Anti-retroviral (ARV) –based microbicides are one of the strategies pursued to prevent HIV-1 transmission. Delivery of ARV drugs to subepithelial CD4+ T cells at concentrations for protection is likely determined by drug transporters expressed in the cervicovaginal epithelium. To define the role of drug transporters in mucosal disposition of topically applied ARV-based microbicides, these must be tested in epithelial cell line-based biopharmaceutical assays factoring the effect of relevant drug transporters. We have characterised gene expression of influx and efflux drug transporters in a panel of cervicovaginal cell lines and compared this to expression in cervicovaginal tissue. We also investigated the effect of dapivirine, darunavir and tenofovir, currently at advanced stages of microbicides development, on expression of drug transporters in cell lines. Expression of efflux ABC transporters in cervical tissue was best represented in HeLa, Ect1/E6E7 and End1/E6E7 cell lines. Expression of influx OCT and ENT transporters in ectocervix matched expression in Hela while expression of influx SLCO transporters in vagina was best reflected in VK2/E6E7 cell line. Stimulation with darunavir and dapivirine upregulated MRP transporters, including MRP5 involved in transport of tenofovir. Dapivirine also significantly downregulated tenofovir substrate MRP4 in cervical cell lines. Treatment with darunavir and dapivirine showed no significant effect on expression of BCRP, MRP2 and P-glycoprotein implicated in efflux of different ARV drugs. Darunavir strongly induced expression in most cell lines of CNT3 involved in cell uptake of nucleotide/nucleoside analogue reverse transcriptase inhibitors and SLCO drug transporters involved in cell uptake of protease inhibitors. This study provides insight into the suitability of cervicovaginal cell lines for assessment of ARV drugs in transport kinetics studies. The modulatory effect of darunavir and dapivirine on expression of drug transporters involved in transport of tenofovir points to the possibility of combining these drugs to improve retention of individual drugs at target tissues.

Objective Fibrosis is the most characteristic pathological hallmark of SSc, a connective tissue disease characterized by vascular and immunological abnormalities, inflammation and enhanced extracellular matrix production, leading to progressive fibrosis of skin and internal organs. We previously demonstrated that parvovirus B19 (B19V) can infect normal human dermal fibroblasts (NHDFs) and that B19V persists in SSc fibroblasts. In this study, we investigated whether parvovirus B19V is able to activate in vitro NHDFs and to induce in these cells some phenotypic features similar to that observed in the SSc fibroblasts. Methods We preliminarily analysed the time course of B19V infection in cultured NHDFs, then we investigated the ability of B19V to induce cell migration, invasive phenotype and mRNA expression of some profibrotic and/or proinflammatory genes. Results We confirmed our previous findings that B19V infects NHDFs, but the infection is not productive. After incubation with B19V, NHDFs showed a significant increase of both migration and invasiveness, along with mRNA expression of different profibrotic genes (α-SMA, EDN-1, IL-6, TGF-β1 receptors 1 and 2, Col1α2), some genes associated with inflammasome platform (AIM2, IFI16, IL-1β, CASP-1) and genes for metalloprotease (MMP 2, 9 and 12). Conclusion These data suggest that B19V can activate dermal fibroblasts and may have a role in the pathogenesis of fibrosis. B19V-induced fibroblast migration and invasiveness could be due to the B19V-associated MMP9 overexpression and activation. Moreover, the up-regulation of MMP12, typical of SSc, could link the B19V infection of fibroblasts to the anti-angiogenic process.

Objective Emerging evidence demonstrates that excessive accumulation of senescent cells is associated with some chronic diseases and suggests a pathogenic role of cellular senescence in fibrotic processes, such as that occurring in aging or in systemic sclerosis (SSc). Recently, we demonstrated that parvovirus B19 (B19V) activates normal human dermal fibroblasts and induces expression of different profibrotic/proinflammatory genes. This observation prompted us to investigate whether it is also able to induce fibroblast senescence as a potential pathogenetic mechanism in B19V-induced fibrosis. Methods Primary cultures of fibroblasts were infected with B19V and analyzed for the acquisition of senescence markers, such as morphological modifications, senescence-associated beta-galactosidase (SA-β-gal) activity, DNA damage response (DDR) and expression of senescence-associated secretory phenotype (SASP)-related factors. Results We demonstrated that B19V-infected fibroblasts develop typical senescence features such as enlarged and flat-shaped morphology and SA-β-gal activity similar to that observed in SSc skin fibroblasts. They also developed a SASP-like phenotype characterized by mRNA expression and release of some proinflammatory cytokines, along with activation of transcription factor NFkB. Moreover, we observed B19V-induced DNA damage with the comet assay: a subpopulation of fibroblasts from B19V-infected cultures showed a significant higher level of DNA strand breaks and oxidative damage compared with mock-infected cells. Increased level and nuclear localization of ɣH2AX, a hallmark of DNA damage response, were also found. Conclusions B19V-induced senescence and production of SASP-like factors in normal dermal fibroblasts could represent a new pathogenic mechanism of non-productive B19V infection, which may have a role in the fibrotic process.

Background Torquetenovirus (TTV), a widespread anellovirus recognized as the main component of the healthy human virome, displays viremia that is highly susceptible to variations in immune competence. TTV possesses microRNA (miRNA)-coding sequences that might be involved in viral immune evasion. Among TTV-encoded miRNAs, miRNA t1a, t3b, and tth8 have been found in biological fluids. Here, the presence of TTV DNA and TTV miRNAs in the plasma of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected subjects was investigated to monitor the possible association with coronavirus disease 2019 (COVID-19) severity. Methods Detection of TTV DNA and miRNA t1a, t3b, and tth8 was investigated in plasma samples of 56 SARS-CoV-2-infected subjects with a spectrum of different COVID-19 outcomes. TTV DNA and TTV miRNAs were assessed with a universal single step real-time TaqMan PCR assay and miRNA quantitative RT-PCR miRNA assay, respectively. Results The TTV DNA prevalence was 59%, whereas at least one TTV miRNA was found in 94% of the patients tested. miRNA tth8 was detected in 91% of subjects, followed by miRNAs t3b (64%) and miRNAt1a (30%). Remarkably, although TTV DNA was unrelated to COVID-19 severity, miRNA tth8 was significantly associated with the degree of disease (adjusted incidence rate ratio (IRR) 2.04, 95% CI 1.14–3.63, for the subjects in the high severity group compared to those in the low severity group). Conclusions Our findings encourage further investigation to understand the potential role of TTV miRNAs in the different outcomes of COVID-19 at early and late stages.

Despite the availability of vaccines, COVID-19 continues to be aggressive, especially in immunocompromised individuals. Therefore, the development of a specific therapeutic agent with antiviral activity against SARS-CoV-2 is necessary. The infection pathway starts when the receptor binding domain of the viral spike protein interacts with the angiotensin converting enzyme 2 (ACE2), which acts as a host receptor for the RBD expressed on the host cell surface. In this scenario, ACE2 analogs binding to the RBD and preventing the cell entry can be promising antiviral agents. Most of the ACE2 residues involved in the interaction belong to the α1 helix, more specifically to the minimal fragment ACE2(24−42). In order to increase the stability of the secondary structure and thus antiviral activity, we designed different triazole-stapled analogs, changing the position and the number of bridges. The peptide called P3, which has the triazole-containing bridge in the positions 36−40, showed promising antiviral activity at micromolar concentrations assessed by plaque reduction assay. On the other hand, the double-stapled peptide P4 lost the activity, showing that excessive rigidity disfavors the interaction with the RBD.