Table II summarizes in tabular form the major features of the fetal endocrine milieu discussed in the foregoing pages. The mammalian fetus develops in an environment where respiration, alimentation, and excretory functions are provided by the placenta. Fetal tissue metabolism is oriented largely to anabolism; body temperature is modulated by maternal metabolism, and fetal tissue thermogenesis is maintained at a basal level. Tissue and organ growth appear to be regulated by growth factors which probably function by autocrine or paracrine mechanisms during most of gestation (72, 146-148). In this milieu conventional endocrine control systems are largely redundant, and other transient systems more appropriate to the intrauterine environment have evolved. We have developed some insights into these systems, but much more information is necessary before we can truly understand this fascinating environment.
D A Fisher
These studies demonstrate that Staphylococcus aureus Cowan I (SAC), a protein A-positive Staphylococcal strain, is a potent and consistent inducer of IgM rheumatoid factor production by normal human peripheral blood mononuclear cells. The frequency and magnitude of this response greatly exceeded that of parallel cultures stimulated with pokeweed mitogen or the protein A-negative S. aureus Wood strain, although all three agents induced a similar amount of total IgM. Cell fractionation studies indicated that SAC-induced IgM rheumatoid factor is T cell-dependent. The striking ability of SAC to induce IgM rheumatoid factor may relate to its protein A content, since cultures stimulated with protein A-coupled sepharose beads also consistently produced this autoantibody. Thus SAC is a new probe of in vitro IgM rheumatoid factor production and its use has provided further evidence that most healthy individuals harbor precursors of IgM rheumatoid factor secreting cells. Unlike other polyclonal activators, SAC is unique in its capacity to bind immunoglobulin, a property that may account for its prominent anti-IgG inducing capacity.
A I Levinson, L Tar, C Carafa, M Haidar
The relationship between physical fitness and bone mass in the femoral neck, lumbar spine, and forearm was studied in 84 normal women. Femoral neck and lumbar spine bone mineral density and forearm bone mineral content were estimated by absorptiometry. Fitness was quantitated from predicted maximal oxygen uptake. Femoral neck and lumbar bone mineral density were significantly correlated with fitness as well as age and weight. In the 46 postmenopausal subjects, fitness was the only significant predictor of femoral neck bone mineral density, and both weight and fitness predicted the lumbar bone mineral density. These data represent the first demonstration of a correlation between physical fitness, and, by implication, habitual physical activity, and bone mass in the femoral neck; they also support the previous reported correlation between lumbar bone mass and physical activity. The data suggest that increased physical fitness may increase bone mass at the sites of clinically important fractures in osteoporosis.
N A Pocock, J A Eisman, M G Yeates, P N Sambrook, S Eberl
Administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] can lower the serum concentration of 25-hydroxyvitamin (25-OH-D). To determine if 1,25(OH)2D3 lowers serum 25-OH-D by increasing clearance or reducing production, we directly measured the metabolic clearance rate (MCR) of 25-OH-D in rats chronically infused with 1,25(OH)2D3. Chronic 1,25(OH)2D3 administration (0 to 75 pmol/d) reduced, in a time- and dose-dependent fashion, the serum concentrations of 25-OH-D3 and 24,25(OH)2D3 from 18 +/- 2 to 9 +/- 1 ng/ml and from 4.8 +/- 0.7 to 1.3 +/- 0.3 ng/ml, respectively, and increased sevenfold the in vitro conversion of 25-OH-D to 24,25(OH)2D3 by kidney homogenates. The reduction in serum 25-OH-D3 was completely accounted for by an increase in MCR. No change in production occurred. The influence of 1,25(OH)2D3 on serum 25-OH-D3 and 24,25(OH)2D3 was shown not to be dependent on induction of hypercalcemia. These data suggest that chronic 1,25(OH)2D3 administration lowers serum 25-OH-D by increasing the metabolic clearance of 25-OH-D3 and not by decreasing its production.
B P Halloran, D D Bikle, M J Levens, M E Castro, R K Globus, E Holton
Acute thermal injury of rat skin produces an early, acute hemoglobinemia that is associated with the presence in blood of osmotically fragile red cells (RBC) that do not contain on their surfaces measurable amounts of complement components. The hemoglobinemia and the appearance in blood of osmotically fragile RBC appear to be the result of complement activation, which leads to oxygen radical production by neutrophils and damage of RBC. This has been demonstrated in vitro as well as in vivo by the ability of antioxidant interventions or neutrophil or complement depletion procedures to prevent the appearance of osmotically fragile RBC and the release of hemoglobin. These data may be relevant to the complications of hemoglobinemia and hemoglobinuria accompanying thermal injury in humans.
J R Hatherill, G O Till, L H Bruner, P A Ward
This study investigates the proliferative and osteogenic role of marrow stromal/osteoprogenitor cells in the development of the cortical bone deficit in ovariectomized (OVX) female rats. In vitro, clonal growth of marrow stromal cells from OVX rats was significantly impaired (vs. sham-operated controls). Yet in vivo, cells from sham-operated and OVX rats had equal osteogenic potential in several in vivo experimental situations, such as in intraperitoneally implanted millipore diffusion chambers and in intramuscular implants of marrow plus osteoinductive bone matrix (composite grafts). Long-term (6 mo) dihydrotachysterol (DHT) treatment of OVX rats enhanced their in vitro proliferative potential and clonal growth, as well as their osteogenic expression in composite grafts. The observation that the in vivo osteogenic performance of OVX rat marrow stromal cells was normal at extraosseous sites suggests that the mechanisms leading to osteopenia may involve an abnormality in cell-matrix interactions.
C Tabuchi, D J Simmons, A Fausto, J E Russell, I Binderman, L V Avioli
To test whether maternal hypoglycemia can impair organogenesis, we induced brief glucopenia with insulin in conscious pregnant rats during either the headfold stage or the early neural tube closure stage of embryogenesis. At each time, 10 pairs of animals received identical insulin infusions for 1 h. Half the animals were maintained at euglycemia during the infusions, while the others were allowed to become hypoglycemic. Euglycemia was maintained or restored in all animals immediately after the insulin was stopped. Spontaneous activity was diminished during the hypoglycemia but consciousness was preserved. Embryos were removed from mothers and examined 2 d later. This examination revealed that embryos from the hypoglycemic mothers were growth-retarded and displayed a small but significant incidence of gross developmental anomalies compared with embryos from the insulin-infused euglycemic mothers. Thus, brief, mild maternal hypoglycemia during early organogenesis can disrupt normal embryo development in the rat. The effect is due to the hypoglycemia per se rather than to the insulin employed for its induction.
T A Buchanan, J K Schemmer, N Freinkel
Several autoimmune disorders as well as congenital adrenal hyperplasia (CAH) are either associated or closely linked with genetic variants of the fourth component of complement (C4A and C4B) and the enzyme steroid 21-hydroxylase (21-OH). These proteins are encoded by genes that are located downstream from the genes for complement proteins, C2 and factor B (BF) between HLA-B and -DR in the major histocompatibility complex (MHC). Previous studies of variants and null alleles were based on electrophoretic mobility of C4 protein and linkage with disease phenotypes. These data did not permit analysis of the basis for the observed null alleles and duplicated variants. We studied this region of the MHC in 126 haplotypes for a structural analysis of the four adjacent loci, C4A, 21-OHA, C4B, and 21-OHB. About half of the C4 genes typed as C4 null are deleted and several unrecognized homoduplicated C4 alleles were detected. Hence the frequencies of different C4 structural variants must be recalculated based on a direct analysis of the genes. Analysis of the C4/21-OH genes of patients with the classical (salt-wasting) form of CAH showed that some involve a deletion of the C4B and 21-OHB genes; whereas for two only the 21-OHB gene is deleted, i.e., the C4B gene is present. Together, these data provide a better understanding of the mechanisms generating and importance of deleted C4 and 21-OH null alleles in human disease.
P M Schneider, M C Carroll, C A Alper, C Rittner, A S Whitehead, E J Yunis, H R Colten
Remnants produced from rat chylomicrons in hepatectomized rats or from human chylomicrons by incubation in postheparin plasma contained much less C-apolipoproteins, but more lysophosphatidylcholine than the parent chylomicrons. A phospholipid-triglyceride emulsion absorbed C-apolipoproteins during incubation in serum, yet not in postheparin plasma, which led to lipid-hydrolysis and increased in lysophosphatidylcholine. The fraction d = 1.006-1.019 g/ml of human serum comprised more lysophosphatidylcholine and less C-apolipoproteins than the fraction d less than 1.006 g/ml. Injection of heparin induced hydrolysis with an increase in lysophosphatidylcholine and loss of C-apolipoproteins in both fractions. These inverse changes of lysophosphatidylcholine and C-apolipoproteins during lipid-hydrolysis suggest a causal relationship, which is strongly supported by the induction of loss of C-apolipoproteins from rat chylomicrons and human triglyceride-rich lipoproteins by addition of lysophosphatidylcholine in vitro. Apolipoprotein C-II was more affected than C-III. These results may elucidate a mechanism for the regulation of the termination of the triglyceride hydrolysis and the final hepatic uptake of remnants.
E E Windler, S Preyer, H Greten
The selective delivery in vivo of a T lymphocyte-specific monoclonal antibody and immunotoxin conjugates to T cells in lymph node and spleen was assessed in rhesus monkeys. A transient coating of all T lymphocytes in the lymph nodes and spleens of healthy rhesus monkeys could be achieved after infusion of unconjugated anti-T11. Because derivatized antibody is cleared more rapidly than unconjugated antibody, it was necessary to infuse a higher dose of immunotoxin than antibody alone to achieve saturation of the lymphocyte binding sites with anti-T11. When sufficient antibody-toxin conjugate was infused, toxin was readily demonstrable on lymph node and spleen T cells by 16 h after infusion. This demonstration that toxins can be successfully delivered with specificity to target T cell populations in the monkey suggests that killing of restricted cell populations in vivo should be feasible.
N L Letvin, L V Chalifoux, K A Reimann, J Ritz, S F Schlossman, J M Lambert
Vasopressin's role as a vasoconstrictor in chronic heart failure, was examined in rabbits with adriamycin cardiomyopathic congestive heart failure. Chronic adriamycin treatment resulted in a decrease in cardiac output (829 +/- 38-610 +/- 36 ml/min, P less than 0.005) and blood pressure (83 +/- 2-76 +/- 3 mmHg, P less than 0.01), and an increase in peripheral resistance (8,377 +/- 381-10,170 +/- 657 dyn-s-cm-5, P less than 0.05). Plasma renin activity (4.7 +/- 0.6-10.9 +/- 2.8 ng angiotensin I/ml X h) and norepinephrine (0.7 +/- 0.1-1.3 +/- 0.2 pmol/ml, P less than 0.05) increased while plasma vasopressin levels did not change. Vasopressin infusion, however, produced significantly greater increases in peripheral resistance in animals with heart failure than in controls. Moreover, a specific vasopressin vascular antagonist reduced blood pressure (7 +/- 3%) and peripheral resistance (14 +/- 4%) and increased cardiac output (10 +/- 3%) in animals with heart failure but had no cardiovascular effects in normal rabbits. These results suggest that vascular sensitivity to vasopressin is increased in heart failure, and that it contributes significantly to the increased afterload in heart failure despite normal plasma levels. In this model of severe, chronic heart failure the sympathetic, renin-angiotensin, and vasopressin systems all appear to be activated.
L Arnolda, B P McGrath, M Cocks, C I Johnston
We examined the changes in glomerular hemodynamics produced by angiotensin II (AII) in both normal Munich-Wistar rats and rats which were unilaterally renal denervated (measured kidney) 4-6 d prior to the measurement periods. Measurements of glomerular dynamics were performed in a control period after plasma volume expansion and during infusion of 11 ng X 100 g body wt-1 X min-1 of AII. The glomerular hydrostatic pressure gradient increased from 38 +/- 1 to 49 +/- 1 mmHg in denervated rats compared with a lesser response in controls (from 39 +/- 1 to 45 +/- 1 mmHg, P less than 0.05). Single nephron plasma flow decreased from 213 +/- 17 to 87 +/- 4 nl X min-1 X g kidney wt (KW)-1 in denervated kidneys versus a more modest decrease in control kidneys (from 161 +/- 9 to 102 +/- 5 nl X min X gKW-1). These changes were due to a greater increase in both afferent and efferent arteriolar resistance after AII infusion in denervated compared with control kidneys. Glomerular AII receptor maximum binding was 1,196 +/- 267 fmol/mg protein in denervated kidneys compared with 612 +/- 89 fmol/mg protein (P less than 0.01) in controls with no change in receptor affinity. We conclude the subacute unilateral renal denervation results in renal vasodilation, denervation magnifies the vasoconstrictive effect of AII infusion on glomerular hemodynamics, and the observed increased response to AII after denervation is associated with increases in glomerular AII receptors.
B J Tucker, C A Mundy, A R Maciejewski, M P Printz, M G Ziegler, J C Pelayo, R C Blantz
Guinea pigs genetically deficient in the second (C2) or fourth component of complement (C4) generally appear healthy in contrast to humans with a C2 or C4 deficiency. However, upon investigation of these genetic deficiencies in guinea pigs for signs of dysregulation in the humoral immune system and especially autoantibodies, many complement-deficient guinea pigs (greater than 50%) had elevated levels of serum IgM and higher concentrations of anti-hapten (dinitrophenyl) antibodies as signs of polyclonally stimulated antibody synthesis. In addition, a significant number of the complement-deficient animals, on average 30%, had IgM rheumatoid factors in their sera compared with less than 1% of the normal animals. These observations, therefore, indicate that guinea pigs, genetically deficient in C2 or C4, show characteristics of immune complex disease in general.
E C Böttger, T Hoffmann, U Hadding, D Bitter-Suermann
Lymphocytes from the synovial fluid of eight out of eight rheumatoid arthritis (RA) patients had elevated very late activation antigen-1 (VLA-1) expression (10-36% positive cells), whereas peripheral blood lymphocytes (PBL) from RA patients and healthy controls had low VLA-1 expression (0-6% positive cells). During 1-2 wk of in vitro culture, VLA-1 increased on synovial fluid cells but remained low on PBL. In comparison, the interleukin 2 receptor (IL-2 R) was less prominent than VLA-1 on fresh synovial fluid cells, did not increase on cultured synovial fluid T cells, but did increase greatly on cultured PBL. The mitogen PHA reversed or prevented the appearance of VLA-1+, IL-2 R- synovial fluid cells during in vitro culture, thus giving IL-2 R+, VLA-1- cells. These results emphasize that VLA-1+ SF cells are different from resting cells or IL-2 R+ activated PBL T cells, and VLA-1 on synovial fluid T cells may be incompatible with mitogen stimulation. In addition, the VLA-2 heterodimer (165,000/130,000 relative molecular mass [Mr]) was regulated opposite to the VLA-1 heterodimer (130,000/210,000 Mr) on synovial lymphocytes, and thus the VLA-1/VLA-2 ratio is another indicator of the stage of T cell activation.
M E Hemler, D Glass, J S Coblyn, J G Jacobson
Antidiuretic hormone (ADH) increases the osmotic water permeability (Posm) of toad urinary bladder. This increase is believed to be produced by fusion of intracellular vesicles called aggrephores with the granular cell apical plasma membrane. Aggrephores contain intramembrane particle aggregates postulated to be water channels. ADH-stimulated Posm is decreased by osmotic gradient exposure, which is termed flux inhibition. We studied flux inhibition by exposing ADH-stimulated bladders to various osmotic gradients. Osmotic water flow was initially proportional to the applied osmotic gradient, but Posm decreased with time. Ultrastructural and quantitative studies of endocytosis demonstrate that apical membrane retrieval was a direct function of the transepithelial osmotic gradient. Posm remained unchanged when apical membrane retrieval was blocked by incubation of bladders at 2 degrees C, or under low water-flow conditions. These effects were reversed by increases in temperature or the applied osmotic gradient. We conclude that apical membrane retrieval causes the phenomenon of flux inhibition.
H W Harris Jr, J B Wade, J S Handler
Synovial fluid mononuclear cells (SFMC) from patients with active rheumatoid arthritis characteristically respond poorly to mitogens. In this study, mitogenic antibodies reactive with the CD3(T3) antigen on human T lymphocytes were used to analyze the basis for the deficiency. OKT3-induced proliferation and release of interleukin 1 (IL-1) and interleukin 2 (IL-2) from SFMC were depressed in all patients. Purified IL-1 or recombinant IL-2 restored proliferative responses in SFMC and increased IL-2 receptor density. Exogenous IL-1 also enhanced IL-2 release. Fractionation of SFMC supernatants on phosphocellulose columns revealed the presence of IL-1 and a potent IL-1 inhibitor. The monocyte-derived IL-1 inhibitor blocked IL-1-dependent responses of normal peripheral blood lymphocytes to OKT3, but had no effect on IL-2-dependent events. These results suggest that IL-1 inhibitor(s) in SFMC impair(s) OKT3-induced mitogenesis by interfering with the effects of IL-1 on T lymphocytes. The net result is deficient IL-2 secretion, IL-2 receptor expression, and impaired cellular proliferation. This novel inhibitory circuit provides a rational explanation for the diminished function of synovial fluid T lymphocytes in rheumatoid arthritis patients.
M Lotz, C D Tsoukas, C A Robinson, C A Dinarello, D A Carson, J H Vaughan
The metabolism of apolipoproteins B-48 and B-100 (apo B-48 and B-100) in large triglyceride-rich lipoproteins was studied in three adults with familial dysbetalipoproteinemia (F. dys.) and compared to that of normolipidemic subjects. One Caucasian F. dys. subject was apparently homozygous for the common form of apo E-2, (Arg158----Cys), whereas the two Black subjects were homozygous for a different apo E-2 mutant (Arg145----Cys), which displays much less defective binding to cells than apo E-2 (Arg158----Cys). The lipoproteins were labeled with 125I and injected intravenously into fasted recipients. The results indicate that the terminal catabolism of triglyceride-rich lipoproteins of intestinal and hepatic origin is markedly impaired in apo E2/2 homozygotes with alleles Arg158----Cys and Arg145----Cys; despite long residence times, apo B-48 of chylomicrons and apo B-100 of large very low density lipoproteins are not converted appreciably to intermediate or low density lipoproteins in apo E2/2 homozygotes.
A F Stalenhoef, M J Malloy, J P Kane, R J Havel
26-Hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and other C27-steroids was demonstrated in cultured skin fibroblasts from healthy individuals. Activities in skin fibroblasts were approximately 5-10% of those previously found in human liver homogenates, and were inhibited by CO. The apparent Km was lowest for 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (1.3 mumol/liter) and highest for 5-cholestene-3 beta, 7 alpha-diol (12 mumol/liter). The rate of 26-hydroxylation was highest with 7 alpha-hydroxy-4-cholesten-3-one. These characteristics are similar to those of hepatic mitochondrial C27-steroid 26-hydroxylase. In skin fibroblasts from three patients with cerebrotendinous xanthomatosis (CTX), 26-hydroxylation of C27-steroids proceeded at a rate of only 0.2-2.5% of healthy controls. No accumulation of endogenous 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol could be demonstrated in these cells, and the lowered formation of radioactive, 26-hydroxylated products could not be explained by dilution of the labeled exogenous substrate. The present results add strong evidence to the concept that the primary metabolic defect in CTX is a deficiency of C27-steroid 26-hydroxylase.
S Skrede, I Björkhem, E A Kvittingen, M S Buchmann, S O Lie, C East, S Grundy
We determined the plasma kinetics of both actin and complexes of actin with the two high affinity actin-binding proteins of plasma, gelsolin, and vitamin D-binding protein (DBP). Actin is cleared rapidly from the plasma by the liver (half-disappearance time, 0.5 h). Using radiolabeled actin-binding proteins, we found that actin accelerated the clearance of both plasma gelsolin and the vitamin D-binding protein. In separate experiments we found that DBP-actin complexes were cleared more quickly than gelsolin-actin complexes, at a rate comparable to the clearance of actin from the blood. A low affinity interaction (dissociation constant, 2.9 X 10(-4) M) between actin and fibronectin was found, suggesting that little actin will bind to fibronectin in plasma. We conclude that while plasma gelsolin and DBP may both clear actin from the circulation, DBP appears to play a more important role. By so doing, DBP may conserve the filament-severing activity of plasma gelsolin.
S E Lind, D B Smith, P A Janmey, T P Stossel
We studied the effect of DL-3-hydroxybutyrate and acetoacetate on lactate transport into isolated hepatocytes and on lactate removal in the isolated perfused rat liver. Ketone bodies inhibited lactate transport into isolated hepatocytes (maximum, 35% at concentrations of 10-20 mM). Lactate removal and glucose production by perfused livers were examined before and after the introduction of a constant infusion of hydroxybutyrate, acetoacetate, or appropriate control into the portal venous limb. Lactate removal was significantly inhibited within 10 s of the appearance of increasing concentrations of ketone bodies in the effluent. Corresponding decreases in glucose production were observed. The dependence of inhibition on D-3-hydroxybutyrate concentration was documented in isolated perfused livers (maximum inhibition of lactate removal, 58% at 14 mM). This phenomenon could be a factor in the development of lactic acidosis accompanying ketoacidosis, and indicates that plasma membrane lactate transport may determine the rate of hepatic lactate removal.
H K Metcalfe, J P Monson, S G Welch, R D Cohen
We determined the free fraction of 25-dihydroxyvitamin D (25OHD) in the serum of subjects with clinical evidence of liver disease and correlated these measurements to the levels of vitamin D binding protein and albumin. These subjects when compared to normal individuals had lower total 25OHD levels, higher percent free 25OHD levels, but equivalent free 25OHD levels. These subjects also had reduced vitamin D binding protein and albumin concentrations. The total concentration of 25OHD correlated positively with both vitamin D binding protein and albumin, whereas the percent free 25OHD correlated negatively with vitamin D binding protein and albumin. The free 25OHD levels did not correlate with either vitamin D binding protein or albumin. We conclude that total vitamin D metabolite measurements may be misleading in the evaluation of the vitamin D status of patients with liver disease, and recommend that free 25OHD levels also be determined before making a diagnosis of vitamin D deficiency.
D D Bikle, B P Halloran, E Gee, E Ryzen, J G Haddad
The study of the Ig variable region heavy chain (VH) genes used to encode antibodies specific for self-epitopes from murine hybridomas showed that three VH families are primarily utilized: VH J558, the largest family, and VH QPC52 and VH 7183, the families most proximal to the Ig joining region heavy chain genes. These monoclonal autoantibodies express cross-reactive idiotopes shared by rheumatoid factors and antibodies specific for Sm. The expression of these idiotypes is independent of major histocompatibility complex and Ig constant region heavy chain haplotypes, self-antigen specificity, and even the VH gene family utilized. Though the experiments described here are limited to murine autoantibodies, similarities exist between murine and human autoimmune diseases. Studies that aim to investigate the relationship between VH gene expression and the presence of cross-reactive idiotypes among human autoantibodies should enable us to better understand the mechanisms of autoimmunity and self-tolerance.
M Monestier, A Manheimer-Lory, B Bellon, C Painter, H Dang, N Talal, M Zanetti, R Schwartz, D Pisetsky, R Kuppers
Since adenosine and its analogue 2-chloroadenosine prevent neutrophils from generating superoxide anion in response to chemoattractants, we sought to determine whether these agents could inhibit neutrophil-mediated injury of endothelial cells. The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM) enhanced the adherence of neutrophils to endothelial cells twofold (18 +/- 2% vs. 39 +/- 3% adherence, P less than 0.001) and caused substantial neutrophil-mediated injury to endothelial cells (2 +/- 2% vs. 39 +/- 4% cytotoxicity, P less than 0.001). 2-Chloroadenosine (10 microM) not only inhibited the adherence of stimulated neutrophils by 60% (24 +/- 2% adherence, P less than 0.001) but also diminished the cytotoxicity by 51% (20 +/- 4% cytotoxicity, P less than 0.002). Furthermore, depletion of endogenously released adenosine from the medium by adenosine deaminase-enhanced injury to endothelial cells by stimulated neutrophils (from 39 +/- 4% to 69 +/- 3% cytotoxicity, P less than 0.001). Indeed, in the presence of adenosine deaminase, even unstimulated neutrophils injured endothelial cells (19 +/- 4% vs. 2 +/- 2% cytotoxicity, P less than 0.001). These data indicate that engagement of adenosine receptors prevents both the adhesion of neutrophils and the injury they cause to endothelial cells. Adenosine inhibits injury provoked not only by cells that have been stimulated by chemoattractants but also by unstimulated cells. Based on this model of acute vascular damage we suggest that adenosine is not only a potent vasodilator, but plays the additional role of protecting vascular endothelium from damage by neutrophils.
B N Cronstein, R I Levin, J Belanoff, G Weissmann, R Hirschhorn
Nonselective and beta 1-selective adrenergic antagonists were tested for their effects on enzymatic adaptation to exercise training in rats as follows: trained + placebo (TC); trained + propranolol (TP); trained + atenolol (TA); and corresponding sedentary groups, SC and SP. Trained rats ran 1 h/d at 26.8 m/min, 15% grade, 5 d/wk, 10 wk. Both beta-antagonists were given at doses that decreased exercise heart rates by 25%. Training increased skeletal muscle citrate synthase, cytochrome c oxidase (Cyt-Ox), carnitine palmitoyltransferase (CPT), beta-hydroxyacyl coenzyme A dehydrogenase, mitochondrial malate dehydrogenase (MDH), and alanine aminotransferase (ALT) activities significantly in the TC group, but not in TP. These enzyme activities, except Cyt-Ox and CPT, were also significantly increased in TA. Hepatic phosphoenolpyruvate carboxykinase activity did not alter with training or beta-blockade. Fructose 1,6-bisphosphatase activity was lower in TC than in SC, but unchanged in TP or TA. Hepatic mitochondrial MDH and ALT activities increased with training only in TC. It is concluded that beta 2-adrenergic mechanisms play an essential role in the training-induced enzymatic adaptation in skeletal muscle.
L L Ji, D L Lennon, R G Kochan, F J Nagle, H A Lardy
Serum gastrin concentrations and gastric acid secretion were measured during intravenous infusion of gastrin heptadecapeptide (G-17) (0, 7, 22.1, 70, 221, and 700 pmol/kg X h) in 15 duodenal ulcer patients and 15 healthy controls. Ulcer patients developed higher serum gastrin concentrations during G-17 infusion due to nearly twofold slower clearance of gastrin (8.8 vs. 15.7 ml/kg X min; P less than 0.01). Despite slower clearance of G-17, ulcer patients had plasma elimination half-times for G-17 similar to controls (6.0 vs. 6.1 min, respectively). Thus, calculated volume of distribution for G-17 was lower in ulcer patients than controls (78.5 vs. 140.7 ml/kg; P less than 0.025). For any serum gastrin during gastrin-17 infusion, acid secretion (millimoles per hour) was higher in ulcer patients than in controls. However, when acid secretion was expressed as a percentage of peak acid output to G-17 (to correct for differences in parietal cell mass), curves relating acid secretion to serum gastrin were identical in ulcer patients and controls.
A J Blair 3rd, C T Richardson, M Vasko, J H Walsh, M Feldman
B cell hyperactivity and resistance to tolerance induction are well-recognized immunologic abnormalities associated with both human and murine models of systemic lupus erythematosus. Studies evaluating the role of B cells in these defects have been complicated by the difficulties of consistently isolating large numbers of B cells from T cells and other host-derived regulatory factors. We have recently developed continuous cell lines of B lymphocytes with a high degree of specificity for the antigen dinitrophenyl (DNP) from both New Zealand black times New Zealand white F1 hybrid (BWF1) and BALB/c mice, and we used them to study intrinsic B cell defects in autoimmunity. We found that the kinetics of the immune response to the antigen DNP-Ficoll of both the BWF1 and BALB/c B cell lines are not different. In addition, the BWF1 cell lines, like the BALB/c cell lines and normal B cells, require nonspecific T cell-derived factors as well as antigen to produce an immune response. Tolerance was tested in the BWF1 B cells by preincubating them with DNP-murine IgG2a (MGG), which can induce tolerance in BALB/c cell line lymphocytes. The BWF1 B cell lines were resistant to tolerance induction by DNP-MGG and required 50-fold higher dose of DNP-MGG than BALB/c cell lines for suppression. They were also relatively resistant to tolerance with trinitrophenyl-d-glutamyl lysine. Thus, DNP-specific B cells from autoimmune mice have an inherent defect in tolerance induction.
M S Brooks, M Aldo-Benson
Previous studies show that cytotoxic activated macrophages cause inhibition of DNA synthesis, inhibition of mitochondrial respiration, and loss of intracellular iron from tumor cells. Here we examine aconitase, a citric acid cycle enzyme with a catalytically active iron-sulfur cluster, to determine if iron-sulfur clusters are targets for activated macrophage-induced iron removal. Results show that aconitase activity declines dramatically in target cells after 4 h of co-cultivation with activated macrophages. Aconitase inhibition occurs simultaneously with arrest of DNA synthesis, another early activated macrophage-induced metabolic change in target cells. Dithionite partially prevents activated macrophage induced aconitase inhibition. Furthermore, incubation of injured target cells in medium supplemented with ferrous ion plus a reducing agent causes near-complete reconstitution of aconitase activity. The results show that removal of a labile iron atom from the [4Fe-4S] cluster, by a cytotoxic activated macrophage-mediated mechanism, is causally related to aconitase inhibition.
J C Drapier, J B Hibbs Jr
Bone marrow cells obtained from 14 patients with light chain amyloid (AL) deposition were examined by biosynthetic labeling techniques. These analyses identified free monoclonal light chain (L-chain) synthesis even in those patients whose serum or urine contained no M protein or free L-chains or only an intact M protein. The experiments also identified a subset of patients whose plasma cells synthesized polypeptides bearing constant region antigenic determinants that migrated more rapidly than intact L-chains on polyacrylamide gels. Since most AL fibrils contain L-chain fragments rather than intact L-chains, these studies suggested that the genesis of the fibril components may reflect aberrant synthesis, proteolytic processing, or both. We also noted that in some individuals the pattern of Ig synthesis normalized after several courses of cytotoxic therapy. Thus, we could use bone marrow Ig synthesis as a sensitive biochemical parameter for monitoring therapy. Finally, the presence of aberrant synthetic products in these clones raised questions about their origin with respect to the normal processes of transcription, translation, and posttranslational modification in Ig-producing cells.
We investigated the roles of insulin and glucagon as mediators of changes in glucose and alanine kinetics during the hypermetabolic response to injury in 10 burn patients by infusing somatostatin with and without insulin replacement. Glucose and alanine kinetics were measured by primed-constant infusions of 6,6-d2-glucose and [3-13C]alanine. The basal rate of glucose production and alanine flux were significantly elevated in all patients. Lowering both hormones simultaneously caused an insignificant reduction in glucose production, but plasma glucose rose significantly (P less than 0.01), because of reduced clearance. Alanine flux and total plasma amino nitrogen increased significantly (P less than 0.05) above basal. Selectively lowering glucagon concentration decreased glucose production (P less than 0.05), and exogenous glucose was infused to maintain euglycemia. Alanine flux and total plasma amino nitrogen remained unchanged. In severely burned patients hyperglucagonemia stimulates increased glucose production, basal insulin suppression glucose production, stimulates basal glucose clearance, and is important for regulation of plasma amino acid concentrations, and the selective lowering of glucagon while maintaining basal insulin constant normalized glucose kinetics.
F Jahoor, D N Herndon, R R Wolfe
Apolipoprotein E (apoE) is important in modulating the catabolism of remnants of triglyceride-rich lipoprotein particles. It is a polymorphic protein with the three common alleles coding for apoE2, apoE3, and apoE4. ApoE3 is considered the normal isoform, while apoE4 is associated both with hypercholesterolemia and type V hyperlipoproteinemia. We quantitated the kinetics of metabolism of apoE4 in 19 normolipidemic apoE3 homozygotes and 1 normolipidemic apoE4 homozygote, and compared this with the metabolism of apoE3 in 12 normolipidemic apoE3 homozygotes. In the apoE3 homozygous subjects, apoE4 was catabolized twice as fast as apoE3, with a mean plasma residence time of 0.37 +/- 0.01 d (+/- SEM) and 0.73 +/- 0.05 (P less than 0.001), respectively. When plasma was fractionated into the lipoprotein subclasses, the greatest amount of labeled apoE4 was present on very low density lipoproteins, while the largest fraction of labeled apoE3 was associated with high density lipoproteins. The plasma apoE concentration was decreased in an apoE4 homozygote compared with the apoE3 homozygotes (3.11 mg/dl vs. 4.83 +/- 0.35 mg/dl). The reduced apoE4 concentration was entirely due to a decreased apoE4 residence time in the apoE4 homozygote (0.36 d vs. 0.73 +/- 0.05 d for apoE3 in apoE3 homozygotes). These results indicate that apoE4 is kinetically different than apoE3, and suggest that the presence of apoE4 in hypercholesterolemic and type V hyperlipoproteinemic individuals may play an important pathophysiological role in the development of these dyslipoproteinemias.
R E Gregg, L A Zech, E J Schaefer, D Stark, D Wilson, H B Brewer Jr
To clarify sulfobromophthalein (BSP) and bilirubin uptake mechanisms, isolated rat hepatocytes were incubated with [35S]BSP. The initial uptake velocity (V0), determined from the first, linear portion of the cumulative uptake curve, was saturable (Michaelis constant [Km] = 6.2 +/- 0.5 microM; Vmax = 638 +/- 33 pmol X min-1 per 10(5) hepatocytes), maximal at 37 degrees C and pH 7.4, and competitively inhibited by bilirubin, but not by taurocholate, cholate, or oleate. Preloading with unlabeled BSP led to trans-stimulation of V0. Sodium substitution or pretreatment of hepatocytes with ouabain or metabolic inhibitors had no effect on V0; trypsin reduced V0 by 39% (P less than 0.001). A rabbit antiserum to the rat liver plasma membrane (LPM)-BSP/bilirubin binding protein selectively reduced V0 of 5 microM [35S]BSP and [14C]bilirubin by 41 and 42%, respectively (P less than 0.01); uptakes of [3H]oleate, [3H]cholate and [3H]taurocholate were not affected. Hence, the LPM-BSP/bilirubin binding protein plays a role in the carrier-mediated uptake of BSP and bilirubin by hepatocytes.
W Stremmel, P D Berk
We demonstrate here that the exceptionally active maleyl-albumin receptor of human monocytes functions in vitro as a chemoattractant receptor. Chemotaxis of human monocytes occurs at an effective median dose of 3-4 microM maleyl-albumin, a concentration representing 1% of the total albumin in the adult human. Computerized analyses by LIGAND of the saturable binding of maleyl-albumin to human monocytes reveal two classes of binding sites, described by dissociation constants of 37 nM and 5.3 microM with maximal binding of 1.6 and 23 pmol maleyl-albumin/mg cellular protein, respectively. Chemotaxis of human monocytes thus occurs at concentrations of maleyl-albumin promoting binding to the lower-affinity sites. We propose that conformational isomers of albumin that are chemotactic may form in vivo and that albumin, in addition to receptor-independent plasma transport functions, may also play an important role in the receptor-mediated recruitment and accumulation of phagocytic cells at sites of inflammation and injury.
M E Haberland, R R Rasmussen, A M Fogelman
The age-related changes in collagen-linked fluorescence (browning) were investigated in skin from subjects with long-standing type I diabetes. Overall browning rates were 2.4 times higher in diabetics than in controls (P less than 0.02) and slope intercept accurately reflected the mean age of onset of diabetes (11.6 vs. 11.2 yr), suggesting that the browning process has the attributes of a biological clock. Browning rates were not different in controls and diabetics without retinopathy (P greater than 0.05) but were 2.4 (P less than 0.05) and 2.7 (P less than 0.01) times increased in the presence of background and proliferative retinopathy, respectively. Compared with subjects with retinopathy, individual browning rates since onset of diabetes decreased with advancing age in subjects free of retinopathy (P less than 0.001). Extrapolation revealed that they would become identical to that of nondiabetic subjects by the age of 66.4 yr. These results suggest the presence of a mechanism that controls the browning rate of collagen in diabetics who do not develop retinopathy.
V M Monnier, C A Elmets, K E Frank, V Vishwanath, T Yamashita
In this report we compare the bioactivities of pure, human monocyte-derived interleukin 1 (IL-1) alpha and beta in the standard murine thymocyte proliferation assay, a human dermal fibroblast proliferation assay, and in an assay measuring stimulation of prostaglandin E2 (PGE2) release from human rheumatoid synoviocytes. In each case the different species of IL-1 produced saturable stimulation and gave similar dose response curves. Half-maximal stimulation was observed at average IL-1 concentrations of 29 pM in the thymocyte assay, 2 pM in the dermal fibroblast proliferation assay, and 5 pM in the synovial cell assay. Our results show that native, monocyte-derived IL-1 alpha and IL-1 beta are both potent stimulators of connective tissue cells and that the specific bioactivities of these molecules are similar to each other in tests on human connective tissue cells, as well as on murine lymphoid cells.
E A Rupp, P M Cameron, C S Ranawat, J A Schmidt, E K Bayne
The mechanisms that mediate renal "escape" from the sodium-retaining effects of mineralocorticoids are incompletely understood. This study was undertaken to determine whether atrial natriuretic peptide (ANP) may play a role in the escape phenomenon. Immunoreactive ANP in rat plasma increased 2.5-fold above baseline values at 12 and 24 h after a single depot injection of desoxycorticosterone acetate in oil and returned to baseline thereafter. In addition, specific pre-pro-ANP messenger RNA content in rat atria was significantly elevated as early as 12 h after mineralocorticoid administration and remained elevated at 24, 48, and 72 h, indicating a prompt and sustained increase in ANP biosynthesis. Renal glomerular ANP receptor density was down-regulated appropriately with rising plasma ANP levels, and receptor affinity was unchanged. Thus, mineralocorticoid administration in the rat is a powerful stimulus for ANP release and for atrial myocyte ANP synthesis, which suggests a potential role for this hormone in overriding mineralocorticoid-induced renal sodium retention.
B J Ballermann, K D Bloch, J G Seidman, B M Brenner
A previously reported deficiency of "total" pyruvate dehydrogenase complex activity is further characterized. Dihydrolipoyl transacetylase (E2) and lipoamide dehydrogenase (E3) activities in the patient's fibroblasts were normal. Pyruvate dehydrogenase activity (E1) was 33% of that in fibroblasts from an age-matched control. The amounts of each of the components of pyruvate dehydrogenase complex were analyzed using an immunoblot technique and specific antibodies. Levels of components E2 and E3 were the same in fibroblasts from the patient and control, confirming the activity measurements. However, the levels of E1 alpha and E1 beta were reduced markedly in fibroblasts from the patient. Thus, impairment in the pyruvate dehydrogenase complex activity was due to a reduction in the amount of the E1 component of the complex.
L Ho, C W Hu, S Packman, M S Patel
J L Goldstein