Effects of Asphyxia on Lung Fluid Balance in Baby Lambs

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Clinical Investigation and the Clinical Investigator: The Past, Present, and Future

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The glucose paradox. Is glucose a substrate for liver metabolism?

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J Katz, J D McGarry

A coagulation pathway on bovine aortic segments leading to generation of Factor Xa and thrombin.

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Previous studies have demonstrated the binding of Factors IX and IXa to cultured bovine aortic endothelial cells. The present study examines the interaction of Factors IX, IXa, and Xa with the luminal surface of calf aortas, shown by microscopic examination to have a continuous layer of endothelium. Radioimmunoassay of Factor IX showed that 74 fmol/10(6) cells of Factor IX could be eluted from freshly prepared aortic segments. Binding of 3H-Factors IX and IXa to aortic segments was saturable, and comparable to binding in previous studies using cultured endothelial cells. Preincubation of aortic segments with 3H-Factor IXa and von Willebrand factor (VWF)/Factor VIII, followed by washing and addition of Factor X, resulted in formation of Factor Xa. The addition of prothrombin to these activation mixtures resulted in formation of thrombin. Exogenous phospholipid and Factor V were not required for Factor X and prothrombin activation on the intact native endothelium. Incubation of 125I-Factor Xa with the vessel segments resulted in most of the tracer being complexed with antithrombin III originally present on the aortic segment (3.8 pmol antithrombin III/10(6) cells). The Factor Xa-antithrombin III complex was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis exclusively in the supernatants. 125I-Factor Xa not complexed with antithrombin III bound specifically to the vessel segment. The time course of binding was biphasic, consisting of an initial more rapid reversible phase followed by a slower irreversible phase. The latter phase correlated with the formation of a covalent complex (Mr, 76,000) between 125I-Factor Xa and a vessel-localized protein presumably distinct from antithrombin III. The activation of prothrombin by vessel-bound Factor Xa was inhibited by anti-bovine Factor V IgG, suggesting that there is interaction of Factor Xa with a Factor V-like molecule provided by the endothelial cell surface. Addition of antibody to antithrombin III prevented formation of Factor Xa-antithrombin III and thrombin-antithrombin III complexes in the supernatant and increased apparent thrombin activity 30-50-fold. These studies demonstrate that freshly obtained vessels with a continuous layer of native endothelium can support activation of Factor X and prothrombin: vessel-bound Factor IXa can activate Factor X in the presence of VWF/Factor VIII. Factor Xa can also bind to the vessel and participate in the activation of prothrombin. The apparent efficiency of prothrombin activation, however, is dampened by the presence of functional antithrombin III on the vessel wall.

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D M Stern, P P Nawroth, W Kisiel, D Handley, M Drillings, J Bartos

Effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of 5-lipoxygenase pathway products by ionophore-activated human neutrophils.

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Exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) have been compared with exogenous arachidonic acid for their capacity to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human neutrophils and for their suitability as parallel substrates in this pathway. The products from specific 14C- or 3H-labeled substrates were isolated by reverse phase high performance liquid chromatography (RP-HPLC) and were identified by elution of radiolabel at the retention times of the appropriate synthetic standards. Each product was also characterized by its ultraviolet (UV) absorption spectrum, and 7-hydroxy-DCHA was defined in addition by analysis of its mass spectrum. The metabolites, 5-hydroxyeicosatetraenoic acid, leukotriene B4 (LTB4), 6-trans-LTB4 diastereoisomers, 5-hydroxyeicosapentaenoic acid, 6-trans-leukotriene B5 diastereoisomers, leukotriene B5 (LTB5), and 7-hydroxy-DCHA were quantitated by integrated UV absorbance during resolution by RP-HPLC. LTB4 and LTB5 were also quantitated by radioimmunoassay of the eluate fractions, and leukotrienes C4 and C5 (LTC4 and LTC5, respectively) were quantitated by radioimmunoassay alone. None of the unlabeled exogenous fatty acids (5-40 micrograms/ml) altered the release of radioactivity from [14C]arachidonic acid-labeled, ionophore-activated neutrophils. The metabolism of 5 and 10 micrograms/ml of exogenous EPA by ionophore-activated, [14C]arachidonic acid-labeled neutrophils not only generated 5-hydroxyeicosapentaenoic acid, 6-trans-LTB5, LTB5, and LTC5, but also stimulated the formation of 5-hydroxyeicosatetraenoic acid, 6-trans-LTB4 diastereoisomers, and LTC4 from membrane-derived arachidonic acid. In contrast, LTB4 production was diminished throughout the EPA dose-response, beginning at 5 micrograms/ml EPA and reaching 50% suppression at 10 micrograms/ml and 84% suppression at 40 micrograms/ml. The selective decrease in extracellular LTB4 concentrations in the presence of EPA was not due to a change in the kinetic appearance of LTB4 or to an increase in conversion to its omega-oxidation metabolites. DCHA was metabolized to 7-hydroxy-DCHA, did not stimulate metabolism of membrane-derived arachidonic acid, did not appreciably inhibit LTB4 formation, and was not a substrate for leukotriene formation. Incremental doses of exogenous arachidonic acid resulted in increased production of 5-hydroxyeicosatetraenoic acid and 6-trans-LTB4 by ionophore-activated, [14C]arachidonic acid-labeled neutrophils without any change in LTB4 production. 5-hydroxyeicosapentaenoic acid and 7-hydroxy DCHA were inactive as chemotactic factors whereas 5-hydroxyeicosatetraenoic acid exhibited 2% of the potency of LBT4. Thus, exogenous DCHA does not appreciably interfere with the metabolism of membrane-derived arachidonic acid by ionophore-activated, [14C]arachidonic acid-labeled neutrophils and is converted only to a monohydroxy derivative. In contrast, exogenous EPA attenuates the generation of LTB4 and is converted to LTB5, which is a weak and partial agonist as compared with LTB4.

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T H Lee, J M Mencia-Huerta, C Shih, E J Corey, R A Lewis, K F Austen

Hepatic messenger ribonucleic acid activity profiles in experimental azotemia in the rat. Relationship to food intake and thyroid function.

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We have studied the hepatic messenger RNA (mRNA) activity profile in chronically azotemic rats and sought to determine whether the observed changes could be mediated either by reduced food intake or diminished thyroid function at the tissue level. mRNA activity profiles were produced by two-dimensional gel electrophoretic separation of radioactively labeled products of an in vitro reticulocyte lysate system which had been programmed by hepatic RNA. Of the approximately 240 translational products identified in this system, seven sequences were consistently altered in azotemia. In pair-fed animals six of these also decreased, but the alterations in three were depressed to a significantly lesser extent in the pair-fed group. Moreover, analysis of covariance suggested that food intake could account for the differences in only one sequence. The possibility that the mRNA activity profile in azotemia could represent the effects of diminished thyroid function was minimized by the finding that the reductions in plasma thyroxine (T4) and triiodothyronine (T3) levels observed were due largely to reduced plasma protein binding, with maintenance of the mean free T4 and free T3 concentrations within the normal range. The changes in only one mRNA sequence could be related to free T3 levels alone. Our findings, therefore, indicate that although diminished food intake and reduced thyroid function may contribute to some of the observed changes in the mRNA activity profiles, the bulk of alterations in azotemia appear to be mediated by other mechanisms. The striking overlap between the sequences affected by azotemia and pair-feeding raises the speculation that altered gene expression in azotemia may reflect an impaired hepatic response at the pretranslational level to metabolic signals associated with food intake.

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W B Kinlaw, H L Schwartz, C N Mariash, C Bingham, F E Carr, J H Oppenheimer

Chronic hypercapnia stimulates proximal bicarbonate reabsorption in the rat.

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The hyperbicarbonatemia of chronic respiratory acidosis might be maintained by a reduction in filtration rate or an enhancement of tubular bicarbonate reabsorption. To investigate this question, 12 Munich-Wistar rats were exposed to a 10% CO2 atmosphere for 6-8 d. Chronic respiratory acidosis developed, with arterial pH 7.30 +/- 0.01, partial pressure of CO2 (pCO2) 80 +/- 2 mmHg, and total CO2 concentration 45 +/- 1 mM. Single nephron glomerular filtration rate was normal (42 +/- 1 nl/min). Chronic hypercapnia caused absolute proximal reabsorption to be significantly stimulated (1,449 +/- 26 pmol/min) as compared with reabsorption previously observed in normal animals (1,075 +/- 74 pmol/min) or in animals subjected to acute hypercapnia (1,200 +/- 59 pmol/min). This is the first demonstration that proximal bicarbonate reabsorption can be stimulated above normal euvolemic values. When eight animals were subsequently allowed to return toward a normocapnic state (arterial pCO2 46 +/- 1 mmHg) over the course of 1-1.5 h, bicarbonate reabsorption was still significantly higher (1,211 +/- 34 pmol/min) than in similarly alkalotic, normocapnic control groups (994 +/- 45 pmol/min). In conclusion, chronic, but not acute, hypercapnia stimulates absolute proximal bicarbonate reabsorption to exceed the level found in normal euvolemic rats.

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M G Cogan

Inhibition of soluble antigen-induced T cell proliferation by warm-reactive antibodies to activated T cells in systemic lupus erythematosus.

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One of the fundamental immunologic characteristics of systemic lupus erythematosus (SLE) is a depressed T cell proliferative response to various specific and nonspecific stimuli. Both intrinsic cellular defect(s) and inhibitory influences of humoral factors, e.g., antilymphocyte autoantibodies or immune complexes, have been postulated to underly this functional abnormality. Because patient serum can induce SLE-like T cell dysfunction in normal cells, an extrinsic mechanism is probably responsible, but the nature and site of action of this humoral activity has not been defined. This laboratory recently described a novel antibody specific for activated T cells in SLE, which raised the possibility that suppression of T cell proliferation by SLE serum involved antibodies directed to surface determinants expressed during the process of activation. In experiments to examine this concept further, relatively warm-reactive antibodies to T cell blasts were found to inhibit strongly the well-characterized T cell response to tetanus toxoid. These antibodies were distinct from conventional cold-reactive IgM antibodies to resting T cells, which exhibited little inhibitory activity. Inhibition involved noncytotoxic effects on early activation events at the level of the responding T cell, which markedly reduced the expression of receptors for interleukin 2. Inhibitory effects on antigen-pulsed macrophages or on T cells already committed to proliferate were not demonstrable. Anti-T blast antibodies were characteristic of active SLE and were detected only occasionally in patients with inactive disease or non-SLE rheumatic disorders. Although the exact antigenic specificity was not identified, considerable evidence was obtained against the presence of antibodies to Ia and certain other surface determinants of functional relevance. Our observations concerning the suppressive effects of anti-T blast antibodies in SLE serum on the T cell response to tetanus toxoid should provide new insight into mechanisms of in vivo T cell dysfunction in this and other immunologic disorders.

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A Yamada, J B Winfield

Activation of the complement system in patients with porphyrias after irradiation in vivo.

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Irradiation of the forearms of two patients with erythropoietic protoporphyria and one patient with porphyria cutanea tarda resulted in an in vivo activation of the complement system, as assessed by diminution of the hemolytic titers of the third component of complement by 23-57%, and of the fifth component of complement (C5) by 19-47%. Such treatment also generated chemotactic activity for human polymorphonuclear cells; the chemotactic activity was stable at 56 degrees C and antigenically related to human C5. On Sephadex G-75 chromatography the chemotactic activity eluted with an apparent molecular weight of 15,000. These in vivo results extend our previous in vitro observation of photoactivation of complement in sera from patients with erythropoietic protoporphyria and porphyria cutanea tarda, and suggest that the complement system may participate in the pathogenesis of cutaneous phototoxicity in these patients.

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H W Lim, M B Poh-Fitzpatrick, I Gigli

Free 1,25-dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease.

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We measured the free concentration of 1,25-dihydroxyvitamin D (1,25[OH]2D) using centrifugal ultrafiltration, and the level of vitamin D-binding protein (DBP) in 24 normal subjects, 17 pregnant subjects, and 25 alcoholic subjects with liver disease. Our objective was to determine whether the increase in total 1,25(OH)2D levels in pregnant women and the reduction in total 1,25(OH)2D levels in subjects with liver disease reflected a true difference in free 1,25(OH)2D levels or whether such differences were due solely to the variations in DBP levels (and thus, the amount of 1,25[OH]2D bound) in these groups. In subjects with liver disease the mean total 1,25(OH)2D concentration (22.6 +/- 12.5 pg/ml) and the mean DBP concentration (188 +/- 105 micrograms/dl) were nearly half the normal values (41.5 +/- 11.5 pg/ml and 404 +/- 124 micrograms/dl, respectively, P less than 0.001), whereas the mean free 1,25(OH)2D level was similar to normal values (209 +/- 91 fg/ml and 174 +/- 46 fg/ml, respectively). In contrast, in pregnant subjects the mean total 1,25(OH)2D level (82 +/- 21 pg/ml) and mean DBP level (576 +/- 128 micrograms/dl) were significantly higher than normal (P less than 0.001). Although the mean percent free 1,25(OH)2D level in pregnant subjects was below normal (0.359 +/- 0.07% vs. 0.424 +/- 0.07%, P less than 0.001), the mean free 1,25(OH)2D level was 69% higher than normal (294 +/- 98 fg/ml vs. 174 +/- 46 fg/ml, P less than 0.001). When data from all three groups were combined, there was a linear correlation between total 1,25(OH)2D and DBP levels but not between DBP and percent free 1,25(OH)2D levels; the increased DBP levels in the pregnant subjects were associated with less of an effect on percent free 1,25(OH)2D than were the reduced DBP levels in the subjects with liver disease. Our data suggest that (a) free 1,25(OH)2D levels appear to be well maintained even in subjects with liver disease and reduced DBP levels, (b) free 1,25(OH)2D levels are increased during pregnancy despite the increase in DBP levels, and (c) free 1,25(OH)2D levels cannot be inferred accurately from measurements of total 1,25(OH)2D and DBP levels alone in subjects with various physiologic and pathophysiologic conditions.

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D D Bikle, E Gee, B Halloran, J G Haddad

Hypocholesterolemic effects of mevinolin in patients with heterozygous familial hypercholesterolemia.

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We have evaluated the hypolipidemic effects of mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis in 13 patients with heterozygous familial hypercholesterolemia (FH). Patients were maintained on a low-cholesterol diet and received sequentially increasing doses of 5, 10, 20, and 40 mg of mevinolin twice daily for a period of 1 mo on each dose. Plasma concentrations of low density lipoprotein cholesterol decreased by 19.8% on the 5 mg twice daily dose (P less than 0.05 vs. base line), 28.4% on 10 mg of mevinolin twice daily (P less than 0.05 vs. 5 mg twice daily), 35% on 20 mg of mevinolin twice daily (P less than 0.05 vs. 10 mg twice daily), and 37.7% on 40 mg of mevinolin twice daily (not statistically different from 20 mg twice daily). Concentrations of high density lipoprotein cholesterol remained stable on all doses of mevinolin whereas plasma triglyceride levels fell significantly on the 20 mg (-30.7%) and 40 mg (-34.3%) twice daily doses of mevinolin. Mevinolin was well tolerated and all patients completed the study period. Side effects during the period of study were limited to transient insomnia and headaches in two patients, transient increases in alkaline phosphatase in three patients, and a modest but sustained increase in alkaline phosphatase in a fourth patient. These results indicate that mevinolin is an effective hypolipidemic agent in patients with heterozygous FH but that the optimal doses in these patients are greater than those previously reported in normal volunteers. If long-term safety can be satisfactorily established, mevinolin offers considerable promise in the therapy of heterozygous FH.

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D R Illingworth, G J Sexton

Adaptation of Na+-H+ exchange in renal microvillus membrane vesicles. Role of dietary protein and uninephrectomy.

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The ablation of renal mass and institution of a high protein diet both lead to renal cortical hypertrophy and increased glomerular filtration rate (GFR). We studied Na+ transport in rat microvillus membrane vesicles isolated from uninephrectomized or sham operated rats fed 6% (low), 24% (standard), or 40% (high) protein diets. The feeding of high protein, as compared with low protein, was associated with a 50% increase in rates of pH-stimulated 22Na+ transport in isolated vesicles from sham and uninephrectomized animals. Values for the standard protein diet were intermediate to values for high and low protein. At each level of dietary protein intake, vesicular Na+ transport was greater in the uninephrectomized than in sham rats. The high protein diet was also associated with increased vesicular 22Na+ flux inhibitable by 1 mM amiloride. Increases in total and amiloride sensitive flux were also noted in the absence of a pH gradient. Conductive Na+ and H+ transport were not altered, nor were sodium-glucose and sodium-alanine cotransport. Kinetic studies revealed evidence for an increased Vmax of Na+-H+ exchange in uninephrectomized animals fed a 40 vs. a 6% protein diet whereas Km was unchanged. Supplements of NaHCO3 in the 40% protein diet, to adjust for an increased rate of net acid excretion, did not prevent the increased rates of Na+-H+ exchange. However, treatment with actinomycin D (0.12 mg/kg) prevented the increased Na+-H+ activity as well as the increased renal mass and GFR noted 24 h after unilateral nephrectomy. Na+-H+ exchange rate was closely correlated with GFR (r = 0.961; P less than 0.005) and renal mass (r = .986; P less than 0.001). These observations provide evidence for modification of the luminal membrane Na+-H+ exchanger in response to changes in dietary protein content and nephron number.

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R C Harris, J L Seifter, B M Brenner

Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells.

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The effect of thrombin on the release of tissue plasminogen activator from endothelial cells was studied in primary cultures of human umbilical vein endothelial cells. Tissue plasminogen activator concentration in conditioned medium was measured by a two-site radioimmunometric assay. The addition of increasing concentrations (0.01 to 10 U/ml) of thrombin to confluent cultures produced a saturable, dose-dependent increase in the rate of release of tissue plasminogen activator. A sixfold increase in tissue plasminogen activator concentration (from 2 to 12 ng/ml) occurred after the addition of 1 U/ml thrombin (8 X 10(-9) M) to cultures containing 5 X 10(4) cells/cm2. Enhanced release was not observed until 6 h after thrombin addition, reached a maximum rate of 1.3 ng/ml per h between 8 and 16 h, and then declined to 0.52 ng/ml per h after 16 h. The 6-h lag period before increased tPA release was reproducible and independent of thrombin concentration. Thrombin inactivated with diisopropylfluorophosphate or hirudin did not induce an increase in tissue plasminogen activator levels. A 50-fold excess of diisopropylfluorophosphate-treated thrombin, which inhibits binding of active thrombin to endothelial cell high affinity binding sites, did not inhibit the thrombin-induced increase. It is concluded that proteolitically active thrombin causes an increase in the rate of release of tissue plasminogen activator from cultured human endothelial cells. The 6-h interval between thrombin treatment and enhanced tissue plasminogen activator release may reflect a delaying mechanism that transiently protects hemostatic plugs from the sudden increase in the local concentration of this fibrinolytic enzyme.

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E G Levin, U Marzec, J Anderson, L A Harker

Relationship of oxygen and glutathione in protection against carbon tetrachloride-induced hepatic microsomal lipid peroxidation and covalent binding in the rat. Rationale for the use of hyperbaric oxygen to treat carbon tetrachloride ingestion.

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CCl4 exerts its toxicity through its metabolites, including the free radicals CCl3. and CCl(3)00.. Oxygen strongly inhibits the hepatic cytochrome P-450-mediated formation of CCl3. from CCl4 and promotes the conversion of CCl3. to CCl(3)00.. Both these free radicals injure the hepatocyte by causing lipid peroxidation and binding covalently to cell structures. A reduced glutathione (GSH)-dependent mechanism can protect the liver microsomal membrane against CCl4-induced damage under aerobic conditions but not under anaerobic conditions (Burk, R.F., K. Patel, and J.M. Lane, 1983, Biochem. J., 215:441-445). Experiments were carried out using rat liver microsomes to examine the effect of O2 tensions found in the liver and of GSH on CCl4-induced covalent binding and lipid peroxidation. An NADPH-supplemented microsomal system was used. CCl4 or 14CCl4 was added to the sealed flask that contained the system, and after 20 min CHCl3 production, thiobarbituric acid-reactive substances (an index of lipid peroxidation), and covalent binding of 14C were measured. O2 tensions of 0, 1, 3, 5, and 21% were studied. Increases in O2 tension caused a fall in CHCl3 production, which indicated that it decreased CCl3.. GSH had no significant effect on CHCl3 production at any O2 tension. Lipid peroxidation and covalent binding of 14C fell progressively as O2 tension was increased from 1 to 21%. The addition of GSH decreased both lipid peroxidation and covalent binding, but did so better at the higher O2 tensions than at the lower ones. These results indicate that low O2 tensions such as are found in the centrilobular areas of the liver favor conversion of CCl4 to free radical products which cannot be detoxified by the GSH-dependent mechanism. They suggest that hyperbaric O2 might decrease free radical formation in the liver in vivo and promote formation of CCl(3)00. from CCl3.. This should result in diminished CCl4-induced lipid peroxidation and liver damage. Rats given CCl4 (2.5 ml/kg) were studied in metabolic chambers. Production of CHCl3 and ethane, the latter an index of lipid peroxidation, were measured. Rats in two atmospheres of 100% O2 produced much less CHCl3 and ethane than rats in air. This strongly suggests that hyperbaric O2 is decreasing free radical formation from CCl4 and/or promoting the formation of CCl(3)00. from CCl3.. These results provide the rationale for the use of hyperbaric O2 in the treatment of CCl4 ingestion.

Authors

R F Burk, J M Lane, K Patel

Volume-independent reductions in glomerular filtration rate in acute chloride-depletion alkalosis in the rat. Evidence for mediation by tubuloglomerular feedback.

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We have recently described reduced superficial nephron glomerular filtration rate (SNGFR) in chloride-depletion alkalosis (CDA) without volume depletion. To elucidate the mechanism of this phenomenon, we studied three degrees of increasing severity of CDA (groups CDA-1, 2, and 3) produced by one or two peritoneal dialyses against 0.15 M NaHCO3 and electrolyte infusions of different Cl and HCO3 content in Sprague-Dawley rats; control rats (CON) were dialyzed against and infused with Ringers-HCO3. Extracellular fluid (ECF) volume was assessed by blood pressure, hematocrit, plasma protein concentration, and 125I-albumin space; none of these variables differed among the four groups. Micropuncture of the latest proximal and earliest distal convolutions was carried out. As CDA intensified from CON to CDA-3 (plasma tCO2 25 +/- 1 to 43 +/- 1 meq/L; P less than 0.01), distally determined SNGFR declined progressively (40.9 +/- 1.7 to 28.3 +/- 1.8 nl/min; P less than 0.01), while in early distal tubule fluid, flow rate (8.6 +/- 0.7 to 3.4 +/- 0.6 nl/min) and Cl concentration (36 +/- 2 to 19 +/- 3 meq/L) decreased and osmolality (110 +/- 5 to 208 +/- 12 mosmol/kg) increased (P less than 0.01), and, in the loop segment, Cl reabsorption decreased progressively (2,009 +/- 112 to 765 +/- 128 peq/min; P less than 0.01). In early distal tubule fluid, Cl concentration correlated positively and osmolality negatively with distally determined SNGFR (P less than 0.05). Proximally determined SNGFRs did not differ among the four groups. Proximal tubule stop-flow pressure responses to increasing rates of orthograde perfusion of the loop segment from 0 to 40 nl/min did not differ between groups CON and CDA-2. We interpret these data to show that reductions in SNGFR in CDA in the rat can occur by tubuloglomerular feedback (TGF) in the absence of differences in ECF volume or of alterations in TGF sensitivity during metabolic alkalosis. Of the proposed signals for TGF sensed by the macula densa, distal tubule fluid osmolality or some related variable is the signal most compatible with our data.

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J H Galla, D N Bonduris, P W Sanders, R G Luke

Tissue-type plasminogen activator increases the binding of glu-plasminogen to clots.

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Porcine tissue-type plasminogen activator (t-PA) increases the binding of 125I-glu-plasminogen to clots made from human plasma or purified fibrinogen in a time and t-PA concentration dependent fashion. The accumulation of plasminogen was faster and greater on noncrosslinked plasma clots than on clots which had been crosslinked by Factor XIIIa. Furthermore, the uptake of plasminogen to crosslinked fibrin clots occurred at a slower rate in the presence of alpha 2-plasmin inhibitor (alpha 2 PI) than in its absence. The kinetics of the uptake of 125I-plasminogen were analyzed using SDS-polyacrylamide gel electrophoresis and radioautography of solubilized plasma clots formed in the presence of t-PA. During the initial phase there was a decrease of clot-bound glu-plasminogen; simultaneously, there was a slight increase in clot-bound glu-plasmin and in plasmin complexed to alpha 2 PI that was crosslinked to alpha-chain polymers of fibrin. This was followed by a marked increase in clot-bound plasminogen having glutamic acid as NH2-terminal (glu-plasminogen) and gluplasmin. t-PA-induced enhancement of glu-plasminogen uptake appears to be mediated by plasmin but does not require the conversion of glu-plasminogen to plasminogen having lysine or methionine as NH2-terminal. The described mechanism assures an adequate supply of clot-bound plasmin, which is the enzyme ultimately involved in the degradation of fibrin.

Authors

C Tran-Thang, E K Kruithof, F Bachmann

Postprandial lipemia. A key for the conversion of high density lipoprotein2 into high density lipoprotein3 by hepatic lipase.

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In this study, we have investigated the effects of alimentary lipemia in 15 normotriglyceridemic individuals on high density lipoproteins2 (HDL2) with respect to structure, composition, and substrate efficacy for hepatic lipase in vitro. In the study subjects, HDL2 levels ranged widely from 4.7 to 151.7 mg/dl plasma. HDL2 were isolated in the postabsorptive (pa) state and in the postprandial (pp) state, i.e., 7 h after ingestion of a standard fatty meal. In going from the pa state to the pp state, HDL2 exhibited higher flotation rates and lower densities due to a decreased proportion of protein (38.7----36.2%) and a higher abundance in phospholipid (32.5----34.9%). There was a variable increase in triglyceride at the expense of cholesteryl esters; this increase was correlated positively with the magnitude of pp lipemia (r = 0.69, P less than 0.01) and inversely with HDL2 levels (r = -0.72, P less than 0.01). Hdl2 fractions were incubated with human hepatic lipase in vitro. Product lipoproteins formed from lipolysis of pa-HDL2 and triglyceride-poorer pp-HDL2 were reduced in phospholipid content (by 25 and 50%, respectively) but remained in the size and density range of native HDL2. By contrast, a major fraction of triglyceride-richer pp-HDL2 was converted to particles with density, size, and apoprotein composition of native HDL3. Changes consistent with these findings in vitro were observed in vivo also, where 15 h postprandially, individuals with high-level lipemia showed a decrease in HDL2 and rise in HDL3, while those with lower-level lipemia did not. This study indicates that the magnitude of postprandial lipemia determines the proportion of triglyceride in pp-HDL2, which in turn determines whether or not HDL2 are converted to HDL3 by hepatic lipase action.

Authors

J R Patsch, S Prasad, A M Gotto Jr, G Bengtsson-Olivecrona

Direct demonstration of the human parvovirus in erythroid progenitor cells infected in vitro.

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The human parvovirus (HPV), the cause of transient aplastic crisis of hereditary hemolytic anemia, has been shown to be cytotoxic for erythroid progenitor cells and its presence in these cells demonstrated by morphologic techniques. A relatively pure population of progenitors, isolated by removal of immature erythroid bursts from primary culture, was the target of the virus infection. Infected cells failed to proliferate in secondary culture. Using a monoclonal antibody to HPV, specific fluorescence was demonstrated in a minority of cells 24-48 h after infection with virus. Infected cells examined by electron microscopy showed marked toxic ultrastructural alterations and parvovirus-like particles in crystalline arrays in the nucleus.

Authors

N Young, M Harrison, J Moore, P Mortimer, R K Humphries

Detection of protein C activation in humans.

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We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the coagulation system at concentrations considerably less than 1.0 nM.

Authors

K A Bauer, B L Kass, D L Beeler, R D Rosenberg

Dietary menhaden oil lowers plasma prostaglandins and calcium in mice bearing the prostaglandin-producing HSDM1 fibrosarcoma.

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Abstract

The omega 3 class of polyunsaturated fatty acids, particularly eicosapentaenoic acid (EPA, 20:5), has been shown to alter the patterns of arachidonic acid (20:4) metabolism in both in vitro and in vivo systems. To examine further the role of arachidonic acid conversion to prostaglandins (PG) in hypercalcemic mice bearing the PG-producing HSDM1 fibrosarcoma, we have performed experiments in which control and tumor-bearing animals were fed diets either low (0.1-0.2% of total fatty acid) or high (17%) in EPA. In all five experiments performed, tumor-bearing mice eating control diets had markedly elevated (average sixfold above control) plasma concentrations of 13,14-dihydro-15-keto-PGE2 (PGE2-M), while in mice bearing HSDM1 tumors and eating the EPA-enriched menhaden oil diet, the elevation was reduced to only twice control values. The increase in plasma calcium concentration (approximately 2.5 mg/dl above control) in tumor-bearing animals was also reduced significantly (P less than 0.05) to only 1.3 mg/dl above control in mice eating the diet enriched in EPA. Plasma immunoreactive hydroxy fatty acids (i12-HETE) and sulfidopeptide leukotrienes (iSRS) were not elevated in tumor-bearing mice and were unaffected by diet. The contents of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were lower in tumor tissue from animals eating the diet high in EPA, whereas the tissue contents of i12-HETE and iSRS were not altered by diet. Fatty acid analysis of liver and tumor tissue revealed marked increases in certain omega 3 fatty acids (20:5, 22:5, and 22:6) from animals eating the enriched diet. Body weights, tumor weights, and tumor histology were not significantly altered by diet. To determine whether dietary calcium played a role in the elevation of plasma calcium in mice bearing the HSDM1 tumor and the reduction of plasma calcium in animals fed EPA, we compared results in mice fed diets containing 0.80% (normal) and 0.015% (deficient) calcium. The increases in plasma calcium and PGE2-M observed in tumor-bearing mice were the same on both normal and very low calcium intakes. We conclude, in mice of the Swiss albino strain bearing the HSDM1 fibrosarcoma, that consumption of a diet enriched in EPA reduces the production of cyclooxygenase products of arachidonic acid metabolism and thereby reduces the elevation of plasma calcium concentration. Dietary enrichment with EPA did not alter the production of serologically determined lipoxygenase products of arachidonic acid.

Authors

A H Tashjian Jr, E F Voelkel, D R Robinson, L Levine

Carbohydrate moiety of von Willebrand factor is not necessary for maintaining multimeric structure and ristocetin cofactor activity but protects from proteolytic degradation.

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Abstract

To better define the role of carbohydrate in the structure and ristocetin cofactor activity of von Willebrand factor, we have removed up to 83% of total hexose by sequential treatment of the molecule with endo-beta-N-acetyl-glucosaminidase F (endo F), neuraminidase, and beta-galactosidase. Endo F alone removed 69% of total hexose and D-galactose, and 71% of sialic acid. However, there was no discernible loss of large multimers and the ristocetin cofactor activity was decreased by only 11%. The reduced von Willebrand factor subunit migrated more rapidly in polyacrylamide gels containing SDS, consistent with a 10% decrease of molecular mass. All multimers of unreduced carbohydrate-modified von Willebrand factor migrated more rapidly in SDS-agarose, but the triplet pattern of individual multimers was unchanged. This alteration in multimer migration rate did not resemble alterations found so far in von Willebrand disease variants. Further treatment of von Willebrand factor with neuraminidase and beta-galactosidase reduced the D-galactose to 15% and ristocetin cofactor activity to 57%. A similar decrease in ristocetin cofactor activity was seen if von Willebrand factor was treated only with neuraminidase and beta-galactosidase. In contrast, treating von Willebrand factor with neuraminidase and beta-galactosidase in the presence of protease inhibitors (20 mM benzamidine, 20 U/ml aprotonin, 15 micrograms/ml leupeptin) resulted in a comparable removal of carbohydrate with no change in ristocetin cofactor activity. Moreover, the multimeric structure remained intact in spite of 80% removal of D-galactose. This suggested that carbohydrate was protecting von Willebrand factor against traces of one or more protease contaminants. Evidence in support of this hypothesis was obtained by exposing von Willebrand factor to plasmin after pretreatment with neuraminidase alone or with neuraminidase and beta-galactosidase. A loss of large multimers was observed from von Willebrand factor that had been pretreated with neuraminidase, but this was even greater if pretreatment was also with beta-galactosidase. In contrast, the multimeric structure of von Willebrand factor with intact carbohydrate was not affected by plasmin under similar conditions. These studies suggest that carbohydrate protects von Willebrand factor from disaggregation occurring secondarily to proteolytic attack but does not play a direct role in maintaining its multimeric structure or ristocetin cofactor activity.

Authors

A B Federici, J H Elder, L De Marco, Z M Ruggeri, T S Zimmerman

Use of carbon monoxide to measure luminal stirring in the rat gut.

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Abstract

We used carbon monoxide (CO) as a probe to quantitatively measure intestinal unstirred water layers in vivo. CO has several features that make it uniquely well suited to measure the unstirred layer in that its tight binding to hemoglobin makes uptake diffusion limited, and its relatively high lipid solubility renders membrane resistance negligible relative to the water barriers of the unstirred layer and epithelial cell. The unique application of CO was the measurement of the absorption rate of CO both from the gas phase as well as a solute dissolved in saline. Several lines of evidence showed that a gut stripped free of saline and then filled with gas contained a negligible unstirred layer. Thus, absorption of CO from the gas phase measured resistance of just the epithelial cell. Subtraction of this value from the resistance of CO absorption from saline provided a direct measure of unstirred layer resistance. Studies in the rat showed for a 3-min absorption period that the conventionally calculated apparent unstirred layer for the jejunum was 411 micron and for the colon was 240 micron. However, this conventionally calculated unstirred layer resistance did not truly depict the situation in the rat gut, since there was a continuing depletion of CO from outer surfaces of luminal contents throughout the experiment period. This produced a continually increasing diffusion barrier with time. Calculation of expected absorption rate from unstirred cylinders with the dimensions of the rat gut indicated that there was virtually no stirring in the small intestine and minimal stirring in the colon. The technique described in this paper appears to be simpler and to require fewer assumptions for validity than other techniques previously used to measure unstirred layers in vivo.

Authors

M D Levitt, T Aufderheide, C A Fetzer, J H Bond, D G Levitt

Baroreflex impairment precedes hypertension during chronic cerebroventricular infusion of hypertonic sodium chloride in rats.

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Abstract

Osmotic minipumps were implanted chronically for continuous 11-d infusion of hypertonic sodium chloride (NaCl) into the third cerebral ventricle (ICV) of awake rats to determine whether baroreflex sensitivity would be altered. Systolic and mean pressures, recorded from aortic catheters on day 11 while the rats were anesthetized with alpha-chloralose, were significantly higher in rats infused with artificial cerebrospinal fluid (CSF) containing hypertonic NaCl than in controls similarly infused with artificial CSF alone. Reflex changes in heart rate produced by subsequent intravenous infusions of either phenylephrine or sodium nitroprusside were inhibited, but reflex changes in renal nerve activity were unaltered. Magnitude of reflex bradycardia during pressor responses to phenylephrine, as well as of reflex tachycardia during depressor responses to sodium nitroprusside, was consistently smaller in NaCl-infused than in control rats. By contrast, group differences in attendant renal nerve firing were not significant. After sinoaortic denervation, drug-induced blood pressure effects persisted, but reflex responses in heart rate and renal nerve firing were abolished or markedly diminished. Peripheral effects produced by hypertonic NaCl leakage from the infusion site were considered unlikely because after 11 d of ICV infusion, sodium concentration, though appreciably elevated in CSF samples collected from the cisterna magna, was unaffected in corresponding serum samples. When cardiovascular responses to phenylephrine were recorded while chronic ICV infusions were in progress, awake rats receiving hypertonic NaCl were still normotensive on day 2 yet reflex bradycardia was already attenuated. In showing that baroreflex impairment preceded the development of hypertension, our results suggest that by depressing the anterior hypothalamus, chronic ICV infusion of hypertonic NaCl reduces sympatho-inhibition, and the ensuing baroreflex impairment then elevates blood pressure. However, other mechanisms could also be involved.

Authors

R D Buñag, E Miyajima

Modulation of the immune response by gangliosides. Inhibition of adherent monocyte accessory function in vitro.

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Abstract

Gangliosides are potent inhibitors of lymphoproliferative responses. Selectively greater inhibitory effects of gangliosides on antigen-induced (vs. mitogen-induced) proliferation have been documented; e.g., 50 nmol of highly purified bovine brain gangliosides (BBG)/ml caused greater than or equal to 87% inhibition of proliferative responses of human peripheral blood mononuclear cells (PBMC) to three soluble specific antigens (Candida, streptokinase-streptodornase, and tetanus toxoid) vs. less than or equal to 37% inhibition of responses to three nonspecific mitogens (phytohemagglutinin, concanavalin A, and pokeweed mitogen). The possibility that BBG interfere with adherent monocyte accessory function, upon which responses to soluble specific antigens are strictly dependent, was therefore considered. PBMC were separated into the adherent and nonadherent subpopulations, exposed to BBG, recombined, and their proliferative responses were measured. Unseparated PBMC preincubated for 48-72 h with 100 nmol BBG/ml and then washed to remove unbound BBG exhibited 73-76% inhibition of subsequent antigen-induced lymphoproliferation. Separate pretreatment of both adherent and nonadherent cell subpopulations in BBG under the same conditions resulted in similar (72-82%) inhibition, which was reproduced by preincubation of only the adherent cells in BBG. Preincubation of only the nonadherent cells in BBG was not inhibitory. Inhibition (a) was independent of whether gangliosides were added in solution or incorporated into liposomes, (b) was abrogated by adding untreated monocytes to cultures containing adherent cells that were preexposed to BBG (excluding the possibility that BBG was inducing suppression mediated by adherent cells), and (c) was reversible by further incubation of BBG-pretreated adherent cells in control medium. Together, these results delineate a mechanism by which gangliosides modulate lymphoproliferative responses--direct, noncytotoxic, and ultimately reversible inhibition of the accessory function of adherent monocytes.

Authors

S Ladisch, L Ulsh, B Gillard, C Wong

Familial protein S deficiency is associated with recurrent thrombosis.

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Abstract

Recent studies have demonstrated that protein C deficiency is associated with recurrent familial thrombosis. In plasma, activated protein C functions as an anticoagulant. This anticoagulant response requires a vitamin K-dependent plasma protein cofactor, referred to as protein S. Since the anticoagulant activity of activated protein C is dependent on protein S, we hypothesized that patients lacking functional protein S might have associated thrombotic disease. Two related individuals with otherwise normal coagulation tests are described whose plasma is not effectively anticoagulated with activated protein C. Addition of purified human protein S to their plasma restores a normal anticoagulant response to activated protein C. We have developed a rapid one-stage clotting assay for protein S to quantitate the level of protein S in their plasma. Plasma is depleted of protein S by immunoadsorption with immobilized antiprotein S antibodies. The resultant plasma responds poorly to activated protein C, but is effectively anticoagulated in a dose-dependent fashion upon addition of purified protein S or small quantities of plasma. The affected individuals possess less than 5% protein S activity. Using Laurell rockets, protein S antigen was detected in the plasma but was at reduced levels of 13 and 18% in the two individuals. When the barium eluate of the patient plasma was chromatographed on quaternary aminoethyl Sephadex, a single peak of protein S antigen devoid of protein S anticoagulant cofactor activity was detected early in the chromatogram. In contrast, the barium eluate from normal donors separated into two peaks, one emerging early and also devoid of anticoagulant cofactor, and the second peak with anticoagulant activity emerging later. The first peak of protein S antigen, from both the normal donor and the patient, chromatographed in the region of the complement component C4-binding protein-protein S complex. These studies suggest that protein S deficiency may result in recurrent thrombotic disease.

Authors

P C Comp, R R Nixon, M R Cooper, C T Esmon

Decreased expression of human class II antigens on monocytes from patients with acquired immune deficiency syndrome. Increased expression with interferon-gamma.

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Abstract

The expression of HLA-DR (a class II histocompatibility antigen) on monocytes isolated from the peripheral blood of normal individuals and patients with acquired immune deficiency syndrome (AIDS) was investigated by the use of dual fluorescent staining and cytofluorometry. In animal models the absence of class II positive monocytes is linked to a failure of T cells to respond to antigens. We now report that patients with AIDS have a paucity of HLA-DR+ monocytes. The percentage of HLA-DR+ monocytes among eight normal individuals ranged from 49.3 to 95.0%+, and only one individual had less than 50% HLA-DR+ monocytes. HLA-DR expression on monocytes from homosexual male patients with lymphadenopathy was similar to that of normal subjects (range, 58.0 to 97.4%+). In contrast, seven of nine patients with AIDS had less than 50% HLA-DR+ monocytes (range, 13.4 to 78.8%+). The in vitro incubation of monocytes from AIDS patients with cloned human interferon-gamma resulted in an increase of the expression of HLA-DR to near normal levels.

Authors

W Heagy, V E Kelley, T B Strom, K Mayer, H M Shapiro, R Mandel, R Finberg

Iodothyronine 5'-deiodinase in rat kidney microsomes. Kinetic behavior at low substrate concentrations.

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Abstract

The thiol-activated enzymatic outer-ring monodeiodination of iodothyronines by rat kidney microsomes at low (nanomolar) substrate concentrations shows an apparently sequential reaction mechanism and is further characterized by insensitivity to inhibition by dicoumarol, a moderate sensitivity to inhibition by propylthiouracil (Ki = 100 microM) and iopanoic acid (Ki = 0.9 mM), responsiveness to 5 mM glutathione (GSH), and a thermal activation profile that is concave downward with a Td of approximately 20 degrees C. In contrast, the activity at high (micromolar) substrate concentrations shows a ping-pong reaction mechanism, is inhibited by micromolar concentrations of propylthiouracil, iopanoic acid and dicoumarol, is unresponsive to 5 mM GSH, and shows a concave upward thermal activation profile. Analysis of the microsomal deiodinase reaction over a wide range of 3,3',5'-triiodothyronine (rT3) concentrations (0.1 nM to 10 microM) suggested the presence of two enzymatic activities, with apparent Michaelis constants (Km) of 0.5 microM and 2.5 nM. Lineweaver-Burk plots of reaction velocities at nanomolar substrate concentrations in presence of 100 microM propylthiouracil also revealed an operationally distinct enzymatic activity with Km's of 2.5 and 0.63 nM and maximum velocities (Vmax's) of 16 and 0.58 pmol/mg protein per h for rT3 and thyroxine (T4), respectively. These findings are consistent with the presence of a low Km iodothyronine 5'-deiodinase in rat kidney microsomes distinct from the well characterized high Km enzyme and suggest that at circulating levels of free T4 the postulated low Km enzyme could be physiologically important.

Authors

A Goswami, I N Rosenberg

Medullary collecting duct acidification. Effects of potassium, HCO3 concentration, and pCO2.

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Abstract

The medullary collecting duct (MCD) from renal outer medulla possesses significant HCO3 absorptive capacity. In vitro microperfusion studies have shown that HCO3 absorption in this segment is carbonic anhydrase dependent, affected by peritubular and luminal chloride concentrations, is independent of the presence of Na or the presence of Na transport, and is stimulated by mineralocorticoid hormone. The present in vitro microperfusion studies defined regulatory influences on MCD acidification as assessed by acute changes in the extracellular K and HCO3 concentrations and pCO2. These studies showed that acute changes in the peritubular K concentration to either 0 mM K or 50 mM K have no significant effect on HCO3 absorption in MCD. Intracellular voltage recordings showed that elevation of peritubular K concentration from 5 to 50 mM produced only a 2.8 mV depolarization of the basolateral cell membrane of MCD cells. In addition, acute reduction of peritubular K from 5 to 0 mM had no significant effect on intracellular voltage. Studies that were designed to assess the effects of HCO3 concentration and pCO2 on acidification showed that acute reduction of peritubular HCO3 concentration from 25 to 5 mM (pH change from 7.4 to 6.8) increased lumen-positive voltage from 30.2 +/- 3.8 to 40.0 +/- 4.4 mV, and simultaneously increased net HCO3 absorption from 15.6 +/- 1.9 to 22.9 +/- 2.9 pmol X mm-1 X min-1. Elevation of peritubular HCO3 concentration from 25 to 50 mM (pH change from 7.4 to 7.8) significantly decreased lumen-positive voltage from 33.8 +/- 2.4 to 26.7 +/- 1.5 mV and simultaneously decreased net HCO3 absorption from 17.9 +/- 1.2 to 12.8 +/- 1.3 pmol X mm-1 X min-1. In addition, acute reduction of peritubular pCO2 from 40 to less than 14 mmHg (final pH 7.8-7.9) significantly decreased lumen-positive voltage from 31 +/- 4.4 to 15.7 +/- 1.0 mV. Coincidentally, HCO3 absorption decreased significantly from 11.0 +/- 3.7 to 5.3 +/- 0.7 pmol X mm-1 X min-1. We conclude that: alteration of peritubular K concentration from 0 to 50 mM in vitro does not affect HCO3 absorption in the MCD, and that this lack of effect appears to be related to a low basolateral cell membrane K conductance; net HCO3 absorption and the associated lumen-positive voltage can be modulated by in vitro changes in peritubular HCO3 and pCO2 (or pH); and the MCD perfused in vitro appears to be a good model for studying the mechanisms and regulation of distal nephron acidification.

Authors

H R Jacobson

Polyclonal immunoglobulin secretion in patients with common variable immunodeficiency using monoclonal B cell differentiation factors.

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Abstract

B cells from 25 patients with common variable immunodeficiency (CVI) were tested for their ability to differentiate under the influence of B cell differentiation factors (BCDF), derived from T cell hybridomas or T cell clones. 11 patients generated Ig plaque-forming cells in the range comparable to that of normal controls with supernatant from the T cell hybrid MOP 1L. With various hybrid or clone supernatants, differing response patterns emerged. Four patients who failed to respond to MOP 1L responded to T cell clone supernatant RAC. Another who failed to respond to both MOP 1L and RAC responded to T cell hybrid supernatant MTP 7. These results indicate that these supernatants contain different BCDFs and suggest heterogeneity in the differentiation states of B cells in CVI. In addition, three patients demonstrated exaggerated responses to BCDF, and evidence was obtained from B cells of these patients for increased BCDF receptor density. Thus, the accumulated evidence indicates that T cell defects may be a primary pathogenetic mechanism in common variable immunodeficiency, and purified BCDF may be of therapeutic value.

Authors

L Mayer, S M Fu, C Cunningham-Rundles, H G Kunkel

Monocyte function in the acquired immune deficiency syndrome. Defective chemotaxis.

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Abstract

The ineffective immune response in patients with the acquired immune deficiency syndrome (AIDS) contributes to severe and widespread infections and unrestricted growth by certain tumors. To determine whether monocyte dysfunction contributes to this immunosuppressed condition, we investigated monocyte chemotaxis in patients with AIDS. Using three different chemotactic stimuli, N-formylmethionylleucylphenylalanine, lymphocyte-derived chemotactic factor, and C5a des Arg, we studied the chemotactic responses of monocytes from seven homosexual men with AIDS, three homosexuals with lymphadenopathy and an abnormal immunological profile, seven healthy homosexual men, and 23 heterosexual control individuals. Monocytes from each of the AIDS patients with Kaposi's sarcoma and/or opportunistic infection exhibited a marked reduction in chemotaxis to all stimuli compared with the healthy control subjects. The reduced chemotactic responses were observed over a wide range of concentrations for each stimulus. Monocytes from AIDS patients who had clinically apparent opportunistic infection(s) exhibited a greater reduction in monocyte migration to all three stimuli than monocytes from the AIDS patient with only Kaposi's sarcoma. Monocytes from each of three homosexuals with lymphadenopathy and an abnormal immunological profile exhibited decreased chemotactic responses that were intermediate between those of the AIDS patients and the healthy heterosexual control subjects. In contrast to these findings, monocytes from each of seven healthy homosexuals exhibited normal chemotactic responses to the same stimuli. In addition, monocytes from AIDS patients exhibited reduced chemotaxis to soluble products of Giardia lamblia, one of several protozoan parasites prevalent in AIDS patients. Thus the immune abnormality in AIDS, previously thought to involve only the T-, B-, and natural killer lymphocytes, extends to the monocyte-macrophage. Defective monocyte migratory function may contribute to the depressed inflammatory response to certain organisms and to the apparent unrestricted growth of certain neoplasms in patients with AIDS.

Authors

P D Smith, K Ohura, H Masur, H C Lane, A S Fauci, S M Wahl

Polymorphism in the 5' flanking region of the human insulin gene. Relationships with noninsulin-dependent diabetes mellitus, glucose and insulin concentrations, and diabetes treatment in the Pima Indians.

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Abstract

Variations in DNA sequences flanking the insulin gene were studied in relation to noninsulin-dependent diabetes mellitus (NIDDM) in 87 unrelated Pima Indians at least 35 yr of age. DNA was isolated from nuclei of peripheral blood leukocytes and digested with restriction endonucleases. Less variation in this region was found in Pima Indians than in other racial groups previously studied. Only two classes of alleles (classes 1 and 3) were found, and there was virtually no variation within classes. At least one class 3 allele was found in 47% of the 38 nondiabetic subjects and in 37% of the 49 with NIDDM (odds ratio = 0.65, P = 0.4, 95% confidence interval for the odds ratio = 0.25 to 1.67). Homozygosity for class 3 alleles, however, was found only in diabetics. There were no differences according to genotype in obesity, fasting or postload glucose or insulin concentrations, or in the relationships between insulin and glucose concentrations. 61% (11/18) of the diabetics with a class 3 allele were receiving drug treatment for diabetes compared with only 26% (8/31) of diabetics without a class 3 allele (P = 0.03). The insulin gene polymorphism probably plays no important role in the genesis of NIDDM in Pima Indians, nor does it influence the glucose or insulin concentrations or their relationship to each other, but the class 3 allele, especially when homozygous in this population, may influence the severity of the disease as indicated by need for drug treatment.

Authors

W C Knowler, D J Pettitt, B Vasquez, P S Rotwein, T L Andreone, M A Permutt

Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxy-cholecalciferol in uremic patients.

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Abstract

Current evidence suggests that administration of 1,25(OH)2D3 to patients with chronic renal insufficiency results in suppression of secondary hyperparathyroidism only if hypercalcemia occurs. However, since the parathyroid glands possess specific receptors for 1,25(OH)2D3 and a calcium binding protein, there is considerable interest in a possible direct effect of 1,25(OH)2D3 on parathyroid hormone (PTH) secretion independent of changes in serum calcium. Recent findings indicate substantial degradation of 1,25(OH)2D3 in the intestine, therefore, it is possible that while oral administration of the vitamin D metabolite increases intestinal calcium absorption, the delivery of 1,25(OH)2D3 to peripheral target organs may be limited. We therefore compared the effects of orally or intravenously administered 1,25(OH)2D3 on the plasma levels of 1,25(OH)2D3 and the effects of these two modes of treatment on PTH secretion. Whereas oral administration of 1,25(OH)2D3 in doses adequate to maintain serum calcium at the upper limits of normal did not alter PTH levels, a marked suppression (70.1 +/- 3.2%) of PTH levels was seen in all 20 patients given intravenous 1,25(OH)2D3. Temporal studies suggested a 20.1 +/- 5.2% decrease in PTH without a significant change in serum calcium with intravenous 1,25(OH)2D3. In five patients the serum calcium was increased by the oral administration of calcium carbonate, the decrement in serum i-PTH was only 25 +/- 6.65% when compared with 73.5 +/- 5.08% (P less than 0.001) obtained by the administration of intravenous 1,25(OH)2D3. Thus, a similar serum calcium achieved by intravenous 1,25(OH)2D3 rather than calcium carbonate has a greater suppressive effect in the release of PTH. These studies indicate that 1,25(OH)2D3 administered intravenously rather than orally may result in a greater delivery of the vitamin D metabolite to peripheral target tissues other than the intestine and allow a greater expression of biological effects of 1,25(OH)2D3 in peripheral tissues. The use of intravenous 1,25(OH)2D3 thus provides a simple and extremely effective way to suppress secondary hyperparathyroidism in dialysis patients.

Authors

E Slatopolsky, C Weerts, J Thielan, R Horst, H Harter, K J Martin

Arginine, an indispensable amino acid for patients with inborn errors of urea synthesis.

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Abstract

The role of arginine as an essential amino was evaluated in four children with one of the deficiencies of carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinase. Within 15-68 h after arginine deprivation nitrogen accumulated as ammonium or glutamine or both, but glutamine was quantitatively the largest nitrogen accumulation product. Concomitantly plasma and urinary urea levels decreased. Resumption of arginine intake (or citrulline in the case of ornithine transcarbamylase deficiency) promptly led to correction of the hyperammonemia, hyperglutaminemia and hypoargininemia. Ornithine was an unsatisfactory substitute for arginine. Arginine deprivation did not interfere with carbamyl phosphate synthesis as manifested by orotic aciduria. It is concluded that arginine is an indispensable amino acid for children with inborn errors of ureagenesis and its absence results in the rapid onset of symptomatic hyperammonemia.

Authors

S W Brusilow

Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis.

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Abstract

Cultured fibroblasts from a recently described patient with homocystinuria and megaloblastic anemia of infancy without methylmalonic aciduria were previously shown to have normal cobalamin uptake and a specific decrease in the proportion of intracellular methylcobalamin. As in control cells but unlike in those from patients with combined homocystinuria and methylmalonic aciduria (cobalamin C and cobalamin D), accumulated 57Co-labeled cobalamin was bound in appropriate amounts and proportion to intracellular binders which are known to be the two vitamin B12-dependent enzymes, methionine synthetase and methylmalonyl-CoA mutase. Despite the association of a normal quantity of intracellular cobalamin with methionine synthetase, the proportion of intracellular cobalamin which was methyl-B12 was below normal and in the range observed in cobalamin C and D cells. This methyl-B12 was decreased by exposure of fibroblasts in culture to nitrous oxide as was observed with control cells. Exposure of control fibroblasts during culture, but not of fibroblasts from this patient, to nitrous oxide significantly reduced the holoenzyme activity of methionine synthetase assayed in cell extracts. In addition, although methionine synthetase activity in cell extracts of control and cells from the patient were similar in the presence of standard assay concentrations of thiols, at low thiol concentrations, methionine synthetase activity in extracts of cells from the patient was much lower than in control extracts. Mixing of control patient extracts corrected this decreased activity in excess of that explained by addition of the individual activities added. The defect of this patient appears to be in a reducing system required for methionine synthesis.

Authors

D S Rosenblatt, B A Cooper, A Pottier, H Lue-Shing, N Matiaszuk, K Grauer

Dexamethasone regulation of glycosaminoglycan synthesis in cultured human skin fibroblasts. Similar effects of glucocorticoid and thyroid hormones.

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Abstract

The effects of dexamethasone on glycosaminoglycan accumulation were examined in confluent human skin fibroblasts in vitro. The glucocorticoid consistently inhibited the incorporation of either [3H]acetate or [3H]glucosamine into hyaluronate when added to culture medium 72 h before harvest. This effect was half-maximal at approximately 1 nM and maximal at 5-10 nM. Inhibition occurred within 5 h of hormone addition and was near maximal by 25 h. 11 alpha-hydrocortisone (10 nM), deoxycorticosterone (10 nM), and progesterone (100 nM) failed to inhibit this accumulation; however, progesterone (2 microM), a known glucocorticoid antagonist at high concentration, could attenuate the response to dexamethasone by 57%. Cultures were pulse-labeled and then chase incubated for up to 68 h. No difference in the rate of [3H]hyaluronate degradation could be demonstrated in steroid-treated cultures. Triiodothyronine (T3) can also inhibit synthesis of hyaluronate in fibroblasts (Smith, T. J., Y. Murata, A. L. Horwitz, L. Philipson, and S. Refetoff, 1982, J. Clin. Invest., 70:1066-1073). Both T3 and dexamethasone could inhibit glycosaminoglycan accumulation in a dose-dependent manner. Maximal T3 effects were achieved at 1 nM and those of dexamethasone at 10 nM. Saturating concentrations of T3 and dexamethasone added alone inhibited [3H]hyaluronate by 54 and 49%, respectively. When both hormones were added, accumulation was inhibited by 84%. Dexamethasone inhibits [3H]hyaluronate accumulation in a time, dose-dependent, and stereo-specific manner. The rate of glycosaminoglycan degradation was unaffected, and thus, the steroid inhibited the rate of macromolecular synthesis. This effect was likely mediated through glucocorticoid receptors. Hyaluronate synthesis in skin fibroblasts appears to be regulated by both glucocorticoids and T3 through different pathways.

Authors

T J Smith

Apolipoprotein A and B (Sf 100-400) metabolism during bezafibrate therapy in hypertriglyceridemic subjects.

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Abstract

This study describes the effects of bezafibrate, an analogue of clofibrate, on the plasma lipid and lipoprotein profiles of 11 hypertriglyceridemic subjects and on their metabolism of apolipoproteins A-I, A-II, and B. The major action of the drug was to lower plasma triglyceride (by 58%; P less than 0.01). This was accompanied by a reduction in the level of very low density lipoprotein apoprotein B (Svedberg units of flotation [Sf] 60-400), whose mean residence time in the plasma fell threefold (from 3.4 to 1.0 h). Synthesis of the B protein in this fraction was not significantly altered, so the drug acts to accelerate the transit of very low density lipoprotein particles down the delipidation cascade. The metabolism of very low density lipoprotein remnant apoprotein B (Sf 12-100) changed little in response to treatment, although we detected a 30% increment (P less than 0.05) in the plasma concentration of this fraction. The mean residence time of these remnant particles in the plasma did not correlate with that of Sf 100-400 very low density lipoprotein apoprotein B, nor was this parameter altered by the drug. The most consistent and significant perturbation seen in the Sf 0-12 fraction (low density lipoprotein) was a reduction in the fractional catabolism of its apoprotein B moiety (26%; P less than 0.05). In those subjects who were grossly hypertriglyceridemic and who responded well to treatment, the level of this protein rose substantially owing to a combined increase in its synthesis and a reduction in its catabolism. In the group as a whole, high density lipoprotein cholesterol rose 13% (P less than 0.02), and detailed examination showed that this was associated with a small but significant increment in the plasma concentration of the high density lipoprotein subfraction 2. High density lipoprotein subfraction 3 also rose on the average, but this was not a consistent feature in all patients. The plasma concentrations and turnovers of the A proteins (A-I and A-II) were not significantly altered by bezafibrate therapy.

Authors

J Shepherd, C J Packard, J M Stewart, R F Atmeh, R S Clark, D E Boag, K Carr, A R Lorimer, D Ballantyne, H G Morgan

Metabolism of apolipoprotein B in large triglyceride-rich very low density lipoproteins of normal and hypertriglyceridemic subjects.

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Abstract

The metabolic fate of very low density lipoprotein can be examined by following the transit of its apolipoprotein B moiety through the delipidation cascade, which leads to low density lipoprotein. In this study we have used cumulative flotation ultracentrifugation to follow the metabolism of various lipoprotein subclasses that participate in this process in normal, hypertriglyceridemic (Type IV), and dysbetalipoproteinemic (Type III) subjects. Large triglyceride-rich very low density lipoproteins of Svedberg units of flotation (Sf) 100-400 were converted virtually quantitatively in normal subjects to smaller Sf 12-100 remnant particles. Only a minor fraction appeared thereafter in low density lipoproteins (Sf 0-12), most being removed directly from the plasma. Type IV hyperlipoproteinemic individuals converted the larger Sf 100-400 very low density lipoproteins to intermediate particles at approximately 50% of the control rate but thereafter their metabolism was normal (fractional clearance of Sf 12-100 particles in controls, 1.29 +/- 0.23 pools/d; in Type IV hypertriglyceridemics, 1.38 +/- 0.23 pools/d; n = 4 in each case). Since the apolipoprotein B in large triglyceride-rich particles did not contribute significantly to the mass of the low density lipoprotein apoprotein pool, the latter must come largely from another source. This was examined by following the metabolic fate of small very low density lipoproteins of Sf 20-60 or of the total lipoprotein spectrum of d less than 1.006 kg/liter (approximate Sf 20-400). The small particles were rapidly and substantially converted to low density lipoproteins, suggesting that the major precursor of the latter was to be found in this density range. Whereas only 10% of apolipoprotein B in Sf 100-400 lipoproteins reached the low density lipoprotein flotation range, greater than 40% of Sf 20-100 B protein eventually appeared in Sf 0-12 particles; and when very low density lipoprotein of d less than 1.006 kg/liter is used as a tracer of apolipoprotein B metabolism it is primarily this population of small very low density lipoprotein particles in the Sf 12-100 flotation range that is labeled. A detailed examination was made of apolipoprotein B metabolism in three dysbetalipoproteinemic subjects. The plasma clearance curves of their Sf 100-400 lipoproteins were distinctly biphasic. The quickly decaying component converted rapidly into remnants of Sf 20-60 at a near normal rate (0.56 vs. 0.62 pools/d in normal subjects). Its subsequent processing, however, was retarded. The more slowly catabolized fraction, comprising 30% of the total apolipoprotein B radioactivity, had no counterpart in normal or Type IV hyperlipoproteinemic individuals. These data, taken together, suggest that the very low density lipoprotein consists of a complex mixture of particles with different origins and fates. Within the Sf 20-100 flotation range there are at least two subcomponents. One represents remnants of larger triglyceride-rich particles which are catabolized slowly and feeds little apolipoprotein B into low density lipoprotein. The other is apparently secreted directly into this flotation interval and transfers significant amounts of B protein rapidly into Sf 0-12 lipoproteins.

Authors

C J Packard, A Munro, A R Lorimer, A M Gotto, J Shepherd

Single cell origin of multilineage colonies in culture. Evidence that differentiation of multipotent progenitors and restriction of proliferative potential of monopotent progenitors are stochastic processes.

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Abstract

In this paper, we report analysis of differentiation in human hemopoietic colonies derived from a single cell. Cord blood mononulear cells and panned My-10 antigen-positive bone marrow and cord blood cells were plated in methylcellulose medium containing erythropoietin and conditioned medium. Initially, we performed mapping studies to identify candidate colony-forming cells. Subsequently, using a micromanipulator, we transferred single cells individually to 35-mm dishes for analysis of colony formation. Cellular composition of the colony was determined by identifying all of the cells in the May-Grunwald-Giemsa stained preparation. Of 150 single candidate cells replated, 63 produced colonies. The incidences of single lineage colonies included 19 erythroid, 17 monocyte-macrophage, and 9 eosinophil colonies. There were 18 mixed hemopoietic colonies consisting of cells in two, three, four, and five lineages in varying combinations. In some instances, we noted the predominance of one lineage and the presence of very small populations of cells in a second or third lineage. These results provide evidence for the single-cell origin of human multilineage hemopoietic colonies, and are consistent with the stochastic model of stem cell differentiation in man. They also indicate that restriction of the proliferative potential of committed progenitors is a stochastic process.

Authors

A G Leary, M Ogawa, L C Strauss, C I Civin

Selective peripheral dopamine-1 receptor stimulation with fenoldopam in human essential hypertension.

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Abstract

SKF 82526-J, or fenoldopam, a benzazepine derivative, is a selective dopamine-1 (DA-1) agonist devoid of activity at dopamine-2, alpha- or beta-adrenergic receptors. We studied SKF 82526-J in 10 patients with essential hypertension and five normal control subjects on constant 150-meq sodium, 60 meq potassium intake. In the hypertensive patients, during a 6-d placebo period supine blood pressure and heart rate were stable at 156 +/- 6/105 +/- 4 mmHg and 76 +/- 5 beats/min, respectively. In response to a single oral dose of 100 mg of SKF 82526-J, supine blood pressure decreased to a nadir of 141 +/- 5/89 +/- 8 mmHg (P less than 0.0001) at 90 min and remained decreased at 145 +/- 6/99 +/- 3 mmHg (P less than 0.0001) at 4 h. Heart rate increased to 91 +/- 5 beats/min (P less than 0.002), but returned to control levels (82 +/- 5 beats/min) at 4 h. Renal blood flow increased from 371 +/- 57 to a peak of 659 +/- 104 ml/min and renal vascular resistance fell from 34 +/- 5 to 19 +/- 2 dyn sec cm-5 X 10(3) (P less than 0.01). Urine volume, sodium and fractional sodium excretion, and plasma renin activity were increased as a result of SKF 82526-J administration. During the ensuing 3 wk of SKF 82526-J, blood pressure remained decreased and returned to control levels after placebo administration. In contrast, in normal subjects SKF 82526-J administration was associated with a small transient reduction in diastolic pressure only. These results suggest that reduced dopaminergic activity expressed at the peripheral DA-1 receptor may contribute to the pathophysiology and/or maintenance of increased blood pressure in essential hypertension. In addition, the results suggest that peripheral DA-1 receptor stimulation with SKF 82526-J may be efficacious in the treatment of human essential hypertension.

Authors

R M Carey, R M Stote, J W Dubb, L H Townsend, C E Rose Jr, D L Kaiser

Normal human alveolar macrophages obtained by bronchoalveolar lavage have a limited capacity to release interleukin-1.

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Abstract

Interleukin-1 (IL-1) is a mediator released by stimulated mononuclear phagocytes that is thought to play an important role in modulating T and B lymphocyte activation as well as in contributing to the febrile response and other inflammatory processes. Circulating mononuclear phagocytes, blood monocytes, readily release IL-1 when stimulated. However, the ability of lung mononuclear phagocytes, alveolar macrophages, to dispose of the large daily burden of inhaled antigens without stimulating an inflammatory response suggests that the release of IL-1 by alveolar macrophages may differ significantly from that of blood monocytes. To evaluate this hypothesis, normal autologous alveolar macrophages, obtained by bronchoalveolar lavage, were compared with blood monocytes for their ability to release IL-1 in response to a standard stimulus, lipopolysaccharide (LPS). Alveolar macrophages were found to be at least 1,000 times less sensitive to LPS than blood monocytes. Furthermore, alveolar macrophages released significantly less IL-1 than blood monocytes (26 +/- 11 vs. 128 +/- 21 U/10(6) cells X 24 h, respectively, after stimulation with 10 micrograms/ml of LPS, P less than 0.001). This difference was not due to the release of substances by macrophages, which inhibited lymphocyte proliferation in response to IL-1, or to degradation of IL-1 by macrophages. Culturing macrophages in the presence of indomethacin and dialysis of macrophage supernatants did not affect the difference, and culturing macrophages with monocytes did not decrease detectable IL-1 activity from the monocytes. The IL-1 produced by the two cell types was indistinguishable by anion-exchange chromatography, gel filtration, and isoelectric focusing. In addition, consistent with the findings for alveolar macrophages, macrophages generated by the in vitro maturation of blood monocytes were also deficient in their ability to release IL-1. These findings suggest that if the population of alveolar macrophages obtained by bronchoalveolar lavage represents the total in vivo population of alveolar macrophages, although normal human macrophages are capable of IL-1 release, they are relatively limited in this ability, and this limitation seems to be linked to the maturational state of the mononuclear phagocyte. These observations may explain, in part, the ability of alveolar macrophages to clear the airspaces of foreign antigens without extensive activation of other pulmonary inflammatory and immune effector cells.

Authors

M D Wewers, S I Rennard, A J Hance, P B Bitterman, R G Crystal

Studies of Factors V and VIII:C in an animal model of disseminated intravascular coagulation.

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Abstract

An experimental animal model of disseminated intravascular coagulation (DIC) induced by the co-infusion of coagulant-active phospholipid and activated Factor X (Factor Xa) is described. The infusion of Factor Xa at a dose of 6.6 X 10(-12) mol/kg with phosphatidylcholine/phosphatidylserine (PCPS) lipid vesicles at a dose of 4.0 X 10(-8) mol/kg was associated with significant falls in the levels of fibrinogen and Factors V and VIII, and a bleeding diathesis developed. Assays of Factors V and VIII were performed by a one-stage prothrombin time and activated partial thrombin time system, respectively. In additional experiments, the effect of the same dose combination of Factor Xa/PCPS on Factor V kinetics was studied by preinfusing 125I-labeled Factor V. After Factor Xa/PCPS infusion, Factors VIII and V were reduced at 2 min by 90 and 50% of the preinfusion levels, respectively, and at 1 h by 80 and 75%, respectively. During the same period, there was little change in the total circulating radioactivity. Autoradiography indicated small but detectable levels of circulating proteolytic products of Factor V that comigrated with peptides obtained by the incubation of Factor V with Factor Xa and activated protein C. The majority of radioactivity remained associated with the intact single-chain precursor Factor V. These observations suggested maintenance of the precursor pool after the onset of DIC. This was confirmed by performing two-stage assays of Factors V and VIII, whereby each was completely converted to the active cofactor, i.e., Va and VIII:Ca, by preincubation of the test sample with thrombin before assaying in a one-stage system as before. The Factor V levels assayed by the two-stage procedure did not change appreciably over 1 h. The Factor VIII levels fell but corrected within 1 h at a time when the level measured by a one-stage assay remained depressed. These results indicate that in the dog, infusion of Factor Xa/PCPS induces changes characteristic of DIC, and this is associated with the appearance of Factor V peptides characteristic of the expression of Factor Xa and activated protein C-like activities. The differences noted between the one-stage and two-stage assays suggest that the one-stage assay is measuring the activated fraction of each cofactor and not the total level of the available precursor for each activated species. The results suggest a close correlation between the activated fraction of both cofactors and the hemostatic abnormality that occurs in DIC.

Authors

A R Giles, M E Nesheim, K G Mann

Regulation of biliary cholesterol secretion in the rat. Role of hepatic cholesterol esterification.

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Abstract

Although the significance of the enterohepatic circulation of bile salts in the solubilization and biliary excretion of cholesterol is well established, little is known about the intrahepatic determinants of biliary cholesterol output. Studies were undertaken to elucidate some of these determinants in the rat. Feeding 1% diosgenin for 1 wk increased biliary cholesterol output and saturation by 400%. Bile flow, biliary bile salt, phospholipid and protein outputs remained in the normal range. When ethynyl estradiol (EE) was injected into these animals, biliary cholesterol output decreased to almost normal levels under circumstances of minor changes in the rates of biliary bile salt and phospholipid outputs. Similarly, when chylomicron cholesterol was intravenously injected into diosgenin-fed animals, biliary cholesterol output significantly decreased as a function of the dose of chylomicron cholesterol administered. Relative rates of hepatic cholesterol synthesis and esterification were measured in isolated hepatocytes. Although hepatic cholesterogenesis increased 300% in diosgenin-fed animals, the contribution of newly synthesized cholesterol to total biliary cholesterol output was only 19 +/- 9%, compared with 12 +/- 6% in control and 15 +/- 5% in diosgenin-fed and EE-injected rats. The rate of oleate incorporation into hepatocytic cholesterol esters was 30% inhibited in diosgenin-fed rats. When EE was injected into these animals, the rate of cholesterol esterification increased to almost 300%. To investigate further the interrelationship between hepatic cholesterol esterification and biliary cholesterol output, we studied 21 diosgenin-fed rats. Six of them received in addition EE and 10 received chylomicron cholesterol. The relationships between biliary cholesterol output as a function of both microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity and hepatic cholesterol ester concentration were significantly correlated in a reciprocal manner. From these results it is concluded that the size of the biliary cholesterol precursor pool can be rapidly modified through changes in the activity of the hepatic ACAT.

Authors

F Nervi, M Bronfman, W Allalón, E Depiereux, R Del Pozo

Role of counterregulatory hormones in the catabolic response to stress.

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Abstract

Patients with major injury or illness develop protein wasting, hypermetabolism, and hyperglycemia with increased glucose flux. To assess the role of elevated counterregulatory hormones in this response, we simultaneously infused cortisol (6 mg/m2 per h), glucagon (4 ng/kg per min), epinephrine (0.6 microgram/m2 per min), and norepinephrine (0.8 micrograms/m2 per min) for 72 h into five obese subjects receiving only intravenous glucose (150 g/d). Four obese subjects received cortisol alone under identical conditions. Combined infusion maintained plasma hormone elevations typical of severe stress for 3 d. This caused a sustained increase in plasma glucose (60-80%), glucose production (100%), and total glucose flux (40%), despite persistent hyperinsulinemia. In contrast, resting metabolic rate changed little (9% rise, P = NS). Urinary nitrogen excretion promptly doubled and remained increased by approximately 4 g/d, reflecting increased excretion of urea and ammonia. Virtually all plasma amino acids declined. The increment in nitrogen excretion was similar in three additional combined infusion studies performed in 3-d fasted subjects not receiving glucose. Cortisol alone produced a smaller glycemic response (20-25%), an initially smaller insulin response, and a delayed rise in nitrogen excretion. By day 3, however, daily nitrogen excretion was equal to the combined group as was the elevation in plasma insulin. Most plasma amino acids rose rather than fell. In both infusion protocols nitrogen wasting was accompanied by only modest increments in 3-methylhistidine excretion (approximately 20-30%) and no significant change in leucine flux. We conclude: (a) Prolonged elevations of multiple stress hormones cause persistent hyperglycemia, increased glucose turnover, and increased nitrogen loss; (b) The sustained nitrogen loss is no greater than that produced by cortisol alone; (c) Glucagon, epinephrine, and norepinephrine transiently augment cortisol-induced nitrogen loss and persistently accentuate hyperglycemia; (d) Counterregulatory hormones contribute to, but are probably not the sole mediators of the massive nitrogen loss, muscle proteolysis, and hypermetabolism seen in some clinical settings of severe stress.

Authors

R A Gelfand, D E Matthews, D M Bier, R S Sherwin

Mechanism of enhanced fibroblast arachidonic acid metabolism by mononuclear cell factor.

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Abstract

Chronic inflammation is associated with an infiltration of mononuclear cells, fibroblast proliferation, and elevated levels of prostaglandin (PG) E2. Mononuclear cell conditioned factor (MNCF) medium (5%) stimulated a 100-fold increase in basal human dermal fibroblast PGE2 release over 48 h as compared with fibroblasts that were incubated with control medium (conditioned medium prepared without cells). The MNCF-induced PGE2 production was suppressed by protein synthesis inhibitors. Fibroblasts pretreated with control medium released PGE2 only modestly in response to 1 nM bradykinin for 1 h (basal, 50 +/- 7 pg PGE2/micrograms protein; stimulated, 104 +/- 12 pg PGE2/micrograms protein), whereas cells that had been pretreated with MNCF showed a greatly facilitated bradykinin-induced release of PGE2. (basal, 297 +/- 59 pg PGE2/micrograms protein; stimulated, 866 +/- 85 pg PGE2/micrograms protein). The exaggerated agonist response is not specific for bradykinin because platelet-derived growth factor elicits a similar response. Exogenous arachidonic acid conversion to PGE2 was also facilitated (two- to threefold) by MNCF pretreatment as compared with control. Both the enhanced agonist-stimulated and exogenous arachidonic acid-induced PGE2 release from the MNCF pretreated cells were inhibited by actinomyin D or cycloheximide. A kinetic study of microsomal cyclooxygenase prepared from fibroblasts pretreated with MNCF showed a threefold increase in the maximum velocity (Vmax) but the same Michaelis constant (Km) as control-treated cells. This augmented arachidonic acid metabolism and subsequent enhanced PGE2 production may play an important role in macrophage-fibroblast interactions at sites of inflammation.

Authors

P J Whiteley, P Needleman

Ontogenesis of iodothyronine-5'-deiodinase. Induction of 5'-deiodinating activity by insulin, glucocorticoid, and thyroxine in cultured fetal mouse liver.

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Abstract

To elucidate the regulatory mechanism of ontogenetic development of iodothyronine-5'-deiodinase in the fetal and neonatal period, fetal mouse liver of the 19th day of gestation, in which no iodothyronine-5'-deiodinating activity was detectable, was cultured in Dulbecco-Vogt medium supplemented with 10% thyroid hormone-depleted fetal calf serum, insulin, hydrocortisone, and thyroid hormones. Iodothyronine-5'-deiodinating activity of the homogenate was assessed by the amount of iodide released from outer-ring-labeled reverse T3 and expressed as picomoles of 127I- per milligram of protein per minute. The enzyme activity was induced in a dose-dependent manner; optimal concentrations for insulin, hydrocortisone, and thyroxine were 1 microgram/ml, 0.4 microgram/ml, and 10(-6) M, respectively. Without supplementation of either hydrocortisone or thyroxine, no 5'-deiodination was detected. The enzyme activity was observed after 3 d of culture, peaked at days 14-20, and then gradually decreased. Lineweaver-Burk analysis revealed that the increase in activity was primarily due to an increase in Vmax (day 3, 0.2 pmol/mg protein per min; day 20, 2.5 pmol/mg protein per min). Half maximal thyroxine (T4) and triiodothyronine (T3) concentrations were 1 X 10(-7) M (free T4: 4 X 10(-10) M), and 2 X 10(-9) M (free T3: 5.0 X 10(-11) M), respectively, whereas reverse T3 did not elicit any activity at 10(-8)-10(-6) M. These results suggest that ontogenetic development of iodothyronine-5'-deiodinase in the liver of the fetal and neonatal mouse is induced by physiological concentrations of glucocorticoid and thyroid hormones, and that insulin plays a permissive role in enhancing T3 formation from T4 in the liver.

Authors

K Sato, H Mimura, D C Han, T Tsushima, K Shizume

Induction of cytolytic activity by anti-T3 monoclonal antibody. Activation of alloimmune memory cells and natural killer cells from normal and immunodeficient individuals.

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Abstract

The ability of the monoclonal antibody directed at the T3 antigen (anti-T3) to induce cytolytic activity was investigated since several agents that can activate T cells induce the acquisition of cytolytic activity in a variety of test systems. Pretreatment of human alloimmune memory cells, generated in primary long-term mixed lymphocyte cultures, with anti-T3 resulted in the induction of statistically significant specific secondary cytolytic activity and natural killer (NK) cell-like activity. No such augmentation or induction of cytolytic activity was found with anti-T3 pretreatment when syngeneic cells or inappropriate allogeneic cells (HLA-A, B antigens different from the original priming stimulus) were used as target cells and pretreatment of memory cells with anti-T4 or anti-T8 did not induce cytolytic activity to allogeneic or syngeneic target cells. Differential effects were observed when anti-T3 was added to the cytotoxicity assay in which anti-T3 pretreated alloimmune memory cells were effectors. The addition of anti-T3 to the assay prior to the introduction of target cells resulted in 39 +/- 8% inhibition of specific secondary cytolytic activity and only 5 +/- 8% inhibition of NK cell activity. NK cell activity mediated by large granular lymphocyte-enriched fraction of peripheral blood mononuclear cells (PBM) obtained from normal individuals was significantly augmented by anti-T3 when NK-sensitive cell lines MOLT-4 or K-562 were used as target cells. This augmentation in NK cell activity was not associated with nonspecific cytotoxicity to syngeneic or allogeneic PBM, and anti-T3 failed to activate the LGL fraction depleted of T cells. The monoclonal antibodies, anti-T4 or anti-T8, did not increase NK cell activity. NK cell activity mediated by PBM from eight immunodeficient individuals (four with acquired immunodeficiency syndrome and four with renal allografts) was also significantly augmented by anti-T3 pretreatment. Our findings, in addition to providing a rationale for the frequent occurrence of re-rejection episodes in renal graft recipients treated with anti-T3, suggest that anti-T3 might be utilized to enhance the cytotoxic armamentarium of immunodeficient patients.

Authors

M Suthanthiran, P S Williams, S D Solomon, A L Rubin, K H Stenzel

Antiestrogenic action of dihydrotestosterone in mouse breast. Competition with estradiol for binding to the estrogen receptor.

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Abstract

Feminization in men occurs when the effective ratio of androgen to estrogen is lowered. Since sufficient estrogen is produced in normal men to induce breast enlargement in the absence of adequate amounts of circulating androgens, it has been generally assumed that androgens exert an antiestrogenic action to prevent feminization in normal men. We examined the mechanisms of this effect of androgens in the mouse breast. Administration of estradiol via silastic implants to castrated virgin CBA/J female mice results in a doubling in dry weight and DNA content of the breast. The effect of estradiol can be inhibited by implantation of 17 beta-hydroxy-5 alpha-androstan-3-one (dihydrotestosterone), whereas dihydrotestosterone alone had no effect on breast growth. Estradiol administration also enhances the level of progesterone receptor in mouse breast. Within 4 d of castration, the progesterone receptor virtually disappears and estradiol treatment causes a twofold increase above the level in intact animals. Dihydrotestosterone does not compete for binding to the progesterone receptor, but it does inhibit estrogen-mediated increases of progesterone receptor content of breast tissue cytosol from both control mice and mice with X-linked testicular feminization (tfm)/Y. Since tfm/Y mice lack a functional androgen receptor, we conclude that this antiestrogenic action of androgen is not mediated by the androgen receptor. Dihydrotestosterone competes with estradiol for binding to the cytosolic estrogen receptor of mouse breast, whereas 17 beta-hydroxy-5 beta-androstan-3-one (5 beta-dihydrotestosterone) neither competes for binding nor inhibits estradiol-mediated induction of the progesterone receptor. Dihydrotestosterone also promotes the translocation of estrogen receptor from cytoplasm to nucleus; the ratio of cytoplasmic-to-nuclear receptor changes from 3:1 in the castrate to 1:2 in dihydrotestosterone-treated mice. Thus, the antiestrogenic effect of androgen in mouse breast may be the result of effects of dihydrotestosterone on the estrogen receptor. If so, dihydrotestosterone performs one of its major actions independent of the androgen receptor.

Authors

R W Casey, J D Wilson

Effect of monocyte migration on low density lipoprotein transport across aortic endothelial cell monolayers.

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Abstract

Endothelial cell monolayers on polycarbonate filters present a barrier to low density lipoprotein (LDL) and albumin transport. These cells form a relatively tight monolayer as shown by measurements of electrical resistance across the monolayer (15 omega-cm2). Monocytes are able to migrate freely across the monolayers in response to chemotactic stimuli. Monocyte chemotaxis across the monolayer caused a marked increase in LDL and albumin transport across the monolayer in the direction of monocyte migration. However, transport in the opposite direction was not significantly increased. These results suggest that monocyte migration across the endothelium could lead to an increased LDL content of the intima.

Authors

M Territo, J A Berliner, A M Fogelman

Decrease in microviscosity and cholesterol content of rat liver plasma membranes after chronic ethanol feeding.

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Abstract

This investigation was performed to determine whether chronic ethanol feeding alters the lipid composition or the fluidity of liver plasma membranes. Male Sprague-Dawley rats were pair-fed nutritionally adequate liquid diets containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4-5 wk. Contrasting with other membranes, chronic ethanol feeding resulted in an increase in hepatic plasma membrane fluidity as assessed by fluorescence anisotropy. This alteration was associated with a decrease in plasma membrane cholesterol content.

Authors

S Yamada, C S Lieber

L-carnitine therapy in isovaleric acidemia.

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Abstract

Isovaleric acidemia, resulting from isovaleryl-coenzyme A dehydrogenase deficiency, is associated with marked reduction of free carnitine in both plasma and urine. Fast atom bombardment-mass spectrometry, hydrolysis, and gas chromatography/mass spectrometry have unequivocally identified the existence of isovalerylcarnitine, a new metabolite specific for this disorder. Administration of equimolar amounts of glycine or L-carnitine separately with leucine demonstrated that isovaleryl-coenzyme A is removed by supplemental L-carnitine in the form of isovalerylcarnitine as effectively as it is by glycine, in the form of isovalerylglycine. When L-carnitine is given alone, excretion of isovalerylglycine decreases in preference to enhanced excretion of isovalerylcarnitine and hippurate. Treatment with L-carnitine alone has proven effective in preventing further hospitalizations in a patient with this genetic disorder.

Authors

C R Roe, D S Millington, D A Maltby, S G Kahler, T P Bohan

Insulin within islets is a physiologic glucagon release inhibitor.

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Abstract

To determine if glucagon secretion is under physiological control of intra-islet insulin, pancreata from normal rats were perfused at a 100 mg/dl glucose concentration with either guinea pig antiinsulin serum or normal guinea pig serum in a nonrecirculating system. Perfusion of antiserum was followed within 3 min by a significant rise in glucagon that reached peak levels three times the base-line values and assumed a hectic pattern that returned rapidly to base-line levels upon termination of the antiserum perfusion. Nonimmune guinea pig serum had no effect. To gain insight into the probable site of insulin neutralization, 125I-labeled human gamma-globulin was added to antiserum or nonimmune serum and perfused for 3 min. More than 83% of the radioactivity was recovered in the effluent within 3 min after termination of the infusion, and only 0.05 +/- 0.015% of the radioactivity injected was present in the pancreas 10 min after the perfusion. The maximal amount of insulin that could be completely bound to insulin antibody at a dilution and under conditions simulating those of the perfusion experiments was 20 mU/min. It is concluded that insulin maintains an ongoing restraint upon alpha cell secretion and in its absence causes hectic hypersecretion of glucagon. This restraint probably occurs largely in the intravascular compartment. Loss of this release-inhibiting action of insulin may account for initiation of hyperglucagonemia in insulin-deficient states.

Authors

H Maruyama, A Hisatomi, L Orci, G M Grodsky, R H Unger