A method for multiple sequential analyses of macrophage functions using a small single cell sample

Microbial pathogens such as bacillus Calmette-Guérin (BCG) induce the activation of macrophages. Activated macrophages can be characterized by the increased production of reactive oxygen and nitrogen metabolites, generated via NADPH oxidase and inducible nitric oxide synthase, respectively, and by the increased expression of major histocompatibility complex class II molecules (MHC II). Multiple microassays have been developed to measure these parameters. Usually each assay requires 2-5 x 10(5) cells per well. In some experimental conditions the number of cells is the limiting factor for the phenotypic characterization of macrophages. Here we describe a method whereby this limitation can be circumvented. Using a single 96-well microassay and a very small number of peritoneal cells obtained from C3H/HePas mice, containing as little as <=2 x 10(5) macrophages per well, we determined sequentially the oxidative burst (H2O2), nitric oxide production and MHC II (IAk) expression of BCG-activated macrophages. More specifically, with 100 µl of cell suspension it was possible to quantify H2O2 release and nitric oxide production after 1 and 48 h, respectively, and IAk expression after 48 h of cell culture. In addition, this microassay is easy to perform, highly reproducible and more economical.

Application of a modified sequential organ failure assessment score to critically ill patients

The purpose of the present study was to explore the usefulness of the Mexican sequential organ failure assessment (MEXSOFA) score for assessing the risk of mortality for critically ill patients in the ICU. A total of 232 consecutive patients admitted to an ICU were included in the study. The MEXSOFA was calculated using the original SOFA scoring system with two modifications: the PaO2/FiO2 ratio was replaced with the SpO2/FiO2 ratio, and the evaluation of neurologic dysfunction was excluded. The ICU mortality rate was 20.2%. Patients with an initial MEXSOFA score of 9 points or less calculated during the first 24 h after admission to the ICU had a mortality rate of 14.8%, while those with an initial MEXSOFA score of 10 points or more had a mortality rate of 40%. The MEXSOFA score at 48 h was also associated with mortality: patients with a score of 9 points or less had a mortality rate of 14.1%, while those with a score of 10 points or more had a mortality rate of 50%. In a multivariate analysis, only the MEXSOFA score at 48 h was an independent predictor for in-ICU death with an OR = 1.35 (95%CI = 1.14-1.59, P < 0.001). The SOFA and MEXSOFA scores calculated 24 h after admission to the ICU demonstrated a good level of discrimination for predicting the in-ICU mortality risk in critically ill patients. The MEXSOFA score at 48 h was an independent predictor of death; with each 1-point increase, the odds of death increased by 35%.

More than 10 years survival with sequential therapy in a patient with advanced renal cell carcinoma: a case report

Although radical nephrectomy alone is widely accepted as the standard of care in localized treatment for renal cell carcinoma (RCC), it is not sufficient for the treatment of metastatic RCC (mRCC), which invariably leads to an unfavorable outcome despite the use of multiple therapies. Currently, sequential targeted agents are recommended for the management of mRCC, but the optimal drug sequence is still debated. This case was a 57-year-old man with clear-cell mRCC who received multiple therapies following his first operation in 2003 and has survived for over 10 years with a satisfactory quality of life. The treatments given included several surgeries, immunotherapy, and sequentially administered sorafenib, sunitinib, and everolimus regimens. In the course of mRCC treatment, well-planned surgeries, effective sequential targeted therapies and close follow-up are all of great importance for optimal management and a satisfactory outcome.

Effects of sequential enzymatic hydrolysis on structural, bioactive and functional properties of Phaseolus lunatus protein isolate

Significant initiatives exist within the global food market to search for new, alternative protein sources with better technological, functional, and nutritional properties. Lima bean (Phaseolus lunatus L.) protein isolate was hydrolyzed using a sequential pepsin-pancreatin enzymatic system. Hydrolysis was performed to produce limited (LH) and extensive hydrolysate (EH), each with different degrees of hydrolysis (DH). The effects of hydrolysis were evaluated in vitro in both hydrolysates based on structural, functional and bioactive properties. Structural properties analyzed by electrophoretic profile indicated that LH showed residual structures very similar to protein isolate (PI), although composed of mixtures of polypeptides that increased hydrophobic surface and denaturation temperature. Functionality of LH was associated with amino acid composition and hydrophobic/hydrophilic balance, which increased solubility at values close to the isoelectric point. Foaming and emulsifying activity index values were also higher than those of PI. EH showed a structure composed of mixtures of polypeptides and peptides of low molecular weight, whose intrinsic hydrophobicity and amino acid profile values were associated with antioxidant capacity, as well as inhibiting angiotensin-converting enzyme. The results obtained indicated the potential of Phaseolus lunatus hydrolysates to be incorporated into foods to improve techno-functional properties and impart bioactive properties.

Modelo conceitual para avaliação de práticas e estratégias climáticas: resultados de sua aplicação nos setores automotivo e de papel e celulose no Brasil

A alta concentração de gases de efeito estufa está provocando mudanças climáticas com forte impacto, inclusive no ambiente competitivo das empresas. Assim, algumas empresas já estão adotando práticas e estratégias climáticas em função não somente das legislações restritivas ao carbono, mas também para assegurarem sua vantagem competitiva. Contudo, há uma escassez de ferramentas gerenciais para avaliar o quanto tais práticas e estratégias são adequadas a esse novo contexto. Este artigo apresenta um modelo conceitual desenvolvido com base em benchmarks (melhores práticas) internacionais, para auxiliar as empresas na avaliação das suas práticas e estratégias climáticas, e mostra como o modelo foi aplicado no Brasil em dois setores representativos de sua economia: o automotivo e o de papel e celulose. Identifica as práticas e estratégias climáticas que precisam ser melhoradas nesses setores, quando comparadas com as internacionais. Na aplicação do modelo, foram testadas cinco hipóteses referentes às principais variáveis do modelo proposto. Para tanto, realizaram-se testes estatísticos: teste t-student, para comparação com as práticas internacionais, e teste não paramétrico Kruskal-Wallis, para verificar diferenças entre as médias dos dois setores investigados. Com base nos resultados, são feitas recomendações para futuras pesquisas e para outras empresas preocupadas em adotar práticas e estratégias climáticas eficazes.

Impacto da reforma de financiamento de hospitais de ensino no Brasil

OBJETIVO: Avaliar o impacto da reforma de financiamento na produtividade de hospitais de ensino. MÉTODOS: A partir do Sistema de Informações dos Hospitais Universitários Federais, foram construídas fronteiras de eficiência e produtividade em 2003 e 2006 com técnicas de programação linear, por meio de análise envoltória de dados, considerando retornos variáveis de escala e orientação a input. Calculou-se o Índice de Malmquist para identificar mudanças de desempenho ao longo dos anos quanto à eficiência técnica (razão entre os escores de eficiência em tempos distintos) e eficiência tecnológica (deslocamento da fronteira no período considerado). RESULTADOS: Houve aumento do aporte financeiro em 51% e da eficiência técnica dos hospitais de ensino (de 11, passaram a ser 17 na fronteira empírica de eficiência), o mesmo não ocorrendo com a fronteira tecnológica. O uso de análise envoltória de dados estabeleceu os benchmarks para as unidades ineficientes (antes e depois da reforma) e os escores de eficiência mostraram uma possível correlação entre a eficiência técnica encontrada e a intensidade e dedicação de ensino. CONCLUSÕES: A reforma permitiu o desenvolvimento de melhorias gerenciais, mas é necessário maior tempo de acompanhamento para observar mudanças mais efetivas do modelo de financiamento.

Interstitial pneumonitis in canine visceral leishmaniasis

Forty-one naturally infected dogs with visceral leishmaniasis from an urban area of Corumbá (Mato Grosso do Sul-BRAZIL) were studied and three types of lung involvement due to visceral leishmaniasis were characterized; a cellular, a cellular-fibrotic and a fibrotic type. These types seem to represent a sequential evolutive proce'as. Visceral leishmaniasis frequently causes an interstitial pneu monitis in naturally infected dogs (80.5%) as well as in man and experimentally infected hamsters.

Secondary dengue infection in schoolchildren in a dengue endemic area in the State of Rio de Janeiro, Brazil

A seroepidemiologic survey was carried out in schoolchildren from public schools of the Niterói municipality, state of Rio de Janeiro, Brazil, after a period of sequential epidemics by dengue virus type 1 and 2 (DEN-1 and DEN-2). 450 blood samples were obtained by fingertip puncture and collected on filter paper discs. The hemagglutination inhibition (HAI) test was carried out using DEN-1 and DEN-2 antigens. HAI titres were demonstrated in 66% (297/450) of the sera and the geometric means of the titres were 1/182 and 1/71 for DEN-1 and DEN-2, respectively. Secondary infections were observed in 61% (181/297) of positive cases. Among these, 75% (135/181) were under fifteen years old. No dengue haemorrhagic fever (DHF) was reported in these children. Asymptomatic or oligosymptomatic infections were detected in 56% of the studied population. The absolute and relative frequencies of positive tests by age group and sex did not evidence statistically significant difference. The number of individuals infected probably produced a immunologic barrier responsible for the non occurrence of dengue epidemic in the latter years.

Serological diagnosis of toxoplasmosis: usefulness of IgA detection and IgG avidity determination in a patient with a persistent IgM antibody response to Toxoplasma gondii

We report the detection of specific IgA antibodies and the determination of IgG avidity in sequential serum samples from a patient exhibiting significant levels of Toxoplasma-specific IgM antibodies for seven years after the onset of the clinical symptoms of toxoplasmosis. IgM antibodies were detected by an indirect immunofluorescence test and by three commercial enzyme-linked immunosorbent assays (ELISA). Anti-T. gondii IgA was quantified by the a-capture ELISA technique using a commercial kit. As defined by the manufacturer of the IgA ELISA test used, most patients with acute toxoplasmosis have antibody levels > 40 arbitrary units per ml (AU/mL). At this cut-off level, the patient still had a positive ELISA result (45 AU/mL) in a serum sample taken one year after the beginning of clinical manifestations. The IgG avidity-ELISA test was performed with the Falcon assay screening test (F.A.S.T.®) - ELISA system. Avidity indices compatible with a recent Toxoplasma infection were found only in serum samples taken during the first 5 months after the onset of the clinical symptoms of toxoplasmosis. These results show that the interpretation of positive IgM results as indicative of recently acquired toxoplasmosis requires additional laboratory confirmation either by other tests or by the demonstration of a significant rise in the antibody titers in sequential serum samples.

IgG recognizing 21-24 kDa and 30-33 kDa tachyzoite antigens show maximum avidity maturation during natural and accidental human toxoplasmosis

We describe the avidity maturation of IgGs in human toxoplasmosis using sequential serum samples from accidental and natural infections. In accidental cases, avidity increased continuously throughout infection while naturally infected patients showed a different profile. Twenty-five percent of sera from chronic patients having specific IgM positive results could be appropriately classified using exclusively the avidity test data. To take advantage of the potentiality of this technique, antigens recognized by IgG showing steeper avidity maturation were identified using immunoblot with KSCN elution. Two clusters of antigens, in the ranges of 21-24 kDa and 30-33 kDa, were identified as the ones that fulfill the aforementioned avidity characteristics.

Use of selective medium for Burkholderia cepacia isolation in respiratory samples from cystic fibrosis patients

Burkholderia cepacia colonizes cystic fibrosis (CF) patients. We evaluated the impact of the use of a selective medium in the rate of B. cepacia recovery from respiratory samples of CF patients. During a 6-month period, respiratory samples were collected from 106 CF patients and cultivated on selective media including a B. cepacia selective medium. Confirmation of the identity of B. cepacia isolates was carried out by species specific PCR and determination of genomovar status performed by a sequential PCR approach. Results of B. cepacia isolation during this period were compared to the preceding two years, when the sample processing was identical except for the lack of the B. cepacia selective medium. B. cepacia was isolated in 11/257 (4.2%) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58%), p < 0.0001. Identity of all 11 isolates was confirmed by PCR and genomovar determination was accomplished in all but one isolate. These results suggest that the use of a selective medium increases recovery rate of B. cepacia from respiratory samples.

Paracoccidioidomycosis in children: clinical presentation, follow-up and outcome

From February, 1981 to May, 2001, 63 children under 15 y old (ages 2 - 15 y, median = 8 y, mean ± 1 SD = 8 ± 3 y) presenting 70 episodes of Paracoccidioidomycosis were admitted. The main clinical manifestations and laboratory features observed upon admission were: lymph node enlargement (87.1%), fever (75.7%), weakness (48.6%), pallor (41.4%), hepatomegaly (40%), splenomegaly (35.7%), anemia (90%), hypergammaglobulinemia (88.5%), eosinophilia (75.5%) and hypoalbuminemia (72.5%). Moderate to severe malnutrition was detected in 35.7% of the episodes (Gomez's criterion). Radiographic and technetium studies showed bone lesions in 20 of the episodes, most of them being multiple lytic lesions, involving both long (70%) and plain bones (30%). First line treatment consisted of an association of sulfametoxazole-trimethoprin, which was used, exclusively, in 50 episodes. Follow-up of hemoglobin levels, number of eosinophils in the peripheral blood, albumin and gammaglobulin serum levels revealed significant sequential improvement one and six months after hospital admission, being quite useful to evaluate treatment effectiveness. Six patients died (9.3%) and four developed sequelae (6.3%) . In conclusion, the juvenile and disseminated forms can be observed in about 70% of the episodes of PCM occurring in children younger than 15 y old, most of them presenting with a febrile lymphoproliferative syndrome associated to anemia, eosinophilia and hypergammaglobulinemia.

Clinical laboratory assessment of hepatitis C and HIV coinfected patients according to the antiretroviral therapy received

During the year of 2001, a retrospective, descriptive study in order to determine the influence of the antiretroviral therapy received by 111 HIV-HCV coinfected patients who had undergone at least one liver biopsy was conduced, 74 of them were treated with a protease inhibitor regimen (WPI), and 37 with a non-protease inhibitor regimen (NPI). The main characteristics found were: a young patient population (mean age 41 years old in both groups), composed in most part of male individuals (74.3% WPI and 51.4% NPI) with previous risk factors for both infections (WPI 93.2% and NPI 89.2%). The most significant findings included AIDS-defining disease (WPI 18.9% and NPI 13.5% of the cases), elevated hepatic enzyme levels (WPI: SGOT 52.1 and NPI 53.2), absence of liver disease-related symptoms (16.2% for both groups), average CD4 count > 350 for both groups (WPI 362.2 and NPI 378.1), predominantly low-grade fibrosis in both populations (0-2 in 63.6% of WPI patients and in 80% of NPI patients), with necro-inflammatory activity ranging from 5-7 in 51.3% and 42.9% of WPI patients and NPI patients, respectively. It is suggested a sequential biopsy to better evaluate the evolution of the hepatic disease, according to the HAART regimen received.

Rapid turnover of Plasmodium falciparum var gene transcripts and genotypes during natural non-symptomatic infections

The var genes of Plasmodium falciparum code for the antigenically variant erythrocyte membrane proteins 1 (PfEMP1), a major factor for cytoadherence and immune escape of the parasite. Herein, we analyzed the var gene transcript turnover in two ongoing, non-symptomatic infections at sequential time points during two weeks. The number of different circulating genomes was estimated by microsatellite analyses. In both infections, we observed a rapid turnover of plasmodial genotypes and var transcripts. The rapidly changing repertoire of var transcripts could have been caused either by swift elimination of circulating var-transcribing parasites stemming from different or identical genetic backgrounds, or by accelerated switching of var gene transcription itself.

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG

Antigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.