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SUMMARY The HIV reverse transcriptase inhibitor tenofovir, was recently formulated into a vaginal gel for use as a microbicide. In human trials, a 1% tenofovir gel inhibited HIV sexual transmission by 39% and surprisingly herpes simplex virus-2 (HSV-2) transmission by 51%. We demonstrate that the concentration achieved intravaginally with a 1% tenofovir topical gel has direct anti-herpetic activity. Tenofovir inhibits the replication of HSV clinical isolates in human embryonic fibroblasts, keratinocytes, and organotypic epithelial 3D-rafts, decreases HSV replication in human lymphoid and cervical tissues ex vivo, and delays HSV-induced lesions and death of topically treated HSV-infected mice. The active tenofovir metabolite inhibits HSV DNA-polymerase and HIV reverse transcriptase. Tenofovir must be topically administered to achieve concentrations, which are higher than systemic levels after oral treatment, that exert these dual antiviral effects. These findings indicate that a single topical treatment, like tenofovir, can inhibit the transmission of HIV and its co-pathogens.

The role of macrophages in the pathogenesis and progression of human immunodeficiency virus (HIV)-related infection is substantiated by in vitro and in vivo evidence. The unique ability to survive HIV infection and produce viral particles for long periods is postulated. Detailed studies of this phenomenon are lacking. The dynamics of HIV-1 replication and cumulative virus production was studied in long-term cultures of macrophages in the presence or in the absence of antiviral drugs. Multiply spliced and unspliced HIV-RNA production was assessed by quantitative PCR, and the number of infected cells was monitored by FACS analysis. Cumulative HIV-1 production was determined by a trapezoidal equation, including such parameters as times of collection and experimental values of genomic-RNA and p24 gag antigen. Unspliced and multiply spliced HIV-RNA increased linearly after macrophage infection; reached levels of 1.5 x 10(8) and 2.8 x 10(5) copies/10(5) cells, respectively, at day 10; and then remained stable throughout the course of the experiment. Cumulative production of genomic-RNA and p24 gag antigen was 10(10) copies/10(6) cells and 10(7) pg/10(6) cells, respectively, with an average of >200 virus particles produced daily by each macrophage. AZT decreased the cumulative production of both genomic-RNA and p24 gag antigen down to 2.5 x 10(9) copies and 1.1 x 10(6) pg/10(6) cells (73.8% and 88.9% inhibition, respectively) up to day 50 without virus breakthrough. Ritonavir had a limited, but consistent, efficacy on the release of mature virus proteins (about 40% inhibition), but not on HIV-RNA production. In conclusion, the long-term dynamics and the high cumulative virus production that characterize HIV-1 infection of macrophages underscore the peculiar role of these cells as a persistently infected reservoir of HIV.

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Activation of coagulation can be frequently found in horses with colic. However, it has also been demonstrated as a sequela of surgical trauma alone in humans. The purpose of the present study was to determine changes in coagulation and fibrinolysis in horses that underwent colic surgery and to evaluate whether these changes were secondary to the colic or the surgery and wound healing. Thirty horses that underwent colic surgery with uncomplicated recovery were included. Ten horses with a Forssell's procedure served as control group with a standardized surgical trauma. Besides daily physical examinations during the observation period of 10 days, activated partial thromboplastin time (aPTT), prothrombin time and thrombin time as well as fibrin monomer (FM), D-Dimer (DD) and antithrombin (AT) III were determined. Compared with the control group the aPTT was the only standard coagulation test that was significantly prolonged before and after the event of colic surgery. After surgery, hyperfibrinogenaemia occurred in all groups. In colic groups FM and DD concentrations were within reference range at admission,and were significantly greater than in control horses after surgery. AT III activity decreased after colic surgery, but did not change in the control group. It was concluded that an activated coagulation state after colic surgery has to be expected, resulting not only from the colic disease, but also from the event of surgery.

Current highly active antiretroviral therapy (HAART) cannot eliminate HIV-1 from infected persons, mainly because of the existence of refractory viral reservoir(s). Beyond latently-infected CD4+-T lymphocytes, macrophages (M/M) are important persistent reservoirs for HIV in vivo, that represent a major obstacle to HIV-1 eradication. Therefore, a rational therapeutic approach directed to the selective elimination of long-living HIV-infected M/M may be relevant in the therapy of HIV infection. Here we report that HIV-1 chronic infection of human macrophages results in the marked increase of expression and phosphorylation of STAT1, a protein involved in the regulation of many functions such as cell growth, differentiation, and maintenance of cellular homeostasis, thereby providing a new molecular target for drug development. A single and brief exposure to 9-(beta-D-arabinofuranosyl)-2-fluoroadenine 5'-monophosphate (FaraAMP, Fludarabine), a potent antileukemic nucleoside analog active against STAT1 expressing cells, selectively kills macrophage cultures infected by HIV-1 without affecting uninfected macrophages. Furthermore, encapsulation of Fludarabine into autologous erythrocytes (RBC) and targeting to macrophages through a single-18 h treatment with drug-loaded RBC, not only abolishes the Fludarabine-mediated toxic effect on non-phagocytic cells, but also enhances the selective killing of HIV-infected macrophages. As a final result, a potent (>98%) and long-lasting (at least 4 weeks without rebound) inhibition of virus release from drug-loaded RBC-treated chronically-infected macrophages was achieved. Taken together, the evidence of HIV-1-induced increase of STAT1, and the availability of a selective drug targeting system, may prove useful in the design of new pharmacological treatments to clear the HIV-1 macrophage reservoir.

Through this study we evaluated whether the HIV-1 tropism determined by genotypic analysis correlates with HIV-1 markers, such as CD4 cell count and plasma HIV-RNA. The analysis was performed on 1221 HIV-1 B-subtype infected patients with an available V3 sequence (all maraviroc naive). Of them, 532 were antiretroviral therapy (ART) naive and 689 ART experienced. Tropism determination was performed by using the geno2pheno (co-receptor) algorithm set at a false-positive rate (FPR) of 10% and 2%. Potential associations of FPR with CD4 cell count and viraemia were evaluated. Association of V3 mutations with genotypic-determined tropism was also evaluated according to different FPR ranges. About 26% of patients (either ART naive or ART experienced) were infected by X4-tropic viruses (using the classical 10% FPR cut-off). However, a significantly lower proportion of ART-naive patients had FPR ≤ 2% in comparison with ART-experienced patients (4.9% vs. 12.6%, respectively, p <0.001). The risk of advanced HIV-1 infection (with CD4 cell count ≤ 200 cells/mm(3)) was significantly greater in X4-infected patients, either ART-naive (OR (95% CI)), 4.2 (1.8-9.2); p 0.0006) or ART-experienced (2.3 (1.4-3.6); p 0.0003), with FPR set at 2% (but not at 10%). This finding was confirmed by multivariable logistic analysis. No relationship was found between viraemia and FPR ≤2%. Some X4-related mutations were significantly associated with FPR ≤2% (ART-naive patients, S11R, Y21V, G24K and G24R, p ≤0.001; ART-experienced patients, Y7K, S11R, H13Y, p ≤0.002). In conclusion, these findings show that within the context of genotypically-assessed CXCR4 tropism, FPR ≤2% defines (far better than 10%-FPR) a viral population associated with low CD4 rank, with potentially greater cytopathic effect, and with more advanced disease.