<p> <i>Candida albicans</i> is an opportunistic yeast pathogen and can cause life-threatening systemic candidiasis. The cell surface of <i>C. albicans</i> is enriched with mannan that is resistant to complement activation in the absence of antimannan antibodies. To better appreciate antimannan antibody functions in human immunity, our laboratory constructed the human recombinant antimannan Fc-free fragment M1 Fab. M1 Fab was subsequently converted to full-length human recombinant antimannan antibodies: M1g1 (IgG1), M1a1 (IgA1), M1a2 (IgA2). Each retains the identical M1 Fab binding region but differ in the isotype. Previously, our laboratory has established that M1 Fab can increase C3b deposition to <i>C. albicans </i> via the alternative pathway and that M1g1 activates the alternative and classical pathways of complement and increases phagocytosis of <i> C. albicans</i> by murine macrophages. The purpose of this study was to assess the influence of M1a1 and M1a2 on M1g1 mediated complement activation and phagocytosis of <i>C. albicans.</i> M1a1 or M1a2 was found unable to promote C3b-deposition to <i>C. albicans</i> as determined by flow cytometry and immunofluorescence microscopy. The formation of the alternative pathway convertase on <i>C. albicans</i> was promoted by M1 Fab but not by M1-Fab contained within M1a1 or M1a2. Additionally, M1g1 mediated C3b deposition was inhibited by M1a1 or M1a2 in a dose-dependent manner. Finally, M1a1 or M1a2 each significantly increased phagocytosis of <i> C. albicans</i> (<i>P</i> &lt;0.001) by human neutrophils independent of serum. The presence of M1a1 or M1a2 did not inhibit M1g1-mediated phagocytosis, indicating a redundant function of IgG1 and IgA antibodies in opsonophagocytosis. Thus, human antimannan IgA subclass variants hinder complement activation while increasing neutrophil phagocytosis of <i>C. albicans.</i> These results contribute to a more complete understanding of the role of serum IgA in host immunity.</p>

<p> HIV-infected cells can reach the central nervous system (CNS) and release HIV virions, which productively infect macrophages and microglia. Infected cells release both viral and host factors that can induce inflammation in bystander immune cells, to initiate neuropathogenesis beyond the initial site of the infected cell. While antiretroviral therapy can effectively control CNS viral loads, HIV-1 RNA and Tat protein can persist in cerebrospinal fluid of treated patients. Ongoing neurotoxic insults can result in HIV associated neurocognitive disorders (HAND). While the incidence of the severe form of HAND has declined with increasing treatment, milder forms of HAND have increased in prevalence. Delineating the viral determinants of cytotoxicity that can lead to HAND is critical to developing effective neuroprotective agents.</p><p> HIV-1 Tat protein contributes to HIV neuropathogenesis in multiple ways, including direct neurotoxicity and recruitment of HIV infected monocytes to CNS. Tat can promote transcriptional transactivation of cellular proinflammatory cytokines in the infected cells. Tat's ability to be secreted and subsequently taken up by bystander cells can lead to an induction of inflammatory cytokines in the uninfected cells as well. Tat's cellular uptake is mediated by an Arginine-rich region termed the cell penetration peptide (CPP). We now describe the functional consequences of a polymorphism in the Tat basic domain (residues 48-57) that diminishes extracellular Tat's uptake by cells. Earlier work, using lade B HIV-1 Tat CPP containing sixArginines, showed that each of the sixArginines are essential for maximal Tat CPP uptake. However, we observed significant variability in the conservation of R57 residue within group M clades &ndash; while R57 is well conserved (>75%) in many, other clades contain a non-Arginine residue at this position.</p><p> We hypothesized that the absence of R57 reduces Tat uptake. Cells incubated with fluorescently-labeled peptides corresponding to residues 48-57 in Tat, with R57, S57 or G57 residues revealed a 3-4 fold higher level of CPP-R57 uptake compared to the others. In order to further study the biological implication of this variation, we chose to compare clade B (most studied clade) with clade C (globally pre-dominant clade). To test the effects of R57S substitution on full-length Tat protein, we established a transcellular Tat transactivation system, where HeLa cells are transfected with expression constructs for Tat-B (R57), Tat-C (S57) proteins or their R57S or S57R mutants respectively. Media are collected from the transfected cells, relative levels of Tat determined by ELISA and equal amounts of Tat applied to TZM-bl reporter cells. We demonstrated that R57S mutation in Tat-B diminished its transcellular reporter signal response, while the S57R mutation in Tat-C enhanced the transcellular response. Furthermore, we investigated the role of R57S substitution on the establishment of an inflammatory environment by exposing monocytic and microglial cells lines to Tat-containing media and quantifying the inflammatory cytokines gene induction. We found significantly higher levels (~2-3 fold) of expression of several cytokine genes, in cells exposed to the R57-containing Tat variants compared with the S57-variants.</p><p> Based on our results, we conclude that Tat R57 is an important viral determinant for efficient cellular uptake of Tat. It contributes to the intercellular, systemic spread of inflammation, and may contribute to the inflammatory phenotype in the CNS of HIV-infected individuals. Intriguingly, a small data set of Tat sequences from CNS samples in HIV-1 C patients indicated an enrichment for R57 variants, compared with the consensus peripheral sequences (77% vs 13%, respectively), suggesting that the rare CNS HIV disease that is observed in some clade C patients may be associated with a R57-containing Tat. Our studies have identified a novel signature of HIV-1 Tat that enhances the induction of inflammatory signal, and may play an important role in neurovirulence. </p>

Source: Dissertation Abstracts International, Volume: 33-07, Section: B, page: 3214. / Thesis (Ph.D.)--The Florida State University, 1972.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.