Nathan W. Cummins

HIV Replication Alters the Composition of Extrinsic Pathway Coagulation Factors and Increases Thrombin Generation

Background HIV infection leads to activation of coagulation, which may increase the risk for atherosclerosis and venous thromboembolic disease. We hypothesized that HIV replication increases coagulation potentially through alterations in extrinsic pathway factors. Methods and Results Extrinsic pathway factors were measured among a subset of HIV participants from the Strategies for Management of Anti‐Retroviral Therapy (SMART) trial. Thrombin generation was estimated using validated computational modeling based on factor composition. We characterized the effect of antiretroviral therapy (ART) treatment versus the untreated state (HIV replication) via 3 separate analyses: (1) a cross‐sectional comparison of those on and off ART (n=717); (2) a randomized comparison of deferring versus starting ART (n=217); and (3) a randomized comparison of stopping versus continuing ART (n=500). Compared with viral suppression, HIV replication consistently showed short‐term increases in some procoagulants (eg, 15% to 23% higher FVIII; P<0.001) and decreases in key anticoagulants (eg, 5% to 9% lower antithrombin [AT] and 6% to 10% lower protein C; P<0.01). The net effect of HIV replication was to increase coagulation potential (eg, 24% to 48% greater thrombin generation from computational models; P<0.01 for all). The pattern of changes from HIV replication was reversed with ART treatment and consistent across all 3 independent comparisons. Conclusions HIV replication leads to complex changes in extrinsic pathway factors, with the net effect of increasing coagulation potential to a degree that may be clinically relevant. The key influence of changes in FVIII and AT suggests that HIV‐related coagulation abnormalities may involve changes in hepatocyte function in the context of systemic inflammation. Clinical Trial Registration URL: ClinicalTrials.gov. Unique identifier: NCT00027352.

The TRAIL to viral pathogenesis: the good, the bad and the ugly.

Since the discovery of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) in 1995, much has been learned about the protein, its receptors and signaling cascade to induce apoptosis and the regulation of its expression. However, the physiologic role or roles that TRAIL may play in vivo are still being explored. The expression of TRAIL on effector T cells and the ability of TRAIL to induce apoptosis in virally infected cells provided early clues that TRAIL may play an active role in the immune defense against viral infections. However, increasing evidence is emerging that TRAIL may have a dual function in the immune system, both as a means to kill virally infected cells and in the regulation of cytokine production. TRAIL has been implicated in the immune response to viral infections (good), and in the pathogenesis of multiple viral infections (bad). Furthermore, several viruses have evolved mechanisms to manipulate TRAIL signaling to increase viral replication (ugly). It is likely that whether TRAIL ultimately has a proviral or antiviral effect will be dependent on the specific virus and the overall cytokine milieu of the host. Knowledge of the factors that determine whether TRAIL is proviral or antiviral is important because the TRAIL system may become a target for development of novel antiviral therapies.

Heme oxygenase-1 regulates the immune response to influenza virus infection and vaccination in aged mice

Underlying mechanisms of individual variation in severity of influenza infection and response to vaccination are poorly understood. We investigated the effect of reduced heme oxygenase‐1 (HO‐1) expression on vaccine response and outcome of influenza infection. HO‐1‐deficient and wild‐type (WT) mice (kingdom, Animalia; phylum, Chordata; genus/species, Mus musculus) were infected with influenza virus A/PR/8/34 with or without prior vaccination with an adeno‐viral‐based influenza vaccine. A genome‐wide association study evaluated the expression of single‐nucleotide polymorphisms (SNPs) in the HO‐1 gene and the response to influenza vaccination in healthy humans. HO‐1‐deficient mice had decreased survival after influenza infection compared to WT mice (median survival 5.5 vs. 6.5 d, P=0.016). HO‐1‐deficient mice had impaired production of antibody following influenza vaccination compared to WT mice (mean antibody titer 869 vs. 1698, P=0.02). One SNP in HO‐1 and one SNP in the constitutively expressed isoform HO‐2 were independently associated with decreased antibody production after influenza vaccination in healthy human volunteers (P=0.017 and 0.014, respectively). HO‐1 deficient mice were paired with sex‐ and age‐matched WT controls. HO‐1 affects the immune response to both influenza infection and vaccination, suggesting that therapeutic induction of HO‐1 expression may represent a novel adjuvant to enhance influenza vaccine effectiveness.—Cummins, N. W., Weaver, E. A., May, S. M., Croatt, A. J., Foreman, O., Kennedy, R. B., Poland, G. A., Barry, M. A., Nath, K. A., Badley, A. D. Heme oxygenase‐1 regulates the immune response to influenza virus infection and vaccination in aged mice. FASEB J. 26, 2911–2918 (2012). www.fasebj.org

Infected Cell Killing by HIV-1 Protease Promotes NF-κB Dependent HIV-1 Replication

Acute HIV-1 infection of CD4 T cells often results in apoptotic death of infected cells, yet it is unclear what evolutionary advantage this offers to HIV-1. Given the independent observations that acute T cell HIV-1 infection results in (1) NF-κB activation, (2) caspase 8 dependent apoptosis, and that (3) caspase 8 directly activates NF-κB, we questioned whether these three events might be interrelated. We first show that HIV-1 infected T cell apoptosis, NF-κB activation, and caspase 8 cleavage by HIV-1 protease are coincident. Next we show that HIV-1 protease not only cleaves procaspase 8, producing Casp8p41, but also independently stimulates NF-κB activity. Finally, we demonstrate that the HIV protease cleavage of caspase 8 is necessary for optimal NF-κB activation and that the HIV-1 protease specific cleavage fragment Casp8p41 is sufficient to stimulate HIV-1 replication through NF-κB dependent HIV-LTR activation both in vitro as well as in cells from HIV infected donors. Consequently, the molecular events which promote death of HIV-1 infected T cells function dually to promote HIV-1 replication, thereby favoring the propagation and survival of HIV-1.

The Association between Serum Biomarkers and Disease Outcome in Influenza A(H1N1)pdm09 Virus Infection: Results of Two International Observational Cohort Studies

Background Prospective studies establishing the temporal relationship between the degree of inflammation and human influenza disease progression are scarce. To assess predictors of disease progression among patients with influenza A(H1N1)pdm09 infection, 25 inflammatory biomarkers measured at enrollment were analyzed in two international observational cohort studies. Methods Among patients with RT-PCR-confirmed influenza A(H1N1)pdm09 virus infection, odds ratios (ORs) estimated by logistic regression were used to summarize the associations of biomarkers measured at enrollment with worsened disease outcome or death after 14 days of follow-up for those seeking outpatient care (FLU 002) or after 60 days for those hospitalized with influenza complications (FLU 003). Biomarkers that were significantly associated with progression in both studies (p<0.05) or only in one (p<0.002 after Bonferroni correction) were identified. Results In FLU 002 28/528 (5.3%) outpatients had influenza A(H1N1)pdm09 virus infection that progressed to a study endpoint of complications, hospitalization or death, whereas in FLU 003 28/170 (16.5%) inpatients enrolled from the general ward and 21/39 (53.8%) inpatients enrolled directly from the ICU experienced disease progression. Higher levels of 12 of the 25 markers were significantly associated with subsequent disease progression. Of these, 7 markers (IL-6, CD163, IL-10, LBP, IL-2, MCP-1, and IP-10), all with ORs for the 3rd versus 1st tertile of 2.5 or greater, were significant (p<0.05) in both outpatients and inpatients. In contrast, five markers (sICAM-1, IL-8, TNF-α, D-dimer, and sVCAM-1), all with ORs for the 3rd versus 1st tertile greater than 3.2, were significantly (p≤.002) associated with disease progression among hospitalized patients only. Conclusions In patients presenting with varying severities of influenza A(H1N1)pdm09 virus infection, a baseline elevation in several biomarkers associated with inflammation, coagulation, or immune function strongly predicted a higher risk of disease progression. It is conceivable that interventions designed to abrogate these baseline elevations might affect disease outcome.

The HIV-1-Specific Protein Casp8p41 Induces Death of Infected Cells through Bax/Bak

ABSTRACT Casp8p41, a novel protein generated when HIV-1 protease cleaves caspase 8, independently causes NF-κB activation, proinflammatory cytokine production, and cell death. Here we investigate the mechanism by which Casp8p41 induces cell death. Immunogold staining and electron microscopy demonstrate that Casp8p41 localizes to mitochondria of activated primary CD4 T cells, suggesting mitochondrial involvement. Therefore, we assessed the dependency of Casp8p41-induced death on Bax/Bak and caspase 9. In wild-type (WT) mouse embryonic fibroblast (MEF) cells, Casp8p41 causes rapid mitochondrial depolarization (P < 0.001), yet Casp8p41 expression in Bax/Bak double-knockout (DKO) MEF cells does not. Similarly, caspase 9-deficient T cells (JMR cells), which express Casp8p41, undergo minimal cell death, whereas reconstituting these cells with caspase 9 (F9 cells) restores Casp8p41 cytotoxicity (P < 0.01). The infection of caspase 9-deficient cells with a green fluorescent protein (GFP) HIV-1 reporter virus results in cell death in 32% of infected GFP-positive cells, while the restoration of caspase 9 expression in these cells restores infected-cell killing to 68% (P < 0.05), with similar levels of viral replication between infections. Our data demonstrate that Casp8p41 requires Bax/Bak to induce mitochondrial depolarization, which leads to caspase 9 activation following either Casp8p41 expression or HIV-1 infection. This understanding allows the design of strategies to interrupt this form of death of HIV-1-infected cells.

Stability in the cumulative incidence, severity and mortality of 101 cases of invasive mucormycosis in high‐risk patients from 1995 to 2011: a comparison of eras immediately before and after the availability of voriconazole and echinocandin‐amphotericin combination therapies

As invasive mucormycosis (IM) numbers rise, clinicians suspect prior voriconazole worsens IM incidence and severity, and believe combination anti‐fungal therapy improves IM survival. To compare the cumulative incidence (CI), severity and mortality of IM in eras immediately before and after the commercial availability of voriconazole all IM cases from 1995 to 2011 were analysed across four risk‐groups (hematologic/oncologic malignancy (H/O), stem cell transplantation (SCT), solid organ transplantation (SOT) and other), and two eras, E1 (1995–2003) and E2, (2004–2011). Of 101 IM cases, (79 proven, 22 probable): 30 were in E1 (3.3/year) and 71 in E2 (8.9/year). Between eras, the proportion with H/O or SCT rose from 47% to 73%, while ‘other’ dropped from 33% to 11% (P = 0.036). Between eras, the CI of IM did not significantly increase in SCT (P = 0.27) or SOT (P = 0.30), and patterns of anatomic location (P = 0.122) and surgical debridement (P = 0.200) were similar. Significantly more patients received amphotericin‐echinocandin combination therapy in E2 (31% vs. 5%, P = 0.01); however, 90‐day survival did not improve (54% vs. 59%, P = 0.67). Since 2003, the rise of IM reflects increasing numbers at risk, not prior use of voriconazole. Frequent combination of anti‐fungal therapy has not improved survival.

Anti-apoptotic mechanisms of HIV: lessons and novel approaches to curing HIV

Past efforts at curing infection with the human immunodeficiency virus (HIV) have been blocked by the resistance of some infected cells to viral cytopathic effects and the associated development of a latent viral reservoir. Furthermore, current efforts to clear the viral reservoir by means of reactivating latent virus are hampered by the lack of cell death in the newly productively infected cells. The purpose of this review is to describe the many anti-apoptotic mechanisms of HIV, as well as the current limitations in the field. Only by understanding how infected cells avoid HIV-induced cell death can an effective strategy to kill infected cells be developed.

Choice of antiretroviral therapy differentially impacts survival of HIV-infected CD4 T cells

BackgroundHIV eradication strategies are now being evaluated in vitro and in vivo. A cornerstone of such approaches is maximal suppression of viral replication with combination antiretroviral therapy (ART). Since many antiretroviral agents have off target effects, and different classes target different components of the viral life cycle, we questioned whether different classes of ART might differentially affect the survival and persistence of productively HIV-infected CD4 T cells.MethodsIn vitro infections of primary CD4 T cells using clinical isolates of HIV-1 that were either protease inhibitor susceptible (HIV PI-S), or resistant (HIV PI-R) were treated with nothing, lopinavir, efavirenz or raltegravir. Cell viability, apoptosis, and the proportion of surviving cells that were P24 positive was assessed by flow cytometry.ResultsIn HIV PI-S infected primary cultures, all three antiretroviral agents decreased viral replication, and reduced the total number of cells that were undergoing apoptosis (P < 0.01) similarly. Similarly, in the HIV PI-R infected cultures, both efavirenz and raltegravir reduced viral replication and reduced apoptosis compared to untreated control (P < 0.01), while lopinavir did not, suggesting that HIV replication drives T cell apoptosis, which was confirmed by association by linear regression (P < 0.0001) . However since HIV protease has been suggested to directly induce apoptosis of infected CD4 T cells, and HIV PI are intrinsically antiapoptotic, we evaluated apoptosis in productively infected (HIV P24+) cells. More HIV p24 positive cells were apoptotic in the Efavirenz or raltegravir treated cultures than the lopinavir treated cultures (P = 0.0008 for HIV PI-R and P = 0.06 for the HIV PI-S), indicating that drug class impacts survival of productively infected CD4 T cells.ConclusionsInhibiting HIV replication with a PI, NNRTI or INSTI reduces total HIV-induced T cell apoptosis. However, blocking HIV replication with PI but not with NNRTI or INSTI promotes survival of productively HIV-infected cells. Thus, selection of antiretroviral agents may impact the success of HIV eradication strategies.

Casp8p41 generated by HIV protease kills CD4 T cells through direct Bak activation

HIV protease converts procaspase 8 into Casp8p41, which binds and activates Bak to induce cell death in infected CD4 T cells.