To elucidate the bleeding tendency that follows the administration of ticlopidine, we investigated the skin bleeding time and some ex vivo functions of platelets obtained from eight healthy volunteers before and 1 wk after daily administration of 500 mg of ticlopidine. We found the following: ticlopidine significantly (P less than 0.001) prolonged the skin bleeding time and impaired the binding of radiolabeled fibrinogen and von Willebrand Factor, the clot retraction and the aggregation of platelets in response to ADP, epinephrine, thrombin, ionophore A23187, collagen, or arachidonic acid. In contrast, the administration of this drug did not affect intraplatelet levels of cAMP, agglutination and binding of von Willebrand Factor in response to ristocetin, shape change in response to ADP, collagen, thrombin, or arachidonic acid, or binding of prostaglandin E1 to resting platelets. Secretion of ATP in response to ADP or epinephrine was completely inhibited, whereas secretion as well as thromboxane synthesis in response to high concentrations of collagen, arachidonic acid, calcium ionophore A23187, or thrombin was unaffected. Studies with monoclonal antibodies showed that the glycoprotein IIb-IIIa complex (the putative receptor for fibrinogen and von Willebrand Factor on the surface of platelets exposed to naturally occurring aggregating agents) was quantitatively unaffected by ticlopidine. This observation was further confirmed by densitometric scannings of Periodic Acid-Schiff-stained gels of platelet suspensions. The onset, as well as the cessation of the inhibitory effect of ticlopidine on platelets was very slow, and reached a maximum after a 3-5-d administration. In addition, ticlopidine appeared to be a much more potent inhibitor when administered to subjects than when added in vitro to platelets. Finally, abnormalities comparable to those found in volunteers taking ticlopidine were observed when platelets from untreated subjects were incubated in the plasma of ticlopidine-treated subjects. We conclude that ticlopidine induces a thrombasthenic state in normal platelets without affecting the glycoprotein IIb-IIIa complex quantitatively. Furthermore, our data suggest that one or more active metabolites rather than the native drug mediate the abnormalities of platelet function observed in ticlopidine-treated subjects.
G Di Minno, A M Cerbone, P L Mattioli, S Turco, C Iovine, M Mancini
Although platelets contain Factor V, localized primarily in the alpha-granules, the origin of this coagulation cofactor in these cells is not known. We therefore explored whether isolated megakaryocytes could biosynthesize Factor V. Guinea pig plasma Factor V coagulant activity was demonstrated to be neutralized by human monoclonal and rabbit polyclonal antibodies directed monospecifically against human Factor V. These antibodies had been used earlier to purify human Factor V. These antibodies had been used earlier to purify human Factor V and to quantify Factor V antigen concentration, respectively (1983. Chiu, H. C., E. Whitaker, and R. W. Colman. J. Clin. Invest. 72:493-503). As determined by a competitive enzyme-linked immunosorbent assay with guinea pig plasma as a standard, Factor V solubilized from guinea pig megakaryocytes was present at 0.098 +/- 0.018 micrograms/10(5) cells. Each megakaryocyte contained about 500 times as much Factor V as is in a platelet (0.234 +/- 0.180 micrograms/10(8) platelets). The content of Factor V antigen in guinea pig plasma was greater (27.0 +/- 3.0 micrograms/ml) than that of Factor V antigen in human plasma (11.1 +/- 0.4 micrograms/ml). In contrast, human platelets contain ninefold more Factor V antigen (2.01 +/- 1.09 micrograms/10(8) platelets) than do guinea pig were 2.85 +/- 0.30 U/ml plasma, 0.022 +/- 0.012 U/10(8) platelets, and 0.032 +/- 0.03 U/10(5) megakaryocytes, compared with human values of 0.98 +/- 0.02 U/ml plasma and 0.124 +/- 0.064 U/10(8) platelets. Isolated megakaryocytes were found to contain Factor V by cytoimmunofluorescence. The megakaryocytes were incubated with [35S]methionine, and radiolabeled intracellular proteins purified were on a human anti-Factor V immunoaffinity column. The purified protein exhibited Factor V coagulant activity and neutralized the inhibitory activity of a rabbit antihuman Factor V antibody, which suggests that megakaryocyte Factor V is functionally and antigenically intact. These results indicate that Factor V is synthesized by guinea pig megakaryocytes. Nonetheless, megakaryocyte Factor V was more slowly activated by thrombin and in the absence of calcium was more stable after activation than was plasma Factor Va. Electrophoresis in sodium dodecyl sulfate and autoradiography of the purified molecule showed a major band of Mr 380,000 and a minor band of Mr 350,000, as compared with guinea pig and human plasma Factor V, where the protein had an Mr of 350,000. Both forms of Factor V were substrates for thrombin. Possible explanations for the higher molecular weight and different thrombin sensitivity and stability observed are that a precursor of Factor V was isolated or that the megakaryocyte Factor V had not been fully processed before isolation.
H C Chiu, P K Schick, R W Colman
Most human pyelonephritis Escherichia coli isolates express both mannose (MS)- and globoside (Gal-Gal)-binding pili. An ascending E. coli urinary tract infection model was established in the 16-wk-old female BALB/c mouse to compare the pathogenic significance of MS and Gal-Gal pili and their efficacy as vaccines for the prevention of pyelonephritis. The distribution and density of pilus receptor compounds in urogenital tissues and as soluble compounds in urine were determined with antibodies to the synthetic receptor analogues, alpha D-Gal(1----4) beta D-Gal and alpha D-Man(1----2) alpha D-Man. Both carbohydrates were detected in vagina, bladder, ureter, and renal pelvis epithelium and in collecting duct and tubular cells. A pilus receptor compound also was detected in urine. It competitively inhibited the binding capacity of MS pili and was found to be physically, chemically, and immunologically related to Tamm-Horsfall uromucoid. Infectivity and invasiveness were quantitatively and histologically characterized for four E. coli strains: J96, a human pyelonephritis strain that expresses both MS and Gal-Gal pili; two recombinant strains prepared from J96 chromosomal DNA encoding MS pili or Gal-Gal pili; and the nonpiliated K12 recipient. Intravesicular administration of J96 (10(6) colony-forming units [CFU]) resulted in renal colonization and invasion in each of nine mice. The Gal-Gal clone (10(6) CFU) colonized the kidneys in each of 10 mice but did not invade. In contrast, the MS clone (10(6) CFU) did not colonize renal epithelium or invade. This effect was superceded when larger doses (greater than or equal to 10(10) CFU) of the MS clone were administered in volumes that cause acute vesicoureteric reflux. The efficacy was determined of vaccines composed of pure MS or Gal-Gal pili or the lipopolysaccharide containing O somatic antigen of the challenge strain, J96. The Gal-Gal pilus vaccine blocked renal colonization in 19 of 22 mice and renal invasion in 10 of 11 mice. Gal-Gal pili may be useful immunogens for the prevention of pyelonephritis in anatomically normal urinary tracts.
P O'Hanley, D Lark, S Falkow, G Schoolnik
Apoproteins B and E both interact with cellular low density lipoprotein (LDL) apolipoprotein B and E (apo B,E)-receptors, and very low density lipoproteins (VLDL) contain both apo B and apo E. Our aim was to study the relative importance of apo B and apo E in the binding of VLDL subfractions to cells. Two monoclonal anti-LDL-apo B antibodies (464B1B3 and 464B1B6, 2a and 2b, respectively) and two anti-apo E antibodies (1506 A1.4 and 1907 F6.4) were used to inhibit lipoprotein-cell interactions. In confirmation of previous findings, the binding and degradation of 125I-LDL by human fibroblasts were inhibited approximately 90% by antibodies 2a or 2b or the antigen-binding fragments of 2a, whereas the cellular processing of 125I-VLDL3 (Sf20-60), 125I-VLDL2 (Sf60-120), and 125I-VLDL1 (Sf greater than 120) were inhibited by only approximately 50%, approximately 25%, and less than 10%, respectively. The VLDL1-3 and LDL-dependent intracellular esterification of cholesterol with [3H]oleate were inhibited to a similar extent. Other monoclonal anti-human apo B antibodies inhibited lipoprotein-cell interactions much less effectively and nonimmune IgG isolated from mouse serum did not inhibit at all. 20-fold excesses of LDL produced about the same patterns of inhibition of degradation of 125I-VLDL1-3 and LDL by cells as did antibodies 2a and 2b, whereas homologous unlabeled VLDL1-3 in like amounts inhibited the matched 125I-VLDL subfraction more effectively. Two anti-apo E monoclonal antibodies and a polyclonal anti-apo E antibody inhibited cell-mediated degradation of and lipoprotein-dependent cholesterol esterification by VLDL1 but not VLDL3 or LDL. The results suggest that receptor recognition sites on apo E in preference to sites on apo B mediate the cellular binding of hypertriglyceridemic VLDL1. However, the proportion of particles bound via apo B seems to increase as VLDL decreases in size toward LDL, and virtually all of LDL binding is mediated by apo B.
E S Krul, M J Tikkanen, T G Cole, J M Davie, G Schonfeld
Cystine depleted cystinotic fibroblasts incubated in cystine-free medium accumulate lysosomal-free cystine from the degradation of cystine-containing intracellular and extracellular proteins. In this report we have used this characteristic of these cells to study lysosomal proteolysis. We find that inhibitors of protein synthesis (cycloheximide, emetine, and puromycin) inhibit cystine accumulation from endogenous proteins and therefore act to inhibit lysosomal proteolysis of these proteins. However, cycloheximide does not inhibit cystine accumulation derived from the degradation of the extracellular disulfide-rich proteins, albumin and RNase, but lysosomal cystine accumulation derived from insulin is inhibited by cycloheximide. We conclude that a rapidly turning over protein may be required for the lysosomal degradation of intracellular and some extracellular proteins.
J G Thoene, R Lemons, S Boskovich, K Borysko
Deoxyadenosine has been implicated as the toxic metabolite causing profound lymphopenia in immunodeficient children with a genetic deficiency of adenosine deaminase (ADA), and in adults treated with the potent ADA inhibitor deoxycoformycin. However, the biochemical basis for deoxyadenosine toxicity toward lymphocytes remains controversial. The present experiments have examined in detail the sequential metabolic changes induced in nondividing human peripheral blood lymphocytes by incubation with deoxyadenosine plus deoxycoformycin, or with 2-chlorodeoxyadenosine (CdA), an ADA resistant deoxyadenosine congener with anti-leukemic and immunosuppressive properties. The lymphotoxic effect of deoxyadenosine and CdA required their phosphorylation, and was inhibited by deoxycytidine. As early as 4 h after exposure to the deoxynucleosides, strand breaks in lymphocyte DNA began to accumulate, and RNA synthesis decreased. These changes were followed by a significant fall in intracellular NAD levels at 8 h, a drop in ATP pools at 24 h, and cell death by 48 h. Incubation of the lymphocytes with 5 mM nicotinamide, a NAD precursor and an inhibitor of poly(ADP-ribose) synthetase, prevented NAD depletion. The nicotinamide treatment also rendered the lymphocytes highly resistant to deoxyadenosine and CdA toxicity, without altering dATP formation or the accumulation of DNA strand breaks. The poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide exerted a similar although less potent effect. These results suggest that NAD depletion, probably triggered by poly(ADP-ribose) formation, is the principle cause of death in normal resting human lymphocytes exposed to deoxyadenosine plus deoxycoformycin, or to CdA.
S Seto, C J Carrera, M Kubota, D B Wasson, D A Carson
From a total of 22 hypertriglyceridemic subjects tested, 14 subjects were selected on the basis of normal postheparin plasma lipoprotein lipase (LPL) levels and the presence of LPL inhibitory activity in their fasting plasma. The inhibitory activity was detected in both the lipoprotein fraction (d less than 1.25 g/ml) and the lipoprotein-deficient fraction (d greater than 1.25 g/ml). Correlational analyses of LPL inhibitory activity and apolipoprotein levels present in the lipoprotein fraction (d less than 1.25 g/ml) indicated that only apolipoprotein C-III (ApoC-III) was significantly correlated (r = 0.602, P less than 0.05) with the inhibition activity of the lipoprotein fraction. Furthermore, it was found that LPL-inhibitory activities of the plasma lipoprotein fraction and lipoprotein-deficient fraction were also correlated (r = 0.745, P less than 0.005), though the activity in the lipoprotein-deficient plasma was not related to the ApoC-III or apolipoprotein E levels. Additional correlational analyses indicated that the LPL levels in the postheparin plasma of these subjects were inversely related to the levels of plasma apolipoproteins C-II, C-III, and E. To explain some of these observations, we directly examined the in vitro effect of ApoC-III on LPL activity. The addition of ApoC-III-2 resulted in a decreased rate of lipolysis of human very low density lipoproteins by LPL. Kinetic analyses indicated that ApoC-III-2 was a noncompetitive inhibitor of LPL suggesting a direct interaction of the inhibitor with LPL. Results of these studies suggest that ApoC-III may represent a physiologic modulator of LPL activity levels and that the incidence of LPL inhibitory activity in the plasma of hypertriglyceridemic subjects is more common than previously recognized.
C S Wang, W J McConathy, H U Kloer, P Alaupovic
We have previously shown that the incubation of normal rat adipose tissue with sera from nondialyzed, nondiabetic uremic patients reduces the transport and metabolism of glucose, in the absence and presence of insulin. In this study insulin-stimulated glucose metabolism by normal rat adipocytes was used as a bioassay to identify the resistance activity, assess the effect of chemical modification on it, and the clinical states associated with its production. The resistance activity was trypsin-labile and had an apparent isoelectric point between 6 and 7, but was not retained by either protein A or concanavalin A columns. The insulin resistance activity was decreased by coincubation with the protein synthesis inhibitor, cycloheximide. Purification to greater than 200,000-fold was attained by heating (100 degrees C) uremic serum, subjecting the supernatant to Sephadex G-25 chromatography and subsequent adsorption to DEAE at pH 7.8 and elution at pH 6.5. The partially purified resistance activity was retained within dialysis tubing of 1,000-mol wt cutoff but not within 2,000-mol wt cutoff. Hemodialysis of patients over 1 wk to 18 mo reduced significantly the amount of resistance activity in their sera. The resistance activity, present in most uremic patients, was not found in the sera of individuals with normal renal function but who were either obese, fasted, elderly or had type II diabetes mellitus. Thus, a circulating small molecular weight peptide, unique to uremia, induced insulin resistance by a protein synthesis-dependent mechanism.
M L McCaleb, M S Izzo, D H Lockwood
The glucagon receptor and the adenylyl cyclase system of human liver membranes were studied in six non-obese and six obese subjects who had elevated insulin and plasma glucagon levels. Analysis of specific glucagon binding by the method of Scatchard demonstrated a linear (monocomponent) plot with a dissociation constant of 2-3 nM, and the binding at low hormone concentrations was sensitive to guanosine triphosphate (GTP). The molecular weight of the glucagon receptor was 63,000 D as determined by an affinity labeling procedure and sodium dodecyl sulfate gel electrophoresis. Affinity labeling of this structure was specific for glucagon and inhibited by GTP. Glucagon stimulated the production of cyclic adenosine monophosphate (cAMP) by human membranes with half-maximal activation elicited by 6 nM hormone. The human cyclase system required GTP to facilitate an optimal glucagon response. NaF (10 mM) also activated the cyclase system and produced the same magnitude of response as maximum glucagon activation. A comparison of the liver adenylyl cyclase system of non-obese and obese subjects was made using glucagon (5 nM and 1 microM) and NaF (10 mM). No significant differences in cAMP production were noted between the two groups, regardless of the agent used to activate the enzyme. These findings agree with the glucagon binding studies that showed similar amounts of binding activity in the membranes from the two groups. Also, there was no influence of either age or sex of the subjects on the adenylyl cyclase response. In conclusion, human liver membranes contain a glucagon receptor and an adenylyl cyclase system that correspond closely to the well-studied system in animal liver. This system in human obesity is not altered by the approximately twofold elevation in plasma glucagon that occurs in this metabolic disorder.
J N Livingston, K Einarsson, L Backman, S Ewerth, P Arner
Abnormalities in glomerular function have been observed frequently in the early stages of both clinical and experimental diabetes mellitus. Because prostaglandins (PGs) are present in the glomerulus and have profound effects on glomerular hemodynamics, and because abnormalities of PG metabolism have been noted in other tissues from diabetics, we studied PG biosynthesis in glomeruli obtained from rats in the early stages of experimental diabetes mellitus. Streptozotocin, 60 mg/kg, was administered intravenously to male Sprague-Dawley rats. Control rats received an equal volume of the vehicle. Glomeruli were isolated 9-23 d later. Production of eicosanoids was determined by two methods: by direct radioimmunoassay after incubation of glomeruli under basal conditions and in the presence of arachidonic acid (C20:4), 30 microM, and by radiometric high-performance liquid chromatography (HPLC) after incubation of glomeruli with [14C]C20:4. When assessed by radioimmunoassay, mean basal production of both prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) was twofold greater in the diabetic animals whereas production of thromboxane B2 (TXB2) was not significantly greater than control. In response to C20:4, both PGE2 and PGF2 alpha were also greater in the diabetic animals, but these differences were not statistically significant. The increased rate of basal PG production did not appear to be related directly to the severity of the diabetic state as reflected by the degree of hyperglycemia at the time of sacrifice. In fact, the rates of glomerular PG production in the individual diabetic animals correlated inversely with the plasma glucose concentration. The increased rate of PG synthesis did not appear to be due to a nonspecific effect of streptozotocin inasmuch as glomerular PG production was not increased significantly in streptozotocin-treated rats which were made euglycemic by insulin therapy. Furthermore, addition of streptozotocin, 1-10 mM, to the incubation media had no effect on PGE2 production by normal glomeruli. PGE2 production by normal glomeruli was also not influenced by varying the glucose concentration in the incubation media over a range of 1-40 mM. When metabolism of [14C]C20:4 was evaluated by high-performance liquid chromatography conversion to labeled PGE2, PGF2 alpha, TXB2, and hydroxyheptadecatrienoic acid by diabetic glomeruli was two- to threefold greater compared with that in control glomeruli, whereas no significant difference in conversion to 12- and 15-hydroxyeicosatetraenoic acid occurred. These findings indicate that glomerular cyclooxygenase but not lipoxygenase activity was increased in the diabetic animals. A concomitant increase in glomerular phospholipase activity may also have been present to account for the more pronounced differences in PG production noted in the absence of exogenous unlabeled C20:4. These abnormalities in PG biosynthesis by diabetic glomeruli may contribute to the altered glomerular hemodynamics in this pathophysiologic setting.
M Schambelan, S Blake, J Sraer, M Bens, M P Nivez, F Wahbe
A series of new, covalent polyethylene glycol (PEG)-streptokinase adducts were synthesized and characterized. PEGs with average molecular weights of 2,000, 4,000, and 5,000 were activated with carbonyldiimidazole and coupled to the protein under standardized reaction conditions. Steady-state kinetic analysis demonstrated comparable Km values for the activation of plasminogen by streptokinase, PEG-2-streptokinase, and PEG-4-streptokinase. The kcat values were somewhat decreased when PEG-2 or PEG-4 was coupled to the streptokinase. Activation by the PEG-5 adduct did not follow Michaelis-Menten kinetics under the conditions employed in this study. Plasmin activity obtained by incubating streptokinase derivatives with plasminogen also was studied as a function of time with each of the PEG-streptokinase derivatives. By this assay, incubations containing PEG-5-streptokinase and unmodified streptokinase demonstrated comparable activity while reaction mixtures containing PEG-2-streptokinase and PEG-4-streptokinase were slightly more active. Streptokinase incubated with plasminogen at a 1:1 molar ratio was extensively degraded after 30 min whereas PEG-2-streptokinase was resistant to plasmin cleavage. The derivatized proteins were radioiodinated and incubated in plastic microtiter plates that were coated with an immunoglobulin fraction containing antibodies to streptokinase. Binding of the PEG-streptokinase adducts was decreased by greater than 95% compared with unmodified streptokinase. Plasminogen activator complexes were formed by reacting the streptokinases with human plasminogen in vitro and the clearance studied in mice. Radioiodinated plasmin in complex with the PEG-streptokinase adducts cleared at a slower rate than did plasmin complexed with unmodified streptokinase. Catabolism of the protease still occurred by a mechanism that involved reaction with alpha 2-macroglobulin as has been described for nonderivatized streptokinase-plasminogen complex (Gonias, S. L., M. Einarsson, and S. V. Pizzo, 1982, J. Clin. Invest., 70:412-423). When more extensive derivatization procedures were utilized, PEG-2-streptokinase preparations were obtained that further prolonged the clearance of complexed 125I-plasmin; however, these adducts did not uniformly retain comparable activity. It is suggested that PEG-streptokinase complexes with greatly reduced antigenicity may be useful in the treatment of thrombotic disorders.
S Rajagopalan, S L Gonias, S V Pizzo
Alpha adrenergic blockade with phentolamine (10 microM) reduces the glucagon response to severe glucopenia (from 150 to 25 mg/dl) to 22% of the control values in the isolated perfused rat pancreas. Propranolol (10 microM) had no significant effect. Neither alpha nor beta adrenergic blockade reduced the magnitude of glucopenic suppression of insulin secretion, but phentolamine increased insulin levels before and during glucopenia. The pattern of somatostatin secretion in these experiments resembled that of insulin. Depletion of norepinephrine from sympathetic nerve endings by pretreatment with 6-hydroxydopamine lowered the pancreatic norepinephrine content to less than 20% of control values and reduced the glucagon response to glucopenia to 69% of the controls. Combined alpha and beta adrenergic blockade during less severe glucopenia (from 120 to 60 mg/dl) reduced the glucagon response to 21% of controls. However, slight glucopenia (from 100 to 80 mg/dl), which elicited only 11% increase in glucagon in the control experiments, was not altered significantly by combined alpha and beta adrenergic blockade. Morphologic studies of adrenergic nerve terminals labeled with [3H]norepinephrine revealed associations with alpha cells. It is concluded that in the isolated rat pancreas adrenergic mediation accounts for most of the glucagon but not insulin response to glucopenia. It is controlled within the pancreas itself, possibly through a direct enhancement by glucopenia of norepinephrine release from nerve endings.
A Hisatomi, H Maruyama, L Orci, M Vasko, R H Unger
Based on in vitro work with rat liver, we recently suggested that the peroxisomal fraction is most important for the oxidation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid. The cerebro-hepato-renal syndrome of Zellweger is a fatal recessive autosomal disorder, the most characteristic histological feature of which is a virtual absence of peroxisomes in liver and kidneys. This disease offers a unique opportunity to evaluate the relative importance of peroxisomes in bile acid biosynthesis. A child with Zellweger syndrome was studied in the present work. In accordance with previous work, there was a considerable accumulation of THCA, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid (24-OH-THCA), 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid (C29-dicarboxylic acid), and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid in serum. In addition, a tetrahydroxylated 5 beta-cholestanoic acid with all the hydroxyl groups in the steroid nucleus was found. 3H-Labeled 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was administered intravenously together with 14C-labeled cholic acid. There was a rapid incorporation of 3H in THCA and a slow incorporation into cholic acid. The specific radioactivity of 3H in THCA was about one magnitude higher than that in cholic acid. The conversion was evaluated by following the increasing ratio between 3H and 14C in biliary cholic acid. The rate of incorporation of 3H in cholic acid was considerably less than previously reported in experiments with healthy subjects, and the maximal conversion of the triol into cholic acid was only 15-20%. About the same rate of conversion was found after oral administration of 3H-THCA. Both in the experiment with 3H-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and with 3H-THCA, there was an efficient incorporation of 3H in the above unidentified tetrahydroxylated 5 beta-cholestanoic acid. There was only slow incorporation of radioactivity into 24-OH-THCA and into the C29-dicarboxylic acid. From the specific activity decay curve of 14C in cholic acid obtained after intravenous injection of 14C-cholic acid, the pool size of cholic acid was calculated to be 24 mg/m2 and the daily production rate to 9 mg/m2 per d. This corresponds to a reduction of approximately 85 and 90%, respectively, when compared with normal infants. It is concluded that liver peroxisomes are essential in the normal conversion of THCA to cholic acid. In the Zellweger syndrome this conversion is defective and as a consequence the accumulated THCA is either excreted as such or transformed into other metabolites by hydroxylation or side chain elongation. The accumulation of THCA, as well as the similar rate of conversion of 5 beta-cholestane-3 alpha,7 alpha.12 alpha-triol and THCA into cholic acid, support the contention that the 26-hydroxylase pathway with intermediate formation of THCA is the most important pathway for formation of cholic acid in man.
B F Kase, I Björkhem, P Hågå, J I Pedersen
To investigate whether energy derived from glycolytic and oxidative metabolism are preferentially used for different functions in heart, tension, intracellular potential, and extracellular [K+] and pH (using triple barrel K/pH electrodes, tip diameter 0.5 mm) were monitored in isolated arterially perfused rabbit interventricular septa during exposure to hypoxia and metabolic inhibitors. Myocardial content of high energy phosphates, lactate, and glycogen were determined under the same conditions. Inhibiting oxidative metabolism with hypoxia, dinitrophenol (10(-5)M), or Na-azide (10(-3)M) caused marked suppression of tension (by 73 +/- 5, 65 +/- 8, and 50 +/- 14%, respectively) and a small increase in [K+]0 (0.8 +/- 0.4, 0.5 +/- 0.25, and 0.4 +/- 0.2 mM, respectively) after 10 min. Inhibiting glycolysis with iodoacetate (IAA) (10(-3)M) had a much smaller suppressant effect on tension (28 +/- 24%) but markedly increased [K+]0 accumulation (by 1.8 +/- 1.1 mM) at 10 min. These differences, when IAA was compared individually to the other interventions, were highly significant. The shortening of action potential duration was not significantly different for the four interventions. pH0 increased slightly during IAA (+0.04-+0.06 U) and fell during the other interventions (-0.10--0.16 U), but this did not account for the differences in [K+]0 accumulation and tension between inhibition of glycolytic vs. oxidative metabolism. Except for hypoxia, total cellular content of high energy phosphates was not significantly depressed under the various conditions. This data suggests that energy from glycolysis is preferentially used to support sarcolemmal function (as manifested by K+ loss), whereas oxidatively derived energy preferentially supports contractile function. Indexing terms: cardiac metabolism, extracellular pH, metabolic inhibitors, hypoxia, extracellular K+ accumulation, and glycolysis.
J Weiss, B Hiltbrand
A mixture of 7 alpha-3H- and 4-14C-labeled cholesterol was administered intravenously to rats. Cholestanol with 20-30% lower ratio between 3H and 14C than in cholesterol could be isolated from different organs. In a healthy human control, cholestanol isolated from feces had a 3H/14C ratio which was 28% lower than in administered cholesterol. Cholesterol and coprostanol reisolated in these experiments had the same ratio between 3H and 14C as in the precursor. A previously unknown pathway for formation of cholestanol, involving 7 alpha-hydroxylated intermediates, may explain these results. Under normal conditions, this pathway is responsible for at most 30% of the cholestanol synthesized from cholesterol. Intravenous administration of the 7 alpha-3H- and 4-14C-labeled cholesterol to a patient with cerebrotendinous xanthomatosis (CTX) resulted in formation of cholestanol which had 70-75% lower 3H/14C ratio. It is concluded that the novel pathway involving 7 alpha-hydroxylated intermediates is accelerated in patients with CTX. This acceleration may contribute essentially to the accumulation of cholestanol, which is a predominant feature of this disease. 7 alpha-Hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one might be intermediates in the novel pathway to cholestanol. After intravenous administration of 7 beta-3H-labeled 7 alpha-hydroxycholesterol in a patient with CTX, significant amounts of 3H were incorporated into plasma and fecal cholestanol. Only small amounts of 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one are excreted into the intestine, and we therefore conclude that the 7 alpha-dehydroxylation step mainly occurs in the liver. In CTX, the synthesis of cholestanol may be accelerated because the concentrations of 7 alpha-hydroxylated bile acid intermediates in the liver are increased. A possible mechanism for the conversion of a minor fraction of 7 alpha-hydroxycholesterol into cholestanol is suggested.
S Skrede, I Björkhem, M S Buchmann, G Hopen, O Fausa
To study interactions between platelets and the fibrinolytic system, we examined the effects of human plasmin on human platelets washed by gel filtration. Plasmin concentrations that did not affect platelet shape change, release, or aggregation (less than 1.0 caseinolytic units [CU]/ml) caused a dose- and time-dependent inhibition of platelet aggregation in response to thrombin, ionophore A23187, and collagen. Complete loss of aggregation occurred at 0.1-0.5 CU/ml of plasmin. In a parallel dose-dependent manner, plasmin likewise inhibited thrombin, ionophore, and collagen-stimulated thromboxane B2 production. In contrast, neither aggregation nor thromboxane B2 formation induced by arachidonate was inhibited by plasmin pretreatment of the platelets. Plasmin blocked the thrombin-induced release of [3H]arachidonic acid from platelet membrane phospholipids and the thrombin-induced platelet oxygen burst. However, plasmin did not inhibit the arachidonate-induced oxygen burst. Inhibition of arachidonic acid release by plasmin was not mediated by increase in platelet cyclic AMP. These results suggest that plasmin inhibits platelet function, at least in part, by blocking the mobilization of arachidonic acid from membrane phospholipid pools. The effects of plasmin on platelets may contribute to the hemostatic abnormalities seen in pathologic and pharmacologic fibrinolysis.
A I Schafer, B Adelman
We have used a well-differentiated human colonic cell line, the T84 cell line, as a model system to study the pathways of cellular ion transport involved in vasoactive intestinal polypeptide (VIP)-induced chloride secretion. A modified Ussing chamber was used to study transepithelial Na+ and Cl- fluxes across confluent monolayer cultures of the T84 cells grown on permeable supports. In a manner analogous to isolated intestine, the addition of VIP caused an increase of net Cl- secretion which accounted for the increase in short circuit current (Isc). The effect of VIP on Isc was dose dependent with a threshold stimulation at 10(-10) M VIP, and a maximal effect at 10(-8) M. Bumetanide prevented or reversed the response to VIP. Inhibition by bumetanide occurred promptly when it was added to the serosal, but not to the mucosal bathing media. Ion replacement studies demonstrated that the response to VIP required the simultaneous presence of Na+, K+, and Cl- in the serosal media. Utilizing cellular ion uptake techniques, we describe an interdependence of bumetanide-sensitive 22Na+, 86Rb+, and 36Cl- uptake, which is indicative of a Na+,K+,Cl- cotransport system in this cell line. This transport pathway was localized to the basolateral membrane. Extrapolated initial velocities of uptake for each of the three ions was consistent with the electroneutral cotransport of 1 Na+:1 K+ (Rb+):2 Cl-. Our findings indicate that VIP-induced Cl- secretion intimately involves a bumetanide-sensitive Na+,K+,Cl- cotransport system which is functionally localized to the basolateral membrane.
K Dharmsathaphorn, K G Mandel, H Masui, J A McRoberts
Adjuvant arthritis is an experimental disease of rats induced by immunization to antigens of Mycobacterium tuberculosis. Our observation that arthritis could be induced in irradiated rats by the A2 line of T lymphocytes in the absence of mycobacterial antigens suggested that adjuvant arthritis is an autoimmune disease. Moreover, the A2 line could be used to vaccinate unirradiated rats against the subsequent induction of adjuvant arthritis by active immunization to Mycobacteria. In the present study we found that thymus cells obtained from A2 vaccinated rats could transfer resistance to adjuvant arthritis to naive rats. This indicates that the mechanism of resistance induced by A2 vaccination is probably immunological and involves thymus-derived lymphocytes.
J Holoshitz, A Matitiau, I R Cohen
Nonionic diffusion and diffusion equilibrium of ammonia have been generally accepted as the mechanism of urinary ammonium excretion. However, these characteristics have not been examined directly in vitro. In the present studies, nonionic diffusion and diffusion equilibrium of ammonia were examined in rabbit cortical collecting tubules perfused in vitro. Collected fluid ammonium and pH were measured in tubules exposed to chemical gradients of NH3/NH+4. In tubules perfused with an acid perfusate free of ammonia and bathed with solutions containing NH4Cl, collected fluid ammonia failed to equilibrate across the epithelium except at slow flow rates. The estimated apparent permeability coefficient to NH3 was approximately 5 X 10(-3) cm/s. Predominant nonionic diffusion of NH3, rather than transport of NH+4, was indicated by alkalinization of luminal fluid in tubules exposed to peritubular NH4Cl and by the relative influence of peritubular NH+4 and NH3 on ammonia entry. In tubules perfused with an acid solution containing NH4Cl, little loss of ammonium was detectable, indicating a low permeability to NH+4. In contrast to the restricted diffusion of NH3 in cortical collecting tubules, proximal convoluted tubules exhibited a much higher apparent permeability to NH3. In conclusion, nonionic diffusion of NH3 accounted for most ammonium transport in the proximal convoluted tubule and in the cortical collecting tubule. However, there was relatively restricted diffusion in the collecting tubules; this may account for the failure of whole kidney ammonium excretion to obey quantitatively the predictions of nonionic diffusion and diffusion equilibrium of ammonia.
L L Hamm, D Trigg, D Martin, C Gillespie, J Buerkert
C5a is an 11,000-D fragment of the fifth component of complement (C5) with potent anaphylatoxic and leukocyte chemotactic activities. C5a is believed to play an important role in the pathophysiology of certain skin disorders and systemic diseases with cutaneous manifestations. However, there is very little known about the in vivo reactivity of C5a in man. In this study we examined the effects of intradermal injections of human C5a in 17 normal volunteers. C5a was prepared by interacting highly purified human C5 with zymosan bound alternative pathway C5 convertase under conditions resulting in consumption of approximately 90% of the C5 substrate. C5a produced in this manner was chemotactic for human neutrophils and monocytes (0.5 X 10(-7) to 10(-9) M) and caused neutrophil aggregation and myeloperoxidase release (concentrations greater than or equal to 10(-10) M) in vitro. In vivo, C5a produced immediate wheal and flare reactions in all volunteers, and was active in doses as low as 1 ng (10(-13) mol). Intradermal testing with 20 ng of C5a in eight volunteers produced a maximal mean wheal of 11.75 mm (+/- 0.80 mm SEM) 20 min after anaphylatoxin injection, and a maximal mean erythema of 62.50 mm (+/- 3.27 mm SEM) 10 min after C5a administration. Reactions at C5a test sites were dose-related, associated with marked pruritus in some subjects, resolved without lesion formation, and were not associated with late phase reactions. In vivo testing revealed that human C5a was a more potent mediator of wheal and flare reactions than histamine, 48/80, human C3a, or morphine sulfate. Skin biopsies from eight volunteers 20 min after intradermal injection of 20 ng of C5a revealed a neutrophil-predominant perivascular infiltrate, endothelial cell edema, and sites of leukocytoclasis. Mast cell degranulation was observed on both light and electron microscopy of biopsies from C5a test sites. Although erythema at C5a injection sites was reduced by pretreating volunteers with hydroxyzine, whealing reactions and cellular infiltrates in biopsies were unaffected by this H1-antihistamine. Moderate doses of systemic corticosteroids did not alter clinical or histologic reactions at C5a injection sites in two volunteers. This study, using doses within the potential physiologic range of the anaphylatoxin, provides a comprehensive assessment of the effect of human C5a on normal human skin.
K B Yancey, C H Hammer, L Harvath, L Renfer, M M Frank, T J Lawley
Heparin cofactor II is a plasma protein that inhibits thrombin rapidly in the presence of either heparin or dermatan sulfate. We have determined the effects of two glycosaminoglycan-binding proteins, i.e., histidine-rich glycoprotein and platelet factor 4, on these reactions. Inhibition of thrombin by heparin cofactor II and heparin was completely prevented by purified histidine-rich glycoprotein at the ratio of 13 micrograms histidine-rich glycoprotein/microgram heparin. In contrast, histidine-rich glycoprotein had no effect on inhibition of thrombin by heparin cofactor II and dermatan sulfate at ratios of less than or equal to 128 micrograms histidine-rich glycoprotein/microgram dermatan sulfate. Removal of 85-90% of the histidine-rich glycoprotein from plasma resulted in a fourfold reduction in the amount of heparin required to prolong the thrombin clotting time from 14 s to greater than 180 s but had no effect on the amount of dermatan sulfate required for similar anti-coagulant activity. In contrast to histidine-rich glycoprotein, purified platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of either heparin or dermatan sulfate at the ratio of 2 micrograms platelet factor 4/micrograms glycosaminoglycan. Furthermore, the supernatant medium from platelets treated with arachidonic acid to cause secretion of platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of heparin or dermatan sulfate. We conclude that histidine-rich glycoprotein and platelet factor 4 can regulate the antithrombin activity of heparin cofactor II.
D M Tollefsen, C A Pestka
We assessed the structural and functional evolution of small intestinal transplant rejection in a rat model by use of 1-micron section, electron microscopic, and in vitro electrophysiologic techniques to study jejunal mucosa 3, 6, and 9 d posttransplantation. The earliest structural abnormalities detected in jejunal loops transplanted from Lewis X Brown Norway F1 hybrids into Lewis rats occurred within 3 d posttransplantation and consisted of focal endothelial cell injury of the microvasculature and focal injury of crypt epithelial cells. Both alterations were associated with adjacent infiltration of large lymphoid cells, and both markedly progressed and became rather diffuse over the following 6 d. In contrast, villus absorptive cells were not markedly altered in structure until the 9th postoperative day. As compared with host jejuna, allograft jejunal epithelium demonstrated multiple functional abnormalities. Transepithelial resistance declined progressively by days 6 and 9 (both P less than 0.05), although baseline transepithelial spontaneous potential difference was only affected at day 9 (P less than 0.01). Stimulated absorption by allograft jejuna, as assessed by measuring electrical response to mucosal glucose, was not significantly diminished until day 9 (P less than 0.05). In contrast, stimulated secretion assessed by measurement of electrical response to serosal theophylline was diminished by day 6 (P less than .01). These data suggest that the earliest epithelial injury during rejection, as judged both structurally and functionally, occurs in the crypt and is paralleled by endothelial injury at the level of the microvasculature. Thus, the primary targets for rejection are most likely endothelial cells and crypt epithelial cells. In contrast, structural and functional impairment of villus epithelium is detectable only at substantially later times during rejection and are most likely secondary processes related to either ischemia produced by microvascular injury or decreased epithelial regenerative ability secondary to crypt injury. Last, we show that the detrimental structural and functional sequellae of jejunal transplantation across the major histocompatibility complex in this model is strikingly ameliorated with cyclosporine therapy.
J L Madara, R L Kirkman
The synthetic heme analogue Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, the rate-limiting enzyme in heme degradation to bile pigment, and can entirely suppress hyperbilirubinemia in neonatal animals and significantly reduce plasma bilirubin levels in a variety of circumstances in experimental animals and man. To further explore the mechanism by which this metalloporphyrin reduces bilirubin levels in vivo, we have examined its effects on bilirubin production in bile duct-cannulated rats, in which bilirubin derived from heme catabolism is known to be rapidly excreted in bile. The administration of Sn-protoporphyrin (10-50 mumol/kg body weight) was followed by prompt (within approximately 1 h) and sustained (up to at least 18 h) decreases in bilirubin output, to levels 25-30 percent below the levels of bilirubin output in control bile fistula animals. The metalloporphyrin had no effect on bile flow or the biliary output of bile acids. Infusions of heme, which is taken up primarily in hepatocytes, or of heat-damaged erythrocytes, which are taken up in reticuloendothelial cells, resulted in marked increases in bilirubin output in bile in control animals; these increases were completely prevented or substantially diminished by Sn-protoporphyrin administration. By contrast, the metalloporphyrin did not alter the high levels of bilirubin in plasma and bile that were achieved in separate experiments by the constant (16 h) infusion of unconjugated bilirubin to bile duct-cannulated rats. Thus, Sn-protoporphyrin exerts no major effects on the metabolic disposition of preformed bilirubin. Heme oxygenase activities were markedly decreased in microsomal preparations from liver, spleen, and kidneys in these experiments, to a degree comparable to the decreases we have observed in the intact rat. We also demonstrated that a substantial proportion (19-35%) of a dose of Sn-protoporphyrin is promptly excreted in bile and that the time course of biliary excretion of this compound more closely reflects plasma concentrations of the metalloporphyrin, which decline rapidly, rather than concentrations in liver, which are considerably more persistent. These results indicate that Sn-protoporphyrin substantially reduces the in vivo production of bilirubin from the degradation of endogenous as well as exogenous heme in the rat. Moreover, this inhibitory effect of the synthetic metalloporphyrin on bilirubin production occurs in both hepatocytes and reticuloendothelial cells, which are the major tissue sites for bilirubin formation. In other studies, we have established that heme oxygenase blockade by Sn-protoporphyrin leads to a marked and rapid excretion of heme into bile presumably because the synthetic metabolism to bile pigment and making it available for excretion via the biliary system in to the gut, These studies strongly suggest that Sn-protoporphyrin diminishes hyperbilirubinemia in animals and man by inhibiting the production of the bile pigment in vivo, and that its principal mode of action involves a potent and sustained competitive inhibition of heme oxygenase.
C S Simionatto, K E Anderson, G S Drummond, A Kappas
To explore the auto-reactive potential of cells infiltrating the gut mucosa in idiopathic chronic inflammatory bowel disease, intestinal lamina propria mononuclear cells (LPMC) were isolated, characterized morphologically and phenotypically, and evaluated for antigen-specific reactivity. The last was assessed by quantitating LPMC cytotoxic capabilities against purified, aqueous-soluble, organ-specific epithelial cell-associated components (ECAC) characterized previously. Enzyme-isolated inflammatory bowel disease LPMC were constituted primarily by T lymphocytes (57 +/- 12% OKT 3-positive), B lymphocytes (18 +/- 9% surface immunoglobulin-positive), and macrophages (11 +/- 6% esterase-positive), and were responsive to phytohemagglutinin (mean uptake 86,933 cpm/5 X 10(5) cells). LPMC present in abnormal segments from 71% of patients with chronic inflammatory bowel disease were cytotoxic for ECAC isolated from colon (12.5 +/- 8.9% specific lysis) and small bowel (7.1 +/- 6.5%), but not for kidney control antigen (0.8 +/- 1.1%) isolated in a manner analogous to that used for ECAC (P less than 0.02). In contrast, despite comparable responses to phytohemagglutinin (mean uptake 59,200 cpm/5 X 10(5) cells), LPMC from histologically normal mucosa of patients with benign (adult megacolon, Hirshsprung's disease, diverticulosis) or malignant disease failed to lyse indicator cells labeled with colon-derived ECAC (0.3 +/- 0.08%), small bowel-derived ECAC (0.4 +/- 1.11%), or kidney antigen (0.29 +/- 0.79%). LPMC reactivity for individual gel-purified macromolecules of small bowel-derived ECAC (designated as the "P" series of components) was greatest against component P1 (by 2-3-fold), but was detectable against three other purified components as well. The addition of patient's serum did not enhance cytotoxicity to ECAC. Characterization of the cytotoxic cell showed that it was nonadherent to plastic surfaces, bore T lymphocyte-specific markers detectable by OKT 11 and OKT 3 monoclonal antibodies, and could be depleted by removal of cells with receptors for sheep erythrocytes. ECAC-specific reactivity was markedly reduced (greater than 93%) in most experiments when LPMC were preincubated for 1 h with ECAC. These data support the concept that autosensitization to several epithelial cell-associated components has occurred in patients with chronic inflammatory bowel disease, and provide initial evidence that antigen-specific, cell-mediated mechanisms may play a role in the pathogenesis of these disorders.
J K Roche, C Fiocchi, K Youngman
Platelet adhesion to monomeric collagen types I and III, which were purified from human umbilical arteries, was studied in a perfusion chamber under well defined flow conditions. For this purpose, glass coverslips were coated with 20-30 micrograms/cm2 of collagen types I and III by spraying a solution of these collagens with a retouching air brush. Platelet deposition increased with the time of perfusion. Adhesion to both collagen types was similar in the first 3 min, but increased platelet deposition occurred on collagen type III after 3 min due to thrombus formation. Adhesion at a shear rate of 800 s-1 was strongly impaired with plasma of a patient with von Willebrand's disease or with fibronectin-free plasma. Addition of purified fibronectin to fibronectin-free plasma restored adhesion to the level obtained with normal plasma. Platelet deposition in normal plasma increased with increasing shear rates. Platelet deposition in VWD-plasma was normal at 490 s-1, but there was no increase at higher shear rates. Platelet deposition in fibronectin-free plasma was diminished at all shear rates studied from 490 to 1,300 s-1. Perfusion with a human albumin solution (HAS) to which purified Factor VIII-von Willebrand factor complex (FVIII-VWF) and fibronectin had been added gave similar platelet deposition as with normal plasma. Preincubation of collagen with FVIII-VWF and perfusion with HAS containing fibronectin, or, conversely, preincubation with fibronectin and perfusion with HAS containing FVIII-VWF, also resulted in adhesion similar to that observed in normal plasma. Similar adhesion was also observed after preincubation with both FVIII-VWF and fibronectin and subsequent perfusion with HAS alone. Sequential preincubations with first FVIII-VWF and then fibronectin, or with first fibronectin and then FVIII-VWF followed by perfusion with HAS, also gave a similar adhesion as observed with normal plasma. These data indicate that platelet adhesion to monomeric collagen types I and III is dependent on both FVIII-VWF and fibronectin. FVIII-VWF is only required at relatively high shear rates; fibronectin also at relatively low shear rates. Their complementary role in platelet adhesion suggests separate binding sites for FVIII-VWF and fibronectin on collagen. Platelet deposition on performed fibrils of collagen types I and III was also studied. Initial adhesion expressed as percentage surface coverage was similar to that found with monomeric collagen, but thrombus formation was much enhanced. Adhesion on fibrillar collagen at 800 s(-1) was impaired in VWD-plasma and fibronectin-free plasma, and was restored by addition of purified fibronectin to fibronectin-free plasma. When perfusions were performed with HAS, only addition of FVIII-VWF was required for optimal adhesion to fibrillar collagen; addition of fibronectin had no effect. These data are in contrast to the studies with monomeric collagens described above, in which the addition of both FVIII-VWF and fibronectin was required. These data are also in contrast to the observation that in plasma both FVIII-VWF and fibronectin are required for optimal adhesion to fibrillar collagen.
W P Houdijk, K S Sakariassen, P F Nievelstein, J J Sixma
The Tn syndrome is an acquired clonal disorder characterized by the exposure of a normally hidden determinant, the Tn antigen, on the surface of human erythrocytes, platelets, granulocytes, and lymphocytes. Two distinct populations, Tn positive (Tn+) and Tn negative (Tn-), of mature hemopoietic cells are present in Tn patients. To determine whether the Tn antigen is already expressed on erythroid, myeloid, and pluripotent progenitors, light-density mononuclear blood cells from two patients with this syndrome were separated by fluorescent-activated cell sorting and by affinity chromatography into Tn+ and Tn- fractions, using their binding properties to Helix pomatia agglutinin (HPA). Burst-forming-unit erythroid (BFU-E), colony-forming-unit granulocyte/macrophage (CFU-GM), cells were assayed in plasma clot cultures. After 12-14 d of culture, colonies were studied by a double fluorescent labeling procedure. First, a fluorescein-conjugated HPA permitted evaluation of the presence or absence of the Tn antigen at the surface of the cells composing each colony, and second, the binding of a murine monoclonal antibody against either glycophorin A (LICR-LON-R10) or against a myeloid antigen (80H5), revealed by an indirect fluorescent procedure, was used to establish the erythroid or myeloid origin of each cell. The Tn+ fraction obtained by cell sorting gave rise to nearly 100% Tn+ colonies composed exclusively of cells bearing this antigen. The reverse was observed for the Tn- cell fraction. These results demonstrate that in the Tn syndrome, BFU-E, CFU-GM, and CFU-GEMM of the Tn+ clone express the Tn antigen at this early stage of differentiation.
W Vainchenker, G Vinci, U Testa, A Henri, A Tabilio, M P Fache, H Rochant, J P Cartron
The effect of 5-azacytidine on erythroid precursors and progenitors was studied in nine patients with sickle cell anemia or severe thalassemia. Each patient received the drug intravenously for 5 or 7 d. 5-Azacytidine caused a four- to sixfold increase in gamma-messenger RNA concentration in bone marrow cells of eight of the nine patients and decreased the methylation frequency of a specific cytosine residue in the gamma-globin gene promoter in all nine patients. Within 2 d of the start of drug treatment there was a rise in the percentage of reticulocytes containing fetal hemoglobin (HbF; F-reticulocytes) without a significant change in the total number of reticulocytes, which suggested that there was a direct action of 5-azacytidine on erythroid precursors. Late erythroid progenitors (CFU-E), present in bone marrow after 2 d of drug administration, formed colonies containing an increased amount of HbF as compared with control colonies. Moreover, the number of CFU-E derived colonies was not decreased at these early times, which suggested that there was a direct action of 5-azacytidine on erythroid progenitors in the absence of cytotoxicity. Exposure of normal bone marrow cells in tissue culture to 5-azacytidine for 24 h reproduced both of these effects as judged during subsequent colony formation. The combined direct effects of 5-azacytidine on both the erythroid precursor and progenitor compartments resulted in an increase in HbF synthesis that was sustained for 2-3 wk. Toxicity to bone marrow as reflected by cytoreduction was evident after treatment in some patients but was not accompanied by an increase in HbF production. A correlation was found between the effects of 5-azacytidine on bone marrow, as assessed by in vitro measurements, and the hematological response of the individual patients to drug treatment.
R K Humphries, G Dover, N S Young, J G Moore, S Charache, T Ley, A W Nienhuis
We have examined the possibility that changes in glomerular filtration rate (GFR) after changes in dietary protein intake may depend on altered function of the tubuloglomerular (TG) feedback system. We studied male Sprague-Dawley rats after dietary pretreatment for 9.6 +/- 3.6 (SD) d with isocaloric diets containing either 6% or 40% casein. We found that GFR in rats fed the high protein diet was 24-29% higher than in rats fed the low protein diet. Simultaneous measurements of single nephron GFR (SNGFR) in the distal tubule were 6.3 nl/min or 21% higher in the rats fed the high protein diet whereas proximally measured SNGFR was not statistically different in the two groups. The higher distally measured SNGFR of rats receiving the high protein diet was associated with a 4.2 nl/min or 50% smaller suppression of SNGFR by TG feedback (-4.3 vs. -8.5 nl/min, P less than 0.001). Loop perfusion experiments demonstrated that in rats fed the high protein diet the TG feedback mechanism was less sensitive than in rats fed the low protein diet. The TG feedback response in rats fed the low protein diet, as assessed by reductions in stop-flow pressure and SNGFR, was half-maximal at flows of 14-15 nl/min. In contrast, the TG feedback response in rats fed the high protein diet was half-maximal at 22-24 nl/min. Maximal suppression of stop-flow pressure and SNGFR and the slope of the TG feedback response to increasing loop flow rates were not different in the two groups. We conclude that the sensing mechanism of the TG feedback system is rendered less responsive by a high protein intake, and that this change permits GFR to increase.
F D Seney Jr, F S Wright
DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL60-4, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gp150) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain [35S]methionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line. Kinetic and biochemical analyses established that gp130 is a precursor that differs in its carbohydrate moiety from gp150, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gp150 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.
A T Look, S C Peiper, M B Rebentisch, R A Ashmun, M F Roussel, C W Rettenmier, C J Sherr
Ribonucleoprotein particles containing Sm antigen were separated from particles containing both Sm and RNP antigens by ion-exchange chromatography to study the recognition of these antigens by autoimmune sera. By using the separated antigens, anti-Sm and/or anti-RNP antibodies were detected in approximately 60% of sera from systemic lupus erythematosus patients by both enzyme-linked immunosorbent assay and immunoprecipitation of radiolabeled antigens followed by analysis on sodium dodecyl sulfate-polyacrylamide gels. These antibodies were detected in 30% of the same sera using the standard passive hemagglutination technique. Competition experiments demonstrated that all of the sera tested that contained anti-Sm antibodies also had anti-RNP-like reactivity. This latter reactivity usually represented 80% or more of the total Sm and RNP binding activity in lupus sera. The binding to RNP-like determinants by several of the sera was uniquely resistant to treatment of the antigen with snake venom exonuclease. These studies indicate that humoral immunity against Sm and RNP antigens in systemic lupus erythematosus is directed primarily against a single type of ribonucleoprotein particle in which the two antigens are physically associated. The specific binding to a single type of ribonucleoprotein particle suggests that this particle may be especially immunogenic and that it might play an important role in induction of the humoral immune response to Sm and RNP.
W H Reeves, D E Fisher, R G Lahita, H G Kunkel
For many years it has been thought that distal nephron hydrogen ion secretion can be importantly modulated by factors such as sodium delivery, sodium avidity, and potassium stores. Free flow micropuncture studies have also indicated that the rate of bicarbonate delivery may also alter the rate of bicarbonate reabsorption. The present studies were undertaken to examine possible luminal influences on total CO2 reabsorption in microperfused distal tubules in the rat in vivo. Tubules from normal and acidotic rats were perfused with five solutions in a manner that induced changes in bicarbonate load, sodium and potassium fluxes (JNa, JK), and luminal sulfate concentration. in each collected perfusate, simultaneous analyses were undertaken to determine water reabsorption, Na, and K concentrations using graphite furnace atomic absorption spectroscopy and total CO2 by microcalorimetry. Using factorial analysis of covariance to account for confounding effects on total CO2 flux (JtCO2) such as water reabsorption, distal tubules of acidotic rats reabsorbed CO2 in the range of 50-112 pmol X min-1 X mm-1 X These JtCO2 values were not significantly correlated with HCO3 load, JNa, or JK despite changes in the latter from net reabsorption to net secretion. Distal tubules of rats with normal acid-base status had JtCO2 values which were neither significantly different from zero nor correlated with changes in JK and JNa. Further, doubling the load from 250-500 pmol/min (by doubling the perfusion rate of 25-mM HCO3 solutions) did not stimulate JtCO2 in these normal animals. Accordingly, these acute in vivo microperfusion studies indicate for the first time that neither load nor potassium or sodium fluxes are important modulators of distal tubule bicarbonate reabsorption.
D Z Levine
After successful bone marrow transplantation, patient hematopoietic and lymphoid cells are replaced by cells derived from the donor marrow. To document and characterize successful engraftment, host and donor cells must be distinguished from each other. We have used DNA sequence polymorphism analysis to determine reliably the host or donor origin of posttransplant cell populations. Using a selected panel of six cloned DNA probes and associated sequence polymorphisms, at least one marker capable of distinguishing between a patient and his sibling donor can be detected in over 95% of cases. Posttransplant patient peripheral leukocytes were examined by DNA restriction enzyme digestion and blot hybridization analysis. We have studied 18 patients at times varying from 13 to 1,365 d after marrow transplantation. Mixed lymphohematopoietic chimerism was detected in 3 patients, with full engraftment documented in 15. One patient with severe combined immunodeficiency syndrome was demonstrated to have T cells of purely donor origin, with granulocytes and B cells remaining of host origin. Posttransplant leukemic relapse was studied in one patient and shown to be of host origin. DNA analysis was of particular clinical value in three cases where failure of engraftment or graft loss was suspected. In two of the three cases, full engraftment was demonstrated and in the third mixed lymphohematopoietic chimerism was detected. DNA sequence polymorphism analysis provides a powerful tool for the documentation of engraftment after bone marrow transplantation, for the evaluation of posttransplant lymphoma or leukemic relapse, and for the comprehensive study of mixed hematopoietic and lymphoid chimeric states.
D Ginsburg, J H Antin, B R Smith, S H Orkin, J M Rappeport
Hydroxylation of 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha-diol was studied in mitochondrial preparations from human fetal livers. The livers were obtained at legal abortions between weeks 14 and 24. In addition to hydroxylation in the 26-position, 5 beta-cholestane-3 alpha, 7 alpha-diol was hydroxylated in the 12 alpha-position. In one experiment, mitochondrial protein was solubilized and partially purified. Material with such chromatographic properties as those of cytochrome P450 showed 12 alpha-hydroxylase activity when combined with adrenodoxin and adrenodoxin reductase from bovine adrenal mitochondria. Because adrenodoxin and adrenodoxin reductase are components specific for mitochondrial hydroxylase systems, the results exclude microsomal contamination as the origin of this 12 alpha-hydroxylase activity. Further, there was no hydroxylase activity when NADPH-cytochrome P450 reductase from rat liver microsomes was added instead of adrenodoxin and adrenodoxin reductase. The microsomal fraction of fetal liver was also shown to possess 12 alpha-hydroxylase activity. Microsomal and mitochondrial hydroxylase activities per milligram of protein towards 5 beta-cholestane-3 alpha, 7 alpha-diol were of the same order of magnitude. The occurrence of an efficient sterol nucleus hydroxylase activity in liver mitochondria appears to be unique for fetal liver.
The effects of a chronic load of nonabsorbable sugars on intracolonic bacterial metabolism of carbohydrates and on H2 breath excretion are disputed. However, most of the discussion relies on indirect evidence or on results of in vitro studies. Thus, we attempted to assess directly and in vivo the effects on intracolonic metabolism of lactulose of a chronic oral load of this nonabsorbable disaccharide. 20 g of lactulose was given orally twice daily during 8 d to eight normal volunteers. In all, breath H2 concentration was measured on days 1 and 8 after ingestion of the morning lactulose dose. In four subjects, stools were collected during 2 d at the beginning and at the end of the lactulose maintenance period to measure fecal pH and daily outputs of carbohydrates and beta-galactosidase. The four other subjects were intubated on days 1 and 8 to measure the pH and the concentrations of carbohydrates, lactic acid, and volatile fatty acids (VFA) in the distal ileum and cecal contents. Moreover, 14C-lactulose was added to cold lactulose and 14CO2 breath outputs determined. Pulmonary H2 excretion fell from day 1 to day 8 (P less than 0.05), whereas 14CO2 excretion increased (P less than 0.01). Fecal water pH, lactic acid, and VFA concentrations did not vary between the two stool collection periods. 24-h fecal weight, fecal water, and carbohydrate outputs showed a trend to decrease between days 1 and 2 and days 7-8, whereas beta-galactosidase activity rose markedly (P less than 0.01). No significant variations were observed for all parameters measured in ileal fluid. In the cecum, areas under the concentration curves decreased from day 1 to day 8 for lactulose, galactose, and fructose (P less than 0.01), while an increase was found for lactic acid (P less than 0.001), acetic acid (P less than 0.0001), and total VFA (P less than 0.001). Cecal fluid pH dropped faster (P less than 0.05) and to a lower level (P less than 0.05) on day 8 than on day 1. These data clearly show that a chronic load of a nonabsorbable sugar induces changes in colonic bacterial metabolic pathways resulting in a better efficiency of the flora to digest the carbohydrate.
C Florent, B Flourie, A Leblond, M Rautureau, J J Bernier, J C Rambaud
In subjects with hypertriglyceridemia, plasma concentrations of low density lipoprotein (LDL) cholesterol are often normal or reduced. Perturbations that alter plasma very low density lipoprotein (VLDL) concentrations are associated with opposite changes in plasma LDL levels. To determine the mechanisms regulating plasma LDL levels, we used 131I-VLDL and 125I-LDL to measure the fractional catabolic rates (FCR), production rates (PR), and rates of interconversion of apoprotein B (apo B) in VLDL, intermediate density lipoprotein, and LDL in six hypertriglyceridemic subjects pre- and post-weight reduction. [2-3H]glycerol was used to quantitate VLDL triglyceride PR. All data are presented as mean +/- SD. Percent ideal body weight fell from 132 +/- 17.9 to 119 +/- 15.9% in the group, P less than 0.05. After weight loss, plasma VLDL triglyceride (486.0 +/- 364.1 vs. 191.3 +/- 65.4 mg/dl, P less than 0.05) and VLDL apo B (32.2 +/- 12.0 vs. 14.8 +/- 6.8 mg/dl, P less than 0.05) concentrations were reduced. VLDL triglyceride PR also fell after weight reduction (56.6 +/- 39.0 vs. 28.6 +/- 23.1 mg/kg per h, P less than 0.05), as did VLDL apo B PR (47.9 +/- 41.4 vs. 19.0 +/- 14.1 mg/kg per d, P less than 0.05). Pre-weight loss, plasma LDL cholesterol and apo B levels were low-normal or reduced (64.0 +/- 12.6 and 58.4 +/- 11.9 mg/dl, respectively) despite normal or elevated LDL apo B PR (17.4 +/- 7.2 mg/kg per d). The reduced cholesterol and apo B levels were associated with increased FCRs (0.68 +/- 0.29 d-1) and reduced cholesterol/protein ratios (1.01 +/- 0.18) in LDL. The plasma levels of LDL cholesterol and apo B rose after weight reduction (84.8 +/- 24.9, P less than 0.05; and 69.5 +/- 14.3 mg/dl, P less than 0.05, respectively, vs. base line). These increased concentrations resulted from a combination of events. First, the FCR for LDL apo B fell in five of six subjects with a significant reduction for the group as a whole (0.48 +/- 0.11 d-1, P less than 0.05 vs. base line). Second, the cholesterol/protein ratio increased in all six subjects with a significantly greater mean after weight loss (1.25 +/- 0.27, P less than 0.05 vs. base line). In contrast, the LDL apo B PR fell or was essentially unchanged in the six subjects after weight loss (mean, 14.4 +/- 2.8 mg/kg per d; NS vs. pre-weight loss). The changes in LDL catabolism and composition were associated with changes in the source of LDL apo B. Pre-weight loss, 73.3% of LDL was derived from VLDL, while 26.7% was directly secreted into plasma. Post-weight reduction, VLDL-derived LDL fell to 46.8% of total, while direct secretion accounted for 53.2% of LDL production. These changes were significant; P < 0.95. Thus, all subjects had direct secretion of LDL apo B and the magnitude of this source of VLDL triglyceride secretion. These results indicate that the regulation of plasma LDL levels in hypertriglyceridemic subjects is quite complex and that the rise in LDL levels after weight loss results from reduction in the fractional catabolism of this lipoprotein. The fall in the FCR is associated with changes in the source of LDL and in its composition.
H N Ginsberg, N A Le, J C Gibson
We describe a sensitive assay to measure immune activation of human macrophages in cell culture. Freshly isolated blood monocytes from normal subjects lack the ability to endocytose and degrade mannosyl-terminated glycoconjugates via specific receptors, but acquired this activity after cultivation in autologous serum for approximately 3 d. Addition of specific antigen, purified protein derivative, or T cell mitogens to mononuclear cells prevented the appearance of macrophage mannosyl receptor activity and lymphokine, gamma-, and alpha-interferons selectively down-regulated receptor activity in monocyte-macrophage targets. The effects of antigen challenge and gamma-interferon on mannosyl receptors can be prevented by 10(-8) M dexamethasone. Dexamethasone also inhibited release of another macrophage activation marker, plasminogen activator, which was increased by both gamma- and alpha-interferons. These studies show that activation of human macrophages is regulated by opposing actions of lymphokines and glucocorticoids.
T Mokoena, S Gordon
Cytosine arabinoside (araC) has proven efficacy in acute myeloid leukemia (AML), but its place in the treatment of acute lymphoblastic leukemia (ALL) and T lymphoblastic lymphoma is uncertain. The therapeutic potential of araC has been assessed in patients with AML, ALL, and T lymphoblastic lymphoma by measuring the conversion of araC to its active metabolite, the 5'-triphosphate of araC (araCTP), in purified blasts from patients as well as in normal polymorphs and lymphocytes. In all leukemias, araCTP was the major intracellular metabolite of araC. The highest araCTP formation was in blasts from T lymphoblastic lymphoma, which formed threefold more nucleotide than myeloblasts, and in turn myeloblasts formed twofold more araCTP than lymphoblasts from ALL. The mean araCTP formation in myeloblasts was sixfold greater than polymorphs, but in contrast, lymphoblasts and lymphocytes formed low and similar amounts of this nucleotide. Reasons for the sixfold range in araCTP accumulation in the various leukemic blasts were studied. The mean size of myeloblasts was 35-70% larger than lymphoblasts when compared on the basis of protein or intracellular water content, but T lymphoblastic lymphoma blasts and lymphoblasts were the same size. Activities of deoxycytidine kinase, deoxycytidylate deaminase, and pyrimidine nucleoside monophosphate kinase were not different between any of the leukemic cell types. The number of nucleoside transport sites on blasts was estimated by measuring the equilibrium binding of [3H]nitrobenzylthioinosine (NBMPR), which binds with high affinity to the transporter. Scatchard analysis yielded mean values of 27,500 sites/cell for T lymphoblastic lymphoma blasts, 10,000 sites/cell for myeloblasts, and 2,300 sites/cell for lymphoblasts. Our previous work has shown that araC influx correlates with the maximum number of 3H-NBMPR binding sites in leukemic and normal white cells. A strong correlation was observed between the number of nucleoside transport sites per leukemic blast cell and the accumulation of intracellular araCTP from extracellular araC at 1 microM. Membrane transport of araC at the low concentrations (approximately 1 microM), which are achieved therapeutically, is a major rate-limiting step in its conversion to araCTP by leukemic blast cells. Myeloblasts form more araCTP than lymphoblasts because of both higher nucleoside transport capacity and larger cell size. The highest nucleoside transport capacity and largest conversion of araC to araCTP is in T lymphoblastic lymphoma, which suggests that araC may be effective in the treatment of this disease.
J S Wiley, J Taupin, G P Jamieson, M Snook, W H Sawyer, L R Finch
Milrinone is a potent positive inotropic and vascular smooth muscle-relaxing agent in vitro, and therefore, it is not known to what extent each of these actions contributes to the drug's hemodynamic effects in patients with heart failure. In 11 patients with New York Heart Association class III or IV congestive heart failure, incremental intravenous doses of milrinone were administered to determine the dose-response relationships for heart rate, systemic vascular resistance, and inotropic state, the latter measured by peak positive left ventricular derivative of pressure with respect to time (dP/dt). To clarify further the role of a positive inotropic action, the relative effects of milrinone and nitroprusside on left ventricular stroke work and dP/dt were compared in each patient at doses matched to cause equivalent reductions in mean arterial pressure or systemic vascular resistance, indices of left ventricular afterload. Milrinone caused heart rate, stroke volume, and dP/dt to increase, and systemic vascular resistance to decrease in a concentration-related manner. At the two lowest milrinone doses resulting in serum concentrations of 63 +/- 4 and 156 +/- 5 ng/ml, respectively, milrinone caused significant increases in stroke volume and dP/dt, but no changes in systemic vascular resistance or heart rate. At the maximum milrinone dose administered (mean serum concentration, 427 +/- 11 ng/ml), heart rate increased from 92 +/- 4 to 99 +/- 4 bpm (P less than 0.01), mean aortic pressure fell from 82 +/- 3 to 71 +/- 3 mmHg (P less than 0.01), right atrial pressure fell from 15 +/- 2 to 7 +/- 1 mmHg (P less than 0.005), left ventricular end-diastolic pressure fell from 26 +/- 3 to 18 +/- 3 (P less than 0.005), stroke volume index increased from 20 +/- 2 to 30 +/- 2 ml/m2 (P less than 0.005), stroke work index increased from 14 +/- 2 to 21 +/- 2 g X m/m2 (P less than 0.01), and dP/dt increased from 858 +/- 54 to 1,130 +/- 108 mmHg/s (P less than 0.005). When compared with nitroprusside for a matched reduction in mean aortic pressure or systemic vascular resistance, milrinone caused a significantly greater increase in stroke work index at the same or lower left ventricular end-diastolic pressure. Milrinone caused a concentration-related increase in dP/dt (32% increase at maximum milrinone dose), whereas nitroprusside had no effect. These data in patients with severe heart failure indicate that in addition to a vasodilating effect, milrinone exerts a concentration-related positive inotropic action that contributes significantly to the drug's overall hemodynamic effects. The positive inotropic action occurs at drug levels that do not exert significant chronotropic or vasodilator effects.
B E Jaski, M A Fifer, R F Wright, E Braunwald, W S Colucci
One mechanism of histamine-mediated inhibition of the immune response in man is to activate T suppressor cells that bear the Leu 2 (OKT8) marker. The current study was undertaken to characterize the histamine-induced suppressor cell using a monoclonal antibody (9.3) shown previously to distinguish cytotoxic T cells from antigen-specific suppressor T cells. Leu 2+ cells isolated from peripheral blood were further separated with antibody 9.3 into Leu 2+, 9.3+, and Leu 2+, 9.3- subsets and each subset was incubated with different concentrations of histamine before determining their ability to suppress immune responses in vitro. The results indicate that the Leu 2+, 9.3- subpopulation includes all histamine-induced suppressor cells, that 10(-4) M histamine is the optimal concentration for suppressor cell induction, and that exposure of Leu 2+, 9.3- cells to histamine for 30 s is sufficient to initiate the induction process. After treatment with histamine these cells inhibit both phytohemagglutinin-induced T cell proliferation and pokeweed mitogen-induced B cell differentiation. The suppression of phytohemagglutinin-induced proliferation was resistant to x-irradiation with 1,200 rad, either before or after histamine exposure, suggesting that Leu 2+, 9.3- cells need not proliferate to become suppressor cells or exert suppression. Moreover, suppression by these cells was not due to altered kinetics of the response. Finally, a histamine type 2 receptor antagonist (cimetidine) but not a type 1 receptor antagonist (mepyramine) blocked the induction of suppressor cells. On the basis of these results and our previous studies of antigen specific suppressor cells, we conclude that Leu 2+ suppressor cells in man are derived from a precursor pool that is phenotypically distinct from cells that can differentiate into cytotoxic T cells.
P Sansoni, E D Silverman, M M Khan, K L Melmon, E G Engleman
During blood coagulation human polymorphonuclear leukocytes release elastase in amounts that can exceed 100 nmol/liter. We therefore studied the effect of elastase on platelet structure and function. Physiologic concentrations of elastase specifically inhibited thrombin-induced platelet aggregation and ristocetin-induced agglutination of washed platelets in a time- and dose-dependent manner. This was associated with a decrease in the number of high affinity thrombin binding sites on the platelet surface (analysis by "Ligand" program) from 31 per platelet to 12 per platelet (P less than 0.05). As analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, treatment of 3H-labeled platelets with elastase resulted in a decrease in the percent glycoprotein at 130,000-150,000 Mr = and an increase in the percent protein at Mr = 102,000. The supernatant from elastase-treated platelets contained a Mr = 88,000 glycoprotein not found in the supernatant from untreated platelets. Immunoprecipitation studies with monoclonal antiglycoprotein Ib demonstrated that treatment of whole platelets with physiologic concentrations of elastase resulted in proteolytic cleavage of glycoprotein Ib. Elastase treatment of glycoprotein immunoisolated with monoclonal antiglycoprotein Ib antibody resulted in formation of a glycopeptide with the same electrophoretic mobility as the Mr = 102,000 membrane-related glycopeptide. In contrast, analysis by Western blot technique using antiglycoprotein IIb and IIIa antibodies demonstrated that elastase did not degrade glycoproteins IIb or IIIa. We conclude that elastase inhibition of thrombin-induced platelet stimulation is accompanied by (a) a reduction in the number of thrombin binding sites per platelet and (b) proteolysis of glycoprotein Ib.
M S Brower, R I Levin, K Garry
Using purified prostaglandin (PG) H synthase, which synthesizes PGG2 and PGH2 from arachidonic acid, we were able to assay for the presence of peroxide activators in biological tissues. This assay system, capable of detecting both hydrogen peroxide (H2O2) and lipid hydroperoxides, detected a significant amount of synthase activator in plasma. Treatment of the active preparations with catalase and glutathione peroxidase showed that the principal activator in normal human plasma was a lipid hydroperoxide rather than H2O2.
M A Warso, W E Lands
Atrophoderma is a rare dermal disorder characterized by a patchy distribution of areas apparently devoid of elastic fibers. Skin fibroblast cultures were established from the normal and affected dermis of a patient with this disorder. Human tropoelastin was identified in culture medium by use of electroblotting and anti-elastin antisera. An enzyme-linked immunosorbent assay was used to establish that significantly less elastin accumulated in the media of cultured cells from lesional fibroblasts over a 3-d period. Since elastin biosynthesis in most tissues is under pretranslational control, molecular hybridization to a nick-translated genomic elastin probe was performed; however, elastin messenger RNA levels were equivalent in both cell strains. Both strains produced less elastin than did normal skin fibroblasts. Extracellular proteolysis of elastin was evaluated as a possible mechanism. Elastase activity was increased and porcine tropoelastin was degraded four times faster, on a per-cell basis, in lesional fibroblast cultures than in cells derived from an unaffected site. The two cell strains exhibited no significant differences in collagen production or collagenase activity. These results are the first demonstration of elastin production by cultured human skin fibroblasts, and they suggest that the primary defect in atrophoderma may be a result of enhanced degradation of newly synthesized elastin precursors.
M G Giro, A I Oikarinen, H Oikarinen, G Sephel, J Uitto, J M Davidson
Starvation in laboratory rodents results in significant alterations in thyroid hormone economy characterized by decreased circulating levels of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) and a decline in serum thyrotropin (TSH) concentration. To investigate this apparent paradox, we have compared in fasted and hypothyroid animals the intracellular parameters mediating thyroid hormone action in the anterior pituitary gland. In vitro saturation analysis combined with quantitation of nuclear T3 content by radioimmunoassay allowed for characterization of pituitary nuclear T3 receptors and estimation of the endogenous fractional receptor occupancy. In rats, thyroidectomized 4 wk earlier, the 10-fold increase in serum TSH levels and decline in peripheral thyroid hormone concentrations were accompanied by a 61% decrease in pituitary nuclear T3 content and a marked decline in fractional T3 receptor occupancy as compared with control animals. In euthyroid animals subjected to short-term starvation (72 h), serum T3, T4, and TSH levels declined by 52, 43, and 48%, respectively. Despite these marked decreases in circulating thyroid hormone levels, pituitary nuclear T3 content in fasted rats declined by only 15% (P less than 0.05) relative to control levels. This modest decline in nuclear T3 content, combined with a 23% decrease in total T3 receptor number, resulted in an estimated fractional receptor occupancy in fasted animals which was equal to or greater than that noted in controls. The effects of fasting and hypothyroidism on the pituitary were further investigated by quantifying low Michaelis constant (Km) T4 5'-deiodinase activity in the crude cytosol fraction of pituitary homogenates. In thyroidectomized animals, maximum velocity was increased ninefold, whereas fasting resulted in a 37% decrease (P less than 0.025) in this parameter compared with controls. Km values were similar in all experimental groups (4.7 +/- 0.6 nM). These results demonstrate that, despite significant reductions in circulating thyroid hormone concentrations and pituitary T4 5'-deiodinase activity, nuclear T3 levels are maintained at relatively normal levels in the pituitary of the fasted animal and fractional T3 receptor occupancy may actually increase. These findings are in marked contrast to those noted in thyroidectomized animals and suggest that the suppression of TSH secretion accompanying starvation in the rat is mediated, at least in part, by local pituitary mechanisms that serve to maintain and possibly enhance nuclear T3 receptor occupancy.
D L St Germain, V A Galton
The effect of the continuous exposure to ovine corticotropin-releasing factor (oCRF) was measured in adult male rats. The intravenous infusion of 0.75 nmol oCRF/h to intact rats over a 24-h period was accompanied by a peak of ACTH and corticosterone secretion that occurred during the first 90 min of administration of the releasing factor, followed by a decrease to lower, but still above control, values. Additionally, corticotropin-releasing factor (CRF)-treated rats had decreased plasma testosterone levels. The subcutaneous administration of 0.075 or 0.75 nmol oCRF/h to intact rats for 7 d also resulted in elevations of both plasma ACTH and corticosterone levels comparable to those measured after a 24-h exposure to the releasing factor, as well as dose-related hypertrophy of the adrenals and increases in pituitary ACTH content. In these animals, CRF markedly inhibited luteinizing hormone (LH) (but not follicle-stimulating hormone [FSH] ), testosterone, and PRL secretion and decreased seminal vesicle weights. All the effects of CRF were mimicked by exogenously administered ACTH. By contrast, with the exception of FSH secretion, which was slightly elevated by CRF, neither CRF nor ACTH were able to significantly modify reproductive parameters in adrenalectomized animals, which suggests that the elevation of circulating levels of adrenal steroids induced by peripherally administered CRF represents major mediators of CRF-induced inhibition of fertility. These results indicate that in the rat, the continuous stimulation of the pituitary-adrenal axis by peripherally administered CRF causes some degree of desensitization of the pituitary-adrenal axis, but is still accompanied by persistent elevations of the circulating levels of both ACTH and corticosteroids. The increased secretion of adrenal steroids by CRF-treated rats is believed to participate in the disruption of reproductive parameters observed in these rats.
C Rivier, W Vale
A new hemoglobin variant, hemoglobin (Hb) Nunobiki, was detected in a Japanese male with marginal erythrocytosis. The Hb Nunobiki component amounted to 13.1% of the total hemoglobin. Structural analysis of this variant established the substitution of a cysteine for an arginine at the carboxy terminus of the alpha-chain (alpha 141). The oxygen equilibrium curves of Hb Nunobiki revealed extremely high oxygen affinity with a reduced Hill coefficient n, a decreased alkaline Bohr effect, and a decreased 2,3-diphosphoglyceric acid effect. The isoelectric point of the Hb Nunobiki changed during storage, although the oxyhemoglobin state was maintained. These findings could be accounted for by the specific characteristics of a newly introduced cysteinyl residue. Cysteinyl residue at alpha 141 in Hb Nunobiki did not seem to be involved in the formation of either intermolecular or intramolecular disulfide bonds under physiologic conditions. The low proportion of Hb Nunobiki (13.1%) in the propositus was also discussed after it was verified that he exhibited four alpha-globin genes per diploid cell.
Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) are synthetic "C" nucleosides whose antineoplastic activity depends on their conversion to tiazofurin-adenine dinucleotide and selenazofurin-adenine dinucleotide which are analogs of NAD. The present study was conducted to determine whether these nucleoside analogs and their dinucleotide derivatives interfere with NAD metabolism and in particular with the NAD-dependent enzyme, poly(ADP-ribose) polymerase. Incubation of L1210 cells with 10 microM tiazofurin or selenazofurin resulted in inhibition of cell growth, reduction of cellular NAD content, and interference with NAD synthesis. Using [14C]nicotinamide to study the uptake of nicotinamide and its conversion to NAD, we showed that the analogs interfere with NAD synthesis, apparently by blocking formation of nicotinamide mononucleotide. The analogs also serve as weak inhibitors of poly(ADP-ribose) polymerase, which is an NAD-utilizing, chromatin-bound enzyme, whose function is required for normal DNA repair processes. Continuous incubation of L1210 cells in tiazofurin or selenazofurin resulted in progressive and synergistic potentiation of the cytotoxic effects of DNA-damaging agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. These studies provide a basis for designing chemotherapy combinations in which tiazofurin or selenazofurin are used to modulate NAD and poly(ADP-ribose) metabolism to synergistically potentiate the effects of DNA strand-disrupting agents.
N A Berger, S J Berger, D M Catino, S J Petzold, R K Robins
In normal subjects, apolipoprotein E (apo E) is present on very low density lipoproteins (VLDL) (fraction I) and on particles of a size intermediate between VLDL and low density lipoproteins (LDL) (fraction II). The major portion of apo E is, however, on particles smaller than LDL but larger than the average high density lipoproteins (HDL) (fraction III). To investigate the possible role of the vascular lipases in determining this distribution of apo E among the plasma lipoproteins, we studied subjects with primary deficiency of either hepatic lipase or of lipoprotein lipase and compared them with normal subjects. Subjects with familial hepatic triglyceride lipase deficiency (n = 2) differ markedly from normal in that fraction II is the dominant apo E-containing group of lipoproteins. When lipolysis of VLDL was enhanced in these subjects upon release of lipoprotein lipase by intravenous heparin, a shift of the apo E from VLDL into fractions II and III was observed. In contrast, apolipoproteins CII and CIII (apo CII and CIII, respectively) did not accumulate in intermediate-sized particles but were shifted markedly from triglyceride rich lipoproteins to HDL after treatment with heparin. In subjects with primary lipoprotein lipase deficiency (n = 4), apo E was confined to fractions I and III. Release of hepatic triglyceride lipase by heparin injection in these subjects produced a shift of apo E from fraction I to III with no significant increase in fraction II. This movement of apo E from large VLDL and chylomicron-sized particles occurred with little hydrolysis of triglyceride and no significant shift of apo CII or CIII into HDL from triglyceride rich lipoproteins. When both lipoprotein lipase and hepatic triglyceride lipase were released by intravenous heparin injection into normal subjects (n = 3), fraction I declined and the apo E content of fraction III increased by an equivalent amount. Either moderate or no change was noted in the intermediate sized particles (fraction II). These data strongly support the hypothesis that fraction II is the product of the action of lipoprotein lipase upon triglyceride rich lipoproteins and is highly dependent on hepatic triglyceride lipase for its further catabolism. In addition, the hydrolysis by hepatic triglyceride lipase of triglyceride rich lipoproteins in general results in a preferential loss of apo E and its transfer to a specific group of large HDL.
A Rubinstein, J C Gibson, J R Paterniti Jr, G Kakis, A Little, H N Ginsberg, W V Brown
Conditioned media were prepared from human peripheral blood monocytes and human umbilical vein endothelial cells. These media were assayed for erythroid burst-promoting activity (BPA) using human peripheral blood monocyte-depleted mononuclear cells as targets and assessing the stimulatory effect of the conditioned media on growth of early erythroid progenitor cells. Both monocytes and endothelial cells produced modest amounts of detectable BPA. Addition of varying concentrations of media conditioned by monocytes to plateau concentrations (5-10%) of media conditioned by endothelial cells had no additive effect. Endothelial cells incubated in the presence of 50% monocyte-conditioned medium produced 2.5- to 6.6-fold more BPA than did endothelial cells incubated only in control tissue culture medium. In contrast, endothelial cell conditioned medium did not stimulate increased BPA production by monocytes. Neither neutrophil- nor marrow fibroblastoid cell-conditioned medium stimulated BPA production by endothelial cells. Therefore, both monocytes and endothelial cells produce BPA. Moreover, monocytes produce a monokine that, in turn, stimulates the production of BPA by endothelial cells. Inasmuch as a monokine also has been shown to stimulate production of granulocyte-macrophage colony-stimulating activity, we propose that monocytes play a critical role in regulating the production of humoral regulators of the very early stages of hemopoietic cell differentiation.
K S Zuckerman, G C Bagby Jr, E McCall, B Sparks, J Wells, V Patel, D Goodrum
Patients with advanced breast cancer may develop acute, severe hypercalcemia when treated with estrogens or antiestrogens. In this study, we examined the effects of estrogens and related compounds on the release of bone resorbing activity by cultured human breast cancer cells in vitro. We found that the estrogen receptor positive breast cancer cell line MCF-7 releases bone resorbing activity in response to low concentrations of 17 beta-estradiol. Bone resorbing activity was also released in response to the antiestrogen nafoxidine. Other steroidal compounds had no effect on the release of bone resorbing activity. Estrogen-stimulated release of bone resorbing activity occurred with live bone cultures, but not with devitalized bones, indicating that the effect was bone cell mediated. The breast cancer cell line MDA-231, which does not have estrogen receptors, did not release bone resorbing activity in response to 17 beta-estradiol or nafoxidine. Release of the bone resorbing activity by MCF-7 cells incubated with 17 beta-estradiol was inhibited by indomethacin (10 microM) and flufenamic acid (50 microM), two structurally unrelated compounds that inhibit prostaglandin synthesis. Concentrations of 17 beta-estradiol and nafoxidine that caused increased release of bone resorbing activity by the breast cancer cells caused a four- to fivefold increase in release of prostaglandins of the E series by MCF-7 cells. These data may explain why some patients with advanced breast cancer develop acute hypercalcemia when treated with estrogens or antiestrogens, and why bone metastases are more common in patients with estrogen receptor positive tumors.
A Valentin-Opran, G Eilon, S Saez, G R Mundy
Namalva, a human B cell lymphoma line, produced a factor with a molecular weight of approximately 60,000 which enhanced the proliferation of normal activated human B lymphocytes. The factor also enhanced the proliferation of certain B cell lines. It can be distinguished physiologically and biochemically from other lymphokines known to enhance B cell proliferation, namely, interleukin (IL) 1, IL 2, and interferon. The production of B cell growth factor by B cell tumor lines may contribute to their ability to grow autonomously and may reflect an important component of the neoplastic potential of the cell. B cell growth factor produced by tumors may also affect normal cells in vivo.
J L Ambrus Jr, A S Fauci
Contracting muscle cells release K ions into their surrounding interstitial fluid, and some of these ions, in turn, enter venous plasma. Thereby, intense or exhaustive exercise may result in hyperkalemia and potentially dangerous cardiotoxicity. Training not only reduces hyperkalemia produced by exercise but in addition, highly conditioned, long-distance runners may show resting hypokalemia that is not caused by K deficiency. To examine the factors underlying these changes, dogs were studied before and after 6 wk of training induced by running on the treadmill. Resting serum [K] fell from 4.2 +/- 0.2 to 3.9 +/- 0.3 meq/liter (P less than 0.001), muscle intracellular [K] rose from 139 +/- 7 to 148 +/- 14 meq/liter (P less than 0.001), and directly measured muscle cell membrane potential (Em) in vivo rose from -92 +/- 5 to -103 +/- 5 mV (P less than 0.001). Before training, resting Em of isolated intercostal muscle in vitro was -87 +/- 5 mV, and after incubation in 10(-4) M ouabain, Em fell to -78 +/- 5 mV. After training, resting Em of intercostal muscle rose to -95 +/- 4, but fell to -62 +/- 4 mV during incubation in 10(-4) M ouabain. The measured value for the Em was not completely explained by the increased ratio of intracellular to extracellular [K] or by the potassium diffusion potential. Skeletal muscle sarcolemmal Na,K-ATPase activity (microM inorganic phosphate mg-1 protein h-1) increased from 0.189 +/- 0.028 to 0.500 +/- 0.076 (P less than 0.05) after training, whereas activities of Mg2+ -dependent ATPase and 5'nucleotidase did not change. In untrained dogs, exercise to the point of exhaustion elevated serum [K] from 4.4 +/- 0.5 to 6.0 +/- 1.0 meq/liter (P less than 0.05). In trained dogs, exhaustive exercise was associated with elevation of serum [K] from 3.8 +/- 0.3 to 4.2 +/- 0.4 (NS). The different response of serum [K] to exercise after training was not explainable by blood pH. Basal insulin levels rose from 7.0 +/- 0.7 microU/ml in the untrained dogs to 9.9 +/- 1.0 microU/ml (P less than 0.05) after training. Although insulin might have played a role in the acquired electrical hyperpolarization, the reduced exercise-produced hyperkalemia after training was not reversed by blockade of insulin release with somatostatin. Although the fundamental mechanisms underlying the cellular hyperpolarization were not resolved, our observations suggest that increased Na-K exchange across the sarcolemmal membrane, the increase of Na,K-ATPase activity and possibly increased electrogenicity of the sodium pump may all play a role in the changes induced by training.
J P Knochel, J D Blachley, J H Johnson, N W Carter
The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells.
K D Sabbath, E D Ball, P Larcom, R B Davis, J D Griffin
The present study demonstrates the graded effect of in vitro corticosteroids (CSs) on the different phases of B cell activation, proliferation, and differentiation. Early events such as activation and proliferation of high-dose anti-mu or Staphylococcus aureus-stimulated B cells are profoundly suppressed by the presence of in vitro CSs. The suppressed proliferative response may be mediated by a direct effect on B cells and/or modulation of accessory cell function. Later events in the B cell cycle such as the proliferative response to B cell growth factor after either in vivo or in vitro activation are less sensitive to the suppressive effects of in vitro CSs. The final events in the B cell cycle; namely, the differentiation to the immunoglobulin-producing state, is not suppressed by in vitro CSs. Indeed, depending on the systems employed, there is either no effect or enhancement of immunoglobulin secretion by the presence of in vitro CSs. The graded effect of in vitro CSs on the discrete phases of the B cell activation, proliferation, and differentiation cycle provide new insights into the complex nature of CS-induced modulation of human B cell responses.
T R Cupps, T L Gerrard, R J Falkoff, G Whalen, A S Fauci
Interleukin-2 (IL-2) production in vitro is depressed in systemic lupus erythematosus (SLE) patients. It is not known whether this abnormality is caused by a defect in the producer lymphocytes or by excessive suppression. We report that removal of OKT8 (Leu 2a)+ cells increased the IL-2 production by in vitro-stimulated lymphocytes to normal or above normal levels in 19 of 21 SLE patients. This increase was more apparent in those patients with clinically inactive disease and/or receiving less than 7.5 mg of prednisone. Removal of OKT8+ cells from normals did not significantly increase IL-2 activity. SLE, but not normal, OKT8+ cells decreased IL-2 production when added back to autologous OKT8-depleted cells. In some experiments, OKT8+ cells from normal donors also suppressed IL-2 production in SLE. This result suggests that the defect in IL-2 production is complex and may involve multiple cell interactions. Three lines of evidence suggest that the SLE OKT8+ cells actively inhibit the production of IL-2 rather than passively absorb this lymphokine: (a) only 3.2% of SLE lymphocytes expressed IL-2 receptors as detected with anti-Tac; (b) freshly prepared SLE lymphocytes did not absorb IL-2; and (c) cell-free supernatants from SLE OKT8+ cells inhibited IL-2 production, but not IL-2 activity. Double-labeling studies by flow cytometry revealed that 19.3% of SLE OKT8+ cells were also Ia-positive, and approximately 33% co-expressed the natural killer cell marker, HNK-1 (Leu 7). Removal of Leu 7+ cells also significantly elevated IL-2 production in SLE. These studies suggest that one or more circulating mononuclear cell subsets in SLE patients can suppress IL-2 production and that one subset may possibly belong to a non-T, non-B "third mononuclear population."
M Linker-Israeli, A C Bakke, F P Quismorio Jr, D A Horwitz
There has been conflict as to whether crude extracts of atrial natriuretic factor increase renal solute excretion by a hemodynamic mechanism or by direct inhibition of tubular transport. To investigate this issue, seven rats were studied during a euvolemic control period and following continuous administration of pure, synthetic 24 amino acid atrial natriuretic factor. A 10-25-fold increase in urinary sodium and chloride excretion occurred with a brisk kaliuresis but little bicarbonaturia. Atrial natriuretic factor caused whole kidney glomerular filtration rate to increase from 1.17 +/- 0.04 to 1.52 +/- 0.07 ml/min (P less than 0.005). A parallel increase in single nephron glomerular filtration rate, from 34 +/- 1 to 44 +/- 2 nl/min (P less than 0.001), and from 26 +/- 1 to 37 +/- 2 nl/min (P less than 0.005) was measured at the end-proximal and early distal nephron sites, respectively. Appropriate for the higher flows were an increase in absolute proximal and loop reabsorptive rates for bicarbonate, chloride, and water, with a slight decrease in fractional solute and volume reabsorption in proximal and loop segments. To exclude the possibility that atrial natriuretic factor increased filtration rate only in anesthetized animals, eight unanesthetized rats were studied. Glomerular filtration rate increased by 45%, from 2.04 +/- 0.17 to 2.97 +/- 0.27 ml/min (P less than 0.005) without significant change in renal plasma flow, as reflected by 14C-para-aminohippurate clearance (5.4 +/- 0.5-5.6 +/- 0.9 ml/min). The clearance and micropuncture data did not preclude changes in relative blood flow distribution to or in transport by deep nephron segments. In conclusion, atrial natriuretic factor appears to increase renal solute excretion predominantly by a hemodynamic mechanism without directly inhibiting superficial tubular transport.
C L Huang, J Lewicki, L K Johnson, M G Cogan