Statins are cholesterol-lowering medications with immunologic properties. To assess the role of statins on incident immune-mediated conditions, a modified case-cohort study was performed using administrative databases from the Midwest Veterans Administration (VA) region. A comparison sub-cohort was formed by randomly sampling 10,000 subjects with medical and pharmacy benefits during fiscal year (FY) 2002. Cases were identified by inpatient or outpatient medical claims using International Classification of Disease, Ninth Revision (ICD-9) codes between FY 2003-2004. All subjects needed at least one year of medical claims and at least one pharmacy claim. The incident cases (n=28,642) included non-mutually exclusive groups of immune-mediated (n=2,327), infectious (n=8,221), and non-immunologic (n=10,730) diagnoses. Demographic and medical variables were obtained from FY 2001-2004, and pharmacologic data from FY 2002-2004. Cox proportional hazards regression modeling was used to estimate hazard ratios for the current statin use (within the last 180 days) and former statin use, compared to non-users, including time-dependant variables for demographic factors, comorbidity as measured by Elixhauser and Chronic Disease Score variables, medications, and visit rates after initiating statins. Current statin use was associated with decreased diagnoses rates of psoriasis; rheumatoid arthritis; inflammatory bowel diseases, including ulcerative colitis and Crohn disease; diffuse connective tissue diseases, including systemic lupus erythematosus; ankylosing spondylitis; bacterial pneumonia; urinary tract infection; cellulitis; sepsis; candidiasis; osteomyelitis; and tuberculosis. Former statin use was also associated with increased rates of polymyalgia rheumatica, sepsis, and osteomyelitis. Statin use was not associated with other spondylitis, multiple sclerosis, thyroiditis, sarcoidosis, temporal arteritis, influenza, shingles, histoplasmosis, or pyelonephritis. Although current statin use appeared protective for some study conditions, selection bias, misclassification, healthy user effects, adherence bias, confounding by indication, and surveillance bias were considered as possible explanations of the study findings.

Because of the increasing number of serious risk factors which predispose a normally immune competent host to infection, the incidence of systemic fungal infections is steadily increasing. This epidemiological rise has especially become apparent since the onset of the AIDS epidemic. Amphotericin B is the drug of choice for these life-threatening mycotic infections. Complications due to drug toxicity, however, severely limit amphotericin B's clinical usefulness. The major complication associated with the administration of amphotericin B is renal toxicity. Research has indicated, however, that besides its antifungal activities, amphotericin B may act as an immune stimulant of both humoral and cellular responses. A new formulation, liposomal amphotericin B, has been developed which has proven to be significantly less toxic to the kidneys. Research has suggested that liposomal amphotericin B may also act as an immune stimulant. Recent reports have also suggested that its stimulatory capabilities may possibly exceed those of the non-liposomal preparations. The purpose of this study was to quantify the specific effects of amphotericin B and liposomal amphotericin B on the in vitro indices: cellular viability, B- and T-lymphocyte proliferation and macrophage activation as indicators of immune system functions. Spleen cells from immune normal, and immune compromised BALB/c female mice were harvested following euthanasia and incubated in the presence of the two drugs. Drug doses were chosen to correlate with those surrounding clinically relevant plasma concentrations. Cyclosporine and cyclophosphamide were used as immune suppressants to simulate organ transplant patients and patients receiving cancer chemotherapy, respectively. Results indicated that amphotericin B consistently reduces the ability of B-cells and T-cells to proliferate and the ability of macrophages to produce interleukin-1. Though direct cytotoxicity may play a part in these assays, it is probably minor because viability studies show no more than a ten percent reduction due to amphotericin B compared with its liposomal analogue. Liposomal amphotericin B was shown to be non-toxic in each of the immune parameters. It appeared that liposomes may be an important means of delivering more drug to a host infected with a fungal organism without further compromising the patient's already suppressed immune system.

In the well known SIR endemic model, the infection-free steady state is globally stable for R0 < 1 and unstable for R0 > 1. Hence, we have a forward bifurcation when R0 = 1. When R0 > 1, an asymptotically stable endemic steady state exists. The basic reproduction number R0 is the main threshold bifurcation parameter used to determine the stability of steady states of SIR endemic models. In this thesis we study extensions of the SIR endemic model for which a backward bifurcation may occur at R0 = 1. We investigate the biologically reasonable conditions for the change of stability. We also analyse the impact of di erent factors that lead to a backward bifurcation both numerically and analytically. A backward bifurcation leads to sub-critical endemic steady states and hysteresis. We also provide a general classi cation of such models, using a small amplitude expansion near the bifurcation. Additionally, we present a procedure for projecting three dimensional models onto two dimensional models by applying some linear algebraic techniques. The four extensions examined are: the SIR model with a susceptible recovered class; nonlinear transmission; exogenous infection; and with a carrier class. Numerous writers have mentioned that a nonlinear transmission function in relation to the infective class, can only lead to a system with an unstable endemic steady state. In spite of this we show that in a nonlinear transmission model, we have a function depending on the infectives and satisfying certain biological conditions, and leading to a sub-critical endemic equilibriums.

Master of Science / The role of the microbiology laboratory is (1) to provide infection control information, so that highly transmissible isolates may be identified and appropriate control measures instigated as rapidly as possible and (2) to provide adequate information to the clinician enabling correct antibiotic choices to be made, particularly in the critically ill. Microbiological data is by definition slow as it is culture dependent: this study focused on the development of genetic, culture-independent methods for detection of resistance in nosocomial pathogens that could be introduced into the routine microbiology department and would fit into the routine workflow with a consequent reduction in time to result. Initially a duplex real-time polymerase chain reaction was developed for the rapid identification and detection of S. aureus and methicillin-resistance. This was optimised for immediate as-needs testing of positive blood cultures signalling with “Gram positive cocci, possibly staphylococcus” evident on Gram stain, on a random access real-time PCR platform. This technology, allowing early identification of S. aureus and its susceptibility to methicillin, by simple automated methodology, may soon become the standard for all microbiology laboratories servicing the critically ill. The second part of the study involved the development of a selective broth and multiplex PCR for detection of three important nosocomial isolates at this institution, methicillin-resistant S. aureus (MRSA), carbapenem-resistant Enterobacteriaceae, and multi-resistant Acinetobacter baumannii (MRAB). A multiplex PCR using four primer sets was designed to detect low colonisation levels of these isolates after overnight incubation in selective broth, significantly reducing the time to result and associated costs. This potentially useful epidemiological screening tool is practical, reproducible and sensitive with the potential of moving to an automated test (using real-time PCR, for example) in the future. The availability of early negative results judged by simple visual scanning (or by densitometry), means that the result is less operator-dependent, potentially reducing error rate. The last part of the study dealt with an important resistance phenotype, aminoglycoside resistance. There had been no recent comprehensive local surveys performed to determine the frequency of aminoglycoside resistance amongst the Enterobacteriaceae, or to identify the genetic determinants and their transmissibility. The isolates collected for the study were all resistant to at least one of gentamicin, tobramycin or amikacin. Identification of integron cassette arrays and use of specific internal primers identified at least one genetic determinant for gentamicin and tobramycin resistance in 22 of 23 isolates. Three isolates had two aminoglycoside resistance genes, and three isolates had three aminoglycoside resistance genes identified (Table 6.1). Transferable gentamicin-resistant plasmids were predominant amongst Klebsiella spp., but less so amongst Enterobacter spp. and E. coli. Gentamicin-resistant Klebsiella spp. were often ESBL positive, the genetic determinants of which were typically co-transferred on a conjugative plasmid. The importance of screening at a local level was demonstrated by the unexpected predominance of aac(6')-IIc amongst Enterobacter spp. and the detection of a new gene (aac(6')-LT). This part of the study has provided an understanding of the primary aminoglycoside resistance genes present in the local setting and their association with other resistances. This knowledge will allow development of assays for patient screening (clinical isolates and colonising flora), to better understand the epidemiology of aminoglycoside resistance and to allow better choice of antibiotic therapy related to presence or absence of these genes.

Pseudomonas aeruginosa (P. aeruginosa) is a frequently isolated pathogen that causes septicaemia and chronic respiratory infection. It exhibits a higher mortality rate than other gram-negative bacteria and the need for effective immunotherapy is emphasised by the frequency of antibiotic resistance associated with this organism. Mucosal immunisation with a whole killed cell P. aeruginosa vaccine has previously demonstrated a significant immune response in both rodent studies and human trials. This study is a continuation of that research, with the major goal being the identification of a purified protein antigen that could form the basis of a mucosal vaccine against P. aeruginosa. Specifically, the aims of this study were the development of purification protocols for the isolation of previously untested protein antigens, assessment of the efficacy of these antigens to enhance bacterial clearance in an animal model of acute respiratory infection, determination of the immune parameters that are associated with the resolution of P. aeruginosa respiratory infection and finally, cloning of an identified antigen which demonstrated vaccine efficacy. Protocols were established to isolate proteins for use as antigens in immune response studies. The proteins purified in this study were Pa 13, Azurin, acyl carrier protein (ACP), Amidase, Aminopeptidase, KatA and Pa70. These proteins were used to immunise rats by intestinal intra-Peyer's patch (IPP) inoculation and intratracheal (IT) boost. The immunisation protocol employed was designed to target mucosal antigen-specific immune responses where the route of immunisation, Peyer's patch (PP) intestinal inoculation, is akin to the oral delivery of antigens to the gut-associated lymphoid tissue (96). Investigations of a previously uncharacterised antigen, Pa60, later identified this protein as the P. aeruginosa catalase, KatA. This study demonstrated enhanced bacterial clearance of both homologous and heterologous challenge following immunisation with KatA. The level of clearance demonstrated by KatA was promising when compared to that of killed whole cell immunisation. KatA was cloned and studies with the recombinant protein showed enhanced bacterial clearance commensurate with that of the native protein. Immunisations with other proteins identified four additional antigens which enhanced bacterial clearance; Pa13, Pa40, Pa45 and Pa70. Amino acid sequence analysis indicated that Pa13 may be a novel protein, whereas Pa40 was determined to be amidase and Pa45, aminopeptidase. Pa70 was not successfully sequenced. These proteins were effective in significantly enhancing bacterial clearance of homologous P. aeruginosa challenge. For KatA, Pa13 and Pa70, clearance was associated with a marked phagocytic cell recruitment. In contrast, amidase and aminopeptidase demonstrated clearance with a minimal cellular response. Proteins; azurin and ACP were non-protective, failing to clear a live P aeruginosa challenge. Analysis of the antigen-specific responses of these nonprotective proteins and comparison with those antigens which enhanced bacterial clearance were used to determine factors that may contribute to the resolution of an acute pulmonary infection. The study has demonstrated that mucosal immunisation using purified protein antigens can enhance the clearance of pulmonary infection with P. aeruginosa. It has also contributed to the understanding of immune responses to newfound antigens of P. aeruginosa and identified antigen-specific responses which confirm their potential as vaccine candidates.

Pulmonary infection caused by nontypeable Haemophilus influenzae (NTHi) is a significant cause of morbidity and mortality in both industrialised and developing countries. Previous work from this group resulted in the development of a respiratory model in rodents which has precipitated studies into the pathogenesis of infection by NTHi and investigation of the humoral and cellular mechanisms by which the bacteria are cleared from the lung. Comparison of mucosally immunised with non-immunised animals has demonstrated that not only are bacteria cleared more rapidly from the lungs, but there is a more rapid response and resolution of inflammatory factors in the mucosally immunised animals following challenge with NTHi. This inflammatory response is partially regulated by the ability of the mucosally immunised animals to rapidly produce, then control the production of tumour necrosis factor (TNF)-a. The TNF-a is produced by both macrophages and type I pneumocytes in the alveoli and also by the endothelial cells lining the blood vessels in the lungs. Immunocytochemical studies have identified cellular subsets accumulating in the lung at various time points following infection. Marked differences in cellular infiltration into the lung tissue were noted between immunised and non-immunised animals after challenge with NTHi. Immunised animals demonstrated an early influx of macrophages, CD8+ T cells and Y8+ T cells, followed by enhanced expression of the MHC-II marker, cellular infiltration by polymorphonuclear leukocytes and finally an increased number of both B cells and CD4+ T cells. In contrast, non-immunised animals did not demonstrate any proliferation nor extravasation of lymphocytes or increased expression of MHC-II before total bacterial clearance had occurred. Polymorphonuclear leukocyte infiltration occurred in the non-immunised animals, however at a later time than that seen in immunised animals. Challenging rodents to establish persistent infection highlighted the inappropriately aggressive white blood cell response to an initial challenge when bacteria may be masked by other substances, followed by the inability to amplify the polymorphonuclear leukocyte response on repeated challenge with NTHi. This hyporesponsiveness in the macrophage population, shown by lack of detectable TNF-a production, concomitant with low numbers of NTHi resulted in a continuously high number of macrophages in the alveoli and the possibility of increased damage to the lung tissue. The requirement for cell surface TNF-a and CD8+ T cells to enhance the clearance of NTHi from the lungs further strengthens previous in vitro and in vivo findings of the possible significance of cellular invasion as a mechanism of pathogenicity for NTHi. This thesis has contributed to the understanding of both the immune response to and the pathogenicity mechanisms of pulmonary infection with NTHi. Kinetic studies identifying cellular responses and cytokine levels have emphasised the ability of mucosal immunisation to increase the rate of immune response and resolution of inflammation to NTHi infection in the lung. Observations demonstrating a requirement for macrophages and CD8+ T cells in mechanisms associated with enhancing NTHi clearance from the lung will lead to further investigations.

Le champignon filamenteux Aspergillus fumigatus et la bactérie Chlamydophila psittaci sont deux agents pathogènes majeurs chez les oiseaux d'élevage. Bien que phylogénétiquement très distants, ces deux micro-organismes partagent un même mode de transmission par voie aérienne. La mortalité et la morbidité associées aux aspergilloses ou aux chlamydioses ont un impact économique qui n'est pas négligeable, en particulier dans les élevages de dindes. En France, les investigations autour de cas humains de chlamydiose identifient fréquemment un lien avec des élevages de canards, chez lesquels l'infection par C. psittaci s'apparente à un portage intestinal asymptomatique. Pour analyser la circulation des agents pathogènes dans les élevages avicoles, nous avons tout d'abord étudié la diversité génétique d' Aspergillus fumigatus et de Chlamydophila psittaci. Pour cela, deux nouvelles méthodes de typage moléculaire MLVA (Multiple Locus VNTR Analysis) ont été mises au point. Ces méthodes se sont révélées très discriminantes, rapides et faciles à mettre en œuvre. Dans le cas d' A. fumigatus, la méthode MLVA a permis de regrouper les génotypes en fonction de leur origine géographique. Elle a également permis d'analyser les modes de contamination d'un couvoir de dindes. Dans un second temps, nous avons analysé le pouvoir pathogène de ces deux microorganismes chez les oiseaux. Pour cela, deux modèles d'infections expérimentales ont été développés. Le premier modèle a permis de reproduire une aspergillose chez des poussins de 1 jour après inhalation d'un aérosol contenant des conidies d'A. fumigatus. Lors de la mise au point du modèle, l'impact de l'immunodépression et de la lignée de poulet a été évalué. Pour C. psittaci, un modèle reproduisant un portage intestinal de la bactérie chez des canetons mulards de 1 jour a été obtenu.

Phytophthora pini Leonian, recently re-established from P. citricola I, is a pathogen with a wide range of forest and nursery hosts. It causes foliar infections in horticultural nurseries in Oregon, where recirculating irrigation systems are common. Increased use of recirculating irrigation systems may contribute to disease caused by waterborne plant pathogens. Simulated nursery chamber experiments were utilized to investigate the relationship between Phytophthora pini zoospore inoculum dose and disease on Rhododendron. Disease incidence in this system was unexpectedly low despite high inoculum levels tested, so further experiments under lab conditions were conducted to explore possible causes. Detached leaf assays were conducted to determine how inoculum dose, leaf wounding, and agitation of zoospore inoculum affected foliar infection of Rhododendron. Wounded and nonwounded leaves were dipped into suspensions of zoospores that were either untreated, mechanically agitated by vortexing to cause encystment, or pumped through an irrigation sprayer system. Disease severity (lesion area) and incidence (number of lesions per leaf area) were measured over seven days. At inoculum levels of ���10,000 propagules/mL, motile zoospores infected both wounded and nonwounded leaves. Vortexing or pumping resulted in zoospore encystment, and inoculation with these treatments caused disease almost exclusively on wounded leaves. No disease symptoms were observed following inoculation with any inocula at ��� 2,000 propagules/mL. Scanning electron microscopy of leaves inoculated with encysted propagules showed germinated cysts with hyphae growing over and around stomata without entering leaf tissue until reaching a wound site. Nonwounded leaves inoculated with motile spores showed stomata penetrated by hyphae. These findings indicate the importance of zoospore motility in reaching suitable infection sites, and demonstrate the impact of zoospore encystment on disease development. This has implications for disease management in nurseries where pruning wounds are common and the pumping of infested irrigation water may influence zoospore motility and infectivity. / Graduation date: 2013

Thesis (Ph.D. (Nutrition))--North-West University, Potchefstroom Campus, 2007.

Cellular immune responses to Hepatitis C (HCV) epitopes are crucial for successful host response to HCV infection. We investigated a platform to assess specific and global immune responses in HCV infection. We identified 57 HCV peptides from literature (24 of CD4+, 33 of CD8+ specificity) and tested them in two peptide pools to assess specific response in non-transplanted and post-liver transplant (LT) patients. Robust interferon-gamma (IFN) response to CD4+ peptide and mitogen stimulation was seen in sustained virological clearance. IFN response to the CD4+ peptide pool could differentiate between SVR and NR with 82% accuracy. In patients with recurrent HCV post-LT, HCV-specific responses were attenuated, but global immune responses were preserved. Significantly lower specific (CD4+) and global immune responses (mitogen response) were observed in patients with advanced allograft disease (fibrosis score>2). Quantiferon-HCV may identify patients likely to respond to anti-HCV treatment, as well as post-LT patients with aggressive HCV recurrence. / Experimental Surgery