Activated lymphocytes secrete many products including the lymphokine human lymphocyte mitogenic factor (HLMF). In preliminary experiments lymphocytes from peripheral blood and palatine tonsils were evaluated as possible sources of HLMF by evaluating their level of activation through screening their spontaneous and concanavalin A (con A)-induced blastogenic responses. Tonsil lymphocytes (TL) were found to have high spontaneous proliferation as compared to peripheral blood lymphocytes (PBL). Cells from both sources responded to con A by undergoing a typical blastogenic response. Because TL must be obtained septically, they are frequently cultured in the presence of the antimycotic agent, Amphotericin B (Am B). Since the primary and induced blastogenesis of TL were greatly inhibited by even low concentrations of Am B, those lymphocytes were considered unacceptable sources of HLMF. In contrast to TL the induced blastogenic responses of PBL were found to be augmented by concentrations of Am B less than 5 (mu)g/ml, but the drug appeared to provide no beneficial effect on the quantity of HLMF produced by the cells. HLMF appeared to be produced optimally in the first 48 hr of culture by 10('7) PBL/ml, cultured in Neuman-Tytell serumless medium which had been adjusted to 5 x 10('-5) M 2-mercaptoethanol, and 5-35 (mu)g con A/ml. Stability of the HLMF activity could best be maintained by immediate dialysis against 0.05 M NH(,4)HCO(,3) solution, followed by lyophilization and storage of the dried material at -80(DEGREES)C until use. Activity was retained at -80(DEGREES)C for greater than 3 months. The activity was diminished after exposure to 56(DEGREES)C for 30 min, and completely lost after treatment at 80(DEGREES)C for 10 min or 100(DEGREES)C for 5 min. HLMF was insensitive to trypsin and exposure to pH ranges 2-7. Separation of HLMF and con A blastogenic activities was accomplished by addition of ovalbumin followed by Bio-Gel P-100 column Chromatography. HLMF activity eluted in the 12,000-20,000 d and 30,000-50,000 d ranges. The lower molecular weight material was active in the pH range 3.4-4.6 as demonstrated by isoelectric focusing. The larger molecular weight fractions had a pI of 4.14 (+OR-) 0.97. HLMF activated T cells, B cells and unfractionated PBL in assay, with the T cell response being generally, but not always greater. The factor behaved in a dose dependent fashion when assayed against unfractionated PBL.

Macrophages play an important role in host antimicrobial immunity and in non-septic inflammatory reactions. Most studies on macrophage activation have focused on the roles of the T cell-produced cytokine, interferon-$\gamma$ (IFN$\gamma)$ and bacterial product, lipopolysaccharide (LPS). T cell-macrophage interaction is a critical step in initiating both specific and nonspecific immune responses to antigenic stimulation. The current study examines the role of cognate T cell-macrophage interaction in activation of macrophage effector functions and induction of macrophage early activation gene expression. Viable resting T$\sb{\rm H}$2 clone cells can activate IFN$\gamma$-primed macrophages to produce reactive nitrogen intermediates (RNI) or express cytostatic activity. The activating signal is mediated by cognate membrane contact between T cells and macrophages as evidenced by the ability of paraformaldehyde fixed anti-CD3-activated T$\sb{\rm H}$2 cells or plasma membranes isolated from the activated T cells to activate the IFN$\gamma$-primed macrophages. In contrast to the antigen-specific interaction of macrophages with viable resting T$\sb{\rm H}$2 cells, the activation of IFN$\gamma$-primed macrophages by fixed activated T$\sb{\rm H}$2 cells or by membranes from activated T$\sb{\rm H}$2 cells does not display antigen specificity. Fixed resting T$\sb{\rm H}$2 cells or plasma membranes isolated from the resting T cells can not activate the IFN$\gamma$-primed macrophages. Similar results are obtained with use of fresh splenic T cells to induce macrophage RNI production and cytostasis. Monoclonal antibody against CD4, which presumably blocks the interaction between CD4 (a co-receptor of T cell receptor) and class II MHC molecules on macrophages, inhibits significantly the activation of IFN$\gamma$-primed macrophages by viable resting T$\sb{\rm H}$2 cells but does not inhibit the ability of fixed activated T$\sb{\rm H}$2 cells to activate the macrophages. To examine the intracellular events in macrophages initiated by the cognate signaling, the expression of a panel of macrophage early activation genes, c-Myc, c-Fos, JE, IP10, D3, TNF$\alpha$ and IL-$\alpha$, are analyzed by dot blot hybridization. Plasma membranes from activated T$\sb{\rm H}$2 cells induce the expression of all these genes in macrophages stimulated for 1-4 hour. In contrast, the plasma membranes from resting T$\sb{\rm H}$2 cells are unable to induce the expression of most of the genes examined. These results suggest that the T cell-macrophage interaction involves reciprocal activation of both cells--an antigen specific activation of the T cells which results in the acquisition of T cell membrane components involved in antigen nonspecific activation of the macrophages. The nature of those T cell membrane components involved in cognate signaling of macrophage is currently being investigated.

Petroleum is one of the most important natural resources, but can also be problematic to environmental and human health. Petroleum is comprised of thousands of compounds, including polycyclic aromatic hydrocarbons (PAHs) and heterocycles, some of which are toxic and/or carcinogenic. Traditional analytical methods for environmental monitoring of low-level PAHs are time-consuming labor-intensive, and often laboratory-bound. Efforts to achieve timely, sensitive, and accurate analysis of PAHs in the field have become a priority for environmental research and monitoring. Antibody-based biosensors are presently being developed for environmental analysis. Anti-PAH antibody molecules can be coupled with electronic transducers to provide new biosensor technology for the rapid determination and quantification of PAHs. Although PAHs are not immunogenic on their own, advances in immunology have provided the means to develop antibodies to PAHs. Thiophenes, a defined subset of aromatic heterocycles, were selected as the target molecules for antibody development. Characterization of a monoclonal antibody (mAb) to dibenzothiophene revealed specificity for 3 to 5-ring PAHs and heterocycles. Therefore, the goals of antibody development were focused on developing additional antibodies to 2-ring PAHs and to alkylated PAHs. Characterization of antibodies to these novel targets revealed unexpected insights into antibody induction and specificity: namely suitable hapten sizes for small hydrophobic molecule recognition should be larger than one benzene ring, derivatization of the hapten target in immunogen synthesis must preserve structural characteristics, the utility of heterologous assay formats can improve antibody inhibition, and high antibody titers can result in limited assay sensitivity. The anti-dibenzothiophene mAb 7B2.3 was employed, along with a fluorescence-based transducer, for the generation of a new biosensor for PAHs. The biosensor was utilized in a variety of different applications to determine dissolved PAH concentrations including: 1) sampling groundwater at a former wood-treatment (creosote) facility, 2) analyzing estuarine water during the dredging of PAH-contaminated sediments, revealing a plume of PAHs emanating from the dredge site, 3) frequent monitoring of phenanthrene (a 3-ring PAH) concentrations during a laboratory toxicological dosing study, and 4) monitoring PAH concentrations in stormwater runoff into both a retention pond and a river near a roadway. Overall, these applications demonstrated the utility of this biosensor for rapid analysis of PAHs in a variety of aqueous environments. The biosensor was operated on-site for both the estuarine and groundwater monitoring trials. The biosensor could process samples, produce quantitative measurements, and regenerate itself in approximately 10 minutes. Sample volumes of 400 mul could be used with little to no sample pretreatment. Most importantly, PAHs could be quantified down to 0.3 microg/l in the field using the sensor platform. These results were validated with conventional gas chromatography-mass spectrometry and high performance liquid chromatography analytical methods. This system shows great promise as a field instrument for the rapid monitoring of PAH pollution.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children. RSV induces variable disease severities in infected children. Severe cases of RSV-induced disease result in bronchiolitis, with a subset of children going onto develop long-term airway morbidities. The host antiviral T cell response is believed to contribute to the severity of pulmonary disease following acute RSV infection. However recent work has questioned the relative proportion of T cells that migrate into the lung tissue following a respiratory virus infection. Using in vivo intravascular antibody labeling, >80% of antigen-specific effector T cells were found to remain in the pulmonary vasculature following an intratracheal infection with the systemic viral pathogen lymphocytic choriomeningitis virus (LCMV). Therefore, I determined the proportion of RSV-specific CD4 T cells located within the lung tissue following infection. In contrast to recent reports with LCMV-specific CD8 T cells, I found approximately 85% of RSV-specific CD4 T cells were located within the lung tissue, indicating that the vast majority of virus-specific effector CD4 T cells are located within the lung tissue and not in the pulmonary vasculature following an acute RSV infection. Genetic variations can occur in the circulating RSV strains both within and between infectious seasons. Therefore, I questioned if different RSV strains could induce differential CD4 T cell responses. I demonstrate that RSV strains induce differential CD4 T helper responses, which are associated with the differential activation of the innate immune response. The RSV line 19 strain induced the early production of the pro-inflammatory cytokines IL-1Β and IL-6 resulting in an increased Th17 response as compared to the RSV strains A2 and 2-20. Blockade and/or neutralization of IL-1Β and IL-6 inhibited the ability of RSV line 19 to induce a Th17 response. These results demonstrate that RSV strains can differentially activate innate immunity that subsequently influences the type of adaptive immune response. This in part may contribute to differential RSV pathogenesis and the development of long-term airway morbidities observed in humans. IL-10 is a pleotropic cytokine able to suppress the adaptive immune response. Because the host adaptive immune response is believed to contribute to RSV-induced pulmonary disease, I evaluated the role of IL-10 in modulating the RSV-specific immune response. I found that IL-10 protein levels in the lung were increased following acute RSV infection with maximum production corresponding to the peak of the virus-specific T cell response. Multiple populations of CD4 T cells accounted for the majority of IL-10 produced in the lung including Foxp3+ Tregs, Foxp3- CD4 T cells that co-produce IFN-Γ, and Foxp3- CD4 T cells that do not co-produce IFN-Γ. Furthermore, RSV-induced disease severity was increased in both the absence of IL-10 and following IL-10 receptor blockade as compared to control mice. I also observed an increase in the magnitude of the RSV-induced CD8 and CD4 T cell response that correlated with increased disease severity following IL-10 receptor blockade. IL-10 receptor blockade during acute RSV infection altered CD4 T cell subset distribution, resulting in a significant increase in IL-17A-producing CD4 T cells and a concomitant decrease in Foxp3+ regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell-mediated pulmonary inflammation and injury. Overall, my data demonstrate that RSV-specific CD4 T cells migrate into the lung tissue with their differentiation influenced by the strain-specific activation of innate immune response. IL-10 is then produced by CD4 T cells to regulate the RSV-specific T cell responses and inhibit virus-induced immunopathology. My data indicate that there are multiple targets for immunotherapy for individuals with severe RSV-induced disease.

Methylation controlled J protein (MCJ) is a negative regulator of mitochondrial metabolism that has a substantial impact on overall cell metabolism and function. MCJ is highly expressed by naïve CD8+ T cells, however its role in their immune effector functions was unknown. In this dissertation, it will be demonstrated that MCJ restricts the mitochondrial metabolism of CD8+ T cells, in part by reducing respiratory supercomplex formation. MCJ deficiency enhances the immune effector functions and memory responses of CD8+ T cells in a mitochondrial ATP dependent manner. As a consequence, protection to influenza virus infection is substantially improved. Reduced expression of MCJ therefore promotes viral immunity, however the loss of MCJ is not always beneficial. In cancer, decreased MCJ expression is correlated with ATP binding cassette (ABC) transporter mediated chemotherapy resistance and poor patient responses. This dissertation will also address the role of MCJ in chemoresistance. Increased mitochondrial ATP production due to MCJ deficiency is sufficient to fuel ABC transporter activity, thereby directly promoting chemoresistance. This can be reversed by restoration of MCJ function in chemoresistant cells. Overall, the results presented in this dissertation identify MCJ as a potential therapeutic target, as modulating MCJ expression can significantly affect the severity of viral infections and the responses to chemotherapy.

Semi-invariant natural killer T (iNKT) cells are critical components of the host immune response in peripheral tissues such as the lung, liver, and gut, and they play important roles in cancer, bacterial infections, autoimmunity, wound repair, and atherosclerosis. Tissue-resident iNKT cells exert their effects early in the developing immune response by rapidly producing a wide variety of cytokines and chemokines, and it was recently discovered that different tissues possess iNKT cell subsets that preferentially produce IFN-γ (NKT1), IL-4 (NKT2), or IL-17 (NKT17). Despite their critical role in the immune response, the mechanisms that regulate iNKT cell function in the periphery remain unclear. Signaling lymphocyte activation marker (SLAM) proteins are cell surface-expressed molecular switches that are expressed on all hematopoietic cells. The nine SLAM family receptors serve a variety of functions including promotion of cell-cell adhesion, regulation of cytokine production, co-stimulation, and inhibition. Importantly, SLAM family receptors are critical for the development of iNKT cells. Yet, numerous efforts to ascribe discrete roles of SLAM family receptors in iNKT cell function has proven difficult. We conducted a comprehensive analysis of SLAM family receptor co-expression on iNKT cell subsets in the lung, spleen, liver, and thymus and identified co-expression profiles that varied in a tissue and strain-dependent manner. Interestingly, we found that SLAM family receptor expression profiles varied among different iNKT cell subsets. In particular, we noted a close association of SLAMf6 expression with the NKT2 and NKT17 subsets in both the periphery and in the thymus. Further investigation using SLAMf6-deficient mice revealed a critical role for SLAMf6 in NKT2 and NKT17 subset development, and in iNKT IL-4 and IL-17 cytokine production in the periphery. This investigation also revealed that the SLAMf6high NKT2 and NKT17 subsets exhibited significantly higher proliferative capacity than the NKT1 subset and the NKT2 and NKT17 proliferation was dependent, in part, on SLAMf6 expression. Since Slam family genes are highly polymorphic, we next investigated whether these polymorphisms regulated iNKT function. We employed a B6.129 congenic mouse exhibiting impaired NKT cell function, in which a 6.6 Mbp 129/SvJ locus encompassing Slam genes was introgressed onto the C57BL/6 background. To test the hypothesis that Slam gene polymorphisms regulate iNKT cell function, we refined this genetic interval by generating B6.129 subcongenic lines and assessing iNKT cell function. Unexpectedly, we found that while Slam gene polymorphisms in this model do regulate iNKT cell function, the dominant regulator was in a 0.14 Mbp interval centromeric to the Slam genes. Further experimentation revealed that impaired iNKT cell development and function was associated with changes in the expression of Fcgr3 (Fc gamma receptor III) on iNKT cells, suggesting it as a novel candidate gene regulating iNKT cell function. Taken together, these data reveal for the first time a specific role for SLAMf6 on NKT2 and NKT17 subset development and function. In addition, these data identify Fcgr3 as a novel candidate gene that regulates iNKT cell subset development and cytokine production. Cumulatively, these data reveal the presence of discrete regulatory mechanisms at work in different iNKT subsets, a finding that has broad implications for our understanding of iNKT-cell mediated immunity.

CD4 and CD8 T cells play a vital role in mediating the clearance of viral pathogens following infection. Mice deficient- or depleted of their CD4 and/or CD8 T cells exhibit a diminished ability to control viral replication following infection and in some cases develop a persistent viral infection. CD8 T cells upregulate cytotoxic effector molecules such as granzyme B, Fas and TNF-related apoptosis-inducing ligand (TRAIL) that them to directly kill virus-infected cells. Following a systemic virus infection the CD8 T cell response is primed within secondary lymphoid organs, such as the spleen and lymph nodes (LNs). Although, it has been shown that the LNs are important for the generation of optimal CD8 T cell responses following systemic viral infections, the relative role of the spleen versus the LN in priming the CD8 T cell response is unknown. Studies in this thesis demonstrate that LNs, but not the spleen, are critical for the optimal generation of a CD8 T cell response following a systemic intraperitoneal (i.p.) lymphocytic choriomeningitis virus (LCMV) infection. Using adoptively transferred naïve LCMV-specific CD8 T cells, we demonstrate that the mediastinal LN (MedLN) serves as the initial draining LN and is responsible for priming the majority of the virus-specific CD8 T cell response following an i.p. LCMV infection. Moreover, the draining MedLN exhibits an increased frequency of CD62L- effector memory (TEM) CD8 T cells for up to 8 weeks following viral clearance. I demonstrate that the increased frequency of CD62L- TEM CD8 T cells is not due to residual viral antigen. Furthermore, a similar increase in CD62L- TEM CD8 T cells is found in the ipsilateral popliteal LN following a footpad LCMV infection. I demonstrate that the increased frequency of CD62L- TEM CD8 T cells in the draining LN is due to increased recruitment. CD4 T cells promote the generation of both effector and memory CD8 T cells either indirectly through their CD40-CD40L-dependent maturation of dendritic cells or through the production of cytokines such as IL-2 and IFN-γ that directly interact with CD8 T cells. CD4 T cells are also critical for the generation of germinal center B cells and promote the differentiation of activated B cells into memory B cells and plasma B cells. However, CD4 T cells often recognize epitopes derived from a broad array of pathogen-encoded proteins, making it difficult to accurately quantify the magnitude of virus-specific CD4 T cell responses. Therefore, I evaluated a large panel of activation and/or memory markers to determine a combination that could be used to reliably identify antigen-specific CD4 T cells following viral infection. I show that the integrins CD11a and CD49d are upregulated in an antigen-dependent manner on virus-specific CD4 T cells following LCMV infection. Furthermore, memory LCMV-specific CD4 T cells retain their CD11ahiCD49d+ expression pattern. Using CD11a and CD49d as surrogate makers for antigen-specific CD4 T cells, I show that approximately 50% of the CD4 T cells following LCMV infection are virus-specific, indicating that the virus-specific CD4 T cell response is substantially larger than previously recognized. Furthermore, I demonstrate that CD11a and CD49d can be used to accurately track newly-activated CD4 T cells following a heterologous virus challenge. In addition to LCMV, respiratory syncytial virus (RSV)-specific CD4 T cells are CD11ahiCD49d+. The two previously identified RSV CD4 T cell epitopes only account for ~3% of the CD11ahiCD49d+ CD4 T cell population during the peak of RSV infection, indicating that additional RSV-derived epitopes remain to be identified. Therefore, I used an overlapping peptide library spanning each of the RSV-derived proteins to identify novel RSV-specific CD4 and CD8 T cell epitopes. Using this approach, I identified 5 novel RSV-derived CD4 T cell epitopes and 4 novel CD8 T cell epitopes. Furthermore, I demonstrate that stimulation of CD4 T cells with 17-mer peptides results in over a 2-fold increase in the frequency of responding CD4 T cells as compared to stimulation with the commonly used 15-mer peptides. Collectively, the data shown here provides new insight into where and how the CD8 T cell response is initiated following a systemic virus infection, as well as provide a novel approach to track the endogenous CD4 T cell response following viral infections.

Demyelination in the CNS is known to involve several immune effector mechanisms, including complement proteins. For this dissertation project the central hypothesis that C3 and downstream effector complement proteins exacerbate demyelination through activation of glial cells was tested. To investigate the role of C3 and downstream complement proteins in demyelination and remyelination pathology in vivo we utilized the cuprizone model. We used C3 knockout mice (C3-/-), which are lacking the central C3 protein and subsequently all downstream complement effector proteins, and transgenic mice expressing C3a or C5a under the control of the glial GFAP promoter. Interestingly, we found no changes in demyelination or remyelination pathology between C3-/- and control mice. However, C3a and C5a transgenic mice had exacerbated demyelination and slightly delayed remyelination in the corpus callosum compared to WT mice. Transgenic mice had increased cellularity in the corpus callosum due to increased activation and/or migration of microglia. There was also evidence of T cells in the corpus callosum during demyelination in C5a transgenic mice, suggesting C5a may modulate BBB permeability. During early remyelination oligodendrocytes migrated to the corpus callosum in higher numbers in C3a and C5a transgenic mice, thus enabling these mice to remyelinate as effectively as WT mice by the end of the ten week study. To determine the effects of anaphylatoxins on individual glial subsets, we created murine recombinant C3a and C5a proteins. We found that the MAPK pathway proteins JNK1 and ERK1/2 were activated in glia upon stimulation with recombinant anaphylatoxin proteins. When microglia and mixed glial cultures were stimulated with C3a and/or C5a, we observed an increase in the production of proinflammatory cytokines and chemokines. In contrast, anaphylatoxin-treated primary astrocytes had suppressed cytokine and chemokine production compared to untreated astrocytes. In vitro, primary microglia and astrocytes did not significantly migrate in response to stimulation with C3a or C5a proteins, suggesting migration may not be a primary anaphylatoxin-mediated function in the CNS. Overall, our findings show that anaphylatoxin production in the brain plays a negative proinflammatory role during demyelination and that anaphylatoxin proteins can activate individual subsets of glia, initiating the production of inflammatory mediators.

Objective: Globally, about 50 to 100 million patients are infected with dengue fever per year and the average mortality rate is about 3.5 to 5% in Asia. Because of appropriate geographic location and cultural factors, dengue fever has been the important subject of infectious disease that Taiwan faces. In order to control and prevent the spread of dengue fever effectively, how to diagnose the suspected case correctly by the clinical symptoms and to improve the early reporting rates become critical research questions. The purpose of this study is to explore the correlation between clinical symptoms and diagnosis of dengue fever, and the factors associated with being reported at the first outpatient visit among confirmed case by using Dengue Fever Survey Form, which including demographics, clinical symptoms, level of the first outpatient visit and whether the patient is reported at the first outpatient visit. Design: 593 virologically confirmed cases during 2006 Dengue endemics in Kaohsiung city were studied. The data were from Dengue Fever Survey Form, which were collected from January 1 to December 31,2006. Result: The mean age of cases was 46.45¡Ó19.06 years (range 2 years to 89 years). The most common symptoms were fever (97.3%), pain (75.2%), GI symptoms (74.7%), skin rash (49.2%), and thirsty/dry mouth (49.1%). Chi-square tests showed gender, age in group, viral type, whether dengue hemorrhagic fever or not, level of the first outpatient visit, pain and gastrointestinal symptoms were significantly associated with being reported at the first outpatient visit. The result of the analysis of logistic regression indicated that the significant predictors of being reported at the first outpatient visit were gender, age in group, viral type, level of the first outpatient visit, gastrointestinal symptoms and fatigue. Conclusion: Reporting of infectious disease is essential to detection of outbreaks, planning of control program and provision of appropriate treatment. Clinical symptoms of dengue fever and the level of the first outpatient visit will influence rates of being reported at the first outpatient visit. All medical providers involved in diagnosis and treatment of dengue fever should strengthen their knowledge by continuing learning in order to improve early identification rates. In addition, health department could try to improve the detection and reporting systems to make the reporting steps more convenient and advance early reporting rates.

This work comprises three projects that extend previous models to include features of practical significance for the statistical analysis of infectious disease data. In the first, we find from a simulation study how the degree of heterogeneity in the number contacts that individuals have affects the relationship between estimates of a pathogen's effective population size based on coalescent theory and the true prevalence and incidence of that pathogen. In the second, we find that aggregating data from many small outbreaks allows the parameters of stochastic epidemic models to be consistently estimated with a generalized linear model. Application of this method to a set of 77 small norovirus outbreaks reveals interesting differences in the transmission parameters between hospital and nursing-home outbreaks. In the third project, we gain insight into HIV contact networks in the United States by fitting data from a number of surveys to a simple stochastic model of a dynamic network. / text