Lithocholic acid and its taurine, glycine, and sulfate derivatives are potent cholestatic agents. Lithocholate glucuronide is present in the plasma and urine of patients with cholestatic syndromes, but little is known of its metabolism, excretion, and cholestatic potential. [3 beta-3H]lithocholate 3-O-beta-D-glucuronide was synthesized, and chemical and radiochemical purity were established. The aqueous solubility of lithocholate glucuronide was determined and found to be greater than that of lithocholic acid or several of its derivatives. In the range of concentrations examined, calcium ions precipitated lithocholate glucuronide stoichiometrically. The material was administered to rats prepared with an external biliary fistula. When 17-25 micrograms quantities were administered, 89.1 +/- 4.5% (mean +/- SEM) of the radiolabel was secreted in bile within the first 20 h after administration, the major fraction being secreted in less than 20 min. Four-fifths of the radiolabeled material in bile was the administered unaltered parent compound, while a minor fraction consisted of a more polar derivative(s). We showed that increasing biliary concentrations of more polar derivatives were observed with milligram doses of [3H]lithocholate glucuronide, and with time after the administration of these loading doses. Milligram doses of [3H]lithocholate glucuronide resulted in partial or complete cholestasis. When induced cholestasis was partial, secretion in bile remained the primary excretory route (82.5-105.6% recovery in bile), while, when complete cholestasis was induced, wide tissue distribution of radiolabel was observed. Cholestasis developed rapidly during infusion of [3H]lithocholate glucuronide. Bile flow was diminished within 10-20 min of the start of an infusion of 0.05 mumol, 100 g-1 body weight, minute-1, administered concomitantly with an equimolar infusion of taurocholate. The results establish that lithocholate glucuronide exerts cholestatic effects comparable to those exerted by unconjugated lithocholic acid.
D G Oelberg, M V Chari, J M Little, E W Adcock, R Lester
We studied the interaction between Legionella pneumophila, which is principally a pulmonary pathogen, with primate alveolar macrophages (AM), which are the primary pulmonary cellular defense mechanism. For these studies we used L. pneumophila, type I, which were grown in albumin-yeast extract broth, were greater than 80% viable, and were comparable in virulence for guinea pigs to organisms from guinea pig spleen homogenates. For comparison, avirulent agar-passed L. pneumophila, type I, and a strain of Escherichia coli were also used. In the absence of detectable antibody, AM phagocytosed similar numbers of virulent and avirulent Legionella and killed the majority of ingested Legionella in 15-30 min, as determined by two different assays. The virulent and avirulent Legionella appeared to be equally susceptible to the cidal systems of the AM and both were killed more readily than were E. coli under both assay conditions. Phagocytosis of Legionella by AM was associated with a localized respiratory burst, as indicated by nitroblue tetrazolium reduction around ingested organisms. Killing of AM-associated Legionella was inhibited by the hydroxyl radical (OH.) scavenger mannitol (but not by an equiosmolar concentration of sodium sulfate), and by a combination of superoxide dismutase and catalase (but not by either enzyme alone). These findings suggest a contribution by OH., one generated by the metal-catalyzed interaction of superoxide and hydrogen peroxide (Haber-Weiss reaction) in the anti-Legionella activity of AM. The virulent Legionella that survived intracellularly increased in number from 4 X 10(4) at 1 h to 6 X 10(6) at 96 h after infection. In contrast, avirulent Legionella replicated more slowly, increasing in number from 4 X 10(4) to 1 X 10(5) over the same period. Replication of virulent Legionella destroyed the AM monolayers by 120 h, whereas monolayers containing avirulent organisms remained intact. Thus, virulence of Legionella appears not to correlate with its ability to survive early killing by AM, but rather with the ability of the small fraction of surviving organisms to replicate within these cells.
R F Jacobs, R M Locksley, C B Wilson, J E Haas, S J Klebanoff
Experiments were carried out to examine relationships between alveolar macrophage maturity and amounts of tissue factor (Clotting Factor III) in these cells under physiologic conditions and during immunologically induced pneumonitis. Using discontinuous density gradient centrifugation, alveolar macrophages from healthy rabbits were rapidly isolated into five subpopulations at different stages of maturation, as demonstrated by morphologic and morphometric evaluation. Very large amounts of tissue factor activity were found in fully mature cells that were purified in the lowest density subpopulation and assayed without preliminary in vitro stimulation or culture. In the remaining four subpopulations of increasing density, amounts of tissue factor were found to progressively diminish in direct correlation with declines of cell maturity. These differences at mean levels were as great as 35-fold. In addition, blood monocytes had less than 1/219 and less than 1/6 of the activity of the fully mature and the least mature subpopulations, respectively. After 16 h culture of the five isolated subpopulations in the absence of lymphokines or of significant numbers of lymphocytes, tissue factor activity increased in inverse correlation with the preincubation stage of cell maturity (2,387 and 109% in the least mature and most mature subpopulations, respectively). These increases required protein synthesis and were accompanied by morphologic and morphometric changes which indicated cellular maturation during the period of tissue factor activity generation in vitro, thus further demonstrating relationships between macrophage maturity and tissue factor content. In additional experiments, direct correlations between cell maturity and tissue factor activity content were also found in activated alveolar macrophage populations from rabbits with Bacillus Calmette Guering (BCG)-induced granulomatous pneumonitis. However, as compared with controls, the BCG populations had increased total amounts of tissue factor activity due to the presence of large numbers of mature alveolar macrophage forms that had high levels of the procoagulant. Thus, tissue factor activity in alveolar macrophages is a marker of cellular maturation in vivo and in vitro. Increased amounts of this initiator of the extrinsic clotting pathway, as found in alveolar macrophage populations from animals with granulomatous pneumonitis induced by BCG hypersensitivity, suggest that alveolar macrophage tissue factor may contribute to the pathology of immune lung diseases.
H Rothberger, M P McGee, T K Lee
To characterize glucose counterregulatory mechanisms in patients with noninsulin-dependent diabetes mellitus (NIDDM) and to test the hypothesis that the increase in glucagon secretion during hypoglycemia occurs primarily via a paracrine islet A-B cell interaction, we examined the effects of a subcutaneously injected therapeutic dose of insulin (0.15 U/kg) on plasma glucose kinetics, rates of glucose production and utilization, and their relationships to changes in the circulating concentrations of neuroendocrine glucoregulatory factors (glucagon, epinephrine, norepinephrine, growth hormone, and cortisol), as well as to changes in endogenous insulin secretion in 13 nonobese NIDDM patients with no clinical evidence of autonomic neuropathy. Compared with 11 age-weight matched nondiabetic volunteers in whom euglycemia was restored primarily by a compensatory increase in glucose production, in the diabetics there was no compensatory increase in glucose production (basal 2.08 +/- 0.04----1.79 +/- 0.07 mg/kg per min at 21/2 h in diabetics vs. basal 2.06 +/- 0.04----2.32 +/- 0.11 mg/kg per min at 21/2 h in nondiabetics, P less than 0.01) despite the fact that plasma insulin concentrations were similar in both groups (peak values 22 +/- 2 vs. 23 +/- 2 microU/ml in diabetics and nondiabetics, respectively). This abnormality in glucose production was nearly completely compensated for by a paradoxical decrease in glucose utilization after injection of insulin (basal 2.11 +/- 0.03----1.86 +/- 0.06 mg/kg per min at 21/2 h in diabetics vs. basal 2.08 +/- 0.04----2.39 +/- 0.11 mg/kg per min at 21/2 h nondiabetics, P less than 0.01), which could not be accounted for by differences in plasma glucose concentrations; the net result was a modest prolongation of hypoglycemia. Plasma glucagon (area under the curve [AUC] above base line, 12 +/- 3 vs. 23 +/- 3 mg/ml X 12 h in nondiabetics, P less than 0.05), cortisol (AUC 2.2 +/- 0.5 vs. 4.0 +/- 0.7 mg/dl X 12 h in nondiabetics, P less than 0.05), and growth hormone (AUC 1.6 +/- 0.4 vs. 2.9 +/- 0.4 micrograms/ml X 12 h in nondiabetics, P less than 0.05) responses in the diabetics were decreased 50% while their plasma norepinephrine responses (AUC 49 +/- 12 vs. 21 +/- 5 ng/ml X 12 h in nondiabetics, P less than 0.05) were increased twofold (P less than 0.05) and their plasma epinephrine responses were similar to those of the nondiabetics (AUC 106 +/- 17 vs. 112 +/- 10 ng/ml X 12 h in nondiabetics). In both groups of subjects, increases in plasma glucagon were inversely correlated with plasma glucose concentrations (r = -0.80 in both groups, P less than 0.01) and suppression of endogenous insulin secretion (r = -0.57 in nondiabe
G B Bolli, E Tsalikian, M W Haymond, P E Cryer, J E Gerich
To define the factors responsible for the inactivation of the active fragment derived from Factor XII (Factor XIIf ) in plasma, we studied the inactivation kinetics of Factor XIIf in various purified and plasma mixtures. We also analyzed the formation of 125I-Factor XIIf -inhibitor complexes by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In purified systems, the bimolecular rate constants for the reactions of Factor XIIf with C-1-inhibitor, alpha 2-antiplasmin, and antithrombin III were 18.5, 0.91, and 0.32 X 10(4) M-1 min-1, respectively. Furthermore, SDS-PAGE analysis revealed that 1:1 stoichiometric complexes were formed between 125I-Factor XIIf and each of these three inhibitors. In contrast, kinetic and SDS-PAGE studies indicated that Factor XIIf did not react with alpha 1-antitrypsin or alpha 2-macroglobulin. The inactivation rate constant of Factor XIIf by prekallikrein-deficient plasma was 14.4 X 10(-2) min-1, a value that was essentially identical to the value predicted from the studies in purified systems (15.5 X 10(-2) min-1). This constant was reduced to 1.8 X 10(-2) min-1 when Factor XIIf was inactivated by prekallikrein-deficient plasma that had been immunodepleted (less than 5%) of C-1-inhibitor. In addition, after inactivation in normal plasma, 74% of the active 125I-Factor XIIf was found to form a complex with C-1-inhibitor, whereas 26% of the enzyme formed complexes with alpha 2-antiplasmin and antithrombin III. Furthermore, 42% of the labeled enzyme was still complexed with C-1-inhibitor when 125I-Factor XII was inactivated in hereditary angioedema plasma that contained 32% of functional C-1-inhibitor. This study quantitatively demonstrates the dominant role of C-1-inhibitor in the inactivation of Factor XIIf in the plasma milieu.
A de Agostini, H R Lijnen, R A Pixley, R W Colman, M Schapira
Binding of 125I-Factor XIa to platelets required the presence of high molecular weight kininogen, was enhanced when platelets were stimulated with thrombin, and reached a plateau after 4-6 min of incubation at 37 degrees C. Factor XIa binding was specific: 50- to 100-fold molar excesses of unlabeled Factor XIa prevented binding, whereas Factor XI, prekallikrein, Factor XIIa, and prothrombin did not. When washed erythrocytes, added at concentrations calculated to provide an equivalent surface area to platelets, were incubated with Factor XIa, only a low level of nonspecific, nonsaturable binding was detected. Factor XIa binding to platelets was partially reversible and was saturable at concentrations of added Factor XIa of 0.2-0.4 microgram/ml (1.25-2.5 microM). The number of Factor XIa binding sites on activated platelets was estimated to be 225 per platelet (range, 110-450). We conclude that specific, high affinity, saturable binding sites for Factor XIa are present on activated platelets, are distinct from those previously demonstrated for Factor XI, and require the presence of high molecular weight kininogen.
D Sinha, F S Seaman, A Koshy, L C Knight, P N Walsh
University of California, Davis line 200 White Leghorn chickens develop an inherited progressive fibrotic disease that includes the appearance of antinuclear antibodies (ANA). To further characterize these ANA, serial aged line 200 birds were studied. Greater than 50% of line 200 birds develop antinuclear and anticytoplasmic antibodies; fluorescent staining patterns included cytoplasmic spider web, most prevalent at 1 mo of age, and fine speckled patterns, characteristic of chickens 6 mo and older. By enzyme-linked immunosorbent assay, 40.4% of line 200 birds were found to have antibodies to single-stranded DNA (ssDNA). In contrast, antibodies to histones, RNA, or poly A . poly U were not detected. Precipitating antibodies to saline extracts from chicken liver were noted in 33.3% of line 200 birds. Saline extracts from turkey, pheasant, and partridge liver but not rat, rabbit, or mouse tissues were also positive in immunodiffusion testing with these line 200 birds. The antigenicity of chicken liver extracts was sensitive to pronase, protease K. and pH variations greater than 10 and less than 5; however, they were resistant to trypsin. DNase. RNase, and incubation at 37 degrees C and 56 degrees C for 1 h. Cell fractionation in conjunction with column chromatographic techniques revealed that several protein antigens with apparent molecular weights in the range of 62,000-290,000 were present in cytoplasm but not in isolated nuclei. Line 200 sera were not reactive against nuclear ribonucleoprotein, Sm, Scl-70, or SS-B/La antigens. Thus, line 200 chickens develop antinuclear and anticytoplasmic antibodies at an early age, which recognize a unique group of protein antigenic determinants found only in avian species. Moreover, and of particular interest, the presence of autoantibodies to saline-extractable antigens correlated with positive ANA, antibodies in ssDNA, and to the clinical expression of disease.
D C Haynes, M E Gershwin
Collagens extracted from bones, cartilage, dermis, and dura mater of an infant with type II (lethal perinatal) osteogenesis imperfecta were evaluated with respect to chain composition and chemical characteristics of their constituent chains. The results indicated that the various types of collagen were present in the indicated tissues in proportions that approximated normal tissues. Nevertheless, the constituent chains of collagens extracted from dermis, i.e., alpha 1(I), alpha 2(I), alpha 1(III), alpha 1(V), and alpha 2(V), chromatographed on carboxymethyl cellulose as though they possessed substantially lower overall positive charge than the homologous chains of normal tissues. Amino acid analyses of the chains confirmed this observation and showed that the chains lacked five to seven residues of lysine (plus hydroxylysine). It was subsequently shown that the apparent deficiency in lysyl residues was due, at least in part, to the presence of unusually high levels of allysine , a cross-link precursor formed from peptide-bound lysine under the catalytic action of lysyl oxidase. These results, in conjunction with previous results obtained on collagens from type II osteogenesis imperfecta tissues, suggest that aberrant fibril formation in this syndrome allows increased lysyl oxidase activity.
O M Petrovic, E J Miller
During inflammation, the superoxide anion (O-2) and hydrogen peroxide (H2O2) are produced by stimulated polymorphonuclear leukocytes and macrophages. The toxic effects of these reactive oxygen intermediates increase when traces of iron are present, because iron catalyzes the formation of the hydroxyl radical (OH.). Partially saturated iron-binding proteins, such as transferrin and ferritin, are unable to catalyze OH. formation in vitro. Mobilization of iron from these proteins is necessary for iron stimulation of OH. formation. This paper reports that stimulated polymorphonuclear leukocytes mobilize iron from human and horse ferritin, but not from human transferrin. Iron release from ferritin depends on O-2 because it can be prevented by the addition of superoxide dismutase. Catalase and dimethylsulfoxide have no inhibitory effect on iron mobilization. The efficiency of the iron release increases at low levels of O-2 production. Only O-2 produced by granulocytes is sufficient for iron mobilization, because solid potassium superoxide is also able to release iron from ferritin. We propose that this reaction may potentiate the formation of the OH. radical in inflammatory states.
P Biemond, H G van Eijk, A J Swaak, J F Koster
The hyperparathyroidism characteristic of patients with moderate renal insufficiency could be caused by decreases in the plasma concentration of ionized calcium (Ca++) evoked by: (a) recurring increases in the plasma concentration of inorganic phosphorus that may be detectable only in the post-prandial period; (b) a reversible, phosphorus-mediated suppression of renal 25-hydroxyvitamin D-1 alpha-hydroxylase that decreases the plasma concentration of 1,25-dihydroxyvitamin D (1,25-(OH)2D) enough to decrease both gut absorption and bone resorption of Ca++; (c) both of these. In a group of eight children with moderate renal insufficiency, mean glomerular filtration rate (GFR) 45 +/- 4 (SE) ml/min per 1.73 M2, ages 6-17 yr, we tested these hypotheses by determining the effect of short term (5 d) restriction and supplementation of dietary intake of phosphorus on the plasma concentration of 1,25-(OH)2D, the serum concentrations of immunoreactive parathyroid hormone (iPTH) and phosphorus, and the fractional renal excretion of phosphorus ( FEPi ). When dietary phosphorus was normal, 1.2 g/d, the serum concentrations of phosphorus throughout the day were not greater than those of normal control children, and the serum concentrations of carboxyl-terminal iPTH (C-iPTH) were greater, 59 +/- 9 vs. 17 +/- 3 mu leq/ml, and unchanging; the serum concentration of intact-iPTH was also greater, 198 +/- 14 vs. 119 +/- 8 pg/ml. The plasma concentration of 1,25-(OH)2D was lower than that of age-matched controls, 27 +/- 3 vs. 36 +/- 2 pg/ml (P less than 0.01). When dietary phosphorus was restricted to 0.35 g/d, the plasma concentration of 1,25-(OH)2D increased by 60% to a mean value not different from that of normal controls, while serum concentrations of C-iPTH and intact-iPTH decreased by 25%, the latter concentration to a mean value not different from that of controls. FEPi decreased from 31 to 9%. When dietary phosphorus was supplemented to 2.4 g/d, the plasma concentration of 1,25-(OH)2D decreased 32%, while those of C-iPTH and intact-iPTH increased by 131 and 45%, respectively; FEPi increased from 27 to 53%. Plasma concentrations of 25-hydroxyvitamin D remained normal and unchanged, and GFR did not change when dietary phosphorus was manipulated. The data demonstrate that in children with moderate renal insufficiency: (a) A normal dietary intake of phosphorus in attended by a decreased circulating concentration of 1,25-(OH)2D and an increased concentration of iPTH, but not by recurring increases in the serum concentration of phosphorus at any time of the day; (b) Dietary phosphorus is, however, a major determinant of the circulating concentrations of both 1,25-(OH)2D and iPTH, which vary inversely and directly, respectively, with dietary intake of phosphorus, and increase and decrease, respectively, to normal values when phosphorus is restricted for 5 d; (c) Restriction and supplementation of dietary phosphorus induces changes in the serum concentration of iPTH that correlate strongly but inversely with those induced in the plasma concentration of 1,25-(OH)2D (r = -0.88, P < 0.001); and (d) The physiologic responsiveness of the renal tubule to changes in dietary phosphorus is to a substantial extent intact. The data provide support for the second hypothesis stated.
A A Portale, B E Booth, B P Halloran, R C Morris Jr
To identify the temporal changes occurring during progression and regression of atherosclerosis in nonhuman primates, we have studied the physicochemical and histological characteristics of arterial wall lesions during a 30-mo progression period of diet-induced hypercholesterolemia and during a 12-mo period of regression. Three groups of cynomolgous monkeys (Macaca fascicularis) were studied. Control groups were fed a basal chow diet for 18, 24, and 30 mo and were compared with progression groups that were fed a high-cholesterol-containing diet for up to 30 mo. Regression groups were fed a high-cholesterol diet for 18 mo to induce atherosclerosis and then fed monkey chow for up to 12 mo. The progression group monkeys were killed at 6, 12, 18, 24, and 30 mo, and the regression animals were killed at 24 and 30 mo (i.e., after 6 and 12 mo of being fed a noncholesterol-containing chow diet). Histology and morphometry, physical microscopy for cholesterol monohydrate crystals, foam cell and droplet melting points and chemical composition studies were completed on a large number of individual arterial lesions. Control animals had very little cholesterol ester, rare foam cells, and no extracellular cholesterol ester droplets or cholesterol crystals. During progression, the arteries first increased cholesterol ester content to produce high melting (approximately 45 degrees C) foam cell-rich lesions essentially devoid of cholesterol crystals. With time, the number of cholesterol crystals increased so that by 30 mo large numbers were present. Foam cells decreased with time but their melting temperature remained high while that of extracellular droplets fell to approximately 38 degrees C. Between 18 and 30 mo necrosis appeared and worsened. After 6-mo regression, unexpected changes occurred in the lesions. Compared with 24-mo progression, the chemical composition showed a relative increase in free cholesterol, a decrease in cholesterol ester and microscopy revealed large numbers of cholesterol crystals. Concomitantly, foam cells decreased and the melting temperature of both intra- and extracellular cholesterol ester markedly decreased. After 12-mo regression cholesterol decreased, cholesterol crystals and necrosis diminished and collagen appeared increased. Thus, during progression there is initially an increase in the number of foam cells containing very high-melting intracellular cholesterol ester droplets. By 30 mo, cholesterol crystals and necrosis dominate and high-melting foam cells appear only at lesion margins, suggesting that the initial process continues at the lesion edge. The lower melting point of extracellular esters indicates a lipid composition different from intracellular droplets. Thus, the changes observed in these animals generally reflect those predicted for progression of human atherosclerosis. During the initial 6 mo of regression, necrosis remains, the number of foam cell decreases, and cholesterol ester content decreases; however the relative proportion of free cholesterol content increases, and large numbers of cholesterol content are formed. Thus, large and rapid decreases in serum cholesterol concentration to produce regression in fact may result in the precipitation of cholesterol monohydrate and an apparent worsening of the lesions. More prolonged regression (12-mo) tends to return the lipid composition of the artery wall towards normal, partially reduces cholesterol crystals, and results in an improved but scarred intima.
D M Small, M G Bond, D Waugh, M Prack, J K Sawyer
Mononuclear phagocytes, particularly macrophages (M phi) that have been activated by lymphokines, are the principal defense against intracellular pathogens such as Toxoplasma gondii. To determine reasons for the newborns' susceptibility to Toxoplasma infection, we compared: the interaction of Toxoplasma with newborns' mononuclear phagocytes (blood monocytes and two types of newborn M phi, those derived from blood monocytes or from placental tissue) with adults' blood monocytes and monocyte-derived M phi and the production of M phi-activating lymphokines (MAF) by Concanavalin A (ConA)-stimulated newborn and adult blood mononuclear cells (MC). Newborn and adult monocytes killed Toxoplasma with equal efficiency. Similarly, survival and replication of Toxoplasma were comparable in control newborn and adult M phi. Exposure to adult ConA supernatants significantly decreased the survival and replication of Toxoplasma both in adult and newborn M phi. In contrast, exposure to cord blood ConA supernatants failed to affect the survival or the replication of Toxoplasma in newborn M phi and decreased the replication but not the survival of Toxoplasma in adult M phi. Exposure to ConA supernatants of peripheral blood MC from 2-5-d old newborns failed to affect survival or replication of Toxoplasma in newborn or adult M phi. Thus, both generation of MAF by newborn blood MC and response to newborn MAF by newborn M phi were impaired. Generation of MAF by adult blood mononuclear cells was not inhibited by cord blood MC nor was generation of MAF by cord blood MC increased by depletion of OKT8 antibody-binding cells, by depletion of adherent cells with or without addition of adult adherent cells, or by addition of indomethacin. Depletion of OKT4 antibody-binding cells abrogated the generation of MAF both by adult and cord blood MC. The activity of adult ConA supernatants was abrogated by dialysis at pH 2 or by addition of anti-gamma-interferon but not anti-alpha-interferon antibody. However, the correlation between antiviral interferon activity and anti-Toxoplasma activity was weak (r = 0.40). Enhanced M phi anti-Toxoplasma activity was not associated with detectably enhanced superoxide anion generation, nitroblue tetrazolium reduction, or phagolysosome fusion, and was not inhibited by catalase, superoxide dismutase, or mannitol. These results indicate that generation of and response to MAF is decreased in cells from human newborns and that gamma-interferon may be the major MAF under these conditions.
C B Wilson, J E Haas
Marrow blasts from children with B cell precursor acute lymphoblastic leukemia (ALL) were studied for differences in quantitative expression of the common ALL antigen (CALLA). Of 42 untreated patients, 35 had detectable amounts of CALLA by flow cytometric (FCM) analysis of J-5 monoclonal antibody binding. Using an FCM technique that provides correlated measurements of a given cell surface antigen, cell size, and DNA content, we detected increased CALLA expression as lymphoblasts moved from G0/G1 phase through S phase of the cell cycle. The density of the antigen (per unit of blast surface area) remained relatively constant over the same interval, indicating that the change was not due to S phase-specific enhancement of CALLA expression. Eight cases had hyperdiploid cellular DNA content and in seven of these, only cells with clonal abnormalities of DNA content expressed the CALLA marker. Mean amounts of CALLA for each patient ranged widely within the study group, from very high to marginally detectable. This variation had no discernible relation to cell size, stem-line DNA content, percentage of cells in S phase, or the presence or absence of cytoplasmic immunoglobulin. Results of a univariate proportional hazards analysis showed that both quantitative level of CALLA for S phase cells (P = 0.048) and white blood cell count (P = 0.012) had made significant contributions to treatment outcome. Patients with relative amounts of CALLA less than the median value for the entire CALLA+ group had a higher rate of failure, which was virtually identical to that for the seven HLA-DR+ patients whose blasts lacked detectable CALLA. The observed interpatient variation in quantitative expression of CALLA is consistent with recognized steps in B cell precursor differentiation and may be useful in distinguishing patients with a less favorable prognosis.
A T Look, S L Melvin, L K Brown, M E Dockter, P K Roberson, S B Murphy
Chemotaxis and generation of the oxidative burst by phagocytes are among the biological functions thought to require methylation reaction(s) for their expression. The present study investigated the effect of different stimuli of the oxidative burst on lipid methylation by human elutriated monocytes as measured by methyl group incorporation from [methyl-3H]methionine into both phospholipid and neutral lipid extracts. Normal monocytes, incubated at 37 degrees C for 1 h with 2 microM methionine, incorporated 10.2-fmol/10(6) cells and 73.6-fmol/10(6) cells of methyl groups into neutral lipids and phospholipids, respectively. Stimulators of the respiratory burst, such as the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate, and the calcium ionophore, A23187, decreased the incorporation of methyl groups into both neutral lipids and phospholipids in a similar manner. Increasing the concentration of methionine in the medium reversed or attenuated the inhibition achieved at lower levels. An inverse relationship existed between the degree of methylation and the extent of stimulation of the oxidative burst, measured as superoxide anion (O-2) release. Stimulated monocytes oxidized methionine to methionine sulfoxide (which cannot act as a methyl-donor), and this was dependent on activation of the respiratory burst. Elimination of the accumulated methionine sulfoxide by replacement of the medium or by prevention of extracellular methionine oxidation by catalase did not effectively restore the normal level of methylation in stimulated cells, and the reduced methylation was not primarily related to a defective methionine uptake by stimulated monocytes. These data suggest that intracellular events related to activation of the respiratory burst are responsible for the decreased lipid methylation in stimulated cells, possibly by their leading to intracellular formation of methionine sulfoxide and by their limiting the availability of methyl-donor. This mechanism may be of potential relevance for the expression of biological functions where methionine-dependent reactions are involved.
E Bonvini, P Bougnoux, H C Stevenson, P Miller, T Hoffman
The pattern of episodic gonadotropin release was studied in 15 normal female volunteers during the luteal phase of the menstrual cycle with 24 h of blood sampling for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels at 10-min intervals. Six subjects (two in the early, two in the mid-, and two in the late luteal phase) also had each of these specimens processed for progesterone levels. A progressive slowing of LH pulsations was present across the luteal phase with the mean LH pulse frequency declining from 15.2 pulses/24 h in the early to 8.4/24 h in the late luteal phase. A trend towards reduction in the amplitude of LH pulses was also observed (12.3 +/- 2.2 SD mIU/ml in the early vs. 8.6 +/- 3.4 mIU/ml in the late luteal phase; NS). In addition, LH pulses of heterogeneous amplitude were identified during the same 24-h study. The mean +/- SD of the larger and of the smaller LH pulses was 16.9 +/- 4.7 and 2.3 +/- 1.0 mIU/ml, respectively (P less than 0.001). While the slowing of the frequency of all LH pulses correlated well (r = 0.80, P less than 0.001) with the day of the luteal phase and poorly with the actual plasma progesterone levels, the incidence of the small LH pulses was highest in the mid-luteal phase and correlated well with the mean progesterone plasma levels (r = 0.63, P less than 0.01). In the early luteal phase, the pattern of progesterone secretion was stable over the 24-h studies and showed no relationship to episodic LH release. In contrast, in the mid- and late luteal phase, plasma progesterone concentrations rapidly fluctuated during the 24-h studies from levels as low as 2.3 to peaks of 40.1 ng/ml, often within the course of minutes. Progesterone increments closely attended episodes of LH release, as documented by the significant (P less than 0.05) cross-correlation between LH and progesterone levels, at time lags of 25-55 min. The results of this study indicate that in the human luteal phase: (a) the frequency of pulsatile release of LH declines progressively and correlates well with the duration of exposure to progressively and correlates well with the duration of exposure to progesterone; (b) the amplitude of LH pulses varies with the appearance of an increased percentage of smaller pulses correlating well with the acute level of progesterone; (c) in the early luteal phase, the pattern of progesterone secretion is stable; (d) in the mid- and late luteal phase, progesterone secretion is episodic, and correlates with LH pulsatile release; and (e) single progesterone estimations in the mid- and late luteal phase do not accurately reflect corpus luteum adequacy.
M Filicori, J P Butler, W F Crowley Jr
We investigated the effects of alpha and/or beta adrenergic blockade (with phentolamine and/or propranolol) on glucose homeostasis during exercise in six normal subjects and in seven Type I diabetic subjects. The diabetics received a low dose insulin infusion (0.07 mU/kg X min) designed to maintain plasma glucose at approximately 150 mg/dl. In normals, neither alpha, beta, nor combined alpha and beta adrenergic blockade altered glucose production, glucose uptake, or plasma glucose concentration during exercise. In diabetics, exercise alone produced a decline in glucose concentration from 144 to 116 mg/dl. This was due to a slightly diminished rise in hepatic glucose production in association with a normal increase in glucose uptake. When exercise was performed during beta adrenergic blockade, the decline in plasma glucose was accentuated. An exogenous glucose infusion (2.58 mg/kg X min) was required to prevent glucose levels from falling below 90 mg/dl. The effect of beta blockade was accounted for by a blunted rise in hepatic glucose production and an augmented rise in glucose utilization. These alterations were unrelated to changes in plasma insulin and glucagon levels, which were similar in the presence and absence of propranolol. In contrast, when the diabetics exercised during alpha adrenergic blockade, plasma glucose concentration rose from 150 to 164 mg/dl. This was due to a significant increase in hepatic glucose production and a small decline in exercise-induced glucose utilization. These alterations also could not be explained by differences in insulin and glucagon levels. We conclude that the glucose homeostatic response to exercise in insulin-dependent diabetics, in contrast to healthy controls, is critically dependent on the adrenergic nervous system.
D C Simonson, V Koivisto, R S Sherwin, E Ferrannini, R Hendler, A Juhlin-Dannfelt, R A DeFronzo
Although intrarenal infusions of kinins produce diuresis, it is not clear to what extent this response is due to hemodynamically mediated medullary washout and/or to direct epithelial effects of kinins. Recent evidence has shown that bradykinin binds to collecting tubules in vitro. We therefore examined the interactions of lysyl-bradykinin and antidiuretic hormone (ADH) with respect to hydraulic conductivity (Lp) in the rabbit cortical collecting tubule perfused in vitro. To ensure adequate substrate for prostaglandin synthesis, the bath contained 2.5 microM arachidonic acid. Arachidonic acid produced no change in base-line Lp and had no effect on the subsequent response to a supramaximal dose of ADH (100 microU/ml). Therefore, all subsequent experiments were done in the presence of arachidonic acid. Lysyl-bradykinin (10(-9)M) added to either the lumen or bath had no effect on base-line Lp. Collecting tubules which were exposed for 1 h to bath lysyl-bradykinin (10(-9)M) had a significantly diminished subsequent Lp in response to ADH (P less than 0.02). In tubules exposed to bath lysyl-bradykinin plus indomethacin (5 microM), the subsequent ADH response was normal. Lysyl-bradykinin (10(-9)M) added to the lumen had no effect on subsequent ADH response. We conclude that lysyl-bradykinin from the basolateral side inhibits the hydroosmotic response of the cortical collecting tubule to ADH, and that this inhibition is probably prostaglandin-mediated. Lysyl-bradykinin does not affect water flow from the luminal surface. These data indicate that the diuresis seen with kinin infusions may result, at least in part, from a direct epithelial effect. They also suggest a role of the renal kallikrein-kinin system in modulating water transport in vivo.
V L Schuster, J P Kokko, H R Jacobson
Calcium absorption decreases with aging, particularly after age 70 yr. We investigated the possibility that this was due to abnormal vitamin D metabolism by studying 10 normal premenopausal women (group A), 8 normal postmenopausal women within 20 yr of menopause (group B), 10 normal elderly women (group C), and 8 elderly women with hip fracture (group D) whose ages (mean +/- SD) were 37 +/- 4, 61 +/- 6, 78 +/- 4, and 78 +/- 4 yr, respectively. For all subjects, serum 25-hydroxyvitamin D [25(OH)D] did not decrease with age, but serum 1,25-dihydroxyvitamin D [1,25(OH)2D], the physiologically active vitamin D metabolite, was lower (P = 0.01) in the elderly (groups C and D; 20 +/- 3 pg/ml) than in the nonelderly (groups A and B; 35 +/- 4 pg/ml). The increase of serum 1,25(OH)D after a 24-h infusion of bovine parathyroid hormone fragment 1-34, a tropic agent for the enzyme 25(OH)D 1 alpha-hydroxylase, correlated inversely with age (r = -0.58; P less than 0.001) and directly with glomerular filtration rate (r = 0.64; P less than 0.001). The response was more blunted (P = 0.01) in elderly patients with hip fracture (13 +/- 3 pg/ml) than in elderly controls (25 +/- 3 pg/ml). We conclude that an impaired ability of the aging kidney to synthesize 1,25(OH)2D could contribute to the pathogenesis of senile osteoporosis.
K S Tsai, H Heath 3rd, R Kumar, B L Riggs
The elongated alpha-globin chains of hemoglobin Constant Spring (alpha cs chain of HbCS ) are produced in low amounts such that the alpha cs-gene acts as a form of alpha-thalassemia; yet in the homozygous state the pathophysiological effects of this mutant are more severe than in the corresponding conditions that result from alpha-globin gene deletions. In studies designed to examine this discrepancy, we have demonstrated that a significant proportion of red cells produced in an HbCS homozygote has a much reduced red cell life span. Contrary to previous reports, we have been able to demonstrate the expected deficit in alpha-chain production in this condition and have shown that both the cessation of globin chain synthesis in vitro and the destruction of the excess beta-chains occur unusually rapidly. Comparison with various deletion forms of alpha-thalassemia suggests that, in terms of intracellular globin chain precipitates and free beta-chain pool, homozygous HbCS red cells more closely resemble those of HbH disease, with three of the four alpha-genes inactivated, than they do the more comparable alpha-thalassemia carriers with only two genes deleted.
S Derry, W G Wood, M Pippard, J B Clegg, D J Weatherall, S N Wickramasinghe, J Darley, S Fucharoen, P Wasi
To identify the mechanism(s) of the altered glucoregulatory response to a glucose load in subjects with impaired glucose tolerance, we selectively quantitated the components of net splanchnic glucose balance, i.e., splanchnic glucose uptake and hepatic glucose output, as well as peripheral glucose uptake, by combining [3-3H]glucose infusion with hepatic vein catheterization. After intravenous glucose infusion (6 mg X kg-1 X min-1 for 90 min), blood glucose rose to 172 +/- 7 mg/dl in controls and 232 +/- 13 mg/dl in subjects with impaired glucose tolerance (P less than 0.01). The response of plasma insulin did not differ significantly between the two groups (29 +/- 4 vs. 40 +/- 10 microU/ml at 90 min in control and in glucose intolerant subjects, respectively; P = NS). In both groups, glucose infusion caused the net splanchnic glucose balance to switch from the net output of the basal state to a net glucose uptake. However, this effect was more marked in subjects with impaired glucose tolerance than in control subjects (at 90 min: 2.83 +/- 0.53 vs. 1.60 +/- 0.18 mg X kg-1 X min-1, respectively: P less than 0.05). The different pattern of splanchnic glucose balance was entirely accounted for by a greater rise in splanchnic glucose uptake in the group of glucose intolerants , as the suppression of endogenous glucose output by the glucose load was practically complete in both groups. In contrast, glucose uptake by peripheral tissues increased considerably less in subjects with impaired glucose tolerance than in controls (2.2-2.6 vs 3.6-4.1 mg X kg-1 X min-1, respectively, between 60 and 90 min; P less than 0.01-0.001). Furthermore, a net splanchnic lactate uptake was present in the basal state, which was inhibited by the glucose load and switched to a comparable net lactate output in both groups. These results indicate that the mechanism responsible for the altered glucoregulation in subjects with impaired glucose tolerance resides entirely in the peripheral tissues whose ability to dispose of a glucose load is drastically reduced. On the other hand, no defect is detectable in any of the explored mechanisms regulating splanchnic glucose metabolism during the disposal of an exogenous glucose load.
L Saccà, G Orofino, A Petrone, C Vigorito
Hereditary elliptocytosis (HE) is a clinically and biochemically heterogenous group of diseases characterized by elliptically shaped erythrocytes and an autosomal dominant mode of inheritance. Whereas the self-association of spectrin heterodimers to tetramers is defective in a subpopulation of HE patients, designated HE[SpD-SpD], it is normal in others. We have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from patients with HE[SpD-SpD] to determine if the functional defects in spectrin self-association could be correlated with structural changes in the spectrin molecule. Although the peptide pattern produced by limited tryptic digestion of spectrin extracts from those HE patients with normal spectrin self-association was indistinguishable from the pattern from control normal volunteers, digestion of the spectrin extracts from the HE[SpD-SpD] patients showed a reproducible diminution in the 80,000-D domain of the alpha-subunit, which is involved in spectrin dimer self-association. The decrease in the 80,000-D fragment was associated with an increase in a 74,000-D fragment in eight of nine families, or, in one family, with an increase of fragments at 46,000 and 17,000 D. These atypical peptide patterns were similar to those previously reported in two variants of hereditary pyropoikilocytosis (HPP), which also had defective self-association of spectrin. These data indicate that two distinct structural variants of spectrin alpha-subunit are associated with the defective spectrin heterodimer self-association in a subpopulation of HE patients.
J Lawler, S C Liu, J Palek, J Prchal
The regulation of human Factor IXa was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, approximately 60% of the 125I-Factor IXa was bound to antithrombin III (ATIII) by 2 h, with no binding to alpha 2-macroglobulin or alpha 1-proteinase inhibitor, as assessed by gel electrophoresis and IgG- antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the 125I-Factor IXa was bound to ATIII by 1 min. The distribution of 125I-Factor IXa in mouse plasma was similar. The clearance of 125I-Factor IXa was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and alpha 1-proteinase inhibitor-trypsin, but not alpha 2-macro-globulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl - (DIP-) Factor Xa, DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor Xa, or DIP-thrombin. Electrophoresis and IgG- antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the 125I-Factor IXa was bound to ATIII. Organ distribution studies with 125I-Factor IXa demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IXa binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IXa, but not Factor X, Factor Xa, prothrombin, or thrombin. The other site recognizes thrombin, Factor Xa, and Factor IXa, but not the zymogen forms of these clotting factors. After this binding, Factor IXa is bound to ATIII and the complex is cleared from the circulation by hepatocytes.
H E Fuchs, H G Trapp, M J Griffith, H R Roberts, S V Pizzo
Urinary acidification in the mammalian collecting tubule is similar to that in the turtle bladder, an epithelium whose H+ secretion is due to a luminal proton-translocating ATPase. We isolated a fraction from bovine renal medulla, which contains ATP-dependent proton transport. H+ transport was found to be electrogenic in that its rate was reduced by a membrane potential. H+ transport activity was inhibited by N-ethyl maleimide and dicyclohexyl carbodiimide, but not by oligomycin or vanadate; its activity did not depend on the presence of potassium, differentiating this ATPase from the mitochondrial F0-F1 ATPase and the gastric H+-K+ ATPase. H+ transport activity had a specific substrate requirement for ATP, distinguishing this pump from the lysosomal H+ ATPase, which uses guanosine or inosine triphosphate as well. The distribution of this H+ pump on linear sucrose density gradient was different from that of markers of lysosomes and basolateral membranes. These results show that the kidney medulla contains an H+ -translocating ATPase different from mitochondrial, gastric, and lysosomal proton pumps, but similar to the turtle bladder ATPase.
S Gluck, Q Al-Awqati
Adult T cell leukemia (ATL) and Sézary leukemia are malignant proliferations of T lymphocytes that share similar cell morphology and clinical features. ATL is associated with HTLV (human T cell leukemia/lymphoma virus), a unique human type C retrovirus, whereas most patients with the Sézary syndrome do not have antibodies to this virus. Leukemic cells of both groups were of the T3, T4-positive, T8-negative phenotype. Despite the similar phenotype, HTLV-negative Sézary leukemic cells frequently functioned as helper cells, whereas some HTLV-positive ATL and HTLV-positive Sézary cells appeared to function as suppressors of immunoglobulin synthesis. One can distinguish the HTLV-positive from the HTLV-negative leukemias using a monoclonal antibody (anti-Tac) that appears to identify the human receptor for T cell growth factor (TCGF). Resting normal T cells and most HTLV-negative Sézary cells were Tac-negative, whereas all ATL cell populations were Tac-positive. The observation that ATL cells manifest TCGF receptors suggests the possibility that an abnormality of the TCGF-TCGF receptor system may partially explain the uncontrolled growth of these cells.
T A Waldmann, W C Greene, P S Sarin, C Saxinger, D W Blayney, W A Blattner, C K Goldman, K Bongiovanni, S Sharrow, J M Depper
The inflammatory response to intraarticular urate crystals is known to be variable in gouty arthritis. One source of variability may be the modulation of cellular responses by crystal-bound proteins. We have identified three apolipoproteins among the polypeptides bound to urate crystals exposed to plasma. Identification was first based on their coelectrophoresis with polypeptides from isolated lipoproteins and diminution in the protein coat of crystals exposed to lipoprotein-depleted plasma. The apoproteins were immunochemically identified by the Western blotting technique as apoprotein A-I, apoprotein B (apo B), and apoprotein E. Because neutrophils play a central role in acute gout, we investigated the potential effects of lipoproteins on neutrophil-urate crystal interactions. Plasma profoundly inhibited urate crystal-induced neutrophil luminol-dependent chemiluminescence (CL). Lipoprotein depletion by KBr density gradient centrifugation completely abrogated the inhibitory effect of plasma on urate-induced CL. The inhibitory activity of lipoprotein-depleted plasma was restored by adding back the d less than or equal to 1.25 g/cm3 lipoprotein fraction. Plasma also inhibited urate crystal-induced neutrophil superoxide generation and cytolysis (lactic dehydrogenase loss). This inhibition was significantly diminished by lipoprotein depletion, indicating that the lipoprotein effect was not limited to CL. Lipoprotein-depleted plasma reconstituted with very low, intermediate, and low density lipoproteins (LDL) inhibited crystal-induced CL. High density lipoprotein reconstitution was without effect. Immunodepletion from plasma of all apo B lipoproteins by agarose-bound apo B-specific antibody also removed all inhibitory activity for urate-induced CL. Thus, apo B lipoproteins were shown to be the inhibitory species in plasma. Binding of apo B lipoproteins to urate crystals and inhibition of CL was also seen in the absence of other plasma proteins. In addition, the binding of whole lipoprotein particles to the crystals was verified by detection of crystal-associated cholesterol in addition to the apoprotein. The effects of LDL on urate crystal-induced CL were stimulus specific. Coincubation of urate crystals and neutrophils in the presence of 10 micrograms/ml LDL resulted in 83% inhibition. In contrast, CL responses to a chemotactic hexapeptide, opsonized zymosan, and Staphylococcus aureus were not inhibited by LDL. The effects of depletion of apo B lipoproteins on plasma suppression of urate crystal-induced CL appeared to be unique. Plasma or sera depleted of other urate crystal-binding proteins including fibrinogen, fibronectin, C1q, and IgG retained virtually all their CL inhibitory activity. Lipoproteins containing apo B are thus a major regulator of neutrophil responses to urate crystals. These lipoproteins are present in variable concentration in synovial fluid and may exert an important influence on the course of gout.
R Terkeltaub, L K Curtiss, A J Tenner, M H Ginsberg
A single-chain 55,000-mol wt form of urokinase (UK), similar to that previously isolated from urine, was purified from a transformed kidney cell culture medium and characterized; and its fibrinolytic properties were evaluated. The preparation immunoprecipitated with UK antiserum, had a low intrinsic amidolytic activity that was 0.1% of its active derivative, and resisted diisopropyl fluorophosphate treatment and inactivation by plasma inhibitors. The single-chain UK was therefore designated pro-UK. In the presence of plasmin and during clot lysis, activation by conversion to two-chain, 55,000-mol wt UK (TC-UK) was demonstrated. This did not occur during blood clotting nor on incubation with purified thrombin. Clot lysis in plasma consistently occurred in 2-5 h with 50-100 IU per ml of pro-UK, whereas comparable lysis was inconsistently achieved by 500-1,000 IU of UK. Pro-UK, in sharp contrast to UK, caused no fibrinogen degradation at fibrinolytic concentrations. In the absence of a clot, pro-UK in plasma was stable for more than 2 d. When a clot was added after incubation (37 degrees C) for 50 h, activation to full lytic activity took place. The findings in vivo were comparable but the rapid clearance of pro-UK required that it be given by a constant infusion despite its plasma stability. In rabbits, a UK-resistant species, pro-UK was significantly (P less than 0.001) more efficacious than TC-UK but neither induced significant fibrinogen degradation. In dogs, a more sensitive species, the high specificity of thrombolysis by pro-UK contrasted with the defibrinogenation and uncontrollable bleeding that accompanied thrombolysis by UK. It was concluded that clot lysis by pro-UK is more effective and specific than UK. The advantage of pro-UK is in the limitation of its activation to the site of a clot. This can be explained by an activation mechanism that is dependent, under physiological conditions, on fibrin-stabilized plasmin.
V Gurewich, R Pannell, S Louie, P Kelley, R L Suddith, R Greenlee
A new hematologic syndrome with phenotypic features of mild Hb H disease was identified in three children from two unrelated black American families. Erythrocytes from each of these children contained Hb H (beta 4) and Hb Barts (gamma 4), as well as a slowly migrating hemoglobin fraction that made up 7-10% of the total hemoglobin. The parents of the affected children all showed mild thalassemia-like changes, with one of the parents in each family also expressing the variant hemoglobin; in the latter individuals the mutant alpha-chains made up less than 2% of the total, and were present mainly or exclusively in combination with delta-chains in the form of a slowly migrating Hb A2. Purified Hb Evanston showed an increased oxygen affinity, but its Bohr effect, cooperativity, and 2,3-diphosphoglycerate effect were normal. The mutant hemoglobin appeared to have normal stability to heat and to isopropanol, and the stability of its alpha-chain in an extended time course synthesis study also appeared to be similar to that of alpha A. However, the results from short-term globin synthesis studies, and from mRNA translation in vitro, suggest that the two types of alpha-chains were synthesized at relatively equal rates, with a major fraction of the newly synthesized variant alpha-chains undergoing rapid catabolism. The hematologic data taken in combination with DNA hybridization and globin synthesis findings indicate that the proposita in each of these families has the genotype--, alpha A/--, alpha Ev. These observations suggest that two separate mechanisms are contributing to the alpha-thalassemia-like expression of Hb Evanston : the newly synthesized alpha EV-chains are unstable and are subject to early proteolytic destruction; and the mutant alpha-allele is linked to an alpha-globin gene deletion.
G R Honig, M Shamsuddin, L N Vida, M Mompoint, E Valcourt, L J Bowie, E C Jones, P A Powers, R A Spritz, M Guis
We examined the possible mechanisms of local initiation of coagulation in vegetation formation in enterococcal endocarditis by using a rabbit model. Contact activation and tissue factor expression by freshly excised aortic valves were assessed using assays developed for use with cultured cells. Bacteria alone lacked procoagulant activity and contact activation of plasma by excised valves did not occur. 4-d infected but not control valves expressed significant tissue factor activity (231 +/- 17 mU vs. 51 +/- 7 SE), which did not correlate with numbers of bacteria in vegetations. Tissue factor activity was also present in valves from rabbits infected for 1 and 2 d, as well as those from granulocytopenic and monocytopenic animals. Our findings suggest that tissue factor, expressed by host cells in response to infection, is a major stimulus for fibrin deposition in vegetation development.
T A Drake, G M Rodgers, M A Sande
The development of T lymphocyte lines and clones of defined specificity has become an important method for investigating both T cell recognition of foreign antigens as well as T cell influence on B cells. In the present study, human antigen-specific T cell lines and clones have been derived from a patient with a naturally acquired filarial infection. These T cells are of the helper phenotype (Leu 1+, Leu 2-, Leu 3+) and are independent of exogenous interleukin-2. Furthermore, these T cells have been shown to require both antigen-presenting cells and antigen for optimal proliferation. Helper function mediated by these T cells as manifested by the in vitro induction of parasite-specific antibody was antigen-dose dependent, requiring much lower antigen concentrations than those necessary to induce blastogenesis. More importantly, there is an absolute requirement of the T cell line for HLA-DR histocompatible antigen-presenting cells; clones derived from this T cell line show a more specific DR-related restriction--to only one of the two parental DR haplotypes in antigen stimulated proliferative responses. Such parasite antigen specific human helper T cell lines and clones should prove useful in exploring the fine control of the host response to naturally acquired helminth infections. In addition, these long-term T cell lines and clones can provide a potent tool for examining not only the events involved in human T cell responses to parasite antigens, but also into the associated cellular and humoral factors necessary for the B cell responses which follow.
T B Nutman, E A Ottesen, A S Fauci, D J Volkman
Abnormal ductal NaCl absorption has been known as the only defect in cystic fibrosis (CF) sweat glands. We have fortuitously found that the secretory portion of CF sweat glands is also abnormal in that it failed to show a sweating response to beta adrenergic stimulation (isoproterenol, [ISO]) both in vivo and in vitro. For the in vitro sweat test, eccrine sweat glands were isolated from skin biopsy specimens of the forearm, cannulated, and stimulated to secrete sweat. All 14 isolated CF sweat glands failed to respond to ISO + theophylline (TH, as aminophylline), but 17 of 18 control glands responded with a mean rate (SR) of 1.1 nl/min per gland. Cholinergic responsiveness of isolated CF sweat glands was comparable with that of control glands. The in vivo sweat test was performed by intradermal injection in the forearm of 0.2 ml of 2.4 or 8 X 10(-5) M ISO with or without 10(-2) M TH (and 1.4 X 10(-4) M atropine as a necessary anticholinergic agent). The beads of sweat secreted into the oil-filled sweat collection ring glued to the skin were then collected with a glass capillary under a stereomicroscope. Of 28 CF patients, 26 failed to show a secretory response to intradermal injection of ISO + TH, and 2 CF patients gave SR of less than 0.007 nl/min per gland in the first test but no response in the repeat test performed later. In contrast, all 35 age- and sex-matched control subjects responded with the mean SR of 0.72 nl/min per gland. Response of CF patients to epinephrine and phenylephrine was comparable with control, indicating that the alpha adrenergic responsiveness of CF sweat glands is not defective. A preliminary attempt was made to determine tissue cyclic AMP accumulation by radioimmunoassay in isolated sweat glands. No significant difference was observed between CF and control glands in their maximal accumulation of tissue cAMP in response to ISO or ISO + TH, except that the rise time of ISO + TH-induced cAMP accumulation in CF glands was significantly slower during the first 5 min of incubation. The data suggest that beta adrenergic regulation is abnormal in CF sweat glands and justifies further investigations into the mechanism of beta adrenergic regulation of the eccrine sweat gland in both normal and CF subjects.
K Sato, F Sato
Oxygen free radicals released during endotoxemia may contribute to the lung injury of the adult respiratory distress syndrome (ARDS). As this syndrome occurs frequently after gram-negative sepsis in humans, we studied the effect of intravenous N-acetylcysteine (NAC), a free radical scavenger, upon the endotoxin (E)-induced model of ARDS in awake sheep. In vivo studies demonstrated that NAC attenuates the endotoxin-induced rise in pulmonary artery pressure (62 +/- 3 torr with E control vs. 43 +/- 3 torr for E + NAC), and markedly diminishes the rise in lymph flow at 1 h (8.5 +/- 1.2 vs 4.5 +/- 0.6 ml/15 min) and 4 h (5.0 +/- 0.6 vs. 3.3 +/- 0.4 ml/15 min), respectively, for E control vs. E + NAC. NAC also markedly attenuated the alterations in lung mechanics after endotoxemia. Dynamic compliance at 2 h after endotoxemia was 44 +/- 6% of base line for E vs. 76 +/- 10% of base line for E + NAC. Resistance to airflow across the lung at 1 h postendotoxin was 811 +/- 280% of base line for E vs. 391 +/- 233% of base line for E + NAC. NAC substantially reduced the 1 h postendotoxin rise in lymph concentrations of thromboxane B2 (8.29 +/- 3.28 vs. 2.75 +/- 1.93 ng/ml for E vs. E + NAC) and 6-keto-prostaglandin-F1 alpha (0.91 +/- 0.27 vs. 0.23 +/- 0.12 ng/ml for E vs. E + NAC). In addition, in vitro studies were performed which revealed NAC to be a potent free radical scavenger in both biologic and nonbiologic free radical generating systems. NAC decreased phorbol-stimulated granulocyte aggregation in a concentration-dependent manner in vitro. Minimal effects were observed, however, upon leukocyte degranulation at the concentrations of NAC achieved during the in vivo tests. Thus, NAC significantly attenuated all monitored pathophysiologic changes in the endotoxin model of ARDS in sheep, possibly by its ability to scavenge toxic oxygen free radicals. A direct impairment of the ability of inflammatory cells to generate oxygen radicals cannot be ruled out.
G R Bernard, W D Lucht, M E Niedermeyer, J R Snapper, M L Ogletree, K L Brigham
Treatment with L-carnitine greatly enhanced the formation and excretion of short-chain acylcarnitines in three patients with propionic acidemia and in three normal controls. The use of fast atom bombardment mass spectrometry and linked scanning at constant magnetic (B) to electric (E) field ratio identified the acylcarnitine as propionylcarnitine in patients with propionic acidemia. The normal children excreted mostly acetylcarnitine. Propionic acidemia and other organic acidurias are characterized by the intramitochondrial accumulation of short-chain acyl-Coenzyme A (CoA) compounds. The substrate specificity of the carnitine acetyltransferase enzyme and its steady state nature appears to facilitate elimination of propionyl groups while restoring the acyl-CoA:free CoA ratio in the mitochondrion. We suggest that L-carnitine may be a useful therapeutic approach for elimination of toxic acyl CoA compounds in several of these disorders.
C R Roe, D S Millington, D A Maltby, T P Bohan, C L Hoppel
Patients with rheumatoid arthritis (RA) are known to have in vitro regulatory T cell abnormalities relating to Epstein-Barr virus (EBV). In this report, we asked whether patients with RA have more circulating EBV-infected B cells than normals. To address this question, we determined the frequency of spontaneously transforming B cells in the peripheral blood of 18 normals, 15 patients with RA, and 8 patients with systemic lupus erythematosus (SLE). The mean frequency of spontaneously transforming B cells in RA patients was 10.1/10(6) B cells, which was significantly greater than that of the normal controls, 2.8/10(6) B cells (P less than 0.005). The group of patients with SLE did not differ from the normals (P greater than 0.4). In further studies undertaken to investigate as to whether RA B cells are more easily transformed by EBV than normal B cells, we determined that the frequencies of transforming B cells in the presence of exogenous EBV were similar in RA patients and normals. Lymphocytes obtained from patients with RA demonstrate a profound T cell defect in their EBV-specific suppression, as measured in vitro; there was no direct correlation, however, between this in vitro T cell abnormality and the number of circulating EBV-infected B cells. Thus, patients with RA, as a group, have abnormally elevated numbers of circulating EBV-infected B cells, and this abnormality most likely derives from a complex dysregulation of the defense mechanisms for infection with EBV.
G Tosato, A D Steinberg, R Yarchoan, C A Heilman, S E Pike, V De Seau, R M Blaese