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The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders.

SPF (Supernatant Protein Factor) is a cytosolic protein that stimulates at least two enzymes in the cholesterol biosynthetic pathway: squalene monooxygenase and HMGCoA reductase. The mechanism of action has not been established but may be related to lipid transfer between intracellular membranes. There are three human genes for SPF: SEC14L2 (SPF1), SEC14L3 (SPF2) and SEC14L4 (SPF3). The present study differentiates these closely related genes by evaluating their tissue-specific and relative expression levels. SPF1 mRNA was found to be most abundant in liver, mammary gland and stomach. SPF2 showed negligible expression in all tissues tested; SPF3 expression pattern was similar to that of SPF1, but at 10-50-fold lower levels than SPF1. A cDNA to SPF3 was cloned and, upon transfection into rat hepatoma cells, was shown to increase cholesterol synthesis by approximately 50%, similar to that obtained with SPF1. However, in contrast to SPF1, SPF3 did not stimulate squalene monooxygenase activity in microsomal preparations, suggesting that it acts primarily through activation of HMG-CoA reductase. SPF possesses a lipid binding domain (Sec14) and a Golgi dynamics domain (GOLD). SPF resides in the cytosol and requires phosphorylation and the presence of Golgi in order to stimulate cholesterol synthesis. To determine if SPF associates with specific subcellular structures, cellular immunofluorescence studies were carried out. A phosphorylationdefective mutant, a protein lacking the GOLD domain, and the effect of protein kinase A-mediated phosphorylation of endogenous SPF were examined. No change in the subcellular location of SPF could be detected with either the phosphorylation mutant or the native SPF after protein kinase A activation. However, removal of the GOLD domain resulted in a protein that co-localized with large vesicular structures around nucleus. Studies with rat hepatoma cells showed that the expression of the two rat SPF genes is upregulated in response to serum deprivation, and is potentiated by removal of glucose. Lipid/cholesterol availability was demonstrated to be at least one of the serum components that affected SPF transcript levels. The oxysterol receptor LXR was shown not to be involved in SPF gene regulation, implicating SREBP and/or PPARα as the principal regulators of SPF gene transcription.

Breast cancer (BC) is one of the leading causes of cancer-related deaths in Canadian women. Aggressive BCs (e.g. triple-negative subtype; TN) present a clinical challenge as defined biomarkers, particularly those indicative of unique cancer-associated signaling pathways, are needed to improve prognostication and prediction of therapeutic response. Overexpression of Src and its substrates, Ezrin and Tks5, have been associated with poor prognosis in many cancers. We have previously shown that Ezrin regulates proteolytic-independent invasion, while others have shown that Tks5 is associated with proteolytic-dependent invasion. Thus, expression of Ezrin versus Tks5 in BC cases may represent different invasion modalities. Additionally, immunofluorescence (IF)-based technologies may provide a more quantitative and objective approach for analysis of biomarker expression and subcellular compartmentalization compared to immunohistochemistry (IHC). In this study, I hypothesize that expression and subcellular localization of Src, Ezrin and Tks5, have improved prognostic significance in BCs, compared to current clinico-pathological parameters. To assess this, I optimized an IF-based automated quantification analysis (AQUA) system to measure subcellular expression in a 63-patient BC cohort and tested associations with clinico-pathological data. This thesis presents that: 1) Expression of Src and Ezrin increased, but that of Tks5 decreased in breast tumours compared to normal breast. 2) Src and Ezrin localized to the apical regions of normal breast epithelia but shifted to the cytoplasm in breast tumours. Tks5 exhibited a granular basal expression in normal breast epithelia, and is weakly expressed in tumour cellular compartments. 3) In our 63-patient cohort, Src and Ezrin had significant correlations with multiple clinico-pathological parameters, including TN status and lymphovascular invasion. 4) Clinico-pathological associations with IF-based AQUA scoring are directly comparable to conventional manual IHC scoring. Our study supports the role of Src and Ezrin as potential prognostic biomarkers for BC. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-07-09 12:51:09.527

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Objetivo: Avaliar in vivo a resposta tecidual de dentes de cães após perfuração de furca e recobrimento com Biodentine&trade;, em comparação ao MTA e à guta-percha, por meio de análise histopatológica, histoenzimológica, imunohistoquímica e por imunofluorescência. Métodos: Foram utilizados 30 dentes de 3 cães, divididos em 3 grupos: I - Biodentine; II - MTA; e III - Guta-Percha (controle). Após tratamento endodôntico e limpeza da câmara pulpar, perfurações no centro do assoalho foram realizadas intencionalmente em cada dente, as quais foram preenchidas com os diferentes materiais. Após 120 dias, os animais foram eutanasiados e as peças contendo os dentes e tecidos perirradiculares foram submetidas ao processamento histotécnico. Foram realizadas análises histopatológicas semi-quantitativas para avaliação da neoformação de tecido mineralizado e da reinserção de fibras, além de análise imunohistoquímica das proteínas osteopontina (OPN) e fosfatase alcalina (ALP) e imunofluorescência para proteína morfogenética óssea (BMP-2), proteína de adesão do cemento (CAP), sialoproteína óssea (BSP), osteocalcina (OCN) e proteína do cemento 1 (CEMP1) no tecido mineralizado neoformado e na região adjacente. Paralelamente, foi realizada a histoenzimologia para a atividade da TRAP e contagem dos osteoclastos. Os dados foram submetidos aos testes qui-quadrado e Kruskal-Wallis, com nível de significância de 5%. Resultados: Na avaliação do tecido mineralizado neoformado, o grupo controle foi significantemente diferente dos demais grupos (p<0,0001), sendo que não houve formação de tecido mineralizado em nenhum espécime desse grupo. Nos grupos tratados com MTA e Biodentine houve formação de tecido mineralizado em 88% e 92% dos espécimes, respectivamente, sem diferença entre eles (p>0,05). Ainda, o grupo controle apresentou fibras colágenas paralelas à perfuração. Nos grupos tratados com MTA ou Biodentine também houve fibras colágenas paralelas à perfuração, porém com algumas fibras reinseridas perpendicularmente em diferentes áreas do tecido mineralizado neoformado. Todos os tratamentos induziram a expressão de OPN e ALP, porém em menor intensidade no grupo controle e em maior intensidade no grupo tratado com MTA (p<0,05). Apenas o tecido mineralizado formado após o tratamento com MTA expressou BMP-2, BSP, OCN, CAP e CEMP1. Com relação à avaliação dos osteoclastos, não foi possível encontrar diferença estatística entre os grupos (p=0,97). Conclusão: Com base nos parâmetros analisados, pôde-se concluir que o MTA e a Biodentine apresentaram resposta tecidual satisfatória, com formação de tecido mineralizado e reinserção parcial de fibras, podendo ser indicados para o selamento de perfurações de furca. Além disso, o presente estudo elucidou alguns mecanismos de ação pelo quais o MTA e a Biodentine induzem a formação do tecido mineralizado, com expressão dos marcadores da mineralização ALP e OPN, sem interferência na quantidade de osteoclastos. Apenas o MTA estimulou a expressão de proteínas associadas à formação de tecido mineralizado semelhante ao cemento / Aim: This study evaluated in vivo tissue response in dogs teeth after sealing of furcation perforations with Biodentine&trade;, MTA and gutta-percha, by means of histopathological, histoenzymological, immunohistochemical and immunofluorescence analysis. Methods: Thirty teeth of 3 dogs were used, divided in 3 groups: I - Biodentine; II - MTA; and III - Guta-Percha (control). After endodontic treatment, perforations were made on the pulp chamber floor and filled with the different materials. The animals were euthanized after 120 days and the teeth and surrounding tissues were processed for histopathological analysis of new mineralized tissue formation and collagen fiber reinsertion, immunohistochemical analysis of osteopontin (OPN) and alkaline phosphatase (ALP) and immunofluorescence analysis for bone morphogenetic protein (BMP-2), cementum attachment protein (CAP), bone sialoprotein (BSP), osteocalcin (OCN) and cementum protein1 (CEMP1). Histoenzymology was performed for TRAP activity and osteoclast count. Data were submitted to chi-square and Kruskal-Wallis tests (=0.05). Results: Gutta-percha did not induce mineralized tissue formation. MTA and Biodentine formed mineralized tissue in 88% and 92% of specimens, respectively, with no significant difference (p>0.05). In addition, the control group had collagen fibers parallel to the perforation. In the groups treated with MTA or Biodentine there were also collagen fibers parallel to the perforation, but with some fibers reinserted perpendicularly in different areas of the neoformed mineralized tissue. All materials induced OPN and ALP expression, weakest for gutta-percha and strongest for MTA (p<0.05). Only MTA induced BMP-2, BSP, OCN, CAP and CEMP1 expression. Osteoclast count was similar in the groups (p=0.97). Conclusion: Thus, according to the parameters analyzed in this present study, MTA and Biodentine presented satisfactory tissue response, with formation of mineralized tissue and partial reinsertion of fibers, and could be indicated for sealing furcation perforations. In addition, the present study elucidated some mechanisms of action by which MTA and Biodentine induce the formation of mineralized tissue, with expression of ALP and OPN mineralization markers, without interference in number of osteoclasts. Only MTA stimulated the expression of proteins associated with the formation of a cement-like mineralized tissue

O vírus Mayaro (MAYV) é um Alphavirus artritogênico responsável por causar uma doença febril aguda com sintomas parecidos aos de Dengue não-hemorrágica, porém com o agravante, como a febre Chikungunya, de ocorrência de artralgia. Os dados epidemiológicos disponíveis ainda são poucos devido à falta de diagnóstico adequado, pois algumas das técnicas desenvolvidas apresentam dificuldades quanto à coleta de amostra, devido à curta viremia, e ao background que interfere na interpretação dos resultados, subestimando o real número dos casos de infecção. Isso é preocupante principalmente em tempos de co-circulação de outras arboviroses como Dengue, Zika e Chikungunya. Neste trabalho desenvolvemos um método de imunofluorescência indireta dirigido à detecção de anticorpos contra a glicoproteína E2 do vírus Mayaro expressa em células S2 de Drosophila melanogaster. / Mayaro virus (MAYV) is an arthritogenic Alphavirus responsible for causing an acute febrile illness with symptoms similar to non-hemorrhagic Dengue, but with the aggravation, as Chikungunya fever, to develop arthralgia. The epidemiological data available still are few due to lack of proper diagnosis, because some of the techniques developed present difficulties regarding sample collection, due to the short viremia, and background that interferes with the interpretation of the results, underestimating the actual number of cases. This is a concern especially in periods of co-circulation of many other socioeconomic impact arboviruses, such as Dengue, Zika and Chikungunya. In this work we developed an indirect immunofluorescence method to the detection of antibodies against Mayaro E2 glycoprotein expressed in Drosophila melanogaster S2 cells.

Plasmodium malariae é responsável por uma baixa porcentagem de casos clínicos de malária, mas tem uma distribuição global ampla e fragmentada. Humanos podem abrigar o parasita por anos sem produzir sintomas significantes, o que dificulta o diagnóstico e consequente controle da doença. Por esta razão, a incorporação de antígenos de P. malariae nas estratégias em curso para produção de uma vacina é altamente recomendada. Embora a proteína de superfície do merozoíto1 (MSP1) de P. vivax e P. falciparum sejam amplamente estudadas como candidatos a vacinas, potencializando a resposta imune em camundongos e macacos, a MSP1 de P. malariae tem sido negligenciada. Portanto, este estudo visou caracterizar a imunogenicidade de proteínas recombinantes da MSP1 de P. malariae em camundongos BALB/c. Cinco regiões da PmMSP1 (N-terminal, F1; repetições em tandem, F2; central, F3 e C-terminal, F4 e PmMSP119) foram clonadas em vetor pGEX-3Y e expressas em Escherichia coli em fusão com GST. As proteínas recombinantes foram produzidas por fermentação e purificadas, fornecendo um alto rendimento de antígenos solúveis. Essas proteínas recombinantes e GST sozinha (grupo controle) foram usadas para imunizar, pela via intraperitoneal, seis grupos de camundongos BALB/c em 3 doses com intervalos de 14 dias. A especificidade e as subclasses dos anticorpos foram avaliadas por enzyme-linked immunosorbent assay (ELISA), utilizando as proteínas recombinantes como antígenos. A resposta imune celular foi analisada pelo ensaio de linfoproliferação e os níveis de citocinas Th1/Th2 nos sobrenadantes de culturas de esplenócitos foram detectados por citometria. Nossos resultados demonstraram que as regiões F1, F2, F3, F4 e PmMSP119 são imunogênicas em camundongos com a capacidade de produzir respostas imunes humorais e celulares, como mostrado pelos altos títulos de anticorpos (1/809,000) e pela proliferação de linfócitos. As respostas imunes induzidas alcançaram níveis máximos após três doses imunizantes permanecendo altas por 70 dias. Os anticorpos induzidos após imunização com as regiões F1, F2, F3 e F4 mostraram afinidades similares aos antígenos alvo, mas anticorpos induzidos após imunização com PmMSP119 mostraram uma afinidade mais alta com o antígeno alvo. Análise das subclasses de IgG mostrou que todas as proteínas recombinantes induziram padrões similares de anticorpos onde IgG1, IgG2a e IgG2b foram mais predominantes, caracterizando uma resposta mista de Th1/Th2. Além disso, a imunização dos camundongos com as proteínas recombinantes e estimulação com os mesmos antígenos promoveu produção de IL-4. Os níveis das citocinas Th1/Th2 não mostraram diferenças significativas quando comparados entre os grupos. A proliferação dos linfócitos estimulados com F2, F3 e PmMSP119, foi maior que aquela dos estimulados com F1, F4 e GST. Ainda, todos os soros dos animais imunizados com as cinco proteínas recombinantes de PmMSP1, que foram positivos por ELISA, mostraram reatividade com esquizontes de P. brasilianum nos ensaios de imunofluorescência (IFA). As proteínas recombinantes de PmMSP1 reagiram com anticorpos IgG nas amostras de soro de indivíduos expostos a P. malariae com alta especificidade comparado a amostras de soro de indivíduos com doenças não relacionadas. Entretanto, as regiões F1 e F2 e PmMSP119 foram reconhecidas por soros de pacientes com P. malariae com os títulos mais altos e não apresentaram reconhecimento em soros de pacientes com P. falciparum e P. vivax. Esses dados reforçam a utilidade destas proteínas como marcadores diagnósticos de P. malariae em estudos epidemiológicos ou no diagnóstico diferencial da malária causada por esta espécie. No entanto, a região F4 foi reconhecida igualmente em soros homólogos ou heterólogos e, portanto, poderia ser útil para testes pointof- care para o diagnóstico de malária. A imunização com as diferentes proteínas recombinantes de PmMSP1 promove uma resposta imune humoral significante, com altos e específicos níveis de IgG, além de uma distribuição de subclasses balanceada, fornecendo evidências de potenciais candidatos a uma vacina contra P. malariae. / Plasmodium malariae is responsible for a low percentage of clinical malaria cases and has a patchy distribution in the world. Humans can host the parasite for years without presenting significant symptoms, which turns its diagnosis and control a difficult task. For this reason, the incorporation of P. malariae antigens in vaccination strategies is highly recommended. Although the merozoite surface protein 1 (MSP1) of P. vivax and P. falciparum are widely studied as vaccine candidates, potentiating the immune response in mice and monkeys, P. malariae MSP1 has been neglected. Herein we invested in the characterization of the immunogenicity of recombinant proteins of P. malariae MSP1 in BALB/c mice. Five regions of PmMSP1 (N-terminus, F1; tandem repeat, F2; central region, F3 and C-terminus, F4 and PmMSP119) were cloned into vector pGEX-3Y and expressed in Escherichia coli in fusion with GST. Recombinant proteins were produced by fermentation and purified, providing a high yield of soluble antigens. These recombinant proteins and GST alone (control group) were used to immunize, via the intra-peritoneal route, six groups of BALB/c mice in three doses at 14-day intervals. The specificity and subtyping of the antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), using the recombinant proteins as antigens. Cellular immune responses were analyzed by lymphoproliferation assays and the Th1/Th2 cytokine levels in the supernatant of splenocyte cultures were detected by cytometry. Our results demonstrated that F1, F2, F3, F4 regions and PmMSP119 are immunogenic in mice with the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers (1/809,000) and the proliferation of lymphocytes. The induced immune responses reached maximum levels after three doses remaining high for 70 days. Antibodies induced after immunization with F1, F2, F3 and F4 regions showed similar affinities to the target antigens, but antibodies induced after immunization with PmMSP119 showed a higher affinity to the target antigen. Analysis of IgG subclasses showed that all the recombinant proteins induced similar antibody subclass patterns where IgG1, IgG2a and IgG2b were most predominant, characterizing a Th1/Th2 mixed response. Furthermore, immunization of mice with the recombinant proteins upon stimulation with the same antigen secreted IL-4. The levels of Th1/Th2 cytokines did not show significant differences when compared among groups. The proliferation of the stimulated lymphocytes, with F2, F3 and PmMSP119, was greater than those stimulated with F1, F4 or GST. Additionally, all sera from mice immunized with the five PmMSP1 recombinant proteins, which were positive by ELISA, showed reactivity with P. brasilianum schizonts by immunofluorescence assays (IFA). The PmMSP1 recombinant proteins reacted with IgG antibodies in serum samples from individuals exposed to P. malariae infections with a high specificity compared to serum samples from individuals with unrelated diseases. However, the F1 and F2 regions and PmMSP119 showed the highest titers in addition to no recognition by sera from P. falciparum and P. vivax infections. These data strengthen the usefulness of these proteins as diagnostic markers of P. malariae in epidemiological studies or in the differential diagnosis of malaria caused by this species. Nevertheless, the F4 region was recognized equally in homologous or heterologous sera, and thus, could be useful for point-of-care tests for malaria diagnosis. Immunization with the different PmMSP1 recombinant proteins promotes a significant humoral immune response, with high and specific IgG levels, in addition, a balanced subclass distribution, suggesting them as potential candidates for a vaccine against P. malariae.

A transmissão oral ganhou destaque devido aos recentes surtos que ocorreram no Brasil com a ingestão de alimentos contaminados, além dessa via outra de grande importância é a transmissão transplacentária, sendo assim o presente trabalho associou as duas formas de transmissão da doença, juntamente com o tratamento com o benzonidazol único medicamento disponível no Brasil para o tratamento da doença. O medicamente é adotado somente para o tratamento da fase aguda, porém alguns pesquisadores mostraram alguns benefícios da terapia com benzonidazol na fase crônica da doença podendo o medicamento diminuir o avanço da doença e prolongar a soro conversão negativa em pacientes intermediários. Assim o objetivo deste trabalho foi de investigar a eficácia do tratamento tanto na fase aguda quanto na fase crônica da doença, bem como o efeito do tratamento em fêmeas de fase crônica. Utilizou-se camundongos Balb/c infectados com 2 x 105 formas tripomastigotas sanguíneos da cepa RC de T. cruzi administrado via oral. Os animais de fase aguda tratados receberam 50mg/Kg/dia de benzonidazol por 16 dias enquanto que aos animais de fase crônica o tratamento se iniciou ao 3º dia de gestação sendo administrado pelo mesmo período. O curso da parasitemia sanguínea foi verificada bem como ao fim do tratamento a parasitemia tecidual em baço, fígado e coração; placenta e feto, quando havia, empregando-se as técnicas de qPCR, com dois distintos métodos de cálculos matemáticos, imunofluorescência em microscopia confocal e análise histopatológica por coloração HE. A administração de benzonidazol proporcionou a diminuição das cargas parasitárias de baço, fígado e coração elucidando a eficácia do medicamento também em fase crônica. Porém em placenta e feto o medicamento gerou aumento de parasitas teciduais nos grupos que receberam tratamento em detrimento aos que não receberam. Deste modo, podemos sugerir que o tratamento com benzonidazol pode ser indicado para minimizar a carga de parasitas ou até mesmo para sua completa erradicação em indivíduos em fase crônica. Bem como, sugerimos que o medicamento deve ser ofertado por maior tempo aos indivíduos chagásicos tanto na fase aguda quanto na fase crônica visto que apresenta melhores resultados com o prolongamento da terapia. Entrementes dissuadimos o tratamento durante o período gestacional visto que, pode induzir a diminuição das barreiras imunológicas maternas e/ou facilitar a migração parasitária para os tecidos placentários e fetais. / Chagas\' disease affect millions of people around the world and the benznidazole is the only drug for the treatment in Brazil, but it is used basically for the acute phase of the disease. Some researchers showed many benefits of benznidazole therapy in chronic phase like reduction of the progression of the disease. Therefore, the objective of this work was investigate the efficacy of treatment in both acute and chronic phase of this disease, as well as the effect of treatment of female mouse in chronic phase. Balb/c mice were infected with 2 x 105 trypomastigotes forms of RC strain of Trypanosoma cruzi by oral route. Mice in acute phase were treated with 50mg/Kg/day Benznidazole for 16 days. Some chronically-infected mice were treated by 3rd to 18th day of gestation with the same dose. Blood parasitaemias were determined by microscopic examination of tail vein blood amount. Tissues parasitism were determined by qPCR with two different mathematical forms, immunofluorescence confocal microscopy and histopathology by HE staining. Administration of benznidazole promoted decrease of parasites in spleen, liver and heart indicating the efficacy of the drug in the chronic phase. However in placenta and fetus the drug caused tissues parasites increased in the groups that received treatment. Therefore, we suggest that the treatment with benznidazole would be indicated to minimize the number of parasites or eliminate them in individuals in chronic phase. As well, we suggest that the drug should be offered for longer chagasic individuals to both acute and chronic phase because it shows better results with prolonged therapy. However, our results indicate the treatment do not be offered during pregnancy because it can induce the decrease of maternal immunological barriers and facilitate the parasite migration to the placental and fetal tissues.

O pênfigo foliáceo (PF) é a mais comum variante do Complexo Pênfigo na espécie canina. A enfermidade se caracteriza clinicamente pela presença de pústulas, histopatologicamente pela acantólise e imunologicamente pela presença de autoanticorpos (IgG) tanto na pele, quanto no soro sanguíneo dos doentes. Acredita-se que estes autoanticorpos estejam relacionados com a atividade do PF. O presente estudo visa avaliar a exeqüibilidade da reação de imunofluorescência indireta (IFI), método de determinação destes anticorpos séricos, para fins diagnósticos, além de verificar a eventual queda na titulação destes anticorpos séricos face à melhora clínica devido à terapia instituída. Para a IFI utilizou-se de fragmentos de coxim canino como substrato. Considerou-se positivo o padrão de fluorescência intercelular. Utilizou-se soros de 7 cães com diagnóstico histopatológico de PF, material este armazenado à uma temperatura de 70 oC. A IFI foi realizada, inicialmente, no momento de diagnóstico ou, no caso de um animal, de grave recidiva do quadro lesional. Quando de resultado positivo fez-se também a reação nos retornos subseqüentes. O grau de acometimento dos pacientes foi avaliado pelo PEFESI, em todas as ocasiões em que se realizou a IFI. Acompanhou-se estes animais por um período de 101 a 341 dias. Obteve-se positividade em 5 destes animais (71,4%). Dos animais positivos, àqueles com maior escore lesional ao PEFESI apresentaram títulos de maior magnitude. Houve queda nos títulos e gG em todos os animais paralelamente à diminuição do PEFESI, sendo que, em alguns casos, os títulos de IgG permaneceram detectáveis, em menor magnitude, mesmo quando do pleno controle da enfermidade. A IFI, portanto, é um método exeqüível tratando-se de PF canino, com 71% de positividade em relação ao exame histopatológico. O coxim canino se mostrou um substrato eficaz e a queda nos títulos de IgG paralelamente à atividade da doença útil para o monitoramento da atividade do PF. / Pemphigus foliaceus (PF) is the most common variant of Pemphigus Complex in dogs. The disease is characterized clinically by the presence of pustules, histopathologically by the acantholysis and immunologically by the presence of autoantibodies (IgG) in patients skin and blood serum. It is believed that these autoantibodies are related to the PF activity. The aim of this study is to evaluate the feasibility of indirect immunofluorescence (IIF), a determination method of these antibodies for diagnostic purposes, besides to identify a possible titration fall of serum antibodies with the clinical improvement due to therapy. For the IIFs, we used canine pawpads fragments as substrate. It was considered positive an intercellular fluorescence pattern. It was used sera from seven dogs with a histopathologic diagnosis of PF, this material was stored at 70 oC. The IIF was first performed at the time of diagnosis or, in the case of one animal, at a severe lesions relapse. When the IIF was positive it was also made at the subsequent animals evaluations. The clinical score was determined by PEFESI, all the times that the IFI was made. These animals were followed up by a 101-341 days period. Positivity was obtained in 5 of these animals (71.4%). Of the positive animals, those with the highest lesional score PEFESI showed greater titres. There was a decrease at the IgG titles in all animals in parallel with the PEFESI decrease, and in some cases, IgG titles remained detectable at a lesser magnitude, even with the full control of the disease. The IFI is, therefore, a feasible method in the case of canine PF, with 71% positivity in relation to histopathology. The canine pawpad proved to be an effective substrate and the IgG titers fall, in parallel with disease activity, useful for monitoring the Pf activity