Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 4 uppsatser.

Στα έντομα, η κυτταροφαγία, διεργασία ανάλογη με αυτή των θηλαστικών, είναι μια σημαντική ανοσολογική απόκριση στην εισβολή παθογόνων και ρυθμίζεται από μια σειρά σηματοδοτικών μορίων. Στη μύγα της Μεσογείου έχει βρεθεί ότι συμμετέχουν οι κινάσες FAK, Src, MAPK και ο μεταγραφικός παράγοντας Elk-1. Στην εργασία αυτή, δείχτηκε με ανοσοκατακρήμνιση και συνεστιακή μικροσκοπία ότι η φωσφορυλιωμένη μορφή του Elk-1 συμπλοκοποιείται και συνεντοπίζεται μόνο με τη φωσφορυλιωμένη μορφή της FAK στη θέση Tyr925. Με ανάλογα πειράματα διαπιστώθηκε ότι οι κινάσες JNK και p38 συμπλοκοποιούνται και συγκατακρημνίζονται με την FAK, χωρίς όμως να συνεντοπίζονται. Με τον ίδιο τρόπο διαπιστώθηκε ότι η JNK και η p38 συμπλοκοποιούνται με την ERK1/2, χωρίς όμως να συνεντοπίζονται. Προφανώς τα μόρια αυτά δεν βρίσκονται σε άμεση επαφή μεταξύ τους και συμπλοκοποιούνται είτε μέσω της FAK, είτε μέσω τρίτων μορίων και όλα μαζί προσδένονται στην FAK. Τα φαγοκύτταρα των θηλαστικών, μακροφάγα και ουδετερόφιλα, καθώς και τα αιμοκύτταρα των εντόμων, παράγουν δραστικές μορφές οξυγόνου κατά την κυτταροφαγία, οι οποίες δρουν ως δεύτερα μηνύματα. Με κυτταρομετρία ροής διαπιστώθηκε ότι η E. coli επάγει τη σύνθεση Η2Ο2 από τα αιμοκύτταρα της μύγας της Μεσογείου. Ο ρυθμιστικός ρόλος του Η2Ο2 επιβεβαιώθηκε με τη χρήση αναστολέων των ενζύμων παραγωγής Ο2- και Η2Ο2, όπου παρατηρήθηκε αύξηση της κυτταροφαγίας. Με πειράματα ανοσοκατακρήμνισης, ανοσοαποτύπωσης και ανοσοφθορισμού φάνηκε ότι το ένζυμο σύνθεσης του Η2Ο2, η δεσμουτάση του υπεροξεικού ανιόντος (SOD), υπάρχει στο κυτταρόπλασμα και στην πλασματική μεμβράνη. Η αποσιώπηση της SOD με siRNA, αύξησε την κυτταροφαγία. Με ανοσοαποτύπωση διαπιστώθηκε ότι η αναστολή της παραγωγής του Η2Ο2 αύξησε τη φωσφορυλίωση της ERK1/2. Τέλος, με συνεστιακή μικροσκοπία φάνηκε ότι η SOD δεν συνεντοπίζεται με τη β υπομονάδα των ιντεγκρινών. Φαίνεται λοιπόν το Η2Ο2 να εμποδίζει την κυτταροφαγία μέσω ελέγχου της φωσφορυλίωσης της ERK1/2. / Phagocytosis is an important innate immune response against pathogen, with similar mechanisms in insects and mammals and is regulated by many different signalling molecules. In medfly Ceratitis capitata, focal adhesion kinase (FAK), Src, MAP kinases and Elk-1 transcription factor regulate this process. Co-immunoprecipitation and confocal microscopy analysis showed that pTyr925FAK associates and co-localizes with pElk-1, during phagocytosis of E. coli and S. aureus, by medfly haemocytes. Moreover, the physical association between JNK and p38 MAP kinases with FAK, was confirmed by immunoprecipitation, but confocal analysis revealed no co-localisation. Similar experiments for JNK and p38 with ERK1/2, revealed an association between JNK, p38 and ERK1/2 and no co-localisation. Apparently, these molecules do not interact directly but they appear to associate with each other indirectly, via FAK molecule or a third molecule and thus all together associate with FAK. Hydrogen peroxide (Η2Ο2) participates as a second messenger in cell signalling in either macrophages and polymorphonuclear cells or insect haemocytes. In this work, the role of Η2Ο2 was investigated, in E. coli phagocytosis by the medfly haemocytes. Block of H2O2 synthesis by specific enzymic inhibitors, namely N-ethylmaleimide (ΝΕΜ) for NADPH oxidase and diethyldithiocarbamate (DDC) for SOD, resulted in the increase of E. coli phagocytosis. Immunoblot analysis, flow cytometry and confocal microscopy, revealed the constitutive expression of SOD, in the medfly haemocytes. Phagocytosis increased by small interfering RNA (siRNA) for SOD, revealing the active involvement of SOD and Η2Ο2. Immunoblot analysis showed an increase of the ERK1/2 phosphorylation, in the presence of the above H2O2 synthesis enzymic inhibitors. In addition, confocal microscopy showed no co-localization of SOD with β integrin subunit. It appears that SOD participates in the regulation of bacterial phagocytosis, due to involvement of the produced Η2Ο2 in the differential phosphorylation of MAP kinases.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-11-30T20:45:01Z No. of bitstreams: 1 José Geraldo Bomfim Lima Efeito da autofagia... 2009.pdf: 32365208 bytes, checksum: 34504be1e8f6483c263480600d8e1880 (MD5) / Made available in DSpace on 2012-11-30T20:45:01Z (GMT). No. of bitstreams: 1 José Geraldo Bomfim Lima Efeito da autofagia... 2009.pdf: 32365208 bytes, checksum: 34504be1e8f6483c263480600d8e1880 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A autofagia vem sendo alvo de estudos que demonstram sua participação em infecções por diversos patógenos intracelulares. A depender do patógeno, a autofagia pode facilitar a sobrevivência intracelular do patógeno ou pode funcionar como controle da infecção pela célula hospedeira. Pouco se sabe sobre a participação da autofagia na infecção por Leishmania. Foi demonstrado que o vacúolo parasitóforo induzido por L mexicana adquire nutrientes citosólicos por microautofagia. Além disso, recentemente foi demonstrado que a indução de autofagia promove aumento da carga parasitária de L. amazonensis em macrófagos infectados. Esses dados sugerem a participação do processo autofágico no estabelecimento da infecção por Leishmania, como um mecanismo que favorece a sobrevivência intracelular do parasito. Assim, o objetivo desse estudo foi determinar a influência da autofagia na infecção, in vitro, de macrófagos de camundongos CBA/J por L. amazonensis. Macrófagos foram induzidos à autofagia por duas formas, fisiológica ou farmacológica, após ou antes da infecção por L. amazonensis ou exposição a partículas de levedo ou zimosan. O percentual de infecção e de fagocitose foi estimado. Os resultados mostram que a indução de autofagia, após a infecção, não altera o percentual de macrófagos infectados, mas promove o aumento na carga parasitária de macrófagos infectados por L. amazonensis. Além disso, a prévia indução de autofagia promove a inibição da capacidade fagocítica do macrófago murino. Estudos adicionais serão realizados no intuito de esclarecer os mecanismos pelos quais a indução de autofagia favorece a infecção por L. amazonensis e altera a capacidade fagocítica do macrófago murino / Recently, studies to delineate the participation of autophagy in intracellular pathogen infections have been performed. Dependent on pathogen infection, autophagy can facilitate microorganism intracellular survival or can control pathogen infection by host cell. Few works evaluated the role of autophagy in Leishmania infection. Recently, it was demonstrated that L mexicana-'mduced parasitophorous vacuoles acquire cytosolic nutrients by microautophagy. Additionally, it was also demonstrated that autophagy promotes enhancement of L. amazonensis burden on infected cells. Taken together, these data suggest the involvement of autophagic process on Leishmania infection, as a mechanism that favors parasite survival. The present work intent to determine the influence of autophagy in L. amazonensis infection of CBA/J macrophages in vitro. Autophagy was induced after and before L. amazonensis infection or particle addition to macrophage cultures. The percentage of infection and particle phagocytosis was estimated. The results show that autophagy induction after infection does not influence the percentage of L. a/rjazonens/s-infected cells, but enhances L. amazonensis burden on infected cells. In addition, previous autophagy induction inhibited macrophage phagocytic capacity. Further studies will be performed to understand the mechanisms involved in autophagy effect on L. amazonensis infection and on macrophage phagocytic capacity.

Made available in DSpace on 2015-04-09T12:28:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-03Bitstream added on 2015-04-09T12:47:49Z : No. of bitstreams: 1 000814427.pdf: 485988 bytes, checksum: 4d7a0c4b4d27bc7139fd4331cc293653 (MD5) / Estudos sobre os possíveis efeitos de beta-glucanos sobre a resposta imune têm sido realizados há muito tempo, pois a imunomodulação que eles podem causar pode auxiliar na resistência a doenças e, até mesmo, tumores. Assim sendo, foram realizados dois experimentos para avaliar a ação de um beta-glucano derivado da parede celular de levedura sobre parâmetros imunológicos na espécie canina. O experimento 1 avaliou a ação ex vivo do composto, leucócitos caninos receberam 3 doses de beta-glucano e foi encontrado que a substância estimulou, de maneira dose-dependente, a produção de espécies reativas do oxigênio e nitrogênio, em neutrófilos e monócitos. O experimento 2 estudou a ação da substância sobre parâmetros imunes de cadelas submetidas a uma situação de imunossupressão: anestesia e cirurgia de castração. Foram utilizadas 28 cadelas adultas de diferentes raças e divididas em três grupos: controle (CT – n=10), suplementadas com 0,1% de beta-glucano na dieta extrusada (BG-E – n=9) e suplementadas com a mesma dose, mas administrada em cápsulas (BG-C – n=9). As avaliações foram realizadas em quatro períodos, 14 dias antes da cirurgia, pré-operatório imediato, pós-operatório imediato e 14 dias após a cirurgia. Os testes realizados para se investigar os efeitos deste nutracêutico foram avaliação hematológica, imunofenotipagem de leucócitos periféricos, avaliação de fagocitose de leucócitos periféricos, dosagem de citocinas em sobrenadante de cultura de células mononucleares, determinação da produção de intermediários reativos do oxigênio (H2O2) e nitrogênio (NO), e dosagem de proteína-C reativa sérica. O tratamento BG-C levou a uma maior porcentagem de fagocitose de monócitos. Apenas as cadelas do grupo controle tiveram uma tendência a aumento da concentração de proteína C-reativa após a cirurgia. O procedimento cirúrgico foi capaz de alterar o número de células e função ... / Studies evaluating the effects of beta-glucans on the immune response have been made long ago; because of the immunemodulation that they cause can lead to increase resistance to infections and even to tumors. Thus, we conducted two experiments to evaluate the action of a yeast-derived beta-glucan in dogs. In the experiment 1 the ex vivo action was studied, canine leukocytes were treated with 3 different doses of beta-glucan and the results showed that the production of oxygen and nitrogen reactive species by neutrophils and monocytes was stimulated in a dose-dependent manner. In the experiment 2 the action of beta-glucan on immune parameters of bitches undergoing ovary-hysterectomy was evaluated, as a situation of immunosuppression. Twenty-eight bitches of different breeds were divided into three groups: control (CT – n=10), 0.1% beta-glucan in the extruded diet (BG-E – n=9) and supplemented with the same dose, but administered in capsules (BG-C – n=9). Evaluations were performed in four periods, 14 days before the surgery, immediate preoperative and postoperative and 14 days after the surgery. The tests performed included complete blood count, evaluation of leukocyte subsets, phagocytosis test, production of cytokines in supernatant of cell culture, production of oxygen and nitrogen reactive species and serum C-reactive protein. BG-C treatment lead to higher percentage of phagocytosis by monocytes. Only the control group tended to increase the concentration of C-reactive protein after the surgery. Surgical procedure was capable of changing the number of cells and their function. In conclusion, beta-glucan acted on the immunity considering the presented models

Le développement des organismes multicellulaires, la morphologie de leurs organes, la formation de connections nerveuses ou même la réponse immunitaire conduisent à la formation de cellules dont la mort a été programmée, ces cellules sont en apoptose. Les corps apoptotiques générés sont éliminés par phagocytose, que l'on peut ainsi considérer comme l'étape ultime du programme apoptotique. Cette élimination, assurée soit par des phagocytes professionnels ou des cellules environnantes est très efficace et peu de cellules en apoptose sont observées dans les différents tissus et organes. Deux familles de protéines, ELMO et DOCK, sont connues pour être au centre d'une voie de signalisation impliquée dans l'activation de la petite GTPase Rac et le remodelage du cytosquelette d'actine permettant l'internalisation de corps apoptotiques, mais la manière dont ELMO est activée par la reconnaissance des corps apoptotique reste largement méconnue. Récemment, ELMO a été identifiée comme cible de la kinase hématopoïétique Hck. La phosphorylation d'ELMO constitue un signal d'activation de la voie de phagocytose. Nous avons pu montrer que les domaines N et C-terminaux d'ELMO sont impliqués dans l'interaction avec le domaine SH3 de Hck. Contrairement au SH3 de DOCK, l'interaction du domaine C-terminal avec le SH3 de Hck est strictement dépendante de la présence du polyproline. Les données obtenues sur les cellules transfectées suggère l'interaction in cellulo du motif polyproline avec le domaine SH3 de Hck. La différence dans le comportement du domaine C-terminal d'ELMO avec les deux SH3 nous a mené à identifier un complexe ternaire entre les trois protéines. Le motif polyproline d'ELMO est adjacent à l'une des 5 tyrosines phosphorylées par Hck, la tyrosine 720, nos résultats montrent que la phosphorylation de cette tyrosine défavorise l'interaction entre le domaine C-terminal et le SH3 de Hck. Cette phosphorylation pourrait être à l'origine d'un effet régulateur de Hck sur le complexe ELMO/DOCK. / Multicellular organisms development, tissue organization and morphogenesis, nerve connection network or even immune response lead to programmed cell death or apoptosis. Phagocytosis is the mean for the engulfment of the apoptotic corpses and can thus be considered as the final step of the apoptotic pathway. Both non-professional and professional phagocytic cells are implicated to achieve an efficient clearance of the apoptotic cells and debris, and very little dead cells are actually present in tissues under normal circumstances, preventing secondary necrosis and further inflammation. Two family of proteins, ELMO and DOCK have been identified as major characters in the signaling pathway leading to the activation of the small GTPase Rac, and actin remodeling, that eventually leads to engulfment. Nevertheless, very little is yet known about the upstream events leading to ELMO activation. Recently, ELMO has been characterized as a target for the hematopoietic cell kinase Hck, and consequent phosphorylation participates in ELMO activation. In the course of the present work, we demonstrated that both N- and C-terminal domains of ELMO are binding the SH3 domain of Hck. In contrast with the SH3 domain of DOCK, the SH3 domain of Hck binding to the C-terminal of ELMO is strictly dependent upon the polyproline motif. Our data obtained using transfected cells, further suggest the polyproline dependent interaction also occurs in cells. Taking into account the differential behavior of both SH3 domains from DOCK and Hck we next demonstrated a ternary complex formation between ELMO and both partners in vitro. Finally, we investigated the possible role for the Hck phosphorylation of Y720 located at the edge of the polyproline motif of ELMO and demonstrated that this phosphorylation clearly increases the Kd of ELMO/Hck interaction suggesting this phosphorylation could be part of a regulation loop in Hck dependent ELMO activation.

Orientador: Aulus Cavalieri Carciofi / Coorientador: Hélio José Montassier / Banca: Mirela Tinucci Costa / Banca: Silvio Luis de Oliveira / Banca: Lucia Helena Faccioli / Banca: Ricardo Souza Vasconcellos / Resumo: Estudos sobre os possíveis efeitos de beta-glucanos sobre a resposta imune têm sido realizados há muito tempo, pois a imunomodulação que eles podem causar pode auxiliar na resistência a doenças e, até mesmo, tumores. Assim sendo, foram realizados dois experimentos para avaliar a ação de um beta-glucano derivado da parede celular de levedura sobre parâmetros imunológicos na espécie canina. O experimento 1 avaliou a ação ex vivo do composto, leucócitos caninos receberam 3 doses de beta-glucano e foi encontrado que a substância estimulou, de maneira dose-dependente, a produção de espécies reativas do oxigênio e nitrogênio, em neutrófilos e monócitos. O experimento 2 estudou a ação da substância sobre parâmetros imunes de cadelas submetidas a uma situação de imunossupressão: anestesia e cirurgia de castração. Foram utilizadas 28 cadelas adultas de diferentes raças e divididas em três grupos: controle (CT - n=10), suplementadas com 0,1% de beta-glucano na dieta extrusada (BG-E - n=9) e suplementadas com a mesma dose, mas administrada em cápsulas (BG-C - n=9). As avaliações foram realizadas em quatro períodos, 14 dias antes da cirurgia, pré-operatório imediato, pós-operatório imediato e 14 dias após a cirurgia. Os testes realizados para se investigar os efeitos deste nutracêutico foram avaliação hematológica, imunofenotipagem de leucócitos periféricos, avaliação de fagocitose de leucócitos periféricos, dosagem de citocinas em sobrenadante de cultura de células mononucleares, determinação da produção de intermediários reativos do oxigênio (H2O2) e nitrogênio (NO), e dosagem de proteína-C reativa sérica. O tratamento BG-C levou a uma maior porcentagem de fagocitose de monócitos. Apenas as cadelas do grupo controle tiveram uma tendência a aumento da concentração de proteína C-reativa após a cirurgia. O procedimento cirúrgico foi capaz de alterar o número de células e função ... / Abstract: Studies evaluating the effects of beta-glucans on the immune response have been made long ago; because of the immunemodulation that they cause can lead to increase resistance to infections and even to tumors. Thus, we conducted two experiments to evaluate the action of a yeast-derived beta-glucan in dogs. In the experiment 1 the ex vivo action was studied, canine leukocytes were treated with 3 different doses of beta-glucan and the results showed that the production of oxygen and nitrogen reactive species by neutrophils and monocytes was stimulated in a dose-dependent manner. In the experiment 2 the action of beta-glucan on immune parameters of bitches undergoing ovary-hysterectomy was evaluated, as a situation of immunosuppression. Twenty-eight bitches of different breeds were divided into three groups: control (CT - n=10), 0.1% beta-glucan in the extruded diet (BG-E - n=9) and supplemented with the same dose, but administered in capsules (BG-C - n=9). Evaluations were performed in four periods, 14 days before the surgery, immediate preoperative and postoperative and 14 days after the surgery. The tests performed included complete blood count, evaluation of leukocyte subsets, phagocytosis test, production of cytokines in supernatant of cell culture, production of oxygen and nitrogen reactive species and serum C-reactive protein. BG-C treatment lead to higher percentage of phagocytosis by monocytes. Only the control group tended to increase the concentration of C-reactive protein after the surgery. Surgical procedure was capable of changing the number of cells and their function. In conclusion, beta-glucan acted on the immunity considering the presented models / Doutor

Orientadores: Renata de Oliveira Mattos-Graner, Edgard Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-10T08:12:26Z (GMT). No. of bitstreams: 1 Negrini_ThaisdeCassia_M.pdf: 1172330 bytes, checksum: 91a509113d532e152ab6ac6d4232f85f (MD5) Previous issue date: 2008 / Resumo: Streptococcus mutans é o principal patógeno da cárie dentária. Antígenos (Ags) secretados ou associados à superfície celular de S. mutans participam deste processo e incluem a proteína ligante de glucano B (GbpB), a qual pode influenciar na susceptibilidade à infecção por S. mutans. Estudos recentes indicam que a expressão de antígenos de superfície reconhecida por fagócitos é controlada por sistemas de dois componentes (SDC) em espécies relacionadas a S. mutans. O objetivo deste projeto foi investigar o efeito de variações na produção de GbpB e a influência de dois SDC, Cov (de control of virulence) e Vic (de virulence control), no padrão de internalização de S.mutans por macrófagos de camundongos. Foram avaliadas 10 cepas clínicas distintas previamente caracterizadas quanto ao padrão de síntese de GbpB e quatro mutantes knock-out de dois SDC: dois mutantes covR- e dois mutantes vicH-. Para isto, macrófagos murinos da linhagem Balb/c foram cultivados em meio RPMI e expostos a diferentes cepas de S. mutans. Os níveis de internalização bacteriana por macrófagos foram determinados através da contagem de macrófagos com bactérias internalizadas, com auxílio de microscópio óptico. Os mecanismos de internalização foram caracterizados em ensaios semelhantes com macrófagos previamente tratados com inibidores específicos dos processos de fagocitose, macropinocitose e endocitose mediada por clatrina e em análises de microscopia eletrônica de transmissão. Não houve diferença estatisticamente significante nos níveis de internalização das cepas que variavam quanto à produção de GbpB. Os mutantes vicH- foram significativamente menos internalizados quando comparados às cepas selvagens e aos mutantes covR. As análises de MET e ensaios com inibidores de processos específicos de internalização indicam que, na ausência de opsoninas, S. mutans pode ser internalizado por diferentes mecanismos incluindo-se fagocitose, macropinocitose e endocitose mediada por clatrina. Esses dados poderão auxiliar na compreensão dos mecanismos de captação e processamento de S. mutans por células apresentadoras de antígenos e dos fatores que influenciam no padrão de resposta imune adaptativa a estes microrganismos / Abstract: Streptococcus mutans are the main pathogens of dental caries. Antigens (Ags) secreted or associated with the bacterial cell surface may influence in the susceptibility to infection by S. mutans. These include the virulence protein Glucan-binding protein B (GbpB). Recent studies indicate that the expression of surface antigens recognized by phagocytes is controlled by systems of two components (TCS, Two Component System) in several streptococci species. The objective of this project was to investigate the effect of variations in the production of GbpB and the influence of the two-component systems (TCS), Cov and Vic, in the pattern of internalization of S. mutans by macrophages from mice under the absence of opsonins. Thus, levels of bacterial internalization by macrophages were evaluated in 10 different clinical strains of S. mutans previously characterized regarding the patterns of GbpB production, and in four knockout mutants of the TCS, two covR- mutants and two vicH- mutants. To this purpose, murine macrophages of the lineage Balb/c were cultured in RPMI medium and exposed to different strains of S. mutans. The levels of macrophages with internalized bacteria were determined with the help of an optical microscope. Patterns of bacteria internalization were analyzed in similar assays using macrophages previously treated with inhibitors of specific mechanisms of internalization (phagocytosis, macropinocytosis and endocytosis mediated by clathrin) and by electron transmission microscopy (ETM) analyses. There was no statistically significant difference in the efficiency of internalization between the strains that differed regarding production of GbpB. The mutants vicH- was significantly less internalized when compared with the respective wild type strains and mutants covR-. Analyses with specific inhibitors and ETM indicated that S. mutans can be internalized by phagocytosis, macropinocytosis and endocytosis mediated by clathrin. These data may help to understand the mechanisms by which S. mutans is internalized and processed by antigen-presenting cells and the factors affecting patterns of adaptative immune response to these bacteria / Mestrado / Microbiologia e Imunologia / Mestre em Biologia Buco-Dental

O presente estudo teve por objetivo avaliar quantitativa e qualitativamente os fagócitos presentes no leite de búfalas (Bubalus bubalis) hígidas criadas nos Estado de São Paulo. Para tal, o experimento foi subdividido em três etapas, designadas capítulos, conforme os objetivos: (1) determinar e estudar a celularidade do leite de búfalas hígidas; (2) estabelecer metodologia adequada para a recuperação das células somáticas do leite de búfalas, possibilitando a avaliação in vitro; (3) descrever e avaliar as provas de atividade fagocítica das células presentes no leite de búfalas hígidas, através de testes de mensuração direta (espraiamento e fagocitose de partículas de Zymosan) e por testes indiretos, que mensuram os metabólitos de oxigênio gerados na “explosão respiratória”. Foram colhidas 132 amostras de leite de 35 búfalas hígidas (sem alterações ao exame físico da glândula mamária, bem como resultado da prova de CMT e exame microbiológico negativos). Após as avaliações do CMT, da CCS, da contagem diferencial, da viabilidade celular, do exame bacteriológico, dos testes de fagocitose, de espraiamento e da liberação de peróxido de hidrogênio, concluiu-se que: (1) os resultados das contagens de células somáticas, microscópica e automática, foram semelhantes, mas a predominância celular diferiu conforme a técnica empregada, sendo identificadas percentualmente mais células mononucleares na lâmina de suspensão celular citocentrifugada, e mais polimorfonucleares no leite submetido à técnica de Prescott e Breed; o leite diluído e citocentrifugado permitiu melhor avaliação da morfologia celular sendo identificados: 61,1% de monócitos e macrófagos; 32,9% de neutrófilos; 5,3% de linfócitos e 0,7% de eosinófilos; os fagócitos mononucleares apresentaram uma acentuada plasticidade na estrutura, com variados padrões morfológicos; (2) a baixa celularidade do leite necessitou maiores volumes da amostra mas, o aumento do volume da amostra aumenta a concentração celular obtida, mas não de forma ilimitada pois pode passar a comprometer a viabilidade das mesmas pelo excesso de manipulação e tempo exigidos pelas amostras mais volumosas; não foi necessária a elicitação, sendo possível a recuperação de 2 x 106 células viáveis/mL de leite de búfalas hígidas com 500 mL de leite, no mínimo; (3) os macrófagos aderidos espraiaram significativamente, além de apresentarem correlação com outro marcador de ativação celular, no caso, a liberação de peróxido de hidrogênio; mais da metade dos macrófagos aderidos fagocitaram partículas de Zymosan; os fagócitos mantêm sua capacidade de liberar peróxido de hidrogênio, espontaneamente ou não, em grau máximo, com uma significativa variação entre amostras / The aim of this study was to make a quantitative and a qualitative evaluation of phagocytes from healthy buffaloes (Bubalus bubalis) milk bred in Sao Paulo state. For this purpose, the experiment was divided in three parts, called chapters, as specific objectives: (1) establish and study the cellularity of healthy buffaloes milk; (2) establish an adequate method for the recuperation of somatic cells from buffaloes milk, making possible an in vitro evaluation; (3) standardize and evaluate the phagocytic activity tests of cells from healthy buffaloes milk, through direct measurement methods (spreading and phagocytosis of Zymosan particles) and indirect methods, which measure the oxygen metabolites from the respiratory burst. Milk samples (n=132) of healthy 35 buffaloes (without alterations on the physical examination of the mammary gland, and also negative results on CMT and microbiologic exams) were obtained. After the evaluations of CMT, SCC, differential cells count, cellular viability, bacteriologic exam, phagocytic tests, spreading and hydrogen peroxide liberation, the conclusions were: (1) the results of somatic cells count, microscopic and automatic, were similar but the cellular predominance differed with the utilized method, being percentually identified more mononuclear cells on slide of cytocentrifugation cellular suspension and more polymorphonuclear on milk submitted to the Prescott and Breed methods; the diluted and cytology by cytocentrifugation of milk permitted a best evaluation of the cellular morphology, being identified: 61.1% of monocytes and macrophages; 32.9% of neutrophils; 5.3% of lymphocytes and 0.7% of eosinophils; mononuclear phagocytes showed a high plasticity on structure with many morphological patterns; (2) a lower cellularity on milk requires higher quantities of the samples but an increase on sample volume increases cellular concentrations obtained, not on an unlimited way because it can compromise the cells viability with the excess of manipulation and time required by the biggest samples; it was not necessary the elicitation, being possible the recuperation of 2 x 106 viable cells/mL on milk of healthy buffaloes with 500 mL of milk minimum (3) adherent macrophages spreaded and were correlated with hydrogen peroxide release; majority of adherent macrophages phagocyted Zymosan particles; milk phagocytes showered a high oxidative metabolism, undependably of PMA stimulation, but with a great individual variation.