We measured alpha 2-adrenoreceptor density as well as affinity for and sensitivity to agonist on intact platelets of normotensive and hypertensive subjects before and after physiological increases in plasma catecholamines. In normotensives, posture-induced rises in plasma catecholamines correlated with reduced alpha 2-adrenoreceptor agonist affinity and fewer high affinity state receptors. Platelet aggregation and inhibition of adenylate cyclase by L-epinephrine also was reduced. Hypertensive subjects had similar rises in plasma catecholamines with upright posture, but showed no change in receptor affinity or sensitivity. No change in platelet alpha 2-adrenoreceptor number occurred in these studies. In vitro incubation with L-epinephrine revealed that platelets from hypertensives had slower desensitization than those from normotensives. Binding studies at different temperatures and with varying sodium concentrations found no thermodynamic or sodium-dependent differences between normotensive and hypertensive groups. These studies demonstrate that platelets from hypertensive subjects exhibit a defect in the ability of physiological concentrations of agonist to desensitize the alpha 2-adrenoreceptor.
A S Hollister, J Onrot, S Lonce, J H Nadeau, D Robertson
The cultured skin fibroblasts from three patients with lacticacidemia were found to have low rates of 1-[14C]pyruvate oxidation in the face of normal pyruvate dehydrogenase activity. After incubation with 1 mM glucose, these three cell strains also exhibited lactate/pyruvate ratios which were three times greater than those of controls. In two of the patients, both ATP and oxygen consumption in fibroblast mitochondrial preparations was deficient with NAD-linked substrates but normal with succinate and ascorbate/N'N'N'N' tetramethyl phenylene diamine. In the third patient, ATP synthesis in mitochondrial preparations was deficient with all substrates tested. Measurement of Rotenone-sensitive NADH-cytochrome c reductase in mitochondrial preparations from skin fibroblasts showed that two of the patients had 14 and 18%, respectively, of control activity. In the third patient, cytochrome oxidase activity was 15% of that in controls. We conclude that respiratory chain defects can be demonstrated in cultured skin fibroblasts with consistency using a number of different techniques.
B H Robinson, J Ward, P Goodyer, A Baudet
Kawasaki syndrome (KS) is characterized by diffuse vasculitis and marked T cell and B cell activation. In this study, sera from 16 patients with acute KS, 15 patients in the convalescent phase of KS, and 19 age-matched controls were assessed for complement dependent cytotoxic activity against 111In-labeled human umbilical vein endothelial (HUVE) cells, Neither sera from patients with KS nor sera from controls had cytotoxic effects on HUVE cells cultivated under standard conditions. Since activated T cells such as those present in acute KS secrete gamma interferon (gamma-IFN), we also examined the effects of sera from acute KS on HUVE cells preincubated with gamma-IFN. We report here that immunoglobulin M (IgM) antibodies in sera from patients with acute KS cause significant (P less than 0.01) killing of gamma-IFN-treated HUVE cells. Pretreatment with interleukin 2, gamma-IFN, or beta-IFN failed to render HUVE susceptible to lysis with acute KS sera. The observed effects were not mediated via immune complexes. The cytotoxic antibodies in acute KS seem to be directed against inducible monomorphic antigenic determinants present on gamma-IFN-treated HUVE cells but not on control or gamma-IFN treated autologous human dermal fibroblasts (HDF). Similarly, acute KS sera also induced lysis of gamma-IFN-treated human saphenous vein endothelial (HSVE) cells but not gamma-IFN treated human saphenous vein smooth muscle (HSVSM) cells. Since gamma-IFN induces the same level of class I and class II major histocompatibility complex (MHC) antigen expression on HDF, HUVE, HSVE, and HSVSM cells, our results suggest that the anti-endothelial cell antibodies in acute KS are directed to gamma-IFN-inducible molecules other than MHC determinants. These observations are further substantiated by the failure of human B cells or monocytes to absorb the anti-endothelial cell activity. Since most vasculitides, including acute KS, are characterized both by marked immune activation and the secretion of lymphokines, antibodies directed to gamma-IFN-inducible endothelial cell antigens may represent a general mechanism for vascular injury.
D Y Leung, T Collins, L A Lapierre, R S Geha, J S Pober
Peripheral vascular effects of opioid peptides are well known, but direct myocardial effects have not been established. We studied the inotropic response of spontaneously beating cultured chick embryo ventricular cells to the enkephalin analogue [D-Ala2]-enkephalin. Amplitude of cell motion increased in a concentration-dependent manner with 0.53 microM [D-Ala2]-enkephalin producing half-maximal response. The mechanism of this positive inotropic effect was investigated by examining alterations in 45Ca influx, cyclic AMP accumulation and adenylate cyclase activity in response to [D-Ala2]-enkephalin. At maximally inotropic concentrations, the 45Ca influx rate increased 39%, adenylate cyclase was stimulated by 30%, and cyclic AMP content rose more than twofold. Thus, in contrast to neural tissue, receptors for enkephalin in cultured heart cells are coupled to adenylate cyclase in a stimulatory manner. Occupancy of these receptors produces an increase in cyclic AMP levels and exerts a positive inotropic effect via a verapamil-sensitive enhancement of Ca influx.
S Laurent, J D Marsh, T W Smith
As the characteristics of sodium and water balance in heart failure remain undefined, we evaluated the hemodynamic, metabolic, and hormonal effects of balanced sodium intake in 10 patients with chronic congestive heart failure. We discontinued diuretics to avoid their confounding influence, and all patients received 1 wk of 10 meq and 100 meq balanced sodium intake and controlled free water. Comparing sodium intake of 10 with 100 meq, the following observations were made. There was weight gain (2.0 kg) and increased sodium excretion (11 +/- 3 to 63 +/- 15 meq/24 h), unaccompanied by increase of blood volume. Both renin-angiotensin system and sympathetic nervous system activity were greater during the 10 meq diet, and suppressed with the 100 meq sodium diet. For both diets, plasma renin and urinary aldosterone excretion were correlated with urinary sodium excretion (r = -0.768, r = -0.726, respectively; P less than 0.005). Systemic hemodynamics were minimally changed with increased sodium intake. However, reversal of vasoconstriction by captopril during the 10 meq diet, and its ineffectiveness during the 100 meq diet, indicated a renin-dependent mechanism in the former, and a renin-independent mechanism in the latter diet. There were two subgroups of response to the 100 meq diet: one group (n = 5) achieved neutral balance, while the second (n = 5) avidly retained sodium and water. Renin-angiotensin system activity was significantly higher in the latter group, and the mechanism for differences in sodium excretion for the subgroups could not be identified by blood volume or hemodynamic parameters. Orthostatic hypotension during tilt was greater during the 10 meq sodium diet, and in all cases, related to ineffective hemodynamic and hormonal compensatory responses.
R J Cody, A B Covit, G L Schaer, J H Laragh, J E Sealey, J Feldschuh
Effects of intraperitoneal injection of allogeneic lymphocytes on insulin secretion were studied in incubated pancreas slices from BALB/c mice. Injection of allogeneic lymphocytes from C57BL/6J (H2b) mice increased insulin secretion, both in basal and 11-mM glucose-stimulated conditions. This effect was only present when at least 5 X 10(6) or 1 X 10(6) cells were injected (in basal and stimulated conditions, respectively). Glucose-induced insulin secretion (3.3-27.5 mM) was significantly increased in pancreata from mice injected with allogeneic lymphocytes. No effect was observed when glucose was not included in the incubation medium. Intraperitoneal injection of Dextran 70 produced no change in glucose-elicited insulin secretion. There were no differences in glucagon and somatostatin (SRIF) secretion obtained from pancreas of mice injected with allogeneic or syngeneic lymphocytes. Injection of allogeneic cells increases insulin secretion (basal and both phases of 11 mM glucose-stimulated secretion). Puromycin significantly inhibited the second phase of insulin secretion. These results suggest that: Injection of allogeneic lymphocytes raises both basal and glucose-stimulated insulin secretion. This effect seems to be connected with the major histocompatibility complex, and to be related to the number of allogeneic cells injected. Injection of allogeneic lymphocytes seems to sensitize the beta cell response to glucose stimulus. Neither glucagon nor SRIF secretion are altered by alloantigen injection. The stimulatory effect of allogeneic lymphocytes is related, at least in part, to insulin synthesis.
J B García, M C Venturino, E Alvarez, L Fabiano de Bruno, M Braun, O H Pivetta, J C Basabe
Cholesterol-rich very low density lipoproteins (VLDL) from the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbit induced marked cholesteryl ester accumulation in mouse peritoneal macrophages. This WHHL rabbit, an animal model of human familial hypercholesterolemia, has severe hypercholesterolemia, cutaneous xanthomas, and fulminant atherosclerosis due to the deficiency of the low density lipoprotein (LDL) receptor. When incubated with mouse peritoneal macrophages, the VLDL from WHHL rabbit (WHHL-VLDL) stimulated cholesteryl [14C]oleate synthesis 124-fold more than did VLDL from the normal Japanese White rabbit (control-VLDL). The enhancement in cholesteryl ester synthesis and accumulation of WHHL-VLDL was due to the presence of a high affinity binding receptor site on the macrophage cell surface that mediated the uptake and lysosomal degradation of WHHL-VLDL. Competition studies showed that the uptake and degradation of 125I-WHHL-VLDL was inhibited by unlabeled excess WHHL-VLDL and beta-migrating VLDL (beta-VLDL), but not LDL. Furthermore, the degradation of WHHL-VLDL was not blocked by either fucoidin, polyinosinic acid, or polyguanylic acid, potent inhibitors of the acetylated (acetyl)-LDL binding site, or by acetyl-LDL. These results suggest that macrophages possess a high affinity receptor that recognizes the cholesterol-rich VLDL present in the plasma of the WHHL rabbit and that the receptor which mediates ingestion of WHHL-VLDL seems to be the same as that for beta-VLDL and leads to cholesteryl ester deposition within macrophages. Thus the uptake of the cholesterol-rich VLDL from the WHHL rabbit by macrophages in vivo may play a significant role in the pathogenesis of atherosclerosis in the WHHL rabbit.
T Kita, M Yokode, Y Watanabe, S Narumiya, C Kawai
HTLV-I is a transforming human retrovirus that is an etiologic agent of adult T cell leukemia/lymphoma. To investigate the effects of this virus on T cell functions, two OKT3+, OKT4+, OKT8- cytotoxic clones (8.7 and 8.8) specific for allogeneic cells bearing DPw2, a class II histocompatibility antigen, were studied before and after infection with HTLV-I. The clones retained cytotoxic function for up to 70 d after exposure to HTLV-I, even without subsequent antigenic stimulation, but then lost their cytotoxic activity. Prior to infection with HTLV-I, clone 8.8 also lysed OKT3 hybridoma cells; after infection, cytotoxic activity against these OKT3-antibody bearing cells was lost in parallel with the loss of activity against DPw2-bearing target cells. In addition, expression of T3 surface antigen by HTLV-I-infected 8.8 cells was decreased at a time when they lost their cytotoxic activity, possibly contributing to the loss of cytotoxic function. Finally, clone 8.8 could provide help for nonspecific IgG production by autologous B cells when stimulated with irradiated DPw2-bearing non-T cells. After infection with HTLV-I, this helper function became independent of DPw2-stimulation and persisted even when the cytotoxic activity was lost. An OKT4+ T cell clone thus could simultaneously manifest both cytotoxic and helper T cell activities, and these activities were differentially affected after HTLV-I infection.
R Yarchoan, H G Guo, M Reitz Jr, A Maluish, H Mitsuya, S Broder
In this study, carried out in the rat and hamster, the receptor-dependent low density lipoprotein (LDL) transport process in each organ was characterized in terms of its maximal uptake rate (Jm) and Michaelis constant (Km), while the rate of receptor-independent uptake was defined in terms of its proportionality constant (P). The highest Jm values of 50-126 micrograms/h per g were found in the liver and endocrine glands in both species and receptor-dependent uptake also was detected in other organs like spleen, kidney, and intestine. The Km values were essentially the same in all of the organs and equaled approximately 90 mg/dl in both species. The receptor-independent uptake constants also were similar in the two species and were highest in the spleen, liver, and intestine. From these values for Jm, Km, and P, it was possible to construct theoretical curves that predict the plasma LDL-cholesterol concentration and fractional catabolic rate given any alteration in LDL-cholesterol production or the magnitude of receptor-dependent LDL transport in any organ of the rat or hamster.
D K Spady, J B Meddings, J M Dietschy
The development of atherosclerotic changes and thromboembolism are common features in homocystinurics. Hence, we postulate a positive correlation between the level of homocyst(e)ine in the blood and the occurrence of coronary artery disease. Homocysteine is found either as free homocystine, cysteine-homocysteine mixed disulfide, or protein-bound homocyst(e)ine. In nonhomocystinuric subjects, most homocysteine molecules are detectable in the protein-bound form. Thus, protein-bound homocyst(e)ine in stored plasma which reflected total plasma homocyst(e)ine was determined in 241 patients with coronary artery disease (173 males and 68 females). The mean +/- SD total plasma homocyst(e)ine was 5.41 +/- 1.62 nmol/ml in male patients, 4.37 +/- 1.09 nmol/ml in male controls, 5.66 +/- 1.93 nmol/ml in female patients, and 4.16 +/- 1.62 nmol/ml in female controls. The differences between the patients with coronary artery disease and the controls were statistically significant (P less than 0.0005).
S S Kang, P W Wong, H Y Cook, M Norusis, J V Messer
We made longitudinal measurements of bone mineral density (BMD) in 139 normal women (ages 20-88 yr) at midradius (99% cortical bone) and lumbar spine (approximately 70% trabecular bone) by single- and dual-photon absorptiometry. BMD was measured 2-6 (median, 3) times over an interval of 0.8-3.4 yr (median, 2.1 yr). For midradius, BMD did not change (+0.48%/yr, NS) before menopause but decreased (-1.01%/yr, P less than 0.001) after menopause. For lumbar spine, there was significant bone loss both before (-1.32%/yr, P less than 0.001) and after (-0.97%/yr, P = 0.006) menopause; these rates did not differ significantly from each other. Our data show that before menopause little, if any, bone is lost from the appendicular skeleton but substantial amounts are lost from the axial skeleton. Thus, factors in addition to estrogen deficiency must contribute to pathogenesis of involutional osteoporosis in women because about half of overall vertebral bone loss occurs premenopausally.
B L Riggs, H W Wahner, L J Melton 3rd, L S Richelson, H L Judd, K P Offord
The kinetics of entry and release of gentamicin was investigated in fluids and tissues of the inner ear of the rat, as well as in renal cortex, and in organs that do not share susceptibility to the toxic effects of aminoglycosides. Various modes of administration were used to achieve different patterns of drug plasma concentrations. Electrophysiological and histological examinations were performed to correlate pharmacokinetics and ototoxicity. Results show that: the uptake of the drug by the inner ear tissues is dose dependent and manifests a rapid saturation kinetics with a concentration plateau of about 1 micrograms/mg of protein. The low ratio of the perilymph and endolymph to plasma concentrations argues against the concept of an accumulation of the drug in the inner ear over drug levels in plasma, which has been considered as the basic mechanism of ototoxicity. In renal cortex, the kinetics appears similar to that of the inner ear but the concentrations achieved are 10-fold higher than in cochlear tissues. In other organs (liver, heart, lung, and spleen), no saturation could be demonstrated within the duration of the experiment. Ototoxicity seems to be related to the penetration of the drug into compartment(s) from which the half-life of disappearance is extremely slow. Rapid uptake, early saturation, and long exposure of the tissues to the drug may account for the development of toxicity in inner ear and kidney.
P Tran Ba Huy, P Bernard, J Schacht
Different T cell lines, which can be induced to secrete interleukin 2 (IL-2) in vitro, were used to dissect the effect of cyclosporin A (CsA). The T leukemia cell Jurkat requires an increase in cytoplasmic calcium concentration ([Ca++]i) and phorbol myristate acetate (PMA) for the induction of IL-2 production, which is completely blocked by CsA. Another T cell line, HUT 78, also produces IL-2 in response to a rise in [Ca++]i and PMA; however, in HUT 78, PMA alone induces low levels of IL-2 production that is not blocked by CsA. After treatment with 5-azacytidine, HUT 78 cells produced maximal levels of IL-2 in response to PMA alone without requiring [Ca++]i increasing stimuli. In these cells no inhibitory effect of CsA on PMA-induced activation could be demonstrated. In addition, CsA does not inhibit PMA-induced translocation of protein kinase C. These data suggest that CsA does not globally inhibit IL-2 gene expression, but rather interferes with signaling events of T cell activation.
B Manger, K J Hardy, A Weiss, J D Stobo
Because treatment with lithium salts may impair renal concentrating ability, we investigated the possibility of a direct effect of lithium ions on the permeability to water of the collecting duct epithelium. The coefficient of hydraulic conductivity (Lp) of isolated perfused rabbit cortical collecting tubules (CCT) was measured in the presence and absence of arginine-8-vasopressin (AVP), or 8-bromo (Br) cyclic AMP (cAMP) and/or lithium chloride (Li 10 mM). In the absence of AVP, Li in the lumen for 30 min failed to affect basal water permeability; however, in tubules preincubated with Li in the lumen for 80 min, basal water permeability was reduced to 30% of the value found in control tubules (P less than 0.01). In CCT incubated at 25 degrees C with Li in the lumen for 3 h, the hydroosmotic response to 2.5 microU X ml-1 AVP (Lp = 6.88 +/- 1.54 nl X cm-2 X s-1 X atm-1) was significantly lower than that in the control tubules (13.98 +/- 1.59, P less than 0.01); the inhibition was not reversible. When Li was present in the peritubular medium only, the hydroosmotic effect of AVP was not different from that of the controls. The hydroosmotic effect of 25 microU/ml AVP was investigated at 37 degrees C. CCT exposed to Li in the lumen had a 49% inhibition of peak Lp under AVP (Lp = 10.98 +/- 1.17) as compared with control tubules (Lp = 21.39 +/- 1.51; P less than 0.005). In contrast, the hydroosmotic response to 8-Br-cAMP was not affected by lithium. The results are compatible with the view that Li inhibits the action of AVP at the level of the regulating protein or the catalytic unit of the membrane adenylate cyclase and that the site of the interaction can be reached by lithium only from the cytoplasmic side. The Li-antidiuretic hormone (ADH) interaction found here may represent the earliest pathophysiological event underlying the renal concentrating defect observed after Li administration.
E Cogan, M Abramow
To study the effects of alveolar hypoxia on canine bronchopulmonary shunt flow, a biventricular bypass preparation was employed. The preparation allowed a constant and sensitive measure of changes in pulmonary venous blood flow. In 16 of 18 dogs with intact bronchial arteries, alveolar hypoxia caused an increase in pulmonary venous return both under conditions of constant pulmonary arterial inflow and under conditions of no pulmonary arterial inflow, suggesting bronchopulmonary shunting. This effect was accompanied by systemic vasodilation despite vagotomy and ganglionic blockade, and was abolished by division of all bronchial vessels. Ibuprofen, 3 mg/kg, and indomethacin, 15 mg/kg, in dogs with intact bronchial vessels, abolished both the increase in pulmonary venous return and the systemic vasodilatation caused by hypoxia. Thus, alveolar hypoxia directly augments bronchopulmonary flow, most likely through release of one or more vasodilating prostaglandins.
R L Warren, W J Powell Jr
Patients with noninsulin-dependent diabetes mellitus (NIDDM) have both preprandial and postprandial hyperglycemia. To determine the mechanism responsible for the postprandial hyperglycemia, insulin secretion, insulin action, and the pattern of carbohydrate metabolism after glucose ingestion were assessed in patients with NIDDM and in matched nondiabetic subjects using the dual isotope and forearm catheterization techniques. Prior to meal ingestion, hepatic glucose release was increased (P less than 0.001) in the diabetic patients measured using [2-3H] or [3-3H] glucose. After meal ingestion, patients with NIDDM had excessive rates of systemic glucose entry (1,316 +/- 56 vs. 1,018 +/- 65 mg/kg X 7 h, P less than 0.01), primarily owing to a failure to suppress adequately endogenous glucose release (680 +/- 50 vs. 470 +/- 32 mg/kg X 7 h, P less than 0.01) from its high preprandial level. Despite impaired suppression of endogenous glucose production during a hyperinsulinemic glucose clamp (P less than 0.001) and decreased postprandial C-peptide response (P less than 0.05) in NIDDM, percent suppression of hepatic glucose release after oral glucose was comparable in the diabetic and nondiabetic subjects (45 +/- 3 vs. 39 +/- 2%). Although new glucose formation from meal-derived three-carbon precursors (53 +/- 3 vs. 40 +/- 7 mg/kg X 7 h, P less than 0.05) was greater in the diabetic patients, it accounted for only a minor part of this excessive postprandial hepatic glucose release. Postprandial hyperglycemia was exacerbated by the lack of an appropriate increase in glucose uptake whether measured isotopically or by forearm glucose uptake. Thus as has been proposed for fasting hyperglycemia, excessive hepatic glucose release and impaired glucose uptake are involved in the pathogenesis of postprandial hyperglycemia in patients with NIDDM.
R G Firth, P M Bell, H M Marsh, I Hansen, R A Rizza
Lipoteichoic acids (LTA) released by gram-positive bacteria can spontaneously bind to mammalian cell surfaces. In the present study, erythrocytes (E) sensitized with pneumococcal LTA (LTA-E) were used as a model system to determine if LTA could render host cells susceptible to damage by autologous complement. Complement (C)-mediated lysis of LTA-E from normal rats and normal humans occurred when these cells were incubated in their respective autologous sera in vitro. In addition, when LTA-E from a C2-deficient human and from C4-deficient guinea pigs were incubated in their autologous sera, there was significant lysis in vitro, demonstrating a role for the alternative pathway. The in vivo survival of 51Cr-labeled autologous LTA-E was also studied. Only 2.9% of autologous LTA-E remained in the circulation of normal rats after 90 min. In contrast, 31.2% of autologous LTA-E remained in the circulation of rats depleted of C3. Intravascular hemolysis accounted for the clearance of LTA-E in the normal rats, whereas liver sequestration was responsible for clearance in the C3-depleted rats. These results demonstrate that LTA can render the host's cells susceptible to damage by its own complement system, establishing this as a possible mechanism of tissue damage in natural bacterial infections.
D S Hummell, J A Winkelstein
A monoclonal antibody, selected for reactivity with the Epstein-Barr virus (EBV)-encoded antigen EBNA-1, exhibited strong reactivity with the synovial lining cells in joint biopsies from 10 of 12 patients with rheumatoid arthritis (RA) and adherent cells eluted from these tissues. No staining of RA synovial membrane frozen tissue sections or eluted synovial-lining cells was obtained with monoclonal antibodies directed against other EBV-encoded antigens (anti-p160, anti-gp200/350) or with monoclonal antibodies directed against antigens encoded by cytomegalovirus, herpes simplex viruses, or human T cell leukemia virus type I. Among 12 osteoarthritis and normal synovial biopsies only rare reactive cells were noted. Characterization of the antigen(s) in RA synovium by the Western immunoblotting technique revealed a 62,000-molecular-weight (mol-wt) protein, in contrast to the 70,000-85,000-mol-wt EBNA-1 antigen found in EBV-transformed cells. The structural basis for the cross-reactivity of the RA synovial membrane 62,000-mol-wt protein and the EBNA-1 antigen appears to reside in the glycine-alanine rich region of these molecules. A rabbit antibody directed against a synthetic peptide (IR3-VI-2) derived from the glycine-alanine-rich region of EBNA-1 reacted with the 70,000-85,000-mol-wt EBNA-1 antigen in EBV-infected cells and with the 62,000-mol-wt molecule in RA synovial membrane extracts. Since strong antibody responses to EBNA-1 are known to exist in RA patients, these results suggest that immune responses to a cross-reactive antigen may play a role in the pathogenesis of RA.
R Fox, R Sportsman, G Rhodes, J Luka, G Pearson, J Vaughan
We studied somatomedin-C/insulinlike growth factor (Sm-C/IGF-I) binding to human fibroblasts in both adherent monolayers and in suspension cultures. The addition of Sm-C/IGF-I in concentrations between 0.5 and 10 ng/ml to monolayers cultures resulted in a paradoxical increase in 125I-Sm-C/IGF-I binding and concentrations between 25 and 300 ng/ml were required to displace the labeled peptide. The addition of unlabeled insulin resulted in no displacement of labeled Sm-C/IGF-I from the adherent cells. When fibroblast suspensions were used Sm-C/IGF-I concentrations between 1 and 10 ng/ml caused displacement, the paradoxical increase in 125I-Sm-C/IGF-I binding was not detected, and insulin displaced 60% of the labeled peptide. Affinity cross-linking to fibroblast monolayers revealed a 43,000-mol wt 125I-Sm-C-binding-protein complex that was not detected after cross-linking to suspended cells. The 43,000-mol wt complex was not detected after cross-linking to smooth muscle cell monolayers, and binding studies showed that 125I-Sm-C/IGF-I was displaced greater than 90% by Sm-C/IGF-I using concentrations between 0.5 and 10 ng/ml. Because fibroblast-conditioned medium contains the 43,000-mol wt complex, smooth muscle cells were incubated with conditioned medium for 24 h prior to initiation of the binding studies. 125I-Sm-C/IGF-I-binding increased 1.6-fold compared to control cultures and after cross-linking the 43,000-mol wt complex could be detected on the smooth muscle cell surface. Human fibroblast monolayers secrete a protein that binds 125I-Sm-C/IGF-I which can be transferred to the smooth muscle cell surface and alters 125I-Sm-C/IGF-I binding.
D R Clemmons, R G Elgin, V K Han, S J Casella, A J D'Ercole, J J Van Wyk
Human T lymphocytes sensitized to Candida albicans (CA) were shown to proliferate in cultures induced with mannan, a ramified polysaccharide extracted from the cell well of CA. We presently describe that, when we used strongly labeled [3H]mannan, antigen-specific T blast cells were able to bind the labeled mannan on their membrane. The observations that irrelevant blast cells did not bind [3H]mannan, and that mannan-specific blast cells did not bind tritiated pneumococcal polysaccharide SIII, indicate the specificity of mannan binding. Mannan binding was reversible and saturable. Mannan binding on T blast cells was inhibited by preincubation with monoclonal antibodies to T3 but not to other T cell-related molecules. The characteristics of this receptor suggest its identity with the T cell receptor for antigen. The direct binding of mannan could be either due to a cross-linking of the receptor by multivalent mannan or to a recognition of mannan in association with HLA-DQ molecules, as suggested by partial blocking of mannan binding using anti-HLA-DQ monoclonal antibodies.
A Durandy, A Fischer, D Charron, C Griscelli
Recent studies from our laboratory indicate that a high concentration of platelet-derived calcium-activated cysteine protease (calpain) can cleave high molecular weight kininogen (HMWK). On immunodiffusion and immunoblot, antiserum directed to the heavy chain of HMWK showed immunochemical identity with alpha-cysteine protease inhibitor--a major plasma inhibitor of tissue calpains. Studies were then initiated to determine whether purified or plasma HMWK was also an inhibitor of platelet calpain. Purified alpha-cysteine protease inhibitor, alpha-2-macroglobulin, as well as purified heavy chain of HMWK or HMWK itself inhibited purified platelet calpain. Kinetic analysis revealed that HMWK inhibited platelet calpain noncompetitively (Ki approximately equal to 5 nM). Incubation of platelet calpain with HMWK, alpha-2-macroglobulin, purified heavy chain of HMWK, or purified alpha-cysteine protease inhibitor under similar conditions resulted in an IC50 of 36, 500, 700, and 1,700 nM, respectively. The contribution of these proteins in plasma towards the inhibition of platelet calpain was investigated next. Normal plasma contained a protein that conferred a five to sixfold greater IC50 of purified platelet calpain than plasma deficient in either HMWK or total kininogen. Reconstitution of total kininogen deficient plasma with purified HMWK to normal levels (0.67 microM) completely corrected the subnormal inhibitory activity. However, reconstitution of HMWK deficient plasma to normal levels of low molecular weight kininogen (2.4 microM) did not fully correct the subnormal calpain inhibitory capacity of this plasma. These studies indicate that HMWK is a potent inhibitor as well as a substrate of platelet calpain and that the plasma and cellular kininogens may function as regulators of cytosolic, calcium-activated cysteine proteases.
A H Schmaier, H Bradford, L D Silver, A Farber, C F Scott, D Schutsky, R W Colman
We explored the effects of alterations in extracellular and intracellular calcium concentration on arginine vasopressin (AVP)-stimulated cAMP formation in cultured rat inner medullary collecting tubule cells. cAMP formation remains constant at extracellular calcium concentrations between 0.5 and 4.0 mM, which did not change intracellular calcium. Maneuvers that alter intracellular calcium concentration are associated with marked changes in cAMP generation. EGTA decreases intracellular calcium and enhances AVP-stimulated cAMP formation, while increasing cellular calcium with 2 microM A23187 decreases AVP-stimulated cAMP formation in the presence, but not in the absence, of extracellular calcium. The changes in cAMP formation observed when intracellular calcium is altered are associated with reciprocal changes in prostaglandin E2 (PGE2) synthesis. Despite greater than 95% inhibition of PGE2 synthesis with 5 microM meclofenamic acid, the changes in cAMP formation accompanying alterations in intracellular calcium concentration are still evident. These studies suggest that intracellular calcium critically influences AVP-stimulated cAMP formation. It does so by a mechanism independent of PG that is probably mediated by a direct effect of the cation on the adenylate cyclase complex.
I Teitelbaum, T Berl
The occurrence, nature, and pathogenesis of intestinal lesions were studied in a number of graft vs. host reaction (GVHR) conditions in mice, combining variations in the nature of the following: the F1 hosts (newborn or adult, normal or lethally irradiated), the injected parental T cells (mixed or selected subsets of Lyt2+ or L3T4+ cells), and the antigenic stimulus (semi-allogeneic or restricted to class I or II MHC loci). The following conclusions were drawn: Three gut alterations are always associated: donor T cell infiltration, predominating in the crypt region; acceleration of the epithelium renewal; and increased epithelial Ia expression. The initial event is T-cell infiltration, which results from stimulation within the Peyer patches followed by cyclic traffic, i.e., migration into the thoracic duct and then seeding to the whole gut mucosa. Both Lyt2+ and L3T4+ cells can infiltrate the gut wall, the extent of the infiltration by a given subset depending upon the capacity of the donor blasts to circulate in the thoracic duct (higher for L3T4+) and then to home in the gut (much higher for Lyt2+ blasts) and the nature of the alloantigenic stimulation that governs the extent of each donor subset proliferation. Both donor T-cell subsets can induce gut epithelial damage, but for a comparable amount of infiltrating cells, L3T4+ cells induce more lesions. When the antigenic stimulation is restricted to class I or class I MHC loci, gut GVHR is much more easily elicited across class II MHC differences, which stimulate preferentially L3T4+ donor cells. The main mechanism of epithelial damage is not direct cytotoxicity, but more probably lymphokine(s) release.
D Guy-Grand, P Vassalli
The effects of interferons (IFNs) on lymphocyte adhesion to cultured human vascular endothelial cells (EC) were investigated using an in vitro assay. Endothelial cells obtained from umbilical vein were first cultured at a low density with a conditioned medium (CM) from 12-O-tetra decanoylphorbol 13-acetate-concanavalin A (TPA-Con A) stimulated human peripheral blood lymphocytes (PBL), or with recombinant (r) gamma interferon (IFN-gamma) or r alpha interferon (IFN-alpha), and then were incubated with freshly isolated PBL. Natural IFN-gamma in the TPA-Con A CM and rIFN-gamma (12.5-500 U/ml) induced major histocompatibility complex-class II antigens (HLA-DR, HLA-DP, and HLA-DQ) and significant lymphocyte adhesion to the EC, whereas rIFN-alpha did not. The lymphocyte adhesion to the EC and the expression of DR antigens on the EC were well correlated in terms of both kinetics and the dose-response pattern of rIFN-gamma. When EC expressing I region associated (Ia) antigen were preincubated with monoclonal anti-DR antibody before the addition of lymphocytes, the lymphocyte adhesion was significantly inhibited in both allogeneic and syngeneic combinations, whereas anti-HLA-DP, anti-HLA-DQ, and anti-HLA-ABC antibodies did not inhibit the binding at all. Cell fractionation experiments indicated that the majority of lymphocytes adhering to Ia-expressed EC were Leu-3+ T cells, whose binding was again almost completely inhibited by anti-DR antibody. Moreover, anti-Leu-3a, but not anti-Leu-2a, antibody effectively inhibited the T cell adhesion to the EC. These results strongly suggest that the interaction of the Leu-3(T4) receptor of T cells with IFN-gamma-induced DR antigens on EC plays a central role in the selective adhesion of Leu-3+ T cell to EC.
J Masuyama, N Minato, S Kano
To determine the incidence of elevated levels of serum methylmalonic acid in patients with cobalamin deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure methylmalonic acid in the serum of 73 patients with clinically confirmed cobalamin deficiency. Values ranged from 55 to 22,300 ng/ml, and 69 of the 73 patients had values above the normal range of 19-76 ng/ml as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum methylmalonic acid was significantly correlated with the serum folate level and the degree of neurologic involvement. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of methylmalonic acid while free of hematologic and neurologic abnormalities. A cobalamin-deficient patient is described with a normal serum cobalamin and an elevated serum methylmalonic acid. We conclude that the ability to measure methylmalonic acid in human serum will be useful in studies designed to determine the incidence of cobalamin deficiency in various patient populations.
S P Stabler, P D Marcell, E R Podell, R H Allen, J Lindenbaum
We previously demonstrated that human embryonic mesenchymal cells derived from the palate (HEMP cells) retain alkaline phosphatase (ALP) content and capacity for collagen synthesis after long-term culture, and their growth is markedly stimulated by epidermal growth factor (EGF). There was a dramatic decrease in ALP content and capacity to synthesize collagen in HEMP cells (HEMP-RV cells) persistently infected with rubella virus (RV). EGF increased ALP activity and decreased collagen synthesis in HEMP cells, whereas EGF showed no effect on these activities in HEMP-RV cells. Growth of HEMP-RV cells was slightly reduced compared with that of HEMP cells. EGF stimulated growth of HEMP cells and to a lesser extent of HEMP-RV cells. Binding of 125I-EGF to cell-surface receptors in HEMP-RV cells was, to our surprise, twice as much as that in HEMP cells. However, internalization of bound 125I-EGF in HEMP-RV cells was profoundly diminished. Thus, persistent RV infection causes not only changes in HEMP cell growth and differentiation but a decrease in or loss of HEMP cell responsiveness to EGF. The effects of persistent RV infection on palatal cell differentiation as well as growth may be responsible for the pathogenesis of congenital rubella. Furthermore, since HEMP cells appear to be closely related to osteoblasts, these results suggest a mechanism for RV-induced osseous abnormalities manifested in congenital rubella patients.
T Yoneda, M Urade, M Sakuda, T Miyazaki
1,25 dihydroxyvitamin D (1,25(OH)2D) has been demonstrated to inhibit hepatic 25 hydroxyvitamin D (25 OHD) production. Changes in cytosolic calcium have been shown to regulate cellular processes. Using the fluorescent dye Quin 2, we have investigated the effects of 1,25(OH)2D and 24,25(OH)2D on cytosolic calcium levels in hepatocytes. 1,25(OH)2D exposure for 5 min increases cytosolic calcium levels by 24% at a concentration of 100 pg/ml, 39% at a concentration of 1 ng/ml, and 50% at a concentration of 2 ng/ml. The latter increment occurs in both the presence and absence of extracellular calcium, indicating that 1,25(OH)2D is mobilizing intracellular calcium pools. 24,25(OH)2D, 10 ng/ml, does not increase cytosolic calcium levels while the calcium ionophore A23187, 3 microM, increases levels by 52%. Calcium inhibits hepatic 25 OHD synthesis in liver homogenates in a dose-dependent fashion, which can be prevented by chelation of calcium with EGTA. 1,25(OH)2D and A23187 decrease hepatocyte 25 OHD synthesis. The inhibitory effect of A23187 can be prevented by chelation of extracellular calcium. The data demonstrate that 1,25(OH)2D increases hepatocyte cytosolic calcium, and that these increments in cytosolic calcium may regulate some of the hepatic actions of the vitamin D metabolite.
D T Baran, M L Milne
Transformation of human peripheral blood lymphocytes with Epstein-Barr virus and rapid screening on rat insulinoma cells by an enzyme-linked immunosorbent assay were used to identify monoclonal autoantibodies that reacted with human pancreatic islets. Six such monoclonal autoantibodies were isolated and cloned. All six also were found to react with human thyroid. It is concluded that lymphocytes able to make autoantibodies that react with both the pancreas and thyroid are common in the human B cell repertoire.
C Garzelli, F E Taub, M C Jenkins, D W Drell, F Ginsberg-Fellner, A L Notkins
Interferons (IFN) elicit antiviral and antineoplastic activities by binding to specific receptors on the cell surface. The binding characteristics of IFN to human lymphocytes were studied using IFN alpha 2 labeled with 125I to high specific activity. The specific binding curves generated were analyzed by the LIGAND program of Munson and Rodbard to determine receptor numbers. The number of receptors in peripheral blood lymphocytes (PBL) and tonsillar B-lymphocytes (TBL) from normal individuals were 505 +/- 293 (n = 10) and 393 +/- 147 (n = 3) respectively. When these cells were preincubated in vitro with unlabeled IFN alpha 2, the receptor number decreased to 82 +/- 45 and 61 +/- 16 respectively. Receptor binding activities recovered gradually over a period of 72 h when the cells were incubated in IFN-free medium. This recovery of receptors could be blocked by the addition of actinomycin D to the incubation medium. A similar decrease in receptor expression was observed in vivo in PBL from patients being treated daily with 5 X 10(6) units/m2 per d of IFN alpha 2 by subcutaneous injection, for acute lymphoblastic leukemia or papilloma virus infections. Receptor numbers in PBL in vivo were further reduced concurrent with the progression of IFN therapy. Thus the reduction in IFN receptor expression observed in vitro can be demonstrated in vivo. These studies indicate that monitoring IFN receptor expression in vivo can provide information regarding the availability of IFN receptors at the cell surface for the mediation of IFN actions during the course of IFN therapy.
A S Lau, G E Hannigan, M H Freedman, B R Williams
Studies were undertaken to characterize the pattern of proximal tubular fluid (APRH2O) and bicarbonate reabsorption (APRHCO3) in the remnant kidney of euvolemic Munich-Wistar rats. The remnant kidney rats were placed on a diet containing either low or normal protein. Collections were obtained in the early, mid-, and late proximal convoluted tubule. Single nephron glomerular filtration rate (SNGFR) increased from 40.2 nl/min in controls to 58.8 nl/min in low protein remnant kidney and 78.1 nl/min in normal protein remnant kidney rats. The filtered load of bicarbonate was 1,272, 1,641, and 2,013 pmol/min, in the three groups, respectively. APRH2O and APRHCO3 increased nearly in parallel. Most of the increase in reabsorption occurred in the early proximal tubule. Tubular hypertrophy could account for at least 20-40% of the increase in reabsorption, but the majority of the increase appeared to be a delivery-dependent response similar to that observed in normal rats after an acute increase in SNGFR.
D A Maddox, J F Horn, F C Famiano, F J Gennari
The effects of changes in peritubular pH, carbon dioxide tension (PCO2), and HCO3- concentration on net HCO3- transport was examined in in vitro perfused cortical collecting tubules (CCTs) from unpretreated New Zealand white rabbits. Lowering peritubular HCO3- concentration and pH by reciprocal replacement of HCO3- with Cl-, significantly stimulated net HCO3- absorption. Lowering peritubular HCO3- concentration and pH, by substitution of HCO3- with gluconate, while keeping Cl- concentration constant, also stimulated net HCO3- absorption. Raising peritubular HCO3- concentration and pH, by reciprocal replacement of Cl- with HCO3-, inhibited net HCO3- absorption (or stimulated net HCO3- secretion). When the tubule was cooled, raising peritubular HCO3- concentration had no effect on net HCO3- transport, suggesting these results are not due to the passive flux of HCO3- down its concentration gradient. The effect of changes in ambient PCO2 on net HCO3- transport were also studied. Increasing the ambient PCO2 from 40 mmHg to either 80 or 120 mmHg, allowing pH to fall, had no effect on net HCO3- transport. Similarly, lowering ambient PCO2 to 14 mmHg had no effect on net HCO3- transport. Simultaneously increasing peritubular HCO3- concentration and PCO2, without accompanying changes in peritubular pH, i.e., isohydric changes, stimulated net HCO3- secretion to the same degree as nonisohydric increases in peritubular HCO3- concentration. Likewise, isohydric lowering of peritubular HCO3- concentration and PCO2 stimulated net HCO3- absorption. We conclude that: acute changes in peritubular HCO3- concentration regulate acidification in the CCT and these effects are mediated by a transcellular process; acute changes in ambient PCO2 within the physiologic range have no effect on HCO3- transport in the in vitro perfused CCT; and acute in vitro regulation of CCT acidification is independent of peritubular pH.
M D Breyer, J P Kokko, H R Jacobson
We report the beneficial effects of calcium infusions in a child with hereditary resistance to 1,25(OH)2D and alopecia. This patient after transient responsiveness to vitamin D derivatives became unresponsive to all therapy despite serum 1,25(OH)2D concentrations maintained at levels approximately 100-fold normal. A 7-mo trial with calcium infusions led to correction of biochemical abnormalities and healing of rickets. Bone biopsies (n = 3) showed a normal mineralization and the disappearance of the osteomalacia. Cultures of bone-derived cells demonstrated a lack of activation of 25-hydroxyvitamin D 24-hydroxylase and osteocalcin synthesis by 1,25(OH)2D3 (10(-9) and 10(-6) M). These results demonstrate that even in the absence of a normal 1,25(OH)2D3 receptor-effector system in bone cells, normal mineralization can be achieved in humans if adequate serum calcium and phosphorus concentrations are maintained; and calcium infusions may be an efficient alternative for the management of patients with this condition who are unresponsive to large doses of vitamin D derivatives.
S Balsan, M Garabédian, M Larchet, A M Gorski, G Cournot, C Tau, A Bourdeau, C Silve, C Ricour
Young female mice fed a choline-deficient, ethionine-supplemented (CDE) diet rapidly develop acute hemorrhagic pancreatitis. We have observed that pancreatic acini prepared from these mice are unable to secrete amylase in response to addition of the cholinergic agonist carbachol, although they retain the ability to secrete amylase in response to the Ca2+ ionophore A23187. The CDE diet does not alter the binding characteristics (Kd or the maximal number of binding sites) for muscarinic cholinergic receptors as tested using the antagonist [3H]N-methylscopolamine nor the competition for this binding by carbachol. Addition of carbachol to acini prepared from mice fed the CDE diet does not result in as marked an increase in cytosolic free Ca2+ levels as that noted in control samples (evaluated using quin2 fluorescence). These observations indicate that the CDE diet interferes with stimulus-secretion coupling in mouse pancreatic acini at a step subsequent to hormone-receptor binding and prior to Ca2+ release. This conclusion is confirmed by our finding that the hormone-stimulated generation of [3H]inositol phosphates (inositol trisphosphate, inositol bisphosphate, and inositol monophosphate) from acini labeled with [3H]myoinositol is markedly reduced in acini prepared from mice fed the CDE diet. This reduction is not due to a decrease in phosphatidylinositol-4,5-bisphosphate. This communication represents the first report of a system in which a blockade of inositol phosphate generation can be related to a physiologic defect and pathologic lesion.
R E Powers, A K Saluja, M J Houlihan, M L Steer
Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast-like collagenase, an enzyme that is distinct from neutrophil collagenase. Using an immunoassay that utilized antibody to skin fibroblast collagenase, we found that U937 cells secreted barely detectable quantities of enzyme, 10-12 ng/10(6) cells per 24 h, under basal conditions. Upon incubation with 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA), however, collagenase release increased 200-fold, comparable to the amount secreted by phorbol-stimulated human fibroblasts. Metabolic labeling and immunoprecipitation confirmed the enhanced synthesis of U937 cell collagenase upon TPA exposure. This enzyme activity further resembled fibroblast collagenase and differed from neutrophil collagenase by exhibiting preferential cleavage of monomeric type III collagen relative to type I. As previously observed with human alveolar macrophages, U937 cells also released a protein identical to the collagenase inhibitor produced by human skin fibroblasts, a molecule not associated with neutrophils. Release of this inhibitor increased 10-fold with TPA exposure. In contrast to collagenase and collagense inhibitor, TPA-treated U937 cells contained only 10-15% as much elastase and cathepsin G activities as control cells. Thus, TPA-induced differentiation modified the presence of these enzymes in the direction of their content in normal monocytes. Since the neutral proteinase profile of undifferentiated U937 cells resembles that of neutrophils and changes markedly after cellular differentiation to one that is characteristic of monocytes, these data suggest that neutrophilic proteinases may be produced by normal monocytes during the early stages of their differentiation.
H G Welgus, N L Connolly, R M Senior
The renal medullary collecting duct (MCD) secretes protons into its lumen and HCO3 into its basolateral space. Basolateral HCO3 transport is thought to occur via Cl/HCO3 exchange. To further characterize this Cl/HCO3 exchange process, intracellular pH (pHi) regulation was monitored in freshly prepared rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Cells were preincubated in bicarbonate-containing solutions and then abruptly diluted into bicarbonate-free media. The MCD cell pHi response to abrupt removal of CO2/HCO3 included an initial alkalinization due to rapid CO2 efflux, followed by an acidification due to HCO3 efflux and a gradual recovery to the resting pHi of 7.24 +/- 0.06 partly due to the action of a plasma membrane H+-ATPase. The initial alkalinization required a CO2/HCO3 gradient and did not occur in the presence of acetazolamide. The acidification phase required intracellular HCO3 and extracellular Cl, which was consistent with a Cl/HCO3 exchange. MCD HCO3 efflux exhibited saturable kinetics for extracellular Cl, with a Michaelis constant (Km) of 29.9 +/- 7.7 mM. HCO3 efflux also exhibited preference for halides over NO3, SCN, and gluconate, and striking sensitivity to disulfonic stilbene and acetazolamide inhibition, with an apparent K1 of 5 X 10(-7) M for DIDS. The final pHi recovery required intracellular ATP, which indicated that Cl/HCO3 and H+-ATPase activities are present in the same cells in these suspensions. The results provide direct evidence for MCD Cl/HCO3 exchange and describe some of the properties of this transport process.
M L Zeidel, P Silva, J L Seifter
In calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, metabolic abnormalities favoring extracellular inorganic pyrophosphate (PPi) accumulation have been suspected. Elevations of intracellular PPi in cultured skin fibroblasts from a single French kindred with familial CPPD deposition (19) and elevated nucleoside triphosphate pyrophosphohydrolase activity (NTPPPH), which generates PPi in extracts of CPPD crystal-containing cartilages (14) favor this suspicion. To determine whether NTPPPH activity or PPi content of cells might be a disease marker expressed in extraarticular cells, human skin-derived fibroblasts were obtained from control donors and patients affected with the sporadic and familial varieties of CPPD (CPPD-S and CPPD-F) deposition. Intracellular PPi was elevated in both CPPD-S (P less than 0.05) and CPPD-F (P less than 0.01) fibroblasts compared with control fibroblasts. Ecto-NTPPPH activity was elevated in CPPD-S (P less than 0.01) but not CPPD-F. Intracellular PPi correlated with ecto-NTPPPH (P less than 0.01). Elevated PPi levels in skin fibroblasts may serve as a biochemical marker for patients with familial or sporadic CPPD crystal deposition disease; ecto-NTPPPH activity further separates the sporadic and familial disease types. Expression of these biochemical abnormalities in nonarticular cells implies a generalized metabolic abnormality.
L M Ryan, R L Wortmann, B Karas, M P Lynch, D J McCarty
Infrarenal heterotopic cardiac isografts maintain structural and functional integrity. We have used this transplantation model to further explore the mechanisms of thyroid hormone-induced cardiac hypertrophy. Thyroid hormone administration, 1-thyroxine (T4) 10 micrograms/animal per d, led to a significant 30% increase in total heart weight and a 40% increase in the myosin content of the in situ heart when compared with control. In contrast, T4 treatment was without effect on the heart weight, protein content, rate of protein synthesis, or calculated myosin content of the heterotopic, nonworking heart. Heterotopic hearts demonstrated a significant decrease in the percentage of the V1 myosin isoenzyme from 95% to 61%. This shift occurred in euthyroid animals but was prevented by T4 treatment. These results suggest that thyroxine-induced cardiac hypertrophy is mediated indirectly via changes in cardiac work. Myosin isoenzyme expression can be altered by changes in work load but is still responsive to increased levels of thyroid hormone.
I Klein, C Hong
The purpose of this study is to develop an early diagnostic method for familial amyloidotic polyneuropathy (FAP) before clinical manifestations appear around the age of 30 yr. Amyloid fibrils isolated from type I FAP (FAP1) of Portuguese, Swedish, and Japanese origins consist of a variant transthyretin (TTR) that contains a methionine-for-valine substitution at position 30 or a mixture of normal TTR and this variant form. The variant TTR is present in the serum of FAP1 patients and can be measured by a radioimmunoassay (RIA) based on a nonapeptide (positions 22-30) derived from the variant TTR. Serum levels of the variant TTR in 45 Japanese FAP1 patients range from 4.71 to 17.61 mg/dl with a mean value of 9.18 mg/dl. The variant TTR is not present in the serum of 100 normal individuals, in four cases of primary and six cases of secondary amyloidosis, nor in 26 non-inheriting members of families with FAP1. The variant TTR level is measured in 24 children of 15 FAP1 patients as well. The variant TTR is already present in nine symptom-free children with the mean serum level of 11.90 mg/dl, but it is not present in 15 other children. FAP1 patients can be differentiated from non-FAP by this noninvasive diagnostic method even within families. The RIA can be applied worldwide to this intractable disorder for early diagnosis during childhood and for appropriate genetic counseling.
M Nakazato, T Kurihara, S Matsukura, K Kangawa, H Matsuo
The effects of thyroidectomy (Tx) and thyroxine replacement (T4Rx) on pituitary growth hormone (GH) secretion and hypothalamic GH-releasing hormone (GRH) concentration were compared to define the mechanism of hypothyroid-associated GH deficiency. Thyroidectomized rats exhibited a complete loss of pulsatile GH secretion with extensive reduction in GRH responsiveness and pituitary GH content. Cultured pituitary cells from Tx rats exhibited reduced GRH sensitivity, maximal GH responsiveness, and intracellular cyclic AMP accumulation to GRH, while somatostatin (SRIF) suppressive effects on GH secretion were increased. Hypothalamic GRH content was also markedly reduced. T4Rx completely restored hypothalamic GRH content and spontaneous GH secretion despite only partial recovery of pituitary GH content, GRH and SRIF sensitivity, and intracellular cyclic AMP response to GRH. The results indicate multiple effects of hypothyroidism on GH secretion and suggest that a critical role of T4 in maintaining normal GH secretion, in addition to restoring GH synthesis, is related to its effect on hypothalamic GRH.
H Katakami, T R Downs, L A Frohman
Normal blood-derived monocyte/macrophages were found to be susceptible to infection in vitro by human T lymphotropic virus type III (HTLV-III), the etiologic agent of the acquired immunodeficiency syndrome. In addition, HTLV-III was recovered from monocyte/macrophages of patients infected with this virus. The above findings raise the possibility that HTLV-III-infected monocyte/macrophages may serve as a vehicle for the dissemination of virus to target organs and as a reservoir for viral persistence, as has been shown for other lentiviruses including visna virus and caprine arthritis encephalitis virus.
D D Ho, T R Rota, M S Hirsch
The insulin binding characteristics and the structural components of the insulin receptor were studied in the purified liver plasma membranes from seven patients with noninsulin-dependent diabetes (NIDDM) and seven control subjects. In comparison to the controls, diabetic subjects had a 65% reduction in plasma insulin levels in response to an oral glucose load. Specific insulin binding by liver membranes from diabetic patients was, however, twofold greater than the binding activity by membranes from control subjects. This alteration resulted largely from an increase in the number of insulin receptors and a modest increase in receptor binding affinity. Holo (nonreduced) receptor species of similar molecular weights were detected by an affinity labeling technique in the two membrane preparations and sulfhydryl reduction demonstrated an insulin binding subunit of 125,000 mol wt. Overall, these results show that the hepatic insulin resistance of NIDDM cannot be explained by a deficiency in insulin binding.
P Arner, K Einarsson, S Ewerth, J Livingston