Thesis (M.Med.Sc.)--University of Hong Kong, 1982. / Also available in print.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Pathology. Includes bibliographical references.

The bursa of Fabricius is a central lymphoid organ essential for B cell development and generation of humoral immune responses in birds. The bursa is connected dorsally to the cloaca and continually “samples” the intestinal fluid phase for the presence of antigen. It is comprised of folds or plicae, which are seeded with individual follicles, where antigen processing and presentation occurs for B cell development as well as generation of antibody responses. The plicae are separated from the bursal lumen by interfollicular epithelium (IFE) and specific regions of follicle-associated epithelium (FAE). The FAE is comprised of M cell-like cells, which are specialized for transcytosis of antigen from the bursal lumen into underlying follicles. The uptake of particulate and soluble antigen in the bursa of Fabricius has been previously demonstrated, but how particle size affects their internalization within bursal FAE and the transport of particulate antigen into deeper follicles has not been explored. It has been shown that nanoparticles (NPs) ≤40 nm are most efficiently internalized by the epithelium of the murine intestine and vaginal tract. Therefore, we examined the uptake of 0.04 - 2 μm fluorescent polystyrene NPs in bursa at 1 hour or 6 hours after bursal administration to determine whether bursal epithelium is similarly constrained. Using immunofluorescence microscopy (IFM) and spectrofluorophotometry we found that NP uptake is inversely correlated with NP size. NPs ≤40 nm are most efficiently internalized by the bursal epithelium and bursal follicles, while NPs ≥500 nm are not effectively taken up by the bursal epithelium within 6 hours of administration. Moreover, once the size-limited capabilities of the bursal epithelium were established, we also found that bursal priming of chickens with 20 nm NPs conjugated to IMP-1, a protective antigen of an important avian pathogen Eimeria maxima, induces IMP-1-specific serum IgG following sub-cutandous boosting. We induced similar IMP-1-specific serum antibody responses in chickens primed bursally and sub-cutaneously boosted as those primed and boosted sub-cutaneously. Whether this route of immunization is able to elicit long-term protection must be investigated. A number of infectious diseases, including Infectious Bursal Disease Virus (IBDV), which directly targets the bursa of young birds and prevents the development of the immune system and causes mortality, are prevalent in the poultry industry. While vaccines exist for many of these diseases they confer only partial or incomplete protection; therefore, alternative vaccine strategies must be investigated. In addition, the bursa is an ideal in vivo model for the investigation of endocytic mechanisms for uptake of particulate antigen. Therefore, further characterizing the mechanisms of NP uptake at mucosal surfaces and their immunogenicity will be important for the development of NP-based mucosal vaccines for agriculturally relevant species such as poultry.

Resumo: Analisar o efeito do raloxifeno sobre o epitélio vaginal de mulheres pós-menopausa. Métodos: Estudaram-se prospectivamente entre novembro de 2004 a fevereiro de 2006, 80 mulheres na pós-menopausa. Quarenta pacientes receberam 6Omg/dia de raloxifeno (GR) e 40 mulheres compuseram o grupo não tratado (grupo controle, GC), pareado por idade e tempo de menopausa. O grupo tratado foi composto por pacientes com osteoporose de coluna lombar e/ou colo do fêmur. Foram excluídos aquelas com sinais e/ou sintomas de infecção do trato genital inferior e usuárias de terapia hormonal (TH) até seis meses prévios ao estudo. Os esfregaços vaginais foram coletados em dois momentos: inicial (MO) e após seis meses de seguimento (Ml). Para avaliação do epitélio vaginal foi utilizado o valor de maturação, com a contagem de células superficias, intermediárias e parabasais. Os esfregaços foram analisados por único citopatologista, sem conhecimento dos dados das pacientes. Para análise estatística empregou- se o teste t de Student, teste Wilcoxon Mann-Witney e o teste Qui-Quadrado. Resultados: Na comparação estatística inicial os grupos foram homogêneos. Comparando os momentos inicial e final, não foram observadas diferenças estatisticamente sígnífícativas nos valores medianos de maturação do epitélio vaginal e na porcentagem de células superficiais, intermediárias e parabasais entre os grupos. Não foi constatada correlação linear significativa entre o valor de maturação e a idade, o tempo de menopausa, o uso ou não de TH prévia, tabagismo e o índice de massa corpórea, em ambos os grupos. Conclusão: O tratamento com raloxifeno por seis meses não alterou o valor de maturação do epitélio vaginal em mulheres na pós-menopausa. / Abstract: To analyze the effect of raloxifene on the vaginal epithelium of postmenopausal women. Methods: Eighty postmenopausal women were studied prospectively between November of 2004 and February of 2006. Forty patients received 6omglday of raloxifene (GR), and 40 women comprised the non-treated group (control group, CG), paired by age and time of menopause. The treated group was composed of patients with osteoporosis of the lumbar column and / or femur. Those with signs and / or symptoms of infection of the inferior genital tract and users of hormonal therapies (HT) up to six months prior to the study were excluded. Vaginal smears were collected at two moments: initial (MO) and after six months of follow-up (Ml). To evaluate the vaginal epithelium, the maturation value was determined, along with counts of superficial, intermediate and parabasal cells. Smears were analyzed by only one cytopathologist, without knowledge of patient data. For statistical analysis Student's t test, Wilcoxon Mann Witney test and Chi-Squared test were employed. Results: In the initial statistical comparison the groups were homogeneous. Comparing the initial and final moments, no statistically significant differences were observed in median values of vaginal epithelial maturation or in percentage of superficial, intermediate and parabasal cells between the groups. There was no significant linear correlation between value of vaginal epithelial maturation and age, time of menopause, use or not of previous HT, smoking or body mass index, in both groups. Conclusion: Treatment with raloxifene for six months did not alter the maturation value of vaginal epithelium in postmenopausal women. / Orientador: Jorge Nahás Neto / Coorientador: Eliana Aguiar Petri Nahás / Banca: Paulo Traiman / Banca: Lúcia Simões Costa-Paiva / Mestre

Allergen proteases from a number sources including the filamentous fungus A. fumigatus, are thought to be important in the development of severe asthma through protease dependent interactions with the respiratory epithelium. The first aim of the thesis was to determine the effect of a variety of growth substrates on the secretion of proteases from different strains of A. fumigatus. The second aim was to investigate the effects of recombinant allergen proteases Asp f 5 and Asp f 13 expressed in the P. pastoris protein expression system and crude A. fumigatus culture supernatants on airway epithelial cells and determine whether protease induced interleukin 8 (IL-8) release from airway epithelial cells was dependent on the activation of protease activated receptor 2 (PAR-2).Results demonstrated that the AF293 strain of A. fumigatus secreted serine proteases during growth on pig lung homogenate medium and metalloproteases during growth on a casein based medium but suppressed protease secretion in Vogel's minimal medium. Analysis of the secretion and RNA levels of proteases in A. fumigatus showed that the matrix metalloprotease, Asp f 5 and the serine protease, Asp f 13 were up-regulated and secreted during growth in pig lung medium and that the matrix metalloprotease, Lap1 was up-regulated and secreted along with Asp f 5 and Asp f 13 in casein medium. This finding was confirmed using protease inhibitors and by using strains of A. fumigatus in which Asp f 5 and Asp f 13 genes were disrupted. These results suggest that A. fumigatus was able to detect different complex proteins available as substrates in its environment and regulate protease secretion accordingly. Furthermore, in several strains of A. fumigatus, protease activity was not suppressed by growth in Vogel's medium, suggesting differences in the regulation of protease secretion between strains. Both A. fumigatus culture supernatants and recombinant Asp f 5 and Asp f 13 produced in P. pastoris caused epithelial airway cell desquamination, and IL-8 release in a protease and dose-dependent manner. In addition, both recombinant Asp f 5 and Asp f 13 were both shown to cleave PAR-2 at a site that resulted in receptor activation.In conclusion, differences in the secretion of proteases between A. fumigatus strains and during growth of A. fumigatus on different media suggests a requirement for the standardisation of the preparation of A. fumigatus allergen extracts used both in clinical diagnosis of A. fumigatus allergy and in vitro and in vivo research studies. Furthermore, it is proposed that allergen proteases secreted by A. fumigatus may interact with a variety of host proteins including, matrix molecules, enzymes and receptors which may exacerbate allergic airway diseases.

The aim of this thesis was to study the regulation of proteinase secretion by epithelial cells (E-cells) derived from the epithelial cell rests of Malassez. Since these epithelial cell rests are present only in small numbers in-vivo, E-cells derived from porcine cell rests were cultured according to Brunette et al. (1976) and conditions chosen so that detectable amounts of the proteinases, neutral proteinase and plasminogen activator, could be obtained. The regulation of the secretion of these enzymes was investigated by varying the cell population density, adding E.Coli lipopolysaccharide to the cultures and altering the shape of the E-cells by both chemical and physical means. Cell population density modulated both neutral proteinase and plasminogen activator secretion. Neutral proteinase secretion was highest at low cell population densities and the activity decreased with increasing cell population density. Plasminogen activator secretion followed a similar pattern. Escherichia coli lipopolysaccharide (E.coli LPS) stimulated both neutral proteinase and plasminogen activator secretion. LPS extracted by the phenol method and LPS extracted by the trichloroacetic acid method caused similar increases in neutral proteinase activity but the increase in plasminogen activator activity was greater when the trichloroacetic acid extracted LPS was used. These findings support the proposal that bacterial LPS in contact with periapical tissues could stimulate the epithelial cell rests into increased production of proteinases, thereby contributing to the degradation of connective tissue associated with dental cyst formation. E-cell shape was altered by physical and chemical means. Addition of cholera toxin and dibutyryl cAMP caused E-cells to flatten. Phorbol myristate acetate, however, caused the cells to retract slightly. Mechanical stretching was applied to the cells to cause cell flattening, and cell rounding was effected by mechanical relaxation. Another method made use of E-cells grown on a substrate with V-shaped grooves which caused the cells to adopt a rounder shape more frequently than cells grown on a flat substrate. In addition, dishes coated with increasing concentrations of poly(HEMA) solution, which altered dish adhesivity to the cell, caused the cells to become less well-spread. In all experiments, a more flattened cell shape correlated with a reduced level of neutral proteinase and plasminogen activator secretion while a more rounded shape correlated with increased amounts of neutral proteinase and plasminogen activator secretion. / Dentistry, Faculty of / Graduate

An in vitro phagocytosis assay system was developed and characterized for studying the specific receptor-mediated phagocytosis of bovine ROS by bovine RPE cells. The phagocytosis of ROS was detected qualitatively by electron microscopy and quantitatively by treating RPE cells with radioiodinated ROS or by probing ROS-treated RPE cells with a radiolabeled antirhodopsin monoclonal antibody. The binding sites for various antirhodopsin monoclonal antibodies were localized as an essential step in their application as immunochemical probes for analysis of the structure and function of rhodopsin. Five monoclonal antibodies raised against rhodopsin have been shown to be directed against the N-terminal regions on the basis of their reactivity to an immunoaffinity purified 2-39 glycopeptide, a 2-16 tryptic glycopeptide and a 1-16 synthetic peptide as measured by radioimmune competition assays. Limited proteolysis, immunogold-dextran labeling and competitive inhibition studies identified two antirhodopsin monoclonal antibodies which bound to internal cytoplasmic loop regions of rhodopsin. Finally, the binding sites for these and other C-terminal specific antirhodopsin monoclonal antibodies were used to elucidate the proposed transmembrane helical model of rhodopsin. An antirhodopsin monoclonal antibody (rho 4D2), which bound to rhodopsin in glutaraldehyde-fixed ROS plasma membranes, was employed as an immunocytochemical probe in studying the possible role of rhodopsin in the binding and phagocytosis of rod outer segments. An immunoaffinity purified 2-39 N-terminal rhodopsin glycopeptide, a synthetic 1-16 peptide analogue of rhodopsin and phospholipid vesicles reconstituted with rhodopsin were all found to be ineffective in inhibiting the phagocytosis of ¹²⁵I-labeled ROS by RPE cells. In essence, these results provided compelling evidence that rhodopsin in the ROS plasma membrane does not function as the ligand for recognition by RPE cells. The molecular properties of the ROS cell surface ligand(s), which are involved in recognition by bovine RPE cells, were studied by limited-proteolytic digestion in conjunction with quantitative phagocytosis assays. Mildly trypsin-treated ROS were found to be less effectively phagocytized than untreated ROS by bovine RPE cells. Moreover, the glycopolypeptides (34kD and 24kD) released from the ROS cell surface by trypsin were capable of inhibiting ROS phagocytosis. The ROS plasma membrane specific, ricin-binding, 230kD glycoprotein was observed by SDS-gel electrophoresis and western blotting to be highly trypsin sensitive under these conditions. Hence, ricin affinity chromatography and immunoaffinity chromatography were employed in an attempt to purify this 230kD glycoprotein from ROS membranes. Enriched preparations of the 230kD glycoprotein were reconstituted into phospholipid vesicles and effectively used to inhibit the phagocytosis of ROS by RPE cells. In summary, a ROS plasma membrane specific, 230kD glycoprotein has been identified and isolated; this protein may act as a ligand in specific ligand-receptor interactions between ROS and RPE cells. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The cellular mechanism and regulation of KC1 reabsorption across the rectum of the desert locust Schistocerca gregaria has been studied using tracer fluxes, ion-sensitive microelectrodes, and electrophysiological techniques. Serosal addition of 1 mM cAMP stimulates transepithelial short- circuit current (I[sub=SC]) and net Cl absorption (J[sub=net;sup=Cl] ) 10-fold, increases transepithelial potential (V[sub=t]) 4-fold, and reduces transepithelial resistance (R[sub=T]) by 40-65%. The properties of locust Cl transport are not consistent with NaCl cotransport models proposed in other epithelia: i) Cl is absorbed from nominally Na-free saline, ii) there is no correlation between trace amounts of Na contamination and the rate of Cl transport, iii) exposure to cAMP increases ³⁶Cl influx across the apical border into rectal tissue without affecting ²²Na influx, iv) Cl-dependent I[sub=SC] is not inhibited by 1 mM ouabain (2 h) or 1 mN furosemide (1 h), v) J[sub=net;sub=Cl] is not affected when the apical Na electrochemical gradient is reduced by 85%, and vi) there is no relationship between Na and Cl net electrochemical gradients across the apical membrane. Cl/HCO₃ exchange is also unlikely since i) Cl-transport is electrogenic, ii) J[sub=net;sub=Cl] is insensitive to CO₂⁻ and HCO₃⁻ removal, and iii) Cl-dependent I[sub=SC] is not inhibited by 1 mM SITS or 1 mM acetazolamide after 1 h exposure. The cAMP-stimulated system is Cl-selective: Cl >> Br >> I,F,SCN,P0₄,SO₄.C₂H₃O₂,urate. The halide sequence suggests a site having high field strength. Cl-dependent I[sub=SC] is inhibited by low mucosal pH and high osmotic pressure. J[sub=net;sub=Cl] obeys Michaelis-Menten-type kinetics. Mucosal K increases both the K[sub=m] and V[sub=max] of transepithelial Cl absorption (K[sub=a] = 5.3 mM K). The active step in J[sub=net;sub=Cl] is at the apical membrane because net entry of Cl occurs against a large, unfavourable electrochemical gradient. Serosal cAMP and mucosal K directly stimulate the active step since both of these agents cause simultaneous increases in J[sub=net;sub=Cl] and the electrochemical potential opposing CI entry. Passive K transport in the mucosa-to-serosa direction is favoured across apical and basal membranes. Most K absorption (~84%) is electrically coupled to active CI transport under open-circuit conditions, however a small active component is apparent during exposure to cAMP. The response of V[sub=T] to transepithelial salt gradients depends strongly on the direction of the gradients, suggesting that locust rectum is a "tight" epithelium. Intracellular current and fluorescent dye injections reveal strong coupling between rectal cells. Flat-sheet cable analysis indicates that locust rectum becomes "tighter" during cAMP exposure, when transcellular conductance increases from 60 to 95% of the total tissue conductance. cAMP increases apical membrane K conductance and basal membrane CI conductance. K permeability is inhibited by high (physiological) K and osmotic concentrations. The driving force of CI transport is calculated by two independent methods and the results are interpreted in terms of an equivalent electrical circuit model for KCl reabsorption across locust rectum. / Science, Faculty of / Zoology, Department of / Graduate

The bladder urothelium is a stratified epithelium that interconnects with the ureters to form the urinary outflow tract. A defining feature of the urothelium is its luminal population of uroplakin-expressing Superficial cells that function to create a protective, waterproof barrier. As such, proper differentiation of urothelial cell types during development and regeneration is essential for bladder function. Previous work has demonstrated that the urothelium is derived from a transient endodermal progenitor population, termed P-cells. However, two remaining uncertainties are the timing and regulation of P-cell fate. Here we show through lineage tracing that the specification of P-cells into luminal, uroplakin-expressing cell types is temporally related to the timing of endogenous retinoic acid (RA) signaling. Selective inhibition of RA signaling in P-cells through ShhCre-driven expression of the RaraT403 dominant-negative (RaraDN) mutant receptor redirected cells towards an abnormal K14+ basal fate that underwent Notch-mediated stratification into a keratinizing squamous epithelium that largely resembled epidermis. Transcriptome analysis of mutant P-cells identified aberrant expression of transcriptional regulators that have critical roles in squamous differentiation and epidermal commitment. Interestingly, inhibition of RA signaling in P-cells also resulted in improper connections between the ureters and the bladder through reduced Caspase 9-mediated apoptosis and remodeling of the common nephric duct. These observations demonstrate that RA signaling in bladder urothelial progenitor cells is required not only for proper epithelial differentiation, but also for regulating inter-organ signals that orchestrate morphogenesis of the urinary outflow tract.

Sexually transmitted infections (STIs) such as gonorrhea, herpes simplex virus 2 (HSV 2), hepatitis C, human papilloma viruses (HPV) and human immunodeficiency virus type 1 (HIV) are a global health concern affecting millions of lives. Although extensive efforts have been geared towards prevention and treatment of STIs, little progress has been achieved. Recently, efforts to develop microbicides have been focused on the commensal bacterial species that comprise the vaginal microbiome and their role in immunity and disease pathogenesis. The lower FRT which includes the cervix and vagina has endogenous bacterial species that are supported by the mucosal epithelium. Lactobaccilli are the dominant endogenous bacterial species in the vagina of most women; lactobacilli convert glycogen to lactic acid (LA) which maintains a low pH environment in the vagina and serves as a deterrent to infectious organisms. The purpose of this research project was to determine the effects of LA on vaginal integrity and inflammation in a vaginal epithelial cell (VEC) tissue model, and on the ability of the broadly neutralizing anti-HIV antibody, VRCO1, to inactivate HIV in vitro. Effects of LA exposure on the viability and integrity of vaginal epithelium were determined by histology, MTT assay and measurement of transepithelial electric resistance (TEER). In addition, an enzyme-linked immunosorbent assay (ELISA) was used to measure concentrations of cytokines secreted by the VEC epithelial cells in response to different doses of LA. Using TLR agonists to simulate infection in the VEC model, we tested the hypothesis that LA has anti-inflammatory properties that modulate immune responses of the vaginal epithelium. We assessed the effect of LA on the neutralization activity of the anti-HIV-1 monoclonal antibody VRCO1 in the TZM-bl HIV neutralization assay. Tissue morphology and integrity were not affected by exposure to LA. Low concentrations of IL-1β and RANTES were expressed by VEC tissues in response to L-LA treatment. VEC tissues expressed significantly elevated concentrations of IL-1RA (p<0.0001), an anti-inflammatory cytokine, in response to LA regardless of incubation time and LA doses. In addition, treatment of VEC tissues with Poly I: C in the presence of LA dampened upregulated expression of IL-1β, TNF-alpha and IL-6 in response to the TLR 3 agonist. LA also elicited significantly higher IL-1RA concentrations when apically added to the TLR agonist-treated VEC tissues. These data suggest that LA elicits an anti-inflammatory response in the vaginal epithelium. LA acidic conditions as low as pH 3.5 did not affect the ability of VRC01, to prevent HIV infection. We found that LA, at concentrations present in vaginal secretions of normal women, inhibited the inflammatory response to a TLR agonist, possibly due to upregulated Il-1RA synthesis. In addition, the functionality of VRCO1 in an acidic milieu was not diminished, providing evidence that antibodies can function in the low pH vaginal environment. This report highlights the potential use of LA in the vagina as a microbicide due to its virucidal activity and anti-inflammatory properties. It also indicates that monoclonal antibody-based vaginal microbicides will not be neutralized by LA. There is still a need to elucidate the exact mechanisms by which LA confers immuno-modulatory properties within the female reproductive tract.