E M Wright, E Turk, B Zabel, S Mundlos, J Dyer
H Kuivaniemi, G Tromp, D J Prockop
This review has summarized recent information derived from many laboratories on the discovery, characteristics, and properties of a new member of the IL-1 family, IL-1 receptor antagonist. In addition to information, an emphasis has been placed on unanswered questions and new concepts. The existence of this first-described naturally occurring specific cytokine receptor antagonist may lead to a different perspective on the cytokine network. A major unanswered question emphasized throughout this review, that now can be addressed more directly, concerns what are the physiological roles of members of the IL-1 family. Although IL-1 beta is presumed to function primarily as an extracellular cytokine, this molecule lacks a leader peptide, is synthesized and handled by the cells in a manner suggestive of a cytoplasmic (not secretory) protein, and may only be released after cellular injury. Furthermore, although IL-1ra possesses a leader sequence, 50% or more of this protein remains cell associated. Do these observations suggest that members of the IL-1 family possess important intracellular functions, as yet undetermined? IL-1 alpha may play an intracellular role in regulating senescence; an IL-1 alpha antisense oligodeoxynucleotide was shown to prolong the life span of cultured human endothelial cells. Whether intracellular IL-1ra plays a role in influencing life span has not been determined. The discovery of IL-1ra has led to a first level of assumptions that this molecule may be functioning in vivo to regulate the pleiotropic extracellular effects of IL-1 in physiological or pathophysiological processes. Although enticing, these assumptions have not yet been proven to be true. Perhaps we need to look beyond, or within, and consider that IL-1ra and other members of the IL-1 family may have additional roles in normal or abnormal cell growth and development.
W P Arend
Because of the potential importance of interleukin 1 (IL-1) in modulating inflammation and the observations that human blood neutrophils (PMN) express IL-1 receptors (IL-1R) and synthesize IL-1 alpha and IL-1 beta, we studied the IL-1R on blood PMN from a group of patients with the sepsis syndrome. We report a marked enhancement in the sites per cell of IL-1R expressed on sepsis-PMN of 25 consecutively studied patients compared to 20 controls (patient mean = 9,329 +/- 2,212 SE; control mean = 716 +/- 42 SE, respectively). There was no demonstrable difference in the Kd of IL-1R on sepsis-PMN (approximately 1 nM) as determined by saturation curves of 125I-IL-1 alpha binding and the IL-1R on sepsis-PMN had an apparent Mr approximately 68,000, a value like that of normal PMN. Cytofluorographic analysis indicated that the sepsis-PMN phenotype is a single homogeneous population with respect to IL-1R expression. In contrast, expression of the membrane complement receptor CR3 is not increased on sepsis-PMN. Similar increases in expression of IL-1R were not observed in various other inflammatory processes, including acute disseminated inflammation and organ failure not caused by infection, acute infection without organ failure, and immunopathologies such as active systemic lupus erythematosus and rheumatoid arthritis. Enhanced expression of IL-1R was not related simply to the state of myeloid stimulation. Increased expression of IL-1R on normal PMN was induced in vitro by incubating cells with recombinant human granulocyte-macrophage/colony-stimulating factor for 18 h and this response was inhibited by cycloheximide, suggesting the possibility that de novo synthesis of IL-1R might occur in PMN during the sepsis syndrome.
M B Fasano, S Cousart, S Neal, C E McCall
Immunological cross-reactivity among nematodes has hampered the development of specific serodiagnostic assays for onchocerciasis. In the present study, an Onchocerca volvulus adult worm complementary DNA expression library was differentially screened with human sera from patients infected with O. volvulus and with an omnibus anti-nematode serum pool comprised of sera from patients infected with Brugia malayi, Loa loa, Wuchereria bancrofti, Mansonella perstans, Strongyloides stercoralis, Ancylostoma duodenale, Ascaris lumbricoides, and Dracunculus medinensis. Seven Onchocerca-specific clones were identified and screened with individual onchocerciasis patient sera. Additional studies were performed to characterize the most immunoreactive clones, OC 3.6 and OC 9.3. OC 3.6 produced a 152-kD beta-galactosidase fusion protein that was recognized in dot-immunoblots by 54 of 55 sera from onchocerciasis patients (98%). The OC 3.6 DNA insert is 996 bp long with an open reading frame of 627 bp and a 369-bp untranslated 3' end. OC 3.6 is closely related to a previously reported clone (OV 33-3), but it differs from that clone at both the 5' and 3' ends. OC 9.3 contained a novel 565-bp insert and produced a 138-kD fusion protein that was recognized by 46 of 55 sera from onchocerciasis patients (83%). Additional studies are in progress to develop and evaluate immunodiagnostic tests for onchocerciasis based on measurement of antibodies to these promising recombinant antigens.
R Chandrashekar, K Masood, R M Alvarez, A F Ogunrinade, R Lujan, F O Richards Jr, G J Weil
We previously identified a Plasmodium falciparum trophozoite cysteine proteinase (TCP) and hypothesized that it is required for the degradation of host hemoglobin by intraerythrocytic malaria parasites. To test this hypothesis and to evaluate TCP as a chemotherapeutic target, we examined the antimalarial effects of a panel of peptide fluoromethyl ketone proteinase inhibitors. For each inhibitor, effectiveness at inhibiting the activity of TCP correlated with effectiveness at both blocking hemoglobin degradation and killing cultured parasites. Benzyloxycarbonyl (Z)-Phe-Arg-CH2F, the most potent inhibitor, inhibited TCP at picomolar concentrations and blocked hemoglobin degradation and killed parasites at nanomolar concentrations. Micromolar concentrations of the inhibitor were nontoxic to cultured mammalian cells. These results support the hypothesis that TCP is a necessary hemoglobinase and suggest that it is a promising chemotherapeutic target.
P J Rosenthal, W S Wollish, J T Palmer, D Rasnick
It has been reported that the severe complication of dengue virus infection, dengue hemorrhagic fever (DHF) is much more commonly observed during secondary dengue virus infections than primary infections. In order to elucidate the role of T lymphocytes in the pathogenesis of DHF, we attempted to determine whether T lymphocytes are activated in vivo during dengue virus infections, by examining the levels of soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4), soluble CD8 (sCD8), interleukin-2 (IL-2) and interferon-gamma (IFN gamma) in the sera of 59 patients with DHF and 41 patients with dengue fever (DF). The levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma were significantly higher in the acute sera of patients with DHF than in the sera of healthy children (P less than 0.001 for all markers). The acute sera of patients with DF contained higher levels of sIL-2R, sCD4, IL-2, and IFN gamma than the sera of healthy children (P less than 0.001 for sIL-2R, IL-2, and IFN gamma; P less than 0.05 for sCD4), but did not have elevated levels of sCD8. The levels of sIL-2R (P less than 0.05), sCD4 (P less than 0.001), and sCD8 (P less than 0.001) were higher in DHF than in DF on days 3-4 after the onset of fever. The levels of IL-2 and IFN gamma in patients with DHF were highest 1 d before defervescence. There were no significant differences in the levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma among grades 1, 2, and 3 of DHF. These results indicate (a) T lymphocytes are activated and produce IL-2 and IFN gamma in vivo during DHF and DF, (b) CD4+ T lymphocytes are activated in DHF and DF, and the level of activation is higher in DHF than in DF, and (c) activation of CD8+ T lymphocytes is evident in DHF, but not in DF.
I Kurane, B L Innis, S Nimmannitya, A Nisalak, A Meager, J Janus, F A Ennis
To investigate whether the response of atrial natriuretic factor (ANF) to volume expansion is impaired in the early stages of dilated cardiomyopathy, the effects of saline load (SL; 0.25 ml/kg.min for 120 min) were assessed in 12 patients with dilated cardiomyopathy and asymptomatic to mildly symptomatic heart failure (HF) and in nine normal subjects (N). SL increased plasma ANF levels in N (from 14.3 +/- 2 to 19.5 +/- 3 and 26 +/- 4 pg/ml, at 60 and 120 min, respectively, P less than 0.001), but not in HF (from 42.9 +/- 9 to 45.9 +/- 9 and 43.9 +/- 8 pg/ml). Left ventricular end-diastolic volume (LVEDV) and stroke volume were increased (P less than 0.001) by SL in N but not in HF. Urinary sodium excretion (UNaV) increased in N more than in HF during SL, whereas forearm vascular resistance (FVR) did not change in N and increased in HF (P less than 0.001). In five HF patients SL was performed during ANF infusion (50 ng/kg, 5 ng/kg.min) that increased ANF levels from 37.1 +/- 10 to 146 +/- 22 pg/ml. In this group, SL raised both LVEDV (P less than 0.01) and ANF (P less than 0.05), whereas FVR did not rise. In addition, the UNaV increase and renin and aldosterone suppressions by SL were more marked than those observed in HF under control conditions. Thus, in patients with dilated cardiomyopathy and mild cardiac dysfunction, plasma ANF levels are not increased by volume expansion as observed in N. The lack of ANF response is related to the impaired cardiac adaptations. The absence of an adequate increase of ANF levels may contribute to the abnormal responses of HF patients to saline load.
M Volpe, C Tritto, N De Luca, A F Mele, G Lembo, S Rubattu, M Romano, P De Campora, I Enea, B Ricciardelli
This study examined apolipoprotein (apo) B metabolism in normolipemic subjects homozygous for the apo E2 (n = 4), apo E3 (n = 5), or apo E4 (n = 5) phenotype. Radioiodinated very low density lipoprotein (VLDL1) (ultracentrifuge flotation rate [Sf] 60-400) and VLDL2 (Sf 20-60) were injected into volunteers and the conversion of apo B was followed through intermediate density lipoprotein (IDL) to low density lipoprotein (LDL). Subjects homozygous for E3 converted approximately 50% of LVDL2 to LDL, the remainder being lost by direct catabolism. Those with the E2 phenotype produced less VLDL1, but converted more of it to VLDL2 (compared to E3 subjects). They displayed a characteristic dyslipidemia with the presence of slowly catabolized VLDL1 and VLDL2 remnants. LDL levels were low owing to increased direct catabolism of VLDL2 and IDL and a reduced efficiency of delipidation; only 25% of VLDL2 apo B was directed to LDL production. In contrast, E4 subjects converted more VLDL2 apo B to LDL than E3 subjects. About 70% of VLDL2 apo B was found in LDL; direct catabolism of VLDL and IDL was reduced as was the fractional catabolic rate of LDL (0.2 vs. 0.26 in E3 subjects). These changes in the VLDL----IDL----LDL metabolic cascade can in part be explained by alterations in hepatic LDL receptors with E2 subjects having higher and E4 subjects lower activities than those in E3 homozygotes.
T Demant, D Bedford, C J Packard, J Shepherd
Arginine vasopressin (AVP) transiently stimulates Na+ transport in the rabbit cortical collecting duct (CCD). However, the sustained effect of both AVP and its putative second messenger, cyclic adenosine monophosphate (cAMP), on Na+ transport in the rabbit CCD is inhibitory. Because maneuvers that increase [Ca++]i inhibit Na+ transport, the effects of AVP and cell-permeable cAMP analogues, on [Ca++]i were investigated in fura-2-loaded in vitro microperfused rabbit CCDs. Low-dose AVP (23-230 pM) selectively stimulated Ca++ influx, whereas 23 nM AVP additionally released calcium from intracellular stores. 8-chlorophenylthio-cAMP (8CPTcAMP) and 8-bromo-cAMP (8-Br-cAMP) also increased CCD [Ca++]i. The 8CPTcAMP-stimulated [Ca++]i increase was totally dependent on basolateral [Ca++]. In the absence of cAMP, peritubular Na+ removal produced a marked increase in [Ca++]i, which was also dependent on bath [Ca++], suggesting the existence of basolateral Na+/Ca++ exchange. Luminal Na+ removal in the absence of cAMP did not alter CCD [Ca++]i, but it completely blocked the cAMP-stimulated [Ca++]i increase. Thus the cAMP-dependent Ca++ increase is totally dependent on both luminal Na+ and basolateral Ca++, suggesting the [Ca++]i increase is secondary to cAMP effects on luminal Na+ entry and its coupling to basolateral Na+/Ca++ exchange. 8CPTcAMP inhibits lumen-to-bath 22Na flux [JNa(l-b)] in CCDs bathed in a normal Ca++ bath (2.4 mM). However, when bath Ca++ was lowered to 100 nM, a maneuver that also blocks the 8CPTcAMP [Ca++]i increase, 8CPTcAMP stimulated, rather than inhibited JNa(l-b). These results suggest that cAMP formation initially stimulates CCD Na+ transport, and that increased apical Na+ entry secondarily activates basolateral Ca++ entry. The cAMP-dependent [Ca++]i increase leads to inhibition Na+ transport in the rabbit CCD.
M D Breyer
Leishmania must survive despite exposure to the toxic oxidant hydrogen peroxide (H2O2) during phagocytosis by macrophages. We investigated the mechanism of H2O2 toxicity for L. donovani chagasi promastigotes, and factors responsible for their relative H2O2 resistance. There was a dose-dependent toxic effect of H2O2 for promastigotes isolated during logarithmic phase of growth. In contrast, stationary phase promastigotes were less susceptible to H2O2 toxicity, and more infectious for BALB/c mice. By spin trapping we found that hydroxyl radical (.OH) was generated after exposure of promastigotes to H2O2, and the amount of .OH was greater with log-phase than with stationary-phase promastigotes. .OH was generated after the addition of H2O2 to the cytosol but not the membranes of fractionated promastigotes, and the magnitude of .OH was greater in log than in stationary promastigote cytosol. Deferoxamine inhibition suggested that intracellular promastigote iron catalyzes .OH formation via the Fenton reaction. Furthermore, exposure of log-phase promastigotes to heat shock induced a relative H2O2-resistant state, which was not associated with a decrease in .OH formation but which required ongoing transcription. Thus, growth to stationary phase and heat shock both induce a state of relative H2O2 resistance, but these are probably due to different resistance mechanisms.
J H Zarley, B E Britigan, M E Wilson
The autoantigen(s) that we have previously described in human glomeruli, recognized in IgA nephropathy, has (have) been identified as mesangial cell in origin. Cultured mesangial cells expressed 48- and 55-kD components binding IgG isotype autoantibodies (IgG-MESCA) present in sera of patients with both IgA nephropathy and Henoch-Schönlein purpura (HSP). IgG-MESCA were not detected in sera of normals, or patients with other autoimmune-mediated glomerulonephritides: anti-glomerular basement membrane disease, Wegener's granulomatosis, or in IgM-mesangial proliferative disease. Binding specificity was proven by F(ab')2 studies in enzyme-linked immunosorbent assay (ELISA) and Western blotting, and there was no significant affinity of IgA or IgM immunoglobulins. Fluorescein isothiocyanate-conjugated IgG from ELISA-positive sera localized to the mesangium and peripheral capillary loops of glomeruli, supporting the belief that the antigen is expressed in normal human renal tissue. However, only about one third of mesangial cells in culture showed affinity for IgG from ELISA-positive sera, suggesting variable expression of the antigen(s) in vitro. The only autoantigen(s) present in glomeruli, and extractable from whole normal glomeruli by the techniques employed, localized on the mesangial cell. In both IgA nephropathy and HSP, autoimmunity was intermittently present, with fluctuating levels of IgG-MESCA detectable in sera. There were positive correlations with the degree of glomerular injury assessed by erythrocyturia and proteinuria in IgA nephropathy, but significance was reached with only the degree of hematuria in HSP. These findings suggest a contributing role in the pathogenesis of the mesangial proliferative lesions and demonstrate autoimmunity common to both IgA nephropathy and HSP.
D J O'Donoghue, A Darvill, F W Ballardie
Escherichia coli hemolysin (Hly) is a proteinaceous pore-forming exotoxin that probably represents a significant virulence factor in E. coli infections. We investigated its influence on human polymorphonuclear neutrophils (PMN), previously identified as highly susceptible targets. Hly provoked rapid secretion of elastase and myeloperoxidase, generation of superoxide, and synthesis of platelet-activating factor (PAF) and lyso-PAF. Concomitantly, marked phosphatidylinositol (PtdIns) hydrolysis with sequential appearance of the inositol-phosphates, inositol-phosphates, inositol triphosphate, diphosphate, and monophosphate, respectively, and formation of diacylglycerol, occurred. The metabolic responses displayed distinct bell-shaped dose dependencies, with maximum events noted at low toxin concentrations of 0.1-0.5 hemolytic units per milliliter. PtdIns hydrolysis and metabolic responses elicited by Hly exceeded those evoked by optimal concentrations of formylmethionyl-leucyl phenylalanine, PAF, leukotriene B4, A23187, or staphylococcal alpha-toxin. The toxin-induced effects were sensitive toward modulators of PMN stimulus transmission pathways (pertussis toxin, the kinase C inhibitor H7, and phorbol myristate acetate "priming"). We conclude that the marked capacity of low doses of Hly to elicit degranulation, respiratory burst, and lipid mediator generation in human PMN probably envolves signal transduction via PtdIns hydrolysis.
F Grimminger, U Sibelius, S Bhakdi, N Suttorp, W Seeger
Glycogen synthase is activated by protein phosphatase type-1 (PP-1). The spontaneous PP-1 activity accounts for only a small fraction of total PP-1 activity, which can be exposed by trypsin digestion of inhibitor proteins in the presence of Mn2+. We determined total PP-1 activity in muscle biopsies from insulin-sensitive and -resistant nondiabetic Pima Indians. Inhibitor-2 sensitive PP-1 represented 90% of total phosphatase activity. Spontaneous and total PP-1 activities were reduced in insulin resistant subjects (P less than 0.05-0.01), suggesting that the reduced PP-1 activity is not the result of inhibition by trypsin-labile phosphatase regulatory subunits. This difference was further investigated by Western blots using two different antibodies. An antibody raised against the rabbit muscle PP-1 catalytic subunit was used to analyze muscle extracts concentrated by DEAE-Sepharose adsorption. An antibody raised against a peptide derived from the COOH-terminal end of the PP-1 catalytic subunit was used to analyze crude muscle extracts. Both antibodies recognized a PP-1 catalytic subunit of approximately 33 kD, which unexpectedly was more abundant in insulin-resistant subjects (P less than 0.05-0.01). The increase in the tissue PP-1 protein content may be a response to compensate for the impairment in the enzyme activity.
B L Nyomba, D L Brautigan, K K Schlender, W Wang, C Bogardus, D M Mott
Denervation rapidly (within 24 h) induces insulin resistance of several insulin-responsive pathways in skeletal muscle, including glucose transport; resistance is usually maximal by 3 d. We examined the effect of denervation on the expression of two glucose transporter isoforms (GLUT-1 and GLUT-4) in rat hindlimb muscle; GLUT-4 is the predominant species in muscle. 1 d postdenervation, GLUT-1 and GLUT-4 mRNA and protein concentrations were unchanged. 3 and 7 d postdenervation, GLUT-4 mRNA and protein (per microgram DNA) were decreased by 50%. The minor isoform, GLUT-1 mRNA increased by approximately 500 and approximately 100%, respectively, on days 3 and 7 while GLUT-1 protein increased by approximately 60 and approximately 100%. The data suggest that the insulin resistance of glucose transport early after denervation does not reflect a decrease in total glucose transporter number; however, decreased GLUT-4 expression may contribute to its increased severity after 3 d. Parallel decreases in GLUT-4 mRNA and GLUT-4 protein postdenervation are consistent with pretranslational regulation; GLUT-1 expression may be regulated pre- and posttranslationally. The cell type(s) which overexpress GLUT-1 postdenervation need to be identified. Nervous stimuli and/or contractile activity may modulate the expression of GLUT-1 and GLUT-4 in skeletal muscle tissue.
N E Block, D R Menick, K A Robinson, M G Buse
It has been proposed that the mercurial-sensitive water transporter in mammalian erythrocytes is the anion exchanger band 3 (AE1) and/or the glucose transporter, band 4.5 (GLUT1). Using a functional assay for water channel expression in Xenopus oocytes (Zhang, R., K. A. Logee, and A. S. Verkman. 1990. J. Biol. Chem. 265:15375-15378), we compared osmotic water permeability (Pf) of oocytes injected with water, reticulocyte mRNA, AE1 mRNA, and GLUT1 mRNA. Injection of oocytes with 5-50 ng of in vitro-transcribed AE1 mRNA had no effect on Pf, but increased trans-stimulated 36Cl uptake greater than fourfold in a dinitro-disulfonic stilbene (DNDS)-inhibitable manner. Injection with 1-50 ng of in vitro-transcribed GLUT1 mRNA increased 3H-methylglucose uptake greater than 15-fold in a cytochalasin B-sensitive manner and increased Pf from (3.7 +/- 0.4) x 10(-4) cm/s (SE, n = 16, 10 degrees C) in water-injected oocytes up to (13 +/- 1) x 10(-4) cm/s (n = 18). Both the increments in sugar and water transport were inhibited by cytochalasin B (25 microM) and phloretin (0.2 mM); neither was inhibited by 0.3 mM HgCl2. In oocytes injected with 50 ng of rabbit reticulocyte mRNA, the Pf of (18 +/- 2) x 10(-4) cm/s (n = 18) was reduced to (4.0 +/- 0.6) x 10(-4) cm/s (n = 10) by HgCl2, but was not inhibited by DNDS (0.4 mM), cytochalasin B or phloretin. Coinjection of reticulocyte mRNA with antisense oligodeoxyribonucleotides against AE1 or GLUT1 did not affect Pf, but inhibited completely the incremental uptake of 36Cl or 3H-methylglucose, respectively. Expression of size-fractionated mRNA from reticulocyte gave a 2-2.5-kb size for water channel mRNA, less than the 4-4.5-kb size for the Cl transporter. These results provide evidence that facilitated water transport in erythrocytes is mediated not by bands 3 or 4.5, but by distinct water transport protein(s).
R Zhang, S L Alper, B Thorens, A S Verkman
This study examined the contribution of nitric oxide (NO) to the susceptibility or resistance to the hypertensive effects of high sodium chloride (8.0% NaCl) intake in young Dahl/Rapp salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats. Using NG-monomethyl-L-arginine (L-NMMA) as a probe for NO production in vivo, we found that increasing dietary sodium chloride increased NO activity in salt-resistant rats, but not in salt-sensitive rats. Exogenous L-arginine, the substrate for NO synthesis, decreased blood pressure to normotensive levels in salt-sensitive rats made hypertensive for 2 wk from 8.0% NaCl chow. D-arginine had no effect on blood pressure of these rats and L-arginine did not change blood pressure of salt-resistant rats. Intraperitoneal injections of L-arginine and its precursor, L-citrulline, and oral L-arginine, but not D-arginine, prevented the increase in blood pressure in salt-sensitive rats on the high salt chow over 2 wk of observation. In contrast, L-arginine did not alter the development of hypertension in spontaneously hypertensive rats. Mean urinary cGMP levels were higher in salt-sensitive rats on oral L-arginine than salt-sensitive rats on D-arginine. Infusion of L-NMMA acutely decreased, whereas intravenous L-arginine rapidly increased, urinary cGMP in both groups. L-arginine and L-citrulline increased production of NO and prevented salt-sensitive hypertension in Dahl/Rapp rats.
P Y Chen, P W Sanders
The hypothesis that von Willebrand factor (vWF) binding to platelet membrane glycoprotein Ib (GpIb) initiates intracellular pathways of platelet activation was studied. We measured the biochemical responses of intact human platelets treated with ristocetin plus vWF multimers purified from human cryoprecipitate. vWF plus ristocetin causes the breakdown of phosphatidylinositol 4,5-bisphosphate, the production of phosphatidic acid (PA), the activation of protein kinase C (PKC), increase of ionized cytoplasmic calcium ([Ca2+]i), and the synthesis of thromboxane A2. PA production, PKC activation, and the rise of [Ca2+]i stimulated by the ristocetin-induced binding of vWF multimers to platelets are inhibited by an anti-GpIb monoclonal antibody, but are unaffected by anti-GpIIb-IIIa monoclonal antibodies. Indomethacin also inhibits these responses without impairing platelet aggregation induced by vWF plus ristocetin. These results indicate that vWF binding to platelets initiates specific intraplatelet signaling pathways. The mechanism by which this occurs involves an arachidonic acid metabolite-dependent activation of phospholipase C after vWF binding to platelet membrane GpIb. This signal then causes PKC activation and increases of [Ca2+]i, which promote platelet secretion and potentiate aggregation.
M H Kroll, T S Harris, J L Moake, R I Handin, A I Schafer
Glucocorticoids (GC) modulate immune function in a number of ways, including suppression of T cell proliferation and other IL-2-mediated T cell functions. These inhibitory effects are similar to those induced by transforming growth factor-beta 1 (TGF-beta 1), a cytokine with potent T cell inhibiting activities. We examined the hypothesis that GC effects may be at least partially achieved through modulation of the expression of the TGF-beta 1 gene in activated T cells. Normal T cells were cultured with or without purified phytohemagglutinin (PHA-p) and 4 beta-phorbol 12-myristate 13-acetate (PMA) in the presence or absence of the synthetic GC, dexamethasone (100-200 micrograms/ml). The production of latent and active forms of TGF beta by these cells were analyzed by immunoblotting and bioassays. The steady-state levels of TGF-beta 1 mRNA were analyzed in total RNA from these cells by Northern hybridizations using a human TGF-beta 1 cDNA. The results showed that dexamethasone caused an increase in TGF beta production and a dose-dependent two to fourfold increase in TGF-beta 1 mRNA in activated as well as in unstimulated T cells, 1 h after exposure of the cultures to the steroid. The increase in TGF-beta 1 mRNA levels by dexamethasone was further potentiated two to threefold by cycloheximide, suggesting that the steroid effect may be due to inhibition of the synthesis of proteins that decrease TGF-beta 1 gene transcription or the stability of its transcripts. Finally, in vitro nuclear transcription studies indicated the dexamethasone effects on TGF-beta 1 gene expression to be largely transcriptional.
O AyanlarBatuman, A P Ferrero, A Diaz, S A Jimenez
Cardiac hypertrophy triggered by mechanical load possesses features in common with growth factor signal transduction. A hemodynamic load provokes rapid expression of the growth factor-inducible nuclear oncogene, c-fos, and certain peptide growth factors specifically stimulate the "fetal" cardiac genes associated with hypertrophy, even in the absence of load. These include the gene encoding vascular smooth muscle alpha-actin, the earliest alpha-actin expressed during cardiac myogenesis; however, it is not known whether reactivation of the smooth muscle alpha-actin gene occurs in ventricular hypertrophy. We therefore investigated myocardial expression of the smooth muscle alpha-actin gene after hemodynamic overload. Smooth muscle alpha-actin mRNA was discernible 24 h after coarctation and was persistently expressed for up to 30 d. In hypertrophied hearts, the prevalence of smooth muscle alpha-actin gene induction was 0.909, versus 0.545 for skeletal muscle alpha-actin (P less than 0.05). Ventricular mass after 2 d or more of aortic constriction was more highly correlated with smooth muscle alpha-actin gene activation (r = 0.852; P = 0.0001) than with skeletal muscle alpha-actin (r = 0.532; P = 0.009); P less than 0.0005 for the difference in the correlation coefficients. Thus, smooth muscle alpha-actin is a molecular marker of the presence and extent of pressure-overload hypertrophy, whose correlation with cardiac growth at least equals that of skeletal alpha-actin. Induction of smooth muscle alpha-actin was delayed and sustained after aortic constriction, whereas the nuclear oncogenes c-jun and junB were expressed rapidly and transiently, providing potential dimerization partners for transcriptional control by c-fos.
F M Black, S E Packer, T G Parker, L H Michael, R Roberts, R J Schwartz, M D Schneider
Systemic lysis may protect against the platelet activation and ongoing thrombosis associated with coronary thrombolysis. To address this hypothesis, we compared urokinase and tissue-type plasminogen activator (t-PA) given intravenously in a chronic, canine model of coronary thrombosis. T-PA 10 micrograms/kg per min induced reperfusion in 55 +/- 7 min but complete reocclusion occurred in 9/10 animals. Reocclusion was prevented by combining t-PA with 7E3, an antibody to the platelet glycoprotein IIb/IIIa which abolished ex vivo platelet aggregation. A similar time to reperfusion was seen with urokinase 750-1,000 U/kg per min. In contrast to t-PA, complete reocclusion occurred in only 1/20 cases (P less than 0.001 vs. t-PA), despite evidence of continued platelet activation in vivo and platelet aggregation ex vivo. Furthermore, this did not reflect a difference in the clearance of the two plasminogen activators. However, plasma fibrinogen was undetectable after urokinase in contrast with t-PA. Furthermore, in animals treated with prourokinase 20 micrograms/kg per min, reocclusion (4/7) correlated with the degree of systemic lysis. To determine whether platelet activation modified the response to urokinase, it was combined with 7E3. 7E3 0.8 mg/kg reduced the time to reperfusion with t-PA (30 +/- 5, n = 6; P = 0.025), but not with urokinase (56 +/- 8 vs. 62 +/- 6, P = ns). Systemic lysis protects against the propensity of continued thrombosis during coronary thrombolysis to delay reperfusion and induce reocclusion. This may modify the requirement for adjunctive antiplatelet therapy.
D J Fitzgerald, M Hanson, G A FitzGerald
Cytogenetic studies have shown frequent clonal abnormalities in papillary carcinoma (PTC) and follicular carcinoma (FTC). Loss of heterozygosity (LOH) may suggest the presence of tumor suppressor genes and has not been reported in these neoplasms. These studies were undertaken to determine if consistent chromosomal abnormalities are associated with thyroid cancer, to determine likely regions for molecular genetic investigations, and to determine if there is allelic loss in thyroid tumors. Cytogenetic analysis of 26 PTC and 5 FTC showed clonal abnormalities in 9 and included -Y, +5, or inv(10)(q11.2q21.2) in PTC, and -Y or near haploidy in FTC. Using DNA probes specific for chromosomes 1, 3, 10, 16, and 17, we carried out restriction fragment length polymorphism analysis on 6 FTC, 3 follicular adenomas (FA), and 12 PTC. LOH of all informative loci on chromosome 3p was observed in all 6 FTC, but not in FA or PTC. No LOH was observed for loci mapped to chromosome 10 in PTC. Our results suggest: cytogenetic abnormalities of chromosome 10q are associated with PTC; cytogenetic and molecular abnormalities of chromosome 3 are associated with FTC; and a tumor suppressor gene may be present on the short arm of chromosome 3 important for the development or progression of FTC.
M A Herrmann, I D Hay, D H Bartelt Jr, S R Ritland, R J Dahl, C S Grant, R B Jenkins
Neutrophils (PMN) migrate across intestinal epithelia in many disease states. Although such migration serves as a histological index of disease activity, little is known concerning the molecular events underlying PMN-intestinal epithelial interactions. We have studied chemotactic peptide-driven movement of PMN across cultured monolayers of the human intestinal epithelial cell line T84. Using a transmigration microassay, we show that both the decreased transepithelial resistance (76 +/- 3%) and transmigration (4 +/- 0.6 x 10(5) PMN.cm-2, when PMN applied at 6 x 10(6).cm-2) are largely prevented by MAbs which recognize either subunit of the PMN surface heterodimeric adhesion glycoprotein, CD11b/CD18. In contrast, such PMN-epithelial interactions are unaffected by MAbs recognizing either of the remaining two alpha subunits CD11a or CD11c. PMN from a leukocyte adherence deficiency patient also failed to migrate across epithelial monolayers thus confirming a requirement for CD11/18 integrins. By modifying our microassay, we were able to assess PMN transmigration across T84 monolayers in the physiological direction (which, for technical reasons, has not been studied in epithelia): transmigration was again largely attenuated by MAb to CD18 or CD11b (86 +/- 2% and 73 +/- 3% inhibition, respectively) but was unaffected by MAb to CD11a, CD11c. For standard conditions of PMN density, PMN transmigration in the physiological direction was 5-20 times more efficient than in the routinely studied opposite direction.
C A Parkos, C Delp, M A Arnaout, J L Madara
The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Sp1. DNAse protection and gel mobility shift assays demonstrate binding of recombinant Sp1 to both decanucleotide Sp1 binding sequences which are located 49 and 14 base pairs upstream of the transcription start site. The more distal of the two binding sites exhibits a somewhat higher affinity for Sp1. The G-C specific DNA binding drug, mithramycin, binds to both consensus sequences and prevents subsequent Sp1 binding. Promoter-dependent in vitro transcription of a DHFR template is selectively inhibited by mithramycin when compared to the human H2b histone gene. A similar effect is also noted in vivo. Mithramycin treatment of MCF-7 human breast carcinoma cells containing an amplified DHFR gene induces selective inhibition of DHFR transcription initiation, resulting in a decline in DHFR mRNA level and enzyme activity. This selective inhibition of DHFR expression suggests that it is possible to modulate the overexpression of the DHFR gene in methotrexate resistant cells.
S W Blume, R C Snyder, R Ray, S Thomas, C A Koller, D M Miller
Experimental myocardial infarction was induced in rats. The myocardial accumulation of hyaluronan (HA) and water during the development of infarction was measured. The extractable HA content of the infarcted area increased progressively from day 1 and on day 3 reached a threefold increase compared with the HA amounts in myocardium of sham operated controls. The relative water content of infarcted areas also increased progressively reaching a maximum value by day 3 and was strongly correlated with the HA accumulation. Affinity histochemistry visualized a thin rim of HA in the endoperimysium in healthy myocardium. By day 2 an interstitial edema with inflammatory cells was apparent. The widened endoperimysium stained extensively for HA. By its water-binding ability, interstitial accumulation of HA will contribute to the interstitial edema in infarcted myocardial tissue. An interstitial edema is likely to influence the electromechanical characteristics of the myocardium and facilitate reentry phenomena due to a loss of contact between muscle cells. The edema also induces an increased extracellular pressure and an altered myocardial wall compliance that might impair myocardial microcirculation. The findings are relevant to an understanding of the beneficial effect of hyaluronidase treatment in limiting cellular damage during myocardial ischemia.
A Waldenström, H J Martinussen, B Gerdin, R Hällgren
Stromal vascular cells were isolated from adipose tissue obtained from three different anatomical locations: epididymal (EPI), retroperitoneal (RP), and dorsal subcutaneous (SC), and allowed to differentiate in primary tissue culture. Cell number, protein concentration, glycerophosphate dehydrogenase, and lipoprotein lipase activity were similar in cells obtained from the EPI, RP, and SC regions, as were total insulin binding and the affinity of insulin for its receptor. However, both maximal insulin receptor tyrosine kinase activity and insulin-stimulated phosphorylation of the insulin receptor were significantly lower (P less than 0.05) in cells cultured from the SC region. In addition, newly differentiated adipocytes from the SC region were less sensitive to the ability of insulin to stimulate glucose uptake, and maximal insulin-stimulated glucose uptake by these cells was also significantly lower (P less than 0.05) when compared to cells obtained from the two other regions. Since these studies were performed on adipocyte precursor cells, allowed to differentiate to a similar degree in primary culture, the observed differences in insulin receptor phosphorylating activity, as well as the ability of insulin to stimulate glucose uptake appear to be intrinsic to adipose tissue from the three sites.
C Sztalryd, S Azhar, G M Reaven
The renal natriuretic actions of endogenous atrial natriuretic factor are enhanced by neutral endopeptidase inhibition (NEP-I). Recognizing that activation of the renin-angiotensin-aldosterone system in congestive heart failure (CHF) antagonizes the renal actions of atrial natriuretic factor, we hypothesized that angiotensin II antagonism with converting enzyme inhibition would potentiate the renal actions of NEP-I in CHF. To test this hypothesis, the renal responses to a specific NEP-I (SQ 28,603) were assessed in dogs with eight days of experimental CHF produced by rapid ventricular pacing. The renal natriuretic responses to NEP-I in experimental CHF were significant. In the same model of CHF, chronic angiotensin antagonism with converting enzyme inhibition potentiated both renal hemodynamic and excretory responses to NEP-I. The potentiated renal hemodynamic response included significant increases in glomerular filtration rate and filtration fraction. In the CHF group with angiotensin antagonism, an intrarenal infusion of low-dose angiotensin abolished the potentiated renal responses to NEP-I, supporting the concept that intrarenal angiotensin antagonism, rather than improved systemic hemodynamics or potentiation of other peptide systems, mediated the enhanced renal responses to NEP-I in the presence of converting enzyme inhibition.
K B Margulies, M A Perrella, L J McKinley, J C Burnett Jr
Sjögren-Larsson syndrome (SLS) is an inherited disorder associated with impaired fatty alcohol oxidation due to deficient activity of fatty alcohol:NAD+ oxidoreductase (FAO). FAO is a complex enzyme which consists of two separate proteins that sequentially catalyze the oxidation of fatty alcohol to fatty aldehyde and fatty acid. To determine which enzymatic component of FAO was deficient in SLS, we assayed fatty aldehyde dehydrogenase (FALDH) and fatty alcohol dehydrogenase in cultured fibroblasts from seven unrelated SLS patients. All SLS cells were selectively deficient in the FALDH component of FAO, and had normal activity of fatty alcohol dehydrogenase. The extent of FALDH deficiency in SLS cells depended on the aliphatic aldehyde used as substrate, ranging from 62% of mean normal activity using propionaldehyde as substrate to 8% of mean normal activity with octadecanal. FALDH activity in obligate SLS heterozygotes was partially decreased to 49 +/- 7% of mean normal activity using octadecanal as substrate. Differential centrifugation studies in fibroblasts indicated that this FALDH enzyme was largely particulate; soluble FALDH activity was normal in SLS cells. Intact SLS fibroblasts oxidized octadecanol to fatty acid at less than 10% of the normal rate, but oxidized free octadecanal normally, suggesting that the FALDH affected in SLS is chiefly involved in the oxidation of fatty alcohol to fatty acid. These results show that the primary enzymatic defect in SLS is the FALDH component of the FAO complex, which leads to deficient oxidation of fatty aldehyde derived from fatty alcohol.
W B Rizzo, D A Craft
This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.
S Radovick, C M Ticknor, Y Nakayama, A C Notides, A Rahman, B D Weintraub, G B Cutler Jr, F E Wondisford
Synovial fibroblasts freshly isolated from the rheumatoid joint are characterized by their marked connective tissue degradative ability. This phenotype includes the ability to secrete large amounts of the matrix-degrading metalloproteinases, collagenase, and stromelysin. We have found that another aspect of this phenotype is the constitutive expression at both protein and mRNA levels of a 92-kD gelatinolytic metalloproteinase, which is not secreted by normal dermal or lung fibroblasts and is immunologically cross-reactive with a type V collagenase expressed by activated macrophages and neutrophils. Expression of this 92-kD metalloproteinase confers upon the fibroblasts the capacity to degrade collagenase- and stromelysin-resistant interstitial elements, such as collagen types IV, V and XI. In contrast to the 92-kD metalloproteinase, a 68-kD gelatinase (type IV collagenase) was expressed by all fibroblast types studied, indicating that its regulation is distinct from that of the 92-kD gelatinase. To identify what cytokines may be important in the induction of the rheumatoid synovial phenotype, including expression of the 92-kD gelatinase, we exposed normal dermal fibroblasts to a number of cytokines including many known or considered likely to be present in rheumatoid synovial fluid and tissue. Although IL-1 beta, tumor necrosis factor-alpha, lymphotoxin, platelet-derived growth factor, and basic fibroblast growth factor were capable of stimulating fibroblasts to secrete collagenase, only tumor necrosis factor-alpha, lymphotoxin, and IL-1 beta were able to induce expression of the 92-kD gelatinase, demonstrating discordant regulation of the two metalloproteinases. Expression of the 68-kD gelatinase was independent of that of the 92-kD gelatinase, as demonstrated at the protein and mRNA levels. Late passage rheumatoid synovial fibroblasts, which no longer constitutively expressed the 92-kD gelatinase, displayed an accentuated response to IL-1 beta when compared to normal dermal fibroblasts. Thus, in addition to IL-1 beta, tumor necrosis factor-alpha or lymphotoxin may contribute to the expression of a specific rheumatoid synovial phenotype in vivo that is associated with progressive matrix destruction.
E N Unemori, M S Hibbs, E P Amento
Flow-mediated vasodilation is endothelium dependent. We hypothesized that flow activates a potassium channel on the endothelium, and that activation of this channel leads to the release of the endogenous nitrovasodilator, nitric oxide. To test this hypothesis, rabbit iliac arteries were perfused at varying flow rates, at a constant pressure of 60 mm Hg. Increments in flow induced proportional increases in vessel diameter, which were abolished by L,N-mono-methylarginine (the antagonist of nitric-oxide synthesis). Barium chloride, depolarizing solutions of potassium, verapamil, calcium-free medium, and antagonists of the KCa channel (charybdotoxin, iberiotoxin) also blocked flow-mediated vasodilation. Conversely, responses to other agonists of endothelium-dependent and independent vasodilation were unaffected by charybdotoxin or iberiotoxin. To confirm that flow activated a specific potassium channel to induce the release of nitric oxide, endothelial cells cultured on micro-carrier beads were added to a flow chamber containing a vascular ring without endothelium. Flow-stimulated endothelial cells released a diffusible vasodilator; the degree of vasorelaxation was dependent upon the flow rate. Relaxation was abrogated by barium, tetraethylammonium ion, or charybdotoxin, but was not affected by apamin, glybenclamide, tetrodotoxin, or ouabain. The data suggest that transmission of a hyperpolarizing current from endothelium to the vascular smooth muscle is not necessary for flow-mediated vasodilation. Flow activates a potassium channel (possibly the KCa channel) on the endothelial cell membrane that leads to the release of nitric oxide.
J P Cooke, E Rossitch Jr, N A Andon, J Loscalzo, V J Dzau
Hepatitis C virus (HCV) is the major etiologic agent associated with non-A, non-B hepatitis. This study was designed to assess virologic and serologic markers in hemophiliacs exposed to non-heat-treated and/or virus-inactivated plasma derivatives. Serial bleeds from 48 hemophilic patients were analyzed for the presence of HCV viral RNA sequences as detected by polymerase chain reaction (PCR) and antibodies to structural (core) and nonstructural (C-100 and 33C) proteins by specific dot immunoblot assay. All patients exposed to non-heat-treated products, and four of six patients exposed only to virus inactivated products, had evidence of HCV infection. However, over the 5-yr study period, six exposed patients (13%) consistently lacked detectable anti-C-100 and seven (15%) lost this antibody. HCV viremia (PCR positive) was found in 91% of exposed patients, and was significantly more frequent in HIV seropositive hemophiliacs (P less than 0.05). Six patients had high antibody level to HCV and elevated ALT, but appeared to clear viremia. Four hemophiliacs were HCV seropositive but lacked detectable viremia. These data indicate that hemophiliacs remain persistently infected by HCV and that antibody to the core antigen of HCV is a reliable marker of this transfusion transmissible agent.
J P Allain, S H Dailey, Y Laurian, D S Vallari, A Rafowicz, S M Desai, S G Devare
The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50% inactivation of scu-PA occurred in 45 min at 160 ng/ml thrombin in the absence of thrombomodulin and at 4.6 ng/ml thrombin in the presence of thrombomodulin. No difference was found in either the absence or the presence of thrombomodulin between the inactivation rates of high molecular weight scu-PA, and a low molecular weight scu-PA which lacked the growth factor and kringle domains. Enzyme kinetic experiments with varying concentrations of scu-PA showed that thrombomodulin decreased the Km of thrombin for scu-PA from 7.8 to 0.43 microM and increased the kcat from 0.30 to 1.2 s-1, corresponding to a 70-fold increase in the second-order rate constant kcat/Km. SDS-polyacrylamide gel electrophoresis showed that scu-PA was cleaved into two chains upon inactivation by thrombin, and confirmed the acceleration effect of thrombomodulin on inactivation of scu-PA. Thrombomodulin thus not only has anticoagulant properties but is also antifibrinolytic. The acceleration may imply a new mechanism for the regulation of local plasminogen activator activity on the cell surface.
G A de Munk, E Groeneveld, D C Rijken
Factor X (FX) is a vitamin K-dependent plasma protein required for the intrinsic and extrinsic pathways of blood coagulation. FXSanto Domingo is a hereditary FX deficiency which is characterized clinically by a severe bleeding diathesis. The proposita has a FX activity of less than 1% and a FX antigen of less than 5%. We have determined the molecular basis of the defect in the FXSanto Domingo gene by amplification of all eight exons with polymerase chain reaction and subsequent sequence analysis. The patient is homozygous for a G----A transition in exon I at codon -20 (numbering the alanine at the NH2 terminus of the mature protein as +1), resulting in the substitution of arginine for glycine in the carboxy-terminal part of the signal peptide. This amino acid change occurs near the presumed cleavage site of the signal peptidase. We hypothesized that the mutation might prevent cleavage by the signal peptidase which in turn would impair proper secretion of the FX protein. To test this hypothesis, we compared the expression of wild type and mutant FX cDNA in a human kidney cell line. Wild type and mutant constructs in the expression vector pCMV4 were introduced into the human embryonic kidney cell line 293 by calcium phosphate transfection. FX antigen levels in the supernatant of the cells harboring the wild type construct were 2.4 micrograms/10(7) cells per 24 h, whereas antigen levels in media from cells containing the FXSanto Domingo construct were undetectable. No FX antigen was detected in the cell lysates of cells transfected with the mutant construct. To insure that the difference in protein levels was not due to a difference in steady state levels of mRNA, Northern analysis was performed on RNA from the cell lysates of both constructs. The results showed a transcript of the same size, present in roughly equal amounts, in both cases. Thus, the defect in the signal sequence of FXSanto Domingo exerts its effect posttranscriptionally. FXSanto Domingo is the first described example of a bleeding diathesis due to a mutation in the signal sequence.
H H Watzke, A Wallmark, N Hamaguchi, P Giardina, D W Stafford, K A High
We previously reported that platelets become unresponsive to agonists when stimulated in combined suspension with aspirin-treated human umbilical vein endothelial cells. Inhibition occurred concomitant with metabolism of platelet-derived endoperoxides to prostacyclin by endothelial cells. We now demonstrate that if aspirin-treated platelets which fully respond to appropriate doses of agonists are exposed to aspirin-treated endothelial cells, they remain unresponsive despite absence of prostacyclin. Platelet inhibition is due in large part to ecto-ADPase activity on the endothelial cells. This was established by incubating aspirin-treated endothelial cells with 14C-ADP. Radio-thin layer chromatography and aggregometry demonstrated that 14C-ADP and induction of platelet activation decreased rapidly and concurrently. AMP accumulated transiently, was further metabolized to adenosine, and deaminated to inosine. The apparent Km of the endothelial cell ADPase was 33-42 microM and the Vmax 17-43 nmol/min per 10(6) cells, values in the range of antithrombotic potential. Thus, at least three complementary systems in human endothelial cells control platelet responsiveness: a cell-associated, aspirin-insensitive ADPase which functions in parallel with fluid phase autacoids such as the aspirin-inhibitable eicosanoids, and the aspirin-insensitive endothelium-derived relaxing factor.
A J Marcus, L B Safier, K A Hajjar, H L Ullman, N Islam, M J Broekman, A M Eiroa
Placental cells of mesenchymal origin were used to study the regulation of fetal growth at the cellular level. A significant difference in the in vitro growth rates of placental fibroblasts was observed as a function of gestational age. Cells derived from 10-19-wk placentae exhibited proliferative rates two to three times greater than cells derived from 7-9-wk placentae (16-30 h vs. 30-60 h, P less than 0.001). The proliferation rate remained stable throughout multiple passages in culture. Additionally, these two groups of cell strains exhibited marked differences in their responsiveness to mitogenic stimuli. Using maximal effective concentrations, insulin-like growth factor I interacted synergistically with epidermal growth factor and fibroblast growth factor to stimulate DNA synthesis in cells derived from 10-19-wk placentae. By contrast, the interaction of insulin-like growth factor 1 with epidermal growth factor and fibroblast growth factor exhibited significantly less synergy in 7-9-wk cells. These findings argue that the accelerated growth rate of human fetal cells results primarily from developmental events intrinsic to the cells and is associated with enhanced responsiveness to the mitogenic action of peptide growth factors.
M E Fant
Increased Na/H antiporter activity has been demonstrated after in vivo chronic metabolic acidosis as well as in vitro acid preincubation of cultured rabbit renal tubule cells. To study the underlying molecular mechanisms of this adaptive increase in Na/H antiporter activity, the present studies examined the effect of low pH media on Na/H antiporter activity and mRNA abundance in cultured renal tubule cells. Na/H antiporter activity was increased by 60% in a mouse renal cortical tubule cell line (MCT), and by 90% in an opossum kidney cell line (OKP) after 24 h of preincubation in acid (low [HCO3]) media. The ethylisopropylamiloride sensitivity of the Na/H antiporters were different in these two cell lines (MCT IC50 = 65 nM; OKP IC50 = 4.5 microM). In MCT cells, Na/H antiporter mRNA abundance measured by RNA blots increased by two- to fivefold after 24 h in low [HCO3] media. Na/H antiporter mRNA abundance was also increased in MCT cells with high CO2 preincubation as well as in rat renal cortex with in vivo chronic acid feeding. In contrast to renal epithelia, acid preincubation of NIH 3T3 fibroblasts led to suppression of Na/H antiporter activity. RNA blots of 3T3 fibroblasts revealed the same size Na/H antiporter transcript as in MCT cells. However, Na/H antiporter mRNA levels were suppressed by acid preincubation. These studies demonstrate differential regulation of Na/H antiporter activity and mRNA abundance in renal epithelial cells and fibroblasts in response to an acidotic environment.
O W Moe, R T Miller, S Horie, A Cano, P A Preisig, R J Alpern
Urethral obstruction produces increased voiding frequency (0.7 +/- 0.06 to 1.1 +/- 0.08 h-1) and hypertrophy of the urinary bladder (89 +/- 1.7 to 708 +/- 40 mg) with profound increments in the dimensions of afferent (4, 6) and efferent neurons (299 +/- 4.7 to 573 +/- 8.6 microns2) supplying this organ in the rat. We discovered that hypertrophied bladders of rat and human contain significantly more nerve growth factor (NGF) per milligram wet weight, protein, and DNA than normal bladders. The temporal correlation between NGF content, neuronal hypertrophy, and bladder weight was consistent with a role for this growth factor in the neurotrophic effects associated with obstruction. Autoimmunity to NGF abolished the hypertrophy of NGF-sensitive bladder neurons in the pelvic ganglion after obstruction. Relief of urethral obstruction reduced bladder size (349 +/- 78 mg), but neuronal hypertrophy (460.2 +/- 10.2 microns2) and elevated NGF levels were only partially reversed. Bladder hypertrophy (133 +/- 4.3 mg) induced by osmotic diuresis slightly increased ganglion cell area (365.2 +/- 6.1 microns2) and only doubled NGF content of the bladder. These findings provide important new evidence that parenchymal cells in the hypertrophied bladder can synthesize NGF and possibly other molecular messengers that act to alter the size and function of neurons in adult animals and man.
W D Steers, S Kolbeck, D Creedon, J B Tuttle
Generalized recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited autosomal disease characterized by dermolytic blister formation. Enhanced collagenase and/or abnormal collagenase have been reported in skin from affected patients, suggesting that collagenase could be responsible for the absence of anchoring fibrils in this disorder. We used a genetic linkage approach to test the hypothesis that this disease is due to a defect in the collagenase gene in nine affected families. Analysis of amplified genomic DNA fragments of the collagenase gene by means of denaturing gradient gel electrophoresis (DGGE) allowed us to detect intragenic polymorphisms, which were subsequently characterized by direct genomic sequencing. Segregation analysis of these polymorphic sites showed exclusion of linkage between the collagenase gene and generalized RDEB phenotype in a family with consanguineous parents and three affected children. However, the possibility of linkage with the collagenase gene in the other eight families tested could not be excluded. The genetic markers described here provide a tool for investigating genetic linkage in other affected families. Overall, our results show that generalized RDEB can be caused by a defect in a gene other than the collagenase gene, and support the hypothesis that the genetic defect lies in abnormal anchoring fibril formation.
A Hovnanian, P Duquesnoy, S Amselem, C Blanchet-Bardon, M Lathrop, L Dubertret, M Goossens
We have previously described a disorder, normotriglyceridemic abetalipoproteinemia, that is characterized by the virtual absence of plasma low density lipoproteins and complete absence of apoB-100, but with apparently normal secretion of triglyceride-rich lipoproteins containing apoB-48. The patient's plasma lipoproteins were shown on polyacrylamide gels and by antibody mapping to have a new truncated apoB variant, apoB-50, circulating along with her apoB-48. We have found this individual to be homozygous for a single C-to-T nucleotide substitution at apoB codon 2252, which produces a premature in-frame stop codon. Thus, this is a rare example of homozygous hypobetalipoproteinemia. Electron photomicrographs revealed that the diameters of particles in the d less than 1.006 g/ml lipoprotein fraction, in both the postprandial and postabsorptive state, are bimodally distributed. The molar ratio of apoE to apoB in these particles is 3.5:1, similar to normal VLDL. The plasma LDL interval contains both spherical and cuboidal particles. Autologous reinfusion of labeled d less than 1.006 g/ml lipoproteins showed exponential disappearance from plasma, with an apparent half-removal time of 50 min, somewhat slower than for normal chylomicrons but within the normal range for VLDL. The calculated production rate for apoB was within the normal range in this subject. A very small amount of label was found briefly in the IDL fraction, but none at any time in LDL or HDL. Therefore, because LDL particles that contain apoB-50 lack the putative ligand domain of the LDL receptor, we conclude that the very low level of LDL is due to the rapid removal of the abnormal VLDL particles before their conversion to LDL can take place.
D A Hardman, C R Pullinger, R L Hamilton, J P Kane, M J Malloy
A viable autosomal recessive mutation (named fch, or ferrochelatase deficiency) causing jaundice and anemia in mice arose in a mutagenesis experiment using ethylnitrosourea. Homozygotes (fch/fch) display a hemolytic anemia, photosensitivity, cholestasis, and severe hepatic dysfunction. Protoporphyrin is found at high concentration in erythrocytes, serum, and liver. Ferrochelatase activity in various tissues is 2.7-6.3% of normal. Heterozygotes (+/fch) are not anemic and have normal liver function; they are not sensitive to light exposure; ferrochelatase activity is 45-65% of normal. Southern blot analysis using a ferrochelatase cDNA probe reveals no gross deletion of the ferrochelatase gene. This is the first spontaneous form of erythropoietic protoporphyria in the house mouse. Despite the presence in the mouse of clinical and biochemical features infrequent in the human, this mutation may represent a model for the human disease, especially in its severe form.
S Tutois, X Montagutelli, V Da Silva, H Jouault, P Rouyer-Fessard, K Leroy-Viard, J L Guénet, Y Nordmann, Y Beuzard, J C Deybach
Cardiac pressure overload induces a shift towards the fetal form of major proteins expressed by the myocytes, and an accumulation of extracellular matrix proteins. One of them, fibronectin (FN), accumulates soon after the imposition of pressure overload. Because FN exists both as cellular FN (c-FN) locally synthesized by nonmuscle cells and as "plasma-FN" (p-FN) synthesized by the hepatocytes, the first issue of this study was to determine whether FN accumulation within the myocardium in response to pressure overload is paralleled by a local increase in mRNA. The expression of c-FN isoforms being developmentally regulated in a tissue-specific manner, the types of FN exons expressed by cardiac cells were analyzed. Pressure overload was induced in 25-d-old rats by stenosis of the thoracic aorta. Using in situ hybridization, we show that the mRNAs encoding the fetal forms of c-FN are accumulated in the interstitial tissue of fetal rat hearts but are absent in adult. 1-3 d after aortic stenosis, the fetal forms of c-FN mRNAs were found in the wall of coronary arteries and in focal areas of the myocardium. Thus nonmuscle cells and smooth muscle cells, like myocytes, do respond to pressure overload by reexpressing fetal gene transcripts.
J L Samuel, A Barrieux, S Dufour, I Dubus, F Contard, V Koteliansky, F Farhadian, F Marotte, J P Thiéry, L Rappaport
The production by monocytes of interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF alpha) in intensive care unit (ICU) patients with sepsis syndrome (n = 23) or noninfectious shock (n = 6) is reported. Plasma cytokines, cell-associated cytokines within freshly isolated monocytes and LPS-induced in vitro cytokine production were assessed at admission and at regular intervals during ICU stay. TNF alpha and IL-6 were the most frequently detected circulating cytokines. Despite the fact that IL-1 alpha is the main cytokine found within monocytes upon in vitro activation of cells from healthy individuals, it was very rarely detected within freshly isolated monocytes from septic patients, and levels of cell-associated IL-1 beta were lower than those of TNF alpha. Cell-associated IL-1 beta and TNF alpha were not correlated with corresponding levels in plasma. Upon LPS stimulation, we observed a profound decrease of in vitro IL-1 alpha production by monocytes in all patients, and of IL-1 beta, IL-6, and TNF alpha in septic patients. This reduced LPS-induced production of cytokines was most pronounced in patients with gram-negative infections. Finally, monocytes from survival patients, but not from nonsurvival ones recovered their capacity to produce normal amounts of cytokines upon LPS stimulation. In conclusion, our data indicate an in vivo activation of circulating monocytes during sepsis as well as in noninfectious shock and suggest that complex regulatory mechanisms can downregulate the production of cytokines by monocytes during severe infections.
C Munoz, J Carlet, C Fitting, B Misset, J P Blériot, J M Cavaillon
Cell-free HIV RNA in plasma was detected and quantitated after antiviral therapy by the polymerase chain reaction. RNA was extracted from plasma, reverse transcribed to cDNA, amplified by polymerase chain reaction, and quantitated by absorbance based on an enzyme-linked affinity assay. 72 HIV antibody-positive subjects had one plasma sample taken. 39 who were not receiving antiretroviral therapy at the time had a mean plasma HIV RNA copy number of 690 +/- 360 (mean +/- SEM) per 200 microliters of plasma, while 33 subjects who had been receiving zidovudine therapy for a minimum of 3 mo had a mean copy number of 134 +/- 219 (P less than 0.05). 27 additional HIV antibody-positive patients had two plasma samples taken before and 1 mo after initiating dideoxynucleoside therapy. Plasma HIV RNA copy number fell from 540 +/- 175 to 77 +/- 35 (P less than 0.05). Finally, nine of these subjects had two baseline samples obtained before initiating therapy and two posttreatment samples 1 and 2 mo after therapy was begun. Mean plasma RNA copy number declined from 794 +/- 274 to less than 40 (below the lower limit of sensitivity) after 1 mo of therapy, with suppression maintained after 2 mo of therapy. These results suggest that gene amplification can be used to detect and quantitate changes in plasma HIV RNA after dideoxynucleoside therapy. Plasma HIV polymerase chain reaction may be a more sensitive marker to monitor antiviral therapy, particularly in asymptomatic patients where measurement of p24 antigen or quantitative plasma cultures are negative.
M Holodniy, D A Katzenstein, D M Israelski, T C Merigan
To assess the contribution of Factor IX/IXa, to intravascular thrombosis, a canine coronary thrombosis model was studied. Thrombus formation was initiated by applying current to a needle in the circumflex coronary artery. When 50% occlusion of the vessel developed, the current was stopped and animals received an intravenous bolus of either saline, bovine glutamyl-glycyl-arginyl-Factor IXa (IXai), a competitive inhibitor of Factor IXa assembly into the intrinsic Factor X activation complex, bovine Factor IX, or heparin. Animals receiving saline or Factor IX developed coronary occlusion due to a fibrin/platelet thrombus in 70 +/- 11 min. In contrast, infusion of IXai prevented thrombus formation completely (greater than 180 min) at doses of 460 and 300 micrograms/kg, and partially blocked thrombus formation at 150 micrograms/kg. IXai attenuated the accumulation of 125I-fibrinogen/fibrin at the site of the thrombus by approximately 67% (P less than 0.001) and resulted in approximately 26% decrease in serotonin release from platelets in coronary sinus (P less than 0.05). Hemostatic variables in animals receiving IXai, remained within normal limits. Animals given heparin in a concentration sufficient to prevent occlusive thrombosis had markedly increased bleeding, whereas heparin levels that maintained extravascular hemostasis did not prevent intracoronary thrombosis. This suggests that Factor IX/IXa can contribute to thrombus formation, and that inhibition of IXa participation in the clotting mechanism blocks intravascular thrombosis without impairing extravascular hemostasis.
C R Benedict, J Ryan, B Wolitzky, R Ramos, M Gerlach, P Tijburg, D Stern
Fc gamma receptors are important components in the binding and phagocytosis of IgG-sensitized cells. Studies on the role of these receptors have been limited by the fact that most hematopoietic cells express more than one Fc gamma receptor. We studied the role of Fc gamma RIIA in isolation on a human erythroleukemia cell line (HEL) which expresses Fc gamma RIIA as its only Fc gamma receptor. HEL cells were observed to bind and phagocytose IgG-sensitized red blood cells (RBCs) in a dose-dependent manner. We then examined the role of Fc gamma RI and Fc gamma RII in isolation and in combination, in transfected COS-1 cells. Fc gamma RIIA-transfected COS cells also mediated both the binding and phagocytosis of IgG-sensitized RBCs. In contrast, phagocytosis was not observed in Fc gamma RI-transfected cells, although these cells avidly bound IgG-sensitized RBCs. Furthermore, coexpression of both receptors by doubly transfected cells did not affect the phagocytic efficiency of Fc gamma RIIA. These studies establish that Fc gamma RIIA can mediate phagocytosis and suggest that transfected COS-1 cells provide a model for examining this process. Since HEL cells exhibit characteristics of cells of the megakaryocyte-platelet lineage, including expression of Fc gamma RII as the only Fc gamma receptor, Fc gamma RIIA on megakaryocytes and platelets may be involved in the ingestion of IgG-containing immune complexes. Furthermore, these studies indicate that Fc gamma RI and Fc gamma RIIA differ in their requirements for transduction of a phagocytic signal.
Z Indik, C Kelly, P Chien, A I Levinson, A D Schreiber
The functional significance of cardiac ATP-sensitive potassium channels remains controversial because of the discrepancy between the low levels of ATP at which activation of the channels occurs and the much higher levels of ATP maintained during myocardial ischemia. We studied the effects of (+)-lactate, which accumulates in large quantity as a result of increased glycolysis during ischemia, on ATP-sensitive potassium channels in adult guinea pig ventricular myocytes using the whole-cell patch-clamp technique. Lactate at 20-40 mM in the internal solution activated ATP-sensitive potassium channels and shortened action potential duration. Activation of the channels occurred even in the presence of 2-5 mM ATP in the internal solution and was dependent on intracellular free magnesium levels. Our results suggest that intracellular lactate may play a significant role in activating cardiac ATP-sensitive potassium channels and shortening action potential duration even at ATP levels similar to those resulting from moderate to severe myocardial ischemia.
E C Keung, Q Li
Lymphocytes enter lymph nodes by first adhering to high endothelial venules, an adhesive event mediated by a lectinlike lymphocyte receptor (L-selectin). Previously, it was shown with an in vitro assay that lymphocytes preferentially adhere to myelin-rich regions in brain sections. Here, using a recombinant form of L-selectin as an immunohistochemical reagent, we demonstrate potential ligands for L-selectin in myelinated regions of the central but not the peripheral nervous system. Using several antibodies and phorbol ester downmodulation of the receptor, we establish that L-selectin on human lymphocytes has a primary involvement in lymphocyte adherence to the myelinated regions. On mouse lymphocytes, the contribution of L-selectin appears to be partial. These findings raise the possibility that leukocyte targeting to myelin-rich regions, via a L-selectin dependent mechanism, may be a factor in the pathogenesis of certain central nervous system demyelinating diseases.
K Huang, J S Geoffroy, M S Singer, S D Rosen