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Background Mesenchymal stromal cells (MSCs) are attractive for cell-based therapies ranging from regenerative medicine and tissue engineering to immunomodulation. However, clinical efficacy is variable and it is unclear how the phenotypes defining bone marrow (BM)-derived MSCs as well as donor characteristics affect their functional properties. Methods BM-MSCs were isolated from 53 (25 female, 28 male; age: 13 to 80 years) donors and analyzed by: (1) phenotype using flow cytometry and cell size measurement; (2) in vitro growth kinetics using population doubling time; (3) colony formation capacity and telomerase activity; and (4) function by in vitro differentiation capacity, suppression of T cell proliferation, cytokines and trophic factors secretion, and hormone and growth factor receptor expression. Additionally, expression of Oct4 , Nanog , Prdm14 and SOX2 mRNA was compared to pluripotent stem cells. Results BM-MSCs from younger donors showed increased expression of MCAM, VCAM-1, ALCAM, PDGFRβ, PDL-1, Thy1 and CD71, and led to lower IL-6 production when co-cultured with activated T cells. Female BM-MSCs showed increased expression of IFN-γR1 and IL-6β, and were more potent in T cell proliferation suppression. High-clonogenic BM-MSCs were smaller, divided more rapidly and were more frequent in BM-MSC preparations from younger female donors. CD10, β1integrin, HCAM, CD71, VCAM-1, IFN-γR1, MCAM, ALCAM, LNGFR and HLA ABC were correlated to BM-MSC preparations with high clonogenic potential and expression of IFN-γR1, MCAM and HLA ABC was associated with rapid growth of BM-MSCs. The mesodermal differentiation capacity of BM-MSCs was unaffected by donor age or gender but was affected by phenotype (CD10, IFN-γR1, GD2). BM-MSCs from female and male donors expressed androgen receptor and FGFR3, and secreted VEGF-A, HGF, LIF, Angiopoietin-1, basic fibroblast growth factor (bFGF) and NGFB. HGF secretion correlated negatively to the expression of CD71, CD140b and Galectin 1. The expression of Oct4, Nanog and Prdm14 mRNA in BM-MSCs was much lower compared to pluripotent stem cells and was not related to donor age or gender. Prdm14 mRNA expression correlated positively to the clonogenic potential of BM-MSCs . Conclusions By identifying donor-related effects and assigning phenotypes of BM-MSC preparations to functional properties, we provide useful tools for assay development and production for clinical applications of BM-MSC preparations.

5-Lipoxygenase initiates the biosynthesis of leukotrienes, lipid mediators involved in normal host defense and in inflammatory and allergic disorders. Despite an obvious gender bias in leukotriene-related diseases (e.g., asthma), gender aspects have been neglected in studies on leukotrienes and 5-lipoxygenase. Here, we show that leukotriene formation in stimulated whole blood or neutrophils from males is substantially lower compared with females, accompanied by changed 5-lipoxygenase trafficking. This is due to gender-specific differential activation of extracellular signal-regulated kinases (ERKs). The differences are directly related to variant male/female testosterone plus 5α-dihydrotestosterone levels, and addition of 5α-dihydrotestosterone to female blood or neutrophils reduced the high (female) LT biosynthesis capacity to low (male) levels. In conclusion, regulation of ERKs and leukotriene formation by androgens constitutes a molecular basis for gender differences in the inflammatory response, and in inflammatory diseases such as asthma.

Oxidative stress due to intratumoral hypoxia in solid cancer has been shown to be associated with increased mortality. Phosphoglycerate kinase 1 (PGK1) is an enzyme of the glycolytic pathway, which is regulated by hypoxia-inducible factor-1α (HIF-1α) and has been described for its role in tumor progression and metastasis in several malignancies. We investigated whether the expression of PGK1 varies between metastatic and non-metastatic colon cancer. We compared PGK1 expression in colon cancer patients either with or without metastasis via polymerase chain reaction (PCR) and immunohistochemistry. Microarray analysis was performed to test altered gene expression after PGK1 silencing, using isolates from HCT116 cell lines. PCR results showed an increased expression of PGK1 in colon cancer tissue from metastatic patients in comparison to patients with no metastasis (fold change 2.6, p<0.001). Immunohistochemical staining of PGK1 showed stronger staining in metastatic tissue in comparison to non-metastatic cancer tissue according to a semi-quantitative evaluation. Microarray and subsequent pathway analysis provided 4 genes of interest (CYR61, FOS, JUN and EGR1) used for pathway proposal. The results indicate that increased expression of PGK1 in colon cancer tissue is associated with metastasis. Furthermore, we propose several genes induced by PGK1 that could account for cell migration, mainly EGR1 and CYR61 together with the transcription factors FOS and JUN.

The determination of fibrinogen levels is one of the most important coagulation measurements in medicine. It plays a crucial part in diagnostic and therapeutic decisions, often associated with time-critical conditions. The commonly used measurement is the Clauss fibrinogen assay (CFA) where plasma is activated by thrombin reagent and which is conducted by mechanical/turbidimetric devices. As quartz crystal microbalance sensors with dissipation (QCM-D) based devices have a small footprint, can be operated easily and allow measurements independently from sample transportation time, laboratory location, availability and opening hours, they offer a great opportunity to complement laboratory CFA measurements. Therefore, the objective of the work was to (1) transfer the CFA to the QCM-D method; (2) develop an easy, time- and cost-effective procedure and (3) compare the results with references. Different sensor coatings (donor’s own plasma; gold surface) and different QCM-D parameters (frequency signal shift; its calculated turning point; dissipation signal shift) were sampled. The results demonstrate the suitability for a QCM-D-based CFA in physiological fibrinogen ranges. Results were obtained in less than 1 min and in very good agreement with a standardized reference (Merlin coagulometer). The results provide a good basis for further investigation and pave the way to a possible application of QCM-D in clinical and non-clinical routine in the medical field.

Background MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period. Results Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6 . The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation. Conclusions We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation.

For clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSC's biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed. Human MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-g in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs. In our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.

Leukotrienes (LTs) play a central role in asthma. Low- to moderate-intensity aerobic exercise (AE) reduces asthmatic inflammation in clinical studies and in experimental models. This study investigated whether AE attenuates LT pathway activation in an ovalbumin (OVA) model of asthma. Sixty-four male, BALB/c mice were distributed into Control, Exercise (Exe), OVA, and OVA + Exe groups. Treadmill training was performed at moderate intensity, 5×/week, 1 h/session for 4 weeks. Quantification of bronchoalveolar lavage (BAL) cellularity, leukocytes, airway remodeling, interleukin (IL)-5, IL-13, cysteinyl leukotriene (CysLT), and leukotriene B4 (LTB4) in BAL was performed. In addition, quantitative analyses on peribronchial leukocytes and airway epithelium for LT pathway agents: 5-lypoxygenase (5-LO), LTA4 hydrolase (LTA4H), CysLT1 receptor, CysLT2 receptor, LTC4 synthase, and LTB4 receptor 2 (BLT2) were performed. Airway hyperresponsiveness (AHR) to methacholine (MCh) was assessed via whole body plethysmography. AE decreased eosinophils (p < 0.001), neutrophils (p > 0.001), lymphocytes (p < 0.001), and macrophages (p < 0.01) in BAL, as well as eosinophils (p < 0.01), lymphocytes (p < 0.001), and macrophages (p > 0.001) in airway walls. Collagen (p < 0.01), elastic fibers (p < 0.01), mucus production (p < 0.01), and smooth muscle thickness (p < 0.01), as well as IL-5 (p < 0.01), IL-13 (p < 0.01), CysLT (p < 0.01), and LTB4 (p < 0.01) in BAL were reduced. 5-LO (p < 0.05), LTA4H (p < 0.05), CysLT1 receptor (p < 0.001), CysLT2 receptor (p < 0.001), LTC4 synthase (p < 0.001), and BLT2 (p < 0.01) expression by peribronchial leukocytes and airway epithelium were reduced. Lastly, AHR to MCh 25 mg/mL (p < 0.05) and 50 mg/mL (p < 0.01) was reduced. Moderate-intensity AE attenuated asthma phenotype and LT production in both pulmonary leukocytes and airway epithelium of OVA-treated mice.

The reliability of implantable blood sensors is often hampered by unspecific adsorption of plasma proteins and blood cells. This not only leads to a loss of sensor signal over time, but can also result in undesired host vs. graft reactions. Within this study we evaluated the hemocompatibility of isocyanate conjugated star shaped polytheylene oxide—polypropylene oxide co-polymers NCO-sP(EO-stat-PO) when applied to gold surfaces as an auspicious coating material for gold sputtered blood contacting sensors. Quartz crystal microbalance (QCM) sensors were coated with ultrathin NCO-sP(EO-stat-PO) films and compared with uncoated gold sensors. Protein resistance was assessed by QCM measurements with fibrinogen solution and platelet poor plasma (PPP), followed by quantification of fibrinogen adsorption. Hemocompatibility was tested by incubation with human platelet rich plasma (PRP). Thrombin antithrombin-III complex (TAT), β-thromboglobulin (β-TG) and platelet factor 4 (PF4) were used as coagulation activation markers. Furthermore, scanning electron microscopy (SEM) was used to visualize platelet adhesion to the sensor surfaces. Compared to uncoated gold sensors, NCO-sP(EO-stat-PO) coated sensors revealed significant better resistance against protein adsorption, lower TAT generation and a lower amount of adherent platelets. Moreover, coating with ultrathin NCO-sP(EO-stat-PO) films creates a cell resistant hemocompatible surface on gold that increases the chance of prolonged sensor functionality and can easily be modified with specific receptor molecules.

Background Vasoactive intestinal polypeptide secreting tumors(VIPomas) are rare endocrine tumors of the pancreas with an estimated incidence of 0.1 per million per year. The molecular mechanisms that mediate development of VIPomas are poorly investigated and require definition. Methods A genome- and gene expression analysis of specimens of a primary pancreatic VIPoma with hepatic metastases was performed. The primary tumor, the metastases, the corresponding healthy tissue of the liver, and the pancreas were compared with each other using oligonucleotide microarrays and loss of heterozygosity (LOH). Results The results revealed multiple LOH events and several differentially expressed genes. Our finding of LOH and downregulation was conspicuous in the microarray analysis for the mismatch repair gene MSH2 in the primary pancreatic VIPoma tumor, the hepatic metastasis but not in the corresponding healthy tissue. Further a strong overexpression of the chemokine CXCR4 was detected in the hepatic metastases compared to its pancreatic primary. With a review of the literature we describe the molecular insights of metastatic development in VIPoma. Conclusion In VIPoma, defects in the mismatch repair system especially in MSH2 may contribute to carcinogenesis, and increased CXCR4 may be associated with liver metastasis.

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.