To explore the initial steps by which transplanted mesenchymal stem cells (MSCs) interact with the vessel wall in the course of extravasation, we studied binding of human MSCs to endothelial cells (ECs). In a parallel plate flow chamber, MSCs bound to human umbilical vein ECs (HUVECs) similar to peripheral-blood mononuclear cells (PBMCs) or CD34 ؉ hematopoietic progenitors at shear stresses of up to 2 dynes/cm 2 . This involved rapid extension of podia, rolling, and subsequent firm adhesion that was increased when ECs were prestimulated with TNF-␣. MSC binding was suppressed when ECs were pretreated with function-blocking anti-P-selectin antibody, and rolling of MSCs was induced on immobilized P-selectin, indicating that P-selectin was involved in this process. IntroductionIn recent years, mesenchymal stem cells (MSCs) have been characterized as adherent-cell populations originating from bone marrow, capable of expanding in vitro as undifferentiated cells or differentiating into osteocytes, chondrocytes, tenocytes, adipocytes, or smooth muscle cells. [1][2][3] MSCs have been used in a number of preclinical models to mediate the regeneration of muscle, endothelial, neuronal, skin, or renal epithelial tissue. [4][5][6][7] In vitro differentiation studies have demonstrated the potential of MSCs to also form alveolar and airway epithelial cells or cardiac pacemaker cells. 8,9 Moreover, MSCs have been transplanted intravenously and shown to distribute to spleen, bone, lung, and cartilage in several rodent models. [10][11][12] Intravenously injected MSCs have already been used in patients to accelerate hematopoietic reconstitution after hematopoietic stem cell transplantation, to overcome the molecular defect in children with osteogenesis imperfecta, or to alleviate the outcome after myocardial infarction. [13][14][15][16][17] Transplantation experiments in mice and primates have shown that intravenously applied MSCs distribute to several tissues and may accumulate in the lungs. 10,12,18,19 However, currently it is poorly understood to what degree MSCs use specific adhesion mechanisms for egress from the bloodstream and whether they home in a tissue-specific manner. To leave the bloodstream, mature leukocytes and hematopoietic progenitor cells (HPCs) have been shown to undergo a coordinated sequence of adhesion steps, initiated by tethering events, which are mainly mediated by selectins and their ligands. 20,21 Subsequently, the captured cells roll and encounter chemokines, which eventually activate integrins, resulting in firm arrest and subsequent transendothelial migration.To elucidate the potential of MSCs to undergo coordinated steps of interaction with endothelial cells (ECs), we investigated human MSCs under shear flow using a parallel plate flow chamber and by intravital microscopy in mice. We show here that human MSCs home to different tissues and display coordinated rolling and adhesion behavior on ECs. Although P-selectin glycoprotein ligand 1 (PSGL-1) is not expressed by MSCs, MSCs bind to ECs in a P-sele...

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Although several virus-and host-related predictive factors for the response to interferon alfa (IFN-␣) have been defined in patients with chronic hepatitis C, no pretreatment parameter can definitely predict the response to antiviral treatment. Assessment of the initial response by quantification of serum hepatitis C virus RNA before and 4 weeks after initiation of therapy may be a clinically applicable and reliable parameter to predict long-term response. Therefore, the aims of the present study were to test the predictive value of a decline in HCV RNA of at least 3 log in the first 4 weeks of treatment (⌬HCV RNA) in patients treated with 3 ؋ 10 6 units of recombinant IFN-␣2a (rIFN␣2a) three times per week subcutaneously and to compare ⌬HCV RNA with other established predictive factors, such as HCV genotype and pretreatment viremia. Serum HCV RNA was measured by a validated quantitative reverse transcription-polymerase chain reaction (RT-PCR). Geno/ subtyping of HCV was performed by direct sequencing of the nonstructural (NS) 5B region of PCR-amplified isolates and subsequent phylogenetic analysis. Stable HCV RNA levels (⌬HCV RNA I 1 log) within the first 4 weeks of IFN-␣ treatment were present in 42 of 70 patients. A decline in HCV RNA levels between 1 to 3 log and more than 3 log was observed in 9 (13%) and 19 patients (27%), respectively. In 21 of 70 patients (30%), HCV RNA was not detectable at the end of 12 months' treatment. Three of 26 patients (11%) with a pretreatment viremia of I10 6 copies/mL (all HCV subtype 3a) and 6 of 44 patients (14%) with a pretreatment viremia of G10 6 copies/mL (HCV subtypes 1b, 2a, 2c, 3a [two patients], and 4) achieved a virological sustained response to interferon-␣2a treatment. All patients with a virological sustained response had an initial ⌬HCV RNA of more than 3 log. In a stepwise discriminant-function analysis, the initial ⌬HCV RNA was confirmed as the strongest predictor of virological sustained response (P F .0001). In conclusion, the data of the present study suggest that IFN-␣ treatment can be terminated after 4 weeks in patients with a decrease in HCV RNA levels of less than 3 log, when apparent HCV eradication is considered the therapeutic target. The predictive value of ⌬HCV RNA clearly exceeds the significance of HCV genotype and pretreatment viremia as predictors of successful

Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.

Nucleotide sequence analysis of hepatitis C virusHepatitis C virus (HCV) infection often progresses to (HCV) strains showed substantial variability leading to chronic hepatitis, cirrhosis, and possibly hepatocellular carcia classification into several genotypes and subtypes. The noma. [1][2][3] Using phylogenetic analysis of the viral 5-noncoding data correlating HCV genotypes and subtypes with hep-region, a classification of HCV strains into six major types atitis C viremia levels, demographic characteristics of (HCV-1 to -6) has been introduced. Analysis of the more varipatients (age, mode of transmission, duration of infec-able coding regions of the viral genome (core, envelope, nontion), and severity of liver disease are conflicting. The structural [NS]-3, and NS-5) indicated that major genotypes interpretation of several studies is further complicated are composed of two or more distinct subtypes, termed 1a, because the molecular methods used lacked specificity 1b, 2a etc. 4,5 The geographic distribution of HCV genomes for genotyping/subtyping and underestimated viremia differs considerably. In Europe and the United States, sublevels, especially in patients infected with HCV geno-types HCV-1a and -1b, HCV-2a and -2b, and HCV-3a are types 2 and 3. In the present study we investigated 97 most abundant, whereas in Japan HCV subtypes 1a and 3a consecutive patients with chronic hepatitis C using mo-are virtually nonexistent. Type 4 genotypes are common in lecular ''gold standard'' methods. HCV subtyping was patients from Northern Africa and the Middle East, and types performed by sequence and phylogenetic analysis of the 5 and 6 have been identified in serum samples obtained from nonstructural (NS)-5 region and serum HCV-RNA con-South Africa and Hong Kong, respectively. Sequence analysis centration was assessed by a validated genotype-inde-of HCV isolates from Vietnamese blood donors revealed addipendent quantitative reverse-transcription-polymerase tional major genetic groups (HCV-7, -8, and -9).6-8 chain reaction assay using an internal RNA standard.Several 9-12 but not all studies 13,14 have indicated a correlaPatients infected with subtypes HCV-1b, HCV-2a-c, and tion between HCV genotypes/subtypes and serum HCV-RNA HCV-4 were older than patients infected with HCV-1a concentrations. The data associating HCV genotypes/suband HCV-3a. Serum HCV-RNA levels ranged from 1.5 1 types and/or hepatitis C viremia levels with demographic, 10 4 to 1.0 1 10 8 copies/mL with no significant differences biochemical, or histological characteristics of chronically inbetween median serum HCV-RNA concentrations in pa-fected patients are conflicting. [15][16][17][18][19][20][21][22][23][24][25][26] These issues are further tients infected with different genotypes/subtypes. Al-complicated by recent evaluations showing that common gethough patients infected with HCV-1b were older, no notyping methods lack specificity 12,27 and that quantification biochemical or histological evidence was obtained that systems underestimate high virem...

A late sample collection on day 3, day 4 or day 5 after blood donation in combination with a rapid bacterial detection method offers a new opportunity to improve blood safety and reduce errors due to sampling., BacT/ALERT, Bactiflow or 16s ID-NAT are feasible for late bacterial screening in platelets may provide data which support the extension of platelet shelf life in Germany to 5 days.

Background and ObjectivesStem cells play an important role in the pathogenesis and maintenance of most malignant tumors. Acute myeloid leukemia (AML) is a stem cell disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (ATRA) induces CR, but not molecular remission (CMR), because the fusion transcript remains detectable, followed by relapse within a few months. Arsenic induces high rates of CR and CMR followed by a long relapse-free survival (RFS). Here we compared the effects of ATRA and arsenic on PML/RARα-positive stem cell compartments. Design and MethodsAs models for the PML/RARα-positive LSC we used: (i) Sca1 + /lin -murine HSC retrovirally transduced with PML/RARα; (ii) LSC from mice with PML/RARα-positive leukemia; (iii) the side population of the APL cell line NB4. ResultsIn contrast to ATRA, arsenic abolishes the aberrant stem cell capacity of PML/RARα-positive stem cells. Arsenic had no apparent influence on the proliferation of PML/RARα-positive stem cells, whereas ATRA greatly increased the proliferation of these cells. Furthermore ATRA induces proliferation of APL-derived stem cells, whereas arsenic inhibits their growth. Interpretations and ConclusionsTaken together our data suggest a relationship between the capacity of a compound to target the leukemia-initiating cell and its ability to induce long relapse-free survival. These data strongly support the importance of efficient LSC-targeting for the outcome of patients with leukemia. 1 The AML phenotype seems to be maintained by an increased proliferation of the blast cells, which is considered to result from the combination of two components: (i) a differentiation block preventing progenitor cells reaching the post-proliferative stage and subsequently undergoing programmed cell death; 2 (ii) an increased capacity for self-renewal of the leukemic progenitors.3,4 Acute promyelocytic leukemia (APL) is a well characterized subtype of AML, classified as FAB M3.5 It can be distinguished from other AML subtypes based on distinct cytogenetic, biological and clinical features.6 More than 95% of patients with APL harbor the t(15;17) translocation, which encodes the PML/RARα fusion protein.5 In addition, APL is clinically characterized by (i) the achievement of complete remission (CR) in about 90% of patients upon treatment with all-trans retinoic acid (ATRA); 7 and (ii) induction of CR in 72-96% of patients upon exposure to low dose arsenic trioxide (As2O3). 8Treatment with ATRA as a single agent results in CR but not complete molecular remission (CMR), because the t(15;17)-associated PML/RARα fusion transcript remains detectable. In about 29% of patients CMR can be induced by ATRA if double the dosage is administered as a liposomal formulati...

The t(6;9)-positive acute myeloid leukemia (AML) is classified as a separate clinical entity because of its early onset and poor prognosis. The hallmark of t(6;9) AML is the expression of the DEK/CAN fusion protein. The leukemogenic potential of DEK/ CAN has been called into question, because it was shown to be unable to block the differentiation of hematopoietic progenitors. We found that DEK/CAN initiated leukemia from a small subpopulation within the hematopoietic stem cell (HSC) population expressing a surface marker pattern of long-term (LT) HSC. The propagation of established DEK/CAN-positive leukemia was not restricted to the LT-HSC population, but occurred even from more mature and heterogeneous cell populations. This finding indicates that in DEK/CAN-induced leukemia, there is a difference between 'leukemia-initiating cells' (L-ICs) and 'leukemia-maintaining cells' (L-MCs). In contrast to the L-IC cells represented by a very rare subpopulation of LT-HSC, the L-MC seem to be represented by a larger and phenotypically heterogeneous cell population.