Specific adherence of Escherichia coli (strain RDEC-1) to membranous (M) cells of the Peyer's patch in Escherichia coli diarrhea in the rabbit.

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Abstract

The RDEC-1 strain Escherichia coli is an enteroadherent bacterium that produces diarrhea in the rabbit. A histopathologically similar disease has been described in humans. The RDEC-1 bacterium adheres to the epithelium of lymphoid follicles in rabbit ileal Peyer's patches by 4 h postinoculation, 3-4 d before its adherence to absorptive epithelium. The purpose of this study was to determine whether the RDEC-1 bacterium adheres to a specific cell type in the lymphoid follicle epithelium. RDEC-1 bacteria were given in a dose of 2 X 10(6) by the orogastric route to postweanling rabbits. The distal ileal Peyer's patch, taken from 5 control rabbits and 43 rabbits at intervals in the first 24 h postinoculation, was examined by routine and high-voltage electron microscopy. The RDEC-1 bacterium adhered specifically to M (membranous) rather than absorptive epithelial cells of the lymphoid follicle epithelium. Further understanding of how the bacterium attaches to M cells, which transport antigens to intraepithelial lymphocytes, could be useful in designing vaccines to protect mucosal surfaces.

Authors

L R Inman, J R Cantey

Identification of human and rodent thymic epithelium using tetanus toxin and monoclonal antibody A2B5.

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Using a monoclonal antibody (A2B5), which binds to GQ ganglioside, and tetanus toxin, which binds to GD and GT gangliosides, distinct regions of human and rodent thymic epithelial cells have been identified. The lymphoid elements of the thymus do not bind A2B5 or tetanus toxin. The A2B5 and tetanus toxin-binding cells form a network of thymic epithelial cells throughout the thymic subcapsular cortex and thymic medulla and contain thymopoietin and thymosin alpha-1.

Authors

B F Haynes, K Shimizu, G S Eisenbarth

Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin.

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Abstract

We have previously reported that virulent egg yolk-grown Legionella pneumophila, Philadelphia 1 strain, multiplies intracellularly in human blood monocytes and only intracellularly under tissue culture conditions. In this paper, we have investigated the effect of erythromycin and rifampin on L. pneumophila-monocyte interaction in vitro; erythromycin and rifampin are currently the drugs of choice for the treatment of Legionnaires' disease. The intracellular multiplication of L. pneumophila is inhibited by erythromycin and rifampin, as measured by colony-forming units, whether the antibiotics are added just before or just after infection of monocytes with L. pneumophila, or 2 d after infection when L. pneumophila is in the logarithmic phase of growth in monocytes. Intracellular multiplication of L. pneumophila is inhibited by 1.25 microgram/ml but not less than or equal to 0.125 microgram/ml erythromycin and 0.01 microgram/ml but not less than or equal to 0.001 microgram/ml rifampin. These concentrations of antibiotics are comparable to those that inhibit extracellular multiplication of L. pneumophila under cell-free conditions in artificial medium; the minimal inhibitory concentration is 0.37 microgram/ml for erythromycin and 0.002 microgram/ml for rifampin. Multiplication of L. pneumophila in the logarithmic phase of growth in monocytes is inhibited within 1 h of the addition of antibiotics. Intracellular bacteria inhibited from multiplying by antibiotics are not killed. By electron microscopy, the bacteria appear intact within membrane-bound vacuoles, studded with ribosomelike structures. L. pneumophila multiplying extracellularly on artificial medium is killed readily by relatively low concentrations of erythromycin and rifampin; the minimal bactericidal concentration is 1 microgram/ml for erythromycin and 0.009 microgram/ml for rifampin. In contrast, L. pneumophila multiplying intracellularly is resistant to killing by these concentrations of erythromycin and rifampin or by concentrations equal to or greater than peak serum levels in humans. Extracellular L. pneumophila in stationary phase is also resistant to killing by erythromycin and rifampin. These findings, taken together with our previous work, indicate that, in vivo, L. pneumophila is resistant to killing by erythromycin and rifampin. Inhibition of L. pneumophila multiplication in monocytes by antibiotics is reversible; when the antibiotics are removed from infected monocyte cultures after 2 d, L. pneumophila resumes multiplication. This study indicates that patients with Legionnaires' disease under treatment with erythromycin and rifampin require host defenses to eliminate L. pneumophila, and that inadequate host defenses may result in relapse after cessation of therapy.

Authors

M A Horwitz, S C Silverstein

Preferential binding of vasoactive intestinal polypeptide to basolateral membrane of rat and rabbit enterocytes.

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Abstract

Binding of radioiodinated vasoactive intestinal polypeptide (VIP) to intestinal cell membranes of the rabbit ileum and rat jejunum was investigated. Specific binding of 125I-labeled VIP could be demonstrated only on the basolateral membrane and not on the brush border membrane. This corresponded with the lack of an effect on ion transport when VIP was applied to the mucosal side of an in vitro preparation of rabbit ileum. VIP altered ion transport only when it was applied to the serosal side. The binding of 125I-VIP was specific and dependent upon incubation temperature. There was a close correlation between the potency of VIP for inhibition of 125I-VIP binding and that for increasing adenylate cyclase activity. These observations demonstrate that VIP receptors are located on the basolateral membrane.

Authors

K Dharmsathaphorn, V Harms, D J Yamashiro, R J Hughes, H J Binder, E M Wright

Immunomodulatory effect of procainamide in man. Inhibition of human suppressor T-cell activity in vitro.

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Abstract

Procainamide (PA) induces the production of a number of autoantibodies in a high proportion of treated individuals and in some a syndrome closely resembling systemic lupus erythematosus. The mechanism underlying this action of PA is unclear. To examine the possibility that PA might induce autoantibody formation by altering normal immunoregulatory mechanisms, the action of this drug on an in vitro model of antibody formation in man was examined. PA was found to augment the generation of immunoglobulin-secreting cells (ISC) from human peripheral blood mononuclear cells (PBM) in response to pokeweed mitogen but had no effect on pokeweed mitogen-induced tritiated thymidine incorporation. When purified populations of B and T cells were used, PA enhanced the generation of ISC in B-cell cultures supported by untreated T cells but not by T cells treated with mitomycin C. These results indicate that PA augmented B-cell responses by inhibiting suppressor T-cell activity and not by augmenting helper T-cell or B-cell function. N-Acetyl-procainamide had no effect on the generation of ISC in this system. The effect of PA on concanavalin A (Con A)-induced suppressor cell activity was also examined to determine whether PA altered the generation or expression of suppressor T-cell function. PBM were cultured with 30 microgram/ml of Con A for 48 h to generate suppressor cells. When these were co-cultured with fresh PBM, the number of ISC generated was decreased by 58.1 +/- 3.4% (mean +/- SEM, n = 6). Cells that had been similarly incubated without Con A were not inhibitory. The addition of PA to the Con A-stimulated cultures inhibited the generation of suppressor cells as indicated by the fact that the response of fresh cells co-cultured with the Con A-stimulated cells was diminished by only 27.2 +/- 4.3%. In this system too, N-acetyl-procaimamide had no effect. By contrast, adding PA only to the co-culture of Con A-stimulated cells with fresh PBM had a less marked effect on suppressor cell function. These results indicate that the major action of PA is to inhibit the generation of suppressor T-cell activity. Such an effect may explain the capacity of this agent to induce autoantibody formation in treated individuals.

Authors

T Ochi, E A Goldings, P E Lipsky, M Ziff

Regulation of immunoglobulin E antibody synthesis in man by antiidiotypic antibodies.

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The effect of rabbit antiidiotypic antibody raised against human IgG F(ab')2 anti-tetanus toxoid (TT) antibodies on the in vitro synthesis of TT-specific IgE antibody by peripheral blood lymphocytes (PBL) was examined in three subjects. Two of these subjects were allergic twins whose sera persistently contained IgE anti-TT antibodies. The third subject was a nonallergic individual who had a slightly elevated serum IgE (250 IU/ml) and who exhibited a transient serum IgE anti-TT response after booster immunization with TT. After appropriate absorptions rabbit anti-idiotype (Id) IgG reacted with anti-TT antibodies of both IgG and IgE isotypes in an idiotype- and antigen-specific fashion. PBL and B cells from the three subjects studied spontaneously synthesized TT-specific IgE in culture. In all three cases, adsorption of B cells over plastic plates coated with anti-Id before culture specifically decreased the synthesis of IgE antibodies to TT but did not affect the synthesis of IgE antibodies to ragweed antigen E by PBL from the twin allergic subjects. Addition of anti-Id to cultures of PBL from all three subjects specifically inhibited the synthesis of TT-specific IgE. This inhibition was shown to be exerted both at the level of the B cells and via the generation of antigen-specific suppressor T cells from radiosensitive precursors. The present results indicate that the synthesis of antigen-specific IgE in man is subject to regulation by idiotypic anti-idiotypic interactions that can involve both B and T lymphocytes.

Authors

R S Geha, M Comunale

Effect of carbonic anhydrase inhibition on superficial and deep nephron bicarbonate reabsorption in the rat.

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Abstract

The nephron segment responsible for the acetazolamide-insensitive fraction of renal bicarbonate reabsorption has not been clearly delineated. This study compares superficial and deep nephron bicarbonate reabsorption before and after acetazolamide at two dose levels (20 and 50 mg/kg per h) in mutant Munich-Wistar rats employing both cortical and papillary micropuncture and microcalorimetry. Systemic acid-base balance and right whole kidney glomerular filtration rate were similar in all groups examined. The effects of the two doses of acetazolamide were indistinguishable and resulted in a significant increase in whole kidney bicarbonate excretion that compared favorably with the fraction delivered out of the left papillary tip. Acetazolamide inhibited superficial proximal bicarbonate reabsorption by 80.0%, whereas reabsorption up to the deep loop of Henle was decreased by only 52% (P less than 0.001). Bicarbonate reabsorption that was insensitive to acetazolamide occurred in the superficial and deep loop of Henle and between the distal tubule and base collecting duct. Because water reabsorption in these segments could serve to generate transepithelial bicarbonate concentration gradients favorable for reabsorption, we attempted to minimize water abstraction by combined administration of mannitol and acetazolamide. During this condition a significant increase in bicarbonate delivery up to the deep loop of Henle was noted (52 vs. 65%), whereas superficial nephron reabsorption was not altered. Furthermore, an outwardly directed bicarbonate concentration gradient from the deep loop of Henle to vasa recta was demonstrated during acetazolamide (delta tCO2 = 20.9 +/- 3.3 mM), but was abolished during combined mannitol and acetazolamide administration (delta tCO2 = 3.5 +/- 0.9 mM). It is concluded that carbonic anhydrase inhibition results in a disparate effect on nephron bicarbonate reabsorption when juxtamedullary and superficial nephron segments are compared. Our findings suggest that a mechanism for residual bicarbonate reabsorption during acetazolamide administration may be passive reabsorption driven by favorable transepithelial concentration gradients.

Authors

T D DuBose Jr, M S Lucci

Species-related innate resistance to Schistosoma mansoni. Role of mononuclear phagocytes in schistosomula killing in vitro.

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Abstract

Resistance to infection with the multicellular parasite Schistosoma mansoni has been previously demonstrated to vary among several host species. The current investigation was designed to examine the basis for this species-related resistance in vitro. Adherent peritoneal macrophages or peripheral blood mononuclear cells from several species of host animals were incubated with S. mansoni schistosomula for 18-24 h; parasite viability was then assayed by methylene blue exclusion. Peritoneal exudate macrophages from susceptible species, such as mice (C57Bl/6) and hamsters killed, respectively, 6.6 +/- 2 and 8.0 +/- 2% of incubated schistosomula. In contrast, cells from resistant species: rats, guinea pigs, and rabbits, killed 21 +/- 2.3, 15 +/- 4.6, and 17 +/- 5.5%, respectively. Furthermore, blood monocytes from rabbits resulted in a mean of 25.9 +/- 2.8% dead organisms. Schistosomula killing by mononuclear phagocytes obtained from resistant species (rats or rabbits) was dependent on the cell/parasite ratio. Significant schistosomula mortality resulted from culture supernatants of rat macrophages or rabbit monocytes. Killing by cells from both species was significantly reduced upon addition of L-arginine, while catalase reduced killing only by rat macrophages. We conclude that mononuclear phagocytes may play a key role in species-related innate resistance to schistosomiasis; their in vitro schistosomulicidal activity parallels the known in vivo susceptibility of the donor species. Killing is mediated by lysosomal enzymes (arginase) and by products of oxidative metabolism, the predominant mechanism depends on the specific animal species.

Authors

C A Peck, M D Carpenter, A A Mahmoud

Hepatic metabolism of 3 alpha-hydroxy-5 beta-etianic acid (3 alpha-hydroxy-5 beta-androstan-17 beta-carboxylic acid) in the adult rat.

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Abstract

Normal human meconium has been shown to contain short-chain (C20-C22) bile acids and, recently, these compounds have been identified in sera of patients with cholestasis. This suggests that shortchain bile acids may be secreted in bile. We have examined this point by studying the hepatic metabolism and biliary secretion of one naturally occurring C20 bile acid, 3 alpha-hydroxy-5 beta-etianic acid (3 alpha-hydroxy-5 beta-androstan-17 beta-carboxylic acid). [3-3H]-3 alpha-hydroxy-5 beta-etianic acid was prepared and administered intravenously to rats prepared with an external biliary fistula. 85.5 +/- 1.2% of the administered dose was recovered in bile over 20 h with 71.5 +/- 1.3% appearing in the first hour. 11.9 +/- 1.6% of the dose was estimated to be distributed in body water and 0.6 +/- 0.2% was recovered as organic matter in urine. Total recovery of label was 98.0 +/- 2.6%. Administration of milligram quantities of 3 alpha-hydroxy-5 beta-etianic acid produced an increase in bile flow (58.9 +/- 7.1% over basal levels) within 20 min after injection of the steroid. The radiolabeled material in bile was shown by thin-layer chromatography (TLC) to be a polar conjugate which, after beta-glucuronidase hydrolysis, cochromatographed with authentic free 3 alpha-hydroxy-5 beta-etianic acid. After purification, and derivatization, the steroid moiety was proven by gas chromatography-mass spectrometry to be identical to 3 alpha-hydroxy-5 beta-etianic acid. Characterization of the conjugate by TLC and by 3 alpha-hydroxysteroid dehydrogenase assay, before and after beta-glucuronidase hydrolysis, indicated that the steroid was secreted in bile as the 3-O-beta-glucuronide. It is concluded that 3 alpha-hydroxy-5 beta-etianic acid is cleared from the plasma, conjugated with glucuronic acid, and secreted into bile rapidly and in high concentration. The choleretic properties of this shortchain bile acid contrast with the cholestatic effects of lithocholic acid, its C24 analog. Both the form of conjugation of etianic acid and its effect on bile flow suggest that the shortened side chain of this steroid markedly alters its hepatic metabolism and physiology.

Authors

J M Little, J St Pyrek, R Lester

Metabolic and cellular alterations underlying the exaggerated renal prostaglandin and thromboxane synthesis in ureter obstruction in rabbits. Inflammatory response involving fibroblasts and mononuclear cells.

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Abstract

Unilateral ureter obstruction in rabbits produced profound changes in endogenous and exogenous renal arachidonic acid metabolism. Isolated perfused hydronephrotic kidneys (removed after 3 or 10 d of ureter obstruction) responded to bradykinin stimulation with a markedly enhanced release of prostaglandin E2 and thromboxane A2. Reversal (3 or 10 d) of the ureter obstruction resulted in a reduction in the vasoactive peptide-induced release of prostaglandin E2 and thromboxane A2 from the perfused hydronephrotic kidney. However, postobstruction reversal of prostaglandin production by the agonist-stimulated perfused kidney was not reflected in the cortical microsomal cyclooxygenase activity, which is greatly enhanced during ureter obstruction and does not decrease after removal of the obstruction. Histological analysis of the renal cortex in rabbits with ureteral obstruction revealed a proliferation of fibroblast-like cells and the presence of mononuclear cells; removal of the obstruction did not result in a disappearance of cortical fibroblasts but did result in a decrease of monocytes. The critical involvement of mononuclear cells in the exaggerated arachidonate metabolism that occurs during hydronephrosis was exhibited by the demonstration that: (a) only the perfused hydronephrotic rabbit kidney responded to administration of endotoxin with a sustained release of prostaglandin E2 and thromboxane A2; (b) the contralateral rabbit kidney, which is devoid of mononuclear cells, did not respond to endotoxin; and (c) the hydronephrotic cat kidney, which exhibits a fibroblast proliferation with a low number of mononuclear cells, did not respond to endotoxin. Thus, proliferation of fibroblast-like cells and the presence of mononuclear cells appear to be involved in the exaggerated prostaglandin and thromboxane production underlying hydronephrosis. The increase in microsomal cyclooxygenase activity is apparently most closely correlated with the increased fibroblastic activation and cellularity, whereas mononuclear cells (possibly via monokines) seem to be critical for the markedly enhanced prostaglandin and thromboxane release induced by endotoxin and bradykinin.

Authors

T Okegawa, P E Jonas, K DeSchryver, A Kawasaki, P Needleman

Role for intrarenal mechanisms in the impaired salt excretion of experimental nephrotic syndrome.

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Abstract

A unilateral model of puromycin aminonucleoside (PAN)-induced albuminuria was produced in Munich-Wistar rats to examine the mechanisms responsible for renal salt retention. 2 wk after selective perfusion of left kidneys with PAN (n = 8 rats) or isotonic saline (control, n = 7 rats), increases in albumin excretion and decreases in sodium excretion were demonstrated in PAN-perfused but not in nonperfused kidneys of PAN-treated rats although systemic plasma protein concentration remained at control level. Total kidney glomerular filtration rate (GFR) and superficial single nephron (SN) GFR were also reduced selectively in PAN-perfused kidneys, on average by approximately 30%, due primarily to a marked decline in the glomerular capillary ultrafiltration coefficient (Kf), which was also confined to PAN-perfused kidneys. Values for absolute proximal reabsorption (APR) were also selectively depressed in PAN-perfused kidneys, in keeping with a similarly selective decline in peritubular capillary oncotic pressure measured in these kidneys, the latter also a consequence of the fall in Kf. In a separate group of seven PAN-treated rats, however, no differences were detected between PAN-perfused and nonperfused kidneys in the absolute amount of sodium reaching the early (0.77 +/- 0.09 neq/min vs. 0.74 +/- 0.08, P greater than 0.40) and late portions of superficial distal tubules (0.31 +/- 0.02) neq/min vs. 0.32 +/- 0.05, P greater than 0.50), despite the lesser filtered load of sodium in PAN-perfused kidneys. Suppressed sodium reabsorption in both proximal convoluted tubules and short loops of Henle of PAN-perfused kidneys contributed to this equalization of sodium delivery rates to the late distal tubule, as did comparable reabsorption along distal convolutions. In two additional groups of PAN-treated rats, infusion of saralasin (0.3 mg/kg per h, i.v.) led to substantial increases in total kidney GFR and SNGFR in PAN-perfused but not in nonperfused kidneys. Despite these increases in total and SNGFR, urinary sodium excretion by PAN-perfused kidneys remained at a level far below that for nonperfused kidneys, again indicating that the antinatriuresis characterizing the PAN-perfused kidney is due to alterations in sodium handling by the tubules rather than changes in GFR. These results therefore indicate (a) that reductions in Kf and depressed sodium reabsorption by proximal tubules and Henle's loop segments in this model are brought about by intrarenal rather than circulating or systemic factors, and (b) assuming that superficial nephrons are representative of the entire nephron population, renal salt retention in this model is due primarily to intrarenal factor(s) acting beyond the distal convolution.

Authors

I Ichikawa, H G Rennke, J R Hoyer, K F Badr, N Schor, J L Troy, C P Lechene, B M Brenner

Hemodynamic sequelae of regression of experimental atherosclerosis.

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Regression of experimental atherosclerosis is characterized by decreased intimal thickness and luminal enlargement, but intimal fibrosis becomes more dense. We tested the hypothesis that fibrosis of arteries during regression might limit vasodilator capacity and restrict hemodynamic improvement despite luminal improvement. We studied limb, coronary, and cerebral hemodynamics in 11 normal cynomolgus monkeys, 10 monkeys given an atherogenic diet for 20 mo and 8 monkeys given a regression diet for an additional 18 mo. The atherogenic diet induced lesions of moderate severity (50-60% stenosis); owing to characteristic vessel growth during the atherogenic period, luminal size did not decrease correspondingly. Regression monkeys showed typical changes of regression with luminal enlargement but increased fibrosis. The iliac artery was perfused at constant blood flow and maximal vasodilatation was produced with papaverine. Blood flow was measured with microspheres during maximal vasodilatation in the coronary bed (adenosine) and cerebral bed (hypercapnia). In normal monkeys, minimal vascular resistances were 1.95 +/- 0.19 mm Hg/ml/min X 100 g (mean +/- SE) (limb), 0.13 +/- 0.01 (coronary), and 0.44 +/- 0.02 (cerebral). In atherosclerotic monkeys minimal resistance increased (P less than 0.05) 108, 62, and 166% in the limb, coronary, and cerebral beds, respectively. In regression monkeys, minimal resistance increased from values found in atherosclerotic animals in the limb (+22%), decreased inconsistently in the coronary bed (-19%), and decreased significantly in the cerebral bed (-44%, P less than 0.05). Thus morphologic regression was accompanied by significant hemodynamic improvement during maximal dilatation only in cerebral vessels. We conclude that increases in luminal size during regression of atherosclerotic lesions may not be associated with increases in vasodilator capacity, as intimal fibrosis may limit physiologically important hemodynamic improvement.

Authors

M L Armstrong, D D Heistad, M L Marcus, D J Piegors, F M Abboud

Human eosinophil arylsulfatase B. Structure and activity of the purified tetrameric lysosomal hydrolase.

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Arylsulfatase B from human eosinophils was purified free of contaminating proteins by gel filtration and sequential affinity chromatography on Affi-Gel Blue and zinc chelate Sepharose. 50 micrograms of the purified enzyme presented as a single stained band on alkaline disc gel electrophoresis. In both goats and rabbits, the purified enzyme elicited monospecific antisera that yielded single precipitation arcs on Ouchterlony analysis with a human eosinophil extract and the purified enzyme; the immunoprecipitation lines fused in a pattern of identity, providing immunochemical evidence for the homogeneity of the purified enzyme. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a dominant lower molecular weight protein and three other bands with molecular weights approximately two, three, and four times that of the major protein band were resolved. The prominence of the less rapidly migrating protein bands increased relative to the major band if the enzyme was maintained under acidic conditions or was reacted with the cross-linking agent dimethyl suberimidate under alkaline conditions before SDS-polyacrylamide gel electrophoresis, supporting the conclusion that the enzyme consists of four subunits. Two stained bands were present on acid disc gel electrophoresis; they were composed of oligomeric forms of enzyme on analysis by SDS-polyacrylamide gel electrophoresis in a second dimension. A minimum molecular weight of 70,190 was determined from amino acid composition analysis for the tetrameric form of the enzyme. The specific functional activity of the purified arylsulfatase B was concentration and time dependent, compatible with its association or dissociation into subunit forms with differing specific activities. Factors that govern subunit interactions of arylsulfatase B, including local enzyme concentration and pH, provide mechanisms for regulating the enzymatic activity of this lysosomal hydrolase.

Authors

P F Weller, K F Austen

Beta endorphin selectively stimulates aldosterone secretion in hypophysectomized, nephrectomized dogs.

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Abstract

We examined the effects of synthetic human beta-endorphin (beta END) and a stable methionine (Met)-enkephalin analogue on aldosterone and cortisol secretion rates in anesthetized, hypophysectomized, and nephrectomized dogs and compared them to those of (1-39) ACTH. The circulation of the adrenal glands was completely isolated on the arterial and venous sides (Hilton Pouch). The peptides were infused to deliver 3 pmol/min into the aortic "pouch." Blood was collected from the vena caval pouch, which received blood only from the adrenal gland. Secretion rates of aldosterone and cortisol were calculated as the product of adrenal blood flow and venous steroid concentration. Duplicate steroid measurements were obtained during a control period, at 10, 30, and 50 min of peptide infusion and during a postcontrol period. BetaEND increased aldosterone secretion rate from 2.4 +/- 0.5 ng/min (mean +/- SEM) to 3.2 +/- 0.9 ng/ min at 10 min (N.S.), 8.2 +/- 2.5 ng/min (P less than 0.05) at 30 min and 11.0 +/- 3.7 ng/ min (P less than 0.05) at 50 min of infusion. Cortisol secretion rate was not affected by infusion of betaEND. Infusion of the stable Met-enkephalin analogue D-alanine2; Metphenylalanine4, Met(O)-enkephalin-ol or saline alone had no effect on aldosterone or cortisol secretion rates. ACTH infusion increased mean aldosterone secretion rate by approximately 215% and significantly stimulated cortisol secretion rate. These results indicate that beta END selectively stimulates aldosterone secretion with a potency similar to that of an equimolar dose of ACTH.

Authors

H G Güllner, J R Gill Jr

Detection and partial characterization of antibacterial factor(s) in alveolar lining material of rats.

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Abstract

Intracellular killing of Staphylococcus aureus by alveolar macrophages is known to be enhanced by exposure to alveolar lining material. Because this material may have a role in pulmonary host defenses, we have studied its effect on pneumococci and other nonstaphylococcal organisms. Alveolar lining material from rats caused rapid killing and lysis of pneumococci. The antipneumococcal activity was localized to the surfactant-containing fraction of the fluid and was not affected by trypsin. Phospholipid extracts of the surfactant fraction or purified lamellar bodies killed pneumococci. Lysis of pneumococci by the surfactant fraction appeared to be mediated by a detergent-like activation of pneumococcal autolysin, in that bacteriolysis was prevented by substitution of ethanolamine for choline in pneumococcal cell walls, and a pneumococcal transformant that lacked autolysin was not lysed. The surfactant fraction readily killed pneumococci containing ethanolamine or the autolysin-defective transformant, and studies with tritiated methyl-D-glucose loading and release showed that killing was associated with increased bacterial cell membrane permeability. Bactericidal activity (without lysis) was observed with several nonpneumococcal gram-positive bacteria, including Streptococcus viridans, unspeciated respiratory streptococci, Streptococcus pyogenes, Streptococcus bovis, and Bacillus species. Purified diacylphospholipids had no antibacterial activity, however, a lysophospholipid, palmitoyl lysophosphatidylcholine, had many properties resembling the surfactant-containing fraction of lavage, including autolysin-mediated pneumococcal lysis, altered cell membrane permeability, and antibacterial activity against several gram-positive bacteria.

Authors

J D Coonrod, K Yoneda

Role of the 26-hydroxylase in the biosynthesis of bile acids in the normal state and in cerebrotendinous xanthomatosis. An in vivo study.

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Abstract

On the basis of different in vitro studies, we have previously suggested that the basic metabolic defect in the rare inherited disease cerebrotendinous xanthomatosis (CTX) is a lack of a hepatic mitochondrial C27-steroid 26-hydroxylase, involved in the normal biosynthesis of bile acids (1980. J. Clin. Invest. 65: 1418-1430; 1981. J. Lipid Res. 22: 191-200; 22: 632-640). In the present work, this hypothesis was tested in vivo. One patient with CTX and two control subjects received intravenously a mixture of [4-14C]7 alpha-hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha,26-dihydroxy-4-cholesten-3-one, steroids believed to be important precursors of chenodeoxycholic acid. The ratio between 14C and 3H in cholic acid and chenodeoxycholic acid isolated from bile of the CTX-patient was approximately 1/40 and 1/60 of those of the control subjects, respectively. Another patient with CTX and one control subject received a mixture of [4-14C]5 beta-cholestane-3 alpha,7 alpha-diol and [1,2-3H]5 beta-cholestane-3 alpha,7 alpha,26-triol, both possible precursors to chenodeoxycholic acid. In this case the 14C/3H ratio in cholic acid and chenodeoxycholic acid from the patient with CTX was 1/10 and 1/15, respectively, compared with that of the control subject. The most likely explanation for these findings is that very little of the 14C-precursors, i.e. without a 26-hydroxyl group, can be converted into cholic acid and chenodeoxycholic acid because of a defect of the 26-hydroxylase step. The results obtained are in accord with our previous findings in vitro. The results further underline the importance of the 26-hydroxylase pathway in the normal biosynthesis of cholic acid and chenodeoxycholic acid in man.

Authors

I Björkhem, O Fausa, G Hopen, H Oftebro, J I Pedersen, S Skrede

Inactivation of kallikrein in human plasma.

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Abstract

Human plasma kallikrein is inactivated by plasma protease inhibitors. This study was designed to determine the nature of these protease inhibitors and to assess their relative importance in the inactivation of kallikrein. Therefore, the kinetics of kallikrein inactivation and the formation of kallikrein inhibitor complexes were studied in normal plasma and in plasma depleted of either alpha 2-macroglobulin (alpha 2M), C1 inhibitor, or antithrombin (AT III). Prekallikrein was activated by incubation of plasma with dextran sulfate at 4 degrees C. After maximal activation, kallikrein was inactivated at 37 degrees C. Inhibition of kallikrein amidolytic activity in AT III-deficient plasma closely paralleled the inactivation rate of kallikrein in normal plasma. The inactivation rate of kallikrein in alpha 2M-deficient plasma was slightly decreased compared with normal plasma, but in contrast to normal, C1 inhibitor-deficient, and AT III-deficient plasma, no kallikrein amidolytic activity remained after inactivation that was resistant to inhibition by soybean trypsin inhibitor. Suppression of kallikrein activity in C1 inhibitor-deficient plasma was markedly decreased, and this was even more pronounced in plasma deficient in both C1 inhibitor and alpha 2M. The pseudo first-order rate constants for kallikrein inactivation in normal, AT III-deficient, alpha 2M-deficient, C1 inhibitor-deficient plasma, and plasma deficient in both alpha 2M and C1 inhibitor, were 0.68, 0.60, 0.43, 0.07, and 0.016 min-1, respectively. Sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis showed that during inactivation of kallikrein in plasma, high-Mr complexes were formed with Mr at 400,000-1,000,000, 185,000, and 125,000-135,000, which were identified as complexes of 125I-kallikrein with alpha 2M, C1 inhibitor, and AT III, respectively. In addition, the presence of an unidentified kallikrein-inhibitor complex was observed in AT III-deficient plasma. 52% of the 125I-kallikrein was associated with C1-inhibitor, 35% with alpha 2M, and 13% with AT III and another protease inhibitor. A similar distribution of 125I-kallikrein was observed when the 125I-kallikrein inhibitor complexes were removed from plasma by immunoadsorption with insolubilized anti-C1 inhibitor, anti-alpha 2M, or anti-AT III antibodies. These results suggest that only covalent complexes are formed between kallikrein and its inhibitors in plasma. As a function of time, 125I-kallikrein formed complexes with C1 inhibitor at a higher rate than with alpha 2M. No difference was observed between the inactivation rate of kallikrein in high-Mr kininogen-deficient plasma and that in high-Mr kininogen-deficient plasma reconstituted with high-Mr kininogen; this suggests that high-Mr kininogen does not protect kallikrein from inactivation in the plasma milieu. These results have quantitatively demonstrated the major roles of C1 inhibitor and alpha 2M in the inactivation of kallikrein in plasma.

Authors

F van der Graaf, J A Koedam, B N Bouma

Reversal of shortened platelet survival in rats by the antifibrinolytic agent, epsilon aminocaproic acid.

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Abstract

Platelet survival in rabbits and rats is shortened by placing indwelling catheters in the aorta; this shortening appears to be at least partly related to the extent of vessel wall injury and platelet interaction with the repeatedly damaged wall. Treatment of rabbit platelets with plasmin and other proteolytic enzymes in vitro shortens their survival when they are returned to the circulation. Because platelets may be exposed to plasmin and other proteolytic enzymes in rabbits and rats with indwelling aortic catheters, we examined the effect of epsilon-aminocaproic acid (EACA) on platelet survival in rats. At a dose of 1 g/kg every 4 h, EACA significantly reduced whole blood fibrinolytic activity and prolonged the shortened platelet survival in rats with indwelling aortic catheters. Mean platelet survival for untreated rats with indwelling aortic catheters was 38.6 +/- 1.9 h, and for rats treated with EACA, 53.8 +/- 3.8 h. Scanning electron microscopy showed that the injured vessel wall of these animals was mainly covered with platelets and fibrin, whereas in control animals that did not receive EACA, the injured surface was mainly covered with platelets and little fibrin was observed. Thus shortened platelet survival during continuous vessel wall injury may result from the local generation of plasmin or the release of proteolytic enzymes at sites where platelets (and possibly leukocytes) interact with the vessel wall.

Authors

P D Winocour, R L Kinlough-Rathbone, M Richardson, J F Mustard

Cyclosporin A-treated guinea pig responder cells secrete a genetically restricted factor that suppresses the mixed leukocyte reaction.

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Abstract

Cyclosporin A (CY A) is a hydrophobic, undecapeptide, fungal metabolite with potent immunosuppressive effects on T lymphocyte-mediated immune responses. Suppressor T lymphocytes generated during a mixed leukocyte reaction (MLR) performed in the presence of CY A, release a factor that suppresses a primary MLR of responder T cells, which is derived from the same strain as the factor producer but lacks specificity for the stimulator cell. These results suggest a finely regulated pathway by which CY A may induce and maintain a permanent state of transplantation tolerance in vivo.

Authors

G A DosReis, E M Shevach

In vitro culture of cells exfoliated in the urine by patients with diabetes mellitus.

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Abstract

As an approach to facilitate the understanding of the progression of diabetic renal disease, we assessed the urine of diabetic patients and normal volunteers for the presence of cells that could be cultured in vitro. The results suggest that both normal control subjects and diabetic patients, without clinically detectable microangiopathy, exfoliate few culturable cells into the urine. In contrast, diabetics with documented retinopathy but without nephropathy exfoliate substantially higher numbers of culturable cells (5.2 cells/100 ml urine), whereas diabetics with both retinopathy and advanced nephropathy exfoliate even greater numbers of culturable cells (50.8 cells/100 ml urine). The cells that are exfoliated and culturable can be divided into five distinct cell types based on morphology at the light microscope level. The exfoliated cells proliferate at clonal density after isolation from urine and are epithelial in appearance. These data suggest that the culture of cells from urine might have diagnostic value as an early indicator of diabetic renal disease and provide a convenient, noninvasive new source of human kidney epithelial cells.

Authors

C J Detrisac, R K Mayfield, J A Colwell, A J Garvin, D A Sens