Monocyte and granulocyte-mediated tumor cell destruction. A role for the hydrogen peroxide-myeloperoxidase-chloride system.

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Abstract

Human monocytes stimulated with phorbol myristate acetate were able to destroy a T lymphoblast cell target (CEM). Stimulated human granulocytes were also capable of mediating CEM cytotoxicity to a comparable degree as the monocyte. CEM destruction was dependent on the pH and the effector cell number. Both monocyte or granulocyte mediated cytotoxicity were inhibited by the addition of catalase, whereas superoxide dismutase had no inhibitory effect. In addition, CEM were protected from cytolysis by the effector cells by the myeloperoxidase inhibitors, azide and cyanide, or by performing the experiment under halide-free conditions. Glucose oxidase, an enzyme system capable of generating hydrogen peroxide, did not mediate CEM cytotoxicity, while the addition of purified myeloperoxidase dramatically enhanced cytolysis. Hypochlorous acid scavengers prevented CEM destruction by the glucose oxidase-myeloperoxidase-chloride system but neither hydroxyl radical nor singlet oxygen scavengers had any protective effect. These hypochlorous acid scavengers were also successful in inhibiting monocyte or granulocyte-mediated CEM cytotoxicity. Based on these observations we propose that human monocytes or granulocytes can utilize the hydrogen peroxide-myeloperoxidase-chloride system to generate hypochlorous acid or species of similar reactivity as a potential mediator of CEM destruction.

Authors

S J Weiss, A Slivka

Complex formation of platelet membrane glycoproteins IIb and IIIa with fibrinogen.

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Abstract

We have recently reported the isolation of purified platelet membrane glycoproteins IIb and IIIa and the generation of monospecific antisera to these membrane proteins. Using these monospecific antisera in an enzyme-linked immunosorbent assay system, it is no demonstrated that glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a complex with purified human fibrinogen. The formation of this GPIIb-GPIIIa fibrinogen complex is calcium dependent, fibrinogen specific, saturable, and inhibited by specific amino sugars and amino acids. These observations suggest that the GPIIb-GPIIIa macromolecular complex on the platelet surface acts under the proper physiologic circumstances as the fibrinogen binding site required for normal platelet aggregation.

Authors

R L Nachman, L L Leung

Effect of colony stimulating factor on murine macrophages. Induction of antitumor activity.

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Abstract

Colony stimulating factor (CSF) was assessed for its capacity to stimulate antitumor activity in macrophages. Murine peritoneal macrophages incubated with CSF for 48 h inhibited [3H[thymidine (TdR) incorporation by P815 tumor cells to approximately 20% of control values. Inhibition of CSF-stimulated macrophages was significantly greater than inhibition by unstimulated macrophages (P less than 0.001). CSF had little direct effect on the proliferation of either tumor cells or macrophages alone, indicating that the antitumor activity of CSF was mediated by macrophages. it is unlikely that impurities in the CSF preparations were responsible for the effect since CSF that had been purified to homogeneity was as active as crude preparations. Furthermore the activity of CSF on macrophages was blocked by addition of purified anti-CSF antibodies. In addition to being tumoristatic, CSF-stimulated macrophages were tumoricidal as determined by a tumor colony growth assay. Tumor cells that had been incubated with CSF-stimulated macrophages showed a significant reduction in tumor colony-forming units (P less than 0.01). Thus, in addition to its effect on hemopoietic stem cells, CSF induces certain effector functions in mature macrophages that may enhance endogenous antitumor host defenses.

Authors

E J Wing, A Waheed, R K Shadduck, L S Nagle, K Stephenson

Retinoic acid. Inhibition of the clonal growth of human myeloid leukemia cells.

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Abstract

Vitamin A and its analogues (retinoids) affect normal and malignant hematopoietic cells. We examined the effect of retinoids on the clonal growth in vitro of myeloid leukemia cells. Retinoic acid inhibited the clonal growth of the KG-1, acute myeloblastic leukemia, and the HL-60, acute promyelocytic leukemia, human cell lines. The KG-1 cells were extremely sensitive to retinoic acid, with 50% of the colonies inhibited by 2.4-nM concentrations of the drug. A 50% growth inhibition of HL-60 was achieved by 25 nM retinoic acid. Complete inhibition of growth of both leukemia cell lines was seen with 1 microM retinoic acid. Exposure of KG-1 cells to retinoic acid for only 3-5 d was sufficient to inhibit all clonal growth. The all-trans and 13-cis forms of retinoic acid were equally effective in inhibiting proliferation. Retinal, retinyl acetate, and retinol (vitamin A) were less potent inhibitors. Clonal growth of the human K562 and mouse M-1 myeloid leukemic cell lines was not affected by 10 microM retinoic acid. Retinoic acid also inhibited the clonal growth of leukemia cells from five of seven patients with acute myeloid leukemia. Retinoic acid at concentrations of 5 nM to 0.3 microM inhibited 50% clonal growth, and 1 microM retinoic acid inhibited 64-98% of the leukemic colonies. The inhibition of clonal growth of KG-1 and HL-60 cell lines and of leukemic cells from two patients was not associated with the presence of a specific cytoplasmic retinoic acid-binding protein. Our study suggests that retinoic acid may prove to be effective in the treatment of human myeloid leukemia.

Authors

D Douer, H P Koeffler

Mechanisms of Epinephrine-induced Glucose Intolerance in Normal Humans: ROLE OF THE SPLANCHNIC BED

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Abstract

To evaluate the role of the splanchnic bed in epinephrine-induced glucose intolerance, we selectively assessed the components of net splanchnic glucose balance, i.e., splanchnic glucose uptake and hepatic glucose production, and peripheral glucose uptake by combining infusion of [3-3H]glucose with hepatic vein catheterization. Normal humans received a 90-min infusion of either glucose alone (6.5 mg/kg−1 per min−1) or epinephrine plus glucose at two dose levels: (a) in amounts that simulated the hyperglycemia seen with glucose alone (3.0 mg/kg−1 per min−1); and (b) in amounts identical to the control study. During infusion of glucose alone, blood glucose rose twofold, insulin levels and net posthepatic insulin release increased three- to fourfold, and net splanchnic glucose output switched from a net output (1.65±0.12 mg/kg−1 per min−1) to a net uptake (1.56±0.18). This was due to a 90-95% fall (P < 0.001) in hepatic glucose production and a 100% rise (P < 0.001) in splanchnic glucose uptake (from 0.86±0.14 to 1.71±0.12 mg/kg−1 per min−1), which in the basal state amounted to 30-35% of total glucose uptake. Peripheral glucose uptake rose by 170-185% (P < 0.001). When epinephrine was combined with the lower glucose dose, blood glucose, insulin release, and hepatic blood flow were no different from values observed with glucose alone. However, hepatic glucose production fell only 40-45% (P < 0.05 vs. glucose alone) and, most importantly, the rise in splanchnic glucose uptake was totally blocked. As a result, splanchnic glucose clearance fell by 50% (P < 0.05), and net splanchnic glucose uptake did not occur. The rise in peripheral glucose uptake was also reduced by 50-60% (P < 0.001). When epinephrine was added to the same dose of glucose used in the control study, blood glucose rose twofold higher (P < 0.001). The initial rise in splanchnic glucose uptake was totally prevented; however, beyond 30 min, splanchnic glucose uptake increased, reaching levels seen in the control study when severe hyperglycemia occurred. Splanchnic glucose clearance, nevertheless, remained suppressed throughout the entire study (40%-50%, P < 0.01).

Authors

Luigi Saccà, Carlo Vigorito, Marco Cicala, Biagio Ungaro, Robert S. Sherwin

Enhancement of Residual Arylsulfatase B Activity in Feline Mucopolysaccharidosis VI by Thiol-induced Subunit Association

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The molecular pathology of the deficient arylsulfatase B activity in feline mucopolysaccharidosis (MPS) VI was investigated. Compared with the highly purified normal feline hepatic enzyme, the purified MPS VI residual activity had a 100-fold higher Michaelis constant (Km), an altered electrophoretic mobility, half the apparent native molecular weight, and markedly decreased thermo-, cryo-, and pH stabilities. Molecular weight and alkylation studies were consistent with the normal enzyme being a homodimer and the residual MPS VI enzyme a monomer. When incubated with various sulfhydryl reagents, the residual specific activity was enhanced several-fold, whereas the activity of the purified normal enzyme was un-affected or slightly inhibited. In the presence of dithiothreitol (DTT) and cysteamine, a lysosomotropic aminothiol, the residual activity had an electrophoretic mobility and native molecular weight similar to those of the normal feline enzyme. These findings suggested that the monomeric residual enzyme was dimerized in the presence of thiol-reducing agents. To determine if thiol-induced subunit association could therapeutically increase the residual activity and degrade the accumulated dermatan sulfate, in vitro and in vivo experiments were undertaken. When 2 mM DTT or cysteamine was incubated with heparinized whole blood from an MPS VI cat, the leukocyte residual arylsulfatase B activity increased 11- and 20-fold, respectively, and the accumulated dermatan sulfate was degraded in the presence of both thiol reagents. Intravenous administration of DTT (50 mg/kg) effected an immediate, but transient, increase in leukocyte residual activity; however, the substrate levels were not significantly decreased. In contrast, intravenous administration of cysteamine (15 mg/kg) increased leukocyte residual activity more than sixfold 30 min postinfusion; concomitantly, the leukocyte substrate was decreased to 35% of the initial level immediately after infusion and to about 45% of preinfusion values during the 120-min period studied. These results suggest that the defective residual activity in feline MPS VI can be therapeutically manipulated by thiol-induced subunit association. Furthermore, this animal analog provides a prototype for the investigation of human inborn errors of metabolism resulting from enzymatic defects that might be amenable to enzyme manipulation therapy.

Authors

Deborah T. Vine, Margaret M. McGovern, Edward H. Schuchman, Mark E. Haskins, Robert J. Desnick

Safety and immunogenicity of high molecular weight polysaccharide vaccine from immunotype 1 Pseudomonas aeruginosa.

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The safety and immunogenicity of a high molecular weight polysaccharide from immunotype 1 Pseudomonas aeruginosa were tested in a dose response fashion in adult volunteers. The vaccine lacked toxicity and pyrogenicity for experimental animals. Doses of 50, 75, 150, or 250 microgram were given to groups of individuals as a single dose subcutaneous injection. Doses of 150 and 250 microgram were associated with a significant rise in binding and opsonic antibody at 2 wk postimmunization. Titers remained unchanged for up to 6 mo. The vaccine was almost devoid of toxicity, eliciting no more than a slightly sore and tender arm at the site of injection. High molecular weight polysaccharide antigen appears to induce a good immune response following vaccination that is effective in mediating opsonophagocytic killing of live P. aeruginosa organisms.

Authors

G B Pier

Hydroxyproline and passive stiffness of pressure-induced hypertrophied kitten myocardium.

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Passive stiffness and hydroxyproline content of myocardium hypertrophied by pressure-loading were determined in kittens 2, 8-16, and 24-52 wk after pulmonary artery banding, which initially elevated right ventricular systolic pressure by 10-15 mm Hg. Right ventricular mass increased by approximately 75%, three-quarters of which occurred during the first 2 wk after banding. Passive stiffness was assessed from resting length-tension relations of isometrically contracting isolated right ventricular papillary muscles. Stiffness constants, alpha and beta were determined from the relationship sigma = alpha (e beta epsilon - 1) where sigma = stress and epsilon = Lagrangian strain. Elastic stiffness (d sigma/d epsilon) was derived from: d sigma/d epsilon = beta sigma + beta alpha. Right ventricular hydroxyproline increased in proportion to muscle mass so that hydroxyproline concentration remained unchanged after banding. Both alpha, beta, and elastic stiffness-stress relations were similar to values in nonbanded controls. Thus, we did not observe an increase in passive stiffness or hydroxyproline concentration of pressure-stiffness or hydroxyproline concentration of pressure-induced hypertrophied myocardium in contrast to most previous studies.

Authors

J F Williams Jr, R D Potter, D L Hern, B Mathew, W P Deiss Jr

Role of Epinephrine-mediated β-Adrenergic Mechanisms in Hypoglycemic Glucose Counterregulation and Posthypoglycemic Hyperglycemia in Insulin-dependent Diabetes Mellitus

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Abstract

Initially euglycemic (overnight insulin-infused) patients with insulin-dependent diabetes mellitus (IDDM), compared with nondiabetic controls, exhibit similar, but somewhat delayed plasma glucose nadirs, delayed glucose recovery from hypoglycemia, and posthypoglycemic hyperglycemia after the rapid intravenous injection of 0.075 U/kg of regular insulin. These abnormalities are associated with and potentially attributable to markedly diminished glucagon secretory responses, partially reduced epinephrine secretory responses and delayed clearance of injected insulin in the diabetic patients. Because glucagon normally plays a primary role in hypoglycemic glucose counterregulation and enhanced epinephrine secretion largely compensates for glucagon deficiency, we hypothesized that patients with IDDM, who exhibit diminished glucagon secretory responses to hypoglycemia, would be more dependent upon epinephrine to promote glucose recovery from hypoglycemia than are nondiabetic persons. To test this hypothesis, glucose counterregulation during β-adrenergic blockade with propranolol was compared with that during saline infusion in both nondiabetic controls and in patients with IDDM. Glucose counterregulation was unaffected by β-adrenergic blockade in controls. In contrast, glucose recovery from hypoglycemia was significantly impaired during β-adrenergic blockade in diabetic patients. This finding confirms the hypothesis that such patients are more dependent upon epinephrine-mediated β-adrenergic mechanisms to promote glucose recovery from hypoglycemia and indicates that the measured deficiency of glucagon secretion is functionally important in patients with IDDM. Further, in the time frame of these studies, posthypoglycemic hyperglycemia was prevented by β-adrenergic blockade in these patients. There was considerable heterogeneity among the diabetic patients with respect to the degree to which β-adrenergic blockade limited the posthypoglycemic rise in plasma glucose. This rise was directly related to the degree of residual glucagon secretion and inversely related to plasma-free insulin concentrations.

Authors

Dennis A. Popp, Suresh D. Shah, Philip E. Cryer

Dynamics of Renal Histamine in Normal Rat Kidney and in Nephrosis Induced by Aminonucleoside of Puromycin

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Abstract

Histamine is known to have a profound effect on capillary permeability in nonrenal tissues and this effect is presumably mediated by cyclic (c)AMP. Because in our previous experiments we found that histamine stimulates cAMP accumulation in glomeruli (Torres, V. E., T. E. Northryn, R. M. Edwards, S. V. Shah, and T. P. Dousa. 1978. Modulation of cyclic nucleotides in isolated rat glomeruli. J. Clin. Invest.62: 1334.), we now explored whether this amine is formed in renal tissue, namely in glomeruli, and whether its renal metabolism is altered in experimental nephrosis induced by puromycin aminonucleoside (PA) in rats. In normal rats, histamine content was higher (Δ + 240%) in cortex than in medulla. In glomeruli isolated from renal cortex, histamine content was significantly higher (Δ + 260%) than in tubules. Incubation of isolated glomeruli with l-histidine resulted in a time-dependent increase of histamine content in glomeruli, but no change was found in tubules. The increase in glomerular histamine was blocked by the histidine decarboxylase inhibitor bromocresine. In rats with PA nephrosis induced by a single intraperitoneal injection of PA (15 mg/100 g body wt) urinary excretion of histamine was markedly increased (>Δ + 200%), but control rats did not differ from rats with PA nephrosis in urinary excretions of l-histidine and of creatinine. At the peak of proteinuria (day 9 after injection of PA) the plasma level of histamine was slightly elevated, and plasma histidine slightly decreased in animals that developed PA nephrosis. The content of histamine was markedly higher and the level of histidine was significantly lower in the renal cortex of PA-nephrotic rats as compared with controls; PA-nephrotic and control rats did not differ in the content of histidine and histamine in the liver. In addition, the content of histamine was higher in glomeruli isolated from PA-nephrotic rats; lesser difference was found in cortical tubules.

Authors

Hanna E. Abboud, S. L. Ou, J. A. Velosa, Sudhir V. Shah, Thomas P. Dousa

Increased enzymatic activity of the alternative pathway convertase when bound to the erythrocytes of paroxysmal nocturnal hemoglobinuria.

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Abstract

To investigate the greater fixation of C3 to the erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH) upon activation of complement, we have examined the formation and the reaction of the C3 nephritic factor-stabilized alternative pathway convertase made with purified components on normal and PNH erythrocytes. Each convertase complex converts four to five times more fluid-phase C3 to C3b when affixed to a PNH cell than when affixed to a normal cell. The greater activity of the convertase on PNH cells is not due to differences in the intrinsic or extrinsic stability of the convertase complex. The excessive binding of C3 to PNH cell si due to this increased conversion of fluid-phase C3, because the efficiency of binding of nascent C3b was identical for the two cell types. This is the first instance in which the enzyme activity of a complement complex has been shown to be increased by being affixed to an abnormal surface.

Authors

C J Parker, P J Baker, W F Rosse

Uridine Diphosphate-Glucuronic Acid-independent Conversion of Bilirubin Monoglucuronides to Diglucuronide in Presence of Plasma Membranes from Rat Liver is Nonenzymic

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Abstract

Two routes have been proposed for conversion of bilirubin monoglucuronide to the diglucuronide: glucuronyl transfer (a) from UDP-glucuronic acid to bilirubin monoglucuronide, catalyzed by a microsomal UDP-glucuronyltransferase, and (b) from one molecule of bilirubin monoglucuronide to another (transglucuronidation), catalyzed by an enzyme present in liver plasma membranes. The evidence regarding the role of the latter enzyme for in vivo formation of bilirubin diglucuronide is conflicting. We therefore decided to reexamine the transglucuronidation reaction in plasma membranes and to study the conversion of bilirubin monoglucuronide to diglucuronide in vivo. Purified bilirubin monoglucuronide was incubated with homogenates and plasma membrane-enriched fractions from liver of Wistar and Gunn rats. Stoichiometric formation of bilirubin and bilirubin diglucuronide out of 2 mol of bilirubin monoglucuronide was paralleled by an increase of the IIIα- and XIIIα-isomers of the bilirubin aglycone, thus showing that dipyrrole exchange, not transglucuronidation, is the underlying mechanism. Complete inhibition by ascorbic acid probably reflects intermediate formation of free radicals of dipyrrolic moieties. The reaction was nonenzymic because it proceeded independently of the protein concentration and heat denaturation of the plasma membranes did not result in decreased conversion rates. Collectively, these findings show spontaneous, nonenzymic dipyrrole exchange when bilirubin monoglucuronide is incubated in the presence of rat liver plasma membranes. Because bilirubin glucuronides present in biological fluids contain exclusively the bilirubin-IXα aglycone, formation of the diglucuronide from the monoglucuronide by dipyrrole exchange does not occur in vivo. Rapid excretion of unchanged bilirubin monoglucuronide in Gunn rat bile after injection of the pigment provides confirmatory evidence for the absence of a UDP-glucuronic acid-independent process.

Authors

Andreas Sieg, Gustaaf P. Van Hees, Karel P. M. Heirwegh

Sodium Channel and Sodium Pump in Normal and Pathological Muscles from Patients with Myotonic Muscular Dystrophy and Lower Motor Neuron Impairment

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Two sodium transport systems have been analyzed in this work: the voltage-sensitive sodium channel and the (Na+, K+) ATPase pump. The sodium channel has been studied using a tritiated derivative of tetrodotoxin; the sodium pump has been studied using tritiated ouabain. Properties of interaction of tritiated tetrodotoxin and of tritiated ouabain with their respective receptors were observed in normal human skeletal muscle and in muscles of patients with myotonic muscular dystrophy and with lower motor neuron impairment.

Authors

C. Desnuelle, A. Lombet, G. Serratrice, M. Lazdunski

Intestinal Phospholipase, a Novel Enzyme

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We evaluated phospholipase activity in the intestine of rats and other species. Phospholipase activity was assayed by a surface barostat technique or an egg yolk titration system. Mucosal activity was found only by the surface barostat technique with phosphatidylglycerol as substrate; it was not found with phosphatidylcholine as substrate in assays by either technique. In gut luminal fluid activity was found when both phosphatidylcholine and phosphatidylglycerol were used as substrate in assays by the surface barostat technique, and phosphatidylcholine as substrate yielded activity in egg yolk titration. In rats in which pancreatic juice had been diverted, mucosal and gut luminal phospholipase activity was greater than in controls, thus demonstrating that enzyme activity was not due to pancreatic phospholipase. Bacterial origin of phospholipase activity was excluded in that phospholipase activity was found in germ-free rats; gastric and salivary gland origins were excluded in that continued phospholipase activity was found in rats with gastric fistula. The physiological importance of the enzyme was established by the finding that rats with pancreatic fistula absorbed 111 μmol of phosphatidylcholine and that controls absorbed 119 μmol of a 135-μmol load. Activity was found to be three times greater in the distal than in the proximal intestine; in cryptal cells it was 10 times greater than in villus tip cells. 65% of the activity in the gut lumen was tightly bound to particulate matter. We propose that intestinal phospholipase may be important in gut bacterial control, in the digestion of vegetable matter (phosphatidylglycerol is a major phospholipid in both plants and bacteria), and in the digestion of phospholipids in the gut lumen.

Authors

Charles M. Mansbach II

beta-Hydroxy fatty acid production by ischemic rabbit heart.

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Abstract

beta-Hydroxymyristate, -palmitate, and -stearate were produced by and accumulated in isolated rabbit heart when perfused ischemically for 2-10 min by the nonrecirculating langendorff technique with 0.75 mM palmitate and 0.16 mM albumin. Tissue fractionation into mitochondria and cytosol showed that by 2 min of ischemia 44% of beta-hydroxypalmitate and 38% beta-hydroxystearate was located in the cytosol; this percentage increased to greater than 50% by 5 min of ischemia. Lipid fractionation studies showed that by 10 min these two beta-hydroxy fatty acids were distributed approximately as 60% acylcarnitine, 20% acyl-coenzyme A (CoA), and 20% free fatty acids. All three chemical forms of beta-hydroxypalmitate were found in both the mitochondria and the cytosol. After 10 min of ischemia beta-hydroxypalmitoyl-CoA and beta-hydroxystearoyl-CoA constituted at least 16% of the incremental long-chain acyl-CoA, whereas beta-hydroxypalmitoylcarnitine and b-hydroxystearoylcarnitine constituted 8% of the incremental long-chain acylcarnitine. These data suggests that myocardial beta-hydroxyacyl-CoA oxidation is limited during ischemia. Substrate accumulates and is transferred to the cytosol where it accumulates primarily as beta-hydroxyacylcarnitine.

Authors

K H Moore, A E Koen, F E Hull

Elastase of U-937 Monocytelike Cells: COMPARISONS WITH ELASTASES DERIVED FROM HUMAN MONOCYTES AND NEUTROPHILS AND MURINE MACROPHAGELIKE CELLS

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Abstract

As an approach to facilitating the understanding of proteinases associated with monocytes we have studied U-937 monocytelike cells. Elastase activity was identified in U-937 cell extracts and compared to monocyte elastase activity, neutrophil elastase, and the elastase activity from a continuous line of murine macrophagelike cells (P388D1). Serine proteinase activity which solubilized 14C-labeled elastin accounted for >90% of the neutral proteinase activity of both U-937 cells and monocyte extracts. U-937 cell and monocyte elastase activities were similar catalytically, resembling neutrophil elastase. U-937 cells and monocytes showed other similarities: (a) both had activities reacting with [3H]diisopropylfluorophosphate that migrated in sodium dodecyl sulfate (SDS) polyacrylamide gels at ∼30,000 and 60,000 daltons and (b) both contained material that cross-reacted with antiserum raised to neutrophil elastase. Preliminary characterization of U-937 cell elastase activity by affinity chromatography and ion-exchange chromatography suggested the presence of at least two distinct elastases. Minimal elastase activity was found in U-937 cell-conditioned medium, indicating that the activity is not spontaneously released by the cells. In contrast to the elastase activity associated with U-937 cells and monocytes, the elastase activity associated with P388D1 cells was a metalloproteinase and was found principally in the culture medium. These results indicate (a) U-937 cells will be useful for further investigation of proteinases associated with normal monocytes; (b) monocytes and U-937 cells contain material with catalytic and immunologic similarities to neutrophil elastase; (c) monocyte elastase activity differs from elastase activity secreted by murine macrophages and murine macrophagelike cells of the P388D1 line.

Authors

Robert M. Senior, Edward J. Campbell, Jill A. Landis, Fraya R. Cox, Charles Kuhn, Hillel S. Koren

Antibody-independent Classical Pathway-mediated Opsonophagocytosis of Type Ia, Group B Streptococcus

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The opsonophagocytic requirements of human sera containing endogenous complement for a variety of type Ia, and group B streptococcal strains were defined. Significant reduction (≧90%) in colony-forming units was noted after a 40-min incubation for the highly encapsulated, mouse-passed prototype strain 090 by sera containing moderate to high concentrations of antibody to type Ia polysaccharide (mean, 16.5 μg/ml), whereas bacterial growth occurred in 25 sera with low levels of specific antibody (mean, 2.1 μg/ml). This absolute requirement for a critical amount of specific antibody in promoting opsonophagocytic killing of strain 090 was not found when 18 fresh clinical type Ia isolates were tested. In antibody-deficient and agammaglobulinemic sera, respectively, mean reductions in colony-forming units of 94 and 95% were seen for fresh clinical isolates, whereas strain 090 was not killed by polymorphonuclear leukocytes in the presence of these sera. All strains required a considerable amount of specific antibody for alternative pathway-mediated opsonophagocytosis. That opsonophagocytic killing of clinical type Ia isolates was mediated by the classical pathway in a nonantibody-dependent fashion was shown when MgEGTA chelation of agammaglobulinemic serum or use of serum deficient in C2 resulted in bacterial growth. The addition of C2 to C2-deficient serum restored bactericidal activity of this serum. These experiments indicate that substances other than the exposed surface of the type Ia capsular polysaccharide initiate classical pathway-mediated opsonophagocytosis of clinical isolates of type Ia, group B streptococci by human sera in the absence of immunoglobulin. Perhaps, a deficiency in classical complement pathway function is critical to the susceptibility of neonates to type Ia, group B streptococcal disease.

Authors

Carol J. Baker, Morven S. Edwards, Bette J. Webb, Dennis L. Kasper

Functional differences between rat islets of ventral and dorsal pancreatic origin.

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Do functional linkages between islet endocrine cells exist? The effect of differences in frequency and distribution of islet endocrine cells on B cell function was examined in islets from the ventral (ventral islets) and dorsal (dorsal islets) areas of the rat pancreas. Dorsal islets contained 10 times as much glucagon as ventral islets, whereas insulin and total protein contents were similar. Basal rates of insulin secretion and proinsulin biosynthesis were similar in the two types of islet, but, under conditions of glucose stimulation, both insulin secretion and proinsulin biosynthesis were significantly greater in the glucagon-rich dorsal islets. Similarly, glucose utilization rates an ATP levels were greater in dorsal islets. In contrast, the rates of processing of newly synthesized proinsulin were similar in ventral and dorsal islets. That the islet glucagon content may have affected B cell function is inferred from two independent findings. Firstly, basal and glucose-stimulated cyclic AMP contents of glucagon-rich dorsal islets were greater than those of ventral islets. Secondly, in the presence of excess exogenous glucagon (1 microgram/ml), the differences in glucose-induced insulin secretion and proinsulin biosynthesis rates between the two types of islets were eliminated. These results strongly suggest that changes in the relative proportions of the different islet endocrine cells exert marked effects on islet function. In particular, a greater A cell and glucagon content is associated with higher rates of glucose-induced insulin secretion and biosynthesis.

Authors

E R Trimble, P A Halban, C B Wollheim, A E Renold

3,3′,5-Triiodothyronine Administration In Vivo Modulates the Hormone-sensitive Adenylate Cyclase System of Rat Hepatocytes

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The ability of 10 μM epinephrine or isoproterenol to stimulate cyclic AMP accumulation was decreased in hepatocytes isolated from hyperthyroid (triiodothyronine treated) as compared to euthyroid rats. In the presence of methylisobutylxanthine, epinephrine or isoproterenol-stimulated cyclic AMP accumulation was ∼65% lower in hyperthyroid as compared with euthyroid rat hepatocytes. The ability of glucagon to stimulate a cyclic AMP response was also decreased in the hyperthyroid state, when assayed in either the absence or presence of a methyl xanthine. The character of the catecholamine-stimulated cyclic AMP response was beta adrenergic in both the hyperand euthyroid states. No evidence for an alpha2 adrenergic mediated component of catecholamine action on cyclic AMP levels was noted. Cyclic AMP phosphodiesterase activity of hepatocyte homogenates was not altered in the hyperthyroid state. Hormone-stimulated, guanine nucleotide- and fluoride-activatable adenylate cyclase activity was reduced in subcellular fractions obtained from hyperthyroid as compared with euthyroid rat hepatocytes. Beta adrenergic receptor binding was reduced ∼35% and glucagon receptor binding reduced ∼50% in the hyperthyroid as compared with euthyroid rat hepatocyte membrane fractions. The status of the regulatory components of adenylate cyclase were examined by in vitro treatment of subcellular fractions with cholera toxin. The ability of cholera toxin to modulate adenylate cyclase was not altered by hyperthyroidism. Cholera toxin catalyzed AD[32P]ribosylation of hyperthyroid and euthyroid rat hepatocyte proteins separated electrophoretically displayed nearly identical autoradiograms. Studies of the reconstitution of adenylate cyclase activity of S49 mouse lymphoma cyc− mutant membranes by detergent extracts from rat hepatocyte membranes, indicated that hyperthyroidism was associated with a reduced capacity of regulatory components to confer fluoride, but not guanine nucleotide activatability to catalytic cyclase. Thyroid hormones regulate the hormone-sensitive adenylate cyclase system of rat hepatocytes at several distinct loci of the system.

Authors

Craig C. Malbon, Michael L. Greenberg

Colipase and maximally activated pancreatic lipase in normal subjects and patients with steatorrhea.

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Human pancreatic lipase in duodenal secretions was studied under conditions of maximal activation by porcine colipase and maximal inhibition by sodium taurodeoxycholate. In almost all samples, total lipase activity in 4 mM sodium taurodeoxycholate was activated by the addition of porcine colipase. Activation was linear until saturation by cofactor was reached, and maximum activity was greater than that obtained in the absence of bile salts. At pH 8.0 in 4 mM sodium taurodeoxycholate, lipase activity was due to pancreatic lipase in samples from normal and steatorrheic individuals and was proportional to the concentration of endogenous colipase in samples that could be activated by exogenous colipase. In these samples, therefore, colipase activity could be conveniently assayed as the lipase activity at pH 0.8 in 4 mM sodium taurodeoxycholate. Colipase to total pancreatic lipase ratios varied widely from individual to individual and on average were significantly lower in steatorrheic patients. In individual samples, colipase secretion was stimulated by pancreozymin and secretin roughly in parallel with total pancreatic lipase, but some variation in the ratio of the two was often seen in successive collection periods. Because pancreatic lipase is usually unsaturated with respect to cofactor, lipolytic activity in duodenal secretions may be finely controlled by modulation of colipase secretion.

Authors

K J Gaskin, P R Durie, R E Hill, L M Lee, G G Forstner

Intracellular Potassium Activity in Guinea Pig Papillary Muscle during Prolonged Hypoxia

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During prolonged hypoxia, intracellular potassium concentration, [K]i has been reported to fall by 70% with a concomitant decrease in the calculated potassium equilibrium potential, EK. Nevertheless, resting membrane potential, Vm, declined only slightly. Because Vm depolarized very little in relation to the calculated EK, it was hypothesized that electrogenic Na-K pumping contributed up to 40 mV to Vm during prolonged hypoxia. To further test this hypothesis we studied what changes prolonged hypoxia makes in the thermodynamically active fraction of cellular potassium, intracellular potassium activity, αKi, and how change in αKi affects the relationship between Vm, EK and, by inference, the Na-K pump. Using double-barrel K-selective electrodes, Vm and αKi were measured in quiescent guinea pig right ventricular papillary muscles superfused for 8 h with hypoxic Tyrode's solution. Over the 8-h period both Vm and αKi decreased. However, the decline in Vm was paralleled by a decrease in the EK calculated from αKi. At no time was there hyperpolarization of Vm beyond EK.

Authors

Thomas Guarnieri, Harold C. Strauss

Structural Identity of Human Histocompatibility Leukocyte Antigen-B27 Molecules from Patients with Ankylosing Spondylitis and Normal Individuals

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Abstract

Although the association between human histocompatibility leukocyte antigen (HLA) B27 and ankylosing spondylitis is the prototype of HLA-disease association, the mechanism underlying these associations has not been determined. We have investigated the possibility that the B27 molecules from patients with ankylosing spondylitis are different from those of normals, and only the “different” molecules predispose the individual to disease. Biosynthetically radiolabeled HLA-B27 molecules from patients with ankylosing spondylitis and normal individuals were compared by two-dimensional gel electrophoresis and tryptic peptide mapping with high pressure liquid chromatography. Extensive charge heterogeneity in the 45,000-dalton heavy chain was detected when B27 molecules were analyzed by two-dimensional gel electrophoresis; the charge heterogeneity was reduced, but not eliminated, when the B27 molecules were treated with neuraminidase to remove sialic acid residues before analysis. No structural difference in the B27 molecules from an ankylosing spondylitis patient and a normal individual were detected by two-dimensional gel electrophoresis. Analysis of [3H]leucine-labeled and [3H]arginine-labeled tryptic peptides and chymotryptic peptides of the trypsin insoluble material by reverse-phase high pressure liquid chromatography revealed identity of the B27 molecules from ankylosing spondylitis patients and normal individuals. These studies indicate that development of akylosing spondylitis in only some B27 positive individuals is not attributable to those individuals possessing variant B27 molecules.

Authors

Robert W. Karr, Yaffa Hahn, Benjamin D. Schwartz

Induction of Membranous Nephropathy in Rabbits by Administration of an Exogenous Cationic Antigen: DEMONSTRATION OF A PATHOGENIC ROLE FOR ELECTRICAL CHARGE

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Abstract

We examined the role of antigenic electrical charge as a determinant of glomerular immune complex localization in the rabbit. Serum sickness nephritis was induced in groups of New Zealand white rabbits by daily 25-mg intravenous injections of bovine serum albumin (BSA) chemically modified to be cationic (pI > 9.5) or more anionic (pI, 3.5-4.6); an additional group received unmodified native BSA (pI, 4.5-5.1). Factors known to influence immune complex localization, e.g., molecular size of the administered antigen and resulting circulating immune complexes, immunogenicity, and disappearance time from the circulation were examined and found to be similar for both anionic and cationic BSA. Charge modification did increase the nonimmune clearance of cationic and anionic BSA compared with native BSA. Injected cationic BSA was shown in paired label experiments to bind directly to glomeruli compared with native BSA. The renal lesion produced by cationic BSA was markedly different from that found in rabbits given anionic or native BSA. Animals receiving cationic BSA uniformly developed generalized diffuse granular capillary wall deposits of IgG, C3, and BSA detected after 2 wk of injections and increasing until death at 6 wk. Qualitatively similar deposits were produced by the administration of low doses of cationic BSA of only 1 or 10 mg/d. In contrast, the injection of both anionic and native BSA resulted in mesangial deposits at 2 and 4 wk with capillary wall deposits appearing by 6 wk. Ultrastructural examination of animals receiving cationic BSA revealed pure, extensive formation of dense deposits along the lamina rara externa of the glomerular basement membrane whereas such deposits were absent or rare in animals injected with the anionic or native BSA. Albuminuria was significantly greater at 6 wk in the groups receiving cationic BSA with a mean of 280 mg/24 h compared with 53 mg/24 h in the combined groups injected with anionic or native BSA. Blood urea nitrogen values were similar in all groups at 2 and 4 wk but higher in the animals receiving cationic BSA at 6 wk.

Authors

Wayne A. Border, Harry, J. Ward, Elaine S. Kamil, Arthur H. Cohen

Contribution of Plasma Protease Inhibitors to the Inactivation of Kallikrein in Plasma

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Abstract

Although C̄l-inhibitor (C̄l-INH) and α2-macroglobulin (α2M) have been reported as the major inhibitors of plasma kallikrein in normal plasma, there is little quantitative support for this conclusion. Thus, we studied the inactivation of purified kallikrein in normal plasma, as well as in plasma congenitally deficient in C̄l-INH, or artificially depleted of α2M by chemical modification of the inhibitor with methylamine. Under pseudo-first-order conditions, the inactivation rate constant of kallikrein in normal plasma was 0.60 min−1. This rate constant was reduced to 0.35, 0.30, and 0.06 min−1, in plasma deficient respectively in C̄l-INH, α2M, or both inhibitors. Thus C̄l-INH (42%) and α2M (50%) were found to be the major inhibitors of kallikrein in normal plasma. Moreover all the other protease inhibitors present in normal plasma contributed only for 8% to the inactivation of the enzyme. To confirm these kinetic results, 125I-kallikrein (Mr 85,000) was completely inactivated by various plasma samples, and the resulting mixtures were analyzed by gel filtration on Sepharose 6B CL for the appearance of 125I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (Mr 370,000) with C̄l-INH, while 48% formed a complex (Mr 850,000) with α2M. After inactivation by C̄l-INH-deficient plasma, >90% of the active 125I-kallikrein was associated with α2M. A similar proportion of the label was associated with C̄l-INH in plasma deficient in α2M. After inactivation by plasma deficient in both C̄l-INH and α2M, 125I-kallikrein was found to form a complex of Mr 185,000. This latter complex, which may involve antithrombin III, α1-protease inhibitor, and/or α1-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either C̄l-INH or α2M, even after the addition of heparin (2 U/ml). These observations demonstrate that C̄l-INH and α2M are the only significant inhibitors of kallikrein in normal plasma confirming previous predictions based on experiments in purified systems. Moreover, in the absence of either C̄l-INH or α2M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.

Authors

Marc Schapira, Cheryl F. Scott, Robert W. Colman

Regulation of Extraadrenal Steroid 21-Hydroxylase Activity: INCREASED CONVERSION OF PLASMA PROGESTERONE TO DEOXYCORTICOSTERONE DURING ESTROGEN TREATMENT OF WOMEN PREGNANT WITH A DEAD FETUS

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Abstract

We measured deoxycorticosterone (DOC) and progesterone (P) in plasma of 47 women pregnant with a dead fetus and sequentially throughout gestation in 35 women pregnant with a live fetus. When P levels in plasma were low, the plasma levels of DOC in women pregnant with a dead fetus varied but usually were similar to those in women pregnant with a live fetus. However, when P levels were high, the levels of DOC in some women pregnant with a dead fetus were considerably lower than those in women pregnant with a live fetus. To test whether this finding was due to loss of transfer of DOC from fetus to mother or else loss of extraadrenal steroid 21-hydroxylase activity in the mother after death of the fetus, we conducted several studies. The levels of P and DOC in plasma of one woman remained constant from 30 min after fetal death until delivery occurred 13 h later. Estrogen treatment of four women pregnant with a dead fetus brought about an increase in plasma levels of DOC in three of the women. In one woman the ratio of plasma DOC to P was 0.015, a value similar to that found before fetal death, but was 0.003 after fetal death but before estrogen treatment. In two women pregnant with a dead fetus the transfer constants of conversion of plasma P to DOC were 0.011 and 0.005 before, and 0.024 and 0.013, respectively, during estrogen treatment. In one woman pregnant with a deformed fetus with adrenal agenesis, the metabolic clearance rates of DOC before and during estrogen treatment were similar, whereas the plasma production rates of DOC were 2.75 before and 4.31 mg/24 h during estrogen treatment. We suggest that (a) the DOC in plasma of near-term pregnant women arises in part by extraadrenal 21-hydroxylation of plasma P and (b) estrogen stimulates steroid 21-hydroxylase activity in extraadrenal tissues.

Authors

Paul C. MacDonald, Susan Cutrer, Sue C. MacDonald, M. Linette Casey, C. Richard Parker Jr.

Cytosine Arabinoside Influx and Nucleoside Transport Sites in Acute Leukemia

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Abstract

Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [3H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx (<0.4 pmol/107 cells per min) and NBMPR binding (<3,000 sites/cell) for the treated group. In summary, myeloblasts have both higher araC transport rates and more nucleoside transport sites than lymphoblasts and this factor may contribute to the greater sensitivity of AML to this drug. AraC transport varied >10-fold between leukemic blasts and normal leukocytes, but transport capacity related directly to the number of nucleoside transport sites on the cell. Finally, low araC transport rates or few NBMPR binding sites on blasts were observed in a subset of patients with acute leukemia who failed to achieve remission with drug combinations containing araC.

Authors

J. S. Wiley, S. P. Jones, W. H. Sawyer, A. R. P. Paterson

Lipoprotein lipase secretion by human monocyte-derived macrophages.

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Abstract

Human monocyte-derived macrophages in culture produced lipoprotein lipase. Although freshly isolated blood monocytes did not secrete much lipase activity, 1 d in culture was sufficient to trigger measureable enzyme production. During 3 wk in culture, maximal activity was attained after 7 d. At all times, the culture medium contained more enzyme activity than did a serum-heparin eluate or a detergent extract of the cell layer. The lipase activity was stimulated by serum and was inhibited by preincubation with antiserum to bovine lipoprotein lipase or when assayed at a high salt concentration. Furthermore, the enzyme bound to a heparin-Sepharose affinity column at physiological ionic strength. Cells cultured from a subject with primary lipoprotein lipase deficiency secreted no detectable enzyme. Since macrophages are prominent components of atherosclerotic lesions in man, their ability to synthesize and secrete lipoprotein lipase may be important to atherogenesis.

Authors

A Chait, P H Iverius, J D Brunzell

Sulfasalazine inhibits the synthesis of chemotactic lipids by neutrophils.

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Abstract

Neutrophils metabolize arachidonic acid through the liposygenase pathway to 5-hydroxy-6,8,11,14-eicosatetrenoic acid (5-HETE) and 5,12-dihydroxy-6,8,10,14-eicosatraenoic acid (5,12 diHETE). 5-HETE and 5,12diHETE are potent chemotactic agents and are thought to have important roles in the inflammatory response. In this study we demonstrate the sulfasalazine, at concentrations found in the stool of patients being treated for ulcerative colitis, blocks the synthesis of both 5-HETE and 5,12 diHETE by human neutrophils. A sulfasalazine metabolite, 5-aminosalicylate, also blocks the synthesis of 5,12 diHETE.

Authors

W F Stenson, E Lobos