Paul Sandstrom

Oxidative stress as a mediator of apoptosis

Many agents which induce apoptosis are either oxidants or stimulators of cellular oxidative metabolism. Conversely, many inhibitors of apoptosis have antioxidant activities or enhance cellular antioxidant defenses. Mammalian cells exist in a state of oxidative siege in which survival requires an appropriate balance of oxidants and antioxidants. Thomas Buttke and Paul Sandstrom suggest that eukaryotic cells may benefit from this perilous existence by invoking oxidative stress as a common mediator of apoptosis.

Drug Resistance Mutations for Surveillance of Transmitted HIV-1 Drug-Resistance: 2009 Update

Programs that monitor local, national, and regional levels of transmitted HIV-1 drug resistance inform treatment guidelines and provide feedback on the success of HIV-1 treatment and prevention programs. To accurately compare transmitted drug resistance rates across geographic regions and times, the World Health Organization has recommended the adoption of a consensus genotypic definition of transmitted HIV-1 drug resistance. In January 2007, we outlined criteria for developing a list of mutations for drug-resistance surveillance and compiled a list of 80 RT and protease mutations meeting these criteria (surveillance drug resistance mutations; SDRMs). Since January 2007, several new drugs have been approved and several new drug-resistance mutations have been identified. In this paper, we follow the same procedures described previously to develop an updated list of SDRMs that are likely to be useful for ongoing and future studies of transmitted drug resistance. The updated SDRM list has 93 mutations including 34 NRTI-resistance mutations at 15 RT positions, 19 NNRTI-resistance mutations at 10 RT positions, and 40 PI-resistance mutations at 18 protease positions.

No evidence of infection with porcine endogenous retrovirus in recipients of porcine islet-cell xenografts

BACKGROUND The study of whether porcine xenografts can lead to porcine endogenous retrovirus (PERV) infection of recipients is critical for evaluating the safety of pig-to-man xenotransplantation. PERV is carried in the pig germline, and all recipients of porcine tissues or organs will be exposed to the virus. METHODS We studied 10 diabetic patients who had received porcine fetal islets between 1990 and 1993, looking for evidence of PERV infection by using PCR serology, PCR, and reverse transcriptase assays. Prolonged xenograft survival (up to a year) was confirmed in five patients by porcine C-peptide excretion and detection of pig mitochondrial DNA (mtDNA) in serum. FINDINGS Despite the evidence for extended exposure to pig cells and despite concomitant immunosuppressive therapy, we were unable to detect markers of PERV infection in any patient. Screening for two PERV sequences in peripheral blood lymphocytes collected 4-7 years after the xenotransplantation was negative. Markers of PERV expression, including viral RNA and reverse transcriptase, were undetectable in sera from both early (day 3 to day 180) and late (4-7 years) time points. Western blot analysis for antibodies was consistently negative. INTERPRETATION These results suggested the absence of PERV infection in these patients. Also this study establishes a minimum standard for post-transplant surveillance of patients given porcine xenografts.

Minority HIV-1 Drug Resistance Mutations Are Present in Antiretroviral Treatment–Naïve Populations and Associate with Reduced Treatment Efficacy

Background Transmitted HIV-1 drug resistance can compromise initial antiretroviral therapy (ART); therefore, its detection is important for patient management. The absence of drug-associated selection pressure in treatment-naïve persons can cause drug-resistant viruses to decline to levels undetectable by conventional bulk sequencing (minority drug-resistant variants). We used sensitive and simple tests to investigate evidence of transmitted drug resistance in antiretroviral drug-naïve persons and assess the clinical implications of minority drug-resistant variants. Methods and Findings We performed a cross-sectional analysis of transmitted HIV-1 drug resistance and a case-control study of the impact of minority drug resistance on treatment response. For the cross-sectional analysis, we examined viral RNA from newly diagnosed ART-naïve persons in the US and Canada who had no detectable (wild type, n = 205) or one or more resistance-related mutations (n = 303) by conventional sequencing. Eight validated real-time PCR-based assays were used to test for minority drug resistance mutations (protease L90M and reverse transcriptase M41L, K70R, K103N, Y181C, M184V, and T215F/Y) above naturally occurring frequencies. The sensitive real-time PCR testing identified one to three minority drug resistance mutation(s) in 34/205 (17%) newly diagnosed persons who had wild-type virus by conventional genotyping; four (2%) individuals had mutations associated with resistance to two drug classes. Among 30/303 (10%) samples with bulk genotype resistance mutations we found at least one minority variant with a different drug resistance mutation. For the case-control study, we assessed the impact of three treatment-relevant drug resistance mutations at baseline from a separate group of 316 previously ART-naïve persons with no evidence of drug resistance on bulk genotype testing who were placed on efavirenz-based regimens. We found that 7/95 (7%) persons who experienced virologic failure had minority drug resistance mutations at baseline; however, minority resistance was found in only 2/221 (0.9%) treatment successes (Fisher exact test, p = 0.0038). Conclusions These data suggest that a considerable proportion of transmitted HIV-1 drug resistance is undetected by conventional genotyping and that minority mutations can have clinical consequences. With no treatment history to help guide therapies for drug-naïve persons, the findings suggest an important role for sensitive baseline drug resistance testing.

HIV-1 protease and reverse transcriptase mutations for drug resistance surveillance

Objectives:Monitoring regional levels of transmitted HIV-1 resistance informs treatment guidelines and provides feedback on the success of HIV-1 prevention efforts. Surveillance programs for estimating the frequency of transmitted resistance are being developed in both industrialized and resource-poor countries. However, such programs will not produce comparable estimates unless a standardized list of drug-resistance mutations is used to define transmitted resistance. Methods:In this paper, we outline considerations for developing a list of drug-resistance mutations for epidemiologic estimates of transmitted resistance. First, the mutations should cause or contribute to drug resistance and should develop in persons receiving antiretroviral therapy. Second, the mutations should not occur as polymorphisms in the absence of therapy. Third, the mutation list should be applicable to all group M subtypes. Fourth, the mutation list should be simple, unambiguous, and parsimonious. Results:Applying these considerations, we developed a list of 31 protease inhibitor-resistance mutations at 14 protease positions, 31 nucleoside reverse transcriptase inhibitor-resistance mutations at 15 reverse transcriptase positions, and 18 non-nucleoside reverse transcriptase inhibitor-resistance mutations at 10 reverse transcriptase positions. Conclusions:This list, which should be updated regularly using the same or similar criteria, can be used for genotypic surveillance of transmitted HIV-1 drug resistance.

Inhibition of activation-induced death in T cell hybridomas by thiol antioxidants: oxidative stress as a mediator of apoptosis.

N‐Acetylcysteine (NAC) is a well established thiol antioxidant which, after uptake, deacylation and conversion to glutathione functions as both a redox buffer and a reactive oxygen intermediate scavenger. We report here that NAC completely blocks activation induced death and associated DNA fragmentation of myelin basic protein (MBP) specific T cell hybridomas. Conversely, NAC had very little effect on the antigen driven proliferation of a MBP specific T cell line similar to that from which the hybridomas were derived. NAC displayed an analogous absolute inhibition of mitogen mediated activation induced death, even if added up to 3 h post activation. Although glutathione was as efficient as NAC at blocking activation induced death, dithiothreitol displayed minimal inhibition while L‐cysteine had no effect at all. The observation that certain thiol antioxidants such as NAC and glutathione can completely block the activation induced death of T cell hybridomas implicates redox regulation in this process. J. Leukoc. Biol. 55: 221–226; 1994.

Redox Regulation of Programmed Cell Death in Lymphocytes

A redox imbalance caused by an over-production of prooxidants or a decrease in antioxidants seems to play a role in the programmed cell death that occurs in various developmental programs. Such a physiological function for oxidative stress is particularly applicable to the immune system, wherein individual lymphocytes undergo continuous scrutiny to determine if they should be preserved or programmed to die. Following activation, lymphocytes produced increased levels of reactive oxygen species (ROS) which may serve as intracellular signaling molecules. The ultimate outcome of this increased ROS formation, i.e., lymphocyte proliferation versus programmed cell death, may be dictated by macrophage-derived costimulatory molecules that bolster or diminish lymphocyte antioxidant defenses. HIV-1-infected individuals display multiple symptoms of redox imbalance consistent with their being in oxidative stress, and lymphocytes from such individuals are more prone to undergo apoptosis in vitro. It is suggested that oxidative stress, and lymphocytes from such individuals are more prone to undergo apoptosis in vitro. It is suggested that oxidative stress is a physiological mediator of programmed cell death in lymphoid cells, and that HIV disease represents an extreme case of what can happen when regulatory safeguards are compromised.

Lipid hydroperoxide-induced apoptosis: lack of inhibition by Bcl-2 over-expression

Increased membrane lipid peroxidation has recently been implicated as being associated with apoptosis. In the present study the addition of 15‐hydroperoxyeicosatetraenoic acid (15‐HPETE) or 13‐hydroperoxydodecadienoic acid (13‐HPODE) to A3.01 T cells is shown to induce marked chromatin condensation coincident with DNA fragmentation, indicative of apoptosis. 15‐HPETE also evoked an immediate and sustained rise in cytoplasmic calcium which was required for the induction of apoptosis. A3.01 cells transfected with the bcl‐2 proto‐oncogene were 6‐ to 8‐fold more resistant to apoptotic killing by tumor necrosis factor‐α, but only 0.4‐fold more resistant to 15‐HPETE. Thus, Bcl‐2 is not capable of protecting cells from undergoing apoptosis following the direct addition of lipid hydroperoxides.

Human Immunodeficiency Virus (HIV) Type 1 Proviral Hypermutation Correlates with CD4 Count in HIV-Infected Women from Kenya

ABSTRACT APOBEC3G is an important innate immune molecule that causes human immunodeficiency virus type 1 (HIV-1) hypermutation, which can result in detrimental viral genome mutations. The Vif protein of wild-type HIV-1 counteracts APOBEC3G activity by targeting it for degradation and inhibiting its incorporation into viral particles. Additional APOBEC cytidine deaminases have been identified, such as APOBEC3F, which has a similar mode of action but different sequence specificity. A relationship between APOBEC3F/G and HIV disease progression has been proposed. During HIV-1 sequence analysis of the vpu/env region of 240 HIV-infected subjects from Nairobi, Kenya, 13 drastically hypermutated proviral sequences were identified. Sequences derived from plasma virus, however, lacked hypermutation, as did proviral vif. When correlates of disease progression were examined, subjects with hypermutated provirus were found to have significantly higher CD4 counts than the other subjects. Furthermore, hypermutation as estimated by elevated adenine content positively correlated with CD4 count for all 240 study subjects. The sequence context of the observed hypermutation was statistically associated with APOBEC3F/G activity. In contrast to previous studies, this study demonstrates that higher CD4 counts correlate with increased hypermutation in the absence of obvious mutations in the APOBEC inhibiting Vif protein. This strongly suggests that host factors, such as APOBEC3F/G, are playing a protective role in these patients, modulating viral hypermutation and host disease progression. These findings support the potential of targeting APOBEC3F/G for therapeutic purposes.

Simian foamy virus infection among zoo keepers

We investigated 322 North American zoo workers in an anonymous serosurvey for antibodies to simian foamy viruses to establish the potential risk of zoonotic transmission by these retroviruses. 4 of 133 (3%) individuals who worked specifically with mammals including primates were seropositive, primarily with chimp-like viruses, indicating the importance of work practices to reduce exposure to these agents.