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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 (, Bill & Melinda Gates Foundation (OPP1066973), Bill & Melinda Gates Foundation (OPP1032817), National Institutes of Health (U.S.) (R01 AI080289), National Institutes of Health (U.S.) (R01 A102660-01), National Institutes of Health (U.S.) (R37 AI080289-06A1), Ragon Institute of MGH, MIT and Harvard

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., Author Summary The female genital tract (FGT) is a key mucosal surface in the context of HIV transmission. Lactobacillus species are beneficial to the FGT, while Garderella vaginalis and other anaerobic bacteria are detrimental. Bacterial vaginosis (BV) is an inflammatory condition characterized by an outgrowth of G. vaginalis and other anaerobes, which is linked to increased HIV acquisition rates. However, the mechanism behind this remains unknown. Here, we used a novel proteomic approach to simultaneously evaluate host and bacterial functions in the FGT. We found that women with G. vaginalis-dominated FGT bacterial communities always displayed markers of decreased epithelial barrier integrity, and decreased wound healing capacity. We also demonstrated that the abundance of proteins from G. vaginalis associated with these signatures of disrupted epithelial integrity. Finally, we showed that products derived from G. vaginalis prevented healing of wounded cell monolayers while products derived from L. iners maintained the ability of the cell monolayers to close wounds. This study provides novel mechanistic insight into the link between BV and increased HIV acquisition rates.

Angiogenesis is crucial during many physiological processes, and is influenced by various biochemical and biomechanical factors. Two such factors: VEGF and Ang 1 are known to be critical and we demonstrate here their effect of sprout formation in an in vitro microfluidic system. Previously, we have developed a 3D hybrid, agent-field model where individual cells are modeled as sprout-forming agents in a matrix field. We have conducted microfluidic experiments under different concentrations of VEGF and Ang 1 and analyzed the difference in sprout number and sprout lengths using Decision Tree Analysis. We demonstrate that under specific transition probabilities, the model gives us capillary characteristics similar to those seen in experiments (R2 ~ 0.82–0.99). Thus, this model can be used to cluster sprout morphology as a function of various influencing factors and, within bounds, predict if a certain growth factor will affect migration or proliferation as it impacts sprout morphology

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. This journal is, National University of Singapore (R-397-000-137-133), National University of Singapore (Engineering-Medicine Seed Grant R-397-000-152-112), China. Ministry of Energy (Tier-1 R-397-000-153- 112), Singapore-MIT Alliance for Research and Technology (SMART) (Grant R-397-000-146-592)

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.

Angiogenesis is the formation of blood vessels from an existing vessel or a monolayer of endothelial cells. It is crucial during the processes of tissue and organ maintenance, wound healing, and development and also plays a crucial role in tumor growth. An understanding of this process is also critical from a tissue engineering perspective. Several biochemical factors such as VEGF, Angiopoietin I (Ang I), Angiopoietin II (Ang II), PLGF, PDGF, TNF α, TGF β, α-FGF, β-FGF, ENA/VASP [1] and biomechanical factors such as extracellular matrix (ECM) stiffness, interstitial flow, shear stress affect the formation of capillaries. While some of them are proangiogenic, others are anti-angiogenic. A balance in these factors is decisive to the characteristics of the capillaries formed.Copyright © 2009 by ASME

Introduction Untreated sexually transmitted infections (STIs) and bacterial vaginosis (BV) cause genital inflammation and increased risk of HIV infection. WHO-recommended syndromic STI and BV management is limited as large numbers of women with asymptomatic infections go untreated. The purpose of this study was to evaluate genital cytokine profiles as a biomarker to identify women with asymptomatic, treatable infections. Methods Luminex was used to measure the concentrations of 42 cytokines in cervicovaginal lavages (CVL) from 227 HIV-uninfected women from Durban, South Africa, and nine cytokines in endocervical swabs from 264 women from Bondo, Kenya and Pretoria, South Africa. Women were screened for BV and treatable STIs ( Chlamydia trachomatis , Neisseria gonorrhoeae , Trichomonas vaginalis , Mycoplasma genitalium ) using microscopy and molecular assays. Nonparametric receiver operating characteristic curves and logistic regression were used to identify cytokine profiles associated with STIs/BV. Results In women from Durban, concomitant increased IL-1α and IL-1β and decreased IP-10 concentrations in CVLs predicted the presence of a treatable genital condition, correctly classifying 76% of women (sensitivity 72%, specificity 81%, PPV 86% and NPV 64%). In a separate validation cohort of women from Bondo and Pretoria, IL-1α, IL-1β and IP-10 concentrations in endocervical swabs correctly classified 72% of the participants according to STI/BV status. This approach performed substantially better than clinical signs in both cohorts from Durban (sensitivity 19%, specificity 92%, PPV 79% and NPV 40%) and Pretoria and Bondo (sensitivity 29%, specificity 78%, PPV 68%, NPV 39%). Conclusion Across two cohorts of women residing in different regions in sub-Saharan Africa, genital IL-1α, IL-1β and IP-10 together was the best immunological predictor of the presence of an STI or BV. Supplementing syndromic management with point-of-care assessment of biomarkers of genital inflammation may improve STI/BV management for women, enabling more effective treatment of asymptomatic infections and potentially reducing their risk of HIV infection. Disclosure of interest statement This work was supported by a Strategic Health Innovation Partnerships (SHIP) grant from the South African Medical Research Council and grants from the Poliomyelitis Research Foundation (PRF) of South Africa and European and Developing Countries Clinical Trials Partnership (EDCTP). The cohorts were supported by grants from the Comprehensive International Program of Research on AIDS (CIPRA) of the Division of AIDS (DAIDS); National Institute of Allergy and infectious Disease (NIAID); National Institutes of Health (NIH) and US Department of Health and Human Services (DHHS) [grant number U19 AI51794]. FEM-PrEP was conducted under two grants funded by the United States Agency for International Development (USAID): the Contraceptive and Reproductive Health Technologies and Research Utilisation Program (GPO-A-00–05–00022–00), and the Preventive Technologies Agreement (GHO-A-00–09–00016–00). Early support was also provided by the Bill and Melinda Gates Foundation. Gilead Sciences, Inc. donated Truvada® and placebo. LM was supported by the PRF; South African Medical Research Council (MRC); the Carnegie Corporation; the National Research Foundation (NRF) of South Africa and the UCT Clinical Infectious Diseases Research Initiative/Wellcome Trust. No pharmaceutical grants were received in the development of this study.

Mena, an actin regulatory protein, is upregulated in human breast cancer and its expression is associated with increased metastatic progression. An invasion-specific isoform, MenaINV, is upregulated selectively in invasive tumor cells and increases invasion and metastasis by potentiating tumor cell responses to EGF. MenaINV causes dysregulation of the tyrosine phosphatase PTP1B, leading to increased phosphorylation of EGFR and other downstream targets. We investigated whether MenaINV could similarly promote invasion through dysregulation of signaling downstream of other receptor tyrosine kinases (RTKs). The behavior of MDA-MB-231 triple negative breast cancer cells stably expressing MenaINV or the other cannonical, broadly expressed Mena lacking the “INV” exon was measured after growth factor treatment in two migration assays: a 2D protrusion assay on matrigel and collagen coated coverslips and a 3D invasion assay into collagen gels. We found that expression of MenaINV sensitized responses to other EGFR ligands including HB-EGF and amphiregulin. In addition, MenaINV expression enhanced responses mediated by two other RTKs associated with tumor progression, Met and IGFR, both known PTP1B substrates. Cells expressing MenaINV respond to 20-fold lower concentrations HGF and IGF than control cells. However, there was no effect of MenaINV on responses to the ERBB3 ligand neuregulin and the chemokine SDF-1. Recently we reported that Mena can regulate cell motility in fibroblasts via its interaction with α5β1 integrin, which is known to co-traffic with EGFR and other RTKs to regulate invasion in tumor cells expressing mutant p53 or upon inhibition of αvβ3. We asked whether the direct interaction of Mena with α5β1 also contributes to invasion. Disrupting the interaction between the Mena isoforms and α5β1 in MDA-MB-231 cells affected how cells responded to EGF in a 3D collagen gel. In addition, fibroblasts that were null for Mena were unable to respond to a fibronectin gradient in a haptotaxis assay. Overall, these results suggest that the effects of MenaINV on invasion and metastasis could be mediated via different RTKs, as well as through direct interaction with integrins and the extracellular matrix. Experiments are currently underway to investigate how MenaINV regulates signalling downstream of these RTKs and integrins, to understand the molecular mechanism underlying the pro-metastatic effect of MenaINV. Citation Format: Madeleine J. Oudin, Shannon K. Hughes-Alford, Miles Miller, Sreeja B. Asokan, James E. Bear, Doug A. Lauffenburger, Frank B. Gertler. MenaINV dysregulation of receptor tyrosine kinase signaling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C41.