Wave-front curvature as a cause of slow conduction and block in isolated cardiac muscle

We have investigated the role of wave-front curvature on propagation by following the wave front that was diffracted through a narrow isthmus created in a two-dimensional ionic model (Luo-Rudy) of ventricular muscle and in a thin (0.5-mm) sheet of sheep ventricular epicardial muscle. The electrical activity in the experimental preparations was imaged by using a high-resolution video camera that monitored the changes in fluorescence of the potentiometric dye di-4-ANEPPS on the surface of the tissue. Isthmuses were created both parallel and perpendicular to the fiber orientation. In both numerical and biological experiments, when a planar wave front reached the isthmus, it was diffracted to an elliptical wave front whose pronounced curvature was very similar to that of a wave front initiated by point stimulation. In addition, the velocity of propagation was reduced in relation to that of the original planar wave. Furthermore, as shown by the numerical results, wave-front curvature changed as a function of the distance from the isthmus. Such changes in local curvature were accompanied by corresponding changes in velocity of propagation. In the model, the critical isthmus width was 200 microns for longitudinal propagation and 600 microns for transverse propagation of a single planar wave initiated proximal to the isthmus. In the experiments, propagation depended on the width of the isthmus for a fixed stimulation frequency. Propagation through an isthmus of fixed width was rate dependent both along and across fibers. Thus, the critical isthmus width for propagation was estimated in both directions for different frequencies of stimulation. In the longitudinal direction, for cycle lengths between 200 and 500 milliseconds, the critical width was < 1 mm; for 150 milliseconds, it was estimated to be between 1.3 and 2 mm; and for the maximum frequency of stimulation (117 +/- 15 milliseconds), it was > 2.5 mm. In the transverse direction, critical width was between 1.78 and 2.32 mm for a basic cycle length of 200 milliseconds. It increased to values between 2.46 and 3.53 mm for a basic cycle length of 150 milliseconds. The overall results demonstrate that the curvature of the wave front plays an important role in propagation in two-dimensional cardiac muscle and that changes in curvature may cause slow conduction or block.     

Putting occupation into practice: occupation as ends, occupation as means

We have investigated the effect of 5-HT2 receptor agonist or antagonist administration on postsynaptic 5-HT1A receptor sensitivity assessed by two behavioral measures, reciprocal forepaw treading or hypothermia induced by acute injection of the 5-HT1A receptor agonist 8-OH-DPAT. The effectiveness of these drug treatments to downregulate 5-HT2A receptors was confirmed by measuring the binding of [3H]-ketanserin in cortical homogenates, because all of these drug treatments have been shown to result in the downregulation of 5-HT2A receptor sites. Acute or chronic treatment of rats with the 5-HT2 receptor antagonist mianserin, or chronic administration of the 5-HT2A receptor antagonist ketanserin, did not alter 8-OH-DPAT-induced hypothermia or forepaw treading. These data indicate that downregulation of 5-HT2A receptors is not sufficient to alter these postsynaptic 5-HT1A receptor-mediated responses. Chronic treatment of rats with the 5-HT2 receptor agonist DOI, however, resulted in the attenuation of both 5-HT1A receptor-mediated responses measured in separate experimental groups. The apparent desensitization of 5-HT1A receptors following chronic DOI treatment was not accompanied by a change in either the number or affinity of 5-HT1A receptor sites as measured by the binding of [3H]-8-OH-DPAT in hippocampal homogenates. Chronic activation of 5-HT2 receptors may be one mechanism by which the sensitivity postsynaptic 5-HT1A receptors can be regulated.     

Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-beta 1 and endothelin-1 from fibroblasts

We examined the effects of different cytokine combinations and culture conditions on the expansion and modulation of cell surface antigens of CD34+ derived dendritic cells (DCs), the most efficient antigen-presenting cells capable of stimulating resting T cells in the primary immune response. Cells with a dendritic morphology and expressing HLA-DR, CD1a, S100 and CD83 were maximally expanded under serum-free conditions with the addition of SCF, GM-CSF, TNF-alpha, TGF-beta and Flt-3 ligand (fold increase of CD1a+ cells = 102 +/- 32 after 2 weeks of culture). CD34+ cells were also grown under continuous flow conditions in an artificial capillary system: after 14d of culture, the expansion in the total cell number was lower than that of the static cultures (3.3 +/- 2 v 18.9 +/- 4) but the percentage of CD1a+/CD83+/ CD80+ cells was considerably higher, whereas the CD14+ cells were significantly reduced (8.9 +/- 2 v 26 +/- 13). In continuous perfusion cultures, low levels of DC precursors and of LTC-IC were still present up to day 14. The DCs generated under flow conditions stimulated the mixed leucocyte reaction (MLR) more than the cells grown in static cultures. By electron microscopy, cells grown in the continuous flow system showed an increased number of large cells with numerous dendritic processes and abundant multilamellar complexes. The cells expanded under these conditions were sorted on the basis of their light-scatter properties into two fractions: one containing a predominance of CD1a+/S100+/ CD8 3+/CD80+/CD14- 'large cells' with great internal complexity (mature DCs); the second including 'small cells' either CD33+/CD14+, CD33+/CD15+ or CD33+/CD13-/CD14. The DCs generated and selected with this method are therefore particularly well suited for immunotherapeutic protocols.     

CTLA4 mediates antigen-specific apoptosis of human T cells

The regulation of T cell-mediated immune responses requires a balance between amplification and generation of effector function and subsequent selective termination by clonal deletion. Although apoptosis of previously activated T cells can be induced by signaling of the tumor necrosis factor receptor family, these molecules do not appear to regulate T-cell clonal deletion in an antigen-specific fashion. We demonstrate that cross-linking of the inducible T-cell surface molecule CTLA4 can mediate apoptosis of previously activated human T lymphocytes. This function appears to be antigen-restricted, since a concomitant signal T-cell receptor signal is required. Regulation of this pathway may provide a novel therapeutic strategy to delete antigen-specific activated T cells.     

Routine analysis of proteins by Kjeldahl and Dumas methods: review and interlaboratory study using dairy products

The Kjeldahl and Dumas (combustion) methods were compared in 11 laboratories analyzing samples of milk, skim milk powder, whole milk powder, whey protein concentrate, infant formula, casein, caseinate, 2 reference compounds (glycine and EDTA), and a secondary reference skim milk powder. The comparison was conducted by using international standards where applicable. Overall means were 8.818 g N/100 g by the Kjeldahl method and 8.810 g N/100 g by the Dumas method. No evidence was found for a consistent bias between methods that may be of concern in the trading of dairy produce. A review of more than 10 related trials revealed a lack of consensus in the bias between the 2 methods, suggesting that differences in methodology and sources of systematic error may be contributors. For samples containing > 2 g N/100 g, the Dumas relative repeatability and reproducibility standard deviations were consistently about 0.35 and 0.75%, respectively, whereas the corresponding Kjeldahl values declined generally with N content and were significantly larger. The Dumas precision characteristics may be due to the dominance of Leco analyzers in this trials, and in most other recent trials, rather than an inherent method attribute. Protein determination methods for dairy products need to be reviewed and updated. The Dumas method needs Codex Alimentarius status as a recognized test method.     

The Landscape of microRNAs in βCell: Between Phenotype Maintenance and Protection

Diabetes mellitus is a group of heterogeneous metabolic disorders characterized by chronic hyperglycaemia mainly due to pancreatic β cell death and/or dysfunction, caused by several types of stress such as glucotoxicity, lipotoxicity and inflammation. Different patho-physiological mechanisms driving β cell response to these stresses are tightly regulated by microRNAs (miRNAs), a class of negative regulators of gene expression, involved in pathogenic mechanisms occurring in diabetes and in its complications. In this review, we aim to shed light on the most important miRNAs regulating the maintenance and the robustness of β cell identity, as well as on those miRNAs involved in the pathogenesis of the two main forms of diabetes mellitus, i.e., type 1 and type 2 diabetes. Additionally, we acknowledge that the understanding of miRNAs-regulated molecular mechanisms is fundamental in order to develop specific and effective strategies based on miRNAs as therapeutic targets, employing innovative molecules.     

Ivabradine Induces Cardiac Protection against Myocardial Infarction by Preventing Cyclophilin-A Secretion in Pigs under Coronary Ischemia/Reperfusion

In response to cardiac ischemia/reperfusion, proteolysis mediated by extracellular matrix metalloproteinase inducer (EMMPRIN) and its secreted ligand cyclophilin-A (CyPA) significantly contributes to cardiac injury and necrosis. Here, we aimed to investigate if, in addition to the effect on the funny current (I(f)), Ivabradine may also play a role against cardiac necrosis by reducing EMMPRIN/CyPA-mediated cardiac inflammation. In a porcine model of cardiac ischemia/reperfusion (IR), we found that administration of 0.3 mg/kg Ivabradine significantly improved cardiac function and reduced cardiac necrosis by day 7 after IR, detecting a significant increase in cardiac CyPA in the necrotic compared to the risk areas, which was inversely correlated with the levels of circulating CyPA detected in plasma samples from the same subjects. In testing whether Ivabradine may regulate the levels of CyPA, no changes in tissue CyPA were found in healthy pigs treated with 0.3 mg/kg Ivabradine, but interestingly, when analyzing the complex EMMPRIN/CyPA, rather high glycosylated EMMPRIN, which is required for EMMPRIN-mediated matrix metalloproteinase (MMP) activation and increased CyPA bonding to low-glycosylated forms of EMMPRIN were detected by day 7 after IR in pigs treated with Ivabradine. To study the mechanism by which Ivabradine may prevent secretion of CyPA, we first found that Ivabradine was time-dependent in inhibiting co-localization of CyPA with the granule exocytosis marker vesicle-associated membrane protein 1 (VAMP1). However, Ivabradine had no effect on mRNA expression nor in the proteasome and lysosome degradation of CyPA. In conclusion, our results point toward CyPA, its ligand EMMPRIN, and the complex CyPA/EMMPRIN as important targets of Ivabradine in cardiac protection against IR.