Soluble CD40 ligand contributes to dendritic cell-mediated T-cell dysfunction in HIV-1 infection.

OBJECTIVE: Plasma soluble CD40 ligand (sCD40L) is increased during HIV-1 infection, but it is unknown whether it circulates in monomeric or multimeric forms, and whether the circulating forms have differential effects on myeloid dendritic cell function and adaptive regulation. DESIGN: sCD40L forms were measured in plasma samples from HIV-infected donors. The effects of sCD40L forms on dendritic cell function were measured in vitro. METHODS: To delineate which forms of sCD40L are present in plasma from HIV-infected donors, immunoblots were performed following enrichment of plasma for medium and low-abundance proteins. Dendritic cells from seronegative donors were exposed to multiple forms of sCD40L prior to Toll-like receptor stimulation and dendritic cell function and adaptive regulation was assessed in vitro. RESULTS: Monomeric and multimeric forms of sCD40L were identified in plasma from antiretroviral therapy-treated HIV-infected donors. Although monomeric and multimeric forms of sCD40L had differential effects on dendritic cell activation when given alone, both strongly suppressed secretion of the Th1 skewing cytokine, interleukin-12, upon subsequent Toll-like receptor stimulation. Furthermore, dendritic cells exposed to both monomeric and multimeric sCD40L induced regulatory T-cell formation and T-cell anergy. CONCLUSION: Elevated sCD40L during HIV infection impairs dendritic cell function, contributing to innate and adaptive immune dysfunction. Antiretroviral adjunctive therapies that decrease sCD40L may provide immune modulatory benefits.

Reproducibility over time and effect of low-dose aspirin on soluble P-selectin and soluble CD40 ligand.

Platelet markers [soluble CD40 ligand (sCD40L) and soluble p selectin (sPselectin)] are associated with platelet activation and cardiovascular events. We sought to investigate the reproducibility of these markers over time and the effect of low-dose aspirin on sCD40L and sPselectin in plasma and serum. Following an overnight fast, 40 healthy volunteers had weekly phlebotomy and were administered aspirin 81 mg/day between weeks 3 and 4. Reproducibility over time was assessed by coefficient of variation (CV) and inter-class correlation coefficient. Correlation between markers was assessed using Pearson r statistic. Difference between levels pre- and post-aspirin was measured with Wilcoxon signed-rank test. Data are presented as median (interquartile range). sCD40L and sPselectin measurements were reproducible over time in plasma and serum (CV < 10 %). Measurement of sCD40L and sPselectin in plasma correlated with levels in serum before aspirin and after aspirin. There was no significant correlation between sCD40L and sPselectin. After 1-week of aspirin 81 mg/day, there was a reduction in sCD40L and sPselectin in serum and plasma, respectively. Soluble CD40L and sPselectin are independent markers that are reproducible over time in both plasma and sera and are reduced by 1-week of low-dose aspirin.

Mean platelet volume reproducibility and association with platelet activity and anti-platelet therapy.

Some studies suggest that mean platelet volume (MPV) correlates with increased risk for cardiovascular morbidity and mortality. In this study, we aim to assess reproducibility, need for standardized measurements, effect of aspirin, and association with other established markers of platelet activity. Following an overnight fast, 48 healthy volunteers had weekly assessment of platelet activity and were administered aspirin 81 mg daily for 7 d between weeks 3 and 4. We investigated the influence of time between phlebotomy and MPV measurement (n=10). Reproducibility was assessed by coefficient of variation (CV) and intraclass correlation coefficient (ICC). MPV measurements were reproducible (Week 1: 10.6 fL [9.9-11], Week 2: 10.6 fL [10.0-10.9], Week 3: 10.6 fL [9.8-11]). CV was ≤ 4% and ICC>0.85 (p<0.001) for each comparison, indicating excellent reproducibility. There was no effect of aspirin on MPV (10.6 fL [9.8-11] versus 10.5 fL [9.9-11]; p=0.81). MPV significantly increased as time between phlebotomy and MPV measurement increased (Spearman's rho=0.94, p=0.001). Increasing MPV tertiles was associated with collagen- and thrombin receptor-activated peptide-induced platelet aggregation but not with ADP- or arachidonic acid-induced or spontaneous platelet aggregation. In conclusion, when standardized, MPV is a reproducible marker of platelet size and not affected by low-dose aspirin. MPV is modestly associated with some, but not all, markers of platelet activity.

Aspirin attenuates platelet activation and immune activation in HIV-1-infected subjects on antiretroviral therapy: a pilot study.

BACKGROUND: Mechanisms for increased cardiovascular risk in HIV-1-infected adults are incompletely understood, but platelet activation and immune activation leading to a prothrombotic state have been proposed as significant contributors. Aspirin has antiplatelet and immunomodulatory properties. We explored whether 1 week of low-dose aspirin attenuates platelet activation and immune activation in HIV-1-infected and virologically suppressed adults on antiretroviral therapy. METHODS: Platelet activation and immune activation were measured in HIV-1-infected subjects virologically suppressed on antiretroviral therapy and controls before and after 1 week of low-dose aspirin. RESULTS: Compared with control subjects, HIV-1-infected subjects had increased platelet activation, as measured by spontaneous platelet aggregation and aggregation in response to adenosine diphosphate, collagen, and arachidonic acid. After aspirin therapy, percent aggregation decreased similarly in both HIV-1-infected and control subjects to all platelet agonists tested except aggregation in response to arachidonic acid, which remained elevated in the HIV-1-infected group. HIV-1-infected subjects exhibited increased markers of T-cell activation (CD38 and HLA-DR) and monocyte activation (sCD14), which decreased after 1 week of aspirin therapy. Moreover, leukocyte responses to Toll-like receptor stimulation were enhanced after 1 week of aspirin therapy. In vitro studies showed that HIV-1 plasma could activate healthy platelets, which in turn activated monocytes, implicating a direct role for activated platelets in immune activation. CONCLUSIONS: Our data demonstrate that heightened platelet activation and immune activation in treated HIV-1 disease are attenuated by 1 week of aspirin therapy. Aspirin should be further studied for its antithrombotic and immunomodulatory benefits in treated HIV-1 disease.

The Na-K-Cl cotransporter in the brain edema of acute liver failure.

BACKGROUND & AIMS: Astrocyte swelling and brain edema associated with increased intracranial pressure are major complications of acute liver failure (ALF). The mechanism for such astrocyte swelling/brain edema, however, is not well understood. We recently found that ammonia, a key etiological factor in ALF, caused the activation of the Na-K-Cl cotransporter-1 (NKCC1) in cultured astrocytes, and that inhibition of such activation led to a reduction in astrocyte swelling, suggesting that NKCC1 activation may be an important factor in the mechanism of brain edema in ALF. To determine whether NKCC activation is also involved in brain edema in vivo, we examined whether NKCC activation occurs in the thioacetamide (TAA) rat model of ALF and determined whether treatment with the NKCC inhibitor bumetanide reduces the severity of brain edema in TAA-treated rats. METHODS: Brain water content was measured using the gravimetric method. NKCC1 phosphorylation and protein expression were measured by Western blots. NKCC activity was measured in brain cortical slices. RESULTS: NKCC activity was elevated in brain cortical slices of TAA-treated rats as compared to sham animals. Western blot analysis showed significant increases in total as well as phosphorylated (activated) NKCC1 protein expression in the cortical tissue. These findings were associated with a significant increase in brain water content which was attenuated by treatment with the NKCC inhibitor bumetanide. CONCLUSIONS: Our studies suggest the involvement of NKCC in the development of brain edema in experimental ALF, and that targeting NKCC may represent a useful therapeutic strategy in humans with ALF.