UVA-induced metabolic changes in non-malignant skin cells and the potential role of pyruvate as antioxidant.

The exposure to UVA (320-400 nm) irradiation is a major threat to human skin concerning photoaging and carcinogenesis. It has been shown that UVA irradiation can induce reactive oxygen species (ROS) and DNA mutations, such as 8-hydroxydeoxyguanosine. Furthermore, UVA induces the expression of photoaging-associated matrix metalloproteases (MMPs), especially of matrix metalloprotease 1 (MMP 1) and matrix metalloprotease 3 (MMP 3). In addition to this, it was recently shown that UVA-induced ROS also increase glucose metabolism of melanoma cells, however, the influence of UVA on the glucose metabolism of non-malignant cells of the human skin has, so far, not been investigated in detail. Here, we investigated the UVA-induced changes in glucose metabolism and the functional relevance of these changes in primary fibroblasts-normal non-malignant cells of the skin. These cells showed an UVA-induced enhanced glucose consumption and lactate production and changes in pyruvate production. As it has been proposed that pyruvate could have antioxidant properties we tested the functional relevance of pyruvate as protective agent against UVA-induced ROS. Our initial experiments support earlier publications, demonstrating that pyruvate treated with H2O2 is non-enzymatically transformed to acetate. Furthermore, we show that this decarboxylation of pyruvate to acetate also occurs upon UVA irradiation. In addition to this, we could show that in fibroblasts pyruvate has antioxidant properties as enhanced levels of pyruvate protect cells from UVA-induced ROS and partially from a DNA mutation by the modified base 8-hydroxydeoxyguanosine. Furthermore, we describe for the first time, that the interaction of UVA with pyruvate is relevant for the regulation of photoaging-associated MMP 1 and MMP 3 expression.

Plerixafor with and without chemotherapy in poor mobilizers: results from the German compassionate use program.

The CXCR4-inhibitor plerixafor mobilizes hematopoietic stem cells amplifying the effects of granulocyte-CSF (G-CSF). Before approval plerixafor was used in a compassionate use program (CUP) for patients who failed a previous mobilization. In the German CUP 60 patients from 23 centers (median age 56.5 years (2-75)) were given 240 µg/kg plerixafor SC 9-11 h before apheresis. A total of 78.3% (47/60) received G-CSF for 4 days before plerixafor administration; 76.6% of those (36/47) yielded at least 2.0 × 10(6) CD34(+) cells/µL. The median cell yield was 3.35 × 10(6) CD34+ cells/kg (0-29.53). Nine patients received plerixafor alone or with G-CSF for less than 4 days mobilizing a median of 3.30 × 10(6) CD34+ cells/kg (1.6-5.6). There was no significant difference between G-CSF application for 4 days and for a shorter period of time (P=0.157). A total of 47 patients received plerixafor plus G-CSF combined with chemotherapy yielding a median of 3.28 × 10(6) CD34+ cells/kg (0-24.79). In all, 40 of 60 patients (66.7%) proceeded to transplantation, and achieved a timely and stable engraftment. Side effects were rare and manageable. In conclusion, mobilization with plerixafor in poor mobilizers is safe and results in a sufficient stem cell harvest in the majority of patients.

Plerixafor is effective and safe for stem cell mobilization in heavily pretreated germ cell tumor patients.

Up to 10% of germ cell tumor patients require salvage high-dose chemotherapy with stem cell support, achieving cure rates in the range of 10-60%. Stem cell mobilization may be difficult in these patients because of multiple lines of treatment known to seriously hamper stem cell recovery. Plerixafor significantly enhances the success of the CD34+ cell harvest, even in cases where prior mobilization attempts have failed. Six germ cell tumor patients provided informed consent and were included in the compassionate use program. All patients were heavily pretreated, with a median of 3.5 prior lines of therapy. All failed prior mobilization with G-CSF in combination with chemotherapy. Five patients yielded a median of 2.6 × 10(6) CD34+ cells per kg body weight in a median of 4 apheresis days when plerixafor was used. Three patients underwent subsequent high-dose chemotherapy with autologous stem cell support. Median time to leukocyte engraftment was 11 days. Median time to platelet engraftment was 12.5 days, both of which are comparable to previous historical data. Accordingly, plerixafor seems to be safe and effective in germ cell tumor patients who have failed prior mobilization therapy. Larger prospective studies are warranted to further assess its use in germ cell cancer.

Recruitment of PKC-betaII to lipid rafts mediates apoptosis-resistance in chronic lymphocytic leukemia expressing ZAP-70.

ZAP-70 is a key signaling molecule in T cells. It couples the antigen-activated T-cell receptor to downstream signaling pathways. Its expression in leukemic B-cells derived from a subgroup of patients with chronic lymphocytic leukemia (CLL) is associated with an aggressive course of the disease. However, its implication for the pathogenesis of aggressive CLL is still unclear. In this study, we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor, recruiting protein kinase C-betaII (PKC-betaII) into lipid raft domains. Subsequently, PKC-betaII is activated and shuttles from the plasma membrane to the mitochondria. We unravel that the antiapoptotic protein Bcl-2 and its antagonistic BH3-protein Bim(EL) are putative substrates for PKC-betaII. PKC-betaII-mediated phosphorylation of Bcl-2 augments its antiapoptotic function by increasing its ability to sequester more pro-apoptotic Bim(EL.) In addition, the phosphorylation of Bim(EL) by PKC-betaII leads to its proteasomal degradation. These changes confer leukemic cells to a more antiapoptotic state with aggressiveness of the disease. Most importantly, these molecular changes can be therapeutically targeted with the small molecule inhibitor Enzastaurin. We provide evidence that this compound is highly active in leukemic cells and augments the cytotoxic effects of standard chemotherapeutic drugs.

Mechanisms of apoptosis-induction by rottlerin: therapeutic implications for B-CLL.

Constitutively activated signaling pathways contribute to the apoptosis-defect of B-CLL cells. Protein kinase C-delta is a permanently activated kinase and a putative downstream target of phosphatidylinositol-3 kinase in B-CLL. Blockade of protein kinase C-delta (PKC-delta) by the highly specific inhibitor rottlerin induces apoptosis in chronic lymphocytic leukaemia (CLL) cells. By co-culturing bone marrow stromal and CLL cells, we determined that the proapoptotic effect of rottlerin is not abolished in the presence of survival factors, indicating that a targeted therapy against PKC-delta might be a powerful approach for the treatment of CLL patients. The downstream events following rottlerin treatment engage mitochondrial and non-mitochondrial pathways and ultimately activate caspases that execute the apoptotic cell death. Herein we report that the inhibition of PKC-delta decreases the expression of the important antiapoptotic proteins Mcl-1 and XIAP accompanied by a loss of the mitochondrial membrane potential Deltapsi. In addition, we discovered that ZAP-70-expressing cells are significantly more susceptible to rottlerin-induced cell death than ZAP-70 negative cells. We finally observed that rottlerin can augment cell toxicity induced by standard chemotherapeutic drugs. Conclusively, PKC-delta is a promising new target in the combat against CLL.

Platelets induce alterations of chemotactic and adhesive properties of endothelial cells mediated through an interleukin-1-dependent mechanism. Implications for atherogenesis.

Platelets and alterations of chemotactic and adhesive properties of endothelium play an important role in the pathophysiology of atherosclerosis. We investigated the effect of platelets on secretion of monocyte chemotactic protein-1 (MCP-1) and on surface expression of intercellular adhesion molecule-1 (ICAM-1) of cultured endothelium. Pretreatment of cultured monolayers of endothelial cells with alpha-thrombin-activated platelets significantly enhanced secretion of MCP-1 and ICAM-1 surface expression (P<0.01) that could be inhibited by interleukin-1 (IL-1) antagonists by approximately 40%. Activation of transcription factor nuclear factor-kappaB (NF-kappaB) which regulates transcription of early inflammatory response genes such as MCP-1, was significantly increased in endothelial cells treated with activated platelets via an IL-1 mediated mechanism as determined by electrophoretic mobility shift assay (EMSA) and kappaB-dependent transcriptional activity. In trans-well experiments, alpha-thrombin-activated platelets enhanced IL-1-dependent surface expression of vitronectin receptor (alpha(v)beta(3)) on the luminal aspect of endothelial monolayers and promoted alpha(v)beta(3)-mediated platelet/endothelium adhesion that could be inhibited by the antiadhesive peptides GRGDSP and c(RGDfV). We conclude that activated platelets induce significant changes in chemotactic (secretion of MCP-1) and adhesive (surface expression of ICAM-1 and alpha(v)beta(3)) properties of cultured endothelium. These findings imply a potential pathophysiological mechanism of platelets in an early stage of atherogenesis.

Platelet function in septic multiple organ dysfunction syndrome.

OBJECTIVE: Altered platelet function plays a role in the pathophysiology of multiple organ failure in sepsis. The purpose of the present study was to evaluate various aspects of platelet adhesive function in septic patients and its putative relevance for prognosis. DESIGN: Prospective clinical study. SETTING: Intensive Care Unit of the University Hospital. PATIENTS AND METHODS: A total of 41 patients admitted to the medical Intensive Care Unit were studied. On the day of admission, patients were evaluated by intensive care scoring systems (Elebute, APACHE II) to assess the severity of sepsis and multiple organ dysfunction syndrome (MODS), and platelet function tests were performed. All patients were observed for 28 days to assess their clinical outcomes. Eleven patients revealed septicemia without MODS (Elebute > or = 12, APACHE II < 20) and 20 septic patients suffered from MODS (Elebute > or = 12, APACHE II > or = 20). Ten non-septic patients without MODS served as a control group (Elebute < 12, APACHE II < 20). Flow cytometric determination of the activated fibrinogen (fg) receptor GPIIb-IIIa and as well as thrombospondin (TSP) on platelets and platelet-neutrophil adhesion (CD 41 immunofluorescence) ex vivo was performed using monoclonal antibodies. The effect of plasma obtained from patients on normal platelet aggregation in vitro, and adhesion to cultured endothelial cells was evaluated. RESULTS: The surface expression of TSP on platelets was increased in septic patients with MODS compared to controls (p < 0.03). Platelet-neutrophil adhesion was not significantly altered in septicemia (p < 0.09) but decreased significantly in the presence of MODS (p < 0.05) when compared to controls. Logistic regression analysis showed that platelet-neutrophil adhesion was an independent predictor for poor clinical outcome (p < 0.01). Plasma from septic patients sensitized normal platelets to hyperaggregate and to adhere to cultured endothelium (p < 0.01). CONCLUSION: In septic patients platelets become activated and are hyperadhesive to other vascular cells including neutrophils and endothelium. This may induce sequestration of platelets and microcirculatory arrest, thus the development of MODS.

Changes in platelet membrane glycoproteins and platelet-leukocyte interaction during hemodialysis.

Platelet aggregation and interaction with other vascular cells play a key role in hemostatic events during hemodialysis. We studied seven patients with end-stage renal failure on long-term hemodialysis treatment. Flow-cytometric techniques and platelet-specific monoclonal antibodies were used to measure the platelet surface expression of glycoproteins-fibrinogen receptor on glycoprotein IIb-IIIa (GP IIb-IIIa; CD41) and alpha-granule membrane protein (GMP-140; CD62). In addition, adhesion of platelets or platelet microparticles with leukocytes was evaluated by appearance of the platelet-specific antigen (GP IIb-IIIa) on leukocytes. Blood samples were taken before the start of dialysis and 15, 60, and 240 min thereafter. There was a significant increase in fibrinogen receptor activation on circulating platelets after 15 min of dialysis treatment (P < 0.001) and enhanced degranulation of GMP-140 (P < 0.05). In parallel, the interaction of platelets with neutrophils and monocytes also increased with the duration of dialysis and was maximal after 15 min (P < 0.001). We conclude that the platelet fibrinogen receptor on GP IIb-IIIa in circulating platelets is activated during hemodialysis and is associated with increased adhesion of platelets or platelet microparticles with circulating leukocytes. Thus, the phenomenon described here of platelet-leukocyte interaction could be pathophysiologically important for the development of dialysis-associated leukopenia.

Flow-cytometric analysis of mepacrine-labelled platelets in patients with end-stage renal failure.

Platelet dysfunction and increased bleeding tendency has been the most consistently described haemostatic abnormality in patients with renal failure. Besides abnormalities in platelet membrane glycoproteins, a reduced amount of platelet-dense granule content has been demonstrated in patients with end-stage renal failure (ESRD) indicating an acquired storage pool deficiency (SPD) present in uraemia. To study dense granules, platelets were labelled with mepacrine, a fluorescent probe which is specifically incorporated into dense bodies. MepaPlatelets of 13 patients with ESRD and of 11 healthy controls were studied. The results showed that mepacrine-labelled platelets of patients with ESRD reveal a significantly (p < 0.05) reduced fluorescence compared to the control group. This implies a reduced number or content of dense granules present in ESRD platelets. Thus, the current data indicate that ESRD is associated with an acquired platelet SPD which may be a useful and rapid method for screening patients with suspected acquired or inherited SPD.