B W O'Malley
Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune blistering diseases in which antibodies develop to the cell surface of epidermal cells. In this study we sought to determine the antigenic specificity of antibodies in the sera of patients with PV and PF. Sera from 12 patients with PV were used to immunoprecipitate extracts of cultured human epidermal cells that were radiolabeled with 14C-amino acids. Immunoprecipitates were identified by SDS polyacrylamide gel electrophoresis (PAGE) and fluorography. All 12 PV sera precipitated a protein which, when reduced, displayed chains of 130,000 and 80,000 mol wt on SDS-PAGE. Electrophoresis under nonreducing conditions identified a 210,000-mol wt molecule, which was presumably formed by disulfide crosslinking of the 130,000 and 80,000-mol wt chains. Immunoprecipitates of epidermal cell extracts that were labeled with 14C-glucosamine indicated that the 130,000-mol wt chain. Seven of eight PF sera, which were run concurrently with the PV sera in this immunoprecipitation assay, did not precipitate this glycoprotein, nor did they specifically precipitate any protein. To determine if a specific molecule which reacted with antibodies in PF sera could be identified, we used immunoblot analysis of extracts of normal human epidermis. The proteins in these extracts were reduced, separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose sheets or to 2-aminophenylthioether paper. Immunoperoxidase staining of the transferred proteins with PF sera indicated that four of eight PF sera contained antibodies that stained a protein band of 160,000 mol wt. Indirect immunofluorescence, using normal human skin as the substrate, indicated that IgG that was eluted from this protein band stained the epidermis in a cell surface pattern. PV sera did not specifically recognize any bands by immunoblot analysis. Immunoblots performed with PV antigen that was immunoprecipitated from cell culture extracts suggested that, once denatured for SDS-PAGE, PV antigen is no longer immunoreactive. Taken together, these data indicate that: autoantibodies contained in PV sera from various patients have a unique molecular specificity; autoantibodies from most PF sera have a specificity different from that of PV autoantibodies; and autoantibodies from various PF patients may not have identical antigenic specificities. These differences in antigenic specificity between PV and PF sera may account for the clinical and histologic differences between these diseases.
J R Stanley, L Koulu, C Thivolet
The physiological relationship of increased circulating angiotensin II and vasopressin to circulatory changes during combined hypoxemia and hypercapnic acidosis is unclear. To evaluate the role(s) of angiotensin II and vasopressin, seven unanesthetized female mongrel dogs with controlled sodium intake (80 meq/24 h X 4 d) were studied during 40 min of combined acute hypoxemia and hypercapnic acidosis (PaO2, 36 +/- 1 mmHg; PaCO2, 55 +/- 2 mmHg; pH = 7.16 +/- 0.04) under the following conditions: (a) intact state with infusion of vehicles alone; (b) beta-adrenergic blockade with infusion of d,l-propranolol (1.0 mg/kg bolus, 0.5 mg/kg per h); of the vasopressin pressor antagonist d-(CH2)5Tyr(methyl)arginine-vasopressin (10 micrograms/kg); and (d) simultaneous vasopressin pressor and angiotensin II inhibition with the additional infusion of 1-sarcosine, 8-alanine angiotensin II (2.0 micrograms/kg per min). The rise in mean arterial pressure during the combined blood-gas derangement with vehicles appeared to be related to increased cardiac output, since total peripheral resistance fell. Beta-adrenergic blockade abolished the fall in total peripheral resistance and diminished the rise in cardiac output during combined hypoxemia and hypercapnic acidosis, but the systemic pressor response was unchanged. In addition, the rise in mean arterial pressure during the combined blood-gas derangement was unaltered with vasopressin pressor antagonism alone. In contrast, the simultaneous administration of the vasopressin pressor and angiotensin II inhibitors during combined hypoxemia and hypercapnic acidosis resulted in the abrogation of the overall systemic pressor response despite increased cardiac output, owing to a more pronounced fall in total peripheral resistance. Circulating catecholamines were increased during the combined blood-gas derangement with vasopressin pressor and angiotensin II blockade, suggesting that the abolition of the systemic pressor response in the last 30 min of combined hypoxemia and hypercapnic acidosis was not related to diminished activity of the sympathetic nervous system. These studies show that vasopressin and angiotensin II are major contributors to the systemic pressor response during combined acute hypoxemia and hypercapnic acidosis.
C E Rose Jr, R L Godine Jr, K Y Rose, R J Anderson, R M Carey
The expression of B cell associated and restricted antigens on tumor cells isolated from 138 patients with non-T cell acute lymphoblastic leukemia (non-T cell ALL) was investigated by flow cytometric analysis by means of a panel of monoclonal antibodies. Tumor cells from these patients could be assigned to one of four subgroups: human leukocyte antigen-DR-related Ia-like antigens (Ia) alone (4%, stage I); IaB4 (14%, stage II); IaB4CALLA (33%, stage III); and IaB4CALLAB1 (49%, stage IV). The expression of B cell-restricted antigens (B4 and B1) and rearrangements of Ig heavy chain genes provided strong evidence for the B cell lineage of stages II, III, and IV tumors. The lineage of the Ia alone group is still unknown. The B4 antigen was expressed on approximately 95% of all non-T cell ALLs tested, and given its absence on T cell and myeloid tumors, it appears to be an exceptional marker to define cells of B lineage. The demonstration that Ia alone, IaB4, IaB4CALLA, and IaB4CALLAB1 positive cells can be readily identified by dual fluorescence analysis in normal fetal and adult bone marrow provided critical support for the view that these leukemic pre-B cell phenotypes were representative of the stages of normal pre-B cell differentiation. It was interesting that the IaB4+ cell was more frequently identified in fetal bone marrow than in adult marrow, whereas the predominant cell found in adult marrow expressed the IaB4CALLAB1 phenotype. These data suggest that the leukemogenic event may be random, since the predominant pre-B cell leukemic phenotype appears to correspond to the normal pre-B cell phenotype present in these hematopoietic organs. Our observations provide an additional distinction between adult and childhood ALL, since these studies show that most non-T cell ALLs seen in children less than 2 yr old are of stage II phenotype, whereas the majority of non-T ALLs in adults are of stage IV phenotype. Finally, it should be noted that the present study suggests that the analysis of leukemic B cell phenotypes and their normal counterparts can provide a mechanism for the investigation and orderly definition of stages of pre-B cell differentiation in man.
L M Nadler, S J Korsmeyer, K C Anderson, A W Boyd, B Slaughenhoupt, E Park, J Jensen, F Coral, R J Mayer, S E Sallan
We have examined the role of heparinlike molecules in the regulation of coagulation by perfusing rat hindquarters with purified human thrombin and with its plasma inhibitor, antithrombin. Our data indicate that contact of the hemostatic components with the endothelium enhances the rate of thrombin-antithrombin complex formation by as much as 19-fold over the uncatalyzed rate of enzyme-inhibitor interaction. Heparinlike molecules are responsible for the antithrombin accelerating activity. The amount of thrombin-antithrombin complex generated within the hindlimb preparation after pretreatment of the vasculature with purified Flavobacterium heparinase or with addition of platelet Factor IV to the hemostatic components, was equal to the uncatalyzed levels. These heparinlike molecules appear to be tightly bound to the luminal surface of the endothelium, since they could not be detected within the physiologic buffer that was perfused through the animal. The above mucopolysaccharides function in a manner similar to commercial heparin, since modification of antithrombin at a site critical for heparin-dependent acceleration of the protease inhibitor resulted in a level of interaction product identical to the uncatalyzed amount. Finally, addition of diisofluorophosphate-thrombin to the enzyme perfusion stream reduced the amount of thrombin-antithrombin complex formed in the animal by 30-40%, which suggested that thrombin bound to the endothelium as well as enzyme free in solution are accessible to antithrombin that has interacted with heparinlike molecules present on the endothelium.
J A Marcum, J B McKenney, R D Rosenberg
Parabiont rats were used to study the regulation of intestinal cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A [HMG-CoA] reductase activity) and esterification (acylcoenzyme A/cholesterol acyltransferase [ACAT] activity) by lipoproteins and micellar cholesterol. The parabiont rat model offers a unique way to observe changes in cholesterol metabolism in the intestine, independently of luminal factors. In one group of six surgically joined pairs, one animal was fed rat chow and 0.1% propylthiouracil (PTU). Its joined mate was fed rat chow, 0.1% PTU, 5% lard, and 0.3% taurocholic acid. In another group of five pairs, one rat was fed rat chow, 0.1% PTU, 5% lard, 0.3% taurocholic acid, plus 1% cholesterol. Its joined mate was fed the same diet except the cholesterol was deleted. Serum cholesterol changes were equal between members of a given pair, attesting to their common circulation. The administration of PTU itself caused a significant elevation of serum cholesterol. When one parabiont ingested cholesterol, serum cholesterol concentrations increased significantly for both pair members compared with control pairs not ingesting cholesterol. Hepatic and intestinal HMG-CoA reductase activities were significantly decreased in rats fed the diet containing cholesterol. ACAT activities in both organs were markedly increased. This supports previous data that suggest that dietary or luminal cholesterol affects both HMG-CoA reductase and ACAT activity in the small intestine. Moreover, in rats that were hypercholesterolemic but not ingesting dietary cholesterol, hepatic and intestinal reductase activities were decreased and ACAT activities were increased compared with the control animals. Intestinal microsomal cholesterol content was increased only in rats fed cholesterol. No changes in intestinal microsomal cholesterol were observed in the other animals. The data suggest that intestinal HMG-CoA reductase and ACAT activities are regulated by plasma lipoproteins independently of luminal factors. This nonluminal regulatory effect occurs without a measurable contribution to the intestinal microsomal cholesterol pool.
B H Purdy, F J Field
Mouse IgG and IgA, with reactivity to dinitrophenol conjugated to carrier protein, have been isolated from myeloma proteins by means of a variety of affinity techniques. The IgA was predominantly in the dimeric form. The in vitro and in vivo biological activities of IgA-containing immune complexes were assessed in the rat. IgA-containing immune complexes were demonstrated, in a dose-dependent manner in vitro, to activate neutrophils and to generate O.-2. In addition, these immune complexes showed evidence of complement activation in vitro, by the use of immunofixation techniques. When IgA was instilled into the airways of rats and antigen was injected intravenously, acute lung injury occurred, as reflected by increases in lung permeability and morphological changes consisting of blebbing of endothelial cells, intra-alveolar hemorrhage, and fibrin deposition. The lung changes were directly proportional to the amount of IgA instilled into the airways and failed to occur if intravenous injection of antigen was omitted. Lung injury did not occur in animals that received an intravenous injection of antigen in the absence of an airway injection of IgA. Lung injury related to IgA-containing immune complexes was complement dependent but neutrophil independent. In companion studies with mouse IgG-containing immune complexes, acute lung injury also occurred and had morphological features similar to those associated with IgA-induced lung injury except that, in the case of IgG immune complex-induced damage, neutrophils were more evident. Acute lung injury induced by IgG-containing immune complexes, whether of mouse or rabbit origin, was complement and neutrophil dependent. The similarities and differences between IgG- and IgA-associated acute immune complex-induced injury of rat lung were reinforced by the use of morphometry techniques. Studies with another monoclonal IgA antibody-containing antigen-binding activity to phosphorylcholine also demonstrated the ability of IgA antibody to cause acute lung injury in the rat. Neither antibody alone nor antigen (phosphorylcholine linked to bovine serum albumin) alone produced evidence of lung injury. These studies indicate for the first time that immune complexes containing IgA have lung-damaging properties and that the pathogenic mechanisms are different from those associated with IgG-associated immune complex-induced acute lung injury.
K J Johnson, B S Wilson, G O Till, P A Ward
The purpose of this study was to assess the effects of combined hypoxia and hypercapnia and of severe asphyxia on lung water balance and protein transport in newborn lambs. We studied ten 2-4-wk-old anesthetized lambs which were mechanically ventilated first with air for 2-3 h, then with 10-12% oxygen in nitrogen for 2-4 h, and then with 10-12% oxygen and 10-12% carbon dioxide in nitrogen for 2-4 h. Next we stopped their breathing for 1-2 min to produce severe asphyxia, after which we followed their recovery in air for 2-4 h. In 5 of the 10 lambs we intravenously injected radioactive albumin and measured its turnover time between plasma and lymph during the baseline period and after recovery from asphyxia. During alveolar hypoxia alone, mean pulmonary arterial pressure increased 60% and lung lymph flow increased 74%, whereas lymph protein concentration decreased from 3.47 +/- 0.13 to 2.83 +/- 0.15 g/dl. Cardiac output, left atrial pressure, and plasma protein concentration did not change. When carbon dioxide was added to the inspired gas mixture, pulmonary arterial pressure increased 22%, cardiac output increased 13%, lung lymph flow increased 33%, and lymph protein concentration decreased from 2.83 +/- 0.15 to 2.41 +/- 0.13 g/dl. Left atrial pressure and plasma protein concentration did not change. After 60-90 s of induced asphyxia, vascular pressures and lung lymph flow rapidly returned to values the same as those obtained during the baseline period. The turnover time for radioactive albumin between plasma and lymph was the same between the baseline and recovery periods (185 +/- 16 vs. 179 +/- 12 min). The ratio of albumin to globulin in lymph relative to the same ratio in plasma did not change during any phase of these experiments. Five lambs killed after recovery from asphyxia had significantly less blood and extravascular water in their lungs than control lambs had. We conclude that in the newborn lamb both alveolar hypoxia and alveolar hypoxia with hypercapnia increase lung lymph flow by increasing filtration pressure in the microcirculation, but neither hypoxia with hypercapnia nor brief severe asphyxia alters the protein permeability of the pulmonary microcirculation.
T N Hansen, T A Hazinski, R D Bland
This study was undertaken to investigate the mechanism underlying sustained monomorphic ventricular tachycardia (VT) in late experimental canine myocardial infarction. The hypothesis that sustained and "organized" continuous electrical activity (CEA) displaying a reproducible pattern with recurrent components recorded by bipolar endocardial, intramural, or epicardial electrodes in 10 animals during electrically induced sustained monomorphic VT represented reentrant excitation in an anatomically small area of the ventricle, was evaluated in the light of the following observations: Organized CEA always preceded the first monomorphic ventricular complex (QRS) of VT as well as the discrete local electrograms from closely surrounding sites during the initiation of VT. The site of organized CEA corresponded to the site of origin of sustained VT determined by iso-chronous contour map analysis of activation sequence. Ventricular pacing at rates more rapid than that of VT failed to terminate VT despite ventricular capture unless it transformed CEA into discrete local electrograms. VT could be terminated in three animals, with a single, critically timed premature stimulus delivered at a critically located focus close to the site of CEA, which would result in local capture and interrupted CEA. In six animals, surgical ablation of the site of organized CEA effectively prevented the reinitiation of sustained VT by programmed cardiac stimulation. These data showed that organized CEA and sustained VT were closely associated phenomena and suggested that organized CEA probably represented an important component of the tachycardia circuit.
H Garan, J N Ruskin
In the present study we examined the effect of reactive oxygen metabolites (generated by the xanthine-xanthine oxidase system), on adenosine-3',5'-cyclic monophosphate (cyclic AMP) and guanosine-3',5'-cyclic monophosphate (cyclic GMP) content in glomeruli and tubules that were isolated from rat renal cortex. Xanthine (0.1 mM)-xanthine oxidase (0.025 U/ml) significantly increased (P less than 0.001) the cyclic AMP content in glomeruli from 18 +/- 1 to 50 +/- 4 pmol/mg protein (n = 13). The response was dose dependent and was markedly inhibited (delta %-74 +/- 9, n = 3) by allopurinol (10(-3), a specific inhibitor of xanthine oxidase. Cyclic AMP content in the tubules, and the cyclic GMP content in glomeruli and tubules, were not altered by the xanthine-xanthine oxidase system. This lack of response was not due to lack of responsiveness of the tissues because parathyroid hormone caused a marked increase in the cyclic AMP content in tubules, and nitroprusside markedly increased the cyclic GMP content in glomeruli. The increase in cyclic AMP in glomeruli was due to generation of reactive oxygen metabolites rather than of other products (e.g. uric acid) of the xanthine-xanthine oxidase reaction--addition of uric acid to incubations had no effect; using another substrate for xanthine oxidase, acetaldehyde significantly increased (delta % 112 +/- 7, n = 4, P less than 0.001) the cyclic AMP content; and catalase that destroys hydrogen peroxide caused a marked inhibition (delta % -90 +/- 5, n = 4) of the response to xanthine-xanthine oxidase. The marked inhibition by catalase, and the lack of effect of superoxide dismutase (in a concentration that completely scavenged superoxide) suggested hydrogen peroxide as the responsible oxygen metabolite for the observed effect. Glucose-glucose oxidase (a system that directly generates hydrogen peroxide), and direct addition of hydrogen peroxide caused a dose-dependent increase in the cyclic AMP content in glomeruli, which further supports the role of hydrogen peroxide as the responsible species for the observed effect. Additional experiments that used prostaglandin synthesis inhibitors and antagonists of serotonin and histamine suggested that hydrogen peroxide increases cyclic AMP content in glomeruli by enhancing prostaglandin synthesis.
S V Shah
Microsomal UDP-glucuronosyltransferase activity toward the bile acids (chenodeoxycholic, deoxycholic, ursodeoxycholic, lithocholic, and glycolithocholic) has been detected in human specimens of liver, kidney, and intestinal mucosa. The characteristics of hepatic and extrahepatic UDP-glucuronosyltransferase activities toward these bile acids were compared with respect to kinetic parameters and other catalytic properties. Whereas no organ-specific differences in the affinities of individual bile acids to hepatic and extrahepatic UDP-glucuronosyltransferases were observed, the individual bile acids showed reaction rates in liver that were about twice the rates estimated in kidney and about twice to three times the rates observed in duodenal mucosa. In intestinal mucosa the rate of chenodeoxycholic acid glucuronidation exhibited a progressive decrease from duodenum to colon, where it was 30% of the duodenal level. Comparison of the glucuronidation rates that were estimated with different bile acids in hepatic or extrahepatic tissues showed that for each organ a bile acid structure-activity relationship existed, with highest activity observed for lithocholic and ursodeoxycholic acids, which was about twofold higher compared with chenodeoxycholic or deoxycholic acids. Lowest activity was estimated for glycolithocholic acid. UDP-glucuronosyltransferase activity toward chenodeoxycholic acid was studied in biopsy specimens of liver that were obtained from a large group of patients with the following liver diseases: liver cirrhosis, liver fibrosis, granulomatous hepatitis, fatty liver hepatitis, and fatty liver. A significant decrease in enzyme activity was observed in patients with liver cirrhosis and in patients with granulomatous hepatitis compared with patients without liver disease.
S Matern, H Matern, E H Farthmann, W Gerok
Children suffering from cystinosis, a genetic disease characterized by high levels of lysosomal cystine, are currently being treated with cysteamine to lower the cystine levels in their cells. In fibroblasts from these patients, cysteamine and its disulfide, cystamine, are equally effective in lowering cystine levels. We recently reported that pantethine, a dietary precursor of coenzyme A, depletes cystine from cultured, cystinotic fibroblasts as effectively as cystamine. To determine the mechanism of action of pantethine, and of cystamine, we have compared the fate of [35S]cystine-derived metabolites in the presence and absence of these agents. The results indicate that the ability of pantethine to deplete cystine resides in its being a metabolic precursor of cysteamine. Furthermore, both pantethine and cystamine act by generating the mixed disulfide of cysteamine and cysteine in the lysosomes, which is then rapidly excreted from the cells. The fall in intracellular [35S]cystine caused by these agents was not accompanied by a comparable increase in any intracellular metabolite; rather, it could be accounted for by the appearance of mixed disulfide in the medium. There was no accumulation of mixed disulfide in the cells. Radioactivity in cytoplasmic glutathione was, however, increased by cystamine or pantethine. Thus, cysteamine (formed intracellularly in these experiments) undergoes thiol-disulfide exchange with cystine in the lysosomes, producing cysteamine-cysteine mixed disulfide and free cysteine, which enter the cytoplasm. The free cysteine is available to several pathways, including oxidation to the disulfide or the mixed disulfide, and synthesis of glutathione. The mixed disulfide is excreted from the cell, which ultimately depletes the cell of its excess cystine.
J D Butler, M Zatz
Studies were designed (a) to determine whether somatostatin is released into the circulation after meals in sufficient amounts to regulate gastric or pancreatic islet function in humans and (b) to investigate the possible role of somatostatin in the pathogenesis of duodenal ulcer disease. Mean plasma somatostatin-like immunoreactivity (SLI) increased from 6.2 +/- 1.5 pg/ml to a peak level of 13.8 +/- 1.3 pg/ml in eight healthy subjects after a 1,440-cal steak meal (P less than 0.005). When somatostatin-14 was infused intravenously, basal and food-stimulated gastric acid secretion and also basal and food-simulated plasma insulin and glucagon concentrations were reduced significantly at mean plasma SLI concentrations within the range seen after a meal. Thus, the amount of somatostatin reaching the systemic circulation after a steak meal was sufficient to inhibit gastric acid secretion and islet cell function. On the other hand, basal and food-stimulated plasma gastrin concentrations were reduced by intravenous somatostatin only at plasma SLI concentrations that were several-fold greater than post-prandial SLI concentrations. Although duodenal ulcer patients had significantly higher basal, food-stimulated, and peak pentagastrin-stimulated gastric acid secretion rates than healthy controls, duodenal ulcer patients and controls had nearly identical basal and food-stimulated SLI concentrations. Moreover, food-stimulated gastric acid secretion and gastrin release were inhibited by intravenous somatostatin to the same extent in ulcer patients and controls. These studies suggest that duodenal ulcer patients release normal amounts of somatostatin into the circulation and that target cells controlling acid secretion and gastrin release are normally sensitive to somatostatin in these patients.
T J Colturi, R H Unger, M Feldman
In order to evaluate the basis for changes in plasma concentrations of the third component of complement (C3) during inflammation, we injected purified radiolabeled C3 into normal New Zealand White rabbits and into rabbits with turpentine-induced pleurisy. In the normal animals, C3 was distributed between the intravascular compartment (75%) and the extravascular space (25%), with an exchange rate of 1.8 +/- 0.1% of the plasma pool per hour. The fractional catabolic rate (FCR) was 2.7 +/- 0.3% of the C3 plasma pool per hour, the synthesis rate was 1.0 +/- 0.2 mg C3/kg per h, and the plasma concentration was 1.23 +/- 0.3 mg C3/ml. Rabbits with turpentine-induced inflammation showed a shift of the volume of C3 distribution in favor of the extravascular compartment. In addition, the rate by which 125I-C3 was cleared from the circulation increased by 29% and was related to the appearance of 20% of the C3-bound circulating radioactivity in the affected pleural cavity at the zenith of inflammation. The FCR, calculated by measuring urinary excretion of radiolabel, increased by only 9% and was probably related to the C3 degradation that was observed in the pleural fluid during the early stages of inflammation. The plasma C3 concentration reached a peak at 230% of the baseline concentration, owing to an increase in the rate of synthesis by as much as 480%. The latter increase could be blocked by cycloheximide, an inhibitor of protein synthesis. We conclude that the increase of plasma C3 in the acute phase is due to stimulated synthesis, which is partially offset by a rise in FCR and by a shift of protein to the site of inflammation.
U Manthei, R C Strunk, P C Giclas
The hypoproliferative anemia in chronic renal failure has been assumed to be the result of decreased erythropoietin (Ep) production by the damaged kidney and of the shortening of erythrocyte survival. However, many in vitro studies suggest that erythropoietic inhibitors in uremic plasma may contribute to the anemia. To determine the in vivo relevance of uremic inhibitors, increasing amounts of Ep as Ep-rich plasma were infused into six uremic sheep, and their erythropoietic responses were compared with those of nine normal sheep receiving similar amounts of Ep-rich plasma. Three sheep were studied in both normal and uremic states. Ep-rich plasma was obtained from phenylhydrazine- and phlebotomy-induced anemic sheep. Stable uremia was created by subtotal nephrectomy. Erythropoiesis was quantitated by reticulocyte response, ferrokinetics (plasma iron turnover and marrow transit time), and by hemoglobin C synthesis. Ep-rich plasma stimulated erythropoiesis similarly in uremic and normal sheep, regardless of the degree of uremia. Nondialyzed uremic sheep responded as well as dialyzed animals. The anemia was corrected in the uremic dialyzed animals. The anemia was corrected in the uremic sheep after 15-40 daily infusions of Ep-rich plasma, the total dosage depending on the severity of the anemia. Polycythemia was induced when the infusions were continued. Reticulocytes, plasma iron turnover, and erythrocyte mass changes increased as the amount of Ep-rich plasma was increased. These dose-response effects, coupled with the identical erythropoietic response in normal and uremic sheep given the same amount of Ep-rich plasma, imply that there are no physiologically significant erythropoietic inhibitors in uremia.
J W Eschbach, J Mladenovic, J F Garcia, P W Wahl, J W Adamson
We have examined the effect of activated neutrophils on the release of prostacyclin (PGI2) from cultured endothelial cells by radioimmunoassay and thin layer chromatography of its stable metabolite, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). Phorbol myristate acetate-activated neutrophils induced a time- and dose-dependent release of 6-keto-PGF1 alpha from human and bovine endothelial cell monolayers, whereas phorbol myristate acetate alone and neutrophils alone did not. Pretreatment of the endothelial cells with aspirin prevented neutrophil-mediated 6-keto-PGF1 alpha release, indicating that it did not depend upon neutrophil-generated endoperoxides. Phorbol myristate acetate-activated neutrophils from a patient with chronic granulomatous disease failed to induce endothelial 6-keto-PGF1 alpha release. Addition of catalase but not of superoxide dismutase significantly reduced human and bovine endothelial 6-keto-PGF1 alpha release by phorbol myristate acetate-activated neutrophils. Catalase-inhibitable endothelial 6-keto-PGF1 alpha release was also observed after the addition of the hydrogen peroxide-generating system, glucose-glucose oxidase, to bovine and human endothelial cell monolayers. Bovine endothelial 6-keto-PGF1 alpha release induced by exogenously generated hydrogen peroxide was attenuated by the phospholipase inhibitor mepacrine, suggesting that hydrogen peroxide may act by triggering endothelial membrane phospholipase activation. The release of 6-keto-PGF1 alpha by enzymatically or neutrophil-generated hydrogen peroxide was not associated with endothelial cell lysis as assessed by 51Cr release. We conclude that exogenously generated hydrogen peroxide or a hydrogen peroxide-derived product mediates rapid nonlytic release of PGI2 from cultured endothelial cells.
J M Harlan, K S Callahan
We have studied major histocompatibility complex markers in Caucasian patients with type I diabetes mellitus and their families. The frequencies of extended haplotypes that were composed of specific HLA-B, HLA-DR, BF, C2, C4A, and C4B allelic combinations, which occurred more commonly than expected, were compared on random diabetic and normal chromosomes in the study families. We demonstrated that all of the previously recognized increases in HLA-B8, B18, B15, DR3, and perhaps DR4 could be ascribed to the increase among diabetic haplotypes of a few extended haplotypes: [HLA B8, DR3, SC01, GLO2]; [HLA-B18, DR3, F1C30]; [HLA-B15, DR4, SC33]; and [HLA-BW38, DR4, SC21]. In fact, HLA-DR3 on nonextended haplotypes was "protective", with a relative risk considerably less than 1.0. There was a paucity or absence among diabetic patients of several extended haplotypes of normal chromosomes, notably [HLA-B7, DR2, SC31] and [HLA-BW44, DR4, SC30]. The extended haplotype [HLA-BW38, DR4, SC21] is found only in Ashkenazi Jewish patients, which suggests that extended haplotypes mark specific mutations that arise in defined ethnic groups. The data show that no known MHC allele, including HLA-DR3 and possibly HLA-DR4, is per se a marker for or itself a susceptibility gene for type I diabetes. Rather, extended haplotypes, with relatively fixed alleles, are either carriers or noncarriers of susceptibility genes for this disease. Thus, the increased frequency (association) or the decreased frequency (protection) of individual MHC alleles is largely explainable by these extended haplotypes.
D Raum, Z Awdeh, E J Yunis, C A Alper, K H Gabbay
Phagocytosis by neutrophils is accompanied by a burst in O2 consumption and activation of the hexose monophosphate shunt (HMPS). Proton secretion equal to the amount of O2 consumed is an additional feature of the respiratory burst, but its source has not been identified, nor has the source of all electrons donated to O2 in the respiratory burst. We chemically quantitated total CO2 generation in human neutrophils and found that proton secretion elicited by phagocytosis was accompanied by a stoichiometric increase in CO2 generation. Addition of carbonic anhydrase and its inhibitors had no effect on either the quantities of CO2 measured or the quantities of protons secreted. Therefore, the CO2 generated in the respiratory burst of stimulated neutrophils is hydrated to form H2CO3, which then dissociates, accounting for the observed proton secretion. Furthermore, the CO2 generated corresponds to the O2 consumed with a respiratory quotient of nearly 1. We conclude on the basis of this and previous studies that the HMPS activity is the source of both the electrons for the NADPH oxidase and of protons secreted in association with the respiratory burst.
N Borregaard, J H Schwartz, A I Tauber
Within any chronically inflamed tissue, there is an increased number of macrophages, pluripotential phagocytic cells that, while critical to host defenses, are also able to profoundly damage parenchymal structure and function. Because of their central role in the inflammatory response, considerable attention has been focused on the mechanisms resulting in an expansion of the macrophage population within an inflamed tissue. Although recruitment of precursor monocytes from the circulation into inflamed tissues clearly plays an important role in macrophage accumulation, it is also possible that replication of tissue macrophages contributes to the expansion of macrophage numbers in inflammation. Because of the accessibility of tissue macrophages with the technique of bronchoalveolar lavage, the lung provides an ideal opportunity to test this hypothesis in humans. To accomplish this, bronchoalveolar lavage was performed to obtain alveolar macrophages from normals (n = 5) and individuals with chronic lung inflammation (normal smokers [n = 5], idiopathic pulmonary fibrosis [n = 13], sarcoidosis [n = 18], and other chronic interstitial lung disorders [n = 11]). Alveolar macrophage replication was quantified by three independent methods: (a) DNA synthesis, assessed by autoradiographic analysis of macrophages cultured for 16 h in the presence of [3H]thymidine; (b) DNA content, assessed by flow cytometric analysis of macrophages fixed immediately after recovery from the lower respiratory tract; and (c) cell division, assessed by cluster formation in semisolid medium. While the proportion of replicating macrophages in normals was very low, there was a 2- to 15-fold increase in this proportion in patients with chronic lung inflammation. In addition, morphologic evaluation demonstrated that individuals with chronic lung inflammation had alveolar macrophages undergoing mitosis. These results suggest that local tissue macrophage replication may play a role in the expansion of the macrophage population in chronic inflammation.
P B Bitterman, L E Saltzman, S Adelberg, V J Ferrans, R G Crystal
The effects of triglyceridemia on plasma lipoproteins were investigated in 16 hypertriglyceridemic (HTG) subjects (222-2,500 mg/dl) before and after the initiation of bezafibrate therapy. Bezafibrate caused a mean reduction of 56% in plasma triglyceride and increased the levels of lipoprotein and hepatic triglyceride lipases by 260 and 213%, respectively. The natures of very low density lipoprotein (VLDL), isolated at plasma density and of low and high density lipoprotein (LDL and HDL), separated by zonal ultracentrifugation, were determined. HTG-LDL appears as multiple fractions whereas HTG-HDL is seen predominantly as HDL3. HTG-VLDL is relatively poor in apoproteins and triglycerides but enriched in free and esterified cholesterol. HTG-LDL (main fraction) is depleted of free and esterified cholesterol but enriched in apoprotein and triglyceride. It is also denser and smaller than normal. HTG-HDL3 is denser than N-HDL3 and demonstrates compositional abnormalities similar to those of HTG-LDL. With the reduction of the VLDL mass, all abnormalities revert towards normal. This is accompanied by an increase in LDL-apoprotein B and cholesterol levels, which indicates an increased conversion of VLDL to LDL. Significant correlations between plasma triglyceride and the degree of all abnormalities are shown. The data obtained during treatment corroborate these relationships. The observations support the concept that most abnormalities reflect the degree of triglyceridemia. We suggest that plasma core-lipid transfer protein(s) is an effector of the abnormal cholesteryl ester distribution. Its prolonged action on increasingly large and slowly metabolized VLDL populations would entail a correspondingly excessive transfer of cholesteryl ester to VLDL and of triglyceride to LDL and HDL. It is calculated that, in moderate HTG, LDL and HDL contain only 50% of the normal cholesterol load. It is suggested that cholesteryl ester redistribution in HTG might be important in regulating metabolic events.
S Eisenberg, D Gavish, Y Oschry, M Fainaru, R J Deckelbaum
We have found that catecholamines stimulate DNA synthesis and centrosomal separation in 3T3 and bovine aortic endothelial cells cultured in the absence of serum or added growth factors. The mitogenic effect is mediated by an alpha 1-adrenergic receptor, as it is inhibited by phentolamine and prazosin but not by propranolol or yohimbine. The physiological and pathological consequences of this effect remain to be determined.
P Sherline, R Mascardo
Previous studies have shown that anti-idiotypic antibodies can be developed in vivo through animal immunization with idiotype, and that these antibodies can be isolated from other anti-immunoglobulin antibodies by affinity purification. These techniques have relied on large amounts of idiotype, which were produced either by hyperimmunization or by monoclonal antibodies, to serve as the affinity adsorbent. In the present study, we produced anti-idiotypic antibodies to human anti-thyroid-stimulating hormone (TSH) receptor antibodies by first injecting rabbits with (TSH receptor purified) IgG from Graves' patients. The resulting antiserum was then adsorbed with Sepharose-coupled TSH in an attempt to specifically bind and isolate the anti-idiotype. The antibody obtained from this process was shown to bind specifically to TSH receptor-binding antibodies from Graves' patients, and this binding could be inhibited by 56% with the addition of 10(-4) M TSH but not by HCG (10(-2) M). The anti-idiotype also bound to TSH, and this binding could be specifically inhibited by receptor-purified Graves' IgG (60% inhibition at 10 micrograms/ml IgG), but not by IgG from normal subjects (no inhibition at 50 micrograms/ml IgG). In a TSH receptor binding assay, the anti-idiotype could inhibit TSH receptor binding in Graves' sera at a 1,000-fold lower concentration than could anti-kappa/lambda antiserum; the anti-idiotypic antiserum also inhibited in vitro TSH-mediated adenylate cyclase stimulation at an IgG concentration of 5 micrograms/ml, while heterologous anti-TSH antisera and normal IgG at similar concentrations had no effect. Finally, despite being generated against a single patient's TSH receptor binding antibody, the anti-idiotype was able to block TSH receptor binding in the serum of six other Graves' patients, thus suggesting that there may be conformational conservation in the antigen that is recognized by different individuals' TSH receptor-binding immunoglobulins.
J R Baker Jr, Y G Lukes, K D Burman
To determine whether healthy homosexual men are immunologically impaired, peripheral blood leukocytes (PBL) from 20 male homosexuals were compared prospectively with PBL from 14 age-matched male heterosexual donors with respect to: (a) the capacity of their PBL to generate functional T cell immune responses in vitro; and (b) the content of total T cells and T cell subsets in their peripheral blood. The homosexual donors studied indicated moderate sexual life styles in that all but one of the donors had less than five current sexual partners. The percentages of OKT3+, OKT4+, and OKT8+ T cells were similar to those of heterosexual controls. T cell function was assessed by measuring cytotoxic T cell responses to influenza virus and to allogeneic cells. Approximately one-third of the homosexual donors consistently exhibited weak cytotoxic T lymphocyte (CTL) responses to influenza virus, whereas all of the heterosexual donors generated strong CTL responses to influenza. There was no correlation between the strength of CTL responsiveness to influenza virus and the strength of CTL responses to allogeneic cells. These results suggest that the influenza-specific CTL response may be a sensitive indicator of immunologic defects in asymptomatic homosexuals. If acquired immune deficiency syndrome results from an infectious agent, it remains to be seen if such immunosuppression predisposes to the infection, or if it reflects early consequences of infection.
G M Shearer, S M Payne, L J Joseph, W E Biddison
To examine the role of vitamin D in the renal tubular handling of calcium, clearance studies were performed in three groups of rats: group A rats fed a standard vitamin D-deficient diet (Ca 0.45%, P 0.3%) for 6 wk, were hypocalcemic with secondary hyperparathyroidism; group B rats fed the same diet as in group A but with high calcium (Ca 1.4%) and 20% lactose, were normocalcemic and without secondary hyperparathyroidism; group C rats fed the same diet as in group A but supplemented with 25 U of vitamin D3 orally twice a week, were normocalcemic, vitamin D-replete, and euparathyroid. After thyroparathyroidectomy (TPTX), each rat was infused intravenously with an electrolyte solution that contained a fixed concentration of calcium (0-30 mM) with or without parathyroid hormone (PTH; 0.75 or 2.5 U/h) at a rate of 3 ml/h. Urinary calcium excretion and serum calcium concentrations were measured between 16 and 19 h of the infusion, and the apparent threshold of calcium excretion was determined. The threshold of calcium excretion was lower in vitamin D-deficient TPTX rats (groups A and B) than in vitamin D-replete TPTX rats (group C), and not different between group A and group B. Administration of PTH at a dose of 0.75 U/h increased the threshold of calcium excretion by approximately 0.6 mM in group C, but did not alter the threshold either in group A or group B. Administration of a higher dose of PTH (2.5 U/h) raised the threshold similarly in both group A and group B to the extent comparable with that in group C, when it was given 0.75 U/h of PTH. These results demonstrate that the renal threshold of calcium excretion is decreased in the vitamin D-deficient rats independent of the secondary hyperparathyroidism, and that the higher dose of PTH was necessary to raise the calcium threshold in vitamin D-deficient rats. Thus, present study indicates the presence of dual effects of vitamin D on renal tubular handling of calcium; the one is to facilitate renal calcium reabsorption and the other is to enhance the responsiveness of the tubule to PTH.
M Yamamoto, Y Kawanobe, H Takahashi, E Shimazawa, S Kimura, E Ogata
Various arachidonic acid (AA) metabolites are known to regulate immune cell function(s) and dictate the progression of both acute and chronic inflammatory reactions. Using a model of Schistosoma mansoni egg-induced hypersensitivity granulomas, we have delineated the in vivo effects of inhibitors of cyclooxygenase (CO) and lipoxygenase (LO) pathways on granuloma development and granuloma macrophage I-region-associated (Ia) antigen expression. In addition, by high performance liquid chromatography (HPLC) we have profiled the metabolism of AA by macrophages that are isolated from granulomatous foci, and have biochemically characterized the in vitro specificity and activity of selected CO and LO inhibitors. The development of hypersensitivity-type pulmonary granulomas in mice was dramatically suppressed by inhibitors with anti-LO activity (nordihydroguairetic acid (NDGA), nafazatrom, and BW755c) in a dose-dependent manner, while indomethacin, which is primarily CO-selective, had no significant effect. Furthermore, NDGA and nafazatrom profoundly arrested the normal progression of preformed granulomatous lesions. The inhibitors of the LO pathway also suppressed the in vivo kinetics of Ia antigen expression by granuloma macrophages. In contrast, indomethacin augmented Ia-antigen expression. The major AA metabolites that were synthesized by the granuloma macrophages were shown to be leukotriene C4 and mono-hydroxyeicosatetraenoic acids. HPLC analysis of AA metabolites from granuloma macrophages that were treated with the various inhibitors confirmed that indomethacin was most CO-selective and NDGA most LO-selective. Nafazatrom and BW755c inhibited AA metabolism by both pathways. Notably, high concentrations of the compounds (5 X 10(-5) M) tended to suppress all products. Our results suggest that LO products may be important in the generation and maintenance of immune granulomatous inflammatory responses.
S L Kunkel, S W Chensue, C Mouton, G I Higashi
Parameters of secretion of IgA and several other plasma proteins from the jejunal mucosa were investigated in 11 individuals who had a normal distribution of Ig-containing cells in the lamina propria and in one patient who was totally deficient in jejunal IgA and IgM plasmacytes. Jejunal samples were collected during segmental gut perfusion. The following results were obtained: (a) The secretion of polymeric IgA (p-IgA, mean equals 217 micrograms/40 cm per min) exceeded those of albumin (132 micrograms), IgG (35 micrograms), and monomeric IgA (m-IgA, 15 micrograms, or 6.4% of total IgA). About 35% of IgA was IgA2 in the jejunal secretion, compared with approximately 23% in serum. This closely corresponds to the 35 and 24% of IgA2 plasmocytes in jejunal mucosa and peripheral lymph nodes, respectively. (b) For each protein, a relative coefficient of excretion (RCE) was calculated (jejunum to serum concentration ratio expressed relative to that of albumin). RCEs of 1.41 for orosomucoid, 1.0 for albumin, 0.83 for IgG, and 0.74 for IgE and, in the deficient patient, of 0.64 for m-IgA and 0.016 for IgM were obtained. This was inversely related to the molecular weight of these proteins and indicated their predominantly passive transport into the jejunum. However, in normal individuals, the RCE of transferrin (approximately 1.11 greater than 1, P greater than 0.05), alpha 2-macro globulin (approximately 0.77), m-IgA (approximately 1.98), and p-IgA (approximately 218) exceeded the value expected from simple seepage from plasma, thus pointing to an additional role of either local gut synthesis and/or active transepithelial transport. (c) Approximately 98% of p-IgA, approximately 99% of IgM, and approximately 68% of m-IgA in jejunal secretions were derived from local production in the gut wall, as determined by 125I-p-IgA specific activities and/or by comparison between the RCE values of the deficient patient to the values of controls. Therefore, the jejunal production of p-IgA (approximately 312 mg/d per 40 cm vs. approximately 54 mg/d from bile) contributes the majority of upper intestinal IgA in humans. The active transport of plasma p-IgA across the intestinal mucosa (approximately 0.08 mg/40 cm per kg per d) contributes less than 2% of the total amount of p-IgA (4.5 mg/kg per d) that is cleared daily from plasma.
P P Jonard, J C Rambaud, C Dive, J P Vaerman, A Galian, D L Delacroix
Investigations of polymorphonuclear leukocyte (PMN) function were performed in a 5-yr-old white female with delayed umbilical cord separation, impaired pus formation, and a severe defect of PMN chemotaxis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an almost total deficiency of a high molecular weight glycoprotein(s) (GP138) in the granule and membrane fractions of the patient's cells, and NaB3H4-galactose oxidase labeling demonstrated the absence of a major glycoprotein complex on the surface of her PMNs. Monoclonal antibodies (MAb) were employed in flow cytometry experiments to demonstrate that two previously characterized glycoproteins (Mo1 and LFA1) were undetectable on the surface of the patient's PMNs and monocytes. Immunoprecipitation of 125I-labeled patient cells with subunit specific MAbs confirmed that the alpha-subunits of Mo1 (155 kD) and LFA1 (177 kD) and their common beta-subunit (94 kD) were totally deficient. Functional analyses of patient PMNs demonstrated severe impairment of adherence- and adhesion-dependent cell functions including spreading, aggregation, orientation in chemotactic gradients, antibody-dependent cellular cytotoxicity, and phagocytosis of particles (Oil-Red-0-paraffin, zymosan) selectively opsonized with C3-derived ligands. Patient PMNs demonstrated a normal capacity to rosette with IgG or C3b-coated sheep erythrocytes, but rosette formation with C3bi-coated erythrocytes was profoundly diminished. Adhesion-independent functions including shape change, N-formyl-methionyl-leucyl-3H-phenylalanine binding, and O-2 generation or secretion elicited by soluble stimuli were normal. Membrane fluidity, surface charge, and microtubule assembly were also normal. These findings provide new evidence that critical PMN surface glycoproteins are required to facilitate multiple adhesion-dependent cellular functions of the inflammatory response.
D C Anderson, F C Schmalstieg, M A Arnaout, S Kohl, M F Tosi, N Dana, G J Buffone, B J Hughes, B R Brinkley, W D Dickey
The interaction of bradykinin (BK) with its specific receptors on intact cultured human fibroblasts results in production of prostaglandins, including prostacyclin (PGI2), and accumulation of cyclic AMP. Incubation of cells with 1 microM BK for 5 min at 37 degrees C led to a marked reduction (75-90%) in BK-induced PGI2 release and in total number of [3H]BK-binding sites with no change in dissociation constant (6.1 and 7.6 nM for control and BK-treated cells, respectively). The decrease in receptor number did not result from BK transferred from the first incubation into the binding assay. BK-induced receptor loss was temperature dependent; exposure of cells to BK at 4 degrees C had little or no effect on receptor number. After incubation with BK for approximately equal to 15 min, further incubation in the absence of BK for 30 min at 37 degrees C almost completely restored both receptor number and BK-induced PGI2 release. With more prolonged exposure to BK (greater than 1 h), restoration of receptors was inversely related to the length of exposure and the concentration of BK. Recovery was unaffected by cycloheximide. During prolonged incubation without removal of BK, cells began to recover receptors by 5 h; greater than 99% of the bradykinin initially present disappeared by 3 h. Bacitracin greatly retarded BK disappearance and totally prevented recovery. These observations provide direct evidence that the number of BK receptors on cultured human fibroblasts can be regulated by BK itself. In addition, it appears that BK-degrading systems, by influencing local concentrations of the peptide, may play an important role in the autoregulation of BK receptors. The presence of highly active degradation systems might serve to protect target tissues from developing chronic insensitivity to BK and, perhaps, similar peptides.
A A Roscher, V C Manganiello, C L Jelsema, J Moss
The effects of alternating cycle lengths (bigeminal rhythm) on His-Purkinje system refractoriness were studied in 14 patients using His bundle and right bundle recordings. Programmed atrial stimulation at constant cycle length (method I) was scanned using the atrial extrastimulus technique (A2) and compared with an atrial cycle length of identical duration coupled to A2 on alternate beats (method II). The results showed that (a) despite shorter cycle length of the His-Purkinje system with method II due to effect of A2 on atrioventricular nodal conduction (699 +/- 90 vs. 743 +/- 87 ms, P less than 0.001), the relative refractory period of the His-Purkinje system was always longer with method II (463 +/- 52 vs. 440 +/- 43 ms, P less than 0.001). A similar increase also occurred in effective refractory period of the His-Purkinje system; (b) while functional right bundle branch block occurred in eight patients and functional left bundle branch block in two patients with method I, functional right bundle branch block occurred in all 14 patients and left bundle branch block in seven patients with method II; (c) in two patients where both functional right and left bundle branch block occurred with method I this never was manifest at identical degree of abbreviation of His-Purkinje system cycle length but was manifest at identical abbreviation in each of seven patients with method II; (d) site of conduction delay and/or block during functional right bundle branch block was always proximal, i.e., between the His bundle and right bundle recordings with both methods. During method II this resulted in shortening of the subsequent right bundle cycle length relative to the subsequent His bundle (and of necessity left bundle) cycle length. The finding of increased His-Purkinje system refractoriness despite shorter preceding cycle length of the His-Purkinje system during atrial bigeminy has never been previously described and suggests that classical concepts of His-Purkinje system behavior may require revision in this setting. Secondly, during atrial bigeminy the occurrence of alternating functional bundle branch block cannot be accounted for solely by the degree of abbreviation of His-Purkinje system cycle length, but may be explained by a relative shortening of the next ipsilateral bundle branch cycle length in the bundle manifesting block.
S Denker, M Lehmann, R Mahmud, C Gilbert, M Akhtar
Vascular or tissue-type plasminogen activator (TPA) is a key enzyme in physiologic fibrinolysis. To study the role of prostaglandins in modulating the synthesis and release of TPA in vivo, we prospectively studied the effect of aspirin (650 mg/d X 2) on TPA activity in 13 human subjects before and after 10 min of forearm venous occlusion. TPA activity was quantified by a newly developed enzyme-linked immunosorbent assay that both measures and differentiates between TPA and urokinase (UK)-like plasminogen activator activity. This assay is based on the observation that the concentration of alpha 2-plasmin inhibitor-plasmin complexes in Reptilase-clotted plasma increases linearly in proportion to the amount of activator added. Resting TPA activity was higher in women than in men (0.56 +/- 0.59 vs. 0.15 +/- 0.11 U/ml, P = 0.049). Venous occlusion induced an eightfold rise in TPA activity in women (to 4.5 U/ml, P = 0.006) and a 15-fold rise in men (to 2.28 U/ml, P = 0.004), whereas UK activity was not detected. Aspirin inhibited the rise in TPA activity after venous occlusion by 69% in men (P = 0.004) and 70% in women (P = 0.014). In contrast, aspirin had no effect on pre- or post-occlusion hematocrits or Factor VIII-related antigen levels. There was no correlation between plasma salicylate level and percentage inhibition of TPA. Neither exogenous aspirin (0-1 microgram/ml) nor salicylate (0-70 micrograms/ml) inhibited the generation of alpha 2-plasmin inhibitor-plasmin complexes by exogenous TPA or interfered with the assay system. We conclude that aspirin may have an antifibrinolytic effect in man that has not been previously described.
R I Levin, P C Harpel, D Weil, T S Chang, D B Rifkin
We studied the erythrocyte Na,K-pump in chronically hemodialyzed uremic patients, immediately before and after a 4-h period of hemodialysis. Using [3H]ouabain as a probe, the number of Na,K-pump units per erythrocyte did not differ in uremic and control subjects, and hemodialysis had no acute effect on this parameter. In contrast, in these same cells the mean level of Na,K-pump-mediated 86Rb transport was 30% lower in predialysis uremic patients than in controls, and this diminution in the rate of 86Rb transport per pump unit was improved after 4 h of hemodialysis in 17 of 18 subjects. The results of in vitro incubation of normal cells with pre- and post-dialysis sera from uremic patients suggested that a serum factor is responsible for the observed inhibition of Na,K-pump activity. Changes in cell Na concentration during dialysis did not appear to be responsible for the increased rate of Na,K-pump turnover after hemodialysis. However, there was a significant correlation between the extent of rise in pump-mediated 86Rb uptake and the weight loss that occurred during dialysis. We conclude that the ion transport turnover rate of the erythrocyte Na,K-pump is impaired in uremia by a nonouabain like circulating factor. This factor, whose activity is diminished acutely by hemodialysis, may play an important role in the systemic manifestations of the uremic syndrome, and could be an important endogenous regulator of the Na,K-ATPase.
H Izumo, S Izumo, M DeLuise, J S Flier
We have performed a comprehensive study of calcium tracer flux, distribution, content, and ionized cytoplasmic calcium during monocyte activation. A model of monocyte calcium was developed from 45Ca uptake and exodus curves which indicated that cell calcium was partitioned between three compartments. The magnitude of the time constants for each pool lead us to propose cellular locations for these three compartments: a surface plasma membrane pool, a cytoplasmic pool, and an organelle pool. 45Ca uptake and exodus experiments were analyzed using a nonlinear least squares fit of compartmental exchange rates and sizes. The production of superoxide was used as a reflection of the state of activation of the monocytes treated with Concanavalin A (Con A). We found that Con A-treated monocytes have an increase in the calcium exchange rate with the cytoplasmic pool from 0.04 to 0.07/min (P less than 0.05), and an increase in the size of the cytoplasmic pool from 0.08 to 0.13 pmol/cell (P less than 0.05). There were no significant changes in the exchange rates or sizes associated with either of the other two compartments. The cytoplasmic ionized calcium was measured with the fluorescent probe, Quin 2, which indicated a resting level of 83 nM free calcium in unadhered monocytes. Con A stimulation caused a doubling of the cytoplasmic free calcium to 163 nM within 45 s. This increment in cytoplasmic free calcium preceded the onset of superoxide following Con A treatment. These studies indicate that Con A binding to the plasma membrane increases the monocyte plasma membrane permeability to calcium. External calcium enters the cell at an increased rate and contributes to both internally bound and free calcium. The magnitude of the increase in free calcium is proportional to the concentration of Con A and stimulates calcium extrusion via the calcium transport ATPase. Moreover, there is an increased concentration of ionized cytoplasmic calcium which has the potential to interact with other cellular regulators that modulate cell activation and superoxide production.
S P Scully, G B Segel, M A Lichtman
Well-myelinated cultures of mouse dorsal root ganglia incubated for 48 h with sigma-aminolevulinic acid (ALA) showed intense porphyrin fluorescence localized in myelin sheaths but not in axons or neuronal somata. When the cultures were continuously incubated with a high concentration of lead, focal swelling and segmental degeneration of myelin began to develop within 2 wk. Incubation of cultures with ALA after 3 wk of lead treatment revealed markedly decreased porphyrin fluorescence in myelin sheaths compared with untreated controls. After 6 wk of lead treatment, myelin showed severe segmental degeneration. Porphyrin fluorescence from ALA at this time was barely detectable in these cultures. No fluorescence was visible in the demyelinated axons; however, silver-impregnation staining after fixation demonstrated continuity of the axon despite the severe loss of myelin. When cultures were continuously incubated with lead and heme together for 6 wk, the segmental demyelination seen in cultures treated with lead alone did not occur. These findings suggest that the lead-induced segmental demyelination in cultured mouse dorsal root ganglia may be due to toxic effects of the metal on the heme biosynthetic pathway in myelinating cells and that exogenous heme may counteract this toxic effect of lead.
W O Whetsell Jr, S Sassa, A Kappas
Generation of reactive oxygen metabolites, thromboxane increases, and vasoconstriction have been implicated in the pathogenesis of acute edematous lung injury, such as that seen in patients with the Adult Respiratory Distress Syndrome (ARDS), but their interactions are unknown. We hypothesized that reactive O2 products would stimulate arachidonic acid metabolism in lungs and that vasoactive products of arachidonate, such as the potent vasoconstrictor thromboxane A2, might then mediate O2-metabolite-induced pulmonary vasoconstriction. We found that O2 metabolites generated by injection of purine plus xanthine oxidase caused increases in mean pulmonary artery perfusion pressures (27 +/- 4 mmHg) in isolated perfused lungs. In addition, purine plus xanthine oxidase also caused 30-fold increases in perfusate levels of thromboxane B2 (the stable metabolite of thromboxane A2) compared with only twofold increases in 6-keto-PGF1a (the stable metabolite of prostacyclin). Moreover, prior addition of catalase inhibited both vasoconstriction and the thromboxane B2 production seen in isolated lungs following injection of purine plus xanthine oxidase. Similarly, pretreatment with cyclooxygenase inhibitors, either aspirin or indomethacin, also completely blocked thromboxane generation and markedly attenuated pressor responses usually seen after purine plus xanthine oxidase (increase in mean pulmonary artery perfusion pressures, 4.4 +/- 1.5 mmHg). Furthermore, imidazole, a thromboxane synthetase inhibitor, also decreased O2-metabolite-induced thromboxane generation and vasoconstriction. These results suggested that thromboxane generation might participate in O2-metabolite-induced vasoconstriction. However, since a significant correlation between thromboxane levels and the degree of vasoconstriction could not be demonstrated, and since addition of superoxide dismutase reduced thromboxane generation but did not affect the intensity of vasoconstriction, it is possible that thromboxane is not the only vasoactive mediator in this model. We conclude that exposing lungs to O2 metabolites results in thromboxane generation and that thromboxane is a major mediator of oxidant-induced vasoconstriction.
R M Tate, H G Morris, W R Schroeder, J E Repine
To elucidate the mechanism responsible for the bile acid-induced changes of biliary lipid secretion, we evaluated bile flow and biliary output of bile acids, cholesterol, phospholipids, and alkaline phosphatase activity in seven cholecystectomized subjects with a balloon occludable T-tube during two experimental periods: (a) depletion of the endogenous bile acid pool and (b) replacement of the pool by means of duodenal infusion with individual bile acids, such as deoxycholic (DCA), chenodeoxycholic (CDCA), cholic (CA), and ursodeoxycholic (UDCA) acids. Bile flow, cholesterol, and phospholipid output were linearly related to bile acid secretion in all experimental periods. During the replacement periods, the amount of cholesterol and phospholipids coupled to bile acids was significantly different (at 1% level at least) for each individual bile acid secreted; it was the highest during DCA secretion (slope value: 0.209 for cholesterol and 0.434 for phospholipids) followed, in the order, by CDCA (0.078 and 1.794), CA (0.044 and 0.127), and UDCA (0.030 and 0.122). The phospholipid to cholesterol ratio was higher during secretion of CA and UDCA as compared with DCA and CDCA. The secretion of CA seemed to stimulate a greater bile flow than the other bile acids did. The infusion of all bile acids, except UDCA, induced an increase of biliary alkaline phosphatase activity as compared with the values of the depletion period. The mean highest increase (13-fold the pretreatment value) was observed during DCA secretion followed by CDCA (fivefold) and CA (1.5-fold). These results would suggest that the physical chemical properties, namely the lipid-solubilizing capacity, of bile acids could directly contribute to the regulation of biliary lipid secretion. The observed changes in biliary alkaline phosphatase activity lend support to the view that bile acid-induced lipid secretion may be, at least in part, contributed by membrane solubilization.
N Carulli, P Loria, M Bertolotti, M Ponz de Leon, D Menozzi, G Medici, I Piccagli
Antibodies to the Ro/SSA antigen occur in patients with systemic lupus erythematosus and Sjögren's syndrome. An immunoaffinity method for the preparation of electrophoretically homogeneous Ro/SSA antigen is described. Several molecular properties of the antigen have been determined. The native RNA protein particle has a molecular weight of approximately 100,000 D determined by gel filtration. Sodium dodecyl sulfate-analysis of the purified Ro/SSA antigen and analysis by staining of bands with silver and Coomassie Blue, Western blotting, and RNAase treatment leads to a hypothesis for the structure of the particle in which an antigenic 60,000 protein is bound to 24,000-27,000 RNA molecules which are not antigenic. An enzyme-linked immunoabsorbent assay method for assay of anti-Ro/SSA is also described which sensitively measures antigen binding at dilutions of sera containing anti-Ro/SSA precipitins up to 10(7) fold. Normal sera on average have 10(3) less binding activity.
H Yamagata, J B Harley, M Reichlin
Complementary DNA (cDNA) clones corresponding to the major histocompatibility (MHC) class III antigen, complement protein C2, have been isolated from human liver cDNA libraries with the use of a complex mixture of synthetic oligonucleotides (17 mer) that contains 576 different oligonucleotide sequences. The C2 cDNA were used to identify a DNA restriction enzyme fragment length polymorphism that provides a genetic marker within the MHC that was not detectable at the protein level. An extensive search for genomic polymorphisms using a cDNA clone for another MHC class III gene, factor B, failed to reveal any DNA variants. The genomic variants detected with the C2 cDNA probe provide an additional genetic marker for analysis of MHC-linked diseases.
D E Woods, M D Edge, H R Colten
An almost complete amino acid sequence of the carboxyl-terminal 20-kD tryptic heavy chain peptide from bovine cardiac myosin Subfragment-1 (S-1) has been determined by automated sequential degradation of the undigested peptide and subfragments derived by chemical and enzymatic digestion. The fragment contains 169 residues, including two reactive cysteinyl residues which are located nine residues apart. At six positions in the sequence, two amino acid residues were present and two different versions of a chymotryptic peptide were isolated in approximately 53 and 24% yields, suggesting that there are two cardiac myosin beta-type heavy chains in this species. Analysis of the secondary structure of the 20-kD peptide predicts that there are two distinct regions within the fragment. The first region (residues 1-121) contains 12% alpha-helix, 25% beta-sheet, 40% beta-bends, and 19% coil; the second region (residues 122-169) may form an extended alpha-helix. Comparison of the bovine sequence with the deduced amino acid sequence of a recombinant plasmid containing DNA sequences coding for the beta-heavy chain of rabbit cardiac myosin (pMHC beta 174) reveals approximately 86% homology.
I L Flink, E Morkin
Epidermal growth factor (EGF) promotes the growth of cultured benign and malignant cells. Recent studies have demonstrated that the amount of EGF receptor is elevated in squamous cell carcinoma cells in tissue culture when compared with normal epidermal cells. This study demonstrates that elevated levels of EGF receptor are detected in biopsy specimens of human squamous cell carcinomas of the lung with a murine monoclonal antibody, EGF-R1, which binds specifically to the receptor. The increased receptor ranged from 2.5- to 5-fold that of normal skin. These findings have been observed in 11 of 11 squamous carcinomas and two of two epidermoid head and neck cancers. Seven of eight adenocarcinomas, two of two small cell, and four of eight undifferentiated lung cancers had negligible amounts of EGF receptor. The EGF receptor antibody did not bind significantly to normal lung tissues and 35 nonepidermoid tumors. Therefore, EGF receptor may be an excellent marker for epidermoid malignancies.
F J Hendler, B W Ozanne
Hydroxyurea, a widely used cytotoxic/cytostatic agent that does not influence methylation of DNA bases, increases fetal hemoglobin production in anemic monkeys. To determine its effect in sickle cell anemia, we treated two patients with a total of four, 5-d courses (50 mg/kg per d, divided into three oral doses). With each course, fetal reticulocytes increased within 48-72 h, peaked in 7-11 d, and fell by 18-21 d. In patient I, fetal reticulocytes increased from 16.0 +/- 2.0% to peaks of 37.7 +/- 1.2, 40.0 +/- 2.0, and 32.0 +/- 1.4% in three successive courses. In patient II the increase was from 8.7 +/- 1.2 to 50.0 +/- 2.0%. Fetal hemoglobin increased from 7.9 to 12.3% in patient I and from 5.3 to 7.4% in patient II. Hemoglobin of patient I increased from 9.0 to 10.5 g/dl and in patient II from 6.7 to 9.9 g/dl. Additional single-day courses of hydroxyurea every 7-20 d maintained the fetal hemoglobin of patient I t 10.8-14.4%, and the total hemoglobin at 8.7-10.8 g/dl for an additional 60 d. The lowest absolute granulocyte count was 1,600/mm3; the lowest platelet count was 390,000/mm3. The amount of fetal hemoglobin per erythroid burst colony-forming unit (BFU-E)-derived colony cell was unchanged, but the number of cells per BFU-E-derived colony increased. Although examination of DNA synthesis in erythroid marrow cells in vitro revealed no decreased methylcytidine incorporation, Eco RI + Hpa II digestion of DNA revealed that hypomethylation of gamma-genes had taken place in vivo after treatment. This observation suggests that hydroxyurea is a potentially useful agent for the treatment of sickle cell anemia and that demethylation of the gamma-globin genes accompanies increased gamma-globin gene activity.
O S Platt, S H Orkin, G Dover, G P Beardsley, B Miller, D G Nathan
Activated B and T lymphocytes from normal human subjects are known to have the specific high-affinity receptor for 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). In an attempt to determine a functional role for the sterol in such cells, we studied the effect of 1,25-(OH)2-D3 on DNA synthesis and Ig production by normal human peripheral blood mononuclear (PBM) cells activated in vitro by the polyclonal lymphocyte activators pokeweed mitogen and phytohemagglutinin, and the specific antigen dermatophyton O. A dose-dependent inhibition of [3H]thymidine incorporation was observed in cells incubated with 1,25-(OH)2-D3 in concentrations ranging from 10(-10) to 10(-7) M. Production of IgG and IgM, determined by enzyme-linked immunosorbent assay, was similarly inhibited by increasing concentrations of 1,25-(OH)2-D3. Half-maximal inhibition of DNA and Ig synthesis was found at 10(-10) to 10(-9) M 1,25-(OH)2-D3. This suppressive effect was specific for 1,25-(OH)2-D3; of the other vitamin D metabolites examined, only 10(-7) M 24R,25 dihydroxyvitamin D3 (24,25-(OH)2-D3) had a similar inhibitory effect. 1,25-(OH)2-D3 was not cytotoxic and did not affect unactivated PBMs. These data demonstrate that 1,25-(OH)2-D3 is a potent inhibitor of human PBM Ig production in vitro and suggest that this action is mediated through the hormone's antiproliferative effect on Ig-producing B cells and/or helper T cells.
J M Lemire, J S Adams, R Sakai, S C Jordan
An immunohistochemical study using monoclonal antibodies specific for the heavy chains of either human atrial (HC alpha) or ventricular (HC beta) myosin was performed to clarify the distribution of each isozyme in normal as well as pressure-overloaded human hearts. In normal human ventricles, all muscle fibers were stained by a monoclonal antibody (HMC14) specific for HC beta, whereas a small number of fibers reacted with a monoclonal antibody (CMA19) specific for HC alpha. In contrast, in normal human atria, almost all muscle fibers were stained by CMA19, and a relatively larger number of muscle fibers also reacted with HMC14. Furthermore, in pressure-overloaded atria, muscle fibers reactive with HMC14 were strikingly increased while those reactive with CMA19 showed a corresponding decrease. The extent of this isozymic redistribution was in good correlation with atrial pressure. These results not only confirmed the existence of isoforms of myosin heavy chain in human hearts, but also demonstrated that redistribution of iso-myosins could occur as an adaptation to pressure overload.
H Tsuchimochi, M Sugi, M Kuro-o, S Ueda, F Takaku, S Furuta, T Shirai, Y Yazaki