The contractile effects of partially purified slow-reacting substance of anaphylaxis (SRS-A) and histamine were compared on isolated guinea pig tracheal spirals and parenchymal strips. Histamine was equally active on both isolated tissues in a concentration-related fashion. SRS-A (0.1--10.0 U/ml) produced a concentration-related effect on parenchymal strips, whereas the tracheal spiral was 100 times less sensitive to this mediator. The contractile activity of SRS-A on parenchymal strips was diminished by incubation with limpet arylsulfatase and antagonized by FPL 55712, a known SRS-A antagonist. SRS-A, further purified by high pressure liquid chromatography, also demonstrated this preferential activity on guinea pig parenchymal strips. These data are consistent with the hypothesis, based on previous in vivo observations, that SRS-A is a selective peripheral airway constrictor.
J M Drazen, R A Lewis, S I Wasserman, R P Orange, K F Austen
Using the isolated rat kidney perfused with an artificial medium containing glucose as the sole fuel, we studied the renal handling of immunoreactive arginine vasopressin (AVP) and determined the effect of various factors on the ability of the kidney to remove AVP.
Ralph Rabkin, Leonard Share, Paul A. Payne, Judy Young, Joan Crofton
The effect of acute and chronic ethanol intake on hepatic glycerolipid biosynthesis in the hamster was studied by in vivo and in vitro techniques. The results were compared with those from control hamsters receiving isocaloric amounts of glucose. Both chronic and acute ethanol intake elevated serum and hepatic triglyceride concentrations and induced a rapid rise in the capacity of neutral glycerolipid formation from sn[1,3-14C]glycerol-3-phosphate by hamster liver homogenate and microsomal fractions. Ethanol intake also produced a corresponding increase in the incorporation of [1,3-14C]glycerol into hepatic neutral glycerolipids by the intact animal. The ethanol-induced rise in the capacity of neutral glycerolipid production by liver as measured in vivo and in vitro correlated well with an increase in hepatic phosphatidate phosphohydrolase activity. Therefore, the rise in hepatic and serum triglyceride levels associated with ethanol intake may be explained in part by an increase in the activity of the enzyme.
R G Lamb, C K Wood, H J Fallon
The subcellular distribution of the superoxide (O2-)-forming enzyme in human neutrophils was investigated. Cells were activated by phorbolmyristate acetate or by opsonized zymosan, and were then fractionated by zonal-rate sedimentation at two different speeds. At high speed, the specific granules were resolved from the azurophils and the membrane fraction, while at low speed, the azurophil granules could be separated from fast-sedimenting particle aggregates. Under both conditions, the major portion of the O-2--forming activity (60--70% of the total) was found to be associated with the membrane fraction which was characterized by the presence of alkaline phosphatase, alkaline phosphodiesterase I, and acid aryl phosphatase. No significant O-2--forming activity was found in either specific or azurophil granules. Some activity was present in the fastest sedimenting fractions which, as shown by electron microscopy, were heterogeneous and contained aggregated material which included membrane fragments. These fractionation results provide strong additional support for the current view that the activable O-2--forming system is localized in the plasma membrane of human neutrophils.
B Dewald, M Baggiolini, J T Curnutte, B M Babior
To investigate the mechanism of exercise-induced bronchospasm, we measured specific airway conductance before and after exercise in 7 healthy normals, 12 asthmatics with intact carotid bodies, and 5 asthmatics who had had bilateral carotid body resection. The subjects breathed either air or oxygen (randomly assigned) during cycle ergometer exercise. Post-exercise bronchodilation was the usual pattern in normals, whereas post-exercise bronchospasm occurred in all asthmatics who breathed air during exercise. Oxygen breathing during exercise markedly attenuated the post-exercise bronchospasm in those asthmatics with intact carotid bodies, but had no significant effect in those without effect in those without carotid bodies. The attenuation of the bronchospasm with oxygen occurred with either incremental or constant load exercise of high intensity. The degree of attenuation did not correlate significantly with changes in end-tidal PCO2, maximum work rate, maximum exercise ventilation, or maximum heart rate. These studies indicate that oxygen attenuates exercise-induced bronchospasm in asthmatics through its action on the carotid bodies.
P L Schiffman, A Ryan, B J Whipp, J E Hansen, K Wasserman
Cholic acid biosynthesis is defective in individuals with cerebrotendinous xanthomatosis (CTX) and is associated with the excretion of 5β-cholestane-3α,7α, 12α,25-tetrol, an intermediate in the 25-hydroxylation pathway of cholic acid in CTX. To define the enzymatic defect in CTX, two suspected precursors of cholic acid, namely 5β-[7β-3H]cholestane-3α,7α, 12α-triol and 5β-[24-14C]cholestane-3α,7α, 12α,24S,25-pentol were examined by both in vivo and in vitro experiments. A third precursor, 5β-[7β-3H]-cholestane-3α,7α, 12α,25-tetrol, was compared with them in vitro.
Gerald Salen, S. Shefer, F. W. Cheng, B. Dayal, A. K. Batta, G. S. Tint
The normal metabolic turnover of plasma kininogens was studied by measuring the disappearance of intravenously administered radiolabeled human and monkey plasma kininogens from the circulation of healthy adult rhesus monkeys. Curves obtained by plotting log radioactivity against time could be expressed as double exponential equations, with the first term representing diffusion, and the second, catabolism. No significant difference between the turnovers of human and monkey kininogens was observed. The difference between the t1/2 of high molecular weight kininogen (25.95 +/- 1.60 h) (mean +/- SEM) and that of low molecular weight kininogen (18.94 +/- 1.93 h) was only marginally significant (P less than 0.05). In contrast, a highly significant (P less than 0.001) difference in their mean catabolic rates (1.12 +/- 0.08 d-1 for high molecular weight kininogen vs. 2.07 +/- 0.09 d-1 for low molecular weight kininogen) was observed. These differences between the two kininogens were attributed to differences in their distribution between the intra- and extravascular pools. Studies of kininogen turnover will be useful in elucidating the in vivo functions of the various kininogens in health as well as during clinical illness.
T Yamada, D A Wing, J V Pierce, G W Pettit
Actively metabolizing human erythrocytes catalyze the extracellular reduction of ferricyanide to ferrocyanide. Because neither of these anions can enter the cell, reducing equivalents generated in the course of glycolysis must in some manner be transferred across the cell membrane, thereby resulting in ferricyanide reduction. Work described in this paper suggests that the transmembrane reduction is effected by ascorbic acid. This compound in its oxidized form (dehydroascorbate) rapidly enters the cell. Here it obtains reducing equivalents which appear to come from NADH made available at the level of glyceraldehyde 3-phosphate dehydrogenase. Once reduced, it leaves the cell as ascorbic acid and accomplishes the non-enzymatic reduction of ferricyanide.
E P Orringer, M E Roer
We have carried out perfusion studies on hydropenic and bicarbonate-loaded rats to provide direct in vivo observations on bicarbonate accumulation in the short loops of Henle. Analysis of early distal tubular fluid was made during bicarbonate-free saline perfusion from the end proximal to the early distal site, documenting accumulation of "new" bicarbonate. During perfusion in hydropenic rats, steady-state bicarbonate concentrations were suggested by early distal values of approximately equal to mM, which were independent of perfusion rate and virtually indistinguishable from bicarbonate concentration measured during free flow when filtered bicarbonate was allowed to enter the loop. Thus, loop bicarconate accumulation was apparently sufficient to allow new bicarbonate to enter at a rate comparable to that delivered to the early distal site during free flow, recognizing of course that free-flow delivery rates are the result of complex components of filtration and bidirectional fluxes. In bicarbonate-loaded rats, however, bicarbonate accumulation rates although higher than in hydropenia, were much lower than free-flow delivery rates. Furthermore, early distal bicarbonate concentrations during bicarbonate loading fell as perfusion rate increased, presumably because of a limitation to increasing ionic bicarbonate entry.
D Z Levine, M K Byers, R A McLeod, J A Luisello, S Raman
Binding of 125I-leukoagglutinin (LPHA) to lymphocyte membrane receptors at equilibrium generated similar curvilinear Scatchard plots in 20 patients with bursa-derived (B)-cell-type chronic lymphatic leukemia (CLL) and 15 controls. If biphasic plots are assumed, the two linear components show markedly diminished receptor capacity (15 and 137 ng/106 lymphocytes) in CLL as compared to controls (60 and 668 ng). In contrast, affinity was similar in patients (1.0 × 108 M−1 and 2.1 × 106 M−1) and controls (1.8 × 108 M−1 and 1.5 × 106 M−1). Highly purified B cells from patients and controls generated binding data comparable to that obtained from the mixed lymphocyte (ML) suspensions from which they originated. Maximal DNA synthesis of highly purified, normal, thymus-derived (T) and B cells in response to LPHA stimulation was comparable to that of ML (mitotic index [MI] 19.9, 20.1, and 23.4, respectively), though B-cell responses were slightly delayed. In CLL the markedly decreased and delayed DNA synthesis by ML (MI 2.3), and by highly purified T (MI 1.6) and B (MI 1.9) cells seemed out of proportion to their decreased receptor capacity for LPHA. The impaired mitogenic responses of leukemic cells from five patients were not enhanced when cocultured with normal lymphocytes. In contrast, cells from eight patients inhibited cocultured normal lymphocyte responses to LPHA by 94.3%. Sera from these patients and supernates from their cultured cells did not mediate this suppressor effect. These observations indicate that the decreased DNA synthesis observed in CLL is not an attribute of B cells and does not represent the expected response of a few residual normal T lymphocytes, but rather reflects impaired responses by all CLL cells. The defect does not relate to the density or function of membrane receptors for LPHA, to the presence of inhibitors in these patients' sera, or to depletion of helper T cells. Our data strongly suggest that one mechanism for the immunoincompetence observed in CLL reflects excessive suppressor-cell activity.
Guy B. Faguet
The effect of the microtubule inhibitor colchicine on the metabolism of 125I-low density lipoprotein (LDL) by cultured human skin fibroblasts and aortic medial cells was studied in vitro. Colchicine did not alter the binding of LDL to cell surface receptors. However, the rate of LDL endocytosis was reduced to 58% of that expected. Despite diminished endocytosis, LDL was found to accumulate within the cells to 165% of that expected, whereas the release of LDL protein degradation products into the medium was reduced to 34% of control, findings consistent with a reduced rate of intracellular LDL breakdown. Colchicine did not alter cell content of the acid protease which degrades LDL, nor did [3H]colchicine accumulate in lysosomal fractions. However, colchicine did alter the intracellular distribution of both fibroblast lysosomes and endosomes. After colchicine, lysosomes tended to accumulate in the perinuclear region, whereas endosomes were found at the cell periphery. These findings are consistent with the hypothesis that ingested LDL is less available to lysosomal enzymes in the presence of colchicine. The actions of colchicine appear to be a result of destruction of cell microtubules. Lumicolchicine, a mixture of colchicine isomers which (unlike the parent compound) does not bind to the subunit of microtubules, was without effect.
Richard E. Ostlund Jr., Barbara Pfleger, Gustav Schonfeld
The crystal-induced chemotactic factor, a cell-derived chemoattractant for neutrophils, binds specifically to a site on human neutrophils but not to erythrocytes or lymphocytes, suggesting a relationship between the presence of specific binding sites on the neutrophils and the ability to be chemotactically activated. The Scatchard analysis revealed an equilibrium dissociation constant at 37 degrees C of 0.446 micrometer and the presence of approximately equal to 6.44 x 10(5) binding sites for 125I-crystal-induced chemotactic factor per cell. Binding was not displaced by the synthetic chemotactic factors F-Met-Leu-Phe and Gly-His-Gly or by complement-activated plasma providing evidence of the specificity of the receptor.
I Spilberg, J Mehta
After intravenous injection of [125I]-iodo-parathyroid hormone in the rat, uptake of the hormone was greatest in the liver and kidneys. Uptake was rapid, reaching a maximal concentration by 4 and 8 min, respectively. Extracts, prepared from both these organs at intervals soon after the injection of intact hormone, showed three main radioactive peaks when samples were subjected to gel filtration under protein-denaturing conditions. The first peak coeluted with intact hormone. The second eluted at a position corresponding to the carboxy-terminal fragments previously described in plasma, and the last eluted at the salt volume of the column. Microsequence analysis of the radioiodinated fragments, a method that has proved valuable for chemically defining the circulating fragments resulting from metabolism of injected hormone, showed that extracts of liver and kidney, prepared at 4 and 8 min after injection of the intact hormone, contained different fragments. The radioiodinated fragments in liver extracts were identical to those previously reported in the plasma of rats and dogs, fragments resulting principally from proteolysis between positions 33 and 34, and 36 and 37 of the intact hormone. Although the same fragments were also present in the kidneys, they constituted less than 15% of the amount present in the liver. More than 50% of the labeled renal fragments consisted of a peptide whose amino-terminal amino acid was position 39 of the intact hormone, a fragment not present in plasma. The rate of appearance of radioiodinated fragments that were chemically identical to those in plasma was more rapid in the liver than in plasma. Correlation of these chemical analyses with studies of the localization of 125I by autoradiography showed that at the times when the intact hormone and the carboxy-terminal fragments comprised nearly all of the 125I-labeled moieties in the tissues, the proximal convoluted tubules of the kidney and sinusoidal lining cells of the liver, which probably are Kupffer cells, contained the highest concentration of 125I. Preferential localization of immunoreactive parathyroid hormone to these tissue sites also was shown by immunoperoxidase staining in studies with unlabeled hormone. Our results suggest that, unless multiple renal mechanisms are present for release of hormonal fragments, one of which releases the circulating fragments preferentially, the liver, rather than the kidney, is principally responsible for generating the carboxy-terminal fragments in plasma after injection of intact hormone, and the Kupffer cells may contain the enzymes that hydrolyze parathyroid hormone.
Pierre D'Amour, Gino V. Segre, Sanford I. Roth, John T. Potts Jr.
Although numerous interventions have been shown to exert a salutary effect on the ischemic myocardium, the severity of ischemia generally has been measured by indirect techniques. In the present investigation the effect of ischemia on intramural carbon dioxide tension (PmCO2) was measured directly in the open-chest, anesthetized dog with a mass spectrometer during repetitive 10-min coronary artery occlusions separated by 45-min periods of reflow; simultaneously, regional myocardial blood flow in the ischemic area was measured by 127Xenon washout. In all dogs the increase in PmCO2 from before to 10 min after the first occlusion (ΔPmCO2) exceeded that during subsequent occlusions. In those dogs not receiving an intervention (controls), ΔPmCO2 during the third occlusion was similar to that during the second occlusion. When propranolol, hyaluronidase, and nitroglycerin were administered to different groups of dogs before the third occlusion, each caused significantly smaller elevations in ΔPmCO2 than those occurring during the control second occlusion, and the combination of all three interventions induced the smallest increase in ΔPmCO2. Regional myocardial blood flow rose with hyaluronidase and was unchanged with propranolol, nitroglycerin, and the three drugs in combination. In contrast to these beneficial interventions, isoproterenol infused with the third occlusion caused a higher ΔPmCO2 than during the control second occlusion. It is concluded, first, that interventions that modify the severity of ischemia can be evaluated by measuring intramural carbon dioxide tension; second, that propranolol, hyaluronidase, and nitroglycerin reduce ischemic injury, whereas isoproterenol increases it; and third, that the combination of propranolol, hyaluronidase, and nitroglycerin exerts an additive beneficial effect on ischemia.
L. David Hillis, Shukri F. Khuri, Eugene Braunwald, Robert A. Kloner, Donald Tow, Ernest Barsamian, Peter R. Maroko
An association between Graves' disease and the human leukocyte antigen (HLA) system has previously been reported. The disease was more strongly associated with the HLA D locus antigen Dw3 than with HLA B8. Products of the HLA D locus are determined by the interaction of test cells with standard typing lymphocytes, a technically difficult procedure. Recently, it has been possible to type serologically for D locus related (DRw) specificities on peripheral bone marrow-derived (B) lymphocytes. Blood B lymphocytes from 50 unrelated controls and 41 patients with Graves' disease were typed for seven HLA DRw specificities. 28 patients with Graves' disease (68%) were positive for DRw3, in contrast to 14 controls (28%); whereas only 21 patients (50%) were HLA B8 positive, compared with 13 (26%) controls. Thus, positivity for DRw3 afforded a relative risk for Graves' disease of 5.5, whereas that for HLA B8 amounted to 3.0. Additionally, a family with multiple cases of Graves' disease in which the disease was previously shown to be inherited with the haplotype, was linked to DRw2, which suggests that the susceptibility to the disease was inherited in association with that antigen. Two HLA B/glyoxalase recombination events were observed in this family; in both instances HLA DRw followed HLA B. This study thus demonstrates that the disease susceptibility gene for Graves' disease is in strong linkage disequilibrium with DRw3; however, it may be associated with other DRw specificities and inherited within family units in association with them.
Nadir R. Farid, Laura Sampson, Elke P. Noel, John M. Barnard, Robert Mandeville, Bodil Larsen, William H. Marshall
To evaluate the role of anti-insulin hormone actions and interactions in the pathogenesis of stress-induced hyperglycemia, the counterregulatory hormones, glucagon, epinephrine, and cortisol were infused alone as well as in double and triple combinations into normal conscious dogs in doses that were designed to simulate changes observed in severe stress. Infusion of glucagon, epinephrine, or cortisol alone produced only mild or insignificant elevations in plasma glucose concentration. In contrast, the rise in plasma glucose produced by combined infusion of any two counterregulatory hormones was 50-215% greater (P < 0.005-0.001) than the sum of the respective individual infusions. Furthermore, when all three hormones were infused simultaneously, the increment in plasma glucose concentration (144±2 mg/dl) was two- to fourfold greater than the sum of the responses to the individual hormone infusions or the sum of any combination of double plus single hormone infusion (P < 0.001).
Neal Eigler, Luigi Saccà, Robert S. Sherwin
HLA-D typing was performed in 126 patients with juvenile rheumatoid arthritis. HLA-DW4, the antigen found in previous studies to characterize adult rheumatoid arthritis, had a significantly lower frequency in children with arthritis than in normal controls (P less than 0.04). By contrast, in children the antigens HLA-DW7 (P less than 0.03) and HLA-DW8 (P less than 0.01) were increased compared to controls. The antigen TMo, detected with homozygous typing cells from a child with juvenile rheumatoid arthritis, was found to be related to the cross-reactive specificities HLA-DW7 and DW11. 46% of the patients with persistent pauciarticular arthritis of childhood typed for the antigen TMo, compared to only 1% of normal controls. Thus the relative risk for persistent pauciarticular arthritis in relation to the presence of TMo was 67.7 (P less than 0.0001). These results provide evidence of fundamental differences between adult rheumatoid arthritis and arthritis of childhood. The latter group appears to include a population distinguishable clinically and characterized in these studies by the HLA-D determinant TMo.
P Stastny, C W Fink
Although prostaglandins E2 and F2α have been suggested as mediators of the pulmonary hypertension seen after endotoxin infusion or during alveolar hypoxia, their precursors, the endoperoxides (prostaglandins G2 and H2) are much more potent vasoconstrictors in vitro. In this study we compared the effects of prostaglandin (PG)H2, a stable 9-methylene ether analogue of PGH2 (PGH2-A), PGE2, and PGF2α on pulmonary hemodynamics in awake sheep. The animals were prepared to allow for measurement of (a) lung lymph flow; (b) plasma and lymph protein concentration; (c) systemic and pulmonary vascular pressures; and (d) cardiac output. We also determined the effect of prolonged PGH2-A infusions on lung fluid balance and vascular permeability by indicator dilution methods, and by assessing the response of lung lymph. Both PGH2 and PGH2-A caused a dose-related increase in pulmonary artery pressure: 0.25 μg/kg × min tripled pulmonary vascular resistance without substantially affecting systemic pressures. Both were 100 times more potent than PGE2 or PGF2α in this preparation. PGH2-A, as our analysis of lung lymph and indicator dilution measurements show, does not increase the permeability of exchanging vessels in the lung to fluid and protein. It does, however, augment lung fluid transport by increasing hydrostatic pressure in the pulmonary circulation. We conclude: (a) that PGH2 is likely to be an important mediator of pulmonary vasoconstriction; (b) its effects are probably not a result of its metabolites PGE2 or PGF2α.
Ronald E. Bowers, Earl F. Ellis, Kenneth L. Brigham, John A. Oates
We have assessed the effectiveness of transplanted histocompatible fibroblasts as a long-lived source of lysosomal enzymes for replacement therapy in three patients with Hunter's syndrome, over periods ranging from 2.5 to 3.75 yr. The level of Hunter corrective factor excreted by all three patients increased after transplantation, as did the activity of alpha-L-idurono-2-sulfate sulfatase in serum, when measured directly with a radioactive disulfated disaccharide substrate. Sulfatase activity was also raised in leukocyte homogenates from the two patients that we were able to assess. These increases in enzyme activity were accompanied by corresponding increases in catabolism of heparan and dermatan sulfates, as shown by (a) a decrease in sulfate:uronic ratios of urinary oligosaccharides, (b) an increase in iduronic acid monosaccharide, and (c) a normalization of Bio-Gel P-2 gel filtration profiles. Both the increase in enzyme activity and increased catabolism were maintained during the period of study and were not affected by either a gradual decrease or total withdrawal of immunosuppressive therapy.
M F Dean, R L Stevens, H Muir, P F Benson, L R Button, R L Anderson, A Boylston, J Mowbray
Complement-activated human plasma causes generation of tissue factor in human leukocytes. This phenomenon appears to be related to the fifth component of complement (C5) as demonstrated by the use of C5 deficient-plasma and suppression of activity with antibody to C5. Isolation of the chemotactic factor from activated serum or trypsinization of purified C5 reproduces the phenomenon. These data provide evidence for a direct link between complement products and activation of the coagulation system. Because chemotactic peptides from C5 can be generated by a variety of enzymes, our findings suggest a relationship between complement, coagulation, and inflammation.
T W Muhlfelder, J Niemetz, D Kreutzer, D Beebe, P A Ward, S I Rosenfeld
The autologous mixed lymphocyte reaction is impaired in patients with systemic lupus erythematosus. This is because nonthymus-derived (T)-lymphocyte preparations from such patients do not stimulate autologous T-lymphocyte proliferation normally. This defect may explain the impaired generation of suppressor activity in systemic lupus erythematosus and thereby the occurrence of autoantibodies in this disease.
M M Kuntz, J B Innes, M E Weksler
Peripheral blood lymphocytes from multiple sclerosis (MS) patients form substantially greater numbers of rosettes with measles virus-infected human epithelial cells than do lymphocytes from healthy controls or from patients with other diseases. We have previously shown that prostaglandin E1-treated normal lymphocytes exhibit increased lymphocyte adherence, and thus behave like MS lymphocytes in this in vitro system. In this study we describe the effect of prostaglandin synthesis inhibition on lymphocyte adherence in both MS and control patients. Direct addition of aspirin or indomethacin to peripheral blood mononuclear cells from MS patients in vitro reduced lymphocyte adherence to control levels. Ingestion of therapeutic (anti-inflammatory) doses of aspirin (1 g, 4 times daily for 2 d) by MS patients resulted in reduction of lymphocyte adherence to levels seen in healthy controls. A single 325-mg dose of aspirin did not reduce lymphocyte adherence. A dose-dependent reduction in lymphocyte adherence was observed after single doses ranging from 650 mg to 1.3 g; duration of the effect was directly related to the aspirin dose. These observations indicate that treatment of MS patients with aspirin profoundly influences adherence of their lymphocytes to measles virus-infected cells and suggest that the altered cellular response, which results in increased lymphocyte adherence in MS patients, may be mediated by a prostaglandin-sensitive mechanism.
Paula Dore-Duffy, Robert B. Zurier
The effect of aspirin in the primary prevention of diet-induced atherogenesis in cynomolgus monkeys was studied. The diet consisted of 2% cholesterol and 10% butter by weight for 24 wk. Six monkeys received only the atherogenic diet and five monkeys received the diet plus aspirin, 81 mg/monkey per day. Aspirin did not affect plasma cholesterol levels or aortic atherosclerosis. Platelet aggregation to arachidonic acid was almost completely suppressed. Aspirin decreased significantly the number of coronary vessels with atherosclerotic involvement, and the number of coronary vessels narrowed by 20% or more. Thus, aspirin appears to exert a protective effect in the primary prevention of diet-induced coronary atherosclerosis in a primate model.
R Pick, J Chediak, G Glick