Orientador: Paulo César Ciarlini / Banca: Fabiano Antonio Cadioli / Banca: Luciana Del Rio Pinoti / Banca: Ana Claudia de Melo Stevanato Nakamune / Banca: Breno Fernando Martins de Almeida / Resumo: A obesidade apresenta alta incidência e está associada com a hiperleptinemia. Há evidências de que a leptina tem um receptor presente em neutrófilos e é capaz de influenciar a sua capacidade oxidativa. Além disso, sabe-se que a ativação do metabolismo oxidativo nos neutrófilos de humanos obesos contribui para o estresse oxidativo. Até o momento, os mecanismos pelos quais a hiperleptinemia altera a produção de superóxido pelos neutrófilos em cães são desconhecidos. Nesse sentido, foi investigada a hipótese de que cães com hiperleptinemia apresentam uma ativação do metabolismo oxidativo dos neutrófilos. Para tal, um grupo controle (n=24) foi constituído de cães com bom escore corporal e foi comparado com um grupo obeso (n=25) e sobrepeso (n=27). Foram constituídos mais dois subgrupos: cães com hiperleptinemia e sem hiperleptinemia, agrupados conforme o intervalo de confiança de 95% obtido dos valores de leptina plasmática do grupo controle. Foram mensuradas as alterações dos marcadores de obesidade (adiponectina e leptina plasmáticas) e marcadores de estresse oxidativo sistêmico (capacidade antioxidante total, concentração de oxidante total, índice de estresse oxidativo e peroxidação lipídica plasmática). O metabolismo oxidativo dos neutrófilos foi avaliado por citometria de fluxo capilar utilizando as sondas hidroetidina e 2′,7′- diacetato de diclorofluoresceína com ou sem estímulo de acetato miristato de forbol (PMA). Nos animais com hiperleptinemia, assim como no grupo obeso... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Obesity has a high incidence and is associated with hyperleptinemia. It is already known that leptin has a receptor present in neutrophils, regulating its oxidative capacity. In addition, the activation of oxidative metabolism of neutrophils from obese humans contributes to oxidative stress. To date, the mechanisms by which hyperleptinemia alters the production of superoxide by neutrophils in dogs are still unknown. In this sense, we investigated the hypothesis that dogs with hyperleptinemia present an activation of oxidative metabolism of neutrophils. For this, a control group (n=24) was composed of dogs with good body score and was compared with an obese (n=25) and overweight (n=27) groups. Two more subgroups were formed: dogs with and without hyperleptinemia, grouped according to the 95% confidence interval obtained from the plasma leptin levels of the control group. Changes in the markers of obesity (plasma adiponectin and leptin), markers of oxidative stress (total antioxidant capacity, total oxidant concentration, oxidative stress index, and plasma lipid peroxidation) were measured. The oxidative metabolism of neutrophils was evaluated by capillary flow cytometry using the probes hydroetidine and 2',7'-dichlorofluorescein diacetate with or without phorbol myristate acetate (PMA) stimuli. In animals with hyperleptinemia, as well as in obese group, we observed higher superoxide production by neutrophils under PMA stimulation and presence of systemic oxidative stress. This is probably one of the first evidences that a pre-activation of the oxidative metabolism in circulating neutrophils occurs in dog with hyperleptinemia, a condition that favors the systemic oxidative stress in dogs / Doutor

Staphylococcus aureus causes nosocomial- and community- acquired infections. This versatile pathogen expresses virulence factors (VF) that enhance establishment of infection and immune evasion. Our research focused on defining the roles of S. aureus VF on host immune responses during intracellular or extracellular infections. Accessory gene regulator (agr) controls VF expression and intracellular survival. Our goal was to determine mammary epithelial cells (MEC) responses to intracellular infection and subsequent polymorphonuclear leukocyte (PMN) responses. Intracellular S. aureus increased thrombomodulin expression by MEC and activated protein C (APC) production. APC inhibited PMN chemotaxis. Findings depicted an indirect role for VF on PMN responses, so next we determined signaling pathways and cytokine responses of PMN to S. aureus toxins. Live S. aureus infections increased activation of stress signaling pathways and highlighted a role for agr-regulated genes in MAPK p38 phosphorylation and α-hemolysin in ERK phosphorylation and IL-8 expression in PMN. Continuing our studies of VF, chemotaxis inhibitory protein of S. aureus (CHIPS) inhibits monocyte chemotaxis. We hypothesized that CHIPS inhibited C5a receptor (C5aR) signaling. Monocytes pretreated with CHIPS did not inhibit C5aR signaling. Nevertheless, signaling pathways can reduce PMN function in models such as glucocorticoid treatment. Immunosuppressive effects of glucocorticoids on PMN are restored with OmniGen-AF® supplementation. Glucocorticoid receptor and Toll-like receptor signaling potentially crosstalk to restore PMN function. OmniGen-AF® supplementation restored dexamethasone-induced immunosuppression in a MyD88-dependent manner. Overall, this research focused on characterizing immune responses to S. aureus infections and PMN signaling pathways and how it is key to understanding pathogenesis. / Ph. D.

L’élimination des polynucléaires neutrophiles (PN) du site inflammatoire est nécessaire à la résolution de l’inflammation. Lors de cette phase, les PN s’engagent massivement vers l’apoptose et sont phagocytés par les macrophages. Dans certaines pathologies inflammatoires comme le syndrome de détresse respiratoire aiguë (SDRA), un défaut des mécanismes d’apoptose des PN est associé à la persistance de l’inflammation et à un mauvais pronostic. GILZ (Glucocorticoid-induced leucine zipper) est une protéine possédant des propriétés anti-inflammatoires, basées principalement sur l’inhibition des facteurs de transcription, comme NF-кB (nuclear factor-kappa B) et AP-1 (activator protein-1). GILZ module également la survie des cellules hématopoïétiques, en favorisant ou retardant l’apoptose selon le type cellulaire étudié. L’objectif de ce travail a été d’évaluer le rôle de GILZ dans les fonctions et l’apoptose du PN lors de l’inflammation. Dans un premier temps, nous nous sommes intéressés aux situations physiopathologiques dans lesquelles GILZ pouvait être exprimé et plus spécifiquement dans le syndrome de détresse respiratoire aiguë (SDRA). Cette pathologie a retenu notre attention pour le caractère systémique de l’inflammation et surtout pour le rôle prépondérant que jouent les neutrophiles dans ce syndrome. Dans le cadre d’une étude clinique prospective menée à l’hôpital Bichat, nous avons pu montrer une plus forte expression de GILZ au niveau transcriptionnel et protéique dans des neutrophiles circulants de patients souffrant de SDRA.Dans la deuxième partie du travail, nous avons évalué le rôle de GILZ dans les fonctions et le phénotype des PN. En utilisant la lignée PLB-985 différenciée en neutrophiles par addition d’acide rétinoïque (ATRA) et de diméthylformamide (DMF), nous avons mis en évidence in vitro que la surexpression de GILZ dans ce modèle de polynucléaires neutrophiles favorisait l’apoptose de ces cellules. Cette apoptose est dépendante des caspases, implique la voie mitochondriale et s’explique notamment par une diminution de l’expression de Mcl-1 (myeloid cell leukemia-1), alors que l’expression des ARNm de mcl-1 n’est pas affectée. Une activation prolongée de JNK (c-JUN N-terminal kinase) serait impliquée et entraînerait la phosphorylation de Mcl-1 et sa dégradation par le protéasome. Ces deux résultats laissent entrevoir un rôle potentiel de GILZ dans la résolution de l’inflammation in vivo. Pour adresser cette question, nous mettons en place le modèle de l’ALI (Acute Lung Injury) ou inflammation pulmonaire aiguë induite par le LPS (lipopolysaccharide), qui est un modèle murin du SDRA. Nous utilisons des souris dans lesquelles l’expression de GILZ a été invalidée spécifiquement dans les neutrophiles. Cette approche nous permettra de mieux comprendre le rôle de GILZ dans la résolution de l’inflammation. GILZ pourrait ainsi représenter une cible thérapeutique dans les pathologies inflammatoires impliquant le PN. / Elimination of polymorphonuclear neutrophils (PN) from inflammatory site is a necessary step in the resolution of inflammation, where PN undergo massive apoptosis and are phagocyted by macrophages. In some inflammatory disorders, such as acute respiratory distress syndrome (ARDS), defective apoptosis of neutrophils was described and related to persistent inflammation and poor prognosis. GILZ (Glucocorticoid-induced leucine zipper) is a potent anti-inflammatory protein, mainly through inhibition of NF-кB (nuclear factor-kappa B) and AP-1 (activator protein-1) transcription factors. Moreover, GILZ modulates hematopoietic cells survival, either by preventing or inducing their apoptosis. The overall objective of this work has been to evaluate the role of GILZ in neutrophils functions and apoptosis during inflammation. First, we sought pathophysiological situations where GILZ could be expressed. ARDS caught our attention because of the systemic nature of inflammation and the prominent role of neutrophils. A prospective clinical study was conducted at Bichat hospital and showed a higher GILZ expression at protein and transcriptional levels in blood neutrophils of ARDS patients.Secondly, we evaluated the role of GILZ in neutrophils functions and phenotypes. Using the PLB-985 cell line induced towards the neutrophil lineage using all trans retinoic acid (ATRA) and dimethylformamid (DMF), we evidenced that GILZ over-expression promoted exacerbated apoptosis of these cells. This apoptosis was caspase-dependent, involved the mitochondrial pathway and was explained, at least in part, by a diminution of myeloid cell leukemia-1 (Mcl-1) expression, whereas mcl-1 mRNA levels were not affected. A sustained activation of c-Jun N-terminal kinases (JNK) could be involved and lead to Mcl-1 phosphorylation and subsequent degradation by the proteasome machinery.Altogether, these results suggest a potential role of GILZ in the resolution of inflammation in vivo. To address this question, we currently develop an acute lung injury (ALI) model induced by lipopolysaccharides, which mimics the septic component of ARDS. This model will be implemented in mice invalidated for GILZ specifically in neutrophils. This approach should allow a better understanding of the role of GILZ in the resolution of inflammation. In the future, GILZ could represent a therapeutical target in inflammatory diseases involving neutrophils.

Phagocytosis of Bacteroides fragilis and Bacteroides thetaiotaomicron by human polymorphonuclear leukocytes (PMNL) was studied using a modified fluorochrome assay. Bacteria were grown overnight, washed and opsonized in normal, human, pooled serum. Preopsonized bacteria, either in suspension or preadhered onto a glass cover slip, were then incubated with PMNL. Afer appropriate incubation, the mixtures were centrifuged onto the cover glasses. The cover glasses were stained with acridine orange, while duplicate cover glasses were also stained with Giemsa solution. The total number and distribution of bacteria and PMNL, as well as morphological changes in PMNL, were observed with the Giemsa stain. The acridine orange stained only ingested bacteria which provided an accurate indication of phagocytosis. Bacteroides cells adhered to a glass surface were phagocytized significantly more efficiently than Bacteroides in suspension.

Mycobacterium avium subspecies paratuberculosis (map) is an intracellular pathogen that is known to parasitize macrophages and monocytes. Map infiltrates gastrointestinal tract host tissue where it is the known etiological agent of johne's disease in ruminants and implicated in the etiology of crohn's disease in humans. Map's ability to survive within macrophages enables it to disseminate throughout the rest of the host, possibly infecting other circulating blood leukocytes. In this study, the survival and fate of map strain atcc 43015 (human isolate) following phagocytosis was determined using in vitro murine macrophage cell line j774a.1 and polymorphonuclear cells (pmnc's) from five crohn's disease patients. Pmnc's from three healthy individuals and two ulcerative colitis patients, as well as escherichia coli (atcc 11303) and mycobacterium tuberculosis strain h37ra (atcc 25177), were included as controls (moi 10:1). Maturation of the phagosome was determined by evaluating the presence of stage specific markers on the surface of the phagosomal membrane. The endosomal protein, transferrin receptor, and the lysosomal marker, lamp-1, were then immunostained with cy-5 conjugated secondary antibodies, and colocalization of bacteria with each marker was evaluated separately using confocal scanning laser microscopy (cslm). In both tissue models, colocalization of viable map and m. Tuberculosis with the early endosomal marker, transferrin receptor occurred with an estimated five fold higher frequency than did association with the late lysosomal marker, lamp-1, as compared to live e. Coli, and all dead bacterial species. Using differential live/dead staining and fluorescent microscopy, survival of m. Tuberculosis and map was estimated at 85% and 79%, respectively compared to only 14% for e. Coli. The outcome was similar for both tissue culture and pmncs from all patients tested, suggesting that map and m. Tuberculosis can survive readily in both cell types, and regardless of the disease state of the host or the killing power of the cell. Map's survival appears to mimic m. Tuberculosis', suggesting the ability to resist phagolysosome fusion, by maintaining association with the early endosomes. Overall, the data confirms map virulence in host human blood leukocytes.

Orientador: Luciane Alarcão Dias-Melicio / Resumo: A paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica, prevalente na América Latina, que tem por agentes etiológicos diferentes espécies de Paracoccidioides. Entre eles encontra-se o Paracoccidioides brasiliensis (P. brasiliensis), espécie composta por um complexo de agrupamentos geneticamente isolados, classificados como espécies filogenéticas: S1, PS2, PS3 e PS4. O P. brasiliensis possui distribuído pela parede fúngica e dentro de vesículas citoplasmáticas uma glicoproteína de 43KDa, a gp43. Esta molécula é considerada como o principal componente antigênico produzido e secretado pelo fungo, a qual é encontrada no soro de pacientes acometidos pela PCM e, está associada aos fatores de virulência e escape do patógeno. Sabendo disso, tornou-se extremamente importante a avaliação da interação dessa proteína com as células do sistema imune inato e sua consequente atuação sobre os mecanismos moduladores celulares. Assim, nosso estudo avaliou os neutrófilos polimorfonucleares (PMNs) que estão presentes em abundância desde o início e durante a infecção pelo Paracoccidioides, e que apresentam além de suas ações efetoras diretas contra o fungo, uma importante ação moduladora da resposta imune. Estas ações envolvem o reconhecimento do fungo por Receptores de Reconhecimento de Padrão (PRRs) que culminam em ativação celular com consequente produção e liberação de diferentes mediadores inflamatórios. Dessa maneira, o presente trabalho avaliou o envolvimento dos receptores cel... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, prevalent in Latin America and has as etiological agents Paracoccidioides spp. One of them is Paracoccidioides brasiliensis (P. brasiliensis) composed by a complex of genetically isolated clusters, classified as phylogenetic species: S1, PS2, PS3 and PS4. P. brasiliensis present along the cell wall and inside cytoplasmic vesicles a 43 KDa glycoprotein, known as gp43. This molecule is recognized as the main antigenic component produced and secreted by the fungus into sera of PCM patients and is associated with the escape and virulence factors of the pathogen. Thereby, becomes extremely important to evaluate the interaction mechanisms of this protein with cells from the innate immune system and their consequent action on cellular functions. Polymorphonuclear neutrophils (PMNs) has prominent participation in PCM, once these cells are present in a great amount at the beginning and during Paracoccidioides infection. PMNs perform effector actions directly against the fungi as well as an important immune modulatory function. Such actions involve the recognition of fungal components by pattern recognition receptors (PRRs) that leads to cell activation and consequent production of inflammatory mediators. So, the present study evaluated the involvement of TLR2 and TLR4 on cytokines and eicosanoids release by PMNs from healthy human stimulated with gp43. Cells were initially incubated in the presence or absence of monoclo... (Complete abstract click electronic access below) / Doutor

A properdina tem a capacidade de estabilizar o complexo enzimático C3bBb, com função de C3-convertase da Via Alternativa, capaz de clivar moléculas de C3, gerando C3a e C3b. Diferentes trabalhos têm sugerido que a properdina pode se ligar diretamente à superficie de um patógeno independentemente complexo enzimático C3bBb. No que diz respeito à interação de properdina com Leptospira tanto patogênica quanto não patogênica, nada se conhece na literatura. Neste trabalho demostramos que tanto a properdina presente no SHN, quanto purificada e todos os seus oligômeros interagiram com tais espiroquetas. Também observamos que a ligação da properdina pode acontecer diretamente na superfície da bactéria ou após ligação prévia do fragmento C3b. Observamos também, que na presença de SHN estas bactérias foram totalmente eliminadas, no entanto, 70% das bactérias sobreviveram quando incubadas com SHD-P. Já a adição de properdina purificada ao SHD-P provoca uma marcante diminuição no número de leptospiras viáveis. Avaliamos também quais proteínas bacterianas teriam a capacidade de se ligar à properdina. Entre as proteínas recombinantes de L. interrogans, apenas a lipoproteína LIC11087, uma proteína presente unicamente na superfície de leptospiras patogênicas, interagiu com a properdina. Todos os oligômeros de properdina presentes no SHN interagiram com a lipoproteína LIC11087. Determinamos por quantificação do complexo enzimático usando anti-Factor B policlonal que a properdina apresenta uma significativa atividade reguladora quando se depositava na superfície das bactérias não patogênicas, promovendo desta maneira, a formação da C3-convertase da Via Alternativa. Constatamos também nos sorovares patogênicos, pouca atividade reguladora pela properdina quando estas leptospiras foram pré-incubadas com a proteína. Também encontramos que a ligação da properdina na superfície de leptospiras contribui para um aumento da fagocitose por polimorfonucleares humanos de leptospiras, principalmente das não patogênicas. Nossos dados obtidos até o momento sugerem que a properdina liga-se na superfície das leptospiras patogênicas e não patogênicas; participa no processo de eliminação de leptospiras não patogênicas pela Via Alternativa; e, após sua deposição na superfície das bactérias, contribui para a formação de uma C3-convertase nas bactérias não patogênicas, diferente ao modelo tradicional. / Properdin is a positive regulatory protein that stabilizes the C3- and C5-convertases of the alternative pathway. Several studies have suggested that properdin can bind directly to the surface of a pathogen regardless enzyme complex C3bBb. With regard to the interaction of properdin with both pathogenic Leptospira and non-pathogenic, nothing is known in the literature. In this work we demonstrate that both properdin present in SHN and purified and all their oligomers interacted with spirochetes and of properdin can binding directly on the surface of bacteria or after prior binding of C3b fragment. We also observed that the Activation of the alternative pathway of complement is crucial for killing non-pathogenic L. biflexa and properdin acts effectively since this bacterium proliferates in P-depleted human serum. Since the addition of purified properdin the SHD-P causes a marked decrease in the number of viable leptospires. We also evaluated bacterial proteins which have the ability to bind to properdin. Among several recombinant leptospiral membrane proteins tested, lipoprotein LIC11087, present only in pathogenic Leptospira, was the ligand for P, P2, P3 and P4. Determined by quantifying the enzyme complex using polyclonal anti-factor B that properdin presents a significant regulatory activity when deposited on the surface of non-pathogenic bacteria, thereby promoting the formation of C3 convertase Alternative pathway. We found also in pathogenic serovars, little regulatory activity by properdin when they were preincubated leptospires with the protein. We also found that the binding of properdin in leptospiras surface contributes to increased phagocytosis of leptospira by human polymorphonuclear, mainly from non-pathogenic. Our data obtained suggest that properdin binds to Leptospira species and may play an important role to limit the proliferation of non-pathogenic Leptospira; participates in leptospiras elimination process nonpathogenic Via Alternative; and, after deposition on the surface of bacteria, it contributes to the formation of a C3 convertase in non-pathogenic bacteria, different from the traditional model.

O objetivo foi avaliar a atividade funcional dos polimorfonucleares (PMN) do sangue de bezerros neonatos \"in vitro\" - metabolismo oxidativo induzida por: S. aureus, E. coli e PMA e fagocitose para S. aureus e E. coli, em citômetro de fluxo. Quinze animais avaliados nos tempos: t0 (antes da ingestão de colostro/leite); t1(até 48 hs. p.n); t2 (48 - 96 hs. p.n.); t3 (96 - 144 hs. p.n.); t4 (144-192 hs. p.n.); t5 (192-240 hs.p.n.) receberam quatro litros (grupo 1 - G1), dois litros de colostro ( grupo 2 - G2) e somente leite (grupo 3 - G3). O metabolismo oxidativo basal não diferiu em nenhum dos grupos e idades. Entre os grupos este foi menor em G1 do que em G3 (t3). Induzido pela S. aureus foi maior entre t1 e t3 (grupo 1), entre t2 e t3 (grupo 2) e sem diferenças (grupo 3). Entre os grupos G1 foi maior que G3 (t1). Induzido pela E.coli foi maior entre t1 e t3 (grupo 1), maior em t2 (grupo 2) e sem diferenças (grupo 3). Entre os grupos G1 foi maior que G2 e G3 (t1). O PMA estimulou a explosão respiratória em G1, G2 e G3 entre t0 e t5. Entre os grupos G1 foi menor para do que G2 e G3. A comparação entre metabolismo basal e induzido para G1 foi maior para PMA e S. aureus entre t0 e t5 e para E. coli entre t1 e t5; para G2 foi maior para o PMA entre t0 e t5, para S. aureus entre t0 e t3 e para a E. coli entre t2 e t4; para G3 foi maior para o PMA e S.aureus entre t0 e t5 e para a E. coli entre t3 e t5. A fagocitose induzida pela S. aureus foi maior nos tempos t1, t2 e t4 (grupo1) e o percentual de fagocitose foi maior após t0; para o grupo 2, foi maior em t2 e t3 e o percentual foi maior após t0; para o grupo 3, não diferiram entre os tempos. Entre os grupos G1 foi maior que G2 e G3 (t4) e o percentual foi maior para G1 e G2 (t3) do que G3 e maior para G1 do que G2 (t5). A fagocitose induzida pela E. coli e o percentual de fagocitose não diferiram em nenhum grupo. Entre os grupos G1 foi maior do que G3 (t0) e o percentual foi maior para G1 do que G2 (t2). Conclusão: o colostro não interferiu no metabolismo oxidativo basal e estimulado pelo PMA, a S. aureus estimulou o metabolismo oxidativo e a fagocitose antes e após a ingestão de colostro. A E. coli não induziu o metabolismo oxidativo (G3) e não foi eficiente para estimular a fagocitose. Não ficou esclarecida a influência da idade sobre a atividade dos PMNs. O fornecimento de quatro litros de colostro melhora resposta funcional dos PMNs para as bactérias. / The aim of this study was to evaluate \"in vitro\" functional activities of neonatal calves blood polymorphonuclear leukocytes (PMNs) - respiratory burst and phagocytosis induced by S.aureus, E.coli and PMA, measured by flow cytometry. Fifteen calves were tested at times: t0 (before milk/colostrum ingestion); t1 (until 48 hours post partum); t2 (48-96 hrs p.p.); t3 (96 - 144 hrs. p.p.); t4 (144-192 hrs. p.p.); t5 (192-240 hrs. p.p.) and the groups intakes were 4 liters of colostrum (group 1), 2 liters of colostrum (group 2) and only milk (group 3). The basal respiratory burst did not differ among groups but it was lower in G1 than G3 (t3). The burst induced by S.aureus was higher between t1 and t3 (G1) and t2 and t3 (G2) and showed no difference (G3). The G1 values were higher than G3 (t1). E.coli induced higher values between t1 and t3 (G1), higher on t2 (G2) and showed no difference (G3). Among groups G1 was higher than G2 and G3 (t1). The PMA induced the respiratory burst in G1, G2 and G3 between t0 and t5. Among groups G1 was lower in G2 than G3. The induced basal metabolism was compared and G1 was higher to PMA and S.aureus between t0 and t5 and to E.coli between t1 and t5. The G2 showed higher values to PMA between t0 and t5, to S.aureus between t0 and t3 and E.coli between t2 and t4. The G3 showed higher values in PMA and S.aureus between t0 and t5 and E.coli between t3 and t5. The S.aureus induced phagocytosis was higher on t1, t2 and t4 (G1) and the phagocystosis percentage was higher after t0. G2 groups showed higher values on t2 and t3 and higher percentage after t0. G3 had no difference on times. G1 showed higher values than G2 and G3 (t4) and G1 and G2 (t3) had higher percentage than G3 with G1 higher than G2 (t5). The E.coli induced phagocytosis and its percentage did not differ among groups. G1 was higher than G3 (t0) and G1 showed higher phagocytosis percentage than G2 (t2). Conclusion: the colostrum did not affect either, the basal respiratory burst and the one stimulated by PMA. S. aureus stimulated the respiratory burst and phagocytosis before and after the colostrum intake. E.coli did not induce the respiratory burst (G3) and did not stimulate the phagocytosis. The age influence in PMNs activity was not totally clarified. The intake of 4 litters of colostrum increases the PMNs activity against the species of bacteria studied.

Thesis (M.Sc.) -- University of the North, 1982 / Refer to the document

The ability of human polymorphonuclear leukocytes (PMNL; neutrophils), to sense and move to sites of infection is essential for our defense against pathogens. Cell motility is critically dependent on a dynamic remodeling of morphology. The morphological polarization toward chemoattractants, such as N-formyl-Met-Leu-Phe (fMLF), is associated with temporary extension and stabilization of lamellipodia in the direction of movement. The underlying mechanisms of cell motility are, however, still not entirely elucidated. It is therefore an urgent task to extend the present experimental evidence to give solid basis for a comprehensive model. Here it is shown that nitric oxide (NO) stimulates the morphological response of neutrophils, most likely due to transient increases in [Ca2+]i, following addition of NO-donors. This will, hypothetically, activate gelsolin and other actin filament severing proteins, leading to a subsequent decrease in filamentous actin. The incapability to efficiently turnover the actin filament network then blocks all motile activity. It is also shown that N-formyl peptide receptors on polarized neutrophils accumulate non-uniformly towards regions involved in motility. It is suggested that neutrophils use the asymmetric receptor distribution for directional sensing and sustained migration. A model for lamellipodium extension, where water fluxes play a pivotal role is presented. It is suggested that water fluxes through water-selective aquaporin (AQP) channels, contribute to the propulsive force for formation of various membrane protrusions and, thus, cell motility. It is well known that small G proteins of the Rho family GTPases play important roles in the intracellular signaling underlying cell motility. In morphologically polarized neutrophils it is shown that Cdc42, Rac2 and RhoA display spatially distinct distributions, which allows for sequential chemoattractant stimulation of neutrophil motility. The specific localizations of Rac2, Cdc42 and RhoA relative to each other and filamentous actin and fMLF receptors support the hypothesized order of activation and regulation of neutrophil cell motility. In conclusion, the detailed analysis of motility-related issues presented here provide new data allowing further refinement of previous models of neutrophil motility.