A. Human immunodeficiency Virus Imposes Rigidity on Cytokine Network in Semen of Chronically HIV-1 Infected Men HIV-1 infection leads to acquired immunodeficiency syndrome (AIDS) and is associated with changes in the levels of soluble immunoregulators, cytokines. The disruption of the cytokine network is believed to contribute to the immunodeficiency. In the previous cycle of our studies, we found that in semen, upon HIV infection, the levels of individual cytokines are drastically altered. However, the interrelations of cytokines that organize them into a network have been studied only fragmentally. Indeed, although important, the changes in the levels of individual cytokines give no information as to the global pattern of their concerted interactions. To evaluate the latter we determined whether cytokines change in a coordinated way by measuring the correlations between the production of each cytokine and that of each of the other cytokines. We investigated the organization of cytokine networks in semen and blood and their alterations by HIV-1 infection. Specifically, semen was collected at the All-India Institute of Medical Sciences (India) from forty seven therapy-nave HIV-1-infected individuals and HIV-1-uninfected controls patients. We measured the levels of 21 cytokines using a multiplex bead array assay and assessed the linear association of continuous variables using the non-parametric Spearmans rank correlation coefficient. We found that in HIV-1-infected individuals the cytokine networks were more interlocked than in uninfected controls. Indeed, in blood and semen of HIV-1 infected individuals there were, respectively, 68 and 72 statistically significant correlations between cytokines, while in uninfected individuals there were 18 and 21 such correlations. HIV infection resulted in the establishment of new correlations and in the strengthening of pre-existing correlations between different cytokines. In summary, we found that HIV-1 infection, had a counter intuitive effect on cytokines: rather than creating disarray in cytokine network, HIV-1 imposes rigidity on this network, thus decreasing its degree of freedom. The high rigidity imposed by HIV-1 infection on the cytokine network may contribute to the inability of the immune system to adapt to new microbial challenges, leading to the eventual failure of a fixed and ordered immune system. Therefore, the rigidity triggered by HIV-1 in this network may be one of important factors leading to immunodeficiency. B. Potentiation of anti-HIV-1 Activity of an Antiherpetic Drug Acyclovir: Suppression of Primary and Drug-resistant HIV Isolates Several clinical studies have demonstrated that ACV treatment of HIV-1 infected patients co-infected with herpes simplex virus 2 (HSV-2) delays progression to AIDS and suppresses not only HSV-2, but also HIV-1. We found that ACV, which is metabolized into ACV-triphosphate (ACV-TP) in herpesviruses-infected cells, is incorporated as ACV-monophosphate into the nascent DNA chain instead of dGMP resulting in the termination of viral DNA elongation and thus directly inhibits the retrotranscription of HIV-1 laboratory strains. Here, we investigated the anti-HIV-1 activity of ACV not only against laboratory HIV-1 strains, but also against primary HIV-1 clinical isolates of different subtypes and against multidrug-resistant variants. We hypothesize that by increasing the intracellular ACV-TP/dGTP ratio we will increase the probability of incorporation of ACV-TP rather than dGTP by RT, thus enhancing the anti-HIV activity of ACV. We tested this hypothesis by using ribavirin that inhibits a key enzyme in the de novo biosynthesis of GTP thus depleting the pool of dGTP. We first tested the susceptibility of four clinical HIV-1 isolates to concentrations of ACV similar to those achieved in patients treated with oral ACV. In these ex vivo experiments ACV suppressed the replication of all tested HIV-1 primary isolates, including two of clade C, one of clade A, and one of clade B. Also, the inhibition was irrespective of HIV-1 co-receptor usage. In general, there was no difference in the suppression of viral replication in ex vivo tonsillar tissues between clinical HIV-1 isolates and laboratory strains, although one of the two clade C isolates was not as susceptible to low ACV concentration as the laboratory isolate LAI.04. For the future use of ACV or its derivatives in anti-HIV-1 therapy, it is important that ACV remains active against common drug-resistant HIV-1 variants that evolve in the course of regular anti-HIV therapy. We found that ACV efficiently suppressed replication of viruses, resistant to the common drugs, zidovudine (AZT) and lamivudine (3TC). Also, we tested a panel of 6 prototypical infectious multidrug resistant HIV-1 RT molecular clones. Each of the clones carries several mutations that occur most frequently in HIV-infected individuals treated with NRTIs. ACV suppressed equally well the replication of the six multi-drug resistant clones and the laboratory strain HIV-1 LAI.04. However HIV-1 variant that carries K65R RT mutation showed a lesser susceptibility to ACV inhibition. We found that ribavirin significantly potentiated the anti-HIV-1 activity of ACV in human tissues ex vivo and in single cell cultures. In summary, we found that in human lymphoid tissue, ACV suppresses the replication of HIV-1 of different clades, of different coreceptor tropisms as well as HIV-1 variants with mutations conferring resistance to the anti-virals currently used in HIV-1 treatment. The combination of ACV with ribavirin may be envisioned as an addition to commonly used anti-viral cocktails. C. Development of a Universal Nanoparticle Cell Secretion Capture Assay Cytokines play an important role in the coordination of the immune response to various pathogens. The identification and the full characterization of cells, that secrete these cytokines is a paramount to the understanding of the basic mechanisms of immunity. Currently, immunohistochemistry and flow cytometry are the two main techniques allowing the identification of individual cells secreting a particular protein in general and cytokines in particular. However, because both techniques identify secretory proteins inside the cell, they do not distinguish between cells that actually secrete these proteins from cells that rather store them. Also, current methods compromises cell viability. We developed a novel, easy, and versatile method, which allows the identification of living cells actually secreting any protein of interest. Our method is based on the targeting, to the cell surface, of nanoparticles, which capture the secreted protein on the surface of the secreting cell. This method allows further characterization of a secreting cell by flow cytometry and does not compromise cell viability. Specifically, we prepared complexes of goat anti-mouse antibodies bound to magnetic nanoparticles with anti-CD45 as a cell-anchoring moiety. Mouse anti-human antibodies against particular cytokines bound to these complexes to capture cytokines on the cell surface. Using our assay we were able to identify cells secreting IL-2, IFN-gamma, as well as MIP-1alpha, MIP-1beta, and RANTES. The versatility of the technique allows the identification of virtually any cells based on their secreted proteins.
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