Stuart A. Bentley

Long-term engraftment failure after marrow ablation and autologous hematopoietic reconstitution : differences between peripheral blood stem cell and bone marrow recipients

We infused peripheral blood stem cells (PBSC) into 51 patients with various malignant disorders, after myeloablative conditioning. Twenty-four patients also received autologous bone marrow (PBSC + BM). In a multivariate analysis, the only statistically significant predictors of neutrophil engraftment were log-dose CFU-GM (P < 0.001) and the number of prior chemotherapy regimens (P = 0.004). The factors predicting RBC and platelet engraftment were log-dose CFU-GM (P = 0.002), PBSC + BM infusion (P = 0.007) and the absence of neoplastic bone marrow involvement (P = 0.009). Seven patients remained platelet and/or red cell transfusion-dependent for 100 days or more post-transplant after good neutrophil recovery. Six of these seven long-term engraftment failures, as well as five additional patients, received <105 CFU-GM/kg. Of the 11 patients who received <105 CFU-GM/kg (low-dose patients), seven were PBSC recipients, of whom six were long-term engraftment failures. In contrast, there were no long-term engraftment failures among the four low-dose autologous marrow recipients. This difference in long-term engraftment failure rate was significant (P = 0.015). The low-dose PBSC patients all had a diagnosis of lymphoma with bone marrow involvement. The low-dose PBSC + BM group was more heterogeneous, but no patient had malignant involvement of the marrow. The low-dose PBSC patients had also received significantly more prior chemotherapy regimens than the low-dose PBSC + BM patients and a significantly higher proportion received total body irradiation (TBI) as part of their conditioning regimen. We conclude that marrow damage resulting from a combination of neoplastic infiltration, chemotherapy and TBI may result not only in low PBSC yields but also in an impaired capacity of the marrow microenvironment to support transplanted stem cells.

An Expanded Phase I/II Trial of Cyclophosphamide, Etoposide, and Carboplatin Plus Total Body Irradiation with Autologous Marrow or Stem Cell Support for Patients with Hematologic Malignancies

The major cause for failure of autologous stem cell transplantation for hematologic malignancies is the risk of recurrent disease. As a result, new treatment regimens that include novel agents or combinations of agents and approaches are needed. The current report describes a large Phase I/II, single-center trial that includes 60 patients with a variety of hematologic malignancies. These patients received a fixed dose of carboplatin (1 g/m(2)/d x 72 hours by CI) etoposide (600 mg/m(2)/d x 3 days) and cyclophosphamide (2 g/m(2)/d x 3 days), plus escalating doses of total body irradiation (TBI) (at 1000, 1200, and 1295 cGy) over 3 days. Eleven patients received infusion of autologous marrow, 32 received peripheral blood stem cells, and 17 patients received both. The maximum tolerated dose of this regimen was a radiation dose of 1200 cGy given in 200-cGy fractions BID x 3 days. The dose-limiting toxicity was mucositis, with 97% of patients requiring narcotic analgesia for mouth pain. Overall treatment-related mortality was 6.7%, with 2 of the 4 deaths occurring in a group of 9 patients aged 60 and older. Responses were seen in all patient groups, but the most encouraging outcomes were seen in 12 patients with high-risk or advanced acute myelocytic lymphoma (AML), 7 of whom remain alive and free of disease beyond 5 years. This regimen is intensive and causes considerable mucositis but is otherwise well tolerated and has demonstrated activity in a number of hematologic malignancies, especially AML.

Biosynthesis of Proteochondroitin Sulfate by HL-60 Human Promyelocytic Cells

Human promyelocytic cells (HL‐60) were labeled with 35S‐sulfate and either 3H‐glucosamine or 3H‐serine as precursors. Accumulation of 35S‐labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/105 viable cells reached a plateau level after 24 h. Virtually none of the cell‐associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE‐Sephacel chromatography, isopyknic CsCI gradient centrifugation, and gel filtration chromatography. HL‐60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL‐4B and Kav = 0.32 on Sepharose CL‐6B, recovered primarily from the high‐density fractions of a dissociative CsCI gradient (ρ > 1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr = 14.5 kD, yielding virtually 100% 4‐sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL‐60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in 35S‐sulfate incorporation to 45% of control values or 32%, expressed as activity/105 cells. Proteoglycans synthesized by DMSO‐treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.

A cost‐effective and Food and Drug Administration‐approved alternative to tissue culture media in cryopreservation

Overman JR, Lewis AM Jr. Effect of high pressures on influenza viruses. Roc Soc Exp Biol Med 1959;102:329-33. Silva JL, Luan P, Glaser M, et al. Effects of hydrostatic pressure on a membrane-enveloped virus: high immunogenicity of the pressure-inactivated virus. J Virol 1992;66:2111-7. Nakagami T, ShigehisaT, Ohmori T, et al. Inactivation of herpes viruses by high hydrostatic pressure. J Virol Methods