Cardiac Fibroblasts Contribute to Myocardial Dysfunction in Mice with Sepsis: The Role of NLRP3 Inflammasome Activation

Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL)-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs) and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk). We show that treatment of CFs with lipopolysaccharide (LPS) induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation) and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA]) and pharmacological (glyburide) inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-chalenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP) responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.     

Methods of tracing hydrogen in polyketide biosynthesis: High field NMR spectroscopy of citrinin produced in a D2O based medium

Penicillium citrinum cultures have been germinated on an H₂O-based medium, resuspended on a D₂O-based medium and treated with [l,2-¹³C₂] acetate. The resulting citrinin (1) has been analysed by²H and¹³C nuclear magnetic resonance spectroscopy and information about the metabolism of hydrogen in citrinin biosynthesis has been deduced.     

Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

The determination of the local cerebral glucose utilization with the 2-deoxyglucose method

Summary In the adult mammalian brain, the energy metabolism is almost entirely dependent on glucose. Furthermore, a close relationship between the energy metabolism and the functional activity could be shown. Thus, the functional activity of the brain or parts thereof can be quantified by measuring the cerebral metabolic rate for glucose. Studying in vivo the fate of a radioactive labeled analogue of glucose, the 2-deoxy-d-[1-¹⁴C]glucose, and using quantitative autoradiographic techniques, it is possible to estimate the cerebral glucose utilization of every discrete brain region. The advantage of the 2-deoxyglucose method is, that the local cerebral glucose utilization represents a metabolic encephalography (Sokoloff 1982).     

Quantitative receptor autoradiography in the human brain

Summary Quantitative receptor autoradiography on sections of the human brain raises methodical problems of which some are relevant also for studies in animal tissue, but others are unique in studies of human brain tissue. Procedures for the following methodical aspects are discussed image analysis for quantitation of the regional distribution of receptor densities, saturation analysis on autoradiographs, influence of age and post-mortem delay and quenching of -radiation in brain tissue. The solutions proposed to these problems make receptor autoradiography in the human brain to a reliable method for studies of chemical neuroanatomy.     

Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay

We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM₁ in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM₁ were bound to SPA particles and incubated with labelled FucGM₁ and 100 µl test-serum overnight, and counted in a -counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM₁. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.Abbreviations MAb monoclonal antibody - SPA scintillation proximity assay - HPTLC high performance thin layer chromatography - SCLC small cell lung cancer - FucGM₁ Fuc1-2Gal1-3GalNAc1-4(NeuAc2-3)-Gal1-4Glc1-1Cer - ELISA enzyme linked immunosorbent assay - FCS foetal calf serum - PBS phosphate buffered saline     

Limited nonenzymatic glucosylation of low-density lipoprotein does not alter its catabolism in tissue culture

This study examines the effects of various degrees of chemical modification of low-density lipoprotein (LDL) on its catabolism by various cell types. Moderate glucosylation of LDL does not alter its interaction with the high-affinity receptor present on human fibroblasts at concentration of 5-2000 micrograms LDL-cholesterol/ml. Only heavily glucosylated LDL (more than 12 lysine residues glucosylated per apolipoprotein B) or LDL glucosylated in the presence of Na(CN)BH3, i.e., conditions not expected to occur in diabetes, inhibit receptor-mediated internalisation and degradation. Moderately glucosylated LDL is also readily recognized by cultured rat hepatocytes and porcine endothelial cells. Human monocyte-derived macrophages accumulate cholesteryl ester when incubated with acetylated LDL for 12 days but no enhanced cholesteryl ester formation was found when native or glucosylated LDL (3.3 lysines glucosylated per apolipoprotein B) were used.     

Differential distribution of3H dihydrotestosterone and3H estradiol nuclear binding sites in mouse male accessory sex organs

Summary The distribution of specific nuclear binding sites for androgens and estrogens in the male accessory sex organs of the mouse was assessed by autoradiography with³H dihydrotestosterone (³H DHT) and³H estradiol (³H E₂). With³H DHT nuclear labeling differed among the epithelia of the organs. It was high in seminal vesicle and ampullary gland, moderate in ventral prostate, urethral gland, prostatic excretory ducts and the ampulla ductus deferentis, low in dorsal prostate and low or absent in coagulation gland. With³H E₂, in contrast, epithelial nuclear labeling was high only in coagulation gland, moderate or low in seminal vesicle, low or absent in ventral and dorsal prostate and absent in ampullary gland and ampulla ductus deferentis. In the lamina propria of all organs nuclear labeling with³H DHT was generally moderate and existed only in some cells, with the highest number in the ampulla ductus deferentis. With³H E₂, nuclear labeling in the lamina propria showed a high intensity in all organs, except in ventral and dorsal prostate which remained unlabeled. Many labeled cells were found in the deferent duct and its ampulla, while in the other organs only a few cells showed nuclear labeling with³H E₂. In the smooth muscle sheath of all organs, some muscle cells were moderately labeled with³H DHT, but not with³H E₂. The results indicate the presence of nuclear receptors in male accessory sex organs for both dihydrotestosterone and estradiol. The differential patterns of³H DHT and³H E₂ nuclear uptake suggest differential sensitivities of the individual organs and their tissue compartments for androgens and estrogens. Supported by PHS grant NSO9914 to W.E.S. and Deutsche Forschungsgemeinschaft Dr94/4 to U.D. The work of Dr. Schleicher and his stay in Chapel Hill were additionally sponsored by Studienstiftung des Deutschen Volkes and Boehringer-Ingelheim Fonds     

Local cerebral glucose utilization in the hippocampus of old rats

Summary The local cerebral glucose utilization (LCGU) was measured in the different areas and layers of the Ammon's horn and dentate gyrus of young adult (3 to 4-month-old) rats, and of 27-month-old rats with proven cognitive deficits. The LCGU was determined by quantitative [¹⁴C]2-deoxyglucose autoradiography. Compared to young animals, in the old rats the LCGU was significantly reduced by 12% to 15% in the oriens layers of CA1 and CA2, the pyramidal layers of the CA sectors 1–3, the radiatum and lacunosum-molecular layers of CA2 and CA3 and in the lucidum layer of CA3. The LCGU values of all the other layers of the Ammon's horn and the dentate gyrus did not differ significantly between young and old rats. The pattern of the LCGU reduction found in the old rats roughly resembles changes found after fimbra-fornix lesions or systemic administration of scopolamine, suggesting a functionally important deficit in the cholinergic innervation of the old rats' hippocampi.