Exposure of human polymorphonuclear leukocytes (PMN) to chemotactic factor, as well as the migration of PMN through a 5-μm pore-size membrane, results in a PMN population with enhanced chemiluminescence, enhanced capacity for superoxide anion production, and increased Escherichia coli bactericidal activity. The enhanced PMN response resulting from exposure to chemotactic factor was observed with several chemotactic stimuli, including a mixture of casein and autologous serum, chemotactic C5 fragment, and formyl-l-methionyl-l-leucine-l-phenylalanine (f-Met-Leu-Phe). Enhanced levels of chemiluminescence were observed with both soluble stimuli (concanavalin A and phorbol myristate acetate) as well as particulate stimuli (opsonized zymosan).
Dennis E. Van Epps, Mary Lynn Garcia
Using a sensitive, specific immunoprecipitation method, the biosynthesis of human skin collagenase was studied in fibroblast cultures from patients with recessive dystrophic epidermolysis bullosa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of solubilized immunoprecipitates showed two 3H-labeled procollagenase species that comigrated with those harvested from control cultures. Recessive dystrophic epidermolysis bullosa cultures accumulated increased amounts of collagenase. Both the initial rate of accumulation of intracellular enzyme and the rate of secretion were enhanced, suggesting that excessive accumulation is related to increased synthesis. Because the turnover of labeled collagenase was unaltered, the accumulation could not be attributed to diminishing enzyme degradation. No preferential incorporation of [3H]leucine into recessive dystrophic epidermolysis bullosa collagenase occurred. Furthermore, the mutant cultures displayed no alteration in total protein synthesis, the intracellular leucine pool, or the growth kinetics of the cells. Cells from a patient with dominant epidermolysis bullosa did not show enhanced accumulation of collagenase. The levels of collagenase synthesized in vitro correlated with those observed previously in vivo in recessive dystrophic epidermolysis bullosa patients, suggesting that this biochemical trait is pathogenetically significant in the disorder.
K J Valle, E A Bauer
Previous attempts to correlate in vivo pyridoxine-responsiveness with in vitro assays of cystathionine β-synthase activity in synthase-deficient homocystinuric patients have been only partially successful. All such studies, however, have been conducted with extracts of cultured skin fibroblasts grown in medium containing a high concentration (1,000 ng/ml) of pyridoxal. Having recently shown that such growth conditions may obscure important aspects of enzyme-coenzyme interactions by saturating most synthase molecules with their cofactor, pyridoxal 5′-phosphate, we have established conditions for growth of cells in pyridoxal-free medium. Under these conditions, intracellular pyridoxal 5′-phosphate fell by >95%, and saturation of cystathionine β-synthase apoenzyme with pyridoxal 5′-phosphate decreased from a predepletion value of 70% to <10%. When such depleted cells were grown in media containing pyridoxal concentrations ranging from 0 to 1,000 ng/ml, cellular pyridoxal 5′-phosphate reached a maximum of 30 ng/mg cell protein at a medium pyridoxal concentration of 100 ng/ml. Maximal saturation of aposynthase with coenzyme in control cells was reached at a medium pyridoxal concentration of 10 ng/ml. In contrast, maximal saturation of residual aposynthase in cells from an in vivo responsive patient was achieved at a medium pyridoxal concentration of 25-50 ng/ml, whereas that from cells from an in vivo unresponsive patient was reached at 100 ng/ml. Estimates of the affinity of control and mutant cystathionine β-synthase for pyridoxal 5′-phosphate in cell extracts supported the differences observed in intact cells. The apparent Km of cystathionine β-synthase for pyridoxal 5′-phosphate in extracts of depleted cells from four in vivo-responsive patients was two to four times that of control. In contrast, the Km for pyridoxal 5′-phosphate in two lines from in vivo nonresponsive patients was 16- and 63-fold normal. These results suggest that cystathionine β-synthase activity in cells from patients containing a mutant enzyme with a moderately reduced affinity for pyridoxal 5′-phosphate can be increased by pyridoxine supplements in vivo, whereas that from patients whose enzyme has a more dramatically reduced affinity for the coenzyme cannot be so modulated because of limits on the capacity of such cells to accumulate and retain pyridoxal 5′-phosphate.
Mark H. Lipson, Jan Kraus, Leon E. Rosenberg
Two sets of phagocytic cells are available to defend the lung against inhaled bacteria. Both resident alveolar macrophages and granulocytes from the circulation have been observed in pulmonary air spaces after the deposition of bacteria; their functional roles, however, have been defined. We rendered mice selectively granulocytopenic with heterologous antiserum in order to ascertain the relative contributions of these two groups of cells in intrapulmonary bacterial killing. The clearance of Staphylococcus aureus was unimpaired in granulocytopenic animals, confirming the primary role of the alveolar macrophages in the killing of these organisms. In contrast, granulocytopenic animals cleared only 10.0+/-7.0% of an inoculum of Klebsiella pneumoniae compared with 33.0+/-4.0% clearance in normal animals (P < 0.02), and Pseudomonas aeruginosa proliferated to 513% of baseline levels in granulocytopenic animals, whereas normal mice cleared 26.8+/-10.6% of the inoculum. These findings indicate that circulating granulocytes play a major role in the clearance of the latter two organisms. This variation in cellular response to different bacterial species suggests that the defense of the lung against pathogenic bacteria is more complex than has been previously assumed.
S R Rehm, G N Gross, A K Pierce
Immunoincompetency is often seen in patients after various types of trauma and is associated with increased morbidity and mortality from infectious complications. To understand better the immunologic impairment associated with trauma, we have studied this phenomenon in an animal model. Splenocytes from mice traumatized by amputation of their right hind limbs were consistently shown to have a diminished capacity to proliferate in response to alloantigens and to form alloreactive cytolytic cells in mixed lymphocyte cultures. Anesthesia itself had no effect in this system. The immunoincompetency was detected from 2 h to 6 d after surgical trauma and was completely reversed by removing adherent and phagocytic cells from the splenocytes. Furthermore, addition of splenocytes from traumatized mice to mixed lymphocyte cultures from normal mice prevented normal lymphocytes from responding to alloantigens, suggesting the existence of suppressor cells. The suppressor cells were found to adhere to glass and to nylon wool columns, and were contained within an esterase-positive cell population. They were insensitive to treatment with anti-Thy 1.2 and anti-Ig sera in the presence of complement. Therefore, the present results suggest that a Thy 1.2-negative, Ig-negative, esterase-positive cell population capable of adhering to glass and nylon wool, presumably macrophages, was responsible for the inhibition of the responsiveness of lymphocytes to alloantigens in traumatized animals.
B S Wang, E H Heacock, A V Wu, J A Mannick
Experiments were designed to determine the basis for the strong competitive reaction of denatured DNA with systemic lupus erythematosus (SLE) antinative DNA antibodies. Secondary structure in denatured DNA was reflected in hyperchromicity upon heating and in multiphase kinetics of its digestion by S1 nuclease. Partial digestion by S1 nuclease completely eliminated the ability of denatured DNA to react with antidenatured DNA antibodies, but not its ability to react with SLE sera. S1 nuclease-resistant cores were isolated from extensively digested denatured DNA. These cores had secondary structure, including some stable fold-back helical regions. The cores, from 20 to several hundred base pairs in size, competed with native DNA for binding by SLE sera. Other experiments measured reactions of denatured DNA under conditions that affected its secondary structure content. Its competitive activity decreased as temperature was increased from 0° to 37°C, whereas the activity of native DNA was not altered in this temperature range. With DNA pieces of 90-110 base pairs, native fragments were much more effective than the denatured fragments, in which stable helical structure is less likely to occur than in high molecular weight denatured DNA. Competitive assays with mononucleotides, oligonucleotides, homopolymers, and RNA-DNA hybrids also indicated that two strands of polydeoxyribonucleotide were required for optimal reactions with these SLE serum antibodies. The antibodies can measure stable helical regions in denatured DNA; they may also stabilize short helical regions that occur in an equilibrium of conformational forms.
B. David Stollar, Michael Papalian
A rabbit model for the diabetic pregnancy was used to investigate the etiology of delayed pulmonary maturation observed in infants of diabetic mothers. Pregnant rabbit does were made glucose intolerant and insulinopenic by injection of alloxan, a pancreatic beta-cell cytotoxin. At 28 d (term approximately 31 d) fetuses of these animals were hyperglycemic, but were not hyperinsulinemic and did not demonstrate tissue overgrowth. Fetal pulmonary maturity was assessed by measurement of pressure-volume relationships on the fetal lungs. Fetuses of glucose-intolerant does demonstrated less retention of air on deflation. Phospholipid components of pulmonary surfactant were assayed on fluid obtained from lavage of the fetal lungs. Levels of disaturated phosphatidylcholine (per-cent total-lavage phospholipids) were diminished in fetuses of glucose-intolerant does compared to control fetuses (20.5 +/- 4.2 vs. 38.0 +/ 4.3%; P less than 0.01). Lecithin/sphingomyelin ratios were similar in both groups and phosphatidylglycerol was not detected in either group. There was a direct correlation between the percentage of alveolar disaturated phosphatidylcholine and retention of air on lung deflation. These findings suggest that in this model pulmonary instability was a result of diminished alveolar disaturated phosphatidylcholine, and this diminution did not result from fetal hyperinsulinemia.
C L Bose, D N Manne, A J D'Ercole, E E Lawson
We previously developed an in vitro organ culture system in which gluten exerts a toxic effect on intestinal mucosa of patients with active gluten-sensitive enteropathy. Gluten generally inhibits the epithelial cell maturation of intestinal biopsy specimens that otherwise occurs if the tissue is cultured for 24-48 h in a gluten-free medium. However, small intestinal mucosa from 15-20% of patients with proven gluten-sensitive enteropathy fails to manifest the expected gluten-induced damage in vitro. In the present study, we explored the relation between in vitro gluten-induced intestinal damage and the presence of HLA-B8. We determined whether the patients' histocompatibility type (HLA-B8 positive or negative) influenced the ability of gluten protein to inhibit epithelial cell maturation of cultured intestinal biopsy specimens from patients with gluten-sensitive enteropathy.
Z. M. Falchuk, D. L. Nelson, A. J. Katz, J. E. Bernardin, D. D. Kasarda, N. E. Hague, W. Strober
Mineralo- and glucocorticoid-deficient states, such as Addison's disease, are partly characterized by an inability to generate a maximally concentrated urine. The purpose of the present study was to develop a model of adrenal insufficiency and to determine whether changes in the intrinsic function of the collecting duct could partly account for this concentrating defect. Two kinds of experiments were performed: an assessment of the in vivo ability of adrenal-ectomized rabbits to concentrate their urine, and an examination of the intrinsic hydroosmotic responsiveness of in vitro perfused collecting ducts isolated from normal and adrenalectomized rabbits. The present study demonstrates that adrenalectomized rabbits are unable to concentrate their urine maximally, and that the in vivo administration of either deoxycorticosterone, 250 μg/kg, or dexamethasone, 50 μg/kg, restored to or toward normal their concentrating ability. When cortical collecting tubules from adrenalectomized rabbits were perfused in vitro, they demonstrated a markedly blunted hydroosmotic response to antidiuretic hormone (ADH), which was corrected by the in vitro addition of either aldosterone (50 pM) or dexamethasone (50 pM), but not progesterone (50 pM). The steroids by themselves, in the absence of ADH, had no intrinsic effect on the water permeability of the collecting duct. The blunted hydroosmotic response across cortical collecting tubules from adrenal-ectomized rabbits was corrected by the addition of either 8-bromo cyclic AMP or a potent phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. The present studies show that the cortical collecting tubules obtained from adrenalectomized rabbits do not respond normally to ADH. The poor hydroosmotic response to ADH was corrected by exogenous aldosterone, dexamethasone, an analog of cyclic AMP, or a phosphodiesterase inhibitor. In conclusion, the present studies are consistent with the view that the concentrating defect seen in adrenal insufficiency is at least partly the result of the absence of the permissive effect that adrenal steroids exert on the ADH-induced reabsorption of water across the collecting duct. The absence of adrenal steroids results in a diminished rate of cyclic AMP accumulation in the cells of the collecting duct, either as a result of an augmented activity of cyclic AMP phosphodiesterase or a diminished rate of cyclic AMP generation.
Michael J. Schwartz, Juha P. Kokko
Acute bacterial meningitis may be associated with increased intracranial pressure, neurological sequelae such as communicating hydrocephalus, and a slow response to antibiotic therapy. Alterations in cerebrospinal hydrodynamics are at least partially responsible for these complications. Constant, low-flow short-duration manometric infusion studies through a hollow-bore pressure monitoring device in direct continuity with the supracortical subarachnoid space were performed in rabbits with experimental meningitis. Maximal resistance to cerebrospinal fluid (CSF) outflow from the subarachnoid to vascular space was markedly increaed in acute pneumococcal meningitis when compared to control, uninfected animals (6.77 +/- 3.52 vs. 0.26 +/- 0.04 mm Hg/microliter per min, P less than 0.001). Similar elevations (8.93 +/- 4.15 mm Hg/microliter per min were found in experimental Escherichia coli meningitis. Despite eradication of viable bacteria from the CSF by penicillin therapy during the acute stage of pneumococcal meningitis, resistance remained elevated (6.07 +/- 4.68 mm Hg/microliter per min) and had not returned to normal up to 15 d later. Administration of methylprednisolone during the early stages of acute pneumococcal meningitis reduced mean peak outflow resistance towards control values (0.59 mm Hg/microliter per min) and no "rebound" effect was apparent 24 h later. These hydrodynamic alterations in experimental meningitis prevent normal CSF absorption and decrease the ability of the bran to compensate for changes in intracranial volume and pressure.
W M Scheld, R G Dacey, H R Winn, J E Welsh, J A Jane, M A Sande
Dog erythrocytes (RBC) have a system for passive Ca and Na movements that resembles the Ca-Na exchanger first described in cardiac muscle. Amrinone, a new cardiotonic drug active in humans with congestive heart failure, is shown to stimulate net Ca uptake by dog RBC. Amrinone's action is on Ca influx rather than efflux. The influence of Amrinone on Ca uptake is enhanced when the cells are placed in low Na media; raising external Na or lowering intracellular Na both abolish the effect of the drug. The data suggest that amrinone potentiates passive Ca entry into the cells by a Na-dependent pathway. If Ca moves through myocardial sarcolemma as it does through dog RBC membranes, then the inotropic action of amrinone can be explained on the basis that the drug increases intracellular Ca levels.
J C Parker, J R Harper Jr
We have compared the abilities of immunoglobulin (Ig)G, IgM, and IgA to induce either mononuclear cell-mediated (complement-independent) or complement-mediated (cell-free) antibacterial activity against group C meningococci. In each of these assays, immunoglobulins purified from the sera of individuals immunized with meningococcal group C polysaccharide were compared with those purified from sera of patients convalescing from disseminated meningococcal disease. Our data support three conclusions. First, although nonbactericidal in cooperation with complement, IgA can induce cell-mediated antibacterial activity as well as IgG. Second, the amount of IgG required to induce cell-mediated antibacterial activity is similar to the amount required for complement-mediated killing. Third, although the amount of either postimmunization or convalescent IgM required to induce complement-mediated killing is 16- to 20-fold less than the amount of respective IgG required, IgM is inferior to IgG in its ability to induce cell-mediated antibacterial activity because in the cell-mediated system (a) postimmunization IgM is ineffective; (b) the amount of convalescent IgM required for minimal activity is eightfold more than the amount of convalescent IgG required; and (c) the maximal antibacterial index induced by convalescent IgM is 50% less than that which can be induced by IgG. These data suggest that IgG and IgA may play a greater role than IgM in mononuclear cell-mediated antibacterial host immune defense.
George H. Lowell, Lynette F. Smith, J. McLeod Griffiss, Brenda L. Brandt, Richard P. MacDermott
To investigate the biochemical and cellular basis for the rise in polymorphonuclear leukocyte (PMN) count during epinephrine administration, PMN from subjects receiving epinephrine were studied for their capacity to adhere to nylon wool fibers and endothelial cell monolayers. After administration of epinephrine, the PMN count increased by 80% at 5 min, and isolated PMN adherence to nylon fibers fell from a base line of 44±2-18±3%. In contrast, when subjects were infused with the β-antagonist propanolol before receiving epinephrine, the PMN count failed to rise and PMN adherence was normal. Exposure of PMN endothelial cell monolayers to 0.1 μM epinephrine led to diminished PMN adherence that could be blocked by 10 μM propanolol but not by 10 μM phentolamine. Sera obtained from subjects 5 min after receiving epinephrine or from supernates derived from endothelial cell monolayers exposed to 90 nM epinephrine inhibited PMN adherence to nylon fibers. Addition of anticyclic AMP antisera but not anticyclic guanosine monophosphate antisera to the postepinephrine sera or to the postepinephrine supernate derived from the endothelial cell monolayers abolished their inhibitory effect of PMN adherence to nylon fibers. In contrast, direct exposure of PMN to epinephrine failed to affect their adherent properties. Because it has been previously shown that endothelial cells contain β-receptors and respond to catecholamines by raising their intracellular concentrations of cyclic AMP, and that PMN adherence is attenuated by cyclic AMP, it would appear that diminished PMN adherence after epinephrine administration is mediated through endothelial cell β-receptor activity, which in turn impairs PMN margination in vivo and could account for the rise in circulating PMN.
Laurence A. Boxer, John M. Allen, Robert L. Baehner, Vicktoria Amick
Endogenous phospholipid metabolism in stimulated human platelets was studied by phosphorus assay of major and minor components following separation by two-dimensional thin-layer chromatography. This procedure obviated the use of radioactive labels. Extensive changes were found in quantities of phosphatidylinositol (PI) and phosphatidic acid (PA) as a consequence of thrombin or collagen stimulation. Thrombin addition was followed by rapid alterations in the amount of endogenous PI and PA. The decrease in PI was not precisely reciprocated by an increase in PA when thrombin was the stimulus. This apparent discrepancy could be explained by removal of a transient intermediate in PI metabolism, such as diglyceride, formed by PI-specific phospholipase C (Rittenhouse-Simmons, S., J. Clin. Invest.63: 580-587, 1979). Diglyceride would be unavailable for PA formation by diglyceride kinase, if hydrolyzed by diglyceride lipase (Bell, R. L., D. A. Kennerly, N. Stanford, and P. W. Majerus. Proc. Natl. Acad. Sci. U. S. A.76: 3238-3241, 1979) to yield arachidonate for prostaglandin endoperoxide formation. Thrombin-treated platelets also accumulated lysophospho-glycerides. Specifically, lysophosphatidyl ethanolamines accumulated within 15s following thrombin addition. Fatty acid and aldehyde analysis indicated phospholipase A2 activity, with an apparent preference for diacyl ethanolamine phosphoglycerides. In the case of collagen, these changes occurred concomitantly with aggregation and consumption of oxygen for prostaglandin endoperoxide formation.
M. Johan Broekman, Jean W. Ward, Aaron J. Marcus
We examined the role of microtubules in platelet aggregation and secretion (release reaction) induced by the calcium ionophore A23187 (0.8-5 μM). At these concentrations, platelet aggregation was preceded by a lag period of ∼1 min. Colchicine (an agent that disrupts microtubule assembly-disassembly) was shown to bind to platelet microtubules by employing [3H]colchicine at a concentration that is specific for microtubules in other tissues (10 nM). Colchicine prolonged the lag period, inhibited the secondary wave of platelet aggregation, and inhibited the release reaction (release of [14C]serotonin). Platelets were next incubated with 20-60% D2O, an agent that stabilizes microtubules. D2O overcame colchicine-induced inhibition of the lag period, aggregation, and release. D2O alone enhanced platelet aggregation by 59±14% (SEM) and shortened the lag period by 43±10%. We conclude that functioning microtubules are required for platelet aggregation and release, and that microtubules of platelet preparations are functioning submaximally.
D. Menche, A. Israel, S. Karpatkin
We have examined the various axonal transport rates in sciatic nerve of streptozotocin diabetic rats 3 h and 10,25, and 50 d after the injection of tritiated proline into the fifth lumbar dorsal root ganglion. Proline-labeled proteins conveyed by the slow transport system were advanced more slowly in diabetic rats. No compensation for this delay took place in terms of protein synthesis, half-life, or transported amount. The decreased deliverance of slowly transported proteins (structural proteins) to the axons may well account for the reduced axon calibre shown in earlier reports. A hypothesis is proposed suggesting that the primary event in the development of neurological abnormalities in diabetes is an impairment of the retrograde axonal transport, secondarily leading to the abnormality of the anterograde transport of structural proteins.
J Jakobsen, P Sidenius
Hyaluronic acid (HA) stimulated the function of polymorphonucler leukocytes (PMN) both in vitro and in vivo. Stimulation in vitro was achieved by the incubation of PMN and HA in heparinized whole blood at concentrations of HA between 5 and 500 microgram/liter. The stimulation of the PMN function was demonstrated by an increase rate of phagocytosis of complement- and/or immunoglobulin (Ig)G-coated latex particles, increased adherence to nylon wool, increased random migration and chemotactic response, increased chemiluminescence during phagocytosis, and raised levels of intracellular ATP. The effect of HA in vivo was demonstrated, after subcutaneous administration of HA (5-20 mg) to healthy volunteers, by an enhanced rate of phagocytosis of the subsequently isolated neutrophils. The duration of the effect of one administration was approximately 1 wk with maximum effect on days 2-4. HA injections to patients with increased susceptibility to bacterial infections and impaired neutrophil function demonstrated an enhanced neutrophil function also in these individuals. HA may therefore be a new principle by which resistance to infections can be enhanced.
L Håkansson, R Hällgren, P Venge
Patients with coronary vasospasm appear to be supersensitive to the coronary constrictor effects of ergonovine. To determine whether atherosclerosis alters arterial reactivity and sensitizes arteries to ergonovine, contractile responses of isolated aortae from control rabbits and from rabbits fed a high-cholesterol diet were compared. Aortic strips were mounted in a myograph for the monitoring of isometric tension, equilibrated in oxygenated Krebs buffer, and exposed to graded concentrations of agonists and antagonists. The concentration-response relation for ergonovine in atherosclerotic arteries exhibited a markedly depressed constrictor threshold concentration (0.5 pM vs. 0.23 μM in controls), a significantly lowered one-half effective dose (ED50) value, and an augmented maximal response. Furthermore, atherosclerotic arteries showed similar, although less pronounced changes in the concentration-response relation for serotonin. In contrast, responses to 34 mM KCl were virtually identical, and the concentration-response relation for phenylephrine were similar in the two groups. In control arteries, 0.1 μM phentolamine and 0.1 μM prazosin suppressed responses to 1 μM ergonovine by 71 and 90%, respectively. However, in atherosclerotic arteries α-blockers in the same concentration inhibited responses to 0.01 μM ergonovine by less than 10%. On the other hand, 0.1 μM cyproheptadine, a serotonergic antagonist, suppressed these responses by 82%. Thus, the supersensitivity to ergonovine appeared to be mediated predominantly by a serotonergic mechanism. These results indicate that smooth muscle in atherosclerotic arteries may be supersensitive to specific vasoconstricting stimuli, a change that might contribute to arterial dysfunction in vivo.
Philip D. Henry, Mitsuhiro Yokoyama
Selected bacteroides species secreted various amounts of protease and glycosidase into their growth medium. Bacteroides vulgatus, distasonis, and ovatus secreted the most (31-60% of total). The secreted protease was similar in action to the protease within the organism, in that it had a broad pH optimum of 6-9, a Km app. for casein of 0.1 μM, and was inhibited by benzamidine, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate (DIFP), and by an elastase inhibitor, Ac(Ala)3AlaCH2Cl.
Stan P. Riepe, Jeffrey Goldstein, David H. Alpers
It has been suggested that ketone bodies might participate in the nitrogen-sparing process occurring during prolonged starvation by inhibiting the muscular production of alanine and glutamine, which are the main gluconeogenic amino acids. The results of the ketone infusion studies on which this theory is based have been reevaluated in this study by following the plasma levels of ketone bodies, alanine, glutamine, and other substrates during 11.5 h in five groups of normal overnight-fasted subjects. Subjects of groups I, II, and III were infused for 3 h, respectively, with Na acetoacetate, Na bicarbonate, or free acetoacetic acid administered in comparable amounts (about 20 μmol/kg per min), whereas group IV was infused with hydrochloric acid (7.0 μmol/kg per min). A control group (V) received no infusion. Na acetoacetate induced a rise in blood pH (+0.1±0.003) and a fall in the plasma levels of alanine (−41.8±4.6%) and glutamine (−10.6±1.4%), whereas free acetoacetic acid had a barely detectable lowering effect on blood pH and induced a rise in alanine (+22.5±8.0%) and glutamine (+14.6±3.2%) levels. Both infusions were associated with a lowering of plasma glucose, which therefore seems independent of the changes in alanine and glutamine concentrations. Sodium bicarbonate reproduced the alkalinizing effect and the hypoalaninemic action of Na acetoacetate, which seems thus unrelated to hyperketonemia. On the other hand, acidification of blood with hydrochloric acid did not mimic the effects of acetoacetic acid.
Françoise Féry, Edmond O. Balasse
Chronic granulomatous disease in males is familial and its transmission is is usually clearly x-linked. The mode of inheritance in females with the syndrome is unknown and the carrier state difficult to identify. Defective polymorphonuclear leukocyte bactericidal activity in this disease is associated with an absence of the respiratory burst generated in stimulated phagocytes and may be detected by the chemiluminescence assay. Polymorphonuclear leukocytes from three of four females with chronic granulomatous disease had extremely low chemiluminescence production, their asymptomatic mothers had intermediate values, and their fathers were normal. Polymorphonuclear neutrophils of two affected males in these kinships generated no chemiluminescence, whereas two of seven female relatives had intermediate values, and all nonaffected males had normal values. In the three families in which leukocytes were studied by nitroblue tetrazolium reduction, two populations of neutrophils were demonstrated for the female patients and/or their mothers. The wide phenotypic variability for clinical disease, evidence of two leukocyte populations in the patients or their mothers, and low but detectable leukocyte chemiluminescence in the affected females is consistent with the Lyon hypothesis of x-chromosome inactivation in these families. The findings suggest an x-linked inheritance in these females with chronic granulomatous disease.
Elaine L. Mills, Kenneth S. Rholl, Paul G. Quie
These studies describe the calcium dependence of the serotonin-induced changes in active electrolyte transport in rabbit ileum in vitro. In the presence of a standard calcium concentration (1.2 mM) in the serosal bathing fluid, serosal serotonin caused a transient increase in short-circuit current and a prolonged decrease in net Na and Cl fluxes. Removing calcium from the serosal (no calcium plus 1 mM EGTA) but not the mucosal bathing fluid inhibited the serotoin-induced increase in ileal short-circuit current, and also completely blocked the serotonin effects on net Na and net Cl fluxes. This inhibition was rapidly reversed by readding calcium. Removing serosal calcium did not inhibit all active electrolyte transport processes, as the effect of a maximum concentration of theophylline (10 mM) was not altered. Similarly, d,l-verapamil, a calcium channel blocker, inhibited the serotonin-induced changes in short-circuit current and in net Na and net Cl fluxes, but did not alter the theophylline effects. In contrast, d-verapamil, a stereoisomer which does not block calcium channels, did not inhibit the serotonin-induced changes. The calcium dependence of these serotonin effects was associated with increased uptake of 45Ca into rabbit ileum, including increaed 45Ca uptake from the serosal surface. Serotonin also increased the rate of 45Ca efflux from rabbit ileum into a calcium-free solution, compatible with serotonin increasing the ileal plasma membrane permeability to calcium. It is postulated that serotonin affects active intestinal electrolyte transport by a mechanism dependent on serosal but not mucosal calcium that involves an increase in the intestinal plasma membrane permeability to calcium, and perhaps an increase in intracellular calcium.
M Donowitz, N Asarkof, G Pike
We asked whether binding of human immunoglobulin (Ig)G antibody reacting with Raji cells could be distinguished from binding of IgG immune complexes. Using a standard Raji assay employing 125I-IgG goat anti-human Fc gamma, we found that digestion of Raji cells with pronase reduced by 95% their ability to bind complement-fixed aggregated human gamma globulin and complement-fixed tetanus toxoid-antitetanus toxin complexes. However, binding at 37 degrees C of IgG from the sera of 16 patients with systemic lupus erythematosus (SLE) to pronase-digested Raji cells was reduced much less consistently and extensively (9-100% reduction; mean reduction of 51%). In more detailed studies of two SLE sera, sucrose density gradient centrifugation showed that greater than 50% of the IgG binding to undigested Raji cells sedimented in the 7S region. Pepsin digestion of immunoglobulin fractions from four SLE sera caused a reduction in SLE IgG binding to undigested Raji cells when detected with 125I anti-Fc gamma, but an increase when binding was detected with 125I-anti-Fab, suggesting that substantial SLE IgG can bind through F(ab')2 regions. Binding of IgG from SLE sera was not directed at neoantigenic sites induced by pronase digestion because binding activity was adsorbed with undigested cells as readily as with digested cells. Moreover, sera from 10 SLE patients that had negative Raji assays contained no IgG that bound to pronase-digested Raji cells. We conclude that much of the IgG bound at 37 degrees C to Raji cells from the sera of many patients with SLE does not represent immune complexes but is probably antibody directed toward sites on the Raji cell.
C L Anderson, W S Stillman
Two pathways of mevalonate metabolism have been demonstrated: the major (sterol) pathway leads to cholesterol synthesis, whereas the second shunts mevalonate away from sterol production and ultimately results in its oxidation to CO2. Previous studies have demonstrated that the female rat metabolizes circulating mevalonate by the shunt pathway at twice the rate of the male, whereas the male rat converts significantly more circulating mevalonate to cholesterol than the female. The present study extends these observations to humans. Six men and five premenopausal women with normal renal function were injected with R,S-[5-14C]mevalonate, and 14CO2 expired in the breath of the subjects was monitored continuously with an ionization chamber. On an average, the female subjects expired 16.5% and the males 9.8% of the injected R-[5-14C]mevalonate (P less than 0.001). No differences were observed in the plasma and erythrocyte [14C]cholesterol levels. These data demonstrate, in human beings, a sex difference in mevalonate metabolism. The overall impact of the greater mevalonate shunt activity on cholesterol balance in women is unknown.
K R Feingold, M H Wiley, G L Searle, B K Machida, M D Siperstein
The transport of [125I]thyroxine (T4) and [125I]triiodothyronine (T3) into liver was investigated with a tissue sampling-portal vein injection technique in the anesthetized rat. The method allows the investigation of the effects of plasma proteins in human serum on the unidirectional influx of T4 or T3 into liver cells. The percent extraction of unidirectional clearance of T3 and T4 was 77±2% and 43±2%, respectively, after portal injection of a bolus of Ringer's solution. Cell membrane transport of T4 or T3 was nonsaturable because 50-μM concentrations of unlabeled hormone had no effect on transport. The addition of bovine albumin in concentrations of 1, 5, or 10 g/100 ml bound >98% of T4 or T3 in vitro, but had no significant effect on T3 or T4 transport in vivo. Conversely, 10% rabbit antisera specific for T3 or T4, completely abolished the intracellular distribution of thyroid hormone into liver. In the presence of rat serum, which contains albumin and thyroid hormone binding pre-albumin (TBPA), 18 and 81% of total plasma T4 and T3, respectively, were available for transport in vivo. The fraction of hormone available for transport in the presence of normal human serum, which contains albumin, TBPA, and thyroid hormone binding globulin (TBG) was 11% for T4 and 72% for T3. The fraction of hormone transported into liver after injection of serum obtained from pregnant or birth control pilltreated volunteers was 4% for T4 (but this was not significantly different from zero) and 54% for T3.
William M. Pardridge, Lawrence J. Mietus
The adult respiratory distress syndrome is characterized by arterial hypoxemia as a result of increased alveolar capillary permeability to serum proteins in the setting of normal capillary hydrostatic pressures. Because bacterial sepsis is prominent among the various diverse conditions associated with altered alveolar capillary permeability, we studied the effect of bacteremia with attendant complement activation on the sequestration of microorganisms and the leakage of albumin in the lungs of guinea pigs. Pneumococci were injected intravenously into guinea pigs and their localization was studied. Unlike normal guinea pigs, complement-depleted guinea pigs did not localize injected bacteria to the lungs. Preopsonization of organisms did not correct this defect in pulmonary localization of bacteria in complement-depleted animals, suggesting that a fluid-phase component of complement activation was required. Genetically C5-deficient mice showed no pulmonary localization of bacteria. C5-sufficient mice demonstrated the usual pulmonary localization, thus further suggesting that the activation of C5 might be important in this localization. The infusion of activated C5 increased alveolar capillary permeability to serum proteins as assayed by the amount of radioactive albumin sequestered in the lung. Neutropenic animals did not develop altered capillary permeability after challenge with activated C5. Thus, complement activation through C5, in the presence of neutrophils, induces alterations in pulmonary alveolar capillary permeability and causes localization of bacteria to the pulmonary parenchyma. Complement activation in other disease states could potentially result in similar clinical manifestations.
Stephen Hosea, Eric Brown, Carl Hammer, Michael Frank
The functional maturity of T and B lymphocyte populations from human newborns was evaluated using a reverse hemolytic plaque assay to detect immunoglobulin-secreting cells generated in in vitro cultures stimulated with pokeweed mitogen (PWM), a T cell-dependent polyclonal activator, and the Epstein-Barr virus (EBV), a T cell-independent B cell activator. Cord blood lymphocytes failed to produce immunoglobulin in response to PWM, but did respond with immunoglobulin synthesis to stimulation with EBV. Co-culture experiments demonstrated that cord blood T cells would inhibit immunoglobulin production by adult cells stimulated with PWM, but not with EBV. Cord blood T cells did suppress immunoglobulin production by cord blood B cells when stimulated with a mixture of EBV and PWM, indicating that cord blood, in contrast to adult blood, contains a population of suppressor T cell precursors that are easily activated by PWM. Irradiation of the cord blood T cells with 2,000 rad eliminated the suppressor activity and revealed normal helper function for immunoglobulin (Ig) G, A, and M when these T cells were co-cultured with adult B cells. Cord blood B cells co-cultured with adult T cells or irradiated cord blood T cells did produce immunoglobulin in response to PWM, but the response was significantly lower than that of adult B cells, and only IgM was produced in these cultures. These studies demonstrate that both the T and B cells of the human newborn have significant functional differences compared with the functions of T and B lymphocyte populations in adults.
Giovanna Tosato, Ian T. Magrath, Irma R. Koski, Nancy J. Dooley, R. Michael Blaese
Transplantation of histocompatible allogeneic peripheral blood leukocytes resulted in successful reconstitution of an adenosine deaminase (ADA)-deficient, severe combined immune-deficient patient. Erythrocyte transfusions before the transplant were associated with a rise of serum immunoglobulin concentration to normal without improvement in T cell function. The patient received 5 × 107 peripheral blood mononuclear leukocytes/kg obtained from the histocompatible father by leukopheresis. 3 wk after the transplant the lymphocyte count, proportion of E rosetting lymphocytes, and the ADA content of the patient's mononuclear leukocytes became normal while the phytohemagglutinin-stimulated blastogenic responses improved and became normal 52 d after the transplant. Antibody response to diphtheria immunization and response to naturally acquired herpes simplex infection were normal while isohemagglutinins progressively increased. Immunization with a neoantigen, bacteriophage φX 174, resulted in a small but definite antibody response but no amplification of the response after secondary immunization. A positive reaction to a skin test for Candida albicans developed. Erythrocyte deoxy ATP (dATP) concentration decreased during the course of erythrocyte transfusions. 9 mo after the transplant, the erythrocyte dATP was elevated to twice pretransfusion levels while mononuclear leukocyte dATP varied from normal to elevated during the first 4 mo of the posttransplant period, but remained normal during the last 8 mo. The improvement in immune function persisted during the 12-mo posttransplant observation period while the mononuclear leukocyte ADA concentration stabilized at ∼0.25 of normal, which is similar to the enzyme activity of the donor cells. This in vivo study supports the hypothesis that lymphoid precursor cells are present in peripheral blood which may partially reconstitute an immune-deficient recipient.
Kenneth C. Rich, Carol M. Richman, Edwin Mejias, Peter Daddona