The sphingosine kinase-sphingosine-1-phosphate axis is a determinant of mast cell function and anaphylaxis

Sphingosine-1-phosphate, a key mediator in immune cell trafficking, is elevated in the lungs of asthmatic patients and regulates pulmonary epithelium permeability. Stimulation of mast cells by allergens induces two mammalian sphingosine kinases (Sphk1 and Sphk2) to produce sphingosine-1-phosphate (S1P). Little is known about the individual role of these kinases in regulating immune cell function. Here we show that in mast cells, Sphk2 is required for production of S1P, for calcium influx, for activation of protein kinase C, and for cytokine production and degranulation. However, susceptibility to in vivo anaphylaxis is determined both by S1P within the mast cell compartment and by circulating S1P generated by Sphk1 predominantly from a non-mast cell source(s). Thus, sphingosine kinases are determinants of mast cell responsiveness, demonstrating a previously unrecognized relationship with anaphylaxis.     

Relevant incidental findings at abdominal multi-detector contrast-enhanced computed tomography: A collateral screening?

AIM: To investigate the prevalence of relevant incidental findings (RIFs) detected during routine abdominal contrast-enhanced computed tomography (CeCT).METHODS: We retrospectively evaluated the reports of a consecutive series of abdominal CeCT studies performed between January and May 2013. For each report, patients’ age and sex, admission as inpatient or outpatient, clinical suspicion as indicated by the requesting physician, availability of a previous abdominal examination, and name of the reporting radiologist were recorded. Based on the clinical suspicion, the presence and features of any RIFs (if needing additional workup) was noted.RESULTS: One thousand forty abdominal CeCT were performed in 949 patients (528 males, mean age 66 ± 14 years). No significant difference was found between inpatients and outpatients age and sex distribution (P > 0.472). RIFs were found in 195/1040 (18.8%) CeCT [inpatients = 108/470 (23.0%); outpatients = 87/570 (15.2%); P = 0.002]. RIFs were found in 30/440 (6.8%) CeCT with a previous exam and in 165/600 (27.5%) without a previous exam (P < 0.001). Radiologists’ distribution between inpatients or outpatients was significantly different (P < 0.001). RIFs prevalence increased with aging, except for a peak in 40-49 year group. Most involved organs were kidneys, gallbladder, and lungs.CONCLUSION: A RIF is detected in 1/5 patients undergoing abdominal CeCT. Risk of overdiagnosis should be taken into account.     

Superficial implantation in pigs is associated with decreased numbers and redistribution of endometrial NK-cell populations

PROBLEM: We evaluated implantation-associated quantitative changes in endometrial and peripheral natural killer (NK)-cell populations of pigs. METHOD OF STUDY: Natural killer cell populations were investigated in 10, 15, 20, 30 and 40 days pregnant and non-pregnant (NP) sows by flow cytometry, immunohistochemistry and morphometry. RESULTS: The number of endometrial CD16(+) NK cells significantly declined at attachment phase of implantation and remained relatively low over the course of implantation. The CD16(+) NK cells in situ showed implantation-phase dependent density and localization. Prior to implantation, they substantially resided in the subepithelial stroma. As implantation advances, the density of NK cells into subepithelial stroma decreased while that of NK cells into glandular layer increased, suggesting implantation-induced re-location far from the attached conceptus. The number of CD56(+) lymphocytes was the greatest at pre-attachment phase of implantation, dropped at the time of attachment and increased up to end of early pregnancy period. The CD3(-) CD8(+) NK-cell number decreased significantly when the definitive placenta is established. No significant differences in the numbers of peripheral blood CD16(+), CD56(+) and CD3(-) CD8(+) NK cells between pregnant and NP animals as well as relative to the implantation phase were observed. CONCLUSION: Superficial and adeciduate implantation of pigs is associated with decreased numbers of endometrial NK-cell populations and specific spatiotemporal profile of classical NK cells.     

Molecular identification and full genome analysis of an echovirus 7 strain isolated from the environment in Greece

Two enteroviruses from river water and four from sewage treatment plant were isolated in Larissa, Greece, that all shared the same sequence. A full genome analysis was conducted in an attempt to reveal the evolutionary pathways of one of the isolated strains (LR11F7). VP1 nucleotide and phylogenetic analysis revealed that the isolated strain had 78% homology with the echovirus 7 prototype strain Wallace. Full genome analysis revealed that LR11F7 P1 region is related to echoviruses 7 and that P2 and P3 regions are originating from contemporary enteroviruses isolated in South Asia. Two recombination events were shown to be involved into the evolutionary history of LR11F7, the one event concerning 3A, 3B, and 2C, and the other concerning 3D genomic region, both with new types of HEV-B. The contribution of recombination to enterovirus evolution is substantial, giving rise to new genetic lineages with unknown properties.     

Cardiometabolic Effects of Anti-obesity Pharmacotherapy

Purpose of Review

We review recent studies discussing the impact of pharmacologic agents for weight loss on clinical cardiovascular events, as well as cardiometabolic risk factors.

Recent Findings

Pharmacotherapy with current FDA-approved medications for weight loss can significantly improve known risk factors for the development of cardiovascular disease such as hypertension, hyperlipidemia, insulin resistance, inflammatory biomarkers, and the quantity of visceral fat, as well as non-alcoholic fatty liver disease. However, data regarding the actual reduction in clinical cardiovascular events with the use of weight loss medications is scarce.

Summary

Pharmacotherapy for weight loss may have additional benefit in optimizing patient’s cardiometabolic comorbidities and improving their clinical cardiovascular outcomes, but each drug should be carefully selected based upon individual patient characteristics.

     

Postnatal development of neurons expressing NADPH-diaphorase and parvalbumin in the parietal cortex of male and female rats

Expression of the enzyme nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and the calcium-binding protein parvalbumin was studied in the parietal cortex of male and female rats during postnatal development at 20, 60 and 90 days of age. First, localization of the activity of NADPH-d was combined with the immunohistochemical localization of parvalbumin to facilitate recognition of morphological details and distribution patterns of these two types of cortical neurons. Double staining of neurons for parvalbumin and NADPH-d was never found. Second, it was found that NADPH-d is a simple and proper marker for quantitative studies. Morphometric analysis revealed sexual dimorphism in the density of NADPH-d-positive neurons in 20 days-old prepubertal rats. Females showed higher amounts of NADPH-d-positive neurons than males. No sex-dependent differences were detected in 60 days-old pubertal and 90 days-old postpubertal rats. The present results suggest that sex differences in the number of NADPH-d-positive neurons in the rat parietal cortex may be related to epigenetic effects of gonadal hormones in the early prepubertal period of postnatal development.     

Cyclosporine A attenuates mitochondrial permeability transition and improves mitochondrial respiratory function in cardiomyocytes isolated from dogs with heart failure

We used isolated cardiomyocytes to investigate a possible role of mitochondrial permeability transition pore in mitochondrial abnormalities associated with heart failure. Cardiomyocytes were isolated from LV myocardium of normal control dogs and dogs with heart failure produced by intracoronary microembolizations. Mitochondrial permeability transition was measured in isolated cardiomyocytes with intact sarcolemma with and without 0.2 microM cyclosporin A using calcein AM and the fluorometer. State-3 mitochondrial respiration was also measured with the Clark electrode. Mitochondrial membrane potential was measured with JC-1 probe using the fluorometer. Propidium iodide was used to ensure sarcolemma integrity. 200 min after loading with calcein AM, mitochondria of failing cardiomyocytes showed only 50% of maximal level of calcein fluorescence while it remained unchanged in normal cells. The mitochondrial membrane potential in failing cardiomyocytes was significantly decreased by 38% compared to normal cardiomyocytes. Cyclosporine A significantly slowed the exit of calcein from mitochondria of failing cardiomyocytes and increased mitochondrial membrane potential by 29%. State-3 respiration was not affected with cyclosporine A in normal cardiomyocytes while it was significantly increased in failing cardiomyocytes by 20%. Exit of calcein (m.w. 1.0 kDa) from mitochondria of viable failing cardiomyocytes with intact sarcolemma suggests an existence of a reversible transitory permeability transition opening in high conductance mode. Attenuation of calcein exit, DeltaPsi(m) and improvement of state-3 respiration achieved with CsA (0.2 microM) show that permeability transition opening could be a cause of mitochondrial dysfunction described in the failing heart.     

Novel genes differentially expressed in cortical regions during late neurogenesis

Differential gene expression across the embryonic cerebral cortex is assumed to play a role in the subdivision of the cortex into distinct areas with specific morphology, physiology and function. In a search for genes that may be involved in the cortical regionalization during late neurogenesis in mouse, we performed an extensive in-situ expression analysis at embryonic day (E)16 and E18. The examined candidate genes were selected beforehand by a microarray screen by virtue of their preferential expression in the anlagen of the motor, somatosensory, visual and cingulate cortices or hippocampus. We present new information about graded or regionally enriched expression of 25 genes (nine of which are novel genes) across the mouse embryonic cortex, in progenitor cells as well as in the cortical plate. The established differential expression of most of these genes is persistent at both stages studied, suggesting that their expression is regulated by an intrinsic programme. For some of the genes, the concept of intrinsic regulation is further substantiated by the high similarity of the reported expression patterns at E16 and E18 and published data from earlier stages. Few genes with robust expression in the E16 caudal cortex showed a more restricted pattern at E18, possibly because of their response to extrinsic cues. In addition, several genes appeared to be suitable novel markers for amygdalar and diencephalic nuclei. Taken together, our findings reveal novel molecular partitions of the late mouse cortex that are in accordance with the model of a leading role of intrinsic mechanisms in cortical arealization.     

Cytokine gene polymorphism in kidney transplantation--impact of TGF-beta 1, TNF-alpha and IL-6 on graft outcome

Chronic allograft nephropathy (CAN) is one of the main causes of graft loss in renal transplantation. Polymorphisms with functional significance in the promoter and coding regions of cytokine genes have been suggested as a possible factor for graft rejection. The aim of this study was to investigate the impact of cytokine gene polymorphism of pro and anti-inflammatory cytokines on development of CAN in a group of renal transplant patients and donors. Eight single nucleotide polymorphisms (SNPs) including TNFA (-308), TGFB1 (cdns10, 25), IL-10 (-1082, -819, -592), IL-6 (-174) and IFNG (+874) were analyzed in 56 patients with stable graft function (SGF), 10 with CAN and 28 kidney donors by PCR-SSP method. CAN was significantly associated with the recipient TGFB1 cod10 T/T and combination of cods10, 25 T/T G/G genotypes (high producer), (p<0.05). Influence of patient's TNFA genotype correlated with high level of gene expression on the development of CAN was further demonstrated when the patients were stratified according to the HLA mismatches (HLA-DRB MMs). Additionally donor TNFA-308 G/A (high) and IL-6-174 CC (low) genotypes were increased in cases with CAN. No statistically significant differences in distribution of IL-10, IL-6 and IFNG genotypes between recipients with SGF and CAN were found. In conclusion our data suggest that the high producer genotype of profibrogenetic TGF-beta1, pro-inflammatory TNF-alpha and genetically determined low production of immunoregulatory IL-6 cytokine might be risk factors for CAN development.     

Direct extraction and molecular characterization of enteroviruses genomes from human faecal samples

Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several enteroviruses serotypes are not easily isolated in cell cultures system used and routinely more than one passage in cell culture is performed. A total of 54 archived faecal samples were examined. The heterogeneous nature of faecal samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. PCR inhibitors are frequently encountered in stool specimens. From the three methods initially compared for extraction of viral RNA, QIAamp Viral RNA Mini Kit was retained as it yielded the highest amount of viral RNA without the interference of RT-PCR inhibitors. Evaluation of 54 archived stool specimens by RT-PCR and cell culture resulted in a higher frequency of detection by RT-PCR. With the use of RT-PCR we were able to detect two additional samples otherwise considered negative for enterovirus isolation if only the cell culture standard methodology was employed. RNA extraction with QIAamp Viral RNA Mini Kit coupled with RT-PCR in the 5'NCR (subgrouping into distinct genetic clusters of all enteroviruses) and VP1 (reliable serotyping by sequencing) is a rapid and sensitive technique of direct poliovirus/non-polio enteroviruses recovery and molecular characterization from human faecal specimens without further passage in cell culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.