Biomedical subjects
Publications and source records attributed to T R Jones.
Apparent hybridization between human cellular DNA and herpes simplex virus DNA was blocked by the presence of guanine-rich ribo- and deoxyribonucleic acid polymers. The data indicate that the apparent hybridization may very well be artifactual and not represent long stretches of authentic base sequence homology.
This investigation was carried out to study allergic contraction of passively sensitized human airway smooth muscle in response to specific antigen challenge. We attempted to determine the role played by histamine, slow reaction substances (SRSs), and cyclooxygenase products in the mediation of this response in tracheal smooth muscle. Tissues were passively sensitized with serum from ragweed-sensitive patients (15 h, 4 degrees C). Subsequent challenge with ragweed antigen produced a slowly developing contraction. The peak contraction to a dose producing a maximal response was 37 +/- 6% of the carbachol maximum. Mepyramine (5 X 10(-6) M) did not alter the contraction. Methylprednisolone (2 X 10(-5) M) attenuated the response to antigen but had no significant effect on the contractile response to arachidonic acid. Indomethacin (5.6-28 X 10(-6) M) enhanced the peak antigen-induced contractions by 25 +/- 11% whereas 5,8,11,14-eicosatetraynoic acid (6.4 X 10(-5) M) selectively attenuated the antigen-induced contraction by 86 +/- 12%. Nordihydroguarietic acid (6-12 X 10(-6) M) attenuated both the antigen plus arachidonate induced responses. FPL-55712 (1-2 X 10(-6) M) antagonized the contractions to antigen. Compound 48/80 and goat antihuman immunoglobulin E produced similar slowly developing contractions in sensitized and in some nonsensitized tissues. These responses, except for an early component of the response to 48/80, were independent of histamine and were reversed by FPL-55712. These findings suggest that arachidonic acid metabolites mediate (slow reacting substances) and modulate (prostaglandins) allergic contraction of human airway smooth muscle while any histamine released contributes little or nothing to the contraction in the larger airways.
The kinetics of binding to the molybdate-stabilized glucocorticoid receptor of rat thymus cytosol were determined at 0 degrees C for a number of glucocorticoid agonists and antagonists. Equilibrium constants derived from the rate constants for association and dissociation were in good agreement with those determined directly or by competition under equilibrium conditions. Kinetics parameters for the slowly dissociating form of binding detected by a non-equilibrium dextran/charcoal competitive binding assay reflected the nature and extent of functional-group substitution on the steroid nucleus, but bore no relation to the classification of steroids as glucocorticoid agonists or antagonists. It is concluded that the binding of antagonists that is detected by such methods is agonist-like binding, which is not relevant to their antiglucocorticoid actions. Both agonists and antagonists displayed Michaelis--Menten association kinetics, but this behaviour was much more pronounced for antagonists. This is attributed to the existence of a second form of steroid-receptor complex, which escapes detection by the usual assay methods as a result of a high rate of dissociation and which is quantitatively antagonist-specific under equilibrium conditions. Direct evidence for the existence of two forms of antagonist-receptor complex was provided by results showing that the dissociation of the glucocorticoid antagonist progesterone from the receptor was biphasic.
The histogenesis of hemangioblastoma stromal cells is unresolved. Ultrastructural observations suggest that the stromal cells, endothelial cells, and pericytes that compose this neoplasm are all derived from angiogenic mesenchyme. The expression of factor VIII/von Willebrand factor (FVIII/vWF), a specific marker for endothelial cells, and of glial fibrillary acidic protein (GFAP), a specific marker for glial cells, was examined in 16 hemangioblastomas using the peroxidase-antiperoxidase immunohistochemical method. Endothelial cell staining for FVIII/vWF was intense in 14 tumors, weak in one, and absent in another. There was no stromal cell staining in any of the neoplasms. Process-bearing, GFAP-positive cells were observed near the tumor margin in 13 cases, and deeper in the neoplasm in 8. In two of these tumors there were also occasional GFAP-positive cells that lacked processes and had a vacuolated cytoplasm. Virtually all of the GFAP-positive cells were interpreted as trapped astrocytes rather than stromal cells. The lack of expression of FVIII/vWF by the stromal cells indicates that they are antigenically distinct from endothelial cells. Several alternatives for stromal cell histogenesis remain open. The stromal undergone antigenic loss, or from angiogenic mesenchymal cells that do not express FVIII/vWF. Alternatively, the stromal cells may originate from non-angiogenic mesenchymal cells derived from the mesoderm or neuroectoderm.
Leukotriene F4 (LTF4) and LTF4 sulfone have been synthesized and their biological activities determined in the guinea pig. In vitro LTF4 displayed comparable activity to LTD4 on guinea pig trachea and parenchyma but was less active on the ileum. When injected intravenously into the guinea pig, LTF4 induced a bronchoconstriction (ED50 16 micrograms Kg-1) which was blocked by indomethacin and FPL-55712 and was 50-100 X less potent than LTD4 in this assay. LTF4 sulfone was approximately 2-5 times less active than LTF4 in vitro and in vivo. When injected into guinea pig skin with PGE2 (100 ng); LTF4 and LTF4 sulfone (10-1000 ng) induced changes in vascular permeability. The order of potency in this assay was LTE4 sulfone = LTD4 = LTD4 sulfone greater than LTE4 greater than LTF4 = LTF4 sulfone.
Leukotriene D4 (LTD4) when administered intravenously or by aerosol to guinea pigs produced changes in pulmonary mechanics including a decrease in dynamic compliance and an increase in pulmonary resistance. The effects of intravenous LTD4 (0.5 microgram kg-1) were short lived and abolished by pretreatment of the animal with either cyclooxygenase inhibitors, a thromboxane synthetase inhibitor (OKY 1555) or an SRS-A antagonist (FPL 55712). These findings suggest that bronchoconstriction produced by the intravenous infusion of LTD4 at 0.5 microgram kg-1 is due to the release of thromboxane A2. However, in animals treated with indomethacin, LTD4 at higher doses (greater than 0.8 microgram kg-1) still elicited a bronchoconstriction which could be blocked by FPL 55712. Nebulization of 0.1 - 1.0 microgram of LTD4 into the lung produced prolonged changes in pulmonary mechanics which were inhibited by FPL 55712 and were potentiated by indomethacin. LTD4, therefore, when administered by aerosol produced effects on the lung which were not mediated by cyclooxygenase products. Responses to nebulized rather than intravenous LTD4 in the guinea pig may more closely resemble those seen in human tissues.
Synthetic leukotriene C4(LTC4) and leukotriene D4(LTD4) were examined on isolated human tracheal and bronchial smooth muscle. Both LTC4 and LTD4 produced slowly developing contraction of trachealis and bronchus which were submaximal (70-85% of the carbachol maximum) and were not blocked by 1.0 microM atropine, 5.0 microM mepyramine, or 5.6 microM indomethacin. The EC50's for LTC4 and LTD4 were 10.3 and 1.8 nM on trachealis and 1.8 and 1.7 nM on bronchus, respectively. LTD4 was in excess of 20 000 times more potent than acetylcholine on some bronchial and tracheal tissues. Similar differences in potency were obtained in some tissues when LTC4 and LTD4 were compared with histamine. Tissues pretreated for 30 min with 2.0-4.0 microM FPL-55712 were less reactive to LTC4 and LTD4 than untreated control tissues. Established contractions to LTC4 and LTD4 persisted despite washing with fresh Krebs solution, were partially reversed by FPL-55712 (0.5-10 microM) and the Ca2+ channel blocker D-600 (5-10 microM), but were completely reversed by the catecholamine adrenaline (1.0 microM) and by high concentrations of FPL-55712 (greater than 20 microM). The marked potency of the leukotrienes on human tracheal and bronchial smooth muscle suggests an important role for these lipoxygenase products in respiratory disease.
An in vitro study of neural and myogenic control of human tracheal smooth muscle was undertaken. Over 80% of these had active tension and 13% had phasic contractile activity. Tonic and phasic activities were not reversed by indomethacin, 5,8,11,14-eicosatetraynoic acid, methysergide, mepyramine, atropine, or tetrodotoxin (TTX) but were blocked by the calcium antagonist verapamil. In some quiescent strips, tonic and/or phasic activity was induced by exposure to potassium-conductance blockers such as 4-aminopyridine (4-AP) and tetraethylammonium chloride (TEA). Electrical (field) stimulation resulted in frequency-dependent biphasic responses: an initial atropine-sensitive cholinergic contraction followed by a nonadrenergic relaxation. This biphasic response to low stimulus parameters (less than 0.5 ms, less than 15 Hz) was blocked by TTX and scorpion venom and enhanced by 4-AP and TEA, consistent with a neural mechanism. Relaxation responses to longer pulse durations (0.5-1 ms) were not blocked by TTX despite abolition of contraction nor were they enhanced by 4-AP and TEA, suggesting a nonneural mechanism. ATP, adenosine, arachidonate metabolites, histamine, 5-hydroxytryptamine, neurotensin, or vasoactive intestinal polypeptide were ruled out as possible nonadrenergic mediators. The nature and physiological significance of the nonneural inhibitory response remains unknown.
The immunohistochemical detection of factor VIII/von Willebrand factor antigen (FVIII/vWF-AG) in formalin-fixed paraffin-embedded tissues was investigated using the peroxidase-antiperoxidase (PAP) method. Highly purified human FVIII/vWF was used to raise rabbit anti-FVIII/vWF-AG serum. In addition to anti-FVIII/vWF-AG activity, the unabsorbed antiserum had anti-IgG, anti-IgM, and anti-alpha2-macroglobulin specificities. Following exhaustive absorption with these proteins, the antiserum reacted monospecifically for FVIII/vWF-AG in immunodiffusion, immunoelectrophoresis, and PAP immunohistochemistry. Sections of normal tissues from six patients and a total of 43 neoplasms were examined. Treatment of the tissue sections with trypsin prior to application of the antiserum markedly increased the sensitivity of FVIII/vWF-AG detection. The positive staining for FVIII/vWF-AG was restricted to endothelial cells in both neoplastic and nonneoplastic tissue. In general, the hyperplastic endothelia in neoplastic and reactive tissues stained more intensely than those in normal tissues. Expression of FVIII/vWF-AG by nonendothelial neoplastic cells was not observed. FVIII/vWF-AG is a reliable marker for endothelial cells.
The sarcomatous components of most glioma-sarcomas are thought to arise from the neoplastic transformation of hyperplastic endothelial and adventitial vascular cells in a preexisting glioblastoma multiforme. The expression of factor VIII/von Willebrand factor (FVIII/vWF), a marker for endothelial cells, and of glial fibrillary acidic protein (GFAP), a marker for glial cells, was examined in 10 glioblastomas and seven mixed glioma-sarcomas using the peroxidase-antiperoxidase immunohistochemical technique. Hyperplasia of small blood vessels was observed in all 10 glioblastomas; in five, the vascular proliferation had resulted in the formation of prominent glomeruloid structures. FVIII/vWF was detected in the endothelial cells in these vascular structures, but not in the adventitial cells. In the mixed glioma-sarcomas. FVIII/vWF was detected only in endothelial cells; there was no staining for FVIII/vWF in the neoplastic mesenchymal cells. The gliomatous components of the mixed tumors stained intensely for GFAP. These observations indicate that both endothelial and nonendothelial cell types contribute to the small vessel hyperplasia in glioblastomas, and that the sarcomatous components of mixed glioma-sarcomas are derived from either non-endothelial cells or endothelial cells that have undergone antigenic loss following transformation.
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The morphologic and biochemical characteristics of human surgical biopsy specimens taken from 17 patients with anaplastic human gliomas and of athymic mouse-grown tumors derived from them were examined. Fourteen were categorized as glioblastoma multiforme, one as an anaplastic astrocytoma, one as a recurrent glioblastoma multiforme, and one as a gliosarcoma. Fifteen of 17 tumors stained positively immunohistochemically for glial fibrillary acidic protein (GFAP), a glial-specific marker. When portions of the 17 surgical biopsy specimens were injected into the flank subcutaneous space of athymic mice, 16 produced tumors; different portions of a single biopsy specimen were used to establish three separate tumor lines; in toto, 18 tumor lines were established. Mouse-borne tumors contained various proportions of fibrillary and protoplasmic astrocytes, gemistocytes, small anaplastic cells, and multinucleated giant cells. Some were more homogeneous than the human tumors from which they were derived, while others contained a mixed population similar to that of the original biopsy specimen. Of these initial 18 tumors, 16 were stained for GFAP and 14 contained from fewer than 5% to almost 100% GFAP-expressing cells. Ten of the tumor lines were studied in serial passage, several demonstrating increased cellularity with increased passage. GFAP expression was followed through serial passage, and 7 of 10 tumor lines continued to express it, often in reduced amounts, and 2 of 10 ceased expression, one (the gliosarcoma) never having expressed it. These data demonstrate that while athymic mouse-borne human anaplastic gliomas retained some features of the human tumors from which they were derived, they varied from one another morphologically. These mouse-borne tumors also continued to evolve, often changing their levels of GFAP and demonstrating increased cellularity with passage.
Factor VIII/von Willebrand factor (FVIII/vWF), a glycoprotein molecular complex found in human plasma, has been demonstrated by cell membrane fluorescence to be present on the surface of cultured human umbilical cord vein endothelial cells. The endothelial nature of these cells was established by electron-microscopic studies that revealed the presence of Weibel-Palade bodies in virtually all cells cultured. A newly developed radioreceptor assay was used to detect FVIII/vWF in the medium taken from these endothelial cell cultures; FVIII/vWF concentration in the medium samples increased with time in culture. FVIII/vWF binding studies showed no significant FVIII/vWF-specific binding to endothelial cell surfaces and did not corroborate a previous report suggesting a FVIII/vWF-specific receptor on human umbilical cord vein endothelium. The presence of FVIII/vWF on endothelial cell membranes and the lack of receptor-mediated binding suggests that the FVIII/vWF either has been absorbed non-specifically to the cell surface or is an integral part of the endothelial cell membrane.
A preliminary survey of the incidence of histoplasmosis in the eastern Caribbean was carried out. The percentages of positive histoplasmin skin test reactors in the populations studied were as follows: Barbados (4%), Trinidad (42%) and Guyana (29%). In Trinidad 69% of individuals under 60 had positive test. However a lower proportion (4%) had serological evidence of past infection with Histoplasma capsulatum. The organism was not isolated from the soil although it had been found previously in Trinidad. On this island five previously diagnosed cases of histoplasmosis were identified. The study confirmed the presence of the infection in the area and further investigations are suggested.
Nicotine produced cholinergic excitatory and adrenergic and non-adrenergic inhibitory responses in isolated guinea-pig trachea. Responses were blocked by hexamethonium (10 micro M), lidocaine (85 micro M) or tetrodotoxin (0.01 micro M) demonstrating that nicotinic receptors in nervous tissue were being activated. In the presence of atropine (0.1 micro M), inhibitory responses to nicotine were partially blocked by specific, experimentally determined, beta-blocking concentrations of pindolol or sotalol or by pretreatment with 6-hydroxydopamine or reserpine but were completely blocked by the less specific beta-blocking drugs 1- and dl-propranolol. Parallel experiments on guinea pig ileum revealed a marked attenuation by dl-propranolol of the atropine sensitive, cholinergic excitatory response to applied nicotine. The non-beta-adrenoceptor blocking agent d-propranolol, produced qualitatively similar attenuation of all excitatory and inhibitory responses to nicotine on both preparations. The remarkable susceptibility of nicotine-induced, neurally mediated responses to low, beta-blocking concentrations of dl-propranolol and to low concentrations of both of its racemates suggests that the non-specific actions of these compounds may have much more significance than is customarily believed. Such studies on the interaction between nicotine and some beta-adrenoceptor blocking drugs are consistent with the hypothesis that non-beta-blocking so-called 'non-specific membrane depressant actions' of dl-propranolol may in concentrations previously considered 'sub-local anaesthetic', significantly depress physiological transmission induced by activation of nicotinic receptors.
The effects of steroids on the binding of [1,2-3H]dexamethasone and [1,2-3H]progesterone to the glucocorticoid receptor of rat thymus cytosol were studied. Although both glucocorticoid agonists and antagonists competed with [1,2-3H]dexamethasone for binding to the receptor under equilibrium conditions, only glucocorticoid antagonists of partial agonists, at micromolar concentrations, were capable of accelerating the rate of dissociation of previously bound [1,2-3H]dexamethasone from the receptor. Antagonists or partial agonists also enhanced the rate of dissociation of [1,2-3H]progesterone from the glucocorticoid receptor, with identical specificity and concentration--response characteristics. These effects are attributed to the presence on the receptor of a secondary, low-affinity, binding site for glucocorticoid antagonists, the occupancy of which produces negatively co-operative interactions with the primary glucocorticoid-binding site. In contrast with the interactions with the primary site, the interactions of steroids with the negatively co-operative site appear to be primarily hydrophobic in nature, and the site resembles the steroid-binding site of progestin-binding proteins in its specificity, though not its affinity. The results also suggest that the initial interactions of both glucocorticoid agonists and antagonists with the receptor under equilibrium conditions are with one primary site on a receptor existing in one conformation only.