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M Howard

Publications and source records attributed to M Howard.

At least 145 records · Page 8Linked to original sources
PubMed1992

[Value of electrocardiogram in localization of the coronary artery occlusion in acute inferior wall myocardial infarction].

Early EKG changes may contribute to predict the site of coronary artery occlusion during acute inferior myocardial infarction (MI). Its interpretation is relevant to therapeutic clinical decisions. We have prospectively evaluated early EKG changes of 40 consecutive patients with acute inferior MI and correlated them with the site and location of the coronary artery culprit lesion. Proximal right coronary artery occlusion was characterized by negative ST-T wave changes in leads D1 and aVL and ST segment elevation in leads D3 > D2. However the most distinctive EKG pattern of proximal right coronary artery occlusion was ST segment elevation with positive T wave in V4R (specificity 96%, predictive value 89%, p < 0.001). Distal right coronary artery occlusion was characterized by a positive T wave without ST segment elevation in V4R. Finally circumflex coronary artery occlusion was defined by a positive R/S > 1 relationship in V1, ST segment elevation in V5 and V6. Again lead V4R with flat or negative ST-T wave morphology had the highest predictive value for circumflex coronary artery occlusion (100%). Thus early EKG changes may contribute to precise the site and location of coronary artery occlusion and may help to implement clinical therapeutic strategies in patient with inferior MI. Right precordial leads are most useful in the EKG interpretation of inferior MI.

Adult↗
PubMed1992

The Advanced Trauma Life Support course for senior medical students.

The authors conducted the Advanced Trauma Life Support (ATLS) Course for 90 students who were in their 4th year of medicine at the University of Manitoba. The impact of the course was evaluated through questionnaires completed by students, instructors and emergency-room physicians. The students' performances were also compared with those of 96 practising physicians who took the ATLS course in Manitoba. The failure rate for students (3.3%) was not statistically different from that for practising physicians (4.2%). Overall, the students' performances in the written test were better (55% of students scored over 90% on the test compared with 15% of practising physicians). The student-to-faculty ratio was 1.5:1 and included 21 physician-instructors. Ninety-five percent of the faculty and students suggested that this course should be mandatory in the 4th year curriculum of medicine and that the course improves trauma care provided by the students and interns by increasing their confidence and improving communication with specialist surgeons. However, 10% of the faculty suggested that more time should be allocated to the surgical-skills practicum. The authors' experience with this program suggests that the ATLS course should be uniformly incorporated in the Canadian undergraduate 4th year medical curriculum and that techniques used in this course should be considered in other areas of the undergraduate medical curriculum.

Canada↗
PubMed1991

IL-10 inhibits cytokine production by activated macrophages.

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.

Animals↗
PubMed1991

IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells.

Murine IL-10 (cytokine synthesis inhibitory factor) inhibits cytokine production by Th1 cell clones when they are activated under conditions requiring the presence of APC. By preincubating APC with IL-10, we demonstrate that IL-10 acts principally on APC to inhibit IFN-gamma production by Th1 clones. Moreover, IL-10 is not active when Th1 cells are stimulated with glutaraldehyde-fixed APC, which also indicates that its action involves regulation of APC function. Furthermore, IL-10 inhibits cytokine synthesis by Th1 cells stimulated with the super-antigen Staphylococcus enterotoxin B, which does not appear to require processing. Flow microfluorimetry purified splenic or peritoneal B cells and macrophages, and B cell and macrophage cell lines can present Ag to Th1 clones. However, IL-10 acts only on sorted macrophages and the macrophage cell line to suppress IFN-gamma production by Th1 clones. IL-10 does not show this effect when B cells are used as APC. In contrast, IL-10 does not impair the ability of APC to stimulate cytokine production by Th2 cells. IL-10 does not decrease IFN-gamma-induced I-Ad levels on a macrophage cell line. Inasmuch as IL-10 also inhibits IL-2-induced IFN-gamma production by Th1 cells in an Ag-free system requiring only the presence of accessory cells, these data suggest that IL-10 may inhibit macrophage accessory cell function which is independent of TCR-class II MHC interactions.

Animals↗
PubMed1991

Interleukin-4 enhances anti-IgM stimulation of B cells by improving cell viability and by increasing the sensitivity of B cells to the anti-IgM signal.

The lymphokine IL-4 is a potent enhancer of anti-IgM-induced B cell proliferation. Although the mechanism of this enhancement is not known, a commonly held view suggests that IL-4 acts together with anti-IgM as a costimulating factor for the activation of a subpopulation of B cells. To evaluate this hypothesis we examined the effect of IL-4 on the proportion of B cells stimulated to divide by different doses of anti-IgM using flow cytometry in combination with measurements of tritiated-thymidine incorporation. The results suggest a novel and surprisingly simple model for the mode of action of IL-4. Our analysis revealed that at high saturating anti-IgM concentrations, the proportion of live B cells which enter into S phase of the cell cycle is the same (approximately 65%) for cells cultured with or without IL-4. Cultures containing IL-4, however, exhibit a twofold increase in thymidine uptake over cultures without IL-4. This increase can be explained completely by the ability of IL-4 to enhance the viability of small dense B cells over the first 24 hr from approximately 50 to 90% of the starting cell number. Normalizing the maximum response levels obtained with and without IL-4 reveals that B cells incubated with IL-4 exhibit a 10-fold decrease in the concentration of anti-IgM required to stimulate the half-maximum proliferation level. Furthermore, evaluation of the number of cells in S phase by flow cytometry and analysis of the kinetics of cell proliferation revealed that the increased response effected by IL-4 at lower anti-IgM concentrations was due to a greater number of proliferating B cells rather than the same number of cells undergoing a faster division rate. We also found a highly nonlinear relationship between B cell number and proliferative response, implying a requirement for an additional, cell cooperation-mediated, activating signal for maximum B cell proliferation. IL-4 enhanced proliferation by the same proportion at all cell concentrations indicating that it does not replace or alter this requirement for cell cooperation. Taken together these results suggest that anti-IgM in combination with a second unidentified cell-cooperation-dependent signal leads to proliferation of up to 65% of small resting B cells. IL-4 does not provide an essential activation signal but serves to raise the sensitivity of B cells to the anti-IgM-generated signal.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses.

Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.

Animals↗
PubMed1990

Interleukin 10, a novel B cell stimulatory factor: unresponsiveness of X chromosome-linked immunodeficiency B cells.

Highly purified, small dense splenic B cells from unstimulated mice showed increased expression of class II major histocompatibility complex (MHC) antigens and enhanced viability when cultured with affinity-purified recombinant interleukin 10 (rIL-10), compared with B cells cultured in medium alone. These responses were blocked by a monoclonal antibody (mAb) specific for IL-10, but not by an isotype-matched control antibody. IL-10 did not upregulate the expression of Fc epsilon receptors (CD23) or class I MHC antigens on small dense B cells or induce their replication as monitored by [3H]thymidine incorporation. While these B cell-stimulatory properties of IL-10 are also mediated by IL-4, the two cytokines appear to act independently in these assays; anti-IL-10 antibodies blocked IL-10 but not IL-4-mediated B cell viability enhancement, and vice versa. Similarly, since IL-4 upregulates CD23 on small dense B cells, the inability of IL-10 to do so argues against its acting via endogenously generated IL-4. Finally, IL-10 did not upregulate class II MHC antigens on B cells from X chromosome-linked immunodeficiency (XID) mice, while the same cells showed normal upregulation of class II antigens in response to IL-4. This report also extends our understanding of the relationship between IL-10 and the highly homologous Epstein-Barr virus (EBV)-encoded Bam HI fragment C rightward reading frame no. 1 (BCRFI) protein. It has previously been shown that BCRFI protein exhibits the cytokine synthesis inhibitory activity of IL-10. This report indicates that BCRFI protein also enhances in vitro B cell viability, but does not upregulate class II MHC antigens on B cells. One explanation for these data is that IL-10 contains at least two functional epitopes, only one of which has been conserved by EBV.

Animals↗
PubMed1990

Purification of a 130-kDa T cell glycoprotein that binds human interleukin 4 with high affinity.

The interleukin 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (Kd) similar to that measured in human cell lines (Kd = 40-70 pM). Affinity cross-linking of 125I-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately 130, 75, and 65 kDa. Human IL-4-binding sites were solubilized from MLA 144 cells using Triton X-100 and then purified by carboxymethyl chromatography, which removed 50% of the protein without loss of IL-4-binding activity. Then sequential affinity purification over wheat germ agglutinin and a single IL-4 Affi-Gel 10 column resulted in a final 8000-fold purification of the IL-4 receptor. When analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel, the purified receptor migrated as a single molecular species of 130 +/- 5 kDa. Identification of the 130-kDa protein as the IL-4 receptor was demonstrated by cross-linking experiments and specific binding of 125I-IL-4 to nitrocellulose membranes after electrophoretic transfer of the purified receptor on sodium dodecyl sulfate-polyacrylamide gel.

Animals↗
PubMed1990

Interleukin 4 receptors on normal human B lymphocytes: characterization and regulation.

Human interleukin 4 (IL 4) up-regulates the expression of CD23 on both resting and "in vivo" activated B cells but induces proliferation and/or differentiation only on "in vitro" activated B lymphocytes. Resting B cells express 360 high-affinity IL 4 receptors (R) per cell (Kd = 25-75 pM). Activation of resting B cells with anti-IgM antibody or Staphylococcus aureus Cowan I (SAC) results in a two-to-threefold increase of IL 4R number without alteration of IL 4R affinity for IL 4. Flow cytometric analysis of biotinylated IL 4 binding shows that IL 4R expression is up-regulated on virtually all anti-IgM-stimulated B cells, but only on a subpopulation of larger cells among SAC-activated B lymphocytes. Culturing cells for 40 h with optimal concentrations of IL 4 does not significantly affect IL 4R levels on resting and anti-IgM-preactivated B lymphocytes but triples IL 4R levels on SAC-preactivated B cells. Removal of IL 4 from cell cultures results in a two-to-fourfold increase of IL 4R levels 2 h later, suggesting an increase in IL 4R turnover. Resting and activated B cells degrade 125I-labeled IL 4 at 37 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of IL 4 binding molecules on resting, "in vivo" activated and anti-IgM-activated B cells reveals the same three species of 130, 80-75, 70-65 kDa. Thus, IL 4 displays its different biological activities on resting and activated B cells through IL 4R of the same affinity, gross biochemical structure and ability to mediate IL 4 degradation.

B-Lymphocytes↗
PubMed1990

IL-2, IL-6, and IFN-gamma have distinct effects on the IL-4 plus PMA-induced proliferation of thymocyte subpopulations.

We have previously reported complex effects of cytokine-containing T cell supernatants on the interleukin (IL)4 plus phorbol 12-myristate 13-acetate (PMA)-induced proliferative response of murine thymocytes. Here we show that recombinant murine IL-2, IL-6, and IFN-gamma each differentially regulate the IL-4/PMA-driven growth of thymocyte subpopulations. Thymocytes fractionated into four subpopulations on the basis of CD4 and CD8 expression were stimulated to proliferate by IL-4/PMA. Interferon-gamma (IFN-gamma) caused almost complete inhibition of the CD4+/CD8- response but had no measurable effect on the growth of CD4-/CD8+ or CD4-/CD8- populations. This inhibitory effect was also observed on splenic CD4+/CD8- T cells. In contrast, IL-6 strongly enhanced the proliferative response of CD4+/CD8- thymocytes, but showed no effect on peripheral CD4+/CD8- T cells, suggesting that IL-6 may be an important regulator of growth in the thymus. IL-2 also enhanced the proliferation of both CD4-/CD8+ and CD4-/CD8- thymocytes to IL-4 and PMA. To test whether the IL-4/PMA stimulus provided all the signals required to initiate growth in each subpopulation, we titrated cell number and examined the relationship between cell dose and cell response. Growth of CD8+/CD4- cells was cell density independent, indicating that IL-4/PMA is sufficient stimulus to induce growth of these cells. In contrast, growth of CD4-/CD8- and CD4+/CD8- cells is cell density dependent, suggesting a requirement for another signal provided by the cells themselves. These observations suggest that more signals remain to be identified in this thymocyte growth system.

Animals↗
PubMed1990

Epithelial autonomy in the development of the inner ear of a bird embryo.

The epithelium lining the inner ear contains a large number of differentiated cell types, arranged in precise patterns. Once the otocyst has closed, do the cells differentiate according to mechanisms intrinsic to the epithelium or are they dependent on external influences? In particular, are they governed by signals from the surrounding periotic mesenchyme? And is the closed structure of the inner ear or the otocyst fluid that it contains important for pattern formation and differentiation as it is for adult function? We have examined these questions by two types of grafting experiment. In the first, early (E3, stage 17-18, or E2, stage 13-14) undifferentiated quail otocysts were stripped of their mesenchyme and grafted into the wing buds of chick embryos. Although surrounded by a foreign mesenchyme the otic epithelium differentiated into the standard inner ear cell types. The gross morphology was abnormal, and the sensory hair cells were grouped into a few large patches instead of the usual eight smaller patches; locally, however, the spatial relationships between the differentiated cell types appeared normal. In the second experiment, open fragments of early undifferentiated otocyst (with some adhering mesenchyme) were grafted onto the surface of a limb bud. In this exposed in vivo situation, where the apical surface of the epithelium is bathed by amniotic fluid instead of otocyst fluid, differentiation proceeds normally also. Thus differentiation of inner ear epithelium at these stages does not require any specific influence from otic mesenchyme and proceeds independently of whether the otocyst is open or closed. Such epithelial autonomy creates special opportunities for in vitro analysis.

Animals↗
PubMed1990

Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding.

Interleukin 4 (IL-4) is a potent mediator of growth and differentiation for various lymphoid and myeloid cells. To isolate a cDNA encoding the murine IL-4 receptor, we have developed an expression cloning method that uses biotinylated ligand as a probe and that may be generally applicable to cloning of receptor genes. COS-7 cells transiently transfected with the cloned full-length cDNA bind murine IL-4 specifically with a Kd = 165 pM. Crosslinking of 125I-labeled IL-4 to COS-7 cells transfected with the cDNA reveals binding to proteins of 120-140 kDa. IL-4-responsive cells also express IL-4-binding proteins of 120-140 kDa but show additional bands at 60-70 kDa; the relationship of the smaller proteins to the larger ones is unclear. The nucleotide sequence indicates that the full-length cDNA encodes 810 amino acids including the signal sequence. While no consensus sequence for protein kinases is present in the cytoplasmic domain, a sequence comparison with the erythropoietin receptor, the IL-6 receptor, and the beta chain of the IL-2 receptor reveals a significant homology in the extracellular domain, indicating that the IL-4 receptor is a member of a cytokine receptor family.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of a cDNA encoding the human interleukin 4 receptor.

Using the mouse interleukin 4 (IL-4) receptor cDNA as a probe, we isolated a cDNA encoding the human IL-4 receptor (hIL-4 receptor) from a multifactor-responsive human myeloid cell line, TF1. The cDNA encodes for an open reading frame of 825 amino acids including a signal sequence (25 amino acids), the external domain (207 amino acids), a transmembrane domain (24 amino acids), and a large cytoplasmic domain (569 amino acids). The human IL-4 receptor has a 65% identity with the mouse IL-4 receptor at the nucleic acid level and retains the typical structural motif of the previously described cytokine receptor family. COS7 cells transfected with the full-length cDNA expressed high levels (140,000 sites/cell) of IL-4 binding sites, with a Kd = 80 pM, an affinity identical to that of the original TF1 cells. Similar to IL-4 responsive cells, cross-linking of [125I]IL-4 to COS7 cells transfected with the cDNA showed a major protein of 130-150 kd and minor species of 55-85 kd.

Amino Acid Sequence↗
PubMed1990

Transport of biotin in basolateral membrane vesicles of rat liver.

We examined biotin transport across the basolateral membrane (BLM) of rat liver using BLM vesicles (BLMV) technique. The purity and suitability for transport studies of liver BLMV were demonstrated by morphological (electron microscopy), enzymatic, and functional criteria. Orientation of liver BLMV was determined by freeze-fracture electron microscopy and by [3H]ouabain binding methodology and was found to be 65.3-69.7% in the right-side-out orientation. Uptake of biotin by liver BLMV was found by osmolarity and temperature studies to be mostly the result of transport of the substrate into an active intravesicular space with little binding to membrane surfaces. Transport of biotin was found to be Na+ gradient dependent with a distinct "over-shoot" phenomenon. Initial rate of transport of biotin as a function of concentration was found to include a saturable component in the presence of a Na+ gradient (out greater than in) but was linear and lower in the presence of a K+ gradient (no Na+). Kinetic parameters of the saturable Na+ gradient-dependent transport process were 0.39 microM and 1,807 fmol.mg protein-1.20 s-1 for the apparent Km and Vmax, respectively. In the presence, but not the absence, of a Na+ gradient (out greater than in), the addition of structural analogues to the incubation medium caused significant inhibition in the transport of 0.079 microM [3H]biotin. Induction, with the use of valinomycin and an inwardly directed K+ gradient, of a relatively positive intravesicular space caused significant inhibition in the initial rate of biotin transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Failure of platelet-derived growth factor plus insulin to stimulate sustained proliferation of Swiss 3T3 cells. Requirement for hydrocortisone, prostaglandin E1, lipoproteins, fibronectin and an unidentified component derived from serum.

Platelet-derived growth factor (PDGF) has been reported to be a potent mitogen for Swiss 3T3 cells made quiescent by growth to saturation density in high serum, and its activity is further potentiated by high levels of insulin, which alone have little effect. We show here that this is not the case for sparse 3T3 cells made quiescent by plating in low serum. Under these conditions, insulin alone is at least as effective as PDGF and frequently more so. Together, the response is no more than additive at best, and in many cases less than additive, the combined effect being no greater than for insulin alone. Instead, we find that optimal mitogenic stimulation requires the additional presence, besides PDGF and insulin, of hydrocortisone, prostaglandin E1 and an unidentified, non-dialysable component contained in serum treated with dithiothreitol (DTT) to inactivate endogenous growth factors. Interestingly, overnight pretreatment of the cells with hydrocortisone alone potentiates the subsequent response to PDGF + insulin, i.e. pretreatment induces a long-term memory that persists after the removal of the hydrocortisone from the medium. In short-term (24h) thymidine incorporation assays, the combination of PDGF, insulin, hydrocortisone, prostaglandin E1 and DTT-serum, is as effective as optimal levels of whole serum, but is unable to sustain longer-term proliferation (measured over 6 days). For this, high- and low-density lipoproteins, fibronectin and, to some extent epidermal growth factor (EGF), are also necessary.

Alprostadil↗