Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degreesC. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degreesC.Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme.An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Use of Poisson spatiotemporal regression models for the Brazilian Amazon Forest: malaria count data

INTRODUÇÃO: A malaria é uma doença endêmica na região da Amazônia Brasileira, e a detecção de possíveis fatores de risco pode ser de grande interesse às autoridades em saúde pública. O objetivo deste artigo é investigar a associação entre variáveis ambientais e os registros anuais de malária na região amazônica usando métodos bayesianos espaço-temporais. MÉTODOS: Utilizaram-se modelos de regressão espaço-temporais de Poisson para analisar os dados anuais de contagem de casos de malária entre os anos de 1999 a 2008, considerando a presença de alguns fatores como a taxa de desflorestamento. em uma abordagem bayesiana, as inferências foram obtidas por métodos Monte Carlo em cadeias de Markov (MCMC) que simularam amostras para a distribuição conjunta a posteriori de interesse. A discriminação de diferentes modelos também foi discutida. RESULTADOS: O modelo aqui proposto sugeriu que a taxa de desflorestamento, o número de habitants por km² e o índice de desenvolvimento humano (IDH) são importantes para a predição de casos de malária. CONCLUSÕES: É possível concluir que o desenvolvimento humano, o crescimento populacional, o desflorestamento e as alterações ecológicas associadas a estes fatores estão associados ao aumento do risco de malária. Pode-se ainda concluir que o uso de modelos de regressão de Poisson que capturam o efeito temporal e espacial em um enfoque bayesiano é uma boa estratégia para modelar dados de contagem de malária.

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

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

Eritrograma, metabolismo do ferro e concentração sérica de eritropoetina em fêmeas caprinas da raça Saanen nos períodos de gestação, parto e pós-parto

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

Pulmonary tuberculosis: Hematology, serum biochemistry and the relation with the disease duration

The aim of the present study was to analyze the relationship between hematological and biochemical parameters and tuberculosis process activity time according to clinical complaint duration. It was a retrospective study analyzing medical records from 80 pulmonary tuberculosis patients at Botucatu Medical School University Hospital ( Botucatu, São Paulo State, Brazil), who were divided into 2 groups according to clinical complaint duration: Group 1 ( G1) - up to three months; Group 2 ( G2) - over three months. Parameters included: age, gender, bacilloscopy, erythrocyte sedimentation rate ( ESR), platelet count, alpha1-globulin, alpha2-globulin, gamma globulin, mucoprotein, alpha1-acid glycoprotein values, and the presence of risk factors such as smoking, alcoholism, drug addiction, sexual promiscuity, contact with tuberculosis carriers, and previous treatment. Groups were compared by calculating t and p, and Chi-square (X-2) and p. Comparisons revealed a tendency towards smoking with a higher frequency of smokers in G1 ( 0.05< p< 0.10). G1 also tended to present greater platelet values than G2 ( 0.05< p< 0.10) and presented significantly higher ESR values than G2 ( p< 0.05). Other factors did not show any significantly different behavior between groups ( p> 0.05). A correlation was found between ESR, platelet count, smoking and less than three months clinical duration.

Decreased oxidative stress in patients with ulcerative colitis supplemented with fish oil omega-3 fatty acids

OBJECTIVE: the potential pathogenicity of free radicals may have a pivotal role in ulcerative colitis. Fish oil omega-3 fatty acids exert anti-inflammatory effects on patients with ulcerative colitis (UC), but the precise mechanism of the action of fish oil on oxidative stress is still controversial. The aim of the present work was to verify the blood oxidative stress in patients with UC and determine whether the association of sulfasalazine to fish oil omega-3 fatty acids is more effective than isolated use of sulfasalazine to reduce the oxidative stress.METHODS:, Nine patients (seven female and two male; me. an age = 40 +/- 11 y) with mild or moderate active UC were studied in a randomized crossover design. In addition to their usual medication (2 g/d of sulfasalazine), they received fish oil omega-3 fatty acids (4.5 g/d) or placebo for 2-mo treatment periods that were separated by 2 mo, when they only received sulfasalazine. Nine healthy individuals served as control subjects to study the oxidative stress status. Disease activity was assessed by laboratory indicators (C-reactive protein, alpha(1)-acid glycoprotein, alpha(1)-antitrypsin, erythrocyte sedimentation rate, albumin, hemoglobin, and platelet count), sigmoidoscopy, and histology scores. Analysis of oxidative stress was assessed by plasma chemiluminescence and erythrocyte lipid peroxidation, both induced by tert butyl hydroperoxide (t-BuOOH) and by plasma malondialdehyde. Antioxidant status was assayed by total plasma antioxidant capacity (TRAP) and microsomal lipid peroxidation inhibition (LPI). Superoxide dismutase (SOD) and catalase erythrocyte enzymatic activities were also determined.RESULTS: No significant changes were observed in any laboratory indicator or in the sigmoidoscopy or histology scores, with the exception of erythrocyte sedimentation rate, which decreased with both treatments. Oxidative stress was demonstrated by significant decreases in TRAP and LPI levels, increased chemiluminescence induced by t-BuOOH, and higher SOD activity in patients with UC. Treatment with fish oil omega-3 fatty acids reverted the chemiluminescence induced by t-BuOOH and LPI to baseline levels but that did not occur when patients received only sulfasalazine. Levels of plasma malondialdehyde, erythrocyte lipid peroxidation, and catalase were not different from those in the control group.CONCLUSIONS: the results indicated that plasma oxidative stress occurs in patients with UC, and there was a significant decrease when the patients used sulfasalazine plus fish oil omega-3 fatty acids. However, there was no improvement in most laboratory indicators, sigmoidoscopy, and histology scores. The results suggested that omega-3 fatty acids may act as free radical scavengers protecting the patients against the overall effect of oxidative stress. (C)Elsevier B.V. 2003.

Comparison of omega-3 fatty acids and sulfasalazine in ulcerative colitis

Fish oil omega-3 fatty acids exert antiinflammatory effects on patients with ulcerative colitis. However, a comparative study in patients with mild to moderate ulcerative colitis receiving only sulfasalazine or omega-3 fatty acids has not been performed. We sought to detect changes in the inflammatory disease activity with the use of either fish oil omega-3 fatty acids or sulfasalazine in patients with ulcerative colitis. Ten patients (five male, five female; mean age = 48 +/- 12 y) with mild to moderate active ulcerative colitis were investigated in a randomized cross-over design. They received either sulfasalazine (2 g/d) or omega-3 fatty acids (5.4 g/d) for 2 mo. Disease activity was assessed by clinical and laboratory indicators, sigmoidoscopy, histology, and whole-body protein turnover (with N-15-glycine). Treatment with w-3 fatty acids resulted in greater disease activity as detected by a significant increase in platelet count, erythrocyte sedimentation rate, C-reactive protein, and total fecal nitrogen excretion. No major changes in protein synthesis and breakdown were observed during either treatment. In conclusion, treatment with sulfasalazine is superior to treatment with omega-3 fatty acids in patients with mild to moderate active ulcerative colitis. Nutrition 2000;16:87-901 (C) Elsevier B.V. 2000.

Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1

Background: In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs). Objective: The aim of this study was to develop a flow cytometric assay for DEA 1.1 and determine whether DEA 1.1 is present on canine platelets.Methods: Blood was collected from 172 clinically healthy dogs. Platelets and erythrocytes from each dog were tested for DEA 1.1 by flow cytometry using anti-DEA 1.1 blood-typing sera. Erythrocytes from each dog were also assessed for DEA 1.1 using a standard tube-typing test (T1) and using a second tube method (T2), if the flow cytometric and T1 results differed.Results: Using flow cytometry, DEA 1.1 was detected on erythrocytes of all 110 dogs shown by T1 or T2 testing to be DEA 1.1-positive. Initial results of the T1 test had a diagnostic accuracy of 93% (160 correct/ 172 tests). The frequency of erythrocyte DEA 1.1 positivity in previously untyped dogs (n = 118) was 56%. DEA 1.1 expression was not detected on platelets from DEA 1.1-positive dogs.Conclusions: Flow cytometry was a reliable method for detection of DEA 1.1 on canine erythrocytes. The absence of DEA 1.1 on platelets from DEA 1.1-positive dogs suggests that their platelets do not express DEA 1.1 and will not induce production of anti-DEA 1.1 antibodies that might lead to platelet refractoriness or reactions to a subsequent transfusion of DEA 1.1positive erythrocytes.

Somaticell® as a screening method for somatic cell count from bovine milk

Os objetivos do presente estudo foram avaliar a correlação entre a contagem eletrônica de células somáticas (eCCS) com o Somaticell® sob diferentes níveis de contagem de células somáticas (CCS) do leite e patógenos causadores de mastites, além de calcular a sensibilidade, especificidade e valores preditivos do Somaticell® utilizando diferentes limites de CCS estabelecidos pelos diferentes países. Trezentos e quarenta amostras de leite foram coletadas assepticamente após realização do California Mastitis Test (CMT). O Somaticell® e a eCCS foram realizados em todas as amostras de leite. A correlação entre o Somaticell® e a contagem eletrônica foi determinada de acordo com o CMT, patógeno isolado e escore de eCCS. de acordo com os escores de CCS estabelecidos, 26,5% das amostras de leite apresentaram escore 1 (69-166 x10³células mL-1), 26,8% escore 2 (167-418x10³células mL-1), 27,4% escore 3 (419-760x10³células mL-1) e 19,4% escore 4 (761 to 1970x10³células mL-1). A eCCS e o Somaticell® apresentaram correlação positiva em quase todos os escores estudados (exceto escore 2 e 3). O valor de r obtido entre CCS e o Somaticell® foi de 0,32. Observou-se que, quando o limite de CCS estabelecido aumentou, a sensibilidade decresceu e os valores de especificidade aumentaram. Os valores preditivos apresentaram-se constantes em todos os limites. Quando o limite de CCS era baixo (<760,000 células mL-1), Somaticell® forneceu resultados consistentemente mais elevados que os valores de CCS. Já para amostras com CCS elevada, Somaticell® resultou em menores contagens que a eCCS. A correlação entre os dois métodos permaneceu relativamente constante em todas as condições e os valores de sensibilidade e especificidade do teste são altamente dependentes do limite estabelecido. Os resultados deste trabalho sugerem que o Somaticell® não é útil para avaliar a CCS do leite, pois seus resultados são significativamente diferentes da eCCS, no entanto, pode ser utilizado como método de triagem, tal como o CMT, para a detecção do aumento da CCS do leite.

Effect of age and abomasal puncture on peritoneal fluid, hematology, and serum biochemical analyses in young calves

The goals of this study were to evaluate techniques for collection of peritoneal fluid from calves, establish reference ranges for fibrinogen in peritoneal fluid during the 1st month of life, and determine if abomasal puncture would alter peritoneal fluid or hematologic variables. Twenty-two healthy Holstein calves underwent 3 peritoneal fluid collections on day 1, day 15, and day 30 of age. Fibrinogen concentration in peritoneal fluid was 0.20 g/dL and 0.10 g/dL (P < .05) for day 1 and day 30, respectively, and 0.10 at day 15 (P > .05) for calves without abomasal puncture. Plasma fibrinogen concentration was 0.60 g/dL and 0.70 g/dL (P < .05) for days 15 and 30, respectively, in calves without abomasal puncture. There were no significant differences (P <= .05) in peritoneal fluid and peripheral blood total protein and fibrinogen concentrations, specific gravity, total and differential cell count, or erythrocyte counts between calves with or without abomasal puncture. We concluded that the reference ranges established for fibrinogen and total protein concentration are important for accurate evaluation of peritoneal fluid in calves for further comparison with similar-aged animals with gastrointestinal-tract or abdominal-cavity disease. Additionally, accidental abomasal puncture does not alter values of fibrinogen, total protein, and nucleated cell Count in peritoneal fluid and does not cause apparent clinical abnormalities.

Haematological alterations of Leporinus macrocephalus (Osteichtyes: Anostomidae) naturally infected by Goezia leporini (Nematoda: Anisakidae) in fish pond

Estudou-se efeito da infecção por Goezia leporini Martins & Yoshitoshi, 2003 (Nematoda: Anisakidae) sobre as características hematológicas de Leporinus macrocephalus (Osteichthyes:Anostomidae) cultivado. Palidez das brânquias, rins, fígado e coração, pontos negros nos rins e acúmulo de líquido na cavidade visceral, estômago e intestinos foram observados. O conteúdo da vesícula biliar tinha aparência pálida e translúcida. Observaram-se alta e moderada correlações positivas entre número de nematóides e peso do peixe estimadas dentro dos grupos de peixe de 0-100g e 100-200g, respectivamente. As extensões sangüíneas revelaram variações no tamanho (anisocitose) e forma (poiquilocitose) dos eritrócitos, bem como eritrócitos em divisão. Não houve alteração (P>0,05) na contagem total de eritrócitos, de leucócitos, na taxa de hemoglobina e nos percentuais de trombócitos e monócitos. A infecção provocou redução (P<0,05) no percentual de hematócrito, no volume corpuscular médio, na concentração de hemoglobina corpuscular média e no percentual de linfócitos, e aumento (P<0,05) no percentual de neutrófilos e eosinófilos no sangue circulante de peixes infectados. Este é o primeiro relato no Brasil que relaciona hematologia e infecção por nematóides em peixes cultivados.

Hematological values and total protein of Brasileiro de Hipismo and Breton mares during pregnancy

Estudaram-se as variáveis eritrométricas, leucométricas e a concentração de proteína total (PT) de éguas gestantes das raças Brasileiro de Hipismo (BH) e Bretão. Utilizaram-se 175 éguas sadias, com 3 a 13 anos de idade. Dentre as éguas utilizadas, 89 foram da raça BH, divididas em 14 não-prenhes e 75 prenhes, e 86 éguas da raça Bretão divididas em 11 não-prenhes e 75 prenhes. As éguas foram subdivididas em quatro grupos por raça: grupo controle (éguas não-prenhes, n=14 para BH e n=11 para Bretão); grupo I (primeiro período de gestação, 25-110 dias, n=25 para BH e Bretão), grupo II (segundo período de gestação, 111-210 dias, n=25 para BH e Bretão); e grupo III (terceiro período de gestação, 211-340 dias, n=25 para BH e Bretão). Os valores médios de cada variável eritrométrica, leucométrica e de proteína total foram obtidos para cada raça e, posteriormente, realizou-se comparação entre as mesmas. O número de hemácias (He), volume globular (VG) e teor de hemoglobina (Hb) aumentaram no segundo período de gestação e diminuíram no último período tanto para a raça BH, como para a raça Bretão. Além disso, essas mesmas variáveis apresentaram diferença estatística significativa tanto nas éguas não-prenhas como nas prenhes durante os três períodos gestacionais. Os valores médios obtidos de leucócitos e neutrófilos segmentados aumentaram a partir do segundo período de gestação para as duas raças estudadas.