Hepatitis C Virus currently affects up to 3% of the world’s population. There is no effective vaccine yet available and the natural immune response to infection is largely inefficient. Progress has been made in isolating several broad-acting neutralizing antibodies that target the viral envelope protein E2. However, a dominant element of the epitopes targeted is an overlap with the highly conserved CD81 binding sites. Various E2 constructs were investigated as possible targets to be used in phage display panning of a combinatorial library of the phagemid vector pComb3H. HVR2 deletion showed optimal exposure of the CD81 binding sites and D535A disrupted known CD81 epitopes. A selection technique was designed to improve exposure of conserved sites on an E2 construct target molecule that disrupts CD81 epitopes while remaining conformationally correct. Optimisation of the screening methodology was used to assess the quality of enrichment of the library panning along with more efficient selection of specific clones. The approach adopted in this project isolated Fab clones specifically reactive to the protein target, one of which also showed preferential binding in acidic environments. Taken together, the information gathered on E2 and the implementation of the phage display method described will contribute to more effective ways of isolating novel antibodies.

published_or_final_version / Nursing Studies / Master / Master of Nursing

published_or_final_version / China Development Studies / Master / Master of Arts in China Development Studies

This dissertation examines factors associated with healthcare-associated infections (HAIs) in four acute care hospitals located in New York City. Specifically, this investigation focuses on the role that the physical environment plays with regard to patient-to-patient transmission. The initial analyses describe the scope of the problem by reporting the incidence of HAIs and antimicrobial resistance over a seven-year period in the study institutions. In total, 19,052 HAIs were identified among 761,426 discharges. HAI rates fell over time within all hospitals and for all organisms and infection types included in the study, and the odds of acquiring an HAI decreased significantly over time for all organisms. Resistance levels were stable for Enterococcus spp., Staphylococcus aureus, Acinetobacter baumannii, and Streptococcus pneumoniae. Multidrug resistance increased for Pseudomonas aeruginosa and decreased for Klebsiella pneumoniae, though imipenem resistance among K. pneumoniae climbed sharply in 2011. A systematic literature review is presented to summarize what is known and unknown about how patients’ exposure to infected or colonized concurrent roommates and prior bed occupants affects their risk of developing HAIs. Eighteen articles meeting the inclusion criteria were identified. More than half reported at least one statistically significant positive association between the infection/colonization status of a roommate or previous room occupant and the development of HAIs. Only a single article identified a statistically significant negative association. The remainder found no associations that reached statistical significance, though this may be due to the fact that they were insufficiently powered. The dissertation concludes with a matched case-control study designed to quantify the association between having a prior bed occupant or roommate with a positive blood, respiratory, urine, or wound culture and subsequent infection with the same organism. In a multivariable analysis controlling for patient characteristics and mutually controlling for each exposure, the odds of being exposed to a prior bed occupant with the same organism were 5.83 (95% Confidence Interval [3.62, 9.39]) times greater for cases versus controls and the odds of being exposed to a roommate with the same organism were 4.82 [3.67, 6.34] times greater.

The human non-integrin laminin receptor (LamR) is a multifunctional protein which is localised to a number of sub-cellular locations. LamR is a component of the ribosome and has a number of intracellular functions; it also acts as an extracellular receptor for laminin, growth factors, pathogenic microorganisms, prion proteins, toxins and the anticarcinogen epigallocatechin gallate (ECGC). Although LamR is present in most cellular compartments, its overexpression in many types of cancer cells suggests a vital role for LamR in tumor-cell migration and invasion. There are two isoforms of laminin receptor: the monomeric 37-kDa laminin receptor precursor (37LRP) and the mature 67-kDa laminin receptor (67LR). Although the precise molecular nature of 67LR is unclear, accumulating evidence strongly suggest that 37LRP can undergo homo- and/or hetero-dimerization with Galectin-3 (Gal-3) to form mature 67LR. A recent study demonstrated that both homo- and heterodimer LamR forms are present on the cell surface, where they form distinct populations. Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) are major causes of bacterial meningitis. The contribution of LamR in traversal of the blood brain barrier (BBB) by neurotropic viruses is well established and interaction with LamR was recently found to be critical for initiation of bacterial contact with the blood brain barrier (BBB). These bacteria bind LamR via the surface protein adhesins: meningococcal PilQ and PorA; pneumococcal CbpA; and H. influenzae OmpP2. Further investigations showed that the fourth and second extracellular loops of meningococcal PorA and OmpP2 of H. influenzae, respectively, are responsible for LamR binding. The work presented here consists of two complementary projects in which a number of approaches were taken to characterise the ligand-binding domains of LamR. The first project aimed to identify sites on LamR that are critical for binding the ligands of bacterial meningeal pathogens. The second project aimed to identify residues that contribute to the stability of LamR homodimers and the heterodimer with Gal-3. Several mutations were introduced into full-length human LamR, either by deletion mutations within the C-terminal domain (CTD) of LamR using inverse polymerase chain reaction (IPCR), or by single-amino acid substitution in the N-terminal domain (NTD) of LamR using site-directed mutagenesis. Protocols for large-scale expression of full-length and truncated LamR proteins in human cells were developed, as well as non-denaturing purification protocols. We hypothesised that bacteria-binding domains could be located on both the NTD and CTD of LamR. In vivo examination using ELISA assays, in which the interaction of LamR and whole bacteria or purified recombinant PorA or PilQ were tested identified several novel sites on LamR that contributed significantly to binding of the bacterial ligands. These sites include amino acids 206-229 and 263-282, located within the CTD, and Tyrosine 156 in the NTD, each of which contributed to the binding of meningococcal PorA, and more specifically it’s fourth extracellular loop. Furthermore, three sites on LamR comprising amino acids 206-229 and 263-282 within the CTD and Tyrosine 139 in the NTD were shown to contribute to binding pneumococcal CbpA, OmpP2 and Loop two of OmpP2 of H. influenzae. These results indicate that the three neuroinvasive bacteria share the same binding sites on LamR. Bimolecular fluorescence complementation (BifC) coupled with flow cytometry and confocal microscopy revealed the vital contribution of amino acid residues Arginine 155, Tyrosine 156 and Lysine 166 (all within the NTD of LamR) for the homodimerization and heterodimerization of LamR with Gal-3. The dimerization of two meningococcal host receptors, LamR and Gal-3, may help to extend spectrum of their bacterial adhesins, which may act cooperatively or synergistically at different stages of infection. Information about the residues in LamR that contribute to the stabilization of LamR dimers has implications for the role of LamR dimers in the pathogenesis of bacterial meningitis. Identification of bacteria-binding domains on LamR is of particular interest for development of vaccines or therapeutic interventions that provide protection against the three major meningitis-causing bacteria. The aim of the current work was first to localise the domains of LamR responsible for binding with PorA and PilQ of N. meningitidis; CbpA and OmpP2 of S. pneumonia, and OmpP2 of H. influenzae. Also, previous studies have shown conspicuous homodimerisation of 37LRP and heterodimerisation with Gal-3. Our second aim was to identify of amino acid residues involved in 37LRP self-association and heterodimer formation with Gal-3. In current work, several regions of LamR were hypothesised to constitute the binding site for the bacterial ligands; these predictions were based on previous studies on LamR binding domains and the crystal structure of laminin receptor. Also, to investigate both homo and heterodimerisation of LamR, the involvement of several putative amino acid residues within 37LRP in LamR dimerisation was was hypothesised.

No description available.

Homoeoprophylaxis (HP) is the use of homoeopathically prepared substances to prevent targeted infectious diseases in recipients. Its first use in an epidemic of Scarlet Fever was documented in 1801. It has been used throughout the world since then for both short-term and long-term preventative purposes. The effectiveness and safety of Golden�s long-term HP program using homoeopathically prepared substances to prevent targeted infectious diseases in recipients was tested through two research projects. The effectiveness of the program could not be established with statistical certainty given the limited sample size and the low probability of acquiring an infectious disease. However, a possible level of effectiveness of 90.3% was identified subject to specified limitations. Further research to confirm the effectiveness of the program is justified. Statistically significant results were obtained that confirmed the safety of the program both in absolute terms as well as compared to all other methods of disease prevention studied. It also appeared possible that a national immunisation system where both vaccination and HP were available to parents would increase the national coverage against targeted infectious diseases, and reduce the incidence of some chronic health conditions, especially asthma.

The gastric pathogen Helicobacter pylori infects more than half of the population worldwide. H. pylori manage to establish persistent infection, which would be life-long if not treated. In order to establish such an infection, this pathogen has to deal with the host immune system. H. pylori has certain characteristics which make the bacteria less announced to the host immune system. Additionally, for remaining in the harsh and acidic environment of the stomach with peristaltic movements and a high frequency of turnover of epithelial cells, H. pylori has developed different binding modes to structures present both in the mucus and on the surface of gastric cells and also to extracellular matrix proteins. Evidently, adhesion has a determinant role for a successful colonization by H. pylori. It has been shown that a small fraction of the H. pylori infection is in intimate contact and attached to the host epithelium. Despite its small proportion, this group maintains the persistency of infection. As there is no suitable in vitro system to mimic the human stomach for studies of H. pylori infection, we have developed the In Vitro Explant Culture technique (IVEC). By using this model we could show that H. pylori use the Lewis b blood group antigen to bind to the host gastric mucosa, during experimental conditions most similar to the in vivo situation. Furthermore, we could show that the host tissue responses to the bacterial attachment by expression of Interleukin 8 (IL- ), which will guide the inflammatory processes. Interestingly, by inhibition of bacterial adhesion through receptor competition i.e., by use of soluble Lewis b antigen, IL-8 production was hampered in the IVEC system, which further validates the presence of a tight relation between bacterial adhesion and induction of host immune responses. One of the inflammation signaling cursors in vivo is the upregulated sialylated Lewis x (sLex) antigen, an inflammation associated carbohydrate structure well established as a binding site for the selectin family of adhesion molecules. We could show that during chronic gastric inflammation, which is actually caused by the persistent H. pylori infection, the bacterial cells adapt their binding mode, and preferentially bind to sLex, which will provide an even more intimate contact with the host cells. This interaction is mediated by SabA, the H. pylori adhesin for sialylated oligosaccharides/glycoconjugates. By employing red blood cells as a model we could further demonstrate that SabA is identical to the “established” H. pylori hemagglutinin. We could also show that SabA binds to sialylated glycolipids (gangliosides) rather than glycoproteins on cell surfaces. Our result also revealed that SabA also binds to and activates human neutrophils. Such effect was unrelated to BabA and the H. pylori Neutrophil Activating Protein (HP- AP), which were not directly involved in the activation of neutrophils. Furthermore, phagocytosis of bacteria by neutrophils was demonstrated to be mainly dependent on presence of SabA. Interestingly, HP-NAP showed a possible role in guiding the bacterial adhesion during conditions of limited sialylation, i.e. equivalent to mild gastritis, when the tissue would be less inflamed and sialylated. In conclusion, H. pylori adhesion causes host tissue inflammation, then the bacteria will adapt to the new condition and bind to epithelial cells in a tighter mode by synergistic activities of BabA and SabA. Additionally, SabA bind to and activate human neutrophils, which will exacerbate inflammation responses and cause damage to host tissue. Thus, BabA and SabA are potential candidates to be targeted for therapeutic strategies against H. pylori and gastric disease.

Thesis (Ph. D.)--UCLA, 2009. / Vita. Description based on print version record. Includes bibliographical references (leaves 232-267).

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Chernoff, Yury; Committee Member: Bommarius, Andreas; Committee Member: Doyle, Donald; Committee Member: Lobachev, Kirill; Committee Member: Yi, Soojin. Part of the SMARTech Electronic Thesis and Dissertation Collection.