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The number of tissue eosinophils were counted and phospholiphase B activity was assayed in the intestines of nonsensitized and sensitized and sensitized mice infected with Trichinella spiralis.

Ph.d.-afhandling. University of Helsinki, 2005. / Også elektronisk adgang via Internetttet.

The relationship among eosinophils, lysophospholipase activity and the immune response in animals infected with Trichinella spiralis was studied using in vivo and in vitro techniques. In an in vivo experiment, anti-thymocyte serum (ATS) was administered to mice infected with T. spiralis and its effects on intestinal lysophospholipase (EC 3.1.1.5.) activity, peripheral blood, bone marrow and intestinal eosinophilia were measured in the same experimental animal. The ATS caused a significant temporally related suppression of both the tissue lysophospholipase response and eosinophilia, in all three compartments. These findings support the hypothesis that parasite-induced eosinophilia is the cause of the increased lysophospholipase activity of parasitized tissue and that the responses are thymus cell-dependent. In vitro experiments demonstrated that the eosinophil was the primary inflammatory cell source of lysophospholipase among eosinophils, neutrophils macrophages and lymphocytes. The role of other cells and antigen in the production of the enzyme by the eosinophil was also investigated in vitro• Results demonstrated that eosinophils cultured with both T. spiralis antigen and other leukocytes yielded enzyme activities significantly greater than eosinophils cultured alone or with only antigen. More specific experiments showed that T-lymphocytes were the cells responsible for influencing the eosinophils' lysophospholipase activity in the presence of antigen, and that their influence was enhanced by the presence of macrophages. These results suggested that increased lysophospholipase activity present in parasitized tissue was not only due to increased numbers of eosinophils infiltrating parasitized tissue but was also due to each eosinophil synthesizing more of the enzyme. The necessity for antigen and other cells suggests a role for cell cooperation in the production of the enzyme, specifically T-lymphocytes and macrophage interaction with the eosinophil. A lymphocyte soluble factor collected from sensitized lymphocytes stimulated with specific antigen or concanavalin A was found to enhance the eosinophil lysophospholipase activity when added to cultures of eosinophils plus other peritoneal cells. The soluble factor did not stimulate the lysophospholipase activity of pure cultures of eosinophils.

Kit Yu Fu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 99-106). / Chapter I. --- Abstract --- p.vii / Chapter II. --- Acknowledgements --- p.viii / Chapter III. --- List of Figures --- p.ix / Chapter IV. --- Chapter --- p.1 / Introduction --- p.1 / Chapter 1.1 --- Laboratory diagnosis of infectious diseases --- p.1 / Chapter a. --- Culture --- p.1 / Chapter b. --- Direct detection by visualization --- p.2 / Chapter c. --- Direct detection by DNA or RNA hybridization --- p.2 / Chapter d. --- Detection by immunological methods (antigen or antibody detection) --- p.3 / Chapter 1.2 --- Types of antigen preparations / Chapter a. --- Crude antigenic extracts --- p.5 / Chapter b. --- Affinity-purified antigens --- p.6 / Chapter c. --- Recombinant antigens --- p.6 / Chapter 1.3 --- Methods of gene transfer to mammalian cells --- p.8 / Chapter 1.4 --- The immunoglobulins --- p.10 / Chapter 1.4.1 --- Ig structure --- p.11 / Chapter 1.4.2 --- Ig genes --- p.13 / Chapter 1.4.3 --- Ig gene rearrangement --- p.15 / Chapter 1.4.4 --- Recombinant Ig --- p.15 / Chapter 1.4.5 --- Myeloma-derived recombinant Ig (chimeric antibodies) --- p.17 / Chapter 1.4.6 --- Ig expression vectors --- p.19 / Chapter 1.5 --- Trichinella spiralis and trichinosis --- p.20 / Chapter 1.5.1 --- The parasite --- p.21 / Chapter 1.5.2 --- Antigens of T. spiralis --- p.21 / Chapter 1.6 --- Aim of present study --- p.25 / Chapter V. --- Chapter2 / Materials and Methods / Chapter 2.1 --- Chemicals --- p.27 / Chapter 2.2 --- Parasite --- p.27 / Chapter 2.3 --- Cell line and expression vectors --- p.28 / Chapter 2.4 --- Extraction of total RNA from T. spiralis --- p.29 / Chapter 2.5 --- Preparation of cDNA fragment from T. spiralis --- p.29 / Chapter 2.6 --- Characterization of Trichinella cDNA fragment --- p.31 / Chapter 2.6.1 --- By gel electrophoresis --- p.31 / Chapter 2.6.2 --- By restriction enzyme digestion --- p.31 / Chapter 2.7 --- Cloning of Trichinella cDNA fragment to g4R --- p.31 / Chapter 2.7.1 --- Preparation Trichinella cDNA fragment for ligation --- p.32 / Chapter 2.7.2 --- Preparation of g4R vector --- p.32 / Chapter 2.7.3 --- Ligation --- p.32 / Chapter 2.7.4 --- Transformation of Escherichia coli (TG1) / Chapter a. --- Preparation of competent cells for transformation --- p.33 / Chapter b. --- Transformation of competent cells by heat shock --- p.34 / Chapter 2.7.5 --- Screening of recombinant clones --- p.34 / Chapter 2.8 --- Preparation of fusion gene for transfection --- p.36 / Chapter 2.9 --- Introduction of DNA to myeloma cells by electroporation --- p.36 / Chapter 2.10 --- Enzyme-linked immunosorbent assay (ELISA) to detect fusion gene product / Chapter 2.10.1 --- Sandwich ELISA --- p.37 / Chapter 2.10.2 --- Detection of Trichinella antigen in fusion gene product --- p.38 / Chapter 2.10.3 --- Detection of Ig CH2-CH3 domains in fusion gene product --- p.38 / Chapter 2.11 --- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.39 / Chapter 2.12 --- Genomic and transcriptional analysis of transfectants --- p.40 / Chapter 2.12.1 --- Genomic analysis of transfectants / Chapter a. --- DNA isolation --- p.40 / Chapter b. --- PCR amplification of the fusion gene fragment --- p.41 / Chapter 2.12.2 --- Isolation of fusion gene cDNA from transfectants --- p.41 / Chapter 2.12.3 --- Cloning of fusion gene cDNA to M13 mpl9 --- p.43 / Chapter 2.12.4 --- Preparation of single-stranded templates from M13 phage --- p.43 / Chapter 2.12.5 --- Dideoxy sequencing method (Sanger) --- p.44 / Chapter 2.12.6 --- Gel analysis of sequencing products --- p.44 / Chapter 2.13 --- Modification of the g4R expression vector by deletion of the CH2-CH3 exons 3' to the XhoI site / Chapter a. --- Partial EcoRI digestion of g4R --- p.45 / Chapter b. --- Addition of adaptors to the partial Eco RI-digested g4R vector --- p.46 / Chapter c. --- Preparation of modified g4R vector --- p.46 / Chapter 2.14 --- Cloning of the Trichinella P53 gene into the modified g4R vector --- p.46 / Chapter 2.15 --- Detection of Trichinella antigen in the second fusion gene product / Chapter a. --- Preparation of biotinylated mouse anti- Trichinella serum --- p.47 / Chapter b. --- Assay for the activity of biotin-Ts serum --- p.48 / Chapter c. --- "Assay for detection of Trichinella antigen in the fusion gene product from Tc2, Te1 and g4R transfected clones" --- p.48 / Chapter 2.16 --- Northern blot analysis of the RNA of transfected clones --- p.48 / Chapter a. --- RNA gel electrophoresis --- p.49 / Chapter b. --- RNA transfer --- p.49 / Chapter c. --- RNA hybridization --- p.49 / Chapter VI. --- "Chapter3 Construction of Ig-Trichinella fusion gene, Te1" / Chapter 3.1 --- Rationale of the gene construction --- p.51 / Chapter 3.2 --- Isolation of T. spiralis P49 gene by cDNA amplification --- p.55 / Chapter 3.3 --- Cloning of Trichinella P49 cDNA to g4R --- p.58 / Chapter 3.4 --- Screening of recombinant clones --- p.58 / Chapter VII. --- Chapter4 Characterization of Tc1 fusion gene product / Chapter 4.1 --- Transfection of fusion gene to J558L myeloma cells / Chapter 4.2 --- Antigenicity of fusion gene product with respect to Trichinella activity --- p.64 / Chapter 4.3 --- Detection of Ig CH2-CH3 domains in fusion gene product --- p.65 / Chapter 4.4 --- Size determination of fusion gene product --- p.69 / Chapter 4.5 --- Transcriptional and genomic analysis of transfectants producing the fusion gene product --- p.71 / Chapter 4.5.1 --- Genomic analysis --- p.71 / Chapter 4.5.2 --- Sequence analysis of transcript from the CH1 to CH2 exon --- p.71 / Chapter 4.5.3 --- Sequence analysis of transcript from the CH1 to CH3 exon --- p.77 / Chapter VIII. --- "Chapter5 Construction and characterization of second fusion gene, Tc2, using modified g4R vector" --- p.81 / Chapter IX. --- Chapter6 General Discussion / Use of recombinant DNA technology to produceantigen for use in the diagnosis of infectious diseases --- p.89 / Characterization of the fusion gene product --- p.89 / Absence of Trichinella sequence in fusion gene product due to exon skipping --- p.94 / A new strategy for producing Ig fusion proteins: modification of the g4R vector --- p.96 / Prospect of utilizing Ig expression system for producing antigen --- p.97 / Chapter X. --- References --- p.99 / Chapter XI. --- Appendix --- p.107

The production of Heat Shock Protein 72, the inducible for~ of the highly conserved 70 kilodalton heat shock protein family, was investigated in MRL++ mouse intestine during the two weeks of a Trichinella spiralis infection. Within hours of an oral infection using the encysted Trichinella spiralis found in the diaphragm of an infected mouse, the larvae are released from the cyst in the stomach. They travel to the intestine and burrow into the epithelial layer of the intestine. The jejunum is the primary site of the intestinal phase of trichinosis (Despommier 1983). This stage of infection in the jejunum was the focus of this study. Heat shock protein (HSP) synthesis is precipitated by stressful stimuli: in vitro by chemicals such as sodium arsenite and in vivo by cytoskeletal disturbance and/or toxic 02 radicals (Linquist 1986). The latter in vivo studies lend support to the inflammatory response induction of HSPs. Heat shock protein 72 (HSP72) is rarely expressed constitutively especially in non-primates and is a good indicator of various stresses. This study hypothesized that HSP72 would be induced by cells in the jejunum of the MRL++ mouse during a Trichinella spiralis infection due to the stress of the parasitic infection. Different techniques were employed to investigate this hypothesis. Immunohistochemistry and immunoblots facilitated this study. Although immunoblots did not demonstrate HSP72 induction, immunohistochemical analysis suggested the presence of HSP72 in various cells in the lamina propria of the jejunal villi.

La función barrera intestinal constituye la primera línea de defensa del tracto digestivo. Ésta se ve condicionada por el transporte hidroelectrolítico, la permeabilidad y la motilidad intestinales. Alteraciones de estas funciones favorecen la estimulación continua, por antígenos y microorganismos luminales, del sistema inmune local llevando a estados inflamatorios más o menos crónicos. Estos cambios parecen caracterizar diversas patologías inflamatorias y funcionales gastrointestinales, incluyendo la enfermedad inflamatoria intestinal y el síndrome de intestino irritable. Los mastocitos (MCs), a través de un proceso de activación y liberación de mediadores neuroinmunes, participan como células efectoras en la respuesta inmune asociada a estas enfermedades. Este trabajo profundiza en las implicaciones de los MCs en las disfunciones intestinales, haciendo un especial énfasis en la función barrera. Para ello, se empleó un modelo de parasitosis por Trichinella spiralis en ratas. Tras la infección, se observó la evolución temporal (días 2 a 30 post‐infección) en las poblaciones de MCs de mucosa y de tejido conectivo en el yeyuno. Las variaciones en los infiltrados mastocitarios se acompañaron de una sobreexpresión en las proteinasas mastocitarias. Las proteinasas, actuando como enzimas sobre sustratos específicos, incluyendo la activación de receptores activados por proteinasas (PARs), regulan las funciones secretomotoras y sensoriales gastrointestinales. Acompañando estos cambios, se han caracterizado remodelaciones neuroepiteliales post‐infecciosas que resultan en una alteración de la función barrera, en concreto: una alteración de la secreción hidroelectrolítica basal, de la respuesta a secretagogos y en el incremento de la permeabilidad intestinal. Estas disfunciones de la barrera tienen como base cambios temporales específicos en la expresión de proteínas de las uniones estrechas intercelulares. A su vez, la parasitosis produjo un aumento de la actividad motora espontánea intestinal, en parte mediada por los MCs, ya que se revierte parcialmente en animales tratados con el estabilizador mastocitario ketotifeno. Por otro lado, las respuestas secretoras intestinales a la degranulación mastocitaria y a la activación de PAR‐2 se vieron reducidas en la fase post‐infecciosa (día 30 post‐infección). Si bien, ello sugeriría una desensibilización epitelial a los mediadores mastocitarios, la estabilización de los MCs con ketotifeno no tuvo efectos sobre las respuestas secretoras basales o mediadas por PAR‐2 ni sobre los incrementos post‐infecciosos en la permeabilidad. En resumen, hemos definido los cambios en las poblaciones mastocitarias y la función barrera intestinal, así como su relación con la expresión de proteínas de uniones intercelulares y con la expresión de PAR‐2 y sus efectos secretores en un modelo de disfunción intestinal post‐infecciosa. Aunque estos datos apoyan una implicación de los MCs en los cambios morfo‐funcionales asociados a la infección cuestionan una participación directa de los mismos en la función barrera intestinal. Mecanismos de regulación similares pueden operar en enfermedades inflamatorias y funcionales gastrointestinales. / Epithelial barrier function is considered part of the defensive mechanisms of the gastrointestinal tract. Barrier function results from an interplay of at least three components: hydroelectrolytic transport, permeability and intestinal motility. Alterations of these functions favor a state of luminal antigens‐ and microorganismsdependent stimulation of the local immune system, leading to an inflammatory‐like stage. These changes are common to several gastrointestinal pathologies, including inflammatory bowel disease and irritable bowel syndrome. Mast cells (MCs), throught a process of activation and release of endogenous neuroimmune mediators, act as effector cells in the neuroimmune responses associated to these alterations. This work aims the characterization of MCs’ implications in gastrointestinals dysfunctions, with emphasis in barrier function. For this, we used a model of Trichinella spiralis infection in rats. After the infection, time‐related (days 2 to 30 postinfection) changes in mucosal and connective tissue MCs were assessed in the jejunum. Changes in MCs infiltrates occurred together with an up‐regulation of proteinases gene expression. MC‐derived proteinases, throught the enzymatic cleavage of specific substrates, including proteinase‐activated receptors (PARs), regulate gastrointestinal secretomotor and sensory functions. In addition, we characterized postinfectious neuroepithelial remodelations, leading to functional changes in barrier function. In particular, alterations in basal electrolytic secretion, secretory responses to secretagogues and an increase in epithelial permeability were observed. These barrier dysfunctions are associated to time‐related changes in the expression of tight junctions‐related proteins. In parallel, an increase in spontaneous intestinal motor activity was also observed. Treatment with the MC stabilizer ketotifen partially prevented these motor alterations, thus suggesting that MCs are implicated, at least partially, in these responses. In addition, secretory responses to MCs degranulation and PAR‐2 activation were reduced during the postinfectious phase (day 30 postinfection). This might suggest a desensitization of the epithelium to MC mediators. However, treatment with the MC stabilizer ketotifen was without effect, thus suggesting the contribution of additional, MCs‐independent mechanisms. In summary, this work characterizes changes in MCs populations and epithelial barrier function in a model of postinfectious gut dysfunction in rats. Furthermore, the relationship with the expression of tight junction‐related proteins and PAR‐2 and its secretory effects was characterized. Overall, results obtained support an implication of MCs in the morphological and functional alterations associated to the infection. However, they also question a direct role of MCs in the control of epithelial barrier function. Similar mechanisms might operate in inflammatory and functional gastrointestinal disorders.

Thèse de doctorat : Parasitologie : Paris 12 : 2007. / Titre provenant de l'écran-titre. Pagination : 335 p. Bibliogr. p. 301-331.

Thesis (M. Phil.)--University of Hong Kong, 1987.

published_or_final_version / Zoology / Master / Master of Philosophy