Walter Sibrowski

Differential roles for membrane-bound and soluble syndecan-1 (CD138) in breast cancer progression.

The heparan sulfate proteoglycan syndecan-1 (Sdc1) modulates cell proliferation, adhesion, migration and angiogenesis. Proteinase-mediated shedding converts Sdc1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. In breast carcinomas, Sdc1 overexpression correlates with poor prognosis and an aggressive phenotype. To distinguish between the roles of membrane-bound and shed forms of Sdc1 in breast cancer progression, human MCF-7 breast cancer cells were stably transfected with plasmids overexpressing wild-type (WT), constitutively shed and uncleavable forms of Sdc1. Overexpression of WT Sdc1 increased cell proliferation, whereas overexpression of constitutively shed Sdc1 decreased proliferation. Fibroblast growth factor-2-mediated mitogen-activated protein kinase signaling was reduced following small-interfering RNA (siRNA)-mediated knockdown of Sdc1 expression. Constitutively, membrane-bound Sdc1 inhibited invasiveness, whereas soluble Sdc1 promoted invasion of MCF-7 cells into matrigel matrices. The latter effect was reversed by the matrix metalloproteinase inhibitors N-isobutyl-N-(4-methoxyphenylsufonyl) glycyl hydroxamic acid and tissue inhibitor of metalloproteinase (TIMP)-1. Affymetrix microarray analysis identified TIMP-1, Furin and urokinase-type plasminogen activator receptor as genes differentially regulated in soluble Sdc1-overexpressing cells. Endogenous TIMP-1 expression was reduced in cells overexpressing soluble Sdc1 and increased in those overexpressing the constitutively membrane-bound Sdc1. Moreover, E-cadherin protein expression was downregulated in cells overexpressing soluble Sdc1. Our results suggest that the soluble and membrane-bound forms of Sdc1 play different roles at different stages of breast cancer progression. Proteolytic conversion of Sdc1 from a membrane-bound into a soluble molecule marks a switch from a proliferative to an invasive phenotype, with implications for breast cancer diagnostics and potential glycosaminoglycan-based therapies.

The role of caspases in cryoinjury : caspase inhibition strongly improves the recovery of cryopreserved hematopoietic and other cells

Cryopreserved cells and tissues are increasingly used for stem cell transplantation and tissue engineering. However, their freezing, storage, and thawing is associated with severe damage, suggesting the need for better cryopreservation methods. Here, we show that activation of caspase‐3 is induced during the freeze‐thaw process. Moreover, we demonstrate that prevention of caspase activation by the caspase inhibitor zVAD‐fmk strongly improves the recovery and survival of several cryopreserved cell types and hematopoietic progenitor cells. A short preincubation with the caspase inhibitor after thawing also enhances the colony‐forming activity of hematopoietic progenitor cells up to threefold. Furthermore, overexpression of Bcl‐2, but not the blockade of the death receptor signaling, confers protection, indicating that cryoinjury‐ associated cell death is mediated by a Bcl‐2‐controlled mitochondrial pathway. Thus, our data suggest the use of zVAD‐fmk as an efficient cryoprotective agent. The addition of caspase inhibitors may be an important tool for the cryopreservation of living cells and advantageous in cell transplantation, tissue engineering, and other genetic technologies.

The adult stem cell marker Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via Notch-1 and p21WAF1/CIP1

The RNA‐binding protein Musashi‐1 has been proposed to maintain stem cell function during development and regenerative processes as a modulator of the Notch‐1 signaling pathway. Musashi‐1 expression is upregulated in endometrial carcinoma, however, its pathogenetic role in this tumor entity is unknown. Here we investigate the functional impact and mode of action of Musashi‐1 on endometrial carcinoma cell behaviour in vitro. Aldehyde dehydrogenase‐1 activity and side population (SP) measurement by Hoechst dye exclusion revealed that the Ishikawa endometrial carcinoma cell line contains a pool of putative cancer stem cells. Musashi‐1 expression is 20.8‐fold upregulated in SP+ compared to SP‐ and equally distributed between ALDH+ and ALDH‐ cell pools. siRNA‐mediated knockdown of Musashi‐1 mRNA expression lead to an altered expression of the signaling receptor Notch‐1 and its downstream targets, the transcription factor Hes‐1 and the cell cycle regulators p21WAF1/CIP1 and cyclin B1, as determined by Western blotting and quantitative real‐time PCR. Flow cytometric and ELISA analyses revealed that Musashi‐1‐mediated modulation of these factors exerted an antiproliferative effect on the cell cycle, and increased apoptosis in endometrial carcinoma cells. We conclude that Ishikawa cells contain a subpopulation of cells with stem cell‐like properties. Musashi‐1 modulates endometrial carcinoma cell cycle progression and apoptosis via the stemness‐related factors Notch‐1, Hes‐1 and p21WAF1/CIP1, thus emerging as a novel future target for endometrial carcinoma therapy.

The influence of blood group differences in allogeneic hematopoietic peripheral blood progenitor cell transplantation.

BACKGROUND: Severe immunohematologic complications after ABO‐mismatched allogeneic blood peripheral blood progenitor cell (PBPC) transplantation (PBPCT), including pure red cell aplasia and immune hemolysis, have been described. Although several studies have addressed this issue, the clinical influence of blood group differences on transfusion requirements and survival is still discussed controversially, especially in the case of PBPCT.

A New No-Lyse, No-Wash Flow-Cytometric Method for the Determination of CD4 T Cells in Blood Samples

Background: Commonly used flow-cytometric methods for immunophenotyping are based on erythrocyte lysing reagents. It is known from the literature that these reagents result in a significant loss of leucocytes caused by membrane destruction. Although the dual-platform method should compensate this phenomenon, the subset-specific individual differences in sensitivity to lysing reagents lead to incorrect values. In order to overcome this problem we introduce a no-lyse, no-wash procedure in combination with absolute true volumetric counting (TVC) for the enumeration of CD4 T cells. Material and Methods: Whole-blood samples of 50 blood donors and 20 samples of patients with acquired immunodeficiency syndrome (AIDS) were treated with both a lyse, no-wash and a nolyse, no-wash procedure. Then, CD4 T cells were counted with a TVC flow cytometer (CyFlow Counter). 30 blood samples of blood donors were treated with a lyse and wash protocol and measured by the CyFlow Counter and FACS-Calibur system (reference method). A new gating strategy was used for the data analysis for both, the lyse and no-lyse method and compared to the traditional gating procedure. Results: The TVC method showed a good reproducibility for both, the lyse (CV 1.9%) and the no-lyse procedure (CV 1.5%). CD4 counts measured by the no-lyse procedure are on average 10% higher than by using the lysing protocol. The comparison of the CyFlow Counter and FACS-Calibur results showed a correlation of r = 0.961. The simplified gating strategy shows a good correlation to the traditional gating procedure (r = 0.998). Conclusion: Erythrocyte lysing procedures cause substantial cell loss with individual values for every single subclass and patient. Therefore, the use of a no-lyse procedure is recommended. The new gating strategy is fully comparable to the traditional one and simplifies enumeration of CD4 T cells. Using the no-lyse procedure in combination with the new gating strategy, it is possible to reduce the costs per sample from EUR 30.– to below EUR 2.–.

Processing of peripheral blood progenitor cell components in improved clean areas does not reduce the rate of microbial contamination

BACKGROUND: Microbial contamination of peripheral blood progenitor cell components (PBPCs) may cause severe complications in immunosuppressed recipients. Therefore, principles of Good Manufacturing Practice (GMP) are applicable for processing of PBPC components to reduce potential risks of contamination.

2 Platelet Concentrates

2.5 Range of Application, Dosage, Modes of Administration 2.5.1 Platelet Transfusion in Patients with Hematologic-Oncologic Disorders 2.5.1.1 Patients with Chronic Thrombocytopenia (Group A) 2.5.1.2 Patients with Elevated Platelet Turnover (Group B) 2.5.1.3 Patients with Impaired Platelet Production due to Chemotherapy (Group C) 2.5.1.4 Patients with Impaired Platelet Production and Additional Risks Of Bleeding (Group D)

Accelerated kidney transplant rejection and hypertensive encephalopathy in a pediatric patient associated with antibodies against angiotensin type 1 receptor and HLA class II.

This work was supported by the University of Nebraska Medical Center Research Support Fund. J.T.L. has received money for support from Merck & Co., Inc. to study sitagliptin in a subsequent study. All other authors declare no conflicts of interest. Address correspondence to: James T. Lane, M.D., Division of Endocrinology and Diabetes, Department of Internal Medicine, Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, 1000 N. Lincoln Blvd., Suite 2900, Oklahoma City, OK 73104-3252. E-mail: james-lane@ouhsc.edu J.T.L. participated in the design, and implementation of the study, participated in the recruitment and clinical care of patients, completed data acquisition, statistical analysis, and manuscript preparation. D.E.O. participated in statistical analysis, data acquisition, and review of the manuscript. C.E.H participated in the recruitment of patients, clinical follow-up within the clinical research center, data acquisition, and review of the manuscript. D.S.C. participated in the design of the study and review of the manuscript. L.E.W. performed renal transplants and administered clinical care. R.B.S. performed renal transplants, administered clinical care, participated in the design and implementation of the study, recruited patients, and reviewed the manuscript. Received 21 July 2011. Accepted 24 August 2011. Copyright

A novel true volumetric method for the determination of residual leucocytes in blood components

Background and Objectives Accurate determination of residual leucocytes [white blood cells (WBC)] in blood components is of high clinical importance. To date, several labour‐intensive, time‐consuming or expensive techniques have been used for this purpose.

Factors affecting the efficacy of peripheral blood progenitor cells collections by large-volume leukaphereses with standardized processing volumes.

BACKGROUND:  Peripheral blood progenitor cell (PBPC) collections should be safe and efficient. Therefore, the influence and risk factors in large‐volume leukaphereses (LVL) with standardized blood volumes was investigated.