Search

We evaluated progenitor cell proliferation in cultures supplemented by different cytokine combinations in the presence or absence of IL-12. In cultures of low density cells, cytokine combinations including IL-12 were associated to a greater proliferation (up to 6.7 +/- 2.5 CFU-GM fold expansion). However, in cultures of purified CD34+ cells the more efficient cytokine combination (147 +/- 49 CFU-GM fold expansion) was SCF, IL-3, IL-11 and MIP-1 alpha, and the addition of IL-12 did not further enhance expansion of progenitors. These results indicate that accessory cells, lost in CD34+ cell purification, could be in part responsible for IL-12 effect on progenitor cell proliferation. In CD34+ cell cultures the addition of IL-12 led to CD19 mRNA generation, suggesting that IL-12 acts on haemopoietic cells with both myeloid and lymphoid potential.

Different procedures for umbilical cord blood (CB) collection, separation, and cryopreservation were compared to evaluate the feasibility of large-scale CB banking for unrelated transplant. CB collection using an open system was associated with a 12.5% rate of bacterial contamination, whereas this rate fell to 3.3% using a closed collection system. When the umbilical cord was clamped within 30 s after delivery, on 378 occasions it was possible to collect 77 +/- 23 ml of CB without risk to mother or infant. Considering that engraftment can be obtained in recipients of 20 x 10(3) CFU-GM/kg, 86% and 28% of CB samples with a volume larger than 50 ml were found to contain a sufficient number of CFU-GM to engraft patients of 20 and 70 kg, respectively. Both Ficoll and gelatin purification procedures were associated with a recovery of 86-92% of CFU-GM, BFU-E, CFU-GEMM, and HPP-CFC, but the gelatin method appears to be more suitable for large-scale CB banking in vials. After cryopreservation, the recovery of clonogenic progenitors was similar for both CB samples stored as whole blood or as mononuclear cells separated using Ficoll or gelatin. In conclusion, large-scale CB banking seems feasible, and CB cell separation could allow storage of a large number of CB samples in a limited liquid nitrogen space.

We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34+ cells as a target for human multidrug resistance (MDR1) gene transfer. Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, efficiency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 micrograms/ml taxol. In uninfected control, 1-2% of CFU-GM and CFU-GEMM were found to be drug-resistant, while 14-31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progenitors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction. MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls). (3) Flow cytometric analysis of the expression of CD34 and P-glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P-glycoprotein was found on 17-25% of viable CD34+ cells. (4) Cytochemical localization by APAAP staining of P-glycoprotein. No specific localization was found in untransduced controls, whereas transduced and cultured CB-cells expressed P-glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor cells seems a feasible and attractive approach to generate a drug-resistant haemopoiesis.

Cord blood (CB) plasma has previously been found to augment the replating capacity of CB-derived hematopoietic progenitors. In the present study, we observed a 2.8 +/- 0.3 and a 1.8 +/- 0.2-fold expansion of CFU-GM in cultures of CB cells without growth factors but supplemented by CB plasma or maternal blood (MB) plasma, respectively. In the absence of growth factors, CFU-GM expansion did not occur in CB cell cultures supplemented with fetal calf serum (FCS), peripheral blood (PB) plasma, PB plasma plus concentrations of IL-6 similar to those previously reported in CB plasma and in bone marrow (BM) cell cultures supplemented with FCS, CB, MB or PB plasma. In the presence of stem cell factor (SCF), IL-3 and IL-11, an expansion of CFU-GM was observed in CB and BM cell cultures supplemented by either CB, MB or PB plasma or FCS. Nevertheless, progenitor expansion was significantly superior in CB cell cultures supplemented by CB plasma (8.7 +/- 0.7, 2.4 +/- 0.3 and 2.6 +/- 0.3-fold expansions for CFU-GM, BFU-E and CFU-Mix, respectively). In conclusion, an unknown factor(s) present in CB plasma, probably capable of crossing the placenta, has an effect alone and in the presence of growth factors in supporting ex vivo expansion of CB progenitors.

We have considered interesting to determine monoclonal gammopathies incidence, in 2191 serum proteins electrophoresis performed in our laboratory from January to December 1974. We have found 15 cases of monoclonal gammopathies, some cases combined with Mieloma (3 cases), some other with other with non specific diseases. We have considered the relations between type of gammopathy and other laboratory tests useful for any other diagnose: they are: immunochemical analysis, E.S.R., red and white count, total proteins, Bence Jones protein.