Tissue Distribution Dynamics of Human NK Cells Inferred from Peripheral Blood Depletion Kinetics after Sphingosine-1-Phosphate Receptor Blockade.

Human natural killer (NK) cell subsets differentially distribute throughout the organism. While CD56(dim) and CD56(bright) NK cell subsets similarly reside in the bone marrow (BM), the CD56(dim) population predominantly accumulates in non-lymphoid tissues and the CD56(bright) counterpart in lymphoid tissue (LT). The dynamics with which these NK cell subsets redistribute to tissues remains unexplored. Here, we studied individuals newly exposed to fingolimod, a drug that efficiently blocks sphingosine-1-phosphate (S1P)-directed lymphocyte - including NK cell - egress from tissue to blood. During an observation period of 6h peripheral blood depletion of CD56(bright) NK cells was observed 3 h after first dose of fingolimod, with 40-50% depletion after 6 h, while a decrease of the numbers of CD56(dim) NK cells did not reach the level of statistical significance. In vitro, CD56(bright) and CD56(dim) NK cells responded comparably to the BM-homing chemokine CXCL12, while CD56(bright) NK cells migrated more efficiently in gradients of the LT-homing chemokines CCL19 and CCL21. In conjuncture with these in vitro studies, the indirectly observed subset-specific depletion kinetics from blood are compatible with preferential and more rapid redistribution of CD56(bright) NK cells from blood to peripheral tissue such as LT and possibly also the inflamed central nervous system. These data shed light on an unexplored level at which access of NK cells to LT, and thus, for example antigen-presenting cells, is regulated.

Targeting the gastrointestinal tract to develop novel therapies for HIV.

Despite the use of antiretroviral therapy (ART), which delays and/or prevents AIDS pathogenesis, human immunodeficiency virus (HIV)-infected individuals continue to face increased morbidities and mortality rates compared with uninfected individuals. Gastrointestinal (GI) mucosal dysfunction is a key feature of HIV infection, and is associated with mortality. In this study, we review current knowledge about mucosal dysfunction in HIV infection, and describe potential avenues for therapeutic targets to enhance mucosal function and decrease morbidities and mortalities in HIV-infected individuals.

Cataloguing of Potential HIV Susceptibility Factors during the Menstrual Cycle of Pig-Tailed Macaques by Using a Systems Biology Approach.

UNLABELLED: Our earlier studies with pig-tailed macaques demonstrated various simian-human immunodeficiency virus (SHIV) susceptibilities during the menstrual cycle, likely caused by cyclic variations in immune responses in the female genital tract. There is concern that high-dose, long-lasting, injectable progestin-based contraception could mimic the high-progesterone luteal phase and predispose women to human immunodeficiency type 1 (HIV-1) acquisition and transmission. In this study, we adopted a systems biology approach employing proteomics (tandem mass spectrometry), transcriptomics (RNA microarray hybridization), and other specific protein assays (enzyme-linked immunosorbent assays and multiplex chemokine and cytokine measurements) to characterize the effects of hormonal changes on the expression of innate factors and secreted proteins in the macaque vagina. Several antiviral factors and pathways (including acute-phase response signaling and complement system) were overexpressed in the follicular phase. Conversely, during the luteal phase there were factors overexpressed (including moesins, syndecans, and integrins, among others) that could play direct or indirect roles in enhancing HIV-1 infection. Thus, our study showed that specific pathways and proteins or genes might work in tandem to regulate innate immunity, thus fostering further investigation and future design of approaches to help counter HIV-1 acquisition in the female genital tract. IMPORTANCE: HIV infection in women is poorly understood. High levels of the hormone progesterone may make women more vulnerable to infection. This could be the case during the menstrual cycle, when using hormone-based birth control, or during pregnancy. The biological basis for increased HIV vulnerability is not known. We used an animal model with high risk for infection during periods of high progesterone. Genital secretions and tissues during the menstrual cycle were studied. Our goal was to identify biological factors upregulated at high progesterone levels, and we indeed show an upregulation of genes and proteins which enhance the ability of HIV to infect when progesterone is high. In contrast, during low-progesterone periods, we found more HIV inhibitory factors. This study contributes to our understanding of mechanisms that may regulate HIV infection in females under hormonal influences. Such knowledge is needed for the development of novel prevention strategies.

A distinct cytokine and chemokine profile at the genital mucosa is associated with HIV-1 protection among HIV-exposed seronegative commercial sex workers.

The predominance of HIV-1 sexual transmission requires a greater understanding of the interaction between HIV-1 and the mucosal immune system. The study of HIV-1-exposed seronegative (HESN) individuals serves as a model to identify the correlates of protection and to aid in microbicide development. A total of 22 cytokines/chemokines were analyzed at the systemic and mucosal compartments in 57 HESN, 51 HIV-1-negative, and 67 HIV-1-infected commercial sex workers from Nairobi, Kenya. HESN individuals had significantly lower expression of monokine induced by interferon-γ (MIG), interferon-γ-induced protein 10 (IP-10), and interleukin-1α (IL-1α) in their genital mucosa compared with controls. HESN cytokine expression also distinctly correlates with mucosal antiproteases, suggesting that HESN individuals have a unique pattern of mucosal chemokine/cytokine expression, which may result in reduced trafficking at the mucosa. These data support the immune quiescence model of protection, whereby lower T-cell activation/recruitment at the mucosal compartment reduces HIV-1 target cell numbers and is an important component of natural protection from HIV-1.

Comprehensive proteomic study identifies serpin and cystatin antiproteases as novel correlates of HIV-1 resistance in the cervicovaginal mucosa of female sex workers.

Not all individuals exposed to HIV-1 become infected, and evidence from HIV-1 highly exposed seronegative women (HIV-1-resistant) suggests that mucosal factors in the female genital tract, the first site of contact for the virus, are playing a role. To better understand factors mediating protection from HIV-1, we performed a large clinical study using the tools of systems biology to fully characterize the cervicovaginal mucosa proteome in HIV-1-resistant women. Cervicovaginal lavage fluid was collected from 293 HIV-1-resistant, uninfected, and infected sex workers and analyzed by 2D-LC LTQ-FT-MS. Of the more than 360 unique proteins identified, 41 were differentially abundant (>3-fold cutoff) in HIV-1-resistant women. The majority of over-abundant proteins were antiproteases (>40%), some with described anti-inflammatory and anti-HIV-1 activity. Quantification of specific anti-HIV-1 antiproteases Serpin A1, Serpin A3, and Cystatin B and an epithelial antiprotease A2ML1 found them to be significantly over-abundant in HIV-1-resistant women (p = 0.004; p = 0.046; p = 0.0003; and p = 0.04, respectively). Expression levels were not correlated to sexual practices or other epidemiological factors. Mucosal antiprotease levels correlated with pro-inflammatory cytokine concentration (p = <0.0001), but independently of pro-inflammatory cytokine levels in HIV-1-resistant women including TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, and IL-8. This comprehensive systems biology approach identifies mucosal serpins and cystatins as novel correlates of HIV-1-resistance. This represents the first study characterizing these factors in the female genital tract.

Intracellular ATP and total adenylate concentrations are critical predictors of reovirus productivity from Vero cells.

The productivity of reovirus type-3 Dearing was studied in cultures of Vero cells in serum-free media. Viral productivity was dependent upon the metabolic state of the cells rather than the phase of growth at which the cells were infected. Cells at different energy states were established by 24-h incubation in nutrient-depleted media. This resulted in variable intracellular nucleotide concentrations but high cellular viability was maintained. Of the nucleotides analyzed at the time of infection only the intracellular [ATP] and total adenylate nucleotides were positively correlated with viral productivity. The correlated data followed a sigmoidal plot with an equation defined by polynomial regression analysis. Apparent threshold values of 3.2 fmol/cell and 3.3 fmol/cell were established for ATP and total adenylate, respectively, at which the viral production was 50% the maximal value. Cultures with lower ATP and total adenylate levels at the time of infection resulted in as much as a 95% reduction in overall viral titer compared to the control. The adenylate energy charge (AEC) showed a negative correlation with viral production with an AEC value >0.97 resulting in low virus productivity. Intracellular ATP or total adenylate concentration at the point of infection may be used as a predictor of viral yield in bioprocesses designed for virus/vaccine production.

Application of a serum-free medium for the growth of Vero cells and the production of reovirus.

Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were propagated in Vero cells grown in stationary or agitated cultures in a serum-free medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 x 10(6) cells/mL were obtained from an inoculum of 2 x 10(5) cells/mL in 4 days in microcarrier cultures. The growth profile and cell yield was not significantly different from serum-supplemented cultures. The virus titer increased by 3-4 orders of magnitude over a culture period of 150 h. The maximum virus titer in stationary cultures reached >1 x 10(9) pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high viral yields in microcarrier cultures. Both the specific productivity and final viral yield was higher in M-VSFM than serum-supplemented cultures. The high viral productivity suggests that this is a suitable system for the production of reovirus as an oncolytic agent for human therapeutic use.