As células da linhagem macrofágica são fundamentais na infecção por T. cruzi. Além do reconhecimento por PRRs, a interação com anticorpos e/ou complemento facilita a internalização. Avaliamos o papel in vivo e in vitro de IgM e IgG específicas na saída de parasitas Sylvio X10/4 do sangue, um clone miotrópico de T. cruzi que causa infecção sem parasitemia patente. Devido à sua saída espontânea, estudamos a remoção no sexto dia de infecção, momento em que a parasitemia subpatente aumenta. Camundongos foram infectados e tratados com soro normal (NMS), soro crônico (B6) e soro de animais CD28KO crônicos (que produzem somente IgM). O grupo tratado com soro de CD28KO apresentou uma remoção significativa, porém menos eficiente do que com soro crônico (B6). Tais resultados foram reproduzidos em estudo in vitro na invasão de macrófagos (derivados de medula óssea ou do peritônio) com os distintos tratamentos. Concluímos que os macrófagos são fundamentais na remoção dos parasitas e os anticorpos, não somente IgG, mas também os da classe IgM reforçam este processo. / The cells of the macrophage lineage are essential for the infection by T. cruzi. Besides the recognition by PRRs, interaction with antibodies and/or complement facilitates internalization. We evaluated the role in vivo and in vitro of specific IgM and IgG in the blood output of parasites Sylvio X10/4, clone myotropic of T. cruzi which causes infection without patent parasitemia. Due to its spontaneous exit, we studied the removal on the sixth day of infection, when the parasitemia subpatent increases. Mice were infected and treated with normal mouse serum (NMS), chronic serum (B6) and serum from CD28KO chronic mice (which produce only IgM). The group treated with CD28KO serum showed a significant removal, but less efficiently than with chronic serum (B6). These results were reproduced in vitro study on the invasion of macrophages (derived from bone marrow or peritoneum) with these different treatments. We conclude that macrophages are essential in the removal of parasites and antibodies, not only IgG, but also IgM enhance this process.

<p>Natural IgM (nIgM) is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. nIgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. However, the cells that maintain high titers of nIgM in the circulation had not yet been identified. Several studies have linked serum nIgM with the presence of fetal-lineage B cells, and others have detected IgM secretion directly by B1a cells in various tissues. Nevertheless, a substantial contribution of undifferentiated B1 cells to nIgM titers is doubtful, as the ability to produce large quantities of antibody (Ab) is a function of the phenotype and morphology of differentiated plasma cells (PCs). No direct evidence exists to support the claim that a B1-cell population directly produces the bulk of circulating nIgM. The source of nIgM thus remained uncertain and unstudied.</p><p>In the first part of this study, I identified the primary source of nIgM. Using enzyme-linked immunosorbent spot (ELISPOT) assay, I determined that the majority of IgM Ab-secreting cells (ASCs) in naïve mice reside in the bone marrow (BM). Flow cytometric analysis of BM cells stained for intracellular IgM revealed that nIgM ASCs express IgM and the PC marker CD138 on their surface, but not the B1a cell marker CD5. By spinning these cells onto slides and staining them, following isolation by fluorescence-activated cell sorting (FACS), I found that they exhibit the typical morphological characteristics of terminally differentiated PCs. Transfer experiments demonstrated that BM nIgM PCs arise from a progenitor in the peritoneal cavity (PerC), but not isolated PerC B1a, B1b, or B2 cells. Immunoglobulin (Ig) gene sequence analysis and examination of B1-8i mice, which carry an Ig knockin that prohibits fetal B-cell development, indicated that nIgM PCs differentiate from fetal-lineage B cells. BrdU uptake experiments showed that the nIgM ASC compartment contains a substantial fraction of long-lived plasma cells (LLPCs). Finally, I demonstrated that nIgM PCs occupy a survival niche distinct from that used by IgG PCs.</p><p>In the second part of this dissertation, I characterized the unique survival niche of nIgM LLPCs, which maintain constitutive high titers of nIgM in the serum. By using genetically deficient or Ab-depleted mice, I found that neither T cells, type 2 innate lymphoid cells, nor mast cells, the three major hematopoietic producers of IL-5, were required for nIgM PC survival in the BM. However, IgM PCs associate strongly with IL-5-expressing BM stromal cells, which support their survival in vitro when stimulated. In vivo neutralization of IL-5 revealed that, like individual survival factors for IgG PCs, IL-5 is not the sole supporter of IgM PCs, but is likely one of several redundant molecules that together ensure uninterrupted signaling. Thus, the long-lived nIgM PC niche is not composed of hematopoietic sources of IL-5, but a stromal cell microenvironment that provides multiple redundant survival signals.</p><p>In the final part of my study, I identified and characterized the precursor of nIgM PCs, which I found in the first project to be resident in the PerC, but not a B1a, B1b, or B2 cell. By transferring PerC cells sorted based on expression of CD19, CD5, and CD11b, I found that only the CD19+CD5+CD11b- population contained cells capable of differentiating into nIgM PCs. Transfer of decreasing numbers of unfractionated PerC cells into Rag1 knockouts revealed an order-of-magnitude drop in the rate of serum IgM reconstitution between stochastically sampled pools of 106 and 3x105 PerC cells, suggesting that the CD19+CD5+CD11b- compartment comprises two cell types, and that interaction between the two necessary for nIgM-PC differentiation. By transferring neonatal liver, I determined that the early hematopoietic environment is required for nIgM PC precursors to develop. Using mice carrying a mutation that disturbs cKit expression, I also found that cKit appears to be required at a critical point near birth for the proper development of nIgM PC precursors.</p><p>The collective results of these studies demonstrate that nIgM is the product of BM-resident PCs, which differentiate from a PerC B cell precursor distinct from B1a cells, and survive long-term in a unique survival niche created by stromal cells. My work creates a new paradigm by which to understand nIgM, B1 cell, and PC biology.</p> / Dissertation

Thesis (M.Sc. in Microbiology) -- Mahidol University, 1976.

Orientador: Claudio Lucio Rossi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T14:37:04Z (GMT). No. of bitstreams: 1 Nascimento_FernandaSantos_M.pdf: 1195114 bytes, checksum: 8f7292945661258264c33482af428d55 (MD5) Previous issue date: 2008 / Resumo: A toxoplasmose, uma zoonose com ampla distribuição mundial, causada pelo parasita intracelular obrigatório Toxoplasma gondii, é geralmente adquirida por meio da ingestão de cistos ou oocistos viáveis do parasita, presentes, respectivamente, em carne crua ou mal cozida e no solo, alimento ou água contaminados com fezes de gatos infectados. A toxoplasmose pode ser altamente debilitante, e ocasionalmente fatal, em crianças infectadas no útero e em receptores de transplante. O diagnóstico de infecção aguda primária em mulheres grávidas é geralmente baseado em testes sorológicos, visto que, na grande maioria dos casos, a toxoplasmose não é reconhecida clinicamente. A longa persistência dos anticorpos IgM em algumas pessoas e a dificuldade para demonstrar soroconversão ou aumento significativo da concentração de anticorpos específicos, têm complicado a interpretação dos testes sorológicos, quando se suspeita de infecção aguda. Com relação à infecção toxoplámica em pacientes transplantados, em muitos casos o status sorológico do doador não é conhecido e a pesquisa periódica de anticorpos anti-T. gondii no receptor raramente é realizada. O objetivo do primeiro estudo foi determinar o valor da demonstração dos anticorpos IgA anti-T.gondii para o diagnóstico da fase aguda da infecção toxoplásmica. Nossos resultados mostraram que os anticorpos IgA são detectados com alta freqüência em amostras de soros obtidas de mulheres com evidência clínica e/ou sorológica de infecção toxoplásmica aguda. Entretanto, em 19% das mulheres apresentando persistência de anticorpos IgM e alto índice de avidez dos anticorpos IgG, anticorpos IgA anti-T. gondii foram detectados em amostras de soros coletadas mais de 9 meses após o início da infecção, indicando que esses anticorpos não podem ser considerados marcadores confiáveis de infecção aguda primária. No segundo estudo, nós relatamos o diagnóstico de infecção toxoplásmica primária em um paciente com mieloma múltiplo submetido a transplante alogênico não-mieloablativo de células hematopoiéticas, provenientes de doador com sorologia negativa para toxoplasmose. A resposta primária contra o T. gondii foi baseada na soroconversão dos anticorpos IgM, IgG e IgA. O paciente foi prontamente tratado e nenhuma complicação relacionada à toxoplasmose foi observada nos meses subseqüentes. Esse caso ressalta a necessidade da detecção dos anticorpos anti-T. gondii no doador e no receptor antes do transplante e a importância do monitoramento sorológico do receptor durante o seguimento pós-transplante / Abstract: Toxoplasmosis, a cosmopolitan zoonotic disease caused by the intracellular parasite Toxoplasma gondii, is usually acquired through the ingestion of viable parasite cysts or oocysts, present, respectively, in raw or undercooked meat and in soil, food or water contaminated with feces of infected cats. Toxoplasmosis can be highly debilitating and occasionally fatal in children infected in utero and in transplant recipients. The diagnosis of acute primary infection in pregnant women is usually based on serology, because in the great majority of cases primary infection is not recognized clinically. The sustained persistence, in some persons, of specific IgM antibodies and the difficulty in demonstrating seroconversion or a significant increase in specific antibody concentrations, have complicated the interpretation of serological tests when acute infection is suspected. With regard to toxoplasmic infection in transplant patients, in many cases the serological status of the donor is not known and the periodic research of anti-T. gondii antibodies in the receptor is rarely performed. In the first study, we investigated the usefulness of detecting anti-T. gondii IgA for the diagnosis of an acute acquired Toxoplasma infection. Our results showed that anti-T. gondii IgA antibodies are detected with a high frequency in serum samples obtained from women with clinical and/or serologic evidence of acute acquired Toxoplasma infection. However, in 19% of the women presenting a sustained IgM antibody response and a high IgG avidity index, anti-T. gondii IgA antibodies were detected in serum samples collected more than nine months after the beginning of infection, indicating that IgA cannot be considered a dependable marker for acute primary infection. In the second study, we report the diagnosis of a primary toxoplasmic infection in a patient with multiple myeloma following a non-myeloablative allogeneic transplant with hematopoietic stem cells from a donor with negative serology for toxoplasmosis. The primary response to T. gondii was supported by IgM, IgG and IgA seroconversion. The patient was promptly treated and there were no complications related to toxoplasmosis in the subsequent months. This case stresses the importance of detecting anti-T. gondii antibodies in the donor and in the recipient before transplantation, and of serologically monitoring the recipient during long-term follow-u / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Submitted by Ramon Santana (ramon.souza@ufpe.br) on 2015-03-10T14:49:55Z No. of bitstreams: 2 Dissertação Viviane Martha de Morais.pdf: 1592021 bytes, checksum: e29a3eec2c8393142f0c5e43344e1535 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-10T14:49:55Z (GMT). No. of bitstreams: 2 Dissertação Viviane Martha de Morais.pdf: 1592021 bytes, checksum: e29a3eec2c8393142f0c5e43344e1535 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-02-28 / Introdução: O diagnóstico rápido, simples e preciso para confirmar a infecção pelo vírus dengue (DENV) é uma necessidade real, uma vez que a doença pode se manifestar com um amplo espectro de sinais e sintomas, similares a outros quadros febris agudos. Durante a infecção por dengue se verifica também a produção da imunoglobulina A (IgA) específica, que aumenta ao mesmo tempo que a imunoglobulina M (IgM), permanece positiva por um período de tempo mais curto e se apresenta em níveis mais elevados na infecção secundária. Objetivo: O presente estudo teve como objetivo investigar a presença de IgA no soro durante a infecção primária e secundária (sequencial) pelo DENV. Metodologia: Foram avaliadas amostras de soro por meio do teste imunoenzimático de captura da IgA (AAC-ELISA) in house. Resultados: Avaliou-se um total de 445 amostras de soro, sendo 171 caracterizados como infecção primária e 194 secundária; 40 amostras de indivíduos saudáveis negativos para dengue e 40 de vacinados contra febre amarela. As amostras foram distribuídas em 13 grupos. A positividade da IgA foi de 42,2% (154/365), sendo 27,5% (47/171) na infecção primária e 55,2% (107/194) na secundária. Na infecção secundária, a IgA foi detectada do 2º ao 4º dias de sintomas (grupo 1), antes mesmo da IgM, assim como no grupo 11 no qual a IgM não havia sido detectada (infecção secundária). Na infecção primária o maior valor da sensibilidade foi de 60,0 (36,4 - 80,0) no grupo com 30-35 dias de sintomas e na secundária foi de 87,5% (60,4 – 97,8), grupo com 8 dias de sintomas. A especificidade foi de 100% nas duas infecções (94,3 – 100). Ao aplicar o teste em paralelo para ambas técnicas observou-se um aumento global de 6,6% na sensibilidade do diagnóstico; sendo 2,7% para a infecção primária e de 15,2% para a secundária. A IgA não foi detectada nas amostras dos indivíduos saudáveis, nem nas amostras dos indivíduos recentemente vacinados contra febre amarela. Conclusões: A detecção da IgA demonstrou ser útil como forma de diagnóstico sorológico e em conjunto com a detecção da IgM poderá auxiliar na confirmação de casos agudos de dengue e na interpretação dos resultados de casos inconclusivos, permitindo a adoção de medidas preventivas para evitar a ocorrência de epidemias e ocorrência de casos graves e óbitos.

Acute kidney injury (AKI) induced by renal ischaemia reperfusion injury (IRI) is characterised by renal failure, acute tubular necrosis (ATN), inflammation and microvascular congestion. Apoptotic cell administration reduces inflammation in experimental models of acute inflammation in the lung, joints and peritoneum. Preliminary data suggested that administration of 20x106 apoptotic thymocytes to mice 24-hours prior to renal IRI ameliorated renal function without affecting ATN 24-hours following IRI. This thesis attempted to validate these finding and explore underlying hypothetical mechanisms. These studies examined if functional protection was conferred by apoptotic cell modulation of (a) circulating IgM antibodies or (b) coagulation status leading to improved intrarenal microvascular blood flow. Pathogenic IgM antibodies bind ischaemic cardiac or skeletal muscle and the intestine leading to complement activation and worse injury. We examined IgM binding to human renal (HK-2) cells by flow cytometry and to ischaemic murine kidney tissue. H2O2 or Antimycin A treated HK-2 cells incubated with human serum (IgM source) exhibited no IgM binding. Medullary IgM deposition assessed by immunofluorescence was minimal following IRI. We also assessed IgM deposition by immunohistochemistry following hepatic IRI and discovered dramatic deposition. These data suggest that IgM antibodies exhibit differential binding to injured tissues and are not directly involved in renal IRI, but may have a role in hepatic IRI. To support our second hypothesis we studied apoptotic cell modulation of coagulation. A thrombin generation assay revealed that early apoptotic cell-treated mice exhibited delayed thrombin generation. Furthermore, in vitro studies confirmed direct apoptotic cell-platelet binding. To replicate apoptotic cell derived functional protection Balb/c mice underwent 20, 24 or 25-minutes of ischaemia to induce mild, moderate or severe kidney dysfunction. Renal function and injury was determined 24-hours following IRI by plasma creatinine measurement and ATN scoring. Unexpectedly, intravenous pretreatment of mice with apoptotic thymocytes conferred no protection. Indeed, apoptotic thymocytes further impaired renal function depending upon injury severity. Impairment of renal function was not secondary to increased microvascular congestion, inferred by fibrin and platelet deposition, neither increased ATN nor inflammation, assessed by neutrophil infiltration. These data indicate that apoptotic cell administration does not protect from subsequent renal IRI and that apoptotic cells are thus not inherently anti-inflammatory in all models of acute inflammation. Unable to replicate apoptotic cell derived functional protection we explored the binding of IgM antibodies to apoptotic cells which acts to facilitate dead cell clearance. We characterised IgM binding to non-apoptotic and apoptotic murine thymocytes and human Jurkat cells using flow cytometry, confocal and electron microscopy. We demonstrated specific IgM binding to a subset of late apoptotic cells. Electron microscopy indicated that IgM+ apoptotic cells exhibited marked plasma membrane disruption, suggesting that access to intracellular epitopes was required for IgM binding. Binding of IgM to permeabilised non-apoptotic and apoptotic cells suggested that IgM bound epitopes are ‘apoptosis independent’ such that IgM may bind any cell with profound plasma membrane disruption. Interestingly, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognise and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption.

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Mutações no gene CD40LG estão associadas à Síndrome de Hiper-IgM, relacionada ao cromossomo X, que é uma Imunodeficiência Primária. A ausência da interação CD40L/CD40 (linfócitos-DCs) acarreta um aumento da suscetibilidade às infecções fúngicas. Os efeitos do tratamento in vitro com interferon-gama em células dendríticas de pacientes com mutações no CD40L demonstraram potencial sobre a imunologia das DCs e nos remete a fundamentar possíveis ensaios clínicos futuros, que tenham por tema a prevenção e o tratamento de infecções oportunistas. / Mutations in the CD40LG gene are associated with the X-linked Hyper-IgM Syndrome, which is a Primary Immunodeficiency. The absence of the CD40L / CD40 interaction (lymphocytes-DCs) leads to increased susceptibility to fungal infections. The effects of in vitro gamma-interferon treatment on dendritic cells from patients with CD40L mutations have demonstrated potential for DC immunology and suggest that future clinical trials should address the prevention and treatment of opportunistic infections.