Inhibitors of monocyte responses to chemotaxins are present in human cancerous effusions and react with monoclonal antibodies to the P15(E) structural protein of retroviruses.

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Abstract

Individuals with cancer have previously been shown to have abnormal chemotactic responsiveness. Surgical removal of the tumor often resulted in normalization of monocyte function, which suggests that human neoplasms might inhibit monocyte chemotaxis by release of soluble mediators. We therefore examined the effects of cancerous effusions on monocyte polarization, i.e., the rapid change in monocyte morphology from round to a triangular "motile" configuration in response to chemoattractants. All 17 malignant effusions, representing 15 tumor types, inhibited monocyte polarization induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine by 45-89% (mean 55.9 +/- 12.7%, P less than 0.01) in blinded assays. None of 17 benign effusions signigicantly inhibited polarization (0-15%, mean 6.2 +/- 4.2%). Dilutions of cancerous effusions as low as 1:200 produced inhibition that was time, temperature, and dose dependent . Monocyte polarization induced by activated serum or by chemotactic lymphokine was also blocked by cancerous effusions. The inhibitory activity affected the monocyte directly, and did not destroy the chemoattractant or block the polarization of granulocytes to chemotactic factors. High pressure liquid chromatography of five cancerous fluids revealed three peaks of inhibitory activity: greater than or equal to 200,000, 46,000 +/- 13,000, and 21,000 +/- 3,000 daltons. Fractionation of noncancerous effusions revealed only small amounts of the highest molecular weight inhibitory activity. The inhibitory activity in cancerous effusion was heat stable (56 degrees C, 30 min), trypsin sensitive, and could be absorbed by three different monoclonal antibodies reactive to P15(E), a structural component of type C retroviruses. In contrast, six monoclonal antibodies with other specificities had no effect on the inhibitors of polarization. This study demonstrates that human cancerous effusions contain novel proteins that are potent inhibitors of monocyte function and that are recognized by antibodies reactive to the P15(E) component of retroviruses. By producing such factors, tumor cells may subvert monocyte-mediated surveillance.

Authors

G Cianciolo, J Hunter, J Silva, J S Haskill, R Snyderman

Increase of lymphocytes with Fc receptors for IgE in patients with allergic rhinitis during the grass pollen season.

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Abstract

Peripheral blood lymphocytes from 10 nonallergic donors and 7 patients suffering from seasonal allergic rhinitis and receiving desensitization therapy were analyzed by rosette assays for Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) before, during and after the grass pollen season. Six of seven patients had moderately elevated IgE levels (330 +/- 268 IU/ml), all had high titers of skin sensitizing antibodies to grass pollens and serum IgE antibodies as measured by radio-allergosorbent tests (RAST). Seven of the nonallergic donors had 2-30 IU/ml IgE and negative RAST, whereas three had 91-267 IU/ml IgE and two were RAST positive to the grass pollens. In March, when the patients were asymptomatic, the mean +/-SD of the Fc epsilon R+ lymphocytes did not significantly differ from the nonallergic control group: nonallergic Fc epsilon R+ 1.2 +/- 0.9% (29 +/- 20/,mm3), allergic Fc epsilon R+ 2.0 +/- 3.1% (48 +/- 52/mm3). In contrast, during the grass pollen season in May and June, when the patients developed symptoms of allergic rhinitis, they had significantly (P less than 0.01) more Fc epsilon R+ lymphocytes than the controls: nonallergic Fc epsilon R+ 1.7 +/- 1.9% (40 +/- 46/mm3), allergic Fc epsilon R+ 4.7 +/- 1.2% (134 +/- 69/mm3). In the postpollen period, August-October, most of the patients again had low numbers of Fc epsilon R+ lymphocytes: nonallergic Fc epsilon R+ 1.4 +/- 0.9% (26 +/- 13/mm3), allergic Fc epsilon R+ 2.1 +/- 1.9% (62 +/- 82/mm3). The nonallergic control donors with elevated IgE levels and positive RAST always had low numbers of Fc epsilon R+ lymphocytes. In contrast, two other nonallergic donors, who had a 2-7 IU/ml IgE and negative RAST, showed significant increases of Fc epsilon R+ lymphocytes over several weeks during the grass pollen season. No statistically significant changes in Fc gamma R+ lymphocytes occurred in both nonallergic and allergic donors. The total and specific IgE serum levels did not vary much in the nonallergic donors and patients during the period of study and any changes that did occur did not correlate with the changes in Fc epsilon R+ lymphocytes. The data demonstrate that Fc epsilon R+ peripheral blood lymphocytes increase in allergic patients during natural antigen exposure and active disease in the absence of measurable increases of total and specific serum IgE. Because two nonallergic control donors also had temporary increases of Fc epsilon R+ lymphocytes, an increase of peripheral blood Fc epsilon R+ lymphocytes may be a sensitive indicator of an ongoing IgE immune response.

Authors

H L Spiegelberg, R A Simon

Effects of inhibition of RBC HCO3-/Cl- exchange on CO2 excretion and downstream pH disequilibrium in isolated rat lungs.

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Abstract

To determine the effects of the speed of the erythrocyte membrane chloride shift on pulmonary gas transfer, CO2 exchange and the kinetics of pH equilibration were measured with isolated rat lungs perfused with 20% suspensions of human erythrocytes. The lungs were ventilated with room air, and the inflowing perfusate blood gases were similar to those in mixed venous blood in vivo. All experiments were performed at 37 degrees C. Rates of CO2 excretion were determined by measuring the fraction of CO2 in mixed expired gas in a steady state. The time-course of extracellular pH equilibration in the effluent perfusate was measured in a downstream stopflow pH electrode apparatus. CO2 excretion was reduced by approximately 16% when the lungs were perfused with suspensions containing erythrocytes whose HCO-3/Cl- exchange rates was inhibited, compared with CO2 excretion when the lungs were perfused with normal erythrocyte suspensions. A fall of 0.06 in effluent perfusate extracellular pH was noted during perfusion with inhibited erythrocyte suspensions, in contrast to no observable downstream pH change during perfusion with normal erythrocyte suspensions. These results are in close agreement with the predictions of a theoretical model. Our observations suggest that CO2 transfer in capillary beds will be adversely affected in vivo when the rate of the erythrocyte HCO-3/Cl- exchange is abnormally low. Since a number of commonly used drugs are known to inhibit the chloride shift in human erythrocytes, these findings may have important clinical implications, especially in patients with impaired lung function.

Authors

E D Crandall, S J Mathew, R S Fleischer, H I Winter, A Bidani

Abnormal mobility of neonatal polymorphonuclear leukocytes. Relationship to impaired redistribution of surface adhesion sites by chemotactic factor or colchicine.

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Abstract

To determine the mechanism(s) of diminished, stimulated, and directed migration of neonatal (N) polymorphonuclear leukocytes (PMN), chemotactic factor (CF) sensory and PMN effector functions were studied in healthy N and adult or maternal controls (C). N PMN demonstrated high affinity binding for N-formyl-methionyl-leucyl-[3H]phenylalanine (fMLP), which was saturable between 40 and 100 nM as observed with C PMN. The kinetics of binding and the characteristics of dissociation of binding by N PMN were equivalent to control PMN. Both "threshold" and "peak" concentrations (1 and 10 nM, respectively) of fMLP effected comparable PMN chemiluminescence among neonates and controls. An equivalent threshold concentration (0.05 nM) of fMLP effected N and C PMN shape change in suspension, and a maximally effective concentration (5 nM) induced comparable bipolar configuration, although uropod formation was only 38 +/- 8% of N PMN, compared with 73 +/- 11% of C PMN (P less than 0.01). Striking abnormalities of N PMN adherence were identified: mean +/- SD base-line (unstimulated) N adherence values (39 +/- 8%) were equal to C (38 +/- 9%), but diminished increments in response to single CF stimuli were noted among N (fMLP: 42 +/- 7% (N), 70 +/- 11% (C); C5a: 41 +/- 6% (N), 68 +/- 6% (C); BCF: 41 +/- 6% (N), 63 +/- 9% (C), P less than 0.01 for each CF). On sequential exposure to increasing concentrations of CF N PMN failed to demonstrate expected decreased adherence values; sequential stimuli with fMLP (0.1 nM, 10 nM) or C5a (8 microgram protein/ml, 32 microgram protein/ml) effected mean +/- 1 SD values of 51 +/- 9% (N), 30 +/- 9% (C), and 34 +/- 10 (N), 48 +/- 14% (C), respectively. As demonstrated with a latex bead-binding technique, N PMN failed to redistribute adhesion sites to the cell's tail under the same experimental conditions; in 21 N samples studied, restricted unipolar binding occurred in 33 +/- 8% (fMLP) or 37 +/- 7% (C5a) of PMN in contrast to C values of 70% (fMLP), or 71% (C5a), P less than 0.001. Similar findings were observed when PMN were preincubated with colchicine (25 microgram/ml); expected diminished adherence scores (compared with base-line values) were demonstrated with C PMN but not with N cells, P less than 0.01. Additionally colchicine-induced redistribution of adhesion sites as was observed with C samples (72 +/- 14% unipolar binding) was significantly (P less than 0.001) less among N PMN (31 +/- 11% unipolar binding). These investigations indicate that CF sensory mechanisms of N PMN are normal, compared with healthy adult or maternal controls. Diminished stimulated locomotion of the N PMN may be functionally related to reduced modulation of cell adhesiveness by chemotactic stimulation.

Authors

D C Anderson, B J Hughes, C W Smith

Mechanism of the postreceptor defect in insulin action in human obesity. Decrease in glucose transport system activity.

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Abstract

We have studied insulin-stimulated 3-O-methyl glucose transport by isolated adipocytes prepared from 10 normal and 11 obese individuals. The results demonstrated that the insulin-glucose transport dose-response curves were shifted to the right in cells from the obese patients, and that the magnitude of this rightward shift was significantly correlated to the reduction in adipocyte insulin receptors in individual subjects (r = 0.48, P less than 0.01). In three obese patients a rightward shift in the dose-response curve could be demonstrated and there was no decrease in maximal insulin effect. This corresponded to in vivo glucose clamp results showing only a rightward shift in the insulin dose-response curve for overall glucose disposal in these three subjects (1980. J. Clin. Invest. 65: 1272-1284). In the remaining eight obese patients, the in vitro glucose transport studies showed not only a rightward shift in the dose-response curves but also a marked decrease in basal and maximally insulin-stimulated rates of transport, indicating a postreceptor defect in insulin action. Again, this was consistent with the in vivo glucose clamp studies demonstrating a marked postreceptor defect in these individuals. In conclusion, these results indicate that the mechanism of the postreceptor defect in insulin action, which exists in many obese patients, is related to a decrease in the activity of the glucose transport effector system.

Authors

T P Ciaraldi, O G Kolterman, J M Olefsky

Gonococcal pilus vaccine. Studies of antigenicity and inhibition of attachment.

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Abstract

A gonococcal pilus vaccine or placebo was injected subcutaneously or intramuscularly into 71 human volunteers. The vaccine was found to be safe. The principal adverse reaction was a complaint of a sore arm, which was caused, at least in part, to the volume of material injected. 6 of 64 (9%) volunteers receiving the larger doses also complained of malaise. The vaccine was found to be antigenic. All of the volunteers developed an immunoglobulin class-specific antibody response as measured by a solid phase radioimmunoassay. The antibody was capable of blocking the attachment of gonococci to epithelial cells. A slight antibody response was also demonstrated to gonococcal lipopolysaccharide but the antibody responsible for blocking attachment of gonococci was directed entirely at the pilus protein. The stimulated antibodies were shown to crossreact with isolated pili of heterologous gonococcal strains and to block the attachment of heterologous gonococci. Absorption of immune sera by a heterologous pilus reduced the inhibition of attachment antibodies to pre-immune level, suggesting that the immune response was directed at a common pilus determinant.

Authors

E C Tramont, J C Sadoff, J W Boslego, J Ciak, D McChesney, C C Brinton, S Wood, E Takafuji

Antielastases of the human alveolar structures. Implications for the protease-antiprotease theory of emphysema.

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Abstract

The current concepts of the pathogenesis of emphysema hold that progressive, chronic destruction of the alveolar structures occurs because there was in imbalance between the proteases and antiproteases in the lower respiratory tract. In this context, proteases, particularly neutrophil elastase, work unimpeded to destroy the alveolar structures. This concept has evolved from consideration of patients with alpha 1-antitrypsin deficiency, who have decreased levels of serum alpha 1-antitrypsin and who have progressive panacinar emphysema. To directly assess the antiprotease side of this equation, the lower respiratory tract of non-smoking individuals with normal serum antiproteases and individuals with PiZ homozygous alpha 1-antitrypsin deficiency underwent bronchoalveolar lavage to evaluate the antiprotease screen of their lower respiratory tract. These studies demonstrated that: (a) alpha 1-antitrypsin is the major antielastase of the normal human lower respiratory tract; (b) alpha 2-macroglobulin, a large serum antielastase, and the bronchial mucous inhibitor, an antielastase of the central airways, do not contribute to the antielastase protection of the human alveolar structures; (c) individuals with PiZ alpha 1-antitrypsin deficiency have little or no alpha 1-antitrypsin in their lower respiratory tract and have no alternative antiprotease protection against neutrophil elastase; and (d) the lack of antiprotease protection of the lower respiratory tract of PiZ individuals is a chronic process, suggesting their vulnerability to neutrophil elastase is always present.

Authors

J E Gadek, G A Fells, R L Zimmerman, S I Rennard, R G Crystal

Cystic fibrosis pseudomonas opsonins. Inhibitory nature in an in vitro phagocytic assay.

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Abstract

Pseudomonas aeruginosa infection plays a primary pathogenetic role in the chronic respiratory tract disease of cystic fibrosis (CF) patients. Despite pronounced humoral immune responses, reflected by high levels of antibodies against Pseudomonas in serum and in sputum, the antibodies do not eliminate this bacterium. In the present study we have used affinity chromatography with a lipopolysaccharide substituted immunoadsorbent gel to isolate high titers (meanCF = 1:256) of immunotype specific Pseudomonas IgG antibodies from the sera of nine CF subjects, and have evaluated the functional ability of these antibodies to promote phagocytosis and intracellular killing of P. aeruginosa in an in vitro human alveolar macrophage culture system. The phagocytic and intracellular bactericidal kinetics revealed that CF IgG antibodies function in an inhibitory fashion. Both the rate of phagocytosis (rateCF = 204 cpm/unit time) and absolute bacterial uptakes maximal at 120 min (uptakeCF = 18 x 10(3) 14C cpm) were inhibited compared with appropriate positive controls (hyperimmune serum, HIS; [rateHIS = 399; uptakeHIS = 29 x 10(3), P less than 0.005]). The ability of such CF-derived opsonins to potentiate macrophage intracellular bactericidal processes was mildly impaired (bacterial survivalCF = 15 x 10(3) colony forming units (CFU)/min, survivalHIS = 9 x 10(3)). Further characterization of this defect, assessed with functional studies of the Fab and Fc portions of the immunoglobulin molecule, revealed an impairment in the attachment of these specific antibodies to the alveolar macrophage membrane Fc gamma receptors. Preliminary studies of the physical-chemical properties of these immunoglobulins were normal. The expression of this inhibitory activity in vivo may facilitate Pseudomonas colonization and the subsequent established infections in the respiratory tracts of CF subjects.

Authors

R B Fick Jr, G P Naegel, R A Matthay, H Y Reynolds

A new polymorphism in the human beta-globin gene useful in antenatal diagnosis.

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Abstract

A new polymorphism in the beta-globin is described, using the restriction enzyme Asu I. A radioactive probe specifically representing the large intervening sequence (IVS 2) of the beta-globin gene has been used to detect this polymorphism. Normally, a 0.8-kilobase fragment containing beta-IVS 2 is generated by Asu I; however, a 1.0-kilobase fragment is seen in association with 18% of beta A-genes, and 38% of beta-thalassemia genes in an Israeli population studied. By contrast, the Asu I polymorphism has rarely been seen in blacks examined to date. An additional Asu I change is seen the the delta-globin gene with a delta-IVS probe. The beta-Asu I polymorphism is shown to be useful in the antenatal diagnosis of beta-thalassemia.

Authors

M C Driscoll, M Baird, A Bank, E A Rachmilewitz

Characterization of resident glomerular cells in the rat expressing Ia determinants and manifesting genetically restricted interactions with lymphocytes.

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Abstract

The existence of a subpopulation of rat glomerular cells bearing Ia determinants has been demonstrated with the aid of techniques for the enzymatic isolation and culture of glomerular cells. The Ia-positive cell is normally resident in the uninflamed glomerulus. It resembles a mononuclear phagocyte and consists of a functionally heterogeneous cell population with the capacity of Fc receptor display and phagocytosis, both in vivo and in vitro. A new technique for labeling these cells in situ in intact glomeruli has indicated that Ia-positive cells make up approximately 2% of the total glomerular cell population. The isolated glomerular cells can take up antigen and stimulate immune lymphocytes in an I-region-restricted interaction. They are strongly stimulatory in an allogeneic primary mixed lymphocyte culture. Characterization of this cell type suggests potential new insights into the pathogenesis of renal allograft rejection and immunologically mediated glomerulonephritis.

Authors

G F Schreiner, J M Kiely, R S Cotran, E R Unanue

Expression of myeloid differentiation antigens on normal and malignant myeloid cells.

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Abstract

A series of monoclonal antibodies have been characterized that define four surface antigens (MY3, MY4, MY7, and MY8) of human myeloid cells. They were derived from a fusion of the NS-1 plasmacytoma cell line with splenocytes from a mouse immunized with human acute myelomonocytic leukemia cells. MY3 and MY4 are expressed by normal monocytes and by greater than 90% of patients with acute monocytic leukemia or acute myelomonocytic leukemia, but are detected much less often on other types of myeloid leukemia. MY7 is expressed by granulocytes, monocytes, and 5% of normal bone marrow cells. 79% of all acute myeloblastic leukemia (AML) patients tested (72 patients) express MY7 without preferential expression by any AML subtype. MY8 is expressed by normal monocytes, granulocytes, all peroxidase-positive bone marrow cells, and 50% of AML patients. MY3, MY4, and MY8 define myeloid differentiation antigens in that they are not detected on myeloid precursor cells and appear at discrete stages of differentiation. These antigens are not expressed by lymphocytes, erythrocytes, platelets, or lymphoid malignancies. The monoclonal antisera defining these antigens have been used to study differentiation of normal myeloid cells and malignant cell lines.

Authors

J D Griffin, J Ritz, L M Nadler, S F Schlossman

Ouabain effects on intracellular potassium activity and contractile force in cat papillary muscle.

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Abstract

The relationship between the positive inotropic and toxic effects of cardiac glycosides and their effects on intracellular ionic composition is incompletely defined. We measured intracellular potassium activity (alpha ik), extracellular potassium activity (alpha ek), resting potential, action potential duration, and contractile force at 32 degrees C in paired papillary muscles from feline right ventricles exposed to ouabain. Muscles used for electrophysiological measurements were quiescent except for isolated stimulation to confirm impalement and record action potential duration. Muscles used for contractile force measurements were quiescent except for 4-min periods when force was measured at a cycle length of 1,400 ms. Muscle length was adjusted to achieve 50% of maximal tension at this cycle length before each experiment. In four experiments, alpha ik and contractile force were measured in the same muscle. Alpha iK was measured with single and double-barrel K-sensitive electrodes. At 10 nM ouabain, action potential duration is prolonged. Among the concentrations tested, the threshold for a clear positive inotropic effect is 0.1 microM ouabain. The threshold for decrease in alpha iK, increase in alpha eK, and decrease in membrane potential is 1 microM, at which concentration toxic signs develop, including arrhythmias, aftercontractions, and alteration in the staircase response of contractile force to repetitive stimulation. Ouabain need not change alpha iK to effect positive inotropy in ventricular muscle, a relationship different from that reported between [K]i (intracellular potassium concentration) and positive inotropy. Higher ouabain concentrations, which others have shown to clearly inhibit active Na and K transport, are shown to upset intracellular potassium activity homeostasis and to consistently produce toxicity.

Authors

D J Browning, T Guarnieri, H C Strauss

Receptor and postreceptor defects contribute to the insulin resistance in noninsulin-dependent diabetes mellitus.

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Abstract

We have assessed the mechanisms involved in the pathogenesis of the insulin resistance associated with impaired glucose tolerance and Type II diabetes mellitus by exploring, by means of the euglycemic glucose-clamp technique, the in vivo dose-response relationship between serum insulin and the overall rate of glucose disposal in 14 control subjects; 8 subjects with impaired glucose tolerance, and 23 subjects with Type II diabetes. Each subject had at least three studies performed on separate days at insulin infusion rates of 40, 120, 240, 1,200, or 1,800 mU/M2 per min. In the subjects with impaired glucose tolerance, the dose-response curve was shifted to the right (half-maximally effective insulin level 240 vs. 135 microunits/ml for controls), but the maximal rate of glucose disposal remained normal. In patients with Type II diabetes mellitus, the dose-response curve was also shifted to the right, but in addition, there was a posal. This pattern was seen both in the 13 nonobese and the 10 obese diabetic subjects. Among these patients, an inverse linear relationship exists (r = -0.72) so that the higher the fasting glucose level, the lower the maximal glucose disposal rate. Basal rates of hepatic glucose output were 74 +/- 4, 82 +/- 7, 139 +/- 24, and 125 +/- 16 mg/M2 per min for the control subjects, subjects with impaired glucose tolerance, nonobese Type II diabetic subjects, and obese Type II diabetic subjects, respectively. Higher serum insulin levels were required to suppress hepatic glucose output in the subjects with impaired glucose tolerance and Type II diabetics, compared with controls, but hepatic glucose output could be totally suppressed in each study group. We conclude that the mechanisms of insulin resistance in patients with impaired glucose tolerance and in patients with Type II noninsulin-dependent diabetes are complex, and result from heterogeneous causes. (a) In the patients with the mildest disorders of carbohydrate homeostasis (patients with impaired glucose tolerance) the insulin resistance can be accounted for solely on the basis of decreased insulin receptors. (b) In patients with fasting hyperglycemia, insulin resistance is due to both decreased insulin receptors and postreceptor defect in the glucose mechanisms. (c) As the hyperglycemia worsens, the postreceptor defect in peripheral glucose disposal emerges and progressively increases. And (d) no postreceptor defect was detected in any of the patient groups when insulin's ability to suppress hepatic glucose output was measured.

Authors

O G Kolterman, R S Gray, J Griffin, P Burstein, J Insel, J A Scarlett, J M Olefsky

Zero-Flow Pressures and Pressure-Flow Relationships during Single Long Diastoles in the Canine Coronary Bed before and during Maximum Vasodilation: LIMITED INFLUENCE OF CAPACITIVE EFFECTS

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Abstract

The proposal that diastolic coronary flow is regulated by an intramyocardial “back-pressure” that substantially exceeds coronary venous and ventricular diastolic pressures has been examined in an open-chest canine preparation in which instantaneous left circumflex pressure and flow could be followed to cessation of inflow during prolonged diastoles. Despite correlation coefficients consistently >0.90, pressure-flow data during individual diastoles were concave to the flow axis before and during pharmacologically induced maximum coronary vasodilation. Data were better fitted (P < 0.01) by second-order equations than by linear equations in >90% of cases. Second-order pressure-axis intercepts (Pf=0)1 averaged 29±7 (SD) mm Hg before vasodilation and 15±2 mm Hg during vasodilation; left and right atrial pressures were always substantially lower (8±3 and 5±2 mm Hg before vasodilation and 8±2 and 4±1 mm Hg during dilation). Values of Pf=0 before vasodilation varied directly with levels of coronary inflow pressure. A modification of the experimental preparation in which diastolic circumflex pressure could be kept constant was used to evaluate the suggestion that Pf=0 measured during long diastoles are misleadingly high because of capacitive effects within the coronary circulation as inflow pressure decreases. Decreases in Pf=0 attributable to capacitive effects averaged only 5.9±3.0 mm Hg before vasodilation and were smaller during dilation. We conclude that Pf=0 is a quantitatively important determinant of coronary driving pressure and flow, resulting from both factors related to, and independent of, vasomotor tone. Adjustments of flow during changing physiological situations may involve significant changes in Pf=0 as well as in coronary resistance.

Authors

Francis J. Klocke, Irwin R. Weinstein, James F. Klocke, Avery K. Ellis, David R. Kraus, Robert E. Mates, John M. Canty, Ran D. Anbar, Roslyn R. Romanowski, Kenneth W. Wallmeyer, Martin P. Echt

A role for prostaglandins and thromboxanes in the exposure of platelet fibrinogen receptors.

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Abstract

Exposure of fibrinogen receptors by a variety of agonists is a prerequisite for platelet aggregation. Because the synthesis of prostaglandins and thromboxane A2 also occurs during platelet aggregation we wondered whether these agents participate in the exposure of platelet fibrinogen receptors. Therefore, we measured the binding of human 125I-fibrinogen to gel-filtered normal human platelets after prostaglandin and thromboxane synthesis had been inhibited by aspirin or indomethacin. The fibrinogen binding assay was performed at 37 degrees C but without stirring to prevent the formation of platelet aggregates. Platelet secretion, measured with [14C]serotonin, did not occur during the procedure. Aspirin or indomethacin inhibited fibrinogen binding stimulated by 10 microM epinephrine by 53%, and inhibited fibrinogen binding stimulated by 1-2 microM ADP by 37.1%. However, ADP at concentrations greater than 2 microM returned fibrinogen binding toward control values. Scatchard analysis demonstrated that aspirin decreased the number but not the affinity of the exposed fibrinogen receptors. To determine whether prostaglandins are capable of directly exposing fibrinogen receptors, prostaglandin H2 was used to stimulate platelets in the fibrinogen binding assay. Prostaglandin H2 exposed approximately 54,000 fibrinogen receptors/platelet and corrected the deficit in receptor exposure induced by aspirin. These studies demonstrate that platelet prostaglandins or thromboxane A2 can play a direct role in the exposure of platelet fibrinogen receptors. In addition, they suggest that the synthesis of prostaglandins and thromboxane A2 by stimulated platelets may be all that is required for optimal secondary platelet aggregation.

Authors

J S Bennett, G Vilaire, J W Burch

Decreased sensitivity of old and progeric human fibroblasts to a preparation of factors with insulinlike activity.

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Abstract

To determine whether old cells have a reduced response to a preparation of factors from human plasma with insulinlike activity (ILA), we analyzed the response to ILA of early and late passage human fibroblasts from young, old, and progeric donors in the acute stimulation of [3H]2-deoxy-D-glucose (2dG) uptake and the delayed stimulation of [3H]thymidine (TdR) incorporation into DNA. The ILA concentration required to produce equivalent, relative stimulation of TdR incorporation was increased two- to three-fold in late passage cells and cells from old and progeric donors (P less than 0.01). 50 and 95% of maximal stimulation (ILA50, ILA95) was achieved by 0.26 +/- 0.07 and 1.38 +/- 0.13 ng insulin equivalents/ml (mean +/- SD) respectively, in cells from young adults at early passage. Corresponding values were 0.54 +/- 0.05 and 2.90 +/- 0.25 in cells from old donors; greater than 0.9 +/- 0.1 and greater than 3.1 +/- 0.1 in cells from a 9-yr-old progeric donor; and 0.4 +/- 0.05 and 1.1 +/- 0.04 in cells from normal children (9-13 yr). For two cell strains from young adults, ILA50 and ILA95 were 0.30 +/- 0.02 and 1.0 +/- 0.3 ng eq/ml at 30% of their in vitro lifespan completed (%LC) and these values increased at rates of 0.005 ng eq/ml per %LC and 0.04 ng eq/ml per %LC, respectively. The mean stimulation of 2dG uptake ratio (ILA/control) decreased from early to late passage from 2.1 +/- 0.6 to 1.3 +/- 0.1 in young adult donors (P less than 0.05), but there were no significant differences between young and old donors at either early or late passage. The mean stimulation ratio in progeric cells (1.2 +/- 0.2) did not change with in vitro passage, but was significantly lower than that of age-matched normal cells (2.1 +/- 0.8, P less than 0.001). In progeria cells, the reduced stimulation of 2dG uptake upon addition of ILA was due to an increased basal rate of uptake (0.19 +/- 0.01 pmol [3H]2dG/min per mg protein vs. 0.13 +/- 0.01 in age-matched normal cells), and not to a decline in the maximal rate of uptake (0.26 +/- 0.01 vs. 0.27 +/- 0.02, respectively). Similar results were found for in vitro aging in cells from an old donor.

Authors

C B Harley, S Goldstein, B I Posner, H Guyda

Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells.

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Abstract

It has been postulated that lipoprotein lipase, an enzyme important in the uptake of fatty acids into tissues, is bound to the vascular endothelial cell surface and that this binding occurs through attachment to heparinlike glycosaminoglycans. Furthermore, it is thought that heparin releases the enzyme from its attachment to the endothelium into the circulation. These hypotheses have never been tested directly in cell systems in vitro. In the present study we have directly evaluated the interaction of lipoprotein lipase, purified from bovine skim milk with monolayer cultures of endothelial cells, isolated from bovine pulmonary artery. Endothelial cells in primary culture had no intrinsic lipoprotein lipase activity but were able to bind lipoprotein lipase quantitatively. The binding reached equilibrium and was saturable at 0.24 nmol of lipoprotein lipase/mg of cell protein. The concentration of lipoprotein lipase at half-maximal binding was 0.52 microM. Bound lipoprotein lipase could be detached from cultured cells by increasing concentrations of heparin, and at and above 0.6 microgram/ml of heparin, 90% of the cell-bound lipoprotein lipase activity was released. Heparan sulfate and dermatan sulfate released the enzyme to a lesser extent and chondroitin sulfate caused little, if any, release of lipoprotein lipase. The release of lipoprotein lipase with heparin was not associated with a release of [3S]glycosaminoglycans from 35S-prelabeled cells. Reductions of lipoprotein lipase binding to endothelial cells and of cell surface-associated [3S]glycosaminoglycans in 35S-prelabeled cells occurred in parallel both when cells were pretreated with crude Flavobacterium heparinum enzyme before lipoprotein lipase binding and when cells were treated with this enzyme after lipoprotein lipase binding. The removal of heparan sulfate from the cell surface by purified heparinase totally inhibited the binding of lipoprotein lipase by endothelial cells, but the removal of chondroitin sulfate by chondroitin ABC lyase had no effect on this binding. These results provide direct evidence for lipoprotein lipase attachment to endothelial cells through heparan sulfate on the cell surface, and provide evidence for the release of lipoprotein lipase by heparin through a detachment from this binding site.

Authors

K Shimada, P J Gill, J E Silbert, W H Douglas, B L Fanburg

Utilization of arachidonic and linoleic acids by cultured human endothelial cells.

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Abstract

When cultured human umbilical vein endothelial cells are supplemented with linoleic acid, the arachidonic acid content of the cellular phospholipids is reduced approximately 35%. Most of the fatty acid compositional change occurs during the first 24 h. One factor responsible for this effect is the inability of the endothelial cells to convert appreciable amounts of linoleic to arachidonic acid, due to a fatty acid delta 6-desaturase deficiency. By contrast, these endothelial cultures contain delta 5- and delta 9-desaturase activity and are able to elongate long-chain polyunsaturated fatty acids. The other factor that contributes to the decrease in arachidonic acid is that high concentrations of linoleic acid reduce the incorporation of arachidonate into cellular phospholipids. Stearic acid, a long-chain saturate, does not produce any reduction, whereas eicosatrienoic acid is an even more effective inhibitor than linoleic acid. In spite of the fact that high concentrations of these polyunsaturates produced inhibition, the endothelial cells were found to efficiently incorporate exogenous arachidonic acid into cellular phospholipids and triglycerides. This may serve to compensate for the inability of these cells to synthesize arachidonic acid from linoleic acid. These findings suggest that the endothelium obtains arachidonic acid from an extracellular source, that this cannot be provided in the form of linoleic acid and, in fact, that high concentrations of linoleic acid actually may interfere with the ability of the endothelium to maintain an adequate supply of intracellular arachidonic acid.

Authors

A A Spector, T L Kaduce, J C Hoak, G L Fry

beta zero thalassemia in Sardinia is caused by a nonsense mutation.

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Abstract

We report the characterization of a molecular lesion of beta thalassemia in Sardinia. Beta thalassemia in this area is predominantly the beta zero type with low levels of beta-globin mRNA. Translation assay of this messenger RNA in a cell-free system showed beta-globin chain synthesis only with the addition of an amber (UAG) suppressor transfer RNA. Double-stranded complementary DNA prepared from reticulocyte mRNA from a Sardinian patient was cloned in a bacterial plasmid and a beta-globin complementary DNA containing clone was isolated and sequenced. At the position corresponding to amino acid number 39, a single nucleotide mutation converted a glutamine codon (CAG) to an amber termination codon (UAG). We previously reported an amber nonsense mutation at amino acid 17 as a cause of Chinese beta zero thalassemia. Thus, beta zero thalassemia in Sardinia represents the second example of a nonsense mutation, and we predict that other beta zero thalassemias with mutations at various points along the beta-globin chain will be found to form a discrete subgroup of beta zero thalassemia. These experiments further illustrate the heterogeneity of lesions that lead to defective globin chain synthesis in beta thalassemia.

Authors

R F Trecartin, S A Liebhaber, J C Chang, K Y Lee, Y W Kan, M Furbetta, A Angius, A Cao

Direct effects of thyrotropin-releasing hormone, cyproheptadine, and dopamine on adrenocorticotropin secretion from human corticotroph adenoma cells in vitro.

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Abstract

In an attempt to delineate the mechanism and the site of action of cyproheptadine and dopaminergic agonists as well as hormones including thyrotropin-releasing hormone (TRH) and hydrocortisone, the effects of these substances on ACTH secretion from corticotroph adenoma cells in culture were examined. Dispersed cells of pituitary adenomas obtained at surgery from four patients with Nelson's syndrome and one subject with Cushing's disease formed a monolayer and actively secreted ACTH into the medium. When TRH (0.1 microM) was added to the medium, a significant increase in ACTH secretion was demonstrated by adenoma cells from two patients who responded to TRH preoperatively. Moreover, a dose-response relationship between TRH concentrations and ACTH secretion was observed. Incubation of cells with cyproheptadine (1 or 0.1 microM) resulted in a significant decrease in ACTH release, and inhibited stimulation produced by TRH in one experiment. This effect of cyproheptadine was blocked when equimolar concentrations of serotonin was coincubated, whereas serotonin by itself did not affect ACTH secretion. Dopamine (0.1 microM) lowered ACTH accumulation in the medium, which was blocked by the addition of haloperidol. When hydrocortisone was added to the culture, dose-dependent suppression of ACTH secretion was demonstrated. TRH at an equimolar concentration reversed this effect, but, failed to overcome the inhibition induced by a higher concentration of hydrocortisone in cells from one adenoma studied. Cultured normal corticotrophs obtained from a patient with metastatic breast cancer, on the other hand, did not show any response to these substances, except for hydrocortisone. We suggest that TRH, cyproheptadine, dopamine affect ACTH secretion in patients with ACTH-producing pituitary adenomas by their direct action on the adenoma.

Authors

M Ishibashi, T Yamaji

Isolation and properties of an abnormal Hageman factor (Factor XII) molecule in a cross-reacting material-positive hageman trait plasma.

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Abstract

We have previously described two unrelated individuals with homozygous Hageman trait (Factor XII deficiency) whose plasmas contained nonfunctional material immunologically indistinguishable from normal Hageman factor (HF). Abnormal HF from the plasma of one these subjects has now been purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, alkaline disc gel electrophoresis, and immunoelectrophoresis. Purified abnormal HF had no clot-promoting activity, but showed the same specific antigenicity as purified normal HF by an immunoassay. The abnormal HF was of a single chain polypeptide with the same molecular weight (80,000) as normal HF and was positively stained by periodic acid-Schiff reagent. Both normal and abnormal HF had similar amino acid compositions and isoelectric points (pI 6.5 approximately 7.1). When 125I-labeled abnormal HF and 131I-labeled normal HF were mixed with normal plasma and exposed to glass, both HF underwent an identical pattern of cleavage, yielding 52,000- and 30,000-mol wt fragments. Similarly, abnormal HF was fragmented by trypsin in the same way as normal HF, but no prekallikrein-activating activity was generated after cleavage. [3H]Diisopropyl phosphorofluoridate was incorporated into a 29,000-mol wt fragment of the trypsin-cleaved normal HF, but not into that of the trypsin-cleaved abnormal HF. These data suggest that the molecular defect in this abnormal HF resides at or near the active site serine residue in the 30,000-mol wt part of the molecule.

Authors

H Saito, S J Scialla

Comparison of different antinucleic acid antibody spectrotypes in spontaneous, induced, and murine lupus.

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Abstract

Sera from patients with systemic lupus erythematosus and from mice spontaneously developing lupus were subjected to isoelectric focusing by a microsucrose gradient method. The spectrotypes of human antibodies to native DNA, denatured DNA, and polyriboadenylic acid (poly A) were compared. Antibodies to native DNA and denatured DNA focused into two regions whose boundaries were pH 5.0-7.0 and pH 8.5-10.0. Antinative DNA antibodies were more homogeneous than antidenatured DNA antibodies. Anti-DNA antibodies in cryoglobulins showed more restriction than those present in serum. There was no relationship between spectrotype and pattern of disease expression. Murine antibodies to native DNA were more heterogeneous than human anti-DNA antibodies. The spectrotypes of antidenatured DNA antibodies from patients with systemic lupus erythematosus or drug induced lupus, or from an immunized rabbit, were similar. Likewise, antibodies to poly A were similar in both human and murine lupus. In contrast to anti-DNA, antibodies to poly A were restricted and focused only in the alkaline range (pH 9.5-10.0). The spectrotype of antipoly A antibodies induced by lipopolysaccharide were comparable but had an additional small band at pH 6.2. Our results suggest unique antibody spectrotypes with varying degrees of restriction for different nucleic acid antigens. Furthermore, spontaneous and induced autoantibodies have similar spectrotypes. Thus, the B cell clones producing antinucleic acid antibodies may be similar whether they are activated spontaneously, following immunization, or as a consequence of polyclonal stimulation.

Authors

M Fischbach, J Rabbie, N Talal

Follicle-stimulating Hormone and Human Spermatogenesis

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Abstract

The role of follicle-stimulating hormone (FSH) in the control of spermatogenesis is not well established in any species, including man. We studied the effect of an experimentally-induced, selective FSH deficiency on sperm production in normal men. After a 3-mo control period, five normal men received testosterone enanthate (T) 200 mg i. m. weekly to suppress luteinizing hormone (LH) and FSH, until three successive sperm counts revealed azoospermia or severe oligospermia (sperm counts <3 million/ml). Then, while continuing T, human chorionic gonadotropin (hCG) 5,000 IU i. m. three times weekly was administered simultaneously to replace LH activity, leaving FSH activity suppressed. The effect of the selective FSH deficiency produced by hCG plus T administration on sperm production was determined.

Authors

William J. Bremner, Alvin M. Matsumoto, Allen M. Sussman, C. Alvin Paulsen

Prevention of doxorubicin cardiac toxicity in the mouse by N-acetylcysteine.

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Abstract

This study was undertaken to investigate the effect of exogenous sulfhydryl compound administration on the toxicity of doxorubicin in mice. Pretreatment of CDF1 mice with a pharmacologic dose (2,000 mg/kg) of n-acetyl-l-cysteine 1 h before doxorubicin (20 mg/kg, i.p.) decreased lethality from 100% (n = 44) to 37.7% (n = 53), P less than 0.001. Variation in the timing and dose of n-acetylcysteine significantly diminished its protective activity. Pretreatment with n-acetylcysteine also significantly reduced long-term mortality in animals receiving multiple doses of doxorubicin; 10 wk after the third of three doxorubicin doses (5 mg/kg, i.p.) administered at 2-wk intervals, survival in the n-acetylcysteine pretreated group was 51.4% (n = 35) compared with 16.7% (n = 30) for animals receiving saline before doxorubicin, P less than 0.01. In this experiment, n-acetylcysteine pretreatment also diminished doxorubicin-related losses in total body weight and heart wet weight by 55.2% (P less than 0.05), and 60.9% (P less than 0.02), respectively, compared with animals pretreated with saline. N-acetylcysteine pretreatment also ablated electron microscopic evidence of doxorubicin cardiomyopathy without alleviating morphological features of its toxic effects on the liver or small intestinal mucosa. The cardioprotective action of n-acetylcysteine may be partially explained by the 429 +/- 60% increase in cardiac nonprotein sulfhydryl content (P less than 0.01) that was measured one hour after n-acetylcysteine administration; nonprotein sulfhydryl concentration in the liver at the same time was insignificantly different from control levels. Treatment with n-acetylcysteine also increased the nonprotein sulfhydryl content of P388 leukemia cells nearly threefold; however, it did not after the chemotherapeutic activity of doxorubicin against this murine tumor. Whereas n-acetylcysteine blocked doxorubicin cardiac toxicity, it did not affect the uptake or metabolism of doxorubicin in the heart or liver. These results suggest that the concentration of free sulfhydryl groups in the heart may play a role in the development of doxorubicin cardiac toxicity and that augmenting cardiac nonprotein sulfhydryl group content with n-acetylcysteine may provide a means to enhance the chemotherapeutic index of doxorubicin.

Authors

J H Doroshow, G Y Locker, I Ifrim, C E Myers

Effect of quinidine on the digoxin receptor in vitro.

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Abstract

To investigate the basis for a clinically important digitalis-quinidine interaction that is characterized by increases in serums digoxin concentrations when quinidine is administered to digoxin-treated patients, we have studied in vitro the interaction of quinidine with the digoxin receptor. Evidence has been obtained that quinidine is capable of decreasing the affinity for digoxin of cardiac glycoside receptor sites on purified Na,K-ATPase and on intact human erythrocyte membranes. As others have shown, quinidine is capable of inhibiting Na,K-ATPase activity, and evidence has been obtained in the current study that, while quinidine can reduce the affinity of the enzyme for digoxin, it is also capable of acting together with digoxin in inhibiting enzyme activity to a degree greater than the inhibitory effect of digoxin alone. The concentrations of digoxin and quinidine used in this study were considerably greater than their therapeutic serum concentrations. Nevertheless, these observations are consistent with the hypothesis that the increases in serum digoxin concentrations and the decreases in volumes of digoxin distribution observed clinically when quinidine is administered to digoxin-treated patients may reflect, at least in part, a decrease in the affinity of tissue receptors for digoxin. The possibility must also be considered that enhanced cardiac effects of digoxin may occur clinically as the result of an augmentation, by quinidine, of digoxin effects, which more than compensates for the modest reduction in digoxin binding.

Authors

W J Ball Jr, D Tse-Eng, E T Wallick, J P Bilezikian, A Schwartz, V P Butler Jr

Familial dysbetalipoproteinemia. Abnormal binding of mutant apoprotein E to low density lipoprotein receptors of human fibroblasts and membranes from liver and adrenal of rats, rabbits, and cows.

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Abstract

Patients with familial dysbetalipoproteinemia (F. Dys.), also called familial type 3 hyperlipoproteinemia, are homozygous for a mutant allele, Ed, that specifies an abnormal form of apoprotein (apo) E, a prominent constituent of remnant lipoproteins derived from very low density lipoproteins (VLDL) and chylomicrons. Apo E is thought to mediate the removal of remnant lipoproteins from the plasma by virtue of its ability to bind to hepatic lipoprotein receptors. In F. Dys. patients, remnant-like lipoproteins accumulate, apparently because of delayed clearance by the liver. In the current studies, we show that the abnormal protein specified by the Ed allele (apo E-D) from some, but not all, patients with F. Dys. has a markedly deficient ability to bind to low density lipoprotein (LDL) receptors. Apo E was isolated from eight control subjects and nine patients with F. Dys. and incorporated into phospholipid complexes. The complexes were tested for their ability to compete with human 125I-LDL or rabbit 125I-beta-VLDL fo binding to LDL receptors in four assay systems: cultured human fibroblasts, solubilized receptors from bovine adrenal cortex, liver membranes from rats treated with 17 alpha-ethinyl estradiol, and liver membranes from normal rabbits. The apo E-D from six of the nine patients with F. Dys. showed binding affinities for LDL receptors that were reduced by greater than 98% in all receptor assays (group 1 patients). All of these group 1 patients were unequivocally of phenotype apo E-D/D by the criterion of isoelectric focussing. The apo E from the three other F. Dys. patients showed a near normal binding ability in all four of the receptor assays (group 2 patients). One of these group 2 patients appeared to have the apo E-D/D phenotype by isoelectric focussing. In the other two patients in group 2, apo E-D was the predominant protein (phenotype, apo E-D/D), but traces of protein in the region corresponding to normal apo E (apo E-N) were also present. The difference between group 1 and group 2 patients was also apparent when the apo E was iodinated and tested directly for binding to liver membranes from rats treated with 17 alpha-ethinyl estradiol. The 125I-labeled apo E from a group 2 patient, but not a group 1 patient, showed enhanced uptake when perfused through the liver of an estradiol-treated rate, indicating that the receptor binding ability of apo E correlated with uptake in the intact liver. The current studies allow the subdivision of patients with F. Dys. into two groups. In group 1, the elevated plasma level of remnants appears to be due to a diminished receptor binding activity of the abnormal protein specified by the Ed allele; in group 2 patients, the cause of the elevated plasma level of remnants remains to be explained.

Authors

W J Schneider, P T Kovanen, M S Brown, J L Goldstein, G Utermann, W Weber, R J Havel, L Kotite, J P Kane, T L Innerarity, R W Mahley

Effects of disodium dichloromethylene diphosphonate on bone loss in paraplegic patients.

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Abstract

21 paraplegic patients with recent traumatic spinal cord injury were orally administered 400 (n = 7) or 1,600 (n = 7) mg/d of disodium dichloromethylene diphosphonate (Cl2MDP) and compared with a placebo group (n = 7) to test the preventive effects of the drug on acute bone loss and osteoclastic resorption. Cl2MDP therapy was initiated at a mean of 17.6 d after the onset of paraplegia. The study lasted at least 6 mo, consisting of a 3.5-mo treatment period, and a variable follow-up period. The effects of Cl2MDP were assessed by blood and urine biochemistry, bone histomorphometry on transilial samples, photon absorptiometry of the tibia and fibula, and radiomorphometry of the femur. The elevation in serum and urinary calcium and in urine hydroxyproline observed in the placebo group did not appear under treatment. With both doses of Cl2MDP there was no further decrease in the bone mineral content. In the treated groups, a smaller percentage increase in osteoclastic population was also noted when compared with the placebo group, but this difference was not significant. There was no mineralization defect induced by Cl2MDP, as shown by tetracycline double labeling. It thus appears that at doses ranging between 400 and 1,600 mg, given as early as possible, Cl2MDP can prevent or reduce the development of the acute bone loss of paraplegic patients, without adverse side effects, though it does not prevent the development of heterotopic ossification.

Authors

P Minaire, E Berard, P J Meunier, C Edouard, G Goedert, G Pilonchery

Tumor promoter phorbol myristic acetate stimulates immunoglobulin secretion correlated with growth cessation in human B lymphocyte cell lines.

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Abstract

Immunoglobulin production by lymphoblast cell lines was studies using protein A-red blood cell plaque formation to detect individual secreting cells. Immunoglobulin (Ig) secretion by 6 of 12 human B-cell lines tested could be stimulated up to twentyfold by phorbol myristic acetate (PMA) at subtoxic concentrations of 10-1000 ng/ml depending on the line. Stimulation was found with both IgM and IgG cell lines. No switch of Ig class synthesis was found in the cell lines as a result of PMA incubation. Increase in Ig secretion was closely associated with cessation of growth resembling induction of terminal differentiation in the cells. PMA induction of Ig secretion in B lymphocytes from normal peripheral blood requires the cooperation of T cells. PMA stimulation of certain cell lines reported here suggests that the lines are late in the differentiation pathway to plasmacyte and can be easily triggered to secrete Ig by membrane-altering agents.

Authors

P Ralph, T Kishimoto

Pathogenic effects of bullous pemphigoid autoantibodies on rabbit corneal epithelium.

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Abstract

Bullous pemphigoid (BP) is associated with circulating autoantibodies reactive with an antigen(s) of the basement membrane zone (BMZ) of skin and mucosae. The pathogenicity of these autoantibodies, although suspected, is unconfirmed. We have investigated the effects of BP autoantibodies on a closely related tissue, the corneal epithelium of the rabbit. IgG fractions from the sera of seven patients with BP were purified by (a) ammonium sulfate precipitation, (b) ion exchange chromatography, or (c) gel filtration. Control IgG was prepared by ion exchange chromatography of pooled normal human gamma globulins. 32 rabbits received corneal intrastromal injections of BP IgG fractions (50 microliter, 0.95-2.05 mg total dose) in one eye, and control IgG (50 microliter, 1.8 mg) in the contralateral cornea. 28 of 32 BP IgG injections produced corneal inflammatory lesions, 10 of which developed visible blisters. Histologically, lesions showed polymorphonuclear cells clustering along the BMZ, and subepithelial blister formation. Immunofluorescence showed in vivo bound IgG and C3 at the BMZ. The intensity of inflammation was dose dependent and correlated often with in vitro complement fixation titers of the fractions. None of 32 corneas injected with control IgG became inflamed. BP IgG fractions injected intradermally into the ear skin of rabbits failed to produce inflammation. This may be due to slow clearance of IgG in the cornea, and optimal binding by the corneal epithelium. The intracorneal injections of BP IgG reproduce the clinical, histological, and immunological features of BP. This study provides evidence that BP autoantibodies are pathogenic.

Authors

G J Anhalt, C F Bahn, R S Labib, J J Voorhees, A Sugar, L A Diaz

Collagen and Collagen-derived Fragments Are Chemotactic for Tumor Cells

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Abstract

Organs that are rich in collagen such as liver, lungs, and bone are frequently sites of tumor cell metastasis. In this study, we have found that cultured tumor cells of human and rat origin migrated unidirectionally in response to collagen in vitro. Synthetic di- and tri-peptides that contained amino acid sequences found frequently in the collagen helix caused similar effects. These results are consistent with the hypothesis that collagen or collagen fragments released during connective tissue remodeling may be important in tumor cell metastasis.

Authors

Gregory R. Mundy, Sandra Demartino, David W. Rowe

Effect of gastric inhibitory polypeptide on plasma levels of chylomicron triglycerides in dogs.

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Abstract

To determine whether gastric inhibitory polypeptide (GIP) promotes the clearance of chylomicron triglycerides (TG) from the circulation in dogs, chyle collected from donor dogs via a thoracic duct fistula was infused at a rate of 2 ml/min i.v. into normal recipient dogs during an infusion of either porcine GIP (1 microgram/kg per h) or saline as a control. In the GIP-infused dogs the rise in plasma TG was significantly below that of the control animals [mean peak of 36 +/- 4 mg/dl vs. 82 +/- 18 mg/dl (P less than 0.05)]. It is concluded that GIP exerts an effect upon the removal of chylomicron TG from the blood. The results suggest that GIP may play a physiologic role in the disposition of ingested fat.

Authors

T Wasada, K McCorkle, V Harris, K Kawai, B Howard, R H Unger

Salicylate-aspirin interaction in the rat. Evidence that salicylate accumulating during aspirin administration may protect vascular prostacyclin from aspirin-induced inhibition.

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Abstract

Aspirin inhibits cyclooxygenase, thus preventing thromboxane A2 production in blood platelets and prostacyclin in vascular cells. Aspirin is rapidly hydrolyzed to salicylate in the circulation. The objectives of this study were (a) to evaluate whether administration of salicylate, though ineffective by itself, prevents the inhibitory effect of aspirin on platelet and/or vascular cyclooxygenase activity; (b) to verify whether salicylate accumulating in blood after aspirin administration interferes with the pharmacological activity of further doses of aspirin. Pretreatment of rats with sodium salicylate (25-100 mg/kg i.p.) resulted in dose-related prevention of the effect of a subsequent dose of aspirin (2.5-10 mg/kg i.v.) on both platelet and vascular cells. Sodium salicylate appeared to amplify the greater response of platelets to aspirin compared with vessel wall. Pretreatment of rats with repeated high doses of aspirin (200 mg/kg) resulted after 24 h in blood salicylate levels (150-200 microgram/ml) that significantly prevented the inhibitory effect of a subsequent dose of aspirin on newly synthesized vascular prostacyclin. Blood salicylate levels obtained after 36 or 48 h (less than 50 microgram/ml) were too low to blunt aspirin's effect. The interference with aspirin of its major endogenous metabolite should be borne in mind when interpreting results obtained with high dose aspirin or during repeated administration of this drug.

Authors

E Dejana, C Cerletti, C de Castellarnau, M Livio, F Galletti, R Latini, G de Gaetano

Vancomycin prophylaxis of experimental Streptococcus sanguis. Inhibition of bacterial adherence rather than bacterial killing.

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Abstract

Using a strain of Streptococcus sanguis tolerant to vancomycin to infect aortic vegetations in rats, we found that prophylactic intravenous vancomycin given 30 min before bacterial challenge decreased the incidence of endocarditis from 88 to 8% (P less than 10(-5)). Because peak vancomycin serum levels were below the minimal bactericidal concentration, mechanisms of protection other than bacterial killing were investigated. S. sanguis were incubated with inhibitory concentration of vancomycin (50 microgram/ml) for 10 h and washed. 85% of rats (73/86) inoculated with control bacteria developed endocarditis, whereas only 42% (33/78) of those inoculated with vancomycin-exposed bacteria did so (P less than 10(-5)). When rats were killed 30 min after bacterial challenge, S. sanguis were detected by culture of the vegetations in 44% of rats injected with control bacteria, but in only 13% of those challenged with vancomycin-exposed bacteria (P less than 0.03). Enhanced clearance of vancomycin-exposed streptococci was not responsible for this protection because blood cultures showed no difference in the level and duration of bacteremia after injection of control or vancomycin-exposed S. sanguis. Moreover, this protection was not abolished in neutropenic rats injected with vancomycin-exposed bacteria, despite more prolonged bacteremia. These results suggest that vancomycin exerted its protection by lowering adherence of tolerant S. sanguis to vegetations rather than through bactericidal activity or enhanced clearance of bacteria by phagocytic cells. In the choice of antibiotics for prophylaxis of endocarditis, reduction of bacterial adhesion may be a criterion as important as bacterial killing.

Authors

J P Bernard, P Francioli, M P Glauser