Dissertations / Theses on the topic 'Immunology, Experimental'

Induction of collagen-induced arthritis (CIA), an animal model for human rheumatoid arthritis, is dependent on anti-collagen type II (CII) antibodies. The effector mechanism by which autoantibodies contribute to inflammatory reactions in autoimmune diseases is not well understood. In this thesis I have studied the effector pathways used by IgG anti-CII antibodies to initiate arthritis, namely the IgG Fc receptors (FcγRs) and the complement system. We have found that FcγRIII is crucial for development of CIA, as CII-immunized mice lacking this receptor do not develop arthritis and IgG1 and IgG2b anti-CII antibodies require FcγRIII to trigger arthritis when transferred to naïve mice. The antibody-mediated arthritis was further enhanced in mice deficient in the inhibitory FcγRIIB, indicating that FcγRIIB regulates the activation of FcγRIII. Furthermore, we demonstrate that FcγRIII exist as three distinct haplotypes in mice, FcγRIII:H, FcγRIII:V and FcγRIII:T. Mice expressing the FcγRIII:H haplotype are more susceptible to CIA than mice expressing the FcγRIII:V haplotype, indicating that certain FcγRIII haplotype predisposes for CIA. We also show that the most likely FcγRIII-expressing effector cell in CIA is the macrophage, since FcγRIII-expressing macrophages exclusively can induce arthritis in FcγRIII-deficient mice challenged for CIA.

The complement system was also investigated in development of CIA. We found that this effector pathway is also necessary for onset of arthritis, as CIA was inhibited by treatment with anti-complement factor 5 (C5) antibodies. C5-deficient mice could neither develop CIA unless provided with C5-containing sera.

Taken together, the work presented in this thesis indicates that FcγRs and the complement system are crucial for the induction of experimental arthritis. These findings are important for understanding the mechanisms behind rheumatoid arthritis and blocking of these effector pathways may in the future be used as treatment of rheumatoid arthritis.

A murine model of host resistance to infection with Francisella tularensis, live vaccine strain, has been established. This facultative intracellular bacterium causes in mice an acute infection which lasts for approximately 12 days. Resistance to infection is associated with the appearance in the spleens of cells with the ability to transfer anti-tularemic resistance to naive mice, a response which is maximal 7 days following infection. Depletion of B cells does not compromise the ability of immune cells to transfer resistance. Treatment of mice with immune serum prior to infection leads to an increased hepatic and decreased splenic growth of Francisella. This effect is attributable to a serum-induced shift in the organ uptake of Francisella from the spleen to the liver. Acquired anti-tularemic resistance is critically dependent upon T lymphocytes, as evidenced by the inability of T lymphocyte-depleted immune cells to transfer resistance, and by the increased susceptibility to infection in mice treated with the T cell immunosuppressant, cyclosporin A. The passive transfer of resistance is mediated mainly by T cells bearing the L3T4 marker, but Lyt 2$ sp{+}$ cells may also play a minor role. Macrophages are essential to the control of Francisella infection, since inhibition of the functional activity of this cell population by silica injection leads to a decrease in the ability of mice to contain bacterial growth. The interactions between T cells and macrophages leading to the expression of anti-tularemic immunity are restricted by the product of the I-A region of the H-2 complex. Resistance to tularemia is influenced by the genetic background of the host. Among inbred mouse strains, several levels of resistance to infection are evident. Genetic analysis of resistance/susceptibility to tularemia in F$ sb1$ and F$ sb2$ hybrid mice, and in recombinant inbred strains of mice derived from resistant and susceptible progenitor strains, indicated that the inheritance pat

Antigen specific CD4+ T cells adoptively transfer airway inflammation comprised mainly of lymphocytes and eosinophils. The ability of these transferred T cells to induce inflammation is dependent on the cytokines they express particularly Th2 cytokines. In order to better understand the mechanism by which adoptively transferred T cells induce airway inflammation, we chose to modulate the expression (GATA-3) and activity (STAT-6) of two key regulators of Th2 cytokine production. To modify expression of GATA-3, we used a bicistronic retroviral vector encoding GATA-3 and enhanced green fluorescent protein (EGFP). As a control, we used a retrovector encoding EGFP alone. By coupling in vitro antigen stimulation with retroviral transduction we generated antigen specific CD4+ T cells expressing EGFP alone or GATA-3 and EGFP. When transferred into naïve recipients that were subsequently challenged, these transduced CD4+ T cells induced lung inflammatory responses with an increase in both CD4+ lymphocytes and eosinophils. This antigen specific inflammatory response was enhanced in animals receiving T cells overexpressing GATA-3. Analysis of the infiltrating cells also revealed that the EGFP+ T cells were present in the lung following antigen challenge, comprising only a small fraction of the CD4+ T cells recruited to the lung during the antigen response. Thus, GATA-3 amplifies antigen-specific inflammatory responses in the airways by augmenting the ability of antigen specific T cells to recruit inflammatory cells to the lung following antigen challenge. To modify the activity of STAT-6 we used chimeric cell penetrating peptides containing a poly-arginine protein transduction domain (PTD) coupled to a sequence predicted to bind and inhibit STAT-6 activity (SIP-1). Using fluorescein-tagged SIP-1, we demonstrate that the poly-arginine PTD efficiently translocates to the cytoplasm within an hour. In vitro, antigen-induced IL-4 production was inhibited in SIP-1-treated spleno
Les cellules CD4+ T à antigènes spécifiques transfèrent par adoption l'inflammation pulmonaire constituées principalement de lymphocytes et d'éosinophiles. L'habileté de celles-ci à transférer des cellules T pour induire l'inflammation est dépendante de leur expression de cytokines Th2. De manière à mieux comprendre le mécanisme par lequel les cellules T transmises par adoption induisent l'inflammation pulmonaire, nous avons choisi de moduler l'expression de GATA-3) ou l'activité de (STAT-6) des deux régulateurs-clés de production de cytokine Th2. Afin de modifier l'expression de GATA-3 dans les cellules T destinées au transfert par adoption, nous avons utilisé un rétrovirus recombinant concentré avec une filtration par centrifugeuse. Ce procédé a dramatiquement augmenté leurs titres et ainsi leur habileté à transduire les cellules CD4+ T en culture primaire. Nous avons utilisé un rétrovirus recombinant qui encode la GATA-3 et / ou la protéine fluorescente verte (EGFP). En couplant in vitro la stimulation d'antigènes avec la transduction par vecteur viral, nous avons généré des cellules CD4+ T à antigènes spécifiques exprimant de l'EGFP seul ou bien de la GATA-3 et de l'EGFP. Lorsque transféré dans un rat qui avait subséquemment été provoqué avec des antigènes, ces cellules CD4+ T induisent une réaction aux inflammations pulmonaires avec une augmentation des lymphocytes et éosinophiles. Cette réaction inflammatoire fut accrue chez les animaux recevant les cellules T surexprimant la GATA-3. L'analyse des cellules infiltrantes a aussi révélé que bien que les cellules EGFP+ étaient présentes dans les poumons suivant la provocation par antigènes, elles étaient constituées seulement d'une petite fraction de cellules CD4+ T recrutées dans les poumons. Ainsi, la GATA-3 amplifie la réaction inflammatoire des poumons induite par antigènes en augmentant l'habileté des cellules T à antigènes spécifiques à recruter

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by leukocytic infiltration of the central nervous system (CNS) and demyelination and remission/relapse. It is induced by CD4$ sp{+}$ T cells. We used reverse transcriptase/polymerase chain reaction to analyse T cell and cytokine gene expression in the CNS of SJL/J mice with myelin basic protein-induced EAE. Undetectable in normal CNS and cerebrospinal fluid, the expression of CD3, IL-2, IFN-$ gamma$, and TNF$ alpha$ increased in EAE, correlating with disease severity, then dropped to background levels during remission. IL-2 and IFN-$ gamma$ were produced by CD4$ sp{+}$ CD45RB$ sp{ rm low}$ T cells isolated from LN and CNS. In contrast, TNF$ alpha$ was predominately made by macrophages and microglia in the CNS. Purified microglia from normal CNS were induced to express TNF$ alpha$ by activated TH1 supernatant, suggesting that TNF$ alpha$ expression by cells in the CNS could be regulated by cytokines from infiltrating T cells. IL-4 was not detectable in total CNS or in isolated CD4$ sp{+}$ CD45RB$ sp{ rm low}$ cells from the CNS, but was readily amplified from CD4$ sp{+}$ CD45RB$ sp{ rm low}$ LN T cells. This suggests an enrichment of TH1 cells in autoimmune CNS.
To determine the effect of IFN-$ gamma$ expression in the CNS, we produced transgenic mice using an IFN-$ gamma$ cDNA downstream of an MBP promoter. Expression of the transgene was CNS-specific. MHC class I was induced in the CNS of transgenic mice. Transgenic animals that were backcrossed up to 5 generations with SJL/J did not develop spontaneous pathology. However, when they were immunized with MBP in adjuvant, the penetrance of EAE was greater, symptoms were more severe, and the duration of the first episode significantly longer than in non-transgenic littermates, suggesting a role for IFN-$ gamma$ in the amplification and perpetuation of EAE.

To understand why remyelination fails in diseases such as multiple sclerosis (MS), I studied the cells and molecules involved in remyelination. I examined the cellular response to cuprizone-induced demyelination in C57B1/6 female mice. In response to the toxin cuprizone, microglia, macrophages, T cells and oligodendrocyte precursor cells (OPCs), accumulated in the corpus callosum. I have identified a novel population of CD11c-expressing microglia with potent antigen presenting capacity, which localized to the demyelinated corpus callosum following cuprizone treatment. A broad panel of chemokines were upregulated as early as 1 week following cuprizone treatment. Administration of one of them, an oligodendrocyte precursor cell growth factor, CXCL1, via adenoviral gene delivery to the CNS, did not affect OPC dynamics in the demyelinated corpus callosum. Insight into cells and molecules produced during demyelination/remyelination will be of use in designing new therapies for MS.

Schistosomiasis is one of the most debilitating tropical diseases with the potential to cause morbidity and mortality in infected populations unless well controlled. Current control measures are limited to treatment with praziquantel. A rather alarming situation given i) the inability of the drug to directly target the pathogenic eggs, ii) the emergence of praziquantel-resistant schistosomes, iii) the persistence of tissue fibro-proliferative destruction caused by the trapped parasite eggs, even after treatment. Intestinal and liver immunopathology are pathognomonic of schistosomiasis and generally result from the host inadequate Th2 and or Th17 responses to the egg-derived antigens. Failure to control these immune responses causes the most of the detriment to the infected host, highlighting the need for a better understanding of the regulatory mechanisms which might help prevent excessive immune responsiveness and the ensuing immunopathology. A Basic leucine zipper transcription factor ATF-like 2 (BATF2) which belongs to a family of transcription factors critical in the control of inflammatory responses has gained enormous momentum recently as a potential target to immune deregulation during cancer and infectious diseases. We reasoned that an eventual BATF2 influence on the host immune response during schistosomiasis might unveil its anti-disease potency and greatly facilitate the quest for a novel control strategy against the immunopathology during schistosomiasis. Addressing this in our present study, BATF2-deficient mice were used and characterized for immunological and physiological parameters during steady state and Schistosomiasis. Although the liver showed a reduced pro-fibrotic response, there was a notable increase of several pro-fibrotic cytokines (TNF-α, IFN-ƴ, TGF-β and IL-13) Which further translated into an elevated level of granulomatous inflammation and fibrosis in the gut of S. mansoni infected BATF-2-deficient mice when compared to the liver tissues of BATF2-deficient mice and both livers and intestines of infected littermate controls indicating that BATF2 appears to have a tissue-specific role on the regulation of fibrogranulomatous inflammation during schistosomiasis. Therefore, BATF2 has a critical, and hitherto unappreciated, role in mediating the regulation of gut fibrogranulomatous response so as to promote host survival during schistosomiasis.

The immunobiology and the control of experimental visceral leishmaniasis were studied in susceptible (C57BL/6J) and resistant (C57L/J) strains of mice. It was found that the resistant/susceptibility phenotype for infection with L. donovani is expressed only by liver macrophage in vitro; the resistant/susceptible phenotypes were transfered reciprocally by bone marrow radiation chimeras. It was also found that the phagocytic activity of macrophages is reduced by the infection and that liver and peritoneal macrophages reflect their specific resistance/susceptibility phenotype following protective or curative treatment with lymphokines: macrophages from resistant C57L/J mice responded better to lymphokine activation. The production of IL-1 by spleen and peritoneal macrophages is inhibited by the infection. Immunosuppression with cyclosporin A (CsA) exacerbated the infection, without affecting phenotype; both CsA-treated strains of mice heal the infection in the absence of IL-1 and IL-2. There were more infected liver macrophages in normal or CsA-treated susceptible C57BL/6J mice and a greater number of amastigotes per cell than in normal or CsA-treated resistant C57L/J mice. CsA treatment did not affect the responsiveness of macrophages to lymphokine activation. IL-2-treated spleen and blood leucocytes from infected animals reduced infection in macrophages infected in vitro. Adoptive immunotherapy in vivo with IL-2-treated spleen cells from infected animals showed significant specific reduction of the parasite load in the liver; daily injections of IL-2 enhanced cure. T lymphocytes are the cells involved in cure; cure is mediated by soluble factors produced by the treated cells, and is specific to Leishmania infection.

A mouse model of surgically induced renal failure was utilized to investigate the pathogenesis of Staphylococcus epidermidis peritonitis which is a frequent and serious complication of continuous ambulatory peritoneal dialysis (CAPD). Compared to sham-operated controls, chronically uremic mice were more susceptible to intraperitoneal S. epidermidis inoculation, presenting decreased survival time and survival (10$ sp9$ cfu, 10$ sp8$ cfu), delayed bacterial clearance and attenuated peritoneal inflammatory response (10$ sp6$ cfu). In mice bearing a peritoneal catheter implant, the catheter was a preferred site for peritoneal bacterial persistence up to one month after intracatheter inoculation. Despite in vitro cytotoxicity of commercial peritoneal dialysis solutions toward peritoneal leucocytes, repeated peritoneal instillation of dialysis solutions did not influence S. epidermidis recoveries following inoculation. Although the mouse preparation did not undergo peritoneal dialysis, these studies nevertheless demonstrate that chronic uremia and the peritoneal catheter may be important etiological factors in the development and persistence of CAPD peritonitis.

Experimental Allergic Encephalomyelitis (EAE) is an autoimmune demyelinating disease of the central nervous system (CNS) with pathological and clinical features, including patterns of remission and relapse, reminiscent of the human disease Multiple Sclerosis (MS). EAE can be induced by immunization with myelin proteins in genetically susceptible rodents and can be adoptively transferred with CD4$ sp+$ T cells. In this thesis the mechanisms governing EAE induction in SJL/J mice were examined by flow cytometric analysis of the migration into the CNS of CD4$ sp+$ T cells labelled with a lipophilic fluorescent dye (PKH2). Selective retention was demonstrated within the CNS of myelin basic protein (MBP)-reactive CD4$ sp+$ T cell blasts, which were of a CD44$ sp{ rm high},$ CD45RB$ sp{ rm low},$ memory/effector phenotype and which expressed mRNA for IL-2 and IFN-$ gamma$ but not for IL-4. Mechanisms of immunoregulation were examined during remission and in mice preimmunized with irradiated T cells (T-cell-vaccination). A loss of CD4$ sp+$ T cells from the CNS was observed in animals that had remitted, but neither reversion of memory/effector phenotype nor increased numbers of regulatory CNS CD8$ sp+$ cells were observed. In vaccination experiments, evidence was obtained for rescue of background activation levels and enhanced proliferative responses to antigens within lymph nodes of vaccinated animals. The effector and regulatory mechanisms described in this thesis may further facilitate the development of effective T-cell-vaccination protocols for the control of cell-mediated autoimmune disease.

A model for experimental cecal and hepatic amebiasis was developed and characterized in the gerbil (Meriones unguiculatus). Pathogenic and non-pathogenic Entamoeba histolytica strains were shown to cause damage in the cecum, proportional to their previous behavior in humans. Secretion of intestinal mucus, crypt hyperplasia and cytolysis of interglandular epithelium were prerequisites for amebic invasion. Ulcerative lesions with destruction of mucosal and submucosal tissues led to amebic dissemination to the liver. Formation of amebic liver abscesses followed subacute changes in the liver. Liver lesions resulted from the cytolytic effects of the enzymes of destroyed neutrophils. Growth of liver abscesses followed cytolysis of the fibrogranuloma walls. Immunodepression in amebiasis was confirmed by serologic findings and histologic alterations in the lymph nodes and spleen, and by a lowered antiamebic effect of lymphoid cells in vitro. A neutrophil stimulating and chemotactic factor from pathogenic amebic membranes was isolated and characterized. It was shown that both host and parasite factors are involved in the pathogenesis and pathology of amebiasis.

The complex interactions between diet and the microbiota that influence mucosal inflammation and inflammatory bowel disease (IBD) are poorly understood. Experimental colitis models provide the opportunity to control and systematically perturb diet and the microbiota in parallel to quantify the contributions between multiple dietary ingredients and the microbiota on host physiology and colitis. To examine the interplay of diet and the gut microbiota on host health and colitis, we fed over 40 different diets with varied macronutrient sources and concentrations to specific pathogen free or germ-free mice either in the context of healthy, unchallenged animals or the dextran sodium sulfate (DSS) colitis model with follow-up studies for 4 diets in the T cell transfer colitis model. Diet influenced physiology in both health and colitis across all models, with the concentration of protein and psyllium fiber having the most profound effects. Increasing dietary protein elevated gut microbial density and worsened DSS colitis severity. Depleting gut microbial density by using germ-free animals or antibiotics negated the effect of a high protein diet. Psyllium fiber influenced host physiology and attenuated colitis severity through microbiota-dependent and microbiota-independent mechanisms. Combinatorial perturbations to dietary protein and psyllium fiber in parallel explain most variation in gut microbial density, intestinal permeability, and DSS colitis severity, and changes in one ingredient can be offset by changes in the other. Our results demonstrate the importance of examining complex mixtures of nutrients to understand the role of diet in intestinal inflammation.

Because of the well established epidemiologic and biologic interactions between human immunodeficiency virus (HIV) transmission and genital ulcer disease, measures to control chancroid could have a significant impact on the epidemic of HIV. I developed an IgG and IgM antibody serologic enzyme linked immunosorbent assay (ELISA) using pooled sera from clinically and microbiologically proven cases of chancroid as positive controls, and pooled sera from normal individuals without a history of sexually transmitted diseases as negative controls. Cross reactivity was minimized by absorption of serum samples with a sorbent prepared from three other Haemophilus species. Performance of the ELISA was enhanced in the period of early convalescence from acute primary chancroid and was not diminished in the presence of HIV coinfection, reflecting the tempo of the natural serologic response to and acute infection. I also developed an inhibition ELISA to determine antigenicity of potential vaccine candidates in human H. ducreyi infection, using lipooligosaccharide (LOS) as the test antigen. In a panel of 10 sera from cases of primary natural H. ducreyi infection reactive to H. ducreyi 35000 soluble antigen, only 4 samples were identified with reaction to H. ducreyi 35000 LOS. Inhibition ELISA confirmed that pre-adsorption with LOS substantially diminished reactivity of these sera to LOS but not to soluble antigen. Using the ELISA to detect immunogenicity by measuring the serologic response to vaccine candidates in rabbits, we further tested the feasibility of three bacterial antigen preparations to induce protective immunity against infection and disease. LOS carbohydrate and a pilus preparation were purified from H. ducreyi 35000 and used in a booster immunization procedure. Virulence was assayed by intra-epithelial challenge and measurement of disease for homologous strain 35000 or the virulent clinical isolate RO-34. LOS and the pilus preparation both induced humoral responses to the corresponding antigen, but the carbohydrate preparation did not. Immunization with LOS or carbohydrate did not modify virulence of infection with H. ducreyi 35000. Immunization with the strain 35000 pilus preparation significantly reduced the severity of disease, and the duration of infection and disease compared with controls. I conducted passive immunization experiments, and characterized the inflammatory infiltrate of chancroidal lesions. Naive rabbits were passively immunized with 24 or 48 mg of purified polyclonal IgG intravenously and 24 hours after infusion, challenged with the homologous strain 35000. No significant difference in disease resulting from infection with the homologous strain was observed. I then comparatively evaluated the serial immunohistology of lesions produced by infectious challenge with the homologous strain in sham-immunized or rabbits immunized with the pilus preparation. Flow cytometric analysis of rabbit peripheral blood leucocytes with CD5 and CD4 rabbit lymphocyte markers prior to infectious challenge revealed two T lymphocyte populations: CD5+CD4+ and CD5+CD4$-$. Pilus preparation vaccinee lesions showed significant quantitative acceleration and increase in T lymphocyte infiltration, and similar early increased recruitment of the CD4+ T lymphocyte subset preceding early lesion sterilization and early healing without ulceration. (Abstract shortened by UMI.)

The majority of cancer related deaths occur due to the invasive growth of metastatic lesions. In the early stages of metastasis, circulating cell interact with the endothelial cells to establish at a distant site. In inflammation endothelial activation results in induction of adhesion molecules on the endothelium that participate in the homing of leukocytes. Because of the interactions of metastatic cells with the endothelium, the question was whether some of the characteristic molecules of endothelial activation were induced during metastasis. In vivo pulmonary metastatic models were used to characterize the expression profile of endothelial activation. Immunohistochemistry identified VCAM-1 to be induced on the pulmonary endothelium following tumour cell arrest. VCAM-1 upregulation was not observed prior to tumour cells arrest or within the first hours. In contrast, tumour cell arrest appeared to be required for endothelial activation, arguing against a mechanism analogous to leukocyte homing. The upregulation of VCAM-1 upon tumour cell arrest corresponded with the initiation of platelet clot formation around the tumour cell and recruitment of leukocytes to the site, both previously shown to be essential for metastasis. Disruption of both phenomena, either through genetic or pharmacological manipulation, demonstrated that in contrast to the recruited leukocytes, platelets were involved in inducing endothelial activation. Another protein investigated was VAP-1. In contrast to VCAM-1, central to VAP-1 adhesive function is its enzymatic activity. Blocking the functions of either molecule highlighted their role in facilitating the recruitment of the leukocyte population to the tumour cell. Disruption of which led to a significant attenuation of metastasis. While VCAM-1 and VAP-1 function appears critical in the early steps of metastasis, their inhibition had no effect at later stages of pulmonary colonization.

Cysteinyl leukotrienes (cysLTs) are potent inflammatory lipid mediators that play a major role in the pathophysiology of inflammatory diseases. They signal primarily through cysteinyl leukotriene receptor-1 (cysLTR1) and have been reported to drive Th2 immune responses. Initiation and amplification of robust Th2 immune responses is crucial for conferring protective immunity to helminth (Schistosoma mansoni and Nippostrongylus brasiliensis) infection in mice. The role played by cysLTs in the development of protective immune responses to helminth infections is not well documented. Hence in the present study, we investigated the role of cysLTs during helminth infection using cysteinyl leukotriene receptor-1 deficient (cysLTR1-/- ) mice. Under steady state conditions, young naïve cysLTR1-/- mice did not reveal any significant alteration of the cellular, tissue and phenotypic profile although we did observe expansion of central memory T cells (Tcm) in secondary lymphoid organs in cysLTR1-/- as compared to wildtype mice. Primary infection with N. brasiliensis indicated increased worm burden in cysLTR1-/- mice at day 7 post infection and a delay in the resolution of infection by day 9 post infection when compared to wild type mice. Furthermore, we observed reduced Th2 immune responses as well as impaired contractility of the small intestine, which are key features required for protective immunity to N. brasiliensis infection. Furthermore, recall of memory responses to N. brasiliensis was abrogated in cysLTR1 -/- mice, with higher numbers of adult worms recovered at day 5 post re-infection in cysLTR1-/- mice comparison with wild type mice. Additionally, cysLTR1-/- mice exhibited impaired production of IL-13 in the lungs and draining lymph nodes compared with wildtype mice. Finally, there was reduced recruitment of effector CD4+ T cells and central memory CD4+ T cells in the lungs of cysLTR1 deficient mice compared to control mice. Taken together, these data demonstrated an essential role played by cysLTR1 in clearance and resolution of N. brasiliensis infection. CysLTR1-/- mice survived acute S. mansoni infection similarly to wildtype mice. In addition, cysLTR1-/- mice displayed reduced granulomatous inflammation and reduced cellular responses in the liver compared with wildtype mice. Further analysis revealed reduced gut fibrosis but cytokine production, immune cell recruitment in the gut and both type 1 and type 2 antibodies were found to be comparable between wildtype and knockout mice, demonstrating that cysLTs signaling through cysLTR1 contribute to granuloma formation in the liver. Similar to acute schistosomiasis, cysLTR1-/- mice were not susceptible to chronic schistosomiasis and indicated by prolonged host survival. This increased host survival observed in cysLTR1-/- mice was associated with reduced granulomatous inflammation, reduced fibrosis and hepatocellular damage, impaired production of IL-4 in the liver, and reduced intracellular secretion of IL4 by CD4+ T cells and ILC2s in cysLTR1-/- mice compared with wildtype mice. Furthermore, we observed reduced granulomatous inflammation in the lungs of chronically infected cysLTR1-/- mice despite the heightened Th2 immune response in the lungs. Collectively, these data revealed that disruption of cysLTR1 leads to reduced granulomatous inflammation and reduced production of IL-4 in the liver during chronic schistosomiasis. In conclusion, the current study demonstrated both positive and negative roles for cysLTs signaling through cysLTR1 during different helminth infection models. Absence of cysLTR1 during N. brasiliensis leads to delayed expulsion of adult worms and impaired recall of memory responses, indicating that cysteinyl leukotriene signaling via cysLTR1 is essential for orchestrating host protective responses. On the other hand, signaling via cysLTR1 appears to be dispensable for the development of host protective responses during acute schistosomiasis in mice. However, mice deficient of cysLTR1 had reduced liver pathology during chronic schistosomiasis, suggesting that inhibition of this receptor could be a potential therapy for reducing granulomatous liver pathology.

Leukocyte recruitment into tissues in response to infection or injury is a crucial event for the elimination of pathogens to protect the host. However, when leukocytes invade the central nervous system (CNS) and neuromflammatory disorders result, neurological function may be compromised. Infiltration of the CNS, predominantly by T cells and macrophages, characterizes Multiple Sclerosis and its animal counterpart, Experimental Autoimmune Encephalomyelitis (EAE).
Autoreactive T cells that initiate EAE produce Th1 cytokines (e.g., IFNgamma, TNFalpha). Nevertheless, previous studies also indicated an unnecessary or even protective role for IFNgamma in EAE. I have identified a novel role for IFNgamma in my studies using IFNgamma- or IFNgammaR-knockout mice. IFNgamma promotes the expression of the chemokines RANTES, MIP-1alpha, and MCP-1, which recruit mononuclear cells in the CNS to induce a non-lethal remitting EAE. Without IFNgamma, the chemokines MIP-2 and TCA-3, and polymorphonuclear leukocytes prevail, producing an unusually lethal EAE. MIP-1alpha is, however, dispensable in recruiting mononuclear cells, as EAE could still be induced in mice deficient in MIP-1alpha or its CCRS receptor.
To examine how much T cells depend on the cooperation with macrophages in the CNS to induce EAE, selective depletion of peripheral macrophages in mice was achieved by intravenous administration of clodronate-loaded liposomes. Treated mice showed no clinical signs of EAE following adoptive transfer of myelin-reactive T cells, but an altered distribution of leukocytes. These leukocytes were confined within the perivascular or meningeal space, not invading the CNS parenchyma. Levels of TNFalpha and inducible nitric oxide synthase (iNOS) in the CNS were reduced in these asymptomatic macrophage-depleted mice compared to untreated mice with EAE. In these asymptomatic mice, NOS expression was restricted to parenchymal astrocytes. In mice with EAE, however, both macrophages/microglia and astrocytes in infiltrates expressed NOS. Surprisingly, some astrocytes that were distant from infiltrates also expressed NOS, thus suggesting that astrocytes may modulate leukocyte infiltration via release of NO through their foot processes in the blood-brain barrier. Collectively, my data propose a model of a dynamic network in which the interplay among cytokines, chemokines and nitric oxide, may determine the magnitude, the composition, or the resolution of inflammatory infiltrates, as well as the clinical outcome of EAE.

Objective: The goal of this project was to determine the impact of local inflammation on changes in the subgingival biofilm composition in ligature-induced periodontitis in rats using the specialized pro-resolving mediator (SPM), resolvin E1 (RvE1). Materials and Methods: The impact of RvE1 on the microbiota of ligature-induced periodontitis was assessed in two separate experiments; treatment of established periodontitis and prevention of ligature-induced periodontitis. In the treatment study, eighteen rats were separated into four groups comprising no ligature, ligature alone (no treatment), ligature with topical RvE1 treatment (ligature+RvE1) and, ligature with topical vehicle treatment (ligature + Vehicle). 3-0 silk ligatures were tied around maxillary second molars bilaterally for three weeks to induce disease. After three weeks, the treatment phase began with the application of RvE1 or vehicle (ethanol) every other day for an additional three weeks. Subgingival plaque samples were collected every four days throughout the experiment. The composition of the subgingival microbiota was initially screened by checkerboard DNA-DNA hybridization using probes on 40 subgingival species. Definitive, unbiased characterization of the subgingival microbiota was accomplished with next-generation sequencing using the Illumina MiSeq® platform. Six rats were sacrificed on Days 1, 21 and 42 and maxillae were dissected to collect samples for gingival RNA extraction, bone morphometric measurements, and histomorphometric analysis. Local tissue gene expression (Cxcl-1, Ptgs2, Nos2) was detected using qRT-PCR. Tissue specimens were prepared for histology and stained with H&E and tartrate resistant acid phosphatase (TRAP). In the prevention study, sixteen rats were separated into four groups (no ligature, ligature + RvE1 (0.1µg/µl), ligature + RvE1 (0.5 µg/µl), ligature + Vehicle). 5-0 silk ligatures were placed around maxillary second molars bilaterally to induce disease. At the time of ligature placement, animals received assigned treatment thrice weekly (M, W, F) for four weeks. Subgingival plaque samples were collected every four days (M and F). Four rats were sacrificed at baseline (Day 1) and the vehicle and two treatment groups (four each) were sacrificed at day 28 and samples processed as described above. The two-group comparisons were assessed by Student’s t-test. The multiple-group comparison was assessed by one-way ANOVA and post hoc tests. Results: In the first study (treatment), topical application of RvE1 significantly reversed the bone loss associated with periodontitis compared to the vehicle. RvE1 application significantly reduced the expression of Cxcl1 and osteoclast density compared to the vehicle application. In the prevention study, RvE1 treatment significantly prevented the bone loss during the disease progression. RvE1 application significantly reduced the expression of Ptgs2, Nos2 compared to the vehicle application. Osteoclast density and inflammatory cell infiltration in the RvE1 groups were significantly lower than these in the Vehicle group. The cell counts of bacterial species gradually increased and the subgingival microbiota shifted during the disease progression. In the treatment study, RvE1 treatment significantly reduced cell counts compared to the vehicle application at the end of treatment phase. The shift of subgingival microbiota was limited by the RvE1 treatment. In the prevention study, the taxonomic composition and diversity of subgingival microbiota was controlled by the RvE1 application. The change of subgingival microbiota appeared to be associated with the state of inflammation in the periodontal environment. Conclusion: Resolvin E1 treatment of existing ligature-induced periodontitis significantly regenerates lost alveolar bone and prevents alveolar bone loss. Resolvin E1 treatment limits microbial shifts and reduces total bacterial load by inhibiting inflammation of local environment in experimental periodontitis.

L’asma es una malaltia inflamatòria crònica que afecta aproximadament a 300 milions de persones en tot el mon. Aquesta malaltia es caracteritza principalment per un conjunt d’alteracions estructurals en les vies respiratòries, conegudes amb el terme de “remodelació”, el qual s’ha associat amb la severitat de la malaltia. De totes aquestes alteracions, l’augment de la massa de musculatura llisa induït per una hiperplàsia e hipertrofia de cèl·ules musculars llises (ASM), es el factor de major importància degut a la seva influencia sobre la obstrucció irreversible del flux i la hiperreactivitat de les vies respiratòries. Actualment, el tractament de l’asma es basa en el control de la inflamació, sense efecte demostrat sobre aquesta remodelació irreversible. L’únic tractament capaç d’actuar sobre aquesta l’alteració es la termoplàstia invasiva, amb un cost-efectivitat discutit i diversos fracassos terapèutics. En aquest context, les cèl·ules mare mesenquimals (MSC) exerceixen efectes inmunoreguladors de potencial interès terapèutic en la remodelació bronquial. No obstant, la seva infusió com a teràpia crònica s’enfronta a la possibilitat de que a traves del seu reclutament i diferenciació puguin participar en la remodelació bronquial, particularment en l’augment de la musculatura llisa. D’altra banda, dades addicionals obtingudes en asma experimental suggereixen que l’efecte terapèutic anti-remodelador de les MSC estan vehiculats per mediadors. Per tant, existeix d’una banda la necessitat d’entendre els mecanismes immunològics involucrats en el procés de remodelació i d’altra banda el desenvolupament de models animals de malaltia experimental amb capacitat de reproduir els mecanismes biològics amb un detall i profunditat lo mes similar possible als humans i permetre una millora en la comprensió i tractament de l’asma. Per tot això, en el present treball de tesis doctoral es va proposar desenvolupar un model d’asma al·èrgica basat en l’exposició local d’un al·ergen, i estudiar en aquest model la possible implicació de les MSC sobre les ASM in vitro. Addicionalment, es van estudiar els mecanismes immunològics de les MSC com a teràpia supressora sobre l’alteració estructural mitjançant sistemes de co-cultiu in vitro i en models desenvolupats in vivo. Els resultats obtinguts van demostrar un paper inductor de les cèl·ules T CD4+ al·ergen-especifiques en la remodelació de la musculatura llisa mitjançant mecanismes de contacte directe, la qual es va mesurar amb un anàlisis del cicle cel·ular per citometria de flux. Addicionalment, les MSC tenen la capacitat d’inhibir la proliferació de les ASM induïdes per cèl·ules T CD4+ nomes quan estan separades mitjançant una membrana permeable “Transwell” i es troben en ratios elevats (MSC:limfòcit). Es va observar que l’efecte supressor no estava vehiculat per exosomes i era secretat espontàniament sense prèvia senyalització. Així mateix, únicament els factors solubles compresos en un rang 30 a 100 kDa de pes molecular en aquest medi condicionat (CM) tenen la capacitat de induir aquest efecte supressor sobre les ASM. Aquest CM (30-100 kDa) generat, va ser testat a diferents dosis in vivo en el model desenvolupat amb anterioritat, i es va observar un efecte supressor en els mecanismes immunològics i el procés de remodelació únicament quan es tractaven als animals amb dosis baixes repetides de CM. Aquest efecte es generat per una possible desviació de la resposta immunitària d’un fenotip característic Th2 en asma al·èrgic, cap a un fenotip Th1/Th17 en les vies respiratòries. En aquest context, el CM de les MSC es postula com una estratègia terapèutica propicia sobre la reversió de la remodelació bronquial degut a una desviació en la resposta immunitària.
El asma es una enfermedad inflamatoria crónica que afecta aproximadamente a unos 300 millones de personas en todo el mundo. Esta enfermedad se caracteriza principalmente por un conjunto de alteraciones estructurales en las vías respiratorias, conocidos mediante el término de “remodelación”, el cual se ha asociado con la severidad de la enfermedad. De entre estas alteraciones, el aumento de la masa de musculo liso inducido por una hiperplasia e hipertrofia de células musculares lisas (ASM), es el factor considerado de mayor importancia debido a su influencia sobre la obstrucción irreversible del flujo aéreo y a la hiperreactividad de las vías respiratorias. Actualmente, el tratamiento del asma se basa en el control de la inflamación, sin efecto demostrado sobre el mecanismo de remodelación irreversible. El único tratamiento capaz de actuar sobre esta alteración es la termoplastia invasiva, con coste-efectividad discutido y varios fracasos terapéuticos. En este contexto, las células madre mesenquimales (MSC) ejercen efectos inmunoreguladores de potencial interés terapéutico en la remodelación bronquial. Sin embargo, su infusión como terapia crónica se enfrenta a la posibilidad de que a través de su reclutamiento y diferenciación puedan participar en la remodelación de las vías respiratorias, particularmente en el crecimiento del musculo liso. Por otra parte, datos adicionales obtenidos en asma experimental sugieren que el efecto terapéutico anti-remodelador de las MSC esta vehiculados por mediadores. Por tanto, existe por una parte la necesidad de comprender los mecanismos inmunológicos que contribuyen en el proceso de remodelación y por otra parte el desarrollo de modelos animales de enfermedad experimental capaces de reproducir los mecanismos biológicos con un detalle y profundidad los más similar posible en sujetos humanos y permitir una mejor comprensión y tratamiento del asma. Por todo ello, en el presente trabajo de tesis doctoral se propuso desarrollar un modelo de asma alérgica basado en la exposición local de un alérgeno, y estudiar en este modelo la posible implicación de las MSC sobre la remodelación del musculo bronquial in vitro. Adicionalmente, se estudió los mecanismos inmunomoduladores de las MSC como terapia supresora sobre esta alteración estructural mediante sistemas de co-cultivo in vitro y en el modelo desarrollado in vivo. Los resultados obtenidos demostraron un papel inductor de las células T CD4+ alérgeno-especificas en la remodelación del musculo liso mediante un mecanismo de contacto directo, la cual se midió mediante análisis de ciclo celular por citometría de flujo. Adicionalmente, las MSC son capaces de inhibir la proliferación de las ASM inducidas por células T CD4+ solamente cuando están separados mediante una membrana permeable “Transwell” y se encuentran en ratios elevados (MSC:linfocito). Además, se observó que este efecto supresor no estaba vehiculado por exosomas y era secretado espontáneamente sin previa señalización requerida. Asimismo, solamente los factores solubles comprendidos en un rango de 30 a 100 kDa de peso molecular que conforman este medio condicionado (CM) son capaces de inducir este efecto supresor sobre las ASM. Este CM (30-100 kDa) obtenido fue testado a diferentes dosis in vivo en el modelo desarrollado con anterioridad, observándose un efector supresor en los mecanismos inmunológicos y el proceso de remodelación solamente cuando se trataban a los animales con dosis bajas repetidas de CM. Este efecto es generado por una posible desviación de la respuesta inmunitaria de un fenotipo característico Th2 en asma alérgico, hacia un fenotipo Th1/Th17 en las vías respiratorias. Por tanto, el CM de las MSC se postula como una estrategia terapéutica prometedora sobre la reversión de la remodelación bronquial debido a una desviación de la respuesta inmunitaria.
Asthma is a chronic inflammatory disease that affects approximately 300 million people worldwide. During the last decades, its prevalence, morbidity, and mortality have increased especially in industrialized countries. This disease is mainly characterized by a set of structural alterations in the respiratory tract, defined by the term “remodeling”, which has been associated with the severity of the disease. Among these alterations, the increase in smooth muscle mass induced by hyperplasia and hypertrophy of smooth muscle cells are crucial factors due to their influence on irreversible airflow obstruction and airway hyperreactivity. Currently, asthma treatments based on the control of inflammation have not demonstrated relevant effects on the remodeling mechanism. The only treatment with the capacity to act on this alteration is invasive thermoplasty, with controversial cost-effectiveness and therapeutic failures. In this context, mesenchymal stem cells (MSC) have a potential therapeutic interest in bronchial remodeling due to their immunoregulatory effects. However, their infusion as a chronic therapy may induce recruitment and differentiation of these cells, which may then participate in airway remodeling, particularly in the smooth muscle growth. However, data from experimental asthma models suggest an anti-remodeling therapeutic effect of MSC, carried by soluble mediators. There is, therefore, a need to understand the immunological mechanisms that contribute to the remodeling process and develop experimental animal models capable of reproducing the physiopathological mechanisms as closely as possible to humans, to provide a better understanding and treatment of asthma. In the present doctoral thesis, it was proposed to develop an allergic asthma model based on primary airway exposure to allergen, to study the implication of MSC in airway remodeling in vitro. Additionally, the immunomodulatory mechanisms of the MSC were studied as a suppressive therapy on airway smooth muscle remodeling by in vitro co-culture systems, and in the in vivo model developed. The results obtained demonstrate a significant role of allergen-specific CD4+ T cells in smooth muscle proliferation through direct cell contact, which was measured by cell cycle analysis through flow cytometry. Additionally, MSC are able to inhibit smooth muscle cell proliferation induced by CD4+ T cells only when they were separated by “Transwell” permeable membranes, and were at high MSC: lymphocyte ratios. Moreover, it was observed that such suppressive effect was not carried by exosomes but mediated by secreted, soluble molecules without prior signaling. Only soluble factors in a range of 30 to 100 kDa molecular weight of MSC-conditioned medium (CM) are capable of suppress smooth muscle cell proliferation. This 30-100-kDa CM, when tested at different doses in vivo in the asthma model, demonstrated a suppressive effect on inflammation and airway remodeling, provided that the animals were treated with repeated low-dose CM. This effect was possibly generated by a deviation of the Th2 immune response towards a Th1/Th17 phenotype in the respiratory tract. In this context, the MSC CM is postulated as a promising therapeutic strategy to reverse airway remodeling through a deviation of the immune response.

Johne�s disease in ruminants is caused by the pathogenic bacterium Mycobacterium avium subspecies paratuberculosis. An experimental infection model in sheep was developed as a prelude to the testing of new vaccines and the development of improved diagnostic assays for Johne�s disease. The final challenge model developed used four doses of 10⁹ viable organisms given at two to three day intervals. Gross and microscopic lesions were found in a high proportion of sheep (80%) at ten months post challenge. There was considerable variation in immune responses from animals challenged with different strains of M. paratuberculosis. Sheep challenged with a low passage laboratory culture of strain (W) M. paratuberculosis, produced strong lymphocyte transformation responses and Interferon gamma (IFN-γ) production at two months post challenge. Subsequent necropsy and culture from intestinal tissues showed only a low level of infection (25%). In comparison a primary tissue isolate of M. paratuberculosis (JD3) resulted in higher (60-90%) infection rates in orally challenged animals. The immune profile from these animals showed very little reactivity for the first three months post challenge, after which IFN-γ production could be detected. Antibody production and lymphocyte transformation response could not be measured until at least seven months post challenge. Sheep challenged with the primary tissue isolate instilled directly into the tonsil resulted in equivalent levels of Johne�s disease to those obtained with oral challenge. However, intratonsillar challenge resulted in higher levels of immune reactivity than oral challenge. The proprietary Johne�s vaccines; NeoparsecTM and GudairTM and an Aqueous vaccine were tested in sheep. The immunological reactions of the sheep to these vaccines showed some variations between the two separate studies, with the NeoparasecTM and GudairTM vaccines evoking high levels of CMI and humoral reactivity within two months of vaccination. Detailed immunological examination of gut associated lymphoid tissues were carried out on subgroups of animals that were either vaccinated or non-vaccinated and went on to develop disease or were immune to experimental challenge. The results showed that the diseased animals examined had multibacillary lesions and strong CMI and humoral responses. There were decreased proportions of CD4⁺, CD8⁺ and CD25⁺ T cells in peripheral blood and gut associated lymphatics of diseased animals compared with the immune or unchallenged subgroups. Profiles from the immune subgroups showed a stronger lymphocyte transformation response than case matched diseased animals. Tissues from immune animals showed increased proportions of B cells above those seen in diseased or unchallenged animals. This study has resulted in the development of a robust experimental sheep model in which Johne�s disease occurs in a high proportion of challenged animals. Critical time points for the establishment of infection or disease have been determined. It can be used in the future to evaluate protective efficacy of vaccines or to critically chart immunological profiles that are associated with infection, disease or protective immunity. Considerable research is needed to develop improved diagnostic tests to identify patterns of immunity during the early stages of infection or while the animal has subclinical disease.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xv, 95 p.; also includes graphics (some col.) Includes bibliographical references (p. 85-95). Available online via OhioLINK's ETD Center

Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE.

The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project.

The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.

CD5 is a scavenger receptor mainly expressed on lymphoid (T and B1a) cells but also on some minor myeloid (Mϕ and DCs) cell subsets. It is long known to negatively modulate differentiation and activation signals mediated by the clonotypic antigen specific receptor complexes of T (TCR) and B1a (BCR) lymphocytes, both being an identity hallmark of the adaptive immune system (Burgueño‐Bucio et al., 2019). Recently, several reports have also shown its ability to recognise and signal the presence of PAMPs of fungal, viral and parasitic origin (Consuegra-Fernández et al., 2015; Burgueño‐Bucio et al., 2019), which is a formal trait of PRRs expressed by the innate immune system’s components (Salazar and Brown, 2018). In consequence, CD5 can be considered as a relevant immunomodulatory receptor at the interphase between the innate and adaptive immune responses. IFIs have emerged in recent decades as a significant health problem associated with high morbidity, mortality, and economic burden (Klingspor et al., 2015). Nowadays, only a few antifungal drugs are available and their use is limited by their associated side effects, making necessary the development of new alternative or complementary therapeutic strategies (Nami et al., 2019). The discovery by our group that CD5 binds with relative high affinity to and signal the presence of β-glucans (Vera et al., 2009) -a constitutive and highly conserved component of fungal cell walls -motivated our interest on exploring the CD5’s physiological function and/or therapeutic potential in IFIs. In our view, the study of soluble and/or membrane-bound immune receptors involved in antifungal immunity, as it may be the case of CD5, could provide an important source of functional information to be translated into such a novel therapeutic approaches. Based on the above mentioned premises, the specific objectives of this thesis have been the following: - To study the influence of the mouse genetic background on fungal infection by analyzing the antifungal immune response of the inbred (C57) and outbred (CD1) mouse strains most widely used in basic and pharma-industry research. - To study the influence of membrane-bound CD5 on fungal infection by analyzing the antifungal immune response of mice genetically deficient for CD5 (cd5-/-). - To study the therapeutic potential of soluble human CD5 administration (alone or in combination) in experimental models of fungal infection. - To study the therapeutic potential of CD5-based adoptive cell transfer strategies by analysing the influence of immune cells transduced with membrane-bound chimerical CD5 receptors in pre-clinical models of fungal infection.

Células T reguladoras (Treg) são cruciais na tolerância periférica e no controle da inflamação. Nós usamos dois modelos bem estabelecidos de tolerância de mucosas para a asma alérgica e a tolerância inalatória local induzida pela exposição crônica a OVA para estudar o aparecimento e função das Treg. Nós mostramos que a tolerância nasal distinguiu da tolerância oral pela produção sistêmica de IgG1 e desenvolvimento da inflamação alérgica na cavidade peritoneal ou pela indução da inflamação das vias aéreas de camundongos RAG-/- reconstituídos com células T CD4+ após desafios com OVA. Observamos também que Treg Foxp3+ migraram para o pulmão alérgico e expressaram fenótipo de ativação e memória que distinguiu essas células das Treg presentes nos linfonodos drenantes. Células T CD4+CD25+ do pulmão dos animais alérgicos suprimiram a proliferação das células T CD4+CD25-, mas não a produção de citocinas Th2. Finalmente, a exposição crônica a OVA levou ao aumento da apoptose de eosinófilos que infiltraram o pulmão resultando na resolução da inflamação alérgica pulmonar.
Regulatory T cells (Treg) are critical for peripheral tolerance and control of inflammation. We used two well established models of mucosal tolerance to allergic airway disease and the local inhalational tolerance induced by chronic OVA exposure to study the appearance and function of Treg cells. We found that nasal tolerance distinguished from oral tolerance by systemic IgG1 antibody production and development of allergic inflammation in the peritoneal cavity or by induction of airway inflammation in RAG-/- mice reconstituted with CD4+ T cells after OVA challenge. We also found that Foxp3+ T cells migrated to allergic lung and expressed an effector/memory phenotype that distinguished them from Treg cells present in lung draining lymph nodes. Lung infiltrating CD4+CD25+ T cells from allergic mice suppressed CD4+CD25- T cell proliferation but not Th2 cytokines production by these cells. Finally, chronic OVA exposure leaded to increased apoptosis of infiltrating lung eosinophils resulting in the resolution of allergic lung inflammation.

Pacific salmon migrate long distances to spawn as part of their life cycle. During this journey from sea to their natal stream, they undergo major endocrine, physiological and immune changes. Cortisol, the primary stress hormone, gradually increases during the journey. Persistent high cortisol levels have deleterious health effects, including suppression of the antibody response. However, pathogens encountered during their journey may stimulate antibody responses to overcome the infection. My main research question focuses on how salmonids balance the immunosuppressive effects of high cortisol levels with activation of the antibody response. A recent field study from our lab showed a transient increase in abundance of B cells during the spawning run which is suggestive of activation of the immune system during this journey. However, our field study had too many confounding variables. In this study, we investigated the activation of the antibody response under conditions of elevated levels of cortisol in rainbow trout under laboratory-controlled conditions. We looked at the effects of a) cortisol alone, b) fish pathogen Flavobacterium psycrophilum (Fp) alone and c) combined cortisol and Fp challenge on the gene expression of immunoglobulins IgM and IgT using qPCR. We have found that cortisol suppresses the IgM response in the spleens of Fp-susceptible line but not in Fp-resistant line of Rainbow trout. No significant effects on B cell development where observed in the anterior kidney. Taken together, our data suggest that the antibody response in Fp-resistant rainbow trout is less sensitive to increased cortisol levels compared to Fp-susceptible fish, confirming our hypothesis that Fp-resistant fish have in some way evolved to manage stress more successfully.

Orientador: Marco Antonio de Andrade Belo
Resumo: O presente estudo investigou os efeitos da ciclofosfamida (CYP) na modulação de proteínas de fase aguda (APPs) e acúmulo celular em exsudatos presentes na bexiga natatória de tilápias (modelo de peixe de aerocistite) e o recrutamento de leucócitos na lamínula de vidro implantadas no tecido subcutâneo. Foram utilizadas 140 tilápias (Oreochromis niloticus) (± 150 g) distribuídas em 14 aquários com capacidade para 250 L de água (n = 10). Os animais foram distribuídos em dois ensaios: Experimento I: foram utilizados 80 animais distribuídos em quatro tratamentos: T1 - controle NAIVE; T2 - controle negativo + solução salina injetada NaCl; T3- controle positivo + injetado com Aeromonas hydrophila; T4- ciclofosfamida, Isopac ® Sigma (200 mg / kg, dose única, por via intraperitoneal) + injetado com A. hydrophila. As amostras de exsudato e sangue foram coletadas 6 e 24 horas após a infecção (HPI) para a contagem de células e determinação do fracionamento eletroforético das APPs por SDS-PAGE, digestão de proteínas em gel e identificação por espectrometria de massa. Experimento II: os peixes foram alocados (n=60) em 3 grupos experimentais: G1 - controle NAIVE; G2 – controle positivo + implante de lamínula; G3 - ciclofosfamida, Isopac ® Sigma (200 mg / kg, dose única, por via intraperitoneal) + implante de lamínula. Após serem anestesiados, foram submetidos ao implante da lamínula de vidro de 10mm de diâmetro no tecido subcutâneo. As amostras de sangue foram coletadas e as lamínulas foram... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The present study investigated the effects of cyclophosphamide (CYP) on the modulation of acute phase proteins (APPs) and cellular accumulation in exudates present in the swim bladder of tilapia (aerocystitis fish model) and the recruitment of leukocytes on a glass cover slip implanted in the subcutaneous tissue. One hundred forty tilapia (Oreochromis niloticus) (± 150 g) were distributed in 14 aquariums with capacity of 250 L (n = 10), for two trials. Experiment I: For this purpose, 80 fish were distributed in four treatments: T1 – naïve control; T2 - negative control + injected with NaCl solution; T3- positive control + injected with A. hydrophila; and T4- cyclophosphamide (200 mg/kg) + injected with A. hydrophila. Samples of exudate and blood were collected 6 and 24 hours post-inoculation (HPI) for cell counts and determination of the APP electrophoretic fractionation by SDS-PAGE, in-gel protein digestion and mass spectrometric identification. Experiment II: Fish were allocated (n = 60) in 3 experimental groups: G1 – naive control; G2 - positive control + coverlet implant; and G3 - cyclophosphamide (200 mg/kg, single dose intraperitoneally) + cover slip implant. The glass coverslips (10 mm diameter) were implanted after anesthetization in the subcutaneous tissue. Blood samples were collected and the coverslips were carefully removed and washed with 0.9% saline 74 and 144 hours implantation. Then they were fixed in Bouin's solution and stained with hematoxylineosin. The tot... (Complete abstract click electronic access below)
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