Inflammation in the female genital tract (FGT) is associated with increased HIV-1 viral replication, HIV-1 transmission and HIV-1 acquisition. The optimal commensal Lactobacillus bacterial species is associated with reduced inflammation in the FGT and dampened immune responses to non-optimal bacteria in vitro. Using a transcriptomics approach, this research aimed to investigate gene expression patterns in the FGT of HIV-infected women compared to peripheral blood. Furthermore, transcriptomics was used to investigate interactions between different vaginal Lactobacillus species and the host to elucidate its immunomodulatory mechanisms. Cervical cytobrushes and blood samples were collected from chronically HIV-infected South African women. Cervical and peripheral blood mononuclear cells (CMCs and PBMCs) were isolated and mRNA was extracted for microarray analysis using the Illumina HumanHT-12 v3 Expression BeadChip system. Eight Lactobacillus isolates, two of each L. jensenii, L. mucosae, L. crispatus and L. vaginalis species were included in this study. The effects of these lactobacilli on cytokine production by vaginal epithelial (VK2) cells stimulated with Gardnerella vaginalis (ATCC 14018) were tested in vitro, RNA was extracted and used for Affymetrix Genechip whole transcript microarray analysis. This study found that significantly over-expressed genes in CMCs compared to PBMCs were mapped to proinflammatory signaling pathways (including Nuclear factor kappa B (NFκB), Tumor necrosis factor (TNF), Toll-like receptor (TLR) and Nucleotide-binding and oligomerization domain (NOD)-like receptor). Concurrently, a signature of reduced potential for adaptive immunity was observed in CMCs compared to PBMCs, as evidenced by underrepresentation of the T cell receptor signaling and natural killer cell mediated cytotoxicity pathways. G. vaginalis induced a potent proinflammatory cytokine response by VK2 cells in vitro. Over-expressed genes in G. vaginalis-stimulated VK2 cells compared to unstimulated VK2 cells were mapped to inflammatory signalling pathways. In contrast, 3/8 Lactobacillus isolates, including two L. mucosae and one L. vaginalis species, reduced inflammatory cytokine production by VK2 cells in response to G. vaginalis and were thus termed “cytokinesuppressive”. Several genes, 7/8 of which are involved in inflammation, were downregulated in VK2 cells co-cultured with lactobacilli and G. vaginalis in combination compared to coculture with G. vaginalis only. Futhermore, when gene expression changes were investigated in cells cultured with cytokine-suppresive lactobacilli versus non-cytokine-suppressive lactobacilli, it was found that SAMD9L, DDX58, IFIT1 gene expression was downregulated exclusively in VK2 cells co-cultured with cytokine-suppressive lactobacilli and G. vaginalis compared to co-culture with G. vaginalis only. The findings of this study have identified distinct gene expression patterns in the FGT compared to peripheral blood. Furthermore, key genes that may play a critical role in the immunomodulatory effects of vaginal lactobacilli were identified, motivating for further confirmatory research.

Pregnancy is dependent on the development of maternal immune tolerance to the genetically foreign fetus. During pregnancy the mother's immune reactivity and energy metabolism undergoes significant changes and the levels of certain hormones in peripheral blood are significantly increased. Hormones are important regulators of the functional activity of the immune system and immune cells within. Hormones secreted by the placenta, protect the fetus from the maternal immune response of the mother, emphasizing their immunomodulatory effects. Therefore, hormonal regulation is essential for the functional activity of immune cells. There is evidence that the hormone, kisspeptin, plays a role in the development of immune tolerance during pregnancy based on its role in the regulation of the adaptive T regulatory (aTreg)/T-helper 17 (Th17) cells, induction of the enzyme indoleamine 2,3-dioxygenase (IDO) and regulation of monocyte function during pregnancy. In addition, kisspeptin has been implicated in the regulation of specific cytokines during pregnancy. It is crucial to maintain an appropriate cytokine balance at the maternal– fetal interface as well as in circulation. Several pregnancy-related disorders have been associated with a variation in Th1/Th2/Th17 cytokines and aTreg cell subsets. Kisspeptin has been implicated in regulating cytokines IL-10 and IL-17A as well as aTreg and Th17 cells which are significant role players in immune tolerance during pregnancy. However, its effect on other pro- and anti-inflammatory cytokines remain unknown. Therefore, more research is required to better understand the role of kisspeptin in the development of immune tolerance during pregnancy. The hypothesis of this study is that kisspeptin alters the expression of anti-and pro-inflammatory cytokines and may thus influence the establishment of immune tolerance in pregnancy. To test this hypothesis, we used a previously established in vitro peripheral blood mononuclear cell (PBMC) Mycobacterium tuberculosis (Mtb) infection assay model as well as a newly established in vitro infection model using lipopolysaccharide (LPS)-stimulated whole blood. Protein expression analysis of selected pro- and anti-inflammatory cytokines was performed on PBMC infected with Mtb and on whole blood cells stimulated with LPS in the absence and presence of kisspeptin-10 for different times. The cytokines levels were measured by luminex multiplex assay and sandwich ELISA, respectively. Results from the PBMC infection assay showed a varied but not statistically significant effect of kisspeptin-10 on selected pro- and anti-inflammatory cytokine expression at 2 hours post-infection. However, there was a suggestion of an inhibitory effect of kisspeptin-10 on selected pro- and anti-inflammatory cytokine expression, macrophage inflammatory protein (MIP)-1α, MIP-1β, tumour necrosis factor (TNF)-α, granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-10, after 24 hours which was not observed at 6 days post-infection. Results from the whole blood stimulation assay suggested an inhibitory effect of kisspeptin-10 on selected LPS-induced pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) whilst generally not having an effect on selected anti-inflammatory cytokines (IL-10). Overall this study suggests, based on the lack of statistically significant data, a potential immunomodulatory effect of kisspeptin-10 based on the observed inhibition of pro-inflammatory cytokines. Investigating and developing an understanding of key regulators and mechanisms of maternal immune tolerance may help researchers understand the pathophysiological mechanisms underlying certain pregnancy-related disorders. This was a pilot study aimed at characterising the effect of kisspeptin stimulation on cytokines and chemokines responses. Manipulation of regulatory hormones such as kisspeptin could represent a potentially novel approach in the treatment of various pregnancy-related disorders including preeclampsia and unexplained recurrent miscarriage.

A central question of evolutionary biology concerns the study of evolvability. A growing body of theory implicates the pattern of genetic effect interactions underlying a trait, referred to as the genetic architecture of a trait, as an important component of evolutionary dynamics. The model organism, Drosophila melanogaster, belongs to a group of fly lineages, the Acalyptrate Diptera, that exhibit evolutionary stasis in their wing shape. The small variation in wing shape between the member species of such a disparate group, indicates either a uniform stabilizing selection of wing shape for all species or a lack of evolvability in the trait. This thesis investigates the genetic architecture of wing shape in D. melanogaster as a possible constraint on its evolvability. I conduct a line-cross analysis for estimating the genetic effects of various percentages of alleles from a parental population in the genetic background of another parental population; and, a chromosome substitution analysis for estimating the genetic effects of various X-chromosomes substituted into different genetic backgrounds. For both experiments, I use fly populations established by Houle et al. that had undergone thirty generations of artificial selection on an index of wing shape. The line-cross analysis suggests that the two lines selected to increase or decrease the wing shape index score had decanalized genetic architectures with respect to their second generation hybrids. This is consistent with the idea that wing shape evolutionary stasis is due, in part, to low evolvability of the trait. The chromosome substitution experiment was hampered by the extinction of over fifty percent of the substitution lines in the experimental design, and so the results had little power to estimate the genetic architecture of the trait. However, the best estimate of the analysis suggests a negative directional epistasis, which is also consistent with the hypothesis that the wings are canalized. / A Thesis submitted to the Department of Biological Sciences in partial fulfillment of the requirements for the degree of Master of Science. / Spring Semester, 2007. / January 25, 2007. / genetic architecture, epistasis, directional epistasis, evolvability, Drosophila / Includes bibliographical references. / Thomas F. Hansen, Professor Directing Thesis; Richard Bertram, Committee Member; Lloyd M. Epstein, Committee Member; David Houle, Committee Member.

The process of normal wound repair after tissue injury follows a closely regulated sequence involving inflammation, the recruitment, activation and proliferation of fibroblasts and the secretion of extracellular matrix, which culminates in healing and termination of the proliferative and secretory processes. In pathological fibrosis, the normal healing and termination stages are disregulated and fibroblast activity continues unabated. This persistent "activated" state of the fibroblasts is the cause of the excessive accumulation of ECM, predominantly fibrillar collagens type I and III, which results in the disruption of the normal tissue function . In cirrhosis, the end stage of liver fibrosis, type I collagen represents up to 50% of liver proteins. Hepatic stellate cells (HSCs) are the major cell type responsible for collagen synthesis in the liver. In normal liver, quiescent HSCs store vitamin A, but only express trace amounts of type I collagen. Upon a fibrogenic stimulus, HSCs become activated, a process in which they lose vitamin A, proliferate, change morphologically into myofibroblasts, and increase their synthesis of extracellular matrix proteins. In order to understand the pathophysiology of cirrhosis, as well as other fibroproliferative disorders which can effect the heart, lungs, and skin, it is critical to elucidate the molecular mechanisms which regulate the expression and synthesis of type I collagen. In the first aim of this dissertation, we examined the role of the RNA-binding protein RBMS3 in regulating type I collagen expression. RBMS3 expression increases upon activation of HSCs, and is also increased in liver fibrosis. Through our research, we showed that RBMS3 specifically interacts with a conserved 60 nucleotide sequence in the 3' UTR of the homeobox transcription factor Prx1. Prx1, which is also upregulated in activated HSCs and liver fibrosis, transactivates the collagen á1(I) promoter and stimulates transcription of the gene. The binding of RBMS3 to the 3' UTR of the Prx1 mRNA results in the stabilization of the mRNA and increased protein synthesis. Since Prx1 is a transcription factor which increases collagen gene transcription, this mechanism may promote the profibrotic phenotype of HSCs. In the second aim of the dissertation, we focused on the posttranscriptional regulation of type I collagen expression. In the 5' UTR of á1(I), á2(I), and á1(III) collagen mRNA there is a stem-loop structure that encompasses the translation initiation codon. This 5' stem-loop is strongly conserved in evolution differing by only two nucleotides between fish and human collagen mRNAs. In order to study the role of the 5' stem-loop, our collaborators designed a transgenic mouse in which the 5' stem-loop structure of the collagen á1(I) gene was abolished. The collagen á1(I) mRNA stability and protein synthesis in fibroblasts from these transgenic mice was significantly decreased. Since it was clear that the 5' stem-loop was essential for proper type I collagen synthesis, we examined the role that 5' stem-loop binding proteins have in regulating this mechanism. Through the use of 2-D gel SDS-PAGE and MALDI-TOF MS, we identified several 5' stem-loop associated proteins which included LARP6, non-muscle myosin IIb, nucleolin, and vimentin. We discovered that LARP6, through direct binding of the collagen 5' stem-loop, enables the aggregation of type I collagen mRNAs into large complexes. Additional proteins identified in these RNA-protein complexes are components of stress granules, which regulate RNA metabolism. This type I collagen mRNA stress granule formation, along with non-muscle myosin IIb, may facilitate the mechanism by which the coordinated translation of á1(I) and á2(I) collagen mRNAs can occur. Overall, my dissertation research has accomplished two major findings. The first finding is how the transcription of the collagen á1(I) gene is regulated through the binding of RBMS3 to the mRNA encoding transcription factor Prx1. The second finding is how the translation of type I collagen is regulated by the LARP6 mediated aggregation of collagen mRNAs and their association with non-muscle myosin. Elucidation of these mechanisms may help in the development of antifibrotic drugs. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2008. / August 7, 2008. / RNA, Liver Fibrosis, Biomedical Science / Includes bibliographical references. / Branko Stefanovic, Professor Directing Dissertation; Ken Roux, Outside Committee Member; Yanchang Wang, Committee Member; James Olcese, Committee Member.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play key roles in excitatory synaptic transmission and are involved in numerous neurological disorders as well. The number of neuronal surface NMDARs are not static and can be altered in response to neuronal activity and sensory input. Endocytosis is well known to decrease the number of surface receptors and down-regulate receptor-mediated functions. However, whether a subset of receptor endocytosis regulates the activity of remaining surface (non-internalized) receptors remains unknown. It is shown from this study that the dynamic endocytosis of NMDARs induced by either the activation of group 1 metabotropic glutamate receptors (mGluRs) or the stimulation of the glycine binding site of NMDARs inhibits the activity of remaining surface NMDARs. This effect can be prevented by the modulation of the functional properties of surface NMDARs, for example, inactivating the surface NMDARs by application of the NMDAR antagonist AP5 or blocking Na+ influx. Furthermore, it is shown from this study that the endocytosis of NMDARs enhances the serine phosphorylation of surface NMDAR NR2A subunits. Coincidentally, the inactivation of surface NMDARs or the blockade of Na+ influx produces similar enhancement of serine phosphorylation in NMDARs as well. It is identi ed, by site directed mutagenesis, phosphorylation of NR2A serine 1416 as an essential mechanism in mediating NMDAR endocytosis-induced functional changes in surface receptors. In this study, protein kinase D (PKD/PKCu) is identified to be activated following NMDAR endocytosis, and intracellular application of PKD/PKCu successfully inhibits NMDAR-mediated whole-cell currents. Moreover, the blocking Na+ influx essentially eliminates the NMDAR endocytosis-induced down-regulation of NMDA-evoked whole-cell and synaptic responses. Taken together, the data from this study provide clues on understanding that the reduction of whole-cell responses induced by NMDAR endocytosis is attributed to the inhibition of the activity of remaining surface NMDARs. In addition, the modulation of the functional properties of surface receptors is shown to be an effective way to prevent functional change in surface receptors induced by recptor endocytosis. / A Dissertation submitted to the Department of Biomedical Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2011. / December 10, 2010. / Includes bibliographical references. / Xian-Min Yu, Professor Directing Dissertation; Paul Trombley, University Representative; Charles Ouimet, Committee Member; Choogon Lee, Committee Member.

Protein phosphorylation ' a reversible covalent process ' has evolved as one of the major posttranslational modifications that targets numerous transcription factors. Apoptosis (programmed cell death) is an irreversible intrinsic mechanism essential for removing unwanted cells, and maintaining tissue homeostasis in multicellular organisms. Activation of caspases is a major event during programmed cell death by which more than 280 proteins are cleaved. YY1 (Yin-Yang 1) is a multifunctional zinc finger transcription factor that has been implicated in different cellular processes such as proliferation, embryogenesis, differentiation, development, tumorigenesis, and apoptosis. YY1 has been shown to be a phosphoprotein and several studies have reported that phosphorylation regulates its activities. Also, YY1 was found to be one of only a few transcription regulators that is a target for cleavage by caspases in vitro and in vivo as well, but very little is known about the mechanisms that regulate its cleavage during cell death. Here, we identify serine 118 in the N-terminal domain of YY1, as the site of CK2' phosphorylation, proximal to a caspase cleavage site. CK2 inhibitors, as well as knockdown of CK2' by siRNA, reduce S118 phosphorylation in vivo and enhance YY1 cleavage under apoptotic conditions, whereas increasing CK2' activity by overexpression in vivo elevates S118 phosphorylation. A serine to alanine substitution at serine 118 also increases the cleavage of YY1 during apoptosis when compared to wild-type YY1. Taken together, we have discovered a regulatory link between YY1 phosphorylation at serine 118 and regulation of its cleavage during programmed cell death. In response to genotoxic stress, eukaryotic cells activate a set of DNA-damage kinase cascades that are initiated by sensor proteins such as ATM, ATR or DNA-PK. These sensor proteins phosphorylate various targets including Chk1, Chk2, and p53. To study the signaling pathway(s) regulating the cleavage of YY1 under apoptotic conditions, we have used ATR-deficient fibroblasts, as wells as knockout cell lines where Chk2, DNA-PKcs, p53 and ATM are ablated. Cisplatin, x a DNA damaging agent, was added to these cells to induce cell death. Cisplatin-induced YY1 cleavage and apoptosis are found to be independent of DNA-PK and Chk2, delayed in ATM and p53 knockout cell lines, and suppressed in ATR-deficient fibroblasts and DLD-Chk1 heterozygous cells. Consistently, inhibition of ATR with a selective ATR inhibitor prevents cisplatin-induced YY1 cleavage. Together, these results suggest a critical role of ATR-Chk1 in regulating YY1 cleavage by caspases during apoptosis. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2011. / June 24, 2011. / Includes bibliographical references. / Myra M. Hurt, Professor Directing Dissertation; Hank Bass, Outside Committee Member; Akash Gunjan, Committee Member; Yanchang Wang, Committee Member; Cathy Levenson, Committee Member.

In the female rat, secretion of prolactin is tightly controlled by a complex interplay of both stimulatory and inhibitory hypothalamic factors. In addition, the profile of prolactin release in the ovariectomized rat treated with estradiol exhibits circadian rhythmicity, which requires an intact suprachiasmatic nucleus (SCN). This suggests that photic cues influence the hypothalamic factors coordinating the release of prolactin, namely oxytocin (arising from the paraventricular and periventricular nucleus of the hypothalamus) and dopamine (arising from neuroendocrine dopaminergic neurons in the periventricular nucleus and the arcuate nucleus). How photic cues regulate the secretion of prolactin, however, has yet to be explicated in animals treated with estradiol. Recent research has determined that the SCN sends efferents of vasoactive intestinal polypeptide to neuroendocrine dopaminergic neurons as well as to the paraventricular and periventricular nuclei of the hypothalamus. The first purpose of this study was to elucidate the mechanism regulating the rhythmicity of prolactin release. To determine this, vasoactive intestinal polypeptide synthesis was disrupted using vasoactive intestinal polypeptide antisense deoxyoligonucleotides infused into the dorsal border of the SCN. This treatment caused a phase-advance in the pattern of prolactin secretion in estradiol-treated ovariectomized rats. Using immunohistochemistry, oxytocin and dopamine activity was established through double labeling with FOS-related antigens (a marker of neuronal activity). In both populations of neurons (oxytocin neurons and neuroendocrine dopaminergic neurons), a phase advance was also observed. In oxytocin neurons, disruption of vasoactive intestinal polypeptide phase-advanced the increase in activity that normally accompanies the prolactin surge. Whereas, in dopamine neurons, disruption of vasoactive intestinal polypeptide phase-advanced the decrease in activity that is required to allow for the prolactin surge. Furthermore, clock gene expression has been localized to neuroendocrine dopaminergic neurons. In all three populations of neuroendocrine dopaminergic neurons, the expression of the clock gene period 2 oscillates. Disruption of vasoactive intestinal polypeptide disrupted this pattern of activity. Thus, the SCN influences the precise timing of the estradiol-induced prolactin surge via vasoactive intestinal polypeptide projections to oxytocinergic and dopaminergic neurons, and possibly does so by entraining clock gene expression in these neurons. Once entrained, oxytocin exerts its stimulatory effects at a previously unknown site. Using a selective oxytocin antagonist that does not cross the blood brain barrier, the pituitary was determined to be the site of oxytocin stimulation. Interestingly, the oxytocin receptor density is upregulated in response to estradiol; therefore, suckling- and estradiol-induced prolactin surges require higher doses of oxytocin antagonist than cervically-stimulated ovariectomized animals. Furthermore, in the presence of progesterone, the oxytocin antagonist was incapable of blocking the estradiol-induced surge; raising the possibility that progesterone may act through mechanisms independent of oxytocin to stimulate prolactin secretion. Therefore, the work in this dissertation provides additional mechanisms involved in the temporal and physiological control of estradiol-induced prolactin secretion. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2009. / July 3, 2009. / Endocrinology, Hypothalamus, Hormone / Includes bibliographical references. / Marc E. Freeman, Professor Directing Dissertation; Penny J. Gilmer, Outside Committee Member; Debra A. Fadool, Committee Member; Elaine Hull, Committee Member; Michael Meredith, Committee Member.

Although zinc has been implicated in the functionality of the hippocampus since the 1970's, the recent discovery of adult neuronal stem cells in this region of the brain provides the potential for novel zinc-regulated hippocampal processes. The current work addressed the role of zinc in the proliferation, maintenance, and survival of neuronal precursor cells. First, this work employed a genome-wide analysis of the effect of dietary zinc deficiency in the hippocampus. The data revealed that 3 weeks of dietary zinc deficiency resulted in the down-regulation of nearly 400 genes, many of which were associated with synaptic plasticity (stau2, syn1), neurotransmitter receptors (grin1, gabrb3), neurogenesis (iguana, id2, nek9), and cell viability (sod2, stat3). Furthermore, using a candidate gene approach, the current work shows a vital role of zinc in p53-mediated mechanisms governing proliferation and apoptosis of neuronal precursor cells. For example, zinc deficient cells show increased nuclear and mitochondrial translocation of the tumor suppressor p53. Using a dominant negative construct to ensure p53 regulation, this work shows that nuclear p53 is responsible for the downstream target genes responsible for cell cycle arrest (reprimo, lats2). These data coincide with a decrease in BrdU-labeling. The current work also highlights initial protective responses governed by the transcription factor p53. However, zinc deficient neuronal precursors also show a p53-dependent increase in mitochondrial reactive oxygen species which could be mediated by a decreased expression of glutathione peroxidase mRNA and mitochondrial localized p53. If the deficiency is severe or prolonged nuclear p53 regulates expression of apoptotic genes (rb1, tgf-β) whereas, mitochondrial p53 mediates interactions with Bcl-family proteins to initiate a loss in mitochondrial membrane potential. Ultimately, the current work adds to the essential role of zinc in the hippocampus and identifies a novel mechanism for zinc in the regulation of neuronal precursor proliferation, maintenance, and survival. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2010. / June 11, 2010. / Zinc, Neurogenesis, Apoptosis, p53, Proliferation, Stem Cells, Hippocampus / Includes bibliographical references. / Cathy W. Levenson, Professor Directing Dissertation; J. Michael Overton, Committee Member; Charles Ouimet, Committee Member; Shridhar Sathe, Committee Member; Laura Keller, University Representative.

To protect against sexual transmission, successful future HIV vaccines will likely be given to adolescents as a booster subsequent to primary immunization during infancy. In sub-Saharan Africa (SSA), a large proportion of children are chronically infected with a variety of helminths. These infections may suppress the ability of a host to elicit vaccine-induced Th1 responses that are considered important for a successful HIV vaccine. This study investigated the effect of chronic helminthic infection on the boosting capacity of a poxvirus-protein HIV vaccine regimen (SAAVI MVA-C and Env gp140 protein) in a mouse model. Groups of mice were prime-vaccinated with SAAVI MVA-C through an intramuscular injection, and Env gp140 protein formulated in Alum adjuvant which was administered via an intraperitoneal injection. These vaccinations were given concurrently, 2 weeks prior to infection with Schistosoma mansoni (Sm) through a percutaneous route. Control mice were either left uninfected (Naïve) or infected in the same manner (Sm) without vaccination. A booster vaccination was given 8 weeks post helminth infection. HIV-specific immune responses were analysed in the blood and spleens two weeks after booster vaccination. The magnitudes of cumulative IFN-γ ELISPOT responses to HIV Gag, RT and Env peptides were significantly (p<0.05) lower in the vaccinated and Sm-infected (Vaccine+Sm) mice (948 sfu/106) than vaccinated and uninfected (Vaccine) mice (1733 sfu/106), with IFN-γ responses to RT (CD8) being the most dominant for both mouse groups (Vaccine+Sm: 734 ± 221 sfu/106, Vaccine: 521 ± 116 sfu/106). No significant difference was observed in the magnitudes of cumulative IL-2 ELISPOT responses to the vaccine peptides between the Vaccine+Sm and Vaccine groups, however IL-2-producing T cell responses to Env (CD4) dominated in both mouse groups. Vaccine+Sm and Sm groups had similar IFN-γ- and IL-2-producing T cell responses to SEA. Splenocytes from Vaccine+Sm mice secreted less Th1 (IFN-γ, IL-2, TNF- α) and Th2 (IL-4, IL-6, IL-10) cytokines than those from uninfected vaccinated mice in response to HIV vaccine peptides. The total number of activated CD4+ T cells responding to vaccine peptides was greater for Vaccine+ Sm mice than Vaccine mice (p<0.05), however, no such statistical significance was observed in the differences seen between these vaccinated mouse groups for the number of activated CD8+ T cells. The frequencies of central memory activated CD4+ T cells were seen to be greater in Vaccine group (Gag; 34.28 ± 8.35%, Pol; 33.53 ± 6.34%, Env(CD4); 33.92 ± 3.87%, Env (CD8); 38.76 ± 10.52%) as opposed to the Vaccine+Sm group (Gag; 28.09 ± 3.95%, Pol; 26.45 ± 4.66%, Env (CD4); 28.79 ± 6.95%, Env (CD8); 28.65 ± 3.29%). Furthermore, Vaccine+Sm mice had higher titres of HIV-1 gp140- specific IgG1 antibodies (p<0.0001) (a Th2 antibody marker) but significantly less gp140- specific IgG2a (p<0.0001) and IgG2b (p<0.001) (Th1 antibody markers) antibodies. This trend was also observed with total non-Env-specific antibody titres. This study demonstrates that chronic helminthic infection is associated with an attenuated boosting capacity of a poxvirus-protein HIV vaccine in a mouse model, suppressing both T cell cytokine production and Th1-type antibody responses. Since HIV vaccine-induced Th1 responses are considered important for a successful HIV vaccine, these data suggest that chronic helminthiasis may impact negatively on future HIV vaccination outcomes in adolescents living in SSA where helminthic parasites are endemic.

Kaposi sarcoma herpes virus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement-binding protein, KCP) that binds complement proteins and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Kaposi's sarcoma is an angiogenic skin lesion that has been recognized as one of the most abundant tumours found in many parts of Southern Africa and which can occasionally become highly invasive, aggressive and capable of causing death, particularly amongst AIDS patients. It is of major significance to understand how complement control proteins (CCPs) such as KCP perform their biological functions at the molecular and structural levels, because of their potentials as therapeutic agents, their implications in the pathology and importance in the etiology of many disease conditions. This study was therefore undertaken to characterise the structure-function relationship of KCP. Based on primary sequence analysis and comparison to other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by PCR, three regions of the predicted ORF 4 from human herpes virus-8 (llliV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris and to produce separately, the 4 N-terminal Sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane binding domain (KCP-M, medium) and the full-length protein (KCPF, full). Expression of the viral proteins was confirmed by SDS-PAGE and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. All the KCP proteins migrated electrophoretically as higher bands compared to their expected sizes. The lower mobilities of the proteins may be due to g1ycosy1ation since there are potential N-and O-glycosylation sites in the protein's primary sequence. Also, diffused bands were obtained in all the electrophoretic gels and Western blots carried out, which is characteristic of glycoproteins. Furthermore, the antibody recognized several larger and smaller bands that may represent aggregates and/or degradation products respectively. Both partially purified KCP-S and KCP-S directly from expression media were able to inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice more efficient in inhibiting the classical pathway-mediated lysis of erythrocytes than the vaccinia virus complement control protein (VCP), which also contains 4 Sushi domains. The KCP-F and KCP-M proteins did not show any significant complement inhibitory activities. Preliminary immunohistochemical studies using the same antibody were carried out to determine the expression and distribution of KCP proteins in Kaposi's sarcoma.