<p> The potential bioterrorism agents, <i>Francisella tularensis</i> and <i>Burkholderia pseudomallei,</i> are the etiologic agents of two life-threatening diseases, termed tularemia and melioidosis, respectively. An early diagnosis and a timely treatment regimen are crucial for successful therapeutic outcomes. However, bacterial isolation, which is known to be time-consuming and have low sensitivity, remains the 'gold standard' for diagnosis of these infections. Therefore, in the first study in this dissertation, we developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) targeting bacterial surface-expressed lipopolysaccharide (LPS) for rapid <i>F. tularensis </i> detection. A murine monoclonal antibody (mAb) specific to <i> F. tularensis</i> LPS, 1A4 IgG3, and its subclass family (1A4 IgG1 and 1A4 IgG2b, bearing the same antigen-binding site as mAb 1A4 IgG3) were isolated and used for assay development. Surface plasmon resonance (SPR) and competition ELISA were used to assess the binding affinities of the mAbs. We found that the assay developed using 1A4 IgG1 or IgG2b had better assay sensitivity compared to when the IgG3 was used. Interestingly, while the assay sensitivity was improved, we also found a decrease in functional affinity as a result of subclass switching. Direct ELISA and SPR suggested that the higher affinity of 1A4 IgG3 might be related to self-association, which correlated to high assay background and low assay sensitivity. Altogether, we demonstrated that IgG subclass switch could improve assay sensitivity by reduction of the assay background (through elimination of IgG3 self-association). </p><p> As for melioidosis, a rapid diagnostic targeting <i>B. pseudomallei </i> CPS (Active Melioidosis Detect (AMD&trade;) rapid test) has already been developed and is currently being assessed. However, a rapid immunoassay for differentiation between typical and atypical LPS strains has never been developed. This is important to advance our understanding of the epidemiology and pathology of melioidosis. Thus, in the second project, we developed antigen-capture immunoassays for typical and atypical LPS strain typing using CPS-specific mAb 4C4 for bacterial capture, and mAbs 4C7 and 3A2 for detection of typical and atypical LPS strains, respectively. In this study, two atypical LPS-specific mAbs (3A2 and 5B4) were successfully isolated; SPR results suggested that 3A2 is preferable for the assay. <i>B. pseudomallei</i> (174 strains) was used to evaluate the assay, and the results showed the assays have 98.8% accuracy, suggesting that they are effective and applicable for <i>B. pseudomallei</i> LPS typing.</p><p> Additionally, in the present work, we also investigate the <i>in vivo</i> fate of <i>B. pseudomallei</i> CPS using a murine model. The goal of this study was to improve our understanding of the appropriate clinical use of the AMD&trade; test. We found that CPS has a short serum half-life and is eliminated predominantly through the urine, suggesting that i) the presence of CPS in serum and/or urine may be indicative of active melioidosis, ii) CPS may be used as a marker to monitor and assess melioidosis treatment outcome, and iii) in addition to serum, urine (a noninvasive sample) has the potential to be used as a clinical specimen for the diagnosis of melioidosis. </p>

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2058. Adviser: Douglas Nixon.

<p> Lipopolysaccharide (LPS) of <i>Burkholderia pseudomallei</i> is known to be involved in innate immune response. <i>B. pseudomallei </i> expresses one of four LPS types: A, B, B2, or rough, all of which can be found in near-neighbor species. Nitric oxide (NO) and cytokines produced by murine macrophages were used to determine the immunogenicity of the different LPS types from <i>B. pseudomallei</i> and select near neighbor species. We found that type A LPS from <i>B. pseudomallei</i> 1026b produced a weaker immune response compared to type B and B2 LPS from <i>B. pseudomallei </i> MB296 and MB840, respectively, as well as compared to all the near neighbor strains. However, the gene expression levels of TLR2 and TLR4 do not relate to the results obtained from NO and cytokine experiments. There were no differences in the levels of TLR gene expression when comparing between the various LPS types. These results suggest that <i>B. pseudomallei </i> types B and B2 LPS are more immunogenic than <i>B. pseudomallei </i> type A LPS when it comes to NO and cytokine production and not with TLR gene expression.</p>

<p> Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, like scrapie, has been shown to involve the immune system, which most likely contributes to their relatively proficient horizontal and environmental transmission. While CWD prions probably interact with the innate immune system immediately following peripheral exposure, little is known about this initial encounter. In the first chapter of this dissertation we examined initial events in lymphotropic and intranodal prion trafficking by tracking highly enriched, fluorescent CWD prions from infection sites to draining lymph nodes. We observed biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. CWD prions rapidly reached draining lymph nodes in a cell autonomous manner within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) showed a strong dependence on Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells comprised the majority of prion-bearing cells in the mediastinal lymph node by six hours. As most B cells are mainly located in the follicles, acquisition of prions by these cells most likely occurred through interaction with resident DCs, subcapsulary sinus macrophages, or directly from the follicular conduit system. These data highlight a novel mechanism of cell autonomous prion transport, and a vital role for B cells in intranodal prion trafficking. </p><p> Upon entry into the draining lymph nodes, prion accumulation and replication on follicular dendrtic cells (FDCs) is greatly facilitated by the complement system. Complete elimination of CD21/35 significantly delays splenic prion accumulation and terminal prion disease in mice inoculated intraperitoneally with mouse-adapted scrapie prions. In the second chapter of this thesis we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. Ablation of complement receptors CD21/35 greatly diminished splenic prion accumulation and replication throughout the course of disease, similar to CD21/35 deficient murine PrP mice infected with mouse scrapie. Mice with deficiencies in CD21/35 showed a reduction in severity of neuropathology and deposition of misfolded, protease-resistant PrP associated with CWD. Prion infection resulted in translocation of CD21/35 to lipid rafts in B cells, and FDC expression of CD21/35 mediated a strong germinal center response that may be conducive to prion amplification. </p><p> Complement component C3 is a central protein in the complement system whose activation is essential for the elimination of infectious pathogens. C3 is the most abundant complement protein, being found in the blood at physiological concentrations of 1 mg/ml. Among the complement proteins, C3 is perhaps the most adaptable and multifunctional protein identified to date, having evolved structural characteristics that allow it to associate with over 25 different proteins. Previous experiments suggest a vital role of C3 in scrapie prion pathogenesis. In the last chapter of my thesis we showed that lack of C3 expression by 5037 mice either transiently or genetically leads to delays in prion pathogenesis. C3 impacts disease progression in the early stages of disease by slowing the kinetic rate of accumulation and/or replication of Pr^PRES. This slower kinetic increase in Pr^PRES correlates with an increase in survival time in mice deficient in C3. This delay in disease is in sharp contrast to the complete rescue we saw in CWD infected Tg 5037;CD21^-/- mice. This suggests a role for CD21/35 in peripheral prion pathogenesis independent of their endogenous ligands. </p><p> Taken together we show that the innate immune system dictates the course of CWD. We have discovered novel immune cells, trafficking pathways, and complement components important in CWD pathogenesis. These data not only highlight the key role of the innate immune system in CWD, but also provide a strong foundation for future immunological studies of prion diseases.</p>

<p> <i>Mycobacterium marinum</i> is an established surrogate pathogen for <i>Mycobacterium tuberculosis</i> because of <i>M. marinum </i>'s strong conservation of thousands of orthologous genes, lower risk, lower financial burden to researchers, and similar pathology in fish. This pathogen causes TB-like chronic disease in a wide variety of fish species and can mount superficial infection of human tissues. As in human TB, the microbe grows within the host macrophages, can mount life-long chronic infections, and produces granulomatous lesions in target organs. One of the fish species known to manifest chronic "fish TB" is the small laboratory fish, Japanese medaka (<i>Oryzias latipes</i>). Recently, our lab documented the progression of the bacterium from the lumen of the gut to underlying tissues and to target organs to mount infection. Since the bacterium can be observed crossing the epithelia to mount infection, I tested to see if mucosal immunity against a wild-type challenge could be induced by initially priming the fish to a live, attenuated vaccine strain. This thesis demonstrates that inoculation by ingestion is an efficient mode by which medaka can become infected and vaccinated with <i>M. marinum.</i> Furthermore, my thesis shows that orally vaccinating fish with a live, attenuated strain indeed provides protection in the gut, liver, and kidney against a virulent, wild-type challenge. </p>

<p> <i>Clostridium difficile</i> infection (CDI) is a bacterial disease affecting the lower gastrointestinal tract of patients whose normal colonic microbiota are altered, generally by administration of antibiotic therapies. <i>C. difficile</i> produces toxins that cause severe diarrhea, with potentially fatal complications in the immunocompromised. CDI has spread unabated despite the best prevention efforts of clinical practitioners. This dissertation is a broad-based study of several factors of importance in prevention and control CDI. In clinical environments, transport media are used to maintain the integrity of clinical samples for later laboratory testing. A transport medium for enteric bacteria was assessed as a preservative in outpatient fecal samples submitted for CDI testing. The transport medium used preserved <i>C. difficile</i> toxin out to five days. The possible effect of fecal pH and trypsin content on toxin stability was investigated. Fecal pH was ruled out as a factor due to CDI selected samples tending toward neutral pH. Trypsin was found to degrade toxin in controlled experiments; however, results from clinical samples were mixed. Oils and fatty acids, including virgin coconut oil (VCO), have antimicrobial effects on a variety of human pathogens. Use of natural products like VCO may reduce or prevent CDI by killing <i>C. difficile</i> while preserving the protective bowel flora. Virgin coconut oil (VCO) and three of its constituent fatty acids were evaluated for their toxic effect on <i>C. difficile</i> and were found to have bactericidal activity, the most potent of which was lauric acid. The outer surface of <i>C. difficile</i> spores is thought to contain proteins that are critical for attachment of the spores to surfaces, including human hands. This strong attachment assists in transmission of infectious spores; however, the source of this strong attachment in the spore remains unknown. Transmission electron micrography shows that <i>C. difficile </i> lacks a true exosporium, rather, they are coated in the remains of the mother cell. Results from subsequent fluorescence microscopy and ELISA with antibodies raised against spore coat proteins confirm that this residue is not part of the spore coat and can be easily removed by chemical treatment, increasing spore binding to anti-coat antibodies.</p>

<p> <i>Helicobacter</i> is a genus of Gram-negative helical bacteria. Colonization location divides the genus into two groups: gastric and enterohepatic. Many species of Helicobacter have become associated with or cause gastric, intestinal, and hepatic disease. Helicobacter canadensisis an enterohepatic bacterium that has become associated with enteritis and bacteremia. The first part of this study investigated the intestinal inflammatory response to <i>H. canadensis</i> in mouse explants. Colon and cecum explants taken from C57BL/6J mice were incubated with 10</p><p>8 CFU/mL <i>H. canadensis</i> for 24 hours and cytokine ELISAs were conducted on supernatants. IL-6, IL-1alpha, and TNF-alpha were not induced in either colon or cecum explants. The second part of this study involved infecting thioglycollate-elicited macrophages with 10</p><p>8 CFU/mL <i>H. canadensis</i> for 6 or 24 hours followedby cytokine ELISAs of supernatants. At 6 hours, IL-6 secretion was increased in thioglycollate-elicited macrophages infected with <i>H. canadensis </i> while IL-1alpha and TNF-alpha secretion was undetectable. At 24 hours, IL-1alpha secretion was significantly greater in thioglycollate-elicited macrophages treated with H. canadensis while IL-6 and TNF-alpha secretion was undetectable. Colon and cecum explant data support previous findings in our lab that <i>H. canadensis</i> does not induce inflammation, while data collected from thioglycollate-elicited macrophage cultures indicate that <i>H. canadensis</i> stimulates inflammatory cytokines.</p>

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0819. Adviser: Roderick Mackie. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7238. Adviser: Joel D. Ernst.

Thesis (Ph.D.)--University of California, San Francisco, 2007. / Source: Dissertation Abstracts International, Volume: 68-04, Section: B, page: 2232. Adviser: Jason G. Cyster. Includes supplementary digital materials.