Modelling the microelimination of chronic hepatitis C in the canton of Bern, Switzerland: Reaching the Swiss Hepatitis Strategy goals despite the impact of the COVID 19 pandemic.

AIMS OF THE STUDY: Since 2014, the Swiss Hepatitis Strategy (SHS) has targeted the elimination of Hepatitis C Virus (HCV) in Switzerland. The epidemiology of HCV is diverse across Swiss cantons, therefore cantonal-level screening and treatment strategies should be developed. This study aimed to identify scenarios to achieve HCV elimination in the canton of Bern by 2030. METHODS: A preexisting Markov disease burden model was populated with data for Bern, and used to forecast the current and future prevalence of HCV, annual liver-related deaths (LRDs), and incidence of hepatocellular carcinoma and decompensated cirrhosis until 2030. Scenarios were developed to assess the current standard of care and potential long-term impact of the COVID-19 crisis on the HCV infected population. Additionally, potential scenarios for achieving the WHO 2030 targets and the SHS 2025 and 2030 targets (reduction of new cases of HCV, HCV-related mortality and viremic HCV cases) were identified. RESULTS: In 2019, there were an estimated 4,600 (95% UI: 3,330-4,940) viremic infections in the canton of Bern and 57% (n = 2,600) of viremic cases were diagnosed. This modelling forecasted a 10% increase in LRDs (28 in 2020 to 31 in 2030) with the current standard of care and a 50% increase in LRDs in a scenario assuming long-term delays. To achieve the WHO and SHS targets, the canton of Bern needs to increase the annual number of patients diagnosed (from 90 in 2019 to 250 per year in 2022-2024 [WHO], or 500 per year in 2022-2025 [SHS]) and treated (from 130 in 2019 to 340 per year in 2022-2024 [WHO] or 670 per year in 2022-2025 [SHS]). CONCLUSIONS: The SHS goals and the WHO targets for HCV elimination can be achieved in the Swiss canton of Bern by 2030; however, not at the current pace of screening, linkage to care and treatment.

Effect of the oral application of a highly selective MMP-13 inhibitor in three different animal models of rheumatoid arthritis.

OBJECTIVE: To evaluate the decrease of cartilage destruction by a novel orally active and specific matrix metalloproteinase 13 (MMP-13) inhibitor in three different animal models of rheumatoid arthritis (RA). MATERIALS AND METHODS: The SCID mouse co-implantation model of RA, the collagen-induced arthritis (CIA) model in mice and the antigen-induced arthritis model (AIA) in rabbits were used. RESULTS: In the SCID mouse co-implantation model, the MMP-13 inhibitor reduced cartilage destruction by 75%. In the CIA model of RA, the MMP-13 inhibitor resulted in a significant and dose-dependent decrease in clinical symptoms as well as of cartilage erosion by 38% (30 mg/kg), 28% (10 mg/kg) and 21% (3 mg/kg). No significant effects were seen in the AIA model. No toxic effects were seen in all three animal models. CONCLUSION: Although several MMPs in concert with other proteinases have a role in the process of cartilage destruction, there is a need for highly selective MMP inhibitors to reduce severe side effects that occur with non-specific inhibitors. Significant inhibition of MMP-13 reduced cartilage erosions in two of three tested animal models of RA. These results strongly support the development of this class of drugs to reduce or halt joint destruction in patients with RA.

Pre-B cell colony-enhancing factor/visfatin, a new marker of inflammation in rheumatoid arthritis with proinflammatory and matrix-degrading activities.

OBJECTIVE: To study possible mechanisms that mediate induction of the recently described adipocytokine pre-B cell colony-enhancing factor (PBEF) in joints of patients with rheumatoid arthritis (RA), and to analyze whether levels of PBEF correlate with disease severity and whether PBEF itself has the potential to act as a proinflammatory and destructive mediator in RA. METHODS: RA synovial fibroblasts (RASFs) and monocytes were stimulated with Toll-like receptor (TLR) ligands, cytokines, and recombinant human PBEF or were transfected with PBEF expression constructs or with PBEF-specific small interfering RNA. Production of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNFalpha) was measured by enzyme-linked immunosorbent assay, and expression of matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. PBEF expression in synovial tissue, synovial fluid, serum, and SFs was assessed by immunohistochemistry, in situ hybridization, Western blotting, and enzyme immunoassays. RESULTS: In RASFs, PBEF was up-regulated by TLR ligands and cytokines that are characteristically present in the joints of patients with RA. In synovial tissue, RASFs were the major PBEF-expressing cells. A predominance of PBEF was found in the synovial lining layer and at sites of invasion into cartilage. Levels of PBEF in serum and synovial fluid correlated with the degree of inflammation and clinical disease activity. Moreover, PBEF itself activated the transcription factors NF-kB and activator protein 1 and induced IL-6, IL-8, MMP-1, and MMP-3 in RASFs as well as IL-6 and TNFalpha in monocytes. PBEF knockdown in RASFs significantly inhibited basal and TLR ligand-induced production of IL-6, IL-8, MMP-1, and MMP-3. CONCLUSION: Our findings establish PBEF as a proinflammatory and destructive mediator of joint inflammation in RA and identify PBEF as a potential therapeutic target.

Effects of liver growth factors on hepadnavirus replication in chronically infected duck hepatocytes.

BACKGROUND/AIMS: Duck hepatitis B virus (DHBV) replication is up-regulated by cell cycle during the early infection of primary duck but the effect of cell cycle on DHBV replication in chronically infected hepatocyte is not known. METHODS: Hepatocytes obtained from DHBV congenitally infected embryos were used. Cell proliferation was controlled by addition of liver growth factors and the impact on viral replication analyzed. RESULTS: EGF induced cell proliferation is associated with a slight increase in CCC DNA synthesis and a decrease in viral transcription. Conversely, TGFbeta blocked cell cycle progression, diminished CCC DNA synthesis but increased viral transcription. CONCLUSIONS: Cell proliferation decreases DHBV transcription but this effect seems to be compensated by an opposite effect on the synthesis of CCC DNA resulting in a global moderate effect on viral replication. Our results also indicate that after division of chronically infected hepatocytes both daughter cells are infected, confirming that liver regeneration is not sufficient to induce CCC DNA eradication as suggested by the lack of effect of some long term anti-HBV therapies.

The role of Toll-like receptor signalling in the pathogenesis of arthritis.

Recent evidence highlighted the role of Toll-like receptors (TLRs) as key recognition structures of the innate immune system. The activation of TLRs initiates the production of inflammatory cytokines, chemokines, tissue destructive enzymes, and type I interferons. In addition, TLR signalling plays an important role in the activation and direction of the adaptive immune system by the upregulation of costimulatory molecules of antigen presenting cells. Considering the important role of TLR signalling as a critical link between innate and adaptive immunity it has been proposed that a dysregulation in TLR signalling might be associated with autoimmunity. In this review, recent studies on TLR signal transduction pathways activated by corresponding ligands are summarized and evidence for a possible role of TLR signalling in the pathogenesis of rheumatoid arthritis is discussed.

RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3.

OBJECTIVE: To assess the expression of Toll-like receptor 3 (TLR-3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR-3 ligands. METHODS: TLR-3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence-activated cell sorting and real-time polymerase chain reaction techniques. TLR-3 signaling was assessed by incubating RASFs with poly(I-C), lipopolysaccharide, palmitoyl-3-cysteine-serine-lysine-4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon-beta (IFNbeta), CXCL10, CCL5, and interleukin-6 (IL-6) protein production in the culture supernatants was performed by enzyme-linked immunosorbent assays. RESULTS: TLR-3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR-3 expression was localized predominantly in the synovial lining, with a majority of the TLR-3-expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR-3 ligand poly(I-C) resulted in the production of high levels of IFNbeta, CXCL10, CCL5, and IL-6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up-regulation of these cytokines and chemokines in a TLR-3-dependent manner. CONCLUSION: Our findings demonstrate the expression of TLR-3 in RA synovial tissue and the activation of RASFs in vitro by the TLR-3 ligand poly(I-C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR-3 ligand for the stimulation of proinflammatory gene expression in RASFs.

Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA).

BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT) and to compare their efficacy with phosphorothioate oligodeoxynucleotides (S-ODNs). METHODS: The effect of two partly overlapping PNAs targeting epsilon and of analogous S-ODNs was tested in cell-free transcription and translation system for DHBV RT expression. In addition their antiviral effect was investigated in primary duck hepatocytes (PDH). RESULTS: Both PNAs reproducibly inhibited DHBV RT in a dose-dependent manner with IC(50) of 10nM, whereas up to 600-fold higher concentration of S-ODNs was required for similar inhibition. The PNA targeting the bulge and upper stem of epsilon appeared as more efficient RT inhibitor than the PNA targeting only the bulge. Importantly, the inhibition was highly sequence-specific since double-mismatched PNA had no effect on the RT reaction. Moreover, in PDH the PNA coupled to Arg(7) cationic delivery peptide decreased DHBV replication. CONCLUSIONS: We provide the first evidence that PNAs targeting the bulge and upper stem of epsilon can efficiently and in a sequence-specific manner inhibit DHBV RT.

Inhibitory effect of adefovir on viral DNA synthesis and covalently closed circular DNA formation in duck hepatitis B virus-infected hepatocytes in vivo and in vitro.

The elimination of viral covalently closed circular DNA (CCC DNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with adefovir, a new acyclic nucleoside phosphonate, the prodrug of which, adefovir dipivoxil, is in clinical evaluation, is able to suppress viral CCC DNA both in vitro and in vivo using the duck HBV (DHBV) model. First, the effect of adefovir on viral CCC DNA synthesis was examined with primary cultures of DHBV-infected fetal hepatocytes. Adefovir was administered for six consecutive days starting one day before or four days after DHBV inoculation. Dose-dependent inhibition of both virion release in culture supernatants and synthesis of intracellular viral DNA was observed. Although CCC DNA amplification was inhibited by adefovir, CCC DNA was not eliminated by antiviral treatment and the de novo formation of CCC DNA was not prevented by pretreatment of the cells. Next, preventive treatment of experimentally infected ducklings with lamivudine or adefovir revealed that both efficiently suppressed viremia and intrahepatic DNA. However, persistence of viral DNA even when detectable only by PCR was associated with a recurrence of viral replication following drug withdrawal. Taken together, our results demonstrate that adefovir is a potent inhibitor of DHBV replication that inhibits CCC DNA amplification but does not effectively prevent the formation of CCC DNA from incoming viral genomes.

Effects of pyrimidine and purine analog combinations in the duck hepatitis B virus infection model.

To design new strategies of antiviral therapy for chronic hepatitis B, we have evaluated the antiviral activity of the combination of amdoxovir (DAPD), emtricitabine [(-)FTC], and clevudine (L-FMAU) in the duck hepatitis B virus (DHBV) model. Using their triphosphate (TP) derivatives in a cell-free system expressing a wild-type active DHBV reverse transcriptase (RT), the three dual combinations exhibited a greater additive inhibitory effect on viral minus-strand DNA synthesis than the single drugs, according to the Bliss independence model. Both dual combinations with DAPD TP were the most efficient while the triple combination increased the inhibitory effect on the DHBV RT activity in comparison with the dual association, however, without additive effect. Postinoculation treatment of experimentally infected primary duck hepatocytes showed that dual and triple combinations potently inhibited viral DNA synthesis during treatment but did not inhibit the reinitiation of viral DNA synthesis after treatment cessation. Preinoculation treatment with the same combinations exhibited antiviral effects on intracellular viral DNA replication, but it was unable to prevent the initial covalently closed circular DNA (cccDNA) formation. Short-term in vivo treatment in acutely infected ducklings showed that the dual combinations were more-potent inhibitors of virus production than the single treatments, with the L-FMAU and FTC combination being the most potent. A longer administration of L-FMAU and FTC for 4 weeks efficiently suppressed viremia and viral replication. However, no viral clearance from the liver was observed, suggesting that the enhanced antiviral effect of this combination was not sufficient for cccDNA suppression and HBV eradication from infected cells.

Are 5'-O-carbamate-2',3'-dideoxythiacytidine new anti-HIV and anti-HBV nucleoside drugs or prodrugs?

In contrast to 5'-O-carbonate 3TC derivatives (23, 24), which are clearly 3TC prodrugs, the corresponding 3TC carbamates (15-21 and 25), found to be very stable compounds with respect to enzymatic hydrolysis (cellular lysates and culture cell media) and still active on both HIV-1 and HBV infected cells, may not be 3TC prodrugs. The antiviral properties as well as the mechanism of action of 3TC analogues have been studied and evaluated.