Eosinophils form 50% of cells in the host granulomatous response to Schistosoma mansoni eggs, but their functional role in these granulomas and their relation to egg destruction is unknown. We have studied the course of S. mansoni infection in mice treated with normal rabbit serum (NRS) or depleted of their eosinophils by monospecific anti-eosinophil serum (AES). At 6-wk of infection (after 2 wk of egg deposition) the AES-treated animals were similar to NRS-treated controls with the exception that hepatic granulomas in the AES-treated animals were 50% smaller and devoid of eosinophils. At 8 wk of infection, AES-treated mice had significantly higher mortality, spleen weight, portal pressure, and 80% more eggs retained in their livers. These data suggest that eosinophil depletion delayed egg destruction. We subsequently studied destruction of eggs injected into the pulmonary microvasculature of sensitized mice. 2,000 S. mansoni eggs were intravenously injected into the tail veins of mice treated with NRS, anti-neutrophil serum, AES or ATG (anti-thymocyte globulin); at time intervals the remaining eggs were recovered from the lungs by tissue digestion. Egg recovery from NRS- or anti-neutrophil serum-treated mice began to decrease by day 16 and the percent recovery of eggs at day 24 was 55 and 52%, respectively. In contrast, animals treated with AES had smaller lung granulomas that were devoid of eosinophils and a marked delay of egg destruction was seen. It took until day 44 for 50% of the eggs to be destroyed. In ATG-treated animals smaller granulomas were seen that had diminished lymphocytes and also 75% less eosinophils. ATG treatment apparently slowed egg destruction but was not statistically significant. Our data define the role of the eosinophils in destruction of schistosome eggs in vivo and delineates the protective function of these cells within the host granulomatous response.
G. Richard Olds, Adel A. F. Mahmoud
Prostaglandins are important modulators of the action of vasopressin. Others researchers have proposed that vasopressin stimulates prostaglandin synthesis, completing a negative feedback loop and thereby limiting vasopressin's antidiuretic effect. We have re-examined this question, using specific radioimmunoassay and thin-layer radiochromatography to determine prostaglandin synthesis by the toad bladder. Under control conditions, the bladder synthesizes prostaglandin (PG)E2 and thromboxane (TX)B2. There was no evidence for synthesis of PGE1 or PGF2 alpha by radioimmunoassay, or of other prostaglandins by radiochromatography. Furthermore, there was no evidence for metabolism of PGE2 by the bladder. Using a variety of protocols, in isolated epithelial cells as well as intact bladders, we were unable to detect any significant increase in PGE2 or TXB2 synthesis after stimulation with arginine vasopressin (AVP) or deamino-8-D-arginine vasopressin (DDAVP). Arachidonic acid, the specific precursor of prostaglandin synthesis, increased PGE2 synthesis twofold, and significantly inhibited AVP- and DDAVP-stimulated water flow by 60 and 75%, respectively. Naproxen and acetaminophen inhibited prostaglandin synthesis and enhanced water flow in response to AVP and DDAVP (44-54%). Our findings indicate that the toad bladder produces tow prostaglandins, PGE2 and TXB2, and that vasopressin does not alter their rate of synthesis. Because agents such as acetaminophen and naproxen inhibit prostaglandin synthesis and enhance vasopressin- and DDAVP-stimulated water flow, we suggest that it is the inhibitory effect of these agents on the hormone-independent rate of prostaglandin synthesis that is responsible for their enhancement of water flow. Furthermore, because AVP appears to increase prostaglandin synthesis by the intact kidney, we suggest that cells other than those of the collecting tubule are responsible for the increased prostaglandin production.
J E Bisordi, D Schlondorff, R M Hays
These studies examine the inhibitory effects of arsenate on the transport of sodium, phosphate, glucose, and para-aminohippurate (PAH) as well as oxidative metabolism by proximal convoluted tubules from the rabbit kidney. Transport rates were measured with radioisotopes in isolated and perfused segments. Metabolic activity was monitored through oxygen-consumption rates and HADH fluorescence in parallel studies in suspensions of cortical tubules. The addition of 1mM arsenate to the perfusate reduced fluid absorption rates from 1.24 +/- 0.17 to 0.66 +/- 0.19 nl/nm.min (P < 0.01) and lumen-to-bath phosphate transport from 9.93 +/- 3.47 to 4.25 +/- 1.08 pmol/mm.min (P < 0.01). Similar concentrations of arsenate reduced glucose transport only slightly from 66.1 +/- 6.0 to 56.8 +/-4 4.6 pmol/mm.min (P < 0.05) and had no effect of PAH secretion. Removing phosphate from the perfusate did not affect the net transport of sodium or glucose. In suspensions of tubules, arsenate increased oxygen consumption rates by 20.5 +/- 2.9% and decreased NADH fluorescence by 10.8 +/- 1.5%. These effects on metabolism were concentration dependent and magnified in the presence of ouabain. The data indicate that arsenate's main effect is to uncouple oxidative phosphorylation, and that graded uncoupling of oxidative metabolism causes graded reductions in the net transport of both sodium and phosphate. Glucose transport is inhibited only slightly and PAH secretion is not affected. Thus, partial as opposed to complete inhibition of metabolism reveals that different relationships exist between net sodium transport and the transport of phosphate, glucose, and PAH by the proximal renal tubule.
P C Brazy, R S Balaban, S R Gullans, L J Mandel, V W Dennis
The clearance of 125I-thrombin and diisopropylphosphoryl-125I-thrombin (DIP-thrombin) from the circulation in rabbits was studied. When given either intraarterially or intravenously, DIP-thrombin, which is active-site blocked, was ∼90% cleared from the circulation by 1 min, the time of earliest sampling, indicating a large first-pass effect. DIP-thrombin given intravenously is found predominantly in the lungs, whereas DIP-thrombin injected into the aortic arch is distributed diffusely in approximate proportion to the blood supply. Renal artery, femoral artery, ear artery, left atrium, and portal vein infusions demonstrate that kidney, muscle, ear, heart, and liver, respectively, can remove DIP-thrombin from the circulation. These data imply that the clearance of DIP-thrombin is not a function of a specific organ but of the vascular bed per se. The clearance of DIP-thrombin was reversible since injection of 0.5 mg of unlabeled DIP-thrombin 10 min after the injection of a tracer dose of DIP-125I-thrombin resulted in the rapid reappearance of the DIP-125I-thrombin into the circulation. In addition, the clearance of DIP-thrombin was saturable, i.e., clearance of DIP-125I-thrombin was inhibited by unlabeled DIP-thrombin in a dose-dependent fashion. In vivo Scatchard analysis of the saturation of the clearance process demonstrated that DIP-thrombin can be removed by binding to high-affinity binding sites, since dissociation constants (KD) of 10 and 13 nM were obtained for human and bovine DIP-thrombin, respectively.
Pete Lollar, Whyte G. Owen
We studied the effect of cholecystokinin (CCK) and the octapeptide of cholecystokinin (OP-CCK) on the feline gallbladder and sphinecter of Oddi. Both CCK caused a dose-dependent gallbladder contraction and sphincter of Oddi relaxation. The half-maximal responses of the sphincter of Oddi were 6 ng/kg for OP-CCK and 0.15 Ivy-dog U/kg for CCK, which were lower than those of the gallbladder with 28 ng/kg and 0.32 Ivy-dog U/kg, respectively. The effect of OP-CCK on the gallbladder was partially blocked by tetrodotoxin (P < 0.02), hexamethonium alone (P < 0.05), or a combination of hexamethonium and atropine (P < 0.01). The gallbladder response to CCK was not blocked by either atropine alone (P < 0.60) or adrenergic antagonists (P > 0.40). The sphincter of Oddi response to OP-CCK was blocked by tetrodotoxin (P < 0.001) but it was not blocked by cholinergic (P < 0.20) or adrenergic antagonists (P < 0.60). After complete denervation with tetrodotoxin, OP-CCK caused sphincter of Oddi contraction. These findings indicate that there are two excitatory receptors for CCK in the gallbladder, one at the cholinergic neurons and the other at the level of the gallbladder muscle. There are also two receptors for CCK in the sphincter of Oddi, one that is inhibitory, and present at the noncholinergic, nonadrenergic neurons, and the other, excitatory, at the circular muscle.
J Behar, P Biancani
This report describes the development and first applications of a sensitive and specific double antibody radioimmunoassay for human apoplipoprotein E (apoE). ApoE was purified from the very low density lipoproteins of hypertriglyceridemic patients by heparin-agarose affinity chromatography, DEAE-cellulose chromatography, and preparative polyacrylamide gel electrophoresis. The purified apoprotein had an amino acid composition characteristic of apoE and resulted in the production of monospecific antisera when injected into rabbits. The radioimmunoassay, which was carried out in the presence of 5 mM sodium decyl sulfate, had a working range of 0.8-12 ng. The withinassay coefficient of variation was 9% and the coefficient of variation for systematic between-assay variability was 3%. Prior delipidation of samples with organic solvents did not alter their immunoreactivity. In 26 normal volunteers, the mean plasma apoE concentration was 36 +/- 13 microgram/ml. Hyperlipidemic patients (n = 68) had higher mean apoE levels. A single patient with type III hyperlipoproteinemia had a plasma apoE level of 664 microgram/ml. The plasma apoE level was independently related to plasma cholesterol and triglyceride levels in a population of 108 normal and nonchylomicronemic hyperlipidemic patients. The multiple correlation coefficient for this relationship was 0.73. Thus, variation in plasma cholesterol and triglyceride concentrations described 53% of the variation in apoE concentrations in this population. The lipoprotein distribution of apoE was investigated by agarose column chromatography and ultracentrifugation of plasma. Agarose column chromatography demonstrated that all or nearly all plasma apoE is associated with lipoproteins. In plasma from normal volunteers and hypercholesterolemic patients, apoE was found in two discrete lipoprotein classes: very low density lipoproteins and a set of lipoprotein particles with size and density characteristics similar to HDL2. In hypertriglyceridemic patients, nearly all apoE was associated with the triglyceride-rich lipoproteins.
C B Blum, L Aron, R Sciacca
The effects of thromboxane B2 and the stable prostaglandin endoperoxide analogs (15Z)-hydroxy - 9α - 11α - (epoxymethano)prosta - 5Z,13E - dienoic acid (U44069) and (15Z)-hydroxy -11α,9α-(epoxymethano) prosta-5Z,13E-dienoic acid (U46619) were tested on water flow across the toad urinary bladder. In the presence of indomethacin or meclofenamic acid, inhibitors of prostaglandin and thromboxane A2 synthesis, thromboxane B2 stimulated water flow in a dose-dependent manner. U44069 (1 μM) stimulated water flow from 3.6±0.8 to 12.4±1.2 mg/min per 10 cm2 hemibladder surface area, while U46619 (1 μM) stimulated water flow from 2.8±1.0 to 21.8±2.0 mg/min per 10 cm2. The prostaglandin endoperoxide/thromboxane A2 antagonist trans- 13-azaprostanoic acid, an inhibitor of vasopressin-stimulated water flow, inhibited thromboxane B2- and U46619-stimulated water flow in a dose-dependent manner. The inactive cis-13-azaprostanoic acid did not inhibit vasopressin-stimulated water flow in untreated hemibladders and had no effect on U46619-stimulated water flow in indomethacin or meclofenamic acid pretreated hemibladders. U46619 (1 μM) enhanced vasopressin-stimulated water flow in indomethacin pretreated hemibladders, producing a significant parallel shift (P < 0.001) in the dose-response relationship to submaximal concentrations of vasopressin (0.1-0.6 mU/ml), while not affecting water flow stimulated by supramaximal concentrations of vasopressin (10 mU/ml). trans-13-Azaprostanoic acid abolished the potentiating effects of U46619 on vasopressin-stimulated water flow. These results show that thromboxane A2-like compounds stimulate water flow in the toad urinary bladder.
Ronald M. Burch, Perry V. Halushka
Monoclonal antibodies were prepared against pyruvate kinase (PyKi; ATP: pyruvate phosphotransferase, EC 126.96.36.199) and used to quantitate PyKi content in L2 lung cells and WI-38 fibroblasts cultivated under hypoxic and normoxic conditions. After 96 h of hypoxic cultivation, PyKi activity was significantly increased in both cell types (L2: normoxia [Po2 = 142 torr], 0.11 +/- 0.01 [SD]; hypoxia [Po2 = 14 torr], 0.25 +/- 0.04 U/microgram DNA, P < 0.01). PyKi content increased proportionately in both cell lines (L2: normoxia, 0.44 +/- 0.13; hypoxia, 0.94 +/- 0.13 microgram enzyme protein/microgram DNA). Specific activity was not significantly different after 96 h (L2: normoxia, 261 +/- 11; hypoxia, 261 +/- 14 U/mg enzyme protein). These results indicate that regulation of glycolysis during chronic hypoxia occurs at the level of enzyme content. Chronic O2 depletion leads to either an increased rate of biosynthesis or a decreased rate of biodegradation of PyKi, causing augmented glycolytic capacity. Monoclonal antibodies provide a highly specific, convenient approach to charcterizing enzymes, as well as quantitating cellular enzyme content.
A J Hance, E D Robin, L M Simon, S Alexander, L A Herzenberg, J Theodore
Previous studies of the biochemistry and physiology of eosinophils have relied upon cells obtained from patients with eosinophilia (EE). It is unknown whether such cells might have been activated or partially exhausted by the pathological state causing eosinophilia. We examined cell surface charge, membrane transport of deoxyglucose, activation of lyso-somal acid phosphatase, and oxidative metabolism to provide a profile to compare EE with purified normal eosinophils (NE) and normal neutrophils.
David A. Bass, William H. Grover, Jon C. Lewis, Pamela Szejda, Lawrence R. DeChatelet, Charles E. McCall
Evidence has been presented suggesting the presence of vitamin D3 3β-glucosiduronate and 1,25-dihydroxyvitamin D3 glucosiduronate in rat bile. To evaluate the role of vitamin D glucosiduronates in calcium and phosphorus homeostasis, we synthesized vitamin D3 3β-glucosiduronate and tested its biological activity in calcium- and vitamin D-deficient rats. After the intravenous administration of vitamin D3 3β-glucosiduronate to rats maintained on a low calcium diet, there was an increase in duodenal calcium transport and an increase in serum calcium. Vitamin D3 3β-glucosiduronate, however, was less active than equimolar amounts of vitamin D3. At doses of less than 0.65-1 nmol per rat, the conjugate exhibited no activity. When vitamin D3 3β-glucosiduronate was administered to vitamin D-deficient rats, 25-hydroxyvitamin D was detected in the serum; the increase in serum 25-hydroxyvitamin D levels was less than that observed after the administration of an equimolar amount of vitamin D3. Vitamin D3 3β-glucosiduronate showed no detectable activity in the induction of calcium binding protein in chick embryonic duodena, a system in which no endogenous steroid β-glucuronidase activity is detectable. These data demonstrate that vitamin D3 3β-glucosiduronate is biologically active in vivo and that the observed activity is due to hydrolysis of the conjugate to vitamin D3. As vitamin D3 3β-glucosiduronate is excreted in the bile of rats, it is possible that this conjugate is reutilized in vivo after hydrolysis to free vitamin D3. These results suggest the existence of a mechanism for reutilization of the biliary products of vitamin D3.
Sreeramulu Nagubandi, Rajiv Kumar, James M. Londowski, R. A. Corradino, Pamela S. Tietz
Dual parameter flow cytometry studies using Coulter volume and cell DNA content were carried out in monodisperse cell suspensions of 64 samples of human lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, and benign lymphoid proliferations. Differences in mean Coulter volume among the lymphomas were due both to the intrinsic differences in mean G1 cell Coulter volume and to the presence of increased fractions of larger S and G2 cells, especially among the large B cell lymphomas. However, the relative contribution of large non-G1 cells to the overall population Coulter volume distribution was a relatively minor one; the presence of cells in S did not increase mean Coulter volume by more than 10%, even in samples with high S fractions. There was a good correlation between mean G1 cell Coulter volume and the log of the fraction of cells in S among the B cell lymphomas (r = 0.55). Evidence is presented that within individual samples, large cells proliferate more rapidly than small cells. This was seen in every case, both in the normal samples and in the lymphomas, and in the T cell lymphomas as well as in the B cell lymphomas. Aneuploidy was detected by flow cytometry in 11 cases; in 7 cases the aneuploid cell component could be analyzed separately from the diploid cell component on the basis of cell Coulter volume differences. The aneuploid components of diploid-aneuploid mixtures had higher S fractions than the diploid components in six of seven cases (0.16 +/- 0.04 [SE] vs. 0.08 +/- 0.02). These findings are considered in relation to the histopathological classification of the lymphomas, and in relation to the concept of clonal selection and clonal evolution of tumors.
S E Shackney, K S Skramstad, R E Cunningham, D J Dugas, T L Lincoln, R J Lukes
To investigate the contribution of branched-chain amino acids as a nitrogen source for alanine in vivo, dogs were infused with l-[15N]leucine, l-[U-14C]leucine, l-[2,3,3,3-2H4]alanine, and d-[6,6-2H2]-glucose. 14C and 15N isotopic equilibrium in plasma leucine, and deuterium enrichment in arterial and femoral plasma glucose and alanine were achieved within 3 h of initiation of the respective isotope infusion in all animals. The average flux of leucine determined by [15N]leucine was 5.4 μmol·kg−1·min−1, whereas using [14C]leucine it was 3.7 μmol·kg−1·min−1. Turnover rates for alanine and glucose were 11.0 and 17.2 μmol·kg−1·min−1, respectively.
E. Ben Galim, K. Hruska, D. M. Bier, D. E. Matthews, M. W. Haymond
In this study we show that patients with primary biliary cirrhosis (PCB) have a marked deficiency in the ability to generate an autologous mixed lymphocyte reaction (AMLR) but have a normal ability to generate an allogeneic mixed lymphocyte reaction (MLR). This deficiency is not due to differences in the time-course of the proliferative response or to an altered response to variable numbers of stimulator cells. The deficiency was consistently found irrespective of the methods used to isolate autologous stimulator cells. Both responder cells and stimulator cells obtained from patients with PBC were similar to normal cells in their ability to generate an MLR in allogeneic normal human serum. In addition, serum from patients with PBC inhibited the ability of normal lymphocytes to generate both the AMLR and MLR to a similar degree, suggesting that the defect of the AMLR in PBC is not due to a serum factor. It has been shown that the responder cell population in the AMLR contains a subpopulation of cells that mediate suppression. Therefore, it is possible that the deficiency of the AMLR may be related to previously described abnormalities of suppressor function in patients with PBC.
S P James, C O Elson, J G Waggoner, E A Jones, W Strober
We used the hybridoma technique to characterize further the platelet glycoprotein abnormality in Glanzmann's thrombasthenia. Spleen cells from Balb/c mice immunized with human platelets were fused to mouse myeloma cell line Sp2/0-Ag14. Hybridoma lines producing a variety of antiplatelet antibodies were isolated by hypoxanthine-aminopterin-thymidine selection and cloned, and purified monoclonal IgG from six lines was prepared. One of these lines, 8aB5-9, produced an antibody, Tab, that binds to a protein on normal but not thrombasthenic platelets. We isolated this protein from Triton X-100 solubilized normal platelet membranes by affinity chromatography on Tab-Sepharose. As determined by SDS polyacrylamide gel electrophoresis, the isolated protein is a complex of glycoproteins IIb and IIIa, because the two subunits comigrate with glycoproteins IIb and IIIa of whole platelets and show identical changes in mobility after disulfide bond reduction. We prepared 125I-Tab to determine the number of glycoprotein IIb-IIIa complexes on normal and thrombasthenic platelets by a direct binding assay. Platelets from 17 normal donors bound 39,000±4,600 (SD) Tab molecules/platelet. Platelets from four patients with thrombasthenia lacked Tab binding sites (<5%). Five obligate and four presumed heterozygotes for thrombasthenia bound 24,500±5,800 Tab molecules/platelet. The platelet alloantigen, PlAl, is not that recognized by Tab, because platelets from three PlAl-negative subjects bound Tab normally. Studies with the Tab antibody have (a) enabled quantitation of the number of glycoprotein IIb-IIIa complexes on normal platelet membranes, (b) demonstrated that thrombasthenic homozygotes lack and heterozygotes have a partial deficiency of this complex, and (c) made possible the isolation of this membrane protein which may be required for normal platelet aggregation and clot retraction.
Rodger P. McEver, Nancy Lewis Baenziger, Philip W. Majerus
The alpha-thalassemia-2 (alpha-thal-2) genotype or mild alpha-thalassemia gene consists of a single structural alpha-globin gene on the chromosome that normally bears two alpha-globin genes. We used blot hybridization to investigate variation in the molecular organization of this genotype and to determine the distributions of these variations in the world population. Two different patterns of gene organization responsible for the alpha-thal-2 genotype were found: the first was the result of a 4.2-kilobase pair deletion involving the normal 5' alpha-globin gene (leftward deletion alpha-thal-2 genotype), and the second probably the result of a crossover deletion of a DNA fragment bridging the two normal alpha-globin genes (rightward deletion alpha-thal-2- genotype). The rightward deletion was found in all 9 Black subjects, all 8 Mediterranean subjects, and 4 of 13 Chinese subjects. The leftward deletion was found in four and the nondeletion alpha-thalassemia lesion was found in five of the nine remaining Chinese subjects. It is likely that these deletions are related to specific DNA sequences that determine DNA recombinational events.
S H Embury, J A Miller, A M Dozy, Y W Kan, V Chan, D Todd
To determine whether the small intestine normally secretes fluid, it would be necessary to reduce or inhibit the greater absorptive processes that would otherwise mask such secretion if present. To do this, we perfused bicarbonate-free solutions in the jejunum of normal subjects, because it has been shown that active absorption from this part of the human small intestine is dependent on luminal bicarbonate. We found that the jejunum did secrete sodium chloride and water when isotonic bicarbonate-free solutions were perfused. Further studies revealed that the sodium secretion was passive, but that chloride was secreted against an electrochemical gradient and that observed chloride flux ratios did not agree with the flux ratios calculated for passive chloride movement. We conclude, therefore, that the normal jejunum actively secretes chloride, but that this is masked by greater absorptive processes when balanced electrolyte solutions are perfused. The rate of this active chloride secretion may be one of the factors that regulate the rate of fluid absorption in the normal human intestine.
G R Davis, C A Santa Ana, S Morawski, J S Fordtran
To study the possible role of the secretion vesicle inligant-receptor interaction, somatostatin binding was measured in islets in the presence of various substances known to promote secretion vesicle migration and fusion with the plasma membrane and insulin release. Rat islets were incubated with glucose, 30 and 300 mg/dl, for 60 min. After inculation, somatostatin binding was measured. In islets preincubated with glucose, 300 mg/dl, somatostatin binding was increased 250% when compared with glucose, 30 mg/dl (P < 0.001). Concomitant with enhanced somatostatin binding, insulin secretion was increased. Galactose, 300 mg/dl, did not stimulate insulin release, and somatostatin binding was unchanged from control levels. The increase in somatostatin binding with glucose was accounted for by a 186% increase in receptor concentration with no change in receptor affinity. Tolbutamide increased somatostatin binding by more than twofold, accompanied by a similar increase in insulin release. Secretion vesicles isolated from the islet exhibited somatostatin binding. We conclude that, first, somatostatin binding is increased concomitantly with the migration and fusion of the secretion vesicle with the plasma membrane and/or the release of insulin; second, enhanced somatostatin binding occurs as a consequence of an increased receptor concentration; and third, augmented somatostatin binding occurring with hormone release may provide a critical constraint in the regulation of secretory events.
P S Mehler, A L Sussman, A Maman, J W Leitner, K E Sussman
The only established role for complement in mediating immunologic renal disease involves elaboration of leukochemotactic factors and neutrophil-dependent glomerular injury. In the passive Heymann nephritis (PHN) model of experimental membranous nephropathy, rats injected with sheep antibody to rat proximal tubular brush border antigen (Fx1A) form subepithelial deposits of sheep IgG and rat complement (C3), and develop heavy proteinuria after 5 d without glomerular inflammatory changes. To study the role of complement in mediating proteinuria in PHN, 16 rats were treated daily with cobra venom factor from before antibody injection to maintain C3 levels at < 10% of pretreatment values and compared to 16 untreated controls. Proteinuria at 5 d was abolished in C3-depleted rats (4 +/- 1, controls 70 +/- 15 mg/d, P < 0.001), although renal deposition of 125I-labeled antibody ws the same in both groups (188 +/- 35 vs. 191 +/- 22 microgram IgG/2 kidneys, P > 0.5). Nephritogenic doses of both the noncomplement-fixing F(ab')2 portion and the gamma 2 subclass of anti-Fx1A IgG produced subepithelial deposits of immunoglobulin without C3, but proteinuria did not occur despite glomerular deposition of up to 70 microgram/2 kidneys of gamma 2. However, glomerular deposition of as little as 60 microgram of gamma 1 produced C3 fixation in vivo and heavy proteinuria. No neutrophil exudate could be detected histologically in PHN from the time of antibody injection through development of proteinuria. Proteinuria in five PHN rats depleted of neutrophils to < 200/mm3 with antineutrophil serum was not reduced compared to six controls with normal neutrophil counts (34 +/- 9.6 vs. 25 +/- 10.4 mg/d, P > 0.5). These results demonstrate that proteinuria in the PHN model of membranous nephropathy is complement-dependent and strongly suggest a neutrophil-independent mechanism. Thus a new role for the complement system in mediating immunologic glomerular injury is identified.
D J Salant, S Belok, M P Madaio, W G Couser
A radioimmunoassay for apolipoprotein E in human blood serum has been developed that measures equally the major polymorphic species of the protein (apolipoproteins E-1, E-2, E-3, and E-4) and the apo E in the dimer of apolipoproteins E and A-II. The assay is specific and yields values for apolipoprotein E in very low density lipoproteins that agree closely with those obtained by a quantitative electrophoretic method. Apolipoprotein E is also present in at least one species of high density lipoprotein, but the content of apolipoprotein E in the lipoprotein fractions of plasma is uncertain owing to dissociation during ultracentrifugation. The concentration of apolipoprotein E is higher in serum of normolipidemic, premenopausal women than in men of comparable age and is a direct function of the serum triglyceride level. Apolipoprotein E levels are increased out of proportion to triglyceride levels in hyperlipidemic patients with familial dysbetalipoproteinemia (homozygotes for lack of apolipoprotein E-3). Heterozygous relatives of homozygotes have significantly higher apolipoprotein E levels in serum than unaffected relatives. The concentration of partially degraded (remnant) triglyceride-rich lipoproteins also appears to be increased in heterozygotes, who comprise about 15% of the population.
R J Havel, L Kotite, J L Vigne, J P Kane, P Tun, N Phillips, G C Chen
Further in vitro studies of hematopoietic regulation were carried out in two patients with polycythemia vera who were also heterozygotes (GdB/GdA) for glucose-6-phosphate-dehydrogenase (G-6-PD). While only G-6-PD type A was detectable in circulating erythrocytes, granulocytes and platelets, cultures of peripheral blood and marrow from one patient revealed a substantial number of G-6-PD type B erythroid burst-forming units (BFU-E) and granulocyte/macrophage colony-forming units. Detailed analysis demonstrated: (a) where detectable, normal BFU-E and granulocyte/macrophage colony-forming units were found with similar frequencies; (b) the same frequencies for normal progenitors characterized cultures of peripheral blood and marrow; (c) inhibition of normal erythroid differentiation between BFU-E and the more mature erythroid colony-forming unit; (d) a decline in the prevalence of normal colony-forming units with time, suggesting that disease progression is associated with further suppression of normal hematopoiesis by products of the abnormal clone.
John W. Adamson, Jack W. Singer, Pat Catalano, Scott Murphy, Nancy Lin, Laura Steinmann, Connie Ernst, Philip J. Fialkow
The purpose of this study was to examine the dynamics of left ventricular ejection in patients with obstructive and nonobstructive hypertrophic cardiomyopathy (HCM). 30 patients with HCM and 29 patients with no evidence of cardiovascular disease were studied during cardiac catheterization. Using a single multisensor catheter, electromagnetically derived ascending aortic flow velocity and high fidelity left ventricular and aortic pressures were recorded during rest (n = 47) and provocative maneuvers (n = 23). Dynamic ventricular emptying during rest was also analyzed with frame-by-frame angiography (n = 46). Left ventricular outflow was independently derived from both flow velocity and angiographic techniques. The HCM patients were subdivided into three groups: (I) intraventricular gradients at rest (n = 9), (II) intraventricular gradients only with provocation (n = 12), and (III) no intraventricular gradients despite provocation (n = 9). During rest, the percentage of the total systolic ejection period during which forward aortic flow existed was as follows (mean +/- 1 SD): group I, 69 +/- 17% (flow), 64 +/- 6% (angio); group II, 63 +/- 14% (flow), 65 +/- 6% (angio); group III, 61 +/- 16% (flow), 62 +/- 4% (angio); control group, 90 +/- 5% (flow), 86 +/- 9% (angio). No significant difference was observed between any of the HCM subgroups, but compared with the control group, ejection was completed much earlier in systole independent of the presence or absence of intraventricular gradients. These results suggest that "outflow obstruction," as traditionally defined by the presence of an abnormal intraventricular pressure gradient and systolic anterior motion of the mitral valve, does not impede left ventricular outflow in HCM.
J P Murgo, B R Alter, J F Dorethy, S A Altobelli, G M McGranahan Jr
To elucidate the significance of β-endorphin in human cerebrospinal fluid (CSF), CSF levels of β-endorphin-like immunoreactivity (β-EP-LI) in various diseases were determined by a specific radioimmunoassay and compared with simultaneously determined ACTH-like immunoreactivity (ACTH-LI) levels in CSF. CSF β-EP-LI and ACTH-LI in the control group, consisting of 5 normal subjects and 19 patients with nonendocrine diseases, were 22.2±1.3 and 14.6±0.4 fmol/ml, respectively. CSF levels of these peptides in patients with schizophrenia (n = 19) and acromegaly (n = 10) were not significantly different from those in the control group. Patients with Cushing's disease (n = 7) had significantly lower CSF β-EP-LI and ACTH-LI levels than those in the control group. Four of them showed a parallel increase in CSF β-EP-LI and CSF ACTH-LI levels after the complete removal of pituitary microadenomas (P < 0.05).
Kazuwa Nakao, Shogo Oki, Issey Tanaka, Kazuko Horii, Yoshikatsu Nakai, Tomoo Furui, Masanori Fukushima, Akio Kuwayama, Naoki Kageyama, Hiroo Imura
Using the in vitro DNA labeling technique of nick translation on purified plasma DNA, we have estimated the plasma DNA concentration in three normal individuals to be 266 +/- 57 ng/ml (mean +/- SD). This was not significantly different in three patients with a chronic inflammatory disease (209 +/- 14 ng/ml) or in five patients with steroid-inactivated systemic lupus erythematosus (SLE) (293 +/- 57 ng/ml). In two untreated, newly diagnosed, active SLE patients, however, the plasma DNA concentration was considerably higher (4,024 and 2,437 ng/ml, respectively). Characterization of these in vitro labeled DNA preparations by neutral sucrose-gradient sedimentation analysis showed a sedimentation coefficient of 6-8S, corresponding to a molecular weight of similar to or approximately 0.2-0.45 x 10(6). No difference was observed between normal subjects or patients. In addition, the relative size uniformity of these DNA molecules might suggest some form of specific protection of the DNA from blood DNAases. Further characterization in terms of buoyant density in cesium chloride did not reveal a difference between normal or SLE plasma and the human (HEp-2 cell) DNA used as marker. Taking into account the limitations of the method, no indication of a possible exogenous origin of the DNA circulating in SLE patients could be found. The physiological or pathophysiological role of this plasma DNA remains to be determined.
L Raptis, H A Menard
Human bronchial airways obtained after surgical resection were maintained in tissue culture for 24-48 h. Incorporation of [3H]- or [14C]-glucosamine, [14C]threonine, or Na2[35S]O4 to the culture media resulted in biosynthesis of two radiolabeled glycoproteins—one filtering in the exclusion volume of Sepharose 2B, and the other filtering with an approximate molecular weight of 400,000. Both fractions had similar elution patterns from DEAE-cellulose anion exchange chromatography. [3H]Glucosamine was incorporated equally into the two fractions.
James H. Shelhamer, Zvi Marom, Michael Kaliner
Phorbol myristate acetate activated in normal human neutrophils a single enzymatic entity that was dormant in unstimulated cells, optimally active at pH 7.0, and capable of oxidizing either NADH or NADPH, producing NAD(P)+ and superoxide (O27). Comparative fluorometric and spectrophotometric measurements supported the stoichiometry NAD(P)H + 20(2) leads to NAD(P)+ + 20(27) + H+. the seemingly considerable NAD(P)+ production at pH 5.5 and 6.0 was due largely to nonenzymatic oxidation of NAD(P)H by chain reactions initiated by HO27 (perhydroxyl radical), the conjugate acid of O27. This artifact, responsible for earlier erroneous assignments of an acid pH optimum for NAD(P)H oxidase, was prevented by including superoxide dismutase in fluorometric assays. NAD(P)H oxidase was more active towards NADPH (Km = 0.15 +/- 0.03 mM) than NADH (Km = 0.68 +/- 0.2 mM). No suggestion that oxidase activity was allosterically regulated by NAD(P)H was seen. Phorbol myristate acetate-induced O27 production was noted to be modulated by pH in intact neutrophils, suggesting that NAD(P)H oxidase is localized in the plasma membrane where its activity may be subject to (auto) regulation by local H+ concentrations.
Y Suzuki, R I Lehrer
A patient with symptoms of easy fatigability, postexercise myalgias, and delayed recovery of muscle strength after activity is described. Skeletal muscle from this patient had <1.0% normal myoadenylate deaminase activity and NH3 was not released from muscle after ischemic exercise. In association with this enzyme deficiency, exercise led to a >90% reduction in muscle content of adenine nucleotides. No inosine monophosphate accumulated after exercise and total purine content of the muscle fell to 21% of control. Repletion of the adenine nucleotide pool in this patient was delayed compared to controls, and ATP content had only returned to 68% of control at 165 min after exercise. These studies demonstrate that disruption of the purine nucleotide cycle as a consequence of myoadenylate deaminase deficiency results in marked alterations in ATP content of muscle, and potentially, these changes in ATP content could account for muscle dysfunction in this patient.
Richard L. Sabina, Judith L. Swain, Bernard M. Patten, Tetsuo Ashizawa, William E. O'Brien, Edward W. Holmes
Treatment of human placenta membranes with dithiothrietol (DTT) followed by N-ethylmaleimide results in a 60% reduction in insulin binding. Treatment with N-ethylmaleimide alone has little effect. The decrease in insulin binding that results from DTT treatment is due to a decrease in affinity for insulin, with little change in total receptor number. DTT has similar effects on receptor solubilized from placenta membranes with Triton X-100, indicating that its effects are not attributable to changes in the arrangement of receptors in the membrane. In contrast to placenta membranes, treatment of liver membranes with DTT does not decrease insulin binding. These results suggest that reduction of a critical disulfide bond in insulin receptors from human placenta converts the receptor to a low affinity form.
S Jacobs, P Cuatrecasas
We examined the effects of K+ and angiotensin II, the major regulators of aldosterone secretion, on phospholipid metabolism during incubation of glomerulosa-rich, adrenal capsules. Addition of increasing amounts of K+ and angiotensin II to the incubation media elicited progressive increases in corticosterone production and capsular concentrations of phosphatidic acid, phosphatidyl-inositol, and polyphosphoinositides. These effects are similar to those previously reported for ACTH in the whole adrenal cortex. A common mechanism, i.e., activation of the phosphatidate-polyphosphoinositide cycle, may be operative in the action of steroidogenic agents in their target tissues.
R V Farese, M A Sabir, R E Larson
To further characterize the IgA deposits found in glomeruli of patients with IgA nephropathy, anaphylactoid purpura nephritis, and systemic lupus erythematosus, renal biopsies from patients with these disorders were stained by immunofluorescence with monoclonal anti-IgA subclass reagents, anti-light chain reagents and anti-J chain. The mesangium and peripheral capillary were brightly stained for IgA1 and were negative for IgA2. IgA1 and, to a lesser extent, IgA2 were contained in tubular casts. Both kappa and lambda light chains were found in all deposits. The intensity of J chain staining correlated with the intensity of IgM and not IgA staining. Biopsies brightly stained for IgA but negative for IgM were negative for J chain. These results indicate that glomerular IgA deposits in these disorders consist predominantly of monomers of IgA1.
M E Conley, M D Cooper, A F Michael
A high incidence of autoantibody against the neutral glycolipid "asialo GM1" was observed in sera from patients with systemic lupus erythematosus (SLE) with neurological disorders, using an immunoflocculation test. The sera from 14 out of 17 cases of SLE with neurological disorders showed antibody activity against asialo GM1 but not against the following glycolipids: asialo GM2 GM1, and galactocerebroside. In another 87 cases of SLE without any history of seizures, as well as 61 cases of other autoimmune diseases (rheumatoid arthritis, progressive systemic sclerosis, mixed connective tissue disease, etc.) and 20 cases of various neurological diseases (epilepsy, multiple sclerosis, etc.), no antibody could be detected. In general, the antibody titer was high several months, even years, before and/or after the seizure, though the titer was low at the time that patients showed definite neurological symptoms. Immunochemical characterization with Sephadex G-200 chromatogrphy and protein A-Sepharose CL-4B affinity column indicated that the antiasialo GM1 was probably an autoantibody belonging to the immunoglobulin G class. The above results suggest that this newly found autoantibody plays a role in the pathogenesis of neurological disorders accompanying SLE.
T Hirano, H Hashimoto, Y Shiokawa, M Iwamori, Y Nagai, M Kasai, Y Ochiai, K Okumura