Spect perfusion imaging versus CT for predicting radiation injury to normal lung in lung cancer patients.

Objectives In non-small cell lung cancer (NSCLC) patients, to establish whether the fractional volumes of irradiated anatomic or perfused lung differed between those with and without deteriorating lung function or radiation associated lung injury (RALI).Methods 48 patients undergoing radical radiotherapy for NSCLC had a radiotherapy-planning CT scan and single photon emission CT lung perfusion imaging (99mTc-labelled macroaggregate albumin). CT defined the anatomic and the single photon emission CT scan (co-registered with CT) identified the perfused (threshold 20 % of maximum) lung volumes. Fractional volumes of anatomic and perfused lung receiving more than 5, 10, 13, 20, 30, 40, 50 Gy were compared between patients with deteriorating (>median decline) vs stable (vs stable FEV1 ( p = 0.005, 0.005 and 0.025 respectively) but did not differ for higher doses of radiation (>30, 40, 50 Gy). Fractional volumes of anatomic and perfused lung receiving > 10 Gy best predicted decline in FEV1 (Area under receiver operating characteristic curve (Az = 0.77 and 0.76 respectively); sensitivity/specificity 75%/81 and 80%/71%) for a 32.7% anatomic and 33.5% perfused volume cut-off. Irradiating an anatomic fractional volume of 4.7% to > 50 Gy had a sensitivity/specificity of 83%/89 % for indicating RALI (Az = 0.83).Conclusion A 10-20 Gy radiation dose to anatomic or perfused lung results in decline in FEV1. A fractional anatomic volume of >5% receiving >50 Gy influences development of RALI.Advances in knowledge Extent of low-dose radiation to normal lung influences functional respiratory decline

Efeito da solução hipertônica de NaCl 7,5% na resposta inflamatória em modelo de choque hipovolêmico*

A solução hipertônica de cloreto de sódio 7,5% (SSH) é eficaz em restaurar os parâmetros hemodinâmicos e reduzir a inflamação em modelos experimentais de choque hemorrágico. Assim, foi nosso objetivo investigar a ação da SSH sobre os mecanismos envolvidos na lesão de isquemia e reperfusão (I/R) em um modelo de choque hemorrágico controlado. Ratos Wistar (280-350 g) foram submetidos à hemorragia controlada, mantendo-se a pressão arterial média em 40 mmHg por 1 h. Após esse período, os animais foram randomizados e receberam SSH (4 ml/kg) ou solução salina isotônica (SSI-34 ml/kg). Não foram observadas diferenças na resposta hemodinâmica nos dois grupos. Também não foram observadas diferenças na geração espécies reativas de oxigênio (medida indiretamente pela concentração de malondialdeído) ou das citocinas IL-6 e IL-10 (medidas por ELISA). A análise histológica qualitativa dos pulmões mostrou que os animais do grupo SSH apresentaram um menor influxo tecidual de neutrófilos. Esses animais também mostraram maior expressão de proteínas do choque térmico 70 (HSP70). Assim, concluímos que o tratamento do choque hemorrágico com SSH pode diminuir o processo inflamatório pulmonar e aumentar a proteção celular, devido ao aumento da expressão de HSP70.Hypertonic saline solution (HSS - NaCl 7,5%) was shown to restore hemodynamic parameters in hemorrhagic shock and to decrease the inflammation triggered by ischemia reperfusion injury (I/R). Therefore, our objective was to investigate the effects of HSS on the mechanisms involved in I/R, in an experimental model of controled hemorrhagic shock. Wistar rats (280-350 g) were submitted to controled bleeding, keeping the mean arterial pressure around 40 mmHg, for 1 hour. After that, rats were randomized and treated with HSS (4 ml/kg)or normal saline (ISS) (34 ml/kg). There were no differences in hemodynamic parameters between the two groups for at least 2 h after shock. No difference either was observed in reactive oxygen species generation (measured indirectly by malondialdehyde concentration)or cytokines (IL-6 and IL-10) production (measured by ELISA). Qualitative analysis of lung tissue showed a smaller neutrophil infiltrate in animals that received HSS. Also, the animals in the HSS group showed an increased expression of heat shock protein 70 (HSP70). Therefore,we conclude that treatment of hemorrhagic shock with hypertonic saline solution can decrease pulmonary inflammation and increase cellular protection by up-regulating HSP70 expression

Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins

Interections between CD40/CD40L and PPARs signaling pathways

O receptor CD40 e seu ligante CD40L possuem um papel importante na interface entre a resposta imune inata e a adaptativa. Disfunções desta via de sinalização são descritas em doenças de origem inflamatória e autoimunes. Em Lúpus eritematoso sistêmico (LES) foi descrito um aumento nos níveis séricos de CD40L solúvel, que participa na produção de autoanticorpos. Receptores ativados por proliferadores de peroxisomos (PPARs) são fatores de transcrição que inicialmente foram descritos como envolvidos apenas no metabolismo lipídico, mas que atualmente são também descritos como atuantes no controle da resposta imune. Com isso, nosso objetivo é determinar se a ativação dos PPARs modula o processo inflamatório através da interação com CD40/CD40L in vitro ou in vivo. Células de linhagem monocítica humana THP-1 foram tratadas por 24 horas com forbol-éster (PMA, 40 nM) e posteriormente estimuladas com CD40L recombinante (rhCD40L, 1 g/ml) por diferentes períodos. Transcritos de mRNA foram analisados por real time PCR e os resultados expressos como razão da expressão do gene housekeeping GAPDH. As células THP-1 apresentam um aumento na expressão de PPAR e após 16 e 2 horas de estímulo com rhCD40L, respectivamente. Estas células também foram estimuladas com LPS (10 g/ml) e LPS+rhCD40L para sabermos se a resposta obtida anteriormente era específica ao estímulo com rhCD40L. O resultado mostra que há uma diminuição na expressão de PPAR e após o estimulo com LPS ou LPS+rhCD40L, indicando que nessas condições a modulação da expressão de PPARs é especifica para a via de sinalização CD40/CD40L. Foi medida também a expressão de CD36, que é descrito na literatura como um indicador da atividade de PPARs. O resultado mostra que o estímulo com CD40L promove um aumento de CD36, o que indica indiretamente que o PPAR estava ativo neste modelo experimental. Para mostrar a interação direta destas duas vias de sinalização, silenciamos o gene de PPAR por siRNA e posteriormente anlisamos a expressão de CD80, cuja expressão encontra-se aumentada logo após a ativação do CD40 de acordo com a literatura. O resultado mostra que, com o silenciamento de PPAR , há um aumento de CD80 logo após a ativação do CD40, evidenciando assim a interação entre essas duas vias de sinalização. A fim de verificar se os achados encontrados in vitro poderiam ser observados in vivo, foi isolada a fração mononuclear de sangue periférico de pacientes com LES com a doença em atividade (n=17), a doença inativa (n=21) ou doadores saudáveis (n=12) e foi medida a expressão de PPAR e por real time PCR. PPAR apresenta um aumento em pacientes com a doença ativa ou inativa em comparação aos doadores saudáveis. Já a expressão de PPAR apresenta aumento apenas em lúpicos em atividade quando comparados com lúpicos inativos ou doadores saudáveis. Quando considerado nesta análise o efeito do tratamento dos pacientes com corticosteróides nos níveis de PPAR, obsevou-se que a expressão de PPAR apresenta o mesmo padrão anterior. Estes resultados sugerem a hipótese de que PPAR seja um possível marcador de atividade de LES. Para confirmar esta especificidade, foram adicionadas à analise células mononucleares retiradas de pacientes com tuberculose e com infecções agudas. Os dados mostram que os níveis elevados de PPAR se mantém apenas em pacientes com lúpus ativo, o que confirma nossa hipótese. Nossos achados sugerem que PPAR e são regulados especificamente em reposta a ativação da via do CD40/CD40L, em monócitos em cultura e em células obtidas de pacientes com LES. Podemos também sugerir que PPAR possa ser um marcador para a atividade de LES. Estes resultados podem representar um novo mecanismo de controle da via de sinalização do CD40/CD40L, participando no controle da resposta inflamatória em cultura e em células de pacientes lúpicosThe membrane receptor CD40 and its ligand CD40L play an important role in the interface between innate and acquired immunity. Dysfunction of this signaling pathway was described in inflammatory and autoimmune diseases. In systemic lupus erythematosus (SLE), increased serum levels of soluble CD40L have been detected, where it plays a significant role in the generation of auto-antibodies. Peroxisome proliferator activator receptors (PPARs) are transcription factors originally described in lipid metabolism. More recently, they were also characterized as inflammatory modulators. Therefore, our objective was to determine whether the activation of PPARs may modulate the inflammatory process through interaction with the CD40/CD40L signaling pathway in vitro and in vivo. Macrophages derived from the human monocytic cell line THP-1 by 24h-treatment with PMA (40 nM) were stimulated with human recombinant CD40L (rhCD40L, 1 g/ml) for different periods. Messenger RNA (mRNA) transcripts for PPAR , and were determined by real time PCR and expressed as a ratio of the housekeeping gene GAPDH transcripts. THP-1 cells express a basal level of PPAR and gene transcription, which is increased 16 and 2 hours after exposure to rhCD40L, respectively. We also stimulated the THP-1 cells with LPS (10 g/ml) and LPS+rhCD40L to see if the increase of PPAR was a response specific to the rhCD40L stimuli. The data show that there is a decrease in PPAR and genes expression upon LPS or LPS+rhCD40L stimulation, indicating that in these times (2 and 16 hours) the response is specific for the CD40/CD40L signaling pathway. Increased expression of CD36 is known as an indicator of PPARs activity. We measured CD36 and saw an increase of this receptor after rhCD40L stimulus, indicating indirectly that PPARs were active in this experimental model. To prove the direct interaction between CD40/CD40L and PPAR , we silenced the PPAR gene by siRNA and analyzed the expression of CD80, which is known to increase after CD40 activation. The results show an increase in CD40L-stimulated CD80 expression upon silencing of PPAR , showing that there is an interaction between these signaling pathways. To confirm whether these findings also occur in vivo, mononuclear cells were isolated from whole blood samples from SLE patients with active (n=17) and inactive disease (n=21), and healthy donors (n=12). The mRNA transcripts for PPARs were detected by real time PCR. In both active and inactive SLE patients, monocytes show an increase in PPAR mRNA expression, as compared to healthy donors. PPAR mRNA is increased only in active patients when compared to healthy donors and inactive lupus patients. Further in this analysis, when we separated the patients with and without the administration of corticosteroids, PPAR displayed the same pattern as above. These results suggested that PPAR may be a marker for lupus activity. To validate this hypothesis, we compared the results obtained from patients with tuberculosis and acute infections. Results showed that only active-lupus patients have an increase in PPAR , confirming the specificity of this phenomenon and hence our hypothesis Our findings suggest that PPAR and are up-regulated specifically in response to CD40/CD40L activation, in both cultured macrophages and in monocytes obtained from SLE patients. We could also suggest that PPAR may be marker for lupus activity. Our results may represent a new control mechanism of the CD40/CD40L signaling pathway and seem to be implicated in the control of the inflammatory response in both human macrophages in vitro and SLE patient