La tesi è organizzata in 4 capitoli: -nel primo vengono brevemente riferite le patologie associate all’infezione da PCV2 con particolare riferimento all’iter diagnostico ed al ruolo rivestito dall’esame istologico e dalla identificazione dell’agente eziologico in situ contestualmente alle lesioni istologiche; -nel secondo viene presentato un iter diagnostico originale da applicare in condizioni di campo, qualora si voglia accertare la presenza del PCV2 nei tessuti dei prodotti di natimortalità/aborto del suino. In specifico si riferisce all’applicazione del protocollo in 2 aziende ed i risultati vengono analizzati per una revisione critica del protocollo impiegato; -nel terzo vengono presentati i risultati di un protocollo di infezione con PCV2 per via genitale tramite seme infetto. Scrofe convenzionali sono state sincronizzate per l’estro e fecondate con un’unica dose di seme PCV2 negativo alla PCR (gruppo controlli) o sperimentalmente esposto al PCV2 (gruppo infette). I risultati vengono analizzati in funzione delle ripercussioni che l’infezione precoce in gravidanza può produrre sulla scrofa (mancata gravidanza, ritorno in calore), sui feti e sugli invogli fetali. Viene stabilito il ruolo protettivo degli anticorpi circolanti al momento dell’infezione, stante l’evenienza che un basso titolo anticorpale si associa a viremia prolungata e maggiore numero di feti positivi al virus; -nel quarto viene presentato un esperimento sovrapponibile a quello riferito nel capitolo 3, però con la presenza anche di un gruppo di soggetti convenzionali vaccinati ed infettati con PCV2 durante la fecondazione artificiale usando seme sperimentalmente esposto al virus. Nella discussione dei risultati vengono enfatizzati 2 aspetti importanti nell’epidemiologia dell’infezione da PCV2: la eliminazione di virus è fortemente ridotta dalla vaccinazione, con conseguenze verosimilmente positive sulla circolazione del virus negli effettivi dell’allevamento; l’esposizione uterina è protetta dalla vaccinazione, stante la bassa percentuale di placente infette nel gruppo dei soggetti vaccinati rispetto a quelli non vaccinati e nei controlli. / The thesis is organized into 4 chapters: -in the first chapter, it is briefly overviewed the association of PCV2 with several diseases with particular emphasis to the diagnostic protocols and to the in situ identification of the virus in histological lesions; -in the second chapter, it is presented an original diagnostic protocol to be applied in field conditions, to check for the presence of PCV2 in piglet tissues obtained from stillbirth/abortion. It refers to the application of the protocol in 2 herds and the results are analyzed for a critical review of the used protocol; -in the third chapter, it is presented an experimental trial aimed to infect gilts during artificial insemination by PCV2 infected semen. Conventional gilts were synchronized for oestrus and inseminated with a single dose of semen PCV2 PCR-negative (control group) or experimentally exposed to PCV2 (infected group). The results are analyzed to evaluate the impact that infection in early pregnancy may have on the sow (no pregnancy, return to oestrus), foetuses and foetal membranes. It emphasizes the protective role of circulating antibodies at the time of infection, given the possibility that a low antibody titre is associated with prolonged viremia and increased number of PCV2 positive foetuses; -in the fourth chapter, it is presented a protocol similar to that of Chapter 3, but with the presence of a third group of animals: gilts vaccinated and infected with PCV2 using semen experimentally exposed to the virus. In the discussion 2 important aspects are emphasized: the shedding of the virus is greatly reduced by vaccination, with positive effects on the reduction of the circulation of the virus in the herds; uterine exposure is protected by vaccination, given the low percentage of infected placentas in the vaccinated group compared with not vaccinated and control groups.

In veterinary medicine, the ability to classify mammary tumours based on the molecular profile and also determine whether the immunophenotype of the regional lymph node and/or systemic metastases is equal to that of the primary tumor may be predictive on the estimation of the effectiveness of various cancer treatments that can be scheduled. Therefore, aims, developed as projects, of the past three years have been (1) to define the molecular phenotype of feline mammary carcinomas and their lymph node metastases according to a previous modified algorithm and to demonstrate the concordance or discordance of the molecular profile between the primary tumour and lymph node metastasis, (2) to analyze, in female dogs, the relationship between the primary mammary tumor and its lymph node metastasis based on immunohistochemical molecular characterization in order to develop the most specific prognostic-predictive models and targeted therapeutic options, and (3) to evaluate the molecular trend of cancer from its primary location to systemic metastases in three cats and two dogs with mammary tumors. The studies on mammary tumours, particularly in dogs, have drawn gradually increasing attention not exclusively to the epithelial component, but also to the myoepithelial cells. The lack of complete information on a valid panel of markers for the identification of these cells in the normal and neoplastic mammary gland and lack of investigation of immunohistochemical changes from an epithelial to a mesenchymal phenotype, was the aim of a parallel research. While investigating mammary tumours, it was noticed that only few studies had focused on the expression of CD117. Therefore, it was decided to further deepen the knowledge in order to characterize the immunohistochemical staining of CD117 in normal and neoplastic mammary tissue of the dog, and to correlate CD117 immunohistochemical results with mammary histotype, histological stage (invasiveness), Ki67 index and patient survival time.

This was a retrospective study including ninety samples of dogs with a histological diagnosis of intermediate grade cutaneous mast cell tumour (MCT). The objectives of the study were to validate Minichromosome Maintenance Protein 7 (MCM7) as a prognostic marker in MCTs and to compare the ability of mitotic index (MI), Ki67 and MCM7 to predict outcome. The median survival for the entire population was not reached at 2099 days. The mean survival time was 1708 days. Seventy-two cases were censored after a median follow up of 1136 days and eighteen dogs died for causes related to the MCT after a median of 116 days. For each sample MI, Ki67 and MCM7 were determined. The Receiver Operating Characteristic (ROC) curve was obtained for each prognostic marker to evaluate the performance of the test, expressed as area under the curve, and whether the published threshold value was adequate. Kaplan-Meier and corresponding logrank test for MI, Ki67 and MCM7 as binary variables was highly significant (P<0.0001). Multivariable regression analysis of MI, Ki67 and MCM7 corrected for age and surgical margins indicated that the higher risk of dying of MCT was associated with MCM7 > 0.18 (Hazard Ration [HR] 14.7; P<0.001) followed by MI > 5 (HR 13.9; P<0.001) and Ki67 > 0.018 (HR 8.9; P<0.001). Concluding, the present study confirmed that MCM7 is an excellent prognostic marker in cutaneous MCTs being able to divide Patnaik intermediate grade tumours in two categories with different prognosis. Ki67 was equally good confirming its value as a prognostic marker in intermediate grade MCTs. The mitotic index was extremely specific, but lacked of sensitivity. Interestingly, mitotic index, Ki67 and MCM7 were independent from each other suggesting that their combination would improve their individual prognostic value.

Made available in DSpace on 2014-06-11T19:27:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-24Bitstream added on 2014-06-13T19:36:16Z : No. of bitstreams: 1 brianezi_g_me_botfm.pdf: 2870031 bytes, checksum: 61bf38a3fd318eca76bd3e6eab217c9f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O teste do cometa é utilizado para estimativa de dano ao DNA nos protocolos de avaliação do risco toxicológico. É rápido, de baixo custo, de fácil realização, seguro e pode ser aplicado em diversos tecidos. Corantes fluorescentes são os mais empregados para o ensaio. É também descrita a utilização de sais de prata com vantagens operacionais, carecendo de sistemas automatizados validados para esta análise. Neste trabalho, desenvolveram-se dois algoritmos automatizados para a análise do cometa corado pela prata. Foram realizados ensaios do cometa para sete grupos com dois camundongos Balb/c machos cada, submetidos ao tratamento intraperitoneal com soro fisiológico (G1), e três doses crescentes de genotóxicos conhecidos: MNU (N-nitroso-N-metilurea) (G2 – 5 μg/g, G3 – 25 μg/g, G4 – 50 μg/g) e DEN (N-nitrosodietilamina) (G5 – 20 μg/g, G6 – 80 μg/g, G7 – 180 μg/g). As lâminas dos testes do cometa foram confeccionadas a partir do sangue total e coradas pela prata e SYBR Gold. As coradas pela prata foram analisados por três avaliadores e pelos algoritmos automatizados. Aqueles corados pela fluorescência foram analisados pelo software Comet IV®. Os avaliadores apresentaram alta correlação de suas estimativas. Os algoritmos se correlacionaram fortemente com a análise dos avaliadores, principalmente com os escores obtidos pela classificação visual. Houve alta concordância na avaliação da sua repetitividade. Os algoritmos apresentaram resultados semelhantes, não indicando superioridade entre si para a análise do teste. Todos os métodos avaliados se mostraram capazes de diferenciar as concentrações dos genotóxicos utilizados. Porém, os algoritmos desenvolvidos não apresentaram correlação com os resultados obtidos pelo sistema do teste do cometa corado pela fluorescência, sugerindo que os métodos de coloração discriminem diferentes estruturas... / The comet assay is used to estimate DNA damage in the protocols for toxicological risk assessment. It's fast, inexpensive, easily performed, safe and can be applied in most tissues. Fluorescent dyes are frequently used for testing. Silver salts have been reported with some operational advantages; however automated systems for its analysis were not sufficiently validated for this staining. In this study, we have developed two algorithms for automated analysis of the comet assay silver stained. The assay was performed for seven groups of two (Balb/c) mice each, treated with intraperitoneal saline (G1) and three different genotoxic doses: MNU (N-nitroso-N-methylurea) (G2 - 5 μg/g, G3 - 25 μg/g, G4 - 50 μg/g) and DEN (N-nitrosodiethylamina) (G5 - 20 μg/g, G6 - 80 μg/g, G7 - 180 μg/g). The slides of the comet assay were made with total blood and were stained with silver and SYBR Gold. The silver stained slides were analyzed by three evaluators and the automated algorithms. Those stained by fluorescence were analyzed through software Comet IV®. Evaluators showed high correlation of their estimates. Both algorithms were correlated strongly with the evaluators' analysis mainly on the scores obtained by visual classification. Moreover, their agreement was high when assessed by repetitivity. The algorithms showed similar results, demonstrating no superiority to each other for the analysis of the test. All methods evaluated proved able of differentiating genotoxic concentrations used. However, the algorithms were not correlated with the results obtained by the automated system of comet assay fluorescence stained, suggesting that the staining methods linkage different genomic structures or with different affinities. The automated algorithms developed proved valid for the direct analysis of comet assay silver stained, allowing their use in genotoxicity research

Orientador: Hélio Amante Miot / Banca: Paula Helena Ortiz Lima / Banca: Marco Antonio Rodrigues Fernandes / Resumo: O teste do cometa é utilizado para estimativa de dano ao DNA nos protocolos de avaliação do risco toxicológico. É rápido, de baixo custo, de fácil realização, seguro e pode ser aplicado em diversos tecidos. Corantes fluorescentes são os mais empregados para o ensaio. É também descrita a utilização de sais de prata com vantagens operacionais, carecendo de sistemas automatizados validados para esta análise. Neste trabalho, desenvolveram-se dois algoritmos automatizados para a análise do cometa corado pela prata. Foram realizados ensaios do cometa para sete grupos com dois camundongos Balb/c machos cada, submetidos ao tratamento intraperitoneal com soro fisiológico (G1), e três doses crescentes de genotóxicos conhecidos: MNU (N-nitroso-N-metilurea) (G2 - 5 μg/g, G3 - 25 μg/g, G4 - 50 μg/g) e DEN (N-nitrosodietilamina) (G5 - 20 μg/g, G6 - 80 μg/g, G7 - 180 μg/g). As lâminas dos testes do cometa foram confeccionadas a partir do sangue total e coradas pela prata e SYBR Gold. As coradas pela prata foram analisados por três avaliadores e pelos algoritmos automatizados. Aqueles corados pela fluorescência foram analisados pelo software Comet IV®. Os avaliadores apresentaram alta correlação de suas estimativas. Os algoritmos se correlacionaram fortemente com a análise dos avaliadores, principalmente com os escores obtidos pela classificação visual. Houve alta concordância na avaliação da sua repetitividade. Os algoritmos apresentaram resultados semelhantes, não indicando superioridade entre si para a análise do teste. Todos os métodos avaliados se mostraram capazes de diferenciar as concentrações dos genotóxicos utilizados. Porém, os algoritmos desenvolvidos não apresentaram correlação com os resultados obtidos pelo sistema do teste do cometa corado pela fluorescência, sugerindo que os métodos de coloração discriminem diferentes estruturas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The comet assay is used to estimate DNA damage in the protocols for toxicological risk assessment. It's fast, inexpensive, easily performed, safe and can be applied in most tissues. Fluorescent dyes are frequently used for testing. Silver salts have been reported with some operational advantages; however automated systems for its analysis were not sufficiently validated for this staining. In this study, we have developed two algorithms for automated analysis of the comet assay silver stained. The assay was performed for seven groups of two (Balb/c) mice each, treated with intraperitoneal saline (G1) and three different genotoxic doses: MNU (N-nitroso-N-methylurea) (G2 - 5 μg/g, G3 - 25 μg/g, G4 - 50 μg/g) and DEN (N-nitrosodiethylamina) (G5 - 20 μg/g, G6 - 80 μg/g, G7 - 180 μg/g). The slides of the comet assay were made with total blood and were stained with silver and SYBR Gold. The silver stained slides were analyzed by three evaluators and the automated algorithms. Those stained by fluorescence were analyzed through software Comet IV®. Evaluators showed high correlation of their estimates. Both algorithms were correlated strongly with the evaluators' analysis mainly on the scores obtained by visual classification. Moreover, their agreement was high when assessed by repetitivity. The algorithms showed similar results, demonstrating no superiority to each other for the analysis of the test. All methods evaluated proved able of differentiating genotoxic concentrations used. However, the algorithms were not correlated with the results obtained by the automated system of comet assay fluorescence stained, suggesting that the staining methods linkage different genomic structures or with different affinities. The automated algorithms developed proved valid for the direct analysis of comet assay silver stained, allowing their use in genotoxicity research / Mestre

Molecular profiling of Peripheral T-cell lymphomas not otherwise specified Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of tumors that the WHO classification basically subdivides into specified and not otherwise specified (NOS). In Western countries, they represent around 12% of all non-Hodgkin's lymphomas. In particular, PTCL/NOS is the commonest subtype, corresponding to about 60-70% of all T-cell lymphomas. However, it remains a complex entity showing great variety regarding either morphology, immunophenotype or clinical behavior. Specially, the molecular pathology of these tumors is still poorly known. In fact, many alteration were found, but no single genes were demonstrated to have a pathogenetic role. Recently, gene expression profiling (GEP) allowed the identification of PTCL/NOS-associated molecular signatures, leading to better understanding of their histogenesis, pathogenesis and prognostication. Interestingly, proliferation pathways are commonly altered in PTCLs, being highly proliferative cases characterized by poorer prognosis. In this study, we aimed to investigate the possible role in PTCL/NOS pathogenesis of selected molecules, known to be relevant for proliferation control. In particular, we analyzed the cell cycle regulators PTEN and CDKN1B/p27, the NF-kB pathway, and the tyrosin kinase PDGFR. First, we found that PTEN and p27 seem to be regulated in PTCL/NOS as in normal T-lymphocytes, as to what expression and cellular localization are concerned, and do not present structural abnormalities in the vast majority of PTCL/NOS. Secondly, NF-kB pathway appeared to be variably activated in PTCL/NOS. In particular, according to NF-kB gene expression levels, the tumors could be divided into two clusters (C1 and C2). Specially, C1 corresponded to cases presenting with a global down-regulation of the entire pathway, while C2 showed over-expression of genes involved in TNF signaling. Notably, by immunohistochemistry, we showed that either the canonical or the alternative NK-kB pathway were activated in around 40% of cases. Finally, we found PGDFRA to be consistently over-expressed (at mRNA and protein level) and activated in almost all PTCLs/NOS. Noteworthy, when investigating possible causes for PDGFRA deregulation, we had evidences that PDGFR over-expression is due to the absence of miR-152, which appeared to be responsible for PDGFRA silencing in normal T-cells. Furthermore, we could demonstrate that its aberrant activation is sustained by an autocrine loop. Importantly, this is the first case, to the best of our knowledge, of hematological tumor in which tyrosin kinase aberrant activity is determined by deregulated miRNA expression and autocrine loop activation. Taken together, our results provide novel insight in PTCL/NOS pathogenesis by opening new intriguing scenarios for innovative therapeutic interventions.

The project was developed into three parts: the analysis of p63 isoform in breast tumours; the study of intra-tumour eterogeneicity in metaplastic breast carcinoma; the analysis of oncocytic breast carcinoma. p63 is a sequence-specific DNA-binding factor, homologue of the tumour suppressor and transcription factor p53. The human p63 gene is composed of 15 exons and transcription can occur from two distinct promoters: the transactivating isoforms (TAp63) are generated by a promoter upstream of exon 1, while the alternative promoter located in intron 3 leads to the expression of N-terminal truncated isoforms (ΔNp63). It has been demonstrated that anti-p63 antibodies decorate the majority of squamous cell carcinomas of different organs; moreover tumours with myoepithelial differentiation of the breast show nuclear p63 expression. Two new isoforms have been described with the same sequence as TAp63 and ΔNp63 but lacking exon 4: d4TAp63 and ΔNp73L, respectively. Purpose of the study was to investigate the molecular expression of N-terminal p63 isoforms in benign and malignant breast tissues. In the present study 40 specimens from normal breast, benign lesions, DIN/DCIS, and invasive carcinomas were analyzed by immunohistochemistry and RT-PCR (Reverse Transcriptase-PCR) in order to disclose the patterns of p63 expression. We have observed that the full-length isoforms can be detected in non neoplastic and neoplastic lesions, while the short isoforms are only present in the neoplastic cells of invasive carcinomas. Metaplastic carcinomas of the breast are a heterogeneous group of neoplasms which exhibit varied patterns of metaplasia and differentiation. The existence of such non-modal populations harbouring distinct genetic aberrations may explain the phenotypic diversity observed within a given tumour. Intra-tumour morphological heterogeneity is not uncommon in breast cancer and it can often be appreciated in metaplastic breast carcinomas. Aim of this study was to determine the existence of intra-tumour genetic heterogeneity in metaplastic breast cancers and whether areas with distinct morphological features in a given tumour might be underpinned by distinct patterns of genetic aberrations. 47 cases of metaplastic breast carcinomas were retrieved. Out of the 47 cases, 9 had areas that were of sufficient dimensions to be independently microdissected. Our results indicate that at least some breast cancers are composed of multiple non-modal populations of clonally related cells and provide direct evidence that at least some types of metaplastic breast cancers are composed of multiple non-modal clones harbouring distinct genetic aberrations. Oncocytic tumours represent a distinctive set of lesions with typical granular cytoplasmatic eosinophilia of the neoplastic cells. Only rare example of breast oncocytic carcinomas have been reported in literature and the incidence is probably underestimated. In this study we have analysed 33 cases of oncocytic invasive breast carcinoma of the breast, selected according to morphological and immunohistochemical criteria. These tumours were morphologically classified and studied by immunohistochemistry and aCGH. We have concluded that oncocytic breast carcinoma is a morphologic entity with distinctive ultrastructural and histological features; immunohistochemically is characterized by a luminal profile, it has a frequency of 19.8%, has not distinctive clinical features and, at molecular level, shows a specific constellation of genetic aberration.

Made available in DSpace on 2014-06-11T19:23:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-13Bitstream added on 2014-06-13T19:09:41Z : No. of bitstreams: 1 madeira_slm_me_botfm.pdf: 1742994 bytes, checksum: 9e37f76228d67b01bef23b26f0e71b96 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O cigarro e o álcool contêm em suas composições substâncias tóxicas e carcinogênicas que se potencializam com a associação dos hábitos. O câncer da cabeça e pescoço contribui com 4 a 5 % do total de neoplasias malignas do organismo, das quais 30 a 40% situam-se na boca, 25% na laringe e 15% na faringe. Os homens são os mais afetados e o tipo histológico predominante é o carcinoma espinocelular, podendo ser precedido de lesões pré-neoplásicas. Pesquisas experimentais permitem a exposição de cada fator agressor separadamente, possibilitando avaliar os efeitos nocivos de cada um ou de sua associação. Estudar, em ratos, os efeitos da exposição crônica à fumaça de cigarro e à ingestão de etanol sobre as mucosas da língua e da faringe, por meio de análise histopatológica. Foram alojados em gaiolas durante 260 dias 40 ratos adultos Wistar subdivididos em quatro grupos de 10 animais: GI (Controle), ração e água ad libitum; GII (Etilismo), submetidos à ingestão de etanol a 30GL diluído em água destilada e ração ad libitum; GIII (Tabagismo) expostos à inalação de fumaça de 10 cigarros/dia, sete dias/semana durante 30 min. em câmara fechada contendo “aparelho de fumar”; GIV (Etilismo e Tabagismo) submetidos à ingestão de etanol a 30GL e à inalação de fumaça de cigarro, ração e água ad libitum. Após 260 dias os animais foram sacrificados, sendo removido em bloco o segmento de língua e faringe. Biopsias foram realizadas nesses sítios e fixadas em formalina 10% para estudo histopatológico. Nas análises deste utilizou-se escore semi-quantitativo de 0 a 3. Nas biópsias de língua, apenas dois animais de GI (controle) apresentaram aumento de número de vasos na lâmina própria. Nos animais de GII (Etilismo), GIII (Tabagismo), ou GIV (Etilismo e Tabagismo) os parâmetros mais pontuados foram: hiperplasia de células apicais... / Cigarettes and alcohol contain toxic and carcinogenic substances whose effect is potentiated when cigarette smoking and alcohol drinking are associated. Head and neck cancers account for 4-5 % of all malignant tumors, and are located in the mouth, larynx and pharynx in 30-40%, 25%, and 15% of the cases, respectively. Males are the most affected, and spinocellular carcinoma is the most common histological type that may be preceded by preneoplastic lesions. Experimental studies allow evaluating the adverse effects of exposure to different aggression agents both separately and in association. To study in rats, the effects of chronic exposure to cigarette smoke and the ingestion of ethanol on the mucosa of the tongue and pharynx by means of histopathology. Forty adult Wistar rats, kept in cages for 260 days, were allocated into four groups of 10 animals each: GI (Control), fed chow and water “ad libitum”; GII (Alcohol drinking), submitted to the ingestion of 30gl ethanol diluted in distilled water, and receiving chow “ad libitum”; GIII (Smoking) exposed to the smoke of 10 cigarettes/day, seven days/week for 30 min. in a closed chamber housing a “smoking machine”; G4 (Alcohol drinking and smoking) submitted to the ingestion of 30gl ethanol and cigarette smoke exposure, receiving food and water “ad libitum”. After 260 days, the animals were sacrificed, and the tongue/pharynx segment was removed by block dissection. Biopsy specimens were fixed in 10% formalin for histopathological analysis using a 0-3 semiquantitative score system. Tongue biopsies showed an increased number of vessels in the lamina propria in only two GI (control) animals. In GII (Alcohol drinking), GIII (Smoking), or GIV (Alcohol drinking and smoking) the parameters receiving the highest scores were: apical cell hyperplasia (GII-60%, GIII-30%,GIV-20%) and basal cell hyperplasia (GII-60%, GIII-40%)... (Complete abstract click electronic access below)

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Introduction: Visceral leishmaniasis is a serious public health problem in several parts of the developing world. In leishmaniasis, protection and healing correlate with the development of Th1 immune response, and IFN-&#947; is considered a key molecule in this response. In contrast, the Th2 response results in disease progression. It is noteworthy that after contact with Leishmania the production of IFN-&#947; differs between healthy individuals. Some of them are high IFN-&#947; producer (HIFN-&#947;P), while others respond with low IFN-&#947; producer (LIFN-&#947;P). This has been observed in the in vitro response to several species of Leishmania, as well as in the early phase of the disease in patients with leishmaniasis. The activation of T lymphocytes can be modulated by immunomodulatory receptors present on the surface of lymphocytes and antigen presenting cells (APCs). Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein that promotes Th2 differentiation. Evidences support the involvement of this molecule in the immune response against parasites. Objective: To evaluate the role of SLAM signaling pathway in the modulation of immune response of peripheral blood mononuclear cells (PBMC) from individuals low IFN-&#947; producers (LP) against Leishmania chagasi. Methodology: Healthy individualsâ PBMC were stimulated in vitro with L. chagasi and monoclonal anti-SLAM (&#945;-SLAM), in the presence or not of inflammatory cytokines (rIFN-&#947; or rIL-12). The supernatants of the cultures were analyzed by ELISA for the determination of IFN-&#947; and interleukin 10 (IL-10) concentrations. Results: Upon stimulation of PBMC with L. chagasi, the blocking of the SLAM signaling pathway with &#945;-SLAM did not affect the synthesis of IFN-&#947; and IL-10, regardless of treatment with rIL-12. However, after rIFN-&#947; treatment of antigen stimulated cells it occurred a download of IL-10 synthesis and upload IFN-&#947; secretion, regardless of the blockade of SLAM signaling pathway. Conclusions: The blocking of SLAM signaling pathway with &#945;-SLAM at the concentration of 10&#956;g/mL does not interfere significantly in the IFN-&#947; and IL-10 production of PBMC from individuals LP stimulated with Leishmania chagasi promastigotes. Treatment of PBMC with rIFN-&#947; is able to induce a reduction of IL-10 and an increment of IFN-&#947; in the supernates cultures, whereas treatment with rIL-12 enhanced IFN-&#947; production, but does not interfered with IL-10. It is necessary to make further studies to better understand the role of the SLAM signaling pathway in the immune response of LP against Leishmania chagasi. / IntroduÃÃo: Nas leishmanioses, proteÃÃo e cura se correlacionam com o desenvolvimento de resposta imune tipo Th1, e IFN-&#947; à considerada uma molÃcula chave nesta resposta. Ao contrÃrio, a resposta Th2, resulta na progressÃo da doenÃa. Vale ressaltar que a produÃÃo de IFN-&#947;, apÃs o contato com a Leishmania, difere entre indivÃduos sadios, alguns apresentam alta produÃÃo de IFN-&#947; (AP), enquanto outros respondem com baixa produÃÃo (BP). Isto tem sido observado com diversas espÃcies de Leishmania, assim como na infecÃÃo natural, na fase inicial da doenÃa. A ativaÃÃo do linfÃcito T pode ser modulada por coestimuladores, presentes na superfÃcie de linfÃcitos e nas APCs, a exemplo da molÃcula sinalizadora na ativaÃÃo de linfÃcitos T (SLAM). EvidÃncias apontam para o envolvimento dessas molÃculas na regulaÃÃo da resposta imunolÃgica. Objetivo: Avaliar o papel da via de sinalizaÃÃo de SLAM na modulaÃÃo da resposta imune em cÃlulas mononucleares de sangue perifÃrico (CMSP) de indivÃduos BP, frente à estimulaÃÃo in vitro por Leishmania chagasi. Metodologia: CMSP de indivÃduos sadios BP foram estimuladas in vitro com L. chagasi na ausÃncia ou na presenÃa do anticorpo monoclonal anti-SLAM (&#945;-SLAM), com ou sem tratamento por citocinas proinflamatÃrias (rIFN-&#947; ou rIL-12). Os sobrenadantes das culturas foram analisados por ELISA para IFN-&#947; e interleucina (IL)-10. Resultados: Sob estimulaÃÃo de L. chagasi, o bloqueio da via SLAM nÃo modificou a sÃntese de IFN-&#947; e IL-10, independente do tratamento com rIL-12. No entanto, o tratamento com rIFN-&#947; reduziu a sÃntese de IL-10 e elevou a secreÃÃo de IFN-&#947; endÃgeno, independente do bloqueio da via SLAM. ConclusÃes: O bloqueio da via SLAM, com adiÃÃo de &#945;-SLAM (10Âg/mL), nÃo interfere significativamente na produÃÃo de IFN-&#947; e IL-10. O tratamento das CMSP com rIFN-&#947; à capaz de induzir a reduÃÃo da produÃÃo de IL-10 e o aumento de IFN-&#947; de forma significativa, enquanto que o tratamento com rIL-12 aumenta a produÃÃo de IFN-&#947;, mas nÃo interfere na produÃÃo de IL-10 dos indivÃduos baixos produtores. Faz-se necessÃrio ampliar o estudo da aÃÃo imunomoduladora da SLAM frente à Leishmania chagasi, para um melhor entendimento do papel desta via de sinalizaÃÃo na resposta imunolÃgica dos BP.

Immune response to M leprae is determinant to leprosy evolution and NK cells are important constituents of the innate immune response and can contribute to the adaptive response activation. The aim of this study was to evaluate the immunoexpression of NK cells markers CD16+ and CD56+ in skin lesions of patients with leprosy, before any treatment. A total of 54 skin biopsies of patients with leprosy were evaluated by immunohistochemistry using rabbit anti-human CD16a (clone SP189) from Spring Bioscience and mouse anti-human CD56 (clone 123C3) from DakoÂ, together with Envision Flex kit from DakoÂ. Nineteen cases were from tuberculoid (TT) clinical form, 14 were borderline (BT, BB, BL) and 20 were lepromatous (LL), while one case was from indeterminate form (I). Among them 31 (57,4%) were from male gender and 30 (55,5%) presented positive baciloscopy. The samples were collected at the âCentro de Dermatologia Dona LibÃniaâ in Fortaleza, CearÃ, Brazil, and were analysed at the âLaboratÃrio de TÃcnicas Cito-histopatolÃgicas Especiais do Departamento de Patologia e Medicina Legal da Universidade Federal do CearÃâ. Positive controls for the CD16 and CD56 expression were lung tissue and carcinoid tumor, respectively. Fisher, Chi-square, Kruskal-Wallis and Mann-Whitney tests were used to statistical analysis and a p value of <0.05 was considered significant. Differences were observed when expression of CD16+ cells was compared between tuberculoid and borderlines forms (p=0.0329), as also among gender comparisons (p=0.0129). The CD16+ cells in the three different clinical forms groups were also statistically different achieving a p value of 0.0085, and showing a higher median of expression of CD16+ in the TT group. These data suggests that a protective immune response against M. leprae in leprosy requires a balance between NK cells subgroups (CD16+ and CD56+) and that the tuberculoid form presents higher levels of CD16+ cells expression when compared to borderlines and LL forms. In addition, the study suggests that there are possible hormonal or genetic influences on the distribution of NK cell function markers. / A resposta imunolÃgica ao Mycobacterium leprae à componente determinante na evoluÃÃo da doenÃa. As cÃlulas Natural Killer (NK) sÃo constituintes importantes da resposta imune inata, podendo participar da ativaÃÃo da resposta adaptativa. Este estudo objetivou avaliar a imunoexpressÃo de marcadores de cÃlulas NK, CD16+ e CD56+, em lesÃes cutÃneas de pacientes com hansenÃase antes do tratamento, por imunoistoquÃmica. Foram analisadas 54 biÃpsias cutÃneas de lesÃes de paciente com hansenÃase. Sendo 19 casos da forma clÃnica tuberculÃide (TT), 14 da forma borderline (BT, BB e BL) e 20 da forma lepromatosa (LL) e 1 caso da forma clÃnica indeterminada (I). Destes pacientes 31 (57,4%) eram do gÃnero masculino, 30 (55,5%) eram baciloscopia positiva. As amostras foram provenientes do Centro de Dermatologia Dona LibÃnia e analisadas no LaboratÃrio de TÃcnicas Cito-histopatolÃgicas Especiais do Departamento de Patologia e Medicina Legal da Universidade Federal do CearÃ. Para a imunistoquÃmica foram utilizados os anticorpos monoclonais primÃrios: rabbit anti-human CD16a (clone SP189) da Spring Bioscience e o mouse anti-human CD56 (clone 123C3) da DakoÂ, juntamente com o kit Envision Flex da DakoÂ. Os controles positivos para CD16 e CD56 foram obtidos a partir de secÃÃes de tecidos de pulmÃes e tumor carcinÃide, respectivamente. Os testes de Fisher, Qui-quadrado, Kruskal-Wallis e Mann-Whitney foram utilizados para anÃlise estatÃstica, sendo considerado significante quando p<0,05. A comparaÃÃo entre a imunoexpressÃo de CD16+ em linfÃcitos teciduais da forma tuberculÃide em relaÃÃo Ãs formas borderlines apresentou diferenÃas estatÃsticas significantes (p=0,0329), como tambÃm em relaÃÃo ao gÃnero (p=0,0129). AtravÃs do teste Kruskal-Wallis, comparando os trÃs grupos das formas clinicas da doenÃa em relaÃÃo ao nÃmero de cÃlulas imunomarcadas com CD16+, foi observado que a mediana do grupo tuberculÃide foi maior, conferindo p=0,0085. Pode-se concluir que o estudo sugere que uma resposta imunolÃgica protetora na hansenÃase requer um equilÃbrio entre subgrupos de cÃlulas NK CD16+ e CD56+, e que a forma tuberculoide cursa com predomÃnio dessas cÃlulas NK citotÃxicas quando comparadas Ãs formas borderline e lepromatosas. Em adiÃÃo, o estudo sugere que hà possÃveis influÃncias hormonais ou genÃticas na distribuiÃÃo destes marcadores de funÃÃo de cÃlulas NK.