STRIP2 Is Indispensable for the Onset of Embryonic Stem Cell Differentiation.

The role of striatin interacting protein 2 (Strip2) in differentiation of embryonic stem cells (ESCs) is still under debate. Strip2-silenced murine (KD) ESCs were differentiated for 4, 8, 12, and 16 days. We show that Strip2 is distributed in the perinucleus or nuclei of wild-type (WT) undifferentiated ESCs, but is localized in high-density nuclear bodies in differentiated cells. CellNet analysis of microarray gene expression data for the KD and scrambled control (SCR) embryoid bodies (EBs), as well as immunostainings of key pluripotent factors, demonstrated that differentiation of KD ESCs is repressed. This occurs even in 16-day-old EBs, which possessed a high tumorigenic potential. Correlated with very high expression levels of epigenetic regulator genes, Hat1 and Dnmt3, enzymatic activities of the histone acetyltransferase type B (Hat1) and DNA (cytosine-5)-methyltransferase 3 beta (Dnmt3b) were higher in differentiated 16-day-old KD EBs than in SCR or WT EBs. The expression levels of let-7, 290, and 302 microRNA families were opposed in KD ESCs, while KD EBs had levels comparable to WT and SCR ESCs during differentiation. Strip2 is critical for the regular differentiation of ESCs. Moreover, Strip2 deficient ESCs showed a dysregulation of epigenetic regulators and microRNAs regulating pluripotency.

Extracorporeal Treatment for the Acute und Long-Term Outcome of Patients with Life-Threatening Acquired Hemophilia.

OBJECTIVES: In acquired hemophilia (AH), autoantibodies (inhibitors) impede blood coagulation factors leading to severe bleedings. Cornerstones of a successful treatment are the control of bleeding and an eradication of autoantibodies. The present study is an update of our previous documentation of the treatment of high-titer AH patients with severe life-threatening bleeding undergoing the modified Bonn-Malmö-Protocol (MBMP). METHODS: 64 AH patients were treated by a standard combination protocol (MBMP) consisting of antibody depletion through immunoadsorption, i.v. immunoglobulin, immunosuppression, and high-dose FVIII substitution. They underwent a long-term follow-up. RESULTS: Primary study endpoints loss of detection of the activity of the inhibitor and FVIII recovery ? 5% were reached in a median time of 3 days (95% CI: 2.6-3.4 days), the median time of FVIII substitution was 13 days (95% CI 10.6-15.3 days), and the median time of immunoadsorption was 16 days (95% CI 13-18.9 days). In 5 patients the AH occurred as paraneoplastic syndrome, and partial remission was achieved. Relapses without bleeding event occurred only in second-line MBMP. Those responded excellently to short time treatment. Overall patients remained in remission over a median follow-up time of 8 years. Conclusion: Except for paraneoplastic AH, MBMP-treated patients have a remarkable prognosis which is confirmed by long-term follow-up with a complete response rate of 93% (53/57) in the first year post MBMP and 100% during long-term follow-up. These outcome in life-threatening AH is unique and until now not achievable via other treatment schedules. In life-threatening bleedings physicians should take into account MBMP as a first line treatment.

Estrogen improves vascular function via peroxisome-proliferator-activated-receptor-γ.

The exact mechanism of estrogen in cardiovascular disease is not fully understood. As estrogen receptors (ERs), the peroxisome-proliferator-activated-receptor-γ (PPARγ) belongs to the family of ligand activated nuclear receptors regulating atheroprotective genes. The aim of this project was to investigate whether vascular effects of estrogen are mediated via PPARγ-regulation in the vascular compartment. Estrogen deficient ovariectomized wildtype-mice (OVX) displayed significant reduction of PPARγ-expression in aortic tissue compared to wildtype-mice with intact ovarian function (Sham). Hormone replacement with subdermal 17ß-estradiol pellets significantly increased vascular PPARγ-expression in ovariectomized female wildtype-mice (OVX/E2). Analogous to wildtype-mice, estrogen-deficient OVX ApoE(-/-)-mice had low vascular PPARγ-expression associated with ROS generation, endothelial dysfunction and atherogenesis. Estrogen replacement (OVX/E2) rescued vascular PPARγ-expression, reduced ROS generation, monocyte recruitment, atherosclerotic lesion formation and improved endothelial function. Inhibition of PPARγ by GW9662, a specific PPARγ-antagonist reduced 17ß-estradiol mediated vascular effects (OVX/E2+GW9662). Finally, despite estrogen deficiency treatment with pioglitazone (OVX+pioglitazone), a selective PPARγ-agonist, compensates deterioration of vascular morphology and function. 17ß-estradiol regulates vascular PPARγ-expression in wildtype- and ApoE(-/-)-mice. The presented data demonstrate the fundamental relevance of PPARγ as downstream target of 17ß-estradiol-related anti-inflammatory and atheroprotective effects within the vascular wall independent of its cardiovascular risk factor modifications.

Immunoadsorption in the treatment of acquired haemophilia.

In acquired haemophilia (AH) healthy humans can suddenly develop severe bleeding due to autoantibodies (inhibitors) against clotting factors, especially factor VIII. The mortality rate of 21 % is considerable, and standardized treatment protocols have not been developed due to the low disease frequency (1-4 per million). Major goals of treatment are the control of bleeding events and rapid inhibitor elimination. Conventional treatment regimens induce immune tolerance via long-term immunosuppression with success rates between 52% and 82%. However, treatment related mortality can rise to 39%. Lack of complete remission, advanced age, underlying malignancies and infections related to immunosuppressive therapy are regarded as principal risk factors for death. The modified Bonn-Malmö Protocol (MBMP), an immune tolerance protocol consisting of antibody depletion through immunoadsorption, i.v. immunoglobulin treatment, immunosuppression and high dose FVIII supplementation, achieves rapid and safe control of acute bleeding. In the largest published single centre study of high risk patients with AH, we previously demonstrated that complete remission (CR) can be achieved in 88.5% of all patients (54/61) within a median time of 3.9 wks (range: 3.2-4.5 wks) and in 97% (54/56) of AH patients without cancer as an underlying condition. Those 5 patients, who suffered also from cancer, achieved partial remission (PR). Mortality or severe treatment-related side effects were not observed. This study confirmed that MBMP is a safe and effective treatment with a high curative potential for severe AH. However, the severity of bleeding, and therefore the cost-effectiveness of the approach, needs to be considered when initiating this treatment protocol.

Aplicación de las tecnologías ómicas a la Fitoterapia / No disponible

La demostración de la eficacia de los preparados fitoterápicos y la determinación de sus mecanismos de acción son retos permanentes para la fitoterapia basada en la evidencia. La genómica, proteómica y metabolómica son tecnologías de alto rendimiento, que permiten detectar simultáneamente un gran número de proteínas/genes, así como relacionar mezclas complejas con efectos complejos en forma de perfiles de expresión génica/proteica. La descripción de perfiles de expresión específicos para un determinado preparado fitoterápico puede ser útil para su estandarización química y farmacológica, así como para la evaluación de su toxicidad. A largo plazo, pueden economizar los estudios de eficacia y de mecanismos de acción, y facilitar la investigación de extractos vegetales carentes de principio(s) activo(s) predominantes. Se ha descubierto que los perfiles de expresión génica inducidos por fármacos individuales o sus combinaciones pueden ser completamente diferentes. La aplicación de las tecnologías “ómicas” puede suponer un cambio de paradigma en cuanto a la aplicación de mezclas complejas en medicina y abrir nuevos campos como la fitogenómica, fitoproteómica y fitometabolómica (AU)

The proof of efficacy of phytopreparations and the determination of their mode of action are permanent challenges for an evidence-based phytotherapy. Genomics, proteomics and metabolomics are high-throughput technologies that allow the simultaneous detection of a high number of proteins/genes and have the potential to relate complex mixtures to complex effects in the form of gene/ protein expression profiles. The development of phytopreparation-specific expression profiles will be useful for its chemical and pharmacological standardization and its toxicological assessment. Over a long-term perspective they may economize the studies on efficacy and mode of action of phytomedicines and allow to investigate herbal extracts without prominent active principle(s). The application of genomics revealed already that the gene expression profiles induced by single drugs and their combinations can be entirely different. The application of the “-omic-” technologies may lead to a change of paradigms towards the application of complex mixtures in medicine and open the new fields of phytogenomics, -proteomics and -metabolomics (AU)

The map kinase ERK regulates renal activity of cyclin-dependent kinase 2 in experimental glomerulonephritis.

BACKGROUND: In vitro, the extracellular signal-regulated kinase (ERK) is an intracellular convergence point of multiple stimuli, which affect the cell cycle. However, the role of ERK in cell cycle regulation in vivo is unknown. METHODS: To address this issue, ERK activity was blocked both in vitro in mesangial cells (MC) and in vivo in experimental glomerulonephritis (GN) by a pharmacological inhibitor (U0126) of the ERK-activating kinase. RESULTS: In stimulated MC, inhibition of ERK reduced cyclin-dependent kinase 2 (CDK2) phosphorylation, CDK2 activity and cyclin E/A expression, whereas downregulation of CDK inhibitor p27(Kip1) expression was inhibited. In vivo, U0126 was given to rats in the acute phase of anti-Thy 1.1 GN. We previously showed that glomerular cell proliferation was reduced by 67% upon treatment with the inhibitor compared to nephritic controls. Now, we detected a significant increase in renal CDK2-activity/phosphorylation in the nephritic controls, that was significantly and dose-dependently reduced by ERK inhibition. CDK2 activation was accompanied by an increase in renal expression of cyclins E/A and the enhanced binding of these cyclins to CDK2 in the nephritic controls. These changes were blunted by U0126 treatment. Finally, we noted an increased expression and CDK2-binding of p27(KIP1) protein in the nephritic controls which was decreased in U0126 treated rats. CONCLUSIONS: Our observations provide the first evidence that ERK is an intracellular regulator of renal CDK2 activity in vivo in a glomerulonephritis model.

Vitamin C promotes human endothelial cell growth via the ERK-signaling pathway.

BACKGROUND: Epidemiological, secondary prevention and small interventional trials suggest a preventive role of vitamin C for cardiovascular diseases (CAD), especially through improving endothelial dysfunction. Large primary prevention trials failed to confirm this. Mechanistic studies may contribute to resolve this discrepancy. AIM OF THE STUDY: We examined whether vitamin C activates mitogen-activated protein kinases (MAPK) in human umbilical cord venous endothelial cells (HUVECs) and whether reactive oxygen species (ROS) play a role in this process. METHODS: Subconfluent quiescent HUVECs were incubated with vitamin C alone or in combination with catalase (CAT) and/or hydrogenperoxide (H2O2). Intracellular MAPK were determined by Western blot, proliferation by cell count and DNA-synthesis by [3H]-thymidine-uptake. RESULTS: HUVECs were incubated with vitamin C (60 microM) for 5-60 min or for 20 min (30-90 microM). A dose-dependent phosphorylation of extracellular signal-regulated-kinases (ERKs)-1 and -2 with a maximum of phosphorylation at 15-20 min was observed and inhibitable by MEK1/2-inhibitor U0126 (5-10 microM). Vitamin C (60 microM) stimulated phosphorylation of ERK5, but not of p38 and c-Jun, demonstrating a different MAPK-activation pattern compared to H2O2. Vitamin C (60 microM) induced proliferation and a dose-dependent [3H]-thymidine-uptake (30-120 microM) within 20 h. CAT (0.3 U/ml) did neither suppress the vitamin C induced [3H]-thymidine-uptake nor ERK1/2-phosphorylation. CAT (0.3 U/ml), but not vitamin C (60 microM) abrogated the inhibitory effects of H2O2 (100 microM) on [3H]-thymidine-uptake. CONCLUSION: Physiological vitamin C-concentrations promote proliferation of subconfluent ECs by activating an ERK1/2 controlled pathway. Targeting MAPK by vitamin C may improve, besides antioxidant mechanisms, endothelial dysfunction by promoting a fast regeneration of the endothelium after tissue injury, particularly required during secondary prevention and early development.

In vivo identification of the mitogen-activated protein kinase cascade as a central pathogenic pathway in experimental mesangioproliferative glomerulonephritis.

Evidence was recently provided for the activation of extracellular signal-regulated kinase (ERK), the best characterized mitogen-activated protein kinase, as an intracellular convergence point for mitogenic stimuli in animal models of glomerulonephritis (GN). In this study, in vivo ERK activity was blocked, with a pharmacologic inhibitor (U0126) of the ERK-activating kinase, in rats with mesangioproliferative GN. After injection of the monoclonal anti-Thy1.1 antibody (OX-7), the rats were treated (days 3 to 6) with low (10 mg/kg body wt) or high (100 mg/kg body wt) doses of U0126 administered intraperitoneally twice daily. On day 6 after induction of the disease, whole cortical tissue and isolated glomeruli were examined by using kinase activity assays, Western blot analyses, and immunohistochemical assays. Treatment with U0126 significantly reduced glomerular stimulation of ERK in anti-Thy1 GN. In the high dose-treated group, this downregulation was accompanied by a reduction in the number of glomerular mitotic figures, back to healthy control levels, and significant decreases in the numbers of total (P < 0.05) and 5-bromo-2'-deoxyuridine-positive (P < 0.05) glomerular cells. Immunohistochemical double-staining of renal sections demonstrated that mesangial cells were the major glomerular targets of U0126 in anti-Thy1 GN. These observations point to ERK as a putative intracellular mediator of the proliferative response in GN and suggest that pharmacologic treatments that interfere with the activation of ERK may be of potential therapeutic interest.