Virus-driven Inflammation Is Associated With the Development of bNAbs in Spontaneous Controllers of HIV.

Background: Understanding the mechanism(s) by which broadly neutralizing antibodies (bNAbs) emerge naturally following infection is crucial for the development of a protective vaccine against human immunodeficiency virus (HIV). Although previous studies have implicated high viremia and associated immune activation as potential drivers for the development of bNAbs, here we sought to unlink the effect of these 2 parameters by evaluating the key inflammatory predictors of bNAb development in HIV-infected individuals who spontaneously control HIV in the absence of antiretroviral therapy ("controllers"). Methods: The breadth of antibody-mediated neutralization against 11 tier 2 or 3 viruses was assessed in 163 clade B spontaneous controllers of HIV. Plasma levels of 17 cytokines were screened in the same set of subjects. The relationship of the inflammatory signature was assessed in the context of viral blips or viral RNA levels in peripheral blood or gastrointestinal biopsies from aviremic controllers (<50 copies RNA/mL) and in the context of viral sequence diversity analysis in the plasma of viremic controllers (<50-2000 copies RNA/mL). Results: A unique inflammatory profile, including high plasma levels of CXCL13, sCD40L, IP10, RANTES, and TNFα, was observed in HIV controllers who developed bNAbs. Interestingly, viral load and tissue viremia, but not intermittent viral blips, were associated with these cytokine profiles. However, viral diversity was not significantly associated with increased breadth in controllers. Conclusion: These results suggest that low antigenic diversity in the setting of a unique inflammatory profile associated with antigen persistence may be linked to the evolution of neutralizing antibody breadth.

Microbiome Composition and Function Drives Wound-Healing Impairment in the Female Genital Tract.

The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity.

Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array.

Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 µL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors.

Ligand/receptor signaling threshold (LIST) model accounts for gp130-mediated embryonic stem cell self-renewal responses to LIF and HIL-6.

We previously demonstrated that embryonic stem (ES) cell self-renewal required sustained signaling by leukemia inhibitory factor (LIF) in a concentration-dependent manner, allowing us to hypothesize that thresholds in ligand-receptor signaling modulate stem cell differentiation control. To test this hypothesis, we have experimentally and computationally compared the abilities of two gp130-signaling cytokines (LIF and Hyper-interleukin-6 [HIL-6]) to sustain ES cell self-renewal. Quantitative measurements of ES cell phenotypic markers (stage-specific embryonic antigen-1 and E-cadherin), functional assays (alkaline phosphatase activity and embryoid body formation efficiency), and transcription factor (Oct-4) expression over a range of LIF and HIL-6 concentrations demonstrated a superior ability of LIF to maintain ES cell pluripotentiality at higher concentrations (> or =500 pM). Additionally, we observed distinct qualitative differences in the ES cell self-renewal dose response profiles between the two cytokines. A computational model permitted calculation of the number of signaling complexes as a function of receptor expression, ligand concentration, and ligand/receptor-binding properties, generating predictions for the degree of self-renewal as a function of cytokine concentration by comparison of these calculated complex numbers to experimentally determined threshold cytokine concentrations. Model predictions, consistent with experimental data, indicated that differences in the potencies of these two cytokines were based primarily on differences in receptor-binding stoichiometries and properties. These results support a ligand/receptor signaling threshold model of ES cell fate modulation through appropriate types and levels of cytokine stimulation. Insights from these results may be more generally applicable to tissue-specific stem cells and could aid in the development of stem cell-based technologies.