The binding of 125I-von Willebrand factor (125I-vWF) to platelets stimulated by thrombin, ADP, and a combination of ADP + epinephrine (EPI) is specific, saturable, and reversible. Active platelet metabolism and divalent cations are required for binding induced by these stimuli, but not by ristocetin, suggesting the existence of different mechanisms involved in the vWF-platelet interaction. A monoclonal antibody directed against an epitope of membrane glycoprotein (GP) Ib had no effect on the binding of 125I-vWF to normal platelets stimulated by thrombin or a combination of ADP + EPI, but completely blocked ristocetin-induced binding. Binding induced by thrombin to GPIb-blocked platelets was specific. Moreover, thrombin-induced binding of 125I-vWF was increased, rather than decreased, in two patients with the Bernard-Soulier syndrome whose platelets lacked GPIb. Conversely, monoclonal antibodies directed against the GPIIb/IIIa complex had no effect on ristocetin-induced binding of 125I-v-WF to normal platelets, but blocked thrombin- and ADP + EPI-induced binding. To exclude effects mediated by the platelet Fc receptor, a monoclonal IgG directed against an epitope present on human B cells and monocytes, but not expressed on resting or stimulated platelets, was used. It did not affect 125I-vWF binding induced by any of the stimuli. These studies show that platelets have more than one binding site for vWF, and that they may be exposed by different stimuli.
Z M Ruggeri, L De Marco, L Gatti, R Bader, R R Montgomery
Breakdown of phosphatidylinositol (PI) has been shown to be increased during Ca2+-mediated stimulation of cellular responses in many systems and has been proposed to be involved in stimulus-secretion coupling. The effects on PI breakdown of insulin secretagogues that alter cellular Ca2+ or cyclic (c)AMP levels were investigated in perifused rat islets of Langerhans. Isolated islets were labeled with myo-[2-3H(N)]inositol and the efflux of 3H-labeled metabolites was monitored. Glucose (16.7 mM) greatly increased 3H release in a manner that paralleled the second phase of the insulin secretory response; by 60 min, the amount of [3H]PI in the islet decreased by 50%. Removal of Ca2+ from the perifusate or blockade of Ca2+ entry through the voltage-dependent channels by D600 (20 microM) abolished the glucose-induced increase in 3H efflux. Depolarization with 47 mM K+, which increases Ca2+ entry, stimulated protracted 3H and insulin release. Glucose-stimulated output of 3H was not prevented by epinephrine (1 microM) even though the insulin response was abolished. In contrast, 3H output was not affected by isobutylmethylxanthine (1 mM), known to raise cellular levels of cAMP, although insulin release was stimulated. These findings indicate that PI breakdown is not related to the exocytotic process since stimulation of insulin release and PI breakdown could be uncoupled, and that it is not associated with cAMP-mediated regulation of insulin release. PI breakdown in islets differs from the immediate, transient phenomenon reported in other systems in both its timing and requirement for Ca2+. It appears to result from the entry of Ca2+ and not to be the mechanism by which glucose initiates Ca2+ influx.
K V Axen, U K Schubart, A D Blake, N Fleischer
To understand the contribution to the pathophysiology of sickle cell anemia of the different erythrocyte density types present in the blood of these patients, we have studied the viscosimetric and hemodynamic characteristics of four major classes of hemoglobin SS erythrocytes. We have isolated reticulocytes, discocytes, dense discocytes, and irreversibly sickled cells (fractions I-IV) on Percoll-Renografin density gradients. Bulk viscosity was studied in a coneplate viscosimeter and the hemodynamic studies were performed on the isolated, artificially perfused mesoappendix vasculature of the rat (Baez preparation). Bulk viscosity measurements at shear rates of 230 S-1 demonstrate that when the cells are oxygenated, fraction I (reticulocyte rich) has a higher viscosity than expected from its low intracellular hemoglobin concentration. The rest of the fractions exhibit moderate increases in bulk viscosity pari-passu with the corresponding increases in density (mean corpuscular hemoglobin concentration). When deoxygenated, all cell fractions nearly doubled their bulk viscosity and the deoxy-oxy differences remained constant. The Baez preparation renders a different picture: oxygenated fractions behave as predicted by the viscosimetric data, but, when deoxygenated, cell fractions exhibit dramatically increased peripheral resistance and the deoxy-oxy difference are directly proportional to cell density, thus, the largest increases were observed for fractions III and IV. The differences between the rheological and the hemodynamic measurements are most probably due to the different sensitivity of the two methods to the extent of intracellular polymerization. These results also demonstrate that the hitherto unrecognized fraction III cells (very dense discocytes that change shape very little on deoxygenation) are as detrimental to the microcirculation as the irreversibly sickled cell-rich fraction IV. They may, however, induce obstruction by a different mechanism. As the extent to which these fractions are populated by erythrocytes varies considerably from patient to patient, the distribution function of cell densities in each sickle cell anemia patient might have consequences for the type of pathophysiological events occurring in their microcirculation.
D K Kaul, M E Fabry, P Windisch, S Baez, R L Nagel
Human peripheral blood monocytes were cultivated for 1-30 d before assay for H2O2 release or challenge with Leishmania donovani promastigotes (LDP) or amastigotes (LDA). 1-d cells readily generated H2O2 in response to both phorbol myristate acetate triggering (1,013 +/- 58 nmol/mg protein . 90 min) and LDP ingestion, and killed 50% of LDP within 6 h, and 90% by 24 h. In contrast, the same cells released little H2O2 during LDA ingestion, killed no LDA at 6 h and less than 30% by 24 h, and supported intracellular LDA replication. Monocyte-derived macrophages (cells first cultivated for greater than or equal to 7 d) generated less than 125 nmol H2O2/mg . 90 min after phorbol myristate acetate triggering, killed neither LDP nor LDA, and permitted both forms to replicate. The addition of mitogen- or antigen-stimulated lymphokines, however, prevented the decline in monocyte oxidative capacity, enhanced macrophage H2O2 release by more than sixfold, and, in parallel, induced 1-d monocytes to kill LDA and cultivated macrophages to display both promastigocidal and amastigocidal activity. In comparison to 1-d monocytes and lymphokine-activated macrophages from normal donors, the same cells from patients with chronic granulomatous disease (CGD) or normal cells whose oxidative activity had been impaired by catalase pretreatment or glucose deprivation exerted considerably less or no antileishmanial activity during the early (6-24 h) postphagocytic period. By 48 h after infection, however, 1-d CGD monocytes and oxidatively impaired normal cells killed 40 and greater than 80% of LDP, respectively. Although a longer period of lymphokine stimulation was required and the resulting antileishmanial effects were not as rapid as with normal cells, activated CGD monocytes and macrophages also eventually achieved promastigocidal and amastigostatic activity. These results indicate that human mononuclear phagocytes utilize both oxygen-dependent and -independent mechanisms to achieve activity against ingested Leishmania, and also demonstrate (a) the differential susceptibilities of the two forms of L. donovani to intracellular killing, (b) the key role of oxygen intermediates in effective mononuclear phagocyte antimicrobial activity, (c) the capacity of lymphocyte products to enhance oxygen-dependent as well as -independent pathways, and (d) the vulnerability of the monocyte-derived macrophage to Leishmania infection in the absence of lymphokine stimulation.
H W Murray, D M Cartelli
This study examines the effects of increased dietary cholesterol (6 eggs/d) on the metabolism of low density lipoproteins in a group of seven healthy volunteers. Egg supplementation raised high density and low density lipoprotein cholesterol levels by 18 and 40%, respectively. The composition of the low density lipoprotein was unaltered and therefore the number of circulating particles must have increased. Kinetic studies indicated that this was due primarily to a 23% rise in the rate of synthesis of the lipoprotein. Catabolism was also affected. The fractional removal rate of native low density lipoprotein fell by 10% (P less than 0.05). However, the clearance of the 1,2 cyclohexanedione-treated lipoprotein remained unchanged (control fractional clearance rate [FCR] = 0.188 pools/d; cholesterol feeding FCR = 0.183 pools/d). Therefore, the reduction in low density lipoprotein catabolism appeared to be due to a fall in receptor activity. Consequently, an increased sterol load (34.2 mumol/kg per d vs. 27.7 mumol/kg per d in the control phase, P less than 0.02) was channelled into the receptor-independent route during egg feeding.
C J Packard, L McKinney, K Carr, J Shepherd
Lymph node (LNL) and salivary gland lymphocytes (SGL) from three patients with pseudolymphoma and primary Sjögren's syndrome (1(0)SS) were characterized with monoclonal antibodies to demonstrate (a) a predominance of T cells (greater than 80%) reactive with anti-T cell antibodies OKT4 (greater than 70%) and OKT8 (less than 20%); (b) a high prevalence of activation antigens (greater than 50% of cells reactive with antibody OKT10 and anti-Ia antibody); (c) polyclonal B cells (8-15% of all cells expressing kappa or lambda); and (d) a specific B cell subset defined by reactivity with antibody B532 that was not present in their peripheral blood. In vitro functional studies showed that both SGL and LNL provided T helper activity for immunoglobulin synthesis and that this activity could be abolished by treatment with antibody OKT4 plus complement. The SGL and LNL exhibited little natural killer, antibody-dependent cellular cytotoxicity, or cytotoxic T cell activity. Normal karyotype was observed in SGL, LNL, and peripheral blood lymphocytes (PBL) from these patients. These findings indicate that pseudolymphoma in 1(0)SS results from the infiltration of salivary glands and extraglandular tissues by nonneoplastic T helper cells. Monoclonal antibodies provide an important tool to distinguish pseudolymphoma from non-Hodgkins (B cell) lymphomas that have a markedly elevated incidence in 1(0)SS patients. Our finding of T helper cells in pseudolymphoma tissues supports the hypothesis that chronic stimulation of B cells by helper T cells leads to eventual escape of a malignant B cell clone.
R I Fox, T C Adamson 3rd, S Fong, C A Robinson, E L Morgan, J A Robb, F V Howell
The effects of Escherichia coli endotoxin on lung mechanics, hemodynamics, gas exchange, and lung fluid and solute exchange were studied in 12 chronically instrumented unanesthetized sheep. A possible role for cyclooxygenase products of arachidonate metabolism as mediators of the endotoxin-induced alterations in lung mechanics was investigated by studying sheep before and after cyclooxygenase inhibition with sodium meclofenamate and ibuprofen. Sheep were studied three times in random order: (a) sodium meclofenamate (or ibuprofen) infusion alone; (b) E. coli endotoxin alone; and (c) meclofenamate (or ibuprofen) and endotoxin. Meclofenamate alone had no effect on any of the variables measured. Endotoxin alone caused early marked changes in lung mechanics: resistance to airflow across the lungs (RL) increased 10-fold, dynamic lung compliance (Cdyn) decreased 80% and functional residual capacity (FRC) decreased by greater than 30%. The alveolar-to-arterial oxygen difference (delta AaPO2) increased markedly following endotoxemia. In the presence of sufficient meclofenamate to inhibit accumulation of thromboxane-B2 and 6-keto-prostaglandin F1 alpha in lung lymph, endotoxin caused no increase in RL, Cdyn decreased by less than 40%, and FRC decreased by only 6%. Meclofenamate significantly attenuated the hypoxemia and early pulmonary hypertension caused by endotoxemia but had no effect on the late increases in lung fluid and solute exchange. Ibuprofen had similar effects to those observed with meclofenamate. We conclude that both the pulmonary hypertension and changes in lung mechanics observed after endotoxemia may be mediated, at least in part, by constrictor prostaglandins or thromboxanes and that gas exchange may be improved by preventing endogenous synthesis of these mediators.
J R Snapper, A A Hutchison, M L Ogletree, K L Brigham
Rabbit medullary collecting duct (MCD) from inner stripe of outer medulla has been identified as a major distal nephron acidification site. The isolated, perfused tubule technique was used to examine the roles of mineralocorticoid and glucocorticoid in regulation of MCD acidification. Surgical adrenalectomy reduced bicarbonate reabsorptive rate (JHCO3, pmol X mm-1 X min-1) from the normal of 9.79 +/- 1.21 to 0.67 +/- 1.1. Chronic administration of deoxycorticosterone acetate (DOCA) increased JHCO3 of MCD significantly to 18.02 +/- 1.62 whereas chronic dexamethasone administration did not affect JHCO3. The direct effects of aldosterone and dexamethasone upon MCD acidification were examined by perfusing tubules harvested from adrenalectomized rabbits in the presence of aldosterone or dexamethasone. Aldosterone, at 5 X 10(-8) M, increased JHCO3 significantly from 1.27 +/- 0.28 to 3.09 +/- 0.34. At 10(-6) M, aldosterone produced a greater increase in JHCO3 from 0.67 +/- 1.1 to 9.39 +/- 1.59. In vitro dexamethasone treatment had no effect on JHCO3. Studies examining the sodium dependence of aldosterone-stimulated acidification demonstrated that JHCO3 in tubules harvested from normal and deoxycorticosterone acetate-treated animals was unaffected by total replacement of sodium with tetramethylammonium. Likewise, luminal amiloride (5 X 10(-5) M) had no effect on JHCO3 in tubules harvested from adrenalectomized and normal animals. Moreover, the acute, in vitro stimulatory effect of aldosterone was seen to occur in the presence of luminal amiloride. These studies define a mammalian distal nephron segment that possesses major acidifying capacity, which is modulated by mineralocorticoid but independent of luminal sodium.
D K Stone, D W Seldin, J P Kokko, H R Jacobson
The effect of reperfusion on regional left ventricular performance following acute myocardial infarction in man was determined. Intracoronary streptokinase was administered in 24 patients within 6 h of the onset of symptoms. 15 patients (62%) were successfully recanalized during the initial study. Mean percent radial shortening (%RS) in both the jeopardized and compensatory regions were determined using 23 radii from the centroid of diastolic and systolic angiographic silhouettes. Sequential measurements were obtained during repeat cardiac catheterization studies at 24 h in 19 patients and before discharge from the hospital (16 +/- 11 d) in 15 patients. At the time of the predischarge study, each acutely reperfused patient showed improvement in %RS in the jeopardized region (P = 0.01) with 56% returning to the normal range. Despite the uniform improvement in the contractile function of the jeopardized region in each reperfused patient, the global ejection fraction showed no improvement or a decrease at the time of the chronic study in 44%. This was due to a decrease in the compensatory wall motion in the uninvolved segments between the acute and chronic study in each case. Neither the %RS nor the ejection fraction changed significantly at the time of the chronic study in the patients who could not be acutely recanalized. These data indicate (a) significant salvage of jeopardized myocardium associated with recovery of contractile function in patients reperfused during the first 6 h of chest pain following acute myocardial infarction; (b) no improvement in regional or global left ventricular performance in patients who could not be reperfused acutely; and (c) the ejection fraction is strongly influenced by changes in the compensatory wall motion of the uninvolved segments and does not accurately reflect changes in the contractile function of the jeopardized myocardium.
R S Stack, H R Phillips 3rd, D S Grierson, V S Behar, Y Kong, R H Peter, J L Swain, J C Greenfield Jr
Anti-gliadin antibody was measured by radioimmunoassay in 30 Caucasians with gluten-sensitive enteropathy (GSE). 22 GSE patients maintained on a gluten-free diet for 1.5 to 20 yr (mean duration 76 mo) had elevated serum concentrations of IgG antigliadin antibody. Among GSE patients on a gluten-free diet, antigliadin antibody was seen only in those having the chromosome 14-encoded IgG immunoglobulin heavy chain allotype marker G2m(n). IgG antigliadin antibody was found in GSE patients with G2m(n) regardless of whether the HLA-B8 and/or -DR3 major histocompatibility complex antigens that occur frequently in GSE were present. No patient lacking G2m(n) had significant levels of antigliadin antibody. The association between antigliadin antibody and the immunoglobulin heavy chain allotype marker G2m(n) in GSE patients likely reflects the presence of Gmn-linked variable region genes or Gmn-linked genes that regulate variable region gene expression.
J B Weiss, R K Austin, M S Schanfield, M F Kagnoff
The myopathy associated with vitamin D deficiency was examined in vitamin D-deficient and vitamin D-supplemented rats. When compared with either vitamin D-supplemented ad lib. or pair-fed rats, weight gain and muscle mass were decreased in vitamin D-deficient hypocalcemic animals. With the exception of a modest decrease in muscle creatine phosphate levels, muscle composition was unchanged by vitamin D deficiency. Muscle protein turnover rates were determined in both in vivo and in vitro studies and demonstrated that myofibrillar protein degradation was increased in vitamin D deficiency. Normal growth rates could be maintained be feeding the rats vitamin D-deficient diets containing 1.6% calcium, which maintained plasma calcium within the normal range. In addition to its role in maintaining plasma calcium, vitamin D-supplemented rats had significantly higher levels of the anabolic hormone insulin. Vitamin D supplementation may affect muscle protein turnover by preventing hypocalcemia, as well as directly stimulating insulin secretion, rather than by a direct effect within skeletal muscle.
S J Wassner, J B Li, A Sperduto, M E Norman
The complement-derived anaphylatoxin C5a and a putative analogue of bacterial chemotactic factor (N-formyl-methionyl-leucyl-phenylalanyl [fMLP]), as well as bacterial lipid A, all stimulate human granulocyte (PMN) adhesiveness and superoxide (O-2) production in a concentration-dependent manner. Since attachment of particulate matter to the PMN membrane is an early event in the triggering of respiratory burst of these cells, we further examined how adherence might modulate the release of O-2 induced by soluble mediators of inflammation. We found that both the quantity and kinetics of O-2 production depend on prior attachment of the cells to a surface. In stirred suspensions of PMN, fMLP induces only a short burst (2.5 min) of O-2 release associated with reversible PMN aggregation. The magnitude, but not the time course, of both these responses depend on the fMLP concentration. Unlike the short respiratory response of cells in suspension, PMN allowed to settle onto stationary petri dishes, then overlaid with fMLP, rapidly spread and attach to the surface where they remain and release O-2 throughout the 60-min test period. Prolonged O-2 release also follows fMLP stimulation in suspensions of PMN pretreated with cytochalasin B, in which case aggregation becomes irreversible during the 20-min burst. If fMLP is slowly infused into a suspension of cells at 37 degrees C or if PMN are challenged at 0 degrees C, and then warmed to 37 degrees C, O-2 release greatly decreases or becomes undetectable. Suspended PMN do not respond to a second challenge by the same stimulus regardless of the rate or temperature at which the first stimulus was added, a phenomenon formerly described as desensitization. However, if PMN challenged with fMLP in suspension undergo the short respiratory response and then are later placed in petri dishes, they adhere and resume production of O-2 without further stimulation. Chemotactic factor-induced adherence and O-2 release of PMN on a surface is entirely independent of either the mode of activation or prior O-2 release during preincubation in suspension. Human C5a also promotes PMN adherence and prolonged O-2 release in petri dishes. Furthermore, lipid A increases O-2 release and adherence of settled PMN, but fails to elicit either response from suspended PMN. These results indicate that cell surface contact plays an essential role in triggering the respiratory burst of PMN activated by soluble stimuli. This long-lasting O-2 release by chemotactic factor-stimulated PMN may play a significant role in inflammatory reactions when PMN become adherent in vivo.
C A Dahinden, J Fehr, T E Hugli
The effects of 5-, 8-, 9-, 11-, 12-, and 15-monohydroxyeicosatetraenoic acid (HETE) (0.1-100 nM) on mucous glycoprotein release from cultured human airways were determined. Each of the HETE was an active secretagogue of mucus at concentrations greater than 1-10 nM with 12- and 15-HETE, the most active. Both 5- and 9-hydroperoxyeicosatetraenoic acid (HPETE) were also active as secretagogues at 100 nM, although of somewhat lower potency. As cultured airways were capable of responding to HETE with mucous glycoprotein release, it was of interest to identify and quantitate airway HETE formation. Accordingly, airways were incubated with tracer quantities of [14C]arachidonate for 16-48 h, and the spontaneous formation of 5-, 12- and 11- and/or 15-HETE was measured by high-pressure liquid chromatography. Indeed, sizeable quantities of 11- and/or 15- greater than 5- greater than 12-HETE were generated. This HETE generation was increased by the addition of 25 micrograms/ml of arachidonate and was reduced somewhat after 18-21 d in continuous tissue culture. Reversed anaphylaxis of human airways using anti-human IgE markedly increased the HETE formation, resulting in the production of micromolar concentrations of 5- and 11- and/or 15-HETE. Thus, human airways not only are capable of responding to the presence of HETE with mucous glycoprotein release, but also generate (both spontaneously and in response to anaphylaxis) at least three species of HETE, and do so in quantities capable of acting as mucus secretagogues.
Z Marom, J H Shelhamer, F Sun, M Kaliner
A reduction in glomerular capillary endothelial pore size and density has been reported in several models of acute renal failure. It has been suggested that these changes underlie the decrease in glomerular filtration rate and altered glomerular capillary hemodynamics measured in various experimental models of acute renal failure. We have thoroughly quantitated the surface characteristics of glomerular capillaries in control rats and in rats with either mercuric chloride-induced acute renal failure (2 mg/kg body wt) evaluated at 6 and 24 h after administration of the nephrotoxin or with gentamicin (G)1-induced acute renal failure evaluated after 8-9 d of 40 mg/kg body wt twice a day. Despite reductions in glomerular filtration rate in the experimental groups, no significant differences were observed between control (C) and any experimental group with respect to percent areas occupied by fenestrated endothelium (C = 53.6 +/- 2.7%; 6 h HgCl2 = 50.9 +/- 1.9%; 24 h HgCl2 = 53.9 +/- 5.7%; G = 56.7 +/- 2.4%), by cytoplasmic ridges (C = 31.2 +/- 1.5%; 6 h HgCl2 = 29.8 +/- 1.9%; 24 h HgCl2 = 30.6 +/- 3.1%; G = 26.5 +/- 1.5%), nonfenestrated endothelium (C = 15.5 +/- 4.0%; 6 h HgCl2 = 19.3 +/- 2.0%; 24 h HgCl2 = 15.6 +/- 4.3%; G = 16.9 +/- 2.3%), in the individual pore area expressed in square nanometers (C = 1,494 +/- 75; 6 h HgCl2 = 1,326 +/- 48; 24 h HgCl2 = 1,559 +/- 130; G = 1,340 +/- 101), or in the percentage of total pore area within fenestrated areas that were measured (C = 12.8 +/- 0.8%; 6 h HgCl2 = 11.2 +/- 0.7%; 24 h HgCl2 = 10.9 +/- 0.8%; G = 10.9 +/- 0.7%). These results provide quantitative data on the normal glomerular capillary endothelial surface characteristics and suggest that reductions of glomerular filtration rate in acute renal failure are not always associated with alterations in glomerular endothelial capillaries.
R E Bulger, G Eknoyan, D J Purcell 2nd, D C Dobyan
Human colonic mucin has been isolated from mucosal scrapings of fresh surgical specimens of normal controls as well as patients with Crohn's colitis and ulcerative colitis. Following sonication and ultracentrifugation, mucin fractions were separated from other soluble colonic glycoproteins by Sepharose 4B chromatography. After nuclease digestion, cesium chloride gradient centrifugation of the excluded material yielded colonic mucin with an average buoyant density of 1.52 g/ml. Subsequent chromatography of the apparently homogeneous colonic mucin on DEAE-cellulose revealed the presence of at least six distinct mucin species (mucin I-VI). Each mucin species was found to have a distinctive hexose, hexosamine, sialic acid, and sulfate content as well as blood group substance activities. Mucin from five patients with Crohn's colitis was found to represent a mixture of at least six discrete species comparable to those isolated from normal colonic specimens. However, in mucin from eight patients with ulcerative colitis there was a marked and selective reduction of one component mucin subclass, designated species IV. Normal mucin and mucin from patients with Crohn's disease contained 48 +/- 17 and 42 +/- 12 mg of species IV/g, while mucin from patients with ulcerative colitis had 5 +/- 3 mg/g solubilized glycoprotein. The selective absence of species IV was found in preparations from both sigmoid (n = 7) and ascending (n = 4) colon and could not be accounted for by an overall decrease in total mucin content. The selective reduction of species IV was also found in mucin isolated from relatively noninflamed colonic mucosa of patients with ulcerative colitis. The carbohydrate composition and blood group activities of the remaining five mucin species were similar to their normal counterparts. Based on the results to date, there appears to be an underlying selective decrease of one colonic mucin subclass in ulcerative colitis.
D K Podolsky, K J Isselbacher
To assess the effect of starvation and to explore the potential interrelationship of starvation and thyroid status at the pretranslational level, we have analyzed by two-dimensional gel electrophoresis, the hepatic translational products of starved and fed euthyroid and hypothyroid rats. 5 d of starvation resulted in a statistically significant change in 27 of 240 products visualized, whereas hypothyroidism caused a change in 20, both in comparison with the fed euthyroid state. Of considerable interest was that 68% of all changing messenger (m)RNA sequences were common to the hypothyroid and starved groups and showed the same directional shift. Further, both starvation and hypothyroidism yielded comparable decreases in total hepatic cytoplasmic RNA content. Although it has been well established that the level of circulating triiodothyronine (T3) and the level of hepatic nuclear receptors fall in starvation, this reduction cannot account for the observed decrease of total hepatic RNA nor for all of the alterations in the concentrations of specific mRNA sequences. Thus, administration of T3 to starved animals in a dose designed to occupy all nuclear T3 receptors fails to prevent the fall in total RNA and the majority of starvation-induced changes in the level of mRNA sequences. Moreover, starvation of athyreotic animals results in a further decrease in total RNA and in a further change in the level of individual mRNA species. We conclude, therefore, that although the reduced levels of circulating T3 and the nuclear T3 receptors can contribute to the observed results of starvation, the starvation-induced changes are not exclusively mediated by this factor. The striking overlap in the genomic response between hypothyroid and starved animals raises the possibility that those biochemical mechanisms regulated at a pretranslational level by T3 are either not helpful or injurious to the starving animal. The reduction in circulating T3 and nuclear receptor sites together with T3-independent mechanisms initiated in the starved animal may constitute redundant processes designed to conserve energy and substrate in the nutritionally deprived organism.
F E Carr, S Seelig, C N Mariash, H L Schwartz, J H Oppenheimer
beta-Adrenergic receptors on human mononuclear leukocytes were assessed using [125I]iodohydroxybenzylpindolol binding. Subjects were studied supine and after being ambulatory, a maneuver that increases plasma catecholamines approximately two-fold. beta-Receptor affinity for agonists, measured by the competition of [125I]iodohydroxybenzylpindolol binding by (-)isoproterenol was significantly reduced with ambulation and this reduction was associated with a reduction in the proportion of beta-receptors binding agonist with a high affinity from a mean (+/- SEM) of 42 +/- 5 to 24 +/- 2% (P less than 0.01). In a parallel series, beta-adrenergic-stimulated adenylate cyclase activity was also reduced with postural change from 4.6 +/- 1.1 to 2.4 +/- 0.6 pmol [32P]cAMP/min per mg protein (P less than 0.05) after ambulation. Similar reductions in the proportion of receptors binding agonist with a high affinity were seen after infusion of norepinephrine. We conclude that the maneuver of ambulation reduces leukocyte beta-receptor responsiveness and affinity for agonists, probably by the effect of increased plasma catecholamines mediating an uncoupling of the beta-receptor-adenylate cyclase complex.
R D Feldman, L E Limbird, J Nadeau, G A FitzGerald, D Robertson, A J Wood
Events that lead to phagocytosis of complement (C3)- or IgG-coated particles after their interaction with specific cell surface receptors are poorly understood. Two mouse monoclonal antibodies (an IgM and an IgG2a) to a human granulocyte-monocyte surface membrane differentiation antigen (Mol) inhibited ingestion by granulocytes both of oil Red O particles opsonized with normal human serum or with IgG and of sheep erythrocytes sensitized with IgG. In addition, they specifically inhibited rosetting between phagocytes and sheep erythrocytes coated with C3bi, a fragment of the complement component C3, generated by cleaving C3b with C3b inactivator and beta IH protein. These monoclonal anti-Mol antibodies did not inhibit IgG Fc, C3b or C3d receptor-mediated binding of erythrocytes coated with the respective proteins. The Fab fragment of the IgG2a monoclonal antibody inhibited noncytotoxic enzyme release from granulocytes when these cells were stimulated with zymosan coated with C3bi. Electrophoretic transfer of polymorphonuclear leukocyte detergent lysates to nitrocellulose, followed by immunofixation with monoclonal antibody, showed that these antibodies were directed to a 155,000-mol wt glycoprotein. This surface membrane structure appears to be involved in Fc and C3 receptor-dependent phagocytosis and closely associated with the C3bi receptor.
M A Arnaout, R F Todd 3rd, N Dana, J Melamed, S F Schlossman, H R Colten
To assess the metabolic recovery of mitochondria after injury, we have monitored, in vivo and noninvasively, changes in the redox state of cytochrome (cyt) a,a3 in 35 rats after tissue hypoxia induced by rapid exsanguination to a mean arterial pressure of 30-35 mmHg. This level of mean arterial pressure was maintained for a shorter period of time in group I (n = 17) and a longer period of time in group II (n = 18), then the shed blood was returned by infusion. The surviving animals were observed for 2 more h before terminating the experiments. During exsanguination, reinfusion and recovery intervals brain tissue parameters of blood oxygenation, relative blood volume, and cyt a,a3 redox state were monitored continuously by spectrophotometry through the closed skull and intact skin. Group I had a high survival rate while group II had a very low survival rate. In both groups, with the onset of hypotension, there was a prompt rapid shift, followed by a slow continued progressive shift, of cyt a,a3 toward a more reduced state. The extent of recovery of cyt a,a3 following reinfusion was different in each group. In group I there was a rapid reoxidation of cyt a,a3 to a level above the base line (16 +/- 12%, mean +/- SEM). In contrast, the extent of reoxidation of cyt a,a3 in group II was significantly lower and stayed 31 +/- 6% below the base-line level. To further evaluate the mechanisms responsible for these observations, another related experiment was performed. 12 rats were subjected to shock and resuscitation as outlined for groups I and II. After death or killing of the animal, we measured, in vitro, oxygen consumption of cerebral cortical slices. Oxygen consumption of cortical tissue slices in subgroup I was significantly higher than in subgroup II. We conclude that, under these experimental conditions, the oxidative response of cyt a,a3 correlates closely with survival or death in the two groups. If in group I animals the greater oxidation of cyt a,a3, in vivo after resuscitation, reflects greater oxygen utilization, as is suggested by the in vitro observations in subgroup I, then we may be observing a useful adaptive response to tissue injury leading to preserved organ function and enhanced survival. Therefore, noninvasively measured cyt a,a3 redox state, reflecting intracellular metabolic activity, seems to indicate both the overall cerebral cellular response to injury and the likelihood of survival.
K Kariman, F F Jöbsis, H A Saltzman
Chemoattractant-receptor coupling triggers several biologic responses in phagocytic cells including activation of the respiratory burst. Prior evidence in intact cells implied that stimulation of the respiratory burst by chemoattractants was by a mechanism different from other soluble agents suggesting the possibility that different oxidative enzymes were responsible. We now show that the chemoattractants N-formyl-methionyl-leucyl-phenylalanine and a split fragment of the fifth component of complement (C5a) stimulate an NADPH oxidase activity, measured in the 50,000-g particulate fraction from human polymorphonuclear leukocytes (PMN). Levels of oxidase activity stimulated by the chemoattractants were both time and dose dependent and required the presence of cytochalasin B during stimulation. In contrast, activation by two nonchemotactic stimuli, the ionophore A23187 and phorbol myristate acetate (PMA), did not require cytochalasin B. Temporal patterns of oxidase activation suggested that different stimuli follow different transductional pathways. Chemoattractant-mediated activation was immediate (no lag); peaked by 45 s and declined rapidly to approximately 50% of maximal by 2 min. In contrast, activation by A23187 or PMA had a 15-30-s lag and increased more slowly. Stimulation by A23187 peaked at 5 min, then declined. Stimulation by PMA plateaued at 20 min and did not decline by 90 min. Comparison of Km values for NADPH and NADH obtained by Lineweaver-Burk analysis of the oxidase activity stimulated by N-formyl-methionyl-leucyl-phenylalanine, A23187, and PMA suggested that the same enzyme was activated by all stimuli. Thus, chemoattractants and other soluble stimuli appear to activate the same respiratory burst enzyme in PMN but they utilize different transductional mechanisms and are regulated differently.
L C McPhail, R Snyderman
Hereditary fructose intolerance (HFI) is a metabolic disorder caused by enzymic deficiency of aldolase B, a genetically distinct cytosolic isoenzyme expressed exclusively in liver, kidney, and intestine. The molecular basis of this enzyme defect has been investigated in three affected individuals from a nonconsanguineous kindred, in whom fructose-l-phosphate aldolase activities in liver or intestinal biopsy samples were reduced to 2-6% of mean control values.
Timothy M. Cox, Martin W. O'Donnell, Michael Camilleri, Arthur H. Burghes
We have examined the relative rates of uptake of several fatty acids into washed, human platelets by measuring incorporation into cellular phospholipids. In the presence of 15 microM fatty acid-free albumin and with radioactive fatty acid concentrations of 5-500 nM, esterification into phospholipid was linear with time and platelet concentration and saturable with respect to fatty acid concentration. Two distinct classes of uptake rate were observed. Arachidonate and 5,8,11,14,17-eicosapentaenoate exhibited high affinity, relatively rapid incorporation into platelet phospholipids at pH 6.5: apparent Michaelis constant (Km) = 30 nM, apparent maximum velocity (Vmax) = 28 pmol/min per 10(9) platelets. Two other eicosanoid precursors, 5,8,11-eicosatrienoate and 8,11,14-eicosatrienoate, exhibited the same Vmax, but Km of 85 and 60 nM, respectively. Under the same conditions, stearate, oleate, and linoleate were incorporated into phospholipids much less efficiently (Vmax approximately 8 pmol/10(9) cells per min, apparent Km greater than or equal to 170 nM). Qualitatively similar results were found at pH 7.4. Uptake of radiolabeled, rapid-uptake fatty acids was not diminished by the presence of excess, unlabeled, slow-uptake fatty acids. Thus, the specificity of this esterification system resembles that of the arachidonate-specific, long-chain acyl-CoA synthetase present in platelets. It may represent the expression in vivo of the synthetase, although the apparent affinity of the synthetase for fatty acid is much less. This esterification system probably represents the physiologic mechanism for platelet arachidonate uptake, whereby arachidonate is collected from plasma, despite the fact that its concentration is considerably lower than that of other plasma fatty acids.
E J Neufeld, D B Wilson, H Sprecher, P W Majerus
Nonhuman primates consuming diets containing cholesterol develop coronary artery atherosclerosis that we have found to be highly correlated with an increase in the size and cholesteryl ester content of plasma low density lipoproteins (LDL). The present studies were designed to determine whether the enlarged plasma LDL are produced directly by the liver of cholesterol-fed monkeys. African green monkeys were fed a diet containing 40% of calories as butter fat and either 0.16 mg cholesterol/kcal (control diet) or 0.78 mg cholesterol/kcal (test diet). The livers of these monkeys were perfused by recirculation with a lipoprotein-free medium for 4 h. The rate of accumulation of perfusate cholesterol was linear and greater in liver perfusates from test diet-fed vs. control diet-fed monkeys and was positively correlated with both the plasma cholesterol concentration and LDL size in the donor animal. All perfusate d less than 1.063 g/ml lipoprotein subfractions from livers of test diet-fed monkeys were enriched in cholesteryl ester severalfold over the corresponding subfractions from control diet-fed monkeys and contained only the larger form of apolipoprotein B typical of plasma LDL. However, the perfusate lipoproteins in the LDL density range did not have an average size or composition typical of LDL from plasma. Rather, they were relatively enriched in phospholipid and unesterified cholesterol and were deficient in cholesteryl esters. In addition, perfusate high density lipoproteins were discoidal particles. These data show that the enzyme lecithin:cholesterol acyltransferase (LCAT) was essentially inactive in these perfusates and, as a result, the dietary cholesterol-induced enrichment of perfusate d less than 1.063 g/ml lipoproteins with cholesteryl esters probably resulted from increased hepatic secretion of cholesteryl esters and not from modification of lipoproteins by LCAT during recirculating perfusion. In spite of this increase, enlarged cholesteryl ester-rich LDL were not found in the perfusate, suggesting that large molecular weight plasma LDL are not directly secreted by the liver but instead probably result from further intravascular metabolism of cholesteryl ester-enriched hepatic precursor lipoproteins.
F L Johnson, R W St Clair, L L Rudel
Effects of human fibroblast (beta) or leukocyte (alpha) interferon (IFN) on differentiations of a human histiocytic lymphoma-derived cell line (U937) or promyelocytic leukemia-derived cell line (HL-60) were studied. When cultured with beta-IFN (400-1,000 U/ml), U937 cells showed gross morphologic and microscopic changes consisting of clumping, increased cytoplasmic-to-nuclear ratio, enhanced prominence of cytoplasmic granules, and membrane ruffling. After culture with beta-IFN, the number of U937 cells reactive with B43.4.1 monoclonal antibody, which is specific for human monocytes, natural killer cells, and neutrophils, increased from less than 10% of U937 cells to 47% beta-IFN treatment also enhanced antibody-dependent cellular cytotoxicity against chicken erythrocytes by U937 cells. The same morphologic, phenotypic, and functional changes were also observed when U937 were treated with recombinant or natural alpha-IFN. The effects of alpha-IFN were totally abolished by anti-alpha-IFN serum. In contrast, HL-60, which differentiates toward cells of the monocyte lineage in response to phorbol 12-myristate 13-acetate (based on the above criteria), and toward granulocytes in response to dimethyl sulfoxide, did not differentiate when cultured with alpha- or beta-IFN. No consistent relationship between induction of differentiation and changes in phospholipid methylation were observed.
T Hattori, M Pack, P Bougnoux, Z L Chang, T Hoffman
Adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN) are related X-linked disorders characterized by adrenal, gonadal, and nervous system dysfunction. While the pathologic finding common to these tissues appears to be the accumulation of excessive amounts of very long chain fatty acids, the mechanism leading to functional impairment in these tissues is unclear. Measurements of fluorescence polarization (P), using the lipid probe diphenylhexatriene, demonstrate a highly significant increase in the microviscosity of erythrocyte membranes in affected patients (P = 0.286 +/- 0.012) vs. normals (P = 0.239 +/- 0.020). Analyses of these membranes by gas-liquid chromatography revealed 1.9-, 1.6-, and 1.3-fold increases above normal values in the C25:0, C26:0, and C27:0 fatty acids, respectively. These observations are compatible with previously obtained data in animals that correlate membrane microviscosity with the number of hormone receptors in target tissues. The present data support the thesis that a decrease in responsiveness to trophic hormones in ALD and AMN is secondary to changes in the membrane microviscosity of the target tissues and suggest a mechanism by which adrenal and gonadal failure occur in such patients.
R A Knazek, W B Rizzo, J D Schulman, J R Dave
Human epidermal growth factor (hEGF) has previously been isolated from urine and probably is identical to human beta-urogastrone (hUG). Immunoreactive hEGF/UG has been found in the plasma of normal subjects. In this study, using immunoaffinity chromatography to extract hEGF/UG from plasma, we found that immunoreactive hEGF/UG in blood was associated with blood platelets. It was present in platelet-rich, but not platelet-poor plasma and serum, and was found predominantly in the platelet fraction of whole blood. Sephadex G-50 Fine gel-exclusion chromatography of an extract of outdated blood bank platelets revealed two hEGF/UG components, one of which eluted in the void volume, and the other of which coeluted with purified standard hEGF/UG. The former hEGF/UG component was a high-molecular weight form that was cleaved into hEGF/UG by incubation with either mouse EGF/UG-associated arginine esterase or trypsin. It appeared to be identical to the high-molecular weight hEGF/UG previously reported in human urine, except for its apparently equal activities in radioimmunoassay and radioreceptor assay. The latter hEGF/UG component was immunologically, biologically, and physiochemically indistinguishable from highly purified hEGF/UG from human urine and was immunologically different from purified human platelet-derived growth factor. Platelet-associated hEGF/UG may account for the mitogenic activity of serum in cell lines in which platelet-derived growth factor is not active. Since hEGF/UG appears to be liberated from platelets during coagulation, platelet-associated EGF/UG may be involved in normal vascular and tissue repair and in the pathogenesis of atherosclerotic lesions. The discovery that the EGF/UG in plasma is associated with blood platelets raises important new possibilities for its role in human health and disease.
Y Oka, D N Orth
The relationship between group B streptococcal (GBS) type-specific antisera and the type II-specific polysaccharide is evaluated from a structural and immunologic viewpoint. Although all GBS type-specific polysaccharides are composed of the same monosaccharides, the type II antigen is more complex structurally and contains these sugars in a molar ratio different from the other antigens. Type II polysaccharide has two side chains. One contains only sialic acid and is less susceptible to acid cleavage than sialic acid residues found on types III, Ia, and Ib polysaccharides. The other side chain is composed of galactose as the only sugar. Immunochemical studies demonstrate that the type II polysaccharide has several immunodeterminants. One of these determinants is likely to be the side-chain galactose, while sialic acid appears to comprise part of another immunodeterminant, more complex than sialic acid alone. A series of cross-reactions is demonstrated between the type II native antigen and antisera to serotypes Ia, III, and Ib by a sensitive radioactive antigen-binding assay, which account for additional, complex immunodeterminants. The strongest of these cross-reactions is with type Ia antiserum and the weakest with Ib antiserum. Since Ia and Ib polysaccharides differ in only one linkage, these findings suggest that the trisaccharide beta D-N-acetyl-glucosamine-p(1 leads to 3) beta D-galactose-p(1 leads to 4) beta D-glucose-p [[beta D-GlcNAcp(1 leads to 3) beta D-Galp(1 leads to 4)beta D-Glcap]] is the likely common site responsible for the interaction of the type II native polysaccharide and type Ia antiserum. Another cross-reaction is observed between type III antiserum and type II native antigen. Inhibition studies indicate that the most likely cross-reactive determinant in this case is [beta D-Galp(1 leads to 4)beta D-GlcNAcp]. Type II polysaccharide has been utilized in a human vaccine trial to test safety and immunogenicity. The polysaccharide is highly immunogenic, inducing an antibody response in 95% of recipients, and nontoxic, with side-effects confined to minimal local reactions. Despite the cross-reactions observed between type-specific antigens and antibody prepared by immunization of rabbits with whole bacteria, which suggest shared immunodeterminants, similar cross-reactions were not detected in human sera after immunization with purified type II polysaccharide.
D L Kasper, C J Baker, B Galdes, E Katzenellenbogen, H J Jennings
The mechanisms of postprandial glucose counterregulation—those that blunt late decrements in plasma glucose, prevent hypoglycemia, and restore euglycemia—have not been fully defined. To begin to clarify these mechanisms, we measured neuroendocrine and metabolic responses to the ingestion of glucose (75 g), xylose (62.5 g), mannitol (20 g), and water in ten normal human subjects to determine for each response the magnitude, temporal relationships, and specificity for glucose ingestion. Measurements were made at 10-min intervals over 5 h. By multivariate analysis of variance, the plasma glucose (P < 0.0001), insulin (P < 0.0001), glucagon (P < 0.03), epinephrine (P < 0.0004), and growth hormone (P < 0.01) curves, as well as the blood lactate (P < 0.0001), glycerol (P < 0.001), and β-hydroxybutyrate (P < 0.0001) curves following glucose ingestion differed significantly from those following water ingestion. However, the growth hormone curves did not differ after correction for differences at base line. In contrast, the plasma norepinephrine (P < 0.31) and cortisol (P < 0.24) curves were similar after ingestion of all four test solutions, although early and sustained increments in norepinephrine occurred after all four test solutions. Thus, among the potentially important glucose regulatory factors, only transient increments in insulin, transient decrements in glucagon, and late increments in epinephrine are specific for glucose ingestion. They do not follow ingestion of water, xylose, or mannitol.
Thomas F. Tse, William E. Clutter, Suresh D. Shah, J. Philip Miller, Philip E. Cryer
The transition from exogenous glucose delivery to endogenous glucose production late after glucose ingestion is not solely attributable to dissipation of insulin and, therefore, must also involve factors that actively raise the plasma glucose concentration--glucose counterregulatory factors. We have shown that the secretion of two of these, glucagon and epinephrine, is specific for glucose ingestion and temporally related to the glucose counterregulatory process. To determine the physiologic roles of glucagon and epinephrine in postprandial glucose counterregulation, we produced pharmacologic interventions that resulted in endogenous glucagon deficiency with and without exogenous glucagon replacement, adrenergic blockade, and adrenergic blockade coupled with glucagon deficiency starting 225 min after the ingestion of 75 g of glucose in normal subjects. Also, we assessed the effect of endogenous epinephrine deficiency alone and in combination with glucagon deficiency late after glucose ingestion in bilaterally adrenalectomized subjects. Glucagon deficiency resulted in nadir plasma glucose concentrations that were approximately 30% lower (P less than 0.01) than control values, but did not cause hypoglycemia late after glucose ingestion. This effect was prevented by glucagon replacement. Neither adrenergic blockade nor epinephrine deficiency alone impaired the glucose counterregulatory process. However, combined glucagon and epinephrine deficiencies resulted in a progressive fall in mean plasma glucose to a hypoglycemic level late after glucose ingestion; the final glucose concentration was 40% lower (P less than 0.02) than the control (epinephrine deficient) value in these patients, and was nearly 50% lower (P less than 0.001) than the control value and approximately 30% lower (P less than 0.05) than the glucagon-deficient value in normal subjects. We conclude (a) the transition from exogenous glucose delivery to endogenous glucose production late after glucose ingestion is the result of the coordinated diminution of insulin secretion and the resumption of glucagon secretion. (b) Epinephrine does not normally play a critical role in this process, but enhanced epinephrine secretion compensates largely and prevents hypoglycemia when glucagon secretion is deficient.
T F Tse, W E Clutter, S D Shah, P E Cryer
The effects of catecholamines on antidiuretic hormone ([Arg8]-vasopressin [AVP])-induced water absorption were evaluated in cortical collecting tubules isolated from the rabbit kidney and perfused in vitro. In the presence of AVP (100 μU/ml), net fluid volume absorption (Jv, nanoliters per minute per millimeter) was 1.14±0.12 and osmotic water permeability coefficient (Pf, X 10-4 centimeters per second) was 217.3±39.9. The addition of the alpha-adrenergic agonist, phenylephrine (PE), in a concentration of 10-6 M resulted in a significant decrease in Jv and Pf to 0.83±0.13 (P < 0.001) and 148.8±41.8 (P < 0.02), respectively. Increasing the concentration of PE to 10-5 M resulted in a further decrease in Jv and Pf to 0.53±0.05 (P < 0.05 vs. PE 10-6 M) and 88.5±9.0 (P 0.05 vs. PE 10-6 M), respectively. In a separate group of tubules, in the presence of AVP (100 μU/ml) and PE (10-5 M), Jv and Pf were 0.35±0.07 and 66.0±17.3, respectively. The addition of the alpha-adrenergic antagonist, phentolamine (PH), in a concentration of 10-6 M resulted in a significant increase in Jv to 1.07±0.19 (P < 0.001) and Pf to 193.3±35.9 (P < 0.005). PH (10-5 M) alone did not significantly affect Jv and Pf in the presence of AVP (100 μU/ml) nor in the presence of 8-bromo adenosine 3',5' cyclic monophosphate (8-BrcAMP). Jv and Pf were 1.20±0.21 and 174.0±25.8, respectively, in the presence of 8-BrcAMP (10-4 M).
Radha K. Krothapalli, W. Brian Duffy, Harry O. Senekjian, Wadi N. Suki
Although the stomach is mainly known for its ability to secrete hydrochloric acid, there is increasing evidence that the gastric mucosa also secretes bicarbonate. A simple method for simultaneous measurement of gastric HCO-3 secretion and H+ secretion was developed from a two-component model of gastric secretion. The method, which is based upon gastric juice volume, H+ concentration, and osmolality, was validated both in vitro and in vivo. In 14 healthy human beings, basal gastric HCO-3 secretion averaged 2.6 mmol/h (range, 0.7-8.7 mmol/h). Basal HCO-3 secretion was approximately 50% of basal H+ secretion and there was a significant correlation between basal HCO-3 and H+ secretion in individual subjects (r = 0.79). HCO-3 was secreted in basal nonparietal secretion at a concentration of approximately 90 mmol/liter. Intravenous pentagastrin infusion markedly stimulated H+ secretion but did not increase HCO-3 secretion. During pentagastrin infusion, the cholinergic agonist, bethanechol, significantly augmented H+ secretion (from 20.2 to 24.7 mmol/h) and increased HCO-3 secretion (from 2.2 to 4.2 mmol/h). A prostaglandin E2 analogue significantly reduced H+ secretion and increased HCO-3 secretion during pentagastrin infusion. The reduction in net gastric juice H+ output following prostaglandin E2 was due more to H+ secretory inhibition than to HCO-3 secretory stimulation. We conclude that the healthy human stomach actively secretes HCO-3 and that gastric HCO-3 secretion can be influenced by cholinergic stimulation and by prostaglandin E2.
Human peripheral blood mononuclear cells from normal donors obtained by separation on a Percoll gradient were incubated with free or liposome-entrapped lymphokines produced from concanavalin A-stimulated lymphocytes and then were tested for cytotoxic activity against tumor cells. The treated monocytes lysed tumorigenic melanoma and glioblastoma target cells, but had no effect on three types of nontumorigenic target cells. The activation of monocytes to become tumoricidal was caused by macrophage-activating factor (MAF) and not by contamination with endotoxins, concanavalin A, or interferon. The endocytosis of liposomes containing MAF, but not of those containing control supernatants, led to the activation of cytotoxic properties in the monocytes. Activation by liposome-encapsulated MAF was very efficient and required less than 1/800th of the amount of free MAF necessary to achieve the same levels of cytotoxicity. Thus, the encapsulation of mitogen-induced MAF in liposomes could provide an effective approach for the activation of blood monocytes in situ.
E S Kleinerman, A J Schroit, W E Fogler, I J Fidler
Deficient activity of the guanine nucleotide regulatory protein (G unit), an integral component of the membrane-bound adenylate cyclase complex, has been implicated as the biochemical lesion in many patients with pseudohypoparathyroidism (PHP) type I. In addition to renal resistance to parathyroid hormone in this disorder, there is decreased responsiveness of diverse tissues to hormones that act via 3',5'-cyclic AMP (cAMP). To assess whether a deficiency of G units could account for impaired adenylate cyclase activity, we studied cAMP production in intact cultured fibroblasts and fibroblast plasma membranes from five patients with PHP in response to several activators of adenylate cyclase.
Michael A. Levine, Charles Eil, Robert W. Downs Jr., Allen M. Spiegel
To define better the role of the fibrinogen receptor in platelet physiology and to characterize it biochemically, a murine monoclonal antibody that completely blocks the binding of fibrinogen to the platelet surface was produced by the hybridoma technique with the aid of a functional screening assay. Purified F(ab')2 fragments and/or intact antibody completely blocked aggregation induced by ADP, thrombin, or epinephrine and the binding of radiolabeled fibrinogen to platelets induced by ADP. The antibody did not block agglutination of formaldehyde-fixed platelets by ristocetin or shape change induced by either ADP or thrombin. ADP- and epinephrine-induced release of ATP was completely inhibited by the antibody, but inhibition of release induced by collagen and thrombin was dose dependent and partial. The antibody also dramatically inhibited platelet retention in glass-bead columns, platelet adhesion to glass, and clot retraction. Thus, the antibody induced a thrombasthenic-like state. Immunofluorescent studies confirmed the specificity of the antibody for normal platelets and megakaryocytes and suggested that there is a marked decrease in detectable antigen in thrombasthenic platelets. Radiolabeled antibody bound to an average of approximately 40,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets of a patient with thrombasthenia. The antibody immunoprecipitated both glycoproteins IIb and IIIa, and both glycoproteins bound to an affinity column of the antibody. These studies indicate that there is probably a single anatomic site that is crucial to the binding of all fibrinogen molecules and that this site is most likely on the glycoprotein IIb/IIIa complex. It also suggests that the thrombasthenic phenotype can be completely accounted for on the basis of the inhibition of fibrinogen binding to platelets.
B S Coller, E I Peerschke, L E Scudder, C A Sullivan
We used a single-pass multiple tracer technique to measure cardiac output, extravascular lung water (EVLW) and lung vascular [14C]urea permeability-surface area (PSu) in 14 patients with acute respiratory failure and pulmonary edema. All patients had increased EVLW, but EVLW in the 10 surviving patients (0.26 +/- 0.06 SE ml/ml total lung capacity [TLC]) was not significantly different from that in the five patients who died (0.22 +/- 0.05). EVLW did not correlate with intravascular pressures or with alveolar-arterial oxygen pressure difference (A-aDO2). PSu was lower in surviving patients (0.50 +/- 0.16 SE ml/s X liter TLC) than in patients who died (3.44 +/- 0.36; P less than 0.05) and also lower than in previously reported data in patients with normal PSu. PSu correlated significantly with A-aDO2. Serial studies showed that PSu returned from a low value toward normal in a patient who survived but remained high in a patient who died. We conclude that the amount of edema in the lungs measured by indicator methods was not the principal determinant of either the magnitude of oxygenation defect or survival in the patients studied. We interpret the low PSu in surviving patients as decreased surface area and infer that the ability of the lung circulation to reduce perfusion of damaged and edematous areas was important in preserving oxygenation. A high PSu, presumably reflecting perfusion of areas with increased permeability, was a sign of especially poor prognosis. Multiple tracer techniques for measuring lung vascular PSu may help to define the pathogenesis and to evaluate therapies of acute lung injury in humans. Such measurements may be a more useful clinical tool than measurements of lung water in patients with acute respiratory failure and pulmonary edema.
K L Brigham, K Kariman, T R Harris, J R Snapper, G R Bernard, S L Young
To define the role of verapamil in the treatment of ventricular tachycardia (VT), we studied 21 patients with chronic recurrent VT. Electrophysiologic studies were performed before and during intravenous infusion of verapamil (0.15 mg/kg followed by 0.005 mg/kg per min). On the basis of the mode of VT initiation and termination, we identified three groups of patients: (a) 11 patients had VT suggestive of reentry, as VT could be initiated with ventricular extrastimulation and terminated with overdrive ventricular pacing. Verapamil did not affect the inducibility and cycle length of VT. (b) 7 patients had VT suggestive of catecholamine-sensitive automaticity as VT could not be initiated with programmed electrical stimulation but could be provoked by isoproterenol infusion. Moreover, the VT could not be converted to a sustained sinus rhythm with overdrive ventricular pacing and it resolved only with discontinuing isoproterenol infusion. Verapamil exerted no effects on VT. (c) 3 patients had VT with electrophysiologic characteristics suggestive of triggered activity related to delayed afterdepolarizations. Characteristically, after attaining a range of cycle lengths, the sinus, atrial or ventricular paced rhythm could initiate VT without ventricular extrastimulation. The first beat of VT invariably occurred late in the cardiac cycle with a premature coupling interval 0-80 ms shorter than the preceding QRS cycle length; the premature coupling interval gradually decreased as the sinus, atrial or ventricular paced cycle length progressively shortened. Of note, verapamil completely suppressed VT inducibility in these three patients. These observations lead us to suggest that verapamil does not affect VT caused by reentry and catecholamine-sensitive automaticity but is effective in suppressing VT caused by triggered activity related to delayed afterdepolarizations in humans.
R J Sung, W A Shapiro, E N Shen, F Morady, J Davis
Previous studies from our laboratory have demonstrated that dietary intake affects the sympathetic nervous system (SNS); carbohydrate intake, in particular, has been shown to stimulate sympathetic activity. The present studies were undertaken to characterize the effect of dietary fat on SNS activity in the rat. Sympathetic activity was assessed by measurement of norepinephrine (NE) turnover in heart, interscapular brown adipose tissue (IBAT), and pancreas and by excretion of NE in the urine. When fed a fat-enriched diet (50% chow, 50% lard), fractional NE turnover in heart (k) increased from 6.3 +/- 0.6% h in ad lib. fed controls to 14.7 +/- 1.3% h in the high-fat group (P less than 0.001); calculated NE turnover rate increased from 24.5 +/- 2.4 ng/heart per h to 36.8 +/- 3.5 (P less than 0.05). Urinary NE excretion more than doubled after 6 d of the same high fat diet (P less than 0.001). Ganglionic blockade produced a greater effect on NE turnover in fat-fed, as compared with chow-fed animals, consistent with increased sympathetic activity in the fat-fed group. When fat absorption was blocked with a bile acid binding resin (cholestyramine), the same high-fat diet did not increase cardiac NE turnover, indicating that fat absorption is required for the stimulatory effect on sympathetic activity. In another series of experiments, in which chow (and hence protein) intake was held constant, the effect of fat and isocaloric sucrose supplements on NE turnover was assessed in heart, IBAT, and pancreas. The caloric value of the supplements was 50, 100, and 335% of the chow in the different experiments. An effect of fat on NE turnover in heart and IBAT was demonstrable at the lowest level of fat supplement. Fat increased pancreatic NE turnover when added in amounts sufficient to double the caloric intake. The stimulatory effect of sucrose and fat on NE turnover in heart and IBAT was similar. These experiments demonstrate that fat increases SNS activity in the rat and that the magnitude of the effect is similar to that of sucrose. The results imply that fat may contribute to dietary thermogenesis in this species.
J H Schwartz, J B Young, L Landsberg
Increasing availability of free fatty acids (FFA) to liver results in enhanced rates of secretion of triglycerides in lipoproteins. However, as FFA uptake increases, triglyceride secretory rates reach a plateau and esterified fatty acids accumulate intracellularly, suggesting that something is limiting lipid transport out of the liver. One possibility could be the limited availability of apoproteins. To test this hypothesis, primary rat hepatocytes in culture were incubated with increasing amounts of FFA (0-2.1 mumol/dish) and the amounts of lipids and apoproteins inside the cells and in culture media were measured; the latter by specific radioimmunoassays. Media also were fractionated on Sepharose 2B and 6B columns and the elution profiles of apoproteins were obtained. With exposure to increasing amounts of free fatty acids, hepatocytes took up more fatty acids and intracellular levels of triglycerides rose (from 71 to 146 micrograms/mg cell protein). Concomitantly, media triglycerides nearly doubled (31 to 51 micrograms/mg). Incorporation of [3H]glyceride into cellular and media triglyceride also rose. However, levels of apoproteins A-I, B, C-III3, and E in cells and media were unchanged. The increasing amounts of triglycerides in media were present in larger particles, as demonstrated on gel permeation chromatography. The elution profiles of apoproteins B, C-III3, and E were altered in that a greater proportion of the apoproteins eluted with larger particles. Similar results were obtained when hepatocytes were preloaded with increasing amounts of FFA over 12 h and analyses of cells and media were carried out 8 and 22 h after removal of fatty acids from the media. During loading of cells, accumulation of cellular triglycerides was directly related to media FFA concentrations. During unloading, triglyceride secretory rates were related to cellular triglyceride levels. At higher triglyceride secretory rates larger particles were secreted and a greater proportion of apoproteins was associated with the larger particles, but total amounts of apoproteins in the system did not change. These data lead us to suggest that enhanced rates of apoprotein synthesis need not occur in the response to acute changes in hepatic lipid transport, rather, increased secretion of lipid is brought about by augmented intracellular lipid apoprotein association.
W Patsch, T Tamai, G Schonfeld
Familial dysbetalipoproteinemia has been reported to be associated uniquely with an apolipoprotein E phenotype (E2/2) that occurs in approximately 1% of all persons. We have observed the typical clinical and biochemical characteristics of this disorder in five members of a family, in all of whom the apolipoprotein E phenotype, as determined by isoelectric focusing electrophoresis, is E3/3. The disorder is present in three generations of the family: the proband, her mother, and three of the proband's five children. The proband's husband, father of all five children, also has apolipoprotein E phenotype E3/3, as do his two unaffected children. As in normal persons with phenotype E3/3, the apolipoprotein E of affected members appears to have a single residue of cysteine. When incorporated with egg lecithin into discoidal complexes, the apolipoprotein E from affected members was taken up normally into perfused livers of estradiol-treated rats, in which a high level of LDL receptors is expressed. When isoelectric focusing electrophoresis was carried out over a narrow range of pH (5-7), each of the apolipoprotein E isoforms of affected members was observed as a doublet, even after reduction of dimers of the protein with 2-mercaptoethanol and treatment with neuraminidase to minimize the content of sialylated forms of the protein. Doublets were also observed in the apolipoprotein E-2 of patients with classical dysbetalipoproteinemia, but only in the affected members of the family with atypical dysbetalipoproteinemia were the components of the doublets equally prominent. As in classical dysbetalipoproteinemia, both apolipoprotein B-100 and B-48 were present in the very low density lipoprotein fraction of plasma obtained in the postabsorptive state, indicating that remnantlike lipoproteins of both hepatic and intestinal origin accumulate. This observation, together with available evidence on the structural and functional heterogeneity of human apolipoprotein E, lead us to suggest that the disorder in this family is caused by one or two structurally abnormal forms of apolipoprotein E that contain a single residue of cysteine.
R J Havel, L Kotite, J P Kane, P Tun, T Bersot
Insulin binds specifically to basolateral renal cortical membranes and modifies tubular electrolyte transport, but the target sites of this hormone in the nephron have not been identified. Using a microassay that permits measurement of hormone binding in discrete tubule segments we have determined the binding sites of 125I-insulin along the rabbit nephron. Assays were performed under conditions that minimize insulin degradation, and specific binding was measured as the difference between 125I-insulin bound in the presence or absence of excess (10(-5) M) unlabeled hormone. Insulin monoiodinated in position A14 was used in all assays. Specific insulin binding (attomol . cm-1 +/- SE) was highest in the distal convoluted tubule (180.5 +/- 15.0) and medullary thick ascending limb of Henle's loop (132.9 +/- 14.6), followed by the proximal convoluted and straight tubule. When expressed per milligram protein, insulin binding capacity was highest along the entire thick ascending limb (medullary and cortical portions) and the distal convoluted tubule, i.e., the "diluting segment" (congruent to 10(-13) mol . mg protein-1), and was lower (congruent to 4 X 10(-14) mol . mg protein-1), and remarkably similar, in all other nephron segments. Binding specificity was verified in competition studies with unlabeled insulin, insulin analogues (proinsulin and desoctapeptide insulin), and unrelated hormones (glucagon, 1-34 parathyroid hormone, prolactin, follicle-stimulating hormone). In addition, serum containing antiinsulin receptor antibody from two patients with type B insulin resistance syndrome markedly inhibited insulin binding to isolated tubules. Whether calculated per unit tubule length or protein content, insulin binding is highest in the thick ascending limb and the distal convoluted tubule, the same nephron sites where a regulatory role in sodium transport has been postulated for this hormone.
R Nakamura, D S Emmanouel, A I Katz
Although recent studies suggest that interferons can increase the number of IgG Fc receptor (FcR gamma) sites on mouse macrophages, direct assessment of similar effects on human mononuclear phagocytes is lacking. We therefore measured the specific binding of 125I- and fluorescein-labeled IgG1 to human monocytes and leukemic cell lines after culture in vitro with highly purified human interferons. We report that natural and recombinant human gamma-interferon causes a dramatic (nearly 10-fold) increase in the number of FcR gamma on normal human monocytes and on the human cell lines HL-60 and U-937. Alpha and beta-interferons cause a modest but significant increase in these receptors. This report demonstrates that gamma-interferon acts directly on human mononuclear phagocytes to increase FcR gamma sites, it identifies a qualitative difference in the physiologic actions of human type I and type II interferons, and it suggests that HL-60 and U-937 cells will be important models for further study of the molecular mechanisms of interferon action. The results reported here could also be the basis for a bioassay to assess the pharmacokinetics and variability of gamma-interferon action on monocytes of individual patients during treatment in vitro and in vivo.
P M Guyre, P M Morganelli, R Miller
The recently described acquired immune deficiency syndrome (AIDS) is characterized by the occurrence of severe opportunistic infections and an aggressive form of Kaposi's sarcoma. A variety of profound defects in cell-mediated immunity have been reported in association with the AIDS, including deficiencies in natural killer (NK) cell activity and cytomegalovirus (CMV)-specific cytotoxicity. In the present study, the in vitro effects of interleukin-2 (IL-2) and interferon beta (IFN Beta) on these abnormalities were examined to assess the potential use of these lymphokines in the immunotherapeutic treatment of this syndrome. The peripheral blood lymphocytes (PBL) from six male homosexuals with AIDS and an active CMV infection exhibited markedly depressed NK cell and CMV-specific cytotoxic lymphocyte responses compared with uninfected, heterosexual control subjects. Incubation of PBL with IFN Beta enhanced the NK cell activity and the CMV-specific cytotoxicity of only one of six and neither of two AIDS patients, respectively, while enhancing the NK cell activity of all six control subjects. In contrast, IL-2 dramatically enhanced both the NK cell and the CMV-specific cytotoxic lymphocyte activities of all of the patients. These results indicate that IL-2 can substantially potentiate the depressed cytotoxic effector functions of PBL from AIDS patients, while IFN Beta has little effect.
A H Rook, H Masur, H C Lane, W Frederick, T Kasahara, A M Macher, J Y Djeu, J F Manischewitz, L Jackson, A S Fauci, G V Quinnan Jr
We have recently found that ketoconazole inhibits adrenal steroidogenesis; in this paper we investigated whether imidazole antimycotic drugs additionally interact with glucocorticoid receptor sites in target tissues. Our approach was to assess the ability of three drugs: ketoconazole, clotrimazole, and RS 49910, to inhibit [3H]dexamethasone binding to hepatoma tissue culture (HTC) cell cytosol. The results indicated dose-dependent, competitive displacement of [3H]dexamethasone binding that was in the potency sequence: clotrimazole greater than ketoconazole greater than RS 49910. We then examined the functional response of this binding by measuring tyrosine aminotransferase (TAT) activity in HTC cells. The antimycotics did not exhibit TAT agonist activity and inhibition of basal enzyme levels was not detected. However, the drugs were potent antagonists of dexamethasone-induced TAT activity and the effect was temporally reversible. This antagonist activity was in the same sequence and closely correlated with the binding potency of the three drugs. We conclude that ketoconazole and other imidazole antimycotic drugs possess glucocorticoid antagonist activity by virtue of occupancy of glucocorticoid receptor sites in target tissues.
D S Loose, E P Stover, D Feldman