Hinnak Northoff

Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells

BackgroundMesenchymal 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.MethodsBM-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.ResultsBM-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.ConclusionsBy 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.

Effect of exhaustive exercise stress on the cytokine response.

Fifteen athletes were investigated 24 h before, 1 h after, and 20 h after an exhaustive exercise stress test (mean duration 68 min). Testing for cytokines was done in serum, urine, and the supernatants of whole blood cell cultures, which were stimulated with lipopolysaccharide (LPS), concanavalin A (Con A), or phythaemagglutinin (PHA). Elevated levels of interleukin 6 (IL-6) and soluble IL-2 receptor (sIL-2R) were found 1 h after the run in both serum and urine samples. TNF-alpha in serum was also increased, whereas IL-2 in urine was decreased after the exercise. All other testings in serum and urine (including IFN-gamma) gave borderline or negative results. In cell cultures, the LPS-induced release of the inflammatory cytokines TNF-alpha, IL-1, and IL-6 was suppressed 1 h after exercise. Also, the Con-A-induced and LPS-induced release of IFN-gamma, and the PHA-induced release of IL-2 were suppressed 1 h after exercise. In contrast, Con-A-induced release of IL-2 was mildly increased after the run. We conclude that exercise of the intensity and duration described here causes an activation of the immune system, which is immediately counter-regulated. Twenty hours after the exercise, most of the observed changes were back to pre-exercise levels, indicating only a short duration for this suppressive counter-regulation.

Both adenosine A1- and A2-receptors are required to stimulate microglial proliferation.

The neuromodulator adenosine is one of the major endogenous inhibitors of overactive excitatory neurotransmission. Adenosine receptors have been identified on neuronal but also on glial surfaces, indicating a role of glial cells in mediation of adenosine effects. Microglia, the immunocompetent cells of the brain, typically respond with proliferation, migration and production of inflammatory substances to viral or bacterial stimuli or to cell damage and degeneration. Since adenosine is released in large amounts in conditions of, for example, hypoxic or ischemic stress, it might be involved in the activation process of microglia. Proliferation of microglia was determined by incorporation of [3H]thymidine into microglial DNA after stimulation with adenosine A1- and A2-receptor agonists. N6-Cyclopentyl adenosine (CPA) and CGS-21680, a specific adenosine A2-receptor agonist had no effect on microglial proliferation. However, combinations of CPA and CGS-21680 as well as the mixed agonist, N6-ethyl-carboxamido adenosine (NECA) increased incorporation of radiolabel above controls. The effect of NECA was inhibited by the adenosine A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). From these results, it is concluded that proliferation of microglia can be increased only by simultaneous stimulation of both adenosine A1- and A2-receptors. Targeted interference with the activation of A1-adenosine receptors by specific drugs appears to be sufficient to reduce microglial activation. The findings may have implications for the treatment of neurodegenerative diseases in which microglial activation is supposed to play a causative role.

HSP expression in human leukocytes is modulated by endurance exercise.

PURPOSE Temperature increase, oxidative stress, and inflammatory reactions after endurance exercise were expected to stimulate the synthesis of heat shock proteins (HSP) in peripheral blood leukocytes. Furthermore, it was of interest whether regular endurance training influences HSP expression. METHODS The expression of HSP27, HSP60, HSP70, constitutive HSC70, and HSP90 in the cytoplasma and surface of lymphocytes, monocytes, and granulocytes of 12 trained athletes was analyzed by flow cytometry before and after (0, 3, and 24 h) a half marathon. Twelve untrained persons at rest were included as control. RESULTS After the race, there was a significantly greater percentage of leukocytes expressing cytoplasmic HSP27, HSP60, and HSP70 (P < 0.01), whereas HSC70 and HSP90 remained unchanged. The fluorescence intensity increased significantly in monocytes for HSP27 (0 and 3 h) and HSP70 (0, 3, and 24 h) and in granulocytes, only 24 h postexercise for HSP70. The percent values of trained athletes at rest were significantly lower compared with untrained persons (P < 0,01). CONCLUSIONS Strenuous exercise increased HSP expression in blood immediately after the run, indicating a protective function of HSP in leukocytes of athletes to maintain function after heavy exercise. The downregulation of HSP-positive cells in trained athletes at rest seems to be a result of adaptation mechanisms to regular endurance training.

Differential gene expression in peripheral blood of patients suffering from post-traumatic stress disorder

Differential gene expression in peripheral blood of patients suffering from post-traumatic stress disorder

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes.

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence‐activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml−1 haemolysin within minutes induces a non‐selective cation permeability. Moreover, haemolysin activates clotrimazole‐sensitive K+ channels, pointing to stimulation of Ca2+‐sensitive Gardos channels. Haemolysin (1 U ml−1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+‐free saline. Erythrocytes treated with haemolysin (0.1 U ml−1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml−1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml−1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.

Curcumin blocks prostaglandin E2 biosynthesis through direct inhibition of the microsomal prostaglandin E2 synthase-1

Prostaglandin E2 (PGE2) plays a crucial role in the apparent link between tumor growth and chronic inflammation. Cyclooxygenase (COX)-2 and microsomal PGE2 synthase-1, which are overexpressed in many cancers, are functionally coupled and thus produce massive PGE2 in various tumors. Curcumin, a polyphenolic β-diketone from tumeric with anti-carcinogenic and anti-inflammatory activities, was shown to suppress PGE2 formation and to block the expression of COX-2 and of microsomal PGE2 synthase-1. Here, we identified microsomal PGE2 synthase-1 as a molecular target of curcumin and we show that inhibition of microsomal PGE2 synthase-1 activity is the predominant mechanism of curcumin to suppress PGE2 biosynthesis. Curcumin reversibly inhibited the conversion of PGH2 to PGE2 by microsomal PGE2 synthase-1 in microsomes of interleukin-1β–stimulated A549 lung carcinoma cells with an IC50 of 0.2 to 0.3 μmol/L. Closely related polyphenols (e.g., resveratrol, coniferyl alcohol, eugenol, rosmarinic acid) failed in this respect, and isolated ovine COX-1 and human recombinant COX-2 were not inhibited by curcumin up to 30 μmol/L. In lipopolysaccharide-stimulated human whole blood, curcumin inhibited COX-2–derived PGE2 formation from endogenous or from exogenous arachidonic acid, whereas the concomitant formation of COX-2–mediated 6-keto PGF1α and COX-1–derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was suppressed only at significant higher concentrations. Based on the key function of PGE2 in inflammation and carcinogenesis, inhibition of microsomal PGE2 synthase-1 by curcumin provides a molecular basis for its anticarcinogenic and anti-inflammatory activities. [Mol Cancer Ther 2009;8(8):2348–55]

Identification of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 as functional targets of the anti-inflammatory and anti-carcinogenic garcinol

Garcinol (camboginol) from the fruit rind of Guttiferae species shows anti-carcinogenic and anti-inflammatory properties, but the underlying molecular mechanisms are unclear. Here we show that garcinol potently interferes with 5-lipoxygenase (EC 7.13.11.34) and microsomal prostaglandin (PG)E2 synthase (mPGES)-1 (EC 5.3.99.3), enzymes that play pivotal roles in inflammation and tumorigenesis. In cell-free assays, garcinol inhibited the activity of purified 5-lipoxygenase and blocked the mPGES-1-mediated conversion of PGH2 to PGE2 with IC50 values of 0.1 and 0.3 microM, respectively. Garcinol suppressed 5-lipoxygenase product formation also in intact human neutrophils and reduced PGE2 formation in interleukin-1beta-stimulated A549 human lung carcinoma cells as well as in human whole blood stimulated by lipopolysaccharide. Moreover, garcinol interfered with isolated cyclooxygenase (COX)-1 (EC 1.14.99.1, IC50 = 12 microM) and with the formation of COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and thromboxane B2 in human platelets. In contrast, neither Ca2+-ionophore (A23187)-induced arachidonic acid release in neutrophils nor COX-2 activity in A549 cells or whole blood, measured as formation of 6-keto PGF1alpha, or isolated human recombinant COX-2 were significantly affected by garcinol (< or = 30 microM). Together, the high potency of garcinol to selectively suppress PGE2 synthesis and 5-lipoxygenase product formation provides a molecular basis for the anti-inflammatory and anti-carcinogenic effects of garcinol and rationalizes its therapeutic use.

Expression of the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes: Acute and adaptational responses to endurance exercise

Inducible heme oxygenase (HO-1) is an antioxidant stress protein, that is mainly induced by reactive oxygen species (ROS), cytokines and hyperthermia. By using flow cytometry the present investigation demonstrated a rise in the cytoplasmic expression of HO-1 in lympho- (L), mono- (M) and granulocytes (G) of 9 endurance-trained male subjects after a half marathon run. The expression was more pronounced in M (median: 98.3% HO-1 positive cells/4.31 mfc) and G (94.8%/1.93 mfc) than in L (80.1%/1.51 mfc) when measured 3 h post-exercise. Additionally the exercise protocol caused a rise in the plasma levels of myeloperoxidase, TNF alpha and interleukin-8 (IL-8), indicating an inflammatory response. We could detect a correlation between IL-8 and HO-1, directly after exercise, that was apparent in G (r = 0.67, p < .05) and L (r = 0.80, p < .05), but did not reach significance in M (r = 0.65, p = 0.06). An additional detection of HO-1 at rest in 12 untrained subjects showed a higher baseline expression of HO-1 compared to the athletes. The regulatory pathways leading to an increased expression of HO-1 after endurance exercise are not completely clear, but a causal involvement of a cytokine-mediated generation of ROS must be discussed. We supposed that the down-regulation of the baseline expression of HO-1 in athletes reflects an adaptional mechanism to regular exercise training.

Labeling of human mesenchymal stromal cells with superparamagnetic iron oxide leads to a decrease in migration capacity and colony formation ability

BACKGROUND AIMS Labeling of stem cells is crucial to allow tracking of stem cell homing and engraftment after transplantation. In this study we evaluated the influence of cell labeling procedures using clinically approved small particles of iron oxide (SPIO) with or without transfection reagents (TA) on functional parameters of human mesenchymal stem cells (MSC). METHODS The study was approved by the institutional review board of the University of Tubingen, Germany. Seven populations of bone marrow (BM)-derived human mesenchymal stem cells (MSC) were labeled with SPIO alone or in combination with various TA. Directly after labeling and two passages after labeling migration assays, quantification of colony-forming units and quantitative evaluation of the differentiation potential were performed. Quantification of the cellular total iron load (TIL), determination of the cellular viability and electron microscopy were also performed. RESULTS Labeling of mesenchymal stem cells with SPIO with or without TA did not affect cell viability and differentiation potential significantly. SPIO in combination with TA coated the cellular surface directly after labeling but was incorporated into the cells after two passages. Labeling of mesenchymal stem cells with TA led to a significant decrease of migration capacity. This effect was abolished after two passages. Labeling with and without TA led to a significant decrease in colony formation ability. This effect could also be observed after two passages. CONCLUSIONS The observed decrease of migration capacity and colony-formation ability was not associated with either TIL or localization of particles of iron oxide. SPIO labeling with and without TA had functional effects on human mesenchymal stem cells by decreasing the migration capacity and colony-formation ability of the stem cells.