To determine the temporal relationship between changes in contractile performance and flux through the citric acid cycle in hearts oxidizing acetoacetate, we perfused isolated working rat hearts with either glucose or acetoacetate (both 5 mM) and freeze-clamped the tissue at defined times. After 60 min of perfusion, hearts utilizing acetoacetate exhibited lower systolic and diastolic pressures and lower cardiac outputs. The oxidation of acetoacetate increased the tissue content of 2-oxoglutarate and glutamate and decreased the content of succinyl-CoA suggesting inhibition of citric acid cycle flux through 2-oxoglutarate dehydrogenase. Whereas hearts perfused with either acetoacetate or glucose were similar with respect to their function for the first 20 min, changes in tissue metabolites were already observed within 5 min of perfusion at near-physiological workloads. The addition of lactate or propionate, but not acetate, to hearts oxidizing acetoacetate improved contractile performance, although inhibition of 2-oxoglutarate dehydrogenase was probably not diminished. If lactate or propionate were added, malate and citrate accumulated indicating utilization of anaplerotic pathways for the citric acid cycle. We conclude that a decreased rate of flux through 2-oxoglutarate dehydrogenase in hearts oxidizing acetoacetate precedes, and may be responsible for, contractile failure and is not the result of decreased cardiac work. Further, anaplerosis play an important role in the maintenance of contractile function in hearts utilizing acetoacetate.
R R Russell 3rd, H Taegtmeyer
Because polycythemia vera (PV) is a clonal hematopoietic stem cell disease with a trilineage hyperplasia, and interleukin-3 (IL-3) stimulates trilineage hematopoiesis, we have studied the response of highly purified PV blood burst-forming units-erythroid (BFU-E) to recombinant human IL-3 (rIL-3). Whereas the growth of normal blood BFU-E in vitro rapidly declined by 40 and 60% after 24 and 48 h of incubation without 50 U/ml of rIL-3, the growth of PV BFU-E declined by only 10 and 30% under the same conditions, demonstrating a reduced dependence on rIL-3. A reduced dependence of PV BFU-E on recombinant human erythropoietin (rEP) was also present. Dose-response experiments showed a 117-fold increase in PV BFU-E sensitivity to rIL-3, and a 6.5-fold increase in sensitivity to rEP, compared to normal BFU-E, whereas blood BFU-E from patients with secondary polycythemia responded like normal BFU-E. Endogenous erythroid colony (EEC) formation, which is independent of the addition of rEP, was reduced by 50% after erythroid colony-forming cells were generated from PV BFU-E in vitro without rIL-3 for 3 d, whereas rEP-stimulated erythroid colonies were unaffected. These studies demonstrate a striking hypersensitivity of PV blood BFU-E to rIL-3, which may be the major factor in the pathogenesis of increased erythropoiesis without increased EP concentrations.
C H Dai, S B Krantz, R T Means Jr, S T Horn, H S Gilbert
Isolated hepatocytes incubated with [35S]-methionine were examined for the time-dependent accumulation of [35S]-glutathione (GSH) in cytosol and mitochondria, the latter confirmed by density gradient purification. In GSH-depleted and -repleted hepatocytes, the increase of specific activity of mitochondrial GSH lagged behind cytosol, reaching nearly the same specific activity by 1-2 h. However, in hepatocytes from ethanol-fed rats, the rate of increase of total GSH specific radioactivity in mitochondria was markedly suppressed. In in vivo steady-state experiments, the mass transport of GSH from cytosol to mitochondria and vice versa was 18 nmol/min per g liver, indicating that the half-life of mitochondrial GSH was approximately 18 min in controls. The fractional transport rate of GSH from cytosol to mitochondria, but not mitochondria to cytosol, was significantly reduced in the livers of ethanol-fed rats. Thus, ethanol-fed rats exhibit a decreased mitochondrial GSH pool size due to an impaired entry of cytosol GSH into mitochondria. Hepatocytes from ethanol-fed rats exhibited a greater susceptibility to the oxidant stress-induced cell death from tert-butylhydroperoxide. Incubation with glutathione monoethyl ester normalized the mitochondrial GSH and protected against the increased susceptibility to t-butylhydroperoxide, which was directly related to the lowered mitochondrial GSH pool size in ethanol-fed cells.
J C Fernández-Checa, C García-Ruiz, M Ookhtens, N Kaplowitz
In a baboon graft model of arterial intimal thickening, smooth muscle cells (SMC) have been observed to proliferate underneath an intact monolayer of endothelium and in the absence of platelet adherence. Because platelets are not present and therefore cannot be a major source of growth stimulus, we have proposed that the vascular wall cells in the graft intima express mitogens and regulate SMC proliferation. To test this hypothesis, we assayed the grafts for mitogenic activity and expression of growth factor genes. Segments of healing graft and of normal artery, when perfused ex vivo, released mitogenic activity into the perfusate. The graft released more mitogen than the normal arterial segment, and some of the activity was inhibitable with an antibody to human platelet-derived growth factor (PDGF). In addition, Northern analysis of total RNA demonstrated higher expression of PDGF-A chain mRNA in the graft intima compared to normal artery. PDGF-B chain mRNA was barely detectable in both tissues. PDGF mRNA levels within the graft interstices were not measured. In situ hybridization of 7.5- or 12-wk grafts indicated that some luminal endothelial cells and adjacent intimal SMC contained PDGF-A chain mRNA. By thymidine autoradiography, intimal SMC were observed to be proliferating in the inner third of the intima. These data demonstrate a difference in the pattern of PDGF transcript expression and luminal perfusate activity in graft as compared with control arteries. The association of intimal smooth muscle cell proliferation with intimal PDGF mRNA expression and release of PDGF-like protein supports the hypothesis that factors from cells that have grown into the graft or populated its surface rather than platelets may regulate intimal smooth muscle cell proliferation in this model.
M A Golden, Y P Au, T R Kirkman, J N Wilcox, E W Raines, R Ross, A W Clowes
We investigated the major products of proglucagon (PG) processing in plasma in the fasting state, after intravenous arginine and after an oral glucose load in noninsulin-dependent diabetics (NIDDM) and in weight matched controls using specific radioimmunoassays and analytical gel filtration. In the fasting state the glucagonlike peptide-1 (GLP-1) immunoreactivity was significantly elevated in the NIDDM group compared with the control group. Both after intravenous arginine and after an oral glucose load a rise in the plasma concentrations of all immunoreactive moieties measured was seen. All integrated incremental responses after intravenous arginine were identical in the two groups. After oral glucose the insulin concentrations in plasma were lower and the concentrations of all proglucagon products were higher in the NIDDM group compared to the control group. The gel filtration analysis showed that arginine stimulated the secretion of pancreatic glucagon (PG 33-61), major proglucagon fragment (PG 72-158) and probably GLP-1 (PG 72-107 amide) in both groups, whereas oral glucose stimulated the secretion of glicentin (PG 1-69) and intestinal GLP-1 (PG 78-107 amide), an insulinotropic hormone. The elevated levels of immunoreactive GLP-1 in diabetics in the fasting state were mainly due to an increased concentration of major proglucagon fragment.
C Orskov, J Jeppesen, S Madsbad, J J Holst
It has been widely proposed that conversion of xanthine dehydrogenase (XDH) to its free radical-producing form, xanthine oxidase (XOD), underlies ischemic/reperfusion injury, although the relationship of this conversion to hypoxia and its physiologic control have not been defined. This study details the time course and control of this enzymatic interconversion. In a functionally intact, isolated perfused rat liver model, mean % XOD activity increased as a function of both the duration (25 to 45% in 3 h) and degree (r = 0.97) of hypoxia. This process was markedly accelerated in ischemic liver by an overnight fast (45 vs. 30% at 2 h), and by imposing a short period of in vivo ischemia (cardiopulmonary arrest 72%). Moreover, only under these conditions was there a significant rise in the XOD activity due to the conformationally altered XDH molecule (XODc, 18%), as well as concomitant morphologic injury. Neither circulating white blood cells nor thrombosis appeared to contribute to the effects of in vivo ischemia on enzyme conversion. Thus, it is apparent that conversion to the free radical-producing state, with high levels of XOD activity and concurrent cellular injury, can be achieved during a relatively short period of hypoxia under certain well-defined physiologic conditions, in a time course consistent with its purported role in modulating reperfusion injury. These data also suggest that the premorbid condition of organ donors (e.g., nutritional status and relative state of hypoxia) is important in achieving optimal organ preservation.
C A Brass, J Narciso, J L Gollan
Nitric oxide (an endothelium-derived relaxing factor) induces smooth muscle relaxation and is an important mediator in the regulation of vascular tone. Advanced glycosylation end products, the glucose-derived moieties that form nonenzymatically and accumulate on long-lived tissue proteins, have been implicated in many of the complications of diabetes and normal aging. We demonstrate that advanced glycosylation products quench nitric oxide activity in vitro and in vivo. Acceleration of the advanced glycosylation process in vivo results in a time-dependent impairment in endothelium-dependent relaxation. Inhibition of advanced glycosylation with aminoguanidine prevents nitric oxide quenching, and ameliorates the vasodilatory impairment. These results implicate advanced glycosylation products as important modulators of nitric oxide activity and endothelium-dependent relaxation.
R Bucala, K J Tracey, A Cerami
Ultradian "oscillations" or "pulses" of insulin secretion with periods around 120 min occur in man. It is not known whether glucose plays an active role in generating these oscillations, or if an intrapancreatic pacemaker generates oscillations in insulin secretion that entrain glucose passively. To determine if the frequency of pulses of insulin secretion could be modified by oscillatory glucose infusion, seven normal men were studied on three separate occasions. The first study involved a constant glucose infusion administered at a rate of 6 mg/kg per min for 28 h. During the two subsequent studies, the subjects received an oscillatory glucose infusion for 28 h with the same mean rate, an amplitude of 33% above and below the mean infusion rate, a sinusoidal waveshape and a period either 20% longer ("slow oscillatory infusion") or 20% shorter ("rapid oscillatory infusion") than the periodicity observed during constant glucose infusion. Samples for insulin, C-peptide, and glucose were drawn at 10-min intervals during the last 24 h of each study. Insulin secretion rates were calculated by deconvolution of C-peptide levels. During constant glucose infusion, the respective periods of oscillation of glucose and insulin secretion averaged 126 +/- 5 min and 118 +/- 3 min (mean +/- SEM). During the slow oscillatory infusion, the period of infusion was 155 +/- 7 min and the periods of insulin secretion and glucose were, respectively, 155 +/- 7 min and 150 +/- 5 min. During rapid oscillatory infusion, the period of infusion was 103 +/- 5 min and the period of both insulin secretion and glucose was 105 +/- 5 min. Thus the periodicity of both insulin secretion and plasma glucose changed in parallel with the exogenous periodicity, indicating complete entrainment of the secretory oscillations. These results suggest that the ultradian oscillations of insulin secretion are caused by the feedback loop linking glucose and insulin.
J Sturis, E Van Cauter, J D Blackman, K S Polonsky
Much of the clinically important pathology associated with IgE-dependent disorders is thought to reflect the actions of the blood-borne leukocytes recruited during these responses. To evaluate the extent to which mast cells are responsible for the leukocyte infiltration associated with IgE-dependent cutaneous reactions, we attempted to elicit these responses in normal mice, genetically mast cell-deficient W/Wv mice, and in W/Wv mice selectively repaired of their mast cell deficiency by the intradermal injection of cultured mast cells derived from the congenic normal (+/+) mice. We found that the tissue swelling associated with IgE-dependent passive cutaneous anaphylaxis reactions developed rapidly and diminished markedly from 2 to 4 h after antigen challenge, but remained detectable for at least 24 h after elicitation of the responses. Infiltration of leukocytes (predominantly neutrophils) also occurred at these sites, but reached maximal levels 6-12 h after antigen challenge, persisted at high levels for 24 h, and largely waned by 48 h. Virtually all of the tissue swelling and leukocyte infiltration associated with IgE-dependent cutaneous reactions was mast cell dependent. Intradermal injection of 40 U of recombinant murine TNF-alpha (rmTNF-alpha) elicited neutrophil infiltration similar in magnitude and kinetics to that observed after IgE-dependent mast cell degranulation. A rabbit anti-rmTNF-alpha (R anti-rmTNF-alpha) antiserum, which was able to inhibit 84% of the neutrophil infiltration observed after i.d. injection of rmTNF-alpha, inhibited IgE-, and mast cell-dependent leukocyte infiltration by 47 +/- 7% in three separate experiments. These findings indicate that TNF-alpha contributes to mast cell-dependent recruitment of leukocytes during IgE-dependent cutaneous late phase reactions, but suggest that other mast cell-associated mediators probably also contribute to this response.
B K Wershil, Z S Wang, J R Gordon, S J Galli
We have previously shown that Na(+)-coupled transport of glucose and amino acids across the apical membrane of intestinal absorptive cells is accompanied by alterations in cytoskeletal structure and altered sieving of small hydrophilic solutes by tight junctions. Here we report that in response to the essential amino acid L-tryptophan at lumenal concentrations likely to be supraphysiological (1 mM or greater), these responses are so exaggerated as to induce disruption of tight junctions and transepithelial macromolecular leaks. Since these effects of L-tryptophan are energy and Na+ dependent and occur with mucosal but not serosal exposure to L-tryptophan, it appears they are triggered by activation of a Na(+)-nutrient cotransporter in the apical membrane of absorptive cells rather than by the presence of an unidentified trace contaminant. Our findings suggest the possibility that dietary supplementation by L-tryptophan may result in loss of the intestinal epithelial barrier to dietary antigens. We speculate that such a response to supraphysiologic tryptophan may contribute, in part, to the recently recognized curious tryptophan-induced eosinophilia myalgia syndrome.
J L Madara, S Carlson
Production of the neutrophil-activating peptide (NAP)-1/IL-8 by mononuclear phagocytes from patients with RA and from control subjects was studied under various conditions. Mononuclear cells from bone marrow (BMMC), PBMC, and synovial fluid (SFMC) were cultured for up to 48 h in the absence or presence of Escherichia coli LPS, different interleukins, interferon-gamma, zymosan, or immune complexes, and the neutrophil-stimulating activity released into the culture medium was determined. As shown by neutralization with an antiserum raised against human recombinant NAP-1/IL-8, over 90% of this activity could be attributed to NAP-1/IL-8. In unstimulated mononuclear cells from control individuals and BMMC from RA patients, the production of NAP-1/IL-8 was very low and was enhanced moderately by stimulation with LPS. By contrast, the spontaneous production of NAP-1/IL-8 was 3- to 10-fold higher in PBMC and even much higher in SFMC from RA patients. In all instances, the yield of NAP-1/IL-8 could be enhanced by stimulation in culture. In addition to LPS, rheumatoid factor-containing immune complexes, zymosan, and IL-1 were highly effective in inducing NAP-1/IL-8 production, while IL-3, GM-CSF, tumor necrosis factor (TNF), and IL-2 were somewhat less potent. An inhibitory effect was obtained with IFN-gamma, which significantly decreased the spontaneous NAP-1/IL-8 release from SFMC and the IL-1- and LPS-induced NAP-1/IL-8 from RA and control PBMC. Inhibition was also observed with glucocorticoids. The production of NAP-1/IL-8 was markedly reduced by dexamethasone in phagocytosis-stimulated PBMC, and almost totally inhibited in SFMC obtained from joints after intraarticular administration of betamethasone. By contrast, the cyclooxygenase inhibitor, indomethacin, tended to increase the NAP-1/IL-8 yield from PBMC in culture.
M Seitz, B Dewald, N Gerber, M Baggiolini
The effects of the nephrotic syndrome in rats on the cholesterol content and the biosynthesis of apolipoprotein E (apoE) by resident peritoneal macrophages have been investigated. Since the nephrotic syndrome has been associated with an increased risk of coronary atherosclerosis, we hypothesized that macrophages from nephrotic rats would accumulate cholesterol and undergo transformation into foam cells, with a concomitant increase in apoE biosynthesis. The nephrotic syndrome was induced in rats with puromycin aminonucleoside. Peritoneal macrophages exposed in vivo for 7-21 d to ascites fluid derived from plasma containing sixfold elevations of lipoproteins did not accumulate unesterified or esterified cholesterol. Nevertheless, immunoprecipitation assays after incubation of the isolated cells with [35S]methionine, or immunoblot analysis of the incubation medium demonstrated a 2.6-fold increase in apoE secretion compared with normal macrophages. This increase was accompanied by 5- to 10-fold increases in cellular apoE messenger RNA as determined by quantitative solution hybridization assay. Peritoneal macrophages cultured from nephrotic rats during the period of hypercholesterolemia also showed distinct and highly reproducible morphologic changes. The dissociation between apoE biosynthesis and macrophage cholesterol content provides new insight into the response of peritoneal macrophages in vivo to endogenous hyperlipemia.
J Bass, E A Fisher, M M Prack, D L Williams, J B Marsh
The human hepatoma cell line, HepG2, secreted an activity that degrades platelet-activating factor (PAF) by the hydrolysis of the sn-2 acetyl group. This activity was Ca++ independent, inhibited by diisopropylfluorophosphate but not by p-bromophenacyl bromide, and resistant to treatment with trypsin or pronase. Separation of HepG2-conditioned medium by gel filtration disclosed that the activity was associated with lipoproteins. An antiserum against PAF acetylhydrolase immunoprecipitated this activity. It was not recognized by an antibody against lecithin:cholesterol acyltransferase (LCAT), which also is secreted by HepG2 cells. Therefore the phospholipase A2 activity of LCAT was excluded as a source of the observed activity. PAF added to the culture medium stimulated the secretion of the PAF-degrading activity by HepG2 cells, while lyso-PAF was inactive. Maximal stimulation was observed with 5 ng/ml PAF, which induced a fivefold increase. The presence of 5 ng/ml PAF, enhanced the secretion of [35S]methionine-labeled PAF acetylhydrolase and cycloheximide inhibited both the basal and PAF-stimulated secretion of the labeled enzyme. We conclude that HepG2 cells produce PAF acetylhydrolase. The liver may be a major source of plasma PAF acetylhydrolase, and PAF may induce the production of its inactivating enzyme by the liver.
K Satoh, T Imaizumi, Y Kawamura, H Yoshida, M Hiramoto, S Takamatsu, M Takamatsu
Secretory leukoprotease inhibitor (SLPI), a 12-kD nonglycosylated serine antiprotease with a high capacity for inhibiting neutrophil elastase (NE), is produced by cells of mucosal surfaces including the human lung. The molar concentrations of SLPI in total respiratory tract epithelial lining fluid (ELF) were 56 +/- 10% that of alpha 1-antitrypsin, suggesting SLPI may be more important for the anti-NE protection of the pulmonary epithelial surface than previously thought. However, evaluation demonstrated that SLPI in respiratory ELF was only one-third functional. Studies aerosolizing recombinant SLPI (rSLPI) to sheep demonstrated that in the short term, neither aerosolization and alveolar deposition nor the lavage procedure inactivated the SLPI molecule. In vitro studies with rSLPI demonstrated that exposure to oxidants did not modify the form of the molecule, while exposure to oxidants and NE caused the molecule to be cleaved from 12 to 8 kD. Consistent with this, evaluation of SLPI in lavage fluid of individuals with cystic fibrosis (a condition with oxidants and NE on the respiratory epithelium) showed that the SLPI was degraded. However, evaluation of SLPI in normal ELF by molecular sieve analysis and Western analysis demonstrated an intact 12-kD molecule, suggesting that the partial inactivation of SLPI in normals in vivo is not because it is complexed to NE or exposed to oxidants + NE. Together, these observations demonstrate that SLPI is present in large amounts in respiratory ELF, but since the majority of the SLPI is inactive, it likely does not play a significant role in protecting the normal respiratory epithelium, except perhaps in the upper airways where the levels of SLPI are the highest.
C Vogelmeier, R C Hubbard, G A Fells, H P Schnebli, R C Thompson, H Fritz, R G Crystal
To define the mechanisms of impaired muscle glycogen synthase and reduced glycogen formation in non-insulin dependent diabetes mellitus (NIDDM), glycogen synthase activity was kinetically analyzed during the basal state and three glucose clamp studies (insulin approximately equal to 300, 700, and 33,400 pmol/liter) in eight matched nonobese NIDDM and eight control subjects. Muscle glycogen content was measured in the basal state and following clamps at insulin levels of 33,400 pmol/liter. NIDDM subjects had glucose uptake matched to controls in each clamp by raising serum glucose to 15-20 mmol/liter. The insulin concentration required to half-maximally activate glycogen synthase (ED50) was approximately fourfold greater for NIDDM than control subjects (1,004 +/- 264 vs. 257 +/- 110 pmol/liter, P less than 0.02) but the maximal insulin effect was similar. Total glycogen synthase activity was reduced approximately 38% and glycogen content was approximately 30% lower in NIDDM. A positive correlation was present between glycogen content and glycogen synthase activity (r = 0.51, P less than 0.01). In summary, defects in muscle glycogen synthase activity and reduced glycogen content are present in NIDDM. NIDDM subjects also have less total glycogen synthase activity consistent with reduced functional mass of the enzyme. These findings and the correlation between glycogen synthase activity and glycogen content support the theory that multiple defects in glycogen synthase activity combine to cause reduced glycogen formation in NIDDM.
A W Thorburn, B Gumbiner, F Bulacan, G Brechtel, R R Henry
Generalized resistance to thyroid hormone (GRTH) is a syndrome characterized by impaired tissue responsiveness to thyroid hormone. Two distinct point mutations in the hormone binding domain of the thyroid hormone receptor (TR) beta have recently been identified in two unrelated families with GRTH. One, Mf, involves a replacement of the normal glycine-345 for arginine in exon 7 and another, Mh, replaces the normal proline-453 for histidine in exon 8. To probe for the presence of the Mf and Mh defect in 19 unrelated families with GRTH, we applied separate polymerase chain reactions using allele-specific oligonucleotide primers containing the normal and each of the two mutant nucleotides at the 3'-position. A total of 24 affected subjects and 13 normal family members were studied. The mode of inheritance was dominant in 13 families, was unknown in 5 families, and was clearly recessive in 1 family in which only the consanguineous subjects were affected. Primers containing the substitutions specific for Mf and Mh amplified exons 7 and 8, respectively, only in affected members of each of the two index families. Primers containing the normal sequences amplified exons 7 and 8 of the TR beta gene in all subjects except affected members of one family. In this family with recessively inherited GRTH, neither exon could be amplified using any combinations of primers and DNA blot revealed absence of all coding exons. These results indicate a major deletion of the TR beta gene, including both DNA and hormone binding domains. Since heterozygous members of this family are not affected, the presence of a single normal allele is sufficient for normal function of the TR beta. These data also support the hypothesis that in the dominant mode of GRTH inheritance the presence of an abnormal TR beta interferes with the function of the normal TR beta. Distinct mutations are probably responsible for GRTH in unrelated families.
K Takeda, S Balzano, A Sakurai, L J DeGroot, S Refetoff
We wanted to establish an in vitro human model for AIDS-associated dementia and pursue the hypothesis that this disease process may be a result of soluble factors produced by HIV-infected macrophages. Human brain aggregates were prepared from nine different brain specimens, and were treated with supernatants from in vitro HIV-infected macrophages (SI), uninfected macrophages (SU), infected T cells, or macrophage-conditioned media from four AIDS patients. Seven of nine treated brains exposed to SI showed peripheral rarefaction after 1 wk of incubation that by ultrastructural analysis showed cytoplasmic vacuolation. Aggregates from two of three brain cultures treated with SI for 3 wk became smaller, an approximately 50% decrease in size. The degree of apparent toxicity in brains exposed to patient-derived macrophage supernatants paralleled the proportion of macrophages found to be expressing HIV p24. Ultrastructural abnormalities were not observed in brains treated with supernatants from HIV-infected T cells, uninfected macrophages, or LPS-activated macrophages. Levels of five neurotransmitter amino acids were decreased in comparison to the structural amino acid leucine. These findings suggest that HIV-infected macrophages, infected both in vitro as well as derived from AIDS patients' peripheral blood, produce factors that cause reproducible histochemical, ultrastructural, and functional abnormalities in human brain aggregates.
L Pulliam, B G Herndier, N M Tang, M S McGrath
The lysosomal storage disorder glycogenosis type II is caused by acid alpha-glucosidase deficiency. In this study we have investigated the possible applicability of mannose 6-phosphate receptor-mediated enzyme replacement therapy to correct the enzyme deficiency in the most affected tissues. Bovine testes acid alpha-glucosidase containing phosphorylated mannose residues was intravenously administered to mice and found to be taken up by heart (70% increase of activity) and skeletal muscle (43% increase); the major target organs. The uptake of nonphosphorylated human placenta acid alpha-glucosidase by heart and skeletal muscle appeared to be significantly less efficient, whereas uptake of dephosphorylated bovine testes enzyme was not detectable. The phosphorylated bovine testes acid alpha-glucosidase remained present in mouse skeletal muscle up to 9-15 d after administration, with a half-life of 2-4 d. Besides being measured in skeletal muscle and heart, uptake of phosphorylated bovine testes and nonphosphorylated human placenta acid alpha-glucosidase was measured in several other organs, but not in brain. The increase of acid alpha-glucosidase activity was highest in liver and spleen. We concluded that application of mannose 6-phosphate receptor-mediated enzyme replacement therapy may offer new perspectives for treatment of glycogenesis type II.
A T Van der Ploeg, M A Kroos, R Willemsen, N H Brons, A J Reuser
Human cancers have an apparent low growth fraction, the bulk of cells presumed to being out of cycle in a G0 quiescent state due to the inability in the past to distinguish G0 from G1 cells. The allosteric M1 subunit of ribonucleotide reductase (M1-RR) is constitutively expressed by cycling cells (i.e., G1, S, G2-M). It is acquired during transition from G0 to G1, lost during exit to G0 and thus distinguishes G0 from G1 cells. To estimate the proportion of G0 and G1 cells in primary human breast (n = 5) and colorectal (n = 12) adenocarcinomas, we used both analytical DNA flow cytometry (ADFC) and immunoperoxidase staining of sections with the monoclonal antibody to M1-RR (MAb M1-RR). ADFC of fresh tumors revealed a low percentage of cells in the S phase (4.0 +/- 3.4%) but immunoperoxidase staining for M1-RR revealed an unexpectedly high proportion of positive cells (52.4 +/- 12.7%) in the G1, S, G2-M phases indicating a high G1 content of primary human tumors. Thus, human cancers are blocked in transition in G1 and are not predominantly in a G0 or quiescent differentiated state. This block was interpreted to mean that human cancers are responding to putative regulatory events at a restriction point in the G1 phase, such as relative growth factor deficiency, density inhibition, antiproliferative cytokines, or gene products. Using flow cytometry for both DNA and M1-RR content we found that human colon cancer cell lines arrest in the G1 but not G0 phase upon serum deprivation or density inhibition. Similarly, human breast cancer cell lines are arrested in G1 but not G0 phase by medroxyprogesterone acetate (MPA) or tamoxifen exposure. These findings match our in situ observations, and support the concept of a restriction point block in primary human tumors.
D L Tay, P S Bhathal, R M Fox
We studied the receptors on human cultured macrophages (MO-M phi) responsible for binding encapsulated and isogenic mutant acapsular strains of Cryptococcus neoformans, and whether such binding leads to a phagocytic event. Both strains required opsonization with complement components in normal human serum in order for binding to occur. Binding of the acapsular, but not the encapsulated, strain led to phagocytosis. MAb directed against any of the three defined complement receptors (CR) on MO-M phi (CR1, CR3, and CR4) profoundly inhibited binding of serum-opsonized encapsulated (and to a lesser extent acapsular) organisms to MO-M phi. Immunofluorescence studies demonstrated migration of CR to the area of the cryptococcal binding site. Trypsin and elastase inhibited binding of encapsulated and, to a lesser extent, acapsular yeasts to MO-M phi. Binding of encapsulated C. neoformans was profoundly inhibited by incubation in the cold or by inhibitors of receptor capping and actin microfilaments. Thus, multiple CR appear to contribute to binding of serum-opsonized encapsulated C. neoformans by MO-M phi. Binding is an energy-dependent process that requires conformational changes in actin yet does not lead to phagocytosis of the organism. In contrast, energy is not required for binding of acapsular yeasts by MO-M phi and binding triggers phagocytosis.
S M Levitz, A Tabuni
Low HDL-cholesterol (HDL-C) levels may elevate atherosclerosis risk, and often associate with hypertriglyceridemia (HTG); however, the metabolic causes of low HDL-C levels with or without HTG are poorly understood. We studied the turnover of radioiodinated HDL apolipoproteins, apo A-I and apo A-II, in 15 human subjects with low HDL-C, six with normal plasma TG levels (group 1) and nine with high TG (group 2), and compared them to 13 control subjects with normal HDL-C and TG levels (group 3). The fractional catabolic rate (FCR) was equally elevated in groups 1 and 2 vs. group 3 for both apo A-I (0.313 +/- 0.052 and 0.323 +/- 0.063 vs. 0.245 +/- 0.043 pools/d, P = 0.003) and apo A-II (0.213 +/- 0.036 and 0.239 +/- 0.037 vs. 0.185 +/- 0.031 pools/d, P = 0.006). Thus, high FCR characterized low HDL-C regardless of the presence or absence of HTG. In contrast, transport rate (TR) of apo A-I did not differ significantly among the groups and the apo A-II TR differed only between groups 2 and 3 (2.15 +/- 0.57, 2.50 +/- 0.39, and 1.83 +/- 0.48 mg/kg per d for groups 1 to 3, respectively, P = 0.016). Several HDL-related factors were similar in groups 1 and 2 but differed in group 3, as with FCR, including the ratio of lipoprotein lipase to hepatic lipase activity (LPL/HL) in post-heparin plasma, the ratio of the HDL-C to apo A-I plus apo A-II levels, and the percent of tracer in the d greater than 1.21 fraction. In linear regression analysis HDL-C levels correlated inversely with the FCR of apo A-I and apo A-II (r = -0.74, P less than 0.0001 for both). Major correlates of FCR were HDL-C/apo A-I + apo A-II, LPL/HL, and plasma TG levels. We hypothesize that lipase activity and plasma TG affect HDL composition which modulates FCR, which in turn regulates HDL-C. Thus, HTG is only one of several factors which may contribute to elevated FCR and low HDL-C. Given the relationship of altered HDL composition with high FCR and low HDL-C levels, factors affecting HDL composition may increase atherosclerosis susceptibility.
E A Brinton, S Eisenberg, J L Breslow
Neonatal T cell-B cell collaboration was investigated utilizing a system of T cell-dependent polyclonal B cell activation and Ig secretion. In this system, T cells activated by immobilized anti-CD3 provide a potent stimulus for Ig production by adult lymphocytes. By contrast, anti-CD3 stimulation of cord blood lymphocytes generated minimal numbers of Ig-secreting cells. Ig production by neonatal lymphocytes was enhanced by the addition of Staphylococcus aureus or secreted factors from mitogen-stimulated adult T cells. Supplementation with IL-2 resulted in the production of large amounts of IgM and small amounts of IgG and IgA, with less Ig produced than by comparable cultures of adult lymphocytes. Neonatal T cells proliferated and produced IL-2 in response to immobilized anti-CD3, and supported B cell proliferation and Ig secretion by adult B cells, although not as effectively as adult T cells. Supernatants from activated neonatal T cells were markedly limited in their capacity to support Ig production by adult B cells. Neonatal B cells could be induced to differentiate in response to anti-CD3-stimulated adult T cells. However, the amounts of IgG and IgA secreted were small compared to adult levels. These studies indicate a relative, but not absolute, functional deficiency of both neonatal B and T cells.
J B Splawski, D F Jelinek, P E Lipsky
The regulation of protein metabolism in the human heart has not previously been studied. In 10 postabsorptive patients with coronary artery disease, heart protein synthesis and degradation were estimated simultaneously from the extraction of intravenously infused L-[ring-2,6-3H]phenylalanine (PHE) and the dilution of its specific activity across the heart at isotopic steady state. We subsequently examined the effect of branched chain amino acid (BCAA) infusion on heart protein turnover and on the myocardial balance of amino acids and branched chain ketoacids (BCKA) in these patients. In the postabsorptive state, there was a net release of phenylalanine (arterial-cardiac venous [PHE] = -1.71 +/- 0.32 nmol/ml, P less than 0.001; balance = -116 +/- 21 nmol PHE/min, P less than 0.001), reflecting protein degradation (142 +/- 40 nmol PHE/min) in excess of synthesis (24 +/- 42 nmol PHE/min) and net myocardial protein catabolism. During BCAA infusion, protein synthesis increased to equal the degradation rate (106 +/- 24 and 106 +/- 28 nmol PHE/min, respectively) and the phenylalanine balance shifted (P = 0.01) from negative to neutral (arterial-cardiac venous [PHE] = 0.07 +/- 0.36 nmol/ml; balance = 2 +/- 25 nmol PHE/min). BCAA infusion stimulated the myocardial uptake of both BCAA (P less than 0.005) and their ketoacid conjugates (P less than 0.001) in proportion to their circulating concentrations. Net uptake of the BCAA greatly exceeded that of other essential amino acids suggesting a role for BCAA and BCKA as metabolic fuels. Plasma insulin levels, cardiac double product, coronary blood flow, and myocardial oxygen consumption were unchanged. These results demonstrate that the myocardium of postabsorptive humans is in negative protein balance and indicate a primary anabolic effect of BCAA on the human heart.
L H Young, P H McNulty, C Morgan, L I Deckelbaum, B L Zaret, E J Barrett
Evidence is emerging for a direct role of glucose, independent of changes in insulin, in the regulation of cellular glucose transport and glucose utilization in vivo. In this study we investigate potential cellular and molecular mechanisms for this regulatory effect of glucose by determining how normalization of glycemia without insulin therapy in diabetic rats influences 3-O-methylglucose transport and the expression and translocation of two genetically distinct species of glucose transporters (GTs) in adipose cells. These results are compared with alterations in glucose disposal in vivo measured by euglycemic clamp. In rats rendered diabetic by 90% pancreatectomy, insulin-stimulated glucose transport in adipose cells is decreased 50% in parallel with reduced insulin-mediated glucose disposal in vivo. Levels of adipose/muscle GTs measured by immunoblotting are decreased in adipose cell subcellular membrane fractions, as are the corresponding mRNA levels assessed by Northern blotting of total adipose cell RNA. Normalization of blood glucose in diabetic rats with phlorizin, which impairs renal tubular glucose reabsorption and thus enhances glucose excretion, restores insulin-stimulated glucose transport in adipose cells and insulin-mediated glucose disposal in vivo. Importantly, levels of the adipose/muscle GT protein remain 43% reduced in the low-density microsomes in the basal state and 46% reduced in the plasma membranes in the insulin-stimulated state. Adipose/muscle GT mRNA levels remain approximately 50% depressed. Levels of the HepG2/brain GT protein and mRNA are unaltered by diabetes or phlorizin treatment. Thus, changes in ambient glucose independent of changes in ambient insulin can regulate the glucose transport response to insulin in isolated adipose cells and changes in responsiveness parallel alterations in glucose uptake in vivo. Since this effect can occur without alteration in the expression of the two species of glucose transporters present in adipose cells or in their translocation to the plasma membrane in response to insulin, it may result from changes in GT functional activity.
B B Kahn, G I Shulman, R A DeFronzo, S W Cushman, L Rossetti
Erythrocytes are known to influence hemostasis. Bleeding times are prolonged in anemia and corrected by normalizing the hematocrit. We now demonstrate that intact erythrocytes modulate biochemical and functional responsiveness of activated platelets. A two-stage procedure, permitting studies of cell-cell interactions and independently evaluating platelet activation and recruitment within 1 min of stimulation, was developed. Erythrocytes increased platelet serotonin release despite aspirin treatment, enzymatic adenosine diphosphate removal, protease inhibition, or combinations thereof. The data suggested that erythrocyte enhancement of platelet reactivity can reduce the therapeutic effectiveness of aspirin. Erythrocytes metabolically modified platelet arachidonate or eicosapentaenoate release and eicosanoid formation. They promoted significant increases in cyclooxygenase and lipoxygenase metabolites upon platelet stimulation with collagen or thrombin. However, with ionophore, erythrocytes strongly reduced platelet lipoxygenation. These erythrocyte modulatory effects were stimulus-specific. Activated platelet-erythrocyte mixtures, with or without aspirin, promoted 3-10-fold increases in extracellular free fatty acid, which would be available for transcellular metabolism. Erythrocyte-induced increases in free eicosapentaenoate may contribute to antithrombotic and anti-inflammatory effects of this fish oil derivative. These results provide biochemical insight into erythrocyte contributions to thrombosis and hemostasis, and support the concept of thrombus formation as a multicellular event.
M T Santos, J Valles, A J Marcus, L B Safier, M J Broekman, N Islam, H L Ullman, A M Eiroa, J Aznar
To assess the role of increased cytosolic free calcium (Caf) in the pathogenesis of acute proximal tubule cell injury and the protection afforded by exposure to reduced medium pH or treatment with glycine, fura-2-loaded tubules were studied in suspension and singly in a superfusion system. The Ca2+ ionophore, ionomycin, increased Caf to micromolar levels and rapidly produced lethal cell injury as indicated by loss of lactate dehydrogenase to the medium by suspended tubules and accelerated leak of fura and failure to exclude Trypan blue by superfused tubules. Decreasing medium Ca2+ to 100 nM prevented the ionomycin-induced increases of Caf and the injury. Reducing medium pH from 7.4 to 6.9 or adding 2 mM glycine to the medium also prevented the cell death, but did not prevent the increase of Caf to micromolar levels. Cells treated with 1799, an uncoupler of oxidative phosphorylation which produced severe adenosine triphosphate (ATP) depletion, did not develop increases of Caf until just before loss of viability. Preventing these increases of Caf with 100 nM Ca2+ medium did not protect 1799-treated cells. Reduced pH and glycine protected 1799-treated cells without ameliorating the increases of Caf. These data demonstrate the toxic potential of increased Caf in the proximal tubule and show that Caf does sharply increase prior to loss of viability in an ATP depletion model of injury, but this increase does not necessarily contribute to the outcome. The potent protective actions of decreased pH and glycine allow the cells to sustain increases of Caf to micromolar levels in spite of severe, accompanying cellular ATP depletion without developing lethal cell injury.
J M Weinberg, J A Davis, N F Roeser, M A Venkatachalam
The plasma concentration of the atherogenic low density lipoproteins (LDL) increases with age. To clarify the mechanism of this change, we studied the kinetics of autologous 125I-LDL apolipoprotein B (apo B) in 41 normolipidemic, nonobese healthy males. For comparison, they were divided into three age groups: young, 21-39 yr (n = 18), middle-aged, 40-59 yr (n = 11), and old, 60-80 yr (n = 12). The levels of plasma LDL cholesterol and LDL apo B increased from respectively 3.4 +/- 0.1 (SEM) mmol/liter and 86 +/- 2 mg/dl in the young to 4.1 +/- 0.1 mmol/liter and 95 +/- 3 mg/dl in the old (P less than 0.01), and this increase was linked to a progressively decreased (r = -0.38, P less than 0.02) fractional catabolic rate of LDL apo B (0.348 +/- 0.010 pools per day in the young vs. 0.296 +/- 0.009 pools per day in the old, P less than 0.01). The production rate of LDL apo B did not differ significantly between the groups. The reduced fractional catabolic rate of LDL apo B in the old was not associated with a decrease in binding affinity of the LDL particle to its receptor, as judged from its ability to compete for 125I-LDL fibroblast binding. When hepatic LDL receptor expression was stimulated by cholestyramine treatment in six old males, their LDL apo B fractional catabolic rate increased to the levels observed in the young subjects. We conclude that the increase in LDL which normally occurs with age is explained by a reduced capacity for its removal, and hypothesize that this is mediated via a reduced hepatic LDL receptor expression.
S Ericsson, M Eriksson, S Vitols, K Einarsson, L Berglund, B Angelin
Several lines of evidence indicate that the oxidative modification of low density lipoproteins (LDL) may provide an important link between plasma LDL and the genesis of the atherosclerotic lesion. Ascorbate is an important water-soluble, chain-breaking antioxidant in humans. Probucol, a lipid-soluble antioxidant drug has been shown to retard the progression of atherosclerosis. The aim of the present study was to compare the effects of probucol and physiologic levels of ascorbate on the oxidative modification of LDL in both a cell-free (2.5 microM Cu++ in phosphate-buffered saline) and cellular system (human monocyte macrophages in Ham's F-10 medium). Both ascorbate and probucol inhibited the oxidative modification of LDL in both systems to a similar degree as evidenced by the thiobarbituric acid-reacting substance activity, electrophoretic mobility, and degradation by macrophages. However, whereas co-incubation with physiologic levels of ascorbate resulted in a substantial preservation of the alpha-tocopherol, gamma-tocopherol, and beta-carotene of the LDL, probucol in concentrations ranging from 10 to 80 microM failed to protect these antioxidants. Thus, in addition to being as potent as probucol in inhibiting the oxidation of LDL, ascorbate in contrast preserves the endogenous antioxidants in the LDL.
I Jialal, S M Grundy
The cytokine interleukin 1 (IL-1) inhibits contractile responses in rat aorta by causing endothelium-independent and prolonged activation of soluble guanylate cyclase. The present study tested whether IL-1 activates guanylate cyclase by inducing prolonged production of nitric oxide in cultured rat aortic vascular smooth muscle cells (VSMC). IL-1 induced a marked time-dependent increase in cyclic guanosine monophosphate (cGMP) in VSMC which was significant at 6 h, and increased progressively for up to 36 h. This effect of IL-1 was abolished when protein synthesis was inhibited with cycloheximide or actinomycin D, suggesting that the effect of IL-1 involves new protein synthesis. IL-1-induced cGMP accumulation was inhibited by the soluble guanylate cyclase inhibitors, methylene blue, LY83583, and hemoglobin and by the L-arginine analogue NGmonomethyl-L-arginine (L-NMMA). The inhibitory effect of L-NMMA was reversed by a 10-fold excess of L-arginine, but not by D-arginine. Nitrite, an oxidation product of nitric oxide, accumulated in the media of VSMC incubated with IL-1 for 24 h in the presence of L-arginine, whereas both IL-1-induced cGMP accumulation and nitrite production were attenuated in VSMC incubated in L-arginine-deficient medium. In L-arginine-depleted VSMC, IL-1-induced cGMP accumulation was restored to control levels by a 15-min incubation with L-arginine. These results demonstrate that IL-1 activates guanylate cyclase in rat VSMC by inducing production of nitric oxide via a pathway dependent on extracellular L-arginine.
D Beasley, J H Schwartz, B M Brenner
Monoclonal antibodies recognizing CD18, CD11a, CD11b, and neutrophil lectin adhesion molecule 1 (LECAM-1), i.e., the human homologue of the murine MEL-14 antigen, were used to assess the relative contribution of these glycoproteins to neutrophil-endothelial adhesion. Under static conditions, the adhesion of neutrophils to IL-1-stimulated human umbilical vein endothelial cell (HUVEC) monolayers was inhibited by antibodies to CD18, CD11a, and the neutrophil LECAM-1, and the effect of combining anti-LECAM-1 and anti-CD11a was almost additive. Under flow at a wall shear stress 1.85 dyn/cm2, a condition where CD18-dependent adhesion is minimal, anti-LECAM-1 inhibited adhesion by greater than 50%. Chemotactic stimulation of neutrophils induced a rapid loss of LECAM-1 from the neutrophil surface, and the level of neutrophil surface LECAM-1 was closely correlated with adhesion under flow. Neutrophils contacting the activated endothelial cells for 30 min lost much of their surface LECAM-1, a phenomenon induced by a soluble factor or factors released into the medium by the stimulated monolayers, and a high percentage migrated through the HUVEC monolayer. This migration was almost completely inhibited by anti-CD18, but was unaffected by antibodies to neutrophil LECAM-1. These results support the concept that LECAM-1 is a neutrophil adhesion molecule that participates in the adherence of unstimulated neutrophils to cytokine-stimulated endothelial cells under conditions of flow, and is then lost from the neutrophil surface coincident with the engagement of CD18-dependent mechanisms leading to transendothelial migration.
C W Smith, T K Kishimoto, O Abbassi, B Hughes, R Rothlein, L V McIntire, E Butcher, D C Anderson, O Abbass
Mast cells are resident in tissues, particularly in association with endothelial and epithelial cell basement membranes, and increase at sites of inflammation, injury, and fibrosis. Although mast cells are known to both release and generate proinflammatory molecules in response to inflammatory stimuli, little is known about their normal biologic function. Here we demonstrate that IL-3-dependent mouse PT18 mast cells, mouse bone marrow-derived mast cells, and rat basophilic leukemia cells express large amounts of mRNA for collagen IV, laminin, and heparan sulfate proteoglycan. Western blot analysis confirmed that mast cells synthesize and secrete significant amounts collagen IV and laminin B1 and B2 chains. These data suggest that mast cells may contribute to normal tissue repair and/or the early overproduction of basement membrane components seen in a variety of fibrotic conditions.
H L Thompson, P D Burbelo, G Gabriel, Y Yamada, D D Metcalfe
These experiments were conducted to determine whether point mutations activating K-ras or H-ras oncogenes, induced by the procarcinogen 1,2-dimethylhydrazine (DMH), were detectable in preneoplastic or neoplastic rat colonic mucosa. Rats were injected weekly with diluent or DMH at 20 mg/kg body wt for 5, 10, 15, or 25 wk, killed, and their colons dissected. DNA was extracted from diluent-injected control animals, histologically normal colonic mucosa from carcinogen-treated animals, and from carcinomas. Ras mutations were characterized by differential hybridization using allele-specific oligonucleotide probes to polymerase chain reaction--amplified DNA, and confirmed by DNA sequencing. While no H-ras mutations were detectable in any group, K-ras (G to A) mutations were found in 66% of DMH-induced colon carcinomas. These mutations were at the second nucleotide of codons 12 or 13 or the first nucleotide of codon 59 of the K-ras gene. The same type of K-ras mutations were observed in premalignant colonic mucosa from 2 out of 11 rats as early as 15 wk after beginning carcinogen injections when no dysplasia, adenomas, or carcinomas were histologically evident, suggesting that ras mutation may be an early event in colon carcinogenesis.
R F Jacoby, X Llor, B B Teng, N O Davidson, T A Brasitus
In the intact rat kidney, bicarbonate reabsorption in the early proximal tubule (EP) is strongly dependent on delivery. Independent of delivery, metabolic acidosis stimulates EP bicarbonate reabsorption. In this study, we investigated whether systemic pH changes induced by acute or chronic respiratory acid-base disorders also affect EP HCO3- reabsorption, independent of delivery (FLHCO3, filtered load of bicarbonate). Hypercapnia was induced in rats acutely (1-3 h) and chronically (4-5 d) by increasing inspired PCO2. Hypocapnia was induced acutely (1-3 h) by mechanical hyperventilation, and chronically (4-5 d) using hypoxemia to stimulate ventilation. When compared with normocapneic rats with similar FLHCO3, no stimulation of EP or overall proximal HCO3 reabsorption was found with either acute hypercapnia (PaCO2 = 74 mmHg, pH = 7.23) or chronic hypercapnia (PaCO2 = 84 mmHg, pH = 7.31). Acute hypocapnia (PaCO2 = 29 mmHg, pH = 7.56) did not suppress EP or overall HCO3 reabsorption. Chronic hypocapnia (PaCO2 = 26 mmHg, pH = 7.54) reduced proximal HCO3 reabsorption, but this effect was reversed when FLHCO3 was increased to levels comparable to euvolemic normocapneic rats. Thus, when delivery is accounted for, we could find no additional stimulation of proximal bicarbonate reabsorption in respiratory acidosis and, except at low delivery rates, no reduction in bicarbonate reabsorption in respiratory alkalosis.
R N Santella, D A Maddox, F J Gennari
To develop an animal model for sickle cell anemia, we have created transgenic mice that express a severe naturally occurring human sickling hemoglobin, Hb S Antilles. Due to its low solubility and oxygen affinity, Hb S Antilles has a greater propensity to cause red cell sickling than Hb S. To make transgenic animals that express a high level of Hb S Antilles, the erythroid-specific DNAse I hypersensitive site II from the human beta-globin cluster was linked independently to the human alpha 2-globin gene and to the beta S Antilles gene. Embryos were injected with both constructs simultaneously and seven transgenic mice were obtained, three of which contained both the human alpha and the human beta S Antilles transgene. After crossing the human transgenes into the mouse beta-thalassemic background a transgenic mouse line was derived in which approximately half the beta-globin chains in the murine red cells were human beta S Antilles. Deoxygenation of the transgenic red cells in vitro resulted in extensive sickling. An increase of in vivo sickling was achieved by placing these transgenic mice in a low oxygen environment. This murine model for red cell sickling should help to advance our understanding of sickle cell disease and may provide a model to test therapeutic interventions.
E M Rubin, H E Witkowska, E Spangler, P Curtin, B H Lubin, N Mohandas, S M Clift
Neuroblastoma is an embryonal tumor that typically arises in cells of the developing adrenal medulla. IGF-II mRNA is expressed at high levels in the adrenal cortex before birth but it is not detectable until after birth in the adrenal medulla. Neuroblastoma cell lines corresponding to early adrenal medullary precursors did not express IGF-II, although all three cell lines we tested were growth stimulated by IGF-II. Cell lines corresponding to more mature adrenal medullary cells expressed IGF-II, and one, SK-N-AS, grows by an IGF-II autocrine mechanism (J. Clin. Invest. 84:829-839) El-Badry, Romanus, Helman, Cooper, Rechler, and Israel. 1989. An examination of human neuroblastoma tumor tissues for IGF-II gene expression using in situ hybridization histochemistry revealed that IGF-II is expressed by tumor cells in only 5 of 21 neuroblastomas, but is detectable in cells of nonmalignant tissues including adrenal cortical cells, stromal fibroblasts, and eosinophils in all 21 tumors. These findings indicate that IGF-II may function as an autocrine growth factor for some neuroblastomas and as a paracrine growth factor for others. They suggest that the growth regulatory pathways utilized by neuroblastoma mimic those used in the precursor cell type from which individual tumors arise.
O M El-Badry, L J Helman, J Chatten, S M Steinberg, A E Evans, M A Israel
The ability to engraft human PBMC or fetal tissue immune cells in the severe combined immunodeficient (SCID) mouse has created a need for characterization of these systems and their application to disease models. We demonstrate that SCID mice reconstituted with PBMC support the growth and differentiation of a restricted set of B cells. Human IgG levels of 1-2 mg/ml (10-20% of normal human serum levels) were routinely achieved in spite of a serum half life of only 12 d. Ig levels peaked around 50 d and Ig production was maintained for greater than 100 d. The Ig was greater than 85% IgG though some IgM, IgA, IgD, and even IgE could be detected. However, the human IgG produced in hu-PBL-SCID mice was pauci-clonal when analyzed by isoelectric focusing and by kappa/lambda light chain usage. Using a new polymerase chain reaction based analysis capable of monitoring individual VH family utilization, we found that the engrafted B cells showed skewed and restricted human VH subfamily utilization. These parameters were markedly variable among hu-PBL-SCID mice reconstituted from the same donor cell population at both early (21-50 d) and late stages (greater than 100 d). Hu-PBL/CVI-SCID mice constructed with cells from patients with common variable immunodeficiency with an in vitro block in terminal B cell differentiation produced human Ig responses that were quantitatively the same as those produced by hu-PBL-SCID mice from normal donors. The hu-PBL-SCID system using PBMC appears to lead to growth and Ig production by a small number of B cells and results in a restricted B cell repertoire.
A Saxon, E Macy, K Denis, M Tary-Lehmann, O Witte, J Braun
Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation.
P Kanavaros, J P Farcet, P Gaulard, C Haioun, M Divine, J P Le Couedic, M P Lefranc, F Reyes
Insulin-stimulated glycogen synthase activity in human muscle correlates with insulin-mediated glucose disposal and is reduced in insulin-resistant subjects. Inhibition of the cyclic AMP-dependent protein kinase (A-kinase) is considered as a possible mechanism of insulin action for glycogen synthase activation. In this study, we investigated the time course of insulin action on human muscle A-kinase activity during a 2-h insulin infusion in 13 insulin-sensitive (group S) and 7 insulin-resistant subjects (group R). Muscle biopsies were obtained from quadriceps femoris muscle at times 0, 10, 20, 40, and 120 min. Insulin infusion resulted in significant inhibition of A-kinase activity at 20 and/or 40 min using 0.2, 0.6, and 1.0 microM cyclic AMP in group S. A-kinase activities both before and after insulin administration were lower in group S than in group R using 0.6 microM cyclic AMP. The decrease in apparent affinity for cyclic AMP during insulin infusion was larger for group S compared with group R. Glycogen synthase activity increased significantly after insulin infusion in both groups and was higher in group S compared with group R. The data suggest that a defective response of A-kinase to insulin in insulin-resistant subjects could contribute to their reduced insulin stimulation of skeletal muscle glycogen synthase.
Y Kida, B L Nyomba, C Bogardus, D M Mott
Familial glucocorticoid resistance is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations in the absence of stigmata of Cushing's syndrome. Our previous studies of the first reported kindred showed a two- to threefold reduction in glucocorticoid receptor-ligand binding affinity in the propositus, and a lesser reduction in affinity in his mildly affected son and nephew. Glucocorticoid receptor cDNA from these three patients was amplified by polymerase chain reaction and sequenced. The cDNA nucleotide sequence was normal, except for nucleotide 2054, which substituted valine for aspartic acid at amino acid residue 641. The propositus was homozygous while the other relatives were heterozygous for the mutation. COS-7 monkey kidney cells were cotransfected with expression vectors for either wild type or Val 641-mutant receptors, together with the reporter plasmid pMMTV-CAT. Dexamethasone increased chloramphenicol acetyltransferase activity in cells expressing wild type receptor, but had no effect in cells expressing Val 641-mutant receptors, despite similar receptor concentrations, as indicated by Western blotting. The binding affinity for dexamethasone of the Val 641-mutant receptor was threefold lower than that of the wild type receptor. These results suggest that glucocorticoid resistance in this family is due to a point mutation in the steroid-binding domain of the glucocorticoid receptor.
D M Hurley, D Accili, C A Stratakis, M Karl, N Vamvakopoulos, E Rorer, K Constantine, S I Taylor, G P Chrousos
To investigate the role of mast cells in transport abnormalities during intestinal anaphylaxis, we examined responses to antigen in isolated intestinal preparations from ovalbumin-sensitized genetically mast cell-deficient WBB6F1-W/Wv (W/Wv) mice and congenic normal WBBGF1(-)+/+ (+/+) mice. Changes in ion transport (primarily secretion of chloride ions) were indicated by increases in short-circuit current (Isc). In tissues from +/+ mice, antigen caused increases in Isc which were significantly inhibited by antagonists to histamine (diphenhydramine) and serotonin (ketanserin), by a cyclooxygenase inhibitor (piroxicam) and by a neurotoxin (tetrodotoxin). In preparations from W/Wv mice, antigen-stimulated responses were approximately 30% of that in +/+ mice and were inhibited only by piroxicam. Responses to electrical transmural stimulation of nerves were approximately 50% in W/Wv versus +/+ mice, and were inhibited by antagonists of mast cell mediators in +/+ but not W/Wv mice. Reconstitution of mast cells in W/Wv mice by intravenous injection of +/+ bone marrow cells restored the normal responses to both antigen and nerve stimulation. Our results indicate that mast cell-dependent mechanisms are primarily responsible for the ion secretion associated with intestinal anaphylaxis, but that other cells are also involved. In addition, our data provide evidence for the functional importance of bidirectional communication between nerves and mast cells in the regulation of ion transport in the gastrointestinal tract.
M H Perdue, S Masson, B K Wershil, S J Galli
The roles of polypeptide growth factors in promoting wound healing and in directing the specificity and sequence of responses of different tissues in wounds are little understood. We investigated the influence of four growth factors on the rates of healing of a novel full thickness dermal ulcer placed on an avascular base in the rabbit ear. The wound model precludes significant wound contraction and requires new granulation tissue and epithelial cells for healing to originate centripetally. 5 micrograms (7-31 pmol/mm2) of platelet-derived growth factor-B chain (PDGF-BB), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) applied locally at the time of wounding resulted in a twofold increase in complete reepithelialization of treated wounds (PDGF-BB, P = 0.02 chi square analysis; bFGF, P = 0.04; EGF, P = 0.05); transforming growth factor (TGF)-beta 1 significantly inhibited reepithelialization (P = 0.05). Both PDGF-BB and TGF-beta 1 uniquely increased the depth and area of new granulation tissue (P less than 0.005), the influx of fibroblasts, and the deposition of new matrix into wounds. Explants from 7-d old PDGF-BB-treated wounds remained metabolically far more active than controls, incorporating 473% more [3H]thymidine into DNA (P = 0.05) and significantly more [3H]leucine and [3H]proline into collagenase-sensitive protein (P = 0.04). The results establish that polypeptide growth factors have significant and selective positive influences on healing of full thickness ulcers in the rabbit.
T A Mustoe, G F Pierce, C Morishima, T F Deuel
Normal dogs were treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) at 10 micrograms/kg/day for 30 d, which caused an initial neutrophilia, followed by a prolonged period of chronic neutropenia. A control dog treated with recombinant canine G-CSF (rcG-CSF) showed persistent neutrophilia over 3 mo. Serum from dogs during neutropenia contained an antibody to rhG-CSF, which neutralized the stimulatory effects of both rhG-CSF and rcG-CSF on dog marrow neutrophilic progenitor cell growth and on NFS-60 cell proliferation. 4 mo after discontinuation of rhG-CSF, the dogs' neutrophil counts returned to the normal range. Rechallenge with the rhG-CSF re-induced severe neutropenia in 1 wk. Neutropenia was transferred by plasma infusion from a neutropenic dog to a previously normal dog. These data suggest that human rhG-CSF immunizes normal dogs and thereby induces neutralization of endogenous canine G-CSF and neutropenia. This model system should allow more precise definition of the in vivo role of G-CSF.
W P Hammond, E Csiba, A Canin, H Hockman, L M Souza, J E Layton, D C Dale
We examined effects of priming with recombinant human interferon-gamma (IFN) or tumor necrosis factor-alpha (TNF) on neutrophil responses to Candida albicans hyphae. Both cytokines increased early superoxide generation after hyphal stimulation. The more pronounced effects of TNF were accompanied by an augmented surface membrane depolarization rate and were insensitive to both pertussis toxin and calcium ion chelation, but were negated by concomitant incubation with puromycin or cycloheximide during priming. IFN augmented hyphal killing despite its only minor enhancement of early respiratory burst responses, but TNF reduced neutrophil fungicidal activity to nearly 40% below those by unprimed control cells even though it enhanced early superoxide responses more dramatically. Though TNF-primed neutrophils killed hyphae at normal initial rates, IFN-primed or even unprimed cells manifested more fungicidal sustained activity. These disparate consequences of cytokine priming on hyphal destruction were paralleled by differences in late generation of potentially candidacidal oxidants, hydrogen peroxide, and hypochlorous acid. IFN added during priming failed to correct TNF-associated functional defects in neutrophil anti-Candida responses. Thus, augmentation of early respiratory burst responses to oxidant-sensitive organisms need not necessarily reflect concomitant salutary effects on microbicidal activity.
R D Diamond, C A Lyman, D R Wysong
Recent studies in nonobese diabetic mice have implicated the autoimmune destruction of pancreatic islet cells with immunity to a beta cell protein cross-reactive to Mycobacterium tuberculosis heat shock protein 65 (hsp 65). Therefore, our studies examined serological immunity to islet cell hsp in humans with insulin-dependent diabetes (IDD). Heat shock of human islet cells in vitro markedly increased the synthesis of proteins of 72,000, 75,000, and 90,000 Mr. No autoantibodies reactive to these hsp, nor to the constituently expressed islet cell hsp 65 protein (identified as 60,000 Mr) were observed in IDD patients. The islet cell 64,000-Mr autoantigen and hsp 65 proteins were physiologically and immunocompetitively distinct. These experiments do not support the hypothesis that IDD in humans is associated with autoimmunity to islet cell heat shock proteins.
M A Atkinson, L A Holmes, D W Scharp, P E Lacy, N K Maclaren
To elucidate the molecular mechanism of familial central diabetes insipidus (FDI), we sequenced the arginine vasopressin-neurophysin II (AVP-NPII) gene in 2 patients belonging to a pedigree that is consistent with an autosomal dominant mode of inheritance. 10 patients with idiopathic central diabetes insipidus (IDI) and 5 normals were also studied. The AVP-NPII gene, locating on chromosome 20, consists of three exons that encode putative signal peptide, AVP, NPII, and glycoprotein. Using polymerase chain reaction, fragments including the promoter region and all coding regions were amplified from genomic DNA and subjected to direct sequencing. Sequences of 10 patients with IDI were identical with those of normals, while in 2 patients with FDI, a single base substitution was detected in one of two alleles of the AVP-NPII gene, indicating they were heterozygotes for this mutation. It was a G----A transition at nucleotide position 1859 in the second exon, resulting in a substitution of Gly for Ser at amino acid position 57 in the NPII moiety. It was speculated that the mutated AVP-NPII precursor or the mutated NPII molecule, through their conformational changes, might be responsible for AVP deficiency.
M Ito, Y Mori, Y Oiso, H Saito
Gamma delta (gamma delta) T cell receptor (TCR) expressing T cells comprise 3% of human peripheral blood lymphocytes, yet their role in the immune response remains largely unknown. There is evidence both in humans and in animal models that these cells participate in the immune response to mycobacterial antigens. In mice, exposure to mycobacterial antigens leads to the expansion of gamma delta T cells in draining lymph nodes and lungs. In humans, gamma delta T cell lines with reactivity to mycobacterial antigens have been derived from synovial fluid of a rheumatoid arthritis patient, skin lesions of leprosy patients, and peripheral blood of a healthy tuberculin reactor. Very little is known, however, about the factors which induce human gamma delta T cells to expand. In studies comparing the human T cell response to live and heat-killed Mycobacterium tuberculosis (MT), we have found that monocytes infected with live MT are very effective inducers of human gamma delta T cell expansion. After 7 d of exposure to live MT, gamma delta T cells were greatly increased in all healthy tuberculin reactors (PPD+) tested and frequently were the predominant T cell population. In contrast, heat-killed MT or purified protein products of MT induced a CD4+, alpha beta TCR+ T cell response with very little increase in gamma delta T cells. Furthermore, a similar selective induction of gamma delta T cells was observed when monocytes infected with live Salmonella were used to stimulate T cells. Heat-killed Salmonella, like heat-killed MT, induced a predominantly CD4+ alpha beta TCR+ T cell response. These findings suggest that human gamma delta T cells are a major reactive T cell population during the early stages of infection with living intracellular bacteria and are therefore likely to exert an important role in the initial interaction between host and parasite.
D V Havlir, J J Ellner, K A Chervenak, W H Boom
Bullous pemphigoid (BP) is an autoimmune disease characterized by subepidermal vesicles and the presence of autoantibodies directed against the epidermal basement membrane zone. Previous studies have identified two protein components of the hemidesmosome, BP180 and BP230, as the primary antigenic targets of BP autoantibodies. We have recently reported the isolation of a 1.0-kb BP180 cDNA. Sequence analysis presented in this report reveals that this partial BP180 cDNA encodes two protein domains which have primary structures that are characteristic of the triple helical domains of collagens, i.e., glycine appears at every third position and over one-third of the remaining residues are proline. The two collagen domains have lengths of 242 and 30 amino acids and are separated by a noncollagen stretch of 12 amino acids. Collagenase digestion of the BP180 cDNA-encoded fusion protein generated a peptide fragment with a size that was consistent with the predicted locations of the collagenase digestion sites. A possible physiological function for the collagen domains of the BP180 hemidesmosomal protein may be to form stable interactions with constituents of the extracellular matrix of the cutaneous basement membrane zone. Such interactions may provide the molecular framework for the adhesion between the basal keratinocyte and the basal lamina.
G J Giudice, H L Squiquera, P M Elias, L A Diaz
Experimental studies in vitro suggest that cytokines are important mediators in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM). However, there is little evidence for the role of cytokines in vivo, either in humans or in the spontaneous animal models of IDDM such as the NOD mouse or BB rat. To address this question, we used the model of cyclophosphamide (CYP)-induced autoimmune diabetes in the NOD/Wehi mouse to examine for (a) the production of IFN-gamma and IL-6 from isolated islets, and (b) the effect of anti IFN-gamma or anti IL-6 monoclonal antibodies on the development of diabetes. After cyclophosphamide, the majority of these mice develop of mononuclear cell infiltrate (insulitis) which by 10-14 d is associated with beta cell destruction. IFN-gamma activity at low levels (2.7 +/- 0.3 U/ml) could be detected only in culture supernatants from islets isolated at day 7 post-cyclophosphamide. In contrast, IL-6 activity progressively increased from 457 +/- 44 U/ml at day 0 to 6,020 +/- 777 U/ml at day 10. Culture of islets with anti-CD3 monoclonal antibody resulted in a significant increase in IFN-gamma activity from 41 +/- 7 U/ml at day 0 to 812 +/- 156 U/ml at day 10. Mice given either anti-IFN-gamma or anti-IL-6 antibody had a significantly reduced (P less than 0.001) incidence of diabetes and especially with IFN-gamma, decreased severity of insulitis. We conclude that IFN-gamma and IL-6 have essential roles in the pathogenesis of pancreatic islet beta cell destruction in this model.
I L Campbell, T W Kay, L Oxbrow, L C Harrison
To determine the mechanism of action of an intestinal secretagogue, serotonin, we have isolated crypt and villus cells and demonstrated Na:H and Cl:HCO3 exchange activity using the intracellular pH-sensitive fluorescent dye, 2,7-bis (carboxy-ethyl)-5,6-carboxy-fluorescein. Serotonin alkalinized both crypt and villus cells. Alkalinization in villus cells was HCO3 dependent and Na independent. In contrast, alkalinization in crypt cells was HCO3 independent and Na dependent. In villus cells, recovery from an alkaline load induced by Cl removal, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid or propionate pulse, known to occur via the Cl:HCO3 exchange, is inhibited by serotonin. In contrast, in crypt cells, recovery from an acid load induced by Na removal, amiloride and NH4Cl pulse, known to occur via Na:H exchange, is stimulated by serotonin. These data suggest that serotonin is inhibiting Cl:HCO3 exchange in villus cells and stimulating Na:H exchange in crypt cells. These effects of serotonin would be expected to inhibit coupled Na and Cl absorption by villus cells and stimulate HCO3 secretion by crypt cells in the intact ileum.
U Sundaram, R G Knickelbein, J W Dobbins
The mammalian proximal tubule is an important mediator of the renal adaptive response to systemic acidosis. In chronic metabolic and respiratory acidosis the bicarbonate reabsorptive (or proton secretory) capacity is increased. This increase is mediated, at least in part, by an increase in Vmax of the luminal Na/H antiporter. To determine whether this adaptation involves increased mRNA expression, Na/H antiporter mRNA levels were measured by Northern analysis in renal cortex of rats with metabolic (6 mmol/kg body wt NH4Cl for 2 or 5 d) and respiratory (10% CO2/air balanced for 2 or 5 d) acidosis and of normal, pair-fed rats. Na/H antiporter mRNA levels were unchanged after 2 d of both metabolic and respiratory acidosis. After 5 d, however, Na/H antiporter mRNA expression was increased 1.76 +/- 0.12-fold in response to metabolic acidosis (P less than 0.005, n = 8), but was not different from normal in response to respiratory acidosis: 1.1 +/- 0.2 (NS, n = 8). Thus, the renal adaptive response to metabolic acidosis involves increased cortical Na/H antiporter mRNA levels. In contrast, the enhanced proximal tubule Na/H antiporter activity and bicarbonate reabsorption in respiratory acidosis seem to involve mechanisms other than increased Na/H antiporter gene expression.
R Krapf, D Pearce, C Lynch, X P Xi, T L Reudelhuber, J Pouysségur, F C Rector Jr