Despite the significant need for prevention education and updated disease curricula in elementary schools, there is a deficit of model units, lesson plans, and activities at the fourth grade level. An infectious disease and prevention teaching unit has been developed, following guidelines specified by the Centers for Disease Control and Prevention and a format consistent with proven pedagogical methods. This curriculum was tested in five classrooms with a total of 94 students. Prior to implementation, an assessment of all fourth grade teachers in the district examined their perceived knowledge of infectious diseases and their perceived self-efficacy in teaching such content. Evaluation of student progress included student pre and post-tests to assess changes in knowledge. Upon completion of the unit, teachers evaluated the unit to determine its relevance, effectiveness, and ease of implementation, and completed a post-test on their own knowledge and efficacy. Results indicate that the unit was effective in increasing student comprehension and interest in infectious disease prevention, and teacher efficacy in delivery of the material. This model curriculum can serve as a foundation to increase school health education in critical public health areas such as infectious diseases and preparedness, and provide an early introduction to public health careers.

Previous studies have demonstrated that the increased incidence of invasive disease caused by serogroup C Neisseria meningitidis in the United States during the 1990s was attributed primarily to strains belonging to the ST11 clonal complex. Subcapsular genotyping of a subset of isolates from Maryland identified distinct early and late clones defined by antigenic shift at the FetA outer membrane protein. Representational difference analysis (RDA) was used to identify additional genetic differences that may have contributed to the emergence of the late clone. A collection of serogroup C isolates representative of the early and late clone was subjected to pulsed field gel electrophoresis (PFGE) to determine genetic relatedness among the isolates and to identify a candidate tester/driver pair for RDA. RsaI-digested tester genomic DNA (late clone) was ligated to specific adaptors followed by two rounds of subtractive hybridization with RsaI-digested driver genomic DNA (early clone). PCR amplification of subtracted tester DNA with adaptor specific primers generated at least three late clone-specific bands that were absent from the early clone. These products were cloned and sequenced and confirmed by Southern blotting with tester and driver digoxigenin-labeled genomic DNA probes to be tester specific. A BLAST search of late clone-specific sequences identified homology to either IS1301 or pJS-B plasmid N. meningitidis sequences. PCR with primers specific to either IS1301 or pJS-B plasmid sequences amplified these elements from late clone isolates but not from early clone isolates. Thus, RDA successfully identified two unique genetic elements present in an emergent N. meningitidis serogroup C ST-11 clone that had undergone antigenic shift at FetA. Further investigation is required to determine the potential role of these elements in clonal emergence and N. meningitidis pathogenesis. The public health significance of this project stems from increased incidence of meningococcal disease being a major concern: morbidity and mortality increase, outbreaks produce panic and disruption in communities, public health agencies must respond for control and prevention, and mass immunization and antibiotic prophylaxis are often required.

Human immunodeficiency virus (HIV), which progresses into the disease commonly referred to as Acquired Immunodeficiency syndrome (AIDS), has become one of the world’s most destructive epidemics since its discovery in the early 80's. To date, the virus has killed more than 25 million people, with an average of 5 million newly infected cases last year alone. The HIV-1 genome is comprised of structural and enzymatic polyproteins as well as regulatory/accessory, which are essential for viral replication. Viral protein R (Vpr), which is identified as one of the regulatory/accessory genes, is responsible for carrying out several of the virus life functions, including virus replication, cell cycle regulation, apoptosis, and immune dysregulation. Through research of the virus, the disease has been divided into two very distinctive categories: Rapid Progressors (RPs) and Long Term Non-Progressors (LTNPs). The differences between these categories are due to the varying quasispecies, which infect the population, and ultimately disease progression. Several well-known mutations that occur within vpr have been associated with disease progression, linking them to one of the category types. Using a population from the Multicenter AIDS Cohort Study (MACS), patient vpr genotypes were analyzed and compared with current findings in research. Several of the patients deduced amino acid sequences revealed different gene variants, truncations, as well as a number of point mutations. Functional analysis revealed a decrease in cell apoptosis, which could have been caused by the observed point mutations. Further analysis is needed in order to determine if any other functions of the virus are disrupted due to the observed mutations. Because the virus has the ability to make changes within, as of right now the only hope in counteracting the effects of HIV is through the use of antiviral medication, such as HAART. But studies have shown that not everyone has the same positive effect when these drugs are administered. By understanding the virus and its pathogenesis, researchers will be able to develop new targets for therapeutic interventions. The public health significance of this project is to provide the valuable research that will lead towards such viable HIV-1 therapeutic interventions.

Abstract HIV/AIDS among students at Historically Black Colleges and Universities (HBCU) in the rural South is a growing public health concern. Lack of basic HIV/AIDS knowledge, underestimating risky behaviors, and lack of discussions relating to sexuality are some factors that contribute to the spread of HIV/AIDS within the HBCU population. Objectives of the study included the following: 1) To examine how the issue of HIV/AIDS is viewed by student leaders and organizations on campus; 2) To examine what student organizations and administrators are doing to educate the student body on HIV/AIDS; 3) To identify barriers that student organizations and administrators face when providing education to students; and 4) To examine how student leaders rate their leadership influence when interacting with their peers. This study utilized a qualitative research design in which student leaders and administrators were interviewed and asked a series of questions related to HIV/AIDS education on their campus. Student leader participants were recruited from a university site located in the rural South. Interviews were collected through a 30 minute tape-recorded session on campus. Interview data were analyzed using principles of grounded theory. The findings of the study suggest that student organizations could be a useful vehicle for HIV/AIDS peer-led interventions if their members are well trained and first address underlying issues such as cultural homophobia, sexuality, and stigma relating to HIV/AIDS. Administrators of the university should encourage students to be creative when addressing their peers about issues surrounding HIV/AIDS. Researchers and public health officials must create appropriate interventions to address issues surrounding HIV/AIDS before effective education of HIV/AIDS can take place. Public health significance: Improving HIV education among HBCU students presents a potentially effective strategy that addresses the larger issue of HIV/AIDS among African Americans by focusing their efforts and targeting a smaller sub-population first. The public health relevance of improving education among HBCU campuses is evident when considered in light of this promising possibility. This sub-population is particularly important because many of these individuals will become leaders of the African American community, and influence community behavior and attitudes towards HIV/AIDS.

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.