A HEp-2 cell culture model was used to investigate the invasive properties of Campylobacter species. Two of twenty-five Campylobacter isolates did not invade HEp-2 cells, and one of these isolates did not adhere to the epithelial cells. Penetration of HEp-2 epithelial cells by C. jejuni was significantly (P < 0.05) inhibited with C. jejuni lysates and a MAb (1B4) in competitive inhibition studies. Immunogold electron microscopic studies revealed that the 1B4 MAb bound to the flagella and cell surface of low passage (invasive) C. jejuni M 96, whereas only the flagella of high passage (non-invasive) C. jejuni were labelled. Western blot analysis revealed that the 1B4 MAb identified an epitope on antigens ranging in size from 66 to 44 kDa in invasive and non-invasive organisms. Antigens were also recognized in lysates prepared only from invasive strains from 42 to 38 kDa. Sodium meta-periodate chemical treatment of C. jejuni lysates significantly (P < 0.05) affected its inhibitory capacity. Additionally, proteinase K and sodium meta-periodate treatment of lysates changed the mobility of antigens recognized by the 1B4 MAb. This suggests that the antigens required for epithelial cell penetration by C. jejuni may be glycoprotein in nature and that the functional binding site is dependent upon an intact carbohydrate moiety. Co-infection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, polio virus (LSc type 1), porcine enterovirus and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of C. jejuni was significantly increased for HEp-2 cells pre-infected with echovirus 7, coxsackievirus B3, and UV-inactivated (non-infectious) coxsackievirus B3 particles. Polio and porcine enterovirus had no effect on C. jejuni adherence and invasiveness. C. hyointestinalis and C. mucosalis, two non-invasive isolates, did not invade virus-infected HEp-2 cells. The increase of invasiveness of C. jejuni appears to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter but are not sufficient to allow internalization by non-invasive bacteria.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Chan Wan Lui Wendy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 158-162). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Abbreviations --- p.ix / List of Figures --- p.xii / List of Tables --- p.xvi / Table of Contents --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.2 --- History of malaria --- p.4 / Chapter 2.3 --- Malaria parasites --- p.4 / Chapter 2.4 --- Life cycle --- p.5 / Chapter 2.5 --- Potential use of malaria vaccine --- p.6 / Chapter 2.6 --- Merozoite surface protein 1 (MSP1) --- p.7 / Chapter 2.7 --- Potential use of MSPl --- p.8 / Chapter 2.8 --- Significance of MSPl C-terminal fragments --- p.9 / Chapter 2.8.1 --- Significance of MSP142 --- p.9 / Chapter 2.8.2 --- Significance of MSP119 --- p.11 / Chapter 2.9 --- Production of MSPl C-terminal fragments --- p.12 / Chapter 2.10 --- Plants as bioreactors --- p.12 / Chapter 2.11 --- Expression of MSPl C-terminal fragments in transgenic plants --- p.14 / Chapter 2.12 --- Phaseolin and its sorting signal --- p.19 / Chapter 2.13 --- Protein targeting signals --- p.20 / Chapter Chapter 3 --- Material and methods --- p.23 / Chapter 3.1 --- Introduction --- p.23 / Chapter 3.2 --- Chemical and enzymes --- p.23 / Chapter 3.3 --- Cloning --- p.24 / Chapter 3.3.1 --- MSP142 and MSP119 constructs --- p.24 / Chapter 3.3.2 --- Protein targeting fusion constructs --- p.24 / Chapter 3.3.3 --- GUS fusion Constructs --- p.30 / Chapter (a) --- Particle bombardment --- p.30 / Chapter (b) --- GUS fusion constructs for plant transformation --- p.32 / Chapter (c) --- Modified GUS fusion constructs --- p.38 / Chapter 3.4 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.39 / Chapter 3.4.1 --- Cloning of pSUNl --- p.40 / Chapter 3.4.2 --- Primer sequence --- p.45 / Chapter 3.5 --- Bacterial strains --- p.46 / Chapter 3.6 --- Particle bombardment --- p.46 / Chapter 3.6.1 --- Plant materials --- p.46 / Chapter 3.6.2 --- Microcarrier preparation and coating DNA onto microcarrier --- p.46 / Chapter 3.6.3 --- GUS assay --- p.48 / Chapter 3.7 --- Transgenic expression in Arabidopsis thaliana --- p.49 / Chapter 3.7.1 --- Plant materials --- p.49 / Chapter 3.7.2 --- Agrobacterium transformation --- p.49 / Chapter 3.7.3 --- Vacuum infiltration Arabidopsis transformation --- p.49 / Chapter 3.7.4 --- Selection of successful transformants --- p.50 / Chapter 3.7.5 --- Selection for homozygous plants --- p.51 / Chapter 3.8 --- Transgenic expression in tobacco --- p.51 / Chapter 3.8.1 --- Plant materials --- p.51 / Chapter 3.8.2 --- Agrobacterium transformation --- p.52 / Chapter 3.8.2.1 --- Preparation of Agrobacterium tumefaciens LBA4401 competent cells --- p.52 / Chapter 3.8.3 --- Leaf discs method for tobacco transformation --- p.53 / Chapter 3.8.4 --- GUS staining --- p.54 / Chapter 3.9 --- DNA analysis --- p.55 / Chapter 3.9.1 --- Genomic DNA extraction --- p.55 / Chapter 3.9.2 --- Genomic PCR --- p.55 / Chapter 3.9.3 --- Southern blot --- p.55 / Chapter 3.10 --- RNA analysis --- p.56 / Chapter 3.10.1 --- RNA extraction --- p.56 / Chapter 3.10.2 --- Northern blot --- p.56 / Chapter 3.11 --- Protein analysis --- p.57 / Chapter 3.11.1 --- Protein extraction --- p.57 / Chapter 3.11.2 --- Western blot --- p.58 / Chapter 3.11.3 --- Western blot analysis --- p.58 / Chapter Chapter 4 --- Results --- p.60 / Chapter 4.1 --- Transient assay of gene expression of MSP142 and MSPl19 --- p.60 / Chapter 4.1.1 --- Construction of the GUS fusion constructs --- p.60 / Chapter 4.1.2 --- Particle Bombardment --- p.63 / Chapter 4.2 --- Transgenic analysis of MSP142 and MSPl19 expression --- p.70 / Chapter 4.2.1 --- MSPl42 and MSPl19 constructs and transformation --- p.70 / Chapter 4.2.2 --- Selection of transgenic plants --- p.71 / Chapter 4.2.3 --- Southern analysis --- p.75 / Chapter 4.2.4 --- Northern analysis --- p.77 / Chapter 4.2.5 --- Western analysis --- p.79 / Chapter 4.3 --- Expression of the protein-targeting and GUS fused modified MSP1 constructs --- p.81 / Chapter 4.3.1 --- Construction of the fusion constructs --- p.81 / Chapter (A) --- Protein-targeting constructs --- p.81 / Chapter (B) --- GUS fusion constructs --- p.90 / Chapter B1. --- Constructs for transient assay --- p.90 / Chapter B2. --- Modification of GUS sequence --- p.96 / Chapter B3. --- Constructs for tobacco transformation --- p.100 / Chapter 4.4 --- Transient assay of GUS fused MP42 and MP19 constructs by particle Bombardment --- p.107 / Chapter 4.4.1 --- The GUS fusion constructs --- p.107 / Chapter 4.4.2 --- Modification of GUS --- p.112 / Chapter 4.5 --- Generation of transgenic tobacco --- p.116 / Chapter 4.6 --- Southern analysis --- p.120 / Chapter 4.7 --- Northern analysis --- p.126 / Chapter (A) --- Protein-targeting constructs --- p.126 / Chapter (B) --- GUS fusion constructs --- p.130 / Chapter 4.8 --- Western analysis --- p.133 / Chapter (A) --- Protein-targeting constructs --- p.133 / Chapter (B) --- GUS fusion constructs --- p.139 / Chapter Chapter 5 --- Discussion --- p.146 / Chapter Chapter 6 --- Conclusion --- p.157 / References --- p.158

by Lau On Sun. / Thesis submitted in: November 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 117-127). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Abbreviations --- p.viii / Table of Contents --- p.x / List of Figures --- p.xiii / List of Tables --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.1.1 --- Global situation --- p.3 / Chapter 2.1.2 --- Malaria parasite and its life cycle --- p.4 / Chapter 2.1.3 --- Need for a malarial vaccine --- p.5 / Chapter 2.2 --- Merozoite surface protein 1 and its fragments - the advanced malaria vaccine candidate --- p.7 / Chapter 2.2.1 --- Basic research on MSP1 --- p.7 / Chapter 2.2.2 --- Vaccine research on MSP1 --- p.8 / Chapter 2.3 --- Transgenic plants as recombinant protein production systems --- p.11 / Chapter 2.3.1 --- Characteristics --- p.11 / Chapter 2.3.2 --- Plant-based vaccine --- p.13 / Chapter 2.4 --- Expression of MSP 1 C-terminal fragments in transgenic plants --- p.15 / Chapter 2.4.1 --- Previous studies --- p.15 / Chapter 2.4.2 --- Plant-optimized MSP142 cDNA --- p.18 / Chapter 2.5 --- Phaseolin: its promoter and vacuolar-sorting signal --- p.20 / Chapter 2.6 --- Sorting of soluble protein to vacuoles in plants --- p.22 / Chapter 2.7 --- Winged bean lysine-rich protein and translational fusion strategy --- p.24 / Chapter 2.8 --- Hypotheses and aims of study --- p.26 / Chapter Chapter 3: --- Materials and Methods --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Chemicals --- p.28 / Chapter 3.3 --- Bacterial strains --- p.28 / Chapter 3.4 --- Chimeric gene construction --- p.29 / Chapter 3.4.1 --- Construction of the lysine-rich protein fusion constructs --- p.33 / Chapter 3.4.2 --- Construction of the phaseolin-targeting constructs --- p.37 / Chapter 3.4.3 --- Confirmation of sequence fidelity of chimeric genes --- p.42 / Chapter 3.4.4 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.42 / Chapter 3.5 --- Transgenic expression in Arabidopsis and tobacco --- p.44 / Chapter 3.5.1 --- Plant materials --- p.44 / Chapter 3.5.2 --- Agrobacterium transformation --- p.44 / Chapter 3.5.3 --- Arabidopsis transformation and selection --- p.45 / Chapter 3.5.4 --- Tobacco Transformation and Selection --- p.47 / Chapter 3.5.5 --- Genomic DNA isolation --- p.49 / Chapter 3.5.6 --- Southern blot analysis --- p.49 / Chapter 3.5.7 --- Total silique RNA isolation --- p.50 / Chapter 3.5.8 --- Northern blot analysis --- p.50 / Chapter 3.5.9 --- Protein extraction and SDS-PAGE --- p.51 / Chapter 3.5.10 --- Western blot analysis --- p.52 / Chapter 3.5.11 --- Enterokinase digestion of recombinant LRP fusion protein --- p.53 / Chapter 3.5.12 --- Deglycosylation studies of recombinant MSP142-AFVY --- p.54 / Chapter 3.6 --- Confocal immunoflorescence studies of MSPl42-AFVY in tobacco --- p.55 / Chapter 3.6.1 --- Preparation of sections --- p.55 / Chapter 3.6.2 --- Labeling of fluorescence probes --- p.55 / Chapter 3.6.3 --- Image collection --- p.56 / Chapter 3.7 --- Bacterial expression of MSP 142 and anti-serum production --- p.57 / Chapter 3.7.1 --- pET expression in E. coli --- p.57 / Chapter 3.7.2 --- Purification of recombinant His-MSPl42 --- p.58 / Chapter 3.7.3 --- Immunization of rabbits --- p.59 / Chapter Chapter 4: --- Results --- p.60 / Chapter 4.1 --- Transgenic analysis of lysine-rich protein fusion constructs --- p.60 / Chapter 4.1.1 --- Construction of the lysine-rich protein fusion constructs --- p.60 / Chapter 4.1.2 --- Selection of transgenic plants --- p.62 / Chapter 4.1.3 --- Southern analysis --- p.65 / Chapter 4.1.4 --- Northern analysis --- p.69 / Chapter 4.1.5 --- Western analysis --- p.71 / Chapter 4.1.6 --- Western analysis with anti-LRP --- p.75 / Chapter 4.1.7 --- Enterokinase digestion of recombinant LRP fusion protein --- p.76 / Chapter 4.2 --- Transgenic analysis of phaseolin vacuolar-sorting signal constructs --- p.80 / Chapter 4.2.1 --- Construction of the phaseolin vacuolar-sorting signal constructs --- p.80 / Chapter 4.2.2 --- Selection of transgenic plants --- p.82 / Chapter 4.2.3 --- Southern analysis --- p.85 / Chapter 4.2.4 --- Northern analysis --- p.89 / Chapter 4.2.5 --- Western analysis --- p.91 / Chapter 4.2.6 --- Deglycosylation studies of recombinant MSPl42-AFVY --- p.96 / Chapter 4.2.7 --- Human serum detection of MSP142-AFVY --- p.100 / Chapter 4.3 --- Confocal immunofluorescence studies of MSP142-AFVY in tobacco --- p.102 / Chapter 4.4 --- Bacterial expression of MSPl42 and anti-serum production --- p.105 / Chapter 4.4.1 --- Expression and purification of recombinant His-MSPl42 in E. coli --- p.105 / Chapter 4.4.2 --- Titer and specificity of the anti-serum --- p.107 / Chapter Chapter 5 --- Discussion --- p.109 / Chapter Chapter 6 --- Conclusion --- p.116 / References --- p.117

Abstract not available

T cell surface antigens are thought to play significant roles in immunological functions. They are involved in cellular interactions and T cell activation and proliferation. Characterization of T cell antigens is important in understanding the molecular machanisms underlying immune responses. The subject of this thesis is to characterize a novel murine T cell surface antigen called YE1/48. YE1/48, defined by two rat monoclonal antibodies YE1/48.10.6 and YE1/32.8.5, is a dimeric glycoprotein with molecular size and charge resembling the murine T cell antigen receptor α/β. It was initially detected at high levels on two T cell lymphomas, EL-4 and MBL-2. In my thesis studies, the YE1/48 antigen was characterized biochemically, a cDNA clone was isolated, and its expression in lymphoid cell populations was determined. The YE1/48 antigen was found to be distinct from the T cell receptor based on direct comparisons of their primary sequences as well as immunological analyses. It is likely a homodimer with similar or identical subunits. No homology with any known proteins could be detected, including the human T cell activation antigen CD28 (T44) which also has a similar dimeric structure as YE1/48. No function of the YE1/48 antigen could be derived from its primary sequence or with the use of the two monoclonal antibodies because the antibodies do not appear to bind to the surface of intact normal T lymphocytes. Some intriguing characteristics of the YE1/48 antigen were observed in the current studies. The YE1/48 antigen belongs to a rare group of type II membrane proteins with orientation of the amino-terminus inside the cell and the carboxy-terminus outside. The YE1/48 gene may have two alleles among different mouse strains and may belong to a multigene family. YE1/48 is expressed at low levels on a wide range of T cells with no restriction to their differentiation stages, and on spleen B cells as well as bone marrow cells. Its expression on lymphocytes is not related to activation or proliferation. However, YE1/48 expression appears to be induced at high levels by Abelson Murine Leukemia Virus-transformation of pre-B cells. Moreover, the epitopes defined by the YE1/48.10.6 and YE1.32.8.5 antibodies seem to be exposed only on three T lymphomas but not on normal T cells. It is thus tantalizing to speculate a correlation of the high level expression of YE1/48 antigen and its epitope exposure on transformed lymphocytes with cellular transformation. In summary, YE1/48 was found to be a novel T cell surface antigen which has similar dimeric structure as the murine T cell receptor α/β and human CD28 (T44). It has now been characterized biochemically, molecularly cloned, and its expression on lymphoid cells has been determined. Although the function of YE1/48 antigen remains unknown, a number of intriguing characteristics observed in the current studies have certainly called for further studies on the antigen and the determination of its function. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

New Zealand Black/White F₁ hybrid (NZB/W) and MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop a Systemic Lupus Erythematosus-like autoimmune disease. While the primary immunologic defect in the NZB/W is due to B cells, in the MRL/lpr it is a result of T cell abnormalities. Diethyldithiocarbamate (DTC), an agent suggested to enhance T cell function, was used to treat both strains. Weekly treatment of NZB/W mice with 25 mg/kg DTC had no significant effect upon survival or autoantibody levels but did induce changes in cell surface antigen expression. MRL/lpr mice treated with DTC displayed normalization of cell surface antigen expression (particularly increased expression of Lyt-2, macrophage markers and Lyt-2⁺/L3T4⁺ thymocytes), decreased lymphoproliferation and thymic atrophy, decreased serum autoantibody levels and kidney deposition of C3 and IgM, restored responses to mitogens and significantly prolonged survival. To determine both the influence of MRL background and lpr genes and to better understand on what cell populations DTC effects, changes in cell surface antigen expression were examined in DTC treated MRL-+/+, Balb/c, and Balb/lpr strains. The only consistent similarities observed between all strains tested were DTC induced changes in Mac-1 splenocyte surface antigen expression. In vitro studies showed DTC to have variable effects upon the mitogenic responses of lymphoid cells to phytohemagluttinin, but DTC alone stimulated both MRL/lpr and Balb/lpr lymphocytes. DTC stimulated the null cell population that predominates in lpr gene-bearing mice, but all observed in vitro effects of DTC were dependent upon the adherent cell population included in culture. DTC had no apparent direct effects upon adherent cells alone however. These studies have shown that DTC is capable of positive effects upon one autoimmune murine strain, the MRL/lpr, but not the NZB/W. DTC appears to affect macrophages, but other cell populations are required to obtain full activity of this compound. The variable effects of DTC emphasize the need to define the immunopathology of individual patients with autoimmune disease before initiating treatment with immunomodulative therapy.

A total of 338 infants with suspected clinical sepsis were investigated, 115 of whom were found to be clinically infected. Twenty-one healthy term neonates were recruited as control subjects. The expression of CD64 on neutrophils in infected infants was significantly elevated at both 0 h and 24 h, compared with those of noninfected infants or controls (both p &lt; 0.0005). The calculated optimal cutoff value for CD64 was 6136 antibody-phycoerythrin molecules bound/cell. CD64 has a very high sensitivity (96%) and NPV (97%) at 24 h. The use of CRP in combination with CD64 as predictive markers only marginally enhanced the sensitivity and NPV (97% and 98%, respectively). There was no statistical difference in the expression of monocyte HLA-DR among infected, noninfected, and control subjects. As a result, the optimal cutoff value for HLA-DR could not be determined. The technology of flow cytometry has potential applications for use in the diagnosis of neonatal sepsis because the measurement is quantitative, requiring only a minimal amount of whole blood and a short duration (within 3 h) for the provision of results. (Abstract shortened by UMI.) / Term newborns in whom infection was suspected when they were &lt;72 h of age were recruited into the study. The expressions of CD64 on neutrophils and HLA-DR on monocytes were measured by flow cytometry at 0 h (the time of sepsis evaluation) and 24 h after the onset of presentation. A full sepsis screen, including complete blood count, serial C-reactive protein (CRP), blood culture, cerebrospinal fluid culture, and chest radiograph were performed. The demographic and clinical data were documented. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of neutrophil CD64, monocyte HLA-DR and the combination of markers for predicting neonatal sepsis were determined. / This prospective study aimed to evaluate the diagnostic utilities of two cell surface markers, neutrophil CD64 and monocyte HLA-DR, for the identification of early-onset clinical infection and pneumonia in term infants. The optimal cutoff value of each marker was defined according to the Receiver Operating Characteristic curve so that it could be used as a reference with which future studies can be compared. / Li Geng. / "May 2005." / Adviser: Pak Cheung Ng. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0174. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 129-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Abstract not available