Increased platelet activation in HIV patients is not altered with reported cocaine use.

<p>Blood samples were collected from HIV-negative, healthy subjects (HIV-, N = 61), HIV-positive individuals without cocaine use (HIV+Coc-, N = 37), and HIV-positive individuals with reported cocaine use within one year (HIV+Coc+, N = 16), and were fixed and stained as described in the methods section. Platelet CD62P expression was significantly higher in HIV+Coc- and HIV+Coc+ samples as compared to HIV- subjects (* p = 0.038, ** p = 0.008, respectively). CD62P expression levels between HIV+Coc- and HIV+Coc+ samples were statistically similar (ns, p = 0.492). Data are represented as fold change in CD62P MFI as compared to HIV- subjects and are shown as mean ± SEM.</p

Platelet Activation in Human Immunodeficiency Virus Type-1 Patients Is Not Altered with Cocaine Abuse

<div><p>Recent work has indicated that platelets, which are anucleate blood cells, significantly contribute to inflammatory disorders. Importantly, platelets also likely contribute to various inflammatory secondary disorders that are increasingly associated with Human Immunodeficiency Virus Type-1 (HIV) infection including neurological impairments and cardiovascular complications. Indeed, HIV infection is often associated with increased levels of platelet activators. Additionally, cocaine, a drug commonly abused by HIV-infected individuals, leads to increased platelet activation in humans. Considering that orchestrated signaling mechanisms are essential for platelet activation, and that nuclear factor-kappa B (NF-κB) inhibitors can alter platelet function, the role of NF-κB signaling in platelet activation during HIV infection warrants further investigation. Here we tested the hypothesis that inhibitory kappa B kinase complex (IKK) activation would be central for platelet activation induced by HIV and cocaine. Whole blood from HIV-positive and HIV-negative individuals, with or without cocaine abuse was used to assess platelet activation via flow cytometry whereas IKK activation was analyzed by performing immunoblotting and <i>in vitro</i> kinase assays. We demonstrate that increased platelet activation in HIV patients, as measured by CD62P expression, is not altered with reported cocaine use. Furthermore, cocaine and HIV do not activate platelets in whole blood when treated <i>ex vivo</i>. Finally, HIV-induced platelet activation does not involve the NF-κB signaling intermediate, IKKβ. Platelet activation in HIV patients is not altered with cocaine abuse. These results support the notion that non-IKK targeting approaches will be better suited for the treatment of HIV-associated inflammatory disorders.</p></div

Cocaine and HIV do not activate platelets in whole blood when treated <i>ex vivo</i>.

<p>Whole blood from healthy subjects (3 to 4 donors) was left untreated (NT) or was treated with cocaine (A), infectious NL4-3 HIV (B), ADP or Tat (C) (D), for 30, 60, and 30 minutes respectively, or as indicated, following which CD62P expression was assessed as a marker of platelet activation via flow cytometry. Treatment with cocaine, HIV, and Tat did not result in increased platelet activation. In each experiment, as indicated, treatment with 1 μL dH₂O for 30 minutes was used as a vehicle (Veh) control. Data are represented as fold change in CD62P MFI as compared to NT samples and are shown as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.</p

Cocaine indirectly activates platelets <i>in vivo</i>.

<p>Wild-type C57BL/6 mice were injected intraperitoneally (IP) with either PBS (N = 12), 5mg/kg cocaine (N = 6), or 50mg/kg cocaine (N = 6) three times per week for three weeks. Following the final injection, blood was collected into tubes containing acid citrate dextrose, and platelet activation in whole blood was assessed via CD62P staining. Treatment with both concentrations of cocaine resulted in increased platelet activation as compared to the PBS treated mice (* p<0.05, ** p<0.01). Data are represented as fold change in CD62P MFI as compared to PBS treated samples and are shown as mean ± SEM.</p

Cocaine sensitizes platelets from HIV+, but not HIV-, donors to activation by ADP.

<p>Whole blood from healthy subjects (HIV-, N = 4) or HIV-positive subjects (HIV+, N = 4) was either left untreated (NT) or was treated with the indicated concentrations of cocaine alone or together with the indicated concentrations of ADP for 30 minutes. Platelet activation in whole blood was assessed via CD62P staining. In the HIV+ samples there was a significant increase in platelet activation following treatment with 10 μM ADP plus cocaine (*** p<0.001 compared to 10 μM ADP alone in the HIV+ samples, ^### p<0.001 compared to 10 μM ADP plus cocaine in the HIV- samples). Treatment with 1 μL DMSO for 5 minutes followed by 1 μL dH₂O for 30 minutes was used as a vehicle (Veh) control. Data are represented as fold change in CD62P MFI as compared to NT samples and are shown as mean ± SEM.</p

HIV-induced platelet activation does not involve NF-κB signaling mechanism.

<p>(A) Platelet lysates collected from HIV-negative subjects (HIV-, N = 2) and HIV-positive subjects (HIV+, N = 2) were subjected to electrophoretic mobility shift assays followed by supershift with anti-p50 and anti-RelA antibodies. Incubation with pre-immune serum was included as a control. Both p50 and RelA antibodies altered the mobility of bands, suggesting the presence of these two molecules in NF-κB/DNA complexes, however, no apparent differences were found between HIV- and HIV+ samples. (B) Platelet lysates collected from HIV-negative, healthy subjects (HIV-, N = 11), HIV-positive patients without cocaine use (HIV+Cocaine-, N = 10), and HIV-positive patients with reported cocaine use within one year (HIV+Cocaine+, N = 8) were subjected to immunoblot analysis with antibodies against IKKα, IKKβ, IκBα, or α-Tubulin. Expression levels of IKKα and IKKβ were noted to be similar between the groups. There was an increase in IκBα phosphorylation as evidenced by the appearance of a less mobile, upper band (*), which was confirmed by densitometric analysis of this upper phospho-IκBα band as shown in (C). (D) Platelet lysates collected from HIV- subjects (N = 15), HIV+Coc- (N = 10), and HIV+Coc+ (N = 5) were subjected to immunoprecipitation with an anti-IKKβ antibody followed by an IKKβ <i>in vitro</i> kinase assay. Incorporation of [³²P] into the recombinant IκBα substrate was measured by densitometric analysis of autoradiograms. No significant differences in IKKβ activity between the sample groups were noted. Data are represented as fold change in IKKβ activity compared to the HIV- samples and are shown as mean ± SEM.</p

Demographic and clinical characteristics of study participants.

<p>HIV-negative, healthy controls (HIV-, N = 61), HIV-positive individuals without cocaine use (HIV+Coc-, N = 37), and HIV-positive individuals with reported cocaine use within the previous one year (HIV+Coc+, N = 16) were enrolled in the study. All HIV-positive individuals were on antiretroviral therapy at the time of the blood draw. Unless otherwise stated, <i>N</i> indicates sample number with percentage of total sample group in parentheses. NA indicates not applicable.</p><p>Demographic and clinical characteristics of study participants.</p

Pre-existing neutralizing antibody mitigates B cell dysregulation and enhances the Env-specific antibody response in SHIV-infected rhesus macaques

<div><p>Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig) purified from previously infected animals (SHIVIG) or control Ig prior to intra-rectal challenge with SHIV_SF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased <i>de novo</i> Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2–3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host’s ability to eliminate HIV.</p></div

High-resolution longitudinal B cell phenotypic profile.

<p>Heatmap shows data corresponding to the change (log2) from baseline value for each subset frequency at each time point. Paired two tailed t-test for all post-baseline time points vs. baseline was computed independently for NIgG and SHIVIG groups; populations shown had at least one significant time point p<0.01 compared to baseline. Subsets were clustered hierarchically based on Euclidean distance and complete linkage. Magenta subset label indicates a significant difference of at least one time point for the log2 change from baseline between NIgG and SHIVIG groups at p<0.01 as determined by two-tailed unpaired t-test. [X] indicates subset is measured as the frequency of X parent population, otherwise parent population is total CD19+CD20+ B cells. Rectangle outline is used to emphasize select subsets.</p

SHIVIG treatment enhances plasma HIV-1 SF162 envelope binding antibody response.

<p>(<b>A</b>) Binding antibody (BAb) titer was determined by IgG ELISA against HIV-1 SF162 gp140 envelope protein. (<b>B</b>) Neutralization antibody (NAb) titer against HIV_SF162 pseudovirus was determined by TZM-bl neutralization assay. Symbols represent group mean±SEM. * indicates significant difference (p<0.05) between groups at indicated time point as determined by two-tailed t-test.</p