Granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor in the treatment of acute myeloid leukemia and acute lymphoblastic leukemia

To explore the role of perfectionism across anxiety disorders, 175 patients with either panic disorder (PD), obsessive compulsive disorder (OCD), social phobia, or specific phobia, as well as 49 nonclinical volunteers, completed two measures [Frost, R. O., Marten, P., Lahart, C., & Rosenblate, R., (1990). The dimensions of perfectionism. Cognitive Therapy and Research, 14, 449-468; Hewitt, P. L., & Flett, G. L., (1991). Perfectionism in the self and social contexts: Conceptualization, assessment and association with psychopathology. Journal of Personality and Social Psychology, 60, 456-470.] that assess a total of nine different dimensions of perfectionism. Relative to the other groups, social phobia was associated with greater concern about mistakes (CM), doubts about actions (DA), and parental criticism (PC) on one measure and more socially prescribed perfectionism (SP) on the other measure. OCD was associated with elevated DA scores relative to the other groups. PD was associated with moderate elevations on the CM and DA subscales. The remaining dimensions of perfectionism failed to differentiate among groups. The clinical implications of these findings are discussed.     

Tumor growth alters T cell and macrophage production of and responsiveness to granulocyte-macrophage colony-stimulating factor: partial dysregulation through interleukin-10

Tumor growth induces phenotypic and functional changes among splenic T cells and macrophages (M phi) that contribute to the immunosuppression observed in tumor-bearing hosts (TBH). These changes partly arise through alterations in immune cell production of and responsiveness to cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important T cell- and M phi-derived cytokine that is produced during normal host immunogenic challenge, but it's involvement during cancer is poorly defined. In contrast, interleukin-10 (IL-10) is an inhibitory cytokine that is produced by immune cells as a deactivation factor. IL-10 can disrupt GM-CSF synthesis and may be associated with tumor-induced changes in cytokine synthesis. We determined if tumor growth alters T-cell and M phi synthesis of and responsiveness to GM-CSF, and if these alterations occur because tumor growth heightens immune cell sensitivity to IL-10. Tumor growth significantly decreased T-cell synthesis of GM-CSF during activation by concanavalin A, and TBH T cells were more susceptible to GM-CSF synthesis inhibition by IL-10 than their normal host (NH) counterparts. This suppression was observed using both unseparated splenic lymphocyte preparations and purified CD4+ and CD8+ T cells. Similarly, TBH M phi (both splenic and peritoneal) produced less GM-CSF than NH M phi during activation by lipopolysaccharide. Tumor growth also altered major histocompatibility complex (MHC) class II- M phi GM-CSF synthesis. TBH M phi were more susceptible to GM-CSF synthesis inhibition by IL-10 than their NH counterparts. Although TBH T cells demonstrate less proliferation than NH T cells during activation, tumor growth did not compromise T-cell responsiveness to GM-CSF. However, tumor growth did increase TBH T-cell susceptibility to inhibition of proliferation by IL-10. Tumor growth suppressed M phi responsiveness to GM-CSF, and IL-10 further decreased M phi responsiveness to GM-CSF. Collectively, these results suggest that T cell and M phi production of and responsiveness to GM-CSF is disrupted during tumor growth, and that TBH T cells and M phi are more susceptible to the suppressor activity of IL-10 than their NH counterparts.     

Cooperative effects of interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor: a new myeloid cell line inducible to eosinophils

The cytokines interleukin-3 (IL-3); IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to contribute to the proliferation and differentiation of eosinophil progenitors. Recently, it was determined that the cellular receptors for these three cytokines share a common beta-chain while having unique alpha-chains. Thus, there is considerable interest in how these cytokines and their receptors interact in promoting production of eosinophils. We have established a cell line (AML14) from a patient with acute myelogenous leukemia that will consistently exhibit eosinophilic differentiation in suspension in response to IL-3, IL-5, and GM-CSF. Proliferation with only modest differentiative effects was observed in response to a single cytokine. Combinations of two cytokines gave variable results, with GM-CSF + IL-3 and IL-3 + IL-5 causing more proliferation than a single cytokine but little more differentiation. The combination of GM-CSF + IL-5 caused marked enhancement of eosinophilic differentiation with only modest augmentation of proliferation. The combination of all three cytokines was most effective in stimulating both proliferation and eosinophilic differentiation (up to 70% of cells) of AML14 cells. Specific binding of GM-CSF and IL-5 to AML14 cells can be conveniently studied by flow cytometric methods, and cross-competition of these two cytokines for their respective receptors was demonstrated. IL-3 was shown to partially compete for IL-5 binding on AML14 cells. Although specific IL-3 binding could not be demonstrated by flow cytometry, mRNA for the alpha-chains of the IL-3, IL-5, and GM-CSF receptors and the beta-chain common to all three receptors was detected in AML14 cells. The AML14 cell line may be a useful model for the study of cooperative interactions of IL-3, IL-5, GM-CSF, and their respective receptors in the promotion of eosinophil progenitor growth and differentiation.     

Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow

Expression of the 210-kD bcr/abl fusion oncoprotein can cause a chronic myelogenous leukemia (CML)-like disease in mice receiving bone marrow cells transduced by bcr/abl-encoding retroviruses. However, previous methods failed to yield this disease at a frequency sufficient enough to allow for its use in the study of CML pathogenesis. To overcome this limitation, we have developed an efficient and reproducible method for inducing a CML-like disease in mice receiving P210 bcr/abl-transduced bone marrow cells. All mice receiving P210 bcr/abl-transduced bone marrow cells succumb to a myeloproliferative disease between 3 and 5 weeks after bone marrow transplantation. The myeloproliferative disease recapitulates many of the hallmarks of human CML and is characterized by high white blood cell counts and extensive extramedullary hematopoiesis in the spleen, liver, bone marrow, and lungs. Use of a retroviral vector coexpressing P210 bcr/abl and green fluorescent protein shows that the vast majority of bcr/abl-expressing cells are myeloid. Analysis of the proviral integration pattern shows that, in some mice, the myeloproliferative disease is clonal. In multiple mice, the CML-like disease has been transplantable, inducing a similar myeloproliferative syndrome within 1 month of transfer to sublethally irradiated syngeneic recipients. The disease in many of these mice has progressed to the development of acute lymphoma/leukemia resembling blast crisis. These results demonstrate that murine CML recapitulates important features of human CML. As such, it should be an excellent model for addressing specific issues relating to the pathogenesis and treatment of this disease.     

The kinetics and extent of engraftment of chronic myelogenous leukemia cells in non-obese diabetic/severe combined immunodeficiency mice reflect the phase of the donor's disease: an in vivo model of chronic myelogenous leukemia biology

In vitro studies have provided little consensus on the kinetic abnormality underlying the myeloid expansion of chronic myelogenous leukemia (CML). Transplantation of human CML cells into non-obese diabetic mice with severe immunodeficiency disease (NOD/SCID mice) may therefore be a useful model. A CML cell line (BV173) and peripheral blood cells collected from CML patients in chronic phase (CP), accelerated phase (AP), or blastic phase (BP) were injected into preirradiated NOD/SCID mice. Animals were killed at serial intervals; cell suspensions and/or tissue sections from different organs were studied by immunohistochemistry and/or flow cytometry using antihuman CD45 monoclonal antibodies (MoAbs), and by fluorescence in situ hybridization (FISH) for the BCR-ABL fusion gene. One hour after injection, cells were sequestered in the lungs and liver, but 2 weeks later they were no longer detectable in either site. Similar short-term kinetics were observed using 51Cr-labeled cells. The first signs of engraftment for BV173, AP, and BP cells were detected in the bone marrow (BM) at 4 weeks. At 8 weeks the median percentages of human cells in murine marrow were 4% (range, 1 to 9) for CP, 11% (range, 5 to 36) for AP, 38.5% (range, 18 to 79) for BP, and 54% (range, 31 to 69) for BV173. CP cells progressively infiltrated BM (21%) and spleen (6%) by 18 to 20 weeks; no animals injected with the cell line or BP cells survived beyond 12 weeks. The rate of increase in human cell numbers was higher for BP (7.3%/week) as compared with CP (0.9%/week) and AP (0. 5%/week). FISH analysis with BCR and ABL probes showed that some of the human cells engrafting after injection of CP cells lacked a BCR-ABL gene and were presumably normal. We conclude that CML cells proliferate in NOD/SCID mice with kinetics that recapitulate the phase of the donor's disease, thus providing an in vivo model of CML biology.     

Characterization of the microheterogeneities of PIXY321, a genetically engineered granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein expressed in yeast

PIXY321, a human cytokine analog genetically engineered by the fusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), was expressed in yeast under the control of the alcohol dehydrogenase 2 (ADH2) promoter and the alpha-mating factor expression system. To provide the material necessary for the evaluation of PIXY321 in clinical trials, the production was scaled up to the 1200-1 scale and the PIXY321 molecule isolated by four successive steps of ion-exchange chromatography. Multiple heterogeneities, due to the presence of different patterns of glycosylation as well as multiple amino acid sequences at both N and C termini, were characterized on the purified molecule using complementary analytical techniques including electrophoresis, liquid chromatography and electrospray mass spectrometry. Four different N-terminal sequences were identified but simplified to a reproducible ratio of two sequences, the mature form and a form starting at Ala3, by adjustment of the process conditions. Molecules lacking 1-6 residues at the C-terminus were identified and their relative frequencies quantified. Amino acid modifications, such as three oxidized Met residues at positions 79, 141 and 187 and one deamidated Asn residue at position 176, were detected at low level. Microheterogeneities in glycosylation were characterized on four different sites, one located in the GM-CSF portion and three in the IL-3 portion of the molecule. The sites were shown to be differentially occupied and to carry 0-10 mannose residues according to their location in the sequence. Precise measurement of the heterogeneities at the molecular level were used to tune the process conditions and ensure reproducibility of the clinical product between lots.     

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.     

Granulocyte-macrophage colony-stimulating factor and interleukin-3 potentiate interferon-gamma-mediated endothelin production by human monocytes: role of protein kinase C

Monocytic cells have been shown to produce endothelin, a potent vasoconstrictor molecule with immune modulating properties. The signalling mechanisms involved in this response are presently unclear. Monocytes are also believed to play an important role in inflammatory bowel disease (IBD). The objective of this study was to characterize the role of various cytokines, bacterial lipopolysaccharide (LPS) and colony-stimulating factors on the production of endothelin (ET) by freshly isolated human monocytes. Compelling circumstantial evidence exists for the conditions being investigated occurring in inflamed bowel mucosa to where monocytes migrate. Whereas LPS stimulated the release of 7 pg ET/2x106 cells in 40 hr, interferon-gamma (IFN-gamma) stimulated 45 pg ET/2x106 cells in 40 hr. There was an additive response when the two stimuli were employed together. Significantly the addition of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) effected a two- to threefold, dose-dependent increase in the production of ET. Production of endothelin was reproducibly blocked by the addition of the protein kinase C (PKC) inhibitors staurosporine and H7, as well as by the protein synthesis inhibitor cycloheximide. Assessment of the activities of the alpha and beta isoforms of conventional protein kinase C (PKC), as determined by MonoQ column fractionated calcium and lipid activatible phosphotransferase activity towards myelin basic protein (MBP) revealed an additive effect of using LPS, IFN-gamma and GM-CSF, which was even greater than that demonstrated for phorbol myristate acetate (PMA). Additionally the secretion of ET by monocytes from Crohn's disease patients (in remission) was analysed and compared with an age-matched control group. There was no significant difference between the two. These results: (1) demonstrate an important synergistic role for GM-CSF and IL-3 in the predominantly IFN-gamma-mediated ET production by normal human monocytes; (2) indicate a possible role for the protein kinase C signalling pathway in this response; and (3) argue against a primary abnormality of ET production in peripheral monocytes from patients with Crohn's disease.     

Granulocyte colony-stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage CSF, interleukin-3, and interleukin-6 levels in sera from children undergoing blood stem cell autografts

Endogenous production of granulocyte colony-stimulating factor (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin-3 (IL-3), and interleukin-6 (IL-6) was investigated in 10 children who underwent a total of 12 courses of autologous peripheral blood stem cell transplant (PBSCT) by measuring their serum levels using immunoassay kits. The serum G-CSF level increased immediately following infusion of PBSC graft, peaked between days 3 and 7 posttransplant and then declined by the time the granulocyte count rose. No definitive association was found between the continuous high levels of G-CSF and infective episodes, the number of infused nucleated cells, monocytes, CFU-GM, or the number of days required to achieve greater than 0.5 x 10(9)/L granulocyte, greater than 1.0 x 10(9)/L leukocyte, or greater than 50 x 10(9)/L platelet counts. After PBSCT, IL-6 levels tended to be elevated. No detectable serum level of GM-CSF or IL-3 (< 50 pg/mL) was observed before PBSCT and 4 patients showed a transient increase in the GM-CSF level after PBSCT. No significant change was observed in the post-transplant serum levels of IL-3 or M-CSF. The role of endogenously secreted cytokines in early hematopoietic recovery after PBSCT needs further clarification, but, at present, routine use of exogenous G-CSF therapy is not recommended.     

Cloning and characterization of the human interleukin-3 (IL-3)/IL-5/ granulocyte-macrophage colony-stimulating factor receptor betac gene: regulation by Ets family members

High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific chain and a common beta chain (betac). Whereas the mouse has two homologous beta subunits (betac and betaIL-3), in humans, only a single beta chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor beta subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine betac/betaIL-3 genes. Surprisingly, we also found the remnants of a second betac chain gene directly downstream of betac. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from -103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5' element specifically binds PU.1, whereas the 3' element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances betac promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.     

Mast cells enhance eosinophil survival in vitro: role of TNF-alpha and granulocyte-macrophage colony-stimulating factor

Mast cell-eosinophil interactions in allergy have not yet been completely defined. To determine whether mast cells influence eosinophil survival, human peripheral blood eosinophils were incubated with rat peritoneal mast cell sonicate. After 3 days, viable eosinophils in medium were 21.3% compared with 44% with mast cell sonicate. Like sonicate, supernatants of compound 48/80-activated mast cells enhanced eosinophil survival, demonstrating that the factor(s) involved is stored preformed and rapidly released. Increased eosinophil survival was due to an inhibition of apoptosis (morphologic analysis; annexin V/PI). Neutralizing Abs to granulocyte-macrophage CSF (GM-CSF), but not to IL-3 or IL-5, decreased by 61.7% the enhancing effect on eosinophil viability. Eosinophils are the source of GM-CSF since its release in the culture medium was inhibited by their incubation with the mast cell sonicate together with dexamethasone. In addition, eosinophils incubated with the sonicate expressed mRNA for GM-CSF. To partially characterize the mast cell-derived factor(s) increasing eosinophil survival, the sonicate was heated (56 degrees C/30 min or 100 degrees C/10 min) or preincubated with antihistamines or with anti-TNF-alpha-neutralizing Abs. Most of the activity was heat labile. TNF-alpha was found to be predominantly (70%) responsible, while histamine had no role. Mast cell sonicate also caused eosinophils to release eosinophil peroxidase and to display morphologic signs of activation. In conclusion, we have demonstrated that mast cells enhance eosinophil survival in part through their activation to produce and release the autocrine survival cytokine GM-CSF.     

Cryptosporidium parvum infection of human intestinal xenografts in SCID mice induces production of human tumor necrosis factor alpha and interleukin-8

The protozoan parasite Cryptosporidium parvum invades intestinal epithelial cells and can cause life-threatening diarrhea in immunocompromised individuals. Despite the clinical importance of this organism, much remains to be learned about the pathogenesis of C. parvum-induced diarrhea. To explore the role of the intestinal inflammatory response in C. parvum disease, using C. parvum oocysts we infected human intestinal xenografts in severe combined immunodeficient (SCID) mice. Seven days after infection, we found levels of human tumor necrosis factor alpha and interleukin-8 in C. parvum-infected human intestinal xenografts that were significantly higher than those seen in uninfected control xenografts. These results demonstrate that human intestinal cells produce proinflammatory cytokines in response to C. parvum infection and establish SCID-HU-INT mice as a model system to study the interactions of C. parvum with the human intestine.     

Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine

The necessity for prolonged tissue culture manipulations limits the clinical application of many form of gene therapy in patients with malignancies. We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a plasmid expression vector could be effectively introduced into resting tumor cells, without the need for tissue culture propagation prior to or following transfection, and that efficient expression of transgenic GM-CSF by the transfected tumor cells would confer an effective immune response against tumors. GM-CSF cDNA in expression vectors was coated onto gold particles and accelerated with a gene gun device into mouse and human tumor cells. Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice. Transfected and irradiated murine B16 melanoma cells produced > or = 100 ng/ml murine GM-CSF/10(6) cells per 24 hr in vitro for at least 10 days. The antitumor efficacy of this nonviral approach was tested using irradiated B16 tumor cells that were transfected with mGM-CSF cDNA and injected into mice as tumor "vaccine". Subsequent challenge of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals wer protected from the tumor by the prior vaccine treatment. In contrast, only 2% of control animals were protected by prior treatment with irradiated B16 cells transfected with the vector containing the luciferase gene. These results suggest that particle-mediated transfection of fresh tumor explants with cytokine cDNA is an effective and clinically attractive approach for cancer therapy.     

In vivo effect of granulocyte-macrophage colony-stimulating factor and interferon combination on monocyte-macrophage and T-lymphocyte functions in chronic hepatitis B leukocytopenic patients

BACKGROUND/AIMS: Both rhGM-CSF and IFN-gamma have antiviral and immunoregulatory effects. Furthermore, GM-CSF has the advantage of increasing WBC in leukocytopenic patients. METHODOLOGY: We investigated a) the antiviral effects of rhGM-CSF and INF-alpha combination treatment in 12 chronic hepatitis B patients with leukocytopenia as a result or not of previous interferon therapy, b) the in vivo effects of these agents on monocyte-macrophage and T-lymphocyte functions and, c) their correlation to HBV infection outcome. RESULTS: Combination therapy caused a significant fall in HBV-DNA levels (p<0.0002), accompanied by significant reductions in transaminase levels and in histological activity index (p<0.0001, in each case). In parallel, rhGM-CSF induced a 2.7- to 5-fold weekly increment in WBC. Moreover, treatment caused a significant increase in all monocyte-macrophage parameters (p<0.0001 for random, directed migration and phagocytosis index) and in peripheral blood lymphocyte parameters (p<0.0001 for IL-2r and HLA-DR expression) studied. A similar picture was also obtained from cytokine levels (IL-2 and GM-CSF) in the supernatants from PHA-cultured T-lymphocytes (p<0.0003, <0.001, respectively). CONCLUSIONS: The combined therapy achieves the initial treatment aim of increasing WBC and exerting an antiviral effect. In addition, the observed changes in immunological parameters probably reflect a Th1 pattern of immune response that could be responsible for the fate of HBV infection. Finally, cytokine levels (IL-2 and GM-CSF) in the supernatants might serve to monitor viral activity and outcome of the HBV infection.     

Cytokine production and function in c-mpl-deficient mice: no physiologic role for interleukin-3 in residual megakaryocyte and platelet production

Mice lacking thrombopoietin (TPO), or its receptor c-Mpl, display defective megakaryocyte and platelet development and deficiencies in progenitor cells of multiple hematopoietic lineages. The contribution of alternative cytokines to thrombopoiesis in the absence of TPO signalling was examined in mpl-/- mice. Analysis of serum and organ-conditioned media showed no evidence of a compensatory overproduction of megakaryocytopoietic cytokines. However, consistent with a potential role in vivo, when injected into mpl-/- mice, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) retained the capacity to elevate megakaryocytes and their progenitors in hematopoietic tissues and increase circulating platelet numbers. However, double mutant mice bred to carry genetic defects both in c-Mpl and IL-3 or the alpha chain of the IL-3 receptor, displayed no greater deficiencies in megakaryocytes or platelets than mpl-deficient animals, suggesting absence of a physiologic role for IL-3 in the residual megakaryocytopoiesis and platelet production in these mice.     

Increased risk of acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) in a county of Hesse, Germany

The incidence of acute and chronic myelogenous leukemia has been compared for the two neighbouring regions of Marburg and Giessen in Hesse (Germany). The investigation was based on the incident cases of the years 1983-1989 which have been diagnosed in the hematological departments of the universities of the two regions. The epidemiological evaluation of the data has been carried out in terms of a historical follow-up study, and shows an increased relative risk for the region around Marburg with a particular elevation for one community within this region. Potential determinants are discussed and focus on trinitrotoluene (TNT) and decomposition products which are known to contaminate the soil of this community, in some places severely, due to insufficient removal of remnants of the TNT production in large underground plants during World War II.     

Extracellular truncations of h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5, lead to ligand-independent activation

The hypothesis that extracellular truncation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 (h beta c) can lead to ligand-independent activation was tested by infecting factor-dependent hematopoietic cell lines with retroviruses encoding truncated forms of h beta c. A truncation, resembling that in v-Mpl, and retaining 45 h beta c-derived extracellular residues, led to constitutive activation in the murine myeloid cell line, FDC-P1. However, infection of cells with retrovirus encoding a more severely truncated receptor, retaining only 7 h beta c-derived extracellular residues, did not confer factor independence on these cells. These experiments show that truncation activates the receptor and define a 37-amino acid segment of h beta c (H395-A431) which contains two motifs conserved throughout the cytokine receptor superfamily (consensus Y/H XX R/Q VR and WSXWS), as essential for factor-independent signaling. The mechanism of activation was also investigated in less severe truncations. A receptor that retains the entire membrane-proximal domain (domain 4) also conferred factor independent growth on FDC-P1 cells; however, a retrovirus encoding a truncated form of h beta c having two intact membrane proximal domains did not have this ability, suggesting that domain 3 may have an inhibitory role in h beta c. The ability of these receptors to confer factor independence was cell specific as demonstrated by their inability to confer factor-independent growth when introduced into the murine IL-3-dependent pro-B cell line BaF-B03. These results are consistent with a model in which activation requires unmasking of an interactive receptor surface in domain 4 and association with a myeloid-specific receptor or accessory component. We suggest that in the absence of ligand intramolecular interactions prevent inappropriate signaling.     

Elevated expression of differentiation inhibitory factor nm23 mRNA in monoblastic crisis of a patient with chronic myelogenous leukemia

OBJECTIVE: To adapt desktop computer software to objectively grade facial movement. DESIGN: The criteria of the facial nerve grading system by House and Brackmann, the current "gold standard," are prone to ambiguous interpretation. Proposed objective grading systems compare the movement of points on each side of the face or use subtraction and thresholding of digitized images of the face to yield images that represent moving areas of the face. The movement of a point on the face and the area of motion determined by digital subtraction were compared during an increasing smile in healthy subjects. The Nottingham system (calculated using measurement of the movement of 4 points on the face) using desktop computer software (Adobe Photoshop 3.0, Adobe Systems Inc, Mountain View, Calif) to measure movement of the points was compared with the system by House and Brackmann. The computer software was used to subtract digitized images and derive a facial movement score, which was compared with the scores of the systems by Nottingham and House and Brackmann. SETTING: Academic otologic practice. STUDY PARTICIPANTS: Nine patients with varying degrees of facial nerve disability and 7 individuals with normal facial nerve function. RESULTS: The movement of the oral commissure compared with the apparent area of movement of the face determined by digital subtraction had high intersubject variability. In patients with facial weakness, the Nottingham score had a correlation coefficient of -0.97 compared with the House and Brackmann grade, and the digital subtraction score had a correlation coefficient of -0.62 (paired Student t test). CONCLUSIONS: The desktop computer software can be used to calculate the Nottingham score. Digital subtraction as a measure of facial function warrants further study.