R W Colman
Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. [3H]thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.
M Rozenberg-Arska, J A van Strijp, W P Hoekstra, J Verhoef
The present study examined the role of cell-mediated immunity (CMI) in the production of experimental autoimmune glomerulonephritis (EAG) in chickens deficient in humorally mediated immunity (HMI). Cyclophosphamide bursectomized (Bsx) and normal control chickens were used. Bsx chickens were used only if they had severe depression of HMI, which was evidenced by marked reduction in bursal weights (0.89 +/- 0.23 vs. 2.92 +/- 0.9 g), decreased serum IgG to less than or equal to 10% of normal, and total lack of HMI to immunization with sheep red blood cells. EAG was produced by immunizing chickens with bovine glomerular basement membrane (GBM) in complete Freund's adjuvant. CMI manifested by wattle thickness increments to PPD was not different, 3.89 +/- 0.45 mm for Bsx compared with 3.73 +/- 0.75 mm for controls. No circulating antibodies to GBM developed in 68% of Bsx chickens, and the anti-GBM titers were less than 1:312 in those Bsx chickens positive for antibody compared with greater than 2,000 for controls. GBM deposits of IgG by fluorescence were much decreased, 0.53 +/- 0.16 compared with 2.19 +/- 0.32 for controls, and were absent in 64% of Bsx chickens. Nonetheless, proliferative nephritis with crescents was present and was even more severe in Bsx chickens than in controls, with glomerular sizes of 20.8 +/- 0.6 U for Bsx-GBM, 19.8 +/- 1.2 for control-GBM, 14.9 +/- 1.5 for Bsx, and 13.6 +/- 0.8 for normal chickens. Nephritic eluates did not produce disease in normal chickens, while administration of sensitized cells with [H3]thymidine to naive birds was associated with increased mesangial grain counts by autoradiography. These findings suggest that CMI plays a major role in the pathogenesis of EAG in chickens in the absence of HMI. By implication, CMI may be crucial in the development of other types of glomerulonephritis as well.
W K Bolton, F L Tucker, B C Sturgill
The circumstantial evidence that indicates that glucocorticoids (GC) may stimulate osteoclastic resorption in vivo has recently found support in observations that demonstrate that these compounds effectively increase the activity of isolated resorptive cells (osteoclasts, macrophage polykaryons, and elicited macrophages [MO] ) in vitro. Data are presented here that indicate that this stimulation by GC is due to an enhancement of the initial stage of the resorption process, the attachment of cells to bone, and that this is caused by alterations of cell surface oligosaccharides. Specifically, dexamethasone and cortisol enhance by 80% the attachment of MO to bone surfaces in a dose dependent manner but do not alter or reduce the binding of these cells to other surfaces (plastic, collagen, and hydroxyapatite crystals). The effect of GC on cell-bone attachment is blocked by the glycosylation inhibitor, tunicamycin, and the glycosylation modifier, swainsonine; this demonstrates that asparagine-linked oligosaccharides are involved in the stimulatory process. Flow cytometric analysis of GC-treated cells using a panel of fluoresceinated lectins confirms this by indicating a selective, enhanced exposure of plasma membrane-associated N-acetylglucosamine and N-acetylgalactosamine residues, sugars we have previously shown to be pivotal in MO-bone binding. Finally, progesterone, a known GC antagonist, blocks GC-stimulated resorption, macrophage-bone binding, and membrane oligosaccharide modification, presumably by competing for the GC receptor. Progesterone alone alters none of these processes. Thus, GC stimulates the resorptive activity of macrophages by enhancing the initial events in the degradative process (cell-bone binding) and does so, apparently, via receptor-mediator alteration of cell surface glycoproteins.
Z Bar-Shavit, A J Kahn, L E Pegg, K R Stone, S L Teitelbaum
A newly synthesized human big gastrin (G34) that was prepared according to the revised structure and that contained less than 3% oxidized methionine residues was compared with synthetic human little gastrin (G17) for acid-stimulating activity and clearance in human subjects. Prolonged infusions of each type of gastrin revealed that the time required to approach stable plasma concentrations was much longer for G34 than for G17. The time course of plasma gastrin concentration could be described by one-compartment models with half-lives of 44 min for G34 and 8 min for G17. After rapid intravenous infusion, G34 produced a much larger total acid response than did an equimolar dose of G17, and the responses were directly proportional to the integrated plasma gastrin increments. During the third hour of prolonged intravenous infusions of G34 and G17, the exogenous dosage of G34 required to produce the same blood concentration of gastrin was only one-fourth that of G17. Equivalent blood concentrations of G34 and G17 were associated with similar rates of acid secretion. These results suggest that G34 is more potent than has been thought, that it has an activity similar to that of G17 and that it must not be ignored in estimating total acid-stimulating activity of circulating gastrins. The measurement of total carboxyl-terminal immunoreactive gastrin can produce a good estimate of total acid-stimulating activity.
V E Eysselein, V Maxwell, T Reedy, E Wünsch, J H Walsh
To study the value of 4-aminopyridine as an antidote to verapamil intoxication, we subjected 12 adult cats to verapamil poisoning by administering doses of 4.0-25.0 mg/kg verapamil by intravenous infusion. Six animals were given 4-aminopyridine 2 X 0.5 mg/kg i.v. after the verapamil infusion was stopped and the other six animals (the control group) were not. Verapamil caused profound cardiovascular depression and also partial neuromuscular block, both of which were completely reversed by 4-aminopyridine within 50 min, in spite of extremely high serum verapamil concentrations (ranging between 3,700 and 13,500 ng/ml). The six animals that received 4-aminopyridine survived the verapamil intoxication, whereas four of the six animals in the control group died. The results suggest that 4-aminopyridine may be useful in the treatment of verapamil intoxication.
S Agoston, E Maestrone, E J van Hezik, J M Ket, M C Houwertjes, D R Uges
Triggered neutrophils rapidly degraded labeled matrices secreted by cultured, venous endothelial cells via a process dependent on elastase but not oxygen metabolites. In the presence of high concentrations of alpha-1-proteinase inhibitor, the ability of the stimulated neutrophil to solubilize the matrix was impaired. However, at lower concentrations of alpha-1-proteinase inhibitor the neutrophil could enhance the degradative potential of its released elastase by a H2O2-dependent process. Coincident with this increase in matrix damage, the stimulated neutrophil destroyed the elastase inhibitory activity of the alpha-1-proteinase inhibitor via a catalase-inhibitable process. The ability of the triggered neutrophil to solubilize the matrix in the presence of alpha-1-proteinase inhibitor was unaffected by superoxide dismutase or hydroxyl radical scavengers but was markedly impaired by catalase, azide, or hypochlorous acid scavengers. We conclude that neutrophils can cooperatively use an oxidant with characteristics similar, if not identical, to hypochlorous acid and the lysosomal proteinase elastase to negate the protective effects of alpha-1-proteinase inhibitor in order to attack the subendothelial matrix.
S J Weiss, S Regiani
The metabolic clearance rate (MCR) and plasma disappearance rate (t1/2) of human pancreatic tumor growth hormone releasing factor [hpGRF(1-40)] was determined in normal adult male subjects by single injection and constant infusion techniques. Single injections of 1, 3.3, and 10 micrograms/kg hpGRF(1-40) were administered intravenously, plasma immunoreactive (IR) GRF levels were measured during the subsequent 180 min, and biexponential curve analysis was performed. Graded, dose-constant infusions of hpGRF(1-40) at rates of 1, 3.3, 10, and 33 ng/kg per min were administered and the MCR was calculated from measurement of steady state plasma IR-GRF levels at each infusion rate. The postinfusion disappearance rate was determined by linear regression analysis of plasma IR-GRF levels during the 120-min period after cessation of the infusion. The calculated MCR during the single injection study was 194 +/- 17.5 liters/m2 per d and was not significantly different from the calculated value during the constant infusion study (202 +/- 16 liters/m2 per d). The disappearance rate during the single injection study was subdivided into two linear phases: an initial equilibration phase (7.6 +/- 1.2 min) and a subsequent elimination phase (51.8 +/- 5.4 min). The latter was similar to the linear disappearance rate observed (41.3 +/- 3.0 min) after cessation of the constant infusion. The chromatographic and biologic characteristics of plasma IR-GRF, 30 min after injection, were similar to those of synthetic hpGRF(1-40). The results have been discussed in relation to the MCR of other hypothalamic hormones and have been used to extrapolate secretion rates of GRF in patients with ectopic GRF production.
L A Frohman, J L Thominet, C B Webb, M L Vance, H Uderman, J Rivier, W Vale, M O Thorner
Certain strains of Staphylococcus aureus associated with toxic shock syndrome elaborate material that induces human blood monocytes to secrete interleukin-1 (IL-1). IL-1 was detected both by its ability to cause fever in rabbits using the leukocytic pyrogen (LP) assay and by its mitogenic activity towards thymocytes in the so-called lymphocyte-activating factor (LAF) assay. Anti-human IL-1 prevents the manifestation of both activities. Filtrates of control strains of S. aureus manifest neither activity. Thus, culture filtrates derived from toxic shock syndrome (TSS)-associated strains cause biphasic fever in rabbits when injected intravenously. The fever lasts several hours. Plasma taken at the peak of the fever and injected into a second set of rabbits produces a brief monophasic fever typical of LP. Further, human monocytes release LP when incubated with TSS filtrates in vitro. The monocyte products also stimulate the proliferation of mouse thymocytes in the presence of phytohemagglutinin in a manner characteristic of LAF. A bacterial filtrate is much less effective without an intermediate incubation with monocytes. The stimulation of monocyte IL-1 production is easily quantified, provides a simple method of assaying the TSS toxin, and since it involves human cells, is directly relevant to the human disease. The assay was used to monitor the purification of TSS toxin. Only 0.1 ng/ml of the purified material is required to induce monocyte IL-1 production. It is thus more potent than endotoxin. In contrast to endotoxin, its effect is not blocked by polymyxin B. We conclude that in TSS the sudden fever and probably other components of the acute phase response may be attributed to a massive release of IL-1.
T Ikejima, C A Dinarello, D M Gill, S M Wolff
The pathophysiology of reduced maximum expiratory flow in a canine model of pulmonary emphysema was studied, and the results interpreted in terms of the wave-speed theory of flow limitation. According to this theory, maximum expiratory flow is related both to the cross-sectional area and compliance at an airway site where a critical gas velocity is first reached ("choke-point") and to gas density. Pulmonary emphysema was produced by the repeated instillations of the enzyme papain into the airways of six dogs. In five control dogs, a saline solution was instilled. During forced vital capacity deflation, in an open-chest preparation, maximum expiratory flow, choke-point locations, and the response to breathing an 80:20 helium/oxygen gas mixture were determined at multiple lung volumes. To locate choke-points, a pressure measuring device was positioned in the airway to measure lateral and end-on intrabronchial pressures, from which the relevant wave-speed parameters were obtained. In general, the reduced maximum expiratory flow in emphysema can be explained by diminished lung elastic recoil pressure and by altered bronchial pressure-area behavior, which results in a more peripheral location of choke-points that have smaller cross-sectional areas than controls. With respect to the density dependence of maximum expiratory flow, this response did not differ from control values in four dogs with emphysema in which frictional pressure losses upstream from choke-points did not differ on the two gas mixtures. In two dogs with emphysema, however, upstream frictional pressure losses were greater on helium/oxygen than on air, which resulted in a smaller cross-sectional area on helium/oxygen; hence density dependence decreased.
S N Mink
Decreased activity of the guanine nucleotide regulatory protein (N) of the adenylate cyclase system is present in cell membranes of some patients with pseudohypoparathyrodism (PHP-Ia) whereas others have normal activity of N (PHP-Ib). Low N activity in PHP-Ia results in a decrease in hormone (H)-stimulatable adenylate cyclase in various tissues, which might be due to decreased ability to form an agonist-specific high affinity complex composed of H, receptor (R), and N. To test this hypothesis, we compared beta-adrenergic agonist-specific binding properties in erythrocyte membranes from five patients with PHP-Ia (N = 45% of control), five patients with PHP-Ib (N = 97%), and five control subjects. Competition curves that were generated by increasing concentrations of the beta-agonist isoproterenol competing with [125I]pindolol were shallow (slope factors less than 1) and were computer fit to a two-state model with corresponding high and low affinity for the agonist. The agonist competition curves from the PHP-Ia patients were shifted significantly (P less than 0.02) to the right as a result of a significant (P less than 0.01) decrease in the percent of beta-adrenergic receptors in the high affinity state from 64 +/- 22% in PHP-Ib and 56 +/- 5% in controls to 10 +/- 8% in PHP-Ia. The agonist competition curves were computer fit to a "ternary complex" model for the two-step reaction: H + R + N in equilibrium HR + N in equilibrium HRN. The modeling was consistent with a 60% decrease in the functional concentration of N, and was in good agreement with the biochemically determined decrease in erythrocyte N protein activity. These in vitro findings in erythrocytes taken together with the recent observations that in vivo isoproterenol-stimulated adenylate cyclase activity is decreased in patients with PHP (Carlson, H. E., and A. S. Brickman, 1983, J. Clin. Endocrinol. Metab. 56:1323-1326) are consistent with the notion that N is a bifunctional protein interacting with both R and the adenylate cyclase. It may be that in patients with PHP-Ia a single molecular and genetic defect accounts for both decreased HRN formation and decreased adenylate cyclase activity, whereas in PHP-Ib the biochemical lesion(s) appear not to affect HRN complex formation.
J A Heinsimer, A O Davies, R W Downs, M A Levine, A M Spiegel, M K Drezner, A De Lean, K A Wreggett, M G Caron, R J Lefkowitz
The purpose of these experiments was to estimate insulin biosynthesis in vivo in a rat model for non-insulin-dependent diabetes. Insulin biosynthesis rates were determined in 4-wk-old animals that had been injected with 90 mg/kg of streptozotocin 2 d postpartum. Control and diabetic animals did not differ in body weight or fasting plasma glucose. Fed plasma glucose was significantly elevated (186 +/- 13 micrograms/dl vs. 139 +/- 7 mg/dl, P less than 0.05) and pancreatic insulin content was reduced (41 +/- 2 micrograms/g vs. 63 +/- 8 micrograms/g, P less than 0.05) in the diabetic rats. Insulin biosynthesis was estimated in vivo by measuring and comparing [3H]leucine incorporation into proinsulin with that into total pancreatic protein 45 min after injection. Insulin biosynthesis was 0.391 +/- 0.07% of pancreas protein synthesized in control rats and 0.188 +/- 0.015% (P less than 0.05) in diabetic rats. In animals of the same age, the fractional and absolute rate of pancreatic protein synthesis were determined. Total pancreatic protein synthesis was not reduced in streptozotocin treated animals (185.5 +/- 14.1%/d vs. 158.6 +/- 14.9%/d, NS) but was markedly reduced in control rats after a 48-h fast (to 70.8 +/- 5.5%/d, P less than 0.01). Because total pancreatic protein synthesis was not decreased in the diabetic rats, the decrease in the fraction of radiolabel incorporated into insulin seems to represent an absolute decrease in the rate of insulin biosynthesis in this animal model for diabetes. Through RNA blot hybridization with 32P-labeled cloned rat insulin complementary DNA, proinsulin messenger RNA (mRNA) was estimated as the rate of insulin biosynthesis in control and diabetic animals. There was a 61% reduction in proinsulin mRNA at 4 wk and an 85% reduction at 7 wk (P less than 0.001) in the diabetic animals. After streptozotocin injection in neonatal rats, there is marked beta-cell damage and hyperglycemia. Beta-cell regeneration occurs with return to normoglycemia, but with age hyperglycemia develops. The reduction in insulin synthesis and proinsulin mRNA seemed disproportionate with the more modest reduction in beta-cell number. The importance of these observations is that, in this animal model, diabetes is associated with a limited ability to regenerate beta-cell mass and to synthesize insulin. The relationship between the defect in glucose-stimulated insulin release and impaired insulin biosynthesis has yet to be determined.
M A Permutt, K Kakita, P Malinas, I Karl, S Bonner-Weir, G Weir, S J Giddings
We have already demonstrated that a hyperinsulinemic, diabetic subject secreted an abnormal insulin in which serine replaced phenylalanine B24 (Shoelson S., M. Fickova, M. Haneda, A. Nahum, G. Musso, E. T. Kaiser, A. H. Rubenstein, and H. Tager. 1983. Proc. Natl. Acad. Sci. USA. 80:7390-7394). High performance liquid chromatography analysis now shows that the circulating insulin in several other family members also consists of a mixture of the abnormal human insulin B24 (Phe----Ser) and normal human insulin in a ratio of approximately 9.5:1 during fasting. Although all affected subjects show fasting hyperinsulinemia, only the propositus and her father are overtly diabetic. Analysis of the serum insulin from two nondiabetic siblings revealed that normal insulin increased from approximately 2 to 15% of total serum insulin after the ingestion of glucose and that the proportion of the normal hormone plateaued or fell while the level of total insulin continued to rise. Animal studies involving the graded intraportal infusion of equimolar amounts of semisynthetic human [SerB24]-insulin and normal human insulin in pancreatectomized dogs (to simulate the secretion of insulin due to oral glucose in man) also showed both a rise in the fraction of normal insulin that reached the periphery and the attainment of a brief steady state in this fraction while total insulin levels continued to rise. Separate experiments documented a decreased hepatic extraction, a decreased metabolic clearance rate, and an increased plasma half-life of human [SerB24]-insulin within the same parameters as those determined for normal human insulin. These results form a basis for considering (a) the differential clearance of low activity abnormal insulins and normal insulin from the circulation in vivo, and (b) the causes of hyperinsulinemia in both diabetic and nondiabetic individuals who secrete abnormal human insulins.
S E Shoelson, K S Polonsky, A Zeidler, A H Rubenstein, H S Tager
This study assesses the relationship between the distribution of thallium-201 and myocardial blood flow during coronary vasodilation induced by intravenous dipyridamole in canine models of partial and complete coronary artery stenosis. 10 dogs were chronically instrumented with catheters in the left atrium and aorta and with a balloon occluder and electromagnetic flow probe on the proximal left circumflex coronary artery. Regional myocardial blood flow was measured during control conditions with radioisotope-labeled microspheres, and the phasic reactive hyperemic response to a 20-s transient occlusion was then recorded. Dipyridamole was then infused intravenously until phasic coronary blood flow increased to match peak hyperemic values. The left circumflex coronary artery was either partially occluded to reduce phasic blood flow to control values (group 1) or it was completely occluded (group 2), and thallium-201 and a second microsphere label were injected. 5 min later, the animals were sacrificed, the left ventricle was sectioned into 1-2-g samples, and thallium-201 activity and regional myocardial blood flow were measured. Curvilinear regression analyses between thallium-201 localization and myocardial blood flow during dipyridamole infusion demonstrated a slightly better fit to a second- as compared with a first-order model, indicating a slight roll-off of thallium activity as myocardial blood flow increases. During the dipyridamole infusion, the increases in phasic blood flow, the distributions of regional myocardial blood flow, and the relationships between thallium-201 localization and regional blood flow were comparable to values previously observed in exercising dogs with similar occlusions. These data provide basic validation that supports the use of intravenous dipyridamole and thallium-201 as an alternative to exercise stress and thallium-201 for evaluating the effects of coronary occlusive lesions on the distribution of regional myocardial blood flow.
A E Mays Jr, F R Cobb
Nine insulin-dependent diabetics and six healthy controls were studied at rest, during, and after 60 min of bicycle exercise at a work load corresponding to 45% of their maximal oxygen intake. The catheter technique was employed to determine splanchnic and leg exchange of metabolites. FFA turnover and regional exchange was evaluated using [14C]oleate infusion. Basal glucose (13.8 +/- 1.1 mmol/l), ketone body (1.12 +/- 0.12 mmol/l), and FFA (967 +/- 110 mumol/l) concentrations were elevated in the diabetics in comparison with controls. In the resting state, splanchnic ketone acid production in the diabetics was 6-10-fold greater than in controls. Uptake of oleic acid by the splanchnic bed was increased 2-3-fold, and the proportion of splanchnic FFA uptake converted to ketones (61%) was threefold greater than in controls. In contrast, splanchnic fractional extraction of oleic acid was identical in diabetics and controls. A direct relationship was observed between splanchnic uptake and splanchnic inflow (plasma concentration X hepatic plasma flow) of oleic acid that could be described by the same regression line in the diabetic and control groups. During exercise, splanchnic ketone production rose in both groups. In the control group the increase in ketogenesis was associated with a rise in splanchnic inflow and in uptake of oleic acid, a rise in splanchnic fractional extraction of oleate, and an increase in the proportion of splanchnic FFA uptake converted to ketone acids from 20-40%. In the diabetic group, the increase in ketogenesis occurred in the absence of a rise in splanchnic inflow or uptake of oleic acid, but was associated with an increase in splanchnic fractional extraction of oleic acid and a marked increase in hepatic conversion of FFA to ketones, so that the entire uptake of FFA was accountable as ketone acid output. Splanchnic uptake of oleic acid correlated directly with splanchnic oleic acid inflow in both groups, but the slope of the regression line was steeper than in the resting state. Plasma glucagon levels were higher in the diabetic group at rest and during exercise, while plasma norepinephrine showed a twofold greater increment in response to exercise in the diabetic group (to 1,400-1,500 pg/ml). A net uptake of ketone acids by the leg was observed during exercise but could account for less than 5% of leg oxidative metabolism in the diabetics and less than 1% in controls. Despite the increase in ketogenesis during exercise, a rise in arterial ketone acid levels was not observed in the diabetics until postexercise recovery, during which sustained increments to values of 1.8-1.9 mmol/l and sustained increases in splanchnic ketone production were observed at 30-60 min. The largest increment in blood ketone acids and in splanchnic ketone production above values observed in controls thus occurred in the diabetics after 60 min of recovery from exercise. We concluded that: (a) In the resting state, increased ketogenesis in the diabetic is a consequence of augmented splanchnic inflow of FFA and increased intrahepatic conversion of FFA to ketones, but does not depend on augmented fractional extraction of circulating FFA by the splanchnic bed. (b) Exercise-induced increases in ketogenesis in normal subjects are due to augmented splanchnic inflow and fractional extraction of FFA as well as increased intrahepatic conversion of FFA to ketones. (c) When exercise and diabetes are combined, ketogenesis increases further despite the absence of a rise in splanchnic inflow of FFA. An increase in splanchnic fractional extraction of FFA and a marked increase intrahepatic conversion of FFA to ketones accounts for the exaggerated ketogenic response to exercise in the diabetic. (d) Elevated levels of plasma glucagon and/or norepinephrine may account for the increased hepatic ketogenic response to exercise in the diabetic. (e) Ketone utilization by muscle increases during exercise but constitutes a quantitatively minor oxidative fuel for muscle even in the diabetic. (f) The accelerated ketogenesis during exercise in the diabetic continues unabated during the recovery period, resulting in an exaggerated postexercise ketosis.
J Wahren, Y Sato, J Ostman, L Hagenfeldt, P Felig
Normal volunteers received either initial or booster immunization with tetanus toxoid. Bone marrow and peripheral blood mononuclear cells were obtained for up to 28 d after immunization and were analyzed for synthesis of total Ig and specific antibodies to tetanus toxoid. Cells were cultured in vitro for 3 or 7 d with or without pokeweed mitogen (PWM). Synthesis of IgG and IgM antibodies to tetanus (IgG-Tet and IgM-Tet) and total IgG and IgM was determined by radioimmunoassay. Four functional B cell subpopulations were detected in the bone marrow after booster tetanus immunization: (a) B cells that spontaneously synthesized IgG-Tet appeared on day 7 after immunization but were undetectable by day 21; (b) B cells that synthesized IgG-Tet after stimulation with PWM appeared after day 21 and persisted for greater than 1 mo; (c) B cells that synthesized IgM-Tet in the presence of PWM were detectable before and after immunization; and (d) B cells that spontaneously synthesized IgM-Tet appeared on day 7 and were undetectable by day 21. In contrast to the other three types of bone marrow B cells described, this fourth subpopulation of PWM-independent IgM-Tet-synthesizing B cells was not detected in the peripheral blood. After primary immunization, no spontaneous antibody-producing cells were detected in the blood or bone marrow, although there was a small rise in IgM-Tet in two of three subjects. In the bone marrow, only IgM-Tet PWM-inducible cells were seen, although mitogen-responsive IgM and IgG-Tet cells were detected in the circulation. The IgM-Tet PWM-reactive cells were present even before primary antigen exposure and appear to represent the initial B cells involved in the antibody response. These data indicate that there are specific times after immunization when different functional classes of anti-Tet-synthesizing B cells and memory B cells appear in human bone marrow. Knowledge of these data may be important in developing a strategy for the transfer of immune memory from donors to recipients in the setting of bone marrow transplantation.
H Kodo, R P Gale, A Saxon
The mechanism of hypoxemia during hemodialysis was investigated by the multiple inert gas elimination technique in anesthetized, paralyzed, mechanically ventilated dogs. Profound leukopenia occurred in the first hour of a 2-h hemodialysis with a cuprophan membrane and dialysate that contained acetate. Arterial partial pressure of O2 and CO2 and oxygen consumption remained unchanged during dialysis. Pulmonary carbon dioxide elimination and lung respiratory exchange ratio decreased with the initiation of dialysis, remained depressed throughout the duration of dialysis, and returned to predialysis levels after the cessation of dialysis. Cardiac output diminished during dialysis but did not return to base-line levels after dialysis. Multiple indices calculated from inert gas analysis revealed no ventilation-perfusion mismatching during dialysis. The shunt and perfusion to regions of low alveolar ventilation-to-perfusion ratio (VA/Q) were unchanged during dialysis. There was no change in the mean or standard deviation of the profile of the percentage of total perfusion to regions of the lung that had VA/Q near 1.0; nor was there any increase in the directly calculated arterial-alveolar partial pressure differences for the inert gases during dialysis. Dead space became mildly elevated during dialysis. These results show that during dialysis with controlled ventilation there is no ventilation-perfusion mismatching that leads to hypoxemia. During spontaneous ventilation any hypoxemia must occur due to hypoventilation secondary to the CO2 exchange by the dialyzer and subsequent reduction in pulmonary CO2 exchange.
D D Ralph, S M Ott, D J Sherrard, M P Hlastala
The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX by a modification of a method described for bovine Factor IX activation by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707). Initial rates of trichloroacetic acid-soluble 3H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl2 (5 mM) and with pure (greater than 98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of 3H preceded the appearance of Factor IXa activity, and the percentage of 3H released remained constant when the mole fraction of 3H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r greater than 0.98, P less than 0.001) was observed between initial rates of 3H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include Km (0.49 microM) of about five- to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (kcat) (7.7/s) is approximately 20-50 times higher than that reported by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707) for Factor IX activation by Factor VIIa plus tissue factor. Therefore, depending on the relative amounts of Factor XIa and Factor VIIa generated in vivo and other factors which may influence reaction rates, these kinetic parameters provide part of the information required for assessing the relative contributions of the intrinsic and extrinsic pathways to Factor IX activation, and suggest that the Factor XIa catalyzed reaction is physiologically significant.
P N Walsh, H Bradford, D Sinha, J R Piperno, G P Tuszynski
To examine the mechanism of mitral flow deceleration in diastole and its potential influence on the genesis of third (S3) and fourth (S4) heart sounds, we simultaneously recorded left atrial and left ventricular pressures (micromanometers), mitral flow velocity (electromagnetic catheter-tip flow velocity meter), and internal and external phonocardiograms in 25 open-chest dogs. Diastolic time intervals, transmitral pressure gradients (planimetry), maximum mitral flow velocity, and acceleration and deceleration of flow were measured under different loading conditions. It was found that deceleration of mitral flow in early and late diastole is always caused by a negative transmitral pressure gradient. After volume loading, diastolic pressures, positive (forward) and negative (backward) transmitral pressure gradients, and acceleration and deceleration of flow increased, and an S3 or S4 appeared (20:25 dogs). These sounds occurred during the phase of flow deceleration and could be recorded from the chest wall, inside the left ventricle, and directly from the epicardial surface of the freely exposed left ventricular wall. After balloon occlusion of the inferior vena cava (17:25 dogs), the opposite changes were observed and gallop sounds disappeared. The results indicate that the left ventricular pressure rise in response to filling reverses the transmitral pressure gradient and decelerates flow. Deceleration of inflow by the left ventricular wall in early and late diastole may represent a key mechanism in the genesis of S3 and S4.
F Van de Werf, J Minten, P Carmeliet, H De Geest, H Kesteloot
A platelet aggregometer was adapted for the simultaneous measurement of perpendicular light scattering in addition to light transmission. The addition of chemoattractants to polymorphonuclear leukocyte suspensions evoked a single wave of increased light transmission, whereas the perpendicular scattering measurement demonstrated a previously unrecognized biphasic response. The first perpendicular scattering response had no detectable latency and peaked at 10 +/- 1 s, then decayed rapidly. The second response peaked at 40 +/- 5 s, and decayed over several minutes. The dose-response curve of chemoattractants for inducing the rapid (10 +/- 1 s) perpendicular scattering peak corresponded to that which initiated chemotaxis. Initiation of the slow (40 +/- 5 s) peak required 10-fold higher amounts of chemoattractants, and the dose-response curve correlated with the induction of lysosomal enzyme secretion and superoxide anion production. Low doses of aliphatic alcohols, which have been shown to enhance chemotaxis but to inhibit secretion and superoxide anion production, abolished the slow perpendicular light-scattering response but left the fast response intact. Stimulants of secretion induced only slow and prolonged responses that were best observed in transmission measurements. In an attempt to resolve the origin of the light-scattering responses, the morphological changes of polymorphonuclear leukocytes were examined microscopically. Neither aggregation nor morphological whole cell polarization could be correlated with changes in light transmission or perpendicular scattering, which suggested that the source of scattering is of subcellular dimensions. The rapid perpendicular light-scattering response of polymorphonuclear leukocytes to chemoattractants appears to record an initial event in the stimulus-response coupling, and its measurement should provide a useful new tool for the study of leukocyte function. The biphasic nature of the light-scattering responses to chemoattractants, moreover, correlates with the dual regulation of the chemotactic and secretory responses of leukocytes.
I Yuli, R Snyderman
To assess the role of the early postnatal surge in plasma thyroid hormone concentrations on cardiovascular and metabolic adaptations, we measured cardiac output, total oxygen consumption, and plasma triiodothyronine (T3) concentrations in three groups of lambs in the first 6 h after delivery. 15 fetal lambs were prepared at gestational ages of 128-129 d by placing catheters in the brachiocephalic artery, descending aorta, distal inferior vena cava, left atrium, and pulmonary artery so that measurements could be made soon after delivery. They were divided into three groups: Group I comprised five control animals; Group II consisted of five fetuses in which thyroidectomy was performed at surgery at 129 d gestation; and Group III consisted of five animals in which thyroidectomy was performed at term gestation during delivery by caesarian section, prior to severing the umbilical cord. The lambs in Group I exhibited a rapid postnatal rise in T3 concentrations, similar to that described previously, reaching a peak value of about 5 ng/ml. Although the postnatal surge in T3 concentration was arrested in Group II and III animals, Group II had no detectable plasma T3, while the Group III animals had T3 concentrations of about 0.8 ng/ml, which were within the range previously reported for term lamb fetuses. The lambs in group II showed 40-50% lower left ventricular outputs (190 vs. 297 ml/kg per min), systemic blood flows (155 vs. 286 ml/kg per min), and oxygen consumptions (9.8 vs. 20.2 ml/kg per min) as compared with Group I animals over the entire 6-h period. The lambs in Group II also had significantly lower heart rates (131 vs. 192 beats/min) and mean systemic arterial pressures (56 vs. 72 torr). However, there were no significant differences for any of these measurements between the Group III and Group I lambs. The reduction in cardiac output in the Group II animals were reflected in a significantly lower blood flow to the peripheral circulation, but there were no significant differences in blood flow to other organs in the three groups. These studies indicate that plasma thyroid concentrations in the 2-3 wk prior to delivery and not the increase in thyroid hormone concentrations which occur after birth are important for postnatal cardiovascular and metabolic adjustments. We speculate that lack of circulating triiodothyronine in late gestation may affect postnatal cardiovascular adaptation by modifying normal beta adrenergic receptor development.
J A Breall, A M Rudolph, M A Heymann
The effects of insulin on basal and hydrocortisone-induced growth hormone (GH) secretion were studied in rat pituitary tumor cells (GH3). Cells were grown in monolayer culture and exposed to exogenously added insulin for up to 8 d. Basal GH secretion was inhibited by insulin (0.7 nM) after a 48-h lag period by approximately 50% (P less than 0.01, vs. untreated control cells). The suppression of GH secretion was reversible, as removal of added insulin resulted in return of GH secretion to normal levels after 24 h. Maximal suppression of basal GH secretion was achieved by 0.7 nM insulin, and these effects were prevented by simultaneous exposure of the cells to guinea pig anti-insulin serum (1:2,000). No effects of insulin on cell replication were evident, and glucose concentration in the medium did not differ in control or insulin-treated wells. Insulin (7 nM) significantly suppressed the fivefold hydrocortisone-induced GH stimulation during 5 d of incubation with up to 1,000 nM of the steroid (P less than 0.001). These inhibitory effects were similarly observed in glucose- and pyruvate-free medium, and in the presence of 2-deoxyglucose. Insulin also reversed the suppression of prolactin (PRL) secretion induced by hydrocortisone (1 uM), and actually stimulated basal PRL secretion by over 50%. Insulin did not alter the inhibitory effect of hydrocortisone on GH3 cell proliferation. Although higher doses (13 nM) of insulin-like growth factor (IGF-I) also suppressed basal GH secretion, IGF-I did not alter the GH and PRL secretory changes induced by hydrocortisone. The results show that insulin exerts a direct, specific inhibitory effect on basal and hydrocortisone-induced GH secretion by GH3 cells unrelated to glucose utilization by the cells.
The receptors in the fundic mucosa that mediate gastrin stimulation of acid secretion have been studied. Synthetic human gastrin-17-I (G17) with a leucine substitution in the 15th position ( [Leu15]-G17) was iodinated by chloramine T; high saturable binding was found to enzyme-dispersed canine fundic mucosal cells. 127I-[Leu15]-G17, but not 127I-G17, retained binding potency and biological activity comparable with uniodinated G17. Fundic mucosal cells were separated by size by using an elutriator rotor, and specific 125I-[Leu-15]-G17 binding in the larger cell fractions was highly correlated with the distribution of parietal cells. There was, however, specific gastrin binding in the small cell fractions, not accounted for by parietal cells. Using sequential elutriation and stepwise density gradients, highly enriched parietal and chief cell fractions were prepared; 125I-[Leu15]-G17 binding correlated positively with the parietal cell (r = 0.98) and negatively with chief cell content (r = -0.96). In fractions enriched to 45-65% parietal cells, specific 125I-[Leu15]-G17 binding was rapid, reaching a steady state at 37 degrees C within 30 min. Dissociation was also rapid, with the rate similar after 100-fold dilution or dilution plus excess pentagastrin. At a tracer concentration from 10 to 30 pM, saturable binding was 7.8 +/- 0.8% per 10(6) cells (mean +/- SE) and binding in the presence of excess pentagastrin accounted for 11% of total binding. G17 and carboxyl terminal octapeptide of cholecystokinin (26-33) were equipotent in displacing tracer binding and in stimulating parietal cell function ( [14C]aminopyrine accumulation), whereas the tetrapeptide of gastrin (14-17) had a much lower potency. Proglumide inhibited gastrin binding and selectively inhibited gastrin stimulation of parietal cell function. Canine parietal cells have specific receptors for gastrin that mediate stimulation of parietal cell function. Gastrin receptors were undetectable on chief cells, and yet present on another smaller mucosal cell(s).
A H Soll, D A Amirian, L P Thomas, T J Reedy, J D Elashoff
Micropuncture and/or morphologic studies were performed in seven groups of uninephrectomized (UNX) adult male Munich-Wistar rats. Control groups 1, 3, and 6 received standard (24% protein) chow and tap water. Groups 2, 4, and 5 received weekly injections of desoxycorticosterone pivilate (DOC) and 1% saline for drinking, groups 2 and 4 were fed standard chow, and Group 5 a diet containing 6% protein. Group 7 received DOC, salt, and standard chow for 3 wk followed by withdrawal of DOC and salt for an additional 6 wk. 10-14 d after UNX, groups 1 and 2 exhibited similar single nephron glomerular filtration rates (SNGFR) and initial glomerular plasma flow rates (QA). Group 2 had higher mean arterial pressure (AP) and glomerular capillary hydraulic pressure (PGC) than group 1. 3-4 wk after UNX, group 4 exhibited further elevations in AP and PGC as compared with groups 2 and 3. SNGFR and QA were similar in groups 3 and 4, but these average values were greater than typical for normal rats. Group 4 also demonstrated increased urinary protein excretion. Morphologic evaluation of glomeruli in groups 2 and 4 revealed mesangial expansion and focal intraglomerular hemorrhage whereas glomeruli of groups 1 and 3 were essentially normal. Values for AP and PGC in group 5 were not different than group 3 but significantly lower than group 4. QA and SNGFR were lower in group 5 (low protein) than in groups 3 and 4. Furthermore, proteinuria and glomerular structural lesions were abolished in group 5. Morphologic studies performed in groups 6 and 7 showed that early DOC-SALT lesions progress to focal glomerular sclerosis. These studies suggest that continued elevations in glomerular capillary flows and pressures predispose to glomerular injury in this model of systemic arterial hypertension.
L D Dworkin, T H Hostetter, H G Rennke, B M Brenner
Previous study strongly suggests that silicotic fibrosis is mediated by macrophages and their soluble mediators. The biochemical properties of the mediators involved in silicotic fibrosis, however, are as yet ill defined. The current study, therefore, determined whether human monocyte-macrophages treated with fibrogenic silica dust released factors capable of activating fibroblasts as measured by an increase in fibroblast proliferation. Silica, but not nonfibrogenic diamond dust, stimulated the release of fibroblast proliferation factors. Moreover, the level of fibroblast proliferation activity was comparable with the level of thymocyte proliferation (interleukin-1) activity in the same culture supernatants. The factors responsible for these seemingly diverse activities were found to behave identically when analyzed by gel filtration chromatography, size exclusion chromatography, isoelectrofocusing, ion exchange chromatography, and hydrophobic chromatography. Moreover, the response of these factors to four different proteases and heat (56 degrees C) was also identical, which shows that their comigration on various separation media could not be explained by noncovalent interaction between otherwise unrelated species. The data demonstrate that a monocyte-derived thymocyte proliferation factor having the molecular properties of interleukin 1 is capable of regulating fibroblast proliferation. In silicosis and other fibrotic diseases, the local release of interleukin 1 may contribute to abnormal connective tissue deposition by stimulating fibroblast proliferation, and thereby, amplifying other signals stimulating the synthesis of connective tissue components.
J A Schmidt, C N Oliver, J L Lepe-Zuniga, I Green, I Gery
Pressure overload left ventricular (LV) hypertrophy was produced by banding the ascending aorta of puppies and allowing them to grow to adulthood. LV free wall weight per body weight increased by 87% from a normal value of 3.23 +/- 0.19 g/kg. Hemodynamic studies of conscious dogs with LV hypertrophy and of normal, conscious dogs without LV hypertrophy showed similar base-line values for mean arterial pressure, heart rate, and LV end-diastolic pressure and diameter. LV systolic pressure was significantly greater, P less than 0.01, and LV stroke shortening was significantly lss, P less than 0.01, in the LV hypertrophy group. In both normal and LV hypertrophy groups, increasing bolus doses of norepinephrine or isoproterenol produced equivalent changes in LV dP/dt. beta-adrenergic receptor binding studies with [3H]-dihydroalprenolol ( [3H]DHA) indicated that the density of binding sites was significantly elevated, P less than 0.01, in the hypertrophied LV plasma membranes (111 +/- 8.8, n = 8), as compared with normal LV (61 +/- 5.6 fmol/mg protein, n = 11). The receptor affinity decreased, i.e., disassociation constant (KD) increased, selectively in the LV of the hypertrophy group; the KD in the normal LV was 6.8 +/- 0.7 nM compared with 10.7 +/- 1.8 nM in the hypertrophied LV. These effects were observed only in the LV of the LV hypertrophy group and not in the right ventricles from the same dogs. The plasma membrane marker, 5' -nucleotidase activity, was slightly lower per milligram protein in the LV hypertrophy group, indicating that the differences in beta-adrenergic receptor binding and affinity were not due to an increase in plasma membrane protein in the LV hypertrophy group. The EC50 for isoproterenol-stimulated adenylate cyclase activity was similar in both the right and left ventricles and in the two groups. However, maximal-stimulated adenylate cyclase was lower in the hypertrophied left ventricle. Plasma catecholamines were similar in the normal and hypertrophied groups, but myocardial norepinephrine was depressed in the dogs with LV hypertrophy (163 +/- 48 pg/mg) compared with normal dogs (835 +/- 166 pg/mg). Thus, severe, but compensated LV hypertrophy, induced by aortic banding in puppies, is characterized by essentially normal hemodynamics in adult dogs studied at rest and in response to catecholamines in the conscious state. At the cellular level, reduced affinity and increased beta-adrenergic receptor number characterized the LV hypertrophy group, while the EC50 for isoproterenol-stimulated adenylate cyclase activity was normal. By these mechanisms, adequate responsiveness to catecholamines is retained in conscious dogs with severe LV hypertrophy.
D E Vatner, C J Homcy, S P Sit, W T Manders, S F Vatner
Systemic infection with Streptococcus pneumoniae produced atrophy, decreased twitch and tetanic tension, and altered intracellular electrolyte composition in rat skeletal muscle. Cathepsin B activity was selectively elevated early in the course of illness. Luepeptin, a cathepsin B inhibitor, and indomethacin, a prostaglandin synthesis inhibitor, prevented muscle atrophy and impaired contractility. Indomethacin, but not leupeptin, prevented the intracellular electrolyte changes. Acetaminophen reduced fever but did not prevent muscle atrophy, impaired contractility, or altered intracellular electrolytes. Muscle wasting and impaired contractility associated with sepsis may involve selective prostaglandin stimulation of cathepsin B activity. Intracellular electrolyte changes may involve prostaglandin synthesis but do not require cathepsin B activation.
R L Ruff, D Secrist
Approximately two-thirds of patients who receive the anticancer drug gallium nitrate develop mild hypocalcemia. To evaluate the mechanism of drug-induced hypocalcemia, we tested the effects of gallium nitrate upon in vitro release of 45Ca++ from explanted fetal rat bones. The drug significantly inhibited 45Ca++ release in response to stimulation with both parathyroid hormone and a lymphokine preparation with osteoclast activating factor activity. The inhibitory effects on bone resorption were both time- and dose-dependent. Later, in a pilot study, we treated 10 patients who had cancer-related hypercalcemia with gallium nitrate administered by continuous infusion. All patients responded by a reduction of total serum calcium to normal or subnormal concentrations (13.8 +/- 1.05 mg/dl, mean +/- SD pretreatment, to 8.03 +/- 1.03 mg/dl, mean posttreatment nadir). Our results indicate that gallium nitrate effectively treats cancer-related hypercalcemia and that it probably acts by inhibiting calcium release from bone.
R P Warrell Jr, R S Bockman, C J Coonley, M Isaacs, H Staszewski
The cloned complementary DNA for coagulation Factor IX (FIX) detects a frequent restriction fragment length polymorphism (RFLP) in human genomic DNAs digested with the restriction endonuclease Taq I. This genetic marker was used, in parallel with coagulation and immunological assays, to follow the segregation of an abnormal FIX gene in a large Hemophilia B family. Among the six potential female carriers, functional assays showed that four had a high probability, and two a low probability of being carriers. Analysis at the DNA level with the cDNA probe was informative in five of the six cases, and in all these five the diagnosis of carrier state was definitively confirmed. This demonstrates the feasibility of using linkage analysis at the DNA level for the genetic screening of Hemophilia B. This method has the advantages over conventional assays of giving a diagnosis of certainty, and of being applicable to early prenatal diagnosis using biopsies of trophoblast villi. At present, the single known polymorphism associated with the FIX gene restricts the application of linkage analysis to informative cases (40%), but findings of additional RFLPs in this region should improve this figure.
L Grunebaum, J P Cazenave, G Camerino, C Kloepfer, J L Mandel, P Tolstoshev, M Jaye, H De la Salle, J P Lecocq