Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology

Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of alpha-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of alpha-toxin, and triggered limited tissue damage. alpha-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure alpha-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of alpha-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of alpha-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against alpha-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of alpha-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia.

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8(+) T Cells

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+) and CD8(+) T cells secreting gamma interferon (IFN-gamma) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis. We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

Toxicity and imaging of multi-walled carbon nanotubes in human macrophage cells.

Multi-walled carbon nanotubes (MWNTs) have been proposed for use in many applications and concerns about their potential effect on human health have led to the interest in understanding the interactions between MWNTs and human cells. One important technique is the visualisation of the intracellular distribution of MWNTs. We exposed human macrophage cells to unpurified MWNTs and found that a decrease in cell viability was correlated with uptake of MWNTs due to mainly necrosis. Cells treated with purified MWNTs and the main contaminant Fe(2)O(3) itself yielded toxicity only from the nanotubes and not from the Fe(2)O(3). We used 3-D dark-field scanning transmission electron microscopy (DF-STEM) tomography of freeze-dried whole cells as well as confocal and scanning electron microscopy (SEM) to image the cellular uptake and distribution of unpurified MWNTs. We observed that unpurified MWNTs entered the cell both actively and passively frequently inserting through the plasma membrane into the cytoplasm and the nucleus. These suggest that MWNTs may cause incomplete phagocytosis or mechanically pierce through the plasma membrane and result in oxidative stress and cell death.

Valoración de la situación sanitaria del camarón litopenaeus vannamei en Puerto Morazán, Chinandega

En vista de la situación de enfermedades en la explotación camaronera, se propuso la realización de esta monografía, cuyo titulo es: Valoración de la situación sanitaria del camarón Litopenaeus vannamei en Puerto Morazán, Chinandega, para lo cual se procedió a establecer objetivos como la determinación de las enfermedades que afectaban mayormente a las explotaciones camaroneras de Litopenaeus vannamei tanto silvestres como cultivados. Para ello se concentraron 6 meses en trabajo de campo desarrollando las actividades de siembra y cosecha en las granjas camaroneras de Puerto Morazán así como también la información de las diferentes industrias camaron eras de Nicaragua. Con este trabajo monográfico se logró concretar que las patologías más frecuentes en la explotación camaronera para L. vannamei silvestre y cultivado son: mancha blanca, virus del síndrome de Taura, virus de la necrosis hipodérmica y hematopoyética infecciosa, Vibriosis, Hepatopancreatitis necrotizante, bacterias del genero Leucothrix, parásitos como las gregarinas. Identificando únicamente que la diferencia entre los dos tipos: silvestres y cultivados consiste únicamente en que los silvestres presentan mayor resistencia a las condiciones ambientales.

Osteoblast and monocyte responses to 444 ferritic stainless steel intended for a Magneto-Mechanically Actuated Fibrous Scaffold

The rationale behind this work is to design an implant device, based on a ferromagnetic material, with the potential to deform in vivo promoting osseointegration through the growth of a healthy periprosthetic bone structure. One of the primary requirements for such a device is that the material should be non-inflammatory and non-cytotoxic. In the study described here, we assessed the short-term cellular response to 444 ferritic stainless steel; a steel, with a very low interstitial content and a small amount of strong carbide-forming elements to enhance intergranular corrosion resistance. Two different human cell types were used: (i) foetal osteoblasts and (ii) monocytes. Austenitic stainless steel 316L, currently utilised in many commercially available implant designs, and tissue culture plastic were used as the control surfaces. Cell viability, proliferation and alkaline phosphatase activity were measured. In addition, cells were stained with alizarin red and fluorescently-labelled phalloidin and examined using light, fluorescence and scanning electron microscopy. Results showed that the osteoblast cells exhibited a very similar degree of attachment, growth and osteogenic differentiation on all surfaces. Measurement of lactate dehydrogenase activity and tumour necrosis factor alpha protein released from human monocytes indicated that 444 stainless steel did not cause cytotoxic effects or any significant inflammatory response. Collectively, the results suggest that 444 ferritic stainless steel has the potential to be used in advanced bone implant designs. © 2011 Elsevier Ltd.

Efecto de siete niveles de nitrógeno, fósforo y zinc sobre algunos componentes de rendimiento del frijol variedad 510-51 (Phaseolus vulgaris L.)

El frijol es uno de los cultivos mas importantes en Nicaragua. Datos estadísticos indican que este cultivo ocupa el segundo lugar en importancia como alimento básico y el quinto lugar en cuanto al valor nutricional. Durante los últimos años se han obtenido bajos rendimientos y para 70-71 se obtuvo un rendimiento promedio de 537.2 kilogramos por hectárea (5). Como se puede observar el rendimiento promedio nacional es muy bajo. Para incrementar este rendimiento ademas de sembrar con variedades mejoradas se debe hacer necesario buscar los niveles mas adecuados de Fertilización. Se ha observado en la zona de Masatepe una aparente deficiencia de ciertos elementos menores y en especial del Zinc manifestándose con síntomas de necrosis general y deformaciones semejantes a rosetas en las yemas foliar en el cultivo de frijol (13). Este ensayo se llevo a efecto el 4 de octubre de 1973 en la Estación Regional de Diversificación Agrícola Campos Azules. El objetivo de este trabajo fue evaluar el efecto del Nitrógeno, Fósforo y Zinc respectivamente. En la evaluación de los datos de rendimiento se empleo el diseño Guadalupe y Bloque al azar con dos repeticiones. De acuerdo a los resultados para altura de planta, el efecto de los tratamientos fue estadisticamente significativo al nivel de 1 por ciento de probabilidad correspondiendo las mayores alturas para las dosis de 75-125-9, 75-150-9 y 150-150-18 kg/ha, de N-P-Zn respectivamente. El análisis de numero de vainas por planta muestra diferencias significativas entre tratamientos lo mismo que para el rendimiento de grano siendo los niveles adecuados para rendimiento en la zona de Masatepe el 150-150-0 y 75-75-0 kg/ha, de N-P-Zn con 1 101 y 1 044 kg/ha respectivamente.

Inflammatory Mediators of Hepatic Steatosis

7 p.

Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

RCeveierwamide and ceramide 1-phosphate in health and disease

Sphingolipids are essential components of cell membranes, and many of them regulate vital cell functions. In particular, ceramide plays crucial roles in cell signaling processes. Two major actions of ceramides are the promotion of cell cycle arrest and the induction of apoptosis. Phosphorylation of ceramide produces ceramide 1-phosphate (C1P), which has opposite effects to ceramide. C1P is mitogenic and has prosurvival properties. In addition, C1P is an important mediator of inflammatory responses, an action that takes place through stimulation of cytosolic phospholipase A2, and the subsequent release of arachidonic acid and prostaglandin formation. All of the former actions are thought to be mediated by intracellularly generated C1P. However, the recent observation that C1P stimulates macrophage chemotaxis implicates specific plasma membrane receptors that are coupled to Gi proteins. Hence, it can be concluded that C1P has dual actions in cells, as it can act as an intracellular second messenger to promote cell survival, or as an extracellular receptor agonist to stimulate cell migration.

Design, synthesis, and biological activity of rhodium metalloinsertors

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells.

Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle and induction of necrosis, which occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents.

In addition, ten distinct metalloinsertors with varying lipophilicities are synthesized and their mismatch binding affinities and biological activities studied. While they are found to have similar binding affinities, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show that all of these metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. Furthermore, metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cytotoxic and antiproliferative activities that are selective for cells deficient in MMR.

To explore further the basis of the unique selectivity of the metlloinsertors in targeting MMR-deficient cells, experiments were conducted using engineered NCI-H23 lung adenocarcinoma cells that contain a doxycycline-inducible shRNA which suppresses the expression of the MMR gene MLH1. Here we use this new cell line to further validate rhodium metalloinsertors as compounds capable of differentially inhibiting the proliferation of MMR-deficient cancer cells over isogenic MMR-proficient cells. General DNA damaging agents, such as cisplatin and etoposide, in contrast, are less effective in the induced cell line defective in MMR.

Finally, we describe a new subclass of metalloinsertors with enhanced potency and selectivity, in which the complexes show Rh-O coordination. In particular, it has been found that both Δ and Λ enantiomers of [Rh(chrysi)(phen)(DPE)]²⁺ bind to DNA with similar affinities, suggesting a possible different binding conformation than previous metalloinsertors. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than the FDA-approved anticancer drugs cisplatin and MNNG. Moreover, these activities are coupled with high levels of selectivity for MMR-deficient cells.

Determination of optimum stocking density of Macrobrachium rosenbergii larvae using multiple feed in a commercial hatchery at Cox’s Bazar, Bangladesh

Experimental stocking density of Macrobrachium rosenbergii in larval rearing was conducted in A.G. Aqua Hatchery, Chakaria, Bangladesh to study the effect of different stocking densities on growth, survival rate and diseases stress under hatchery condition. The research work was conducted using six cemented rectangular tanks having 3m3 capacity (1.5mX2mX1m) each. Stocking density were maintained in three experimental setup as 200, 150 and 100ind/L of the T1, T2 and T3 respectively with one replicate each. The larvae were fed with Artemia nauplii, Custard, Maxima and brine shrimp flakes. Water quality was maintained by exchanging 20-30% (12ppt saline water) daily. During the study period, temperature, pH, DO, salinity, nitrite-nitrogen, ammonia and alkalinity were maintained from 28.5-31.5ºC, 7.5-7.8, 5.8-5.9mg/L, 12-13ppt, 0.14-0.2 mg/L, 0.22-0.3mg/L, and 140-160mg/L respectively. The growth rates of larvae at 11th stage were recorded in terms of body length 0.115, 0.136, and 0.169 mm/day whereas body weight were observed 0.000115, 0.000180, and 0.000240g/day. The survival rate of larvae were found 21.8%, 30.4% and 51.3% in treatments T1, T2 and T3 respectively. PL was obtained as 43, 45, and 51PL/L and days required of 41, 38 and 34 days in stocking density of 200, 150, and 100ind/L respectively. It was found that the minimum of 34 days was required to attain the PL (12th stage) using the stocking density of 100 individuals/L. Cannibalism, Zoothamnium, Exuvia Entrapment Disease (EED), and Bacterial Necrosis (BN) were found to be the threat to the commercial hatchery operation that might responsible for potential larval damages which can be reduced by lowering the stocking densities in larval rearing tank that also increased the survival and growth rate.

Associação entre polimorfismos dos genes IL1B, IL1RN e TNFA e a periodontite agressiva e a periodontite crônica severa

A periodontite é um processo inflamatório crônico de origem bacteriana mediado por citocinas, em especial, interleucina-1 (IL1) e fator de necrose tumoral (TNFα). Polimorfismos genéticos de IL1 e TNFA têm sido associados com a variação de expressão dessas proteínas, o que poderia justificar as diferenças interindividuais de manifestação da doença. O objetivo do presente estudo foi investigar possíveis associações entre os genes IL1B, IL1RN e TNFA e a suscetibilidade à periodontite agressiva e à periodontite crônica severa. Foram selecionados 145 pacientes do Estado do Rio de Janeiro, 43 com periodontite agressiva (PAgr) (33,1 4,8 anos), 52 com periodontite crônica severa (PCr) (50,6 5,8 anos) e 50 controles (40,1 7,8 anos). Os DNAs genômicos dos integrantes dos grupos PAgr, PCr e controle foram obtidos através da coleta de células epiteliais bucais raspadas da parte interna da bochecha com cotonete. Os SNPs IL1B -511C>T, IL1B +3954C>T e TNFA -1031T>C foram analisados pela técnica de PCR-RFLP, utilizando as enzimas de restrição Ava I Taq I e Bpi I, respectivamente. O polimorfismo de número variável de repetições in tandem (VNTR) no intron 2 do gene IL1RN foi feita pela análise direta dos amplicons. Todos os polimorfismos foram analisados por eletroforese em gel de poliacrilamida 8%. As frequências alélica e genotípica do polimorfismo IL1B +3954C>T no grupo PCr foram significativamente diferentes das observadas no grupo controle (p=0,003 e p=0,041, respectivamente). A freqüência do alelo A2 do polimorfismo IL1RN VNTR intron2 no grupo PAgr foi significativamente maior do que no grupo controle (p=0,035). Não houve associação entre os polimorfismos IL1B -511C>T e TNFA -1031T>C e as periodontites agressiva e crônica. A presença dos alelos 2 nos genótipos combinados de IL1RN VNTR intron2 e IL1B +3954C>T no grupo PCr foi significativamente maior quando comparada ao grupo controle (p=0,045). Entretanto, não se observou associação entre as combinações genotípicas IL1B -511C>T / IL1B +3954C>T e IL1RN VNTR / IL1B -511C>T e a predisposição à doença periodontal. De acordo com os nossos resultados podemos sugerir que, para a população estudada, o polimorfismo IL1B +3954C>T interfere no desenvolvimento da periodontite crônica, enquanto a presença do alelo A2 do polimorfismo IL1RN VNTR intron2 pode ser considerado como indicador de risco para a periodontite agressiva. O presente estudo também nos permite sugerir que a ausência de homozigose dos alelos 1 nos genótipos combinados de IL1RN VNTR intron2 e IL1B +3954C>T pode representar maior suscetibilidade à periodontite crônica severa.

Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates

The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.

Here, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.

We have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.

We further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (γH2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.

Efeitos cardiovasculares da transferência adotiva de linfócitos T reguladores em camundongos submetidos à infusão crônica de angiotensina II e de aldosterona

A angiotensina (Ang) II e aldosterona induzem hipertensão arterial por mecanismos em parte mediados pela imunidade adaptativa, envolvendo linfócitos T auxiliares respondedores (Tresp). Os linfócitos T reguladores (Treg) são capazes de suprimir os efeitos próinflamatórios do sistema imune. O presente estudo avaliou se a transferência adotiva de Treg é capaz de prevenir a hipertensão e a lesão vascular induzidas pela Ang II ou pela aldosterona, em dois protocolos distintos. No protocolo com Ang II, camundongos machos C57BL/6 sofreram a injeção endovenosa de Treg ou Tresp, sendo depois infundidos com Ang II (1μg/kg/min), ou salina (grupo controle) por 14 dias. No protocolo com aldosterona, um outro conjunto de animais sofreu injeções de Treg ou Tresp, sendo depois infundido com aldosterona (600μg/kg/d) ou salina (grupo controle), pelo mesmo intervalo de tempo. O grupo tratado com aldosterona recebeu salina 1% na água. Tanto o grupo Ang II como aldosterona apresentaram elevação da pressão arterial sistólica (43% e 31% respectivamente), da atividade da NADPH oxidase na aorta (1,5 e 1,9 vezes, respectivamente) e no coração (1,8 e 2,4 vezes, respectivamente) e uma redução da resposta vasodilatadora à acetilcolina (de 70% e 56%, respectivamente), quando comparados com os respectivos controles (P<0,05). Adicionalmente, a administração de Ang II proporcionou um aumento rigidez vascular (P<0,001), na expressão de VCAM-1 nas artérias mesentéricas (P<0,05), na infiltração aórtica de macrófagos e linfócitos T (P<0,001) e nos níveis plasmáticos das citocinas inflamatórias interferon (INF)-γ, interleucina (IL)-6, Tumor necrosis factor (TNF)-α e IL-10 (P<0,05). Ang II causou uma queda de 43% no número de células Foxp3+ no córtex renal, enquanto que a transferência adotiva de Treg aumentou as células Foxp3+ em duas vezes em comparação com o controle. A administração de Treg preveniu o remodelamento vascular induzido pela aldosterona, observado na relação média/lúmen e na área transversal da média das artérias mesentéricas (P<0,05). Todos os parâmetros acima foram prevenidos com a administração de Treg, mas não de Tresp. Estes resultados demonstram que Treg são capazes de impedir a lesão vascular e a hipertensão mediadas por Ang II ou por aldosterona, em parte através de ações antiinflamatórias. Em conclusão, uma abordagem imuno-modulatória pode prevenir o aumento da pressão arterial, o estresse oxidativo vascular, a inflamação e a disfunção endotelial induzidos por Ang II ou aldosterona.

Atividade pró-trombótica da toxina ExoU de Pseudomonas aeruginosa detectada em modelos experimentais in vivo e in vitro

Pseudomonas aeruginosa é um importante agente de pneumonia, particularmente em pacientes submetidos à ventilação mecânica, que pode evoluir para sepse, com elevadas taxas de letalidade. Na sepse, o processo inflamatório sistêmico exacerbado favorece o desequilíbrio entre as vias de coagulação e fibrinólise e a instalação de um estado pró-coagulante, com o aparecimento de trombose microvascular, coagulação intravascular disseminada e falência de múltiplos órgãos. Conhecendo a potente atividade pró-inflamatória da toxina ExoU produzida por P. aeruginosa, decorrente de sua atividade fosfolipásica A2, o objetivo desta tese foi investigar seu potencial de indução de alterações hemostáticas relacionadas à patogênese da sepse. Utilizando modelo de sepse em camundongos inoculados, por via intratraqueal, com suspensões de P. aeruginosa produtora de ExoU (PA103) ou de cepa com deleção do gene exoU, não produtora da toxina, foi mostrado que ExoU determinou maior gravidade da infecção, maior taxa de letalidade, leucopenia, trombocitose, hiperpermeabilidade vascular e transudação plasmática, evidenciadas, respectivamente, pela maior concentração de proteínas nos lavados broncoalveolares (LBAs) e acúmulo do corante Azul de Evans, previamente inoculado nos animais, por via endovenosa, no parênquima renal. ExoU favoreceu, também, a ativação plaquetária, confirmada pela maior concentração de plaquetas expressando P-selectina em sua superfície, maior número de micropartículas derivadas de plaquetas e maior concentração plasmática de tromboxano A2. A histopatologia dos pulmões e rins dos animais infectados com PA103 confirmou a formação de microtrombos, que não foram detectados nos animais controles ou infectados com a cepa mutante. Nos pulmões, a produção de ExoU determinou intensa resposta inflamatória com maior concentração de leucócitos totais e polimorfonucleados, interleucina-6 e fator de necrose tumoral-α nos LBAs. A análise imunohistoquímica mostrou intensa deposição de fibrina nos alvéolos e septos interalveolares. A atividade pró-coagulante dependente do fator tissular detectada nos LBAs dos camundongos infectados com PA103 foi independente da produção do inibidor da via de ativação do fator tissular (TFPI), mas associada ao aumento da produção do inibidor do ativador do plasminogênio-1 (PAI-1). Para investigar a participação do fator de ativação plaquetária (PAF) na liberação de PAI-1, foi pesquisada a atividade da enzima PAF-acetil-hidrolase (PAF-AH) nos LBAs dos camundongos. A atividade de PAF-AH apresentou-se significativamente elevada nos LBA dos camundongos infectados com PA103. O tratamento dos animais com um inibidor do PAF, antes da infecção, resultou na diminuição significativa das concentrações de PAI-1 e de leucócitos totais, bem como da atividade pró-coagulante dos LBAs. In vitro, ExoU induziu maior expressão do RNA mensageiro de PAI-1 e maior liberação da proteína PAI-1 nos sobrenadantes de células epiteliais respiratórias da linhagem A549. O tratamento das células A549 com um anticorpo anti-receptor de PAF, antes da infecção, reduziu significativamente a concentração de PAI-1 nos sobrenadantes de células infectadas com a cepa selvagem. Estes resultados demonstraram um novo mecanismo de virulência de P. aeruginosa através da atividade pró-trombótica de ExoU e a possibilidade de utilização da identificação de ExoU em isolados clínicos de pacientes graves como um marcador prognóstico para estes pacientes.