This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering. The variable domain genes were isolated from the hybridoma cell line producing the monoclonal antibody and successfully joined by PCR for the construction of the scFv, named anti-GPE. Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fab version of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the short CDR2 was due to deletions generated during the somatic mutation process and a model for this is described. A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I. A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPE and NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-binding chimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling to increase the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as a selection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identified and are discussed.

Common measures of egg quality have been survival to specific developmental stages, higher hatching rate of fertilized eggs and final production of fry. Determinants of egg quality are variable among and between teleost species and no common unified criteria have been established. Maternally inherited genes influence egg quality and early embryo development is partially programmed by the messenger ribonucleic acid (mRNA). Among the genes, the insulin family is important for growth functions and the presence of their transcripts in the ovary, oocytes and embryos implies their involvement during the reproductive process and their relevance to egg quality. The insulin-like growth factor (IGF) system has three components, the ligands IGF-I and II, the IGFBPs (insulin-like growth factor binding proteins) and the IGF receptors that mediate biological activity of the ligands. Vitellogenin (Vtg) is the major source of nutrients for the developing embryo and elevated levels in female fish plasma signals gonadal development preceding spawning. In oviparous fish where the developing embryo is dependent on the stored food in the yolk, vitellogenin levels in the egg could indicate its capability to support embryonic growth. This study aimed to develop molecular tools, specifically probes for IGF-I, IGF-II and IGF-IR, for the evaluation of fish egg quality. These probes would be used to determine expression levels of IGF-I, IGF-II and IGF-IR during egg development to assess their potential as molecular indicators for egg quality. In addition, this study also aimed to establish an enzyme-linked immunoassay (ELISA) for quantifying Vtg in fish eggs and determine if differences in Vtg levels could be linked to fertilization and hatching success. Through reverse-transcription polymerase chain reaction (RT-PCR) putative complementary deoxyribonucleic acid (cDNA) fragments of IGF-I, IGF-II and IGF-IR were cloned and sequenced from mullet (Mugil cephalus) and grouper (Epinephelus coioides). The relative expression ratio of the three genes in the eggs of mullet and grouper were assayed by quantitative PCR (QPCR) and calculated using the Pfaffl method (Pfaffl, 2001). Levels of vitellogenin in different batches of mullet eggs were quantified by ELISA. Spawned eggs of grouper were grouped into low (<60%) or high (>60%) fertilization rate (FR) and the fertilized eggs that were incubated until hatching were grouped into medium (>90%) or high (>90%) hatching rate (HR). Samples were categorized into sinking eggs, late embryo and hatched larvae. Relative expression ratio of IGF-II was significantly high (P<0.01) compared to IGF-I and IGF-IR in all samples examined. All three genes were strongly expressed in sinking eggs compared to either late embryo or hatched larvae. However, there was no significant interaction effect between the genes and the samples analyzed. Mullet samples all came from a high FR and high HR group and were categorized into sinking, multicell stage, blastula, gastrula, late embryo and hatched larvae. There was a significant interaction effect (P<0.01) between gene and stage, showing that genes are differentially expressed during embryonic development. IGF-II was strongly expressed relative to the other genes in all stages examined and was highest during the gastrula stage. Vtg levels were examined in mullet oocytes and egg samples that were grouped into 4; oocytes from females that subsequently spawned, had fertilized eggs which hatched (Group A); oocytes from females that did not spawn, therefore no fertilization and no hatching (Group B); eggs that were stripped, artificially fertilized but no hatching (Group C); and eggs that were spawned, assumed to be fertilized but did not hatch (Group D). Group A showed a trend of higher Vtg levels than the other three but this result was not statistically significant.

Cell-envelope associated and secreted proteins of Salmonella are integral for host-pathogen interactions, and for the induction of protective immune responses. An array of exported proteins of S. Brandenburg was identified through constructing an expression library using alkaline phosphatase gene technology. A partial digest of S. Brandenburg strain S59 was cloned into the vector pJEM11, and expressed in E. coli. The DNA inserts from randomly selected alkaline phosphatase positive clones were sequenced, and the sequences were analysed using public databases to find the ones that may play a role in host immune cell activation. The phase-1 flagellin (fliC) gene identified from an alkaline phosphatase positive phenotype was chosen for further studies. The complete nucleic acid sequence of the fliC gene was obtained by PCR amplification. The complete ORF, part of the variable region (V456) and region IV (V4) of the fliC gene were cloned into the pET14b vector for the expression of N-terminal histidine-tagged fusion proteins. The proteins were purified through metal affinity chromatography, and were evaluated for their humoral immunogenic properties by Western blotting with sera collected from 81 sheep naturally infected with S. Brandenburg. All 81 naturally infected sheep had IgG antibodies against recombinant FliC, V456, and V4 proteins. Furthermore, Western blotting of sera from 6 salvexinTM+B-vaccinated sheep (Trial 2004) had IgG antibodies against the 3 recombinant proteins. Whole blood cells of vaccinated sheep did not show interferon-gamma production upon stimulation with recombinant FliC and V456 proteins. Western blotting of sera from sheep vaccinated with salvexinTM and salvexinTM+B (Trial 1999), and those from rabbits vaccinated with S. Brandenburg, S. Hindmarsh and S. Typhimurium suggested that recombinant V4 contains epitopes specific for S. Brandenburg. Therefore, V4 was used to develop a novel indirect enzyme-linked immunosorbent assay (ELISA) for the detection of serum IgG antibodies in S. Brandenburg infected sheep. The ELISA showed a specificity of 100%, and a sensitivity of 93.8%. Furthermore, a new PCR assay was developed targeting rfbJ(B) gene in a single reaction, and genes invA, fliC and fljB in a multiplex reaction for the identification of S. Brandenburg from pure cultures. The sensitivity and specificity of the PCR assay was calculated to be 100%.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-27T12:04:45Z No. of bitstreams: 1 marcelle-bral-de-mello.pdf: 1087389 bytes, checksum: 74eb4d07ffe50812d0cea3fd71cdf47a (MD5) / Made available in DSpace on 2012-11-27T12:04:45Z (GMT). No. of bitstreams: 1 marcelle-bral-de-mello.pdf: 1087389 bytes, checksum: 74eb4d07ffe50812d0cea3fd71cdf47a (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Bio-Manguinhos produz, desde a década de 80, Kits para diagnóstico, inclusive para o diagnóstico da doença de Chagas. O kit EIE-Recombinante-Chagas, que utiliza na sua composição as proteínas recombinantes CRA e FRAdo T. cruzi, foi registrado como produto junto à ANVISA no ano de 1999. No período de renovação do registro, o Kit passou por uma avaliação prévia no INCQS, onde não foi possível alcançar condição satisfatória quanto aos níveis de sensibilidade. O presente trabalho teve por objetivo retomar a padronização deste Kitno âmbito do desenvolvimento tecnológico. Para tanto foi estabelecido a padronização de três modelos de protótipos, utilizando variações das construções disponíveis das proteínas recombinantes CRA e FRA, na forma de apresentação como antígeno e como conjugado. Posteriormente, foi avaliado o desempenho de cada protótipo, em três diferentes fases. Na primeira fase, o melhor desempenho foi apresentado pelo protótipo 3, cujos níveis de sensibilidade e especificidade foram respectivamente de 99% e 99,5% (IC 95%). Na segunda fase o nível de sensibilidade do protótipo 3 foi de 95.8% e o de especificidade 100%, mantendo a melhor condição de desempenho em relação aos demais. Na última fase, somente o protótipo 3 foi avaliado em função da indisponibilidade de volume das amostras selecionadas e os níveis de sensibilidade e especificidade encontrados de 100%.O desempenho do protótipo 2 foi insatisfatório nas duas primeiras fases de avaliação.Tendo em vista o bom desempenho alcançado pelo protótipo 3, propomos a continuação deste modelo na elaboração futura de lotes-piloto, de estudos multicêntricos e de validação, em ações conjuntas com o controle e a garantia da qualidade para a elaboração de documentação visando pedido de registro desse produto junto aos órgãos regulatórios. / Bio-Manguinhos produces, since the 80’s, a variety of kits for diagnosis, including the kit for Chagas’ disease diagnostic. The EIE-Recombinant-Chagas’ kit, which has in its constitution the recombinant proteins CRA and FRA from T. cruzi, was registered at ANVISA as a product in 1999. During the renewal of the registration period, the kit was submitted to a previous registration analysis by The National Institute for Quality Control in Health (INCQS), when it did not show satisfactory sensitivity levels. The present work aimed to remake the kit standardization process under the scope of the technological development. In order to do it, we established the standardization of three prototype models, each one using variations of available constructions from CRA and FRA recombinant proteins in the form of presentation as antigen and as conjugate. Afterwards, we evaluated the performanceof each prototype, in three different stages. In the first one, prototype 3 showed the best performance with 99% sensitivity and 99.5% specificity levels (CI 95%). In the second stage, prototype 3 levels sensitivity and specificity were 95.8% and 100% respectively, keeping the best performance condition as compared to the other two prototypes. In the last stage, only prototype 3 was assessed due to the lack of sample volumes. The reported sensitivity and specificity levels forsuch prototype were equal to 100%. The performance of prototype 2 was unsatisfactory in the first two assessment stages. Taking into account the performance achieved by prototype 3, we propose the continuation of this model for future development of pilot lots, multicenter and validation studies, in joint actions with quality assurance and quality control for the arrangement of the documents requested by the National Regulatory Agency for the registration of the product.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-12-04T11:37:37Z No. of bitstreams: 1 fernanda-martins.pdf: 786034 bytes, checksum: e059b45f6a12387864511efbf2ecfdbe (MD5) / Made available in DSpace on 2012-12-04T11:37:37Z (GMT). No. of bitstreams: 1 fernanda-martins.pdf: 786034 bytes, checksum: e059b45f6a12387864511efbf2ecfdbe (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil. / A combinação de vacinas é uma estratégia de grande relevância para o Programa Nacional de Imunizações. Através dela, é possível aumentar a proteção a múltiplas doenças em uma única vacina, bem como diminuir as constantes visitas ao posto de saúde. Contudo, uma das desvantagens em relação a esse tipo de estratégia é a possibilidade de ocorrer interferência antigênica entre os seus componentes,o que pode resultar na diminuição da resposta imunológica. Devido a este fato, foi realizada uma combinação com vacinas já presentes no calendário brasileiro de imunizações (DTP-Hib) a vacinas experimentais em desenvolvimento em Bio-Manguinhos (meningocócica B e meningocócica C conjugada), com a finalidade de apresentar uma nova perspectivade produto a esta unidade bem como estabelecer a correlação antigênica entre esses componentes, comparando metodologias já padronizadas para este fim à metodologia alternativa (ELISA), além de avaliar a pirogenicidade e a interferência entre os componentes vacinais utilizados na combinação. A resposta imunológica aos componentes vacinais foiavaliada em camundongos suíços, NIH e cobaias Short-Hair pelo ELISA (VME, polissacarídeo C, PRRP, Bordetella pertussis) e os testes de soroneutralização in vivo(componentes tetânico e diftérico). Todos os componentes vacinais avaliados pelo ELISA induziram soroconversão nos animais 30 dias após a última imunização. Quando comparadas à vacina combinada completa, somente a resposta imunológica ao polissacarídeo C sofreu interferência de algum componente vacinal. Após novas combinações davacina meningocócica C conjugada às outras vacinas, pode-se concluir que avacinas DTP e Hib interagem positivamente na resposta daquela vacina. Em relação à soroneutralização in vivo, houve uma diminuição da potência dos componentes tetânicoe diftérico quando cobaias Short-Hair foram imunizadas com a vacina DTP-Hib combinada às vacinas meningocócicas B e C conjugada. Em contrapartida, na quantificação de IgG total em camundongos suíços imunizados com as duas combinações (DTP-Hib e DTP-Hib/B/C), não ocorreu diferença significativa entre os dois grupos. O teste de pirogenicidade realizado em coelhos comprovou que, quando combinadas entre si, às vacinas são capazes de aumentar a temperatura destes animais, provavelmente, devido àpresença de Bordetella pertussise VME de Neisseria meningitidisgrupo B. Apesar de não ter sido possível à comparação com ostestes padronizados, o ELISA mostrou-se muito satisfatório na pesquisa da resposta imunológica em camundongos. Embora preliminares, os resultados são muito importantes, pois introduzem novas perspectivas para a realização de outras combinações que atendam as demandas requisitadas pelo Programa Nacional de Imunizações. / The combination of vaccines is a great relevance strategy to the National Immunization Program. It enables increase protection to multiple diseases in a single injection, as well as reduces constant visits to health care. However, a disadvantage of this strategy is antigenic interference among vaccine components, resulting in immune response decreased. Due to this fact, a combination between vaccines of Brazilian immunization calendar (DTP-Hib) and experimental vaccines developed in Bio-Manguinhos (meningococcal B and meningococcal C conjugate) wasperformed, in order to present a new perspective of product to this unit and establish the antigenic correlation of these components, comparing standardized methodologies with alternative methodology (ELISA), besides evaluating pyrogenicity and interference ofcombined vaccine components. The immune response to vaccine components was evaluated in Swiss and NIH mice and Short-Hair guinea pigs by ELISA (OMV, polysaccharide C, PRP, Bordetella pertussis) and in vivoneutralization test (tetanus and diphtheria components). All vaccine components assessed by ELISA induced seroconversion rates 30 days after the last immunization in animals. The complete combined vaccine, interfered in the immune response to polysaccharide C. After new combinations of meningococcal C conjugate vaccine to other vaccines, we concluded that DTP and Hib vaccines induce a positive interaction in immune response to that vaccine. Regarding in vivoneutralization, there was a decrease of tetanus and diphtheria components potency when Short-Hair guinea pigs were immunized with DTP-Hib combined to B and C meningococcal conjugate vaccines. In contrast, when total IgG in Swiss mice immunizedwith the two combinations (DTP-Hib and DTP-Hib/B/C) was quantified, no significant difference was observed. Pirogenicity test in rabbits proved that complete combined vaccine increase the temperature of these animals, probably due to the presence of Bordetella pertussisand Neisseria meningitidisgroup B outer membrane vesicle. Although it was not possible comparision with standardized test, ELISA was a satisfactory test in studing immune response in mice. Although preliminary, the results are important because introduce new perspectives for other combinations could be done to atempt the required demands of National Immunization Program.

Made available in DSpace on 2014-06-11T19:27:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-19Bitstream added on 2014-06-13T18:56:29Z : No. of bitstreams: 1 watanabe_s_me_araca.pdf: 310690 bytes, checksum: cd933b6e1170509831a34ff5afc29200 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A necessidade de melhorar a qualidade dos materiais utilizados no tratamento endodôntico tem estimulado o estudo de vários materiais, buscando melhores condições de trabalho, biocompatibilidade e propriedade de estimular a ocorrência do selamento biológico. Este trabalho teve a finalidade de avaliar quantitativamente e qualitativamente a resposta tecidual frente ao implante de tubos de polietileno preenchidos pelos cimentos Endo-CPM-Sealer®, Sealapex® e Ângelus MTA® em tecido subcutâneo de ratos e a citotoxicidade e a produção de citocinas (IL-1β e IL-6) por fibroblastos de camundongos estimulados por estes cimentos. Para os testes de biocompatibilidade foram utilizados 30 ratos, os quais receberam individualmente três implantes de tubos de polietileno contendo os cimentos a serem testados e mais um tubo vazio como controle. Após 7, 15, 30, 60 e 90 dias do período de implantação, os animais foram sacrificados e os tubos de polietileno juntamente com o tecido que o circunda foram removidos, fixados e processados para análise em microscopia de luz. Após inclusão em glicol metacrilato, foram realizados cortes seriados de 3mm, corados com hematoxilina-eosina e cortes de 10mm, corados com Von Kossa e sem coloração para serem analisados sob luz polarizada. Os resultados foram analisados estatisticamente pelo teste de Kruskal Wallis. Os materiais Endo-CPM-Sealer®, Sealapex® e Ângelus MTA® causaram reações moderada no tempo experimental de 7 dias que diminuiu com o tempo. A resposta foi similar entre os grupos Controle, Endo- CPM-Sealer® e Ângelus MTA® no tempo experimental de 30 dias. Aos 60 dias todos os grupos foram semelhantes ao Controle. Mineralizações e granulações birrefringentes à luz polarizada foram observadas com todos os materiais. Foi possível concluir que o Endo-CPM-Sealer® foi biocompatível e estimulou a mineralização. Para o teste... / The need of improving the quality of materials used in endodontic treatment has stimulated the study of various materials seeking better working conditions, biocompatibility and property to stimulate the occurrence of biological sealing. The aim of this study was to evaluate quantitatively and qualitatively the tissue response to the implantation of polyethylene tubes filled with Endo-CPM-Sealer®, Sealapex® and Ângelus MTA® in subcutaneous tissue of rats and cytotoxicity and production of cytokines (IL-1β and IL-6) by mouse fibroblasts stimulated by these sealers. Thirty rats were used for the biocompatibility tests. Each animal received three implants of polyethylene tubes containing the cement to be tested and one empty polyethylene tube was used as control. After 7, 15, 30, 60 and 90 days from the implantation time, the animals were killed and the tubes with surrounding tissues were removed, fixed and processed for analysis using a light microscope. After inclusion in glycol methacrylate, they were serially sectioned into 3 μm or 10 μm cuts and stained with hematoxylineosin, Von Kossa or remained without staining to be observed under polarized light. The results were statistically analyzed by Kruskal Wallis test. The materials Endo- CPM-Sealer®, Sealapex® and Ângelus MTA® caused moderate reactions at 7 days which decreased with time. The response was similar among the control group, Endo- CPM-Sealer® and Ângelus MTA® at the 30th day. At 60th day all groups were similar to control group. Mineralization and granulations birefringent to the polarized light were observed with all materials. It was possible to conclude that Endo-CPM-Sealer® was biocompatible and stimulated mineralization. To test the cytotoxicity of mouse fibroblast (L929) were incubated in plate of 24 wells and stimulated with the sealers tested. Cell culture... (Complete abstract click electronic access below)

The enzyme 3-Hydroxy-3- Methylglutaryl Coenzyme A Reductase catalyzes the rate limiting step of hepatic cholesterol biosynthesis and is unique among the enzymes in the early part of the pathway in that it is membrane bound. This gives rise to potential regulation of the enzyme through interactions with the endoplasmic reticulum membrane. A purification procedure has been developed which consistently produces enzyme of high specific activity. In order to fully characterize the interactions between HMG-CoA reductase and the lipids in its immediate environment, HMG-CoA reductase was purified to homogeneity and shown to be a protein-lipid complex.

Orientador: Carmino Antonio de Souza / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T13:20:46Z (GMT). No. of bitstreams: 1 TiconaPerez_FanyVeronica_M.pdf: 4562191 bytes, checksum: dfed269ba3213d41785041f56823dd90 (MD5) Previous issue date: 2007 / Resumo: Este estudo prospectivo avalia os níveis séricos e as especificidades dos anticorpos anti-HLA em receptores de rim de doador cadáver, além de estimar sua influência na etiologia e gravidade das crises de rejeição. Entre os meses de junho de 2004 a agosto de 2006 foram analisados 40 receptores de transplante renal com prova cruzada pré-transplante negativa e acompanhados, clinicamente, no mínimo por 90 dias após o transplante. Os pacientes foram estratificados por sintomatologia clínica de rejeição bem sucedido e mau sucedido, sendo o grupo A composto de 26 casos com rejeição e o grupo B com 14 casos sem complicações. As amostras de soro para a pesquisa de anticorpos anti-HLA, aplicando os testes imunoenzimáticos (One Lambda Inc.), foram obtidas antes e até 60 dias depois do transplante, pois coincide com o aumento dos níveis de creatinina sérica, nos pacientes com má evolução do enxerto. O resultado dos anticorpos anti-HLA antes do transplante foi negativo. Enquanto o número médio de incompatibilidade HLA (mismatched) entre receptor e doador, considerando os loci HLA-A, B e DR, foi 4/6 em ambos os grupos, sendo que no grupo A as mais freqüentes foram 4/6 e 3/6 e do grupo B somente 4/6. Dentre os 26 pacientes do grupo A, 3 (11,5%) desenvolveram anticorpos anti-HLA detectados nos dias 16, 28 e 46 após o transplante. Os anticorpos desenvolvidos foram específicos aos antígenos do doador, sendo apenas um caso pertencente ao grupo de reação cruzada, no qual se incluía também o antígeno do doador. Estes pacientes desenvolveram rejeição aguda do tipo vascular. Os demais pacientes deste grupo apresentaram crises de rejeição reversíveis com a administração da terapia imunossupressora (ciclosporina, micofenolato e tacrolimus) estabelecida pelos protocolos do Centro Integrado de Nefrologia. Enquanto o grupo B não desenvolveu anticorpos anti-HLA. Embora a casuística seja pequena, os resultados sugerem a importância de desenvolver anticorpos que combatam os antígenos HLA do doador perante a gravidade da crise de rejeição e por conseguinte, na perda do enxerto / Abstract: This prospective study evaluates the seric levels and anti-HLA antibodies¿ specificities of kidney receptor from dead donor, besides to esteem its influence in the etiology and its severity in the rejection crises. From June 2004 to August 2006, 40 transplanted kidney receptors had been analyzed with negative crossed test before of transplant and followed, clinically, at least 90 days after transplant. Patients were grouped by positive and negative rejection of clinical symptoms, being the group A made up of 26 positive cases and group B 14 negative cases. The serum samples for researching anti-HLA antibodies, applying the immunoenzimatic tests (One Lambda Incorporation), had been gotten just before and up to 60 days after the transplant, so that it coincides with increasing creatinine serum levels in the patients with bad evolution of engraftment. The anti-HLA antibodies results before transplant were negative. While the average number of HLA incompatibility (mismatched) between receptor and donor, considering HLA-A, B and DR loci, was 4/6 in both groups. Being in the group A the most frequent had been 4/6 and 3/6 and in the group B only 4/6. Among 26 patients of group A, 3 (11.5%) had developed detected anti-HLA antibodies in days: 16, 28 and 46 after transplant. The developed antibodies had been specific to donor¿s antigens, being only one case belongs to the crossed reaction group, which it had also the donor¿s antigens. These patients had an acute rejection of the vascular type. The others of this group presented reversible crises of rejection using immunosuppressive therapy (cyclosporine, mycophenolate and tacrolimus) determined by the Institution protocols. Group B did not develop anti-HLA antibodies. Although this sample is small, the results suggest the importance of creating antibodies that battle with donor¿s HLA antigen due to severity of rejection crisis and therefore, its loss / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

INTRODUÇÃO: As dermatoses bolhosas autoimunes são um grupo heterogêneo de afecções da pele e/ou mucosas associadas à produção de autoanticorpos dirigidos às moléculas de adesão epitelial. Podem ser classificadas em dermatoses bolhosas intraepidérmicas (pênfigos) ou subepidérmicas (penfigóides, epidermólise bolhosa adquirida). Nos últimos anos, a transição entre dermatoses bolhosas autoimunes ou coexistência de autoanticorpos de diferentes dermatoses têm sido relatadas em alguns pacientes e atribuída ao fenômeno de epitope spreading (ES): a diversificação de epítopos reconhecidos pelo sistema imune evocaria uma reação secundária a antígenos distintos e não relacionados aos da doença primária. Neste trabalho avaliamos a ocorrência de fenômenos de ES em pacientes portadores de pênfigo. CASUÍSTICA E MÉTODOS: Inicialmente, foi realizada análise de dados clínicos e laboratoriais (exame histopatológico, de imunofluorescência direta-IFD, indireta IFI e ELISA) de 351 pacientes portadores de pênfigos acompanhados no Ambulatório de dermatoses bolhosas autoimunes do Departamento de Dermatologia da Faculdade de Medicina da Universidade de São Paulo no período de dezembro de 2002 a dezembro de 2012. Foram selecionados pacientes com quadro sugestivo de conversão à dermatose bolhosa distinta da doença primária. RESULTADOS: Nove pacientes apresentaram sinais sugestivos de fenômeno de ES e foram incluídos no estudo: 8 com a conversão de Pênfigo vulgar (PV) a foliáceo (PF) 2,3% (grupo1) e um de PF a Epidermólise bolhosa adquirida (EBA) 0,3% (grupo 2). No grupo 1 o intervalo mediano para a conversão foi de 3,5 anos. Cinco pacientes apresentaram modificação histopatológica de clivagem intraepidérmica na camada suprabasal para clivagem na camada subcórnea durante a suspeita de ES; 2 apresentaram clivagem na camada epidérmica média durante a transição e um manteve clivagem suprabasal, apesar de quadro clínico sugestivo de PF. Todos os pacientes apresentavam depósitos intercelulares de IgG e/ou C3 durante o diagnóstico de PV e PF à IFD. Títulos de IFI variaram de 1:160 a 1:5120. Os valores de ELISA para Dsg1 variaram de 22 a 319; e para Dsg3 de 0.4 a 224 (positivo se > 20). A relação Dsg1/Dsg3 correspondeu à mudança PV-PF. No grupo 2, o ES para EBA ocorreu sete anos após o diagnóstico de inicial de PF. No momento da suspeita de ES o paciente apresentava-se em remissão clínica do quadro de pênfigo folíaceo. A avaliação laboratorial mostrou clivagem subepidérmica neutrofílica, IFD com IgG intercelular intraepidérmica e depósitos de IgM, IgA, IgG e C3 na zona da membrana basal. IFI com técnica de salt split skin revelou depósitos de IgG do lado dérmico. Ao immunobloting houve reconhecimento de colágeno VII e ELISA para Dsg1 foi positivo. CONCLUSÃO: A frequência de ES em pacientes portadores de pênfigo foi de 2,6%. Estudos serão necessários para elucidar a patogênese deste evento e sua importância na progressão dos pênfigos / BACKGROUND: Autoimmune bullous skin diseases represent a heterogeneous group of disorders of skin and mucosa associated with autoantibodies against distinct adhesion molecules. They can be classified, based on the level of loss of adhesion in intraepidermal and sub epidermal dermatosis. The shift from an autoimmune blistering disease to another has been recently described and attributed to the \"epitope spreading\" (ES) phenomena. It occurs when a primary inflammatory/autoimmune process releases \"hidden\" epitopes which are recognized by the lymphocytes and evoke a secondary reaction to antigens distinct from, and non-cross-reactive, with the disease causing-epitope. This study attempted to characterize the occurrence of ES in pemphigus patients. METHODS: We analyzed data from 351 pemphigus patients treated ambulatorially at the Department of Dermatology, Faculty of Medicine, University of São Paulo, from December 2002 to December 2012. A careful search for clinical and laboratorial (histopathology, direct-DIF and indirect-IIF immunofluorescence, ELISA) changes suggestive of shift to a secondary bullous disease was performed. RESULTS: Nine out of 351 patients presented clínical shift and were included in the study: eight from pemphigus vulgaris (PV) to foliaceus (PF) 2.3% (group 1) and one from PF to epidermolysis bullosa acquisita (EBA) 0.3% (group 2). In group 1, median interval of disease shift was 3.5 years. Of 8 patients with clinical PF, five showed change of histopathology pattern from suprabasilar cleavage to subcorneal acantholysis, two had cleavage within the middle epidermal layer, and one sustained the suprabasilar acantholysis. One shifted back to PV after clinical and histopatological changes of PF. All patients showed intercellular IgG and/or C3 deposits during PV and PF diagnosis by DIF. IIF titers varied from 1:160 to 1:5120. ELISA index for Dsg1 varied from 22 to 319; and for Dsg3 from 0.4 to 224 (positive if > 20). Dsg1/Dsg3 indexes corresponded to the clinical PV-PF changes. In group 2, onset of PF occurred at the age of 25, and ES to EBA 7 years later in the absence of PF lesions. Laboratory evaluation showed sub epidermal cleavage with neutrophils, IgG intercellular staining in the epidermis and IgM, IgA, IgG and C3 deposits at BMZ by DIF, IgG deposits by indirect salt-split, recognition of collagen VII by immunoblotting, and positive ELISA for Dsg1. CONCLUSIONS: Intermolecular ES occurred in 2.6% (9/351) of pemphigus patients. Futures studies will be necessary to elucidate the pathogenesis of this event and its significance in pemphigus progression