Pacientes com doença reumática cardíaca (DRC) desenvolvem lesões valvares mediadas por linfócitos T CD4+, capazes de reconhecer cruzadamente proteínas cardíacas e estreptocócicas pelo mecanismo de mimetismo molecular. Neste trabalho empregamos uma biblioteca peptídica de Phage Display para identificar auto-antígenos cardíacos capazes de serem reconhecidos por duas linhagens intralesionais de linfócitos T e um clone derivado de uma das linhagens isolados de pacientes com DRC. A análise dos peptídeos dos fagos em banco de dados de proteínas revelou novos epitopos da miosina cardíaca, laminina, vimentina e outras proteínas coiled-coil, provavelmente involvidos no processo auto-imune da DRC. Outras moléculas inflamatórias como citocinas, integrinas e fatores de crescimento também foram identificadas / Rheumatic heart disease (RHD) patients develop valvar lesions with CD4+ T lymphocytes infiltrating the heart. Molecular mimicry between streptococcal and cardiac proteins recognized by these T cells may explain these auto-aggressive lesions. In the present work we used a Phage Display peptide library to identify cardiac antigens which could be recognized by two heart infiltrate T cell lines and by a T cell clone derived from one of the lines which were isolated from RHD patients. Using the protein data bank to analyse the phage peptides, we observed that many sequences showed homology with cardiac myosin, laminin, vimentin and other coiled-coil proteins, suggesting the involvement of these proteins in the autoimmune process of RHD. Other inflammatory molecules such as cytokines and integrins were also identified

Background: Type 1 Diabetes mellitus (TIDM) is a chronic disorder in which the insulin producing beta cells are selectively self-destroyed. Although the etiology of the disease has not been determined, genetic dispositions such as SLC11A1 polymorphism in suffering patients have been reported. The role of pathogenic microorganisms such as Mycobacterium avium subspecies paratuberculosis (MAP) in TIDM has also been recently debated. MAP is already known to cause paratuberculosis in cattle and now it is a strong suspect of causing autoimmune diseases in humans such as Crohn’s disease, multiple sclerosis, autoimmune Thyroiditis, rheumatoid arthritis and autoimmune diabetes. We hypothesis that molecular mimicry between MAP Heat shock protein 65K (Hsp65) and human Glutamic Acid Decarboxylase 65K (GAD65) can be the trigger which leads to the autoimmune destruction of beta cell in patients exposed to MAP . Method: To test the hypothesis, peptide sequences of MAP Hsp65 and human GAD65 were investigated using BLAST and PyMOL bioinformatics tools. Moreover, 18 blood samples from humans with TIDM and controls, and 100 sera samples from cattle with paratuberculosis and controls were evaluated for the presence of MAP, MAP DNA and its antibodies. Glucose, insulin and GAD65 antibodies were also determined in some of the clinical samples. Results: Peptide BLAST analysis revealed 44% identity between the two proteins with 75% positive identities in a 16 amino acid region. PyMOL structural analysis identified possible shared epitope regions of the proteins in its 3D conformation. Immunoblot analysis revealed a strong cross reactivity between lysate of E.coli recombinant of MAP Hsp65 and plasma from human subject with TIDM. A weak cross reactivity was also observed between healthy rat pancreatic homogenate and rabbit anti MAP IgG. Nested PCR using IS900-specific iv oligonucleotide primers did not detect MAP DNA in peripheral blood from 18 subjects with Type I Diabetes, Type II Diabetes and non-diabetic controls. Long term culture of leukocytes from blood samples from same subjects resulted in the presence of MAP in 3/10 (30%) TIDM and 4/8 (50%) control subjects. However anti MAP IgG were detected in 5/10 (50 %) TIDM samples compared to 3/8 (37.5 %) controls. Insulin level was measured in sera from paratuberculosis cattle and controls. In MAP infected cattle, insulin level ranged from below 0.1ng/ml to 2.456 ng/ml with an average of 0.36 +/- 0.57ng/ml compared to below 0.1ng/ml to 13.47ng/ml with an average of 2.86 +/- 3.00ng/ml in healthy cattle. Conclusion: Bioinformatics analysis between MAP Hsp65 and human GAD65 through BLAST and PyMOL analysis revealed a homology of 16 amino acid motif and possible shared epitope regions; immunohistochemistry analysis revealed a cross reactivity between rabbit antiMAP IgG and pancreatic cell homogenate from a healthy rat. Moreover, plasma from patient with TIDM (TD8), who was confirmed to be positive for MAP DNA and MAP IgG, reacted strongly with MAP Hsp65 in MAP protein lysate and MAP Hsp65 recombinant clone pmptb20. Culture of MAP from human leukocytes is significant despite the lack of correlation between MAP in samples from TIDM and controls. It is worth noting that some of the control subjects have not been evaluated for other autoimmune diseases possible MAP role. Additionally, antiMAP IgG levels in TIDM subjects compared to controls have raised a suspicion on the involvement of MAP in TIDM. The poor correlation of MAP in blood versus either the antiMAP IgG or the insulin level may be related with the fastidious nature of MAP and in vitro cultivation. Since MAP is the sole causative agent of Johne’s disease, it is significant that the insulin level is 8 folds less in MAP infected cattle compared to MAP free cattle. Overall, the data v is mixed and suggest that further study is needed to investigate the intriguing question to whether MAP is involved in TIDM or not.

A febre reumática é um bom exemplo de uma doença auto-imune deflagrada por um processo infeccioso. Num prazo de uma a quatro semanas após a resolução de uma faringite não tratada por cepas reumatogênicas de S. pyogenes, o organismo de um hospedeiro susceptível desencadeia uma resposta imune contra grandes articulações, coração, tecidos subcutâneos e cérebro. Acredita-se que elementos presentes na bactéria e reconhecidos durante a infecção na orofaringe, sejam confundidos com estruturas próprias do organismo, num processo denominado mimetismo molecular. Entre as proteínas envolvidas na reação cruzada, encontram-se a miosina cardíaca, pelo lado do hospedeiro, e a proteína M do microorganismo invasor. Esta última (proteína M) tem sido extensamente estudad. É a base da classificação das cepas de S. pyogenes e importante fator de virulência. Também tem sido explorada como imunógeno em várias estratégias vacinais. O estudo desta patologia tem sido dificultado pela ausência de um modelo animal que reproduza aspectos fundamentais da patologia humana, entre estes as lesões cardíacas. Uma das razões é o fato de que animais não contraem infecção pelo S. pyogenes. Portanto, produzimos a proteína M1 recombinante e mostramos que a imunização de 28 ratos Lewis por um período de 21 dias ou 14 ratos por 41 dias, com esta proteína foi capaz de induzir resposta inflamatória na maioria dos animais com intensidade variável. Células similares aos nódulos de Aschoff e células de Anitschkow, sugestivas das lesões patognomônicas da febre reumática foram observadas em dias e também de um em quatro dos animais controles que receberam PBS e adjuvantes. Estes resultados sugerem a presença de células auto-reativas no miocárdio dos animais imunizados. Em conclusão, o uso de proteína M1 recombinante como imunógeno em modelo animal de ratos Lewis é capaz de desencadear reação inflamatória em miocárdio e tecido valvular e lesões similares às da febre reumática. O modelo do rato Lewis é até o momento o que apresenta maior semelhança com a doença humana e pode ajudar a esclarecer a imunopatologia da febre reumática. Além disso, certamente será importante para a avaliação do potencial de proteção e de segurança em modelos de vacinas contra o S. pyogenes. / Rheumatic fever is a good example of an autoimmune disease triggered by an infectious process. One to four weeks after the resolution of a non treated pharyngitis caused by rheumatogenic strains of S. pyogenes, the susceptible host unravels an immune response targeting joints, heart, conective tissues and brain. It is thought that molecules present in the bacteria and recognized during the infection at the pharynx are confounded with the organism self structure in a process called ?molecular mimicry?. Amongst the proteins involved in the cross reaction, it may be found cardiac myosin, on the host side, and M protein on the invading organism?s side. The latter (Mprotein) has been extensively studied. It is the basis of the S. pyogenes strains classification, and also an important virulence factor. It has also been explored as an immunogen in several vaccine strategies. The nderstanding of this disease has been hampered by the absence of an animal model that reproduces fundamental aspects of the human pathology, specially cardiac lesions. One of the reasons is the fact that animals do not get infected by S. pyogenes. Hence we have produced the recombinant M1 protein and shown that either the immunization of 28 Lewis rats for a period of 21 days or 14 rats for a period of 41 days, was capable of inducing an inflammatory response in most of the animals with variable intensity. Aschoff nodules-like or Anitschkow-like cells resembling rheumatic fever pathognomonic lesions were seen in 50% of the animals immunized subcutaneously and sacrificed on day 21. We have observed an humoral and cellular response (spleen and lymph node derived cells) specifically targeting M1 protein and the amino (M1AB) and carboxy (M1C) terminus of the protein. However, cross reactions with cardiac myosin were not observer. We have derived T lymphocyte lineages obtained from myocardium infiltrating mononuclear cells from 6 of the 10 animals immunized with M1ABC protein subcutaneously and sacrificed on day 41 and also from one out of four PBS - adjuvant immunized animals. These results suggest the presence of autoreactive cells in the myocardium of the immunized animals. In conclusion, the use of the M1 protein as an immunogen on the Lewis rat model is capable of triggering an inflammatory reaction in the myocardium and valvular tissue and it can produce rheumatic fever like lesions. The Lewis rat model is up to this moment the one to present the highest similarity with human disease. Besides, it will certainly be important on the evaluation of the protection and safety of S. pyogenes vaccines.

A febre reumática é um bom exemplo de uma doença auto-imune deflagrada por um processo infeccioso. Num prazo de uma a quatro semanas após a resolução de uma faringite não tratada por cepas reumatogênicas de S. pyogenes, o organismo de um hospedeiro susceptível desencadeia uma resposta imune contra grandes articulações, coração, tecidos subcutâneos e cérebro. Acredita-se que elementos presentes na bactéria e reconhecidos durante a infecção na orofaringe, sejam confundidos com estruturas próprias do organismo, num processo denominado mimetismo molecular. Entre as proteínas envolvidas na reação cruzada, encontram-se a miosina cardíaca, pelo lado do hospedeiro, e a proteína M do microorganismo invasor. Esta última (proteína M) tem sido extensamente estudad. É a base da classificação das cepas de S. pyogenes e importante fator de virulência. Também tem sido explorada como imunógeno em várias estratégias vacinais. O estudo desta patologia tem sido dificultado pela ausência de um modelo animal que reproduza aspectos fundamentais da patologia humana, entre estes as lesões cardíacas. Uma das razões é o fato de que animais não contraem infecção pelo S. pyogenes. Portanto, produzimos a proteína M1 recombinante e mostramos que a imunização de 28 ratos Lewis por um período de 21 dias ou 14 ratos por 41 dias, com esta proteína foi capaz de induzir resposta inflamatória na maioria dos animais com intensidade variável. Células similares aos nódulos de Aschoff e células de Anitschkow, sugestivas das lesões patognomônicas da febre reumática foram observadas em dias e também de um em quatro dos animais controles que receberam PBS e adjuvantes. Estes resultados sugerem a presença de células auto-reativas no miocárdio dos animais imunizados. Em conclusão, o uso de proteína M1 recombinante como imunógeno em modelo animal de ratos Lewis é capaz de desencadear reação inflamatória em miocárdio e tecido valvular e lesões similares às da febre reumática. O modelo do rato Lewis é até o momento o que apresenta maior semelhança com a doença humana e pode ajudar a esclarecer a imunopatologia da febre reumática. Além disso, certamente será importante para a avaliação do potencial de proteção e de segurança em modelos de vacinas contra o S. pyogenes. / Rheumatic fever is a good example of an autoimmune disease triggered by an infectious process. One to four weeks after the resolution of a non treated pharyngitis caused by rheumatogenic strains of S. pyogenes, the susceptible host unravels an immune response targeting joints, heart, conective tissues and brain. It is thought that molecules present in the bacteria and recognized during the infection at the pharynx are confounded with the organism self structure in a process called ?molecular mimicry?. Amongst the proteins involved in the cross reaction, it may be found cardiac myosin, on the host side, and M protein on the invading organism?s side. The latter (Mprotein) has been extensively studied. It is the basis of the S. pyogenes strains classification, and also an important virulence factor. It has also been explored as an immunogen in several vaccine strategies. The nderstanding of this disease has been hampered by the absence of an animal model that reproduces fundamental aspects of the human pathology, specially cardiac lesions. One of the reasons is the fact that animals do not get infected by S. pyogenes. Hence we have produced the recombinant M1 protein and shown that either the immunization of 28 Lewis rats for a period of 21 days or 14 rats for a period of 41 days, was capable of inducing an inflammatory response in most of the animals with variable intensity. Aschoff nodules-like or Anitschkow-like cells resembling rheumatic fever pathognomonic lesions were seen in 50% of the animals immunized subcutaneously and sacrificed on day 21. We have observed an humoral and cellular response (spleen and lymph node derived cells) specifically targeting M1 protein and the amino (M1AB) and carboxy (M1C) terminus of the protein. However, cross reactions with cardiac myosin were not observer. We have derived T lymphocyte lineages obtained from myocardium infiltrating mononuclear cells from 6 of the 10 animals immunized with M1ABC protein subcutaneously and sacrificed on day 41 and also from one out of four PBS - adjuvant immunized animals. These results suggest the presence of autoreactive cells in the myocardium of the immunized animals. In conclusion, the use of the M1 protein as an immunogen on the Lewis rat model is capable of triggering an inflammatory reaction in the myocardium and valvular tissue and it can produce rheumatic fever like lesions. The Lewis rat model is up to this moment the one to present the highest similarity with human disease. Besides, it will certainly be important on the evaluation of the protection and safety of S. pyogenes vaccines.

As Hsp60 acham-se conservadas em todos os organismos, participando da estruturação de proteínas e em processos crônico-degenerativos. Foi avaliada a ação das Hsp65r WT e sua mutante K409A de M. leprae no envelhecimento. Análises do Tempo Médio de Sobrevida (TMS), titulação dos isótipos, ensaios de avidez e análises histopatológicas foram realizadas em camundongos das linhagens HIII e LIII inoculados intraperitonealmente aos 120 ou 270 dias de vida com 2.5µg/mL das Hsp65r. Verificou-se redução no TMS de fêmeas HIII, envelhecidas e adultas, inoculadas com WT. A inoculação da Hsp65r WT em fêmeas LIII envelhecidas resultou no aumento do TMS. A dosagem dos anticorpos não revelou alterações marcantes na produção dos isótipos, e a avidez de IgG foi menor nas fêmeas HIII envelhecidas inoculadas com WT. A análise histopatológica mostrou inflamação nos rins de fêmeas HIII velhas tratadas com Hsp65r WT. Os resultados indicaram a interferência da Hsp65r WT na imunidade durante o processo de senescência de fêmeas envelhecidas constitutivamente boas produtoras de anticorpos. / The Hsp60 are conserved in all organisms, involved in proteins folding and chronic-degenerative processes. We evaluated the action of M. leprae rHsp65 WT and its mutant K409A in aging. Analyses of mean survival time (MST), antibody titration, avidity assays and histopathological examinations were performed in mice from HIII and LIII lines intraperitoneally inoculated at 120 or 270 days of life with 2.5µg/mL of rHsp65. There was a decrease in MST in adult and aged HIII females mice inoculated with WT protein. WT rHsp65 treatment in aged LIII females resulted in the increase of MST. The antibodies titration showed no marked changes in the production of isotypes and IgG avidity was lower in aged HIII females inoculated with WT. Histopathology showed inflammation in the kidneys of old HIII females treated with WT rHsp65. The results indicated the interference of WT rHsp65 in immunity during the senescence of aged females from HIII line constitutively producing good antibodies.

As Hsp60 acham-se conservadas em todos os organismos, participando da estruturação de proteínas e em processos crônico-degenerativos. Foi avaliada a ação das Hsp65r WT e sua mutante K409A de M. leprae no envelhecimento. Análises do Tempo Médio de Sobrevida (TMS), titulação dos isótipos, ensaios de avidez e análises histopatológicas foram realizadas em camundongos das linhagens HIII e LIII inoculados intraperitonealmente aos 120 ou 270 dias de vida com 2.5µg/mL das Hsp65r. Verificou-se redução no TMS de fêmeas HIII, envelhecidas e adultas, inoculadas com WT. A inoculação da Hsp65r WT em fêmeas LIII envelhecidas resultou no aumento do TMS. A dosagem dos anticorpos não revelou alterações marcantes na produção dos isótipos, e a avidez de IgG foi menor nas fêmeas HIII envelhecidas inoculadas com WT. A análise histopatológica mostrou inflamação nos rins de fêmeas HIII velhas tratadas com Hsp65r WT. Os resultados indicaram a interferência da Hsp65r WT na imunidade durante o processo de senescência de fêmeas envelhecidas constitutivamente boas produtoras de anticorpos. / The Hsp60 are conserved in all organisms, involved in proteins folding and chronic-degenerative processes. We evaluated the action of M. leprae rHsp65 WT and its mutant K409A in aging. Analyses of mean survival time (MST), antibody titration, avidity assays and histopathological examinations were performed in mice from HIII and LIII lines intraperitoneally inoculated at 120 or 270 days of life with 2.5µg/mL of rHsp65. There was a decrease in MST in adult and aged HIII females mice inoculated with WT protein. WT rHsp65 treatment in aged LIII females resulted in the increase of MST. The antibodies titration showed no marked changes in the production of isotypes and IgG avidity was lower in aged HIII females inoculated with WT. Histopathology showed inflammation in the kidneys of old HIII females treated with WT rHsp65. The results indicated the interference of WT rHsp65 in immunity during the senescence of aged females from HIII line constitutively producing good antibodies.

Les anticorps à réactivité croisée sont caractérisés par leur capacité à reconnaitre des épitopes différents de ceux qui ont causé leur induction. Cela se produit lorsque des similitudes structurales entre les deux déterminants antigéniques deviennent suffisantes pour permettre une liaison spécifique. Nous rapportons ici pour la première fois la présence, à une haute fréquence, d'anticorps dans des échantillons de sang provenant de sujets vivant en France avec une protéine de Trypanosoma cruzi. Nous avons appelé ces anticorps ''Trypanosoma cruzi Cross-reactive antibodies'' ou TcCRA. Nos résultats montrent une forte séroprévalence des anticorps à réaction croisée, suggérant qu'ils sont induits par un immunogène largement répandu, acquis dès l’enfance et qui ne semble pas être associé à des agents pathogènes communs en clinique humaine. Les recherches effectuées in silico orientent vers un virus de la famille des Herpesviridae. Cette hypothèse est renforcée par la documentation d’un profil sérologique de séroconversion chez un patient qui a subi une transplantation de cellules souches allogéniques. Ce premier travail va servir de base à la mise en oeuvre d’investigations cliniques rétrospectives et prospectives destinées à élucider l’étiologie et l’importance clinique du biomarqueur TcCRA / Cross-reactive antibodies are characterized by their recognition of antigens that are different from the trigger immunogen. This happens when the similarity between two different antigenic determinants becomes adequate enough to enable a specific binding. Here, we report for the first time the presence, at an ‘‘abnormal’’ high frequency in blood samples from French human subjects, of antibodies that cross-react with a protein of Trypanosoma cruzi. We called these antibodies ‘‘Trypanosoma cruzi Cross-Reactive Antibodies’’ or TcCRA. Our findings show a large seroprevalence of cross-reactive antibodies and suggest that they are induced by a widely spread immunogen, acquired during childhood. Furthermore TcCRA serology does not seem to be associated with commonly known pathogens in clinical routine. Our hypothesis of the implication of a viral agent in the induction of TcCRA was further put forward when we documented a seroconversion pattern in a patient after allogenic stem cell transplantation. This initial exploratory work will serve as the basis for organizing prospective and retrospective clinical investigations, where we will pursue the analysis of TcCRA in order to elucidate its etiology and clinical importance

Previous nutritional physiology research using L-histidine and zinc in American lobster intestine (Homarus americanus) has suggested that these solutes can be co-transported as complexes (Histidine-Zinc-Histidine) across the intestine using a peptide transporter. Furthermore, transport of L-leucine was shown to be inhibited by high calcium concentrations. Dipeptide and bis-complex transport and the role of calcium were investigated in the perfused intestines of lobster and Atlantic white shrimp (Litopenaeus setiferus). Following trans-intestinal transport, serosal medium was analyzed for amino acid composition by gas chromatography. In lobster, the transport of glycylsarcosine (Gly-Sar) from mucosa to serosa was stimulated two-fold with luminal pH 8.5, compared to the pH 5.5 control. Mucosa to serosa and serosa to mucosa fluxes of Gly-Sar were measured; the dipeptide was transported intact in both directions, but the net flux was from mucosa to serosa. The use of 0.5mM calcium chloride stimulated Gly-Sar transport two-fold, compared to 25 mM. In shrimp, the addition of 50 µM zinc chloride increased the rate of L-histidine transport, while Gly-Sar inhibited histidine transport in the presence of zinc. The rate of histidine transport was significantly higher with 1mM calcium chloride than with 25mM. These results suggest that shrimp transport bis-complexes in a manner similar to lobster. High calcium concentration had an inhibitory effect on both amino acid and dipeptide transport. Proposed mechanisms accounting for the effects of metals and calcium on trans-intestinal transports of both amino acids and dipeptides by lobster and shrimp digestive tracts are discussed.

No description available.