Autophagy inhibitor 3-methyladenine could not modulate rotenone neurotoxicity in primary mesencephalic cell culture / El inhibidor de la autofagia 3-metiladenina no logra modular la neurotoxicidad de la rotenona en el cultivo primario de células mesencefálicas

Dysregulated autophagy, whether excessive or downregulated, has been thought to be associated with neurodegenerative disorders including Parkinson's disease. Accordingly, the present study was carried out to investigate whether 3-methyladenine, an autophagy inhibitor, can modulate the effects of rotenone on dopaminergic neurons in primary mesencephalic cell culture. Cultures were prepared from embryonic mouse mesencephala at gestation day 14. Four groups of cultures were treated on the 10th DIV for 48 h as follows: the first was kept as an untreated control, the second was treated with 3-methyladenine alone (1, 10, 100, 200 mM), the third was treated with 20 nM rotenone and the fourth was co-treated with 20 nM rotenone and 3-methyladenine (1, 10, 100, 200 mM). On the 12th DIV, cultured cells were stained immunohistochemically against tyrosine hydroxylase and culture media were used to measure the levels of lactate dehydrogenase. 3methyladenine had no effects on both the survival of dopaminergic neurons and the release of lactate dehydrogenase. Rotenone significantly decreased the number of dopaminergic neurons and increased the levels of lactate dehydrogenase in the culture media. When cultures concomitantly treated with 3-methyladenine and rotenone, 3-methyladenine had no effect against rotenone-induced dopaminergic cell damage and lactate dehydrogenase release into the culture medium. In conclusion, the autophagy inhibitor 3-methyladenine could not modulate rotenone-induced dopaminergic cell damage in primary mesencephalic cell culture.

Se estima que la autofagia desregulada, ya sea excesiva o con baja regulación, está asociada con trastornos neurodegenerativos, incluyendo la enfermedad de Parkinson. En consecuencia, el se realizó este estudio para investigar si la 3metiladenina, un inhibidor de la autofagia,puede modular los efectos de la rotenona en las neuronas dopaminérgicas en el cultivo primario de células mesencefálicas. Los cultivos se prepararon a partir de mesencéfalo de ratón embrionario el día 14 de gestación. Cuatro grupos de cultivos se trataron en el 10º DIV durante 48 h de la siguiente manera: el primer grupo se mantuvo como un control no tratado, el segundo se trató con 3-metiladenina sola (1, 10, 100, 200 mM), el tercer grupo se trató con rotenona 20 nM y el cuarto se trató conjuntamente con rotenona 20 nM y 3-metiladenina (1, 10, 100, 200 mM). En el 12º DIV; las células cultivadas fueron tratadas mediante tinción inmunohistoquímica en tirosina hidroxilasa y se usaron medios de cultivo para medir los niveles de lactato deshidrogenasa. La 3-metiladenina no tuvo efectos tanto en la supervivencia de las neuronas dopaminérgicas como en la liberación de lactato deshidrogenasa. La rotenona disminuyó significativamente el número de neuronas dopaminérgicas y se observó un aumento de los niveles de lactato deshidrogenasa en los medios de cultivo. Cuando los cultivos tratados concomitantemente con 3-metiladenina y rotenona, la 3metiladenina no tuvo efecto contra el daño celular dopaminérgico inducido por la rotenona y la liberación de lactato deshidrogenasa en el medio de cultivo. En conclusión, el inhibidor de la autofagia 3-metiladenina no moduló el daño celular dopaminérgico inducido por la rotenona en el cultivo celular mesencefálico primario.

Protective effect of extract of the Camellia japonica L. on cerebral ischemia-reperfusion injury in rats / Efeito protetor do extrato da Camellia japonica L. na lesão de reperfusão isquêmica cerebral em ratos

ABSTRACT Objective: We investigated the protective effect of the extract of the Camellia japonica L. flower on cerebral ischemia-reperfusion injury in rats. Methods: The rat ischemia-reperfusion injury was induced by middle cerebral artery occlusion for 90 minutes and reperfusion for 48 hours. The animals received an intravenous injection once a day of 20, 40, 80 mg/kg extract of C. japonica for three consecutive days before the ischemia reperfusion. The learning and memory function, the infarct volume, serum malondialdehyde (MDA) level and lactate dehydrogenase activity, and extravasation of immunoglobulin G (IgG) into cerebral parenchyma were assessed as the cell damage index. Results: Pretreatment with extract of C. japonica markedly reduced the infarct volume, serum malondialdehyde level and lactate dehydrogenase activity, and markedly inhibited the extravasation of IgG. Moreover, pretreatment with extract of C. japonica may also inhibit the learning and memory deficits induced by an ischemia-reperfusion injury. Conclusion: It was concluded that pretreatment with extract of C. japonica has a protective effect on cerebral ischemia-reperfusion injury in rats.

RESUMO Objetivo: Investigamos o efeito protetor do extrato da flor de Camellia japonica L. (ECJ) na lesão de reperfusão isquêmica cerebral (I/R) em ratos. Métodos: A lesão de I/R de rato foi induzida por uma oclusão da artéria cerebral média por 90 minutos e reperfusão por 48 horas. Os animais receberam uma injeção intravenosa uma vez ao dia de 20, 40, 80 mg/kg de ECJ por três dias consecutivos antes da I/R. A função de aprendizagem e memória, o volume do infarto, o nível sérico de malondialdeído (MDA), a atividade da desidrogenase láctica e o extravasamento de imunoglobulina (IgG) no parênquima cerebral foram avaliados como índices de dano celular. Resultados: O pré-tratamento com ECJ reduziu acentuadamente o volume do infarto, o nível sérico de MDA e a atividade da desidrogenase láctica, e inibiu marcadamente o extravasamento de IgG. Além disso, o pré-tratamento com ECJ também poderia inibir os déficits de aprendizado e memória induzidos pela lesão de I/R. Conclusão: O pré-tratamento com ECJ tem um efeito protetor contra lesão cerebral de I/R em ratos.

Insulin resistance is increased in adult patients with dermatomyositis / Resistência insulínica aumentada em pacientes com dermatomiosite

OBJECTIVE: To evaluate insulinemia in glucocorticoid naïve patients with dermatomyositis and to evaluate insulin resistance using the homeostatic model assessment of insulin resistance (HOMA2-IR). METHODS: This cross-sectional study included 25 dermatomyositis, non-diabetic glucocorticoid naïve patients. The control group consisted of 50 volunteers matched for age, gender, ethnicity, weight and height. The HOMA2-IR index was calculated from baseline insulin and glucose data. The International Myositis Assessment & Clinical Studies Group (IMACS) parameters were used to evaluate disease status. RESULTS: Mean age of the patients was 43.5 years and these were predominantly females. Patients had low disease activity according to IMACS parameters. Higher body mass index and waist circumference were observed in the dermatomyositis group compared to the control group. Insulin level and HOMA2-IR were also higher in patients with dermatomyositis. Moreover, analyzing dermatomyositis alone, the HOMA2-IR index correlated positively with weight, body mass index and waist circumference and was independent on disease status parameters. CONCLUSIONS: Patients with dermatomyositis had higher values for basal insulinemia, insulin resistance, body mass index and waist circumference. Moreover, HOMA2-IR moderately correlated with these anthropometric parameters. These metabolic abnormalities are related to the development of metabolic syndrome, one of the main comorbidities observed in dermatomyositis.

OBJETIVO: Avaliar a insulinemia em pacientes com dermatomiosite virgens de glicocorticoide e avaliar a resistência insulínica, utilizando o modelo de avaliação da homeostase de resistência insulínica (HOMA2-IR). MÉTODOS: Este estudo transversal incluiu 25 pacientes com dermatomiosites, não-diabéticos e sem uso prévio de glicocorticoides. Para o grupo de controle, 50 voluntários foram pareados por idade, gênero, etnia, peso e estatura. O índice HOMA2-IR foi calculado a partir de dados basais de insulina e glicose. Os parâmetros do International Myositis Assessment & Clinical Studies Group (IMACS) foram utilizados para avaliar o status da doença. RESULTADOS: A méda de idade dos pacientes foi de 43,5 anos, predominantemente do sexo feminino. Os pacientes apresentaram baixa atividade de doença de acordo com os parâmetros do IMACS. O índice de massa corporal e a circunferência da cintura foram maiores no grupo da dermatomiosite em comparação com o grupo controle. O nível de insulina e o HOMA2-IR também foram maiores em pacientes com dermatomiosite. Além disso, analisando a dermatomiosite isoladamente, o índice HOMA2-IR correlacionou-se positivamente com o peso, o índice de massa corporal e a circunferência da cintura e foi independente dos parâmetros de status da doença. CONCLUSÕES: Pacientes com dermatomiosite apresentam valores mais elevados de insulinemia basal, resistência à insulina, índice de massa corporal e circunferência da cintura. Além disso, o HOMA2-IR está moderadamente correlacionado com esses parâmetros antropométricos. Essas anormalidades metabólicas estão relacionadas ao desenvolvimento da síndrome metabólica, uma das principais comorbidades observadas na dermatomiosite.

The Effect of Sleep Deprivation on Cardiac Function and Tolerance to Ischemia-Reperfusion Injury in Male Rats / O Efeito da Privação de Sono na Função Cardíaca e Tolerância à Lesão de Isquemia-Reperfusão em Ratos

Abstract Background: Sleep deprivation (SD) is strongly associated with elevated risk for cardiovascular disease. Objective: To determine the effect of SD on basal hemodynamic functions and tolerance to myocardial ischemia-reperfusion (IR) injury in male rats. Method: SD was induced by using the flowerpot method for 4 days. Isolated hearts were perfused with Langendorff setup, and the following parameters were measured at baseline and after IR: left ventricular developed pressure (LVDP); heart rate (HR); and the maximum rate of increase and decrease of left ventricular pressure (±dp/dt). Heart NOx level, infarct size and coronary flow CK-MB and LDH were measured after IR. Systolic blood pressure (SBP) was measured at start and end of study. Results: In the SD group, the baseline levels of LVDP (19%), +dp/dt (18%), and -dp/dt (21%) were significantly (p < 0.05) lower, and HR (32%) was significantly higher compared to the controls. After ischemia, hearts from SD group displayed a significant increase in HR together with a low hemodynamic function recovery compared to the controls. In the SD group, NOx level in heart, coronary flow CK-MB and LDH and infarct size significantly increased after IR; also SD rats had higher SBP after 4 days. Conclusion: Hearts from SD rats had lower basal cardiac function and less tolerance to IR injury, which may be linked to an increase in NO production following IR.

Resumo Fundamento: A privação de sono (PS) acha-se fortemente associada a alto risco cardiovascular. Objetivo: Determinar o efeito da PS nas funções hemodinâmicas basais e tolerância à lesão miocárdica de isquemia‑reperfusão (IR) em ratos machos. Métodos: A PS foi induzida com o método da plataforma única por 4 dias. Utilizou-se o modelo de perfusão de coração isolado de Langendorff, medindo-se os seguintes parâmetros nas condições basais e após IR: pressão desenvolvida no ventrículo esquerdo (PDVE), frequência cardíaca (FC) e taxa máxima de aumento e redução da pressão do ventrículo esquerdo (±dp/dt). O nível cardíaco de NOx, o tamanho do infarto e os níveis de CK-MB e LDH no efluente coronário foram medidos após IR. A pressão arterial sistólica (PAS) foi medida no início e no final do estudo. Resultados: No grupo PS, os valores basais de PDVE (19%), +dp/dt (18%) e-dp/dt (21%) foram significativamente mais baixos (p < 0,05), e a FC (32%) significativamente mais alta em comparação aos dos controles. Após isquemia, os corações do grupo PS apresentavam um significativo aumento da FC além de uma menor recuperação da função hemodinâmica em comparação aos dos controles. No grupo PS, os níveis de NOx no coração e de CK-MB e LDH no efluente coronário, além do tamanho do infarto, foram significativamente maiores após IR. O grupo PS também apresentou maior PAS após 4 dias. Conclusão: Os corações do grupo PS apresentaram menor função cardíaca basal e menor tolerância à lesão de IR, o que pode estar relacionado ao aumento da produção de NO após IR.

Development of a Novel Quality Improvement Indicator Based on the Hemolysis Index

Hemolysis frequently causes preanalytical errors in laboratory measurements. We aimed to develop a quality improvement indicator for evaluating the extent of inappropriate procedures causing hemolysis in clinical samples collected in medical care units. We defined the threshold value of the hemolysis index (H index) causing significant interference with analyte measurement and analyzed the H index values of clinical samples in relation to the threshold. The H index threshold value causing a 10% bias in the measurement of lactate dehydrogenase was found to be 25. The monthly mean H index and monthly frequency of samples with an H index >25 were significantly different among the types of ward (P=0.001, respectively), and significantly decreased after replacement of a laboratory centrifuge lacking temperature control (20.6±0.58 vs 23.30±1.08, P=0.01; 23.4±1.69% vs 32.6±1.78%, P=0.01). The monthly mean H index and the monthly frequency of samples with an H index above a threshold value may be useful quality improvement indicators for detection of inappropriate procedures in the acquisition and handling of blood samples in medical care units.

Pleural fluid characteristics of pleuropulmonary paragonimiasis masquerading as pleural tuberculosis

BACKGROUND/AIMS: Pleuropulmonary paragonimiasis produces no specific symptoms or radiologic findings, allowing for the possibility of misdiagnosis. We evaluated the specific clinical and pleural fluid features of pleuropulmonary paragonimiasis masquerading as pleural tuberculosis. METHODS: We retrospectively analyzed the clinical and radiologic characteristics of 20 patients diagnosed with pleuropulmonary paragonimiasis between 2001 and 2011. RESULTS: In total, 17 patients presented with respiratory symptoms, including dyspnea (30%), hemoptysis (20%), cough (20%), and pleuritic chest pain (15%). Chest radiographs revealed intrapulmonary parenchymal lesions, including air-space consolidation (30%), nodular opacities (20%), cystic lesions (15%), ground-glass opacities (10%), and pneumothorax (5%). A pleural f luid examination revealed eosinophilia, low glucose levels, and high lactate dehydrogenase (LDH) levels in 87%, 76%, and 88% of the patients, respectively. These traits helped to distinguish pleuropulmonary paragonimiasis from other pleural diseases such as parapneumonic effusion, malignancy, and pleural tuberculosis. CONCLUSIONS: Pleuropulmonary paragonimiasis is often initially misdiagnosed as other pleural diseases. Therefore, it is important to establish the correct diagnosis. In patients with unexplained pleural effusion living in paragonimiasis-endemic areas, pleural fluid obtained by thoracentesis should be examined to distinguish pleuropulmonary paragonimiasis. When marked eosinophilia, high LDH levels, and low glucose levels are identified in pleural fluid, physicians could consider a diagnosis of pleuropulmonary paragonimiasis.

Valor pronóstico de las isoenzimas de LDH en pacientes con linforma no hodgkin / Prognostic value of LDH isoenzymes in patients with non-Hodgkin linforma

La enzima lactato deshidrogenasa (LDH) es un factor pronóstico en Linfoma No Hodgkin (LNH). El objetivo del trabajo consistió en evaluar prospectivamente el valor pronóstico de las isoenzimas de LDH en pacientes con LNH. Se estudiaron 67 pacientes de primera consulta con diagnóstico de LNH, sin tratamiento previo, VIH negativo y sin otras enfermedades, tiempo promedio de seguimiento 30 meses (rango 3-48 meses). Las muestras de suero se recolectaron previas al tratamiento. La LDH total (LDHT) e isoenzimas de LDH se determinaron respectivamente por método cinético y electroforesis de proteínas en gel de agarosa. Se procesaron muestras de 122 controles sanos para establecer los valores de referencia de las isoenzimas de LDH. 49(73%) LNH agresivos y 18(27%) LNH indolentes y según el Índice Pronóstico Internacional (IPI), 60 (90%) bajo riesgo y 7(10%) alto riesgo. Las isoenzimas LDH1, LDH2, LDH3, LDH4 y LDH5 presentaron niveles absolutos significativamente elevados en 25 (37%), 29 (43%), 32 (48%), 20 (39%) y 11 (16%) de los casos respectivamente (p<0,0001). La actividad porcentual de LDH4 en los pacientes con LNH agresivos fue significativamente superior respecto al grupo de LNH indolentes (p=0,01). En el análisis univariado, valores absolutos elevados de LDH1 se asociaron significativamente con una sobrevida global disminuida (p=0,0064) en el grupo total de pacientes. LDH1 conservó su valor pronóstico aún en el grupo de pacientes con valores normales de LDHT (p=0,04). En pacientes con LNH agresivos, valores elevados de LDHT e IPI alto riesgo se asociaron significativamente con una menor sobrevida global (p<0,05). En el análisis multivariado la LDHT e IPI resultaron factores pronósticos independientes de la sobrevida. Alteraciones específicas del patrón de isoenzimas de LDH sugieren la relación de LDH4 con la biología del tumor y su actividad proliferativa en LNH agresivos y el valor pronóstico de LDH1 como factor adverso de la sobrevida en el análisis univariado.

Lactate dehydrogenase (LDH) is a prognostic factor in non-Hodgkin lymphoma (NHL). Our objective was to evaluate prospectively the prognostic value of LDH isoenzymes in patients with NHL. We studied 67 newly diagnosed NHL patients, previously untreated, HIV-negative and free from other disease, median follow-up of 30 month (range 3-48 month). Before starting treatment serum samples were collected for the determination of total LDH (LDHT) and LDH isoenzymes that were respectively assayed by kinetic method and protein electrophoresis in agarose gel. In order to set reference values of LDH isoenzymes samples from122 healthy controls were processed. Results: 49(73%) of the patients were aggressive NHL and 18(27%) indolent NHL and according to the International Prognostic Index (IPI), 60(90 %) low risk and 7(10%) high risk. High absolute values of LDH1, LDH2, LDH3, LDH4 and LDH5 isoenzymes were significantly elevated in 25 (37%), 29 (43%), 32 (48%), 20 (39%) and 11 (16%) of cases respectively (p<0,0001). The percentage value of LDH4 activity in aggressive NHL patients was significantly higher compared to indolent NHL group (p=0,01). In univariate analysis increased LDH1 absolute values were significantly associated with decreased overall survival in the total group of patients (p = 0.0064). LDH1 remained a prognostic factor for survival even when considering the group of patients with normal serum LDHT values (p = 0.04). In patients with aggressive NHL increased values of LDHT and high risk IPI were significantly associated with decreased overall survival (p<0.05). In a multivariate analysis LDHT and IPI score were independent prognostic factor for survival.

Cathepsin B is activated as an executive protease in fetal rat alveolar type II cells exposed to hyperoxia

Alveolar type II cells are main target of hyperoxia-induced lung injury. The authors investigated whether lysosomal protease, cathepsin B (CB), is activated in fetal alveolar type II cells in the transitional period from the canalicular to saccular stages during 65%-hyperoxia and whether CB is related to fetal alveolar type II cell (FATIIC) death secondary to hyperoxia. FATIICs were isolated from embryonic day 19 rats and exposed to 65%-oxygen for 24 h and 36 h. The cells exposed to room air were used as controls. Cell cytotoxicity was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry. CB activity was assessed by colorimetric assay, qRT-PCR and western blots. 65%-hyperoxia induced FATIIC death via necrosis and apoptosis. Interestingly, caspase-3 activities were not enhanced in FATIICs during 65%-hyperoxia, whereas CB activities were greatly increased during 65%-hyperoxia in a time-dependent manner, and similar findings were observed with qRT-PCR and western blots. In addition, the preincubation of CB inhibitor prior to 65%-hyperoxia reduced FATIIC death significantly. Our studies suggest that CB activation secondary to hyperoxia might have a relevant role in executing the cell death program in FATIICs during the acute stage of 65%-hyperoxia.

Dosagem de proteínas totais e desidrogenase lática para o diagnóstico de transudatos e exsudatos pleurais: redefinindo o critério clássico com uma nova abordagem estatística / Determination of total proteins and lactate dehydrogenase for the diagnosis of pleural transudates and exudates: redefining the classical criterion with a new statistical approach

OBJETIVO: Propor um novo critério de classificação para a diferenciação entre exsudatos e transudatos pleurais através da dosagem de proteínas totais no líquido pleural (PT-LP) e de desidrogenase lática no líquido pleural (DHL-LP) exclusivamente, assim como comparar o rendimento diagnóstico entre esse novo critério com o critério clássico. MÉTODOS: Estudo observacional, transversal de tipo individualizado, no qual foram selecionados 181 pacientes com derrame pleural tratados em dois hospitais universitários no estado do Rio de Janeiro (RJ) entre 2003 e 2006. Os parâmetros diagnósticos incluídos no critério clássico, assim como os do novo critério, foram determinados. RESULTADOS: Dos 181 pacientes, 152 e 29 foram diagnosticados, respectivamente, com exsudato pleural e transudato pleural. A sensibilidade, especificidade e acurácia do critério clássico para o diagnóstico de exsudato pleural foram, respectivamente, de 99,8 por cento, 68,6 por cento e 94,5 por cento, enquanto, para o diagnóstico de transudato pleural, essas foram de 76,1 por cento, 90,1 por cento e 87,6 por cento. Utilizando-se os pontos de corte de 3,4 g/dL para a dosagem de PT-LP e de 328,0 U/L para aquela de DHL-LP (novo critério), a sensibilidade, especificidade e acurácia foram de, respectivamente, 99,4 por cento, 72,6 por cento e 99,2 por cento, para o diagnóstico de exsudato, e de 98,5 por cento, 83,4 por cento e 90,0 por cento, para o diagnóstico de transudato. A acurácia do novo critério proposto para o diagnóstico de exsudato pleural foi significativamente maior que aquela do critério clássico (p = 0,0022). CONCLUSÕES: O rendimento diagnóstico dos dois critérios estudados foi semelhante. Portanto, esse novo critério de classificação pode ser utilizado na prática diária.

OBJECTIVE: To propose a new classification criterion for the differentiation between pleural exudates and transudates-quantifying total proteins in pleural fluid (TP-PF) and lactate dehydrogenase in pleural fluid (LDH-PF) exclusively-as well as to compare this new criterion with the classical criterion in terms of diagnostic yield. METHODS: This was an observational, cross-sectional study with a within-subject design, comprising 181 patients with pleural effusion treated at two university hospitals in the state of Rio de Janeiro, Brazil, between 2003 and 2006. The diagnostic parameters included in the classical criterion were identified, as were those included in the new criterion. RESULTS: Of the 181 patients, 152 and 29 were diagnosed with pleural exudates and pleural transudates, respectively. For the classical criterion, the sensitivity, specificity, and accuracy for the diagnosis of pleural exudates were, respectively, 99.8 percent, 68.6 percent, and 94.5 percent, whereas the corresponding values for the diagnosis of pleural transudates were 76.1 percent, 90.1 percent, and 87.6 percent. For the new criterion (cut-off points set at 3.4 g/dL for TP-PF and 328.0 U/L for LDH-PF), the sensitivity, specificity, and accuracy for the diagnosis of exudates were, respectively, 99.4 percent, 72.6 percent, and 99.2 percent, whereas the corresponding values for the diagnosis of transudates were 98.5 percent, 83.4 percent, and 90.0 percent. The accuracy of the new criterion for the diagnosis of pleural exudates was significantly greater than was that of the classical criterion (p = 0.0022). CONCLUSIONS: The diagnostic yield was comparable between the two criteria studied. Therefore, the new classification criterion can be used in daily practice.

Isolamento, diferenciação e aspectos bioquímicos de células-tronco de líquido amniótico / Isolation, differentiation and biochemical aspects of amniotic fluid stem cell

As células-tronco mesenquimais (MSCs) são células com grande potencial de diferenciação e estão sendo recentemente introduzidas na clínica para tratamento de várias doenças. Possuem várias vantagens incluindo sua estabilidade fenotípica in vitro. OBJETIVO: isolamento das MSCs de líquido amniótico, sua expansão e a demonstração da sua capacidade de se diferenciar em células miogênicas e adipogênicas, sem alterar a estabilidade cromossomal em meio de cultura. MÉTODOS: a fim de avaliar a mudança funcional destas células, foram avaliados parâmetros bioquímicos nas células adipogênicas já diferenciadas e antes da diferenciação através da dosagem de triglicérides. A diferenciação em células musculares foi avaliada comparando os níveis de creatinofosfoquinase - CK, desidrogenase lática - LDH e aldolase produzidas por estas células antes e após diferenciação. RESULTADOS: os níveis de triglicérides foram significativamente maiores nas células diferenciadas, mostrando ainda a formação de grânulos intracitoplasmáticos. Todos os outros valores obtidos foram significativamente maiores nas células miogênicas diferenciadas quando comparadas às não diferenciadas. CONCLUSÃO: os resultados sugerem que estes protocolos podem ser usados para avaliar diferenciação de células-tronco em células adipogênicas e miogênicas, e que o líquido amniótico pode ser uma fonte para obtenção destas células.

The mesenchymals stem cells (MSCs) are cells with the great potential of differentiation are being introduced in the clinic for treatment of several diseases. Mesenchymal stem cells have several advantages including the stability of their phenotype in vitro. BACKGROUND: isolation of MSCs in amniotic fluid, its expansion and the demonstration of the capacity of these cells to differentiate in adipogenic and miogenic cells, without to change the chromosomal stability of the MSCs in culture. METHODS: in order to evaluate the functional change of these cells, were gotten values of the differentiated adipogenic cells and not differentiated through the dosage of triglycerides. The miogenic nature of the differentiated cells was analyzed comparing the creatine kinase - CK, lactic dehydrogenase - LDH and aldolase produced by the cells. RESULTS: the values of triglycerides were significantly higher in differentiated cells, showing intracitoplasmatic granule form after differentiation. All the biochemical characters were significantly higher in differentiated miogenic cells. CONCLUSIONS: this study suggests that the standardized protocol of differentiation can be used in the attainment of cells with characteristics of adipogenic and muscular cells, from amniotic fluid.

Pleural fluid analysis with traditional and additional parameters.

36 patients with pleural effusion were studied clinically with pertinent investigations and pleural fluid analysis. The pleural fluid analysis was done with routine protocol including study of traditional parameters like protein and cell count and additional parameters like pleural LDH, GOT(AST) and their ratio with serum LDH and GOT respectively. The utility of these parameters was studied critically. Amongst the isolated parameters cell count enjoyed highest (100%) specificity and positive predictive value. LDH ratio had highest (79.1%) sensitivity. Pleural LDH showed highest (52.6%) negative predictive value and diagnostic accuracy (69.4%). Amongst the combination of two parameters protein with LDH ratio had highest (87.5%) sensitivity, cell count with LDH ratio showed highest specificity (100%), positive predictive value (75%) and diagnostic accuracy (88.9%). Amongst the combination of three parameters pleural protein and cell count with LDH ratio and GOT ratio respectively enjoyed best sensitivity (87.5%), specificity and positive predictive value (100%), negative predictive value (80%) and diagnostic accuracy (90.6%). Similarly competent were the combinations offour parameters namely p rotein and cell count with LDH and its ratio and GOT and its ratio respectively. Combination of all parameters exhibited similar degree of utility.

Comportamiento de la deshidrogenasa lactica en niños con enfermedades neoplasicas: Consulta de Oncología infantil: Hospital Central de San Cristóbal, abril a octubre del año 2003 / Behavior of the lactic deshydrogenases in children with disease neoplasic infant: Oncology Consultation in the Central Hospital of San Cristobal: april-october 2003

La incidencia de cáncer infantil es de unos 120 casos anuales por cada millón de niños y conocer el diagnóstico oportuno mejora la sobrevida. Estudiar las concentraciones de la deshidrogenasa láctica (LDH) en niños con neoplasias que asisten a la consulta de Oncología Infantil del Hospital Central de San Cristóbal. Estudio prospectivo, descriptivo, longitudinal y observacional, diseño no experimental y longitudinal panel. 34 pacientes, 22 (65 por ciento) sexo masculino, 12 (5 por ciento) sexo femenino, preescolar 19 (56 por ciento), procedencia del medio urbano 23 (68 por ciento), 11 (32 por ciento) del medio rural, 17 (50 por ciento) leucemias, 5 (14,7 por ciento) tumor de wilms, 3 (8,82 por ciento) retinoblastoma, 3 (8,82 por ciento) linfoma no Hodgkin, 2 (5,88 por ciento) linfoma Hodgkin, 2 (5,88 por ciento) meduloblastoma, 1 (2,94 por ciento) sarcoma y 1 (2,94 por ciento) nueroblastoma. 9 (26 por ciento) consultan por tumoración abdominal y 28 (50 por ciento) tienen paraclínica de anemia. Leucemias en inicio y recaída la LDH elevada mayor de 285UI/L y normaliza en tratamiento, el tumor de wilms en tratamiento y seguimiento normal y en recaída elevada para luego normalizar, el retinoblastoma es normal en seguimiento y elevada en recaída y luego normaliza, el linfoma no Hodgkin elevada en inicio y luego normaliza y en tratamiento es normal, el linfoma Hodgkin normal en inicio, tratamiento y seguimiento, el meduloblastoma y neuroblastoma normal en seguimiento y el sarcoma normal en tratamiento. La LDH aumenta en fase de inicio y recaída y disminuye en fase de tratamiento y se normaliza en fase de seguimiento.

Determinação das atividades séricas de creatina quinase, lactato desidrogenase e aspartato aminotransferase em eqüinos de diferentes categorias de atividade / Determination of serum activities of creatine kinase, lactate dehydrogenase, and aspartate aminotransferase in horses of different activities classes

The creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) seric activities in horses of different activity classes (athlete, traction, and reproduction), were compared. Fifty-eight horses were alloted into three groups - group 1 with 20 athletes, "vaquejada" competitors; group 2 with 20 breeding horses; and group 3 with 18 draft horses, averaging 10 working hours daily. The average values for CK serum activity were 80.2, 83.9, and 94.4 U/l in groups 1, 2, and 3, respectively. Result of group 3 was significantly different from the other groups. The averages values for LDH were 102.5, 98.6, and 112.8 U/l in groups 1, 2, and 3, respectively, with no statistical difference between groups. The AST averages were 56.8, 33.0, and 50.1 U/l in groups 1, 2, and 3, respectively, with group 2 significantly differing from the others. Clinical biochemistry values of muscular function in horses varied according to activity category.

Predictive model for the diagnosis of tuberculous pleural effusion

This study developed a predictive model to identify pleural tuberculosis. A consecutive cases study of patients investigating the cause of pleural effusion, in an area of high prevalence of tuberculosis (Rio de Janeiro, Brazil). Clinical and laboratory variables were compared among patients with tuberculosis (TB) and without tuberculosis (NTB), individually and using logistic regression. The performance was described as diagnostic accuracy, compared to a gold standard in a masked way. We have studied 104 TB patients, 41 with malignant, 29 transudates, 28 parapneumonic, 13 with miscellaneous diseases. After identification of individual discrimination power aided by clinical, radiological and laboratory variables, the following ones were included in a multivariate analysis: ADA, total leukocytes, percentile of lymphocytes, protein, lactate dehydrogenase, duration of disease, age and gender. A logistic regression model to predict pleural tuberculosis including the five first variables showed the best performance. A receiver operating characteristic curve identified the best cutoff at 0.7, resulting in a sensitivity and specificity of more then 95 percent. The predictive model improved the specificity of ADA alone, keeping its sensitivity. This model seems helpful when a microbiological or histological diagnosis of pleural tuberculosis could not be established. External validation of these results is necessary before recommendation for routine application.

Clinical and laboratory parameters in the differential diagnosis of pleural effusion secondary to tuberculosis or cancer

PURPOSE: To evaluate the clinical and laboratory characteristics of pleural effusions secondary to tuberculosis (TB) or cancer (CA). METHODS: A total of 326 patients with pleural effusion due to TB (n=182) or CA (n=144) were studied. The following parameters were analyzed: patient gender, age and pleural effusion characteristics (size, location, macroscopic fluid aspect, protein concentration, lactate dehydrogenase (DHL) and adenosine deaminase activity (ADA) and nucleated cell counts). RESULTS: Young male patients predominated in the tuberculosis group. The effusions were generally moderate in size and unilateral in both groups. Yellow-citrine fluid with higher protein (p < 0.001) levels predominated in effusions from the tuberculosis group (5.3 + 0.8 g/dL) when compared to the CA group (4.2 ± 1.0 g/dL), whereas DHL levels were more elevated in CA (1,177 ± 675 x 1,030 ± 788 IU; p = 0.003) than in TB. As expected, ADA activity was higher in the TB group (107.6 ± 44.2 x 30.6 ± 57.5 U/L; p < 0.001). Both types of effusions presented with high nucleated cell counts, which were more pronounced in the malignant group (p < 0.001). TB effusion was characterized by a larger percentage of leukocytes and lymphocytes (p < 0.001) and a smaller number of mesothelial cells (p = 0.005). Lymphocytes and macrophages were the predominant nucleated cell in neoplastic effusions. CONCLUSION: Our results demonstrate that in lymphocytic pleural exudate obtained from patients with clinical and radiological evidence of tuberculosis, protein and ADA were the parameters that better characterize these effusions. In the same way, when the clinical suspicion is malignancy, serous-hemorrhagic lymphocytic fluid should be submitted to oncotic cytology once this easy and inexpensive exam reaches a high diagnostic performance (approximately 80 percent). In this context, we suggest thoracocentesis with fluid biochemical...

OBJETIVO: Avaliar as características clínicas e laboratoriais de derrames pleurais secundários à tuberculose ou câncer. MÉTODOS: Um total de 326 pacientes com derrame pleural por tuberculose (n=182) ou câncer (n=144) foi avaliado. Os seguintes parâmetros foram analisados: sexo e idade dos pacientes e características do líquido pleural (tamanho, localização, aspecto macroscópico, concentração de proteínas, atividade da desidrogenase lática (DHL) e da adenosina deaminase (ADA) e contagem de células nucleadas). RESULTADOS: A tuberculose pleural predominou nos pacientes mais jovens e do sexo masculino. Em ambos os grupos, os derrames pleurais foram de tamanho moderado e unilaterais. Derrames com aspecto amarelo-citrino com níveis mais elevados de proteínas predominaram na tuberculose (5,3 ± 0,8 g/dL), quando comparados aos neoplásicos (4,2 ± 1,0 g/dL), enquanto que níveis mais elevados de DHL foram observados nos derrames neoplásicos (1.177 ± 675 x 1.030 ± 788 UI; p = 0,003). Conforme esperado, a atividade da ADA foi maior na tuberculose que no câncer (107,6 ± 44,2 x 30,6 ± 57,5 U/L; p < 0,001). Ambos os derrames apresentaram alta celularidade, embora mais pronunciada no grupo neoplásico (p < 0,001). Os derrames de etiologia tuberculosa se caracterizaram por apresentar uma maior percentagem de leucócitos e de linfócitos (p < 0,001) e um pequeno número de células mesoteliais (p = 0,005). Linfócitos e macrófagos foram as células nucleadas que predominaram nos derrames pleurais malignos. CONCLUSÃO: Nossos resultados demonstram que em exsudatos pleurais linfocíticos de pacientes com evidências clínicas e radiológicas de tuberculose, os níveis de proteína e de ADA foram os parâmetros que melhor caracterizaram esses derrames. Da mesma maneira, quando a suspeita clínica é câncer, um líquido serohemorrágico e linfocítico deve ser submetido à citologia oncótica, uma vez que este exame laboratorial de fácil...

Study of biochemical parameters of ascitic fluid in exudative ascites with special reference to tuberculous peritonitis.

In an attempt to differentiate between three important but clinically similar conditions of exudative ascites like tuberculous peritonitis, spontaneous bacterial peritonitis (SBP) and malignant ascites, we evaluated the biochemical parameters of ascitic fluid as a diagnostic aid. The serum ascitic albumin gradient (SAAG), lactate dehydrogenase (LDH), pH, adenosine deaminase(ADA), carcino-embryonic antigen (CEA) and carbohydrate antigen (CA-125) levels were measured in 36 patients with tuberculous peritonitis, 30 patients with SBP and 30 patients with ascites due to malignant disorders. The LDH level was significantly lower in tuberculous peritonitis patients than in malignant and SBP groups. A value of < 110 U/l gave the assay a sensitivity of 94% and a specificity of 93%, positive predictive value of 89% and negative predictive value of 96% for tuberculous peritonitis. The ADA activity was significantly higher in tuberculous peritonitis group than in the other two groups. A cut off value > 33 U/l gave the ADA test a sensitivity of 89%, specificity of 100%, positive predictive value of 100 % and a negative predictive value of 94% for tuberculosis. A pH value of <7.26 with high SAAG (>11 g/l) predicted SBP with reasonable accuracy. Elevated ascitic fluid CEA (>2 ng/ml) and CA - 125(> 35 U/l) was found exclusively in cases of malignant ascites with a single case of tuberculous peritonitis showing CA-125 value > 35 U/l. All these tests are rapid, non-invasive, and easily reproducible and offer good predictive accuracy which is comparable to that of more invasive procedures like peritoneoscopy and biopsy.

Diagnostic and prognostic utility of rapid strip (OptiMal and Paracheck) versus conventional smear microscopy in adult patients of acute, uncomplicated P. falciparum malaria in Mumbai, India.

OBJECTIVES: The present study compared the diagnostic and prognostic utility of two rapid tests the (Paracheck and OptiMal) versus conventional smear microscopy. METHODS: Using two independent microscopists we carried out the three tests in 31 adult cases of smear positive, acute, uncomplicated Plasmodium falciparum malaria. All three tests were done pretreatment, and on Days 8, 15 and 29. RESULTS: Compared to microscopy, the Paracheck had a sensitivity of 100%, while the OptiMal had a sensitivity of 83.7%. The lower sensitivity of OptiMal resulted from misidentification by both microscopists of 6/31 cases as Plasmodium vivax. As a follow up tool, the OptiMal was better than Paracheck, due to the earlier disappearance of the parasite LDH. Also in the Paracheck, between microscopists, there was a significant difference in reading the tests, on Days 8 and 15. CONCLUSION: Our study reiterates, the continued utility of conventional smear microscopy.

Lactate dehydrogenase isoenzymes in dental pulp of rats according to stage of root development

The objective of this study was to present a classification of the root development stage of female rat molar teeth and to evaluate the variation in the lactate dehydrogenase (LDH) activity and electrophoretic isoenzyme profile according to the stage of root development of the molar teeth. We also studied the LDH activity and isoenzymes of the pulp of incisor teeth. The stage of development of the rat first molar at the age of 15 days and that of the second molar at the age of 18 days was classified as the beginning of root formation. At the age of 15 days, the electrophoretic profile of the isoenzymes for the first molar showed a prevalence of LDH-1 followed by LDH-2. However, for the maxillary second molar there was a prevalence of LDH-4 followed by LDH-1, while for the mandibular second molar LDH-1 predominated followed by LDH-2 and LDH-4. From 18 days of age, the prevalence was always of LDH-1. The electrophoretic profile of LDH isoenzymes from the pulp of the incisor teeth at the ages studied (25 and 60 days) showed the following order of prevalence: LDH-1 > LDH-2 > LDH-3 > LDH-4 > LDH-5. These results suggest that there are variations in the prevalence of the various forms of LDH isoenzymes in the dental pulp of rats according to the developmental stage of the root

New Helicoverpa armigera Hbn cell line from larval hemocyte for baculovirus studies.

A new cell line from the larval hemocytes of H. armigera was established in Grace's medium modified by adding lactalbumin hydrolysate and yeastolate (3.3g/l), and supplemented with fetal bovine serum (20%). The cell line was designated as NIV-HA-1195. The cell population at P-78 consisted mainly of epithelial-like cells (89.36%), fibroblast-like cells (8.31%) and giant cells (2.13%). The population doubling time was 96hr at P-8, 60hr at P-43. The chromosome number ranged from 45 to 200. The cell line is susceptible to the baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV), Spodoptera litura NPV and the homologous HaNPV. Isoenzyme profile and results of 16S rRNA heteroduplex analysis clearly indicated the species specificity of the new cell line.

Comparison of the OptiMAL rapid test with routine microscopic examination of Giemsa-Stained Thick Blood Film for diagnosis of malaria.

The OptiMAL is a rapid immunodiagnostic test developed by Flow Inc., Portland, Oreg. for diagnosis and differentiation of P. falciparum and non P. falciparum malaria infection. It has been based on detection of circulating parasite lactate dehydrogenase enzyme (pLDH), produced by live Plasmodium parasites. The purpose of this study was to compare the efficacy of the OptiMAL test with routine microscopic examination of Giemsa-Stained Thick Blood Film (routine GS-TBF) for the diagnosis of malaria at a local malaria clinic in a hyperendemic area of Thailand by using a standard GS-TBF (standard GS-TBF) as reference. One hundred and seventy five patients attending the clinic were recruited; 50, 42 and 83 were falciparum malaria, vivax malaria and non-malaria patients, respectively. Compared with the reference, the OptiMAL test had sensitivities of 92 per cent and 97.6 per cent, whereas, the routine GS-TBF had sensitivities of 81.3 per cent and 81 per cent for the detection of P. falciparum and P. vivax, respectively. Both tests showed no false positive resulting in 100 per cent specificities. However, the OptiMAL test was able to detect only 20 per cent of infection with less than 200 parasitaemia/microlitre. It was also shown in our study that the OptiMAL test was advantageous in follow-up of the treatment outcome. No false positive occurred among 40 follow-up cases. The OptiMAL test detected malaria infection more accurately than the routine GS-TBF (p < 0.05) and was simple, easy to perform and rapid. It is an alternative tool for the diagnosis of malaria in a hyperendemic area where experienced microscopists are not available.