The acquired immunodeficiency syndrome (AIDS).

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Authors

F S Rosen

Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

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Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha.

Authors

M J Griffith, L Breitkreutz, H Trapp, E Briet, C M Noyes, R L Lundblad, H R Roberts

Interaction between fibrinogen and cultured endothelial cells. Induction of migration and specific binding.

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It has been suggested that fibrinogen (fg) or its physiological derivatives influence the motility and growth of endothelial cells (ECs), but direct support for this concept is still lacking. In the present study, the capacity of fg to interact with ECs and induce the migration of ECs was examined. The capacity of fg to induce EC migration was studied by means of a modification of the Boyden chamber technique. fg in the lower compartment of the chamber caused a time- and concentration-dependent migration of ECs across filters. fg present in equal concentrations above and below the filter increased EC migration, but the maximal effect invariably occurred in the presence of a gradient between the lower and the upper compartments. Trypsin or plasmin digestion of fg and preincubation of fg with Fab fragments from specific antibody completely abolished fg-induced EC migration. Dialysis of fg to eliminate small peptides that might contaminate the preparation did not modify fg-induced migration. Plasma obtained from healthy donors induced EC migration, but plasma from an afibrinogenemic patient was completely ineffective. The addition of purified fg to afibrinogenemic plasma restored plasma-induced EC migration. Plasmin degradation fragments D and E, of 100,000 and 50,000 mol wt, respectively, did not induce EC migration. However, fragment E caused dose-related inhibition of fg-induced EC migration Direct interaction of highly purified radioiodinated human fg with cultured human and bovine Ecs was observed. The binding was time dependent and plateaued at 10 min. Nonlabeled fg in a large molar excess inhibited the interaction, but unrelated proteins, including fibronectin, ovalbumin, and myoglobin, did not. Monospecific Fab fragments directed to fg inhibited binding by 38% at a 50 to 1 molar ratio whereas nonimmune Fab caused only 2% inhibition at a similar concentration. The binding of 125I-fg with ECs was saturable, and an apparent dissociation constant of 0.23 x 10(-6) M was estimated from binding isotherms. After 30 min of incubation the interaction between 125I-fg and the cells was completely reversible and displaceable by a large molar excess of unlabeled fg. Autoradiography of the display of EC-bound 125I on polyacrylamide gel showed the constitutive B beta- and gamma-chains of the fg molecule, with a partial loss of the A alpha-chain. Purified fragment E and E were tested for their capacity to inhibit fg binding. At a 1 to 400 125I-fg-to-fragment molar ratio, fragment E, which also inhibited migration, competed for binding by 44%, but fragment D was completely ineffective. These data show that fg may specifically associate with ECs and induce migration of these cells; it also appears that the structural requirement of this activity is located in the N-terminal part of the molecule.

Authors

E Dejana, L R Languino, N Polentarutti, G Balconi, J J Ryckewaert, M J Larrieu, M B Donati, A Mantovani, G Marguerie

Asialo von Willebrand factor interactions with platelets. Interdependence of glycoproteins Ib and IIb/IIIa for binding and aggregation.

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Asialo von Willebrand factor (AS-vWf) binds to and aggregates normal human platelets in the absence of ristocetin. Maximal specific binding of AS-vWf is 1-2 micrograms vWf protein/10(8) platelets. Despite the specificity of the binding, only 60% of the bound AS-vWf can be dissociated after equilibrium has been reached. We investigated the site of binding and the mechanism of aggregation of platelets by AS-vWf by (a) pre-incubating platelets with either of two monoclonal antibodies, one against glycoprotein Ib (GPIb) or a second against the glycoprotein IIb/IIIa complex (GPIIb/IIIa), and (b) varying the concentration of fibrinogen in the medium. The results of our studies indicate that AS-vWf binds initially to GPIb. This binding then results in the exposure of receptors for AS-vWf on GPIIb/IIIa. In the presence of plasma fibrinogen, both AS-vWf and fibrinogen bind to GPIIb/IIIa. In the presence of plasma fibrinogen, 50% more AS-vWf binds to the platelet, and this additional AS-vWf binds almost exclusively to GPIIb/IIIa. Despite this enhanced binding of AS-vWf in the absence of fibrinogen, platelet aggregation is much less than that which occurs in the presence of plasma fibrinogen. Comparative studies of AS-vWf binding to normal platelets and the platelets of patients with Glanzmann's thrombasthenia reveal decreased binding to the thrombasthenic platelets and a marked decrease in the extent of platelet aggregation. These studies indicate that AS-vWf binding to, and ensuing aggregation of, platelets is different from that observed with intact vWf protein when platelets are stimulated with either ristocetin or thrombin. The AS-vWf binds to GPIb which, in turn, makes additional AS-vWf receptors available on GPIIb/IIIa. If plasma fibrinogen is present, it competes with the AS-vWf for binding to GPIIb/IIIa and causes aggregation of platelets. In the presence of plasma fibrinogen, more of the AS-vWf binds to GPIIb/IIIa, but this AS-vWf is much less effective than fibrinogen in supporting platelet aggregation.

Authors

H R Grainick, S B Williams, B S Coller

Immunoglobulins in the hyperimmunoglobulin E and recurrent infection (Job's) syndrome. Deficiency of anti-Staphylococcus aureus immunoglobulin A.

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Patients with the hyperimmunoglobulin E and recurrent infection syndrome (HIE) characteristically have frequent skin and respiratory infections caused by Staphylococcus aureus. We have developed a set of enzyme-linked immunosorbent assays that use whole S. aureus (Wood's strain) immobilized on 0.22-micrometers filters and highly specific, affinity-purified enzyme conjugates of goat anti-human IgE, anti-human IgD, anti-human IgG, anti-human IgA, and anti-human IgM. These reagents were used to determine S. aureus-specific immunoglobulin (Ig) levels. As previously published, 10 patients with HIE had markedly higher levels of anti-S. aureus IgE than did 5 patients with eczema and recurrent superficial S. aureus infections (P less than 0.001). The HIE patients were also found to have a deficit of anti-S. aureus serum IgA as compared with 12 normal subjects, 12 patients with chronic granulomatous disease, 5 patients with chronic eczema and recurrent superficial S. aureus infections, and 3 patients with the Chediak-Higashi syndrome (P less than 0.01 for each comparison). In addition the HIE patients had an excess of anti-S. aureus IgM as compared with normal subjects (P less than 0.01). An expected excess of anti-S. aureus IgG was absent. These abnormalities cannot be explained by variations of total serum Ig levels or by a general inability to produce antigen-specific IgA because levels of naturally occurring IgA antibody against Escherichia coli lipopolysaccharide and the antigens of the pneumococcal vaccine are normal. Parotid saliva from patients with HIE contained less salivary IgA per milligram of protein (P less than 0.01) and less salivary anti-S. aureus IgA per milligram of protein (P less than 0.05) than did normal controls. The incidence of infection at mucosal surfaces and adjacent lymph nodes correlated inversely with serum anti-S. aureus IgA (r = -0.647, P = 0.034), serum anti-S. aureus IgE (r = -0.731, P = 0.016), total serum IgE (r = -0.714, P = 0.020), and total serum IgD (r = -0.597, P = 0.049). These findings are evidence of a previously undescribed immunoregulatory defect in patients with HIE, which may contribute to the increased susceptibility to infection in this syndrome.

Authors

S C Dreskin, P K Goldsmith, J I Gallin

Reconstruction of platelet proteins into phospholipid vesicles. Functional proteoliposomes.

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Platelet membrane glycoproteins IIb and IIIa were reconstituted into liposomes containing phosphatidylcholine. The reconstituted vesicles bound antiplatelet antibodies and showed specific binding to thrombin-activated platelets. Prostacyclin, a known inhibitor of thrombin-activated platelet aggregation, inhibited the binding of the proteoliposomes to thrombin-activated platelets. The reconstituted vesicles also specifically bound 125I-labeled fibrinogen. This binding was insensitive to ADP but dependent on calcium ions. These data indicate that platelet glycoproteins IIb and IIIa have been successfully reconstituted into phospholipid vesicles such that their behavior is similar to that in intact platelets.

Authors

J J Baldassare, R A Kahn, M A Knipp, P J Newman

Biochemical and neuropsychological effects of elevated plasma phenylalanine in patients with treated phenylketonuria. A model for the study of phenylalanine and brain function in man.

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Phenylketonuria provides a human model for the study of the effect of phenylalanine on brain function. Although irreversible mental retardation is preventable through newborn diagnosis and dietary phenylalanine restriction, controversy exists regarding the effects of increased concentrations of phenylalanine in older patients. We have studied ten older, treated, phenylketonuric patients using a triple-blind, multiple trials, crossover design. Each patient was tested at the end of each of three 1-wk periods of high or low phenylalanine intakes. Tests included a repeatable battery of neuropsychological tests, analysis of plasma amino acids, and measurement of urine amino acids, phenyl organic acids, dopamine, and serotonin. In all 10 patients plasma phenylalanine rose (900-4,000 microM). In 9 of 10 patients there was an inverse relationship between plasma phenylalanine and urine dopamine excretion. When blood phenylalanine was elevated, these patients had prolonged performance times on neuropsychological tests of higher but not lower integrative function. Urinary serotonin fell during phenylalanine loading in six patients. The concentration of phenylacids in the urine was not proportional to the plasma phenylalanine at concentrations below 1.5 mM. In one patient, neither performance time nor dopamine excretion varied as blood phenylalanine rose or fell. We interpret these data as follows: blood phenylalanine above 1.3 mM impairs performance on neuropsychological tests of higher integrative function, this effect is reversible, and one mechanism may involve impaired biogenic amine synthesis.

Authors

W Krause, M Halminski, L McDonald, P Dembure, R Salvo, D Freides, L Elsas

Regulation of parathyroid hormone release and cytosolic calcium by extracellular calcium in dispersed and cultured bovine and pathological human parathyroid cells.

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Alterations in parathyroid glandular sensitivity to calcium may contribute to the hypersecretion of PTH in hyperparathyroidism. Since the cytosolic calcium concentration may mediate the effects of extracellular calcium on PTH release, we have employed the calcium-sensitive intracellular dye QUIN-2 to examine the relationship between extracellular calcium, cytosolic calcium, and PTH secretion in adult, neonatal, and cultured bovine as well as pathological human parathyroid cells. PTH release was measured using C- and N-terminal radioimmunoassays. Neonatal bovine parathyroid cells showed a greater set-point for secretion (the Ca++ concentration causing half of the maximal inhibition of PTH release) than adult cells (1.27 +/- 0.11 vs. 1.06 +/- 0.11 mM extracellular calcium, P less than 0.01), and a slightly higher extracellular calcium was necessary to raise the cytosolic calcium concentration to a given level in neonatal than in adult bovine parathyroid cells. In individual neonatal and adult cell preparations, there was a close correlation between the set-point for secretion and the "set-point" for cytosolic calcium (r = 0.832, P less than 0.001). In cells from five human parathyroid adenomas, which had an increase in set-point for secretion, the extracellular calcium concentration necessary to raise the cytosolic calcium concentration to a given level was slightly greater than in the neonatal cells. In four preparations of human parathyroid cells there was a significant correlation between the set-points for secretion and cytosolic calcium (r = 0.856, P less than 0.01). Because neonatal bovine and pathological human parathyroid glands show cellular hyperplasia, we studied the temporal relationship between cellular proliferation and the regulation of PTH release and cytosolic calcium concentration in cultured bovine parathyroid cells. Cellular proliferation, estimated by 3H-thymidine incorporation, increased significantly in culture from 104 +/- 10.1 counts/well on day 1 (first 24 h in culture) to 588 +/- 188 and 6,156 +/- 649 counts/well on days 2 and 4, respectively. In cultured cells on day 1, highly Ca++ (2-3 mM) inhibited maximal PTH release by 58.8 +/- 3.2%, which decreased significantly (P less than 0.001) to 38.2 +/- 1.9 and 17.1 +/- 3.7% on days 2 and 4, respectively. The cytosolic calcium observed at 3 mM calcium on day 1 was 701 +/- 43 nM, which declined to 466 +/- 60 and 314 +/- 14 nM on days 2 and 4 (P less than 0.05). There was a close correlation between this progressive decrease in maximal inhibition of PTH release and the cytosolic calcium at high extracellular calcium in cultured cells (r = 0.99, P < 0.001). Thus, during active proliferation of cultured cells, there is an alteration in the regulation of cytosolic calcium at a given extracellular calcium concentration, and changes in the regulation of PTH release and cytosolic calcium by extracellular calcium may be related to enhanced cellular proliferation.

Authors

M S LeBoff, D Shoback, E M Brown, J Thatcher, R Leombruno, D Beaudoin, M Henry, R Wilson, J Pallotta, S Marynick

Characterization of iron-mediated peroxidative injury in isolated hepatic lysosomes.

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Peroxidative degradation of the lysosomal membrane and the resultant release of hydrolytic enzymes may be responsible for hepatocellular injury in iron toxicity. In this study, highly purified hepatic lysosomes were exposed to iron salts in vitro; the nature of this iron-mediated process of injury and the susceptibility of the lysosomal integrity were studied. Native hepatic lysosomes from rats were isolated by free flow electrophoresis. Incubation of the lysosomes at 37 degrees C with Fe3+-ADP in the presence of ascorbate resulted in rapid generation of malondialdehyde, which approached a plateau at 20 min. Subsequently, the loss of lysosomal latency, determined as an increased percentage free activity of N-acetyl-beta-glucosaminidase, also occurred and reached a maximum loss at 30 min. The half-maximal level of ascorbate, required to promote the Fe3+-ADP mediated lysosomal peroxidation, was approximately 10 microM; high concentrations of ascorbate were inhibitory and half-maximal inhibition was achieved at a concentration of 2 mM. The iron-mediated lysosomal peroxidation was not inhibited by most active oxygen scavengers and appeared to depend solely on the generation of Fe2+ species. When a fresh solution of Fe2+ was incubated with the lysosomes, both the extent of lipid peroxidation and the degree of latency loss increased as a function of increasing Fe2+ concentration. High concentrations of Fe2+ stimulated lysosomal lipid peroxidation instantaneously and reached the highest level within 10 min; whereas the subsequent maximum loss of latency was achieved within 20 min. Both the MDA formation and the loss of latency in either the Fe3+-ADP + ascorbate or the Fe2+ system were effectively prevented by the presence of vitamin A or vitamin E.

Authors

I T Mak, W B Weglicki

Locus assignment of alpha-globin structural mutations by hybrid-selected translation.

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The two human alpha-globin genes, alpha 1 and alpha 2 located 3.4 kilobases apart on chromosome 16, encode identical alpha-globin proteins. A mutation in either gene could result in a structural hemoglobinopathy. It has only recently become possible to assign an alpha-chain mutant to one of these two loci by using recombinant DNA technology. While definitive, this approach has necessitated the cloning and sequencing of the specific gene in question. We present an alternative approach which results in rapid and definitive assignment of an alpha-globin mutation to its encoding genetic locus. This approach uses the technique of hybrid-selected translation. Reticulocyte RNA from individuals with alpha-globin mutations can be fractionated into beta-, alpha 9 (total)-, alpha 1-, and alpha 2-globin mRNA by selective hybridization of each mRNA species to its respective complementary DNA (cDNA) immobilized on nitrocellulose paper. Each mRNA purified in this way can be translated in vitro, and the mRNA species (and hence gene locus) encoding the globin mutant can then be directly identified by gel analysis of the radiolabeled translation products. This procedure can be used to identify globin mutants as alpha or beta and to localize alpha-globin mutants to the alpha 1 or alpha 2 gene. We have used this technique to localize the two alpha-globin mutants, alpha 125Pro (Hb Quong Sze) and alpha 47HIS (Hb Hasharon), to the alpha 2 locus. This approach could potentially be expanded to serve as an alternative to peptide analysis for the initial characterization of all globin structural mutants.

Authors

S A Liebhaber, F E Cash

Identification of carriers of a variant plasma prealbumin (transthyretin) associated with familial amyloidotic polyneuropathy type I.

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A method is described for detecting carriers of a variant plasma prealbumin that is associated with familial amyloidotic polyneuropathy (FAP) type I. It is based on the finding of an extra methionine in the variant prealbumin, at position 30 from the amino terminals. Since normal prealbumin has only one methionine (position 13), treatment with cyanogen bromide (CNBr), which cleaves only at methionines, results in two peptides. CNBr treatment of the variant prealbumin gives three peptides. The extra can then be detected in two ways: by HPLC using a reverse phase C18 column, and by sequential Edman degradation. Each method can detect as little as 1% variant prealbumin in isolated plasma prealbumin, and therefore, can identify carriers of the gene for the variant protein. Since FAP type I usually is not manifest until after the childbearing years, this method to identify carriers of the gene offers a new approach for genetic counseling of families with this disease. To date, kindreds with hereditary amyloidosis that could benefit from these studies include those with FAP type I of Swedish, Japanese, and Portuguese origins.

Authors

M D Benson, F E Dwulet

Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

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Abnormal factor IX variant proteins were isolated from the plasmas of three unrelated severe hemophilia-B families that had been previously shown to contain functionally impaired molecules immunologically similar to normal factor IX. The families studied were: (1) a patient with markedly prolonged ox brain prothrombin time, designated factor IX Bm Lake Elsinore (IXBmLE); (b) three patients (brothers) with moderately prolonged ox brain prothrombin time, designated factor IX Long Beach (IXLB); and (c) a patient with normal ox brain prothrombin time designated factor IX Los Angeles (IXLA). Each variant molecule comigrates with normal factor IX (IXN) both in the sodium dodecyl sulfate and in the nondenaturing alkaline gel electrophoresis. All three variant proteins are indistinguishable from IXN in their amino acid compositions, isoelectric points, carbohydrate distributions and number of gamma-carboxyglutamic acid residues. Each variant protein undergoes a similar pattern of cleavage by factor XIa/Ca2+ and by factor VIIa/Ca2+/tissue factor, and is activated at a rate similar to that observed for IXN. All of the three variant proteins also react with an anti-IXN monoclonal antibody that interferes with the binding of activated IXN(IXaN) to thrombin-treated factor VIIIC. However, in contrast to IXaN, the cleaved IXBmLE has negligible activity (approximately 0.2%), and cleaved forms of IXLA and IXLB have significantly reduced activity (approximately 5-6%) in binding to antithrombin-III/heparin, and in activating factor VII (plus Ca2+ and phospholipid) or factor X (plus Ca2+ and phospholipid) +/- factor VIII. These data, taken together, strongly indicate that the defect in these three variant proteins resides near or within the latent catalytic site. This results in virtually a complete loss of catalytic activity of the cleaved IXBmLE molecule and approximately 95% loss of catalytic activity of the cleaved IXLA and IXLB molecules.

Authors

P Usharani, B J Warn-Cramer, C K Kasper, S P Bajaj

Coronary thrombolysis and infarct size reduction after intravenous infusion of recombinant tissue-type plasminogen activator in nonhuman primates.

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Occlusive thrombus was produced by thrombin-induced coagulation in the left anterior descending coronary artery (LAD) of 16 open-chest baboons. In six control animals, occlusive thrombosis persisting over a period of 4 h as evidenced by coronary arteriography resulted in large transmural infarction (63.1 +/- 3.5% of the perfusion area). In 10 animals, tissue-type plasminogen activator obtained by recombinant DNA technology (rt-PA) was infused systemically at a rate of 1,000 IU (10 micrograms)/kg per min for 30 min after 30-80 min of coronary thrombosis. Reperfusion occurred within 30 min in nine animals. In one animal, intravenous infusion was followed by an intracoronary infusion at the same rate, which resulted in thrombolysis within 8 min. In the rt-PA group, mean duration of occlusion before reperfusion was 77 +/- 24 min. Reocclusion occurred in one animal. Recanalization resulted in an overall reduction of infarct size (37.8 +/- 5.9%, P less than 0.05 versus controls). Residual infarction was related to the duration of occlusion (r = 0.80, P less than 0.01). Reperfusion was associated with reduced reflow. Myocardial blood flow in the perfusion area of the LAD was only 70% of normal after 4 h despite perfect angiographic refilling. The infusion of rt-PA was not associated with systemic activation of the fibrinolytic system, fibrinogen breakdown, or clinically evident bleeding. It is concluded that intravenous infusion of rt-PA may recanalize thrombosed coronary vessels without inducing systemic lysis. The extent of residual infarction is closely related to the duration of coronary artery occlusion before thrombolysis.

Authors

W Flameng, F Van de Werf, J Vanhaecke, M Verstraete, D Collen

Origin of leukemic relapse after bone marrow transplantation detected by restriction fragment length polymorphism.

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Bone marrow transplantation has become an accepted modality in the treatment of acute leukemia. With this therapy, it is possible to obtain long-term disease-free survival. However, leukemia recurs occasionally. In most cases, leukemic relapse is of recipient origin. There have been several reports, though, of leukemia developing in donor cells. These cases have been limited to instances in which there is an easily identifiable chromosome difference or abnormality, usually a sex chromosome. In this paper we describe the use of restriction fragment-length polymorphism analysis to determine the origin of recurrent leukemia cells in which no identifying chromosome was present. We found that the leukemia had recurred in recipient cells. We also were able to demonstrate the presence of normal hemopoietic cells of donor origin.

Authors

M D Minden, H A Messner, A Belch

Increased glomerular thromboxane synthesis as a possible cause of proteinuria in experimental nephrosis.

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Altered glomerular metabolism of arachidonic acid (AA) has already been demonstrated in experimental nephrotoxic nephritis. The enhanced synthesis of thromboxane A2 (TxA2) in isolated glomeruli that has been found may mediate changes in renal hemodynamics. The objectives of this investigation were: to check whether glomerular AA metabolism is also altered in a model of glomerulopathy in which no leukocyte infiltration or platelet deposition could be demonstrated; to establish a correlation between the altered AA metabolism and proteinuria; and to explore whether the alteration of the prostaglandin (PG) pathway found in isolated glomeruli is an in vitro artifact or reflects a modification in vivo. We used a model of glomerular damage characterized by heavy and persistent proteinuria, which was induced in the rat by a single intravenous injection of adriamycin. At light microscopy, minimal glomerular abnormalities were found in this model. Electron microscopy showed profound alterations of glomerular epithelial cells with extensive fusion of foot processes and signs of epithelial cell activation. Electron microscopy of numerous glomeruli showed no platelet deposition or macrophage and leukocyte infiltration in this model. Isolated glomeruli from nephrotic rats studied 14 or 30 d after a single intravenous injection of adriamycin (7.5 mg/kg) when animals were heavily proteinuric generated significantly more TxB2, the stable breakdown product of TxA2, than normal glomeruli. No significant changes were found in the other major AA metabolites formed through cyclooxygenase. Urinary excretion of immunoreactive TxB2 was also significantly higher in nephrotic than in normal animals. Administration of a selective Tx synthetase inhibitor, UK-38,485, from day 14 to day 18 after adriamycin resulted in a significant reduction of proteinuria compared with pretreatment values. Glomerular synthesis and urinary excretion of TxB2 were normal during the UK-38,485 treatment. Additional experiments showed that elevated glomerular synthesis and urinary excretion of TxB2 were not a consequence of increased substrate availability. Maximal stimulation of the renin-angiotensin axis with furosemide increased glomerular TxB2 synthesis in normal rats, which was significantly lower than in nephrotic animals. Finally, experiments using a unilateral model of adriamycin nephrosis indicated that the enhancement of glomerular TxB2 synthesis is not simply a consequence of the nephrotic syndrome. We conclude that: there is an abnormality of glomerular AA metabolism in nephritic syndrome, which leads to increased TxA2 production; the increased Tx generation correlates with protein excretion and might be responsible for altering the glomerular basement membrane permeability to protein; and the alteration found in isolated glomeruli probably reflects a modification in vivo, in that urinary excretion of immunoreactive TxB2 is also consistently increased in adriamycin nephrosis.

Authors

G Remuzzi, L Imberti, M Rossini, C Morelli, C Carminati, G M Cattaneo, T Bertani

Dynamics of interaction between complement-fixing antibody/dsDNA immune complexes and erythrocytes. In vitro studies and potential general applications to clinical immune complex testing.

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Soluble antibody/3H-double-stranded PM2 DNA (dsDNA) immune complexes were briefly opsonized with complement and then allowed to bind to human erythrocytes (via complement receptors). The cells were washed and subsequently a volume of autologous blood in a variety of media was added, and the release of the bound immune complexes from the erythrocytes was studied as a function of temperature and time. After 1-2 h, the majority of the bound immune complexes were not released into the serum during blood clotting at either 37 degrees C or room temperature, but there was a considerably greater release of the immune complexes into the plasma of blood that was anticoagulated with EDTA. Similar results were obtained using various conditions of opsonization and also using complexes that contained lower molecular weight dsDNA. Thus, the kinetics of release of these antibody/dsDNA immune complexes differed substantially from the kinetics of release of antibody/bovine serum albumin complexes that was reported by others. Studies using the solution phase C1q immune complex binding assay confirmed that in approximately half of the SLE samples that were positive for immune complexes, there was a significantly higher level of detectable immune complexes in plasma vs. serum. Freshly drawn erythrocytes from some SLE patients exhibiting this plasma/serum discrepancy had IgG antigen on their surface that was released by incubation in EDTA plasma. Thus, the higher levels of immune complexes observed in EDTA plasma vs. serum using the C1q assay may often reflect the existence of immune complexes circulating in vivo bound to erythrocytes.

Authors

R P Taylor, C Horgan, M Hooper, J Burge

Regional distribution and alterations of lectin binding to colorectal mucin in mucosal biopsies from controls and subjects with inflammatory bowel diseases.

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Glycoconjugate composition of colorectal goblet cell mucin was characterized according to the anatomical distribution of lectin-binding sites in mucosal biopsies from 35 control subjects and 55 patients with inflammatory bowel disease. 24 of the controls had mucosal inflammation on biopsy, without clinical evidence of inflammatory bowel disease. These inflamed controls showed a similar rate of presence of lectin-binding sites as the normal noninflamed group. In the controls, the frequency of binding of Ricinus communis agglutinin I to galactosyl residues was consistently higher than that found with either Ulex europaeus agglutinin I to fucosyl or Dolichus biflorus agglutinin to N-acetyl galactosyl groups. A significant proximal to distal gradient for Ulex europaeus agglutinin I binding sites was identified in the controls group. These binding sites were present four times more often in the proximal colon than in the distal colon (P less than 0.025). In the ulcerative and Crohn's colitis groups, this gradient effect was lost, predominantly as a result of decreased availability of fucosyl residues in the proximal colon. In the descending colon of Crohn's colitis tissues, there was a complete absence of Dolichus biflorus agglutinin binding sites compared with the 62.5% incidence in the control group (P less than 0.05). These results demonstrate that the expression of lectin-binding sites in human large intestinal goblet mucin is specifically altered in inflammatory bowel disease, indicating that there are changes in glycosylation of colorectal mucin consistent with alterations in goblet cell differentiation.

Authors

L R Jacobs, P W Huber

Antineutrophil autoantibodies in Graves' disease. Implications of thyrotropin binding to neutrophils.

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The hyperthyroidism of Graves' disease may be caused by autoantibodies to thyrotropin (TSH) receptors. We have found that patients with this disease have autoantibodies to neutrophils as well, which can be displaced by TSH. Using a radiochemical opsonic assay, we found serum antibodies against homologous neutrophils in 6 of 11 Graves' patients. With a staphylococcal protein A-binding assay, we detected circulating antibodies to homologous neutrophils in 10 of 20 patients, while finding cell-bound antibody on autologous neutrophils in 7 of 8 (including 2 with negative serum tests). Use of human 125I-TSH in a radioligand binding assay revealed that TSH bound to neutrophils rapidly (maximum binding within 10 min at 22 degrees C, pH 7.4), specifically (less than 20% nonspecific binding), and reversibly. Adding TSH to the radiochemical assay resulted in a dose-dependent inhibition of opsonic antibody activity in serum from patients with Graves' disease. In contrast, TSH did not inhibit antibody activity of serum from patients with immune neutropenia not associated with thyroid disease. Our findings suggest a basis for the association of Graves' disease with neutropenia. Furthermore, the discovery of such antineutrophil antibodies in Graves' disease permits detection of cell-bound antibody when free antibody is not present.

Authors

S A Weitzman, T P Stossel, D C Harmon, G Daniels, F Maloof, E C Ridgway

Variability in purified dysfunctional C1(-)-inhibitor proteins from patients with hereditary angioneurotic edema. Functional and analytical gel studies.

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C1(-)-inhibitor (C1(-)-INH) proteins from normal persons and members of eight different kindred with dysfunctional C1(-)-INH proteins associated with hereditary angioneurotic edema (HANE) were compared with respect to their inhibitory activity against purified preparations of C1s-, plasma kallikrein, activated forms of Hageman factor, and plasmin. Each dysfunctional C1(-)-INH protein showed a unique spectrum of inhibitory activity against these enzymes. Although none of the dysfunctional C1(-)-INH proteins significantly impaired amidolysis by plasmin, all but one inhibited activated Hageman factor. One purified dysfunctional C1(-)-INH (Ta) inhibited purified C1s- to a normal degree. Another C1(-)-INH (Za) had almost seven times as much inhibitory activity as normal C1(-)-INH against activated Hageman factor, but had decreased activity against C1s- and no activity against plasmin. Analyses of mixtures of plasmin and C1(-)-INH proteins in SDS gel electrophoresis revealed variability in the patterns of complex formation and cleavage of dysfunctional proteins after exposure to C1s- and plasmin. Some bound to plasmin and were cleaved, even though none significantly impaired the amidolytic activity of plasmin. Two were cleaved by C1s-, whereas neither normal or other dysfunctional C1(-)-INH were cleaved. Dysfunctional C1(-)-INH proteins from patients with HANE are thus heterogeneous in their inhibitory properties and there must be different structural requirements for the inhibition of the various plasma enzymes that can be regulated by normal C1(-)-INH. The data suggest that in addition to common sites of interactions between these proteases and C1(-)-INH, there are also points of contact that are specific for each protease. Genetic mutations leading to structural changes at some of these sites may have differing effects on the interaction between individual proteases and abnormal C1(-)-INH proteins. These alterations may allow these proteins to serve as probes for structural requirements for inhibitory actions of normal C1(-)-INH.

Authors

V H Donaldson, R A Harrison, F S Rosen, D H Bing, G Kindness, J Canar, C J Wagner, S Awad

Feline glucose-6-phosphate dehydrogenase cellular mosaicism. Application to the study of retrovirus-induced pure red cell aplasia.

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Abstract

Neoplasms result from the uncontrolled proliferation of abnormal or transformed cells. The early stages of this process are difficult to study because of the lack of sensitive and specific markers of clonal evolution in an experimental system. We have developed a cat model using cellular mosaicism for glucose-6-phosphate dehydrogenase (G-6-PD). Our findings confirm that the structural locus for feline G-6-PD is on the X-chromosome and demonstrate that it is randomly inactivated in somatic cells. Heterozygous cats have balanced ratios of G-6-PD enzyme types in peripheral blood cells and hematopoietic progenitors that remain stable over time. In our initial studies, we used the model to analyze the events surrounding marrow failure experimentally induced by selected strains of feline leukemia virus (FeLV). Two G-6-PD heterozygous cats, one F1 male hybrid and one domestic cat were infected with FeLV (C or KT) and developed pure red cell aplasia (PRCA). Colonies arising from the more mature erythroid colony-forming cell were not detected in marrow culture of anemic animals although erythroid bursts persisted, suggesting that the differentiation of early erythroid progenitors (BFU-E) was inhibited in vivo. The ratio of G-6-PD types in hematopoietic progenitors and peripheral blood cells from the heterozygous cats did not change when the animals developed PRCA. Thus, the anemia did not result from the clonal expansion of a transformed myeloid stem cell. With this experimental approach, one may prospectively assess clonal evolution and cellular interactions in other FeLV-induced diseases.

Authors

J L Abkowitz, R L Ott, J M Nakamura, L Steinmann, P J Fialkow, J W Adamson

Effect of uracil arabinoside on metabolism and cytotoxicity of cytosine arabinoside in L5178Y murine leukemia.

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Abstract

Pretreatment of L5178Y murine leukemia cells with uracil arabinoside (ara-U) enhances the cytotoxicity of cytosine arabinoside (ara-C). This effect is mediated by the cytostatic effect of ara-U, which causes a delay of cell progression through S-phase. Consequently, the specific activity of enzymes that peak during S-phase increases, and deoxycytidine kinase increases 3.6-fold over untreated controls. This allows enhanced anabolism of ara-C to nucleotides, as well as increased incorporation into DNA with ultimate synergistic cytotoxicity. It is postulated that the systemic metabolism of high-dose ara-C to sustained high levels of ara-U in patients with acute leukemia may enhance the activity of subsequent doses of ara-C, and thus contribute to a means for pharmacologic self-potentiation, contributing to the unique therapeutic activity of high-dose ara-C.

Authors

J L Yang, E H Cheng, R L Capizzi, Y C Cheng, T Kute

Stereospecific transport of triiodothyronine from plasma to cytosol and from cytosol to nucleus in rat liver, kidney, brain, and heart.

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Abstract

We have investigated the transport of L- and D-triiodothyronine (T3) from plasma to cellular cytoplasm and from cytoplasm to nucleus by estimating the concentration of free hormone in these compartments in rat liver, kidney, brain, and heart. We assessed the distribution of T3 in various tissues and its metabolism by standard isotopic techniques and measured plasma and cytosolic tissue T3 by radioimmunoassay. In addition, we determined the fraction of radiosensitive T3 associated with the cytosol in individual tissues and estimated the cytosolic volume per gram of tissue. Equilibrium dialysis allowed us to determine the binding power of cytosols and plasma, and in vitro saturation techniques provided values for the affinity (ka) for L- and D-T3 of isolated nuclei in aqueous solution at 37 degrees C. We calculated the free cytosolic hormone from the product of cytosolic T3 and the binding power of cytosol for T3, and the free intranuclear T3 from the ka and previously determined ratio of occupied-to-unoccupied binding sites under steady state conditions in euthyroid animals. Our results showed that the free cytosolic/free plasma concentrations for L-T3 and D-T3, respectively, were: liver 2.8, 21.6; kidney 1.17, 63.3; heart 1.31, 1.58; brain 0.86, 0.24. The free nuclear/free cytosolic ratios for L-T3 and D-T3, respectively, were: liver 58.2, 3.70; kidney 55.9, 1.54; heart 80.6, 24.9; and brain 251, 108.6. Our findings suggest that stereospecific transport occurs both from plasma to cytosol and from cytosol to nucleus. The high gradients from cytosol to nucleus imply that there is an energy-dependent process and appear to account for the differences in the nuclear association constant determined in vivo and in vitro.

Authors

J H Oppenheimer, H L Schwartz

Vitamin D status regulates 25-hydroxyvitamin D3-1 alpha-hydroxylase and its responsiveness to parathyroid hormone in the chick.

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Abstract

We asked this question: Under normal or near-normal metabolic conditions, does the prevailing normal or near-normal vitamin D status dampen the activity of 25-hydroxyvitamin-D3-1 alpha-hydroxylase (1 alpha-hydroxylase) such that it determines not only its "basal" activity but also its responsiveness to stimulation by increased circulating concentrations of parathyroid hormone (PTH)? To answer this question, we measured the activity of 1 alpha-hydroxylase in chicks, with and without administration of PTH, immediately before and during deprivation of vitamin D. Before deprivation of vitamin D, 1 alpha-hydroxylase activity increased only slightly with administration of PTH. With deprivation of vitamin D for 5 and 10 d, while the plasma concentrations of calcium and phosphorus persisted normal and unchanged, 1 alpha-hydroxylase activity not only increased progressively but also became sharply and increasingly responsive to stimulation by administration of PTH. But after 15 d of vitamin D deprivation, and the supervention of hypocalcemia, 1 alpha-hydroxylase activity was not further increased by the administration of PTH. With deprivation of vitamin D, the progressive increase in 1 alpha-hydroxylase correlated inversely with circulating levels of 1,25-dihydroxyvitamin D (1,25-[OH]2D), and the decreasing calcemic response to PTH correlated inversely with the responsiveness of 1 alpha-hydroxylase to PTH (in chicks deprived of vitamin D for 1-10 d). These results demonstrate that: under normal metabolic conditions, the normal vitamin D status regulates the activity of 1 alpha-hydroxylase so as to dampen both its "basal" activity and its responsiveness to stimulation by PTH; and vitamin D deprivation insufficient to cause hypocalcemia enhances both the "basal" activity of 1 alpha-hydroxylase and its responsiveness to stimulation by PTH. The results suggest that the normal dampening of 1 alpha-hydroxylase and both of the demonstrated enhancements of its activity are mediated by normal and reduced levels of circulating 1,25-(OH)2D, respectively. The finding that PTH fails to further stimulate 1 alpha-hydroxylase when vitamin D deprivation is sufficient in duration to cause hypocalcemia confirms the findings of other investigators and again demonstrates that observations made during abnormal metabolic circumstances may bear little on the physiologic regulation of 1 alpha-hydroxylase under normal or near-normal metabolic circumstances.

Authors

B E Booth, H C Tsai, R C Morris Jr

Effect of estradiol and steroid analogues on the clearance of immunoglobulin G-coated erythrocytes.

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Abstract

Although the disproportionate frequency of several immunologic disorders among women is well recognized, the effect of sex steroids on immunologic processes is unclear. We used an animal model, which has helped to elucidate the effect of corticosteroids in vivo, to quantitatively assess the effect of estradiol and steroid analogues on the immune clearance of IgG-coated erythrocytes. While corticosteroids impaired the clearance of IgG-coated erythrocytes, estradiol, in doses comparable to those achieved during pregnancy, significantly enhanced the clearance. Estradiol, however, did not enhance the splenic clearance of heat-altered erythrocytes. Splenic macrophages isolated from estradiol-treated animals expressed enhanced receptor affinity for the Fc portion of immunoglobulin G [Fc(IgG)], an effect probably responsible for the enhanced in vivo clearance. No consistent effect of estradiol on the splenic macrophage C3 receptors was observed. The synthetic androgen danazol, the mineralocorticoid deoxycorticosterone, and the cortisol metabolite tetrahydrocortisone did not alter the clearance of IgG-coated cells after 7 d of therapy. The estrogen antagonist/agonist tamoxifen enhanced the clearance of IgG-coated cells, but to a lesser extent than estradiol. An effect of estrogens on macrophage Fc (IgG) receptor-mediated clearance may explain in part the variation in clinical expression of several autoimmune disorders during changes in hormonal state, such as pregnancy.

Authors

D Friedman, F Netti, A D Schreiber

Serum prostacyclin binding defects in thrombotic thrombocytopenic purpura.

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Abstract

To understand the pathophysiologic significance of abnormal serum prostacyclin (PGI2) binding activities in thrombotic thrombocytopenic purpura (TTP), we evaluated the PGI2 binding characteristics in three chronic TTP sera and 19 normal sera. PGI2 binding by serum was rapid and reversible. The binding activity in TTP sera (22.1 +/- SD, 4.4%) was significantly lower than that of normal sera (42.2 +/- 6.2%). Moreover, the antiaggregating activity and 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) content in the gel filtrates representing the binding peak was proportionally lower in a TTP serum than normal serum. Although normal and TTP sera bound [14C]arachidonate with similar activity, and neither bound [3H]6KPGF1 alpha, there was a difference in prostaglandin E1 (PGE1) binding. Binding of [3H]PGE1 was subnormal in two TTP sera (W.J. and T.G.) and normal in the third (H.S.). Normal serum corrected the binding defects of TTP serum. Interestingly, the mixture of two TTP sera (W.J. and H.S.) mutually corrected their PGI2 binding defects. In addition, although in vivo plasma transfusions improved the PGI2 binding activity of W.J. and H.S., there existed a striking difference in the nature of their response. These observations indicate that there is at least two types of PGI2 binding defects in TTP. Our data indicate that TTP is associated with diminished serum binding of PGI2. This defect may reduce the availability of PGI2 to damaged vascular sites and decrease an important modulator of platelet thrombus formation at times of severe vascular insult.

Authors

K K Wu, E R Hall, E C Rossi, A C Papp

Relationships between the cytotoxicity of tiazofurin and its metabolism by cultured human lung cancer cells.

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Abstract

The antitumor activity of the antineoplastic agent, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), has previously been shown to require intracellular anabolism of the drug to a nicotinamide adenine dinucleotide (NAD) analog (2-beta-D-ribofuranosylthiazole-4-carboxamide adenine dinucleotide or "tiazofurin adenine dinucleotide"), which then acts as a potent inhibitor of the target enzyme inosine monophosphate (IMP) dehydrogenase. Inhibition of the latter enzyme in turn brings about a profound depletion of intracellular guanosine nucleotides essential for tumor cell growth and replication. In the present study, the cytotoxicity and metabolism of tiazofurin have been examined in six human lung cancer cell lines. At the pharmacologically attainable drug concentration of 100 microM, colony survival was less than 1.5% in three cell lines ("sensitive"), while survival in the remaining three was greater than 50% ("resistant"). The metabolism of tritiated tiazofurin was examined at concentrations ranging from 0.5 to 100 microM following both brief (6 h) and protracted (14 d) exposures. The sensitive lines accumulated concentrations of tiazofurin adenine dinucleotide that were approximately 10 times those achieved by the resistant lines at both time points. We also observed tendencies for the sensitive cell lines to exhibit: (a) higher specific activities of NAD pyrophosphorylase, the enzyme required for the synthesis of tiazofurin adenine dinucleotide, (b) significantly lower levels of a phosphodiesterase which degrades the latter dinucleotide, (c) greater inhibition of the target enzyme IMP dehydrogenase, and (d) greater depressions of guanosine nucleotide pools after drug treatment. By contrast, the basal levels of IMP dehydrogenase and purine nucleotides in these six lines did not correlate in any obvious way with their responsiveness or resistance. The accumulation and monophosphorylation of parent drug were also not prognostic variables. These studies thus suggest that the extent of accumulation of tiazofurin adenine dinucleotide, as regulated by its synthetic and degradative enzyme activities, is the single most predictive determinant of the responsiveness of cultured human lung tumor cells to tiazofurin.

Authors

D N Carney, G S Ahluwalia, H N Jayaram, D A Cooney, D G Johns

Uncoupling of the membrane skeleton from the lipid bilayer. The cause of accelerated phospholipid flip-flop leading to an enhanced procoagulant activity of sickled cells.

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Abstract

We have previously reported that the normal membrane phospholipid organization is altered in sickled erythrocytes. More recently, we presented evidence of enhanced transbilayer movement of phosphatidylcholine (PC) in deoxygenated reversibly sickled cells (RSC) and put forward the hypothesis that these abnormalities in phospholipid organization are confined to the characteristic protrusions of these cells. To test this hypothesis, we studied the free spicules released from RSC by repeated sickling and unsickling as well as the remnant despiculated cells. The rate of transbilayer movement of PC in the membrane of deoxygenated remnant despiculated cells was determined by following the fate of 14C-labelled PC, previously introduced into the outer monolayer under fully oxygenated conditions using a PC-specific phospholipid exchange protein from beef liver. The rate of transbilayer movement of PC in the remnant despiculated cells was significantly slower than in deoxygenated native RSC and was not very much different from that in oxygenated native RSC or irreversibly sickled cells. The free spicules had the same lipid composition as the native cells, but were deficient in spectrin. These spicules markedly enhanced the rate of thrombin formation in the presence of purified prothrombinase (Factor Xa, Factor Va, and Ca2+) and prothrombin, indicating the exposure of a significant fraction of phosphatidylserine (PS) in the outer monolayer. This effect was not observed when the spicules in this assay were replaced by normal erythrocytes, deoxygenated native RSC, or a deoxygenated sample of RSC after repetitive sickling/unsickling. The results are interpreted to indicate that the destabilization of the lipid bilayer in sickled cells, expressed by the enhanced flip-flop of PC and the exposure of PS in the outer monolayer, occurs predominantly in those parts of the membrane that are in spicular form.

Authors

P F Franck, E M Bevers, B H Lubin, P Comfurius, D T Chiu, J A Op den Kamp, R F Zwaal, L L van Deenen, B Roelofsen

Human monocyte-derived mucus secretagogue.

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Abstract

Human peripheral monocytes were stimulated with opsonized zymosan or protein A-containing Staphylococcus aureus to examine whether factors might be released that were capable of stimulating mucous glycoprotein release from cultured human airways, as has recently been described with human pulmonary macrophages. While the supernatant from monocytes exposed to opsonized zymosan or protein A-containing S. aureus caused an impressive activity was found in the control samples that were cultured in parallel and exposed to nonactivated zymosan or S. aureus that was deficient in protein A. The responsible factor was termed monocyte-derived mucus secretagogue (MMS). The maximum MMS release was reached 4-8 h after stimulation, and the amount of MMS released was dependent on the dose of opsonized zymosan added. Chromatographic analyses of MMS indicate that its molecular weight was approximately 2,000 and that the isoelectric point (pI) was 5.2, with a smaller second peak of 7.4 on isoelectric focusing. MMS itself was not detected in monocyte lysates, nor was it formed by monocytes treated with the protein synthesis inhibitor, cycloheximide, before exposure to activating particles. MMS was not a prostaglandin, could not be extracted into organic solvents, and is probably not an eicosanoid. Based on these observations, we conclude that stimulated human peripheral monocytes synthesize a small, acidic molecule, termed MMS, that is capable of stimulating human airways to secrete mucus and in nearly every respect is identical to pulmonary macrophage-derived MMS.

Authors

Z Marom, J H Shelhamer, M Kaliner

Regulation of lipoprotein lipase in primary cultures of isolated human adipocytes.

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Abstract

To study the regulation of adipose tissue lipoprotein lipase (LPL) in human adipocytes, omental adipose tissue was obtained from healthy subjects and digested in collagenase. The isolated adipocytes thus obtained were suspended in Medium 199 and cultured at 37 degrees C. Cell viability was demonstrated in adipocytes cultured for up to 72 h by constancy of cell number, cell size, trypan-blue exclusion, and specific 125I-insulin binding. In addition, chloroquine induced an increase in cell-associated 125I-insulin at 24, 48, and 72 h after preparation. Thus, isolated adipocytes retained their ability to bind, internalize, and degrade insulin. LPL was measured as activity secreted into the culture medium (CM), released from cells by heparin (HR), and extracted from cell digests. A broad range of heparin concentrations produced a prompt release of LPL from a rapidly replenishable pool of cellular activity. When cells were cultured in medium containing 10% fetal bovine serum, there was a marked stimulation of CM and HR. The secretory response to serum (CM) correlated strongly with HR 24 h after preparation (rs = 0.731, P less than 0.001). In addition, HR was found to correlate logarithmically and inversely with body mass index (r = -0.731, P less than 0.001). Insulin, at 400 ng/ml only, increased HR by 36 +/- 10%, an effect simulated by lower concentrations of insulin-like growth factor-1 (IGF1). Thus, LPL is produced and regulated in isolated human adipocytes. The degree of adiposity and serum are important regulators of HR activity, whereas insulin is stimulatory only at a pharmacologic concentration. This effect of insulin may be mediated through the IGF1 receptor. Isolated human adipocytes represent a novel and useful system for the study of LPL and lipid metabolism as well as for other aspects of adipocyte biology.

Authors

P A Kern, S Marshall, R H Eckel

Isolation of high density lipoproteins from rat intestinal epithelial cells.

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Abstract

Previous studies have defined forms of high density lipoproteins (HDL) in rat mesenteric lymph, suggesting that they have a secretory origin. This study describes the isolation and characterization of intestinal intracellular HDL. Two preparations were made as follows: (a) Rat enterocytes were isolated and a Golgi organelle fraction was prepared. (b) Cell homogenates were subjected to nitrogen cavitation and a cytoplasmic fraction was prepared. Lipoproteins were isolated from both preparations by sequential ultracentrifugation. When the HDL fraction (1.07-1.21 g/ml) was subjected to isopyknic density gradient ultracentrifugation, a peak of apoproteins A-I and B (apoA-I and apoB, respectively) was found at a density of 1.11-1.14 g/ml. Electron microscopy of the fraction showed spherical particles ranging in size from 6 to 13 nm. Immunoelectrophoresis revealed a precipitin arc in the alpha region against apoA-I which extended into the pre-beta region where a precipitin arc against apoB was also seen. ApoB antisera depleted the pre-beta particles whereas the alpha migrating particles remained. Lipid analysis of the whole HDL fraction revealed phospholipid, cholesteryl ester, and triglyceride as the major lipids. [3H]leucine was then administered into the duodenum and a radiolabeled intracellular HDL fraction was isolated. The newly synthesized apoproteins of the HDL fraction, as determined by gel electrophoresis, were apoB, apoA-I, and apolipoprotein A-IV (ApoA-IV). Immunoprecipitation of the apoB particles revealed apoA-I and apoA-IV in the supernatant. These data demonstrate that there are at least two intracellular intestinal forms of HDL particles, one of which contains apoB. The other particle contains apoA-I and apoA-IV, has alpha mobility, is spherical, and resembles a particle found in the lymph.

Authors

A M Magun, T A Brasitus, R M Glickman

Hydraulic water permeability and transepithelial voltage in the isolated perfused rabbit cortical collecting tubule following acute unilateral ureteral obstruction.

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Abstract

Ureteral obstruction affects the kidney's ability to conserve water and sodium. Using the isolated perfused tubule technique, we studied cortical collecting tubules (CCT) taken from rabbits subjected to a sham operation or to 4 h of unilateral ureteral obstruction (UUO). Tubules were perfused in the presence of an osmotic gradient directed to promote water movement from lumen to bath, and volume flux (Jv), hydraulic water permeability (Lp), and transepithelial voltage (V1) were determined. In tubules from sham-operated and UUO animals, basal (before exposure to vasopressin) J, and Lp were not different from zero. After addition of 200 microU . ml-1 of arginine vasopressin (aVP) to the bath, Jv and Lp increased to 1.64 +/- 0.23 nl . mm-1 . min-1 and 127.9 +/- 19.8 cm . s-1 . atm-1 x 10(7), respectively, in tubules from sham-operated animals, but not only 0.27 +/- 0.09 nl . mm-1 . min-1 an 18.8 +/- 6.2 cm . s-1 . atm-1 . 10(7) in tubules from UUO animals. Pretreatment with desoxycorticosterone acetate (DOCA) or indomethacin in vivo did not prevent the blunted vasopressin response seen in tubules taken from UUO animals. The Jv and Lp responses to the cyclic AMP (cAMP) analogue, 8-Br-cAMP, were also diminished in tubules taken from UUO animals compared with shams. V1, measured during the basal period, was diminished in tubules from UUO kidneys (-5.0 +/- 2.1 mV) compared with shams (-21.9 +/- 4.1 mV), and pretreatment with DOCA did no prevent the effects of UUO on V1. In contrast, tubules taken from animals that received indomethacin prior to UUO developed voltages not different from voltages in tubules taken from sham-operated animals (-17.3 +/- 1.7 mV). We conclude that, although CCT from UUO animals can maintain osmotic gradients, their ability to respond to vasopressin by increasing Lp is impaired by an intrinsic defect located at a step beyond the generation of cAMP, and that prostaglandin inhibition or DOCA pretreatment do not reverse the decreased responsiveness of Lp to aVP. UUO also diminished V1, and this abnormality was prevented by previous treatment with indomethacin, suggesting that prostaglandins may mediate the effect of UUO on V1.

Authors

H T Campbell, E Bello-Reuss, S Klahr

Evolution of recurrent herpes simplex lesions. An immunohistologic study.

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Abstract

We performed immunoperoxidase stains on skin biopsies taken from nine patients with recurrent peripheral herpes simplex lesions at 12 h to 6 d after onset of signs of symptoms to phenotype the inflammatory infiltrate, to detect cells producing interferons alpha and gamma, and to locate herpes simplex virus antigen-containing cells. Viral glycoprotein antigen was located in the nuclei and cytoplasm of necrotic epidermal cells, often within vesicles, in biopsies taken between the first and third day. Histologically, biopsies of all stages showed intradermal focal perivascular and diffuse mononuclear inflammatory infiltrates. The cells constituting the infiltrates were predominantly T lymphocytes with lesser numbers of histiocytes; Leu 7+ (most natural killer/killer) cells and B cells were rare in the biopsy specimens. Leu 3a+ ("helper") T lymphocytes predominated in both subepidermal and perivascular regions of early lesions (12-24 h). Tissue helper/suppressor ratios ranged from 6.3 to 3.4 compared with 1.9-1.0 in blood. In later lesions (after 2 d), monocytes/macrophages were more prominent in tissue sections and the helper/suppressor ratios (2.3-2.5) more nearly approximated those of blood (1.6-2.7). The negative correlation of tissue ratios with time was significant (P less than or equal to 0.02). A large proportion of the infiltrated T lymphocytes expressed DR antigens. There was also diffuse strong DR expression on epidermal cells in five cases (all of two or more days). In six biopsies, scattered macrophages and small cells, presumably lymphocytes, demonstrated cytoplasmic or membrane staining for a substance which copurifies with interferon gamma. We identified such stained cells within vessels, suggesting that these cells circulate. Gamma interferon might have an important role within the herpetic lesions, possibly inducing macrophage activation and cytotoxic T lymphocytes and increasing DR expression on monocyte and epidermal cells.

Authors

A L Cunningham, R R Turner, A C Miller, M F Para, T C Merigan

Abnormalities of myeloid progenitor cells after "successful" bone marrow transplantation.

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Abstract

We studied recovery of peripheral blood- and bone marrow-derived myeloid progenitor cells (CFU-G,M) in 29 patients who received bone marrow transplants 2 mo to 8.5 yr previously. All patients had normal levels of peripheral blood neutrophils, normal bone marrow cellularity, and a normal myeloid-erythroid ratio. Both peripheral blood- and bone marrow-derived CFU-G,M were markedly reduced compared with normal controls and bone marrow donors [5 +/- 1/10(6) vs. 37 +/- 4/10(6) (P less than 0.001) and 23 +/- 5/2 x 10(5) vs. 170 +/- 21/2 x 10(5) (P less than 0.001)]. Five patients had no detectable CFU-G,M even when 10(6) bone marrow cels were plated. These abnormalities of CFU-G,M were unrelated to age, sex, diagnosis, conditioning regimen, dose of bone marrow cells transplanted, and presence or absence of graft-vs.-host disease. Patients who received either autotransplants or transplants from identical twins also had decreased or absent CFU-G,M indicating that allogeneic factors and posttransplant immune suppressor with methotrexate or corticosteroids were not major determinants of this abnormality. Co-culture of normal or donor peripheral blood or bone marrow mononuclear cells with recipients peripheral blood or bone marrow mononuclear cells, purified T cells, or serum failed to show any evidence of active CFU-G,M suppression. Furthermore, the abnormality of CFU-G,M could not be corrected by the addition of normal syngeneic (donor) hematopoietic cells or serum. Depletion of T-cells from recipient bone marrow by physical techniques resulted in marked increase in CFU-G,M (36 +/- 13 vs. 138 +/- 36; P less than 0.05). The abnormality could be reproduced in vitro by readdition of autologous T cells. In contrast to results with T cell depletion by physical techniques, T cell depletion with a monoclonal anti-T antibody (B7) and complement had no effect. These data indicate that most-transplant recipients have a marked abnormality in CFU-G,M when these cells are cultured in vitro. In at least some of these patients, the decreased cloning efficiency of CFU-G,M appears to be mediated by a suppressive effect of autologous T cells.

Authors

S Li, R Champlin, J H Fitchen, R P Gale

Platelet C1- inhibitor. A secreted alpha-granule protein.

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Abstract

In order to characterize which proteins of the contact phase of coagulation interact with platelets, human platelets were studied immunochemically and functionally to determine if they contain C1- inhibitor. By means of monospecific antibody to C1- inhibitor, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to measure directly platelet C1- inhibitor. With the CELISA, from 33 to 115 ng of C1- inhibitor antigen per 10(8) platelets from 15 normal donors was quantified in lysates of washed human platelets solubilized in nonionic detergent. The mean concentration in 10(8) platelets was 62 +/- 33 ng (SD). Plasma C1- inhibitor either in the platelet suspension medium or on the surface of the platelets could account for only from 6.5 to 16% of the total antigen measured in the solubilized platelets. Upon functional studies, platelets contained 84 +/- 36 ng (SD) of C1- inhibitor activity in 10(8) platelets. As assessed by the CELISA, platelet C1- inhibitor antigen was immunochemically identical to plasma and purified C1- inhibitor. In contrast, the mean concentration of platelet C1- inhibitor antigen in platelets from four patients with classical hereditary angioedema was 8.3 ng/10(8) platelets (range, 5.3 to 11.3 ng/10(8) platelets). 25 and 31% of the total platelet C1- inhibitor was secreted without cell lysis from normal platelets after exposure to collagen (20 micrograms/ml) and thrombin (1 U/ml), respectively, and this secretion was blocked by metabolic inhibitors. Platelet subcellular fractionation showed that platelet C1- inhibitor resided mostly in alpha-granules, similar to the location of platelet fibrinogen. Thus, human platelets contained C1- inhibitor, which became available by platelet secretion. The identification of platelet C1- inhibitor suggests that platelets may modulate the activation of the proteins of early blood coagulation and the classical complement pathways.

Authors

A H Schmaier, P M Smith, R W Colman

Hyperuricemia in glycogen storage disease type I. Contributions by hypoglycemia and hyperglucagonemia to increased urate production.

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Abstract

Studies were performed to determine whether hypoglycemia or the glucagon response to hypoglycemia increases uric acid production in glycogen storage disease type I (glucose-6-phosphatase deficiency). Three adults with this disease had hyperuricemia (serum urate, 11.3-12.4 mg/dl) and reduced renal clearance of urate (renal urate clearance, 1.1-3.1 ml/min). These abnormalities were improved in one patient by intravenous glucose infusion for 1 mo, suggesting a role for hypoglycemia and its attendant effects on urate metabolism and excretion. A pharmacologic dose of glucagon caused a rise in serum urate from 11.4 to 13.0 mg/dl, a ninefold increase in urinary excretion of oxypurines, a 65% increase in urinary radioactivity derived from radioactively labeled adenine nucleotides, and a 90% increase in urinary uric acid excretion. These changes indicate that intravenous glucagon increases ATP breakdown to its degradation products and thereby stimulates uric acid production. To observe whether physiologic changes in serum glucagon modulate ATP degradation, uric acid production was compared during saline and somatostatin infusions. Serum urate, urinary oxypurine, radioactivity, and uric acid excretion increased during saline infusion as patients became hypoglycemic. Infusion of somatostatin suppressed these increases despite hypoglycemia and decreased the elevated plasma glucagon levels from a mean of 81.3 to 52.2 pg/ml. These data suggest that hypoglycemia can stimulate uric acid synthesis in glucose-6-phosphatase deficiency. Glucagon contributes to this response by activating ATP degradation to uric acid.

Authors

J L Cohen, A Vinik, J Faller, I H Fox

Sinusoidal efflux of glutathione in the perfused rat liver. Evidence for a carrier-mediated process.

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Abstract

Turnover of hepatic glutathione in vivo in the rat is almost entirely accounted for by cellular efflux, of which 80-90% is sinusoidal. Thus, sinusoidal efflux play a major quantitative role in homeostasis of hepatic glutathione. Som preliminary observations from our laboratory (1983. J. Pharmacol. Exp. Ther. 224:141-147.) and circumstantial evidence in the literature seemed to imply that the raising of the hepatic glutathione concentration above normal was not accompanied by a rise in the rate of sinusoidal efflux. Based on these observations, we hypothesized that the sinusoidal efflux was probably a saturable process and that at normal levels of hepatic glutathione the efflux behaved as a zero-order process (near-saturation). We tested our hypothesis by the use of isolated rat livers perfused in situ, single pass, with hemoglobin-free, oxygenated buffer medium at pH 7.4 and 37 degrees C. Preliminary experiments established a range of perfusion rates (3-4 ml/min per g) for adequacy of oxygenation, lack of cell injury, and minimization of variability contributed by perfusion rates. Hepatic glutathione was lowered to below normal by a 48-h fast, diethylmaleate (0.1-1.0 ml/kg i.p.), and buthionine sulfoximine (8 mmol/kg i.p.), and raised to above normal by 3-methylcholanthrene (20 mg/kg x 3 d i.p.) and cobalt chloride (0.05-0.27 g/kg-1 subcutaneously). Steady state sinusoidal efflux from each liver was measured over a 1-h perfusion, during which the coefficient of variation of glutathione in perfusates stayed within 10%. Hepatic glutathione efflux as a function of hepatic concentration was characterized by saturable kinetics with sigmoidal (non-hyperbolic) features. The data were fitted best with the Hill model and the following parameter values were estimated: Vmax = 20 nmol/min per g, Km = 3.2 mumol/g, and n = 3 binding/transport sites. The efflux could be inhibited reversibly by sulfobromophthalein-glutathione conjugate but was not affected by the addition of glutathione to the perfusion medium. The results support our hypothesis that sinusoidal efflux of glutathione is near saturation (approximately equal to 80% of Vmax) at normal (fed and fasted) liver glutathione concentrations. The phenomenon of saturability coupled with the ability to inhibit the efflux leads us to propose that sinusoidal efflux from hepatocytes appears to be a carrier-mediated process. Some recent studies by others, using sinusoidal membrane-enriched vesicles, also support these conclusions.

Authors

M Ookhtens, K Hobdy, M C Corvasce, T Y Aw, N Kaplowitz

Molecular defect in the sickle erythrocyte skeleton. Abnormal spectrin binding to sickle inside-our vesicles.

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Abstract

Although functional abnormalities of the sickle erythrocyte membrane skeleton have been described, there is little quantitative data on the function of the proteins that compose the skeleton. We have examined the association of spectrin, the major skeletal protein, with ankyrin, its high-affinity membrane binding site, and found sickle erythrocytes to have markedly reduced binding. Binding is assayed by incubation of purified 125I-spectrin with spectrin-depleted inside-out vesicles (IOVs) and measurement of the label bound to IOVs. Sickle IOVs bind approximately 50% less ankyrin than do controls IOVs (P less than 0.001). Control experiments show that this reduced binding is not a function of faulty composition or orientation of sickle IOVs, or of reticulocytosis per se. Our least symptomatic patient has the highest binding capacity, suggesting that this abnormality may be related to clinical severity. This trend is supported by experiments showing that asymptomatic subjects with sickle trait, sickle cell anemia and high fetal hemoglobin, and sickle beta +-thalassemia have normal binding, whereas a symptomatic patient with sickle beta zero-thalassemia has abnormal binding. In contrast to what we see with ankyrin in situ on the IOV, when isolated and studied in solution, sickle ankyrin binds normally to spectrin. This discrepancy may be related to preferential purification of the normal ankyrin species or to an abnormal topography of the membrane near the spectrin attachment site. We hypothesize that sickle hemoglobin or perhaps the metabolic consequences of sickling damage the protein skeleton. This damage may alter the surface of the erythrocyte and result in abnormal cell-cell interactions which may be related to clinical severity.

Authors

O S Platt, J F Falcone, S E Lux

Interaction of antithrombin III with bovine aortic segments. Role of heparin in binding and enhanced anticoagulant activity.

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Abstract

Bovine antithrombin III (AT III) interaction with the luminal surface of bovine aortic segments with a continuous layer of endothelium was examined. Incubation of 125I-AT III with vessel segments, previously washed free of endogenous AT III, demonstrated specific, time-dependent binding to the protease inhibitor to the endothelium. Half-maximal binding was observed at an added AT III concentration of 14 nM. Binding of 125I-AT III to the vessel wall was reversible (50% dissociated in 4 min), and addition of either heparin or Factor Xa accelerated displacement of 125I-AT III from the vessel segment. Dissociation of 125I-AT III from the vessel segment in the presence of factor Xa coincided with the formation of a Factor Xa-125I-AT III complex. Inactivation of Factor IXa and Factor Xa by AT III was facilitated in the presence of vessel segments. Pretreatment of vessel segments with highly purified Flavobacterium heparinase precluded the vessel-dependent augmentation of AT III anticoagulant activity as well as specific binding of 125I-AT III to the vessel endothelium. In contrast, pretreatment of the vessel segments with chrondroitinases (ABC or AC) had no detectable effect on 125I-AT III binding or on AT III anticoagulant activity. AT III binding to vessel segments was competitively inhibited by increasing concentration of platelet factor 4. Binding of the protease inhibitor to vessel segments was inhibited by chemical modification of AT III lysyl or tryptophan residues. These AT III derivatives retained progressive inhibitory activity. These data suggest that heparin-like molecules are present on the aortic vessel wall and mediate binding of AT III to the vessel surface, as well as enhancing the anticoagulant activity of AT III at these sites.

Authors

D Stern, P Nawroth, J Marcum, D Handley, W Kisiel, R Rosenberg, K Stern

Comparison of hepatic elimination of different forms of cholecystokinin in dogs. Bioassay and radioimmunoassay comparisons of cholecystokinin-8-sulfate and -33-sulfate.

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Abstract

The influence of hepatic transit on the ability of exogenous cholecystokinin-8-sulfate and -33-sulfate (CCK-8 and CCK-33, respectively) to stimulate gallbladder contraction and exocrine pancreatic secretion, as well as on the peripheral plasma concentration of each agent, was evaluated in five conscious dogs with pancreatic and gallbladder fistulas and complete portacaval transposition. The gallbladder pressure increments after portal administration of CCK-8 (0.125, 0.25, 0.50, and 1.0 microgram/kg per h for 5 min) were diminished by 36, 45, 39 and 25%, respectively, in comparison with those obtained with systemic administration of identical doses of CCK-8 (P less than 0.05). In a subsequent experiment, the integrated pancreatic juice volume, bicarbonate, and protein secretion were diminished by 22, 32, and 48%, respectively, during a 30-min infusion of CCK-8 (0.10 micrograms/kg per h) into the portal venous system, in comparison with the results obtained with systemic administration of CCK-8 (P less than 0.05). In contrast, the gallbladder pressure and pancreatic exocrine secretory responses to portal administration of CCK-33 did not differ significantly (P greater than 0.05) from the results obtained with systemic administration of CCK-33. Radioimmunoassay for CCK-8 in plasma showed that the integrated CCK-8 value during portal administration was significantly lower (P less than 0.05) than it was during systemic administration. The results for CCK-33, however, did not vary, whether it was given by a systemic or portal route (P greater than 0.05). Thus, the present study demonstrates that CCK-8 is partially inactivated by the liver whereas CCK-33 is not, which suggests that CCK-3 in the circulation may play a significant role in the physiologic regulation of the gallbladder and exocrine pancreas.

Authors

T Sakamoto, M Fujimura, J Newman, X G Zhu, G H Greeley Jr, J C Thompson

Platelet-induced neurogenic coronary contractions due to accumulation of the false neurotransmitter, 5-hydroxytryptamine.

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Abstract

The purpose of this study was to determine if 5-hydroxytryptamine released from aggregating platelets could be accumulated and released by canine coronary adrenergic nerves, and if the false neurotransmitter resulted in an abnormal response of the smooth muscle to nerve stimulation. Isometric tension was measured in rings of epicardial coronary suspended in organ chambers filled with physiological salt solution. The response to electrical stimulation or exogenously added norepinephrine was elicited after contraction with prostaglandin F2 alpha. Electrical stimulation and exogenous norepinephrine caused beta-adrenergic relaxation of control rings. However, after rings were exposed for 2 h to aggregating platelets or 5-hydroxytryptamine, electrical stimulation caused frequency-dependent contractions. These contractions were prevented by the serotonergic antagonists, cyproheptadine or ketanserin, or by the neuronal uptake inhibitor, cocaine. The relaxation caused by exogenously added norepinephrine was unchanged after exposure to platelets or 5-hydroxytryptamine, indicating that smooth muscle alpha- and beta-adrenergic responsiveness was unchanged. The electrically stimulated overflow of radiolabeled norepinephrine from superfused strips of coronary artery was not altered by prior exposure to 5-hydroxytryptamine, indicating that the effect of exposure on the response to electrical stimulation is primarily at smooth muscle serotonergic receptors. Canine coronary arteries accumulated and metabolized radiolabeled 5-hydroxytryptamine in vitro. The accumulation of 5-hydroxytryptamine was inhibited by cocaine or by adrenergic denervation with 6-hydroxydopamine but unaffected by removal of endothelium, indicating that the adrenergic nerves were the primary site of accumulation. Electrical stimulation of superfused strips of coronary artery preincubated with radiolabeled 5-hydroxytryptamine caused the release of the intact indoleamine; this was blocked by the neurotoxin, tetrodotoxin. These studies suggest that 5-hydroxytryptamine liberated from aggregating platelets may be accumulated by coronary adrenergic nerve endings. Upon its release from the nerves as a false transmitter, the amine can activate serotonergic receptors on the smooth muscle and reverse the action of the adrenergic nerves from dilator to constrictor.

Authors

R A Cohen

Enhanced alveolar macrophage-mediated antigen-induced T-lymphocyte proliferation in sarcoidosis.

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Abstract

Expansion of T-lymphocyte numbers is a characteristic feature of the alveolitis of pulmonary sarcoidosis. One mechanism that may influence the numbers of T-lymphocytes in the lung is the process of antigen presentation in which alveolar macrophages, in the presence of antigen, induce T-lymphocytes to replicate. To evaluate this process in sarcoidosis, alveolar macrophages were obtained by bronchoalveolar lavage, pulsed with tetanus toxoid, and co-cultured with purified autologous T cells. Strikingly, antigen-pulsed alveolar macrophages from sarcoid patients induced more than a twofold increase in autologous T-lymphocyte proliferation compared with the response seen using cells from normal or patients with idiopathic pulmonary fibrosis (P less than 0.001), all comparisons). In contrast, when monocytes were used as the antigen presenting cell, no significant differences were observed in T cell proliferation induced by antigen among the three groups. The enhanced T-lymphocyte proliferation induced by sarcoid alveolar macrophages was not dependent on the compartment from which the T cells were derived, and was independent of the specific antigen used. One possible explanation for augmented antigen presentation seen in sarcoid is that an increased percentage of sarcoid alveolar macrophages express HLA-DR or HLA-DS surface antigens. However, most normal and sarcoid alveolar macrophages express HLA-DR and HLA-DS surface antigens, and the percentage of macrophages expressing these antigens was not significantly different in the two groups. Thus, while the mechanisms of the enhanced antigen presentation in the sarcoid lung are unknown, the process of antigen-driven, alveolar macrophage-modulated lung T cell proliferation may explain, at least in part, the expansion of lung T-lymphocyte numbers that characterizes this disease.

Authors

A Venet, A J Hance, C Saltini, B W Robinson, R G Crystal

Sn-protoporphyrin rapidly and markedly enhances the heme saturation of hepatic tryptophan pyrrolase. Evidence that this synthetic metalloporphyrin increases the functional content of heme in the liver.

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Abstract

Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, the rate-limiting enzyme in heme degradation to bile pigment, and has been successfully utilized to suppress hyperbilirubinemia in a variety of experimental and naturally occurring forms of jaundice in animals and man. The compound is presumed to act in vivo primarily by inhibiting heme oxidation; thus it would be reasonable to expect that preservation of some functional moiety of cellular heme from degradation by heme oxygenase would occur after Sn-protoporphyrin administration. We have examined this question in liver by studying the heme saturation of tryptophan pyrrolase, the heme-dependent enzyme which controls the first and rate-limiting step in the catabolism of L-tryptophan. Sn-protoporphyrin, in doses (10 mumol/kg body wt) which entirely suppress neonatal hyperbilirubinemia in the experimental animal, leads to a very rapid (approximately 30-60 min) increase in the heme saturation of tryptophan pyrrolase from normal levels of approximately 50-60% to nearly 100%. The effect peaks at 1-2 h and lasts for at least 12 h. Sn-protoporphyrin is also able to block the rapid and marked decline in heme saturation of tryptophan pyrrolase elicited by inorganic cobalt, a potent inducer of heme oxygenase in liver. These findings establish clearly that after the administration of Sn-protoporphyrin in the whole animal, a functionally active heme pool, the one related to tryptophan pyrrolase, is rapidly increased in liver, confirming that the metalloporphyrin inhibits the degradation of endogenous heme by heme oxygenase.

Authors

A Kappas, G S Drummond, M K Sardana

Gastrin releasing peptide is a selective mitogen for small cell lung carcinoma in vitro.

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Abstract

Human small cell lung carcinoma (SCLC) cells have been shown to contain significant levels of a bombesin-immunoreactive peptide. The 27-amino acid peptide, gastrin releasing peptide (GRP), has recently been shown to be responsible for the bombesin-like immunoreactivity found in SCLC cells. Among four lung cancer cell lines examined in vitro, GRP exhibited mitogenic activity for two SCLC subtypes, but not for a squamous carcinoma or adenocarcinoma lung cell line. The mitogenicity of the GRP molecule has been isolated to the carboxyterminal fragment, designated GRP 14-27, which is in part homologous to bombesin. The aminoterminal fragment, GRP 1-16, is no homologous to bombesin and exhibits no mitogenic activity. Thus, GRP may be an important growth regulating or autocrine factor in human SCLC.

Authors

S Weber, J E Zuckerman, D G Bostwick, K G Bensch, B I Sikic, T A Raffin

Fab-mediated binding of drug-dependent antibodies to platelets in quinidine- and quinine-induced thrombocytopenia.

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Abstract

Platelets coated with quinine- or quinidine-induced antibodies form rosettes around protein A-Sepharose beads and normal platelets form rosettes about protein A-Sepharose beads coated with these antibodies. These reactions occurred only in the presence of sensitizing drug. Platelets also formed rosettes about protein A-Sepharose beads coated with an anti-PIA1 antibody, but drug was not required. Formation of rosettes between antibody-coated platelets and protein A-Sepharose was inhibited by F(ab')2 fragments of goat antibody specific for the Fc portion of human IgG, while rosette formation between antibody-coated protein A-Sepharose and platelets was inhibited by F(ab')2 fragments directed against the F(ab')2 portion of the IgG molecule. Since binding of IgG to protein A is known to occur via the Fc region, these findings suggest that binding of drug-induced antibodies to platelets occurs at the Fab domains of the IgG molecule.

Authors

D J Christie, P C Mullen, R H Aster

Binding of a monoclonal anti-DNA autoantibody to identical protein(s) present at the surface of several human cell types involved in lupus pathogenesis.

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Abstract

A monoclonal anti-DNA antibody PME77, spontaneously produced in autoimmune B/W mice, has been found to recognize identical protein(s) present at the surface of several human cell types involved in the pathogenesis of systemic lupus erythematosus: glomeruli, platelets, erythrocytes, T and B cells, and neuronal tissue. Data indicate that protein(s) could represent a major stimulus or the target of anti-DNA autoimmunity and could account for tissue lesions observed in this disease.

Authors

L Jacob, M A Lety, D Louvard, J F Bach