional analysis of kinin receptors coupled to the intracellular signaling pathways of Sf9 cells revealed differential patterns of ligand-induced phosphorylation for the two kinin receptors. The B1 receptor failed to undergo ligand-induced phosphorylation. However the B2 receptor showed selective phosphorylation of a minor 38 kDa band and lack of phosphorylation of a dominant 33 kDa band, indicating that only a fraction of the receptor protein is functionally linked to the kinase pathway. A striking discrepancy between the number of binding sites and the amount of receptor protein per cell (molar ratio of 1:20 to 1:1000) indicated that a significant portion of kinin receptors is associated with the intracellular compartments of Sf9 cells. Pulse-chase and immunoprecipitation experiments demonstrated that the heterogeneity of recombinant receptors is not due to proteolytic processing but likely reflects incomplete or lacking N-glycosylation. We conclude that the baculovirus/Sf9 system is suitable for the recombinant expression and functional analysis of kinin receptors though limitations of the system have to be considered.

Predicting outcome of group counseling with severely disabled patients.

Client characteristics associated with outcome of group counseling were identified in a sample of physically disabled persons using standardized self report inventories. Thirty-eight subjects were tested before and after an 8 week treatment interval for signs of emotional disorder, inactivity, and social problems related to being severely disabled. Treatment consisted of group conference phone calls using self determined task assignments as a focus for discussion. Change in report of life satisfaction was predicted reliably from pretest data. Loneliness accounted for 73% of the variance in posttreatment life satisfaction scores. Other significant predictors included signs of depression, alcohol use, and constructs related to social support. Using regression analysis, persons with adjustment problems could be identified and prognosis for treatment predicted with acceptable reliability.