Este estudo avaliou a imunidade humoral e celular em cães naturalmente infectados com L. (L.) chagasi correlacionando com a transmissibilidade para o vetor. Soros e biópsias de baço, linfonodo e pele foram coletados de 120 cães provenientes do Centro de Controle de Zoonoses do município de Araçatuba, São Paulo, Brasil. Os soros foram processados por ELISA para detecção de IgG, IgG1, IgG2 e IgE; e as biópsias foram processadas por técnicas histológicas usuais coradas pelo HE e imunoistoquímica para a detecção de parasito, macrófago e células T CD3. De acordo com os sinais clínicos, 65/120 (54%) cães foram classificados como sintomáticos e 55/120 (46%) como assintomáticos. O diagnóstico parasitológico foi confirmado em 71% dos sintomáticos e em 40% dos assintomáticos. A correlação dos sinais clínicos com parasitismo mostrou que a carga parasitária estava diretamente associada com cães sintomáticos (p<0.05). Em relação aos anticorpos específicos anti-L.(L.)chagasi, cães de área endêmica com diagnóstico parasitológico positivo mostraram maiores níveis de IgG total comparado com ambos os controles (p<0.05), sem diferença entre cães sintomáticos e assintomáticos. IgG1 esteve presente em baixos níveis e foi mais intensa no grupo sintomático parasito-positivo (p<0.05). Níveis mais elevados foram observados para IgG2 em cães de área endêmica (p<0.05), mas sem correlação com o parasitismo e sinais clínicos. IgE também esteve presente em baixos níveis, mas mostrou diferenças entre cães de área endêmica e cães de área não endêmica; e cães com diagnóstico parasitológico positivo mostrou níveis mais elevados que cães com diagnóstico parasitológico negativo (p<0.05). Histopatologicamente, linfonodos mostraram hiperplasia e hipertrofia de macrófagos na área medular e em muitos casos linfadenite granulomatosa. Na polpa branca do baço, hiperplasia folicular foi observada; e a polpa vermelha mostrou granulomas. As lesões de pele foram caracterizadas por infiltrado inflamatório crônico na derme formado por macrófagos, linfócitos e plasmócitos; variando de discreto a intenso, assim como de focal a difuso. Foi evidente a presença de granulomas epitelióides na pele de alguns animais. Imunoistoquímica mostrou presença de células marcadas pelo anticorpo anti-macrófago e anti-CD3 em 100% dos baços e linfonodos variando de intensidade entre discreto e intenso. Na pele, macrófagos foram positivos em 90% e células CD3 em 39% dos casos. Houve associação direta entre baixa expressão de células CD3 e alto parasitismo na pele. Animais assintomáticos mostraram baixa expressão de macrófagos junto com baixo parasitismo na pele. Em relação ao xenodiagnóstico, no 4o dia depois da alimentação, as fêmeas dos vetores foram dissecadas e examinadas para observação de parasitos no intestino. Formas promastigotas foram observadas em 27% das fêmeas que se alimentaram em cães sintomáticos e em 42% das fêmeas que se alimentaram em cães assintomáticos. A técnica da PCR foi também utilizada para avaliar as fêmeas positivas depois do xenodiagnóstico. DNA de Leishmania foi detectado em 24% das fêmeas que se alimentaram em cães sintomáticos e em 34% das fêmeas que se alimentaram em cães assintomáticos. Os dados mostraram que a imunidade humoral e celular não teve correlação direta com as formas clínicas de leishmaniose canina. A alta porcentagem de vetores infectados na alimentação em cães assintomáticos mostra a importância destes animais na transmissibilidade para o vetor. / These studies evaluate humoral and cellular immunity in dogs naturally infected with L. (L.) chagasi correlating to the transmissibility to the vector. Serum and biopsy from spleen, lymph node and skin were collected from 120 dogs referred to the Center of Zoonosis Control of Araçatuba city, São Paulo, Brazil. The sera were processed by ELISA for IgG, IgG1, IgG2 and IgE detection; and the biopsies were processed usual histological techniques stained by HE and immunohistochemistry for parasite, macrophage and T CD3 cells detection. According to the clinical signs, 65/120 (54%) dogs were classified as symptomatic and 55/120 (46%) as asymptomatic. Parasitological diagnosis was confirmed in 71% of symptomatic and in 40% of asymptomatic dogs. The correlation of clinical signs and parasitism showed that parasite burden was directly associated with symptomatic dogs (p<0.05). Concerning to L.(L.)chagasi-specific antibodies, dogs from the endemic area with positive parasitological diagnosis showed high levels of total IgG compared to both controls (p<0.05), without difference between symptomatic and asymptomatic dogs. IgG1 was present at low levels and was more intense in the parasite-positive symptomatic group (p<0.05). More elevated levels were observed for IgG2 in dogs from endemic area (p<0.05), but with no correlation to parasitism and clinical signs. IgE was also present at low levels, but showed differences between dogs from non-endemic and endemic areas; and dogs with positive parasitological diagnosis showed higher levels than dogs with negative parasitological diagnosis (p<0.05). Histopathologically, lymph nodes showed macrophage hyperplasia and hypertrophy in the medullary area and in many cases granulomatous lymphadenitis. In the white pulp of the spleen, follicular hyperplasia was observed; and the red pulp showed granulomas. The skin lesions were characterized by dermal chronic inflammatory infiltrate formed by macrophages, lymphocytes and plasma cells; it varied between descreet to intense, as well as focal to diffuse. The epithelioid granulomas were evident in the skin of some animals. Immunohistochemistry showed presence of labeled cells by anti-macrophage and anti-CD3+ antibodies in 100% of spleen and lymph nodes varying the intensity between mild to intense. Macrophage was positive in 90% of the skin and CD3 cells in 39%. There was a direct association between lower CD3 cells expression and higher parasite burden in the skin. Asymptomatic animals showed lower macrophage expression together with lower parasitism in the skin. Concerning to the xenodiagnosis, on the 4th day after the blood meal, female flies were dissected and examined for visible parasites in the gut. Promastigotes forms were observed in 27% of female which fed in symptomatic dogs and in 42% of female which fed in asymptomatic dogs. PCR technique was also used to evaluate the positive females after the xenodiagnosis. Leishmania DNA was detected in 24% of female which fed in symptomatic dogs and in 34% of female which fed in asymptomatic dogs. The data showed that the humoral and cellular immune response not has direct correlation to the clinical form of canine leishmaniasis. The high percentage of sand flies female infected by feeding in the asymptomatic dogs show the importance these animals on the parasite transmissibility to the vector.

Este estudo avaliou a imunidade humoral e celular em cães naturalmente infectados com L. (L.) chagasi correlacionando com a transmissibilidade para o vetor. Soros e biópsias de baço, linfonodo e pele foram coletados de 120 cães provenientes do Centro de Controle de Zoonoses do município de Araçatuba, São Paulo, Brasil. Os soros foram processados por ELISA para detecção de IgG, IgG1, IgG2 e IgE; e as biópsias foram processadas por técnicas histológicas usuais coradas pelo HE e imunoistoquímica para a detecção de parasito, macrófago e células T CD3. De acordo com os sinais clínicos, 65/120 (54%) cães foram classificados como sintomáticos e 55/120 (46%) como assintomáticos. O diagnóstico parasitológico foi confirmado em 71% dos sintomáticos e em 40% dos assintomáticos. A correlação dos sinais clínicos com parasitismo mostrou que a carga parasitária estava diretamente associada com cães sintomáticos (p<0.05). Em relação aos anticorpos específicos anti-L.(L.)chagasi, cães de área endêmica com diagnóstico parasitológico positivo mostraram maiores níveis de IgG total comparado com ambos os controles (p<0.05), sem diferença entre cães sintomáticos e assintomáticos. IgG1 esteve presente em baixos níveis e foi mais intensa no grupo sintomático parasito-positivo (p<0.05). Níveis mais elevados foram observados para IgG2 em cães de área endêmica (p<0.05), mas sem correlação com o parasitismo e sinais clínicos. IgE também esteve presente em baixos níveis, mas mostrou diferenças entre cães de área endêmica e cães de área não endêmica; e cães com diagnóstico parasitológico positivo mostrou níveis mais elevados que cães com diagnóstico parasitológico negativo (p<0.05). Histopatologicamente, linfonodos mostraram hiperplasia e hipertrofia de macrófagos na área medular e em muitos casos linfadenite granulomatosa. Na polpa branca do baço, hiperplasia folicular foi observada; e a polpa vermelha mostrou granulomas. As lesões de pele foram caracterizadas por infiltrado inflamatório crônico na derme formado por macrófagos, linfócitos e plasmócitos; variando de discreto a intenso, assim como de focal a difuso. Foi evidente a presença de granulomas epitelióides na pele de alguns animais. Imunoistoquímica mostrou presença de células marcadas pelo anticorpo anti-macrófago e anti-CD3 em 100% dos baços e linfonodos variando de intensidade entre discreto e intenso. Na pele, macrófagos foram positivos em 90% e células CD3 em 39% dos casos. Houve associação direta entre baixa expressão de células CD3 e alto parasitismo na pele. Animais assintomáticos mostraram baixa expressão de macrófagos junto com baixo parasitismo na pele. Em relação ao xenodiagnóstico, no 4o dia depois da alimentação, as fêmeas dos vetores foram dissecadas e examinadas para observação de parasitos no intestino. Formas promastigotas foram observadas em 27% das fêmeas que se alimentaram em cães sintomáticos e em 42% das fêmeas que se alimentaram em cães assintomáticos. A técnica da PCR foi também utilizada para avaliar as fêmeas positivas depois do xenodiagnóstico. DNA de Leishmania foi detectado em 24% das fêmeas que se alimentaram em cães sintomáticos e em 34% das fêmeas que se alimentaram em cães assintomáticos. Os dados mostraram que a imunidade humoral e celular não teve correlação direta com as formas clínicas de leishmaniose canina. A alta porcentagem de vetores infectados na alimentação em cães assintomáticos mostra a importância destes animais na transmissibilidade para o vetor. / These studies evaluate humoral and cellular immunity in dogs naturally infected with L. (L.) chagasi correlating to the transmissibility to the vector. Serum and biopsy from spleen, lymph node and skin were collected from 120 dogs referred to the Center of Zoonosis Control of Araçatuba city, São Paulo, Brazil. The sera were processed by ELISA for IgG, IgG1, IgG2 and IgE detection; and the biopsies were processed usual histological techniques stained by HE and immunohistochemistry for parasite, macrophage and T CD3 cells detection. According to the clinical signs, 65/120 (54%) dogs were classified as symptomatic and 55/120 (46%) as asymptomatic. Parasitological diagnosis was confirmed in 71% of symptomatic and in 40% of asymptomatic dogs. The correlation of clinical signs and parasitism showed that parasite burden was directly associated with symptomatic dogs (p<0.05). Concerning to L.(L.)chagasi-specific antibodies, dogs from the endemic area with positive parasitological diagnosis showed high levels of total IgG compared to both controls (p<0.05), without difference between symptomatic and asymptomatic dogs. IgG1 was present at low levels and was more intense in the parasite-positive symptomatic group (p<0.05). More elevated levels were observed for IgG2 in dogs from endemic area (p<0.05), but with no correlation to parasitism and clinical signs. IgE was also present at low levels, but showed differences between dogs from non-endemic and endemic areas; and dogs with positive parasitological diagnosis showed higher levels than dogs with negative parasitological diagnosis (p<0.05). Histopathologically, lymph nodes showed macrophage hyperplasia and hypertrophy in the medullary area and in many cases granulomatous lymphadenitis. In the white pulp of the spleen, follicular hyperplasia was observed; and the red pulp showed granulomas. The skin lesions were characterized by dermal chronic inflammatory infiltrate formed by macrophages, lymphocytes and plasma cells; it varied between descreet to intense, as well as focal to diffuse. The epithelioid granulomas were evident in the skin of some animals. Immunohistochemistry showed presence of labeled cells by anti-macrophage and anti-CD3+ antibodies in 100% of spleen and lymph nodes varying the intensity between mild to intense. Macrophage was positive in 90% of the skin and CD3 cells in 39%. There was a direct association between lower CD3 cells expression and higher parasite burden in the skin. Asymptomatic animals showed lower macrophage expression together with lower parasitism in the skin. Concerning to the xenodiagnosis, on the 4th day after the blood meal, female flies were dissected and examined for visible parasites in the gut. Promastigotes forms were observed in 27% of female which fed in symptomatic dogs and in 42% of female which fed in asymptomatic dogs. PCR technique was also used to evaluate the positive females after the xenodiagnosis. Leishmania DNA was detected in 24% of female which fed in symptomatic dogs and in 34% of female which fed in asymptomatic dogs. The data showed that the humoral and cellular immune response not has direct correlation to the clinical form of canine leishmaniasis. The high percentage of sand flies female infected by feeding in the asymptomatic dogs show the importance these animals on the parasite transmissibility to the vector.

Development of combination products such as tissue engineered constructs which combine biomaterials with biologics has prompted the need to clarify the role of biomaterial in potentiating the immune response towards the biological component due to adjuvant effect. In tissue engineering applications, immune responses are to be minimized while vaccine strategies seek to enhance the protective immune response. Thesis project presented herein showed that adjuvant effect of poly(lactic-co-glycolic acid) (PLGA) is mediated in part by maturation of dendritic cells (DCs), immune cells that orchestrate adaptive immune response. Maturation of human peripheral blood monocyte-derived DCs in response to PLGA contact was demonstrated in vitro and in vivo by increased co-stimulatory and MHC molecule expression, mixed lymphocyte reaction, cytokine release, and delayed type hypersensitivity reaction. In contrast to PLGA, agarose did not induce DC maturation, in accordance with its low inflammatory effect. Roles of various receptors involved in DC maturation and recognition of biomaterials were assessed by in vitro receptor blocking studies. In particular, role of Toll-like receptors were further investigated using DCs derived from bone marrows of murine model of Toll-like receptor 4 deficiency (C3H/HeJ). While PLGA induced maturation of DCs from C57BL6 mice, maturation was not observed in DCs from C3H/HeJ strain or control strain, C3H/HeOuJ, perhaps due to particular haplotypes of these animals. Collectively, these results establish the differential adjuvant effects of agarose and PLGA on the level of DC maturation, and begin to elucidate the mechanisms of biomaterial adjuvant effect. In addition, assays developed herein provide methods to screen for biomaterials to be used in combination products, such that biomaterials with desired levels of adjuvanticity as measured by DC maturation effects may be selected for given application.

The host response toward biomaterial component of tissue-engineered devices has been extensively investigated. The objective of this research was to understand the response of dendritic cells (DCs) to different biomaterials upon contact and identify biomaterials suitable for use in tissue engineering constructs for rheumatoid arthritis (RA) applications. Differential levels of functional DC maturation were observed depending on the type of biomaterial in 2-dimensional films or 3-dimensional scaffolds used to treat immature DCs; Poly(lactic-co-glycolic acid) (PLGA) or chitosan supported higher levels of DC maturation, as compared to immature DCs. Alginate supported moderate levels of DC maturation. Agarose did not support DC maturation whereas hyaluronic acid inhibited DC maturation. Further, these DCs treated with different biomaterials induced differential phenotype and polarization of autologous T cells upon co-culture of DCs and T cells; DCs treated with PLGA induced T helper type I with immunogenic response while DCs treated with agarose did T helper type II with tolerogenic response. Effect of different biomaterials (PLGA and agarose) was assessed in vivo upon implantation of them into the knee joint of RA-induced rabbit. Total leukocyte concentrations in the peripheral blood or in the joint lavage of the left knees (untreated control) were observed in differential levels depending on the biomaterial implant, possibly due to the systemic circulation of the peripheral blood. Furthermore, cartilage and bone healing progression was differentially observed in the osteochondral defect of the knee joint of RA-induced rabbit, depending on type of biomaterial scaffold implanted into the defect. Collectively, these results demonstrate the multifunctional impacts of inherently different biomaterials on in vitro immunomodulation of phenotype and polarization of DCs and autologous T cells. Furthermore, taken together with these immunomodulatory impacts of biomaterials, in vivo effects of different biomaterial scaffolds on RA environment shown in this study can suggest the criteria of selection and design of biomaterials for orthopedic tissue engineering, which may ultimately be best integrated into the diseased cartilage and bone.

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

A cromoblastomicose é uma infecção fúngica subcutânea causada por fungos da família Dematiceae sendo o principal agente etiológico o fungo Fonsecaea pedrosoi (F. pedrosoi). Estes fungos induzem uma lesão crônica na pele de freqüente recidivas. O objetivo do trabalho foi avaliar alguns aspectos imunológicos na cromoblastomicose experimental através de dois modelos de infecção pelas vias: intraperitoneal (i.p.) e subcutânea (s.c.). No primeiro modelo de infecção pela via s.c. em camundongos BALB/c infectados com 106 conídios de F. pedrosoi, ocorreu a cura espontânea da infecção em aproximadamente 4 semanas. Na subtipagem de linfócitos T em linfonodos regionais ocorreu um predomínio de células T CD4+ que foi constante até a 4ª semana de infecção, no entanto, observamos aumento significativo de linfócitos T CD8+ ao longo da infecção sugerindo que essa população tenha também uma importante participação no controle da doença. Os ensaios de linfoproliferação demonstraram, na 1ª semana de infecção, elevado índice de proliferação celular quando as células de linfonodos foram estimuladas in vitro com antígenos de F. pedrosoi, além da liberação principalmente da citocina IFN-&#947;, já na 4ª semana de infecção não foi detectado proliferação celular. Esses resultados sugerem que no início da infecção a resposta celular seja mediada principalmente por linfócitos T CD4+ produtores de IFN-&#947;, o que nos sugere, que neste modelo experimental, polarize uma resposta de células T do tipo Th1. No segundo modelo de infecção, via intraperitoneal (i.p.), camundongos BALB/c infectados com 106 conídios de F. pedrosoi mostraram desenvolvimento de infecção crônica com preservação da imunidade celular mesmo após a 8ª semana. Ainda pela via i.p., os camundongos C57BL/6 nocautes de T CD4+ apresentaram uma maior carga fúngica no início da infecção e em tempos mais tardios a carga fúngica foi semelhante aos camundongos controles (C57BL/6); esses mesmos animais nocautes não apresentaram uma ativação da resposta celular medida pelo teste de HTT (Hipersensibilidade do Tipo Tardio). Quando avaliamos o padrão de citocinas, a citocina IFN-&#947; produzida pelos órgãos baço e fígado apresentou menores níveis no início da infecção quando comparado ao camundongos controle. Já os níveis de IL-10 aumentaram gradativamente ao longo da infecção e IL-4 não apresentou diferenças em relação ao controle. Nos camundongos nocautes para coa (C57BL/6 CD8 \"KO\"), a carga fúngica, os níveis de citocinas e o teste de HTT foram semelhantes aos animais controle. Esses resultados mostraram que pela via i.p. os linfócitos T, principalmente células T CD4+ são importantes no controle inicial da infecção. Em tempos mais tardios a infecção foi controlada mesmo em camundongos deficientes de linfócitos TCD 4+ ou T CD8+, sugerido que outras células como macrófagos ou NK, estariam atuando de forma mais efetiva no controle da infecção. / Abstract not available.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-03T11:38:10Z No. of bitstreams: 1 flaviamarciadecastroesilva.pdf: 2659537 bytes, checksum: 1c8331ace53624c1553f76ae25681622 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-03T15:37:23Z (GMT) No. of bitstreams: 1 flaviamarciadecastroesilva.pdf: 2659537 bytes, checksum: 1c8331ace53624c1553f76ae25681622 (MD5) / Made available in DSpace on 2016-06-03T15:37:23Z (GMT). No. of bitstreams: 1 flaviamarciadecastroesilva.pdf: 2659537 bytes, checksum: 1c8331ace53624c1553f76ae25681622 (MD5) Previous issue date: 2015-04-10 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A asma e a obesidade são doenças inflamatórias crônicas de perfis imunológicos opostos. Contudo, estudos clínicos e epidemiológicos demonstram uma associação entre as duas patologias, através da observação de que indivíduos obesos asmáticos representam um fenótipo clínico distinto da asma alérgica clássica, apresentando aumento na gravidade dos sintomas e resistência a terapias convencionais. Entretanto, os mecanismos imunológicos envolvidos na associação obesidade e asma não estão esclarecidos, devido à escassez de estudos e a uma heterogeneidade nos dados encontrados em modelos experimentais. Portanto, o objetivo do presente estudo foi avaliar a influência da obesidade sobre a inflamação alérgica pulmonar. Para isso, a obesidade foi induzida por dieta com alto teor de gordura durante dez semanas nos animais dos grupos OB e OB/AP, enquanto os animais dos grupos CN e AP foram alimentados com a dieta padrão. Da sexta a décima semana do protocolo de indução da obesidade, os animais dos grupos AP e OB/AP foram submetidos a subsequentes sensibilizações e desafios com a ovalbumina. As análises foram realizadas em 24 e 48 horas após o último desafio com a OVA. Os resultados demonstraram que após os desafios com o alérgeno, os animais do grupo AP apresentaram características marcantes da resposta imune alérgica, com elevado número de eosinófilos no LBA, no tecido pulmonar e na medula óssea, correlacionando com os níveis elevados de CCL11 e peroxidase eosinofílica, além de citocinas de eperfil Th2 como IL-4, IL-5, IL-9, IL-13, IL-25, IL-33 e TSLP e de IgE sérica anti-OVA. Contudo, foi observado em 48 horas um declíneo na resposta de perfil Th2 nos animais deste grupo. Já os animais do grupo OB/AP apresentaram em 24 horas, um menor número de eosinófilos no lavado broncoalveolar, no tecido pulmonar e na medula óssea, associado a menores níveis de CCL11, EPO e de IL-4, IL-5, TSLP e IL-25 assim como de IgE sérica anti-OVA. Em 48 horas, as análises de citocinas no grupo OB/AP demonstraram um aumento nos níveis de IL-1β, IL-4, IL-6, IL-9, IL-12, IL-13, IL-17A, TNF-α e IFN- associado ao maior influxo de macrófagos M1. Surpreendentemente, em 48 horas após o último desafio com a OVA, houve um aumento significativo de neutrófilos na medula óssea e de mieloperoxidase no tecido pulmonar. Paralelamente, os animais do grupo OB/AP, apresentaram um número maior de mastócitos e células caliciformes em ambos os tempos analisados, quando comparado aos animais do grupo AP. Conclusão: Somados estes resultados sugerem que a obesidade desenvolvida em camundongos BALB/c, foi capaz de influenciar a resposta imune no pulmão dos animais após as sensibilizações e os desafios com alérgeno, interferindo no desenvolvimento da resposta imune Th2 clássica e acarretando um atraso no desenvolvimento da resposta imune inflamatória. Adicionalmente, os animais obesos asmáticos apresentaram exacerbada resposta imune Th2, Th9 e altos níveis de IL-17A associada a um maior influxo de neutrófilos para o pulmão e a uma intensa produção de muco, sugerindo que estes animais apresentaram um perfil inflamatório mais grave de alergia pulmonar. / Asthma and obesity are chronic inflammatory diseases with opposite immune profiles. Although, clinical and epidemiological studies reveal the association between them, as obese asthmatic individuals represent a distinct phenotype from the classic allergic asthma. However, the immune mechanisms involved in this association are not established yet, due to the lack of studies and the heterogeneity of the data obtained in experimental models. Therefore, the present study aimed to evaluate the influence of obesity over the immune response pulmonary allergic. Female Balb/c mice were fed with high fat diet during ten weeks so as to induce obesity. From the sixth to the tenth week of the protocol, PA and PA/OB groups were sensitized and challenged with ovalbumin. The following analyses were performed 24 and 48 hours after the last OVA challenge. Striking features of the allergic immune response were observed in the PA group, as elevated eosinophil count in BAL, lung tissue bone marrow, in association with high IL-4, IL-5, IL-9, IL-13, IL-25, IL-33, TSLP and anti-OVA IgE levels. There was also elevated production of CCL11 and EPO correlated with the eosinophilia. In contrast, IL-4, IL-5, TSLP and IL-25 levels were diminished in PA/OB group. In association with the reduced eosinophil count, low levels of CCL11, EPO and Anti-OVA IgE were detected. However, 48 hours after the last challenge, IL-1β, IL-4, IL-6, IL-9, IL-12, IL-13, IL-17A, TNF-α and IFN- level were higher in the PA/OB, the was also an increased M1 macrophage influx. There was also more neutrophils in the bone marrow and MPO in the lung tissue, indicating their increased influx to the lung of PA/OB animals. Mast cells and goblet cells count was increased in this group, 24 and 48 hours after the last challenge. Taken together these results suggest that obesity developed in BALB/c mice was able to influence the immune response in the lungs of animals after sensitization and challenge with allergen, interfering with the immune response classical Th2 and causing a delay in the development inflammatory immune response. Additionally, asthmatic obese animals showed exaggerated Th2 immune response, Th9 and high IL-17A levels associated with an increased influx of neutrophils into the lung and an intense mucus production, suggesting that these animals showed an allergy more severe inflammatory profile lung.

Chapter 1: Introductionn Staphylococci are gram positive rotund bacteria that grow in clusters; and hence get their name. The genus of Staphylococcus comprises of over 30 species of which S. epidermidis and S.aureus are significant in their interaction with humans and are known to cause diseases. S.aureus invades various soft tissues and causes a vast multitude of diseases spanning from simple boils and abscesses to osteomyelitis and endocarditis, which can become fatal upon the onset of bacteremia and toxic shock. S. aureus has also been established as one of the leading causes of nosocomial infections especially because of their multi-drug resistant traits and their ability to colonize prosthetic devices and catheters. The increasing incidence of the multi-drug resistant strains and the rising prevalence of community acquired S. aureus infections mandates a comprehensive understanding of the pathogen and its biology, its intracellular fate and the defense mechanisms in the host. Towards this end, we have attempted to delineate some aspects of the pathogen’s virulence and the host responses to them. S. aureus normally inhabits the skin and mucosal surfaces as a commensal. Upon the onset of permissive circumstances it turns into an opportunistic pathogen. Immuno-compromised conditions or breach of skin can serve as the portals of entry for the pathogen. Upon entry, the bacteria encounter macrophages as the first line of defense in the host. Macrophages appear at the site of infection and phagocytose the bacteria, subjecting the pathogen to phagolysosomal degradation which facilitates antigen presentation and pathogen clearance. As part of their immune evasion mechanism, various pathogens are known to adopt a multitude of strategies to subvert this fate and survive in the host cells. This dissertation work aims at gaining insight into the staphylococcus-macrophage interaction in the ongoing host-pathogen duel, to gain better understanding about the pathophysiology and etiology of the disease. Chapter 2: Intracellular Trafficking of Staphylococcus aureus in Macrophages Successful targeting of the pathogen necessitates a comprehensive understanding of its biology and physiology in its interactions with the host. With this objective we undertook a study to uncover the intracellular niche of S. aureus in RAW264.7 murine macrophage-like cells. Any invading pathogen once internalized by the macrophage is contained in a phagosome, which undergoes progressive acidification and maturation from the early endosome to late endosome and ultimately fuses with the phagolysosome, where where the invading pathogen is subject to degradation. Through exhaustive electron microscopy of the infected macrophages, we show that S. aureus is present as a single bacterium per vacuole through the entire period of infection. We have further monitored the intracellular trafficking of the bacteria in the macrophage through confocal studies with endosomal markers which serve as indicators of vesicle maturation. Soon after the onset of the infection, the bacteria were found to be present in the early endosome (EEA-1 positive vesicles) which gradually matured into LAMP1 positive, late endosomal vesicles. However, only a small fraction of the bacteria containing vesicles were found to fuse with the lysosomes, suggesting that the bacteria prevented phagolysosomal fusion. We further observed that the bacteria did not prevent the acidification of the vesicles they resided in, but only limited their fusion with the lysosome. Taken together, our studies delineating the intracellular niche of S. aureus in RAW macrophages revealed that the pathogen has successfully evolved immune evasion mechanisms to overcome its phagolysosomal relegation. Chapter 3: Staphylococcus aureus Succumbs to the Hepcidin in Murine Macrophage We have further attempted to study the intracellular fate of the bacteria in macrophages towards gaining greater insight into its biology. Our studies on the intracellular fate of S. aureus in RAW264.7 cells revealed a distinct biphasic fate of the bacteria. The pathogen was found to replicate initially and this proliferative phase was subsequently followed by a gradual fall in its numbers. Interestingly however, the pathogen is never found to be cleared from the system suggesting the presence of a residual infective pool in the macrophages. We thus explored the possible mechanisms which could attribute to this biphasic intracellular fate of the bacteria. Macrophages come armed with a rich repertoire of defense mechanisms to incapacitate the invading pathogens. They have in their arsenal, reactive oxygen (ROS) and nitrogen species (RNS) and many potent anti-microbial peptides, apart from the lysosomal machinery, to degrade the invading pathogen. Upon investigation, we find that the RAW macrophages do not mount a ROS/RNS response when infected with S. aureus. Induction of these responses in the macrophage by alternate means further reveals that the pathogen is recalcitrant to death by these oxidative/nitrosative bursts. Of the antimicrobial peptides (AMPs) harbored by macrophages, we find that Hepcidin is up-regulated upon infection with S. aureus. Hepcidin is a peptide which is known to have a key regulatory role in iron homeostasis in addition to its potent antimicrobial functions. Since Hepcidin is known to be induced upon increased iron availability; we pre-treated the host cells with iron and monitored the effect of the same on bacterial fate. As expected, we observed that Hepcidin induction by pre-treatment with iron equips the macrophage to counter the pathogen better and thus leads to hastened and heightened clearance of the bacteria. This induction of hepcidin is significant at the mRNA and protein levels and is also corroborated by increased co-localisation of the bacteria with the anti-microbial peptide. Our studies thus identify hepcidin as a key line of the host defense towards countering the bacterial infection thus explaining the near complete bacterial clearance observed. Chapter 4: Global gene expression studies offering insight into potential immune evasion strategies of S.aureus in countering host offences. The interactions between host and the pathogen are multi-layered with the involvement of numerous players and many signaling cascades. In this light, we have attempted to get a holistic view of the host-pathogen interplay through microarray studies. These global profiling studies were aimed at identifying the important players in bacterial virulence and the macrophage response factors involved in countering the same in the context of S. aureus infection. The array was uniquely designed to incorporate both bacterial and host probes so as to facilitate parallel analysis of the host and pathogen gene expression profiles in the same sample. The expression profiling studies were carried out at three time points which represent the key phases of the bacterial infection viz. internalization, replication and clearance. A comprehensive analysis of the bacterial and host gene expression profiles under these phases provided insights into bacterial virulence and the host’s strategies to counter the same. We observe a large scale metabolic shut down in S. aureus subsequent to its internalization. We find the distinct up-regulation of a small subset of genes, majority of which are as yet uncharacterized. Amongst these were a few well-characterized virulence genes which remained active, representing the bacterial strategies to subvert the host immune response. The large scale down-regulation of gene expression can be possibly explained as the adaptation of the bacteria to the available metabolites and its submission to a quiescent phase of existence in the macrophage. In parallel, the host system exhibits the induction of TNF-α and up regulation of TLR2 and Nod2, which are typically triggered by a gram-positive infection. But simultaneously, we also observed a marked increase in the expression of anti-apoptotic and anti-inflammatory responses. This was re-iterated by a significant down-regulation in some of the pro-inflammatory, pro-apoptotic and antigen presentation involved genes and processes. We further find that the time course of the infection did not largely influence the gene expression kinetics. The macrophages were influenced and committed to a fate conducive for the bacteria fairly early in the infection regime. Thus, our studies of the expression profiles of the pathogen and the host under the different phases of the infection provide us with a comprehensive understanding the strategies of bacterial offense and host defenses thereby offering a window into this fascinating world of host-pathogen interactions. Chapter 5: Conclusion To summarize, we have attempted to study the intracellular fate of the S. aureus pathogen in macrophages. Our studies suggest that the bacterium attempts to evade clearance by the host immune system by actively preventing fusion with the lysosomal vesicles. We also find that despite these defenses, the pathogen appears to succumb to the host immune system as it is targeted by Hepcidin, an anti-microbial peptide. The lack of complete bacterial clearance under these conditions is however suggestive of an underlying strategy by the pathogen, possibly to maintain a chronic infective state in the host system. The microarray studies, in addition, shed light on the other possible immune evasion strategies that S.aureus might be employing to escape the host offences. The results are indicative of the bacteria influencing anti-apoptotic, anti-inflammatory and antigen presentation responses and thereby prolonging its survival in the macrophage. In conclusion, given the fact that the macrophages are itinerant cells with a long life span, the light thrown by our findings of the various immune evasion strategies that S.aureus is adopting; it suggests that the macrophages could serve as potential carriers which could account for the dissemination of the infection to new sites, which has perpetually been a major concern for any Staphylococcal infection.

L'opinion générale est que les ulcères sont rares pendant l'enfance, les lésions provoquées par Helicobacter pylori (H. pylori) ne se produisant que des décennies après l'acquisition de l'infection. L’infection par cette bactérie est en outre moins fréquente chez les enfants dans les pays développés par rapport aux adultes. Par ailleurs, l’usage chronique de médicaments gastro-toxiques est peu fréquent dans cette tranche d’âge. Cependant, plusieurs études ont montré qu’environ 1/10 des enfants référés pour des symptômes de dyspepsie en Europe et infectés par H. pylori présentent un ulcère gastrique ou duodénal, mais aussi que la fréquence de ces lésions chez les enfants non infectés n’est pas nulle.<p>Afin de déterminer la fréquence des ulcères gastriques et duodénaux et des érosions, nous avons commencé par réaliser une étude prospective avec la participation de 19 centres répartis dans 14 pays d'Europe. Tous les enfants référés pour une endoscopie haute ont été recrutés durant une brève période de 1 mois. Parmi les 694 enfants inclus, 56 (8,1%) avaient soit des ulcères (ulcère gastrique 17/56, 30% - ulcère duodénal 7/56, 13%) soit des érosions (érosions gastriques 21/56, 37% - érosions duodénales 9/56, 16% - érosions gastriques et duodénales 2/56, 4%). Cette étude a permis de confirmer que la fréquence des lésions augmente avec l’âge, les enfants atteints de lésions étant significativement plus âgés que les témoins. En effet, les lésions ont surtout été observées chez les enfants dans la deuxième décade de vie. Une infection par H. pylori était présente seulement chez 15 des 56 enfants (27%), un médicament gastro-toxique avait été utilisé chez 13/56 (23%), une maladie inflammatoire chronique de l’intestin était présente chez 7/56 (13%) et une polyarthrite juvénile chez 2/56 (4%, plus d'un facteur de risque présent dans la plupart des cas). Aucun facteur de risque n’a pu être démontré chez 24/56 enfants (43%), une proportion beaucoup plus élevée que celle initialement attendue.<p>Nous avons ensuite réalisé une étude cas-témoins prospective et multicentrique (12 centres participants). Tous les patients avec une lésion érosive ou ulcérée de la muqueuse gastroduodénale ont été inclus avec deux témoins appariés pour l’âge, le centre et la période. Sept cent trente-deux patients (244 cas dont 153 avec seulement des érosions et 91 avec un ou des ulcères, 488 témoins) ont été inclus. Les enfants qui avaient reçu un antibiotique, un inhibiteur de la pompe à proton ou un anti-H2 durant les 4 semaines précédant l’endoscopie ont été exclus de l’analyse statistique parce que ces médicaments influencent la détermination<p>7<p>du statut H. pylori et la gravité des lésions (42 cas et 98 témoins). Nos résultats montrent que, chez les enfants, l'infection à H. pylori est un facteur de risque pour les ulcères duodénaux et les érosions duodénales, mais pas pour les lésions gastriques. Le sexe masculin, la consommation d'AINS, les maladies rénales chroniques et le tabagisme sont d'autres facteurs de risque indépendants de lésions érosives ou d’ulcères gastroduodénaux. Cependant, aucun facteur de risque identifiable n’a été retrouvé dans une grande proportion d'enfants (97/202, 48.0%) ce qui confirme les résultats de notre première étude.<p>Chez les adultes également la proportion d’ulcères sans infection à H. pylori et sans prise d’AINS est en augmentation ces dernières années tout en restant plus faible que chez l’enfant. La fréquence des ulcères gastriques et duodénaux avec un diamètre d’au moins 5 mm a été comparée, dans notre centre et dans un centre d’endoscopie adulte situé dans la même région de Bruxelles, sur une période de deux ans. Ces données montrent que les ulcères sont moins fréquents chez les enfants que chez les adultes (20/1279 enfants avec endoscopie haute - 1,6% vs adultes 58/1010 - 5,7%, OR 0,30, 95%CI 0,10-0.86, p = 0,02) et surtout moins fréquemment associés à une infection par H. pylori (8/20 vs 40/58, OR 0,26, 95%CI 0,16- 0.78, p <0,0001).<p>Comme l’activation de la réponse immunitaire locale est inefficace pour éliminer l’infection par H. pylori et serait plutôt impliquée dans la pathogenèse des lésions de la muqueuse, nous avons comparé la réponse immunitaire muqueuse des lymphocytes T et les réponses naïves chez les enfants et chez les adultes infectés par H. pylori ainsi que chez des témoins non infectés appariés pour l’âge.<p>Dans une première étude, nous avons obtenu des biopsies de la muqueuse antrale chez 43 patients dyspeptiques (12 enfants, 31 adultes). Les concentrations de cytokines libérées dans le milieu de culture et la densité de cellules CD3+, CD25+ et CD69+ ont été évaluées par cytométrie en flux. Le nombre de cellules sécrétant de l’interféron-γ (IFN-γ), de l’interleukine-4 (IL-4) et de l’IL-10 a été mesuré par ELISPOT. Les données obtenues montrent que l’augmentation de la sécrétion d'IFN-γ et l’élévation du nombre de cellules secrétant de l’IFN-γ au niveau de la muqueuse antrale lors d’une infection par H. pylori sont plus faibles chez les enfants que chez les adultes.<p>8<p>Dans une seconde étude, nous avons comparé l’infiltrat inflammatoire de la muqueuse antrale dans différents groupes d’âge (moins de 8 ans, 8 à 17 ans, 18 à 55 ans) de patients successifs infectés par H. pylori et des témoins appariés pour l’âge. Nous avons montré une corrélation entre l'âge et la densité de neutrophiles, de cellules CD3+ et de CD8+, mais pas de cellules CD20+. Le recrutement des neutrophiles dans la muqueuse antrale est plus faible chez les enfants et apparaît corrélé avec une plus faible activation du facteur de transcription NF-kB (déterminé par immunohistochimie et par EMSA) dans cette même muqueuse. L’infiltrat inflammatoire et l’activation du NF-kB sont légèrement (mais non significativement) plus intenses en cas d’infection par une souche plus virulente (facteur de virulence cagA). Ces souches cagA+ sont retrouvées en proportion équivalente dans les différents groupes d’âge. Par contre, la charge bactérienne, mesurée par un score semi-quantitatif en histologie, n’influence pas l’intensité de l’infiltrat inflammatoire.<p>En conclusion :H. pylori reste un facteur étiologique majeur pour les ulcères et les érosions duodénales chez l’enfant, mais pas pour les lésions gastriques dans les pays à faible prévalence de l'infection et la proportion de lésions associées à une infection est plus faible que chez les adultes. Aucun facteur d’exposition connu ne peut être associé aux lésions endoscopiques dans la moitié des cas, ce qui justifiera des études ultérieures pour identifier d’autres causes exogènes ou endogènes à ces lésions.<p>La réponse immunitaire de l’hôte est impliquée dans la pathogenèse des lésions gastroduodénales associées à une infection par H. pylori. Or il a été démontré dans les travaux faisant l’objet de cette thèse que cette réponse immunitaire est plus faible chez l’enfant que chez l’adulte pour certains facteurs (cytokines Th1, immunité humorale, recrutement des polynucléaires et des lymphocytes au niveau muqueux, activation du facteur de transcription NF-κB). D’autres études confirment la plus faible réponse humorale et Th1, mais également Th17 ainsi qu’une activation plus intense des Treg. Les cytokines ou les voies de signalisation responsables de cette réponse immunitaire plus faible restent inconnues, ce qui ouvre la voie à d’autres investigations. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished