Christopher B. Brown

High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector

An expression cassette for continuous high-level expression of secreted glycoproteins by transformed lepidopteran insect cells has been developed as an alternative to baculovirus and mammalian cell expression systems. The expression cassette utilizes the promoter of the silkmoth cytoplasmic actin gene to drive expression from foreign gene sequences, and also contains the ie-1 transactivator gene and the HR3 enhancer region of BmNPV to stimulate gene expression. Using an antibiotic-resistance selection scheme, we have cloned a Bm5 (silkmoth) cell line overexpressing the secreted glycoprotein juvenile hormone esterase (JHE-KK) at levels of 190 mg/L in batch suspension cultures. A baculovirus (AcNPV) expressing the same gene under the control of the p10 promoter of AcNPV produced only 4 mg/L active JHE in static cultures of infected Sf21 cells. A cloned Bm5 cell line overexpressing a soluble isoform of the alpha-subunit of the granulocyte-macrophage colony stimulating factor receptor (solGMRalpha) was also generated and produced five times more solGMRalpha in static cultures than a cloned BHK cell line obtained by transformation with a recombinant expression cassette utilizing the human cytomegalovirus (CMV) enhancer-promoter system. Finally, we show that recombinant protein expression levels in transformed Bm5 cells remain high in serum-free media, that expression is stable even in the absence of antibiotic selection, and that lepidopteran cells other than Bm5 may be used equally efficiently with this new expression cassette for producing recombinant proteins.

High Busulfan Exposure Is Associated with Worse Outcomes in a Daily i.v. Busulfan and Fludarabine Allogeneic Transplant Regimen

Low plasma busulfan (Bu) area under the concentration-time curve (AUC) is associated with graft failure and relapsed leukemias, and high AUC with toxicities when Bu is used orally or i.v. 4 times daily combined with cyclophosphamide in myeloablative hematopoietic stem cell transplantation (SCT) conditioning regimens. We report Bu AUC and its association with clinical outcomes in 130 patients with hematologic malignancies given a once-daily i.v. Bu (3.2 mg/kg days -5 to -2) and fludarabine (Flu, 50 mg/m(2) days -6 to -2) regimen. Total-body irradiation (TBI) 200 cGy x 2 was added for 51 patients with acute leukemias. Plasma AUC varied 3.6-fold (2184-7794 microM.min, median 4699 microM.min). Patients with an AUC >6000 microM.min had lower overall survival (OS) than those with AUC < or =6000 microM.min at 12 months (38% versus 74%) and 36 months (23% versus 68%, P < .001). This effect was apparent in patients with standard-risk and high-risk disease, and persisted when potential confounders were considered (hazard ratio 3.2, 95% confidence interval 1.7-6.3). Nonrelapse mortality (NRM) at 100 days (6% versus 19%) and progression free survival (PFS; 58% versus 16%) at 3 years were better with AUC < or =6000 microM.min. These data support a role for therapeutic dose monitoring and dose adjustment with daily i.v. busulfan.

Lipopolysaccharide-stimulated or granulocyte-macrophage colony-stimulating factor-stimulated monocytes rapidly express biologically active IL-15 on their cell surface independent of new protein synthesis.

Although IL-15 shares many of the biological activities of IL-2, IL-2 expression is primarily under transcriptional regulation, while the mechanisms involved in the regulation of IL-15 are complex and not completely understood. In the current study, we found that CD14+ monocytes constitutively exhibit both IL-15 mRNA and protein. IL-15 protein was found stored intracellularly and stimulation of CD14+ monocytes with either LPS or GM-CSF resulted in mobilization of IL-15 stores to the plasma membrane. This rapidly induced surface expression was the result of a translocation of preformed stores, confirming that posttranslational regulatory stages limit IL-15, because it was not accompanied by an increase in IL-15 mRNA and occurred independent of de novo protein synthesis. After fixation, activated monocytes, but not resting monocytes, were found to support T cell proliferation, and this effect was abrogated by the addition of an IL-15-neutralizing Ab. The presence of preformed IL-15 stores and the ability of stimulated monocytes to mobilize these stores to their surface in an active form is a novel mechanism of regulation for IL-15.

Unrelated donor BMT recipients given pretransplant low-dose antithymocyte globulin have outcomes equivalent to matched sibling BMT: A matched pair analysis

Fifty-seven patients receiving unrelated donor (UD) BMT were matched for disease and stage with 57 recipients of genotypically matched related donor (MRD) BMT. All UD recipients were matched serologically for A and B and by high resolution for DR and DQ antigens. All patients received CsA and ‘short course’ methotrexate with folinic acid. Unrelated donor BMT patients also received thymoglobulin 4.5 mg/kg (6 mg/kg if <30 kg) in divided doses over 3 days pretransplant. For UD and RD BMT, respectively, incidence of acute GVHD grade II–IV was 19 ± 6% vs 36 ± 8%, grade III–IV 10 ± 6% vs 18 ± 7%, chronic GVHD 44 ± 8% vs 51 ± 8%, non-relapse mortality 15 ± 5% vs 8 ± 4% at 100 days, 28 ± 8% vs 36 ± 7% at 3 years. At 3 years, relapse was 45 ± 7% vs 42 ± 7%, and disease-free survival 39 ± 7% vs 37 ± 7%. None of these differences are significant. Three-year overall survival was identical at 42 ± 7%. For 29 patients with low/intermediate risk leukemia, disease-free survival was 68 ± 10% after UD BMT vs 59 ± 9% for RD BMT recipients (P = NS). Corresponding figures for high risk patients were 14 ± 7% and 21 ± 8%, respectively. We conclude that UD BMT recipients matched as above and given pretransplant ATG have similar outcomes to recipients of MRD BMT using conventional drug prophylaxis. Unrelated donor BMT should be considered in any circumstance where MRD BMT is routine.

Early outcomes after allogeneic stem cell transplantation for leukemia and myelodysplasia without protective isolation: A 10-year experience

Although it is common practice to use some form of isolation to protect allogeneic stem cell transplant patients from infection, the necessity for these practices in all environments has not been demonstrated. The current study evaluated patterns of infection and 100-day transplant-related mortality in 288 patients with myelodysplasia and leukemia transplanted without isolation. Patients were allowed out of hospital at any time within constraints of the medication schedule. Fever, foci of infection, and positive cultures within 28 days and death within 100 days because of the transplant procedure were recorded. Fever occurred in 57% of patients, and 10% had a clinical or radiographic focus of infection. Most infections were apparently endogenous; blood cultures from 24% of recipients grew organisms, 87% of which were gram-positive bacteria. Four patients (1%) died with aspergillus infection in circumstances indicating that isolation would not have been helpful. Twenty percent of patients remained without evidence of infection throughout. Transplant-related mortality at 100 days was 1% for 108 patients with early leukemia receiving transplants from matched siblings. For patients at higher risk, by virtue of donor and/or disease status, mortality was 21%. These figures compare favorably with those reported to the International Bone Marrow Transplant Registry, the majority of patients having been subjected to some form of isolation. We conclude that allogeneic stem cell transplantation can be safely performed in some environments without confining patients continuously to the hospital.

B lymphocytes inhibit human osteoclastogenesis by secretion of TGFβ

The role of B lymphocytes in osteoclast (OC) formation is controversial, because both stimulatory and inhibitory effects of B‐lineage cells on osteoclastogenesis and life span have been reported. In this study, we have investigated the effects of mature B cells on human osteoclastogenesis using cultures of peripheral blood stem cells (PBSC), a system that generates functional OCs in the absence of stromal cells. We report that B cells inhibit the formation of OCs and shorten the life span of mature OCs by secreting transforming growth factor β (TGFβ), a factor that induces apoptosis in these cells. The antiosteoclastogenic effects of B cells are abolished by addition of anti‐TGFβ antibody to osteoclast cultures and mimicked by treatment of B cell‐deprived PBSC cultures with recombinant TGFβ, thus confirming TGFβ as the B cell produced antiosteoclastogenic activity. Thus, the ability of B cells to downregulate osteoclastogenesis by secretion of the apoptotic cytokine TGFβ provides new insights into the ability of immune cells to regulate OC formation under basal and inflammatory conditions. J. Cell. Biochem. 78:318–324, 2000.

Allogeneic blood stem cell and bone marrow transplantation for acute myelogenous leukemia and myelodysplasia: influence of stem cell source on outcome.

We have compared the outcomes of 87 patients with acute myelogenous leukemia (AML) and myelodysplasia (MDS) receiving matched sibling transplants with stem cells from peripheral blood (blood cell transplant, BCT) or bone marrow (BMT). In good risk patients (AML in CR1) granulocytes recovered to 0.5 × 109/l a median of 14 days after BCT compared with 19 days after BMT (P < 0.0001). for patients with poor risk disease (aml beyond cr1 and mds) corresponding figures were 16 vs 26 days (P < 0.0001). platelet recovery to 20 × 109/l was also faster after BCT (good risk 12 vs 20 days, P < 0.0001; poor risk 17 vs 22 days, P = 0.04). Red cell transfusions were unaffected by cell source, but BCT recipients required less platelet transfusions (good risk 1 vs 5, P = 0.002; poor risk 5 vs 11, P = 0.004). Blood cell transplants resulted in more chronic GVHD (86% vs 48%, P = 0.005) and a significantly higher proportion of recipients with KPS of 80% or less (48% vs 5%, P = 0.004). Disease-free survival at 4 years was 23% for both groups of poor risk patients but outcome in good risk patients was better after BCT (93% vs 62%, P = 0.047) related mainly to less relapse. While disease-free survival may be better after BCT than BMT for AML in CR1, quality of life may be relatively impaired.

Transplantation from Matched Siblings Using Once-Daily Intravenous Busulfan/Fludarabine with Thymoglobulin: A Myeloablative Regimen with Low Nonrelapse Mortality in All But Older Patients with High-Risk Disease

Two hundred patients received hematopoietic stem cell transplantation (HSCT) from matched sibling donors (MSD) after myeloablative conditioning including fludarabine (Flu) and once-daily intravenous busulfan (Bu). Thymoglobulin (TG) was added to methotexate (MTX) and cyclosporine (CsA) as graft-versus-host disease (GVHD) prophylaxis. For low-risk (acute leukemia CR1/CR2, CML CP1) patients projected 5-year nonrelapse mortality (NRM) and overall survival (OS) were 4% and 76% for those <or=45 years old (n = 54) and 6% and 83% for those >45 (n = 31). For high-risk (HR) patients NRM was 6% versus 27% (18% at 1 year) (P = .04) and OS 64% versus 37% (P = .47) in younger (n = 40) and older (n = 75) patients, respectively. To correct for imbalance in HR diagnoses each of 17 younger HR patients were matched with 2 older HR (OHR) patients by diagnosis and details of stage, and thereafter for other risk factors. For the younger HR and OHR patients, respectively, OS was 70% versus 37% (P = .02) and NRM 0 versus 34% (P = .02). When outcomes of OHR patients were compared with the other 3 groups combined NRM was 27% versus 5%, respectively (P = .002). Incidence of acute graft-versus-host disease (aGVHD) grade II-IV, aGVHD grade III-IV, and chronic GVHD (cGVHD) was 23% versus 10% (P = .02), 4% versus 2% (P = ns), and 66% versus 41% (P = .001), respectively. Nine of 14 nonrelapse deaths in the OHR group were related to GVHD or its treatment compared with 3 of 6 in all others (P value for GVHD related death = .01). Multivariate analysis of OS and DFS correcting for potentially confounding pretransplant factors identified only the OHR patients as having significantly increased risk (relative risk [RR] 3.32, confidence interval [CI] 1.71-6.47, P < .0001, and RR 3.32, CI 1.71-6.43, P < .0001, respectively). The effect of age on NRM is only apparent in HR patients, and is not explained by heterogeneity in diagnoses. Older HR patients experience more GVHD and more GVHD-related death than others, but NRM is no higher than reported with many nonmyeloablative regimens.

Cholesterol depletion inhibits src family kinase‐dependent calcium mobilization and apoptosis induced by rituximab crosslinking

The monoclonal antibody (mAb) rituximab produces objective clinical responses in patients with B‐cell non‐Hodgkins lymphoma and antibody‐based autoimmune diseases. Mechanisms mediating B‐cell depletion by rituximab are not completely understood and may include direct effects of signalling via the target antigen CD20. Like most but not all CD20 mAbs, rituximab induces a sharp change in the solubility of the CD20 protein in the non‐ionic detergent Triton‐X‐100, reflecting a dramatic increase in the innate affinity of CD20 for membrane raft signalling domains. Apoptosis induced by rituximab hypercrosslinking has been shown to require src family kinases (SFK), which are enriched in rafts. In this report we provide experimental evidence that SFK‐dependent apoptotic signals induced by rituximab are raft dependent. Cholesterol depletion prevented the association of hypercrosslinked CD20 with detergent‐insoluble rafts, and attenuated both calcium mobilization and apoptosis induced with rituximab. CD20 cocapped with the raft‐associated transmembrane adaptor LAB/NTAL after hypercrosslinking with CD20 mAbs, regardless of their ability to induce a change in the affinity of CD20 for rafts. Taken together, the data demonstrate that CD20 hypercrosslinking via rituximab activates SFKs and downstream signalling events by clustering membrane rafts in which antibody‐bound CD20 is localized in a high‐affinity configuration.

The Addition of 400 cGY Total Body Irradiation to a Regimen Incorporating Once-Daily Intravenous Busulfan, Fludarabine, and Antithymocyte Globulin Reduces Relapse Without Affecting Nonrelapse Mortality in Acute Myelogenous Leukemia

A combination of fludarabine (Flu) and daily i.v. busulfan (Bu) is well tolerated and effective in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia (AML). The addition of rabbit antithymocyte globulin (ATG) may reduce morbidity and mortality from graft-versus-host disease (GVHD), but lead to increased relapse. To compensate for this effect, we added 400 cGy of total body irradiation (TBI) to the Flu/Bu regimen in 89 patients, and compared outcomes with those achieved in 90 patients who received the drug combination alone. Although nonrelapse mortality (NRM) at 3 years did not differ between the groups, the inclusion of TBI significantly reduced relapse (hazard ratio [HR] = 0.29; 95% confidence interval [CI] = 0.15-0.54; P = .0001). Consequently, both overall survival (OS; HR = 0.50; 95% CI = 0.3-0.84; P = .009) and disease-free survival (DFS; HR = 0.43; 95% CI = 0.26-0.72; P = .001) were improved with the inclusion of TBI. This study confirms the importance of regimen intensity in allogeneic HSCT for AML. The combination of daily i.v. Bu, Flu, 400 cGy TBI, and ATG provides a well-tolerated regimen with antileukemic activity in AML comparable to that of other, conventional myeloablative (MA) regimens.