Leukotrienes Produced in Allergic Lung Inflammation Activate Alveolar Macrophages

It has been well-documented that leukotrienes (LTs) are released in allergic lung inflammation and that they participate in the physiopathology of asthma. A role for LTs in innate immunity has recently emerged: Cys-LTs were shown to enhance Fc gamma R-mediated phagocytosis by alveolar macrophages (AMs). Thus, using a rat model of asthma, we evaluated Fc gamma R-mediated phagocytosis and killing of Klebsiella pneumoniae by AMs. The effect of treatment with a cys-LT antagonist (montelukast) on macrophage function was also investigated. Male Wistar rats were immunized twice with OVA/alumen intraperitoneally and challenged with OVA aerosol. After 24 h, the animals were killed, and the AMs were obtained by bronchoalveolar lavage. Macrophages were cultured with IgG-opsonized red blood cells (50: 1) or IgG-opsonized K. pneumoniae (30: 1), and phagocytosis or killing was evaluated. Leukotriene C(4) and nitric oxide were quantified by the EIA and Griess methods, respectively. The results showed that AMs from sensitized and challenged rats presented a markedly increased phagocytic capacity via Fc gamma R (10X compared to controls) and enhanced killing of K. pneumoniae (4X higher than controls). The increased phagocytosis was inhibited 15X and killing 3X by treatment of the rats with montelukast, as compared to the non-treated group. cys-LT addition increased phagocytosis in control AMs but had no effect on macrophages from allergic lungs. Montelukast reduced nitric oxide (39%) and LTC(4) (73%). These results suggest that LTs produced during allergic lung inflammation potentiate the capacity of AMs to phagocytose and kill K. pneumonia via Fc gamma R. Copyright (C) 2010 S. Karger AG, Basel

A combination of techniques to evaluate photodynamic efficiency of photosensitizers

Photodynamic therapy, used mainly for cancer treatment and microorganisms inaction, is based on production of reactive oxygen species by light irradiation of a sensitizer. Hematoporphyrin derivatives as Photofrin (R) (PF) Photogem (R) (PG) and Photosan (R) (PF), and chlorin-c6-derivatives as Photodithazine (R)(PZ), have suitable sensitizing properties. The present study provides a way to make a fast previous evaluation of photosensitizers efficacy by a combination of techniques: a) use of brovine serum albumin and uric acid as chemical dosimeters; b) photo-hemolysis of red blood cells used as a cell membrane interaction model, and c) octanol/phosphate buffer partition to assess the relative lipophilicity of the compounds. The results suggest the photodynamic efficient rankings PZ > PG >= PF > PS. These results agree with the cytotoxicity of the photosensitizers as well as to chromatographic separation of the HpDs, both performed in our group, showing that the more lipophilic is the dye, the more acute is the damage to the RBC membrane and the oxidation of indol, which is immersed in the hydrophobic region of albumin.

eNOS T-786C polymorphism affects atorvastatin-induced changes in erythrocyte membrane fluidity

Statins have pleiotropic effects, including endothelial nitric oxide synthase (eNOS) upregulation and increased nitric oxide formation, which can be modulated by a genetic polymorphism in the promoter region of the eNOS gene (T-786C). Here, we report our investigation of whether this polymorphism modulates the effects of atorvastatin on the fluidity of erythrocyte membranes. We genotyped 200 healthy subjects (males, 18-60 years of age) and then randomly selected 15 of these with the TT genotype and 15 with the CC genotype to receive placebo or atorvastatin (10 mg/day oral administration) for 14 days. Cell membrane fluidity was evaluated by electron paramagnetic resonance (EPR) and spin-labeling method. The EPR spectra were registered on a VARIAN-E4 spectrometer. Thiobarbituric acid-reactive species (TBA-RS) and plasma membrane cholesterol were determined in the erythrocytes. Atorvastatin reduced membrane fluidity in CC subjects (P < 0.05) but not in those with the TT genotype (P > 0.05). While no significant differences were found in plasma membrane cholesterol concentrations, higher TBA-RS concentrations were found in the CC subjects than in the TT subjects (P < 0.05). These findings suggest that a short treatment with atorvastatin is disadvantageous to subjects with the CC genotype for the T-786C polymorphism compared to those with TT genotype, at least in terms of the hemorheological properties of erythrocytes.

Multiple stages of detergent-erythrocyte membrane interaction-A spin label study

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate- and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9 mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5 mM detergent. Between 3.2 and 10 mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10 mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs: the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5 mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111). (C) 2010 Elsevier B.V. All rights reserved.

Similarity between the in vitro activity and toxicity of two different fungizone™ / lipofundin™ admixtures

Amphotericin B (AmB), an antifungal agent that presents a broad spectrum of activity, remains the gold standard in the antifungal therapy. However, sometimes the high level of toxicity forbids its clinical use. The aim of this work was to evaluate and compare the efficacy and toxicity in vitro of Fungizon™ (AmB-D) and two new different AmB formulations. Methods: three products were studied: Fungizon™, and two Fungizon™ /Lipofundin™ admixtures, which were diluted through two methods: in the first one, Fungizon™ was previously diluted with water for injection and then, in Lipofundin™ (AmB-DAL); the second method consisted of a primary dilution of AmB-D as a powder in the referred emulsion (AmB-DL). For the in vitro assay, two cell models were used: Red Blood Cells (RBC) from human donors and Candida tropicallis (Ct). The in vitro evaluation (K+ leakage, hemoglobin leakage and cell survival rate-CSR) was performed at four AmB concentrations (from 50 to 0.05mg.L-1). Results: The results showed that the action of AmB was not only concentration dependent, but also cellular type and vehicle kind dependent. At AmB concentrations of 50 mg.L-1, although the hemoglobin leakage for AmB-D was almost complete (99.51), for AmB-DAL and AmB-DL this value tended to zero. The p = 0.000 showed that AmB-D was significantly more hemolytic. Conclusion: The Fungizon™- Lipofundin™ admixtures seem to be the more valuable AmB carrier systems due to their best therapeutic index presented

Análise in vitro da atividade antifúngica e de toxicidade da anfotericina B pré-aquecida

The aim of this work was to evaluate how an aqueous micellar system containing Amphotericin B (AmB) and sodium deoxycholate (DOC) can be rebuilt after heating treatment. Also a review of the literature about the new physicochemical and biological properties of this new system was carried out. Afterwards, heated (AmB-DOC-H) and unheated (AmB-DOC) micelles were subsequently diluted at four different concentrations (50mg.L-1, 5mg.L-1, 0.5mg.L-1 and 0.05mg.L-1) to perform the physicochemical study and, then, the pharmacotoxicity assay, in which two cell models were used for the in vitro experiments, Red Blood Cells (RBC) from human donors and Candida parapisilosis (Cp). While potassium (K+) and hemoglobin leakage from RBC were the used parameters to evaluate the acute and chronic toxicity, respectively, the efficacy of AmB-DOC and AmB-DOC-H were assessed by K+ leakage and cell survival rate from Cp. The spectral study revealed a slight change on the aggregate peak from 327nm to 323nm for AmB-DOC-H compared to AmB-DOC. Concerning the toxicity, although AmB-DOC and AmB-DOC-H presented different behavior for hemoglobin leakage, AmB-DOC produced higher leakage than AmB-DOC-H at high concentrations (from 5mg.L-1) with values tending to zero. However, concerning K+ leakage, both AmB-DOC and AmB-DOC-H, showed similar profile for both cell models, RBC and Cp (p<0,05). AmB-DOC-H and AmB-DOC also revealed similar profile of activity against Cp with equivalent survival rate. In short, the AmB-DOC-H showed much less toxicity than AmB-DOC, but remained as active as the late one against fungal cell. Therefore, the results highlight the importance of this new procedure as a simple, inexpensive and safe alternative to produce a new kind of micelle system for treatment of systemic fungal infections

Biological indicators of stress in pacu (Piaractus mesopotamicus) after capture

Os efeitos da captura (perseguição, contenção em puçá e exposição aérea) no perfil sanguíneo do cortisol, glicose, cloreto, sódio, potássio, cálcio e na osmolaridade, hematócrito, hemoglobina, número de células vermelhas (CV) e volume corpuscular médio (VCM) foram investigados no pacu (Piaractus mesopotamicus). Um total de 132 peixes (49,7 ± 11,7 g) foi submetido à captura com 3 ou 5 minutos de exposição aérea. Nove peixes de cada tratamento foram amostrados 5, 15, 30, 60 minutos e 24 horas depois e outros nove peixes foram amostrados antes da captura e considerados controle. A captura resultou em aumento do cortisol e glicose no sangue 30 e 5 minutos depois da captura, respectivamente, independente do tempo de exposição aérea. Ambos os indicadores recuperaram os valores controle em 24 horas. Nos dois grupos de peixes, o cloreto plasmático diminuiu 60 minutos após captura e não recuperou os valores controle, enquanto o sódio sérico aumentou entre 15 e 30 minutos recuperando a condição controle em 24 horas. Não houve alteração significativa nos valores de potássio, cálcio, osmolaridade ou no hematócrito, hemoglobina, CV e VCM como consequência da captura. Os estressores sequenciais aplicados no pacu durante a captura ativaram o eixo cérebro-pituitária-interrenal (respostas do cortisol e glicose), mas a ativação do eixo cérebro-sistema simpático-células cromafins foi aparentemente moderada (respostas iônicas e hematológicas).

Parâmetros hematológicos de jumentas gestantes em diferentes períodos

The hematological parameters and total protein concentration in healthy donkeys during pregnancy, under grass handling conditions in tropical weather, were evaluated. Fifth-two Marchador Brasileiro breed healthy donkeys, ranging from 8 to 16 years old, were evaluated, 36 of which were pregnant. The animals were separated in four groups: non pregnant (control), pregnancy from 25 to 110 days (first phase), from 111 to 210 days (second phase), and from 211 to 340 days (third phase). Total protein, erythrocyte, and leukocytes counts; packed cell volume; hemoglobin concentration; hematimetric absolute rates of mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); and mean corpuscular hemoglobin concentration (MCHC) were evaluated. Total protein and eosinophils count decreased in the first phase. Red blood cells, hemoglobin concentration, and MCHC increased in the third phase of pregnancy. However, MCV and MCH decreased in the same phase.

T lymphocyte subsets of the umbilical cord blood of dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antibody and blood leukocyte response in Rhipicephalus sanguineus (Latreille, 1806) tick-infested dogs and guinea pigs

The dog is considered to be the natural host of Rhipicephalus sanguineus and is unable to develop appreciable resistance even after repeated feedings. The guinea pig develops strong resistance after one infestation with adult ticks. Antibody (IgG) titres against tick salivary gland antigens (SGAs) and blood leukocyte numbers in dogs and guinea pigs undergoing experimental R. sanguineus tick infestations were measured to detect a possible correlation with susceptibility or resistance of hosts. Since infested dogs develop an immediate hypersensitivity reaction to R. sanguineus antigens, total and anti-R. sanguineus SGA IgE levels were also measured in this host species. IgG and IgE antibody levels were determined by an enzyme-linked immunosorbent assay (ELISA) along three consecutive infestations of both hosts. Most dogs and guinea pigs displayed low IgG levels against R. sanguineus SGAs, though marked differences in individual response were observed. Although dog's total serum IgE levels increased significantly after infestations, no change in the amount of anti-salivary gland IgE was detected. Total and differential blood cell counts were determined in dogs and guinea pigs during primary and secondary infestation. In dogs, a tertiary infestation and a subsequent higher infestation level were also evaluated. Infested dogs did not display any alteration in blood leukocyte counts throughout the experiment. Guinea pigs, on the other hand, developed a significant basophilia during primary infestation which increased further during secondary infestation. These data reveal similarities and differences in the reactions of resistant and non-resistant hosts to ticks. They contribute for the understanding of such host-parasite relationships and will hopefully aid in the development of immune control of ticks. (C) 2003 Elsevier B.V. All rights reserved.

Feed restriction on performance and blood parameters of broilers fed diets with different sodium levels

The experiment was conducted to evaluate the effect of feed restriction and different sodium levels in the diet on the performance and hematological values (erythrocyte, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration and white blood cell) of broilers from 22 to 42 days of age. A completely randomized design was used in a 4 x 2 x 2 factorial (levels of sodium: 0.16%, 0.19%, 0.22% and 0.25%; with or without feed restriction; male and female), and two replicates of thirty broilers each. Sodium levels did not affect performance (p>0.05). Feed restriction resulted in increased feed intake and affected weight gain and feed conversion negatively (p<0.05). Better weight gain and feed conversion (p<0.05) were seen in male broilers. Hematological values were not affected by sodium levels, feed restriction or sex (p>0.05).

How can micelle systems be rebuilt by a heating process?

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Lack of clastogenic/genotoxic effects of Baccharis dracunculifolia extract on Swiss mouse peripheral blood cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lavado traqueobrônquico por via nasotraqueal como metodologia de colheita de células do trato respiratório de ovinos sadios e portadores de afecções pulmonares

Os estudos das secreções traqueobrônquicas são amplamente utilizados nas pesquisas de doenças pulmonares nas diversas espécies animais, inclusive no homem. Os objetivos desta pesquisa foram a viabilização da técnica de colheita de lavado traqueobrônquico na espécie ovina e o estudo da relação clínico-citológica do lavado de ovinos portadores de broncopneumonia e sadios. Foram utilizados 33 ovinos, 18 sadios e 15 portadores de enfermidade respiratória com sinais clínicos de envolvimento das vias aéreas, divididos nos respectivos grupos, GS e GD. Após o exame físico foi realizado o lavado traqueobrônquico por via nasotraqueal. A colheita do lavado foi feita com a inoculação e aspiração de solução fisiológica estéril. As amostras foram processadas citologicamente através de citocentrifugação e coradas pelos métodos Wright-Giemsa e Shorr. Tanto a contagem total de células epiteliais quanto o número de hemácias por mililitro foi maior no grupo de animais com broncopneumonia. Nos animais sadios notou-se predomínio de macrófagos, seguido por células epiteliais cilíndricas, neutrófilos e linfócitos. No grupo de animais doentes havia menor número de macrófagos, e predomínio da população de neutrófilos. Por ser de fácil realização, pouco dispendiosa e pela obtenção representativa de material, a técnica estudada mostrou-se eficaz na obtenção de fluidos traqueobrônquicos e, portanto um bom método de colheita de células para uso nas pesquisas de vias aéreas.

Fish ladders select fish traits on migration - still a growing problem for natural fish populations

We investigated the potential selective effect of fish ladders on physiological and morphological profiles of the curimbata, Prochilodus lineatus, during reproductive migration in Brazil. We registered sex, body weight and length, plasma glucose, hepatosomatic and gonadosomatic indices (HSI and GSI, respectively), hematocrit, leucocrit, blood cell and nucleus areas, and the diameter of white and red muscle fibers in fish sampled at the bottom (downstream) and at the top (upstream) of a fish ladder at a hydroelectric dam. Males and females at the top of the ladder showed higher size (weight and length), white muscle fiber diameters, plasma glucose levels and lower hematocrit when compared with those at the bottom. These size and muscle traits assist fish to overcome the ladder barrier and bypass the dam, an effort that might be reflected in the glucose levels. Females also showed higher GSI at the top of the fish ladder, a trait possibly facilitating their reproduction upstream. These results indicate that a dam system favors fish with specific morphological-physiological profile. This may have a strong influence upon upstream fish populations over generations and implies the presence of artificial selective pressure.