A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.

Although both are caused by Chlamydia trachomatis, Lymphogranuloma Venereum (LGV) presents differently from the infections caused by Oculogenital (OG) strains. The endothelium of blood and lymph vessels allows passage of cells to the site of infection. Endothelial cells also secrete chemokines and cell adhesion molecules which act as attractants and binding sites for various cellular immune components. Since LGV biovar affect the lymphoid tissue we studied the effect of C. trachomatis on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were infected with C. trachomatis LGV serovars L1, L2, L3 and the OG strain E at multiplicity of infection (MOI) of 1 and incubated for 24 hours. Stimulation of Interleukin-8 (IL-8) and monocyte chemokine protein-1 (MCP-1) chemokines and the intercellular adhesion molecule -1 (ICAM-1) were quantified by enzyme linked immunosorbent assays (ELISA). Transendothelial migration of neutrophils and monocytes was carried out in transwells. The lactate dehydrogenase (LDH) release assay was used to measure cell necrosis. Apoptotic cell death was analysed using the BioVisionTM CaspGLOW Fluorescein Caspase Staining Kit and DeadEndTM Colorimetric Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) system with the C. trachomatis Culture Confirmation kit as a counter stain. All Chlamydia trachomatis serovars (L1, L2, L3 and E) successfully infected and replicated in HUVEC after 24 hours of infection. Only L3 stimulated significantly higher production of IL-8, MCP-1 and ICAM-1 by HUVEC as compared to the negative control and mock-infected cells. However, the remaining LGV serovars (L1 and L2) and the OG serovar E showed no significant difference in the stimulation of IL-8, MCP-1 and ICAM-1 when compared to the controls. Comparison of LGV and OG serovars showed no significant difference between these two biovars in inducing production of IL-8 and MCP-1, but L3 stimulated ICAM-1 at a significantly higher level than E. There was no significant difference in the number of migrated neutrophils between untreated HUVEC, mock infected HUVEC and HUVEC infected with Chlamydia serovars. L2 and L3 had significally higher amount of migrated monocytes than the controls with L3 being the highest. L3 was the only serovar that had a significant level of cell death by necrosis. Apototic cells were observed in both uninfected and infected HUVEC which is due to normal cell turn over. None of the infected cells showed TUNEL positive nuclei. It can be concluded that L3 is more virulent than the other serovars during the first 24 hours of infection. Infection with C. trachomatis serovars does not seem to cause any cell death by apoptosis 24 hours post infection. The only cell death that occurs is by necrosis and only on serovar L3 infected cells. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.

Graduate

This thesis is focused on determining the structures of two anti-carbohydrate antibodies to understand how they achieve their specificity toward antigen. First, the structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide (LPS) has been determined by x-ray diffraction to 2.6 Å resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo (3-deoxy-α-D-manno-oct-2-ulopyranosonic acid) residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater avidity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high avidity and specificity independently of CDR H3. Second, the structure of a rabbit, single chain variable fragment against terminal mannose-6-phosphate (Man6P) residues, termed scFv M6P-1, has been determined by x-ray diffraction to 2.7 Å resolution with Man6P in the binding site. The Man6P pathway is the predominant pathway that transports acid hydrolases from the trans-Golgi to endosomes. Newly synthesized hydrolases first require the generation of Man6P markers before they can be transported. Maintaining a full complement of hydrolases within lysosomes is essential as failure to do so results in a number of different lysosomal storage diseases. Due to its specificity, scFv M6P-1 is able to diagnose lysosomal storage diseases mucolipidosis II and mucolipidosis III. scFv M6P-1 is also able to purify Man6P containing proteins which may be useful for enzyme replacement therapies. Additionally, scFv M6P-1 is one of the first structures of an antibody fragment that exhibits high specificity for a single carbohydrate residue and is one of the first structures of a rabbit antibody fragment. The specificity of scFv M6P-1, which gives it these unique attributes, is revealed in the structure where multiple hydrogen bonds are seen between the antibody’s heavy chain and the mannose ring while two salt bridges are observed between the antibody’s light chain and the phosphate moiety. Finally, scFv M6P-1 binds in such a way as to allow binding to proteins possessing terminal Man6P residues. Crystallographic challenges that arose during this research included poor crystal growth as well as twinning and these are explored while the structure of scFv M6P-1 complex with Man6P is analysed. / Graduate / 0487 / 0982 / 0307 / dyl.w.evans@gmail.com

Transmissible diseases are re-emerging as a global problem, with Sexually Transmitted Diseases (STDs) becoming endemic. Chlamydia trachomatis is the leading cause of bacterially-acquired STD worldwide, with the Australian cost of infection estimated at $90 - $160 million annually. Studies using animal models of genital tract Chlamydia infection suggested that the hormonal status of the genital tract epithelium at the time of exposure may influence the outcome of infection. Oral contraceptive use also increased the risk of contracting chlamydial infections compared to women not using contraception. Generally it was suggested that the outcome of chlamydial infection is determined in part by the hormonal status of the epithelium at the time of exposure. Using the human endolmetrial cell line ECC-1 this study investigated the effects of C. trachomatis serovar D infection, in conjunction with the female sex hormones, 17β-estradiol and progesterone, on chlamydial gene expression. While previous studies have examined the host response, this is the first study to examine C.trachomatis gene expression under different hormonal conditions. We have highlighted a basic model of C. trachomatis gene regulation in the presence of steroid hormones by identifying 60 genes that were regulated by addition of estradiol and/or progesterone. In addition, the third chapter of this thesis discussed and compared the significance of the current findings in the context of data from other research groups to improve our understanding of the molecular basis of chlamydial persistence under hormonal different conditions. In addition, this study analysed the effects of these female sex hormones and C. trachomatis Serovar D infection, on host susceptibility and bacterial growth. Our results clearly demonstrated that addition of steroid hormones not only had a great impact on the level of infectivity of epithelial cells with C.trachomatis serovar D, but also the morphology of chlamydial inclusions was affected by hormone supplementation.

Proefschrift Maastricht. / Lit. opg. - Met een samenvatting in het Nederlands.

Dissertation (Ph.D.)--Auburn University, / Abstract. Vita. Includes bibliographic references (p.139-152).

Dissertation (Ph.D.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references (p.85-113).

Thèses (M.A.)--Université de Sherbrooke (Canada), 1997. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Zugl.: Berlin, Humboldt-Univ., Diss., 2009