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Biomedical subjects

G R Johnson

Publications and source records attributed to G R Johnson.

At least 145 records · Page 8Linked to original sources
PubMed1989

Mycobacteriosis in yellow perch (Perca flavescens) from two lakes in Alberta.

Granulomatous skin lesions were identified in a population of yellow perch (Perca flavescens) from Sandy Lake (Alberta, Canada) in 1985. Severe granulomatous peritonitis and hepatitis was identified subsequently in a separate population of yellow perch from Wolf Lake (Alberta, Canada). Mycobacterium chelonae was isolated from affected fish in both epizootics. The source and route of infection were not determined in either case. The nature of the inflammatory reaction in both cases suggested a depressed immunity.

Alberta↗
PubMed1988

Evidence for interactions between monocytes and natural killer cells in the regulation of in vitro hemopoiesis.

The role of accessory cells in modulation of in vitro hemopoiesis was studied using C-mediated lysis with mAb of differing specificities. One such antibody, 25E11, which bound to all NK cells and a subset of monocytes, consistently led to enhancement of in vitro hemopoiesis when 25E11+ cells were depleted by complement or the FACS. In cultures maximally stimulated by erythropoietin and human placental conditioned medium, depletion of 25E11+ cells from beta-thalassemic PBMC resulted in up to a fourfold enhancement of multi-potential and pure erythroid colonies and a twofold enhancement of non-erythroid colonies when compared with C-only treated cells. This enhancement of in vitro hemopoiesis was also observed with cells from normal bone marrow, cord blood, and peripheral blood. The enhancement of in vitro hemopoiesis was abrogated by removal of either adherent cells or monocytes using Leu M3 antibody and C lysis. The data presented herein suggest that NK cells and a subset of monocytes may exert a negative regulatory control on both granulopoiesis and erythropoiesis. Consequently, the number of progenitor and multipotential cells in cultures of unfractionated cell populations may be greatly underestimated.

Adult↗
PubMed1988

Transformation of single myeloid precursor cells by the malignant histiocytosis sarcoma virus (MHSV): generation of growth-factor-independent myeloid colonies and permanent cell lines.

Direct single-cell assays for oncogenic transformation are available for fibroblasts but not for other cell types. Using malignant histiocytosis sarcoma virus (MHSV), a member of the ras family of retroviruses, in vivo-infected granulocyte/macrophage and macrophage precursor cells lost the requirement for externally added hematopoietic growth factors. Factor-independent growth was demonstrated by colony-transfer experiments. More than 25% of the independent colonies were established as permanent macrophage cell lines following a phase of adaptation to tissue culture conditions. Factor-independent colony growth was also obtained by in vitro infection of single cells. As many as 50% of all myeloid precursor cells were target cells for MHSV as measured by this assay. About 2 x 10(-3) of these colony-forming cells acquired growth factor independence and immortality after in vitro infection. Cell lines derived from these colonies did not require adaptation to tissue culture conditions.

Animals↗
PubMed1988

Mojave toxin: rapid purification, heterogeneity and resistance to denaturation by urea.

This report establishes that purified Mojave toxin prepared from the snake venom of Crotalus scutulatus scutulatus contains multiple heterogeneous dimers (isoforms) differing slightly in isoelectric points. This conclusion is based upon chromatographic, immunological, sodium dodecyl sulfate--polyacrylamide gel electrophoretic and polyacrylamide isoelectric focusing experiments. The Mojave toxin-related proteins were rapidly purified from venom via a single chromatography step. Generation of Mojave toxin-related proteins from isolated subunits and immunoblots of these proteins subsequent to electrophoretic separation demonstrate that each of the proteins consists of acidic and phospholipase basic subunits. The analysis of venom in narrow range polyacrylamide isoelectric focusing gels at varying concentrations of urea, in conjunction with immunoblots utilizing antibodies specific to the basic subunit, demonstrates that the isoforms of Mojave toxin are native and not artifacts from isolation procedures. Analyses of venoms from Crotalus scutulatus scutulatus individuals indicate that each snake produces multiple isoforms of the neurotoxin. Additionally, the same predominant isoform of Mojave toxin is present in both individual and commercial venoms. The heterogeneity of the Mojave toxin-related proteins is largely due to differences in the acidic subunits and some of the forms may reflect post-translational processing of the protein. The Mojave toxin-related proteins demonstrate a resistance to urea denaturation by characteristically entering and focusing in polyacrylamide isoelectric focusing gels containing 0-6 M urea, but dissociating to constituent subunits in 8 M urea. Experimental evidence suggest that salt bridges may be important in stabilization of the Mojave toxin complex.

Animals↗
PubMed1988

In vitro-derived leukemic erythroid cell lines induced by a raf- and myc-containing retrovirus differentiate in response to erythropoietin.

In vitro infection of murine fetal liver cells with a retrovirus containing v-raf and v-myc oncogenes has produced continuous lines of immature erythroid cells that are leukemogenic. These cells synthesized a factor that stimulated their growth in vitro before autonomous variants emerged. Approximately 1000 high-affinity erythropoietin receptors could be detected per cell, and the hormone induced terminal differentiation in these cells. The lines were generated at an extremely low frequency (approximately 1 in 10(7) cells), suggesting that the combination of raf and myc is insufficient to develop erythroid cell lines and that additional events are necessary for transformation.

Abelson murine leukemia virus↗
PubMed1988

Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes.

In order to identify factors that influence expression by retroviral vectors in hemopoietic cells, we have compared viral RNA levels in cells infected with several different recombinant viruses. All of the vectors tested carry the neomycin resistance gene and provide for the insertion of a second gene which, in these studies, comprised sequences from the myc or myb oncogenes or the gene encoding granulocyte-macrophage colony-stimulating factor. The vectors utilize two different strategies for the coexpression of the two genes: alternate splicing and the use of a separate internal promoter. We found that expression in hemopoietic cells could be increased by substituting sequences from the myeloproliferative sarcoma virus long terminal repeat for those of the Moloney murine leukemia virus long terminal repeat. However, none of the vectors examined was able to express a second gene at levels equivalent to those achieved by the parental vectors carrying only the neomycin resistance gene. The reasons for this varied with the different vectors and included inefficient splicing and/or a reduction in the level of unspliced transcripts upon insertion of a second gene. Although the basis of the latter phenomenon is not clear, it is probably related to the position--near the 5' long terminal repeat--at which the second gene was inserted, since insertion of the same genes near the 3' end of another vector had no effect on viral RNA levels. In an attempt to circumvent some of these problems, we constructed a vector that employs an internal beta-actin promoter. Although this vector could express granulocyte-macrophage colony-stimulating factor sequences in a responsive hemopoietic cell line, the level of granulocyte-macrophage colony-stimulating factor produced was disappointingly low. The results from these studies suggest approaches to the design of improved vectors for effective expression of genes in hemopoietic cells.

Animals↗
PubMed1988

Recombinant retrovirus-mediated gene transfer to normal haemopoietic cells.

Several of parameters seeking to increase the infection frequency of normal haemopoietic cells by recombinant retroviruses containing a drug selectable marker and/or a haemopoietic growth factor gene have been compared. By using bone marrow cells pretreated with 5-fluorouracil, cocultivation of cells for at least 4 days with virus producing fibroblasts, and addition of pokeweed mitogen stimulated spleen cell conditioned medium, from 30 to 100% of haemopoietic spleen colony forming cells (CFU-S) could be infected. Because drug (G-418) selection was found to select subsets of CFU-S, a retrovirus lacking the drug selectable marker Neor, but containing a GM-CSF gene, was used to study the effects of endogenous GM-CSF production on CFU-S differentiation. Infected CFU-S produced equivalent numbers of erythroid and neutrophil-macrophage committed progenitor cells to control uninfected CFU-S. However, the progeny of infected CFU-S produced GM-CSF, some cells grew autonomously and neutrophil numbers were greatly increased at the expense of erythroblasts. These results suggest that unregulated GM-CSF production, although not altering commitment, alters the amplication phase following commitment.

Animals↗
PubMed1988

The effectiveness of shock-absorbing insoles during normal walking.

This paper describes a study of the effectiveness of commercially available shock absorbing insoles when used in four different pairs of shoes during normal walking. The measurement method was based on the use of the Fourier Transform of the axial acceleration of the leg measured by an accelerometer mounted at the ankle. The magnitude of shock was measured by the "Shock Factor" which has been defined as the rms acceleration between 50 Hz and 150 Hz expressed as a proportion of that between 10 Hz and 150 Hz. Nine insoles were tested in each pair of shoes and the Shock factor for each combination was compared with the value obtained for the shoes alone. Statistically significant reductions of Shock Factor were noted in 58% of cases; the largest improvement (30% reduction in Shock Factor) was achieved by lightweight Sorbothane. The experimental technique has now been further developed to allow the measurement of Shock Factor by a portable Shock Meter.

Biomechanical Phenomena↗
PubMed1988

Effects of excess granulocyte-macrophage colony stimulating factor (GM-CSF) in mice infected with a GM-CSF retrovirus.

To study the effects of dysregulated granulocyte-macrophage colony stimulating factor (GM-CSF) production, a retroviral vector (MPZen) containing a GM-CSF cDNA has been used to infect murine bone marrow cells prior to their transplantation into lethally-irradiated recipients. Animals transplanted with 1 x 10(6) bone marrow cells 14 to 21 days after transplantation displayed shortness of breath, trembling to the touch, and dragging of the hind limbs. These animals were shown to have increased peripheral blood neutrophil counts (x 200), decreased marrow cellularity (but with increased neutrophil and macrophage frequency) and increased spleen cellularity (with up to a 40-fold increase in neutrophil, macrophage and eosinophil frequency). Examination of sections of tissues from infected mice showed massive infiltration of neutrophils and macrophages in muscle, lungs, liver, and variable infiltration of these cells in lymphoid tissues and the eye. Haemopoietic tissues produced high levels of GM-CSF (10(5) Units/10(6) cells) and the serum contained up to 5.5 x 10(5) Units of GM-CSF per ml.

Animals↗
PubMed1988

Enhancement of in vitro beta-thalassemic and normal hematopoiesis by a noncytotoxic monoclonal antibody, 9.1C3: evidence for negative regulation of hematopoiesis by monocytes and natural killer cells.

The enhancement of in vitro human hematopoiesis by the addition of a noncytotoxic monoclonal antibody, 9.1C3, is described. Enhancement of all aspects of in vitro hematopoiesis was observed on addition of 9.1C3 antibody to cultures of mononuclear cells from normal bone marrow, cord blood, and peripheral blood from beta-thalassemia major patients. In cultures with no exogenous colony-stimulating factor (CSF), the addition of 9.1C3 resulted in a two- to eightfold increase in nonerythroid colony formation. Similarly, for cultures maximally stimulated with CSF, the addition of 9.1C3 antibody resulted in a one- to fourfold increase in colony formation. These effects were abrogated by the removal of either adherent, Leu-M3+ or Leu-7+ cells. Colony-forming cells were shown to be present among the 9.1C3-negative cells when mononuclear cells were sorted by flow cytometry. Media conditioned in the presence of 9.1C3 and mononuclear cells were able to enhance colony formation in vitro for normal nonadherent bone marrow cells beyond that achieved with supramaximal amounts of human placental-conditioned medium and erythropoietin. The data suggest that natural killer cells interact with monocytes to exert a negative regulatory control on in vitro granulopoiesis and erythropoiesis. Consequently, the number of progenitor and multipotential cells in cultures of unfractionated cell populations may be greatly underestimated.

Adjuvants, Immunologic↗
PubMed1988

Fractionation of subsets of BFU-E from normal human bone marrow: responsiveness to erythropoietin, human placental-conditioned medium, or granulocyte-macrophage colony-stimulating factor.

Normal human bone marrow mononuclear cells were fractionated by differential adherence, immunomagnetic separation, and fluorescence-activated cell sorting (FACS). The resultant fractionated cells were cultured in semisolid medium to monitor the presence of BFU-E, Mix-CFC, and nonerythroid CFC. Two populations of cells were recovered on the basis of binding by the monoclonal antibody (MoAb) RFB-1. One of these populations contained BFU-E that were stimulated only by erythropoietin (Epo), whereas the second population contained BFU-E responsive to Epo, Epo and recombinant human granulocyte-macrophage colony-stimulating factor (rHGM-CSF), or Epo and human placental-conditioned medium (HPCM). Prior enrichment of clonogenic cells by removal of adherent and Leu-M3+ve, Leu-4+ve, Leu-7+ve, B1+ve, WEMG1+ve, and Glycophorin A+ve cells, followed by FACS fractionation on the basis of RFB-1 binding, consistently resulted in recoveries of BFU-E, Mix-CFC, and nonerythroid CFC of greater than 100% (up to 800%). These procedures also resulted in enrichment of up to 200-fold and frequencies of 1:6 for BFU-E, 1:5 for CFC, and 1:130 for Mix-CFC.

Antibodies, Monoclonal↗
PubMed1988

Clonal proliferation in vitro of individual murine and human hemopoietic cells after fluorescence-activated cell sorting.

A modification of the fluorescence-activated cell sorter (FACS) was used to rapidly and reliably study the clonal proliferation of single hemopoietic cells. Murine FDC-P1 and human cord blood progenitor cells were examined for their ability to proliferate from single cells in 96-well microtiter plates containing agar medium and appropriate stimuli. FACS-sorted FDC-P1 single cells formed colonies in 345 out of 558 wells (62%), which compared favorably with control cultures (53%) and micromanipulated single cells (55%). Similarly, the frequency and type of day-14 colonies arising from cord blood progenitor cells when sorted as single cells by the FACS compared favorably with those grown from micromanipulated single cells or in control cultures.

Animals↗
PubMed1988

High haloperidol concentrations in a traffic suicide.

High concentrations of haloperidol are seen in a psychiatric patient who ran from a health care facility into traffic. Haloperidol concentrations were found to be 1.2 mg/L in heart blood, 2.7 mg/L in brain, and 10.8 mg/L in liver. No other drugs were detected.

Accidents, Traffic↗
PubMed1987

Macrophage cell lines transformed by the malignant histiocytosis sarcoma virus: increase of CSF receptors suggests a model for transformation.

The malignant histiocytosis sarcoma virus (MHSV) contains Ha-v-ras-related oncogenic sequences and rapidly transforms myeloid cells in vivo and in vitro. Myeloid cell lines can be derived which do not require growth factor for continued proliferation. We initiated this work to define the process of transformation leading to autonomy of cell growth in transformed myeloid cells. Five established cell lines were examined. All express macrophage-specific cell-surface antigens and exhibit several other properties typical for mature macrophages. Growth properties, growth factor release, and growth factor receptor presentation were examined: Release of growth factors is not a consistent feature. All cell lines show cell-density-independent colony formation and do not release self-stimulating factors, thus excluding autocrine stimulation as a model leading to transformation. All cell lines express unusually high levels of granulocyte-macrophage (GM)- and multi-CSF receptors and, except for one, M-CSF receptors. The high increase in GM-CSF and other growth factor receptors may be causally related to the transformed state of the cells. MHSV can be used as a tool to easily derive cell lines of the macrophage pathway as a model to study myeloid transformation, differentiation, and macrophage function.

Animals↗
PubMed1987

Dynamic back movement measured using a three-dimensional television system.

A technique for the measurement of rotational human back movements in three dimensions has been developed. The spatial coordinates of reflective markers on rigs attached to the back surface were calculated using a calibrated television/computer system. A mathematical analysis of the change in these coordinates, as subjects performed set manoeuvres, was developed to provide unique descriptions of the rotational movements in terms of clinical angles related to anatomical frames of reference. The technique produced angles of rotation with maximum errors about any axis of +/- 2 degrees, with a root mean square error of less than 1 degree. The analysis of back movements using surface markers was shown to be feasible from studies of six volunteers who demonstrated consistent patterns of movement which were similar to previously reported patterns of spinal movement measured radiographically.

Back↗
PubMed1987

In vitro maintenance of hemopoietic stem cells with lymphoid and myeloid repopulating ability by a cloned murine adherent bone marrow cell line.

Colonies containing blast cells (greater than 95%) have been characterized in 14-day cultures of normal murine bone marrow or spleen cells, stimulated by an adherent bone marrow cell line, B.Ad. These blast cell colonies occurred at a frequency of 1-2 per 2.5 X 10(4) bone marrow or 2.5 X 10(5) spleen cells and contained from 0.7 to 14.7 X 10(4) cells. Assay of individual blast cell colonies showed that they contained day 8 and day 13 colony-forming units-spleen (CFU-S) and from 11 to 10,434 in vitro CFC. Chromosomally marked blast colony cells were transplanted into lethally irradiated recipients and shown to produce both myeloid and lymphoid progeny in the recipients' bone marrow, spleen, mesenteric lymph node, and thymus.

Animals↗