Purpose: Osteoarthrosis, the degeneration of the articular cartilage, is a severe health problem of the aging population, but the mechanisms regulating the differentiation of embryonic chondrocytes into mature articular cartilage of the synovial joints are still not sufficiently understood. Several lines of evidence indicate that the differentiation of embryonic into permanent articular cartilage is guided by epigenetic reprogramming. Additionally, recent data provide evidence that changes in enhancer accessibility are linked to osteoarthrosis progression. Nevertheless, a systematic analysis of the epigenetic profile of the articular chondrocytes is still lacking. Methods: The distribution of activating and repressive histone marks of chondrocytes dissected from femoral heads of early postnatal mice was established by ChIPseq and compared to that of proliferating and hypertrophic chondrocytes. Chromatin states, representing combinations of different histone marks at the same genomic location, were defined using ChromHMM. In parallel, gene expression was assessed by RNAseq and integrated with the epigenetic information using Pareto optimization. Results: Computational comparisons of RNAseq and ChIPseq data identified similar numbers of expressed genes and genes carrying activating histone marks in all chondrocyte populations. In contrast, we detected a specific enrichment of the repressive mark H3K27me3 in articular and hypertrophic chondrocytes, with different sets of genes becoming repressed in either hypertrophic or articular cartilage. These findings revealed a common mechanism by which genes are turned off during differentiation along different lineages. In the next step, we analyzed the number and location of enhancer elements, marked by H3K27ac. Distal enhancers were enriched at lineage specific genes in proliferating and articular chondrocytes and at genes that regulate metabolic pathways and angiogenesis in hypertrophic chondrocytes. Interestingly, many genes relevant for articular chondrocyte differentiation already carried H3K27ac at enhancers in proliferating chondrocytes, although gene expression was low. With increased expression, the number of H3K27ac peaks was increased in articular chondrocytes. To translate this data to human, we compared the epigenetic profile of murine articular chondrocytes to human articular chondrocytes from healthy donors and found a highly similar epigenetic pattern at conserved sites. This conservation not only applied to active enhancer marks, but also to the repressive histone marks, which were associated with a similar set of genes in both species. Conclusions: With this data, we provide new insight into the mechanisms guiding the differentiation of embryonic chondrocytes into different chondrocyte subtypes, terminal hypertrophic and permanent articular chondrocytes. Both differentiation paths appear to be regulated by selective epigenetic repression.

Hepatitis C virus infection is causing chronic liver disease, cirrhosis, and hepatocellular carcinoma. By combining direct-acting antivirals (DAAs), high sustained virologic response rates (SVRs) can be achieved. Resistance-associated substitutions (RASs) are commonly observed after DAA failure, and especially nonstructural protein 5A (NS5A) RASs may impact retreatment options.1-3 Data on retreatment of DAA failure patients using first-generation DAAs are limited.4-7 Recently, a second-generation protease- and NS5A-inhibitor plus sofosbuvir (voxilaprevir/velpatasvir/sofosbuvir [VOX/VEL/SOF]) was approved for retreatment after DAA failure.8 However, this and other second-generation regimens are not available in many resource-limited countries or are not reimbursed by regular insurance, and recommendations regarding the selection of retreatment regimens using first-generation DAAs are very important. This study aimed to analyze patients who were re-treated with first-generation DAAs after failure of a DAA combination therapy.

OBJECTIVE:Osteoarthritis (OA) is a progressive degenerative disease of the articular cartilage caused by an unbalanced activity of proteases, cytokines and other secreted proteins. Since heparan sulfate (HS) determines the activity of many extracellular factors, we investigated its role in OA progression.METHODS:To analyze the role of the HS level, OA was induced by anterior cruciate ligament transection (ACLT) in transgenic mice carrying a loss-of-function allele of Ext1 in clones of chondrocytes (Col2-rtTA-Cre;Ext1e2fl/e2fl). To study the impact of the HS sulfation pattern, OA was surgically induced in mice with a heterozygous (Ndst1+/-) or chondrocyte-specific (Col2-Cre;Ndst1fl/fl) loss-of-function allele of the sulfotransferase Ndst1. OA progression was evaluated using the OARSI scoring system. To investigate expression and activity of cartilage degrading proteases, femoral head explants of Ndst1+/- mutants were analyzed by qRT-PCR, Western Blot and gelatin zymography.RESULTS:All investigated mouse strains showed reduced OA scores (Col2-rtTA-Cre;Ext1e2fl/e2fl: 0.83; 95% HDI 0.72-0.96; Ndst1+/-: 0.83, 95% HDI 0.74-0.9; Col2-Cre;Ndst1fl/fl: 0.87, 95% HDI 0.76-1). Using cartilage explant cultures of Ndst1 animals, we detected higher amounts of aggrecan degradation products in wildtype samples (NITEGE 4.24-fold, 95% HDI 1.05-18.55; VDIPEN 1.54-fold, 95% HDI 1.54-2.34). Accordingly, gelatin zymography revealed lower Mmp2 activity in mutant samples upon RA-treatment (0.77-fold, 95% HDI: 0.60-0.96). As expression of major proteases and their inhibitors was not altered, HS seems to regulate cartilage degeneration by affecting protease activity.CONCLUSION:A decreased HS content or a reduced sulfation level protect against OA progression by regulating protease activity rather than expression.

Hepatitis D virus (HDV) super-infection of hepatitis B virus (HBV)-infected patients is associated with rapid progression to liver cirrhosis and hepatocellular carcinoma. Treatment options are limited, and no vaccine is available. Although HDV-specific CD8+ T cells are thought to mediate viral control, little is known about the repertoire of targeted epitopes, and it remains unclear why HDV-specific CD8+ T cells ultimately fail during persistent infection. We aimed to define how viral escape impacts the efficacy of HDV-specific CD8+ T-cells.

The CD8 T cell response plays a central role for spontaneous immune control of acute Hepatitis B Virus (HBV) infection. In turn, patients with chronic infection typically have a weak and often exhausted T cell response. Accordingly, rescue of such dysfunctional T cell responses by therapeutic vaccination or transfer of adaptive cytotoxic T cells has been suggested as potential treatment strategies of patients with chronic infection. Importantly, these therapies rely on presentation of viral epitopes in the context of HLA class I molecules. Although selection of viral escape mutations has been well defined in chronic infections with HCV or HIV, the influence of viral sequence variants on the T cell response in HBV is less clear. Here, we studied viral sequence diversity in the HLA-A*02-restricted immunodominant HBV CTL epitope core18 – 27 (FLPSDFFPSV) and its impact on the CD8 T cell response in detail.

Background: HIV Gag mutations have been reported to confer PI drug resistance. However, clinical implications are still controversial and most current genotyping algorithms consider solely the protease gene for assessing PI resistance.Objectives: Our goal was to describe for HIV infections in Switzerland the potential role of the C-terminus of Gag (NC-p6) in PI resistance. We aimed to characterize resistance-relevant mutational patterns in Gag and protease and their possible interactions.Methods: Resistance information on plasma samples from 2004-12 was collected for patients treated by two diagnostic centres of the Swiss HIV Cohort Study. Sequence information on protease and the C-terminal Gag region was paired with the corresponding patient treatment history. The prevalence of Gag and protease mutations was analysed for PI treatment-experienced patients versus PI treatment-naive patients. In addition, we modelled multiple paths of an assumed ordered accumulation of genetic changes using random tree mixture models.Results: More than half of all PI treatment-experienced patients in our sample set carried HIV variants with at least one of the known Gag mutations, and 17.9% (66/369) carried at least one Gag mutation for which a phenotypic proof of PI resistance by in vitro mutagenesis has been reported. We were able to identify several novel Gag mutations that are associated with PI exposure and therapy failure.Conclusions: Our analysis confirmed the association of Gag mutations, well known and new, with PI exposure. This could have clinical implications, since the level of potential PI drug resistance might be underestimated.

Background: Chronic infections with the hepatitis B virus (HBV) are worldwide an enormous public health problem. Effective suppression of viral replication can be achieved with inhibitors of the viral polymerase, however, in most cases lifelong treatment is required to avoid recurrence of viremia. Activation of HBV-specific CD8 T cells by therapeutic vaccination may promote sustained control of viral replication by clearance of cccDNA from infected hepatocytes. Importantly, little is known about the exact targets of the CD8 T cell response and the extent of selection pressure on the virus. Here, it was hypothesized that CD8 T cell responses associated with strong selection pressure on the virus can be identified by viral sequence analysis.

(CXCL9),RANTES and SCGF-b were differentially influenced by PEG-IFNa therapy and increased or decreased until week 12 of treatment. Most of these changes were maintained until the end of therapy but nor all altered levels returned to baseline after treatment was stopped. Of note, none of the 50 factors analyzed before therapy including IP-10 differentiated patients with and without virological responses. However, induction of IP-10 was more pronounced during treatment in patients who became HDV RNA negative. Moreover, a more pronounced breadth of differentially regulated cytokines and chemokines at week 12 was evident in patients being HDV RNA negative during therapy than virological nonresponder. Biochemical flares during therapy were associated with distinct changes in cytokine/chemokine patterns. CONCLUSION: These data suggest that (i), in contrast to HCV infection, pre-treatment serum cytokines or chemokines including IP-10 do not predict virological response to PEG-IFNa in hepatitis delta, (ii) that PEG-IFNa therapy induces a profound change in the overall inflammatory milieu of hepatitis delta patients which does not necessarily reverse to pre-treatment levels when treatment is stopped, and (iii) that detection of HDV RNA is associated with altered IFNa-response patterns possibly indicating differential effects of HDV on ISG induction.

Different diagnostic parameters may affect the tropism prediction reliability. The impact of usage of FPR cut‐offs<20%, use of viral RNA versus proviral DNA samples, single versus triple amplification, and presence of MVC resistance mutations on tropism prediction at baseline were analysed on 101 patients receiving maraviroc (MVC) and correlated with their clinical outcome. This was a non‐interventional, retrospective study. 82 RNA and 54 DNA samples from the 101 patients receiving MVC were obtained. The V3 region was sequenced and the tropism predicted using the geno2pheno[coreceptor] and T‐CUP tools with FPR cut‐offs of 5%, 7.5%, 10%, 15% and 20%. Additionally, 27/82 RNA and 28/54 DNA samples were analysed in triplicate and 34/82 samples with the ESTA assay. The influence of 16 MVC resistance mutations on clinical outcome was studied. The genotypic susceptibility score (GSS) of the concomitant drugs was mapped to numerical values: susceptible to 1 (or 0.5 for NRTIs), intermediate to 0.5 (0.25 for NRTIs) and resistant to 0. Detection of baseline R5 viruses in RNA (by geno2pheno[coreceptor] and T‐CUP) or DNA (by T‐CUP) samples correlated with MVC‐treatment success. Both tools performed very similarly, with PPVs close to 90%, even with FPR cut‐offs as low as 5%. The use of triple amplification did not improve the prediction value but reduced the number of patients elegible for MVC treatment. No influence of the GSS or MVC resistance mutations on the clinical outcome was detected. Genotypic tropism testing from viral RNA and proviral DNA using the geno2pheno[coreceptor] and T‐CUP systems is valid to select candidates for MVC treatment. Our data suggest that the use of FPR cut‐offs of 5–7.5% and single amplification from RNA or DNA would assure a safe administration of MVC without excluding many patients who could benefit from this potent antiretroviral drug.

Aims: Mutational escape from immune pressure exacted by CD8 T cells has been described during infection with hepatitis C virus (HCV). However, the extent to which this immune adaptation process contributes to viral evolution at the population level is less clear. Here, we took advantage of a large HCV genotype 1b outbreak after inoculation of contaminated immunoglobulins in 1977/78 to study this adaptation process.

POSTERS(102 with MC(MC+) and 108 (MC-) without any evidence of lymphoproliferative/autoimmune disorders).The BAFF promoter polymorphism -871C/T, inducing a higher transcription of the BAFF gene, was also analyzed.A significantly higher prevalence of both T/T homozygosis (p < 0.001) and the presence of a T-allele (T/T and T/C, p = 0.004) at site -871 of BAFF promoter was shown in MC+ patients, resulting in a significant increase of BAFF serum concentration (4.12±1.29 vs. 2.09±0.81ng/ml, p < 0.0001).Real-time-PCR analysis of the BAFF gene expression showed a 2.8-fold higher transcription in MC+.By contrast, no significant difference in prevalence of different FcgRs genotypes was observed.However, a trend towards a higher prevalence of the FCgR3A-F/F genotype -showing a reduced affinity for IgG -was shown in MC+patients (p < 0.09).Interestingly, in 19 MC+patients undergoing anti-CD20 antibody treatment, the response was strictly related to the F-allele presence, being strongly reduced in F/F homozygous patients.These results underline the importance of the host genetic background in the development of HCV-related MC, suggesting that mechanisms enhancing Ig production and B-cell survival have a more relevant role than genetic defects in cryoglobulin clearance.This latter mechanism, however seems to be very important in the effectiveness of biological therapies, frequently used in HCV-related MC.