Whether an individual's state of mind can influence the body's immune system has been studied for several decades. Historical notions of a homeostatic, self-contained, and self-monitored system have been discarded. Studies have explored conditioning effects and cognitive behavioral methods to affect the immune response. This study is based on the assumption that relaxation imagery can be used as an endogenous means to produce specific physiological change in the immune function. Subjects were instructed to make a directional change in the absolute number of peripheral lymphocytes using relaxation imagery.

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Lactic acid bacteria produce pediocin-like bacteriocins designated as Class Ha. These antimicrobial peptides are antagonistic against Listeria monocytogenes and other closely related Gram-positive bacteria Self-protection of the producer organism is attributed to the immunity proteins, encoded by genes that are eo-transcribed with the structural gene that encode the bacteriocin. The lactic acid bacterium, Leuconostoc gelidum UAL 187-22 is immune to its own bacteriocin, leucocin A. This is accredited to its immunity protein and the possible absence of a receptor on its cytoplasmic membrane. Leucocin A was purified from the supernatant of 1. gelidum to 90% purity by ion-exhange chromatography and C18 reverse phase High Pressure Liquid Chromatography (RP-HPLC) eluted with an acetonitrile, 0.1% Triflouroacetic acid (TFA) gradient. The immunity gene was isolated from the same producer using the polymerase chain reaction from the recombinant plasmid pJF 5.5 using primers EAL-2 and EAL-3. The amplicon was truncated into versions A and B by removing the C- and N-terminals, with HaeIII and ClaI restriction enzymes, respectively. The amplicon and the truncated fragments A and B were cloned into pMALc2 to construct recombinant plasmids pKPl, pKPIA and pKPIB, correspondingly, which were transformed into Escherichia coli (E. coli) strain JMI03. Clones were confirmed by colony PCR and Southern blot hybridization. The recombinant clones were subsequently expressed as MBP-IP, MBP-IPA and MBP-IPB fusion proteins that were verified by Western blot using the anti-MBP antibody. Factor Xa protease was used to cleave MBP from the proteins of interest. The resulting pure immunity protein versions had an approximate molecular weight of slightly more that 10 kDa. The binding interactions of the purified immunity protein constructs and leucocin A were compared on the Biacore 2000 instrument with surface plasmon resonance. None of the immunity constructs interacted with leucocin A, however, the N-terminal region of the immunity protein interacted with the cytoplasmic extract. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

La tuberculose demeure un problème majeur de santé publique. Mycobacterium tuberculosis infecte un tiers de la population mondiale et environ 3 millions de décès, des suites de l’infection, sont recensés chaque année dans le monde. La mise au point d’une stratégie vaccinale efficace constitue dès lors la solution idéale pour tenter d’éradiquer la bactérie. Le système immunitaire humain répond à l’infection par l’induction d’une réponse cellulaire, caractérisée essentiellement par la sécrétion de médiateurs pro-inflammatoires, comme l’interféron-gamma (IFN-?). A la fois les lymphocytes T CD4+ et T CD8+ produisent cette cytokine, mais les seconds sont également doués de propriétés cytotoxiques, entraînant la mort de la cellule infectée et du bacille. L’évaluation du potentiel de nouveaux candidats vaccins implique dès lors la caractérisation exhaustive de la réponse immunitaire induite. <p>La « heparin-binding hemagglutinin (HBHA) » est une protéine de 28-kDa, sécrétée et exprimée à la surface de M. tuberculosis et de M. bovis BCG. Montrant une affinité importante pour les gycoconjugués sulfatés, elle favorise la dissémination hématogène du bacille de Koch. <p><p>Nos résultats démontrent que la HBHA stimule l’immunité à médiation cellulaire humaine avec, toutefois, des différences selon que le sujet infecté souffre ou non de tuberculose active. En effet, les cellules mononuclées circulantes, de la majorité des individus infectés mais non-malades, secrètent de l’IFN-? en réponse à la HBHA, alors qu’une minorité de sujets malades produit de faibles quantités d’IFN-? après stimulation in vitro avec l’antigène. Lors de l’infection naturelle par le bacille de Koch, la HBHA devient dès lors une cible pour le système immunitaire, et plus particulièrement au sein des sujets, généralement considérés, comme protégés.<p>L’analyse de la réponse cellulaire, spécifique à l’adhésine, démontre que les lymphocytes T CD4+, mais également T CD8+, des sujets infectés mais non-malades, produisent de l’IFN-? L’antigène est effectivement présenté aux lymphocytes T grâce aux glycoprotéines du complexe majeur d’histocompatibilité de classe I et de classe II. Le phénotypage des cellules productrices d’IFN-? témoigne également la participation des cellules « natural killer (NK) » dans la réponse immunitaire contre la HBHA. En l’absence des lymphocytes T restreints à l’antigène, les cellules NK se montrent toutefois incapables de secréter de l’IFN-? au contact de la HBHA. Les interactions entre les lymphocytes T, spécifiques à l’antigène, déterminent également la production de cytokines. Alors que la déplétion des cellules T CD8+ diminue légèrement la production d’IFN-? l’absence des lymphocytes T CD4+ abolit toute sécrétion résiduelle d’IFN-? lors de la stimulation avec la HBHA. Par contre, les lymphocytes T CD8+, pré-stimulés avec l’antigène en présence de cellules T CD4+, répondent secondairement à la présentation de la HBHA par des macrophages. Ce résultat suggère une coopération entre ces deux sous-populations cellulaires, afin de produire de l’IFN-? à l’encontre de la HBHA. Grâce à un contact cellulaire, les lymphocytes T CD4+ spécifiques à la HBHA soutiennent effectivement l’activation des cellules T CD8+.<p>Outre la production de cytokines, la participation des lymphocytes T CD8+ à la lutte contre M. tuberculosis, se traduit également par leurs fonctions cytotoxique et bactéricide. La caractérisation des cellules T CD8+, spécifiques à la HBHA, s’est dès lors poursuivie par l’évaluation de leur potentiel cytolytique. Après expansion clonale, les lymphocytes T CD8+ induisent la mort des macrophages présentant la HBHA. Le mécanisme cytotoxique engage la libération du contenu des granules cytoplasmiques, comme le montre l’augmentation de la synthèse de perforine et de granzyme A, lorsque les cellules T CD8+ sont stimulées avec la HBHA. Privés de ces médiateurs solubles, les lymphocytes T CD8+, spécifiques à la HBHA sont alors incapables de lyser les cellules cibles. En définitive, l’activité microbicide constitue actuellement le meilleur corrélat de protection. La culture de macrophages infectés par M. bovis BCG, en présence de cellules T CD8+ spécifiques à la HBHA, limite partiellement la croissance de la bactérie phagocytée, soulignant le pouvoir anti-mycobactérien de l’immunité cellulaire induite par la HBHA, chez le sujet infecté mais non-malade.<p>D’autre part, l’analyse biochimique, menée à l’Institut Pasteur de Lille, démontre que la HBHA subit une modification post-traductionnelle, lors de sa synthèse. Il s’agit d’une méthylation des multiples résidus lysine, qui composent son extrémité C-terminale. La comparaison des formes native méthylée et recombinante non-méthylée de la HBHA démontre que la méthylation détermine l’immunogénicité et le pouvoir protecteur de la HBHA. En effet, contrairement à la HBHA native, la forme recombinante stimule faiblement la production d’IFN-? chez les individus infectés mais non-malades, et ne protège pas la souris contre l’infection par le bacille de Koch. La sécrétion d’IFN-? est, par ailleurs, partiellement restaurée lorsque la HBHA est artificiellement méthylée in vitro. Les splénocytes murins se comportent également différemment, selon qu’ils ont été immunisés avec la forme méthylée ou non. Alors que la HBHA recombinante est immunogène chez la souris et chez l’homme, l’immunité cellulaire murine induite demeure impassible face à l’infection des phagocytes par les mycobactéries, ce qui se traduit par l’absence de protection.<p><p>En conclusion, la HBHA se compose d’épitopes protecteurs, qui dépendent de la présence des groupements méthyls, associés à son domaine C-terminal. Il s’agit, à notre connaissance, de la première mise en évidence de l’implication de la méthylation dans la réponse d’immunité cellulaire à l’encontre d’une protéine. De plus, l’immunité adaptative spécifique à la HBHA, chez le sujet infecté mais non-malade, se caractérise par les trois principaux corrélats de protection, actuellement décrits chez l’homme. Le potentiel vaccinal de cette adhésine mycobactérienne est donc bien réel. / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

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The present study was undertaken to explore the relationship of cellular immunity to total serum ICE in an unselected sample of 278 healthy infants. Statistical analyses of infant history profiles and cellular immune and serum IgE determinations from cord blood and 10 month samples were performed using SPSSx. During the first year of life, the percent CD3, CD4, CD8, ERFC, and B-cell counts significantly increased and the CD4/CD8 ratio and responses to ConA and PW mitogens significantly decreased. Boys had lower percent CD8 cell counts and decreased responses to ConA and PW mitogens in cord blood. Cellular immune variable values were not associated with total serum ICE in any analysis. Early feeding method had no effect on 10 month serum IgE levels or cellular immune variables. Cord blood percent CD3 and CD4 cell counts were significantly decreased in infants with parental histories of asthma. In summary, (1) boys had a lower percent of suppressor T-cells and lower mitogen responses in cord blood (2) boys and girls reached similar levels for all variables by 10 months of age (3) infants with familial histories of asthma had fewer total and helper T-cells in cord blood.

INTRODUÇÃO: A paracoccidioidomicose (PCM), causada pelo fungo dimórfico Paracoccidioides brasiliensis, é uma doença sistêmica granulomatosa, endêmica em toda América Latina. Estudos anteriores no modelo experimental e em humanos relacionaram a resposta imune tipo Th1 à proteção, com imunodepressão antígenoespecífica transitória nas formas aguda e crônica, e recuperação após cura clínica. As células dendríticas de origem mielóide (DCs) são as mais potentes apresentadoras de antigeno, sendo capazes de induzir resposta Th1 em linfócitos T, porém seu papel na PCM humana ainda não foi descrito. OBJETIVO: Investigar as características in vitro de DCs derivadas de monócitos de pacientes com PCM. MÉTODOS: DCs de pacientes com PCM ativa (AP) e tratada (TP), e de indivíduos sadios (CO) foram geradas a partir de monócitos do sangue periférico aderidos ao plástico e tratados com IL-4 e GM-CSF. Após 6 dias, as DC diferenciadas (CD11c+, CD1a+, CD14-) foram ativadas com TNF- (DC+TNF) ou deixadas sem estímulo (nDC) por 48 horas. As moléculas de superfície CD11c, CD1a, HLA-DR, CD86, CD80 e DCSIGN das DCs foram analisadas por citometria de fluxo e as citocinas IL-10, IL- 12p40 e CCL18 foram dosadas nos sobrenadantes das culturas por ELISA. As DCs de TP e CO também foram geradas a partir de monócitos obtidos por seleção positiva em coluna magnética (micropérolas) e comparadas às células obtidas pelo método de aderência. Os sobrenadantes das DCs de TP foram utilizados como estímulo das DCs de CO e as moléculas de superfície e a secreção de citocinas foram analisadas novamente. RESULTADOS: As DCs dos grupos AP e TP apresentaram expressão de CD11c e CD1a similar à observada nas DCs de CO. No grupo TP, foi observada maior expressão de HLA-DR, CD86 e DC-SIGN nas nDC e HLA-DR e CD86 nas DC+TNF que nos grupos AP e CO que, por sua vez, apresentaram percentual de DCs positivas e a expressão dessas moléculas similares. As nDC do grupo AP secretaram significantemente menos IL-10 que as DCs de CO, sendo que a ativação com TNF- reduziu a produção dessa citocina em todos os grupos. O efeito oposto foi observado na secreção de IL-12p40, sendo que as DC+TNF do grupo TP apresentaram os maiores níveis dessa citocina. Tanto nDC quanto DC+TNF do grupo AP secretaram mais CCL18 que as correspondentes do grupo TP. Os sobrenadantes de TP tiveram pouco ou nenhum efeito sobre as DCs de CO. CONCLUSÕES: DCs derivadas de monócitos do sangue periférico de pacientes com PCM podem ser diferenciadas e ativadas in vitro com TNF-a, expressando as moléculas de superfície características. DCs de pacientes com PCM tratada tem alta expressão de HLA-DR, CD86 e DCSIGN e secreção aumentada de IL-12p40, e DCs de pacientes com a doença ativa apresentam expressão de moléculas similar às de DCs de indivíduos sadios. DCs obtidas por aderência de monócitos resultaram em maior freqüência de DCs CD86+, com maior secreção de IL-12p40 que células obtidas por micropérolas em amostras de indivíduos sadios e de pacientes tratados. Neste trabalho demonstrou-se a plasticidade in vitro de DCs derivadas de monócitos de pacientes com PCM, revelando o potencial dessas células na geração de uma resposta imune protetora / INTRODUCTION: Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis, being a granulomatous systemic disease, endemic in Latin America. Previous studies on experimental and human PCM have established the relation between Th1 immune response and protection, antigenspecific transitory immunodepression in acute and chronic forms and recovery after clinical treatment. Myeloid dendritic cells (DCs) are the most potent antigen presenting cells, capable of inducing Th1 response on T lymphocytes. Their role on human PCM has not yet been described. OBJECTIVE: To investigate in vitro characteristics of monocyte-derived DCs from PCM patients. METHODS: DCs from active (AP) and treated PCM (TP) and from healthy individuals (CO) were generated from plastic adhered monocytes treated with IL-4 and GM-CSF. After 6 days, differentiated DCs (CD11c+, CD1a+, CD14-) were activated with TNF- (DC+TNF) or untreated (nDC) for 48 hours. Surface molecules CD11c, CD1a, HLA-DR, CD86, CD80 and DC-SIGN were analyzed by flow citometry and cytokines IL-10, IL- 12p40 and CCL18 were assayed on cultures supernatants by ELISA. DCs from the TP and the CO groups were also generated from positive selection of monocytes on magnetic column (microbeads) and compared to adhered monocyte-derived DCs. DC from the CO group were stimulated with supernatants from the TP cultures followed by flow cytometric analysis of surface molecules and production of cytokines. RESULTS: DCs from the AP and the TP groups showed similar expression of CD11c and CD1a in comparison to the CO group. On the TP group, nDC showed higher expression of HLA-DR, CD86 and DC-SIGN and DC+TNF of HLA-DR and CD86 than the AP and the CO groups which had similar percentage of positive cells and expression of these molecules. nDC from the AP group showed significantly lower levels of IL-10 than the CO group and decreased levels of this cytokine were observed with TNF- activation. The opposite effect was observed on IL-12p40 production in which DC+TNF from the TP group showed the highest levels. Both nDC and DC+TNF from the AP group had increased levels of CCL18 compared to the TP cells. Supernatants from TP had little or no effect on DCs from the CO group. CONCLUSIONS: Peripheral blood monocyte-derived DCs from patients with PCM can be differentiated and activated with TNF- in vitro with expression of typical molecules. DCs from patients with treated PCM showed up-regulation of HLA-DR, CD86 and DC-SIGN and the highest production of IL-12p40. DCs from patients with active PCM had similar expression of these molecules compared to control group. Adherence of monocytes resulted in higher frequency of CD86+ DCs with upregulation of IL-12p40 than microbeads positive selection method on samples from healthy donors and treated patients. This study demonstrated the in vitro plasticity of monocyte-derived DCs from patients with PCM, revealing the potential of these cells in generating a protective immune response