Dendritic cells from the human female reproductive tract rapidly capture and respond to HIV.

Dendritic cells (DCs) throughout the female reproductive tract (FRT) were examined for phenotype, HIV capture ability and innate anti-HIV responses. Two main CD11c+ DC subsets were identified: CD11b+ and CD11blow DCs. CD11b+CD14+ DCs were the most abundant throughout the tract. A majority of CD11c+CD14+ cells corresponded to CD1c+ myeloid DCs, whereas the rest lacked CD1c and CD163 expression (macrophage marker) and may represent monocyte-derived cells. In addition, we identified CD103+ DCs, located exclusively in the endometrium, whereas DC-SIGN+ DCs were broadly distributed throughout the FRT. Following exposure to GFP-labeled HIV particles, CD14+ DC-SIGN+ as well as CD14+ DC-SIGN- cells captured virus, with ∼30% of these cells representing CD1c+ myeloid DCs. CD103+ DCs lacked HIV capture ability. Exposure of FRT DCs to HIV induced secretion of CCL2, CCR5 ligands, interleukin (IL)-8, elafin, and secretory leukocyte peptidase inhibitor (SLPI) within 3 h of exposure, whereas classical pro-inflammatory molecules did not change and interferon-α2 and IL-10 were undetectable. Furthermore, elafin and SLPI upregulation, but not CCL5, were suppressed by estradiol pre-treatment. Our results suggest that specific DC subsets in the FRT have the potential for capture and dissemination of HIV, exert antiviral responses and likely contribute to the recruitment of HIV-target cells through the secretion of innate immune molecules.

Fc receptor-mediated phagocytosis in tissues as a potent mechanism for preventive and therapeutic HIV vaccine strategies.

Although the development of a fully protective HIV vaccine is the ultimate goal of HIV research, to date only one HIV vaccine trial, the RV144, has successfully induced a weakly protective response. The 31% protection from infection achieved in the RV144 trial was linked to the induction of nonneutralizing antibodies, able to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), suggestive of an important role of Fc-mediated functions in protection. Similarly, Fc-mediated antiviral activity was recently shown to play a critical role in actively suppressing the viral reservoir, but the Fc effector mechanisms within tissues that provide protection from or after infection are largely unknown. Here we aimed to define the landscape of effector cells and Fc receptors present within vulnerable tissues. We found negligible Fc receptor-expressing natural killer cells in the female reproductive and gastrointestinal mucosa. Conversely, Fc receptor-expressing macrophages were highly enriched in most tissues, but neutrophils mediated superior antibody-mediated phagocytosis. Modifications in Fc domain of VRC01 antibody increased phagocytic responses in both phagocytes. These data suggest that non-ADCC-mediated mechanisms, such as phagocytosis and neutrophil activation, are more likely to play a role in preventative vaccine or reservoir-eliminating therapeutic approaches.

The quantitative assay of 3-hydroxy-3-methylglutaryl coenzyme A reductase: comparison of a thin-layer chromatographic assay with a rapid chloroform extraction assay.

Two assays for the quantitative measurement of HMG-CoA reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis, are described. The first of these procedures employs thin-layer chromatography (TLC) of the reaction product on plastic-backed silica gel G strips impregnated with ammonium carbonate. The TLC strip is then cut into 14 segments and each segment is assayed for radioactivity. Extraction efficiency and exact chromatographic mobility are monitored by the use of authentic [4-(3)H]mevalonolactone as an internal reference. A wide and complete separation is achieved between HMG and mevalonolactone. Also the radiochemical purity of biosynthetic [3-(14)C]mevalonolactone can be assessed by measurement of the (3)H/(14)C ratio across the mevalonolactone peak. While the TLC assay is accurate and sensitive, it is laborious. Therefore a second assay procedure was developed using chloroform extraction of an incubation mixture saturated with solid equal molar KH(2)PO(4)-K(2)HPO(4) at pH 6.8. Extraction efficiency was monitored by the addition of authentic [4-(3)H]mevalonolactone as an internal reference. The chloroform assay procedure was compared with the TLC procedure over a wide range of enzyme activities, both for rat liver microsomal HMG-CoA reductase and for solubilized enzyme. Excellent correspondence (r = 0.999) between the two assays was observed. TLC performed on the chloroform extract demonstrated that biosynthetic [3-(14)C]mevalonolactone was the only (14)C-labeled compound present in the extract. The chloroform extraction assay is rapid; 20-30 samples can be processed in 2-3 hr. This procedure should facilitate studies concerning the nature and regulation of HMG-CoA reductase.