Erythropoietin receptor. Subunit structure and activation.

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Authors

A D D'Andrea, L I Zon

Accumulation of apolipoprotein E-rich high density lipoproteins in hyperalphalipoproteinemic human subjects with plasma cholesteryl ester transfer protein deficiency.

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This study characterized the plasma lipoproteins of familial hyperalphalipoproteinemic patients with or without deficiency of cholesteryl ester transfer protein (CETP) activity. The subjects with CETP deficiency have increased levels of apolipoprotein (apo) E. The increased concentration of apo E in these subjects was correlated to the appearance of apo E-rich high density lipoproteins (HDL). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed that these lipoproteins contained predominantly the apo E (82%) and little amount of apo A-I (18%). These apo E-rich HDL displayed a much higher affinity than human LDL in binding to LDL receptors on human fibroblasts. Furthermore, 3.5 times fewer apo E-rich HDL than LDL were required to saturate the receptors on fibroblasts. These data indicated that the apo E-rich HDL in CETP-deficient human subjects contained multiple copies of apo E and bound to the LDL receptor through multiple interactions. The apo E-rich HDL, with similar properties as cholesterol-induced apo E HDLc, were not detectable in normal human subjects or in hyperalphalipoproteinemic subjects with normal CETP activity. The apo E-containing HDL in the latter subjects were smaller and contained only small amounts of apo E (14%). The difference in apo E-containing HDL in these subjects suggests a correlation between CETP level and the appearance of apo E-rich HDL.

Authors

S Yamashita, D L Sprecher, N Sakai, Y Matsuzawa, S Tarui, D Y Hui

Human very low density lipoproteins and chylomicrons can protect against endotoxin-induced death in mice.

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Endotoxemia stimulates many physiologic responses including disturbances in lipid metabolism. We hypothesized that this lipemia may be part of a defensive mechanism by which the body combats the toxic effects of circulating endotoxin. We tested the effects of mixtures of endotoxin, lipoproteins, and lipoprotein-free plasma and determined the ability of varying concentrations of human very low density lipoproteins (VLDL) and chylomicrons, as well as low density lipoproteins (LDL) and high density lipoproteins (HDL), and of the synthetic lipid emulsion SOYACAL to prevent endotoxin-induced death in mice. This study demonstrates that the triglyceride-rich VLDL and chylomicrons, as well as cholesterol-rich LDL and HDL, and cholesterol-free SOYACAL can protect against endotoxin-induced death. Protection required small amounts of lipoprotein-free plasma, and depended on the incubation time and the concentration of lipoprotein lipid. Despite stringent techniques to prevent exogenous endotoxin contamination eight of ten duplicate VLDL preparations contained endotoxin (5,755 +/- 3,514 ng endotoxin/mg triglyceride, mean +/- SEM) making the isolation of endotoxin-free VLDL difficult. In contrast, simultaneous preparations of LDL and HDL were relatively free of endotoxin contamination (3 +/- 3 and 320 +/- 319 ng/mg total cholesterol, respectively), suggesting that the contamination of VLDL occurs in vivo and not during the isolation procedure. These observations suggest a possible role for increased triglyceride-rich lipoproteins in the host's defense against endotoxemia and infection.

Authors

H W Harris, C Grunfeld, K R Feingold, J H Rapp

Regulation of human monocyte/macrophage function by extracellular matrix. Adherence of monocytes to collagen matrices enhances phagocytosis of opsonized bacteria by activation of complement receptors and enhancement of Fc receptor function.

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In inflammation monocytes emigrate from the peripheral circulation into an extravascular area rich in extracellular matrix proteins. In this milieu, phagocytes ingest and kill invading pathogens. In the present studies, we found that monocytes adhered to type I collagen gels phagocytized 2.5-12-fold more opsonized Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae than plastic-adherent monocytes. The rate of phagocytosis and the number of bacteria ingested by collagen-adherent monocytes was equal to, or greater than, the number of bacteria ingested by 7-d cultured macrophages (M phi). Although both collagen- and plastic-adherent monocytes were bactericidal for E. coli and S. aureus, more bacteria were killed by collagen-adherent monocytes by virtue of their enhanced phagocytic capacity. Cultured M phi only were bacteriostatic. Adherence of monocytes to collagen gels activated C receptors (CR) types 1 and 3 for phagocytosis, and enhanced Fc receptor (FcR)-mediated phagocytosis. Collagen- and plastic-adherent monocytes produced equivalent amounts of superoxide anion in response to phorbol myristate acetate and opsonized zymosan. Thus, the enhanced phagocytosis and killing of opsonized bacteria by collagen-adherent monocytes appear to be by regulation of the function of membrane CR and FcR, without apparent enhancement of the respiratory burst. These data suggest that adherence of monocytes to the extracellular matrix during inflammation may rapidly activate these cells for enhanced phagocytic bactericidal activity.

Authors

S L Newman, M A Tucci

Enzymolysis of glomerular immune deposits in vivo with dextranase/protease ameliorates proteinuria, hematuria, and mesangial proliferation in murine experimental IgA nephropathy.

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The therapeutic effects of saccharolytic and proteolytic enzymes were investigated in models of IgA nephropathy. Mesangial glomerulonephritis was induced in mice by intravenous injection of preformed soluble immune complexes of dextran sulfate and either IgA (J 558) or IgM (MOPC 104 E) anti-dextran MAb (passive model) or by immunization with DEAE dextran (active model). In the passive model, only 30-40% of dextranase-treated mice given IgA or IgM immune complexes had mesangial Ig or dextran deposits, compared with 100% of saline-treated controls (P less than 0.01). There was no significant difference in mice given only protease. In the active model, dextranase and protease separately each reduced glomerular dextran and C3 deposits, and hematuria (P less than 0.01). Dextranase also reduced the glomerular IgA deposits (20 vs. 100% of saline-treated mice) and the frequency and severity of mesangial matrix expansion (both P less than 0.02), but did not reduce the modest IgG or IgM codeposits. Protease reduced IgG and IgM deposits, proteinuria and mesangial hypercellularity compared with saline (P less than 0.02), but did not diminish IgA, and had no effect on mesangial matrix expansion. The combination of dextranase plus protease attenuated all components of glomerular injury as judged by clinical and pathological parameters, but inactivated dextranase plus inactivated protease had no effect on any parameter. We conclude that enzymatic digestion of antigen and antibody can reduce immune deposits, mesangial proliferation, proteinuria, and hematuria in experimental glomerulonephritis.

Authors

L Gesualdo, S Ricanati, M O Hassan, S N Emancipator, M E Lamm

Thyroid-stimulating antibody activity between different immunoglobulin G subclasses.

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To investigate the distribution of thyroid-stimulating antibody (TSAb) activity between IgG subclasses, sera from 11 patients with Graves disease (including the National Institute of Biological Standards and Control (NIBSC) Research Standard, long acting thyroid stimulator-B) were fractionated by chromatography on affinity columns of monoclonal IgG subclass antibodies or protein A to deplete all but a single subclass. The resulting fractions were 98% or more pure for a single subclass. In all 11 patients, TSAb activity appeared to be confined to the IgG1 fraction as determined by cAMP production on addition of the fractions to the FRTL-5 rat thyroid cell line. In all of eight specimens from seven patients so tested, the whole serum activity was recovered in the IgG1 fraction, after adjusting for the recovery of the isotype from the column. TSAb activity in one serum comprised both lambda and kappa light chains but was IgG1 restricted. This IgG subclass restriction was not found when the same fractions were tested for thyroglobulin, microsomal/thyroid peroxidase, or tetanus toxoid antibody activity. Together with previous results showing marked restriction of both light chain usage and isoelectric point of TSAb, these results support the idea that Graves' disease may be the result of an oligo- or possibly monoclonal response at the B cell level.

Authors

A P Weetman, M E Yateman, P A Ealey, C M Black, C B Reimer, R C Williams Jr, B Shine, N J Marshall

A missense mutation at codon 188 of the human lipoprotein lipase gene is a frequent cause of lipoprotein lipase deficiency in persons of different ancestries.

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Lipoprotein lipase (LPL) plays a crucial role in the regulation of lipoprotein metabolism by hydrolysing the core triglycerides of circulating chylomicrons and VLDL. Human, bovine, mouse, and guinea pig complementary DNA clones have recently been isolated and the organization of the human LPL gene is now known to comprise 10 exons spanning approximately 30 kb. Here we report a similar mutation on 21 alleles from 13 unrelated affected probands with LPL deficiency of French Canadian, English, Polish, German, Dutch, and East Indian ancestry. We show that an identical missense mutation within exon 5, resulting in an amino acid substitution of glutamic acid for glycine at position 188, is responsible for LPL deficiency in 21 of 88 LPL alleles assessed. This mutation alters an Ava II restriction site in exon 5 and will allow a rapid screening test for this mutation in patients with LPL deficiency. This mutation has occurred on the same haplotype in all the unrelated affected persons suggesting a common origin. The amino acid substitution lies within the longest segment of homology for LPL in different species and results in a protein that is catalytically defective.

Authors

M V Monsalve, H Henderson, G Roederer, P Julien, S Deeb, J J Kastelein, L Peritz, R Devlin, T Bruin, M R Murthy

Phenotypic expression of heterozygous lipoprotein lipase deficiency in the extended pedigree of a proband homozygous for a missense mutation.

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Familial lipoprotein lipase (LPL) deficiency is a rare genetic disorder accompanied by well-characterized manifestations. The phenotypic expression of heterozygous LPL deficiency has not been so clearly defined. We studied the pedigree of a proband known to be homozygous for a mutation resulting in nonfunctional LPL. Hybridization of DNA from 126 members with allele-specific probes detected 29 carriers of the mutant allele. Adipose tissue LPL activity, measured previously, was reduced by 50% in carriers, but did not reliably distinguish them from noncarriers. Carriers were prone to the expression of a form of familial hypertriglyceridemia characterized by increased plasma triglyceride, VLDL cholesterol and apolipoprotein B, and decreased LDL and HDL cholesterol concentrations. These manifestations were age modulated, with conspicuous differences between carriers and noncarriers observed only after age 40. Several noncarriers exhibited similar lipid abnormalities, but without the inverse relationship between VLDL cholesterol and LDL cholesterol noted among carriers. In addition to age and carrier status, the potentially reversible conditions, obesity, hyperinsulinemia and lipid-raising drug use were contributory. Thus heterozygous lipoprotein lipase deficiency, together with age-related influences, may account for a form of familial hypertriglyceridemia.

Authors

D E Wilson, M Emi, P H Iverius, A Hata, L L Wu, E Hillas, R R Williams, J M Lalouel

Skeletal muscle response to exercise training in congestive heart failure.

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To examine the ability of the skeletal muscle of congestive heart failure (CHF) patients to adapt to chronic exercise, five patients performed localized nondominant wrist flexor training for 28 d. Inorganic phosphate (Pi) and phosphocreatine (PCr) were monitored by magnetic resonance spectroscopy in both forearms at rest and during submaximal wrist flexion exercise at 6, 12, 24, and 36 J.min-1 before and after exercise training. Simultaneous measurements of limb blood flow were made by plethysmography at 12, 24, and 36 J.min-1. Forearm muscle mass and endurance were measured by magnetic resonance imaging and wrist flexion exercise before and after training. The Pi/PCr ratio and pH were calculated from the measured Pi and PCr. Exercise cardiac output, heart rate, plasma norepinephrine, and lactate measured during training were not elevated above resting values, confirming that training was localized to the forearm flexor muscles. After training, muscle bioenergetics, as assessed by the slope of the regression line relating Pi/PCr to submaximal workloads, were improved in the trained forearm of each patient, although muscle mass, limb blood flow, and pH were unchanged. Forearm endurance increased by greater than 260% after training. In the dominant untrained forearm, none of the measured indices were affected. We conclude that localized forearm exercise training in CHF patients improves muscle energetics at submaximal workloads in the trained muscle, an effect which is independent of muscle mass, limb blood flow, or a central cardiovascular response during training. These findings indicate that peripheral muscle metabolic and functional abnormalities in CHF can be improved without altering cardiac performance.

Authors

J R Minotti, E C Johnson, T L Hudson, G Zuroske, G Murata, E Fukushima, T G Cagle, T W Chick, B M Massie, M V Icenogle

Rapid and reversible secretion changes during uncoupling of rat insulin-producing cells.

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To determine whether insulin secretion is affected by a blockage of gap junctions between B cells, we have studied the secretion of rat pancreatic islets of Langerhans, primary dispersed islet cells, and cells of the RINm5F line, during short-term exposure to heptanol. Within minutes, this alkanol blocked gap junctions between the B cells of intact islets and abolished their normal secretory response to glucose. These two changes were rapidly and fully reversible after return of the islets to control medium. We further found that heptanol had no significant effect on the glucose-stimulated secretion of single B cells but inhibited that of B cell pairs. In the clone of RINm5F cells, whose junctional coupling and D-glyceraldehyde-induced stimulation of insulin release by aggregated cells were also inhibited by heptanol, this alkanol did not perturb intracellular pH and Ca2+ and the most distal steps of the secretion pathway. In summary, a gap junction blocker affected the secretion of insulin-producing cells by a mechanism which is dependent on cell contact and is not associated with detectable pleiotropic perturbations of the cell secretory machinery. The data provide evidence for the involvement of junctional coupling in the control of insulin secretion.

Authors

P Meda, D Bosco, M Chanson, E Giordano, L Vallar, C Wollheim, L Orci

Therapeutic treatment of New Zealand mouse disease by a limited number of anti-idiotypic antibodies conjugated with neocarzinostatin.

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-81 and NE-1 idiotypes (Id) of human nephritogenic anti-DNA antibodies are interspecies Id expressed also in NZB/W F1 mice. We tried to manipulate the synthesis of spontaneously occurring anti-DNA antibody using monoclonal anti-Id antibodies (D1E2 and 1F5) conjugated with a cytotoxic agent, neocarzinostatin (NCS). In vivo administration of anti-Id antibodies conjugated with NCS brought about an improvement in the survival rate of female NZB/W F1 mice. It also caused a retardation of development of lupus nephritis and decreased the numbers of anti-DNA-producing cells. The suppression of anti-DNA antibody synthesis was specific and Id-mediated. The results indicate that the use of a limited number of anti-Id antibodies in combination with a cytotoxic agent may be applicable therapeutically to autoimmune diseases.

Authors

N Harata, T Sasaki, H Osaki, T Saito, S Shibata, T Muryoi, O Takai, K Yoshinaga

Alterations in phospholipid N-methylation of cardiac subcellular membranes due to experimentally induced diabetes in rats.

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Phosphatidylethanolamine N-methylation was examined in cardiac subcellular membranes after inducing chronic experimental diabetes in rats (65 mg streptozotocin/kg, i.v.). The incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine in diabetic sarcolemma was significantly depressed at all three catalytic sites (I, II, and III) of the methyltransferase system. An increase in methyl group incorporation was evident at site I without any changes at sites II and III in diabetic sarcoplasmic reticulum and mitochondria. Similar changes were also seen for the individual N-methylated lipids (monomethyl-, dimethylphosphatidylethanolamine, and phosphatidylcholine) specifically formed at each catalytic site in all cardiac membranes from diabetic animals. These alterations in N-methylation were reversible by a 14-d insulin therapy to the diabetic animals. In the presence of 10 microM ATP and 0.1 microM Ca2+, N-methylation was maximally activated at site I in both control and diabetic sarcolemma and sarcoplasmic reticulum, but not in mitochondria. Incubation of cardiac membranes with of S-adenosyl-L-methionine showed that Ca2(+)-stimulated ATPase activities in both sarcolemma and sarcoplasmic reticulum were augmented; however, the activation of diabetic sarcolemma was lesser and that of diabetic sarcoplasmic reticulum was greater in comparison with the control preparations. These results identify alterations in phosphatidylethanolamine N-methylation in subcellular membranes from diabetic heart, and it is suggested that these defects may be crucial in the development of cardiac dysfunction in chronic diabetes.

Authors

V Panagia, Y Taira, P K Ganguly, S Tung, N S Dhalla

Distinct abnormalities in the interaction of purified types IIA and IIB von Willebrand factor with the two platelet binding sites, glycoprotein complexes Ib-IX and IIb-IIIa.

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We have studied the interaction of the congenitally abnormal type IIA and IIB von Willebrand factor (vWF) molecules, both lacking the larger multimeric forms, with the two vWF binding sites on platelets, the glycoprotein (GP) Ib-IX and GP IIb-IIIa complexes. Variant as well as normal (N) vWF were purified from plasma. Estimates for binding of subunit molecules per platelet at saturation (Bmax) and dissociation constant in moles/liter (Kd), respectively, were obtained from binding isotherms of 125I-labeled vWF, with the following results. In the presence of ristocetin (binding to GP Ib-IX): N, 25,693 and 0.5 x 10(-8); IIA, both parameters not measurable; IIB, 17,708 and 0.87 x 10(-8). After thrombin stimulation (binding to GP IIb-IIIa): N, 17,059 and 1.12 x 10(-8); IIA, 23,751 and 4.87 x 10(-8); IIB, 19,890 and 2.52 x 10(-8). Distinct experiments based on measuring the ability of the variant species (from the same patients and one additional IIB patient) to inhibit the binding of normal 125I-vWF to platelets gave results in agreement with those reported above. Other studies showed that only IIB vWF bound to platelets in the absence of any mediating substance (Kd = 5.21 x 10(-8) mol/liter and Bmax = 9,599 subunits per platelet) and induced aggregation at a concentration of 10 micrograms/ml (3.6 x 10(-8) M). Thus, IIB vWF binds to GP Ib-IX with high affinity and induces platelet aggregation, whether with or without ristocetin, in spite of the absence of larger multimers. In contrast, the binding of IIA vWF to GP Ib-IX occurs with very decreased affinity, and this defective function may result from specific structural abnormalities rather than just being a reflection of the absence of larger multimeric forms. Both IIA and IIB vWF exhibit decreased affinity for GP IIb-IIIa. In this case, the extent of the defect correlates with the absence of larger multimers.

Authors

L De Marco, M Mazzucato, D De Roia, A Casonato, A B Federici, A Girolami, Z M Ruggeri

Erythrocyte sequestration and anemia in severe falciparum malaria. Analysis of acute changes in venous hematocrit using a simple mathematical model.

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Microvascular erythrocyte sequestration, the characteristic pathological feature of falciparum malaria, was evaluated using a mathematical model in 46 patients with severe infections. From admission radioisotopic circulating red cell volumes and simultaneous venous hematocrits, the model-derived sequestrum hematocrit (mean [95% confidence limits]: 0.70 [0.43-0.97], n = 29) was twice that of peripheral blood (0.33 [0.30-0.36]). Serial reticulocyte and radiolabeled erythrocyte counts indicated that small numbers of cells enter the circulation during initial therapy. The mean fall in hematocrit over 84 h in 26 nontransfused patients conformed to a three-term equation. A first-order decline (t1/2 2.0 h [0.6-3.4]) suggested an average 7.5% plasma volume expansion through rehydration. A zero-order 6.3% (3.1-9.5) fall (t1/2 25.7 h [21.2-30.2]) occurred contemporaneously with a fall in mean parasitemia from 4.5% (3.6-5.4); from these data the model-derived average sequestered erythrocyte volume (4.8% of the admission hematocrit) was similar to the peripheral parasite burden. A second, first-order fall (t1/2 1,047 h [278-1,816]) indicated loss of uninfected erythrocytes with mean lifespan 62 d. Predicted total plasma volume expansion during initial therapy (21.2%) was similar to radioisotopic estimates in 11 patients (17.3% [2.0-33.1]). Application of the model to individual patient data showed wide variations in relative proportions of circulating and sequestered parasitized cells. The model provides evidence of the nature and fate of all parasitized erythrocytes in malaria.

Authors

T M Davis, S Krishna, S Looareesuwan, W Supanaranond, S Pukrittayakamee, K Attatamsoonthorn, N J White

Lack of effect of lovastatin therapy on the parameters of whole-body cholesterol metabolism.

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The effects of lovastatin therapy on the parameters of body cholesterol metabolism were explored in nine hypercholesterolemic patients. Long-term cholesterol turnover studies were performed before therapy, and were repeated after 15 mo of lovastatin therapy (40 mg/d) while continuing on therapy. The major question addressed was whether a reduction in plasma cholesterol level with lovastatin would be associated with a reduction in the whole-body production rate of cholesterol or with the sizes of exchangeable body cholesterol pools as determined by the three-pool model of cholesterol turnover. The mean plasma cholesterol level decreased 19.4% (from 294 to 237 mg/dl), and low-density lipoprotein cholesterol decreased 23.8% (from 210 to 159 mg/dl) with lovastatin therapy. Changes in high-density lipoprotein cholesterol level were not significant. The cholesterol production rate did not change significantly with therapy (1.09 +/- 0.10 [mean +/- S.D.] vs. 1.17 +/- 0.09 g/d). By comparison, colestipol and niacin treatment in three other subjects more than doubled the cholesterol production rate (1.14 +/- 0.28 vs. 2.42 +/- 0.34 g/d). Thus, hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibition by lovastatin at the therapeutic dose used here did not change the steady-state rate of whole-body cholesterol synthesis. Despite the changes in plasma cholesterol levels, no significant changes were seen in the values of M1, of M3 or of Mtot, the sizes of the pools of rapidly, of slowly, and of total body exchangeable cholesterol. Conclusion: lovastatin therapy to lower plasma cholesterol does not lead to corresponding reductions in body cholesterol pools or to a reduction in the rate of whole-body cholesterol synthesis. In the new steady state that exists during long-term lovastatin therapy, along with increased expression of the genes for HMG-CoA reductase and the LDL receptor, the body compensates for the effects of the drug so that cholesterol production rate and tissue pool sizes are not changed from pretreatment values.

Authors

I J Goldberg, S Holleran, R Ramakrishnan, M Adams, R H Palmer, R B Dell, D S Goodman

Analysis of thymic endogenous retroviral expression in murine lupus. Genetic and immune studies.

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Inbred mouse genomes contain two subclasses of proviruses related to mink cell focus-forming (MCF) retroviruses: polytropic (Pmv), and modified polytropic (Mpmv). To determine whether one of these subclasses is associated with murine lupus, oligonucleotide probes specific for Pmv or Mpmv sequences were used in Northern analyses. Thymus 8.4 kb Mpmv RNA was expressed in five of five lupus-prone strains and crosses and this expression was not affected by genes that retard or accelerate development of lupus. Two of four leukemia-prone strains expressed low levels of such thymic transcripts, but none of 11 control strains did. 8.4 kb Mpmv RNA expression was not induced in thymuses of control mice by the lpr/lpr or gld/gld genotypes (which cause polyclonal immune activation) nor by treatment with mitogens. In contrast to Mpmv, thymic 8.4 kb Pmv expression was poorly associated with autoimmunity: it was easily detected in nearly all strains, and was increased by polyclonal activation in control mice. These studies indicate that the organ-specific thymic 8.4 kb Mpmv expression (a) is characteristic of several genetic backgrounds which predispose to murine lupus, (b) precedes and does not correlate with disease development, (c) is not due to polyclonal activation, and (d) is regulated independently of 8.4 kb Pmv expression.

Authors

A M Krieg, A D Steinberg

An immunoprotective molecule, the major secretory protein of Legionella pneumophila, is not a virulence factor in a guinea pig model of Legionnaires' disease.

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We have examined whether a molecule that is capable of inducing immune protection, the major secretory protein (MSP) of Legionella pneumophila, is required for virulence in a guinea pig model of Legionnaires' disease. To do so, we have compared the virulence in guinea pigs of an isogenic pair of L. pneumophila, Philadelphia 1 strain, one of which produces MSP (MSP+) and one of which does not (MSP-). Both the MSP- strain and the MSP+ strain of L. pneumophila are highly virulent for guinea pigs, inducing similar signs and progression of illness. Both strains are lethal and have comparable LD50s and LD100s. Both strains multiply in the lungs of guinea pigs at a similar rate, and both strains produce indistinguishable pathological lesions in the lungs. Both strains maintain a stable phenotype with guinea pig passage, i.e., the MSP- strain does not regain the capacity to secrete MSP and the MSP+ strain retains its capacity to secrete MSP after lung passage. Although vaccination with MSP induces strong protective immunity in the guinea pig against lethal aerosol challenge with L. pneumophila, this protective immunogen is not required in its intact proteolytically active form for the expression of virulence by the intracellular pathogen L. pneumophila. This demonstrates that a protective immune response need not necessarily be directed against a virulence determinant and suggests that any molecule that allows the host immune system to detect and act against an intracellularly sequestered pathogen may potentially serve as a protective immunogen against such a pathogen.

Authors

S J Blander, L Szeto, H A Shuman, M A Horwitz

Vasoactive intestinal peptide in human nasal mucosa.

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Vasoactive intestinal peptide (VIP), which is present with acetylcholine in parasympathetic nerve fibers, may have important regulatory functions in mucous membranes. The potential roles for VIP in human nasal mucosa were studied using an integrated approach. The VIP content of human nasal mucosa was determined to be 2.84 +/- 0.47 pmol/g wet weight (n = 8) by RIA. VIP-immunoreactive nerve fibers were found to be most concentrated in submucosal glands adjacent to serous and mucous cells. 125I-VIP binding sites were located on submucosal glands, epithelial cells, and arterioles. In short-term explant culture, VIP stimulated lactoferrin release from serous cells but did not stimulate [3H]glucosamine-labeled respiratory glycoconjugate secretion. Methacholine was more potent than VIP, and methacholine stimulated both lactoferrin and respiratory glycoconjugate release. The addition of VIP plus methacholine to explants resulted in additive increases in lactoferrin release. Based upon the autoradiographic distribution of 125I-VIP binding sites and the effects on explants, VIP derived from parasympathetic nerve fibers may function in the regulation of serous cell secretion in human nasal mucosa. VIP may also participate in the regulation of vasomotor tone.

Authors

J N Baraniuk, J D Lundgren, M Okayama, J Mullol, M Merida, J H Shelhamer, M A Kaliner

Distribution of 11 beta-hydroxysteroid dehydrogenase along the rabbit nephron.

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It has been recently proposed that 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is responsible for aldosterone tissue specificity. A 11 beta-OHSD deficiency has been invoked as a cause of the syndrome of apparent mineralocorticoid excess, and 11 beta-OHSD inhibition by liquorice has been invoked to explain the hypertension induced by this drug. Since the renal tubule is composed of aldosterone-sensitive and insensitive segments, we determined the distribution of 11 beta-OHSD along the rabbit tubule. Pools of tubular segments isolated by microdissection were incubated for 2 h at 37 degrees C in the presence of [3H]corticosterone (3H-B, 8.10(-9) M). Afterwards, the amounts of 3H-B and of the metabolite 11-dehydrocorticosterone (3H-A) were determined using HPLC analysis. In the proximal tubule, in either its convoluted or straight portion, and in the medullary thick ascending limb, the amount of 3H-A was 19.6 +/- 3.8% (n = 12), 17.9 +/- 3.4 (n = 8), and 15.0 +/- 2.2 (n = 4), respectively, of the sum of 3H-A + 3H-B. In the cortical ascending limb and the collecting tubule in its cortical and medullary parts, it was 74.7 +/- 6.8% (n = 4), 74.1 +/- 4.9 (n = 9) and 64.6 +/- 14.1 (n = 3), respectively. In both proximal and cortical collecting tubule, addition of carbenoxolone 8.10(-4) M, an inhibitor of 11 beta-OHSD, almost completely inhibited the conversion of 3H-B to 3H-A. Thus, 11 beta-OHSD activity was high in the aldosterone-sensitive segments, and low in the aldosterone-insensitive segments. These results strongly favor the hypothesis that 11 beta-OHSD is a key enzyme in mineralocorticoid tissue specificity along the rabbit nephron. They reinforce the notion that a defect in 11 beta-OHSD plays a major role in the syndrome of apparent mineralocorticoid excess and liquorice-induced hypertension.

Authors

J P Bonvalet, I Doignon, M Blot-Chabaud, P Pradelles, N Farman

Increased pulmonary neuroendocrine cells with bombesin-like immunoreactivity in adult patients with eosinophilic granuloma.

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Cigarette smoking is associated with hyperplasia of pulmonary neuroendocrine cells and variably increased levels of bombesin-like peptides in the lower respiratory tract. Because the neuropeptide bombesin is a chemoattractant for monocytes and a mitogen for 3T3 fibroblasts, we hypothesized that an excess of neuroendocrine cells and bombesin-like peptides could contribute to lung inflammation and fibrosis in certain cigarette smokers. Eosinophilic granuloma is a fibrotic lung disease of unknown etiology that in adults occurs almost invariably in cigarette smokers. We quantitated neuroendocrine cells with bombesin-like immunoreactivity in open lung biopsies from patients with eosinophilic granuloma (n = 6) and compared these with cigarette smokers (n = 6) who underwent lung resection for reasons other than primary lung disease. In addition, we compared them with patients with idiopathic pulmonary fibrosis (n = 8), a disease not associated with cigarette smoking. Finally, we also examined the mitogenic effect of bombesin on cultured human adult lung fibroblasts. The patients with eosinophilic granuloma exhibited a 10-fold increase in neuroendocrine cells with bombesin-like immunoreactivity compared to both smokers (P = 0.005) and patients with idiopathic pulmonary fibrosis (P = 0.005). In addition, bombesin produced a significant mitogenic effect on cultured human adult lung fibroblasts at concentrations of 1 nM and above. We conclude that increased numbers of pulmonary neuroendocrine cells with bombesin-like immunoreactivity are commonly found in patients with eosinophilic granuloma and, since bombesin-like peptides are chemotactic for monocytes and mitogenic for human lung fibroblasts, we speculate that neuroendocrine cell hyperplasia may be important in the pathogenesis of eosinophilic granuloma in adult cigarette smokers.

Authors

S M Aguayo, T E King Jr, J A Waldron Jr, K M Sherritt, M A Kane, Y E Miller

Effects of interleukin 2 on cardiac function in the isolated rat heart.

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Adoptive immunotherapy with IL 2 is associated with severe cardiovascular toxicities including peripheral and pulmonary edema, hypotension decreased systemic vascular resistance, increased heart rate, and an increased cardiac index. The purpose of this investigation was to determine whether IL 2 alone or in combination with lymphokine-activated killer cells (LAK) cells depress cardiac function using the isolated, perfused, working rat heart preparation. Male Sprague-Dawley rats (250-350 g) were anesthetized and the hearts were removed and placed on the perfusion apparatus. Hearts were perfused with oxygenated Krebs-Henseleit buffer (KHB), or oxygenated KHB containing IL 2 alone, IL 2-Media (cell culture media supplemented with 1,500 U IL 2/ml), LYMPH (cell culture media from cultured mononuclear cells from healthy volunteers), or LAK (cell culture media from cultured lymphocytes harvested from patients receiving IL 2/LAK in the presence of 1,500 U/ml IL 2). The cells were removed before perfusion (n = 9). Cardiac output and coronary flow were measured at 20-min intervals with preload constant (afterload varied or afterload constant (preload varied). The results indicate a significant depression in cardiac function in hearts treated with LAK. This depression was evident at 20 min and was more pronounced at 60 min. Washout of the KHB plus LAK reversed this depression. Thus, IL 2-stimulated/cultured human mononuclear cells produce a soluble factor that produces a reversible severe depression of cardiac function.

Authors

P A Sobotka, J McMannis, R I Fisher, D G Stein, J X Thomas Jr

Ascitic fluid from human ovarian cancer patients contains growth factors necessary for intraperitoneal growth of human ovarian adenocarcinoma cells.

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Abstract

Human ovarian cancer, the leading cause of death from gynecologic malignancy, tends to remain localized to the peritoneal cavity until late in the disease. In established disease, ascitic fluid accumulates in the peritoneal cavity. We have previously demonstrated that this ascitic fluid is a potent source of in vitro mitogenic activity including at least one unique growth factor. We now report that the human ovarian adenocarcinoma line, HEY, can be induced to grow intraperitoneally in immunodeficient nude mice in the presence (23/28 mice), but not absence (0/21 mice) of ascitic fluid from ovarian cancer patients. Ascitic fluid from patients with benign disease did not have similar effects on intraperitoneal growth of HEY cells (1/15 mice). Once tumors were established by injections of exogenous ascitic fluid, they could progress in the absence of additional injections of ascitic fluid. The mice eventually developed ascitic fluid which contained potent growth factor activity, suggesting that the tumors eventually produced autologous growth factors. This nude mouse model provides a system to study the action of ovarian cancer growth factors on tumor growth in vivo and to evaluate preclinically, therapeutic approaches designed to counteract the activity of these growth factors.

Authors

G B Mills, C May, M Hill, S Campbell, P Shaw, A Marks

Interleukin 1 inhibits insulin secretion from isolated rat pancreatic islets by a process that requires gene transcription and mRNA translation.

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Abstract

Recombinant human IL 1 beta inhibits glucose-induced insulin secretion from isolated pancreatic islets and from purified beta-cells obtained by fluorescence-activated cell sorting (FACS) of dispersed islet cells. Brief (1 h) exposure of isolated islets to IL 1 produces sustained inhibition of insulin secretion for at least 17 h after the IL 1 has been removed from the culture medium. An inhibitory effect of IL 1 on insulin secretion is not observed when islets are coincubated with an inhibitor of DNA transcription (actinomycin D). This finding indicates that the inhibitory effect of IL 1 on insulin secretion requires transcription of one or more genes during the first hour of exposure of islets to IL 1. The inhibitory effect of IL 1 on insulin secretion also requires mRNA translation, because three structurally distinct inhibitors of protein synthesis (cycloheximide, anisomycin, and puromycin) prevent IL 1-induced inhibition of insulin secretion when added to islets after the 1-h exposure to IL 1. Two-dimensional gel electrophoresis of islet proteins metabolically labeled with [35S]methionine demonstrates that IL 1 augments the expression of a 65-kD (pl approximately 6.5) protein by greater than 2.5-fold. These findings indicate that biochemical events occurring within 1 h of exposure of islets to IL 1 lead to an inhibition of insulin secretion that persists for at least 17 h after the removal of IL 1. One of the early biochemical effects of IL 1 on islets is gene transcription (0-1 h), which is followed by mRNA translation (after 1 h). Our results suggest that the inhibitory effect of IL 1 on insulin secretion is mediated by protein(s) whose synthesis is induced by IL 1.

Authors

J H Hughes, J R Colca, R A Easom, J Turk, M L McDaniel

Cloning and characterization of a second complementary DNA for human tryptase.

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Abstract

A second cDNA for human tryptase, called beta-tryptase, was cloned from a mast cell cDNA library in lambda ZAP. Its nucleotide sequence and corresponding amino acid sequence were determined and compared with those of a previously cloned tryptase cDNA, now called alpha-tryptase. The 1,142-base sequence of beta-tryptase encodes a 30-amino acid leader sequence of 3,089 D and a 245-amino acid catalytic region of 27,458 D. The amino acid sequence of beta-tryptase is 90% identical with that of alpha-tryptase, the first 20 amino acids of the catalytic portions being 100% identical. This identity, together with recognition of each recombinant protein by monoclonal antibodies directed against purified tryptase validate the tryptase identity of both alpha-tryptase and beta-tryptase cDNA molecules. Modest differences between the nucleic acid sequences of alpha- and beta-tryptase occurred throughout the cDNA molecules except in the 3' noncoding regions, which were identical. Although most highly conserved regions of amino acid sequence in the trypsin superfamily are conserved in both tryptase molecules, beta-tryptase has one carbohydrate binding site compared to two in alpha-tryptase, and one additional amino acid in the catalytic sequence. Regions of the substrate binding pocket in beta-tryptase (DSCQ, residues 218-221; SWG, residues 243-245) differ slightly from those in alpha-tryptase (DSCK, residues 217-220; SWD, residues 242-244). The presence of both alpha- and beta-tryptase sequences in each haploid genome was indicated by finding alpha- and beta-tryptase specific fragments after amplification by PCR of genomic DNA in 10 unrelated individuals. Localization of both alpha- and beta-tryptase sequences to human chromosome 16 was then performed by analysis of DNA preparations from 25 human/hamster somatic hybrids by PCR. It is now possible to assess the expression of each tryptase cDNA by mast cells and the relationship of each gene product to the active enzyme.

Authors

J S Miller, G Moxley, L B Schwartz

Pituitary-like proopiomelanocortin transcripts in human Leydig cell tumors.

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Abstract

Proopiomelanocortin is a polypeptide precursor molecule, the processing of which generates ACTH, beta-endorphin, the beta- and gamma-lipotropins, the joining peptide, and the NH2-terminal fragment. Anterior pituitary corticotrophs are the major site of proopiomelanocortin gene expression in man and the predominant, if not sole source of circulating ACTH. Recent data have established that proopiomelanocortin gene expression also occurs in various normal nonpituitary tissues, one of the best studied being the testis. In this latter organ the dominant gene products are short transcripts of approximately 800 nucleotides, which lack the first two exons of the gene and cannot encode a complete proopiomelanocortin molecule. In this report we show that the mode of proopiomelanocortin gene expression is occasionally modified in human Leydig cell tumors: a 1,200-nucleotide mRNA species identical to that in the pituitary is produced. It results from the usual (pituitary) start site of transcription and thus can encode the complete proopiomelanocortin molecule. In two out of six tumors, large amounts of the 1,200-nucleotide transcript led to a dramatic increase of approximately 1,000-fold in proopiomelanocortin peptide concentrations as compared with the normal and peritumoral testis. Proopiomelanocortin processing in these tumors generates various peptide fragments including ACTH. These results may help to understand the mechanism of proopiomelanocortin expression in nonpituitary tumors and have implications for the more general phenomenon of ectopic hormone secretion.

Authors

Y de Keyzer, F Lenne, J F Massias, D Vieau, J P Luton, A Kahn, X Bertagna

Differential regulation of insulin-like growth factor binding protein secretion from human decidual cells by IGF-I, insulin, and relaxin.

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Abstract

Several growth hormone-independent 25-31,000 kD insulin-like growth factor binding proteins (IGF-BPs) have been identified in plasma, extravascular fluids, and various cell-conditioned media. Cultured human decidual cells release three IGF-BPs with 24,000, 30,000, and 34,000 Mr. Using ligand blot analysis and an RIA for the 30,000-Mr form (IGF-BP-1), we examined the effects of IGF-I (10-1,000 ng/ml), insulin (10-10,000 ng/ml), and relaxin (10-250 ng/ml) on decidual cell IGF-BP release after 120 h of hormone exposure. IGF-I inhibited release of both IGF-BP-1 and the 24,000 Mr form. Inhibition of IGF-BP-1 release was noted after 48 h of treatment and was progressive throughout the subsequent 120 h. Insulin stimulated a fourfold increase in release of the 24,000-Mr protein while inhibiting IGF-BP-1 release comparable to IGF-I, alpha-IR3, a monoclonal antibody to the IGF-I receptor, blocked approximately 33% of the IGF-I response but had no effect on insulin-mediated IGF-BP-1 inhibition. Relaxin stimulated a 2.4-fold increase in release of the 24,000-Mr form and a 16-fold increase in the 30,000-Mr protein after 120 h. Stimulation of the 30,000-Mr protein was inhibited by the addition of cycloheximide (50 micrograms/ml). Both IGF-I and insulin also blocked the relaxin-mediated increase in IGF-BP-1. These studies suggest that three structurally related proteins differentially regulate IGF-BP secretion possibly via activation of distinct receptor subtypes.

Authors

K M Thraikill, D R Clemmons, W H Busby Jr, S Handwerger

Two distinct abnormalities in patients with C8 alpha-gamma deficiency. Low level of C8 beta chain and presence of dysfunctional C8 alpha-gamma subunit.

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Abstract

The sera from three C8 alpha-gamma deficient patients previously reported to have a selective C8 alpha-gamma defect were analyzed by SDS-PAGE and Western blot using two polyclonal antisera to C8 alpha-gamma and a monoclonal antibody to C8 alpha. All three sera exhibited C8 alpha-gamma bands that dissociated into alpha and gamma chains under reducing conditions. Quantitation of the alpha-gamma subunit in these sera by a sensitive ELISA revealed an amount approximately 1% of that found in normal human serum. A similar assay performed with a specific antiserum to C8 beta showed unexpectedly low levels of C8 beta in these sera, which were confirmed by hemolytic titration of C8 beta. The remarkable differences between C8 alpha-gamma and C8 beta in the C8 alpha-gamma deficient sera was that in spite of their comparable immunochemical levels, C8 beta still exhibited functional activity whereas C8 alpha-gamma was totally inactive. That the residual C8 alpha-gamma was inactive was also proved by its inability to show lytic bands in an overlay system after SDS-PAGE and subsequent removal of SDS. The implications of these findings for a novel concept of C8 deficiency are discussed.

Authors

F Tedesco, L Roncelli, B H Petersen, V Agnello, J M Sodetz

Congenital hypothyroidism, as studied in rats. Crucial role of maternal thyroxine but not of 3,5,3'-triiodothyronine in the protection of the fetal brain.

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Abstract

To study the protective effects of maternal thyroxine (T4) and 3,5,3'-triiodothyronine (T3) in congenital hypothyroidism, we gave pregnant rats methimazole (MMI), an antithyroid drug that crosses the placenta, and infused them with three different doses of T4 or T3. The concentrations of both T4 and T3 were determined in maternal and fetal plasma and tissues (obtained near term) by specific RIAs. Several thyroid hormone-dependent biological end-points were also measured. MMI treatment resulted in marked fetal T4 and T3 deficiency. Infusion of T4 into the mothers increased both these pools in a dose-dependent fashion. There was a preferential increase of T3 in the fetal brain. Thus, with a T4 dose maintaining maternal euthyroidism, fetal brain T3 reached normal values, although fetal plasma T4 was 40% of normal and plasma TSH was high. The infusion of T3 pool into the mothers increased the total fetal extrathyroidal T3 pool in a dose-dependent fashion. The fetal T4 pools were not increased, however, and this deprived the fetal brain (and possibly the pituitary) of local generation of T3 from T4. As a consequence, fetal brain T3 deficiency was not mitigated even when dams were infused with a toxic dose of T3. The results show that (a) there is a preferential protection of the brain of the hypothyroid fetus from T3 deficiency; (b) maternal T4, but not T3, plays a crucial role in this protection, and (c) any condition which lowers maternal T4 (including treatment with T3) is potentially harmful for the brain of a hypothyroid fetus. Recent confirmation of transplacental passage of T4 in women at term suggests that present results are relevant for human fetuses with impairment of thyroid function. Finding signs of hypothyroidism at birth does not necessarily mean that the brain was unprotected in utero, provided maternal T4 is normal. It is crucial to realize that maintainance of maternal "euthyroidism" is not sufficient, as despite hypothyroxinemia, the mothers may be clinically euthyroid if their T3 levels are normal.

Authors

R Calvo, M J Obregón, C Ruiz de Oña, F Escobar del Rey, G Morreale de Escobar

Acetylcholinesterase and butyrylcholinesterase genes coamplify in primary ovarian carcinomas.

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Abstract

The genes for acetylcholinesterase (ACHE) and butyrylcholinesterase (CHE) are expressed in multiple tumor tissues, including ovarian carcinomas. Both CHE and ACHE genes coamplify in leukemias. To examine the relationship of gene amplification to the expression of these genes in tumors, ACHE and CHE genes and their expression were studied in primary ovarian carcinomas. DNA blot hybridization demonstrated a significant amplification and mutagenesis of both genes in 6 of 11 malignant tumors studied. This was greater or of the same order of magnitude as the amplification of the oncogenes c-rafi, v-sis, and c-fes in these tumors. No amplification was found in normal ovarian tissues or benign ovarian cysts. Xenopus oocyte microinjections, blot and in situ hybridizations, and immuno- and cytochemical staining revealed translatable CHEmRNA and its active protein product in discrete tumor foci. The frequent coamplification in ovarian carcinomas of ACHE and CHE genes implicates cholinesterases in neoplastic growth and/or proliferation.

Authors

H Zakut, G Ehrlich, A Ayalon, C A Prody, G Malinger, S Seidman, D Ginzberg, R Kehlenbach, H Soreq

Point mutation in the beta-spectrin gene associated with alpha I/74 hereditary elliptocytosis. Implications for the mechanism of spectrin dimer self-association.

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Abstract

alpha I/74 hereditary elliptocytosis (HE) is a subgroup of HE in which patients exhibit an impaired self-association of spectrin dimers and an abnormal proteolytic cleavage of the alpha I domain of spectrin. We studied a family in which the proband presented with a severe neonatal hemolytic anemia with poikilocytosis. Biochemical analysis of erythrocytes from the proband and his family members allowed us to ascertain a diagnosis of homozygosity for alpha I/74 HE in the proband and heterozygosity in his parents and several of their offspring. Results of polymorphism linkage analysis suggested that the defect in this family was located in beta rather than alpha spectrin. We analyzed the 3' end of the beta-spectrin gene of the proband and detected a mutation that changes a codon for alanine to one for proline. Allele-specific oligomer hybridization on slot blots of DNA from other family members confirmed the presence of the mutation only in members heterozygous for the disorder. This is the first example of a point mutation in the beta-spectrin chain that is associated with defective spectrin dimer self-association and an abnormal proteolytic cleavage of the alpha chain. Based on this finding, we propose a model for the mechanism of interaction between the alpha- and beta-spectrin chains.

Authors

W T Tse, M C Lecomte, F F Costa, M Garbarz, C Feo, P Boivin, D Dhermy, B G Forget

Co-localization of transforming growth factor beta 2 with alpha 1(I) procollagen mRNA in tissue sections of patients with systemic sclerosis.

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Abstract

The role of transforming growth factor beta 2 (TGF-beta 2) in the pathogenesis of systemic sclerosis (SSc) was investigated by in situ hybridization of skin biopsies from six patients with SSc. Two patients with acute systemic lupus erythematosis (SLE), one with acute dermatomyositis (DM), and three healthy individuals were used as controls. TGF-beta 2 mRNA was found to be co-localized with pro alpha 1(I) collagen expression around dermal blood vessels in all patients with the inflammatory stage of SSc, whereas there was no expression of either gene in the dermis of patients in the fibrotic stage, the SLE patients or the normal controls. These findings provide evidence that TGF-beta 2 released by inflammatory cells around blood vessels may play a role in mediating the collagen gene disregulation in fibrosis.

Authors

M Kulozik, A Hogg, B Lankat-Buttgereit, T Krieg

A molecular defect in hepatic cholesterol biosynthesis in sitosterolemia with xanthomatosis.

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Abstract

We examined the relationship between cholesterol biosynthesis and total and high affinity LDL binding in liver specimens from two sitosterolemic and 12 healthy control subjects who died unexpectedly and whose livers became available when no suitable recipient for transplantation was identified. Accelerated atherosclerosis, unrestricted intestinal sterol absorption, increased plasma and tissue plant sterol concentrations, and low cholesterol synthesis characterize this disease. Mean total microsomal HMG-CoA reductase (rate-control controlling enzyme for cholesterol biosynthesis) activity was sevenfold higher (98.1 +/- 28.8 vs. 15.0 +/- 2.0 pmol/mg protein per min) and microsomal enzyme protein mass was eightfold larger (1.43 +/- 0.41 vs. 0.18 +/- 0.04 relative densitometric U/mg protein) in 11 controls than the average for two sitosterolemic liver specimens. HMG-CoA reductase mRNA probed with pRED 227 and pHRED 102 was decreased to barely detectable levels in the sitosterolemic livers. In addition, there was a 50% decrease in the rate [2-14C]mevalonic acid was converted to cholesterol by sitosterolemic liver slices compared with controls (112 vs. 224 +/- 32 pmol/g liver per h). In contrast, average total LDL binding was 60% greater (326 vs. 204 +/- 10 ng/mg), and high affinity (receptor-mediated) binding 165% more active (253 vs. 95.1 +/- 8.2 ng/mg) in two sitosterolemic liver membrane specimens than the mean for 12 controls. Liver morphology was intact although sitosterolemic hepatocytes and microsomes contained 24 and 14% less cholesterol, respectively, and 10-100 times more plant sterols and 5 alpha-stanols than control specimens. We postulate that inadequate cholesterol biosynthesis is an inherited abnormality in sitosterolemia and may be offset by augmented receptor-mediated LDL catabolism to supply cellular sterols that cannot be formed.

Authors

L B Nguyen, S Shefer, G Salen, G C Ness, G S Tint, F G Zaki, I Rani

Cytokine-dependent long-term culture of highly enriched precursors of hematopoietic progenitor cells from human bone marrow.

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Abstract

Human marrow cells positive for the CD34 antigen but not expressing HLA-DR, CD15, or CD71 antigens were isolated. In a liquid culture system supplemented with 48-hourly additions of recombinant interleukins IL-1 alpha, IL-3, IL-6, or granulocyte/macrophage colony-stimulating factor (GM-CSF), these cells were capable of sustaining in vitro hematopoiesis for up to eight weeks. The establishment of an adherent cell layer was never observed. Cultures containing no exogenous cytokine produced clonogenic cells for only 1 wk. IL-1 alpha and IL-6 were alone able to support hematopoiesis for 2 or 3 wk. Cells maintained with GM-CSF proliferated and contained assayable colony-forming cells for 3 or 4 wk, while maximal cellular expansion and generation of assayable progenitor cells occurred in the presence of IL-3 for 4-5 wk. When IL-3 was combined with IL-1 alpha or IL-6, hematopoiesis was sustained for 8 wks. Basophil numbers were markedly increased in the presence of IL-3. These studies indicate that marrow subpopulations can sustain hematopoiesis in vitro in the presence of repeated additions of cytokines. We conclude that a major function of marrow adherent cells in long-term cultures is that of providing cytokines which promote the proliferation and differentiation of primitive hematopoietic cells.

Authors

J Brandt, E F Srour, K van Besien, R A Briddell, R Hoffman

Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

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Abstract

The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections.

Authors

M Hermann, M E Jaconi, C Dahlgren, F A Waldvogel, O Stendahl, D P Lew

Regulation of binding proteins for insulin-like growth factors (IGF) in humans. Increased expression of IGF binding protein 2 during IGF I treatment of healthy adults and in patients with extrapancreatic tumor hypoglycemia.

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Abstract

Insulin-like growth factors (IGFs) in blood form two complexes with specific binding proteins (BPs): a large, growth hormone (GH)-dependent complex with restricted capillary permeability, and a smaller complex, inversely related to GH, with high turnover of its IGF pool and free capillary permeability. The distribution of BPs and of IGFs I and II between these complexes was studied in sera from healthy adults treated with IGF I or/and GH and from patients with extrapancreatic tumor hypoglycemia. Like GH, IGF I administration raises IGF I and two glycosylation variants of IGFBP-3 in the large complex, but unlike GH drastically reduces IGF II. During IGF I infusion, IGFBP-3 appears in the small complex whose IGFBP-2 and IGF I increase three- to fivefold and fivefold, respectively. GH treatment, associated with elevated insulin levels, suppresses IGFBP-2 and inhibits its increase owing to infused IGF I. The small complex of tumor sera contains increased amounts of IGFBP-2 and -3, and two- to threefold elevated IGF II. Conclusions: low GH and/or insulin during IGF I infusion and in extrapancreatic tumor hypoglycemia enhance expression of IGFBP-2 and favor partition of IGFBP-3 into the small complex. Free capillary passage and high turnover of its increased IGF I or II pools may contribute to compensate for suppressed insulin secretion during IGF I infusion or to development of tumor hypoglycemia.

Authors

J Zapf, C Schmid, H P Guler, M Waldvogel, C Hauri, E Futo, P Hossenlopp, M Binoux, E R Froesch

Antioxidant macromolecules in the epithelial lining fluid of the normal human lower respiratory tract.

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Abstract

We hypothesized that the alveolar structures may contain extracellular macromolecules with antioxidant properties to defend against oxidants. To evaluate this 51Cr-labeled human lung fibroblasts (HFL-1) and cat lung epithelial cells (AKD) were exposed to a H2O2-generating system and alveolar epithelial lining fluid (ELF) from healthy nonsmokers was tested for its ability to protect the lung cells from H2O2-mediated injury. The ELF provided marked antioxidant protection, with most from a H2O-soluble fraction in the 100-300-kD range. Plasma proteins with anti-H2O2 properties were in insufficient concentrations to provide the antioxidant protection observed. However, catalase, a normal intracellular antioxidant, was present in sufficient concentration to account for most of the observed anti-H2O2 properties of ELF. Depletion of ELF with an anticatalase antibody abolished the anti-H2O2 macromolecular defenses of ELF. Since catalase is not normally released by cells, a likely explanation for its presence in high concentrations in normal ELF is that it is released by lung inflammatory and parenchymal cells onto the epithelial surface of the lower respiratory tract during their normal turnover and collects there due to the slow turnover of ELF. It is likely that catalase in the ELF of normal individuals plays a role in protecting lung parenchymal cells against oxidants present in the extracellular milieu.

Authors

A M Cantin, G A Fells, R C Hubbard, R G Crystal

Interleukin 1 (IL-1) gene expression, synthesis, and effect of specific IL-1 receptor blockade in rabbit immune complex colitis.

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Abstract

Interleukin 1 (IL-1) may be a key mediator of inflammation and tissue damage in inflammatory bowel disease (IBD). In rabbits with immune complex-induced colitis, IL-1 alpha and beta mRNA levels were detectable at 4 h, peaked at 12 but were absent at 96 h after the induction of colitis. Colonic IL-1 tissue levels were measured by specific radioimmunoassays. IL-1 alpha was significantly elevated at 4 h (9.4 +/- 1.5 ng/g colon), progressively increased at 48 h (31 +/- 5.8 ng/g) and then decreased by 96 h (11.5 +/- 3.4 ng/g). IL-1 beta levels were 2.0 +/- 0.5 ng/g colon at 4 h, 5.0 +/- 1.6 ng/g at 48 h and undetectable by 96 h. By comparison, colonic levels of PGE2 and LTB4 were unchanged during the first 12 h and did not become elevated until 24 h. IL-1 alpha levels were highly correlated with inflammation (r = 0.885, P less than 0.0001), edema (r = 0.789, P less than 0.0001) and necrosis (r = 0.752, P less than 0.0005). Treatment with a specific IL-1 receptor antagonist (IL-1 ra) before and during the first 33 h after the administration of immune complexes markedly reduced inflammatory cell infiltration index (from 3.2 +/- 0.4 to 1.4 +/- 0.3, P less than 0.02), edema (from 2.2 +/- 0.4 to 0.6 +/- 0.3, P less than 0.01) and necrosis (from 43 +/- 10% to 6.6 +/- 3.2%, P less than 0.03) compared to vehicle-matched colitis animals. These studies demonstrate that (a) IL-1 gene expression and synthesis occur early in the course of immune complex-induced colitis; (b) are significantly elevated for 12 h before the appearance of PGE2 and LTB4; (c) tissue levels of IL-1 correlate with the degree of tissue inflammation and; (d) specific blockade of IL-1 receptors reduces the inflammatory responses associated with experimental colitis.

Authors

F Cominelli, C C Nast, B D Clark, R Schindler, R Lierena, V E Eysselein, R C Thompson, C A Dinarello

Autoreactive T lymphocytes in multiple sclerosis determined by antigen-induced secretion of interferon-gamma.

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Abstract

Multiple sclerosis (MS) is a disease with unknown cause characterized by inflammation and demyelination in the central nervous system. Although an autoimmune pathogenesis has been suggested, there are no conclusive data on the number of T cells autoreactive with myelin antigens in MS compared to controls. We showed that T lymphocytes secreting interferon-gamma in response to possible target autoantigens are severalfold more common among PBL mononuclear cells in patients with MS than in patients with aseptic meningitis and tension headache. On average T cells reactive with myelin basic protein (MBP), two different MBP peptides, or with proteolipid protein amounted to 2.7-5.2/10(5) PBL from MS patients. MBP-reactive T cells were still more frequent among mononuclear cells isolated from the cerebrospinal fluid (CSF; 185/10(5) CSF cells). We concluded that T cells reactive with myelin autoantigens are strongly increased in MS. This approach to detect them could allow definition of immunodominant T cell epitopes in individual MS patients, and thereby enable further development towards specific immunotherapy.

Authors

T Olsson, W W Zhi, B Höjeberg, V Kostulas, Y P Jiang, G Anderson, H P Ekre, H Link

Restricted expression of the erythroid/brain glucose transporter isoform to perivenous hepatocytes in rats. Modulation by glucose.

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Abstract

The "erythroid/brain" glucose transporter (GT) isoform is expressed only in a subset of hepatocytes, those forming the first row around the terminal hepatic venules, while the "liver" GT is expressed in all hepatocytes. After 3 d of starvation, a three- to fourfold elevation of expression of the erythroid/brain GT mRNA and protein is detected in the liver as a whole; this correlates with the expression of this GT in more hepatocytes, those forming the first three to four rows around the hepatic venules. Starvation-dependent expression of the erythroid/brain GT on the plasma membrane of these additional hepatocytes is lost within 3 h of glucose refeeding; however, by immunoblotting we show that the protein is still present. Its loss from the surface is possibly explained by internalization.

Authors

M Tal, D L Schneider, B Thorens, H F Lodish

Familial hypertrophic cardiomyopathy is a genetically heterogeneous disease.

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Abstract

We demonstrate that familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disorder of heart muscle, is a genetically heterogeneous disease. The locus responsible for FHC in members of one large kindred was recently mapped to chromosome 14q11-12 (FHC-1). We have characterized three additional unrelated families in which the gene for FHC segregates as an autosomal dominant trait to determine if these disease loci also map to FHC-1. All family members were clinically studied by physical examination, electrocardiogram, and two-dimensional echocardiography. Genetic studies were performed using DNA probes which are derived from loci that are closely linked to FHC-1. In one family the genetic defect maps to the previously identified FHC-1 locus. However, the loci responsible for FHC in two other families were not linked to FHC-1. We conclude that FHC can be caused by defects in at least two loci and is a genetically heterogeneous disorder.

Authors

S D Solomon, J A Jarcho, W McKenna, A Geisterfer-Lowrance, R Germain, R Salerni, J G Seidman, C E Seidman

Molecular basis of medium chain acyl-coenzyme A dehydrogenase deficiency. An A to G transition at position 985 that causes a lysine-304 to glutamate substitution in the mature protein is the single prevalent mutation.

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Abstract

We sequenced polymerase chain reaction (PCR)-amplified variant medium chain acyl-CoA dehydrogenase (MCAD) cDNAs in cultured fibroblasts from three MCAD-deficient patients. In all three patients, an A to G transition was identified at position 985 of the coding region. Since no appropriate restriction sites for detecting this point mutation were found, we devised a PCR method that amplifies an 87-bp fragment from position 955. In the 5' primer encompassing positions 955 to 984, A-981 was artificially substituted with C. With the presence of C-981 and G-985, an Nco I restriction site is introduced in the mutant copies. When cDNA or genomic DNA from fibroblasts of nine MCAD-deficient patients were tested with this method, the copies from all of them completely cleaved into two shorter fragments by Nco I, indicating their homozygosity for the A----G-985 transition. In contrast, the copies from all eight controls remained intact. Thus, this A----G-985 transition is the single prevalent mutation causing MCAD deficiency, a highly unusual feature for any genetic disorder. The PCR/Nco I digestion method is suitable for the diagnosis of MCAD deficiency.

Authors

I Yokota, Y Indo, P M Coates, K Tanaka

Enhanced expression of an exocrine pancreatic protein in Alzheimer's disease and the developing human brain.

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Abstract

Pancreatic thread protein (PTP) is a major exocrine secretory protein that in vitro forms filamentous bundles reminiscent of the paired helical filaments of Alzheimer's disease (AD). We previously described increased PTP immunoreactivity in AD brains and now report high levels in the developing human brain. Using a full-length cloned bovine PTP cDNA and synthetic oligonucleotides corresponding to human PTP cDNA, which is identical to human islet cell regeneration factor, we analyzed the expression of PTP in pancreas and brain. A major 0.9-kb as well as several minor transcripts were identified in human pancreas. In AD brain, the same size transcripts were detected by Northern analysis, primer extension assay, or polymerase chain reaction amplification of cDNAs generated by reverse transcriptase assay. There were significantly higher levels of PTP mRNA in brains with AD compared with aged controls, with increased amounts of 1.2-, 0.6-, and 0.4-kb transcripts by Northern analysis. In situ hybridization localized expression to pyramidal neurons in the cerebral cortex, the same population that contains neurofibrillary tangles and high levels of immunoreactive PTP. These findings suggest that AD is associated with enhanced expression of PTP-related transcripts with intraneuronal accumulation of PTP-like proteins.

Authors

S M de la Monte, M Ozturk, J R Wands