A.Mosaics of spikes on HIV-1: Analysis of individual Envs on individual HIV-1 visions. The HIV envelope plays a critical role in HIV infection. Functional HIV envelope proteins (Envs) or spikes are trimers consisting of three homodimers of gp120 and trans-membrane gp41 subunits linked to each other via non-covalent bonds.The correct conformation of Env is critical for the virus to bind to cell receptors (CD4) and coreceptors (CCR5/CXCR4) and to undergo the complex conformational change required for plasma membrane fusion and viral entry. Malfunctioning of this machinery renders virions incapable of binding/fusion with cells. This malfunctioning may be mediated by incorporation of dysfunctional forms of Env into the virion membrane or by the instability of incorporated functional Env. Indeed a significant proportion of virions within any viral population are thought to be non-infectious. Several ways by which the HIV Env can be dysfunctional include uncleaved Env , dimeric or monomeric Env and gp41 stumps. Each HIV-1 virion carries 10 -14 spikes and in principle, it is possible that on a given virion all the spikes are either defective or all are functional, rendering the former virion defective and the latter virion infectious. Alternatively, virions may carry both functional and non-functional Envs in different conformations, as it is currently thought. To answer this question it is necessary to analyze the conformation of individual Env proteins on individual virions. To date, however, the majority of the biochemical and immunochemical analysis of HIV surface proteins is performed in bulk so that the diversity of individual virions is lost. Recently we developed a nanoparticle-based technique, flow virometry, that permits the analysis of antigenicity of individual viral particles. Here, we applied flow virometry to investigate the functional and non-functional conformations of Envs on individual virions using a panel of antibodies that discriminate between various gp120 conformations. We found that viruses exhibit little mosaicism: on the majority of virions either all the spikes are functional or all are defective. This all-or-nothing viral strategy is likely to aid immune evasion by subverting the focus of humoral responses to generate multiple non-neutralizing antibodies at no cost to infectious visions. Therefore only the induction of antibodies that target functional Envs and thus target predominantly infectious viruses seem to be critical for the development of effective prophylactic strategies. B. Maturity of individual Dengue virions Maturation of Dengue virus (DENV) is determined by the cleavage of prM proteins into M. Although based on bulk analysis it is possible to evaluate the presence of prM on DENV virions and thus to determine the average degree of maturation of a viral preparation, only the analysis of individual viral particles would allow us to discriminate whether all virions are mosaic and carry both prM and M or there is a fraction of fully mature viruses, that do not carry prM. In our paper using flow virometry we performed such an analysis. We captured DENV with magnetic nanoparticles coupled to antibodies against E protein, (present on all DENV viral envelopes), and stained virions with fluorescent antibodies against prM protein, present on the surface of immature/partially mature virions . To validate our technology, we compared maturation of DENV produced, BHK-21 and LoVo cells. The latter are deficient of furin, that is required for prM cleavage. We found that in DENV produced in BHK-21 cells prM is present on about 50% of DENV particles, while in DENV produced in LoVo cells about 85% of DENV virions carry uncleaved prM on their surface, indicating their not fully mature state. C. Cytokines as mediators of immunoactivation by human pathogens. The change of cytokine network as the response to the pathogen invasion is common for various infections. Since the cytokines are incorporated in the complex and redundant network, monitoring of individual cytosine changes is not sufficient but rather the global analysis of the network pattern changes is necessary. Earlier we performed such an analysis for HIV infection. Here we extend and further develop such an analysis to the intra-amniotic infection/inflammation that may lead to spontaneous preterm labor/delivery. The goal of this study was to characterize the cytokine network in the amniotic fluid in patients with preterm labor. We found that patients with preterm labor and intact membranes who had microbial-associated intra-amniotic inflammation had a higher amniotic fluid cytokine concentration correlation than those without intra-amniotic inflammation. and patients with sterile intra-amniotic inflammation had different correlation patterns of inflammatory-related proteins when compared to those without intra-amniotic inflammation. Also, there were more coordinated inflammatory-related protein concentrations in the amniotic fluid of women with microbial-associated intra-amniotic inflammation than in those with sterile intra-amniotic inflammation. IL-4 and IL-33 had the largest number of perturbed correlations with other inflammatory-related proteins in the differential correlation network. In general, our observations show that the cytokine network connectivity in women with microbial-associated intra-amniotic inflammation is denser and coordinated than in those with sterile inflammation or without intra-amniotic inflammation.Our network analysis provides deeper insight into understanding the pathophysiological mechanisms of intra-amniotic infection/inflammation in preterm labor, as well as identifying potentially relevant modules of cytokines that correspond to distinct disease pathways in preterm labor. Also, such an approach may help to minimize the number of individual cytokines measured in order to characterize pathologic states, since some elements of the network may have key roles in the regulation of the entire network/modules. A similar network analysis can be now applied to other pathogens. In particular we studied thcytokine perturbation in cohorts of HIV-infected individuals receiving antiretroviral therapy we found that a particular cytokine pattern associated with confection with the cytomegalovirus may predispose these individuals to syphilis acquisition. D. Immunoactivation as a common pattern for various human diseases. We undertook a broad analysis of the biomedical literature and reviewed our own earlier results to determine the possible role of immunoactivation in human disease. This analysis led us to conclude that immunoactivation, which evolved as a system of host defense against pathogens, can become dysregulated and promote the pathogenesis of diverse diseases with both known and unknown etiologies (e.g., AIDS, age-related macular degeneration, cancer) as well as aging. Immunoactivation seems to be a common denominator or general mechanism of pathogenesis, and may explain the association and similarities in pathology among otherwise unrelated human diseases. Thus, immunoactivation, which evolved as a system of host defense against pathogens, can become dysregulated and promote the pathogenesis of diverse diseases with both known and unknown etiologies and even be an important factor in general aging.Identification of general mechanisms of immunoactivation may lead to the development of new therapeutic strategies applicable to many diseases even before detailed knowledge of their specific etiology and pathogenesis may be available
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