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Biomedical subjects

G R Johnson

Publications and source records attributed to G R Johnson.

At least 163 records · Page 9Linked to original sources
PubMed1987

Purification and properties of bacterially synthesized human granulocyte-macrophage colony stimulating factor.

Human granulocyte-macrophage colony stimulating factor (GM-CSF) has been synthesized in high yield using a temperature inducible plasmid in Escherichia coli. The human GM-CSF is readily isolated from the bacterial proteins because of its differential solubility and chromatographic properties. The bacterially synthesized form of the human GM-CSF contains an extra methionine residue at position 1, but otherwise it is identical to the polypeptide predicted from the cDNA sequence. The specific activity of 2.9 X 10(7) units/mg of protein for purified bacterially synthesized human GM-CSF indicates that despite the lack of glycosylation, the molecule is substantially in its native conformation. This molecule stimulated the same number and type of both seven- and 14-day human bone marrow colonies as the CSF alpha preparation from human placental conditioned medium. Human GM-CSF had no activity on murine bone marrow or murine leukemic cells. There was no detectable, direct stimulation of adult human erythroid burst forming units (BFU-E) by the bacterially synthesized human GM-CSF. Although impure preparations containing native human GM-CSF (eg, human placental conditioned medium) stimulated the formation of mixed colonies, even in the presence of erythropoietin, the bacterially synthesized human GM-CSF failed to stimulate the formation of mixed colonies from adult human bone marrow cells. The bacterially synthesized human GM-CSF increased N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced superoxide production and lysozyme secretion. Antibody-dependent cytotoxicity and phagocytosis by human neutrophils was stimulated by the bacterially synthesized human GM-CSF and eosinophils were also activated in the antibody-dependent cytotoxicity assay.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1987

Generation of murine hematopoietic precursor cells from macrophage high-proliferative-potential colony-forming cells.

High-proliferative-potential colony-forming cells (HPP-CFC) have been described as primitive murine macrophage progenitors. We have previously demonstrated the existence of two populations of HPP-CFC: one population, termed HPP-CFC-1, is stimulated by the combination of macrophage colony-stimulating factor (CSF-1) plus haemopoietin-1 (H-1) and actively generates a second population of HPP-CFC, termed HPP-CFC-2. HPP-CFC-2 are stimulated by CSF-1 plus interleukin-3 and generate macrophage CFC that differentiate to form mature macrophages. In this study, we have demonstrated that HPP-CFC-1, when stimulated by CSF-1 plus H-1, generate colony-forming cells (CFC) for the megakaryocyte and granulocyte lineages in addition to HPP-CFC-2 and M-CFC. No CFC were detected with erythroid potential. In addition, HPP-CFC-1 generated cells that formed day-13 spleen colonies, cells that repopulated the bone marrow, cells with platelet-repopulating ability, and cells with erythroid-repopulating ability in lethally irradiated mice. These data support previous data that the HPP-CFC-1 represent a primitive hemopoietic cell population and demonstrate the multipotentiality but not totipotentiality of these cells.

Animals↗
PubMed1987

Quantitative responsiveness of murine hemopoietic populations in vitro and in vivo to recombinant multi-CSF (IL-3).

Purified, bacterially synthesized, recombinant Multi-CSF (rMulti-CSF) exhibited in vitro proliferative effects identical to those of native Multi-CSF on a broad range of progenitor cells from normal murine bone marrow and on the continuous cell line FD-CP1. The injection into normal adult mice of 6-200 ng purified rMulti-CSF three times daily for six days induced dose-related rises in peritoneal macrophages, neutrophils, and eosinophils, and in the spleen weight and content of megakaryocytes, mast cells, and progenitor cells, some responses being detectable with the lowest dose injected. No Multi-CSF was detected in the serum, organ extracts, or organ-conditioned medium from normal or endotoxin-injected mice. While the data indicate that even small doses of injected rMulti-CSF can produce detectable hemopoietic changes in normal adult mice, the pattern of these changes and the failure to detect Multi-CSF in vivo raise doubts that Multi-CSF plays a significant role as a regulator of hemopoiesis in normal adult life.

Animals↗
PubMed1987

Characterization of hemopoietic activities in media conditioned by a murine marrow-derived adherent cell line, B.Ad.

The hemopoietic activities present in medium conditioned by a murine bone marrow-derived adherent cell line (B.Ad) have been studied. B.Ad-conditioned medium stimulated neutrophil, neutrophil-macrophage, and macrophage colonies in agar cultures of bone marrow cells and 90% of this activity was neutralized by antimacrophage colony-stimulating factor (anti-M-CSF). The conditioned medium supported the generation and/or maintenance of spleen colony-forming units (CFU-S) in liquid cultures and synergized with multilineage colony-stimulating factor (Multi-CSF; IL-3) to stimulate colony formation by day-3 post-5-fluorouracil (FU)-treated bone marrow cells. When used as feeder layers, B.Ad cells stimulated erythroid colony-forming units (CFU-E) and markedly enhanced erythroid burst-forming units (BFU-E) stimulation more than did maximal Multi-CSF (IL-3) and Epo stimulation. No CFU-E- or BFU-E-stimulating activities were detected in medium conditioned by B.Ad cells. Similarly, B.Ad-conditioned medium was unable to stimulate Multi-CSF (IL-3) or granulocyte-macrophage (GM)-CSF-dependent cell lines. The data suggest that medium conditioned by this bone marrow-derived adherent cell line contains M-CSF and other factors not detectable as CSFs that either directly or by means of a synergistic mechanism are able to stimulate CFU-S and colony-forming cells (CFC).

Animals↗
PubMed1987

Prediction of the range of hand positions available to a patient with movement restrictions at the joints of the upper limb--a mathematical model.

A mathematical model has been constructed to predict the workspaces available to patients with reduced ranges of motion at the joints of the upper limb. The model uses the inverse kinematic method of Benati et al. (1982) together with simplified anatomical data. Comparisons with experimental data (Dempster, 1955) show significant discrepancies which are believe to be due to differences in scapular constraint and to simplified anatomical modelling. It is intended to improve the anatomical model by the use of the published data of Eyclesheimer and Shoemaker (1911). Despite the discrepancies, the model already detects significant differences between normal workspaces and those of patients with restriction at the joints of the upper limb. It is believed that this model could, when incorporated with empirical data, form the basis of an "expert" system to assist the therapist and clinician in the planning of rehabilitation of the upper limb.

Activities of Daily Living↗
PubMed1987

Transfusion-dependent beta-thalassemia: in vitro characterization of peripheral blood multipotential and committed progenitor cells.

Conditions for the in vitro culture of multipotential and committed progenitor cells obtained from the peripheral blood of transfusion-dependent beta-thalassemic individuals have been determined. When stimulated by either human placental-conditioned medium (HPCM), phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), or U5637-conditioned medium and erythropoietin, colonies containing neutrophils, neutrophils and macrophages, macrophages, eosinophils, erythroid cells, or mixtures of erythroid cells and neutrophils, eosinophils, or macrophages were obtained in cultures of beta-thalassemic mononuclear cells. Removal of adherent cells from beta-thalassemic peripheral blood demonstrated the presence of cells capable of the autostimulation of all of the above colony types in the presence of erythropoietin. In addition, adherent cells also appeared to inhibit erythropoiesis in cultures stimulated with HPCM. In cultures of nonadherent beta-thalassemic mononuclear cells, one population of BFU-E was stimulated by erythropoietin and a second required both erythropoietin and HPCM. Titration of erythropoietin, in the presence of optimal stimulation by HPCM, showed that beta-thalassemic peripheral blood BFU-E were more responsive to erythropoietin than was normal peripheral blood BFU-E. Stimulation of multipotential cells in cultures of nonadherent mononuclear cells was dependent upon HPCM. Using these culture conditions, the studies show that, compared with normals, the peripheral blood of transfusion-dependent beta-thalassemic patients contain elevated numbers of multipotential and committed progenitor cells, and that splenectomy did not account for the elevated numbers of these cells.

Cells, Cultured↗
PubMed1987

Expression of genes transferred to haemopoietic stem cells by recombinant retroviruses.

We have compared several recombinant retroviruses carrying the selective marker NeoR for gene delivery to murine haemopoietic stem cells. Provirus content and expression was assessed in animals reconstituted with infected bone marrow cells. With viruses bearing only a NeoR gene, viral RNA was readily detectable both in spleen colonies and in the haemopoietic tissues of long-term reconstituted mice. Thus, the progeny of infected primitive multipotential cells are competent to express integrated proviruses. The level of expression was significantly greater for proviruses carrying a modified long terminal repeat in which the Moloney enhancer had been replaced with that from the myeloproliferative sarcoma virus. With viruses bearing the c-myc gene in addition to NeoR, however, no viral RNA could be detected in spleen colonies harbouring proviral inserts, even though these viruses are expressed in vitro within haemopoietic cells. The implications of these results for gene transfer to haemopoietic stem cells are discussed.

Animals↗
PubMed1986

In vitro actions on hemopoietic cells of recombinant murine GM-CSF purified after production in Escherichia coli: comparison with purified native GM-CSF.

Recombinant murine GM-CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM-CSF. Both recombinant and native GM-CSF stimulated granulocyte and/or macrophage colony formation by adult and fetal mouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM-CSF (25 X 10(8) U/mg) was similar to that of the native form (15 X 10(8) U/mg). At high concentrations (greater than 200 U/ml), both forms of GM-CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (greater than 800 U/ml), megakaryocyte and some erythroid and mixed-erythroid colony formation. Recombinant GM-CSF was as effective in stimulating the proliferation of the GM-CSF-dependent cell line FD as the native molecule. Both recombinant and native GM-CSF were able to induce partial differentiation in colonies of WEHI-3B myeloid leukemic cells. Recombinant GM-CSF competed effectively for the binding of 125I-labeled native GM-CSF to hemopoietic cells, and antiserum to recombinant GM-CSF also neutralized the biological activity of native GM-CSF. The bacterially synthesized GM-CSF was a slightly more effective stimulus for megakaryocyte colony formation than the native molecule. The demonstration that purified bacterially synthesized GM-CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.

Animals↗
PubMed1986

Hemopoietic effects of purified bacterially synthesized multi-CSF in normal and marrow-transplanted mice.

Normal adult mice and irradiated, marrow-transplanted mice were injected three times daily for 6 days with 200 ng purified bacterially synthesized recombinant Multi-CSF. Normal mice responded with increased peritoneal macrophages, eosinophils and neutrophils, spleen weight increases and major increases in spleen mast cells and hemopoietic progenitor cells. Irradiated, marrow-transplanted mice failed to develop large increases in peritoneal cell numbers but did show rises in spleen weight, marrow cellularity and spleen and marrow progenitor cell levels. The results confirm data indicating a regulatory role for Multi-CSF in vivo and suggest that Multi-CSF may be of some value in accelerating hemopoietic regeneration following marrow transplantation.

Animals↗
PubMed1986

Veterans Administration cooperative study on hepatitis and dentistry.

Personnel in the VA dental facilities were screened for the detection of viral hepatitis and identification of factors implicating infectivity. A total of 963 personnel from 126 dental facilities throughout the United States voluntarily participated in the study. The rate of seroconversion for any hepatitis B markers was approximately 1% per year. Serial positive tests for antibody to hepatitis B core antigen or antibody to hepatitis B surface antigen (or both) were present in 16.2% of dentists and 13.0% of dental auxiliary personnel. Oral and maxillofacial surgeons composed the highest prevalence occupation (24.0%), and clinical personnel composed the lowest prevalence occupation (8.9%). There was a significant association between years in dental environment and serological positivity for viral B infection. The dentists and dental auxiliary personnel had significant linear trends of increasing serological positivity with years in the dental environment. Although a majority of personnel reported wearing gloves while treating high-risk patients or performing invasive procedures, inadequate prophylactic measures were exercised for most patients undergoing a variety of less invasive procedures. The results of the study show the need for an active immunization program against type B viral infection for dental and dental auxiliary personnel, preferably before the initial exposure to the professional environment.

Dental Auxiliaries↗
PubMed1986

Effects of purified bacterially synthesized murine multi-CSF (IL-3) on hematopoiesis in normal adult mice.

Normal adult C57BL, BALB/c, and C3H/HeJ mice were injected intraperitoneally three times daily for up to 6 days with 102,000 U (200 ng) per injection of purified, bacterially synthesized, Multipotential colony-stimulating factor (CSF) (Interleukin-3) (rMulti-CSF) and compared with control mice injected with serum/saline with or without added endotoxin (1 ng/mL). Mice injected with rMulti-CSF exhibited tenfold rises in blood eosinophil and twofold to threefold rises in neutrophil and monocyte levels. The spleens from mice injected with rMulti-CSF showed a 50% increase in weight, elevated levels of maturing granulocytes, eosinophils, nucleated erythroid cells and megakaryocytes, and up to 100-fold rises in mast cells. Progenitor cell frequencies in the spleen were elevated sixfold to 18-fold. No significant changes were observed in the marrow. Sixfold to 15-fold rises were observed in peritoneal cell populations of mice injected with rMulti-CSF with evidence of increased peritoneal macrophage phagocytic activity. Livers of C57BL mice, but not of the other strains, exhibited increased numbers of infiltrating hematopoietic cells whereas rises in mast cell numbers were observed in the mesenteric lymph node, skin, and gut in BALB/c and C3H/HeJ mice. Endotoxin was excluded as being responsible for the observed changes except possibly those involving peritoneal macrophage phagocytic activity. The results indicate that the injection of normal mice with rMulti-CSF significantly stimulates the same types of hematopoietic populations as are stimulated in vitro by Multi-CSF and indicate that this and other CSFs should be useful in stimulating hematopoietic repopulation and functional activity in vivo.

Animals↗
PubMed1986

Biologic properties in vitro of a recombinant human granulocyte-macrophage colony-stimulating factor.

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM-CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM-CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.

Animals↗
PubMed1985

Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse spleen cell conditioned medium.

A factor able to stimulate the proliferation and differentiation of multipotential stem cells and progenitor cells of the granulocyte-macrophage, eosinophil, and erythroid lineages as well as being able to maintain factor-dependent cell lines in culture has been purified from pokeweed mitogen-stimulated mouse spleen cell-conditioned medium. The factor was purified over 2 million-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-Sepharose, reverse-phase high performance liquid chromatography on a phenyl-silica column, and gel permeation high performance liquid chromatography. All of the biological activities ascribed to the multipotential colony-stimulating factor co-fractionated through all steps, and the other known mouse-active hemopoietic regulator in pokeweed mitogen-stimulated mouse spleen cell-conditioned medium, granulocyte-macrophage colony-stimulating factor, was separated at the ion exchange step. Two protein species having Mr = 24,000 and 19,000 were visualized by silver-staining of sodium dodecyl sulfate-polyacrylamide gels of the purified factor. Both species migrated coincidently with the biological activities. The factor was active at a half-maximal concentration of 1 X 10(-13) M when assayed on a factor-dependent cell line.

Animals↗
PubMed1985

Effects of a murine mammary tumor on in vivo and in vitro hemopoiesis.

The effects of an autologous transplanted mammary tumor (RIII-T3) on hemopoiesis in RIII mice are described. Tumor-bearing animals died 30 to 40 days after inoculation and displayed splenomegaly, extreme neutrophilia, and moderately increased monocyte levels in the spleen, peripheral blood, and bone marrow. The precursors of neutrophils and monocytes, granulocyte/macrophage colony-forming cells (GM-CFC) were elevated in the spleen, bone marrow, and peripheral blood. RIII-T3-conditioned medium stimulated bone marrow GM-CFC and caused the myelomonocytic cell line, WEHI-3B, to differentiate in vitro. The conditioned medium did not stimulate erythroid, megakaryocyte, or eosinophil colony formation. When conditioned medium was fractionated, two peaks of activity corresponding to GM-CSF and G-CSF were observed, suggesting that the extreme neutrophilia observed in tumor-bearing animals may result from chronic exposure of the hemopoietic system to these hemopoietic hormones.

Animals↗
PubMed1985

Evaluation of aortic stenosis by spectral analysis of the murmur.

A relation between the peak transaortic pressure gradient and the frequency content of the murmur (r = 0.79) was demonstrated in a prospective "test" set of 50 patients with the clinical diagnosis of aortic stenosis. After heart sounds were recorded and digitized, three segments of the systolic murmur were isolated and analyzed by fast Fourier transform technique. An average frequency spectrum was quantitated by a previously described empiric spectral estimator. Clinical data and spectral ratio were correlated with the transaortic pressure gradient and aortic valve area was calculated from cardiac catheterization data. The best prediction of the transaortic pressure gradient was obtained when a 170 ms murmur segment was analyzed and when the predictive algorithm also included the aortic dimension (r = 0.87). The aortic valve area was poorly predicted (r = -0.48) unless estimates of blood flow and valvular calcification were included in the algorithm (r = 0.84). Further refinement of this technique may provide a non-invasive and clinically useful method for the estimation of aortic valve stenosis.

Adult↗