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Citrobacter rodentium is a gut pathogen, which infects the distal colon of mice. It has many similarities to human Enteropathogenic and Enterohemorrhagic E.coli in terms of mechanisms of pathogenicity and methods of transmission. Like many other gram negative bacteria, C. rodentium has developed a complex and highly specialised protein secretion system, known as type three (T3SS), to deliver bacterial proteins into eukaryotic cells. By injecting effector proteins into host cell cytoplasm, the pathogens are able to modulate host cellular functions to facilitate their own survival and replication. There is growing evidence that Attaching Effacing (AE) pathogens can inject effector proteins into gut epithelial cells, which dampen pro-inflammatory responses. There is also evidence that EPEC, Yersinia and Shigella can inject effectors into immune cells and also modulate their function. The objective of this work was to visualise and identify the host cells targeted for type III secretion by C. rodentium, and consequently determine the effect on host immune responses. The method chosen to detect cells targeted for effector protein delivery was the β-lactamase reporter system, where cells loaded with the fluorogenic substrate CCF2-AM emit a green FRET signal upon excitation by UV light, but emit a blue signal when cleaved by β-lactamase. By creating reporter strain of C.rodentium expressing fusion proteins between NleD effector and β-lactamase, I was able to show that C.rodentium is capable of injecting NleD in a wide variety of murine cell lines including Swiss 3T3 fibroblasts, J774 macrophages, CMT93 epithelial cells and BW715 T cells in a dose and time dependent manner in vitro. In addition, I found that C.rodentium has the ability to inject proteins into the cytoplasm of immune cells isolated from mouse lymphoid tissues including the spleen, mesenteric lymph nodes and Peyer's patches. Detailed analysis of the types of cells injected with effectors in vitro showed that NleD- injected cells represented B cells, dendritic cells and T cells. After inoculation of mice with the reporter strain of CitropACYCnleD, the plasmid encoded reporter fusion remained stable throughout infection and was able to inject cells in vitro after passage through the mouse gut. Unfortunately under the conditions described in this study, we were unable to visualise any gut cells targeted for protein delivery by C. rodentium in vivo, thus highlighting the complex nature of the host pathogen relationships in the gut. Although there is a need to develop better strategies to visualise effector translocation in vivo, our study has demonstrated, for the first time, the ability of C. rodentium to target immune cells for effector injection in vitro.

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The general problem of alteration in viral susceptibility by the irradiation of monolayers of tissue cells in culture was examined in this study; specifically an increased susceptibility of HeLa (an established cell line which was derived from an epidermaid carcinoma of the cervix) to destruction by herpes simplex virus (the virus commonly associated with cold sores or fever blisters). The experimental procedures included the study of the radiosensitivity of the cell line, survival curve analyses expressed as the efficiency of plating, that is the per cent of viable cells capable of forming colonies visible to the unaided eye within twelve days, and finally infectivity studies. Tissue culture has proved to be a very useful tool in the study of radiation effects on tissues of higher animals since the effects of radiation can apparently be explained on the cellular level. Many types of cells have been studied but in all cases the most striking characteristic in irradiated populations is the increased cell size. Ionizing radiation effects both the reproductive and synthesizing capacity of cells with the former being the more sensitive. Some irradiated cells never divide while others divide several times before reproduction stops. After the cells stop dividing, they continue to grow in size forming giants because synthesis of the cellular constituents continues. Giant cells resulting from x-irradiation are more readily destroyed by the action of viruses than are non-irradiated cells. PUCK & MARCUS reported that NDV when plated on a mixture of giant and normal cells, destroyed more of the giants than normal cells. An enhancement of cell susceptibility following irradiation was also demonstrated for two enteroviruses by HSIUNG. The increased susceptibility of x-ray-induced giant cells to CPE of virus and the earlier release of virus by such cells was also demonstrated by LEVINE in studies with the Leon strain of type 3 polio. Many tissue culture-virus systems have been used to demonstrate alterations in susceptibility induced by x-irradiation, but few investigators have used a HeLa-HSV system to study this altered susceptibility using low levels of irradiation (50 roentgens to 500 roentgens) [TRUNCATED] / 2031-01-01

Malaria has been a major driving force in the evolution of the human genome. In sub-Saharan African populations, two neighbouring polymorphisms in the Complement Receptor 1 (CR1) gene, named Swain-Langley (Sl2) and McCoy (McCb), occur at high frequencies, consistent with selection by malaria. This thesis investigates the association between these two polymorphisms and severe malaria. Previous studies into this area have produced conflicting findings. Using a large case-control study of severe malaria in Kenyan children and statistical models adjusted for confounders, I found that the Sl2 polymorphism was associated with markedly reduced odds of cerebral malaria and death, while the McCb polymorphism was associated with increased odds of cerebral malaria. I also identified an interaction between Sl2 and α+thalassaemia, with the protective association of Sl2 greatest in children with normal α-globin. Following these epidemiological findings, I explored potential biological hypotheses which might explain them. The first approach examined whether the Sl2 and McCb polymorphisms affected how CR1 forms clusters on erythrocyte membranes, a process which is key in the binding and transfer of immune complexes from erythrocytes to macrophages. Using erythrocytes from Kenyan children, I performed immunofluorescence assays (IFAs) with confocal microscopy to quantify CR1 cluster number and volume. I found no association between the Sl2 and McCb polymorphisms and either the number or volume of CR1 clusters formed. The second approach investigated whether the cerebral malaria-specific associations seen with Sl2 and McCb might be due to expression of CR1 by human brain endothelial cells (HBEC). The immortalised cell line HBEC-5i was investigated for expression of CR1 using IFA, flow cytometry, western blotting, functional C3b degradation assays, mass spectrometry, immunoprecipitation and siRNA knockdown experiments. A pool of α-CR1 monoclonal antibodies recognised an intracellular antigen in permeabilised HBEC-5i cells which was a similar molecular weight to CR1 on western blotting. However, when the α-CR1 monoclonal antibodies were tested individually, only E11 recognised an HBEC-5i antigen. Further investigative approaches did not support the presence of CR1 on HBEC-5i cells, instead suggesting that E11 was not specific for CR1 and was instead recognising a protein in the Golgi apparatus. The final approach was to examine whether the Sl2 and McCb polymorphisms might influence the binding of the complement components mannose binding lectin, C1q and L-ficolin to the LHR-D region of CR1. I aimed to generate recombinant proteins of the LHR-D region which included the polymorphisms. Site-directed mutagenesis of the region was successful and subcloning and expression of the mutant amplicons will be performed at a later date. In summary, I have identified opposing associations between the Sl2 and McCb polymorphisms and cerebral malaria, which do not appear to be due to differences in CR1 clustering or expression of CR1 by human brain endothelial cells. My investigation into whether the polymorphisms might influence complement component binding is ongoing.

Perkinsus marinus is an alveolate protozoan parasite of the eastern oyster (Crassostrea virginica) which is responsible for much of the decline in United States oyster populations. Perkinsus marinus can be cultured in vitro, but is rapidly attenuated in the process. Supplementation of a protein-free medium with oyster products altered proliferation, changed protease expression in the parasite extracellular products (ECP), induced morphological forms typically seen in vivo, and partially reversed parasite attenuation. Supplements derived from dissected oyster tissues were used to determine if these changes could be differentially elicited. These supplements, with the exception of adductor muscle, reduced proliferation. Whole oyster and digestive gland/gonad supplements favored palintomic, rather than binary, fission. The total ECP protease activity was generally decreased in supplemented cultures, though gill/mantle supplements may have induced proteases. A low molecular weight subset of proteases was upregulated most effectively by heart- and adductor muscle-derived supplements. Serine proteases and other ECP proteins may be virulence factors. Attempts to create antibodies to study P. marinus cells and ECP have been largely unsuccessful due to poor immune responses and crossreactivity. Ultrafiltration-concentrated P. marinus ECP were poorly immunogenic and toxic to experimental animals. Immunogenicity was not substantially affected by heat denaturation or proteolytic inhibition. Co-administration of ECP with oyster plasma caused a suppression in the anti-plasma antibody response with restriction of epitope recognition. Analysis of medium constituents revealed that a surfactant, Pluronic F-68 (PF68), was immunosuppressive. Although isolated protein antigens from the ECP remained immunosuppressive, separation of the antigens from PF68 enabled antibody production. Five monoclonal antibodies were created against ECP from unsupplemented medium and were used to study ECP function, regulation, and mechanism of storage and release. ECP are secreted by release from the cell wall and from two morphologically distinct intracellular compartments. A sandwich ELISA allowed quantification of an ECP protein with significantly reduced expression in supplemented cultures. Another antibody, which specifically bound to trophozoite and tomont walls, was used to investigate morphological and antigenic changes during thioglycollate-induced formation of prezoosporangia, and confirm supplement-induced formation of prezoosporangia. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner.

Human palatine tonsil lymphocytes, when compared to peripheral blood lymphocytes (PBL), were in an activated state even though there was no in vitro stimulation. When these tonsil lymphocytes were cultured in the absence of serum and polyclonal mitogens or antigens, the supernatant fluid often inhibited the proliferative response of target PBL to con A. The extent of this suppression ranged from 22% to 84%, and target cell viability was 90% or greater. There was no evidence for the presence of immunoglobulins or (alpha)2-macroglobulin in whole supernatant fluids. The suppressor was partially denatured at 80(DEGREES)C and was rendered completely inactive upon exposure to 100(DEGREES)C for 5 min. It was trypsin sensitive, and had an apparent molecular weight of 100,000 or greater. The protein adhered strongly to DE-52 cellulose, and the most active material eluted with 0.4-0.6 M NaCl. The suppressor was active in the pH range 5.0 (+OR-) 0.6 as demonstrated by isoelectric focusing. Occasionally, supernatant fluids comprised material which augmented the expected response of con A stimulated PBL. The augmentor was 30,000 in molecular weight and was eluted from DE-52 cellulose in the 0.15-0.25 M NaCl range. Nearly all supernatant preparations tested contained a mitogenic substance which stimulated naive allogeneic human PBL without the necessity of co-stimulation by a mitogen. The mitogenic factor (MF) behaved in a dose dependent fashion and was evidently different from the augmentor since the MF stimulated PBL independently of lectin co-stimulation.

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.