Recombinant Human Granulocyte-macrophage Colony Research Articles

Study on rhGM-CSF as adjuvant in revaccination among adults of non-and hyporesponders to hepatitis B vaccine

To investigate the effect of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) as adjuvant on immune response in adults of non-and hyporesponders to hepatitis B vaccine. Those who were once immunized with recombined yeast gene hepatitis B vaccine more than one standard scheme in two years and negative for hepatitis B markers were randomly sorted as group A and group B. 33 adults of group A were given hepatitis B vaccine 10 microg each time. The immune procedure was 0, 1 and 6 month. 34 adults of group B were given rhGM-CSF 300 microg for the first day, then 10 microg each time for routine immune. The blood samples were collected before the first injection and in 1, 2 and 8 months (T1, T2, T8) following the first injection to test Anti-HBs. Anti-HBs positive conversion rates of group A and B at T8 was 39.39% and 64.71% respectively (P = 0.038). Anti-HBs levels of group B at T1, T2, T8 were (113.85 +/- 198.56) mIU/ml, (312.40 +/- 349.44) mIU/ml, (427.74 +/- 411.58) mIU/ml (P = 0.001). There was significant difference between group A and B in T8 Anti-HBs levels (P = 0.010). Better immune response was found in the group of rhGM-CSF with hepatitis B vaccine. So rhGM-CSF can induce the immune respond to hepatitis B vaccine.

Subcutaneous Four-Week Repeated Dose Toxicity Studies of Rice Cell-Derived Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor in Rats.

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a glycoprotein and hematopoietic growth factors that regulates the proliferation of myeloid precursor cells and activates mature granulocytes and macrophages. In a previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study, we examined the repeated dose toxicity of rhGM-CSF in SD rats. The repeated dose toxicity study was performed at each dose of 50 and 200 µg/kg subcutaneous administration of rhGM-CSF everyday for 28-days period. The results did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the tested groups. The hematological and blood biochemical parameters were statistically not different in all groups. These results suggest that rhGM-CSF may show no repeated dose toxicity in SD rats under the conditions.

Temperature Reduction in Cultures of hGM-CSF-expressing CHO Cells: Effect on Productivity and Product Quality

We have demonstrated that temperature reduction from 37 to 33 degrees C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO-K1-hGM-CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO-K1-hGM-CSF cells were cultured in a biphasic mode: first, a 37 degrees C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 degrees C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.7 days in the culture kept at the lower temperature, when compared to the control culture maintained at 37 degrees C. Furthermore, the total rhGM-CSF production increased 6 times in the culture shifted to 33 degrees C. Because the quality and hence the in vivo efficacy of a recombinant protein might be affected by numerous factors, we have analyzed the N- and O-glycosylation of the protein produced under both cell culture conditions using high-pH anion-exchange chromatography and complementary mass spectrometry techniques. The product quality data obtained from the purified protein preparations indicated that decreasing temperature had no significant effect on the rhGM-CSF glycosylation profiles, including the degree of terminal sialylation. Moreover, both preparations exhibited the same specific in vitro biological activity. These results revealed that the employed strategy had a positive effect on the cell specific productivity of CHO-K1-hGM-CSF cells without affecting product quality, representing a novel procedure for the rhGM-CSF production process.

Co-expression of proteinase inhibitor enhances recombinant human granulocyte–macrophage colony stimulating factor production in transgenic rice cell suspension culture

The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte–macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production.

Antitumor Effect of Lung Cancer Vaccine with Umbilical Blood Dendritic Cells in Reconstituted SCID Mice

Dendritic cells (DCs) are important cells in initiating an immune response. A generation of functional DCs has potential clinical use in treating cancer. However, the source of DCs and patient immunodeficiency with cancer have been hindrances in clinical therapy. We generated DCs from human umbilical cord blood mononuclear cells (UBMCs) with recombinant human granulocyte-macrophage colony stimulating factor, recombinant human interleukin-4, and recombinant human tumor necrosis factor-alpha. The mature DC-A549 lung cancer vaccine (AgL-DC) was prepared through loading A549 lysate, treating with lipopolysaccharide (LPS) and positive selecting with CD83 magnetic beads. AgL-DC can secrete interleukin (IL)-12 and IL-1. Further in vitro analysis showed that AgL-DC notably induced human UBMC lymphocyte proliferation (p < 0.01) by 3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, increased the cytotoxic T-lymphocyte (CTL) activity of UBMC lymphocytes against A549 cells (p < 0.05, at effector cells:target cells ratios of 50:1 and 100:1) by lactate dehydrogenase (LDH) cytotoxic assay, and improved production of IL-6 and tumor necrosis factor-beta (p < 0.01, p < 0.05) by enzyme-linked immunosorbent assay. Subsequently, the reconstitute immunity model in severe combined immunodeficiencies (SCID) mice has been established using human UBMC transplantation, and similar trends to results of UBMC in vitro experiments have been shown in lymphocyte proliferation, CTL activity, and IL-6 and tumor necrosis factor-beta secretion levels in these models. AgL-DC also significantly (p < 0.01) increased the antitumor effect in vivo. The tumor infiltrating immunocytes were positively expressed human CD83 and CD3 molecules, and they were negatively expressed in tumor tissue treated with control. These results have demonstrated that umbilical cord DCs are a useful source of vaccine cells for augmenting CTL-mediated cytotoxicity and have potential usefulness in cellular therapy for human cancer in a new vaccination strategy.

Oral administration of recombinant human granulocyte-macrophage colony stimulating factor expressed in rice endosperm can increase leukocytes in mice

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is used clinically to treat leucopenia typically caused by cancer chemotherapy or radiotherapy. This study used multiple strategies to obtain very high expression levels of OsrhGM-CSF (14 microg/seed) in rice endosperm. Electron micrographs of immunogold-labeled transgenic endosperm showed that rhGM-CSF was not only localized in protein bodies but was also distributed in the apoplast. A biological activity assay indicated that OsrhGM-CSF stimulated the growth of TF-1 cells in vitro. In addition, the transgene was used to effectively treat leucopenia by oral administration of the unprocessed transgenic grains. In cyclophosphamide-induced leucopenic mice, transgenic seeds produced a 27% (t=0.021) gain in leukocytes after 14 days feeding. Even in non-leucopenic mice, leukocyte gain was 37% (t=0.002) more than that of mice fed non-transgenic seeds. This study provides a novel approach to the use of oral unprocessed transgenic OsrhGM-CSF seeds to treat leucopenia.

A New Population of Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Induces CD4+CD25+Foxp3+ T Cells

Several studies have shown that development of hepatocellular carcinoma (HCC) generates a number of immune suppressive mechanisms in these patients. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that have been shown to inhibit T-cell responses in tumor-bearing mice, but little is known about these cells in humans owing to a lack of specific markers. In this study, we have investigated the frequency and function of a new population of MDSC denoted here as CD14(+)HLA-DR(-/low) in HCC patients. We have also identified a novel, MDSC-mediated immune regulatory pathway in these patients. We have directly isolated and characterized MDSCs for phenotype and function from peripheral blood (n = 111) and tumor (n = 12) of patients with HCC. The frequency of CD14(+)HLA-DR(-/low) cells in peripheral blood mononuclear cells (PBMC) from HCC patients was significantly increased in comparison with healthy controls. CD14(+) HLA-DR(-/low) cells were unable to stimulate an allogeneic T-cell response, suppressed autologous T-cell proliferation, and had high arginase activity, a hallmark characteristic of MDSC. Most important, CD14(+)HLA-DR(-/low) cells from HCC patients induced a CD4(+)CD25(+)Foxp3(+) regulatory T-cell population when cocultured with autologous T cells. CD14(+)HLA-DR(-/low) cells are a new population of MDSC increased in blood and tumor of HCC patients. We propose a new mechanism by which MDSC exert their immunosuppressive function, through the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells in cocultured CD4(+) T cells. Understanding the mechanism of action of MDSC in HCC patients is important in the design of effective immunotherapeutic protocols.

Intravenous Single and Two Week Repeated Dose Toxicity Studies of Rice Cells-derived Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor on Rats

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) regulates proliferation and differentiation of hematopoietic progenitor cells and modulates function of the mature hematopoietic cells. In the previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study we examined the single and repeated dose toxicity of rice cells-derived hGM-CSF in SD rats. During single dose toxicity study for 7 days, there were no any toxic effects at any dose of from 10 to <TEX>$1000{\mu}g/kg$</TEX>. The lethal dose (<TEX>$LD_{50}$</TEX>) was not found in this range. Moreover, repeated dose toxicity study of 14-days period and at the doses of 50 and <TEX>$200{\mu}g/kg$</TEX> (i. v.) of rhGM-CSF did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the test groups. The hematological and blood biochemical parameters were statistically not different in all the groups. These results suggest that rhGM-CSF has no toxicity in SD rats.

Polysialylated Neuropilin-2 Is Expressed on the Surface of Human Dendritic Cells and Modulates Dendritic Cell-T Lymphocyte Interactions

Polysialic acid (PSA) is a unique linear homopolymer of alpha2,8-linked sialic acid that has been identified as a posttranslational modification on only five mammalian proteins. Studied predominantly on neural cell adhesion molecule (NCAM) during development of the vertebrate nervous system, PSA modulates cell interactions mediated by NCAM and other adhesion molecules. An isoform of NCAM (CD56) on natural killer (NK) cells is the only protein known to be polysialylated in cells of the immune system, yet the function of PSA in NK cells remains unclear. We show here that neuropilin-2 (NRP-2), a receptor for the semaphorin and vascular endothelial growth factor families in neurons and endothelial cells, respectively, is expressed on the surface of human dendritic cells and is polysialylated. Expression of NRP-2 is up-regulated during dendritic cell maturation, coincident with increased expression of ST8Sia IV, one of the key enzymes of PSA biosynthesis, and with the appearance of PSA on the cell surface. PSA on NRP-2 is resistant to digestion with peptide N-glycosidase F but is sensitive to release under alkaline conditions, suggesting that PSA chains are added to O-linked glycans of NRP-2. Removal of polysialic acid from the surface of dendritic cells or binding of NRP-2 with specific IgG promoted dendritic cell-induced activation and proliferation of T lymphocytes. Thus, this newly recognized polysialylated protein on the surface of dendritic cells influences dendritic cell-T lymphocyte interactions through one or more of its distinct extracellular domains.

Renaturation and Purification of rhGM-CSF with Ion-Exchange Chromatography

The renaturation and purification of recombinant human granulocyte macrophage colony stimulation factor (rhGM-CSF) expressed in Escherichia coli with strong anion-exchange chromatography (SAX) were studied. The effects of pH values, ratios of concentrations of GSH/GSSG, and urea concentrations in the mobile phase on the renaturation and purification of rhGM-CSF with SAX were investigated, respectively. The results show that the above three factors have remarkable influences on the efficiency of renaturation and mass recovery of rhGM-CSF. The addition of GSH/GSSG in the mobile phase can improve the formation of correct disulfide bonds in rhGM-CSF so that its renaturation yield increases. In addition, to enhance the mass recovery of rhGM-CSF with SAX, the low concentration of urea was added in the mobile phase to prevent denatured protein aggregation. Under the optimal conditions, rhGM-CSF was renatured with simultaneous purification on SAX column within 30 min only by one step. After that its specific bioactivity, mass recovery, and purity reached 1.66 x 10(7) IU x mg, 58.8%, and 96.2%, respectively.

Production and purification of recombinant human granulocyte–macrophage colony stimulating factor (GM-CSF) from high cell density cultures of Pichia pastoris

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor, which has been used as a therapeutic agent in clinical cases like neutropenia. In this study, we report the production of recombinant human GM-CSF in the methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. Recombinant P. pastoris GS115 cells were grown in fed batch cultures to obtain a biomass density of 55.6 gDCW L(-1) and a high volumetric activity of 131 mg L(-1) of GM-CSF. The protein migrated as a diffuse band on SDS-PAGE at the range of 28-35 kDa indicating differential glycosylation. The secreted protein was purified to 95% in two steps using cation exchange and size exclusion chromatography.

Recombinant human granulocyte macrophage colony stimulating factor following alternating non cross resistant chemotherapy in hodgkin's disease

Fourteen patients with Hodgkin's disease (two previously untreated, 12 following relapse or with refractory disease) were treated with a combination chemotherapy regimen comprising chlorambucil, vinblastine, procarbazine, prednisolone, etoposide, vincristine and adriamycin administered on days 1-8. Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (mammalian glycosylated, Sandoz/Schering-Plough) was administered after alternate cycles of chemotherapy from day 10 for 7 days by continuous intravenous (i.v.) infusion in 12 patients in a dose finding study (dose: 2 micrograms/kg/day in four patients, 4 micrograms/kg/day in four patients and 8 micrograms/kg/day in four patients) and by daily subcutaneous (s/c) injections in two patients (8 micrograms/kg/day). There was a rapid peripheral leucocytosis following the rhGM-CSF, reaching a peak at 1-2 days in 12/14 patients. The initial leucocytosis was composed of neutrophils followed by a rise in immature myeloid cells. There was no difference observed in the duration or depth of the nadir following chemotherapy or in the rate of recovery of peripheral white cell counts between cycles with and without rhGM-CSF in patients treated with 2 and 4 micrograms/kg/day. At the dose of 8 micrograms/kg/day, 3/6 patients had a shorter nadir duration in the cycle with rhGM-CSF, compared with cycle without rhGM-CSF. There was no difference in frequency of infection in cycles with and without rhGM-CSF. Following chemotherapy, six patients achieved clinical remission, six partial remission and two had progressive disease.

Phase II trial of subcutaneous recombinant human interleukin 11 with subcutaneous recombinant human granulocyte-macrophage colony stimulating factor in patients with acute myeloid leukemia (AML) receiving high-dose cytarabine during induction: ECOG 3997

Randomized trials of substituting high-dose cytarabine (HiDAC) for standard dose cytarabine (SDAC) during induction therapy for newly diagnosed AML have not demonstrated an improvement in the complete remission (CR) rate. Phase II trials of the scheduled administration of HiDAC after SDAC suggest an improved outcome. The hematological complications of intensification are considerable. GM-CSF after chemotherapy improved the survival of older patients in a randomized trial. Recombinant human interleukin 11, a thrombopoietic cytokine, reduced the incidence of chemotherapy-induced thrombocytopenia in patients with solid tumors. Therefore, 34 patients were treated, with newly diagnosed AML less than 56 years of age, with daunorubicin 45 mg/m 2 on days 1–3, cytarabine 100 mg/m 2 days 1–7 and cytarabine 2 g/m 2 for 12 h on days 8–10 (7 + 3 + 3). rhIL-11 (50 μg/kg/day,) and GM-CSF (250 μg/kg/day) were administered subcutaneously from day 11 until recovery. The complete remission rate was 59% (90% C.I. 43–73%). The median time to recovery of neutrophils to >500 and platelets to ≥20,000 μl −1 was 27 days (95% C.I. 27–30 days) and 25 days (95% C.I. 24–29 days), respectively. The trial does not confirm the high CR rate observed in phase II trials, despite optimal supportive care.

Bone Marrow–Derived Dendritic Cell Vaccination of Dogs with Naturally Occurring Melanoma by Using Human gp100 Antigen

Canine malignant melanoma (CMM) is a common and aggressive form of cancer in dogs. Established therapeutic approaches such as surgery, chemotherapy, and radiation therapy (RT) have not proven curative. As a coadjuvant of RT and to enhance the antimelanoma immune response, we characterized dendritic cells (DCs) from the bone marrow (BM) of dogs with CMM, ex vivo, for use in therapeutic vaccines. BM mononuclear cells from 3 dogs with melanoma and from 1 healthy dog were cultured for 12 days in media supplemented with recombinant human granulocyte-macrophage colony stimulating factor, stem cell factor, tumor necrosis factor, and Flt-3 ligand. On day 11, DCs were transduced with an adenovirus vector encoding a xenoantigen, human melanoma antigen gp100. Each dog received 3 subcutaneous vaccinations over a 4-month period. Phenotypic analysis of the expanded DC population demonstrated expression of CD11c/CD18 and major histocompatibility complex class II surface markers, and ultrastructural features characteristic of DCs were observed on electron microscopy. On functional analysis, these DCs were able to stimulate allo-reactivity and capture and express gp100. One dog demonstrated antigen-specific cytotoxic T lymphocyte (CTL) activity in peripheral blood lymphocytes. This dog has displayed no clinical signs, either locally or systemically, of recurrent melanoma 48 months after initial DC injection. However, another dog, which was CTL negative, relapsed 22 months after vaccination. Ex vivo DC expansion is feasible for immunotherapy of spontaneous cancers in outbred dogs.

Bone Marrow‐Derived Dendritic Cell Vaccination of Dogs with Naturally Occurring Melanoma by Using Human gp100 Antigen

Canine malignant melanoma (CMM) is a common and aggressive form of cancer in dogs. Established therapeutic approaches such as surgery, chemotherapy, and radiation therapy (RT) have not proven curative. As a coadjuvant of RT and to enhance the antimelanoma immune response, we characterized dendritic cells (DCs) from the bone marrow (BM) of dogs with CMM, ex vivo, for use in therapeutic vaccines. BM mononuclear cells from 3 dogs with melanoma and from 1 healthy dog were cultured for 12 days in media supplemented with recombinant human granulocyte‐macrophage colony stimulating factor, stem cell factor, tumor necrosis factor, and Flt‐3 ligand. On day 11, DCs were transduced with an adenovirus vector encoding a xenoantigen, human melanoma antigen gp100. Each dog received 3 subcutaneous vaccinations over a 4‐month period. Phenotypic analysis of the expanded DC population demonstrated expression of CD11c/CD18 and major histocompatibility complex class II surface markers, and ultrastructural features characteristic of DCs were observed on electron microscopy. On functional analysis, these DCs were able to stimulate allo‐reactivity and capture and express gp100. One dog demonstrated antigen‐specific cytotoxic T lymphocyte (CTL) activity in peripheral blood lymphocytes. This dog has displayed no clinical signs, either locally or systemically, of recurrent melanoma 48 months after initial DC injection. However, another dog, which was CTL negative, relapsed 22 months after vaccination. Ex vivo DC expansion is feasible for immunotherapy of spontaneous cancers in outbred dogs.

A simple purification procedure of biologically active recombinant human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of 80.1% by ammonium sulfate precipitation and a single chromatographic step involving FPLC-anion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from 21.5∼29 kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.

Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells

Recently, we demonstrated that a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to partially reconstitute the missing link between glycolysis and TCA, increasing the flux of glucose into the TCA and achieving higher yields of recombinant erythropoietin. In the present study, a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor was used to evaluate the impact of PYC2 expression and reduced culture temperature. Temperature reduction from 37 to 33 °C revealed a reduced growth rate, a prolonged stationary phase and a 2.1-fold increase of the cell specific rhGM-CSF production rate for CHO-K1-hGM-CSF cells. The PYC2-expressing cell clones showed a decreased cell growth and a lower maximum cell concentration compared to the control expressing rhGM-CSF but no PYC2. However, only 65% lactate were produced in PYC2-expressing cells and the product yield was 200% higher compared to the control. The results obtained for CHO cells compared to BHK cells reported previously, indicated that the PYC2 expression dominantly reduced the lactate formation and increased the yield of the recombinant protein to be produced. Finally, the growth and productivity of PYC2-expressing CHO-K1-hGM-CSF cells under both temperature conditions were investigated. The average cell specific rhGM-CSF production increased by 3.2-fold under reduced temperature conditions. The results revealed that the expression of PYC2 and a reduced culture temperature have an additive effect on the cell specific productivity of CHO-K1-hGM-CSF cells.

Partitioning of recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) from plant cell suspension culture in PEG/sodium phosphate aqueous two-phase systems

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andKhGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% andKhGM-CSF of 7.64 after 2 h of incubation at room temperature.

Hematopoiesis

Chronic myeloid leukemia (CML) is a hematological neoplasia that results from the transformation of a hematopoietic stem cell. It is characterized by the expansion of the myeloid lineage, which results in the accumulation of mature and immature granulocytes in peripheral blood and bone marrow. However, when CML marrow cells are cultured in Dexter-type long-term cultures (LTMC) hematopoiesis is defective and can be sustained for only a few weeks. One possible explanation for the deficient growth of hematopoietic cells in CML LTMC is that some factors that act as key regulators of hematopoiesis are absent in this experimental system. Thus, we tested this hypothesis by adding recombinant cytokines to these cultures. As a first approach, we added recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), rhGranulocyte-CSF (rhG-CSF) and rhErythropoietin (rhEPO); each factor was added individually once a week. Addition of rhGM-CSF and rhG-CSF resulted in a significant increase in the levels of nucleated cells and myeloid progenitors; the highest effects were seen in the presence of rhGM-CSF. Interestingly, such a cytokine also induced a significant decrease in the levels of erythroid progenitors. Recombinant hEPO had no significant effects on nucleated cells or myeloid progenitors, however, it induced a significant, although transient, increase in the levels of erythroid cells. The above results indicate that the hematopoietic regulators used here (rhGM-CSF, rhG-CSF and rhEPO) are capable of stimulating the growth of hematopoietic cells in LTMC from CML patients. Thus, this study demonstrates that it is, indeed, possible to manipulate CML LTMC by the addition of recombinant cytokines; this observation may be of particular relevance, since this in vitro experimental system has already been used as a method for purging of leukemic cells in autologous transplant settings. By using specific recombinant hematopoietic modulators it might be possible to make LTMC a more efficient system for such a clinical purpose.

Choice of the adequate quantification method for recombinant human GM-CSF produced in different host systems

Three enzyme-linked-immunosorbent assays (ELISA) were developed and compared with a bioassay to quantify the recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF). These assays were suitable to quantify the non-glycosylated rhGM-CSF present in mixtures with variable protein content, and therefore useful for determining concentrations of the cytokine in production processes. Among these assays, the competitive ELISA, developed with a MAb that recognises an epitope not involved in glycosylation, was the only one suitable to quantify the glycosylated form of rhGM-CSF produced in mammalian cell cultures.