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.

Orientador: Carlos Rossa Júnior / Resumo: Os macrófagos representam uma das principais pontes entre imunidade inata e adaptativa e estão presentes em grande número tanto no estroma circundando tumores sólidos quanto no interior da massa tumoral (TAMs, tumor-associated macrophages), onde podem representar até 50% da massa da lesão. Nos carcinomas espinocelulares de cabeça e pescoço (HNSCC), TAMs apresentam predominantemente o perfil M2 (pró-tumoral), e a quantidade de TAMs é inversamente relacionada ao prognóstico. A efetividade do tratamento não-cirúrgico (quimio/radioterapia ou imunoterapia) dos tumores sólidos é diretamente relacionada à indução de morte das células neoplásicas. No processo de reparo, as células apoptóticas são removidas por fagocitose por outros tipos celulares, num processo denominado eferocitose. Embora seja um processo importante para o reparo e homeostasia tecidual, a eferocitose pode afetar o fenótipo dos macrófagos, favorecendo a polarização para o perfil M2, associado à progressão de HNSCC. A galanina é um peptídeo de 29 aminoácidos amplamente distribuída no organismo e com amplo espectro de efeitos biológicos. A expressão constitutiva de galanina por células de OSCC está associada à maior agressividade do tumor e tem sido estudado como marcador prognóstico de agressividade do tumor e também como um possível alvo terapêutico de HNSCC. O objetivo do presente trabalho é determinar a influência dos produtos secretados pelas células de carcinoma espinocelular de cabeça e pescoço, bem como da ga... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Macrophages are a major link between innate and adaptive immunity and are present both in the surrounding stroma and within various solid tumors (when they are denominated tumor-associated macrophages, TAMs), where these cells may represent up to 50% of the tumor mass. In head and neck squamous cell carcinomas (HNSCC), TAMs are predominantly of the M2 phenotype and the prognosis is inversely correlated with their abundance. The effectiveness of non-surgical treatment of solid tumors (chemotherapy/radiotherapy) is directly related to the induction of neoplasic cell death. In the repair process, apoptotic/dead cells must be removed by phagocytosis by other cell types (particularly macrophages), in a process called efferocytosis. Although this is a critical step in the repair and reestablishment of tissue homeostasis, efferocytosis itself may affect the phenotype of macrophages, skewing the cells towards an M2 phenotype, which is deemed as a 'pro-tumoral' phenotype. Galanin is a 29 aminoacid peptide of 29 produced by various cell types and with a broad spectrum of biological effects. The constitutive expression of galanin by head and neck cancer cells is associated with the increased of tumor aggressiveness and has been proposed as a biomarker of tumor aggressiveness and also as a possible therapeutic target in HNSCC. Thus, the goal of the present study was to determine the role of secreted products from head and neck cancer cells, and of galanin independently, on macrophage phe... (Complete abstract click electronic access below) / Mestre

As doenças periodontais afetam os tecidos de suporte dos dentes e são desencadeadas por microrganismos que possuem a capacidade de invadir os tecidos periodontais. A evolução desta doença é influenciada pela resposta inflamatória e imunológica do hospedeiro e envolve a participação de diversos tipos celulares. Atualmente, existem evidências de que os mastócitos, além de outras funções, possuem a capacidade de eliminar bactérias, através da fagocitose. Assim sendo, este estudo teve por objetivo avaliar a capacidade fagocítica dos mastócitos frente ao periodontopatógeno A. actinomycetemcomitans, além de comparar sua capacidade fagocítica com a dos macrófagos, considerados fagócitos profissionais. Para este fim, foram realizados ensaios fagocíticos in vitro utilizando mastócitos e macrófagos murinos, desafiados ora com A. actinomycetemcomitans ora com Escherichia coli, opsonizados ou não, sob diferentes proporções célula: bactérias. Após 1 hora de desafio, as células foram coradas com laranja de acridina e avaliadas qualitativamente utilizando-se microscópio de varredura confocal a laser e quantitativamente através do microscópio de fluorescência convencional. Nossos resultados demonstraram que os mastócitos murinos se mostraram eficientes quanto a sua capacidade fagocítica frente a A. actinomycetemcomitans. Os valores percentuais de mastócitos com A. actinomycetemcomitans internalizados, na ausência de opsonização com complemento, foram maiores que aqueles na presença da opsonização, sugerindo a participação de receptores opsoninas-independentes no reconhecimento deste patógeno pelos mastócitos, além do receptor de complemento tipo 3 (CR3). Comparando os dois tipos celulares, verificou-se que ambas as células apresentaram importante atividade fagocítica contra A. actinomycetemcomitans, porém os valores percentuais de mastócitos com bactérias internalizadas sem complemento foram maiores que aqueles de macrófagos com bactérias internalizadas com complemento, em uma das proporções (1:10). Os resultados deste trabalho sugerem o papel dos mastócitos como fagócitos profissionais na patogênese da doença periodontal induzida por placa dentobacteriana. / Periodontal diseases affect the supporting tissues of the teeth and are triggered by microorganisms which are capable of invading periodontal tissues. The evolution of this disease is influenced by inflammatory and immune response of the host and involves the participation of different cell types. Currently, there is evidence that mast cells, among other functions, have the ability to eliminate bacteria by phagocytosis. Thus, this study aimed to evaluate the phagocytic ability of mast cells against the periodontopathogens A. actinomycetemcomitans, and compare with the phagocytic capacity of macrophages, which are considered professional phagocytes. Therefore, in vitro phagocytic assays were conducted using murine mast cells and macrophages, challenged with A. actinomycetemcomitans or Escherichia coli, at the same time, opsonized or not, under different proportions cell: bacteria. After 1 hour of challenge, cells were stained with acridine orange and qualitatively assessed by using a confocal laser scanning electron microscope and quantitatively by the conventional scanning fluorescence microscope. The results demonstrated that phagocytic ability of murine mast cells was effective against A. actinomycetemcomitans. The percentages of mast cells with A. actinomycetemcomitans internalized in the absence of opsonization with complement, were higher than those in the presence of opsonization, suggesting the involvement of opsonin-independent receptors in recognition of this pathogen by mast cells, as well as complement receptor type 3 (CR3). Comparing the two cell types, it was observed that both cells showed significant phagocytic activity against A. actinomycetemcomitans, however, the percentages of mast cells with internalized bacteria without complement were higher than those of macrophages with internalized bacteria with complement, in one of the proportions (1:10). The results suggest the role of mast cells as professional phagocytes in the pathogenesis of periodontal disease induced by dental plaque.

Macrophages are sentinels present in most tissues of the body, where they recognise and respond to biological dangers. Recognition and uptake of particles is mediated through phagocytic receptors which upon activation induce appropriate responses. These responses need to be tightly regulated in order to destroy pathogens but prevent uncontrolled inflammation. Phagocytosis is an evolutionarily conserved process required for host defence and homeostasis. During phagocytosis, particles are recognised by cell surface receptors that trigger rearrangement of the actin cytoskeleton and internalization of the bound particle into a de novo, membranous organelle known as the phagosome. Regulation of phagocytosis and phagosome maturation can be achieved through changes in transcription/translation and differential recruitment of proteins but also through their non-translational modifications. Here I explored the role of ubiquitylation in the phagosome biogenesis of Interferon-gamma (IFN-ɣ) activated macrophages. Ubiquitylation is a diverse, reversible post-translational modification which is not only involved in protein degradation but also in vesicle trafficking and immune signalling. My data shows that phagosomes are enriched in polyubiquitylation, which is further enhanced by IFN-ɣ. I applied a targeted AQUA peptide approach by which we quantified ubiquitin chain linkage peptides from phagosome samples by PRM. This data shows that all chain linkages apart from M1/linear chains are present on phagosomes. Furthermore, IFN-ɣ activation enhanced K11, K48 and K63 chains significantly. In order to identify the molecular function of this polyubiquitylation, I characterized the ubiquitinome of phagosomes of IFN-γ activated macrophages and can demonstrate that ubiquitylation is preferentially attached to proteins involved in vesicle trafficking, thereby delaying fusion with late endosomes and lysosomes. I demonstrated that most ubiquitin chains are on the cytoplasmic site of the phagosome enabling an interaction of ubiquitin chains with cytosolic proteins such as Rab7. Rab7 a major regulator of vesicle trafficking could be shown to be ubiquitylated on phagosomes. I further showed that phagosomal recruitment of the E3 ligase RNF115 is enhanced upon IFN-γ stimulation and RNF115 is responsible for most of the increase of K63 polyubiquitylation of phagosomal proteins. Knock-down of RNF115 promotes phagosome maturation and induces an increased pro-inflammatory response to Toll-like receptor (TLR) agonists, indicating that RNF115 is a negative regulator of vesicular trafficking to the lysosome and disruption of this pathway induces pro-inflammatory responses in macrophages. In conclusion, this is the first study showing unbiasedly that ubiquitylation plays an important role in vesicle trafficking to the lysosome.

An increase in attachment of SRBCs to Procambarus clarkii hemocytes has been shown after the crayfish were injected with a live or killed Pseudomonas strain RS2b. The increase in attachment occurred at 8 hours post injection and peaked at 24 hours for both experimental groups. The population of hemocytes with receptors for LPS and mannose also increased at 8 hours post injection and peaked at 24 hours for both experimental groups. At 96 hours post injection the number of receptor bearing hemocytes and hemocytes bound to SRBCs began to decrease to the level of the control for both groups. The protein concentration of hemolymph from the experimental groups remained stable at 8 and 24 hours post injection and increased at 96 hours. The correlation of the protein concentration increase at 96 hours with the decrease of receptor bearing hemocytes may be due to the degranulation of the receptor bearing hemocytes.

Yersinia pseudotuberculosis is a foodborne pathogen that is the ancestral strain to Yersinia pestis, the causative agent of Plague. Y. pseudotuberculosis invades a host through the intestinal epithelium. The bacteria resist mucosal innate immune defenses including antimicrobial chemokines and phagocytic cells, and replicate in local lymph nodes. They cause Tuberculosis-like symptoms, including necrosis of local tissue and granuloma formation. Like all bacteria, Y. pseudotuberculosis has a net negative charge, which contributes to its susceptibility to some cationic antimicrobial peptides. Y. pseudotuberculosis is able to reduce this negative charge by adding 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A portion of lipopolysaccharide. The production and addition of the L-Ara4N is coded for by the pmrHFIJKLM (pmrF) operon. A previous study has shown that the Y. pseudotuberculosis pmrF operon is important for resistance against polymyxin, but is not important for virulence in mice. Several previous reports have shown a strong influence of growth temperature on resistance to antimicrobial peptides and pmrF expression in pathogenic Yersinia species, but these studies also suggest significant variability between species, and even between strains of individual species. In particular, the regulation of the Y. pseudotuberculosis pmrF operon and its effect on bacterial interactions with mucosa-associated antimicrobial chemokines and neutrophils is not understood. In these studies, we investigated the environmental influences on pmrF expression in Y. pseudotuberculosis. We found that the promoter activity of the pmrHFIJKLM operon is increased at lower temperatures (21ºC) and in the presence of human serum. A ΔpmrI mutant strain of Y. pseudotuberculosis defective for addition of L-Ara4N was found to be more susceptible to killing by the antimicrobial chemokine CCL28 compared to wild-type. This suggests that this gene is important in the bacterial defense against antimicrobial chemokines. However, when the ΔpmrI mutant strain was exposed to human neutrophils, there was a decrease in phagocytosis as compared to wild-type bacteria. Our results suggest that the regulation of L-Ara4N modifications in Yersinia is more complex than previously appreciated and varies between species. Addition of L-Ara4N to Y. pseudotuberculosis appears to enhance resistance to some antimicrobial peptides like CCL28 and promote greater phagocytic engulfment by neutrophils. These opposing effects may partly explain why there is no net apparent survival defect in mutants lacking the pmrF operon during infection.

Tularemia is a potentially fatally illness caused by the facultative intracellular Gram-negative bacterium Francisella tularensis. Virulent strains of F. tularensis can cause a fatal disease after inhalation of a few as ten organisms. Due to the highly pathogenic features of Francisella, it has been designated as a Tier 1 select agent, meaning that its possession and handling is highly restricted. Macrophages are phagocytes that play a central role in the innate immune response to infection that can be used by certain pathogens, including Francisella, as a niche for bacterial replication and dissemination during infection. After infection of macrophages Francisella escapes from the phagosome and replicates in the cytosol, however the bacterial factors required for these aspects of virulence are incompletely defined. Here we describe the isolation and characterization of F. tularensis subspecies tularensis strain Schu S4 mutants in iglI, iglJ, and pdpC, three genes located in the Francisella Pathogenicity Island. Our data demonstrate that these mutants were unable to replicate in macrophages due to a defect in phagosome escape. However, a small percentage of pdpC mutants were able to reach the cytosol and replicate moderately. Both iglJ and pdpC mutants were highly attenuated for virulence in a mouse intranasal infection model, however pdpC but not iglJ mutants, were able to disseminate from the lung before eventual clearance. These data demonstrated that the FPI genes tested were essential for F. tularensis Schu S4 virulence, but suggest that they may have different functions due to the unique phenotype observed for pdpC mutants. Our studies also characterized the role of F. tularensis O-antigen and capsule to facilitate interactions with components of the serum complement system; demonstrating that the O-antigen is required for binding of IgM to the bacteria in order to initiate complement opsonization. IgM dependent complement opsonization of both F. tularensis Schu S4 and LVS strains facilitated enhanced phagocytosis of the bacteria by complement receptors 3 and 4 of human macrophages. In addition, we examined the mechanisms of macrophage cytotoxicity and proinflammatory cytokine secretion that was induced after infection with a Schu S4 LPS O-antigen and capsule mutant. The response to the mutant was dependent on phagosome escapes, suggesting a cytosolic pattern recognition receptor was involved in recognition of the bacteria. We found that the cytotoxic and proinflammatory responses had both similar and distinct requirements between human and murine macrophages. Infection with the O-antigen mutant induced robust proinflammatory cytokine secretion that was dependent on caspase-1, cathepsin B, and ASC while cytotoxicity was partially dependent on these molecules. Importantly, we demonstrated that wild-type Schu S4 predominately activated apoptotic caspases, and not inflammatory caspases, during infection and had a blunted cytotoxic response. This was in contrast to the robust cytotoxicity and activation of inflammatory caspases after infection with the non-virulent strain LVS. Together, these studies demonstrated that the Schu S4 LPS O-antigen and capsule are required for evasion of macrophage cytosolic host defense mechanisms.

The work detailed in this dissertation has an overarching theme of modulating microglia activation in both in vivo and in vitro models relevant to AD. The premise is that understanding microglia function in this context may lead to a better understanding of AD pathogenesis and thus to effective therapeutic interventions. In chapter 3, we employ a well-defined model of microglia activation whereby the intraperitoneal delivery of LPS results in CNS microglia activation and TNF-α production. Having previously identified that CD45 signaling pathways antagonized microglia TNF-α production in vitro and given that immunotherapy with anti-CD45 antibodies are already in clinical trials for both the treatment of malignant disorders as well as for tolerance induction following organ transplants, we investigated whether microglial CD45 could be a relevant molecular target in the opposition of microglia activation in vivo. Given that a number of epidemiological studies have shown an inverse correlation between the use of statins (a class of drugs that were initially described as specific inhibitors of cholesterol biosynthesis) and the incidence of Alzheimer's disease (Wolozin, 2000; Simon 2002; Zamrini, 2004); in chapter 4, we employed one of the most prescribed statins, lovastatin in our microglia activation paradigm. Interestingly, we show that statins only inhibit the enzyme HMG-CoA (the rate limiting step of cholesterol biosynthesis) but that they also display pleiotrophic effects including the inhibition of CD40 expression. Given the role of CD40 in microglia activation and its potential role in AD pathogenesis, we investigated whether lovastatin's protection in AD might be derived from effects on microglia function as governed by the CD40 pathway. In chapter 5, we provide evident for a model of microglia activation that addresses some of the current controversies concerning the role of microglia cells in AD pathogenesis. The model suggests that microglia cells exist in a number of distinct activation states, in one such state that we denote the "phagocytic state"; microglia function to clear cellular debris or foreign invaders or in the case of AD to remove β-amyloid/Aβ peptides. However, in response to certain co-stimuli (i.e. CD40 activation) these microglia take the form of an "antigen presenting cell" (APC) whereby they lose their phagocytic capacity in lieu of cytokine production and thus potential contributing to AD pathogenesis.

<p>Lipopolysaccharide (LPS) is a structure of the gram-negative bacteria that protect from chemicals and works as a stabilization component for the membrane. Studies show that LPS also may have a function to avoid immune defense. In this project we investigate two <em>Salmonella enterica</em> variants with different LPS conformation. The wild-type Salmonella got an originally LPS structure and the mutant form had a defect one. The bacteria were transfected with a green fluorescent protein (GFP) to allow measuring of phagocytosis. Monocytes were isolated from human blood and were incubated for several days with cytokines to give dendritic cells. The cells were exposed to each type of <em>Salmonella</em> and incubated for different times. After labeling with phalloidin and studies with fluorescent microscopy, phagocytosis and F-actin were measured. The results show that it is a difference in phagocytosis and F-actin depending on LPS conformation. That means that LPS may have a decisive role for the pathogenicity of <em>Salmonella</em>.</p>

Lipopolysaccharide (LPS) is a structure of the gram-negative bacteria that protect from chemicals and works as a stabilization component for the membrane. Studies show that LPS also may have a function to avoid immune defense. In this project we investigate two Salmonella enterica variants with different LPS conformation. The wild-type Salmonella got an originally LPS structure and the mutant form had a defect one. The bacteria were transfected with a green fluorescent protein (GFP) to allow measuring of phagocytosis. Monocytes were isolated from human blood and were incubated for several days with cytokines to give dendritic cells. The cells were exposed to each type of Salmonella and incubated for different times. After labeling with phalloidin and studies with fluorescent microscopy, phagocytosis and F-actin were measured. The results show that it is a difference in phagocytosis and F-actin depending on LPS conformation. That means that LPS may have a decisive role for the pathogenicity of Salmonella.