Guanine nucleotide-binding regulatory proteins and dual control of adenylate cyclase.

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Abstract

Authors

A G Gilman

Complex formation of platelet thrombospondin with histidine-rich glycoprotein.

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Abstract

Thrombospondin and histidine-rich glycoprotein are two proteins with diverse biological activities which have been associated with human platelets and other cell systems. Using an enzyme-linked immunosorbent assay, we have demonstrated that purified human platelet thrombospondin formed a complex with purified human plasma histidine-rich glycoprotein. The formation of the thrombospondin-histidine-rich glycoprotein complex was specific, concentration dependent, and saturable. Significant binding was detected when histidine-rich glycoprotein was incubated with thrombospondin immobilized on anti-thrombospondin IgG-coated plates, indicating that the observed complex formation was not due to a thrombospondin interaction with the plastic surface. Sucrose-density-gradient ultracentrifugation of a mixture of thrombospondin and histidine-rich glycoprotein also revealed the formation of fluid-phase complexes, with an estimated stoichiometry of 1 thrombospondin: 3.5 histidine-rich glycoprotein. Fibrinogen, which has been previously shown to bind to absorbed thrombospondin, did not inhibit the formation of the thrombospondin-histidine-rich glycoprotein complex. Histidine-rich glycoprotein complexed with thrombospondin was capable of binding heparin and neutralizing the anticoagulant activity of heparin in plasma. Specific complex formation between thrombospondin and histidine-rich glycoprotein may play a significant role in influencing platelet blood vessel wall interactions as well as modulating the association of various cells with the extracellular matrix.

Authors

L L Leung, R L Nachman, P C Harpel

Regulation of renal Na-K-ATPase in the rat. Role of the natural mineralo- and glucocorticoid hormones.

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Both mineralo- and glucocorticoids stimulate renal Na-K-ATPase, but their relative role in the regulation of the enzyme remains controversial. In this study we measured Na-K-ATPase activity in the cortical collecting tubule (CCT) of adrenalectomized rats replaced with either the native mineralocorticoid (aldosterone) or glucocorticoid (corticosterone) in doses calculated to yield previously determined physiologic concentrations of these hormones (5 ng X dl-1 and 5 micrograms X dl-1, respectively). This was achieved by continuous delivery of aldosterone (1 microgram X 100 g-1 X d-1) from an osmotic minipump or of corticosterone (2 pellets of 20 mg each), implanted subcutaneously either at adrenalectomy or 7 d later, when Na-K-ATPase activity reached its nadir. Adrenalectomized rats not receiving hormone replacement and adrenal-intact animals served as controls. The CCT was chosen because it contains the highest concentration of binding sites for both hormones. Na-K-ATPase activity declined 52% in the CCT of untreated adrenalectomized rats after 7 d, and remained unchanged thereafter. Physiologic replacement doses of aldosterone prevented this decline and restored the activity of the enzyme after it had been allowed to decrease maximally following adrenal ablation, whereas similar replacement of corticosterone was without effect. These observations suggest that under physiologic conditions Na-K-ATPase in the CCT, a probable target nephron segment of both hormones, is under mineralocorticoid rather than glucocorticoid control.

Authors

S K Mujais, M A Chekal, W J Jones, J P Hayslett, A I Katz

Mechanism of acute depletion of plasma fibronectin following thermal injury in rats. Appearance of a gelatinlike ligand in plasma.

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Plasma fibronectin was depleted within 15 min following sublethal burn, followed by partial recovery at 8 h and complete restoration by 24 h in anesthetized rats. Radiolabeled 75Se-plasma fibronectin, injected intravenously before burn, was rapidly sequestered in burn skin as well as the liver. Fibronectin levels at 2 h postburn as detected by immunoassay vs. 75Se-plasma fibronectin indicated that more fibronectin was in the plasma than detected by electroimmunoassay. Crossed immunoelectrophoretic analysis of fibronectin in early postburn plasma demonstrated a reduced electrophoretic mobility of the fibronectin antigen. Addition of heparin or fibrin, both of which have affinity for fibronectin, to normal plasma was unable to reproduce this altered fibronectin electrophoretic pattern. In contrast, addition of gelatin or native collagen to normal plasma reproduced the abnormal electrophoretic pattern of fibronectin seen in burn plasma. Extracts of burned skin, but not extracts of normal skin, when added to normal plasma, elicited a similar altered electrophoretic pattern for fibronectin. By gel filtration, fibronectin in burn plasma had an apparent molecular weight approximately 40% greater than that observed in normal plasma. These data suggest the release into the blood of a gelatinlike ligand from burned skin, which complexes with plasma fibronectin. Thus, fibronectin deficiency acutely postburn appears mediated by (a) its accumulation at the site of burn injury; (b) its removal from the circulation by the liver; and (c) its presence in the plasma in a form that is less detectable by immunoassay.

Authors

D C Deno, M H McCafferty, T M Saba, F A Blumenstock

Biliary proteins. Unique inhibitors of cholesterol crystal nucleation in human gallbladder bile.

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Abstract

The onset time for cholesterol crystal nucleation of supersaturated normal human gallbladder biles is consistently prolonged when compared with biles from patients with cholesterol gallstone disease. Investigation of the factor(s) responsible for the suspended supersaturation (metastability) of normal human biles revealed that model bile solutions of cholesterol saturation index (CSI) and molar lipid composition identical to individual gallbladder bile specimens had much shorter crystal nucleation times, i.e., exhibited decreased metastability. Unsaturated normal biles, after supplementation with lecithin, cholesterol, and sodium taurocholate to a 'standard' supersaturated lipid composition, also demonstrated nucleation times three- to 15-fold longer than the comparable standard model bile. Total lipid extracts of normal biles, however, when similarly supplemented, did not differ in nucleation time from the control model solution. Gallbladder biles were fractionated by gel chromatography and the eluted fractions were pooled into two fractions. The fractions eluting in about the first 25% of the included volume when mixed with the supersaturated standard model bile induced a modest increase in nucleation time of approximately 1.5 times the control value. The fractions eluting in the second 25% of the included volume and which contained all of the bile lipids, were concentrated and supplemented with lipids to the standard composition. The nucleation times of these supplements were 3-10 times longer than the control nucleation times. Delipidated bile protein mixtures, purified by discontinuous sucrose gradient centrifugation, were recombined with purified lipids at the standard composition used previously. The nucleation times of these mixtures were significantly prolonged to the same extent as those associated with the second chromatographic fraction. These observations demonstrate that the delayed onset (inhibition) of cholesterol crystal nucleation observed in normal human gallbladder bile is produced by a factor(s) present in the biliary protein fraction.

Authors

R T Holzbach, A Kibe, E Thiel, J H Howell, M Marsh, R E Hermann

Energetics of sodium transport in the urinary bladder of the toad. Effect of aldosterone and sodium cyanide.

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Experiments were designed to determine whether the stimulatory effect of aldosterone on sodium transport involves an increase in tissue ATP. Urinary bladders that were removed from toads presoaked in 0.6% saline for 48-72 h, mounted as sacs, and maintained in open circuit except for brief observation of short circuit current every 30 min responded to 100 nM aldosterone added to the serosal bath with an increase in short circuit current to 170% of control hemibladders, which plateaus at 2-3 h. Tissue (ATP)/(ADP) X (Pi) measured in perchloric acid extracts increased to a maximum of 208% of controls (P less than 0.001) and ATP increased to 116% of controls (P less than 0.01) at 180 min. The short circuit current response to aldosterone paralleled the increase in ATP and (ATP)/(ADP) X (Pi) measured at 75, 120, 180, and 240 min. In bladders clamped at -150 mV, the short circuit current response to aldosterone was greater: 280% of controls (P less than 0.001) and tissue (ATP)/(ADP) X (Pi) increased to 191% of controls (P less than 0.001). In continuously short circuited bladders and bladders clamped at +75 mV, the short circuit current response to aldosterone and the change in ATP, ADP, or Pi were markedly diminished. 100 microM amiloride added to mucosal bath decreased the short circuit current to zero and inhibited the short circuit current response to aldosterone, whereas tissue ATP increased to 141% (P less than 0.05). 100, 250, and 500 microM NaCN dropped the short circuit current to 59, 35, and 24% of control values, respectively. Concurrently, tissue ATP measured at 60 min after the addition of NaCN dropped to 79, 66, and 56% of control values, respectively, and tissue ATP/ADP dropped to 68, 50, and 40%, respectively. The data revealed significant correlation between the change in the rate of sodium transport produced by aldosterone or NaCN as measured by the short circuit current and the concentration of ATP (r = 0.96, P less than 0.001), as well as ATP/ADP (r = 0.95, P less than 0.001). In conclusion, these results support the view that the stimulatory effects of aldosterone on sodium transport involve an increase in ATP or (ATP)/(ADP) X (Pi).

Authors

N Cortas, E Abras, M Arnaout, A Mooradian, S Muakasah

Immunosuppression by D-penicillamine in vitro. Inhibition of human T lymphocyte proliferation by copper- or ceruloplasmin-dependent generation of hydrogen peroxide and protection by monocytes.

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Abstract

It has been suggested that D-penicillamine is active in rheumatoid arthritis because of its capacity to function as a selective inhibitor of T lymphocyte function. The basis for the immunosuppressive action of this drug as well as mechanisms whereby the effect of D-penicillamine could be modified by elements of rheumatoid synovial tissue were examined. As previously reported, D-penicillamine, in the presence of copper ions markedly inhibited mitogen-induced human T lymphocyte DNA synthesis. Since the vast majority of copper in the body exists as an integral part of the ceruloplasmin molecule, the capacity of this cuproprotein to augment D-penicillamine-mediated inhibition of T cell function was examined. The requirement for copper ions could be entirely replaced by purified ceruloplasmin, which had been depleted of nonspecifically bound copper by passage over Chelex-100 columns. The mechanism by which D-penicillamine in the presence of either copper ions or ceruloplasmin caused inhibition of T lymphocyte responsiveness was examined. Partial protection from this inhibitory effect was accomplished by sodium borohydride. While superoxide dismutase had no protective effect, catalase was found to protect lymphocyte responsiveness totally from the inhibitory action of D-penicillamine and either copper ions or ceruloplasmin. Similarly, horseradish peroxidase and myeloperoxidase also protected responsiveness from these inhibitors while boiled catalase was without effect. These results indicate that inhibition of T lymphocyte responsiveness resulted from the generation of hydrogen peroxide. Since a number of cells likely to be present at chronic inflammatory sites, such as mononuclear phagocytes, contain enzymatic mechanisms to degrade hydrogen peroxide, the modulatory influence of these cells on the inhibition of T cell function caused by D-penicillamine and copper was examined. Monocytes, whose function was not suppressed by D-penicillamine and copper, were found to protect T lymphocyte responsiveness from the inhibitory effects of either the mixture of D-penicillamine and CuSO4 or of hydrogen peroxide. By contrast, endothelial cells, fibroblasts, or cells obtained from enzyme-digested noninflamed synovium could not protect T cells from the inhibitory effects of D-penicillamine and copper. Protection of T cells was afforded by means of a heat labile, azide-sensitive soluble factor present in lysates of human monocytes. These results indicate that the mechanism whereby D-penicillamine in the presence of copper or ceruloplasmin inhibits T lymphocyte responsiveness involves the generation of hydrogen peroxide and that other neighboring cells likely to be found w

Authors

P E Lipsky

Direct effects of catecholamines, thyrotropin-releasing hormone, and somatostatin on growth hormone and prolactin secretion from adenomatous and nonadenomatous human pituitary cells in culture.

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Abstract

To determine the mechanism and the site of action of catecholamines as well as hormones including thyrotropin-releasing hormone (TRH)1 and somatostatin on pituitary hormone release in patients with acromegaly and in normal subjects, the effects of these substances on growth hormone (GH) and prolactin (PRL) secretion from adenomatous and nonadenomatous human pituitary cells in culture were examined. When dopamine (0.01-0.1 microM) or bromocriptine (0.01-0.1 microM) was added to the culture media, a significant inhibition of GH and PRL secretion from adenoma cells from acromegalic patients was observed. This inhibition was blocked by D2 receptor blockade with metoclopramide or sulpiride, but not by D1 receptor blockade. Similarly, dopamine suppressed GH and PRL release by nonadenomatous pituitary cells in a dose-dependent manner, which was again blocked by D2 receptor blockade. The minimum effective concentration of dopamine required for a significant inhibition of PRL secretion (0.01 microM) was lower than that for GH release (0.1 microM). Norepinephrine, likewise, caused a suppression of PRL secretion from adenomatous and nonadenomatous pituitary cells. This effect was blocked by sulpiride, phentolamine, however, was ineffective. When TRH was added to the media, both GH and PRL secretion were enhanced in adenoma cells, while only the stimulation of PRL release was observed in nonadenomatous pituitary cells. Coincubation of TRH and dopamine resulted in variable effects on GH and PRL secretion. Somatostatin consistently lowered GH and PRL secretion in both adenomatous and nonadenomatous pituitary cells and completely blocked the TRH-induced stimulation of GH and PRL secretion from adenoma cells. Opioid peptides (1 microM) failed to affect hormone release. These results suggest that no qualitative difference in GH and PRL responses to dopaminergic agonists or to somatostatin exists between adenoma cells of acromegalic patients and normal pituitary cells, and that the direct effect of catecholamines on GH and PRL secretion from human pituitary cells is mediated mainly through dopamine receptor activation.

Authors

M Ishibashi, T Yamaji

Mechanism of decreased vascular reactivity to angiotensin II in conscious, potassium-deficient rats.

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Abstract

Chronic potassium deficiency in the rat results in a decrease in the pressor sensitivity to exogenous angiotensin II (AII). To define the mechanism of this resistance to AII, studies were performed in conscious rats after 14-21 d of dietary potassium deficiency. The pressor response to graded doses of AII was 50% less in potassium-deficient than control animals. In contrast, the pressor response to graded doses of norepinephrine was preserved in potassium-deficient rats; therefore, the decreased response to AII was not due to a generalized defect in vascular reactivity. Pretreatment with either the converting enzyme inhibitor, teprotide, or the prostaglandin synthesis inhibitor, indomethacin, failed to normalize the response to AII. Thus, neither prior receptor occupancy with endogenous AII nor the presence of vasodilatory prostaglandins caused the decreased AII response in potassium deficiency. Since the pressor response to AII involves angiotensin interaction with its vascular receptor, binding studies of mesenteric artery and uterine smooth muscle AII receptors were performed. Scatchard analysis showed that potassium deficiency resulted in a decrease in binding affinity (50% increase in Kd) in both uterine (6.00 vs. 3.82 nM; P less than 0.05) and vascular (1.39 vs. 0.973 nM; P less than 0.005) smooth muscle. Furthermore, despite increased circulating AII, there was an increase in AII receptor number in potassium-deficient uterine (308 vs. 147 fmol/mg protein; P less than 0.005) and vascular (470 vs. 316 fmol/mg protein; 0.05 less than P less than 0.1) smooth muscle. Although potassium deficiency resulted in alterations in receptor-binding parameters, the changes in binding affinity and number were directionally opposite, so that in potassium deficiency there was either no change or an increase in total AII binding. We conclude that the decrease in angiotensin pressor sensitivity in potassium-deficient rats is mediated by a postreceptor defect since it occurs subsequent to the binding of AII to its vascular smooth muscle receptor.

Authors

M S Paller, J G Douglas, S L Linas

Protection against oxygen toxicity by intravenous injection of liposome-entrapped catalase and superoxide dismutase.

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Abstract

Survival of rats exposed to 100% oxygen was increased from 69.5 +/- 1.5 to 118.1 +/- 9.9 h (mean +/- SEM, P less than 0.05) when liposomes containing catalase and superoxide dismutase were injected intravenously before and during exposure. The increased survival time in 100% oxygen was also associated with significantly less fluid in the pleural cavity. Rats injected with catalase- and superoxide dismutase-containing liposomes, which had increased survival in 100% oxygen, had increased lung wet weight upon autopsy compared with saline-injected controls (2.9 +/- 0.2 g/lung vs. 4.8 +/- 0.4 g/lung, mean +/- SE, P less than 0.05). Intravenous injection of control liposomes along with catalase and superoxide dismutase in the suspending buffer decreased the mean pleural effusion volume 89% and had no significant effect on survival time. Lung catalase and superoxide dismutase activities were increased 3.1- and 1.7-fold, respectively, 2 h after a single intravenous injection of liposomes containing catalase or superoxide dismutase. Superoxide dismutase activity was also significantly greater than controls in both air- and 100% oxygen-exposed rat lungs, when enzyme activity was assayed 24 h after cessation of injection of control and oxygen-exposed rats with enzyme-containing liposomes every 12 h for 36 h. Free superoxide dismutase and catalase injected intravenously in the absence of liposomes did not increase corresponding lung enzyme activities, affect pleural effusion volume, lung wet weight, or extend the mean survival time of rats exposed to 100% oxygen. The clearance of liposome-augmented 125I-labeled catalase from lung and plasma obeyed first order kinetics according to a one-compartment model. When clearance of liposome-augmented catalase activity or radioactivity were the parameters used for pharmacokinetic studies, the half-life of augmented lung catalase was 1.9 and 2.6 h, respectively. The half-life of liposome-entrapped catalase and superoxide dismutase activity in the circulation was 2.5 and 4 h, respectively, while intravenously injected catalase and superoxide dismutase had a circulation half-life of 23 and 6 min, respectively.

Authors

J F Turrens, J D Crapo, B A Freeman

Segmental chloride and fluid handling during correction of chloride-depletion alkalosis without volume expansion in the rat.

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To determine whether chloride-depletion metabolic alkalosis (CDA) can be corrected by provision of chloride without volume expansion or intranephronal redistribution of fluid reabsorption, CDA was produced in Sprague-Dawley rats by peritoneal dialysis against 0.15 M NaHCO3; controls (CON) were dialyzed against Ringer's bicarbonate. Animals were infused with isotonic solutions containing the same Cl and total CO2 (tCO2) concentrations as in postdialysis plasma at rates shown to be associated with slight but stable volume contraction. During the subsequent 6 h, serum Cl and tCO2 concentrations remained stable and normal in CON and corrected towards normal in CDA; urinary chloride excretion was less and bicarbonate excretion greater than those in CON during this period. Micropuncture and microinjection studies were performed in the 3rd h after dialysis. Plasma volumes determined by 125I-albumin were not different. Inulin clearance and fractional chloride excretion were lower (P less than 0.05) in CDA. Superficial nephron glomerular filtration rate determined from distal puncture sites was lower (P less than 0.02) in CDA (27.9 +/- 2.3 nl/min) compared with that in CON (37.9 +/- 2.6). Fractional fluid and chloride reabsorption in the proximal convoluted tubule and within the loop segment did not differ. Fractional chloride delivery to the early distal convolution did not differ but that out of this segment was less (P less than 0.01) in group CDA. Urinary recovery of 36Cl injected into the collecting duct segment was lower (P less than 0.01) in CDA (CON 74 +/- 3; CDA 34 +/- 4%). These data show that CDA can be corrected by the provision of chloride without volume expansion or alterations in the intranephronal distribution of fluid reabsorption. Enhanced chloride reabsorption in the collecting duct segment, and possibly in the distal convoluted tubule, contributes importantly to this correction.

Authors

J H Galla, D N Bonduris, S L Dumbauld, R G Luke

Adenosine triphosphate-dependent calcium pump in the plasma membrane of guinea pig and human neutrophils.

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Changes in cytosolic free Ca may function as a second messenger in neutrophils. Since the plasma membrane seems to be a major regulator of intracellular Ca in many cells, we characterized an energy-dependent Ca transport system in plasma membrane-enriched fractions ("podosomes") from phorbol myristate acetate-stimulated guinea pig and human neutrophils. The active Ca transport system in guinea pig podosomes exhibited a high affinity for Ca (Michaelis constant [Km]Ca 280 +/- 120 nM) and a maximum velocity of 0.83 nmol Ca/mg protein per min. Uptake showed an absolute requirement for Mg ATP (Km ATP 67 microM), whereas other trinucleotides were inactive. Ca uptake was optimal at pH 7, was azide insensitive and temperature dependent. Vanadium, an inhibitor of the Ca/Mg ATPase of heart sarcolemma, inhibited Ca pump activity by 50% at 1 microM. Ca transport was not affected in a NaCl-containing medium, an observation arguing against the presence of a Na/Ca exchange system. Calmodulin at 0.5-10 micrograms/ml stimulated the Ca pumping activity of EGTA-washed podosomes. Calmodulin depletion decreased the affinity of the Ca pump for Ca (Km Ca 2.07 microM) and its readdition restored it (Km Ca 0.55 microM). ATP-dependent Ca transport by podosomes and phagocytic vesicles was inactivated by exposure to trypsin or to the nonpenetrating sulfhydryl reagent rho-chloromercuribenzene sulfonate. Human podosomes had a Ca uptake system with properties similar to those of the guinea pig. These findings demonstrate the presence of a Ca pump in the neutrophil plasma membrane, which is active at physiological concentrations of free cytosolic Ca. By changing Ca concentrations at the cell periphery, this pump could control various motile functions of the neutrophil, such as locomotion or degranulation.

Authors

H Lagast, P D Lew, F A Waldvogel

Concurrent sickle cell anemia and alpha-thalassemia. Effect on pathological properties of sickle erythrocytes.

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The concurrence of sickle cell anemia and alpha-thalassemia results in less severe hemolytic anemia apparently as a result of reduced intraerythrocytic concentration of hemoglobin S and its retarded polymerization. We have evaluated the effect of alpha-globin gene number on several interrelated properties of sickle erythrocytes (RBC) that are expected to correlate with the hemolytic and rheologic consequences of sickle cell disease. The irreversibly sickled cell number, proportion of very dense sickle RBC, and diminished deformability of sickle RBC each varied directly with alpha-globin gene number. Sickle RBC density was a direct function of the mean corpuscular hemoglobin concentration (MCHC). Even in nonsickle RBC, alpha-globin gene number varied directly with RBC density. Despite differences in alpha-globin gene number, sickle RBC of the same density had the same degree of deformability and dehydration. These data indicate that the fundamental effect of alpha-thalassemia is to inhibit the generation of sickle RBC having high density and MCHC, and that the other beneficial effects of sickle RBC are secondary to this process. The less consistent effect on overall clinical severity reported for subjects with this concurrence may reflect an undefined detrimental effect of alpha-thalassemia, possibly on the whole blood viscosity or on sickle RBC membrane-mediated adherence phenomena.

Authors

S H Embury, M R Clark, G Monroy, N Mohandas

Biliary excretion of glutathione and glutathione disulfide in the rat. Regulation and response to oxidative stress.

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Abstract

Regulation of the biliary excretion of reduced glutathione (GSH) and glutathione disulfide (GSSG) and responses to selected model toxins were examined in male Sprague-Dawley rats. In control and phenobarbital-pretreated rats in which the intrahepatic concentration of GSH was modulated by the administration of diethyl maleate or acetaminophen, the biliary concentration of GSH was consistently lower than, but directly proportional to, the intrahepatic concentration of GSH. Furthermore, increments in bile flow produced by the infusion of sulfobromophthalein (BSP)-glutathione were associated with proportional increases in the biliary excretion of GSH, suggesting that GSH passes into bile passively along a concentration gradient. In contrast, GSSG appears to be secreted into bile against a steep concentration gradient. An increased hepatic production and biliary excretion of GSSG resulted from the administration of t-butyl hydroperoxide. Measurement of biliary GSSG and BSP during a constant infusion of the GSH adduct of BSP indicated that GSSG shares a common excretory mechanism with GSH adducts. Diquat, nitrofurantoin, and paraquat also markedly stimulated the biliary excretion of GSSG. On a molar basis, these compounds generated much more GSSG than a direct substrate for glutathione peroxidase such as t-butyl hydroperoxide, indicating that the compounds undergo redox-cycling with concomitant production of hydrogen peroxide. Aminopyrine (0.8 mmol/kg) also significantly increased biliary GSSG. This increase, however, was associated with a proportional increase in bile flow and in the biliary excretion of GSH such that the GSSG/GSH ratio in bile did not change. Acetaminophen and chloroform, two compounds generating electrophilic metabolites that deplete intrahepatic GSH, led to a progressive decrease in the biliary excretion of GSH and GSSG. Furosemide and dimethylnitrosamine, the electrophilic metabolites of which do not deplete hepatic GSH, minimally altered biliary GSH and GSSG. Similarly, carbon tetrachloride and iproniazid, which yield organic radical metabolites that can peroxidize membrane lipids, did not increase the biliary excretion of GSSG. This finding indicates that membrane-bound lipid hydroperoxides may not be good substrates for glutathione peroxidases. The measurement of the biliary excretion of GSSG and of the GSSG/GSH ratio in bile is a sensitive index of oxidative stress in vivo and thus complements other in vivo parameters for the study of reactive intermediates of xenobiotics such as the determination of covalent binding, the formation of lipid hydroxy acids, and the depletion of intracellular GSH.

Authors

B H Lauterburg, C V Smith, H Hughes, J R Mitchell

Calcium is a competitive inhibitor of gentamicin-renal membrane binding interactions and dietary calcium supplementation protects against gentamicin nephrotoxicity.

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The divalent cations, Ca++ and Mg++, are known to competitively inhibit a large number of aminoglycoside-membrane interactions, so that Ca++ prevents both the neurotoxic and ototoxic effects of these antibiotics acutely in vitro. Since gentamicin-induced plasma and subcellular membrane damage appear to be critical pathogenetic events in gentamicin nephrotoxicity, Ca++ may play a similar protective role in gentamicin-induced acute renal failure. To test this possibility in vivo, rats (group 2) were given a 4% calcium (in the form of CaCO3) supplemented diet to increase delivery of Ca++ to the kidney and administered single daily subcutaneous injections of gentamicin, 100 mg/kg, for 10 d. Compared with a simultaneously studied group (group 1) of rats receiving identical gentamicin dosages and normal diets, Ca++ supplementation ameliorated gentamicin-induced acute renal failure. After 10 doses of gentamicin, blood-urea nitrogen values in group 1 averaged 213 +/- 15 (SE) and 25 +/- 3 (P less than 0.001) in group 2. The progressive decline in renal excretory function, as measured by BUN, in group 1 animals was accompanied by simultaneous declines in renal cortical mitochondrial function and elevations in renal cortex and mitochondrial Ca++ content, quantitative indices of the degree of renal tubular cell injury. Oral Ca++ loading markedly attenuated these gentamicin-induced derangements. After eight and 10 doses of gentamicin, mitochondria isolated from the renal cortex of group 2 rats had significantly higher rates of respiration supported by pyruvate-malate, succinate and N,N,N',N'-tetramethyl-p-phenyldiamine-ascorbate, higher rates of dinitrophenol-uncoupled respiration and greater acceptor control ratios than those measured in mitochondria isolated from the renal cortex of group 1 animals. Similarly, after 8 and 10 doses, renal cortex and renal cortical mitochondrial Ca++ content of group 2 was significantly lower than values observed in group 1. Thus, dietary calcium supplementation significantly protected against gentamicin-induced renal tubular cell injury and, consequently, gentamicin-induced acute renal failure. The mechanism for this protective effect of Ca++ may relate to the manner in which this polycationic antibiotic interacts with anionic sites, primarily the acidic phospholipids of renal membranes. In this regard, Ca++ was found to be a competitive inhibitor both of 125I-gentamicin binding to renal brush border membranes, the initial site of interaction between gentamicin and renal proximal tubule cells, with a composite inhibition constant (Ki) of 12 mM and of 125I-gentamicin binding to phosphatidic acid, an important membrane acidic phosph

Authors

H D Humes, M Sastrasinh, J M Weinberg

Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media.

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Abstract

Actin of smooth muscle cells of rat and human aortic media shows a predominance of the alpha-isoform. In experimental rat aortic intimal thickening, in human atheromatous plaque, and in cultured aortic smooth muscle cells, there is a typical switch in actin expression with a predominance of the beta-form and a noticeable amount of gamma-form. This pattern of actin expression represents a new reliable protein-chemical marker of experimental and human atheromatous smooth muscle cells.

Authors

G Gabbiani, O Kocher, W S Bloom, J Vandekerckhove, K Weber

Deficiency of a surface membrane glycoprotein (Mo1) in man.

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Deficiency of a granulocyte surface glycoprotein of 150,000-D had been associated with defective C3- and IgG-dependent phagocytosis in a patient with recurrent bacterial infections. By using monoclonal antibodies, we found that this patient's granulocytes, monocytes, and null cells were deficient in Mo1 (equivalent to OKM1 and Mac-1), a cell surface molecule consisting of two noncovalently linked glycoproteins of 155,000 and 94,000 D. The 155,000-D subunit is closely associated with the human complement receptor that recognizes C3bi and/or a further degradation product termed C3dg (C3bi receptor); the 94,000-D subunit has been shown to be shared, on normal cells, by two other surface membrane glycoproteins: lymphocyte function-associated antigen-1 (LFA-1) and P-150, 95. Both subunits of Mo1 were deficient on the patient's granulocytes as determined by immunoprecipitation with subunit-specific monoclonal antibodies as well as fluorescence analysis. Mol-deficient monocytes, like granulocytes, had defective C3-and IgG-dependent phagocytosis. Natural killing activity by the patient's peripheral blood leukocytes was normal. Mo1-deficient granulocytes and monocytes rosetted normally with sheep erythrocytes coated with C3bi. This rosetting was totally inhibited by a mixture of anti-Mo1 and anti-C3b (the major fragment of C3) receptor antibodies but not by either antibody alone. Since monoclonal antibodies to the 155,000-D subunit of Mo1 can inhibit C3bi receptor binding, immune phagocytosis, opsonized zymosan-induced degranulation, and superoxide generation by normal phagocytes (functions which are defective in Mo1-deficient cells), it appears likely that Mo1 deficiency may in part underlie the functional aberrations leading to recurrent bacterial infections in man.

Authors

N Dana, R F Todd 3rd, J Pitt, T A Springer, M A Arnaout

Development and application of an enzyme-linked immunosorbent assay for the quantitation of alternative complement pathway activation in human serum.

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Abstract

We have developed a new, specific, and highly sensitive enzyme-linked immunosorbent assay (ELISA) which quantitates activation of the alternative pathway in human serum, plasma, or on the surface of activators. The ELISA detects the third component of complement (C3b), proteolytic fragment of complement Factor B (Bb), and properdin (P) complex or its derivative product, C3b,P. In the method, activator-plasma mixtures, plasma containing an activated alternative pathway, or other samples are added to the wells of microtitration plates precoated with antibody to P. C3b, Bb,P or C3b,P complexes which become bound are quantitated by subsequently added, enzyme-labeled, anti-C3. The resulting hydrolysis of the chromogenic substrate is expressed as nanograms of C3b by reference to a C3 standard curve. In addition to absolute specificity for activation of the pathway because of the nature of the complex detected by the assay, the ELISA is highly sensitive and able to reproducibly detect 10-20 ng/ml of C3b,P complexes in serum. This value corresponds to 0.0015% of the C3 in serum. In a series of studies to validate the parameters of the ELISA, reactivity was found to be dependent on the presence of alternative pathway proteins, the functional integrity of the pathway, and on the presence of magnesium. Sheep erythrocytes were converted to activators by treatment with neuraminidase. By using a variety of activators, the kinetics of activation and the numbers of bound C3b molecules quantitated by the ELISA were very similar to those measured by C3b deposition. The ELISA also detected identical activation kinetics when MgEGTA-serum and a mixture of the purified alternative pathway proteins were used as sources of the pathway. ELISA reaction kinetics also correlated with the restriction index, a measure of alternative pathway-activating ability. These studies cumulatively validate the ELISA as a direct and quantitative assay for alternative pathway activation. The sensitivity of the ELISA has permitted its use to detect direct alternative pathway activation by several viruses. The ELISA has also shown that certain classical pathway activators trigger the amplification loop of the alternative pathway while others do not. In addition, stable ELISA reactive complexes appeared in the supernatant of mixtures of serum with certain, but not other activators. The ability of the ELISA to detect activation which has already occurred and the stability of the reactive complexes permits studies of clinical sera. Normal human sera (20) contained low levels (5-20 ng/ml) of ELISA-reactive complexes. A proportion of sera from individuals with the adult respiratory distress syndrome (9-10), typhoid fever (8-10), malaria (3-5), gram-negative sepsis (9 of 47), acute trauma and shock (6 f 25), and systemic lupus erythematosus (3 of 29) showed elevated levels of complexes reactive in the alternative pathway ELISA. In contrast, nine sera from patients with circulating C3 nephritic factor were not reactive in the ELISA.

Authors

J T Mayes, R D Schreiber, N R Cooper

Short-term aluminum administration in the rat. Effects on bone formation and relationship to renal osteomalacia.

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Abstract

Aluminum may be pathogenic in the osteomalacia observed in some patients receiving hemodialysis. To study the early effects of Al on bone growth, bone formation, mineralization, and resorption were measured during short-term Al exposure in the tibial cortex of pair-fed control (C, n = 10), aluminum-treated (AL, n = 9), subtotally nephrectomized control (NX-C, n = 7), and subtotally nephrectomized aluminum-treated (NX-AL, n = 8) rats using double tetracycline labeling of bone. Animals received 2 mg/d of elemental Al intraperitoneally for 5 d/wk over 4 wk. Total bone and matrix (osteoid) formation, periosteal bone and matrix formation, and periosteal bone and matrix apposition fell by 20% in AL from C, P less than 0.05 for all values, and by 40% in NX-AL from NX-C, P less than 0.01 for all values. Moreover, each measurement was significantly less in NX-AL than in AL, P less than 0.05 for all values. Osteoid width did not increase following aluminum administration in either AL or NX-AL. Resorption surface increased from control values in both AL and NX-AL; also, resorptive activity at the endosteum was greater in NX-AL than in NX-C, P less than 0.05. Thus, aluminum impairs new bone and matrix formation but does not cause classic osteomalacia in the cortical bone of rats whether renal function is normal or reduced. These findings may represent either a different response to aluminum administration in cortical bone as contrasted to trabecular bone or an early phase in the development of osteomalacia. Aluminum may increase bone resorption and contribute to osteopenia in clinical states associated with aluminum accumulation in bone.

Authors

W G Goodman, J Gilligan, R Horst

Selective vulnerability of the medullary thick ascending limb to anoxia in the isolated perfused rat kidney.

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Abstract

A specific anatomical lesion sharply localized to the cells of the medullary thick ascending limbs (mTAL) and characterized by mitochondrial swelling progressing to nuclear pyknosis and cell death is elicited reproducibly in isolated rat kidneys perfused for 15 or 90 min with cell-free albumin-Ringer's medium gassed with 5% CO2, 95% O2 (O2 content, 1.5 vol/100 ml). The lesion, involving about half of mTALs, appears first in mTALs removed from vascular bundles and near the inner medulla, areas most likely to be anoxic. Hypoxic perfusion (O2 content 0.12 vol/100 ml) exaggerates the lesion, wiping out gradations of damage and extending it to all mTALs. O2-enriched perfusions using rat erythrocytes (O2 content 7.1 vol/100 ml) completely eliminates the lesion (unless gassed with carbon monoxide). Similarly, supplementation of the perfusion medium with a purified hemoglobin (O2 content 5.8 vol/100 ml) prevents mTAL injury. Perfusion with a fluorinated hydrocarbon blood substitute, Oxypherol (O2 content 4.3 vol/100 ml) also attenuates the lesion. These findings suggest that the mTAL is exquisitely susceptible to anoxic damage because of low O2 supply imposed by the medullary vascular system and the high rate of metabolism mandated by active reabsorption of sodium chloride. The vulnerability of the mTAL to anoxic injury could play a key role in the pathogenesis of ischemic renal injury.

Authors

M Brezis, S Rosen, P Silva, F H Epstein

Immunoregulatory subsets of the T helper and T suppressor cell populations in homosexual men with chronic unexplained lymphadenopathy.

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Abstract

Unexplained, generalized lymphadenopathy in homosexual men, which can be a prodrome to the acquired immunodeficiency syndrome, is associated with impaired cell-mediated immunity, a low ratio of T helper-inducer to T suppressor-cytotoxic cells (defined by the T4 and T8 monoclonal antibodies), and hypergammaglobulinemia. We performed double-marker studies on T cells by using a panel of monoclonal antibodies (Ia, T17, TQ1, and Leu-8), which reportedly detect activation or functional subsets of the T4 and T8 T cell populations. The T4:TQ1- or T4:Leu-8- subset, which is the major helper subset for B cell responses, is normally represented in lymphadenopathy patients. A depression in the reciprocal subset, T4:TQ1+ or T4:Leu-8+, accounts for the T4 T cell defect. Similarly, the TQ1 and Leu-8 markers delineate the abnormality of T8 T cells: the T8:TQ1- or T8:Leu-8- subset is elevated, whereas the T8:TQ1+ or T8:Leu-8+ subset is normally represented. We found no evidence of excessive activation of T4 T cells by using the T17 or Ia monoclonal antibodies. We did find an overall increase in Ia-positive T cells; however, this was due to increased T8:Ia+ cells. In functional studies, immunoglobulin production induced by pokeweed was subnormal. Most lymphadenopathy patients had normal T helper cell function when combined with normal B cells. The dampened pokeweed responses could be partially explained by depression of the T4:TQ1+ (or T4:Leu-8+) subset (which has minor help-associated function) and/or greater than expected suppression. However, subnormal pokeweed responses could not be totally explained by immunoregulatory T cell abnormalities because we also found an intrinsic defect in the B cell responses of lymphadenopathy patients.

Authors

J K Nicholson, J S McDougal, T J Spira, G D Cross, B M Jones, E L Reinherz

Human granulocyte/pollen-binding protein. Recognition and identification as transferrin.

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Abstract

Normal human serum was found to contain a heat-stable protein which promoted the binding of granulocytes to timothy grass pollen (granulocyte/pollen-binding protein [GPBP]). GPBP was purified by gel filtration, anion exchange, and affinity chromatography. Virtually all of the granulocyte/pollen-binding activity was associated with a beta-1-protein having a molecular mass of approximately 77,000 D and an isoelectric point of between 5.5 and 6.1. By immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was identified as transferrin. Monospecific antisera raised against either GPBP or transferrin removed biological activity from GPBP preparations, and GPBP and transferrin gave lines of identity with these two antisera. The apparent heterogeneity in the molecular size and charge of GPBP observed during progressive purification was minimal when GPBP was saturated with ferric ions before the separation procedures. These experiments indicate that granulocyte/pollen binding is a hitherto unrecognized property of transferrin which appears to be unrelated to iron transport and raises the possibility that transferrin might have a physiological role in the removal of certain organic matter.

Authors

S P Sass-Kuhn, R Moqbel, J A Mackay, O Cromwell, A B Kay

Arthritis induced in rats by cloned T lymphocytes responsive to mycobacteria but not to collagen type II.

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Abstract

We have been studying the pathogenesis of adjuvant arthritis in rats using a long-term cell line of T lymphocytes, the A2 line, which can induce polyarthritis and can also be used to vaccinate rats against adjuvant arthritis. Although line A2 was selected for its proliferative response to mycobacteria, it also responded to collagen type II. To elucidate its role of responsiveness to collagen type II and the relationship between arthritogenicity and vaccination, we cloned A2 and selected a subline A2b. We now report that subline A2b, which bore a marker of helper/delayed hypersensitivity T lymphocytes, was strongly arthritogenic, but could not vaccinate against arthritis. Moreover, A2b showed no response to collagen type II. Therefore, reactivity to collagen type II is not a requisite for arthritogenicity, and mediation of arthritis and vaccination can be distinct properties of different populations of T lymphocytes.

Authors

J Holoshitz, A Matitiau, I R Cohen

Contribution of the pancreas to circulating somatostatin-like immunoreactivity in the normal dog.

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Abstract

These studies were performed to assess the contribution of the pancreas to the somatostatin-like immunoreactivity (SLI) circulating in arterial and portal venous plasma. Basal SLI concentrations in arterial, pancreatic venous, and portal venous plasma were 95 +/- 9, 277 +/- 32, and 130 +/- 12 pg/ml, (means +/- SEM), respectively. Measurement of pancreatic and portal venous blood flow (5 +/- 1 vs. 365 +/- 46 ml/min) and hematocrit allowed calculation of net, base-line SLI output from the right lobe of the pancreas (521 +/- 104 pg/min) and from the gastrointestinal tract (8,088 +/- 1,487 pg/min), which suggested that the contribution of the pancreas to circulating SLI was minor when the D cells were not stimulated. To stimulate the secretion of SLI from both pancreatic and nonpancreatic sources, isoproterenol, a beta-adrenergic agonist, was infused intravenously for 1 h into six anesthetized dogs. Arterial SLI increased by 52 +/- 9 pg/ml; superior pancreatico-duodenal venous SLI increased by 380 +/- 95 pg/ml; portal venous SLI increased by 134 +/- 14 pg/ml. Pancreatic venous blood flow remained unchanged at 5 +/- 1 ml/min, but portal venous blood flow increased to 522 +/- 62 ml/min. SLI output from the right lobe of the pancreas increased by 684 +/- 227 pg/min and that from the gastrointestinal tract increased by 23,911 +/- 3,197 pg/min, again suggesting that the pancreas was a minor source of circulating SLI even when the D cells were stimulated. We conclude that the measurement of arterial-venous SLI concentrations, in the absence of measurements of organ blood flow, can give a false impression of the organ's contributions of circulating SLI. To verify that the contribution of the pancreas was negligible, six dogs received an acute pancreatectomy and then an intravenous infusion of isoproterenol at the same rate. In these dogs, both the base-line level of SLI in arterial plasma (109 +/- 12 pg/ml) and the increment during isoproterenol (56 +/- 8 pg/ml) were similar to those of normal dogs. Likewise, in pancreatectomized dogs both the base-line level of SLI in portal venous plasma (129 +/- 16 pg/ml) and the increment during isoproterenol (174 +/- 34 pg/ml) were similar to those of normal dogs. We conclude that, in normal dogs, the pancreas makes a negligible contribution to the basal and stimulated level of SLI in arterial and portal venous plasma and therefore that these levels should not be used as an index of secretory activity of the pancreatic D cells.

Authors

G J Taborsky Jr, J W Ensinck

Characterization of a virus that causes transient aplastic crisis.

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Abstract

Transient aplastic crisis in children with congenital hemolytic anemias has been linked epidemiologically to infection with a serum parvovirus-like virus (SPLV). The virus is found in the blood in the early stages of the crisis, and serum containing SPLV inhibits erythroid colony formation in vitro. After sedimentation of virus-containing sera through a sucrose density gradient, colony inhibitory activity is present in the particulate fraction and separate from serum immunoglobulins. No inhibitory activity can be recovered from convalescent-phase sera after similar fractionation procedures. Inhibition of erythroid colony formation in vitro is not a feature of sera from other viral infections. The pattern of resistance of SPLV activity to chemicals and enzymes is compatible with it being a parvovirus. By using replating techniques, a target of SPLV has been identified as a late erythroid progenitor cell. Neither SPLV antigen nor anti-SPLV IgM was present in the sera of patients with other forms of bone marrow failure.

Authors

N S Young, P P Mortimer, J G Moore, R K Humphries

Utilization of individual lecithins in intestinal lipoprotein formation in the rat.

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Abstract

To determine the molecular species composition of lecithins of different nascent lipoproteins, high density lipoproteins (HDL), very low density lipoproteins (VLDL), and chylomicrons (CM) were isolated from the mesenteric lymph of rats. Lymph was collected at 0 degrees C with 5,5'-dithiobis-2-dinitrobenzoic acid added to inhibit lecithin-cholesterol acyl transferase. CM were separated by ultracentrifugation and HDL from VLDL by dextran SO4-MG+2 precipitation. Molecular species of lecithin were directly isolated by reverse phase high performance liquid chromatography. In fasted animals, the lecithin compositions of lymph HDL and VLDL were virtually the same and closely resembled the lecithin composition of intestinal mucosa. When bile lecithin was eliminated (by bile diversion), there was a marked change in lecithin composition of all lipoprotein and mucosal samples, which was most notable for a reduction in 16:0-species (which are predominant in bile) and a relative increase in the corresponding 18:0-species. Feeding unsaturated triglycerides (triolein, trilinolein, or a combination of triolein and trilinolein) also resulted in a change in HDL and VLDL lecithin composition. The effect was similar whether bile lecithin was present or eliminated and was notable for a reduction in 16:0-species, an increase in 18:0-species, and the emergence of large amounts of diunsaturated lecithins that corresponded to the fatty acid composition of the triglycerides fed (i.e., 18:1-18:1, 18:2-18:2, and 18:1-18:2 lecithins). When bile-diverted rats were infused via the duodenum with a mix of [14C]choline-labeled lecithins (isolated from the bile of other rats), the incorporation of infused lecithins into different lymph lipoproteins was distinctly different. Individual lecithins were incorporated to a variable extent into each lipoprotein. In fasted rats the specific activities of all major molecular species of lecithin were relatively greater in VLDL than HDL, indicating that HDL derived proportionately more of its lecithins from an endogenous pool than did VLDL. Feeding triolein changed the specific activities of more of the lecithin species of VLDL than of HDL. The specific activities of lecithins in CM were more similar to VLDL than to HDL after triolein feeding. Results thus indicate that, although the lecithins of different mesenteric lymph lipoproteins are similar and may be derived from membrane sites with the same lecithin composition, lecithins incorporated into different lipoproteins originate from different metabolic pools and/or by different mechanisms.

Authors

G M Patton, S B Clark, J M Fasulo, S J Robins

Reflex influence of selective coronary artery occlusion on the total capacitance vasculature in the dog.

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Abstract

While the reflex influence of selective coronary arterial occlusion on the resistance vasculature has been well delineated, the reflex influence of coronary occlusion on the total capacitance vasculature has not been examined. Thus, selective coronary occlusions were performed in 65 anesthetized dogs. Blood was drained from the vena cavae and returned to the right atrium at a constant rate so that changes in total intravascular volume could be recorded as reciprocal changes in extracorporeal reservoir volume. In 10 animals, 2.5 min of left anterior descending occlusion was associated with only an insignificant total volume increase of 6 +/- 4 ml (SEM), whereas 2.5 min of left circumflex occlusion was associated with a 27 +/- 4 ml (P less than 0.001) increase in volume, which was significantly attenuated (P less than 0.001) to only a 7 +/- 3 ml increase after cervical vagectomy. Epicardial lidocaine in four animals reduced the volume increment associated with circumflex occlusion from 30 +/- 3 to 11 +/- 4 ml (P less than 0.025). The volume increase was attenuated from 45 +/- 6 to 24 +/- 5 ml with propranolol administration (P less than 0.001) (seven animals) and from 26 +/- 5 to 17 +/- 6 ml with atropine (P less than 0.025) (eight animals), but was not attenuated with phenoxybenzamine (28 +/- 7 ml before and 25 +/- 2 ml after phenoxybenzamine) (five animals). Double blockade with propranolol and atropine reduced the volume increase to 3 +/- 2 ml (NS) in four of these animals. In order to compare the influences of selective beta-1 adrenergic blockade and combined beta-1 and beta-2 blockade, volume responses were assessed before and after administration of metoprolol or propranolol in doses that produced the same amount of beta-1 blockade (15 animals). The volume increase associated with circumflex occlusion was not attenuated after beta-1 blockade (20 +/- 4 ml before and 18 +/- 5 ml after metoprolol) (eight animals) but was attenuated from 30 +/- 5 to 14 +/- 5 ml after propranolol (P less than 0.05) (seven animals). To examine further the efferent limb of the observed reflex, circumflex occlusions were performed before and after either vagectomy at the level of the diaphragm or section of the sympathetic splanchnic nerves in 12 animals. The volume increment was significantly attenuated after either procedure. In four animals undergoing prior arterial baroreceptor denervation, volume still increased 30 +/- 6 ml (P less than 0.001) with circumflex occlusion. Thus, inferior myocardial ischemia is associated with an autonomic reflex that acts to increase total intravascular volume. The afferent limb is mediated through the vagi, and the efferent limb, throug

Authors

D L Rutlen, R S Underwood

Interferon enhances prostacyclin production by cultured vascular endothelial cells.

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Abstract

The effects of interferon (IFN) on the arachidonate metabolism and physiological functions of cultured endothelial cells and blood platelets have been examined. Cultured bovine aortic endothelial cells were found to be sensitive to the antiviral and antiproliferative activities of human leukocyte (alpha) IFN and to increase their capacity to synthesize prostacyclin (PGI2) upon exposure to IFN. Several observations indicate that IFN stimulates PGI2 synthesis at the level of the enzymes phospholipase A2 and cyclooxygenase: (a) PGI2 production was dependent upon the supply of exogenous arachidonic acid or the liberation of endogenous cellular arachidonate by ionophore A23187, but was not observed when IFN-treated cells were exposed to the endoperoxide prostaglandin H2. (b) IFN had no effect on the spontaneous release of PGI2 into the culture medium during the incubation period (24-72 h). (c) The stimulatory effect of IFN on PGI2 production was inhibited by both glucocorticoids and indomethacin. The effect of IFN on platelet prostaglandin metabolism was also investigated. Incubation of platelet-rich plasma with IFN had no effect on platelet aggregation and thromboxane A2 production. The biological significance of the findings presented in this paper may be considered in view of the protective role of PGI2 in the vessel wall and the fact that infection with certain viruses induces endothelial damage both in man and experimental animal models.

Authors

A Eldor, R Fridman, I Vlodavsky, E Hy-Am, Z Fuks, A Panet

Pancytopenia as a clonal disorder of a multipotent hematopoietic stem cell.

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Abstract

Hematopoiesis was investigated in a 14-yr-old girl who had a 2-yr history of stable asymptomatic pancytopenia and who was also heterozygous at the structural locus for glucose-6-phosphate dehydrogenase (G-6-PD). There was no morphologic or cytogenetic evidence for preleukemia and no suggestion of Fanconi anemia. In the skin and sheep erythrocytes-rosetted T lymphocytes, the ratio of G-6-PD A/B activities was 1:1. However, only type B activity was found in peripheral blood erythrocytes, granulocytes, and platelets. Most erythroid bursts and all granulocyte/macrophage colonies formed in methylcellulose culture were derived from the abnormal clone. These findings demonstrate that (a) some cases of pancytopenia are stem cell diseases that apparently develop clonally; (b) circulating differentiated cells originate from this clone; (c) despite a hypoproliferative anemia, the in vivo expression of presumably normal (nonclonal) progenitors is suppressed. In this patient, the relationship between clonal dominance and possible malignancy may be assessed prospectively.

Authors

J L Abkowitz, P J Fialkow, D J Niebrugge, W H Raskind, J W Adamson

A luteinizing hormone-releasing hormone agonist decreases biological activity and modifies chromatographic behavior of luteinizing hormone in man.

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Abstract

The effect of the luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6,Pro9-NEth]LHRH (LHRHA), on luteinizing hormone (LH) bioactivity was assessed with a rat interstitial cell assay in four men during a 14-d treatment period. Biologic/immunologic (B/I) ratios were unchanged initially with treatment but by day 12 had fallen to levels lower than basal values. Frequent sampling on day 12 revealed blunted gonadotropin responsiveness to LHRHA and absence of spontaneous LH pulsations. Despite continued administration of LHRHA, human chorionic gonadotropin administration resulted in elevated B/I ratios and testosterone levels. Further characterization of the serum immunoreactive LH by Sephadex chromatography revealed a later elution profile during treatment with LHRHA. Thus, LHRHA appears to act, in part, by modification of the bioactivity of LH in man.

Authors

R M Evans, G C Doelle, J Lindner, V Bradley, D Rabin

HLA-DR histocompatibility leukocyte antigens permit cultured human melanoma cells from early but not advanced disease to stimulate autologous lymphocytes.

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Abstract

HLA-DR histocompatibility antigens are commonly expressed by the melanocytes of melanoma and its precursors, but not by the melanocyte of normal skin. Further, the primary lesion of biologically early melanoma is commonly infiltrated with host T cells. Advanced disease is characterized by a paucity of such cells. To investigate the interaction of melanoma cells and autologous lymphocytes and its dependence on HLA-DR expression, we have established cell lines from biologically early (4 lines) and advanced disease (11 lines) and examined their capacity to stimulate blastogenesis of autologous T cells in vitro. Melanocytes from early disease expressed HLA-DR antigens and stimulated autologous T cells. Those from advanced disease, irrespective of DR expression, were nonstimulatory. To determine whether expression of DR was required for melanoma cells to be stimulatory, we first treated a stimulating cell line of DR3 allospecificity with anti-DR3-specific serum and demonstrated marked inhibition of its capacity to provoke blastogenesis. Next we used fluorescence-activated flow cytometry to sort a stimulating line heterogeneous for DR expression into DR-enriched and -depleted populations. When such cells were examined in the lymphocyte proliferation assay, their stimulatory capacity was proportional to their quantitative expression of HLA-DR. These studies indicate that cell lines may reflect important biological differences between early and advanced melanoma. HLA-DR expression may be an early event in neoplasia of melanocytes. These antigens are able to interact directly with autologous T cells; and their expression is necessary, but not sufficient, for melanoma cells to induce lymphocyte proliferation.

Authors

D Guerry 4th, M A Alexander, M F Herlyn, L M Zehngebot, K F Mitchell, C M Zmijewski, E J Lusk

Dietary omega-3 fatty acid deficiency and visual loss in infant rhesus monkeys.

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Abstract

Linolenic acid (18:3 omega 3) is a dietary precursor of docosahexaenoic acid (22:6 omega 3), the major fatty acid in the photoreceptor membranes of the retina. We hypothesized that rhesus monkeys deprived of dietary omega-3 fatty acids during prenatal and postnatal development would show plasma depletion of these fatty acids and visual impairment. Semipurified diets low in omega-3 fatty acids were fed to one group of adult female rhesus monkeys throughout pregnancy and to their infants from birth. A control group of mothers and infants received similar diets but supplying ample linolenic acid. In the plasma phospholipids of deficient infants, linolenic acid was generally undetectable and 22:6 omega 3 levels became progressively depleted, falling from 42% of control values at birth to 21% at 4 wk, 9% at 8 wk, and 6% at 12 wk of age. In the other plasma lipid classes, 22:6 omega 3 was undetectable by 12 wk. The visual acuity of the deprived infants, as measured by the preferential looking method, was reduced by one-fourth at 4 wk (P less than 0.05) and by one-half at 8 and 12 wk (P less than 0.0005) compared with control infants. These results suggest that omega-3 fatty acids may be an essential nutrient, and that 22:6 omega 3 may have a specific function in the photoreceptor membranes of the retina.

Authors

M Neuringer, W E Connor, C Van Petten, L Barstad

Immunoreactive beta-endorphin in a subpopulation of mouse spleen macrophages.

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Abstract

Using radioimmunoassay and immunofluorescence with antibodies to beta-endorphin (beta EP) and ACTH, we have shown that a subpopulation of mouse spleen cells, expressing Mac-1, a marker of macrophage differentiation, contains immunoreactive (ir)-beta EP, ir-ACTH, and smaller amounts of presumptive higher molecular weight forms of both. Neither nonadherent spleen cells, nor adherent or nonadherent cells from peripheral blood, contained detectable levels of these peptides. These findings suggest that beta EP and ACTH may be synthesized in a subpopulation of spleen macrophages, and are consistent with the possibility that these or related peptides may modulate lymphocyte function in the specific microenvironment of the spleen.

Authors

S J Lolait, A T Lim, B H Toh, J W Funder