High capacity for leukotriene biosynthesis in peripheral blood during pregnancy.

Pregnancy is accompanied by major immunological changes to maintain both tolerance for the fetus and immune competence. Leukotrienes are powerful 5-lipoxygenase-derived inflammatory mediators and the characteristics of leukotriene-related diseases (e.g., asthma, allergic rhinitis) change during pregnancy. Here, we show that pregnancy affects leukotriene synthesis in human blood and leukocytes. 5-Lipoxygenase product formation in stimulated blood of pregnant women was significantly higher than in non-pregnant females. Although a pregnancy-related increase in neutrophil and monocyte counts may explain these observations, granulocytes of pregnant donors have lower leukotriene-synthetic capacities. On the other hand, granulocytes from non-pregnant woman produced more leukotrienes when resuspended in plasma of pregnant women than of non-pregnant females. Together, we show that leukotriene biosynthesis in maternal blood is increased by the interrelations of higher leukocyte numbers, lower cellular capacity for leukotriene synthesis and stimulatory effects of plasma. This bias may affect leukotriene-related diseases during pregnancy and their pharmacological treatment.

On the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo.

BACKGROUND AND PURPOSE: Leukotrienes (LTs) are pro-inflammatory mediators produced by 5-lipoxygenase (5-LO). Currently available 5-LO inhibitors either lack efficacy or are toxic and novel approaches are required to establish a successful anti-LT therapy. Here we provide a detailed evaluation of the effectiveness of the plant-derived alkaloid tryptanthrin as an inhibitor of LT biosynthesis. EXPERIMENTAL APPROACH: We analysed LT formation and performed mechanistic studies in human neutrophils stimulated with pathophysiologically relevant stimuli (LPS and formyl peptide), as well as in cell-free assays (neutrophil homogenates or recombinant human 5-LO) and in human whole blood. The in vivo effectiveness of tryptanthrin was evaluated in the rat model of carrageenan-induced pleurisy. KEY RESULTS: Tryptanthrin potently reduced LT-formation in human neutrophils (IC(50) = 0.6µM). However, tryptanthrin is not a redox-active compound and did not directly interfere with 5-LO activity in cell-free assays. Similarly, tryptanthrin did not inhibit the release of arachidonic acid, the activation of MAPKs, or the increase in [Ca(2+) ](i) , but it modified the subcellular localization of 5-LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC(50) = 10µM) and reduced LTB(4) levels in the rat pleurisy model after a single oral dose of 10mg·kg(-1) . CONCLUSIONS AND IMPLICATIONS: Our data reveal that tryptanthrin is a potent natural inhibitor of cellular LT biosynthesis with proven efficacy in whole blood and is effective in vivo after oral administration. Its unique pharmacological profile supports further analysis to exploit its pharmacological potential.

Wound fluid lactate concentration: a helpful marker for diagnosing soft-tissue infection in diabetic foot ulcers? Preliminary findings.

AIMS: To investigate the impact of wound fluid lactate concentration on diagnosing soft-tissue infection in diabetic foot ulcers. METHODS: Lactate concentration in wound fluid obtained from diabetic foot ulcers was determined using a lactate analyser and compared with clinical examination findings. RESULTS: Overall median wound fluid lactate concentration was 21.03 mm (5.58-80.40 mm). Wound lactate levels were significantly higher in infected compared with non-infected diabetic foot ulcers (P=0.001). Non-infected diabetic foot ulcers that healed within 6 months of treatment showed a significantly lower wound fluid lactate concentration at baseline as opposed to those that did not heal (P=0.007). CONCLUSIONS: Non-healing diabetic foot ulcers are characterized by high wound fluid lactate levels. Assessment of wound fluid lactate concentration might be helpful for confirming the suspicion of soft tissue infection, particularly when clinical signs are atypical.

Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense.

BACKGROUND AND PURPOSE: Frankincense, the gum resin derived from Boswellia species, showed anti-inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed to be responsible for these effects but their anti-inflammatory efficacy in vivo and their molecular modes of action are incompletely understood. EXPERIMENTAL APPROACH: A protein fishing approach using immobilized BA and surface plasmon resonance (SPR) spectroscopy were used to reveal microsomal prostaglandin E(2) synthase-1 (mPGES1) as a BA-interacting protein. Cell-free and cell-based assays were applied to confirm the functional interference of BAs with mPGES1. Carrageenan-induced mouse paw oedema and rat pleurisy models were utilized to demonstrate the efficacy of defined BAs in vivo. KEY RESULTS: Human mPGES1 from A549 cells or in vitro-translated human enzyme selectively bound to BA affinity matrices and SPR spectroscopy confirmed these interactions. BAs reversibly suppressed the transformation of prostaglandin (PG)H(2) to PGE(2) mediated by mPGES1 (IC(50) = 3-10 µM). Also, in intact A549 cells, BAs selectively inhibited PGE(2) generation and, in human whole blood, ß-BA reduced lipopolysaccharide-induced PGE(2) biosynthesis without affecting formation of the COX-derived metabolites 6-keto PGF(1α) and thromboxane B(2) . Intraperitoneal or oral administration of ß-BA (1 mg·kg(-1) ) suppressed rat pleurisy, accompanied by impaired levels of PGE(2) and ß-BA (1 mg·kg(-1) , given i.p.) also reduced mouse paw oedema, both induced by carrageenan. CONCLUSIONS AND IMPLICATIONS: Suppression of PGE(2) formation by BAs via interference with mPGES1 contribute to the anti-inflammatory effectiveness of BAs and of frankincense, and may constitute a biochemical basis for their anti-inflammatory properties.

Exercise in fibromyalgia and related inflammatory disorders: known effects and unknown chances.

Fibromyalgia (FM) is characterised by chronic widespread pain and allodynia (pain from stimuli which are not normally painful with pain that may occur other than in the area stimulated) of more than 3 months duration. The current hypothesis of the aetiology of FM includes inflammatory and neuroendocrine disorders. The biophysiology of this syndrome, however; remains still widely elusive, and there are no formally approved therapies. Non-pharmacological interventions in FM patients include habitual exercise programs which improve physical function and quality of life of patients and may even reduce pain. However the mechanisms through which exercise benefits FM symptoms needs to be elucidated. In this article we firstly review the main topics and characteristics of the FM syndrome, while focusing our attention on the inflammatory hypothesis of FM, as well as on the beneficial effects of habitual exercise as a co-therapy for FM patients. In this context, the latest developments in research on anti-inflammatory effects of exercise are also reviewed and discussed. To find out what is known about the connection between benefits of exercise for FM and anti-inflammatory effects of exercise, we carried out a PubMed search using the term "fibromyalgia" and "exercise" together with "inflammation", and no more than ten published articles were found (six of them reviews), which are also discussed. In the second part of the article we present a pilot investigation on a group of 14 female FM patients with a diagnosis of FM by a rheumatologist. They took part in a pool-aquatic program in warm water over a period of fourth months (three weekly 60-min sessions). Circulating inflammatory (IL-1beta, IL-2, IFNgamma, TNFalpha, IL-8, IL-6, IL-4, IL-10 and CRP) and neuroendocrine (NA and cortisol) markers were determined. FM patients showed higher circulating levels of IL-8, IFNgamma and CRP as well as cortisol and NA than age-matched healthy control women. After the exercise program, a significant decrease in IL-8, IFNgamma, and CRP were found, in parallel with a decrease in circulating concentrations of cortisol and increased levels of NA. The results confirm an elevated "inflammatory status" in the FM syndrome and strengthen the hypothesis that the benefits of exercise in FM patients are mediated, at least in part, by its anti-inflammatory effects. A better regulation of the cytokine-HPA axis feedback may be also involved.

Survival, neuron-like differentiation and functionality of mesenchymal stem cells in neurotoxic environment: the critical role of erythropoietin.

Mesenchymal stem cells (MSCs) can ameliorate symptoms in several neurodegenerative diseases. However, the toxic environment of a degenerating central nervous system (CNS) characterized by hypoxia, glutamate (Glu) excess and amyloid beta (Abeta) pathology may hamper the survival and regenerative/replacing capacities of engrafted stem cells. Indeed, human MSC (hMSC) exposed to hypoxia were disabled in (i) the capacity of their muscarinic receptors (mAChRs) to respond to acetylcholine (ACh) with a transient increase in intracellular [Ca(2+)], (ii) their capacity to metabolize Glu, reflected by a strong decrease in glutamine synthetase activity, and (iii) their survival on exposure to Glu. Cocultivation of MSC with PC12 cells expressing the amyloid precursor protein gene (APPsw-PC12) increased the release of IL-6 from MSC. HMSC exposed to erythropoietin (EPO) showed a cholinergic neuron-like phenotype reflected by increased cellular levels of choline acetyltransferase, ACh and mAChR. All their functional deficits observed under hypoxia, Glu exposure and APPsw-PC12 cocultivation were reversed by the application of EPO, which increased the expression of Wnt3a. EPO also enhanced the metabolism of Abeta in MSC by increasing their neprilysin content. Our data show that cholinergic neuron-like differentiation of MSC, their functionality and resistance to a neurotoxic environment is regulated and can be improved by EPO, highlighting its potential for optimizing cellular therapies of the CNS.

Myrtucommulone, a natural acylphloroglucinol, inhibits microsomal prostaglandin E(2) synthase-1.

BACKGROUND AND PURPOSE: The selective inhibition of prostaglandin (PG)E(2) formation via interference with microsomal PGE(2) synthase (mPGES)-1 could have advantages in the treatment of PGE(2)-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1. EXPERIMENTAL APPROACH: The effect of MC on PGE(2) formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1beta-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF(1alpha) formation. KEY RESULTS: MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH(2) to PGE(2) (IC(50) = 1 micromol x L(-1)). PGE(2) formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 micromol x L(-1), and only moderate inhibition of cellular or cell-free COX-1 was evident (IC(50) > 15 micromol x L(-1)). CONCLUSIONS AND IMPLICATIONS: MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE(2) formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.

Basic research and clinical applications of non-hematopoietic stem cells, 4-5 April 2008, Tubingen, Germany.

From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.

Randomized phase III study of granulocyte transfusions in neutropenic patients.

Despite antibiotics, antifungals and haematopoietic growth factors, infections remain a major threat to neutropenic patients. To determine the role of granulocyte transfusions (GTs) in anti-infective therapy during neutropenia, GT administration was randomized in 74 adults with haematological or malignant diseases, febrile neutropenia and pulmonary or soft-tissue infiltrates after conventional or high-dose chemotherapy, a majority of them after allo-SCT (n=39). Neutrophil reconstitution was equal in the treatment and control arm. GT toxicity was minimal. The probability of 28-day survival after randomization was >80% in both groups, and no effect of GT on survival until day 100 could be detected in patients with fungal (n=55), bacterial or unknown infection (n=17) and various levels of neutropenia (ANC <500 vs >500 x 10(6)/l). These findings can be attributed primarily to procedural obstacles, such as long delay from randomization to first GT, low cell content and slow sequence of GT, difficulties in randomizing a safe and potentially life-saving treatment in severely endangered individuals, and a large proportion of rapidly recovering patients in both arms. The requirement of another trial in a more specific patient population with daily transfusions of sufficient numbers of granulocytes to support or refute the empirically acknowledged benefits of GT is discussed.

Cardioprotection and cardiac regeneration by mesenchymal stem cells.

Mesenchymal stem cells (MSC) are promising candidates in the emerging field of regenerative medicine. MSC have been applied in numerous experimental and preclinical studies evaluating their therapeutical potential in various models of diseases. For cardiac applications, especially myocardial ischemia, MSC have been shown to provide an interesting therapeutical potential. However, to date the mechanisms of their beneficial effects on the cardiac tissue are only faintly elucidated. On the one hand we do not completely understand the biology of the MSC, on the other hand their interactions with the complex in vivo situation e. g. after myocardial infarction and the following remodeling processes are difficult to study. Initially, with respect to their in vitro differentiation capacity, it has been assumed that transplanted MSC may, promoted by the local microenvironment, differentiate in vivo into cells of cardiomyogenic phenotype or even cardiomyocytes, integrate themselves into the myocardium and help to regenerate the damaged tissue. The finding that after transplantation only a minor percentage of the MSC showed long term survival and persistent lack of evidence for in vivo differentiation make the original mechanistic concept of in vivo cardiomyogenic differentiation questionable. More recent studies suggest that the transplanted MSC may interact with the local tissue releasing paracrine factors and may hereby support the regenerative process. In this article, referring to experimental and preclinical studies, characteristics, sources, differentiation and paracrine activity of MSC contributing to cardioprotection and cardiac regeneration will be discussed.

A closed system for islet isolation and purification using the COBE2991 cell processor may reduce the need of clean room facilities.

During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502 +/- 253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest was cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation, and expelling were repeated several times. Islets in pellet form were then purified by adding a gradient (bottom loading). Using this closed system 1098 +/- 489 IEQ per gram pancreas were purified with a total cell viability of 67 +/- 10% and a beta-cell viability of 41 +/- 13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of beta-cells was still 56 +/- 10% and was only reduced after the addition of proapoptotic IL-1 and TNF-alpha to 40 +/- 4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islet preparations to a minimum and may open the way for islet preparations without clean room demand.

Aptamer-based isolation and subsequent imaging of mesenchymal stem cells in ischemic myocard by magnetic resonance imaging.

PURPOSE: Mesenchymal stem cells (MSC) seem to be a promising cell source for cellular cardiomyoplasty. We recently developed a new aptamer-based specific selection of MSC to provide "ready to transplant" cells directly after isolation. We evaluated MRI tracking of newly isolated and freshly transplanted MSC in the heart using one short ex vivo selection step combining specific aptamer-based isolation and labeling of the cells. MATERIALS AND METHODS: Bone marrow (BM) was collected from healthy pigs. The animals were euthanized and the heart was placed in a perfusion model. During cold ischemia, immunomagnetic isolation of MSC from the BM by MSC-specific aptamers labeled with Dynabeads was performed within 2 h. For histological identification the cells were additionally stained with PKH26. Approx. 3 x 10(6) of the freshly aptamer-isolated cells were injected into the ramus interventricularis anterior (RIVA) and 5 x 10(5) cells were injected directly into myocardial tissue after damaging the respective area by freezing (cryo-scar). 3 x 10(6) of the aptamer-isolated cells were kept for further characterization (FACS and differentiation assays). 20 h after cell transplantation, MRI of the heart using a clinical 3.0 Tesla whole body scanner (Magnetom Trio, Siemens, Germany) was performed followed by histological examinations. RESULTS: The average yield of sorted cells from 120 ml BM was 7 x 10(6) cells. The cells were cultured and showed MSC-like properties. MRI showed reproducible artifacts within the RIVA-perfusion area and the cryo-scar with surprisingly excellent quality. The histological examination of the biopsies showed PKH26-positive cells within the areas which were positive in the MRI in contrast to the control biopsies. CONCLUSION: Immunomagnetic separation of MSC by specific aptamers linked to magnetic particles is feasible, effective and combines a specific separation and labeling technique to a "one stop shop" strategy.

[Tissue engineering and stem cell research in urology for a reconstructive or regenerative treatment approach]. / "Tissue engineering" und Stammzellforschung in der Urologie für den rekonstruktiven bzw. regenerativen Therapieansatz.

With the involvement of clinical reconstructive urology in the field of tissue engineering, outstanding results have been achieved in basic research as well as in some clinics. Stem cell research has even opened up possibilities for regenerative aspects. In close cooperation with various disciplines, the Department of Urology at the University of Tübingen investigates different clinical aspects with regard to reconstructive and regenerative urology. The regeneration of the external urethral sphincter requires functionally integrated muscle cells. In addition stricture reconstruction with multilayer urothelium should become less invasive and the re-stricture rate reduced. After the application of differentiating stem cells was proven, the clinical setting needed to be set for legal issues. In addition to the specification of culture media and verification in the animal model, the possibility to harvest omnipotent stem cells out of human testis and to differentiate those into the three germ layers was demonstrated. With the reduced invasiveness of harvesting the urothelium cells by a bladder wash using specific culture fluids, the cell culture was significantly improved enabling successful creation of urothelium by stratification. In addition urothelial cells in a matrix are further improved for endoscopic application. The close cooperation of different disciplines shortens the time to develop therapeutic approaches with a close clinical relationship in reconstructive and regenerative urology.

[Value of stem cell therapy for the treatment of stress incontinence. Current status and perspectives]. / Stellenwert der Stammzelltherapie für die Behandlung der Belastungsinkontinenz. Aktueller Stand und Ausblick.

Parallel to a fundamental change in the therapeutic approach to managing stress incontinence, an increasing number of patients ask for reconstruction of the outer, striated urethral sphincter as therapy for urinary stress incontinence. Regenerative medicine is starting to offer solutions using stem cells as a part of oncological therapy or in reconstructive surgery. In addition to the many auspicious experimental approaches, one published study reports the effective therapeutic use of myogenic stem cells in urinary stress incontinent patients. Before this procedure is adopted into general clinical practice, further studies with validated evaluations and a sound legal basis are needed.

Exercise intensity and duration affect blood soluble HSP72.

Soluble heat shock protein 72 (sHSP72) is suggested to play a role as a signalling molecule in the immune response to exercise. We were interested in whether duration and intensity of endurance running affect the level of inducible sHSP72 in the plasma/serum of endurance athletes. In the first part of the study, the influence of a continuous treadmill run of 60 min (CR) with an intensity of 75 % VO2max, a long treadmill run of 120 min (LR) with an intensity of 60 % VO2max, an extensive interval training program (IT; 10 x 1000 m, ca. 35 min, VO2max 88 %), and a competitive marathon run (MA) within 260 +/- 39 min (VO2max ca. 65 %) on the release of sHSP72 into the peripheral blood was tested. Blood samples were drawn before and directly after exercise, as well as 0.5, 1, 3, 24 h after exercise to determine sHSP72 levels. Secondly, we compared the effects of two exercise bouts with identical duration (23.7 +/- 7 min) but different intensities (Exhaustive exercise (ET) at 80 % VO2max vs. moderate exercise (MT) at 60 % VO2max) on sHSP72 concentration. The sHSP72 levels in plasma/serum were analyzed using an enzyme immunoassay specific for inducible HSP72 (Stressgen,Victoria, Canada). Early, significant increases of sHSP72 were detected immediately after all types of exercise with highest levels after MA. ET induced significantly higher levels of sHSP72 compared with MT. Long-lasting, competitive endurance exercise induced a more pronounced response of sHSP compared with more intensive but shorter exercise. Exercise intensity was also an important influencing factor. A duration- and intensity-dependent role for sHSP72 in the exercise-induced changes of the immune response may be assumed.

Antioxidant intervention does not affect the response of plasma erythropoietin to short-term normobaric hypoxia in humans.

Recent research has demonstrated that reactive oxygen species (ROS) participate in intracellular signaling processes initiated during hypoxia. We investigated the role of ROS in the response of plasma erythropoietin (Epo) to short-term normobaric hypoxia in humans. Twelve male subjects were exposed twice to 4 h of normobaric hypoxia (H; inspired oxygen fraction 12.5%) with a period of 6 wk between both experiments (H1 and H2). With the use of a randomized placebo-controlled crossover design, the subjects received orally a combination of the antioxidants all-rac-alpha-tocopherol (800 mg/day for 3 wk) and alpha-lipoic acid (600 mg/day for 2 wk) or placebo before H1 and H2, respectively. Three weeks before H1, the subjects underwent one control experiment in normoxia (N; inspired oxygen fraction 20.9%) without any treatment. Serum alpha-tocopherol was significantly higher after treatment with antioxidants compared with placebo. Capillary Po(2) declined during H without significant differences between antioxidants and placebo. Plasma peroxide levels were lower under antioxidant treatment but not affected by hypoxia. The response of Epo to H did not show significant differences between antioxidant [maximum increase (means, 95% confidence interval): +121%, +66 to +176%] and placebo conditions (+108%, +68 to +149%). Similarly, hypoxia-induced increase of Epo corrected for diurnal variations, as revealed during N, did not differ between antioxidants and placebo. Individual variability of Epo in response to H was not related to the individual degree of hypoxemia during H. Our results do not support the assumption that ROS play a major modulating role in the response of Epo to short-term normobaric hypoxia in humans.

Microarray technology--the future analyses tool in exercise physiology?

Microarray analysis offers a set of analytical platforms that provide rapid, affordable and substantial information at the DNA, RNA or protein level. It enables the analysis of thousands of genes simultaneously in a parallel manner across samples derived from various biological sources and treatment regimens. This development became possible as a result of the combination of three technological advances achieved at the beginning of the 1990's, namely parallelism, miniaturization and automation. In regular physical checkups the microarray technology could be used to supplement the current spectrum of tests and therefore enhance the quality of the data obtained. Arrays for analyses of RNA expression will allow gene expression profiling also in exercise physiology. DNA chips may also be used for genetic screening and diagnostics to analyze polymorphisms and mutations which may underlie genetic diseases or interindividual variations between subjects. Using microarrays in exercise physiology will provide new insights into the complex molecular mechanisms of the exercise-induced stress response, adaptation to training and modulation of immune function. Gene expression profiling and genetic screening will probably help to characterize and predict the individually variable response to and efficiency of training.