Whereas ANT has been widely regarded as being essential for mitochondrial depolarization and consequent cytochrome launch, apoptosome formation, and apoptosis, a recent statement directly difficulties that part. and consequent immunodeficiency is usually one such disease state in which excessive apoptosis has been implicated. Current therapies for HIV not only reduce HIV Cardiogenol C HCl replication but may also directly impact apoptosis; indeed, many groups have now reported that HIV protease inhibitors (PIs) can inhibit apoptosis at concentrations much Cardiogenol C HCl like those that are commonly seen in the plasma of patients receiving such treatments (examined in ref. 1). Paradoxically, such brokers may also induce apoptosis, particularly of transformed cells, when used at higher doses (2C5). Studies by several groups have investigated the potential mechanisms by which PIs may inhibit apoptosis, yielding different results. Proposed mechanisms include altered transcriptional regulation of important apoptosis regulatory proteins (5C7) and/or direct inhibition of the apoptosis enzyme ICE (8, 9) and/or calpain (10). Such theories cannot fully account for the ability of PIs to inhibit diverse apoptotic stimuli (examined in ref. 1) or the lack of enzymatic inhibition of recombinant caspases in vitro (11). According to another proposed mechanism to account for apoptosis inhibition, PIs alter the propensity of mitochondria to transduce apoptotic signals. This latter model is supported by the findings that PIs are able to block Fas-induced apoptosis including mitochondrial signaling but not Fas-induced apoptosis that is mitochondria impartial (11) and that PIs are able to rescue cells from apoptosis induced by mitochondriotoxic brokers (5, 12). Despite these in vitro findings, it remains controversial whether PI therapy for HIV-infected patients offers additional benefits in terms of CD4 T cell reconstitution compared with non-PICcontaining regimens of equivalent antiviral ITGA6 potency (13, 14). Most studies that demonstrate enhanced CD4 T cell improvements in patients receiving PI therapy were retrospective, post-hoc analyses (15), which raises issues about the methodologies used. Consequently, at least one study was designed to compare CD4 T cell number, activation profile, memory and naive Cardiogenol C HCl T cell subsets, Cardiogenol C HCl and apoptosis between patients receiving PI-continuing or PI-sparing regimens (16). No differences were observed between groups regarding CD4 T cell number, activation, or memory or naive subsets; however, within the first week of therapy, significantly less apoptosis was seen in CD4 T cells of patients receiving PI therapy than in patients who did not receive PI therapy (16). Such data are consistent with the postulated antiapoptotic effects of PIs. The objectives of this study were, first, to evaluate whether PIs were antiapoptotic in vivo by evaluating apoptotic changes in animal models of disease that are associated with excessive apoptosis but that do not depend upon viral replication and, second, to evaluate the mechanisms involved. Results As mouse metabolism of PIs differs from that of humans, we first performed pharmacokinetic studies of mice treated with nelfinavir (NFV) at doses used in human therapy. Within 1 hour of dosing, mice experienced undetectable levels of NFV. Therefore, we co-dosed mice with ritonavir (RIT), another PI known to increase PI levels in humans (17). Ultimately, a dose of 125 mg/kg NFV and 13 mg/kg RIT resulted in drug levels much like those of humans treated with NFV alone (see Methods). This dose was utilized for in vivo screening. First, we evaluated the impact of NFV/RIT treatment on CD95/Fas-induced hepatic failure (18C20). Mice received NFV/RIT or vehicle control pretreatment for 24 hours followed by treatment with Cardiogenol C HCl 6 or 12 g of IV Jo-2 anti-Fas antibody. Control animals died in a dose-dependent manner, whereas NFV/RIT-pretreated animals displayed superior survival compared with controls (Physique ?(Figure1A).1A). Moreover, survival of mice treated with RIT (13 mg/kg) was comparable to that of controls, which indicates that NFV was responsible for the observed improved survival. Importantly, all mice that died did so within 72 hours, which indicates that NFV/RIT truly prevents rather than delays Jo-2Cinduced hepatotoxicity and death. In parallel, groups of 10 mice received 2.5, 5, or 7.5 g of IV Jo-2 with or without NFV/RIT pretreatment. Mice were sacrificed at 4 or 24 hours and analyzed for serum biochemistry, H&:E histology, and apoptosis by TUNEL staining. Serum glucose, blood urea nitrogen, creatinine, phosphorus, total protein, albumin, globulin,.