Immunological abnormalities of aging: an analysis of T lymphocyte subpopulations of Werner's syndrome.

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Abstract

Two T lymphocyte-specific antisera, i.e. naturally-occurring auto-antibody to T cells of systemic lupus erythematosus patients (natural T cell toxic autoantibody) and heterologous antiserum against human brain tissue (antibrain-associated T-cell antigen), were used to detect cell surface antigens of human peripheral T lymphocytes. Nylon column-purified T cells from normal aged individuals and patients with Werner's syndrome (a premature aging syndrome) were reacted with these auto- and heterologous antibodies followed by staining with appropriate fluorescence reagents. The cells were subjected to the automated analysis with fluorescence-activated cell sorter. Fluorescence profiles to T cells of both aged individuals of over 90 yr and Werner's syndrome showed a very similar pattern, with a drastic decrease in the population that had high fluorescence intensity stained with either antiserum accompanied by the relative increase in the cell population that had low fluorescence intensity. Natural T cell toxic autoantibody comparable to that detected in systemic lupus erythematosus patients was found in the serum of six out of seven patients with Werner's syndrome, whereas normal aged individuals produced no such an autoantibody. The results suggest that Werner's syndrome has a change in the lymphocyte population very similar to old individuals, and that such a change is caused by the production of autoantibodies reactive to T lymphocytes.

Authors

M Goto, Y Horiuchi, K Okumura, T Tada, M Kawata, K Ohmori

Glucose Memory of Pancreatic B and A₂ Cells: EVIDENCE FOR COMMON TIME-DEPENDENT ACTIONS OF GLUCOSE ON INSULIN AND GLUCAGON SECRETION IN THE PERFUSED RAT PANCREAS

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The influence of previous exposure to glucose on the subsequent B- and A2-cell secretory responses to arginine was investigated in the perfused pancreas of the rat. Arginine (8 mM) was administered in two brief (9 min) pulses separated by a period of 66 min. In pancreata from 18-h-fasted animals the two pulses of arginine elicited biphasic glucagon secretory responses, while stimulation of insulin release was barely detectable. When 27.7 mM glucose was administered for 30 min during the intervening period up to 20 min before the second pulse of arginine, the glucagon response to arginine was diminished by 55% while the insulin release was markedly increased in comparison with the first pulse. 8.3 mM glucose, when administered before the second pulse of arginine, exerted effects that were smaller but otherwise similar to those of 27.7 mM glucose.

Authors

Valdemar Grill, Ulf Adamson, Margareta Rundfeldt, Sten Andersson, Erol Cerasi

Plasma acetate turnover and oxidation.

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Plasma acetate turnover and oxidation were determined in 11 healthy subjects by the constant infusion of a trace amount of [1-14C]acetate for 6 h. The subjects ages ranged from 22 to 57 yr. There was a positive correlation (P less than 0.001) between plasma acetate concentration and turnover rate, and a negative correlation (P less than 0.001) between turnover and age. The plasma acetate concentration in the subjects 22--28 yr old was 0.17 vs. 0.13 mM (P less than 0.02) in subjects 40--57 yr old. The plasma acetate turnover rate was also greater in the younger age group (8.23 +/- 0.66 vs. 4.98 +/- 0.64 mumol/min . kg, P less than 0.01). Approximately 90% of the plasma acetate turnover was immediately oxidized to CO2 in both age groups, however, 13.2 +/- 0.89% of the CO2 output in the younger group was derived from plasma acetate oxidation compared to 7.9 +/- 0.94% in the older group (P less than 0.01). The mean plasma acetate concentration, turnover, and oxidation in six cancer patients 47--63 yr old were similar to the values observed in the age-matched healthy subjects. Uptake or output of acetate by various tissues was measured by arterial-venous plasma acetate concentration differences. In seven of eight subjects undergoing elective surgery, the arterial-portal venous concentration difference was negative, which indicated that the gastrointestinal tract can contribute to plasma acetate production. Uptake of plasma acetate by both the leg and liver appeared to be dictated by the arterial acetate concentration. Net production of acetate by both the leg and liver was most often observed at arterial plasma acetate concentrations less than 0.08 mM.

Authors

C L Skutches, C P Holroyde, R N Myers, P Paul, G A Reichard

Lymphocytes with Immunoglobulin E Fc Receptors in Patients with Atopic Disorders

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Abstract

Lymphocytes from normal nonallergic donors and patients with atopic disorders were analyzed for subpopulations bearing Fc receptors for immunoglobulin (Ig)E (Fcε) and IgG (Fcγ), surface IgM (sIgM) and IgD (sIgD), and for T cells forming spontaneous rosettes with sheep erythrocytes (E). The patients were divided into three groups according to serum IgE concentrations and systemic corticosteroid treatment. Group I consisted of 12 atopic patients with either normal or moderately increased IgE levels up to 4,000 U/ml. Four patients of group II and three of group III had 10,500-31,000 U/ml and severe atopic dermatitis. Patients of group III, but not I and II, were receiving corticosteroids systemically. The percentage (mean ±SD) and total number of Fcε+ lymphocytes were 1.2±0.5%, 41±24/mm3 in 12 normals; 1.6±0.9%, 59±43/mm3 in patients of group I: 7.0±2.0%, 187±67/mm3 in group II; and 0.3±0.1%, 13±5/mm3 in patients of group III. The increase in group II and decrease in group III of Fcε+ cells were statistically significantly different from the normal persons and patients of group I. In contrast, the patients did not differ significantly from the donors in sIgM+, sIgD+, Fcγ+, and E+ cell populations. As shown by depletion of sIg+ cells in four patients with atopic disorders, the great majority of the Fcε+ lymphocytes were B cells. However, two patients with elevated Fcε+ cell numbers had small numbers of mixed E- and Fcε-rosetting cells, presumably T cells. Two patients of group II were examined during an acute herpes simplex infection. Both showed an ≅80% decrease of Fcε+ cells at that time. No apparent correlation between numbers of Fcε+ cells and IgE level existed in patients of group I. Injection of an IgE myeloma protein into two monkeys did not significantly change their percentages of Fcε+ lymphocytes.

Authors

H. L. Spiegelberg, R. D. O'Connor, R. A. Simon, D. A. Mathison

Properdin factor D: effects on thrombin-induced platelet aggregation.

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Factor D, when preincubated with platelet suspensions, at concentrations as low as 1.2 micrograms/ml, inhibited thrombin-induced platelet aggregation. No inhibition of collagen or arachidonic acid-induced platelet aggregation was found. Inhibition occurred, but to a lesser extent, when thrombin and factor D were added to platelets at the same time. No inhibition occurred when factor D was added after thrombin. Thrombin was able to overcome inhibition by factor D by increasing its concentration. Diisopropyl-phosphorofluoridate-inactivated factor D also inhibited thrombin-induced platelet aggregation so that enzymatic activity of factor D was not required for inhibition. Factor D absorbed with hirudin coupled to Sepharose 6B showed no decrease in inhibitory capacity. 125I-Factor D bound to platelets in a manner suggesting an equilibrium reaction similar to thrombin. At low factor D input, binding was linear, whereas at higher input, binding began to approach saturation. Binding of 125I-labeled thrombin to platelets was inhibited by factor D. Analysis of these data show that factor D does not alter the total number of thrombin molecules which bind to the platelet surface at saturation. However, the dissociation constant for thrombin is altered from 2.78 to 6.90 nM in the presence of factor D (20 micrograms/ml). Factor D is thus a competitive inhibitor of thrombin binding, although the affinity of factor D for the platelet thrombin receptor is much less than that of thrombin. These phenomena occur at physiologic concentrations of factor D. Therefore, factor D may function in vivo as an inhibitor of platelet aggregation.

Authors

A E Davis 3rd, D M Kenney

Intestinal Calcium Absorption and Serum Vitamin D Metabolites in Normal Subjects and Osteoporotic Patients: EFFECT OF AGE AND DIETARY CALCIUM

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Intestinal calcium absorption assessed by a double-isotope method, decreased significantly with aging in 94 normal subjects (r = −0.22, P < 0.025). In 52 untreated patients with postmenopausal osteoporosis, calcium absorption was significantly lower than normal when either age or habitual calcium intake was used as a covariable (P < 0.001). Serum 25-hydroxyvitamin D (25-OH-D) and 1,25-dihydroxyvitamin D (1,25(OH)2D) were measured in 44 normal subjects and 27 osteoporotic patients. For all normals, calcium absorption and serum 1,25(OH)2D were positively correlated (r = 0.50, P < 0.001). In nonelderly normal subjects (ages 30-65 yr), dietary calcium intake correlated inversely with both calcium absorption (r = −0.39, P < 0.01) and with serum 1,25(OH)2D (r = −0.50, P < 0.01). Both osteoporotic patients and elderly normal subjects (ages 65-90 yr) differed from nonelderly normals in that these correlations were not present. In addition although serum 25-OH-D was normal, serum 1,25(OH)2D was significantly decreased in both osteoporotic patients and elderly normals (P < 0.001). In osteoporotic patients, calcium absorption increased significantly (P < 0.001) after 7 d administration of a small dose (0.4 μg/d) of synthetic 1,25(OH)2D3. In osteoporotics mean serum immunoreactive parathyroid hormone was either normal (COOH-terminal assay) or low (NH2-terminal assay) relative to age-matched controls, and mean serum phosphate was increased.

Authors

J. C. Gallagher, B. Lawrence Riggs, John Eisman, Alan Hamstra, Sara B. Arnaud, Hector F. Deluca

Measurement, Characterization, and Source of Somatostatin-like Immunoreactivity in Human Amniotic Fluid

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Somatostatin-like immunoreactivity (SLI) is widely distributed in tissues and biological fluids. To determine whether SLI is also present in amniotic fluid, samples obtained by amniocentesis from 30 normal and 27 abnormal pregnancies were studied by radioimmunoassay. Direct incubation of [125I-Tyr1]tetradecapeptide somatostatin (SRIF) with amniotic fluid resulted in 89% tracer degradation. Damage was reduced to <5% when samples were acidified and boiled before the assay. With this technique, SLI was detectable in all normal amniotic fluid samples; the mean level at 15-20 wk of gestation (320±55 pg/ml, n = 15) being 4.5 times higher than the mean at 32-43 wk (70±12 pg/ml, n = 15) (P < 0.001). In cases of preeclampsia (n = 6), gestational diabetes (n = 5), anencephaly (n = 1), and meningomyelocele (n = 1), SLI values were in the normal range, but in one juvenile diabetic and one patient with chronic renal failure, SLI was undetectable (<10 pg/ml). In a pair of monochorionic diamniotic twins, SLI levels were very different (33 and 197 pg/ml), which suggests that fetal factors are more important than materno-placental ones in determining amniotic fluid SLI. Serial dilutions of amniotic fluid showed parallelism with standard SRIF. When concentrates of pooled amniotic fluid were chromatographed on Sephadex G-25 columns, all SLI eluted in the void volume ahead of SRIF even after treatment with 8 M urea and dithiothreitol. This “big” SLI incubated in amniotic fluid showed 100% stability over 24 h at 37°C, whereas SRIF was rapidly inactivated (t½ ≅ 7 min). Extracts of placenta and fetal membranes contained no SLI, but small amounts (6-20% of total amniotic fluid SLI) were found in cells from fresh fluid. Radioimmunoassay of SLI in extracts of seven paired cord arterial and venous plasma samples showed no arteriovenous gradient consistent with fetal origin of cord blood SLI. It is concluded that (a) amniotic fluid contains SLI which is of fetal origin and (b) normal levels vary with gestational age. The SLI has a higher molecular weight (≥5,000) and is more stable in amniotic fluid than SRIF.

Authors

David Fitz-Patrick, Yogesh C. Patel

Altered Metabolism (In Vivo and In Vitro) of Plasma Lipoproteins after Selective Chemical Modification of Lysine Residues of the Apoproteins

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Chemical modification of lysine residues by acetoacetylation of the apoproteins of iodinated canine and human low density lipoproteins (LDL) and canine high density lipoproteins (HDL) resulted in a marked acceleration in the rate of removal of these lipoproteins from the plasma after intravenous injection into dogs. Clearance of the lipoproteins from the plasma correlated with their rapid appearance in the liver. Acetoacetylated canine 125I-LDL (30-60% of the lysine residues modified) were essentially completely removed from the plasma within an hour, and > 75% of the activity cleared within 5 min. Reversal of the acetoacetylation of the lysine residues of the LDL restored to these lipoproteins a rate of clearance essentially identical to that of control LDL. Identical results were obtained with modified human LDL injected into dogs. At 10 min, when ≅ 90% of the acetoacetylated human 125I-LDL had been removed from the plasma, 90% of the total injected activity could be accounted for in the liver. Furthermore, it was possible to demonstrate an enhancement in uptake and degradation of acetoacetylated LDL by canine peritoneal macrophages in vitro. The mechanism(s) responsible for the enhanced removal of the LDL and HDL in vivo and in vitro remains to be determined. By contrast, however, acetoacetylation of canine 125I-apoE HDLc did not accelerate their rate of removal from the plasma but, in fact, retarded their clearance. Control (native) apoE HDLc were removed from the plasma (64% within 20 min) and rapidly appeared in the liver (39% at 20 min). At the same time point, only 45% of the acetoacetylated apoE HDLc were cleared from the plasma and <10% appeared in the liver. Acetoacetylation of the apoE HDLc did not enhance their uptake or degradation by macrophages. The rapid clearance from the plasma of the native apoE HDLc in normal and hypercholesterolemic dogs suggests that the liver may be a normal site for the removal of the cholesteryl ester-rich apoE HDLc. The retardation in removal after acetoacetylation of apoE HDLc indicates that the uptake process may be mediated by a lysine-dependent recognition system.

Authors

Robert W. Mahley, Thomas L. Innerarity, Karl H. Weisgraber, Suk Y. Oh

Heterogeneity of DNA fragments associated with the sickle-globin gene.

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Abstract

We have examined the genetic polymorphism previously reported to be associated with the sickle-cell (beta s) gene. The polymorphism involves an alteration of the DNA sequence 3' to the beta-globin gene as detected with the restriction endonuclease, Hpa I. In normal individuals, the beta-globin gene is contained within a DNA fragment of 7.6 kilobases (kb), whereas 87% of individuals with sickle-cell anemia have been reported to have the beta s-gene associated with a 13.0-kb Hpa I fragment. We have studied this polymorphism in 31 New York Black individuals homozygous for sickle-cell anemia to ascertain its genetic and biochemical significance and to evaluate its potential use in the prenatal diagnosis of sickle-cell disease. Our results show only a 58% association of the beta s-gene and the 13.0-kb Hpa I fragment, as well as the presence of additional variants involving the Hpa I site. In addition, the 13.0-kb fragment is also found associated with the beta c- and beta A-genes. Thus, the Hpa I polymorphism probably represents a change in DNA not specifically associated with the beta s-gene, and appears to antedate the beta s-and beta c-mutations.

Authors

J Feldenzer, J G Mears, A L Burns, C Natta, A Bank

Elevated Cholesterol and Bile Acid Synthesis in a Young Patient with Homozygous Familial Hypercholesterolemia

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Abstract

Cholesterol balance studies were carried out twice in a young male patient with homozygous familial hypercholesterolemia. At 13 mo, cholesterol balance in this patient averaged 31.3 mg/kg per d, and bile acid excretion was 12.0 mg/kg per d; at 3 yr, results were similar, 27.3 and 15.5 mg/kg per d for cholesterol balance and bile acids, respectively. A normal boy of 3 yr was also studied for comparison with the second study in our patient. Cholesterol balance and bile acid outputs in the normal child were 11.5 and 3.3 mg/kg per d, respectively. Thus, in comparison with the normal child, the patient with homozygous familial hypercholesterolemia had a marked increase in synthesis of cholesterol and bile acids. Although synthesis of bile acids was high in this patient, the fraction of newly synthesized cholesterol converted into bile acids (40-56%) was in the normal range; this suggests that the enhanced output of bile acids was secondary to an increased synthesis of cholesterol and not to malabsorption of bile acids, which likely would have produced a higher fractional conversion. Although our patient has been studied at a younger age than any reported in the literature, two similar children 5 and 6 yr of age were also observed to have elevated cholesterol synthesis. This finding contrasts with those in older children with the homozygous as well as heterozygous forms of this disease who appear to have normal synthesis of cholesterol and bile acids. Therefore, increased synthesis of cholesterol seems to be characteristic of early homozygous familial hypercholesterolemia, and may be a manifestation of a loss of feedback inhibition of cholesterol synthesis secondary to an absence of specific cell-surface receptors for low density lipoproteins. However, as children with this disease grow older, other mechanisms may come into play to restore cholesterol synthesis to normal levels.

Authors

Kathleen B. Schwarz, Joseph Witztum, Gustav Schonfeld, Scott M. Grundy, William E. Connor

Human Plasma Protein C: ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTIVATION BY α-THROMBIN

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Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity.

Authors

Walter Kisiel

Relationship between alveolar PO2 and the rate of p-nitroanisole O-demethylation by the cytochrome P-450 pathway in isolated rabbit lungs.

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The relationship between alveolar PO2 and the rate of O-demethylation of p-nitroanisole, a model substrate for cytochrome P-450 -linked mixed-function oxidation, was evaluated in the isolated rabbit lung perfused with Krebs-Ringer bicarbonate buffer. The appearance of the product, p-nitrophenol, in the pulmonary perfusate was measured spectrophotometrically, The PO2 of the ventilating gas was varied with an accurate gas mixing pump and measured with an electrochemical O2 analyzer. In control lungs ventilated with 5% CO2 in air, the rate of p-nitrophenol production was approximately equal to 3.1 +/- 0.04 (mean +/- SE; n = 9) mumol/h per g dry wt. p-Nitrophenol production was unaltered when O2 in the ventilating gas was decreased to 1%, but it was depressed reversibly when alveolar O2 WAS 0.1% OR LESS AND WAS ABOLISHED DURING VENTILATION WITH 0.005% O2. The rate of the reaction was inhibited by 50% when alveolar PO2 was 0.3 mm Hg representing and intracellular [O2] OF approximately equal to muM. In the presence of metyrapone (0.1--1 mM), an inhibitor of cytochrome P-450-dependent reactions, p-nitrophenol production was 0.07--0.17 mumol/h per g dry wt. Ventilation of lungs with varying CO concentration in 20% O2 resulted in 50% inhibition of p-nitrophenol production when CO concentration was 10% (CO/O2 = 0.5). These results indicated that O-demethylation of p-nitroanisole by the lung is a cytochrome P-450-dependent reaction and that its rate is not affected until alveolar PO2 is less than 1 mm Hg.

Authors

A B Fisher, N Itakura, C Dodia, R G Thurman

Mechanism of Action of Colchicine in Acute Urate Crystal-Induced Arthritis

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Phagocytosis of urate crystals by human or rabbit neutrophils induces the synthesis and release of a glycoprotein, the crystal-induced chemotactic factor (CCF), which is chemotactically active both in vitro and in vivo. It has been proposed that CCF is a prime mediator of the acute gouty attack. Colchicine has been shown to decrease the production and release of this factor in vitro. In these studies, colchicine, at nonleukopenic doses, is shown to abrogate the acute arthritis induced by monosodium urate crystals in rabbits, but to have no effect upon the arthritis induced by the injection of the purified cell-derived chemotactic factor. Serum colchicine levels were 0.48-0.58 μM at 30 min and 0.12-0.3 μM at 90 min after intravenous injection of 0.2 mg/kg colchicine. Peripheral blood polymorphonuclear leukocytes obtained from colchicine-treated animals migrated normally towards a chemotactic stimulus but failed to produce CCF after phagocytosis of monosodium urate crystals. The dialyzed synovial fluid from rabbits injected with microcrystalline sodium urate contained chemotactic activity that was not present when animals were also given intravenous colchicine or injected intra-articularly with the chemotactic factor formyl-methionyl-leucyl-phenylalanine. Furthermore, the synovial fluid from rabbits injected with microcrystalline sodium urate significantly decreased 125I-CCF binding to neutrophils. The binding of 125I-CCF to its neutrophil receptor was not significantly reduced by the synovial fluid of colchicine-treated rabbits nor by the synovial fluid of control rabbits injected with the chemotactic factor formyl-methionyl-leucyl-phenylalanine. Colchicine (10 and 0.1 μM) was shown to have no effect upon the binding of 125I-CCF to its cell receptor.

Authors

Isaias Spilberg, Brian Mandell, Jagdish Mehta, Louis Simchowitz, David Rosenberg

Cross-linking of fibronectin to collagen by blood coagulation Factor XIIIa.

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Abstract

Soluble fibronectin is found in body fluids and media of adherent cultured cells and binds to fibrin and collagen. Insoluble fibronectin is found in tissue stroma and in extracellular matrices of cultured cells. Fibronectin is a substrate for Factor XIIIa (plasma transglutaminase) and can be cross-linked by Factor XIIIa to itself and the the alpha-chain of fibrin. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to investigate Factor XIIIa-mediated crosslinking of fibronectin to collagen. At O degrees or 37 degrees C, fibronectin could be cross-linked to iodinated cyanogen bromide fragment 7 of the alpha 1(I) chain. At 22 degrees or 37 degrees C, fibronectin could be cross-linked to isolated alpha 1(I) chains of type I collagen. Fibronectin could also be crosslinked to types I and III collagen, but only at 37 degrees C. alpha 1(I)-CB7, alpha 1(I) collagen chains, type I collagen, type III collagen, and fibrin all blocked cross-linking between 125I-alpha 1 (I)-CB7 and fibronectin. alpha 1(I)-CB7 blocked cross-linking between fibronectin and fibrin. These results indicate that the determinants of fibronectin-fibrin and fibronectin-collagen binding and cross-linking are similar. Cross-linking of fibronectin to collagen likely occurs in vivo and may be important for normal wound healing, collagen fibrillogenesis, and embryogenesis.

Authors

D F Mosher, P E Schad

Mechanism of synergistic cell killing when methotrexate precedes cytosine arabinoside: study of L1210 and human leukemic cells.

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Abstract

Synergistic killing of L1210 cells occurs when methotrexate (MTX) is administered just before 1-beta-D-arabinofuranosylcytosine (Ara-C). This pehnomenon is dependent upon both the dose and time of exposure to MTX. Such increased killing of cells can be explained by the enhanced intracellular accumulation of Ara-C in cells exposed to MTX. This enhancement of Ara-C entry into cells was only observed when the dose of MTX was high enough (1, 10, and 100 muM) to result in free intracellular nondihydrofolate reductase-bound MTX. At the highest doses of MTX (10 and 100 muM) Ara-C triphosphate was increased eightfold and deoxycytidine triphosphate was decreased by 50%. Therefore, the maximum synergistic cell kill when MTX precedes Ara-C may be the consequence of greater inhibition of DNA polymerase by th;e increased Ara-C triphosphate in the presence of the decreasing natural substrate of this enzyme, deoxycytidine triphosphate. Enhanced Ara-C accumulation after administration of MTX was also observed in human acute myelogenous leukemia cells.

Authors

E Cadman, F Eiferman

Radioimmunochemical quantitation of human adenosine deaminase.

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Abstract

Markedly reduced or absent adenosine deaminase activity in man is associated with an autosomal recesive form of severe conbined immunodeficiency disease. To further define the genetic nature of this enzyme defect, we have quantitated immunologically active adenosine deaminase (CRM) in the hemolysate of homozygous deficient patients and their heterozygous parents. A highly specific radioimmunoassay was developed capable of detecting 0.05% of normal erythrocyte adenosine deaminase. Hemolysates from nine heterozygotes (five families) showed a wide range in CRM (32--100% of normal) and variable absolute specific activities with several being at least 1 SD BELOW THE NORMAL MEAN. Hemolysates from four unrelated patients showed less than 0.09% adenosine deaminase activity with CRM ranging from less than 0.06 to 5.6% of the normal mean. In conclusion, heterozygote and homozygote hemolysates from five of the eight families analyzed revealed variable levels of CRM suggesting heterogeneous genetic alteration or expression of the silent or defective allele(s) of adenosine deaminase.

Authors

P E Daddona, M A Frohman, W N Kelley

Anaphylatoxin-induced histamine release with human leukocytes: studies of C3a leukocyte binding and histamine release.

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Abstract

Purified human C3a and synthetic COOH-terminal peptides of C3a, i.e., a pentapeptide, Leu-Gly-Leu-Ala-Arg (5R), and an octapeptide, Ala-Ala-Ala-Leu-Gly-Leu-Ala-Arg (8R) induced histamine release from human basophil granulocytes. On a molar basis, 5R was one-tenth and 8R was one-fifth as active as C3a in causing histamine release. It was found that 125I-C3a binds to whole leukocytes, interacting with both mononuclear cells and neutrophils and the binding was inhibited by preincubation of cells with unlabeled C3a, but not by C5a. 5R and 8R also inhibited the binding of 125I-C3a to the cells. However, on a molar basis, 2,000 times more 8R or 6,000 times more 5R is required for 50% inhibition of 125I-C3a binding as compared with native C3a. Autoradiography of cells using 125I-C3a and 125I-C5a showed preferential binding of 125I-C3a to eosinophils and basophils, whereas 125I-C5a binds primarily to neutrophils and eosinophils and to a lesser extent to basophils. The preferential binding of C3a and C5a to different cell types may herald significance related to their physiological functions.

Authors

M M Glovsky, T E Hugli, T Ishizaka, L M Lichtenstein, B W Erickson

Prostaglandin E inhibition of T-lymphocyte colony formation: a possible mechanism of monocyte modulation of clonal expansion.

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Prostaglandin and monocyte modulation of a T-lymphocyte cell capable of undergoing clonal expansion was studied. Circulating human mononuclear cells were isolated by density centrifugation. After 24 h in culture with phytohemagglutinin present, the cells were mixed with 0.3% agar and overlayed onto a 0.5% agar layer that contained media and phytohemagglutinin. At day 6, colonies that contained greater than 50 cells were counted. These colonies represented clonal prolifertion of a phytohemagglutinin-responsive T-lymphocyte precursor. This responder cell accounted for less than 0.3% of the starting cell population. Colonies were comprised of cells which, when isolated, formed E rosettes. These colony cells could be shown to have helper or suppressor function as measured by their ability to promote or inhibit immunoglobulin synthesis. By these latter criteria the colony cells were considered to be mature T lymphocytes. The addition of prostaglandin E to the cultures demonstrated a linear, r = 0.82, dose-dependent inhibition of colony formation with a 50% point of inhibition (I50) = 0.18 muM. Low numbers of normal monocytes when added to the cultures mimicked the effect of synthetic prostaglandin E2. A highly significant correlation could be shown for endogenous prostaglandin E levels and colony counts. It appears that monocytes through their synthesis of prostaglandin E2 can restrict the clonal expansion of a circulating T-lymphocyte precursor.

Authors

R S Bockman, M Rothschild

Characterization of a distinct nuclear acidic protein antigen (MA) and clinical findings in systemic lupus erythematosus patients with MA antibodies.

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Circulating antibodies against certain nuclear acidic protein antigens have been shown to have diagnostic and prognostic importance in connective tissue disease. We describe a new precipitin system found in the sera of patients with systemic lupus erythematosus. The antigen, called MA, was prepared from calf thymus nuclei, and was shown to be distinct from other nuclear acidic protein antigens by physicochemical and immunologic techniques. MA antibodies were detected in the serum of 12 of 66 lupus patients and in none of 554 sera from normal controls or patients with other rheumatic diseases. Lupus patients having MA antibodies had more severe disease than did lupus patients with Sm or native DNA antibodies, manifested by recalcitrant skin rashes and a significantly greater incidence of hypocomplementemia, serious renal disease, hypertension, hepatosplenomegaly, lymphadenopathy, and neurological disease (P values range from 0.025 to 0.005). The presence of circulating MA antigen was demonstrated in three lupus patients immediately before a flare of nephritis. These data suggest that MA is a nuclear acidic protein antigen that may identify a subset of lupus patients with very severe disease. The presence of the antigen in the circulation before clinical flares suggests a possible biologic role for the MA system in an immune complex nephritis.

Authors

D M Winn, J F Wolfe, D Harmon, G C Sharp

Receptor-mediated binding and internalization of leukocyte elastase by alveolar macrophages in vitro.

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Radioiodinated leukocyte elastase was found to bind rapidly and specifically to alveolar macrophages in vitro. In contrast to the binding of pancreatic and bacterial proteases, leukocyte elastase binding did not require the presence of alpha 2 macroglobulin. The binding was inhibited by an excess of unlabeled enzyme and was saturable by increasing elastase concentrations. Leukocyte elastase binding thus met criteria for receptor-mediated binding, with and estimated association constant of 4.97 x 10(5) M-1 and an estimated total of 640 x 10(6) binding sites/cell. It differed from the previously described binding of lysosomal glycosidases to macrophages in that it was insensitive to trypsin pretreatment, did not require calcium ions, and was not inhibited by yeast mannan. High-resolution autoradiography indicated that the cell-associated radiolabeled leukocyte elastase was rapidly incorporated into phagolysosomes. Macrophage binding may have a role in clearance of leukocyte elastase from tissue sites where alpha 2 macroglobulin is absent or present in low concentration. Thus, enzyme uptake by alveolar macrophages may be an important factor in the amelioration of lung tissue injury by extracellular leukocyte elastase.

Authors

E J Campbell, R R White, R M Senior, R J Rodriguez, C Kuhn

Regulation of Sodium and Potassium Transport in Phytohemagglutinin-Stimulated Human Blood Lymphocytes

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Abstract

Phytohemagglutinin (PHA) or concanavalin A treatment of lymphocytes causes an increase in membrane permeability so that the leak rates of Na and K increase 1.5- to 2-fold. Active Na and K transport increase proportionately in response to the increased membrane permeability. We have examined the role of lymphocyte Na concentration in sustaining the increased Na and K transport observed after PHA treatment. Cell Na concentration increases from 14.8 to 20.5 mmol/liter cell water in PHA-treated lymphocytes (P < 0.001). Four lines of evidence suggest that the 5-6 mmol/liter cell water increase in lymphocyte Na accounts for the increase in active Na and K transport in mitogen-treated lymphocytes. First, PHA does not increase directly the maximal Na, K-ATPase activity of isolated lymphocyte membrane vesicles. Second, when the Na concentration is increased by 6 mmol/liter cell water in unstimulated lymphocytes, Na and K transport increase nearly twofold. Third, the cell Na concentration (15 mmol/liter cell water) is near the Km for Na activation of the Na, K-ATPase in lymphocyte membranes. The ATPase activity thus, is capable of increasing as the cell Na rises above normal. Fourth, if lymphocytes are incubated in a medium containing a low Na concentration, K transport does not maintain the internal K concentration and the fall in cell K is accentuated in PHA-treated lymphocytes. These studies indicate that the adaptive acceleration of Na and K transport in mitogen-treated lymphocytes is mediated by a small increase in cell Na.

Authors

George B. Segel, William Simon, Marshall A. Lichtman

Comparison of spontaneous and experimentally induced canine prostatic hyperplasia.

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Abstract

Spontaneous prostatic hyperplasia in the beagle appears to progress with age from a glandular to a cystic histological appearance. Prostatic hyperplasia can be induced in young beagles with intact testes by treatment for 4 mo with either dihydrotestosterone or 5 alpha-androstane-3 alpha, 17 beta-diol, alone, or with either of these steroids in combination with 17 beta-estradiol. In contrast, the induction of prostatic hyperplasia in young castrated beagles, in which the gland had been allowed to involute for 1 mo, requires the administration of both 17 beta-estradiol and either 5 alpha-androstane-3 alpha, 17 beta-diol or dihydrotestosterone. Testosterone and 17 beta-estradiol, either singly or in combination, did not produce the hyperplastic condition in intact or castrated beagles. The experimentally induced prostatic hyperplasia is identical in pathology to the glandular hyperplasia that occurs naturally in the aging dog with intact testes. However, cystic hyperplasia was not produced by any of the treatments tested in young animals.

Authors

D P DeKlerk, D S Coffey, L L Ewing, I R McDermott, W G Reiner, C H Robinson, W W Scott, J D Strandberg, P Talalay, P C Walsh, L G Wheaton, B R Zirkin

Excretion and metabolism of catecholamines by the isolated perfused rat kidney.

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Abstract

The excretion and metabolism of labeled epinephrine and norepinephrine by the isolated, perfused rat kidney were studied. The excretion of both catecholamines significantly exceeded the amount filtered, thus providing direct evidence of net tubular secretion. Renal clearance of epinephrine was significantly greater than that of norepinephrine. Tubular secretion was a linear function of the concentration of unbound catecholamine in the medium with no demonstrable tubular maximum at the concentrations studied. The isolated kidney removed catecholamines from the medium by metabolism as well as excretion in the urine. O-Methylation was the major metabolic route and O-methylated metabolites were rapidly excreted and concentrated in urine. Preferential excretion and metabolism of epinephrine were confirmed in double-label experiments in which [14C]epinephrine and [3H]norepinephrine were perfused together. The ratio of 14C:3H in urine exceeded that in perfusion medium for total radioactivity as well as for catecholamines and O-methylated amines. The present study thus provides direct evidence for (a) net tubular secretion of epinephrine and norepinephrine with a direct relationship between secretion and medium concentration; (b) significant renal metabolism of both epinephrine and norepinephrine with O-methylation as the major metabolic route; and (c) preferential excretion and metabolism of epinephrine.

Authors

P Silva, L Landsberg, A Besarab

Genetic control of the eighth component of complement.

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Abstract

Using isoelectric focusing in polyacrylamide gel and a hemolytic assay for development of patterns, extensive, structural polymorphism in human C8 has been delineated. Two alleles, C8A and C8B, have been identified in orientals, with gene frequencies of 0.655 and 0.345. In blacks, what appears to be a third common allele was found, so that frequencies were 0.692, 0.259, and 0.049 for C8A, C8B, and C8A1. In whites, C8A1 was rare with a frequency of 0.003, and frequencies for C8A and C8B were 0.649 and 0.349. Inheritance was autosomal codominant in family studies and the distribution of types in random unrelated populations fit the Hardy-Weinberg equilibrium in all groups. C8 allotypes have been determined for two previously studied families, each with a homozygous C8-deficient propositus. This study suggests that C8 deficiency is a silent or null allele of the C8 structural locus, and that half normal levels of C8 cannot be used as a single criterion for the establishment of heterozygous C8 deficiency. C8 allotypes, as well as 18 other autosomal markers, were also determined for 24 families. The C8 structural locus is not closely linked to these markers, including the human histocompatibility loci complex.

Authors

D Raum, M A Spence, D Balavitch, S Tideman, A D Merritt, R T Taggart, B H Petersen, N K Day, C A Alper

Deletion of the A gamma-globin gene in G gamma-delta beta-thalassemia.

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Abstract

In an individual homozygous for G gamma-delta beta-thalassemia, a physical alteration in gamma-globin gene organization was detected by restriction enzyme mapping. The data indicated that the absence of A gamma-globin chains resulted from extension of the DNA deletion from the delta beta-globin gene region into the gamma-globin gene region rather than a functional disturbance of gamma-gene expression.

Authors

S H Orkin, B P Alter, C Altay