Recombinant Granulocyte-macrophage Colony Research Articles

Recombinant granulocyte macrophage colony stimulating factor (rhu-GM-CSF) in the treatment of extensive leg ulcers: A case report

Surgery 2000;127:589–92.

Characterization of a bioassay for detection of recombinant and native ovine interleukin-5.

In order to analyse Th2-type immune responses in sheep by the assay of interleukin (IL)-5 in biological fluids, the ovine IL-5 gene was cloned and expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression vector system. The recombinant product was purified as BAC-OV-IL-5 from the supernatant fluid. The ovine IL-5 was biologically active in a bioassay using IL-5-dependent Baf cells, which have been used previously to specifically detect human IL-5. The specificity of Baf cells for ovine IL-5 was examined by two methods. First, Baf cells only proliferated in response to BAC-OV-IL-5 and did not respond to addition of recombinant ovine cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), IL-1beta, IL-2, IL-3, IL-6, IL-8, stem cell factor (SCF) or IFN-gamma at doses from 0.01 to 1 microg/well. Second, the rat monoclonal antibody to murine IL-5, TRFK-5, neutralized murine, but not ovine, IL-5. However, rabbit antisera to BAC-OV-IL-5 neutralized murine and ovine recombinant IL-5 and abolished responses of Baf cells to IL-5 activity in supernatant fluids from mesenteric lymph node cells (MLNC) of parasitized sheep. The bioassay had a sensitivity to detect 8 ng in a 200 microL assay (40 ng/mL). Thus, the specificity of Baf cells to detect human IL-5 also extends to ovine IL-5 and therefore provides a method for monitoring the production of Th2 immune reactivity in sheep.

Marked suppression of T cells by a benzothiophene derivative in patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis.

In a search for new anti-autoimmune agents that selectively suppress activation of autoreactive T cells, one such agent, 5-methyl-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide (CI-959-A), was found to be effective. This compound, which is known to suppress tumor necrosis factor alpha (TNF-alpha)-induced CD54 expression, inhibited the primary proliferative response of the T cell to antigen (Ag)-presenting cells (APCs) including allogenic dendritic cells (DCs), autologous Epstein-Barr virus-infected B cells, and human T lymphotropic virus type I (HTLV-I)-infected T cells. Autoreactive T cells from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) spontaneously proliferate in vitro, and their activation is reported to be associated with CD54 expression. The spontaneous proliferation of T cells from patients with HAM/TSP was entirely blocked by CI-959-A. However, in this study, the T-cell proliferation in 15 patients with HAM/TSP was found to depend more extensively on major histocompatibility complex (MHC) class II and CD86 than on CD54 Ags. Since most important APCs for the development of HAM/TSP are DCs and HTLV-I-infected T cells, the effect of CI-959-A on DC generation and on the expression of surface molecules on activated T cells is examined. CI-959-A suppressed recombinant granulocyte-macrophage colony stimulating factor (GM-CSF)- and recombinant interleukin-4-dependent differentiation of DCs from monocytes and inhibited the expression of CD54 and, more extensively, MHC class II and CD86 Ags. CI-959-A showed little toxicity toward lymphoma or HTLV-I-infected T-cell lines or toward monocytes and cultured DCs. These results suggest that CI-959-A might be a potent anti-HAM/TSP agent.

Granulocyte-macrophage colony stimulating factor promotes prolonged survival and the support of virulent infection by African swine fever virus of macrophages generated from porcine bone marrow and blood.

Long-surviving cultures of non-adherent cells of the monocyte-macrophage lineage were established from the bone marrow and blood of weanling pigs by culturing cells from these tissues in the presence of recombinant porcine granulocyte-macrophage colony stimulating factor (GM-CSF). The cells increased in number, principally during the first 4 weeks of culture, bound monoclonal antibodies recognizing porcine macrophage antigens and avidly phagocytosed latex particles. The GM-CSF generated mononuclear phagocytes were highly infectible [correction of infectable] with a virulent Malawi isolate of African swine fever virus (ASFV) and able to generate levels of virus progeny similar to those produced by freshly isolated pig macrophages. The cultured cells retained their susceptibility to ASFV infection for as long as the cultures survived i.e. for up to 3 months.

247 A phase III trial of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) as corrective treatment in patients (PTS) with neutropenic fever following antineoplastic chemotherapy (CT): Results of an intermediate analysis

Fifty-one patients (pts) with neutropenic fever (fever ≥ 38.5° C at 3 hour-interval and an absolute neutrophil count (ANC) Patients were hospitalized and randomized, in the 12 hours following the beginning of fever, to receive either IV antibiotherapy (control group) or antibiotherapy and GM-CSF, given subcutaneously at a daily dose of 5 μg/kg, to be continued until 2 days after the recovery of ANC ≥ 1.109/1. A stratification was done, before randomization, according to chemotherapy level of hematologic toxicity [i.e: high (HCT) versus moderate (MCT)]. There were 19 male and 32 female, with a median age of 50.8 years old (18–79). Twenty-five pts had received HCT and 26 MCT. Results are summarized in the table below: Treatment arm GM-CSF Control group Wilcoxon's test(p) number of pts 26 25 median days with ANC 3(1–8) 4 (1–9) 0.06 median days with ANC 3 (1–10) 6 (2–12) median days with ANC 3 (1–18) 4 (1–6) 0.66 median days with ANC 4(1–10) 5 (2–12) 0.22 median days with ANC 2 (1–4) 4 (1–9) 0.04 median days with ANC 3 (1–5) 7 (2–10) median days with fever ≥ 38.5° 2.5 (1–7) 2 (1–12) 0.39 One pt, in the control group, died with infection. Side effects related to GM-CSF consisted in mild bone pain in 2 pts. In conclusion, GMCSF, given as corrective treatment, significantly reduced the duration of neutropenia (days with ANC

Oncogenic transformation and growth factor production of a human urothelial cell line

The objective of this study was to demonstrate that a nontumorigenic simian virus 40 (SV40) immortalized human urothelial cell line can be tumorigenically transformed following growth in a serum free, growth factor free, chemically defined culture system. The tumorigenicity of the cells was determined by the generation of tumors after inoculation into athymic nude mice. A cell line was derived from one of the tumors and the origin of the transformed cells was confirmed by immunohistochemical staining and analysis of the DNA fingerprint profile. Conditioned medium derived from the nontumorigenic parent cell line and the tumor line contained an activity that synergized with recombinant granulocytemacrophage colony stimulating factor (rmu GM-CSF) and recombinant macrophage colony stimulating factor (rhu M-CSF) to produce large colonies in bone marrow cultures (HPP-CFC). The concentration of human stem cell factor (SCF) and human Interleukin-I a-like activity produced by the urothelial cells was lower in the medium conditioned by the transformed tumorigenic cells than by the nontumorigenic parental cells. This data suggest that there are differences in the growth factor production of normal urothelial and tumor cells. It is possible that tumors deprived of normal levels of growth factors and nutrients may undergo further tumor progression, thus contributing to the heterogeneity seen in the clinic.

Interleukin-1 beta gene expression in human oral polymorphonuclear leukocytes.

Oral polymorphonuclear leukocytes (PMN) were obtained from 10 adult donors in good oral health using a method employing repeated mouth rinse collection. Interleukin-1 beta (IL-1 beta) mRNA was detected in freshly obtained cells by blot hybridization of total cellular RNA with a biotin labeled cDNA probe. Supernates from oral PMN placed in culture for 3 hours contained substantial amounts of IL-1 beta measured by ELISA. Significantly greater numbers of PMN and amounts of PMN-derived IL-1 beta were obtained from the same donors 2 hours subsequent to an oral sucrose challenge (3.23 x 10(6) vs. 1.57 x 10(6) mean PMN number, P = 0.004; 59.80 vs. 20.05 mean pg/ml IL-1 beta, P = 0.036, respectively). However, the elevated levels of IL-1 beta were due to the higher cell number rather than to increased production by individual cells. Stimulation of oral PMN with recombinant granulocyte-macrophage colony stimulating factor did not enhance their cytokine production. In most instances. IL-1 beta production by oral PMN was dramatically greater than that of their blood counterparts. These findings suggest that oral PMN are an important source of IL-1 beta, which plays a central role in oral immunity and inflammatory disease states.

Autologous peripheral blood progenitor cell transplantation.

Harvesting of autologous peripheral blood stem cells (PBSCs) has been facilitated by using currently available, efficient apheresis technology at the time of rebound from chemotherapy while patients are receiving recombinant growth factors, i.e., granulocyte (G) or granulocyte-macrophage (GM) colony stimulating factor (CSF). Ideally pheresis should be done before patients have had extensive stem cell toxins, i.e., alkylating agents or nitrosoureas. This strategy has facilitated the use of high dose chemoradiotherapy given as a single regimen or in a divided dose for patients with solid tumors or hematologic malignancies and results in more rapid engraftment than bone marrow transplantation (BMT). Although there are no assays which measure repopulating stem cells, enumeration of CD34+ cells within PBSCs is a direct and rapid assay which provides an index of both early and late long-term reconstitutive capacity, since it correlates with colony-forming unit (CFU)-GMs, as well as pre-progenitor or delta assays and long-term culture-initiating cells (LTC-IC). A threshold of > or = 2 x 10(6) CD34+ cells/kg recipient body weight has been reported to be required for engraftment, but may vary depending upon the clinical setting. Strategies for mobilization of normal PBSCs also increase tumor cell contamination within PB in the setting of both hematologic malignancies and solid tumors, but the significance of these tumor cells in terms of patient outcome is unclear. Recently isolation of CD34+ cells from PBSCs has been done using magnetic beads or immunoabsorption on columns or rigid plates in order to enrich for normal hematopoietic progenitors and potentially decrease tumor cell contamination. As for other cellular blood components, standards have been developed to assure efficient collection and processing, thawing, and reinfusion, and to maintain optimal PBPC viability. Finally, future directions of clinical research include expansion of hematopoietic progenitor cells ex vivo; use of umbilical cord or placenta as rich sources of progenitor cells; syngeneic hematopoietic stem cell transplantation; related and unrelated allogeneic hematopoietic stem cell transplantation; treatment of infections, i.e., Epstein Barr virus, or tumor relapse after allogeneic BMT using donor PBSC infusions; and gene therapy approaches.

Human recombinant granulocyte‐macrophage colony stimulating factor (hrGM‐CSF) improves double hemibody irradiation (DHBI) tolerance in patients with stage III multiple myeloma: A pilot study

Double hemibody irradiation (DHBI) is an alternative treatment of stage III multiple myeloma (MM) in patients aged over 55 years. Toxic side-effects such as myelosuppression are a severe limiting factor to its use. We performed DHBI associated with human recombinant granulocyte-macrophage colony stimulating factor (hrGM-CSF) as support therapy in 10 patients with stage III MM to improve the tolerance to this treatment. Ten patients received subcutaneously 5 micrograms/kg/d of hrGM-CSF during 2 weeks after each course of hemibody irradiation. All these patients had stage III MM: eight previously received chemotherapy, six of them were regarded as patients with refractory MM and two with relapse. Two patients received DHBI as first-line treatment. hrGM-CSF increased safety and tolerance of DHBI. GM-CSF support reduced the mean time between upper body irradiation (UBI) and lower body irradiation (LBI): 41 v 108 d in a cohort of 32 patients previously treated without growth factor support. Overall there was no lethal infection with hrGM-CSF or granulocytopenia (5.0 x 10(9)/l v 0.4 x 10(9)/l at day 15 in patients without growth factor). hrGM-CSF also reduced stomatitis grading and thrombocytopenia (90 x 10(9)/l v 45 x 10(9)/l at day 15). Furthermore, hrGM-CSF increased blood colony forming unit-granulocyte macrophage (CFU-GM) and was well tolerated in all but one patient. hrGM-CSF reduces toxic side-effects of DHBI, thus providing an effective treatment in patients with advanced and resistant MM.

Histamine synthesis by cells of the macrophage lineage in bone marrow of mice.

An injection of Escherichia coli lipopolysaccharide (LPS) increased the activity of histidine decarboxylase (HDC) in bone marrow (BM) cells of C3H/HeN mice much more than in C3H/HeJ mice, which are resistant to various effects of LPS. In WBB6/F1 (W/Wv) mice, which are genetically deficient in mast cells, HDC activity increased more than in C3H/HeN mice. Cultured BM cells of W/Wv mice spontaneously synthesized histamine in a HDC-dependent way. LPS caused a slight increase in HDC-associated histamine synthesis by these cells. Treatment of the BM cells with murine recombinant granulocyte-macrophage colony stimulating factor (mrGM-CSF) increased the histamine synthesis. In addition, treatment with mrGM-CSF made the cells respond to LPS by a dose-dependent increase in HDC activity and histamine synthesis. Most dish-adherent BM cells that had been treated with both mrGM-CSF and LPS for 48 h were stained for nonspecific esterase and not for chloroacetate esterase, and had twice as much HDC activity as the nonadherent cells had. Immunocytochemical analysis of the BM cells of W/Wv mice treated with both mrGM-CSF and LPS showed that HDC was in the cytoplasm of cells having Mac-1, a macrophage-differentiation antigen. These results suggest that cells of the macrophage lineage in the BM of mice synthesize histamine.

Analysis of a recombinant granulocyte macrophage colony stimulating factor dosage form by capillary electrophoresis, capillary isoelectric focusing and high-performance liquid chromatography

The analysis of a recombinant granulocyte macrophage colony stimulating factor (GM-CSF) dosage form by free solution capillary electrophoresis (FSCE), capillary isoelectric focusing (cIEF), and high-performance liquid chromatography (HPLC) is described. The quantitative use of capillary electrophoresis, whether FSCE or cIEF, will always be prone to special problems, such as sensitivity to differences in salt concentrations between the standard and sample, and can not match the ruggedness of HPLC. The usable quantitative linear range for both HPLC and FSCE surpass that achieved for cIEF methods by a factor of 10 or greater. The FSCE system, utilizing an octyl bonded/Brij-35 coated capillary, did not work for all proteins examined. This is probably due to an interaction of the protein with the bonded phase or the absorbed Brij-35. In contrast, the cIEF method worked well for all proteins tested thus far, yielding high efficiency and resolution comparable to slab gel isoelectric focusing. This paper addresses the potential for using free solution capillary electrophoresis and capillary isoelectric focusing as a quantitative analytical tool. Also, the effect of salt in the dosage form on quantitation, reproducibility, and efficiency of capillary electrophoresis methods is also discussed.

Divergent regulation of cell surface protease expression in HL-60 cells differentiated into macrophages with granulocyte macrophage colony stimulating factor or neutrophils with retinoic acid.

Taking advantage of the recently demonstrated presence of N-aminopeptidases and the serine protease dipeptidyl aminopeptidase IV (DPP IV) at the surface of human myeloblastic HL-60 cells, the regulation of these protease activities in HL-60 cell differentiation has been assessed using combined spectrophotometric and flow cytometric assays. Addition of human recombinant granulocyte macrophage colony stimulating factor (rHu-GM-CSF) to HL-60 cells to induce differentiation into macrophages led to a time- and dose-dependent increase in both cell surface N-aminopeptidase and DPP IV activities. Protease up-regulation was due to an enhancement in cell surface protease number, associated with a slight rise in apparent affinities of the enzymes for their substrates. In contrast, in HL-60 cells induced to differentiate into neutrophils in the presence of retinoic acid, expression of cell surface N-aminopeptidases was almost completely abolished in a time- and dose-dependent fashion, and this down-regulation was accompanied by a weak but significant decrease in affinity. However, no noticeable difference was seen in serine DPP IV expression between retinoic acid-treated and untreated HL-60 cells. Retinoic acid treatment also reduced soluble protease activity in vitro indicating that down-regulation of membrane aminopeptidases was not due to their proteolytic clip. No modulation in the activity of any of the enzymes tested was seen with human recombinant tumor necrosis factor-alpha or retinol which do not induce HL-60 cell differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Current status and future prospects in the immunotherapy of human immunodeficiency virus (HIV) infection.

I reviewed recently the status of immunotherapy of HIV infection in Trends in Pharmacology (1). To recount briefly, a large number of trials involving acquired immunodeficiency syndrome (AIDS) and pre-AIDS patients have been performed with relatively meager results. Intravenous gamma globulin (IVIG) therapy is thought to be of benefit for children with AIDS and a large scale study has been initiated to document this impression. Similar studies have been proposed for adults with or without azidothymidine (AZT) (2). Recombinant alpha interferon, particularly with AZT, has proven successful for managing Kaposi’s sarcoma in AIDS and is now licensed by the FDA for this use. Similarly, recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) has proven useful and is licensed for leukopenia due to AZT in AIDS. Interleukin II (IL-2) by continuous infusion has been shown to induce T lymphocytosis and combined trials with AZT are in progress. Diethyl dithiocarbamate (DTC; Imuthiol) was reported to reduce the incidence of infection in AIDS patients in one study (3). The other immunotherapeutic agents have proven of no value in AIDS.

Peri- and postnatal study (segment III) of recombinant granulocyte-macrophage colony stimulating factors (LBD-005) in rats.

A perinatal and postnatal study was carried out in Sprague-Dawley rats subcutaneously injected recombinant Granulocyte-Macrophage Colony Stimulating Factors (LBD-005), the growth of stem cell and the development of the hematopoietic cell, at dose levels of 250, 500 and 1000 micrograms/kg/day for a period from day 17 of gestation to day 21 after delivery. All pregnant dams were allowed to litter naturally, and the postnatal development of the offsprings was observed. The incidences of external, internal and skeletal anomalies were not significantly increased in the F1 and F2 fetuses of any treated groups. Recombinant GM-CSF caused no effects on parturition, lactation, postnatal growth and differentiation, behaviour and reproductive ability of the male and female offsprings.

GM-CSF triggers a rapid, glucose dependent extracellular acidification by TF-1 cells: evidence for sodium/proton antiporter and PKC mediated activation of acid production.

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down-regulated by PMA.

Teratogenicity study (segment II) of recombinant granulocyte-macrophage colony stimulating factors (LBD-005) in rats.

A teratogenicity study was carried out in Sprague-Dawley rats subcutaneously injected recombinant Granulocyte-Macrophage Colony Stimulating Factors (LBD-005), the growth of stem cell and the development of the hematopoietic cell, at dose levels of 250, 500 and 1000 micrograms/kg/day for a period of 11 days from day 7 to day 17 of gestation. Two-thirds of the pregnant females in each group were sacrificed on day 20 of gestation and their fetuses were examined. The remaining dams were allowed to litter naturally, and the postnatal development of the offspring was observed. The incidences of external, internal and skeletal anomalies were not significantly increased in the fetuses of any treated groups. Recombinant GM-CSF caused no effects on parturition, lactation, postnatal growth and reproductive ability of the male and female offsprings.

Effects of bryostatin 1 and rGM-CSF on the metabolism of 1-β-d-arabinofuranosylcytosine in human leukaemic myeloblasts

The effects of the protein kinase C activator bryostatin 1, either with or without recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) were examined with respect to the in vitro metabolism of ara-C in leukaemic myeloblasts obtained from 10 patients with acute myelogenous leukaemia (AML). Coincubation of cells with 12.5 x 10(-9) M bryostatin 1 and 10(-5) M ara-C for 4 h resulted in a significant increase in ara-CTP formation (compared to controls) in 6/10 specimens (mean increase 106%; range 38-255%), and no change in the remainder. In contrast, coincubation of cells with 1.25 ng/ml rGM-CSF resulted in a significant increase in only one specimen, and decreases in two. Bryostatin 1 also significantly increased ara-C DNA incorporation in 6/9 evaluable samples, including two which did not display an increase in ara-CTP formation. Coincubation of cells with both bryostatin 1 and rGM-CSF did not lead to a further increase in ara-CTP formation or ara-C DNA incorporation compared to values obtained with either agent alone. Finally, exposure of blasts to bryostatin 1 for 24 h before ara-C led to an increase in ara-CTP formation in 3/8 additional specimens, and a decrease in one sample displaying evidence of bryostatin 1-induced macrophage differentiation. Incubation of cells with both rGM-CSF and bryostatin 1 for this period resulted in ara-CTP levels equivalent to those obtained with bryostatin 1 alone. These studies indicate that while bryostatin 1 exerts a heterogeneous effect on ara-C metabolism in leukaemic myeloblasts, it is capable of potentiating ara-C phosphorylation in a subset of patient samples, including some that do not exhibit an increase in response to rGM-CSF. They also raise the possibility that bryostatin 1-induced potentiation of ara-C metabolism in some leukaemic cells may contribute, at least in part, to the antileukaemic efficacy of this drug combination.

Membrane Expression and Function of Complement Receptors CR1 and CR3 on Neutrophils from HIV‐Infected Subjects: Modulation by rTNF‐α and rGM‐CSF

We evaluated membrane expression and function of complement receptors CR1 and CR3 on neutrophils from 27 HIV-positive (HIV+) subjects (14 in the CDC class III and 13 class IV) as well as their modulation in vitro by recombinant tumour necrosis factor-alpha (rTNF-alpha) and granulocyte-macrophage colony stimulating factor (rGM-CSF). While CR1 was expressed at similar levels on neutrophils from controls and HIV+ subjects, CR3 expression was significantly higher in CDC class IV subjects than in healthy controls. CR1 and CR3 expression was significantly increased after treatment of neutrophils with both cytokines, without differences between controls and HIV+ subjects. Similarly, the superoxide anion (O2-) production in response to C3-coated zymosan (C3zy) was significantly enhanced on neutrophils from CDC class IV subjects when compared with controls. rGM-CSF and rTNF-alpha treatment significantly enhanced the spontaneous as well as C3zy-stimulated O2- production by neutrophils from controls and CDC class III subjects, and induced an upward trend in the CDC class IV group. These results indicate that the neutrophils of HIV+ patients are preactivated in vivo but they also indicate that these cells may correctly respond to a subsequent particulate stimulus as well as to activating cytokines. Our findings suggest that desensitization or functional exhaustion of complement receptors are not implicated in the abnormalities observed on neutrophils from HIV+ patients.

Enhanced marrow recovery by short preincubation of marrow allografts with human recombinant interleukin-3 and granulocyte-macrophage colony- stimulating factor

We studied an alternative method of using hematopoietic growth factors (HGFs) to enhance hematopoietic recovery in patients undergoing bone marrow transplantation (BMT), by short in vitro preincubation. Twenty consecutive patients with leukemia received T-cell-depleted allografts using Campath-1G. Two thirds of the marrow was infused on the scheduled day of transplant and one third of the marrow following preincubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) on day 4. Engraftment parameters and duration of hospitalization were compared by actuarial analysis to those of 40 historical controls. Patients receiving the incubated boost had significantly faster platelet recovery (P = .017) and shorter hospitalization period (P = .001) when compared with the control subjects. Platelet count reached greater than 25 x 10(9)/L on day 17 (median) in the study group and on day 23 in the controls. The median duration of hospitalization was 20 and 36 days, respectively. In the early posttransplantation follow-up, two of four patients in the study group died as a result of graft rejection, while all 13 deaths in the control group resulted from complications associated with marrow suppression. We suggest that pretransplant in vitro activation of bone marrow cells with IL-3 and GM-CSF may prove to be an efficient method for enhancing marrow recovery after BMT.

Production of epithelial cell growth factors by lamina propria mononuclear cells.

The effects of lamina propria mononuclear cell culture supernatant on epithelial cell DNA synthesis were studied using cells isolated from patients with inflammatory bowel disease and normal controls. Supernatants from resting and phytohaemagglutinin stimulated cells were studied and supernatants that strongly promoted DNA synthesis were pooled, and growth factor activity partially characterised. The effects of recombinant interleukins-1 beta,2,3,interferon-gamma, and granulocyte macrophage colony stimulating factor were tested in the same system. Resting lamina propria mononuclear cells produce factors that increase DNA synthesis. Production of these factors is increased by phytohaemagglutinin stimulation. No significant differences were found in production of these factors between patients with inflammatory bowel disease and normal controls. The molecular weight of the active factor(s) lies in the region 31-48 kD. Chromatofocusing produced two peaks of activity, one in the region pk 5.5 and one around pk 6.4. The activity was heat and acid pH labile. Activity was not destroyed, however, by 0.05% trypsin. Recombinant granulocyte macrophage colony stimulating factor was a weak stimulus to epithelial DNA synthesis, interleukin-1 beta was weakly inhibitory but other cytokines tested did not have any effect. Granulocyte macrophage colony stimulating factor is probably important in controlling epithelial cell growth.