Antineutrophil Autoantibodies in Graves' Disease

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Free 1,25-dihydroxy vitamin D levels in Serum from Normal Subjects, Pregnant Subjects, and Subjects with Liver Disease

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Endothelial cell synthesis of von Willebrand antigen II, von Willebrand factor, and von Willebrand factor/von Willebrand antigen II complex.

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von Willebrand antigen II (vW AgII) and von Willebrand factor (vWf) are immunochemically distinct proteins that are deficient in the plasma and platelets of patients with severe von Willebrand's disease. Normal human umbilical vein endothelial cells were cultured in the presence of [35S]methionine. Crossed immunoelectrophoresis of endothelial cell supernates and detergent-solubilized endothelial cells demonstrated specific incorporation of the [35S]methionine into vW AgII. Furthermore, when endothelial cells were lysed in the presence of proteolytic inhibitors, a second, less anodal peak was identified on crossed immunoelectrophoresis. This peak represented a complex of vW AgII and vWf and demonstrated a reaction of complete identity with the vW AgII immunoprecipitate. When plasma, serum, or platelets were evaluated by crossed immunoelectrophoresis, this "complex" peak was not present. When antibodies to vWf, fibronectin, or fibrinogen were present in the first dimension of crossed immunoelectrophoresis, only the antibodies to vWf removed the complex. Radioiodinated polyclonal and monoclonal antibodies to vWf also localized vWf to this complex. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled immunoprecipitates indicated that the molecular weight of vW AgII is 98,000 and that vWf was present as two species of 220,000 and 260,000 mol wt, respectively. Immunofluorescent microscopy of endothelial cells demonstrated colocalization of vW AgII and vWf in endothelial cells with intense immunostaining of the same subcellular granules.

Authors

D R McCarroll, E G Levin, R R Montgomery

Calcium transport in canine renal basolateral membrane vesicles. Effects of parathyroid hormone.

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The effects of parathyroid hormone were studied on Ca2+ fluxes in canine renal proximal tubular basolateral membrane vesicles (BLMV). Efflux of Ca2+ from preloaded BLMV was found to be stimulated by an external Na+ gradient, and this was inhibited by the Na+ ionophore, monensin, and enhanced by intravesicular negative electrical potentials, which indicated electrogenic Na+/Ca2+ exchange activity. There was a Na+ gradient independent Ca2+ flux, but membrane binding of Ca2+ was excluded from contributing to the Na+ gradient-dependent efflux. The Na+ gradient-dependent flux of Ca2+ was very rapid, and even 2- and 5-s points may not fully represent absolute initial rates. It was saturable with respect to the interaction of Ca2+ and Na+ with an apparent (5 s) Km for Na+-dependent Ca2+ uptake of 10 microM, and an apparent (5 s) Vmax of 0.33 nmol/mg protein per 5 s. The Na+ concentration that yielded half maximal Ca2+ efflux (2 s) was 11 mM, and the Hill coefficient was two or greater. Both Na+ gradient dependent and independent Ca2+ efflux were decreased in BLMV prepared from kidneys of thyroparathyroidectomized (TPTX) dogs, and both were stimulated by parathyroid hormone (PTH) infusion to TPTX dogs. BLMV from TPTX dogs exhibited significantly reduced maximal stimulation of Na+ gradient-dependent Ca2+ uptake with an apparent (5 s) Vmax of 0.23 nmol/mg protein per 5 s, but the apparent Km was 8 microM, which was unchanged from normal. The Na+ gradient independent Ca2+ uptake was also reduced in BLMV from TPTX dogs compared with normal. Thus, PTH stimulated both Na+/Ca2+ exchange activity and Na+ independent Ca2+ flux. In vivo, the latter could result in an elevation of cytosolic Ca2+ by PTH, and this might contribute to the observed decrease in solute transport in the proximal tubule.

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J E Scoble, S Mills, K A Hruska

Relationship between insulin-mediated glucose disposal and lipid metabolism in man.

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To assess the possible effects of lipid metabolism on insulin-mediated glucose disposal, 18 nondiabetic Pima Indian women (age 18-35 yr) were studied using 1-14C-palmitate infusion to measure free fatty acid turnover rate followed by a euglycemic clamp (clamp) to measure in vivo insulin-mediated glucose disposal (M). Indirect calorimetry was performed in the basal state and during the clamp. This was used to assess glucose oxidation rate, lipid oxidation rate, and to calculate nonoxidative glucose disposal (storage). Basal and clamp lipid oxidation rate correlated with basal plasma free fatty acid concentration (r = 0.81, P less than or equal to 0.0001, r = 0.67, P less than 0.003, respectively). The fall in lipid oxidation was highly correlated with the increase in glucose oxidation during the insulin infusion (r = 0.96, P less than or equal to 0.0001). The clamp lipid oxidation rate negatively correlated with the glucose oxidation rate (r = -0.85, P less than 0.0001) and with the M value (r = -0.60, P less than 0.01) but was not correlated with the clamp glucose storage (r = -0.2, P = 0.4). On the other hand, glucose storage appeared to make a greater contribution to the difference in M value between the upper and lower extremes of M than did glucose oxidation, as evidenced by an increase in glucose storage of 0.59 mg/kg fat-free mass times minute per 1 mg/kg fat-free mass times minute increase in glucose disposal. The M value was negatively correlated with obesity as measured by percent body fat (r = -0.64, P less than 0.004), but neither basal free fatty acid concentration, basal free fatty acid turnover, basal lipid oxidation, nor clamp lipid oxidation correlated with percent body fat. We conclude that an interaction of lipid and glucose metabolism in a glucose fatty acid cycle, as proposed by Randle et al. (1), may be operative in the regulation of glucose oxidation in man. The disposal of glucose however has two components. The storage component does not appear to be associated with lipid oxidation in the way that the oxidative component is and may be regulated by a different mechanism. Since the results show that the glucose storage component plays a significant role in distinguishing between those with low and high M values, we suggest that the glucose fatty acid cycle can, at best, only partially explain impaired in vivo insulin-mediated glucose disposal. Furthermore, the data suggest that the impact of obesity on in vivo insulin resistance appears to be mediated by factors other than changes in lipid availability or metabolism.

Authors

S Lillioja, C Bogardus, D M Mott, A L Kennedy, W C Knowler, B V Howard

Validation of the difference in urine and blood carbon dioxide tension during bicarbonate loading as an index of distal nephron acidification in experimental models of distal renal tubular acidosis.

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Recent classifications of the several pathophysiologic types of distal renal tubular acidosis (secretory, voltage dependent, and gradient) have been based on the response of acidification parameters to a series of provocative maneuvers in vivo and in vitro. A reduction in the difference in urine and blood CO2 tension during bicarbonate loading (U-B pCO2 gradient), a widely applied parameter, has been employed as an index of reduced distal nephron proton secretion. This study was designed to test the validity of the U-B pCO2 gradient in a variety of experimental models of distal renal tubular acidosis by measuring and comparing disequilibrium pH (a direct technique to detect H+ secretion in situ) with the pCO2 in the papillary collecting duct of the rat in vivo during bicarbonate loading. Chronic amiloride, lithium chloride, and amphotericin-B administration, and the post-obstructed kidney models were employed. Amiloride resulted in an acidification defect which did not respond to sulfate infusion (urine pH = 6.15 +/- 0.08), and was associated with an obliteration of the acid disequilibrium pH (-0.26 +/- 0.05- -0.08 +/- 0.03) and reduction in papillary pCO2 (116.9 +/- 3.2 - 66.9 +/- 2.5 mmHg). The defect induced by lithium administration responded to Na2SO4 (urine pH = 5.21 +/- 0.06) but was similar to amiloride with respect to the observed reduction in disequilibrium pH (-0.04 +/- 0.02) and pCO2 (90.3 +/- 3.0 mmHg). The post-obstructed kidney model was characterized by an abnormally alkaline urine pH unresponsive to sulfate (6.59 +/- 0.06) and a reduction in disequilibrium pH (+0.02 +/- 0.06) and pCO2 (77.6 +/- 3.6 mmHg). Amphotericin-B resulted in a gradient defect as characterized by excretion of an acid urine after infusion of sodium sulfate (5.13 +/- 0.06). Unlike other models, however, amphotericin-B was associated with a significant acid disequilibrium pH (-0.11 +/- 0.05) and an appropriately elevated urine pCO2 (119.8 +/- 6.4 mmHg) which did not differ from the respective values in control rats. Thus, these findings support the use of the U-B pCO2 as a reliable means of demonstrating impaired distal nephron proton secretion in secretory and voltage-dependent forms of distal renal tubular acidosis (RTA) and supports the view that proton secretion is not impaired in gradient forms of distal RTA.

Authors

T D DuBose Jr, C R Caflisch

Plasma and muscle free carnitine deficiency due to renal Fanconi syndrome.

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Plasma and urine free and acyl carnitine were measured in 19 children with nephropathic cystinosis and renal Fanconi syndrome. Each patient exhibited a deficiency of plasma free carnitine (mean 11.7 +/- 4.0 [SD] nmol/ml) compared with normal control values (42.0 +/- 9.0 nmol/ml) (P less than 0.001). Mean plasma acyl carnitine in the cystinotic subjects was normal. Four subjects with Fanconi syndrome but not cystinosis displayed the same abnormal pattern of plasma carnitine levels; controls with acidosis or a lysosomal storage disorder (Fabry disease), but not Fanconi syndrome, had entirely normal plasma carnitine levels. Two postrenal transplant subjects with cystinosis but without Fanconi syndrome also had normal plasma carnitine levels. Absolute amounts of urinary free carnitine were elevated in cystinotic individuals with Fanconi syndrome. In all 21 subjects with several different etiologies for the Fanconi syndrome, the mean fractional excretion of free carnitine (33%) as well as acyl carnitine (26%) greatly exceeded normal values (3 and 5%, respectively). Total free carnitine excretion in Fanconi syndrome patients correlated with total amino acid excretion (r = 0.76). Two cystinotic patients fasted for 24 h and one idiopathic Fanconi syndrome patient fasted for 5 h showed normal increases in plasma beta-hydroxybutyrate and acetoacetate, which suggested that hepatic fatty acid oxidation was intact despite very low plasma free carnitine levels. Muscle biopsies from two cystinotic subjects with Fanconi syndrome and plasma carnitine deficiency had 8.5 and 13.1 nmol free carnitine per milligram of noncollagen protein, respectively (normal controls, 22.3 and 17.1); total carnitines were 11.8 and 13.3 nmol/mg noncollagen protein (controls 33.5, 20.0). One biopsy revealed a mild increase in lipid droplets. The other showed mild myopathic features with variation in muscle fiber size, small vacuoles, and an increase in lipid droplets. In renal Fanconi syndrome, failure to reabsorb free and acyl carnitine results in a secondary plasma and muscle free carnitine deficiency.

Authors

I Bernardini, W B Rizzo, M Dalakas, J Bernar, W A Gahl

Kinetics of rubidium-82 after coronary occlusion and reperfusion. Assessment of patency and viability in open-chested dogs.

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Currently available noninvasive techniques are unable to rapidly assess artery patency and tissue viability during acute myocardial infarction. In prior studies, rubidium-82 (Rb-82), a short-lived positron emitter obtained from a generator, was validated as an indicator of flow with a model that included the rate constants for transfer into and out of the cell. Accordingly, in the current study, 20 open-chested dogs with experimental infarction were studied serially at base line, after coronary occlusion, and at reperfusion. Time-activity curves acquired with beta probes on the epicardial surface were used to measure flow and net transfer of rubidium. Flow decreased to 0.41 +/- 0.08 ml/min per gram during occlusion and increased to 2.73 +/- 0.56 ml/min per gram in potentially viable ischemic tissue, whereas flows were 0.32 +/- 0.08 during occlusion (P less than 0.05 vs. viable) and 1.58 ml/min per gram (P less than 0.002 vs. viable) in irreversibly injured tissue. The transfer rate constant for Rb-82, kT, at base line was +1.22 +/- 0.60 X 10(-3) s-1 and did not change significantly during occlusion in viable vs. nonviable samples (+1.41 +/- 1.27 vs. +0.93 +/- 1.51 X 10(-3) s-1, respectively), except that 4 out of 11 nonviable tissue samples had negative kTs. At reperfusion, viable myocardial samples were all positive (+1.26 +/- 1.58 X 10(-3) s-1), whereas all irreversibly injured tissues had a negative kT, indicating leakage of tracer (-1.50 +/- 1.10 X 10(-3) s-1, P less than 0.001). This study suggests that Rb-82 time-activity curves can be useful to determine patency of an infarct related artery and potential viability after reperfusion during myocardial infarction.

Authors

R A Goldstein

Homology of the NH2-terminal amino acid sequences of the heavy and light chains of human monoclonal lupus autoantibodies containing the dominant 16/6 idiotype.

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The NH2-terminal amino acid sequences have been determined by automated Edman degradation for the heavy and light chains of five monoclonal IgM anti-DNA autoantibodies that were produced by human-human hybridomas derived from lymphocytes of two patients with systemic lupus erythematosus. Four of the antibodies were closely related to the idiotype system 16/6, whereas the fifth antibody was unrelated idiotypically. The light chains of the 16/6 idiotype-positive autoantibodies (HF2-1/13b, HF2-1/17, HF2-18/2, and HF3-16/6) had identical amino acid sequences from residues 1 to 40. Their framework structures were characteristic of VKI light chains. The light chain of the 16/6 idiotype-negative autoantibody HF6-21/28 was characteristic of the VKII subgroup. The heavy chains of the 16/6 idiotype-positive autoantibodies had nearly identical amino acid sequences from residues 1 to 40. The framework structures were characteristic of the VHIII subgroup. In contrast, the GM4672 fusion partner of the hybridoma produced small quantities of an IgG with a VHI heavy chain and a VKI light chain. The heavy chains of the lupus autoantibodies and the light chains of those autoantibodies that were idiotypically related to the 16/6 system had marked sequence homology with WEA, a Waldenstrom IgM that binds to Klebsiella polysaccharides and expresses the 16/6 idiotype. These results indicate a striking homology in the amino termini of the heavy and light chains of the lupus autoantibodies studied and suggest that the V regions of the heavy and light chains of the 16/6 idiotype-positive DNA-binding lupus auto-antibodies are each encoded by a single germ line gene.

Authors

P M Atkinson, G W Lampman, B C Furie, Y Naparstek, R S Schwartz, B D Stollar, B Furie

Cholecystokinin bioactivity in human plasma. Molecular forms, responses to feeding, and relationship to gallbladder contraction.

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A sensitive and specific bioassay for the measurement of cholecystokinin (CCK) in human plasma was developed to determine the molecular forms of CCK in circulation, CCK responses to feeding, and the physiologic role of CCK in gallbladder contraction. First, plasma was quantitatively extracted and concentrated with octadecylsilylsilica, and the extracts were then assayed for their ability to stimulate amylase release from isolated rat pancreatic acini. Acini were highly sensitive to CCK whereas gastrin reacted only weakly in this system. With the assay, plasma levels of cholecystokinin octapeptide (CCK-8) bioactivity as low as 0.2 pM were detectable. CCK bioactivity in plasma was inhibited by the CCK antagonist, bibutyryl cyclic guanosine monophosphate, and was eliminated by immunoadsorption with an antibody directed against the carboxyl terminus of CCK. Detection of fasting levels of CCK was possible in all individuals tested and averaged 1.0 +/- 0.2 pM (mean +/- SE, n = 22) CCK-8 equivalents. Plasma CCK biological activity was normal in patients with gastrin-secreting tumors. After being fed a mixed liquid meal, CCK levels rose within 15 min to 6.0 +/- 1.6 pM. The individual food components fat, protein, and amino acids were all potent stimulants of CCK secretion; in contrast, glucose caused a significant but smaller elevation in plasma CCK levels. Gel filtration studies identified three major forms of CCK bioactivity in human plasma: an abundant form that eluted with CCK-33, a smaller form that eluted with CCK-8, and an intermediate form that eluted between CCK-33 and CCK-8. Ultrasonic measurements of gallbladder volume indicated that this organ decreased 51% in size 30 min after feeding a mixed liquid meal. This contraction occurred coincidentally with the increase in plasma CCK levels. Next CCK-8 was infused to obtain CCK levels similar to postprandial levels. This infusion caused a decrease in gallbladder volume, similar to that seen with a meal. The present studies indicate, therefore, that CCK can be bioassayed in fasting and postprandial human plasma. These studies also suggest that CCK may be an important regulator of gallbladder contraction.

Authors

R A Liddle, I D Goldfine, M S Rosen, R A Taplitz, J A Williams

Characterization of the adhesion of the human monocytic cell line U937 to cultured endothelial cells.

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Adhesion of blood-borne monocytes to the vascular endothelium is the first step in the infiltration of this leukocyte into the vessel wall or the interstitial space during inflammation. A significant role for the monocyte in both wound healing and atherogenesis is now well accepted. The molecular interactions involved in monocyte attachment to the endothelium are unknown. To study this phenomenon we have developed an in vitro system that uses the human monocytic tumor cell line U937 as a model for the blood-borne monocyte. 51Cr-labeled U937 cells were found to adhere with high affinity to cultured endothelial cells (ECs) from several sources. Much less binding was observed to either smooth muscle cells or fibroblasts from several species. Conditioned medium and cocultivation experiments ruled out the possibility that target cells could affect U937 cell binding by secretion of factors. Binding of U937 cells to porcine aortic ECs reached equilibrium after 30 min at 37 degrees C and 90 min at 4 degrees C with similar extent of binding at the two temperatures. Binding of U937 to the endothelium reached saturation at 9-12 U937 per porcine aortic EC (semi-confluent) with half-maximal binding at 1.5 X 10(6) U937 cells/ml. Bound cells dissociated with a half-life of 20 h at 37 degrees C. Adhesion of U937 cells was blocked by prior incubation of ECs with normal monocytes but not with platelets, lymphocytes, or neutrophils. Trypsin treatment or detergent solubilization of ECs inhibited U937 cell binding. A striking effect of EC density on monocytic cell adhesion was observed with bovine, rat, and porcine ECs. Confluent cultures of these cells exhibited negligible binding of U937, but when plated sparsely, the same cells were excellent targets for U937 cell adhesion. In addition, when confluent cultures of bovine aortic ECs were "wounded" with a cotton swab and then allowed to recover for 24 h at 37 degrees C, U937 cells were found to adhere most readily to the ECs migrating into the wound and neighboring the wound but not to ECs in the confluent monolayer away from the wound edge. These latter results may have implications for the focal adhesion of monocytes to the vessel wall in vivo.

Authors

P E DiCorleto, C A de la Motte

In vitro cytotoxicity and transplantation protection by autologous natural and activated killer cells against an in vitro transformed tumorigenic fibroblast line. A case study.

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Cytotoxic immune response by autologous natural killer (NK) cells against a spontaneous in vitro transformed tumorigenic fibroblast line, VIP-F:T, was studied in a 4 h 51Cr-release microcytotoxicity assay and in a tumor cell neutralization technique in vivo in nude mice. Although highly cytotoxic against the NK prototype target K562, the autologous NK cells in their nascent state were only marginally cytotoxic against VIP-F:T and unreactive against the autologous normal fibroblasts, Pen-F2. Autologous NK activity against VIP-F:T could, however, be induced by 2-16-h treatment of the NK cells with several species of interferon and by interferon-free interleukin 2 (IL-2). In vitro co-culture (IVC) in IL-2 of autologous peripheral blood lymphocytes (PBL) against VIP-F:T was shown by fluorescence activated cell sorting and by cold target competition experiments to generate almost exclusively an effector population bearing HNK-1 and Leu-11a phenotypes which exhibited receptor specificity for VIP-F:T distinct from receptors on Pen-F2 or K562 cells. PBL, co-cultured in IL-2 against Pen-F2 or K562, or cultured in IL-2 alone, generated high levels of nonspecific killing and showed no receptor specificity. Identical IVC in IL-2 of autologous PBL against a melanoma line, VIP (PBL and the VIP line derived from the same patient from whom the VIP-F:T line was also derived), and similar IVC in IL-2 of several other autologous PBL against their corresponding target cell lines (established from surgical specimens) generated cytotoxic responses involving cytotoxic populations bearing T8 as well as HNK-1 phenotypes; but the cytotoxic activities in none of these systems showed target receptor specificity. Autologous PBL, co-cultured against VIP-F:T in IL-2, were shown to be capable of rejecting tumorigenic challenge with VIP-F:T.3 (a clone of VIP-F:T) in nude mice at effector to VIP-F:T ratio of 10:1. The protective effect of the co-culture activated PBL was abrogated if the HNK-1+ cells were depleted from the effector population. Our data, thus, demonstrate specificity of cytotoxic reactivity which, by phenotypic markers, can be characterized as HNK-1 and Leu 11a+ cells under these experimental conditions against this particular in vitro transformed VIP-F:T line. In addition, this study shows that similar studies of cytotoxic autologous reactivities against in vitro transformed target cell lines will provide valuable information on the subject of NK-mediated surveillance against human neoplasia.

Authors

S A Wilhelm, B Mukherji

Antithrombotic properties in rabbits of heparin and heparin fragments covalently coupled to human antithrombin III.

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Clinical grade heparin is a very heterogeneous mucopolysaccharide, containing molecules with Mr ranging from 6,000 to 30,000 that have either a high affinity or a low affinity for antithrombin III (AT). In this study, the antithrombotic properties of intact high-affinity heparin (Mr = 15,000) and of two heparin fragments (h16, a 16-monosaccharide fragment, with Mr = 4,300, and h12, a 12-monosaccharide fragment, with Mr = 3,200) and of their functional covalent stoichiometric complexes with human AT were compared in a venous thrombosis stasis model in rabbits. Thrombosis was induced by injection of glass-activated human plasma and measured in a segment of the jugular vein that was isolated between two vascular clamps for 10 min. Injections of 55 micrograms/kg resulted in a clear antithrombotic effect for intact heparin, but not for the two fragments. Equivalent amounts (carbohydrate moiety) of covalent complexes of heparin or of both heparin fragments with human AT resulted in an antithrombotic effect lasting for 45-60 min. Injection of 110 micrograms/kg of heparin and of the heparin fragments yielded an antithrombotic effect, lasting 45-60 min; the corresponding amounts of covalent complexes caused an anti-thrombotic effect for 60-120 min. The free and conjugated fragments produced equal antithrombotic effects at equal plasma levels of anti-Factor Xa activity, but the specific antithrombotic activities of free and complexed intact heparin, on a molar basis, were 10-20-fold greater than those of the free and complexed heparin fragments. The plasma half-life of the covalent complexes of the heparin fragments with AT is, however, 10 times longer than that of the complex between intact heparin and AT and 30 times longer than that of free intact heparin. Covalent complexes between AT and heparin fragments could, therefore, be useful to maintain more stable levels of antithrombotic activity in plasma.

Authors

C Mattsson, M Hoylaerts, E Holmer, T Uthne, D Collen

Purification and partial characterization of a megakaryocyte colony-stimulating factor from human plasma.

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Human plasma obtained from patients with hypomegakaryocytic thrombocytopenia contains a factor that promotes megakaryocyte colony formation by normal human marrow cells. This megakaryocyte colony-stimulating factor was purified from such a plasma specimen. A four-step purification scheme which included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, affinity chromatography on wheat germ lectin-Sepharose 6MB, and reverse-phase high performance liquid chromatography resulted in a recovery of 16.6% of the initial biological activity and an increase in specific activity by 3,489-fold. The purified protein produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified megakaryocyte colony-stimulating factor was capable of promoting megakaryocyte colony formation at a concentration of 7.6 X 10(-8) M. Megakaryocyte colony-stimulating factor was shown to be a glycoprotein and had an apparent 46,000 mol wt. Deglycosylation of megakaryocyte colony-stimulating factor by treatment with trifluoromethane-sulfonate resulted in the loss of its ability to promote megakaryocyte colony formation. Megakaryocyte colony-stimulating factor appears to be an important regulator of in vitro human megakaryocytopoiesis at the level of the colony-forming unit megakaryocyte and may be of importance physiologically.

Authors

R Hoffman, H H Yang, E Bruno, J E Straneva

Platelet antibodies of the IgM class in immune thrombocytopenic purpura.

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The clinical course and response to therapy of patients with immune thrombocytopenic purpura (ITP) are not completely determined by the level of IgG present on the platelet surface. It is possible that antibodies of other immunoglobulin classes also play a role in platelet destruction in some of these patients. Therefore, we studied 175 patients with ITP for the presence of IgM anti-platelet antibodies using radiolabeled polyclonal or monoclonal anti-IgM. We observed that 57% of patients with clinical ITP had increased levels of IgM on their platelets, compared with normal controls and patients with thrombocytopenia who did not have ITP (less than 10%), (P less than 0.01). We obtained similar results using either radiolabeled polyclonal or monoclonal anti-IgM, reagents whose integrity was first characterized using erythrocytes coated with defined amounts of IgM antibody. Among patients with increased platelet-IgM there was a significant correlation both with the presence of increased platelet-C3 as well as the amount of platelet-C3 (P less than 0.01, r = 0.53). We demonstrated the presence of warm-reacting IgM anti-platelet antibodies in the plasma of two of these patients who were further studied. The isolated IgM fraction from these two plasmas was able to activate complement and place 3H-C3 on normal platelets. These studies demonstrate the presence of warm-reacting IgM anti-platelet antibodies in some patients with ITP. They suggest that the binding of complement to platelets by IgM antibodies may initiate platelet clearance as well as enhance the effect of IgG antibodies in ITP.

Authors

D B Cines, S B Wilson, A Tomaski, A D Schreiber

Bone and parathyroid inhibitory effects of S-2(3-aminopropylamino)ethylphosphorothioic acid. Studies in experimental animals and cultured bone cells.

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S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR 2721) is a radio- and chemoprotective agent which produces hypocalcemia in humans. Intravenous injection of 30 mg/kg WR 2721 in rats and 15 mg/kg in dogs lowers serum calcium by 19 and 25%, respectively. Hypocalcemia in dogs is associated with a fall in serum immunoreactive parathyroid hormone (PTH), which suggests that the mechanism of its hypocalcemic effect is acute hypoparathyroidism. Despite this effect on PTH, in eight chronically parathyroidectomized rats on a low phosphate diet, WR 2721 reduced serum calcium from 9.4 +/- 0.6 to 7.7 +/- 0.5 mg/dl (P less than 0.01) at 3 h. Furthermore, in dogs rendered hypercalcemic by continuous infusion of PTH, WR 2721 reduced serum calcium from 11.0 +/- 0.5 to 10.6 +/- 0.5 mg/dl (P less than 0.01). To determine whether WR 2721 causes hypocalcemia by enhancing the exit of calcium from the circulation or inhibiting its entry, the drug was infused 3 h after administration of 45Ca to rats. WR 2721 did not significantly increase the rate of disappearance of 45Ca from the circulation even though serum calcium fell by 19%. However, serum 45Ca specific activity was higher at 1.5 h (P less than 0.01) and 3 h (P less than 0.05) in rats given WR 2721 than in rats given vehicle alone, which suggests that WR 2721 blocks the entry of nonradioactive calcium into the circulation, presumably from bone. In incubations with fetal rat long bone labeled in utero with 45Ca, 10(-3) M WR 2721 inhibited PTH-stimulated, but not base-line release of 45Ca. Bone resorption by primary culture of chick osteoclasts was inhibited by WR 2721 at concentrations as low as 10(-4) M in the absence of hormonal stimulation. These studies suggest that WR 2721 lowers serum calcium predominantly by blocking calcium release from bone. This acute hypocalcemic effect is at least in part independent of its effect on the parathyroid glands, and is most likely a direct effect of the agent on bone resorption.

Authors

M F Attie, M D Fallon, B Spar, J S Wolf, E Slatopolsky, S Goldfarb

Interaction of asialo von Willebrand factor with glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and mediates platelet aggregation.

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von Willebrand factor (vWF) is necessary for the initial attachment of platelets to exposed subendothelium, particularly under flow conditions like those prevailing in the microcirculation. Little is known about its possible participation in subsequent events leading to formation of platelet thrombi at sites of vascular injury. We addressed this question by studying the mechanisms by which desialylated vWF induces platelet aggregation in the absence of any other stimulus. Asialo vWF, unlike the native molecule, does not require ristocetin to interact with platelets. Agglutination induced by ristocetin is largely independent of active platelet metabolism and only partially reflects physiological events. We have shown here that binding of asialo vWF to platelets was accompanied by release of dense granule content and subsequent ADP-dependent fibrinogen binding to receptors on the glycoprotein (GP) IIb/IIIa complex. The initial interaction of asialo vWF with platelets was mediated by GPIb, as shown by blocking obtained with monoclonal antibody. Inhibition of this initial interaction completely abolished platelet aggregation induced by asialo vWF. The same effect was obtained with a monoclonal anti-GPIIb/IIIa antibody. This, however, did not block asialo vWF binding to platelets, but rather inhibited subsequent fibrinogen binding induced by asialo vWF. Therefore, the latter process was also essential for platelet aggregation under the conditions described. At saturation, asialo vWF induced binding of between 3.2 and 27.7 X 10(3) fibrinogen molecules/platelet, with an apparent dissociation constant between 0.28 and 1.18 X 10(-6) M. This study shows that asialo, and possibly native, vWF acts as a platelet agonist after its binding to GPIb and induces aggregation through a pathway dependent on GPIIb/IIIa-related receptors.

Authors

L De Marco, A Girolami, S Russell, Z M Ruggeri

Regulation of myocardial amino acid balance in the conscious dog.

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The effects in vivo of physiologic increases in insulin and amino acids on myocardial amino acid balance were evaluated in conscious dogs. Arterial and coronary sinus concentrations of amino acids and coronary blood flow were measured during a 30-min basal and a 100-min experimental period employing three protocols: euglycemic insulin clamp (plasma insulin equaled 70 +/- 11 microU/ml, n = 6); euglycemic insulin clamp during amino acid infusion (plasma insulin equaled 89 +/- 12 microU/ml, n = 6); and suppression of insulin with somatostatin during amino acid infusion (plasma insulin equaled 15 +/- 4 microU/ml, n = 6). Basally, only leucine and isoleucine were removed significantly by myocardium (net branched chain amino acid [BCAA] uptake equaled 0.5 +/- 0.2 mumol/min), while glycine, alanine, and glutamine were released. Glutamine demonstrated the highest net myocardial production (1.6 +/- 0.2 mumol/min). No net exchange was seen for valine, phenylalanine, tyrosine, cysteine, methionine, glutamate, asparagine, serine, threonine, taurine, and aspartate. In group I, hyperinsulinemia caused a decline of all plasma amino acids except alanine; alanine balance switched from release to an uptake of 0.6 +/- 0.4 mumol/min (P less than 0.05), while the myocardial balance of other amino acids was unchanged. In group II, amino acid concentrations rose, and were accompanied by a marked rise in myocardial BCAA uptake (0.4 +/- 0.1-2.6 +/- 0.3 mumol/min, P less than 0.001). Uptake of alanine was again stimulated (0.9 +/- 0.3 mumol/min, P less than 0.01), while glutamine production was unchanged (1.3 +/- 0.4 vs. 1.6 +/- 0.3 mumol/min). In group III, there was a 4-5-fold increase in the plasma concentration of the infused amino acids, accompanied by marked stimulation in uptake of only BCAA (6.8 +/- 0.7 mumol/min). Myocardial glutamine production was unchanged (1.9 +/- 0.4-1.3 +/- 0.7 mumol/min). Within the three experimental groups there were highly significant linear correlations between myocardial uptake and arterial concentration of leucine, isoleucine, valine, and total BCAA (r = 0.98, 0.98, 0.92, and 0.97, respectively); P less than 0.001 for each). In vivo, BCAA are the principal amino acids taken up by the myocardium basally and during amino acid infusion. Plasma BCAA concentration and not insulin determines the rate of myocardial BCAA uptake. Insulin stimulates myocardial alanine uptake. Neither insulin nor amino acid infusion alters myocardial glutamine release.

Authors

R G Schwartz, E J Barrett, C K Francis, R Jacob, B L Zaret

Transport of urokinase across the intestinal tract of normal human subjects with stimulation of synthesis and/or release of urokinase-type proteins.

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Abstract

Oral administration of clinical-type high molecular weight urokinase (HMW-UK) in a single dose of 30,000-60,000 International Units (IU) in enteric-coated capsules, in a group of normal human subjects, induced a plasma fibrinolytic state suggesting transport of HMW-UK across the intestinal tract. Other groups of human subjects were given a single dose of 120,000 IU daily of pure HMW-UK for 1 d and 7 d together with a placebo dose, all of the ingredients except urokinase (UK), daily for 7 d. UK-type proteins were isolated from the plasma of blood samples drawn 6 h after administration of the final dose, by a sequential two-step affinity chromatography method first with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose and second with a specific rabbit anti-HMW-UK-IgG-Sepharose. The yield of UK-type proteins from the 7-d group, 0.79 mg/dl, was approximately twofold greater than that obtained from either the placebo or 1-d groups. The specific plasminogen activator activity of the 1-d and 7-d groups were similar, 508 and 537 IU/mg protein, respectively; negligible plasminogen activator activity could be detected in the placebo group. The kinetics of activation parameters of human Glu-plasminogen of the UK-type protein, isolated from the 1-d group, were similar to those obtained with urinary HMW-UK. The UK-type proteins isolated only from the 7-d group showed, in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a major protein band of molecular weight 53,000 in the same position as HMW-UK. In addition, two other major protein bands of approximately 140,000 and approximately 120,000 mol wt were found in the 7-d placebo-, and 1-d groups, and also in the 7-d group. The 53,000 mol wt protein, about 50% of the total protein in the 7-d group, was further purified by preparative SDS-polyacrylamide gel electrophoresis, and found to be a two-chain protein with a specific activity of 1,241 IU/mg protein. The protein showed common antigenic determinants with urinary HMW-UK. The oral administration of 120,000 IU HMW-UK to human subjects for 7 d stimulated the synthesis of a UK-type protein of 53,000 mol wt, probably the zymogen, from either the liver or vascular endothelium, which was released into the circulation, and converted into active UK by circulating plasmin. The induction of the fibrinolytic state produced the conversion of native circulating Glu-plasminogen into the degraded Lys-plasminogen form, which was found in large amounts in the plasma of the 7-d group. The new plasma components, e.g., the 53,000-mol wt UK-zymogen and Lys-plasminogen, could play an important role in clot resolution of the fibrin-thrombus in thromboembolic diseases. Oral administration of HMW-UK has been shown to be clinically effective in patients with cerebral thrombosis in a multicenter double blind study.

Authors

N Toki, H Sumi, K Sasaki, I Boreisha, K C Robbins

Stimulation of the proliferation of human bone cells in vitro by human monocyte products with interleukin-1 activity.

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Abstract

The process of induction of bone formation, which follows bone resorption during normal and pathological bone turnover, is well documented. However, the mechanisms responsible for this process are unclear. Mononuclear phagocytes present at the sites of bone remodeling could play a role in this "coupling" of bone formation to bone resorption. This study was designed to investigate such a possibility. By measuring both the increase in [3H]thymidine incorporation and in cell number, we found that human monocytes in culture released factors capable of stimulating the proliferation of osteoblast-like cells derived from human bone. Rapidly dividing cells exhibited a greater response to interleukin 1 (IL-1) than confluent cells. The factors are similar to IL-1 in that they exhibited the same molecular weight and isoelectric point, were present in fractions that contained IL-1 activity after gel filtration chromatography and isoelectric focusing, and showed similar dose-response characteristics. Proliferation was more marked when prostaglandin production by the cells, which was also stimulated by these factors, was inhibited by indomethacin. A factor produced by monocytes that affects osteoblast activity may be important in the coupling of osteoclast and osteoblast actions.

Authors

M Gowen, D D Wood, R G Russell

Isolation and partial characterization of a human alveolar macrophage-derived neutrophil-activating factor.

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Abstract

Human alveolar macrophages (AM) were obtained from eight normal volunteers using fiberoptic bronchoscopic lavage to explore potential interrelationships among leukocytes in pulmonary defense against infection. AM placed in monolayer tissue cultures released material into culture supernatants with the capacity to enhance the bactericidal capacity of human neutrophils. Neutrophils preexposed to supernatants killed Pseudomonas aeruginosa from 70 to 90% more efficiently than control cells (P less than 0.02). AM culture supernatants contained this material by 4 h of incubation, and in vitro stimulation of AM cultures with heat-killed P. aeruginosa further increased its production. Gel filtration of AM culture supernatants with a G-50 Sephadex column allowed isolation of a 6,000-D neutrophil-activating factor (NAF) that was resistant to heat (56 degrees C, 30 min). The isoelectric point of NAF, as determined by chromatofocusing, was approximately 7.6. Enzyme digestion of NAF specimens, prepared sequentially by gel filtration and chromatofocusing, demonstrated 50-70% loss of activity after incubations with trypsin, chymotrypsin, and neuraminidase. NAF was only minimally chemotactic and eluted from Sephadex G-50 with particles of a different molecular size than those of AM-derived chemotactic factors (i.e., approximately 10,000 D and less than 500 D). Preincubation of neutrophils with NAF resulted in greater release of superoxide anion upon their subsequent stimulation by either bacterial phagocytosis or by phorbol myristate acetate, as compared with control neutrophils stimulated in a like manner. These studies indicate that human AM secrete a heat-stable, low molecular weight basic protein, with the capacity to enhance oxidative microbicidal activity of neutrophils.

Authors

J E Pennington, T H Rossing, L W Boerth, T H Lee

Decreased serum triiodothyronine in starving rats is due primarily to diminished thyroidal secretion of thyroxine.

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Abstract

Although thyroxine (T4) 5'-deiodinase activity is diminished in liver homogenates of starved rats, no information is available regarding the effect of starvation on net T4 to triiodothyronine (T3) conversion in the intact rat. It appeared important to clarify this relationship since rat liver homogenates are widely used as a model for the study of the factors responsible for reduced circulating T3 in chronically ill and calorically deprived patients. In contrast to the expected selective decrease in circulating T3 levels in calorically restricted humans due to diminished T4 to T3 conversion, 5 d of starvation of two groups of male Sprague-Dawley rats resulted, paradoxically, in a greater decrease in serum T4 than in serum T3 levels. Kinetic studies show that starvation is associated with no change in the metabolic clearance rate (MCR) of T3, a 20% increase in the MCR of T4, a 67% reduction in turnover rate of T4, but only a 58% reduction in the turnover rate of T3. Moreover, in the first group of rats studied, direct chromatographic analysis of the isotopic composition of total body homogenates after the injection of 125I-T4 showed that 21.8% of T4 is converted to T3 in control rats and 28.8% in starved rats, suggesting that virtually all extrathyroidal T3 in starved and control rats is derived from the peripheral conversion of T4, and that there is little or no direct thyroidal secretion of T3. Our findings strongly point to a reduced thyroidal secretion of T4 as the primary cause of the observed reduction in circulating T3. Since the mechanisms leading to reduced levels of plasma T3 differ in humans and rats, it may be important to reexamine the use of liver homogenate preparations as models for study of the pathogenesis of the "low T3 syndrome" in humans.

Authors

W B Kinlaw, H L Schwartz, J H Oppenheimer

Functional role of thromboxane production by acutely rejecting renal allografts in rats.

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Abstract

We investigated the role of thromboxane in mediating the reduction in renal function and renal blood flow characteristic of acute renal allograft rejection. We transplanted kidneys from Lewis rats to Brown-Norway recipients. By the third day after transplantation, histologic changes that were consistent with cellular rejection occurred in the kidney. These changes were associated with a moderate reduction in renal function. By day 6, histologic changes of rejection were advanced and included interstitial and perivascular infiltration by mononuclear cells. The clearances of inulin and para-aminohippuric acid were also markedly reduced. As renal function deteriorated, thromboxane B2 (TXB2) production by ex vivo perfused renal allografts increased progressively from 2 to 6 d after transplantation. However, prostaglandin (PG) E2 and 6-keto PGF1 alpha production remained essentially unchanged. There was a significant inverse correlation between the in vivo clearance of inulin and the log of ex vivo TXB2 production. Infusion of the thromboxane synthetase inhibitor UK-37248-01 into the renal artery of 3-d allografts significantly decreased urinary TXB2 excretion and significantly increased renal blood flow (RBF) and glomerular filtration rate (GFR). Although renal function improved significantly after the acute administration of UK-37248-01, GFR and RBF did not exceed 33 and 58% of native control values, respectively. In other animals, daily treatment with cyclophosphamide improved the clearances of inulin and para-aminohippuric acid and reduced thromboxane production by 6-d renal allografts. These studies demonstrate that histologic evidence of rejection is associated with increased renal thromboxane production. Inhibition of thromboxane synthetase improves renal function in 3-d allografts. Cytotoxic therapy improves renal function, reduces mononuclear cell infiltration, and decreases allograft thromboxane production. Thus, the potent vasoconstrictor thromboxane A2 may play a role in the impairment of renal function and renal blood flow during acute allograft rejection.

Authors

T M Coffman, W E Yarger, P E Klotman

Effects of cysteamine and antibody to somatostatin on islet cell function in vitro. Evidence that intracellular somatostatin deficiency augments insulin and glucagon secretion.

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Abstract

In this study we have characterized the effects of cysteamine (CHS) on the cellular content and release of immunoreactive somatostatin (S-14 LI), insulin (IRI), and glucagon (IRG) from monolayer cultures of neonatal rat islets. Incubation of cultures with 0.1-10 mM CHS for 1 h led to an apparent, dose-dependent reduction of cellular S-14 LI that was 50% of control at 0.3 mM, 87% at 1 mM, and 95% at 10 mM. IRI content was unaffected by CHS up to 1 mM, but at 10 mM 90% loss of IRI occurred. All concentrations were without effect on IRG content. The loss of S-14 LI and IRI was completely reversible with time, but with different recovery rates for the two hormones (48 h for S-14 LI, and 72 h for IRI). Released S-14 LI rose progressively with increasing doses of CHS from 21 +/- 2.5 pg/ml per hour to 41 +/- 1.4 pg/ml per hour at CHS concentrations of 5 mM and 10 mM. IRI and IRG secretion were both also significantly enhanced (by 55% and 88%, respectively), despite the elevated medium S-14 LI. Since CHS reduced cellular S-14 LI but augmented medium S-14 LI, the relative effects of CHS (1 mM) and immunoneutralization with antibody to S-14 LI on IRI and IRG secretion were tested. Anti S-14 LI alone stimulated basal IRG (67%) but not IRI. Cultures rendered S-14 LI deficient with both CHS and anti-S-14 LI exhibited threefold and 2.3-fold potentiation of IRG and IRI secretions, respectively, greater than that expected from the separate effects of the two agents. Increasing medium glucose from 2.8 mM to 16.7 mM stimulated IRI release by 86% and suppressed IRG by 53%. CHS (1 mM) and anti-S-14 LI further augmented stimulated IRI release, by 30%; although 16.7 mM glucose suppression of IRG was still maintained under these conditions, the quantitative IRG response was significantly greater. These results suggest that CHS induces an apparent loss of islet S-14 LI, and at high doses, of IRI as well, but has no effect on A cells. Complete islet S-14 LI deficiency augments IRI and IRG secretion over a wide range of glucose concentrations, suggesting a physiological role of D cells on B cell and A cell regulation. D cell modulation of B cells requires cellular but not extracellular S-14 LI, being mediated probably though direct intracellular communication, whereas the A cells seem to be regulated by both direct contact as well as through locally secreted S-14 LI.

Authors

Y C Patel, I Pierzchala, M Amherdt, L Orci

Evidence for carrier-mediated chloride/bicarbonate exchange in canalicular rat liver plasma membrane vesicles.

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Abstract

To determine whether anion exchangers might play a role in hepatic bile formation, we looked for the presence of Cl-:OH- and Cl-:HCO3- exchange in highly purified canalicular (c) and basolateral (bl) rat liver plasma membrane (LPM) vesicles. In cLPM vesicles, a pH gradient (7.7 in/6.0 out) stimulated 36Cl- uptake twofold above values obtained during pH-equilibrated conditions (7.7 in = out). When 50 mM HCO3- was also present inside the vesicles, the same pH gradient (7.7 in/6.0 out) resulted in Cl- uptake to levels fourfold above pH- and HCO3--equilibrated controls and two- to threefold above Cl- equilibrium (overshoot). Initial rates of both pH and HCO3- gradient-stimulated Cl- uptake were completely inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). A valinomycin-induced K+ diffusion potential (inside positive) also stimulated Cl- uptake in cLPM, but this conductive Cl- pathway was insensitive to DIDS. The DIDS-sensitive, pH and HCO3- gradient-stimulated Cl- uptake demonstrated: saturation with Cl- (Km approximately 6.3 mM; Vmax approximately 51 nmol X mg-1 X min-1); partial inhibition by bumetanide (26%), furosemide (33%), probenecid (37%), and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (49%); cis-inhibition by chloride and nitrate but not by sulfate and various organic anions, and independence from the membrane potential. These data demonstrate the presence of an electroneutral Cl-:OH- and Cl-:HCO3- exchanger in rat liver canalicular membranes that favors Cl-:HCO3- exchange. In contrast, no evidence was found for the presence of a Cl-:HCO3- (OH-) exchange system in blLPM vesicles. Furthermore, neither blLPM nor cLPM vesicles exhibited Na+-stimulatable Cl- uptake, indicating the absence of a NaCl co-transport system in either LPM subfraction. These findings are consistent with a functional role for a Cl-:HCO3- (OH-) exchanger in canalicular bile formation.

Authors

P J Meier, R Knickelbein, R H Moseley, J W Dobbins, J L Boyer

Evidence for reduced thermic effect of insulin and glucose infusions in Pima Indians.

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Abstract

Several authors have reported a reduced thermic effect of food in obese subjects. The hyperinsulinemic-euglycemic clamp technique has been used to measure one component of the thermic effect of food, insulin and insulin-mediated glucose disposal. We used this technique to measure the thermic responses to insulin and glucose infusions in 120 glucose-tolerant Pima Indians, a population with a high prevalence of obesity. During high-dose insulin infusions (400 mU/m2 per min) the measured increase in energy expenditure (MEE), 150 +/- 6 cal/min, was greater than the predicted increase in energy expenditure (PEE), 72 +/- 2 cal/min, for glucose storage as glycogen. During low-dose insulin infusions (40 mU/m2 per min) the mean MEE, 6 +/- 5 cal/min, was not significantly different from zero and was not greater than the mean PEE, 9 +/- 1 cal/min. These data were in contrast to results obtained from Caucasians by others and suggested a markedly reduced thermic effect of low-dose insulin and glucose infusions in Pima Indians. We also studied 23 glucose-tolerant male Caucasians and compared their results with the results from male Indians matched for glucose storage rates and obesity. The results showed that the thermic response to insulin and glucose infusions was similar in the two racial groups during high-dose insulin infusions but was markedly reduced in the Indians compared with the Caucasians during low-dose insulin infusions.

Authors

C Bogardus, S Lillioja, D Mott, J Zawadzki, A Young, W Abbott

Subclass restriction and polyclonality of the systemic lupus erythematosus marker antibody anti-Sm.

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Abstract

Anti-Sm antibodies are highly specific markers for the diagnosis of systemic lupus erythematosus (SLE). This specificity suggests that the immunoregulation of these autoantibodies would reflect fundamental immune abnormalities in this disorder. As a clue to this immunoregulation, we have investigated the isotype distribution of anti-Sm antibodies by enzyme-linked immunosorbent assays. We have found that the anti-Sm response is markedly restricted to the IgG1 heavy chain isotype. On the other hand, the light chain distribution reflects that in normal serum, while isoelectric focusing analysis fails to show an oligoclonal pattern. The related specificity, anti-ribonucleoprotein, is also restricted to IgG1, while the SLE-specific antibody anti-double-stranded DNA is mostly IgG1 with a lesser contribution by IgG3. These results suggest that antinuclear antibodies that are strongly associated with SLE are produced by a T cell-dependent response, probably driven by antigen. The immunoregulation of the response to several autoantigens may be quite similar.

Authors

R A Eisenberg, K Dyer, S Y Craven, C R Fuller, W J Yount

Identification of three accessory cell populations in human bone marrow with erythroid burst-promoting properties.

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Abstract

Several laboratories have demonstrated a requirement for burst-promoting activity (BPA), a product of T cells, or T cell/monocyte collaboration in the induction of differentiation of peripheral blood erythroid burst-forming units (BFU-E) in vitro. The physiologic significance of this finding is brought into question by patients with severe mature T cell deficiency who have normal in vivo erythropoiesis. The studies described here were designed to determine whether the burst-promoting effects of marrow T cells and adherent cells are similar to those of peripheral blood, to define whether a third population of marrow cells is capable of production of BPA, and to describe the BPA requirements of immature and mature marrow erythroid progenitors. To that end we prepared adherence- and E-depleted low-density peripheral blood mononuclear cells as a source of BFU-E and demonstrated that their optimal erythropoietin-induced differentiation requires BPA. We then determined that both bone marrow and peripheral blood T cells and monocytes could provide the necessary BPA to induce their erythropoietin dependent differentiation. BPA production by T cells was sensitive to irradiation, but that of the whole bone marrow low-density population was considerably less sensitive. This in itself demonstrated that BPA production in marrow is not T cell dependent. We further demonstrated a potent, albeit infrequent, third population of BPA-producing marrow cells. These proved to be nonadherent, E receptor-negative, granulocyte antigen-negative, and gamma-Fc receptor-positive. Finally, we separated all of these BPA-producing cells from marrow erythroid progenitors and concentrated the latter into a population in which they comprised 6% of the cells. With this population we demonstrated that both immature (BFU-E) and mature (colony-forming units [CFU-E]) erythroid progenitors require BPA in addition to erythropoietin to induce them to form erythroid colonies in vitro. These results may explain the normal erythropoiesis found in patients with mature T cell deficiency. Though the differentiation of both BFU-E and CFU-E requires BPA, this need can be met by a special class of nonadherent, radioresistant, E receptor-negative, granulocyte antigen-negative, and gamma-Fc-positive cells.

Authors

D C Linch, J M Lipton, D G Nathan

Correction of abnormal renal blood flow response to angiotensin II by converting enzyme inhibition in essential hypertensives.

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Abstract

In 40-50% of patients with essential hypertension, a high sodium intake does not increase renal blood flow (RBF). These patients have been defined as nonmodulators because sodium intake does not modulate renal and adrenal responsiveness to angiotensin II (AII). To define the role of AII in mediating this altered responsiveness, we assessed the effect of a converting enzyme inhibitor (enalapril) on RBF and its responsiveness to AII in 25 patients with essential hypertension--10 modulators and 15 nonmodulators--and 9 normotensive controls. After 5 d of a 200-meq sodium intake, the nonmodulators did not increase RBF, whereas the normotensives (79 +/- 28 ml/min per 1.73 m2) and modulators (75 +/- 26 ml/min per 1.73 m2) did (P less than 0.025). Arterial blood pressure did not change in the modulators with the salt loading, whereas in the nonmodulators, blood pressure rose (P less than 0.004). After enalapril administration for 66 h, there was a significant difference (P less than 0.01, Fisher Exact Test) in the blood pressure response in the two hypertensive subgroups. In the modulators, there was no change; in the nonmodulators, despite the high salt diet, a blood pressure reduction occurred. In parallel, basal RBF and RBF responsiveness to AII were not changed after converting enzyme inhibition in the normotensive control (n = 9) or the hypertensive modulators (n = 10). Conversely, in the nonmodulators (n = 14), the basal RBF increased significantly (83 +/- 25 ml/min per 1.73 m2; P = 0.01), the increment being indistinguishable from the response to salt loading in normal subjects. Furthermore, renovascular responsiveness to infused AII was also significantly enhanced (P = 0.027) in the nonmodulators, suggesting that enalapril-induced increase in RBF reflected a fall in intrarenal AII levels, and not an increase in prostaglandins or kinins, which would have blunted the renal response to AII. Thus, short-term converting enzyme inhibition corrected abnormalities in sodium-mediated modulation of renal vascular responsiveness to AII. The close quantitative relation of the increase in RBF with sodium loading in normal subjects and modulators, and with converting enzyme inhibition in nonmodulators, viewed in the context of the effectiveness of enalapril only in the latter, and parallel shifts in sensitivity to AII, raises the intriguing possibility that converting enzyme inhibition reversed the failure of the renal blood supply to respond to sodium loading. Thus, converting enzyme inhibitors may reduce blood pressure specifically in this subset of patients with essential hypertension, who are sodium sensitive by way of mechanisms more closely related to local than systemic activity of the renin-angiotensin system.

Authors

J Redgrave, S Rabinowe, N K Hollenberg, G H Williams

Reduced affinity of the androgen receptor for 5 alpha-dihydrotestosterone but not methyltrienolone in a form of partial androgen resistance. Studies on cultured genital skin fibroblasts.

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Abstract

We have studied a child with posterior labial fusion, clitoral phallus, female urethra, and a short, blind vagina born to a mother with decreased axillary and pubic hair. Her karyotype is 46,XY. At 2 yr of age, the child's basal level of plasma testosterone was less than 0.35 nM and after human chorionic gonadotropin stimulation, it rose to 2.6. Testis and epididymis histology were normal. Her cultured genital (labial) skin fibroblasts have normal testosterone 5 alpha-reductase activity, and metabolize 5 alpha-dihydrotestosterone (DHT) normally, but they do not augment (up-regulate) their basal androgen-receptor binding activity during prolonged incubation with DHT. With DHT, the androgen receptor in her genital skin fibroblasts has a normal binding capacity (maximum binding capacity = 25 fmol/mg protein), but an increased rate constant of dissociation (k = 11.6 X 10(-3) min-1; normal, 6 +/- 1.2 (+/- SD)), and a decreased apparent equilibrium binding affinity (Kd = 0.6 nM; normal, 0.22 +/- 0.09) that is evident in the results of 2-h assays but not of those lasting 0.5 h. With the synthetic androgen, methyltrienolone, all three binding properties of the receptor are normal, and her receptor activity up-regulates normally. We interpret these results to mean that the subject has a ligand-selective defect in the time-dependent transformation of initial, low-affinity androgen-receptor complexes to serial states of higher affinity, presumably as the result of a structural mutation at the X-linked locus that encodes the androgen receptor protein.

Authors

L Pinsky, M Kaufman, A E Chudley

Role of granulocyte oxygen products in damage of Schistosoma mansoni eggs in vitro.

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Abstract

The objectives of this study were to describe the ultrastructure of granulocyte-Schistosoma mansoni egg interaction and to determine the role of reduced oxygen products as effectors of cell-mediated damage to the parasite target. Granulocytes attached to the parasites and closely applied their plasma membranes to the microspicules of the egg shell 30 min after mixing in the presence of immune serum. By 4 h, the egg shell was fractured and granulocyte pseudopodia extended toward the underlying miracidium. Granulocyte attachment to eggs resulted in release of O2- (0.30-0.52 nmol/min per 2 X 10(6) cells) and accumulation of H2O2 (0.14-0.15 nmol/min) in the presence of antibody or complement. Granulocytes reduced egg tricarboxylic-acid cycle activity and hatching by 28.3 +/- 0.9 and 35.2 +/- 2.8%, respectively (cell-egg ratio of 1,000: 1). Exogenous superoxide dismutase (10 micrograms/ml) inhibited granulocyte toxicity for egg metabolic activity (3.0 +/- 2.1% reduction in acetate metabolism vs. 28.3 +/- 0.9% decrease in controls without superoxide dismutase, P less than 0.0005) and hatching (12.5 +/- 1.8% reduction, P less than 0.0005), whereas catalase and heparin had no effect. Inhibitors of myeloperoxidase (1 mM azide, cyanide, and methimazole) augmented granulocyte-mediated toxicity of egg tricarboxylic-acid cycle activity (44-58% reduction in activity vs. 31 and 35% reduction in controls), suggesting that H2O2 released from cells was degraded before reaching the target miracidium. Oxidants generated by acetaldehyde (2 mM)-xanthine oxidase (10 mU/ml) also decreased egg metabolic activity and hatching by 62.0 +/- 9.0 and 38.7 +/- 7.3%, respectively. Egg damage by the cell-free system was partially prevented by superoxide dismutase (26.5 +/- 4.2% reduction in egg tricarboxylic-acid activity) and completely blocked by catalase (0% reduction in activity). These data suggest that granulocyte-mediated toxicity for S. mansoni eggs is dependent on release of O2- or related molecules. These oxygen products, unlike H2O2, may readily reach the target miracidium where they may be converted to H2O2 or other microbicidal effector molecules.

Authors

J W Kazura, P de Brito, J Rabbege, M Aikawa

Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells.

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Abstract

It has been postulated that thrombin binds to endothelial cells through, at least in part, cell surface glycosaminoglycans such as heparan sulfate, which could serve as antithrombin cofactor on the endothelium. In the present study, we have directly evaluated the binding of 125I-labeled bovine thrombin to cultured porcine aortic endothelial cells. The thrombin binding to the cell surface was rapid, reversible, and displaced by enzymatically inactive diisopropylphosphoryl-thrombin. The concentration of thrombin at half-maximal binding was approximately 20 nM. Both specific and nonspecific binding of 125I-thrombin to the endothelial cell surface was partially inhibited in the presence of protamine sulfate, after the removal of cell surface heparan sulfate by the treatment of cells with crude Flavobacterium heparinum enzyme or purified heparitinase. The binding as a function of the concentration of thrombin revealed that the maximal amount of specific binding was reduced by approximately 50% with little alteration in binding affinity by these enzymatic treatments. The reversibility and active-site independence as well as the rate of the binding did not change after heparitinase treatment. Whereas removal of chondroitin sulfates by chondroitin ABC lyase treatment of cells did not affect the binding, identical enzymatic treatments of [35S]sulfate-labeled cells showed that either heparan sulfate or chondroitin sulfate was selectively and completely removed from the cell surface by heparitinase or chondroitin ABC lyase treatment, respectively. Furthermore, proteolysis of cell surface proteins by the purified glycosaminoglycan lyases was excluded by the identical enzymatic treatments of [3H]leucine-labeled or cell surface radioiodinated cells. Our results provide the first direct evidence that heparan sulfate on the cell surface is involved in the high-affinity, active site-independent thrombin binding by endothelial cells, and also suggest the presence of thrombin-binding sites that are not directly related to heparan sulfate.

Authors

K Shimada, T Ozawa

Effects of adrenalectomy and chronic adrenal corticosteroid replacement on potassium transport in rat kidney.

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Abstract

Clearance experiments were carried out in pair-fed rats to examine the long-term effects of adrenalectomy and selective adrenal corticosteroid replacement in physiological amounts on renal potassium transport. To this end, clearance studies were conducted in rats that were sham operated, or adrenalectomized (ADX). ADX animals were given either vehicle, aldosterone (0.5 microgram/100 g body wt per day), dexamethasone (1.2 micrograms/100 g body wt per day), or aldosterone and dexamethasone, by osmotic minipump for 7-9 d whereupon clearance experiments were conducted. After chronic hormone treatment, during basal conditions when only Ringers solution was infused, all groups excreted similar amounts of potassium. However, in all ADX animals without mineralocorticoid replacement, the maintenance of urinary potassium excretion at control levels was associated with hyperkalemia, increased urine flow, and natriuresis; all are factors known to stimulate urinary potassium excretion. During acute potassium infusion, the increase in urinary potassium excretion was less in ADX rats than in controls. This functional deficiency in potassium excretion was partially corrected by dexamethasone and was uniformly associated with a significant increase in urine flow. Aldosterone replacement or aldosterone and dexamethasone given together chronically, sharply increased potassium excretion but did not restore excretion to control levels. Only acute aldosterone infusion (0.2 microgram/100 g body wt bolus plus 0.2 microgram/100 g body wt per hour), superimposed upon chronic aldosterone and dexamethasone treatment, fully restored potassium excretion to control levels. This aldosterone induced enhancement of potassium excretion, both chronic and acute, was not associated with hyperkalemia, and increased urine flow or natriuresis. Thus, physiological levels of both classes of adrenal corticosteroids stimulate renal potassium excretion albeit by different mechanisms. Mineralocorticoids stimulate tubular potassium excretion directly, whereas glucocorticoids augment excretion indirectly by increasing fluid and sodium delivery along the distal nephron.

Authors

B Stanton, G Giebisch, G Klein-Robbenhaar, J Wade, R A DeFronzo

Ultrastructure of rat initial collecting tubule. Effect of adrenal corticosteroid treatment.

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Abstract

This study examines the effects of adrenalectomy and physiological replacement of mineralocorticoids and glucocorticoids on the cellular ultrastructure of the rat initial collecting tubule (late distal tubule). Animals were adrenalectomized (ADX) and for 10 d received by osmotic minipump either: vehicle, aldosterone (0.5 micrograms X 100 g-1 X d-1), aldosterone (2.0 micrograms X 100 g-1 X d-1), dexamethasone (1.2 micrograms X 100 g-1 X d-1), or aldosterone (0.5 micrograms X 100 g-1 X d-1) with dexamethasone (1.2 micrograms X 100 g-1 X d-1). Radioimmunoassay revealed that the low dose of aldosterone restored plasma aldosterone to control levels. The higher dose of aldosterone increased plasma levels by threefold. Morphometric techniques were used to measure membrane length of individual principal and intercalated cells in each condition. The basolateral membrane length of principal cells decreased by 35% in ADX animals. Low dose aldosterone replacement (0.5 micrograms X 100 g-1 X d-1) in ADX animals maintained membrane length at control values; at a higher level of aldosterone (2.0 micrograms X 100 g-1 X d-1) membrane length increased by 111% compared with control. Dexamethasone treatment, at a level that restored glomerular filtration rate to normal, had no effect on cellular ultrastructure. Combined aldosterone and dexamethasone replacement had no greater effect on basolateral membrane length than aldosterone alone. The length of the luminal membrane of the principal cell type was not affected by ADX or hormone treatment. Intercalated cell membrane length was not affected by ADX or hormone replacement. Thus, chronic aldosterone levels have an important, selective effect on the basolateral membrane of the principal cell. The correlation between these morphological results and the steroid hormone effects on renal electrolyte excretion, reported in the companion paper (15), suggests that basolateral membrane length is an important factor controlling the rate of sodium and potassium transport by the initial collecting tubule.

Authors

B Stanton, A Janzen, G Klein-Robbenhaar, R DeFronzo, G Giebisch, J Wade

Origin of urinary nonconjugated 19-nor-deoxycorticosterone and metabolism of infused radiolabeled 19-nor-deoxycorticosterone in men and women.

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Abstract

It is known that 19-nor-deoxycorticosterone (19-nor-DOC) is a potent mineralocorticosteroid that is present in urine of rats and humans in a free, i.e., nonconjugated, form. In some forms of hypertension in rats, the levels of free 19-nor-DOC in urine are increased compared with those in urine of normotensive animals. Yet, despite the potential importance of this mineralocorticosteroid in the pathogenesis of certain forms of hypertension, little is known of its site of origin or metabolism. In the present investigation, we evaluated the metabolism of intravenously infused [3H]19-nor-DOC and the possibility that 19-nor-DOC was formed from plasma DOC. We found that the metabolism of [3H]19-nor-DOC infused intravenously in men and women was similar to that of DOC with important exceptions. The majority of the radiolabeled urinary metabolites of intravenously infused [3H]19-nor-DOC were excreted in urine as glucuronosides. Little radioactivity, infused as [3H]19-nor-DOC, was recovered in urine as nonconjugated or sulfoconjugated steroids. There was no free radiolabeled 19-nor-DOC in urine after the simultaneous infusion of [3H]19-nor-DOC and [14C]DOC. A major metabolite of [3H]19-nor-DOC in urine was 19-nor-DOC-21-glucuronoside, whereas little or no intravenously infused radiolabeled DOC was excreted as radiolabeled DOC-glucuronoside. We also found that intravenously infused [14C]DOC was not converted to urinary [14C]19-nor-DOC (glucuronoside) and that other tritium-labeled metabolites of infused [3H]19-nor-DOC contained no carbon-14. The production rate of 19-nor-DOC, computed from the specific activity of urinary 19-nor-DOC (glucuronoside), in one normal man was 16 micrograms/d and in the two women of this study, it was 10 micrograms/d. These findings are supportive of the proposition that free urinary 19-nor-DOC is not formed from plasma DOC; it may be formed in kidney from a precursor other than DOC or it may be formed nonenzymatically in kidney or urine from a precursor such as 19-oic-DOC.

Authors

M L Casey, A Guerami, L Milewich, C E Gomez-Sanchez, P C MacDonald

Regulation of human B cell activation by prostaglandin E2. Suppression of the generation of immunoglobulin-secreting cells.

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Abstract

The role of prostaglandin E2 (PGE2) in the generation of immunoglobulin-secreting cells (ISC) from human peripheral blood B cells was examined. Initial studies demonstrated that monocyte (M phi)-mediated suppression of the generation of ISC in Staphylococcus aureus (SA)-stimulated cultures was mitigated by indomethacin, and thus suggested that the cyclooxygenase pathway products of arachidonic acid played a role in the regulation of B cell activation. The possibility that PGE2, one of the major products of this pathway generated by M phi-affected human B cell responses, was therefore investigated. PGE2 was found to cause concentration-dependent inhibition of the generation of ISC in pokeweed mitogen- or SA-stimulated B cell cultures supported by T cells. Studies were therefore carried out to determine whether PGE2 inhibited the production of necessary T cell factors or directly altered B cell responsiveness. Initially, the effect of PGE2 on the capacity of mitogen-stimulated cells to secrete a factor that supported the differentiation of B cells into ISC was investigated. Excessive numbers of M phi or PGE2 inhibited the production of B cell differentiation factor from mitogen-stimulated T cells. The effect of PGE2 on the capacity of B cells to differentiate into ISC was more complex. PGE2 inhibited the generation of ISC when B cells were stimulated with SA and B cell differentiation factor-containing T cell supernatants. PGE2-mediated inhibition of ISC generation was observed even when addition of PGE2 was delayed until after ISC first were detected in culture. By contrast, PGE2 caused only minimal inhibition of the generation of ISC cultures stimulated by T cell supernatants alone or protein A-free SA and T cell supernatants. These results suggested that SA-responsive B cells were particularly sensitive to inhibition by PGE2. Additional experiments supported the conclusion that B cell sensitivity to inhibition by PGE2 is augmented by the immunoglobulin cross-linking effects of protein A-containing SA. Overall, the results support the conclusion that PGE2 at physiologically relevant concentrations can influence human antibody responses by means of a direct inhibitory action on the responding B cell or an indirect one on the production of necessary T cell factors.

Authors

D F Jelinek, P A Thompson, P E Lipsky

Affinity-labeled plasma somatomedin-C/insulinlike growth factor I binding proteins. Evidence of growth hormone dependence and subunit structure.

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Abstract

By using disuccinimidyl suberate, we have covalently cross-linked 125I-labeled somatomedin-C (Sm-C)/insulinlike growth factor I to specific binding proteins in human plasma. In unfractionated plasma samples from normal and acromegalic donors, 125I-Sm-C binding-protein complexes with relative molecular weights (Mr) of 160,000, 135,000, 110,000, 80,000, 50,000, 43,000-35,000, and 28,000-24,000 were consistently observed. In contrast, the 43,000-35,000-mol wt species were frequently the only specific complexes observed in hypopituitary plasma and were consistently more intensely labeled in such samples. Reduction of samples with beta-mercaptoethanol did not alter the electrophoretic pattern of these 125I-Sm-C binding-protein complexes. All Sm-C binding proteins, with the exception of the 43,000-35,000-mol wt complex, were adsorbed by concanavalin A-Sepharose. When acromegalic or normal plasma was fractionated on a Sephadex G-200 column and affinity labeled, the same complexes that were adsorbed by concanavalin A were found in fractions that eluted near the gamma-globulin peak. On the other hand, the 43,000-35,000-mol wt complex consistently eluted in size-appropriate fractions near the albumin peak. These data suggest that the growth hormone (GH)-dependent Sm-C binding protein, represented by the 160,000-mol wt complex, is in some way composed of smaller species, i.e., the 135,000-, 110,000-, 80,000-, 50,000-, and 28,000-24,000-mol wt complexes. Acid incubation of plasma prior to Sephadex G-200 chromatography results in the elimination of specific 125I-Sm-C binding-protein complexes which elute near gamma-globulin and a concurrent increase in the labeling intensity of the 28,000-24,000-mol wt complexes. We speculate, therefore, that each of the GH-dependent Sm-C binding-protein complexes represents an oligomer composed of 28,000-24,000-mol wt protomers. The 43,000-35,000-mol wt species is not dependent upon GH and appears to represent a different type of Sm-C binding protein.

Authors

J R Wilkins, A J D'Ercole

Determination of the hemoglobin F program in human progenitor-derived erythroid cells.

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Abstract

The absolute adult and fetal hemoglobin (HbF) contents of the erythroid cells derived from the differentiation of normal human and simian erythroid progenitors and of the peripheral blood erythroid burst-forming units (BFU-E) of patients with nondeletion hemoglobinopathies have been measured with a sensitive radioligand immunoassay. The HbF content varied between 0.13 and 2.96 pg/cell, representing between 0.7% and 19.6% of the total hemoglobin with a mean value of 7.0%. The absolute content of HbF was indistinguishable in the well-hemoglobinized progeny of marrow erythroid colony-forming units, marrow or blood BFU-E, or of mixed colony-forming units. The term HbF program refers to this inherent capacity to produce fetal hemoglobin (HbF) in the erythroid cells derived from these progenitors in vitro. The HbF content of marrow erythroblasts as determined by the same radioligand immunoassay was similar to that found in the peripheral blood, suggesting that the switch off of gamma-chain production occurs after the erythroid colony-forming unit stage of maturation. Increasing concentrations of a crude erythropoietin-containing preparation induced higher numbers of erythroid colonies, which were larger in size, but the HbF program was unaffected. In contrast to the hemoglobin accumulation in human progenitor-derived colonies, simian progenitor-derived colonies produced considerably more HbF, and the amount of HbF was strongly influenced by progenitor maturity. Assays of the HbF content of erythroblasts derived from culture of the peripheral blood BFU-E of patients with nondeletion hemoglobinopathies and their parents showed that the HbF program in the progenitors of such patients is highly variable. Some produce only a slight excess of HbF in progenitor-derived erythroblasts, whereas others have extraordinarily high HbF programs. The molecular basis of this variability is presently unknown.

Authors

A D Friedman, D C Linch, B Miller, J M Lipton, J Javid, D G Nathan

In vivo interaction of antibodies with cell surface antigens. A mechanism responsible for in situ formation of immune deposits in the zona pellucida of rabbit oocytes.

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Abstract

It was the aim of this study to test the hypothesis that the interaction of antibodies with antigens expressed on the plasma membrane of cells surrounded by a basement membrane or a basement membrane-like structure results in in situ formation of immune deposits. Ovary was chosen for the experiments because we found that a well-characterized protein, angiotensin-converting enzyme (ACE), is expressed in a diffuse pattern on the plasma membrane of mature oocytes. To investigate the events following the in vivo interaction of oolemma-ACE with its antibody, rabbits were injected with goat anti-rabbit ACE gamma-globulin or with Fab fragments of goat anti-rabbit ACE IgG in an ear vein for a maximum of 4 d; they were followed for up to 20 d thereafter. Ovary tissue was studied by immunofluorescence, and immunoelectron, light, and transmission electron microscopy. The results of this study document two new findings: First, that ACE is expressed on the oolemma of rabbit oocytes. Second, that the in vivo interaction of divalent antibodies to this cell surface antigen induces formation of granular immune deposits in the adjacent zona pellucida through a mechanism of "patching" and "shedding" of immune complexes, similar to that occurring in in vitro systems characterized by interaction of plasma membrane receptors with soluble ligands. This mechanism might have importance in the pathogenesis of Heymann glomerulonephritis and of other immunological diseases involving antigens expressed on the plasma membrane of cells.

Authors

S Matsuo, P R Caldwell, J R Brentjens, G Andres

Human platelet-derived growth factor stimulates prostaglandin synthesis by activation and by rapid de novo synthesis of cyclooxygenase.

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Abstract

Human platelet-derived growth factor (PDGF) stimulated prostaglandin (PG) E2 synthesis in the cell cycle of Swiss 3T3 cells at two distinct time intervals, with a first plateau within 10 min and a second plateau within 2-4 h after addition of PDGF. At 4 h, the concentration of PGE2 in PDGF-stimulated cultures exceeded the quiescent control cells by a factor of 10-15. Quiescent cells incubated with up to 16 microM exogenous arachidonic acid (AA) synthesized only small amounts of PGE2. In contrast, 4 h after addition of PDGF, the concentration of PGE2 synthesized from exogenous AA exceeded that in quiescent cultures by a factor of 28. The effect of PDGF stimulation on PG synthesis from exogenous AA could not be explained by growth factor-mediated increase in the cellular free AA pool as shown in experiments using [14C]AA. PDGF also stimulated synthesis of PGI2 (prostacyclin), thromboxane, and PGF2 alpha from exogenous AA. While inhibition of protein synthesis by 10 micrograms/ml cycloheximide had no effect on the early increase in PGE2 synthesis, the second increase was completely prevented. Additionally, cycloheximide treatment at 6 h after PDGF stimulation resulted in rapid decline of PGE2 synthesis from exogenous AA. Quiescent cultures pretreated with 100 microM aspirin and stimulated by PDGF thereafter recovered from cyclooxygenase inhibition within 180 min. Our results suggest that phospholipase activation and resultant AA release is not sufficient to induce the burst of PG synthesis observed in PDGF-stimulated cells. Instead, PDGF stimulates PG synthesis by direct effects on the PG-synthesizing enzyme system, one involving a protein synthesis-independent mechanism and another that requires rapid translation of cyclooxygenase.

Authors

A J Habenicht, M Goerig, J Grulich, D Rothe, R Gronwald, U Loth, G Schettler, B Kommerell, R Ross

Evidence for the involvement of B lymphoid cells in polycythemia vera and essential thrombocythemia.

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Abstract

Previous studies with the X-chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) as a marker of cellular mosaicism demonstrated that polycythemia vera (PV) and essential thrombocythemia (ET) are clonal disorders of hematopoietic stem cells that can differentiate to erythrocytes, granulocytes, and platelets. To determine if the involved stem cells could also differentiate along the B-lymphoid pathway, we studied one woman with PV and one woman with ET. Of 117 Epstein-Barr virus-transformed B-lymphoblastoid lines expressing a single G6PD derived from the patient with PV, 108 expressed G6PD type A, the type characteristic of the abnormal clone. The ratio of 108:9 was significantly different from the one to one ratio predicted for this patient, which suggested that at least some circulating progenitors for B-lymphoid cell lines differentiate from the stem cell involved by the disease. Results obtained from the patient with ET were similar--104 of the 109 lymphoblastoid lines monotypic for G6PD expression displayed the enzyme type found in the abnormal clone of marrow cells. Therefore, in these patients, PV and ET, like chronic myelogenous leukemia, involve a stem cell pluripotent for the lymphoid as well as the myeloid series.

Authors

W H Raskind, R Jacobson, S Murphy, J W Adamson, P J Fialkow