Upper ocean vertical supply: A neglected primary factor controlling the distribution of neodymium concentrations of open ocean surface waters?

Neodymium (Nd) isotopes are an important geochemical tool to trace the present and past water mass mixing as well as continental inputs. The distribution of Nd concentrations in open ocean surface waters (0�100 m) is generally assumed to be controlled by lateral mixing of Nd from coastal surface currents and by removal through reversible particle scavenging. However, using 228Ra activity as an indicator of coastal water mass influence, surface water Nd concentration data available on key oceanic transects as a whole do not support the above scenario. From a global compilation of available data, we find that more stratified regions are generally associated with low surface Nd concentrations. This implies that upper ocean vertical supply may be an as yet neglected primary factor in determining the basin-scale variations of surface water Nd concentrations. Similar to the mechanism of nutrients supply, it is likely that stratification inhibits vertical supply of Nd from the subsurface thermocline waters and thus the magnitude of Nd flux to the surface layer. Consistently, the estimated required input flux of Nd to the surface layer to maintain the observed concentrations could be nearly two orders of magnitudes larger than riverine/dust flux, and also larger than the model-based estimation on shelf-derived coastal flux. In addition, preliminary results from modeling experiments reveal that the input from shallow boundary sources, riverine input, and release from dust are actually not the primary factors controlling Nd concentrations most notably in the Pacific and Southern Ocean surface waters.

Matrix metalloproteinase inhibition lowers mortality and brain injury in experimental pneumococcal meningitis

Pneumococcal meningitis (PM) results in high mortality rates and long-lasting neurological deficits. Hippocampal apoptosis and cortical necrosis are histopathological correlates of neurofunctional sequelae in rodent models and are frequently observed in autopsy studies of patients who die of PM. In experimental PM, inhibition of matrix metalloproteinases (MMPs) and/or tumor necrosis factor (TNF)-converting enzyme (TACE) has been shown to reduce brain injury and the associated impairment of neurocognitive function. However, none of the compounds evaluated in these studies entered clinical development. Here, we evaluated two second-generation MMP and TACE inhibitors with higher selectivity and improved oral availability. Ro 32-3555 (Trocade, cipemastat) preferentially inhibits collagenases (MMP-1, -8, and -13) and gelatinase B (MMP-9), while Ro 32-7315 is an efficient inhibitor of TACE. PM was induced in infant rats by the intracisternal injection of live Streptococcus pneumoniae. Ro 32-3555 and Ro 32-7315 were injected intraperitoneally, starting at 3 h postinfection. Antibiotic (ceftriaxone) therapy was initiated at 18 h postinfection, and clinical parameters (weight, clinical score, mortality rate) were recorded. Myeloperoxidase activities, concentrations of cytokines and chemokines, concentrations of MMP-2 and MMP-9, and collagen concentrations were measured in the cerebrospinal fluid. Animals were sacrificed at 42 h postinfection, and their brains were assessed by histomorphometry for hippocampal apoptosis and cortical necrosis. Both compounds, while exhibiting disparate MMP and TACE inhibitory profiles, decreased hippocampal apoptosis and cortical injury. Ro 32-3555 reduced mortality rates and cerebrospinal fluid TNF, interleukin-1β (IL-1β) and collagen levels, while Ro 32-7315 reduced weight loss and cerebrospinal fluid TNF and IL-6 levels.

Differential effect of p47phox and gp91phox deficiency on the course of Pneumococcal Meningitis.

Bacterial meningitis is a severe inflammatory disease of the central nervous system and is characterized by massive infiltration of granulocytes into the cerebrospinal fluid (CSF). To assess the role of NADPH oxidase-derived reactive oxygen species (ROS) in pneumococcal meningitis, mice deficient in either the gp91 subunit (essential for functioning of the phagocyte enzyme) or the p47 subunit (essential for functioning of homologous enzymes in nonphagocytic cells) were intracisternally infected with live Streptococcus pneumoniae, and defined disease parameters were measured during the acute stage of infection. While none of the parameters measured (including CSF bacterial titers) were significantly different in gp91(-/-) and wild-type mice, the infection in p47(-/-) mice was associated with significantly increased inflammation of the subarachnoid and ventricular space, disruption of the blood-brain barrier, and the presence of interleukin-1 beta, tumor necrosis factor alpha, and matrix metalloproteinase 9 in the cortex. These changes were associated with approximately 10-fold-higher CSF bacterial titers in p47(-/-) mice than in wild-type mice (P < 0.001). In contrast to infection with live bacteria, the inflammatory response, including CSF leukocytosis, was significantly attenuated in p47(-/-) mice (but not gp91(-/-) mice) challenged with a fixed number of heat-inactivated pneumococci. Impairment of the host defense appeared to be responsible for the higher bacterial titers in p47(-/-) mice. Therefore, these results indicate that ROS generated by a gp91-independent NADPH oxidase(s) are important for establishing an adequate inflammatory response to pneumococcal CSF infection.

Intracisternal application of endotoxin enhances the susceptibility to subsequent hypoxic-ischemic brain damage in neonatal rats.

Perinatal brain damage is associated not only with hypoxic-ischemic insults but also with intrauterine inflammation. A combination of antenatal inflammation and asphyxia increases the risk of cerebral palsy >70 times. The aim of the present study was to determine the effect of intracisternal (i.c.) administration of endotoxin [lipopolysaccharides (LPS)] on subsequent hypoxic-ischemic brain damage in neonatal rats. Seven-day-old Wistar rats were subjected to i.c. application of NaCl or LPS (5 microg/pup). One hour later, the left common carotid artery was exposed through a midline neck incision and ligated with 6-0 surgical silk. After another hour of recovery, the pups were subjected to a hypoxic gas mixture (8% oxygen/92% nitrogen) for 60 min. The animals were randomized to four experimental groups: 1) sham control group, left common carotid artery exposed but not ligated (n = 5); 2) LPS group, subjected to i.c. application of LPS (n = 7); 3) hypoxic-ischemic study group, i.c. injection of NaCl and exposure to hypoxia after ligation of the left carotid artery (n = 17); or 4) hypoxic-ischemic/LPS study group, i.c. injection of LPS and exposure to hypoxia after ligation of the left carotid artery (n = 19). Seven days later, neonatal brains were assessed for neuronal cell damage. In a second set of experiments, rat pups received an i.c. injection of LPS (5 microg/pup) and were evaluated for tumor necrosis factor-alpha expression by immunohistochemistry. Neuronal cell damage could not be observed in the sham control or in the LPS group. In the hypoxic-ischemic/LPS group, neuronal injury in the cerebral cortex was significantly higher than in animals that were subjected to hypoxia/ischemia after i.c. application of NaCl. Injecting LPS intracisternally caused a marked expression of tumor necrosis factor-alpha in the leptomeninges. Applying LPS intracisternally sensitizes the immature rat brain to a subsequent hypoxic-ischemic insult.

Matrix metalloproteinase (MMP)-8 and MMP-9 in cerebrospinal fluid during bacterial meningitis: association with blood-brain barrier damage and neurological sequelae.

To evaluate the spectrum and regulation of matrix metalloproteinases (MMPs) in bacterial meningitis (BM), concentrations of MMP-2, MMP-3, MMP-8, and MMP-9 and endogenous inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were measured in the cerebrospinal fluid (CSF) of 27 children with BM. MMP-8 and MMP-9 were detected in 91% and 97%, respectively, of CSF specimens from patients but were not detected in control patients. CSF levels of MMP-9 were higher (P<.05) in 5 patients who developed hearing impairment or secondary epilepsy than in those who recovered without neurological deficits. Levels of MMP-9 correlated with concentrations of TIMP-1 (P<.001) and tumor necrosis factor-alpha (P=.03). Repeated lumbar punctures showed that levels of MMP-8 and MMP-9 were regulated independently and did not correlate with the CSF cell count. Therefore, MMPs may derive not only from granulocytes infiltrating the CSF space but also from parenchymal cells of the meninges and brain. High concentrations of MMP-9 are a risk factor for the development of postmeningitidal neurological sequelae.

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

CD40 activation induces NREM sleep and modulates genes associated with sleep homeostasis

The T-cell derived cytokine CD40 ligand is overexpressed in patients with autoimmune diseases. Through activation of its receptor, CD40 ligand leads to a tumor necrosis factor (TNF) receptor 1 (TNFR1) dependent impairment of locomotor activity in mice. Here we report that this effect is explained through a promotion of sleep, which was specific to non-rapid eye movement (NREM) sleep while REM sleep was suppressed. The increase in NREM sleep was accompanied by a decrease in EEG delta power during NREM sleep and by a decrease in the expression of transcripts in the cerebral cortex known to be associated with homeostatic sleep drive, such as Homer1a, Early growth response 2, Neuronal pentraxin 2, and Fos-like antigen 2. The effect of CD40 activation was mimicked by peripheral TNF injection and prevented by the TNF blocker etanercept. Our study indicates that sleep-wake dysregulation in autoimmune diseases may result from CD40 induced TNF:TNFR1 mediated alterations of molecular pathways, which regulate sleep-wake behavior.

Decision making in beach volleyball defense: Crucial factors derived from interviews with top-level experts

Objectives: In fast ball sports like beach volleyball, decision-making skills are a determining factor for excellent performance. The current investigation aimed to identify factors that influence the decisionmaking process in top-level beach volleyball defense in order to find relevant aspects for further research. For this reason, focused interviews with top players in international beach volleyball were conducted and analyzed with respect to decision-making characteristics. Design: Nineteen world-tour beach volleyball defense players, including seven Olympic or world champions, were interviewed, focusing on decision-making factors, gaze behavior, and interactions between the two. Methods: Verbal data were analyzed by inductive content analysis according to Mayring (2008). This approach allows categories to emerge from the interview material itself instead of forcing data into preset classifications and theoretical concepts. Results: The data analysis showed that, for top-level beach volleyball defense, decision making depends on opponent specifics, external context, situational context, opponent's movements, and intuition. Information on gaze patterns and visual cues revealed general tendencies indicating optimal gaze strategies that support excellent decision making. Furthermore, the analysis highlighted interactions between gaze behavior, visual information, and domain-specific knowledge. Conclusions: The present findings provide information on visual perception, domain-specific knowledge, and interactions between the two that are relevant for decision making in top-level beach volleyball defense. The results can be used to inform sports practice and to further untangle relevant mechanisms underlying decision making in complex game situations.

Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

Clinical and biochemical profiles suggest fibromuscular dysplasia is a systemic disease with altered TGF-β expression and connective tissue features.

Fibromuscular dysplasia (FMD) is a rare, nonatherosclerotic arterial disease for which the molecular basis is unknown. We comprehensively studied 47 subjects with FMD, including physical examination, spine magnetic resonance imaging, bone densitometry, and brain magnetic resonance angiography. Inflammatory biomarkers in plasma and transforming growth factor β (TGF-β) cytokines in patient-derived dermal fibroblasts were measured by ELISA. Arterial pathology other than medial fibrodysplasia with multifocal stenosis included cerebral aneurysm, found in 12.8% of subjects. Extra-arterial pathology included low bone density (P<0.001); early onset degenerative spine disease (95.7%); increased incidence of Chiari I malformation (6.4%) and dural ectasia (42.6%); and physical examination findings of a mild connective tissue dysplasia (95.7%). Screening for mutations causing known genetically mediated arteriopathies was unrevealing. We found elevated plasma TGF-β1 (P=0.009), TGF-β2 (P=0.004) and additional inflammatory markers, and increased TGF-β1 (P=0.0009) and TGF-β2 (P=0.0001) secretion in dermal fibroblast cell lines from subjects with FMD compared to age- and gender-matched controls. Detailed phenotyping of patients with FMD allowed us to demonstrate that FMD is a systemic disease with alterations in common with the spectrum of genetic syndromes that involve altered TGF-β signaling and offers TGF-β as a marker of FMD.

Increased sleep need and daytime sleepiness 6 months after traumatic brain injury: a prospective controlled clinical trial.

Post-traumatic sleep-wake disturbances are common after acute traumatic brain injury. Increased sleep need per 24 h and excessive daytime sleepiness are among the most prevalent post-traumatic sleep disorders and impair quality of life of trauma patients. Nevertheless, the relation between traumatic brain injury and sleep outcome, but also the link between post-traumatic sleep problems and clinical measures in the acute phase after traumatic brain injury has so far not been addressed in a controlled and prospective approach. We therefore performed a prospective controlled clinical study to examine (i) sleep-wake outcome after traumatic brain injury; and (ii) to screen for clinical and laboratory predictors of poor sleep-wake outcome after acute traumatic brain injury. Forty-two of 60 included patients with first-ever traumatic brain injury were available for follow-up examinations. Six months after trauma, the average sleep need per 24 h as assessed by actigraphy was markedly increased in patients as compared to controls (8.3 ± 1.1 h versus 7.1 ± 0.8 h, P < 0.0001). Objective daytime sleepiness was found in 57% of trauma patients and 19% of healthy subjects, and the average sleep latency in patients was reduced to 8.7 ± 4.6 min (12.1 ± 4.7 min in controls, P = 0.0009). Patients, but not controls, markedly underestimated both excessive sleep need and excessive daytime sleepiness when assessed only by subjective means, emphasizing the unreliability of self-assessment of increased sleep propensity in traumatic brain injury patients. At polysomnography, slow wave sleep after traumatic brain injury was more consolidated. The most important risk factor for developing increased sleep need after traumatic brain injury was the presence of an intracranial haemorrhage. In conclusion, we provide controlled and objective evidence for a direct relation between sleep-wake disturbances and traumatic brain injury, and for clinically significant underestimation of post-traumatic sleep-wake disturbances by trauma patients.

The secretome of induced pluripotent stem cells reduces lung fibrosis in part by hepatocyte growth factor.

INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic lung disease, resulting in respiratory insufficiency and reduced survival. Pulmonary fibrosis is a result of repeated alveolar epithelial microinjuries, followed by abnormal regeneration and repair processes in the lung. Recently, stem cells and their secretome have been investigated as a novel therapeutic approach in pulmonary fibrosis. We evaluated the potential of induced pluripotent stem cells (iPSC) conditioned media (iPSC-cm) to regenerate and repair the alveolar epithelium in vitro and improve bleomycin induced lung injury in vivo. METHODS IPSC-cm was collected from cultured iPSC derived from human foreskin fibroblasts and its biological effects on alveolar epithelial wound repair was studied in an alveolar wound healing assay in vitro. Furthermore, iPSC-cm was intratracheally instilled 7 days after bleomycin induced injury in the rat lungs and histologically and biochemically assessed 7 days after instillation. RESULTS iPSC-cm increased alveolar epithelial wound repair in vitro compared with medium control. Intratracheal instillation of iPSC-cm in bleomycin-injured lungs reduced the collagen content and improved lung fibrosis in the rat lung in vivo. Profibrotic TGFbeta1 and alpha-smooth muscle actin (alpha-sma) expression were markedly reduced in the iPSC-cm treated group compared with control. Antifibrotic hepatocyte growth factor (HGF) was detected in iPSC-cm in biologically relevant levels, and specific inhibition of HGF in iPSC-cm attenuated the antifibrotic effect of iPSC-cm, indicating a central role of HGF in iPSC-cm. CONCLUSION iPSC-cm increased alveolar epithelial wound repair in vitro and attenuated bleomycin induced fibrosis in vivo, partially due to the presence of HGF and may represent a promising novel, cell free therapeutic option against lung injury and fibrosis.

The secretome of endothelial progenitor cells promotes brain endothelial cell activity through PI3-kinase and MAP-kinase

BACKGROUND Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. METHODS Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. RESULTS Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. CONCLUSION The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.