Inflammatory Pre-Conditioning of Adipose-Derived Stem Cells with Cerebrospinal Fluid from Traumatic Brain Injury Patients Alters the Immunomodulatory Potential of ADSC Secretomes.

Immunomodulation by adipose-tissue-derived stem cells (ADSCs) is of special interest for the alleviation of damaging inflammatory responses in central nervous system injuries. The present study explored the effects of cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients on this immunomodulatory potential of ADSCs. CSF conditioning of ADSCs increased messenger RNA levels of both pro- and anti-inflammatory genes compared to controls. Exposure of phorbol-12-myristate-13-acetate-differentiated THP1 macrophages to the secretome of CSF-conditioned ADSCs downregulated both proinflammatory (cyclooxygenase-2, tumor necrosis factor alpha) and anti-inflammatory (suppressor of cytokine signaling 3, interleukin-1 receptor antagonist, and transforming growth factor beta) genes in these cells. Interleukin-10 expression was elevated in both naïve and conditioned secretomes. ADSC secretome treatment, further, induced macrophage maturation of THP1 cells and increased the percentage of CD11b+, CD14+, CD86+, and, to a lesser extent, CD206+ cells. This, moreover, enhanced the phagocytic activity of CD14+ and CD86+ cells, though independently of pre-conditioning. Secretome exposure, finally, also induced a reduction in the percentage of CD192+ adherent cells in cultures of peripheral blood mononuclear cells (PBMCs) from both healthy subjects and TBI patients. This limited efficacy (of both naïve and pre-conditioned secretomes) suggests that the effects of lymphocyte-monocyte paracrine signaling on the fate of cultured PBMCs are strongest upon adherent cell populations.

MicroRNA-451a overexpression induces accelerated neuronal differentiation of Ntera2/D1 cells and ablation affects neurogenesis in microRNA-451a-/- mice.

MiR-451a is best known for its role in erythropoiesis and for its tumour suppressor features. Here we show a role for miR-451a in neuronal differentiation through analysis of endogenous and ectopically expressed or silenced miR-451a in Ntera2/D1 cells during neuronal differentiation. Furthermore, we compared neuronal differentiation in the dentate gyrus of hippocampus of miR-451a-/- and wild type mice. MiR-451a overexpression in lentiviral transduced Ntera2/D1 cells was associated with a significant shifting of mRNA expression of the developmental markers Nestin, ßIII Tubulin, NF200, DCX and MAP2 to earlier developmental time points, compared to control vector transduced cells. In line with this, accelerated neuronal network formation in AB.G.miR-451a transduced cells, as well as an increase in neurite outgrowth both in number and length was observed. MiR-451a targets genes MIF, AKT1, CAB39, YWHAZ, RAB14, TSC1, OSR1, POU3F2, TNS4, PSMB8, CXCL16, CDKN2D and IL6R were, moreover, either constantly downregulated or exhibited shifted expression profiles in AB.G.miR-451a transduced cells. Lentiviral knockdown of endogenous miR-451a expression in Ntera2/D1 cells resulted in decelerated differentiation. Endogenous miR-451a expression was upregulated during development in the hippocampus of wildtype mice. In situ hybridization revealed intensively stained single cells in the subgranular zone and the hilus of the dentate gyrus of wild type mice, while genetic ablation of miR-451a was observed to promote an imbalance between proliferation and neuronal differentiation in neurogenic brain regions, suggested by Ki67 and DCX staining. Taken together, these results provide strong support for a role of miR-451a in neuronal maturation processes in vitro and in vivo.

The UGT1A1*28 gene variant predicts long-term mortality in patients undergoing coronary angiography.

BACKGROUND: Uridine diphosphate glycosyltransferases 1A1 (UGT1A1) plays an essential role in detoxification and excretion of several endogenous and exogenous compounds. A functional polymorphism in the promoter of the UGT1A1 gene (TA repeat insertion, UGT1A1*28, rs3064744) has been associated with reduced UGT1A1 enzyme activity. The purpose of the present study was to investigate the role of UGT1A1 genotypes in mortality. METHODS: UGT1A1 genotypes as well as baseline plasma bilirubin levels were analyzed in participants of the Ludwigshafen Risk and Cardiovascular Health study (n=3316). UGT1A1*28 genotypes were determined on an ABI PRISM 3730 genetic analyzer. RESULTS: As expected, UGT1A1 genotypes were associated with baseline bilirubin levels (*1/*1 genotype: 9.1±4.6 µmol/L; *1/*28 genotype: 10.8±5.3; *28/*28: 16.9±9.2; p<0.001). During a median follow-up of 10.4 years, 995 subjects (30.0%) died. In a multivariate regression analysis adjusting for age, sex, smoking, type 2 diabetes, dyslipidemia, alanine aminotransferase (ALT) levels and bilirubin levels, the UGT1A1*28 variant predicted lower overall mortality (hazard ratio [HR], 0.86; 95% confidence interval [CI], 0.78-0.95; p=0.003). Contrary to expected, higher baseline bilirubin levels predicted increased mortality (HR, 1.014; 95% CI, 1.002-1.025; p=0.019). CONCLUSIONS: The UGT1A1*28 gene variant is associated with lower mortality rates. The protective effect of the UGT1A1*28 variant likely includes mechanism other than bilirubin metabolism.

Effects of the polymeric niche on neural stem cell characteristics during primary culturing.

The polymeric niche encountered by cells during primary culturing can affect cell fate. However, most cell types are primarily propagated on polystyrene (PS). A cell type specific screening for optimal primary culture polymers particularly for regenerative approaches seems inevitable. The effect of physical and chemical properties of treated (corona, oxygen/nitrogen plasma) and untreated cyclic olefin polymer (COP), polymethymethacrylate (PMMA), PP, PLA, PS, PC on neuronal stem cell characteristics was analyzed. Our comprehensive approach revealed plasma treated COP and PMMA as optimal polymers for primary neuronal stem cell culturing and propagation. An increase in the number of NT2/D1 cells with pronounced adhesion, metabolic activities and augmented expression of neural precursor markers was associated to the plasma treatment of surfaces of COP and PMMA with nitrogen or oxygen, respectively. A shift towards large cell sizes at stable surface area/volume ratios that might promote the observed increase in metabolic activities and distinct modulations in F-actin arrangements seem to be primarily mediated by the plasma treatment of surfaces. These results indicate that the polymeric niche has a distinct impact on various cell characteristics. The selection of distinct polymers and the controlled design of an optimized polymer microenvironment might thereby be an effective tool to promote essential cell characteristics for subsequent approaches.

More than cell dust: microparticles isolated from cerebrospinal fluid of brain injured patients are messengers carrying mRNAs, miRNAs, and proteins.

Microparticles are cell-derived, membrane-sheathed structures that are believed to shuttle proteins, mRNA, and miRNA to specific local or remote target cells. To date best described in blood, we now show that cerebrospinal fluid (CSF) contains similar structures that can deliver RNAs and proteins to target cells. These are, in particular, molecules associated with neuronal RNA granules and miRNAs known to regulate neuronal processes. Small RNA molecules constituted 50% of the shuttled ribonucleic acid. Using microarray analysis, we identified 81 mature miRNA molecules in CSF microparticles. Microparticles from brain injured patients were more abundant than in non-injured subjects and contained distinct genetic information suggesting that they play a role in the adaptive response to injury. Notably, miR-9 and miR-451 were differentially packed into CSF microparticles derived from patients versus non-injured subjects. We confirmed the transfer of genetic material from CSF microparticles to adult neuronal stem cells in vitro and a subsequent microRNA-specific repression of distinct genes. This first indication of a regulated transport of functional genetic material in human CSF may facilitate the diagnosis and analysis of cerebral modulation in an otherwise inaccessible organ.

Impact of CYP2C8 and 2C9 polymorphisms on coronary artery disease and myocardial infarction in the LURIC cohort.

AIMS: As data on the cardiovascular risk associated with CYP2C8 and CYP2C9 polymorphisms is controversial, we performed a cross-sectional analysis of subjects enrolled in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. MATERIALS & METHODS: CYP2C8 and CYP2C9 genetic polymorphisms were determined with real-time PCR in 2827 patients. Based on angiography, 1052 of these patients had coronary artery disease (CAD) and 615 did not; 1160 patients had signs or a history of myocardial infarction (MI) in addition to CAD. The association of genotypes with CAD and MI was determined by logistic regression analysis, adjusted for age, sex, dyslipidemia, hypertension, diabetes mellitus and smoking status. RESULTS: Frequencies of CYP2C8 and 2C9 variants were neither significantly different between CAD and control patients, nor between MI and control patients. Men carrying the CYP2C9*3 allele had an increased risk of MI (odds ratio [OR]: 1.67; 95% CI: 1.06-2.63; p = 0.03) and women carrying the CYP2C9*3 allele had a decreased risk of CAD (OR: 0.65; 95%CI: 0.42-0.9; p = 0.05). CONCLUSION: Overall, LURIC data confirmed that there is no major cardiovascular risk associated with CYP2C8 and CYP2C9 variants in a cardiovascular risk population of patients having undergone coronary angiography.

Association of polymorphisms in the chemokine receptor CX3CR1 gene with coronary artery disease.

Two chemokine receptor CX3CR1 gene variants, V249I and T280M, have been implicated in coronary artery diseases (CAD). Currently no consistent effect has been revealed and their role in cardiovascular disease is still conflicting. In the present study the association of CX3CR1 genotypes with CAD and myocardial infarction (MI) was investigated in the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort, including 3316 individuals in whom cardiovascular disease angiographically has been defined or ruled out. Similarly to previous studies, the alleles I249 and M280 were in strong linkage disequilibrium and formed an I(249)M(280) haplotype. However, there was no relationship between CX3CR1 genotypes or corresponding haplotypes and the prevalence of CAD or MI. Adjusted for classical risk factors (age, sex, hypertension, dyslipidemia, diabetes mellitus and smoking), the odds ratio (OR) of V249I for CAD was 0.95 (95% confidence interval (CI)=0.78-1.15, p=0.61). The OR of T280M for CAD was 0.83 (95% CI=0.66-1.04, p=0.11). Furthermore, CX3CR1 variants were not associated with C-reactive protein levels, age at onset of CAD, severity of CAD and MI. In conclusion, present data of LURIC do not support the hypothesis that common variants of the CX3CR1 gene are associated with the presence of CAD or MI.

The fibrinogen gamma (FGG) 10034C>T polymorphism is associated with venous thrombosis.

INTRODUCTION: Thrombin-induced conversion of fibrinogen to fibrin plays an essential role in hemostasis and results in the stabilization of thrombi. Elevated plasma fibrinogen levels have been associated with both increased plasma viscosity and platelet aggregability. Recently, a haplotype-tagging single nucleotide polymorphism characterized by a C to T substitution at nucleotide 10034 of the fibrinogen gamma gene (FGG 10034C>T, rs2066865), has been proposed as a novel risk factor for deep venous thrombosis (DVT). Aim of the present study was to provide further data on the role of the FGG 10034C>T polymorphism for DVT. MATERIALS AND METHODS: FGG genotypes were determined by 5'-exonuclease assay (TaqMan) in 358 patients with documented DVT and a total of 783 control subjects. RESULTS: In a multivariate analysis adjusting for age, sex, presence of factor V Leiden and carriage of prothrombin 20210A, homozygosity for the FGG 10034 TT genotype yielded an odds ratio of 2.01 (95% CI 1.23-3.31; p=0.006) for DVT. CONCLUSIONS: Our data confirm the primary finding that the FGG 10034C>T polymorphism is associated with DVT risk.

G-protein beta3 subunit (GNB3) gene polymorphisms and cardiovascular disease: the Ludwigshafen Risk and Cardiovascular Health (LURIC) study.

A common 825C>T polymorphism in exon 10 of the gene for the beta-3 subunit of heterotrimeric G-proteins, GNB3, has been associated in some studies with traits of the metabolic syndrome as well as coronary artery disease (CAD), but these associations were refuted by other studies. To investigate the role of GNB3 gene variations in CAD and myocardial infarction (MI), we determined five GNB3 polymorphisms (-1429G>A, IVS5 +41G>A, 657T>A, 814G>A and 825C>T) in the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort, including 2575 patients with angiographically documented CAD and 731 individuals in whom CAD had been ruled out by angiography. None of the GNB3 polymorphisms was associated with CAD, MI, diabetes, hypertension, blood pressure, body weight or body mass index. We conclude that a major contribution of GNB3 gene variants to CAD or MI risk is unlikely.