Malaria causes 300-500 million clinical cases and 1-3 million deaths per year, the majority of which occur in young African children. Ingestion of hemozoin by peripheral blood mononuclear cells (PBMC) initiates the cytokine-mediated cascade of immune dysregulation in malaria. Innate immunity plays a critical role in protective responses against infection, which are determined by an appropriate balance between pro- and anti-inflammatory mediators. Pro-inflammatory mediators (TNF-&#945, IL-12, IFN-&#947) that control parasitemia also contribute to pathology. Over-production of immunomodulatory cytokines (TGF-&#946, IL-10) suppresses the protective pro-inflammatory immune response. The effects of hemozoin on hematological outcomes and inflammatory mediator production in children with malarial anemia and the temporal kinetics of hemozoin-induced cytokine dysregulation in cultured PBMC were investigated in this study. Hematological analyses of healthy control (HC), uncomplicated malaria (UM), mild malarial anemia (MlMA), moderate malarial anemia (MdMA), and severe malarial anemia (SMA) groups revealed that age, temperature, and all erythrocyte, leukocyte, and platelet indices were significantly different between clinical categories. Neither parasitemia nor the prevalence of high density parasitemia (HDP) differed significantly between clinical groups. Stratification of study participants according to proportion of pigment-containing monocytes (PCM) demonstrated that parasitemia, HDP, temperature, and most leukocyte, erythrocyte, and platelet indices were significantly different between 0%, &#8804 10%, and > 10% PCM categories. The > 10% PCM category contained children with the lowest hemoglobin, hematocrit, and erythrocyte counts and the greatest proportion of children with SMA. Plasma IFN-&#945, IL-4, IL-6, IL-10, and IL-12p40/p70 levels differed significantly between clinical categories but not between UM and SMA groups. Plasma IFN-&#945, IFN-&#947, TNF-&#945, IL-1&#946, IL-2, IL-4, IL-6, IL-10, and IL-12p40/p70 were not significantly associated with % PCM. &#946-hematin-induced IL-1&#946, IL-2, IL-6, IL-12p35, IL-12p40, IL-18, IFN-&#945, IFN-&#947, TNF-&#945, LT-&#945, NOS-2A, COX-1, COX-2, IL-4, IL-10, and TGF-&#946 expression in cultured PBMC revealed a pro-inflammatory response that varied in magnitude among individuals. Importantly, innate inflammatory mediators modulate the adaptive immune response to Plasmodium parasites. Of public health significance, a better understanding of the molecular mechanisms governing these responses will facilitate the development of more immunogenic vaccines, through inclusion of cytokines or other compounds that activate the innate immune system.

Toll-like receptors (TLRs) are key innate immune receptors that recognize non-self pathogens and trigger host responses. Activation of these receptors results in the release of antimicrobial peptides, inflammatory cytokines, and co stimulatory molecules that initiate adaptive immunity for infections with gram-negative bacteria, lipopolysaccharide is the main source of inflammation, and toll-like receptor 4 (TLR4) is crucial in mediating its effects. TLR4 is expressed on cardiomyocytes, macrophages, airway epithelia, endothelial, smooth-muscle cells and in small amounts in most other tissue. But, uncontrolled activation of TLR signaling molecules may cause auto immune diseases, sepsis, and tissue damage so the activation of TLR4 should be under control. Ubiquitin¨Cdependent receptor degradation as well as stabilization was recently suggested as a novel regulatory mechanism in controlling several TLR activations. We have recently found that an ubiquitin-like protein named protein linking integrin associated protein to cytoskeleton 1 (PLIC-1) interacts with the cytoplasmic domain of TLR4. The interaction between TLR4 and PLIC-1 was verified by western blot and immunoprecipitation. Further mapping of the interacting domain was done and we observed that the N terminal fragment of PLIC-1 is interacting with TLR4. PLIC-1 has been reported to stabilize proteins by interfering with proteosomal degradation. Consistent with this finding, we observed that over expression of PLIC-1 accumulated ubiquitinated TLR4. By flow cytometric analysis we observed that over expression of PLIC-1 is stabilizing TLR4. Reporter studies show that PLIC-1 inhibits the TRIF-dependent IFN-¦Â pathway. When endogenous PLIC-1 was knocked down by RNAi, the activation of TRIF-dependent IFN-¦Â luc was further increased. The same effect was observed in J774 mouse macrophages. Taken together our results suggest that PLIC-1 is a negative regulator of TLR pathway. This knowledge may be applied in immunotherapy as a means to modulate TLR activation in diseases such as septic shock, thus provides benefit for public health.

Gene therapy offers a promise for treating inherited muscle disorders. The advantages of recombinant adeno-associated virus (rAAV) gene delivery vector include nonpathogenicity and long-term gene expression after a single administered dose. However, rAAV predominantly transduces the liver after systemic administration, reducing its efficiency for gene transfer to the heart and skeletal muscle. The question of how to deliver the therapeutic genes into most of the diseased myofibers becomes a challenge. The goal of this project is to develop an efficient and muscle-specific AAV vector for systemic delivery. Here, the muscle-targeting peptide ASSLNIA was incorporated into AAV2 capsid after residue 587 without heparin-binding motif (587 TG MTP vector) or with heparin-binding motif (588 HB MTP vector) since heparan sulfate is the primary cellular receptor of AAV2. The efficiencies and selectivities of muscle targeting of modified rAAVs were evaluated in vitro and in vivo. This study demonstrated that the peptide-modified vectors maintained their myotube transduction ability. Peptide-engineered AAVs decreased their transductions in non-muscle cell lines. In addition, the 587 TG MTP vector did not require the heparin-dependent mechanism for muscular targeting. The C2C12 myotube transductions of 587 TG MTP and 588 HB MTP vectors were inhibited 47%~58% in the presence of free ASSLNIA peptide while unmodified rAAV2 transduction was only suppressed by 25%. To explore the muscle-targeting abilities of modified rAAVs in vivo, mice were injected intravenously via a tail vein with a viral dose containing 9x10^11 genomic particles. After four weeks, mouse organs were harvested for the luciferase assay. The 587 TG MTP vector demonstrated enhanced muscle and heart transduction compared to unmodified rAAV2. Importantly, the 587 TG MTP virus significantly reduced its transduction of liver, lungs, and spleen. The vector biodistribution in organs was also determined by real-time PCR and peptide-modified vectors showed similar targeting effects. Moreover, this study found that both 587 TG MTP and 588 HB MTP vectors were resistant to antibody neutralization. These results indicate that this muscle-targeting peptide facilitates the generation of an efficient and muscle-specific AAV vector for systemic gene delivery in the treatment of muscle diseases to provide clinical and public health benefits.

Human herpesvirus-8 (HHV-8), also known as Kaposis sarcoma-associated herpesvirus, is the most recently identified human herpesvirus. A key question regarding HHV-8 is the location of infected cells within HHV-8 seropositive individuals. Outside of tumor tissues, HHV-8 viral proteins have been detected in saliva, circulating B cells, and semen of some, but not all HHV-8 seropositive individuals. HHV-8 is the causative agent of Kaposis sarcoma (KS) and is associated with two other distinct proliferative disorders: primary effusion lymphoma and some forms of multicentric Castlemans disease. To better understand viral infection including the cellular targets of infection, we have begun a systematic screening of autopsy tissues from HHV-8 seropositive men who died with AIDS. Using immunohistochemistry (IHC), my goals were to determine reservoirs of HHV-8 infection and latency in organ tissues, determine the type of viral infection (lytic and/or latent) of each tissue type, and attempt to identify the infected cell type. In this report, using IHC, we document the presence of HHV-8 infected cells in several organs including kidney, lung, liver, and gastrointestinal tract samples from the Multicenter AIDS Cohort Study (MACS). Both lytic and latent infections have been detected and the infected cells appear to consist of both immune and non-immune cells. These results demonstrate the ability of HHV-8 to establish infections in various organs which may affect the pathogenesis of the virus in infected individuals. Kaposis sarcoma is currently a major public health concern, as it is the most common malignancy found in individuals with AIDS and iatrogenic KS is a key concern in the field of solid-organ transplantation. This study will attempt to identify reservoirs of HHV-8 infection within the body in order to better understand the biology of HHV-8 in infected individuals, and the role HHV-8 plays in disease pathogenesis.

Neisseria meningitidis is a major cause of septicemia and meningitis worldwide. Traditional typing methods like pulsed-field gel electrophoresis (PFGE) for identifying outbreaks are subjective and time consuming. Multi-locus variable number tandem repeats analysis (MLVA) is an objective typing method amenable to automation that has been used to type other bacterial pathogens. This report describes the development of MLVA for outbreak detection of N. meningitidis. Tandem Repeats Finder software was used to identify variable number tandem repeats (VNTRs) from 3 sequenced N. meningitidis genomes. PCR amplification of identified VNTRs was performed on DNA from 7 serogroup representative isolates. PCR products were sequenced and repeats were manually counted. VNTR loci identified by this screen were evaluated on a collection of 46 outbreak and sporadic serogroup C isolates. Alleles at each locus were concatenated to define the MLVA type for each isolate. Minimum spanning tree (MST) analysis was performed to determine the genetic relationships among the isolates. The genetic distance was defined as the summed tandem repeat difference (STRD) between isolates MLVA types. Outbreak clusters were defined by a STRD less than or equal to 3. These data was compared to PFGE data to determine the utility of MLVA for outbreak detection. Twenty-one VNTR loci with variable copy numbers among the sequenced genomes were identified that met the established criteria of short repeat length and consensus sequence > 85%. Seven VNTR loci were reliably amplified among the 7 serogroups tested. These loci had repeat lengths between 4 and 20 nucleotides and exhibited between 10 and 26 alleles among 61 isolates belonging to 7 different serogroups. MST analysis with 7 loci differentiated serogroups, discriminated sporadic isolates and identified 7 out of 8 serogroup C outbreaks. In summary, MLVA with 5 VNTR loci distinguished N. meningitidis isolates from 7 different serogroups and sporadic isolates within each serogroup. In addition, MLVA identified 88% of PFGE-defined serogroup C outbreaks. Further investigation of these and other outbreak-associated isolates is necessary to define the optimal combination of VNTR loci and to evaluate MST analysis criteria in order to determine the utility of MLVA for N. meningitidis outbreak detection.

Severe malarial anemia (SMA), caused by infections with Plasmodium falciparum, is one of the leading causes of childhood mortality in sub-Saharan Africa. Although the molecular determinants of SMA are largely undefined, dysregulation in host-derived inflammatory mediators influences disease severity. Macrophage migration inhibitory factor (MIF) is an important regulator of innate inflammatory responses that has recently been shown to suppress erythropoiesis and promote pathogenesis of SMA in murine models. The role of MIF in childhood malarial pathogenesis was investigated by examining peripheral blood MIF production in children residing in a hyperendemic area of Gabon, and a holoendemic region of western Kenya. The relationship between MIF concentrations and monocytic acquisition of hemozoin, and the effects of MIF on erythropoiesis in vivo and in vitro were investigated. In addition, the influence of genetic variation at MIF -173 (G/C) and -794 (CATT5-8) on MIF production and susceptibility to SMA and high-density parasitemia (HDP) was examined. Circulating MIF concentrations and peripheral blood mononuclear cells (PBMC) MIF production progressively declined with increasing anemia severity and increasing levels of hemozoin-containing monocytes. However, circulating MIF concentrations were not significantly associated with reticulocyte production in children with acute malaria. Additional experiments in malaria-naïve individuals demonstrated that hemozoin caused both increased and decreased MIF production in cultured PBMC based on genetic differences. In addiiton, a novel in vitro model of erythropoiesis was developed and used to demonstrate that treatment with exogenous MIF or blocking endogenous MIF did not signifcantly impact on the efficiency of erythropoiesis. Genetic analyses revealed that the MIF -173 CC genotype was associated with an increased risk of HDP compared to MIF -173 GG. In addition, individuals with the MIF -794CATT6/-173G haplotype were significantly protected from SMA while those with -794CATT7/8/-173C haplotypes were at an increased risk of developing SMA. Taken together, our findings demonstrate that SMA is associated with decreased MIF production and that individuals with high MIF-producing genetic variants are less susceptible to severe malaria. The public health significance of this study is that investigations presented here increase our understanding of protective inflammatory responses to childhood malaria, which is critical in the formulation of an effective malarial vaccine.

The lymphatic endothelium is involved in the drainage of interstitial fluid and in the migration of immune cells like dendritic cells (DCs) from the periphery to draining lymph nodes (LNs). Tuberculosis has been declared a pandemic infectious disease accounting for more than 2 million deaths annually and is caused by the intracellular bacteria, Mycobacterium tuberculosis. The chronic inflammatory response to M. tuberculosis infection is characterized by the formation of granulomatous structures in the pulmonary compartments of infected individuals. These structures contain excess interstitial fluid and are enriched with immune cells including DCs. Therefore, the lymphatic vessels might play important roles in regulating drainage of fluid and migration of immune cells from granulomas to the draining LNs. My hypothesis was that there is an increased concentration of lymphatic vessels in these granulomatous structures and that the inflammatory environment including mycobacterial components present in granulomas and at other sites of infection elicit an inflammatory response from these lymphatic vessels which contribute to the overall immune response to M. tuberculosis infection. To address this hypothesis I have examined the distribution of lymphatic vessels in granulomatous and LN tissues obtained from nonhuman primates infected with M. tuberculosis and analyzed their expression of multiple chemokines and lymphatic markers. In addition, I evaluated the response of LECs to inflammatory mediators that included multiple TLR ligands, M. tuberculosis components and cytokines. I observed an association of lymphatic vessels with granulomas, and found that there was heterogeneity in the expression of chemokines and lymphatic markers by LECs in tissues. I also found that primary human LECs expressed multiple TLR molecules and responded to TLR ligands, cytokines and M. tuberculosis components by increasing expression of inflammatory chemokines, cytokines and adhesion molecules. These LECs also demonstrated phenotypic similarities with DCs. Overall my findings support the involvement of the lymphatic endothelium in the inflammatory immune response to pathogens like M. tuberculosis. From the perspective of public health relevance, these studies provide direction in the development of new therapeutic targets against M. tuberculosis infections and aid in the development of better adjuvants for vaccines for infectious diseases and cancers.

More than 40 million people are living with human immunodeficiency virus-1 (HIV-1). A prophylactic vaccine inducing a 'sterilizing immunity' is desired to prevent further infections, but will require many years to develop. Moreover, prophylactic vaccines will not help the millions of people who are already infected with the virus, and who face life-long treatment with expensive and toxic antiretroviral therapy (ART). This dissertation is based on the proposal that the best strategy for these individuals is a therapeutic vaccine that will attack residual viral reservoirs by expanding HIV-1 specific, primary T cell responses to the persons's own, autologous virus. Previously, this laboratory demonstrated that mature dendritic cells (DC) loaded with immunodominant HIV-1 peptides or HIV-1 infected apoptotic bodies can activate residual HIV-1 specific memory T cell responses. However, such memory T cells are only partially restored during ART. I hypothesized that targeting naive CD8⁺ T cells through a DC-based immunotherapy could elicit a robust and broad T cell response to HIV-1. Furthermore, most immunotherapy studies have used consensus strains of HIV-1 antigens that I believe inadequately represent the host's diverse pool of HIV-1 quasispecies. The current study has provided initial data that support that CD8⁺ T cells can be primed by in vitro engineered DC, even against autologous HIV-1 peptides representing immune escape variants. This study therefore supports the concept of using autologous virus as an antigen in immunotherapy and demonstrates that the use of autologous viral sequences expands both memory and primary T cell responses in vitro. Thus, a potential advantage is that future immunotherapies could use autologous virus representing a large repertoire of the host's diverse HIV-1 antigen pool. This could elicit primary immune responses specific for each patient's quasispecies of HIV-1, as well as activation of residual HIV-1 specific memory T cells, giving the broadest immune control of HIV-1 infection during ART. Such an approach has important public health implications by having a strong positive impact on, and improve the control of, HIV-1 infection in persons on ART. It also serves as an in vitro priming model for development of prophylactic vaccines against HIV-1 and other infectious agents.

Plasmodium falciparum malaria is one of the predominant causes of morbidity and mortality in children under five years of age in sub-Saharan Africa. In malaria endemic regions, the intensity of transmission and the age at which malaria is first acquired are important in conditioning disease outcomes. In addition, inter-individual variability in disease severity among age-matched children (aged <3 yrs) with similar levels of parasite exposure is largely determined by genetic variability. Historical exposure to malaria in endemic populations has exerted tremendous selective pressure on the human genome, particularly in the host-immune response genes that mediate susceptibility and clinical outcomes. Hemoglobinopathies, such as the alpha thalassemia 3.7 kb deletion and sickle-cell trait (HbAS) also confer protection against severe malaria through a mechanism(s) that are yet to be fully elucidated. As such, this study examined the role of alpha thalassemia 3.7 kb deletion and HbAS in protection against severe malaria anemia (SMA) in children (n=468; aged 3-36 months) residing in a holoendemic P. falciparum transmission region of western Kenya. These investigations demonstrated that successful genotyping of the deletion required high-quality genomic DNA from large volumes of whole blood that was unavailable for most of the small, underweight-for-age, severely anemic children in which DNA was isolated from dried blood spots. Results presented here further demonstrated that the HbAS genotype was significantly associated with a reduced burden of both low (<10%; P=0.03) and high (greater than or equal to 10%; P<0.001) pigment-containing monocytes (PCM). In addition, hemoglobin (Hb; P=0.05) and red blood cell (RBC; P=0.04) counts were significantly higher in the HbAS group relative to children with the HbAA genotype. The HbAS genotype was also significantly associated with protection against SMA using both the World Health Organization (i.e., <5.0 g/dL; P=0.04) and modified definitions of SMA (i.e., <6.0 g/dL; P=0.02). Taken together, results presented here suggest that the HbAS genotype confers protection against SMA by reducing the natural acquisition of malarial pigment (hemozoin) in monocytes. This study has significant public health importance by demonstrating that one of the mechanisms by which HbAS provides protection against SMA is through reducing the overall burden of hemozoin in monocytes.

In India, HIV-1 subtype C has been the predominant subtype throughout the course of the HIV-1 epidemic, regardless of geographic region in the country. We hypothesize that the dominance of HIV-1 subtype C compared to other subtypes in India is due to enhanced replication fitness and/or enhanced transmission efficiency of this subtype across the mucosal surface over other subtypes present in India. The specific aims of this project are: (1) to compare the replication fitness between Indian HIV-1 subtype A and subtype C; (2) to evaluate the transmission efficiency of Indian HIV-1 subtype A and subtype C across the mucosa of cervical tissue; and (3) to determine the role of the LTR and env gene in replication fitness and transmission efficiency. Replication fitness was assessed using a dual infection growth competition assay. We observed that primary HIV-1 subtype C isolates had higher overall relative fitness and transmission efficiency than primary subtype A isolates in PBMC and in an ex vivo cervical tissue derived organ culture, respectively. Furthermore, a comparison of replicative fitness between a subtype A/subtype C half genome chimeric virus and parental subtype A virus indicates that the higher replication fitness and transmission efficiency of subtype C virus over subtype A virus from India is not due to the env gene alone. We have also characterized the genetic structure and functional characteristics of subtype A and subtype C LTRs from India. Despite their apparent variability, no significant difference was observed in the transcriptional activity between the LTRs of subtype A and subtype C. Therefore, the LTR region alone is not responsible for higher replication fitness of subtype C over subtype A. The findings presented in this study are significant for public health because an understanding of the mechanism of the asymmetric distribution of HIV-1 subtypes in India is an important component in the development of strategies to control HIV-1 infection in this country.