Combination of Cobe AutoPBSC and Gambro Elutra as a platform for monocyte enrichment in dendritic cell (DC) therapy: clinical study.

Monocytes are a common source for generating dendritic cells (DCs). The aim of the present study was to evaluate the efficiency of a platform for monocyte collection and enrichment in a clinical setting. The platform was based on the combination of two semiautomated devices; the Cobe Spectra Auto PBSC for mononuclear cells (MNC) collection followed by counterflow elutriation for monocyte enrichment (Gambro BCT Elutra). Twenty-four patients with various types of epithelial cancer participated in the study. MNC collections were first performed as large volume leukapheresis (LVL). Subsequently, MNC products were processed with an elutriation system for monocyte isolation. LVL resulted in the collection of MNC at a median of 8.1 x 10(9) cells, containing of 31.4% monocytes. A similar efficacy was also shown in patients with lower peripheral blood counts. Elutriation of the MNC product with the Cobe Elutra device resulted in the enrichment of monocytes at a median of 2.7 x 10(9) cells, with a recovery of 80.2% and a purity of 90.7%. These monocytes were then successfully developed into DCs for clinical therapy after in vitro manipulation. These data suggest that the combination of the Cobe Spectra Auto PBSC and the Gambro BCT Elutra is an effective platform for monocyte enrichment in clinical practice according to GCP standards and GMP guidelines, and can be easily implemented in the clinical routine under current DC protocols.

The effect of intensified extracorporeal photochemotherapy on long-term survival in patients with severe acute graft-versus-host disease.

Acute graft-versus-host disease (GVHD) is a major cause of mortality after allogeneic hematopoietic stem cell transplantation. We performed a phase II study on patients with acute steroid-refractory GVHD grades II to IV given extracorporeal photochemotherapy (ECP) weekly and analyzed response and long-term survival. Complete resolution of GVHD was achieved in 82% of patients with cutaneous involvement, 61% with liver involvement, and 61% with gut involvement. The probability of survival was 59% among patients who responded completely to ECP compared to 11% in patients not responding completely. We conclude that intensified ECP is highly effective in acute GVHD and that sustained responses are associated with over 50% long-term survival.

HMGA2 is associated with invasiveness but not a suitable marker for the detection of circulating tumor cells in breast cancer.

Previously, the human high mobility group protein member HMGA2 mRNA was reported to be expressed in peripheral blood of patients with breast cancer, but not in healthy individuals. Expression of HMGA2 in blood was suggested to be an independent indicator of poor prognosis in metastatic breast cancer. These very promising findings propose HMGA2 as a potential marker for the detection of circulating tumor cells in peripheral blood. Therefore, we analyzed peripheral blood specimens from healthy controls and patients with breast tumors for HMGA2 expression using TaqMan real-time RT-PCR to test if HMGA2 is a suitable marker for the early detection of breast cancer and monitoring therapy response in peripheral blood. Furthermore, we examined the possible involvement of HMGA2 expression in invasion investigated by an in vitro invasion assay using established breast cell lines. HMGA2 expression was detected in peripheral blood of breast cancer patients as well as of healthy individuals. No significant association of HMGA2 expression with any clinical or histopathological data was apparent. However, there was a significant correlation of HMGA2 expression in invasive and non invasive breast cell lines (p=0.0056). Although, HMGA2 obviously contributes to invasion it is not a specific marker for the detection of circulating tumor cells in peripheral blood.

Collection and storage of leukocyte depleted whole blood in autologous blood predeposit in elective surgery programs.

INTRODUCTION: The aim of this investigation was to provide evidence that leukocyte depleted whole blood meets the requirements for transfusion of the European Council and thus may be an alternative to leukocyte and plasma depleted packed red blood cells in autologous blood predeposit for patients undergoing elective surgery programs. MATERIAL AND METHODS: Standard units of 450mL blood were collected from 25 healthy male volunteers. Leukocyte depletion was done via inline filtration 4h after collection. Storage lesion was assessed by measuring the release of K(+), LDH, free hemoglobin, and lactate into the storage medium, as well as by the increase of hemolysis, the decrease of pH and consumption of glucose over a storage period of 35 days. As surrogate marker for red cell quality the intracellular concentrations of adenine nucleotides [ATP, ADP, AMP] were determined. RESULTS: The extent of storage lesion remained within the ranges of standard liquid storage conditions. Hemolysis was far below the threshold of 0.8% in all WB units at the end of their shelf life. Only minor changes of intracellular adenine nucleotide levels were measured indicating a preserved function of red blood cells in leukocyte depleted whole blood. At the end of shelf life 70%+/-18% of initial ATP levels were detected. CONCLUSION: Based on our data we propose that leukocyte depleted whole blood, stored for 35 days can be an option in the autologous blood supply as it meets the requirements for transfusion of the European Council.

The influence of human platelet antigen match on the success of allogeneic peripheral blood progenitor cell transplantation following a reduced-intensity conditioning regimen.

BACKGROUND: Allogeneic transplantation in elderly patients requires a dose-reduced conditioning regimen. Owing to reduced-intensity conditioning, host- and donor-type immune responses may affect the early posttransplant period, whereas only later on donor-derived reactions may ensue. Mismatches in the HLA system are known to be detrimental for the outcome of transplantation. Mismatches between donor and recipient for human platelet antigens (HPAs) may also affect the success of transplantation owing to serving as minor histocompatibility antigens and therefore rendering recipients at risk for graft-versus-host disease (GVHD) or graft rejection and inhibition of thrombopoiesis attributed to platelet (PLT) antibodies. PATIENTS AND METHODS: Therefore, the occurrence of GVHD, incidence of relapse, need of PLT support, and outcome by analysis of 45 donor-recipient pairs for HPA-1, -2, -3, and -5 allotypes and screening for PLT antibodies were evaluated before transplantation and again 1 year thereafter. RESULTS: Mismatches within the HPA system were not associated with an increased occurrence of transplant-related mortality or GVHD, the onset of thrombopoiesis, the frequency of PLT transfusions, or the incidence of relapse. Neither were settings of homozygous donors versus heterozygous recipients (graft-vs.-host direction) nor homozygous recipients versus heterozygous donors (host-vs.-graft direction) associated with any adverse effects on the outcome of the transplantation. CONCLUSION: Thus, the HPA match does not affect the outcome of transplantation after reduced-intensity conditioning.

Response to vWF-coated beads and soluble p-selectin to characterize apheresis platelets: a comparison of three cell separators.

BACKGROUND: Plateletpheresis technologies differ among various cell separators. Differences in centrifugation force, centrifugation time, and platelet concentration in the platelet concentrate may affect platelet activation and function. STUDY DESIGN AND METHODS: In a three-way crossover design, 12 donors were randomly assigned to three types of cell separators, two continuous flow systems (Amicus DN, Fenwall Division, Baxter and Spectra LRS, Gambro BCT) and one intermittent flow system (MCS+, Haemonetics). The response to vWF-coated beads was determined in the peripheral blood and fresh platelet concentrates to obtain information about the initiating step of platelet aggregation, that is, platelet adhesion. Levels of soluble p-selectin were measured as a marker of platelet activation. RESULTS: Platelet yield and concentration of platelets were higher in platelet concentrates obtained with the Amicus DN than with the Spectra LRS or the MCS+. Maximal aggregation was significantly higher in platelets from the Amicus DN than in platelets from the Spectra LRS or the MCS+. Higher concentrations of soluble p-selectin were seen in platelet concentrates obtained with the Amicus DN than in concentrates from the Spectra LRS or the MCS+, but they did not differ after correction for the number of platelets per component. CONCLUSIONS: Different plateletpheresis procedures induced distinct changes of platelet function. Platelets collected using the Amicus DN retained the strongest adhesion capacity irrespective of their activation.

Endothelial precursor cells in the synovial tissue of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: To find evidence for the presence of endothelial precursor cells, which can induce new vessel formation, in the synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Precursor cells in the synovial tissue of 18 RA patients and 15 OA patients were identified by immunohistochemistry, morphometric analysis, and confocal laser scanning microscopy using the following phenotype markers: CD31, CD34, STRO-1, CD133, vascular endothelial growth factor receptor 2 (VEGFR-2), and CXCR4. The presence of CD31, CD34, CD133, VEGFR-2, and CXCR4 messenger RNA in the synovial tissue was determined by reverse transcriptase-polymerase chain reaction, and the message for CXCR4 was quantified by an RNase protection assay. RESULTS: A population of cells that expressed CD34 on their surface but lacked the endothelial cell marker CD31 was found in the synovial tissue of RA and OA patients. CD34+,CD31- cells were detected in close proximity to STRO-1+ and CD133+ cells, forming cell clusters in the sublining area of the synovial membrane. Within these cell clusters, CD34+,CD31- precursor cells were located on the inside surrounded by STRO-1+ cells and with CD133+ cells on the outside. CD34+ precursor cells in the cell layer expressed high levels of the chemokine receptor CXCR4, while VEGFR-2 was expressed on CD34+ and CD133+ cells, and alpha-smooth muscle actin was expressed on STRO-1+ cells. CONCLUSION: The presence of endothelial precursor cells in the synovial tissue of RA and OA patients provides evidence for vasculogenesis induced by precursor cells that arise in situ or from circulating progenitors.