Carbimazole induced agranulocytosis: rescue with human recombinant granulocyte colony stimulating factor

We report on a 25 yr old woman with primary autoimmune hyperthyroidism who was treated with carbimazole. Forty-three days later she developed agranulocytosis (64 x 10(6)/l). With 4 days treatment with recombinant human Granulocyte Colony Stimulating factor the granulocyte count rose to normal--4,350 cells x 10(6)/l. This is considerably faster than the rate of spontaneous recovery which usually takes one to 2 weeks.     

Effects of macrophage colony stimulating factor and granulocyte-macrophage colony stimulating factor on osteoclastic differentiation of hematopoietic progenitor cells

Although the hematopoietic origin of the osteoclast is generally accepted, the precise phenotype of the progenitor and the regulation of its differentiation are unclear. This study compares proliferation and differentiation of progenitors in response to macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Nonadherent progenitor cells from murine long-term bone marrow cultures (LTBMC) (as a source of osteoclast progenitors) demonstrated a significant proliferative response to M-CSF. In addition, M-CSF increased the number of multinucleated cells, only a small percent of which (14-16%) were tartrate-resistant, acid phosphatase (TRAP)-positive. In contrast, cells cultured with GM-CSF generated more TRAP-positive multinucleated cells even at concentrations less stimulatory of proliferation than M-CSF. The osteoclast phenotype of these multinucleated cells was also assessed by ultrastructural characterization of ruffled borders in association with bone fragments. The bone-active hormone 1,25-dihydroxyvitamin D3 inhibited the proliferation of this subset of progenitor cells in the presence of M-CSF or GM-CSF. All of these results show effects on progenitors in the absence of the stromal cell microenvironment in this system. These results provide evidence for a divergence in the biological responsiveness of osteoclast progenitor cells to M-CSF compared with GM-CSF; they support the notion that M-CSF has a "priming" effect on osteoclast progenitors whose subsequent differentiation to osteoclastic multinucleated cells is promoted by GM-CSF.     

Psoriasiform eruption triggered by recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) and exacerbated by granulocyte colony stimulating factor (rG-CSF) in a patient with breast cancer

Colony-stimulating factors (CSFs) are commonly used for the treatment of neutropenia following chemotherapy and for the mobilization of peripheral blood stem cells (PBSC). We recently experienced a rare case of a new onset of psoriasiform eruption by GM-CSF (granulocyte-macrophage colony-stimulating factor) which was exacerbated by G-CSF (granulocyte colony-stimulating factor) in a patient with breast cancer. A 36-year-old woman had received neoadjuvant chemotherapy (cyclophosphamide, epirubicin and 5-fluorouracil), modified radical mastectomy and adjuvant chemotherapy with paclitaxel and mitoxantrone followed by GM-CSF administration for the treatment of locally advanced breast cancer. She had developed a psoriatic skin lesion on face and both upper arms during leukocyte recovery in spite of no previous history of psoriasis. Next, the chemotherapy course was complicated by a flare of mild psoriatic skin lesion, although CSF was changed into G-CSF due to GM-CSF-associated psoriasis. Subsequently, she had had high-dose chemotherapy and autologous peripheral blood stem cell transplantation for consolidation therapy. GM-CSF was administered for the mobilization of PBSC and post-transplant period, but psoriatic skin lesion did not appear. During 6 months after PBSCT, psoriasiform eruption did not appear.     

Production of granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, and tumor necrosis factor alpha during remission and infections in patients with acute leukemia

We measured circulating serum levels of granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating comparable to the levels of these factors in 12 children with acute febrile infections without malignancy or hematological disorders and 15 age matched healthy controls. There were significantly elevated levels of G-CSF, GM-CSF and TNF alpha, in 12 children with infections without leukemia, as compared with controls. Also in 18 leukemic children with infections serum G-CSF and TNF alpha levels were significantly higher than those in the leukemic children without infection and healthy controls, whereas no significant difference was noted in the GM-CSF levels in these groups. Although elevation in TNF alpha levels in response to infections were similar in the children with and without leukemia, in the G-CSF levels lower elevation was noted in the leukemic children with infections as compared to the children with infections without leukemia. Despite leukopenia enhanced the production of G-CSF, even in leukopenic children with leukemia and infections, serum G-CSF levels were still lower than those for the children with infections without leukemia. We concluded that, the production of G-CSF and GM-CSF as a response to infection was deficient in the patients with acute leukemia in remission, probably due to the maintenance and reinforcement chemotherapy. Therefore, the use of recombinant G-CSF may be recommended in the infections of these patients.     

Coordinate and noncoordinate colony stimulating factor formation by human monocytes

The regulation of macrophage colony-stimulating factor (M-CSF) formation by elutriation-purified human monocytes was studied in vitro and compared with that for granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF). The levels of the three CSFs were measured by immunoassay. Lipopolysaccharide (LPS, 100 ng/ml) was able to enhance CSF formation but the levels were significantly influenced by the presence of a cyclooxygenase product(s) in the cultures. In the presence of LPS, both M-CSF and GM-CSF were up-regulated by cyclooxygenase inhibition (indomethacin, 10(-5) M), while G-CSF was down-regulated. Exogenous prostaglandin E2 (PGE2, 10(-7) M) reversed the actions of indomethacin. In LPS-treated cells, in contrast to the different regulation by endogenous eicosanoid of M-CSF and GM-CSF formation and G-CSF formation, both interleukin-4 (IL-4, 250 pM) and the glucocorticoid dexamethasone (10(-7) M) lowered the amounts of all CSFs. When the actions of CSFs were examined for their effects on CSF formation, M-CSF could not stimulate either GM-CSF or G-CSF synthesis, in contrast to literature findings, while GM-CSF enhanced M-CSF formation but not that of G-CSF. These studies indicate that there can be both coordinate and noncoordinate control of CSF expression by human monocytes.     

Characterization of the receptor binding determinants of granulocyte colony stimulating factor

We performed a series of experiments using alanine-scanning mutagenesis to locate side chains within human granulocyte colony-stimulating factor (G-CSF) that are involved in human G-CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G-CSF. The G-CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G-CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild-type G-CSF and the E19A mutant were expressed in E. coli. The re-folded proteins were found to have proliferative activities similar to the analogous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G-CSF. Although G-CSF is likely to signal through receptor homo-dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self-antagonism at high G-CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G-CSF probably does not signal via a pure 2:1 receptor ligand complex. Thus, G-CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high-affinity interaction with its receptor.     

Taxol plus recombinant human granulocyte-colony stimulating factor as initial and as salvage chemotherapy for metastatic breast cancer: a preliminary report

Twenty-eight patients received Taxol as their first chemotherapy for stage IV breast cancer. An additional 51 patients with extensive prior exposure to other chemotherapeutic agents received Taxol as salvage therapy. We found significant activity for the drug in both situations, as well as a strong clinical suggestion of non-cross-resistance with doxorubicin. An excellent response in previously irradiated skin was noted in one case. The routine use of recombinant human granulocyte-colony stimulating factor seemed to ameliorate some of the dose-limiting toxicity of neutropenia. Other toxicity was mild to moderate in most cases. With further development, Taxol should play a significant role in the systemic management of breast cancer.     

Granulocyte colony stimulating factor (G-CSF) producing renal cell carcinoma

A 88-year-old male patient with G-CSF producing renal cell carcinoma is reported. The patient was admitted to our hospital complaining of macrohematureia. The laboratory examination showed marked leukocytosis of 18,200/microliter (neutrophil 92%) in the peripheral blood and high levels of G-CSF (120 pg/ml) in the serum. An abdominal CT scan revealed a right renal tumor. The neutrophil count rose to 38,700/microliter with increasing of tumor size. Histopathological diagnosis was renal cell carcinoma (Grade 3) and immunohistochemical staining using Histo anti-G-CSF antibody demonstrated cancer cells produced G-CSF. This is the second case of G-CSF producing renal cell carcinoma diagnosed by immunohistochemical staining in the literature.     

Expression and purification of a truncated macrophage colony stimulating factor in Kluyveromyces lactis

A truncated human macrophage colony stimulating factor (M-CSF) cDNA encoding amino acid residues from 3 to 149 of the native M-CSF was obtained by using polymerase chain reaction. When inserted into plasmid pCXJ1 and psPHO5 and introduced into Kluyveromyces lactis, it directs the the secretory expression of the biologically active dimeric form of M-CSF. Through a four-step purification protocol, i.e. ammonium sulfate salting out, DEAE-cellulose column chromatography, hydrophobic chromatography on phenyl-sepharose and Mono Q fast protein liquid chromatography, the recombinant truncated M-CSF was purified to homogenerity and show its apparent molecular mass at 21KDa on reduced SDS-PAGE, with a specific activity of 1.21 x 10(7) units/mg protein.     

Use of granulocyte macrophage colony stimulating factor in children after orthotopic liver transplantation

The objective of this study was to present quantitative data concerning prostatic growth during the foetal period (gestational age: 13 to 36 weeks) and to provide normal curves of the growth of the prostatic volume correlated with foetal age and weight, using the allometric method. This study was performed on 45 non-fixed human male foetuses, in a good state of preservation and not presenting any congenital malformations. The gestational age of the foetuses ranged from 13 to 36 weeks. Analysis of correlations showed that growth curves presented an angle less than 45 degrees, indicating that growth of the foetal prostate is slower than that of the individual as a whole. The authors also found a statistically significant correlation (p < 0.001) between prostatic volume and foetal weight during the foetal period.     

Granulocyte-macrophage colony stimulating factor suppresses lipopolysaccharide-induced osteoclast-like cell formation in mouse bone marrow cultures

Lipopolysaccharide (LPS) is a potent bone resorbing factor. We investigated the effect of LPS on osteoclast formation in three types of cultures. LPS inhibited osteoclast formation induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], in a dose-dependent manner, in cultures of whole bone marrow cells without dexamethasone. LPS increased the amount of granulocyte-macrophage colony stimulating factor (GM-CSF) in the culture supernatant, and anti-GM-CSF antiserum almost abolished the inhibition of osteoclast formation by LPS, thereby indicating that GM-CSF generated by treatment with LPS may be responsible for the inhibition of osteoclast formation. In cultures with dexamethasone, the amount of GM-CSF was decreased to one-third of that with 1,25(OH)2D3 alone and was not changed by treatment with LPS. In this culture system, LPS enhanced osteoclast formation. In the coculture system of nonadherent bone marrow cells and a stromal cell line in the presence of 1,25(OH)2D3 and dexamethasone, where no detectable GM-CSF was present in the supernatant, LPS markedly enhanced osteoclast formation, whereas exogenously added GM-CSF (100 pg/ml) almost completely inhibited osteoclast formation. LPS stimulated pit formation on dentin slices by the osteoclast-like cells formed by in vitro culture system.     

Cytokine regulation of granulocyte-macrophage colony stimulating factor and macrophage colony-stimulating factor production in human arterial smooth muscle cells

Smooth muscle cells (SMC) are the major cell type found in the walls of large blood vessels and appear to participate in local immune and inflammatory reactions, as well as in certain vascular diseases. We tested whether human arterial SMC can produce in vitro the colony stimulating factors (CSFs), granulocyte macrophage-CSF (GM-CSF) and macrophage CSF (M-CSF). Untreated internal mammary artery and aortic SMC produced no detectable GM-CSF but constitutively made M-CSF, measured by ELISA and radioimmunoassay, respectively. Interleukin-1 (IL-1) and, to a lesser extent, tumor necrosis factor alpha (TNF alpha) stimulated GM-CSF formation within 3 h; mRNA levels also increased particularly in the presence of the protein synthesis inhibitor, cycloheximide. IL-1, TNF alpha and, in addition, interferon-gamma (IFN-gamma) raised the M-CSF levels within 6 h; cycloheximide potentiated the effects of IL-1 and TNF alpha on mRNA levels. These results suggest that cytokine-stimulated human arterial SMC may be a source of the M-CSF found in atherosclerotic lesions. Since monocytes/macrophages can be activated by GM-CSF and M-CSF, while GM-CSF can also affect granulocyte function, SMC may participate in inflammatory reactions and vascular diseases by releasing these cytokines.     

Circulating levels of the macrophage colony stimulating factor CSF-1 in primary and metastatic breast cancer patients. A pilot study

Earlier results [1], suggesting an autocrine tumor cell stimulation by CSF-1, are in agreement with data by Fildermann et al. [2], showing an enhanced motility and invasiveness in the CSF-1 receptor expressing BT20 breast cancer cell line upon stimulation with recombinant CSF-1. Tumor-cell secreted CSF-1 has also been shown to cause monocyte recruitment, but not cytotoxicity [3]. Down-regulation of monocyte class II antigen expression after exposure to high concentrations of CSF-1 [4] may decrease macrophage-mediated tumor cytotoxicity and favor tolerance. Raised CSF-1 serum levels may thus increase tumor metastatic behavior as well as cause immune suppression in advanced stage disease. We set out to evaluate serum CSF-1 levels in primary and metastatic breast cancer. Serum samples from one hundred and eighteen primary breast cancer patients and seventy-five patients with metastatic disease were assayed by radio-immuno-assay (RIA) for circulating colony-stimulating factor 1. Mean serum levels were significantly higher in the metastatic population (9.7 ng/ml +/- 0.8) as compared to the patients with primary tumors (4.2 +/- 0.2) (p = 0.0001). Patients with early stage tumors (T0/T1/T2) had significantly lower levels than patients with tumors of larger size (T3/T4) (p = 0.0001). Relapse and survival statistics were analyzed using Kaplan-Meier estimates. Samples from 118 primary breast cancer patients were available to study. The median follow up was 85 months (range: 1-108). An elevated CSF-1 concentration (> 6.6 ng/ml or > 550 Units/ml) was associated with a shorter disease free interval (p = 0.03). In a multivariate analysis, including T (clinical tumor size), N (clinical node status), histological grade, and hormone receptor status, CSF-1 remained significantly associated with a poorer outcome (relative risk of relapse: RR: 3.3 [1.3-8.5]), together with tumor size (RR: 2.8[1-8.2]) and clinically involved nodes (RR: 4.1[2.1-8]). These results were not modified following adjustment for type of treatment. We conclude that raised circulating CSF-1 levels may be an indicator of early metastatic relapse.     

Effect of macrophage colony stimulating factor on the advanced atherosclerosis in Watanabe heritable hyperlipidemic rabbits

We have reported that macrophage colony-stimulating factor (M-CSF) prevents atherosclerosis in young WHHL rabbits (Atherosclerosis 93:245, 1993). In the present study, we injected recombinant human M-CSF (250 micrograms/day) into WHHL rabbits aged 11 months 3 times a week after advanced atherosclerosis was established. After 8 months of treatment, we did not find any significant difference in plasma lipid levels, cholesterol ester content in the aorta or macroscopic atherosclerosis lesion area between M-CSF treated and non-treated rabbits. There was, however, a significant difference in the ratio of intimal to medial thickness (1.08 vs 1.7, p < 0.01). Thus, M-CSF may influence vascular smooth muscle cell function and modify the process of atherosclerosis in advance lesions.     

Lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte-macrophage colony stimulating factor

Mice deficient in granulocyte-macrophage colony stimulating factor (GM-CSF) develop pulmonary alveolar proteinosis (PAP). We found that bronchoalveolar lavage fluid (BALF) from 11 patients with idiopathic pulmonary alveolar proteinosis (IPAP) suppressed the growth of peripheral blood monocytes and TF-1 cells, a cell line dependent on either GM-CSF or interleukin-3 (IL-3). The inhibitory effect of PAP-BALF occurred only when TF-1 cells were cultured with GM-CSF but not when cultured with IL-3, suggesting that PAP-BALF contains a factor that specifically interferes with GM-CSF function. 125I-GM-CSF binding to TF-1 cells was prevented in the presence of BALF from IPAP patients. Furthermore, cross-linking of 125I-GM-CSF to IPAP-BALF produced two major bands on SDS-PAGE; these bands were not observed in normal BALF. These data suggest that IPAP is caused by expression of binding factor(s) which inhibit GM-CSF function in the lung.     

Dexamethasone enhances macrophage colony stimulating factor- and granulocyte macrophage colony stimulating factor-stimulated proliferation of bone marrow-derived macrophages

The authors describe a study in which they placed 126 Class V composite resin restorations without mechanical retention, divided into three groups of 42, in 23 patients. They followed the performance of the restorations over a three-year period. For all three groups, restorations were placed using All-Bond 2 dental adhesive and Z100 composite resin; A.R.T. Bond and Brilliant Dentin composite; and Prisma Universal Bond 3 and Variglass VLC polyacid-modified composite resin. The authors evaluated retention as well as color stability, wear resistance, sensitivity, sulcular depth, loss of attachment, bleeding on probing and crevicular fluid flow. Based on their results, the authors propose that restoration of Class V lesions without using mechanical retention could be expected to succeed in seven of 10 restorations over a three-year period using these restorative systems.