Lawrence M. Souza

Effect of granulocyte colony-stimulating factor on neutropenia and associated morbidity due to chemotherapy for transitional-cell carcinoma of the urothelium

We evaluated the ability of human recombinant granulocyte colony-stimulating factor (rhG-CSF) to prevent chemotherapy-induced neutropenia or to accelerate recovery from this complication and thus allow patients to receive full doses of antineoplastic agents on time, according to protocol design. Twenty-seven patients with transitional-cell carcinoma of the urothelium who were undergoing treatment with methotrexate, doxorubicin, vinblastine, and cisplatin were given rhG-CSF (up to 60 micrograms per kilogram of body weight per day) before their first cycle of combination chemotherapy, during the first cycle, or at both points. Treatment with rhG-CSF before chemotherapy resulted in a dose-dependent increase in the absolute neutrophil count. Treatment with rhG-CSF after chemotherapy significantly reduced the number of days (91 percent) per patient on which the absolute neutrophil count was 1000 per microliter or less (P = 0.0039), reduced the number of days (1 vs. 35) on which antibiotics were used to treat fever and neutropenia, and significantly increased the percentage (100 vs. 29 percent) of patients qualified to receive planned chemotherapy on day 14 of the treatment cycle (P = 0.0015). In addition, the incidence of mucositis was significantly decreased (11 vs. 44 percent, P = 0.041), as was its severity. These findings demonstrate that rhG-CSF is a potent stimulus of normal neutrophil proliferation and maturation. In addition, its administration can reduce both the hematopoietic and oral toxicity of chemotherapy.

Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide

Recombinant human granulocyte colony-stimulating factor (G-CSF) by itself was not an effective stimulus for inducing the release of superoxide (O-2) in human granulocytes. However, G-CSF was able to prime human granulocytes, and enhanced O-2 release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). The preincubation with G-CSF for 5-10 min at 37 degrees C was sufficient for priming the cells. The optimal enhancing effect was obtained at 25 ng/ml of G-CSF. The enhancement of O-2 release by G-CSF was observed over the complete range of effective concentrations of FMLP (10(-8)-10(-6) M). These findings indicate that G-CSF is a potent activator of mature granulocyte functions.

Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor.

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.

Effects of recombinant growth factors on radiation survival of human bone marrow progenitor cells

The purpose of this study was to evaluate the individual radioprotective effects of 4 distinct purified recombinant human hematopoietic growth factors, namely recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF), recombinant human granulocyte colony stimulating factor (rG-CSF), recombinant human interleukin 1 (rIL-1), and recombinant human interleukin 2 (rIL-2) on human myeloid (CFU-GM) and erythroid (BFU-E) bone marrow progenitor cells. We demonstrate that (a) preconditioning with rGM-CSF, rG-CSF, or rIL-1 enables CFU-GM to repair sublethal radiation damage and renders CFU-GM less radiosensitive, (b) preconditioning with rGM-CSF or rIL-1 enables BFU-E to repair sublethal radiation damage, and (c) preconditioning with rIL-2 does not increase the radiation survival of CFU-GM or BFU-E. The effects of recombinant growth factors, in particular rGM-CSF, on the radiation damage repair, radiosensitivity, and proliferative activity of bone marrow progenitor cells resulted in a substantial increase in the mean numbers of progenitor cell-derived hematopoietic colonies in irradiated marrow samples. The effects of rGM-CSF on the radiation response of CFU-GM and BFU-E, and the effects of rG-CSF as well as rIL-1 on the radiation response of CFU-GM did not appear to require the presence of T-cells/T-cell precursors, NK-cells, B-cells/B-cell precursors, monocytes, macrophages, MY8 antigen positive non-CFU-GM myeloblasts, promyelocytes, myelocytes, metamyelocytes, granulocytes, or glycophorin A positive erythroid cells since virtually identical results were obtained with unsorted marrow samples or highly purified fluorescence activated cell sorter (FACS) isolated progenitor cell suspensions. To our knowledge, this report represents the first study on recombinant human growth factor-induced modulation of the radiation responses of normal human bone marrow progenitor cells.

Growth acceleration of coho salmon (Oncorhynchus kisutch) following oral administration of recombinant bovine somatotropin

Abstract The growth response of juvenile coho salmon to injected or orally administered recombinant bovine somatotropin (rbst) was examined; rbst was delivered orally either alone or with an antacid and putative penetration enhancer. Oral delivery of 12.5 μg rbst/g body wt per week resulted in accelerated growth in weight, over a 7-week period, when compared to three control treatments. Coadministration of the same dose of rbst with antacid and a penetration enhancer provided enhanced weight gain when compared to unprotected orally delivered rbst, and the growth matched that of fish injected with 2.5 μg rbst/g body wt per week during the study.

Recombinant bovine somatotropin more than doubles the growth rate of coho salmon (Oncorhynchus kisutch) acclimated to seawater and ambient winter conditions

Abstract Sub-adult coho salmon ( Oncorhynchus kisutch ) were tested in a culture situation to assess whether somatotropin therapy would improve growth performance during their first “sea-winter”. Untreated fish and sham-treated fish did not differ significantly in their growth performance. Over the 12-week course of the experiment, the mean increase in fork length for these groups was 9.5% (±6.0 SD) while the mean increase in weight was 29.6% (±24.1 SD). Relative to these control groups, the administration of recombinant bovine somatotropin by injection, cholesterol pellet or mini-osmotic pump resulted in substantially better growth. Mean increases in fork length ranged from 16.5% (±5.5 SD) to 23.0% (±8.7 SD) while mean increases in weight ranged from 52.2% (±31.2 SD) to 65.5% (±44.0 SD). Specific growth rate was elevated 2–4 weeks after the initiation of somatotropin therapy and the effect did not diminish over the 8-week treatment period. Subsequent to hormone withdrawal, the growth rates of somatotropin-treated fish were no longer distinguishble from those of the controls. Somatotropin was noted to decrease the rate of growth depensation, yielding a more uniform group of salmon with respect to size. Condition factor was decreased by the hormone treatment. It was concluded that somatotropin treatment can produce a significant increase in the growth rate of coho salmon, both after they have made the transition to salt water and during the winter months, when environmental conditions are sub-optimal for growth.

Experimental administration of recombinant bovine growth hormone to juvenile rainbow trout (Salmo gairdneri) by injection or by immersion

Abstract The ability of recombinant bovine growth hormone 21K (rbGH-21K) to accelerate the growth of juvenile rainbow trout (Salmo gairdneri) was investigated, and the potential of bath immersion as a method of administration of growth hormone was assessed. Injection of 10 μg/g rbGH-21K every 2 weeks for 8 weeks resulted in a highly significant increase in the rate of growth in both weight and length as well as an increase in condition factor. Administration of rbGH-21K by immersion at a dosage of 100 mg/l for 4 h every 5 days resulted in a significant increase in specific growth rate relative to similarly treated controls during the 8-week treatment period, although the effect was minimal when compared to the injected group. Comparison of growth in immersed versus non-immersed controls indicated that the immersion protocol itself inhibited growth. Modifications to the immersion protocol which may improve its effectiveness are discussed.

Postirradiation treatment with granulocyte colony-stimulating factor and preirradiation WR-2721 administration synergize to enhance hemopoietic reconstitution and increase survival

These studies tested whether WR-2721 could be used to protect hemopoietic stem cells, which after irradiation could be stimulated by granulocyte colony-stimulating factor (G-CSF) to proliferate and reconstitute the hemopoietic system. Female C3H/HeN mice were administered WR-2721 (4 mg/mouse, i.p.) 30 min before 60Co irradiation and G-CSF (2.5 micrograms/mouse/day, s.c.) from days 1-16 after irradiation. In survival studies, saline, G-CSF, WR-2721, and WR-2721 + G-CSF treatments resulted in LD50/30 values of 7.85 Gy, 8.30 Gy, 11.30 Gy, and 12.85 Gy, respectively. At these LD50/30 values, the dose reduction factor (DRF) of 1.64 obtained in combination-treated mice was more than additive between the DRFs of G-CSF-treated mice (1.06) and WR-2721-treated mice (1.44). Bone marrow and splenic multipotent hemopoietic stem cell (CFU-s) and granulocyte-macrophage progenitor cell (GM-CFC) recoveries were also accelerated most in mice treated with WR-2721 + G-CSF. In addition, mice treated with WR-2721 + G-CSF exhibited the most accelerated peripheral blood white cell, platelet, and red cell recoveries. These studies (a) demonstrate that therapeutically administered G-CSF accelerates hemopoietic reconstitution from WR-2721-protected stem and progenitor cells, increasing the survival-enhancing effects of WR-2721 and (b) suggest that classic radioprotectants and recombinant hemopoietic growth factors can be used in combination to reduce risks associated with myelosuppression induced by radiation or radiomimetic drugs.

T-cell activation defect in common variable immunodeficiency: restoration by phorbol myristate acetate (PMA) or allogeneic macrophages.

Common variable immunodeficiency (CVI) represents a group of familial and sporadic diseases characterized by a range of B-cell, T-cell, and macrophage defects. A defect in T-cell activation, involving reduced proliferation and IL-2 production after stimulation with OKT3 antibody, has been described previously. In the present study we found that these defects could be corrected in vitro by adding phorbol myristate acetate (PMA) to OKT3-stimulated peripheral blood mononuclear cells (PBMC) of 14 patients with CVI. PBMC of 6 out of 7 patients with CVI studied also exhibited a profound defect in IL-2 receptor expression when incubated with OKT3 antibody. IL-2 receptor expression after stimulation with PMA alone was normal, indicating that the OKT3- but not the PMA-induced pathway of IL-2 receptor expression was defective. On the RNA level, the genes for IL-2 and IL-2 receptor were expressed after stimulation with OKT3 antibody. IL-2 and IL-2 receptor gene expression were normal, indicating a possible post-transcriptional defect. To investigate whether the defect in T-cell activation was at the macrophage or the T-cell level, we prepared adherent cells and monocyte-depleted T cells (E+) from 3 patients with CVI and from normal blood donors. Incubating CVI E+ cells with normal adherent cells resulted in normal proliferation and IL-2 production in the presence of OKT3, whereas incubation of normal E+ cells with adherent cells from patients with CVI under the same conditions showed reduced IL-2 production and proliferation, suggesting the macrophage as the origin of the failure in T-cell activation in the patients with CVI studied. Inhibition by macrophage-secreted prostaglandins was excluded by failure to correct the IL-2 production and proliferation defects in the presence of indomethacin.

Biological Activities of Recombinant Human Granulocyte Colony Stimulating Factor (rhG-CSF) and Tumor Necrosis Factor: In Vivo and In Vitro Analysis

Within the family of hematopoietic growth factors, murine G-CSF was first recognised as acting predominantly upon mature neutrophils and neutrophil progenitors, stimulating murine neutrophil colonies characterized by small size, maturity, and relative in-frequency [1, 2]. G-CSF is synthesized in many murine tissues and organs, possibly by common populations of macrophages or endothelial cells [3], and the factor has been purified from the lung tissue of endotoxin-treated mice as a 24000–25000 M r glycoprotein [4]. In contrast to GM-CSF and interleukin-3 (IL-3), G-CSF exerts a potent differentiation-inducing action on myeloid leukemic cells such as the WEHI-3 myelomonocytic leukemic cell line [4–6] and the M1 myeloblastic leukemic line [7].