Efficient marking of human cells with rapid but transient repopulating activity in autografted recipients.

Short-term hematopoietic reconstituting cells have been identified in mice, nonhuman primates, and among human cells that engraft xenogeneic hosts. We now present clonal marking data demonstrating a rapid but unsustained contribution of cultured human autografts to the initial phase of hematologic recovery in myeloablated patients. Three patients received transplants of granulocyte colony-stimulating factor-mobilized autologous peripheral blood (PB) cells, of which a portion (8%-25% of the CD34+ cells) had been incubated in vitro with growth factors (5 days) and clinical grade LN retrovirus (3-5 days). More than 9% of the clonogenic and long-term culture-initiating cells harvested were transduced. Semiquantitative and linear amplification-mediated polymerase chain reaction analyses of serial PB samples showed that marked white blood cells appeared in all 3 patients within 11 days and transiently constituted up to 0.1% to 1% of those produced in the first month. However, within another 2 to 9 months, marked cells had permanently decreased to very low levels. Analysis of more than 50 vector insertion sites showed none of the clones detected in the first month were active later. Eighty percent of inserts were located within or near genes, 2 near CXCR4. These findings provide direct evidence of cells with rapid but transient repopulating activity in patients and demonstrate their efficient transduction in vitro.

Evidence of similar effects of short-term culture on the initial repopulating activity of mobilized peripheral blood transplants assessed in NOD/SCID-beta2microglobulin(null) mice and in autografted patients.

OBJECTIVE: Human mobilized peripheral blood (mPB) is known to contain high numbers of cells with rapid but short-term repopulating activity in NOD/SCID-beta2microglobulin(-/-) mice. Here we assessed the effect of short-term culture on these cells and compared the levels of retained activity with the pace of hematologic recovery in myeloablated patients transplanted with similarly cultured autografts of the same cells. PATIENTS AND METHODS: In a phase 1 clinical study, mPB cells were collected from 6 advanced cancer patients. CD34(+) cells were then harvested, cultured for 3 days in the presence of early-acting growth factors, and transplanted, and posttransplant recovery of blood cell parameters monitored. Assays for primitive hematopoietic activity using both in vivo (in NOD/SCID-beta2microglobulin(-/-) mice) and in vitro (CFC and LTC-IC) endpoints were also performed on the cells pre- and posttransplant. RESULTS: All patients showed event-free, timely leukocyte recoveries but slightly delayed platelet recoveries in some cases. During the 3-day period of culture, the CFCs doubled but the LTC-IC activity decreased (twofold), as did the short-term repopulating activity in NOD/SCID-beta2microglobulin(-/-) mice. CONCLUSION: Patients can be transplanted with 3-day cultured autografts with minimal effects on hematologic recovery. This is associated with a variable but, on average, modest loss of short-term repopulating activity detectable in NOD/SCID-beta2microglobulin(-/-) mice.

Comparative neurotoxicity of weekly non-break paclitaxel infusions over 1 versus 3 h.

We evaluated the effects of weekly short infusions of paclitaxel (PAC) on the development of a peripheral neuropathy (PNP) as primary endpoint. Patients with advanced cancer were randomized to a weekly regimen of PAC (100 mg/m2) infused over 1 versus 3 h. PNP was evaluated by a clinical score including sensory symptoms, strength, tendon reflexes and vibratory sense (range 0-12; PNP >3 points). Kaplan-Meier-type curves were calculated. In total, 22 study centers enrolled 121 patients, 92 assessable for analysis. The probability to exceed a PNP score of 3 increased from 0.20 versus 0.30 after six to 0.68 versus 0.47 after 12 administrations (1 versus 3 h: p = 0.66). After 12 weeks of therapy only a quarter of assessable patients were free of PNP. Cox analysis yielded a relative risk of 1.10 for 1-h infusions (p = 0.80). We observed a rapid increasing risk of PNP manifestation in the course of weekly PAC administrations without significant differences between 1- and 3-h infusions. This is in contrast to pharmacokinetic observations indicating that a shortening of infusion time might enhance neurotoxicity by increasing the AUC of Cremophor. A majority of patients experiencing neurotoxic effects require the investigation of potential nerve protectors in future clinical trials accompanying PAC therapy.

Telomere maintenance in human B lymphocytes.

Telomere shortening has been causally linked to replicative senescence in human cells. To characterize telomere-length heterogeneity in peripheral blood cells of normal individuals, we analysed the mean length of telomeric repeat sequences in subpopulations of peripheral blood leucocytes, using fluorescence in situ hybridization and flow cytometry (flow-FISH). Although the telomere length of most haematopoietic subsets was within the same range, the mean telomere length was found to be 15% higher in B compared with T lymphocytes in adult peripheral blood. Whereas telomere loss with ageing corresponded to 33 base pairs (bp) per year in T cells, telomere shortening was slower in B cells, corresponding to 15 bp per year. Separation of adult B-lymphocyte subpopulations based on CD27 expression revealed that telomere length was almost 2 kb longer in CD19+CD27+ (memory) compared with CD19+CD27- (naive) cells. Furthermore, peripheral blood B cells were activated in vitro. Whereas B-cell activation with Staphylococcus aureus Cowan strain (SAC) did not increase telomere length, a striking telomere elongation was observed when cells were stimulated with SAC and interleukin 2 to induce plasma cell differentiation. Our observations support the concept that telomere dynamics in B cells are distinct from other haematopoietic cell lineages and that telomere elongation may play an essential role in the generation of long-term B memory cells.