HLA-DRB1: genetic susceptibility and disability progression in a Spanish multiple sclerosis population.

BACKGROUND AND OBJECTIVE: The association of HLA-DRB1*15 with susceptibility to multiple sclerosis (MS) has been consistently reported although its effect on the clinical phenotype is still controversial. The objectives of this study are to investigate the influence of the HLA-DRB1 alleles on the genetic susceptibility to MS and to study their impact on disability progression in a Spanish population. METHODS: HLA-DRB1 typing was performed by PCR-SSP in 380 patients with sporadic MS and 1088 unrelated healthy controls. Allelic frequencies were compared between groups. We studied the correlation between the different alleles and the progression of MS. RESULTS: The HLA-DRB1*15 allele in patients with MS had a statistically significant higher frequency when compared with controls (18.9% in patients vs. 10.1% in controls, Odds ratio (OR)=2.07, 95% CI=1.64-2.60, P<0.001). In the univariate analysis, the DRB1*01 and DRB1*04 alleles were associated with a worse prognosis when considering the time to reach an EDSS of 6, whereas the DRB1*03 was correlated with a better outcome. In the multivariate analysis, the alleles*01 and *04 were demonstrated to be independent factors to have a worse prognosis. CONCLUSIONS: HLA-DRB1*15 is associated with MS when comparing patients with unrelated healthy controls in a Spanish population. The HLA-DRB1*01 and HLA-DRB1*04 alleles are related to a worse prognosis when considering the time taken to reach severe disability.

Factors modulating circulation of hematopoietic progenitor cells in cord blood and neonates.

BACKGROUND: Hematopoietic progenitor cells (HPC) circulate at high levels at birth and disappear rapidly afterwards, but the underlying mechanism it is not known. The aim of this study was to assess circulating HPC in cord blood at different gestational ages and shortly after birth and concomitantly study the biologic markers involved in this phenomenon. METHODS: All samples were analyzed for CD34(+) cells, colony-forming units (CFU) and cytokines. RESULTS: The results obtained confirmed a slight decrease in HPC concentration during the late stage of fetal life (R(2)=0.41). After birth, CD34(+) cells showed a rapid decline from circulation: 25+/-29% at 3 h, 51+/-42% at 12 h and 80+/-48% at 60 h. CFU cleared following a similar pattern. Cord plasma showed higher concentrations of stem cell factor (SCF), fetal liver tyrosine kinase 3-ligand (FLT3l), erythrpoietin (EPO), granulocyte colony-stimulating factor (G-CSF) and interleukin-11 (IL-11) compared with an adult control. Interestingly, the EPO concentration in newborn plasma correlated with the kinetics of HPC decline after birth. Moreover, we observed an up-regulation of l-selectin and a down-regulation of CXCR4 expression in CD34(+) cells 3 h after birth. DISCUSSION: These data combined suggest that an active homing process results in the clearance of HPC from the circulation immediately after birth.

Washing of cord blood grafts after thawing: high cell recovery using an automated and closed system.

BACKGROUND AND OBJECTIVES: Cord blood (CB) progenitor cells are an alternative source of haematopoietic stem cells for bone marrow reconstitution. The critical importance of cell dose in the clinical outcome has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility. This aim of this study was to evaluate an automated CB washing protocol of thawed cord blood units using the Sepax device. MATERIALS AND METHODS: After an initial 1:1 dilution using a dextran/albumin-containing buffer, the cells were washed in order to obtain a final product ready for transplantation. The automatic method was compared with the conventional manual washing procedure. Blood samples were taken after thawing and after washing. The processing time, viability and mean recovery of nucleated cells (TNC) and progenitors were determined. RESULTS: The automatic procedure resulted in a median recovery of 93% CD34+ cells and 89% TNC; no significant differences were observed between methods. In addition, median viability, as assessed by annexin V and 7-aminoactinomycin D (7-AAD), was 98% and 94%, respectively, within CD34+ cells. CONCLUSIONS: The automatic washing method described is as effective as the manual method in terms of viability and progenitor cells recovery, but faster and easier for the operators to perform. Overall, our data suggest that the automatic method is safe and suitable for the routine washing of thawed CB grafts in the clinic.

Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols.

BACKGROUND: Cord blood is a useful source of HPCs for allogeneic transplantation. HPC ex vivo expansion of a cord blood graft has been proposed as a way to increase the speed of engraftment and thus to reduce the occurrence of transplantation-related complications. OBJECTIVE: The purpose of this study was to optimize a method for CD34+ cell selection of thawed cord blood grafts under clinical grade conditions, intended for application in a static, serum-free expansion culture. MATERIAL AND METHODS: Twelve samples were thawed and washed with dextran, albumin, and rHu-deoxyribo-nuclease I (RHu-DNase) to avoid clumping. CD34+ cells were selected by using a sensitized immunomagnetic bead and 9C5 MoAb complex. A buffer containing rHu-DNase, citrate, albumin, and immunoglobulin in PBS was used during the procedure. CD34+ cells were eluted and detached by using an immunomagnetic cell selection device. Cells from the enriched fraction were cultured for 6 days in serum-free medium supplemented with rHu-SCF, rHu-IL-3, rHu fetal liver tyrosine kinase 3 ligand, and rHu thrombopoietin (50 ng/mL each). Cells were expanded in well plates and in two semipermeable bags. RESULTS: A mean of 1.94 x 10(6) (+/- 1.55) CD34+ cells was obtained, yielding a CD34+ cell recovery of 52 +/- 12 percent. Nonspecific loss of CD34+ cells was 32 +/- 10 percent. CFU-GM and BFU-E/CFU-Mixed recoveries were 33 +/- 15 percent and 27 +/- 12 percent, respectively. CD34+ cells obtained were functionally comparable with fresh CD34+ cells selected for clonogenic potential. The capacity for expansion was not significantly different in the two types of bags studied. HPCs in wells were expanded 33 +/- 14-fold for CD34+ cells and 42 +/- 19-fold for overall colonies. The expansion rates observed in wells were significantly superior to those obtained in bags. CONCLUSION: The feasibility of a clinical-scale cord blood selection procedure based on a direct immunomagnetic method after thawing, followed by an ex vivo expansion culture using semipermeable bags, is shown. After 6 days of expansion, it was possible to generate a 9-fold increase in CD34+ cells, a 6-fold increase in CFU-GM and a 13-fold increase in BFU-E/CFU-Mixed colonies.

Immunomagnetic bone marrow (BM) and peripheral blood progenitor cell (PBPC) purging in follicular lymphoma (FL).

Twenty-nine B cell follicular lymphoma (FL) patients had their BM (n = 12) or PBPC (n = 17) purged using a panel of monoclonal antibodies and immunomagnetic beads (IMB). The median recovery of nucleated cells (NC) and CD34+ cells was 59.3% (40.5-74) and 56.1% (30.8-82.9) in BM and 77.2% (64.7-88.3) and 73.5% (61.5-98.6) in PBPC (P<0.0005). A median of >1.62 and >1.02 log of target cell depletion was achieved as judged by flow cytometry analysis in BM and PBPC, respectively. Of 29% of initial harvests that had a bcl2 PCR-amplified signal, 37.5% became PCR negative in the final purged products. Absorbed cells containing IMB-target cell complexes gave bcl2 rearrangement signal in 20% of samples in which the start and final purged components were negative. Twenty-three of 26 patients receiving an autologous purged product are evaluable for engraftment. Median time to reach an ANC >0.5x10(9)/l and platelet count >20x10(9)/l was 21 (11-43) and 41 days (13-70) for BM (n = 9) and 14 (10-31) and 14 (8-37) for PBPC (n = 14) autografted patients (P = 0.01 and 0.001). One patient did not engraft and was rescued with a back-up BM. These data demonstrate that this indirect immunomagnetic technique is able to achieve a high grade of lymphoma cell depletion in BM and PBPC and that these purged products are capable of rapid engraftment after autologous transplantation.

Peripheral blood CD34+ cell immunomagnetic selection in breast cancer patients: effect on hematopoietic progenitor content and hematologic recovery after high-dose chemotherapy and autotransplantation.

BACKGROUND: Tumor cells have been detected in mobilized peripheral blood of breast cancer patients, and they may contribute to tumor recurrence after the transplantation of peripheral blood progenitor cells. One of the most widespread technologies for tumor purging of the graft is immunomagnetic hematopoietic progenitor cell selection. STUDY DESIGN AND METHODS: The study assessed the effectiveness of a magnetic cell-separation system in selecting functional subpopulations of hematopoietic progenitors from 14 blood-derived harvests of 11 patients with high-risk breast cancer after mobilization following cytotoxic chemotherapy supported by granulocyte--colony-stimulating factor, as well as the feasibility of transplanting these selected subpopulations. RESULTS: CD34(+)-enriched cell fractions had a median purity of 93.0 percent (72.7-98.5%). The procedure yielded 52.6 percent of the CD34+ cell input (39.4-116.8%). Median recoveries of colony-forming units (CFUs) (36.87%) and cobblestone area-forming cells (CAFCs) (152.5%) were, respectively, 0.70 and 2.87 times those of CD34+ cells (52.6%). Moreover, CAFC efficiency in the positive cell fraction was 2.57 times that in the starting cell fraction. Peripheral blood neutrophil counts of 0.5 x 10(9) per L and platelet counts of 20 x 10(9) per L were reached after median times of 9 and 11 days, respectively. The number of transfused CAFCs per kg, CD34+ cells per kg, and postthaw CFU-granulocyte-macrophage per kg was correlated, respectively, with the speed of engraftment of neutrophils, platelets, or both. Tumor cells detected in one patient's peripheral blood were not found after CD34+ cell selection. CONCLUSION: Transplantation of immunomagnetically purified peripheral blood CD34+ cells does not increase transplantation-related morbidity. It induces a selective enrichment of more immature hematopoietic progenitors, which makes it suitable for use in cell expansion and gene therapy protocols.

A new human cell line with pre-B cell phenotype and t(5;12).

A new cell line (LR10.6) with pre-B cell phenotype has been established from bone marrow cells obtained from a child with B lineage acute lymphoblastic leukemia in complete clinical remission. The line expresses nuclear TdT enzyme, cytoplasmic Ig lambda-chain and membrane mu-chain and other B but no T or myeloid markers. The cells also show activation antigens CD69 and CD71, adhesion molecules CD54, CD50 and CD56 and the tyrosine kinase receptor CD117. No expression of multidrug resistance phenotype MDR-1 is observed on these cells which nevertheless express the transcriptional factor p53 protein in a mutant form. Cytogenetic study shows a translocation t(5;12)(q31;p13) involving breakpoints which contain the growth factor interleukin 3 gene (5q31) and the recently identified TEL/ETV6 gene (12p13). Activation of the cells with phorbol-12 myristate 13-acetate (PMA) up-regulates the expression of the CD69 activation antigen and down-regulates the CD117 molecule. In addition, PMA fails to induce the CD20 B cell antigen.

Distinct patterns of urokinase receptor (uPAR) expression by leukemic cells and peripheral blood cells.

The urinary type plasminogen activator, urokinase (uPA) is localized on the cell surface through the binding of a specific receptor, the uPA receptor (uPAR). The uPA localization enhances plasmin formation on the cell surface and facilitates cell migration. The cellular and tissue distribution of uPAR is not fully established. We have analyzed uPAR expression in nine leukemic cell lines of distinct lineages and maturational states and correlated this with expression of plasminogen receptors, tissue-type plasminogen activator (tPA) receptors and LDL receptor-related protein (LRP). The most immature and least differentiated cell line (an erythro-myeloid cell line) and cells of lymphoid lineage, did not express uPAR, whereas cells differentiated along the myelo-monocytic pathway displayed this receptor. Plasminogen and tPA receptors were expressed by all leukemic cell lines and by all nucleated peripheral blood cells but B and T lymphocytes were negative for cell surface expression of both uPAR and LRP while monocytes and neutrophils were positive for expression of both uPAR and LRP. PMA stimulation induced surface expression of uPAR in lymphocytes but did not induce expression of LRP by these cells. In contrast, lymphoid cell lines were negative for uPAR expression even after PMA stimulation, indicating differences in regulation of uPAR expression between lymphocytes and lymphoid cell lines. The pattern of uPAR expression on leukemic cell lines was also studied on bone marrow blast cells from leukemic patients. Only the most mature myeloid cells expressed uPAR on their surfaces. In contrast, M3 leukemic cells and other blast cells displaying lymphoid markers such as TdT (+) and/or CD2 (+) did not express intracellular or cell-surface associated uPAR, indicating an heterogeneity among these promyelocytic cells and suggesting that uPAR may be a useful marker for leukemia typing. Myeloid blast cells from some patients contained intracellular pools of uPAR but displayed no receptor on the cell surface, suggesting that translocation may be a mechanism regulating uPAR expression in these cells. The comparison of uPAR expression between these cell lines and peripheral blood cells and it correlation with plasminogen receptors, tPA receptors and LRP expression offers new insights regarding potential mechanisms for regulation of uPA-uPAR-mediated pericellular proteolysis.

Mobilization of peripheral stem cells with intensive chemotherapy (ICE regimen) and G-CSF in chronic myeloid leukemia.

Seventeen patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) were treated with the ICE regimen plus G-CSF with the aim of mobilizing and collecting Ph-negative peripheral stem cells (PSC) in the setting of an autotransplant program. Fifteen patients had CML in first chronic phase (CP), and two in accelerated phase (AP). Three patients had been previously treated with interferon alpha 2a (IFN). Twelve patients underwent leukaphereses and a mean of 4.7 x 10(8)/kg mononuclear cells were obtained. Four CP patients did not show a significant mobilization peak of CD34+ cells and leukapheresis was not performed; finally, one patient died before apheresis could be performed. Six of the 12 who underwent leukaphereses obtained more than 1.0 x 10(6)/kg CD34+ cells. Eight of the 12 mobilized patients (67%) obtained a major cytogenetic response, including two complete and six partial; in the remaining four patients minimal or absent cytogenetic responses were observed. A higher rate of Ph purging was obtained in patients mobilized early or showing residual Ph-negative cells before mobilization, even if they were in AP. Infectious complications were frequent with a 38% rate of bacteremia recorded and one case of pulmonary aspergillosis resulting in a toxicity similar to that occurring in acute myeloid leukemia-induction chemotherapy. The ICE regimen can promote 'in vivo' purging of the Ph+ cells in 67% of CML mobilized patients (8/12). Failure of mobilization occurs in 65% of patients (11/17), mainly because of poor CD34+ cell yield.