Growth factors increase retroviral transduction but decrease clonogenic potential of umbilical cord blood CD34+ cells.

BACKGROUND AND OBJECTIVE: The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of their stable transduction. The great potential of umbilical cord blood as a source of CD34+ cells combined with the availability of advanced cell purification procedures prompted us to evaluate whether incubation with growth factors might influence the type of cells effectively transduced by retroviral vectors. DESIGN AND METHODS: Isolated, at least 95% pure, CD34+ cells were infected with the LXSN murine retrovirus carrying the neomycin-resistance gene. Different schedules of CD34+ cell infection were performed with or without incubation for different times in the presence of Interleukin-3 (IL-3), Interleukin-6 (IL-6) and stem cell factor (SCF). Efficiency of transduction was evaluated by clonogenic assays, semiquantitative PCR and RT-PCR analyses performed either immediately or after 7 day expansion of CD34+ cells in liquid culture in the presence of erythropoietin (EPO), IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). RESULTS: The results obtained indicated that the amount of transduced cells increased with the lenght of incubation with growth factors, either before or during infections. However, different types of cells were transduced depending on the duration of stimulation and infection. Thus, following one week culture of CD34+ cells in the presence of EPO, IL-3 and GM-CSF the clonogenic potential was affected dyshomogeneously. Precisely, with a single 3-hour infection performed after 12 hours of stimulation with growth factors, the clonogenic potential of the transduced cells greatly increased after one week in culture. In contrast, with a 48 hour infection, the transduced cells completely lost their clonogenic potential after one week in culture. INTERPRETATION AND CONCLUSIONS: These results demonstrate that a reasonably high transduction efficiency of purified CD34+ cells can be achieved with short schedules of incubation/infection in the absence of stroma or extracellular matrix.

von Willebrand factor: biological function and molecular defects.

The human von Willebrand factor (vWF) plays a pivotal role in the mechanisms of blood clotting and platelet thrombus formation; it also binds and stabilizes factor VIII procoagulant protein. The biological functions of vWF are dependent on distinct molecular domains responsible for the specificity and affinity for ligands. The multimeric structure of vWF provides an array of binding sites that allow multivalent interactions, thus supporting the formation of stable platelet aggregates at the site of vascular injury, particularly under flow conditions characterized by high shear stress. Quantitative and qualitative abnormalities of vWF cause the most common congenital bleeding disorder in humans, the von Willebrand disease (vWD). This review will provide an update on the recent advances toward the elucidation of structure-function relationships and the detection of molecular defects leading to vWD and will highlight the revised classification of vWD.

Platelet antibody detection in pediatric immune thrombocytopenic purpura: evaluation of three screening methods.

BACKGROUND AND OBJECTIVES: Immune thrombocytopenic purpura (ITP) is a common hematologic disorder, two forms of which occur in children. The detection of circulating platelet antibodies is helpful in diagnosis. MATERIALS AND METHODS: We evaluated three different immunological methods for detecting platelet antibodies in the serum of children with ITP. These were: a solid-phase red-cell adherence test (SPRCA), an enzyme immunoassay (EIA), and an immunofluorescence test (PSIF). RESULTS: The sensitivity of the methods in detecting IgG antibodies ranged from 28.1 (EIA) to 39.4% (SPRCA). We also looked for IgM antibodies by PSIF, thus raising the sensitivity of this test from 32.0 to 40.0%. A combination of two tests (SPRCA and EIA) allowed us to detect 61.8% positive samples. By doing all three tests, we obtained 71.3% positive samples. Finally, we reached 73.5% by adding PSIF for IgM. We found a higher frequency of circulating antibodies in both acute and chronic ITP at onset than in clinical remission. There were a few positive sera in chronic ITP, but not in the acute form in remission. CONCLUSION: The individual tests each have a relatively low sensitivity, but the combination of all three increases the diagnostic effectiveness. The finding of platelet antibodies during remission may predict evolution toward a systemic autoimmune state.

[Platelet hyperaggregation induced by an antiplatelet monoclonal antibody in a female patient with essential thrombocythemia of childhood]. / Iperaggregazione piastrinica indotta da un anticorpo monoclonale anti-piastrine in una paziente con trombocitemia essenziale in età pediatrica.

Essential thrombocythemia (ET) is a chronic myeloproliferative disease, rarely observed in pediatric age, characterized by a persistently increased platelet count. Abnormalities of platelet function observed in ET patients may be, at least in part, responsible for the thrombohemorrhagic complications. The authors report about a pediatric patient affected by ET, showing an abnormal platelet response following stimulation by anti-platelet monoclonal antibody. Such finding may be attributable to a structural abnormality of the platelet fibrinogen receptor or to post-receptor alterations.

Quantification of theophylline in human plasma by reversed-phase ion-interaction high-performance liquid chromatography and comparison with the TDx fluorescence polarization immunoassay procedure.

A new method for the determination of theophylline (1,3-dimethyl-1H-purine-2,6-dione) in human plasma is described, free from interference by theobromine (3,7-dimethyl-1H-purine-2,6-dione) and caffeine (1,3,7-trimethyl-1H-purine-2,6-dione). The method makes use of ion-interaction reversed-phase high-performance liquid chromatography (octylamine-orthophosphate being the interaction reagent and a C18 reversed-phase column the stationary phase) with spectrophotometric detection at 274 nm. The quantitative results obtained in the analysis of samples of plasma from patients undergoing treatment with theophylline were compared with those obtained for the same samples with the TDx fluorescence polarization immunoassay procedure (using the Abbot Therapeutic Drug Monitoring system), which is generally employed in hospitals and clinical laboratories. Statistical F-test and t-test for multiple samples were applied to the data obtained by the two methods. The results showed no significant difference between the two methods at the 95% confidence level.

Phenotypic variability and abnormal type I collagen unstable at body temperature in a family with mild dominant osteogenesis imperfecta.

Autosomal dominant inheritance of a mild form of osteogenesis imperfecta (osteogenesis imperfecta type I) with different phenotypic expression was found in a family. Phenotypic expression was different for the affected mother and son, in the presence of the same biochemical results. Dermal fibroblast cultures synthesized normal and mutant type I collagen alpha chains. Collagen heterotrimers containing abnormal chains were overmodified along the entire triple helical domain and showed an unusually low denaturation temperature, so far found only in lethal cases. The mild phenotype in the family is probably due to the fact that abnormal type I collagen molecules are more likely to be degraded than utilized in the extracellular matrix.