Structure and function of the T cell antigen receptor.

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Authors

A Weiss

Hyponatremia in rats induces downregulation of vasopressin synthesis.

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Hyponatremia due to inappropriate secretion of vasopressin is a common disorder in human pathophysiology, but vasopressin synthesis during hypoosmolality has not been investigated. We used a new method to quantitate synthesis of vasopressin in rats after 3, 7, and 14 d of hyponatremia induced by administering dDAVP (a vasopressin agonist) and a liquid diet. Vasopressin synthesis was completely turned off by 7 d. Vasopressin mRNA levels in the hypothalamus paralleled the reduction in synthesis and were reduced to levels of only 10-15% of the content in control rats. When hyponatremia was corrected by withdrawal of dDAVP, vasopressin mRNA slowly returned to normal over 7 d. The observation that vasopressin synthesis can be so completely turned off leads to several conclusions: under normal physiological conditions the neurohypophysis is chronically upregulated; there must be an osmotic threshold for initiation of vasopressin synthesis (and release); the large store of hormone in the posterior pituitary is essential for vasopressin to be available during times of decreased synthesis; and, finally, some nonosmolar stimulus for synthesis must be present during clinical disorders when vasopressin is secreted (and synthesized) despite hypoosmolality.

Authors

A G Robinson, M M Roberts, W A Evron, J G Verbalis, T G Sherman

Possible role of bacterial siderophores in inflammation. Iron bound to the Pseudomonas siderophore pyochelin can function as a hydroxyl radical catalyst.

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Tissue injury has been linked to neutrophil associated hydroxyl radical (.OH) generation, a process that requires an exogenous transition metal catalyst such as iron. In vivo most iron is bound in a noncatalytic form. To obtain iron required for growth, many bacteria secrete iron chelators (siderophores). Since Pseudomonas aeruginosa infections are associated with considerable tissue destruction, we examined whether iron bound to the Pseudomonas siderophores pyochelin (PCH) and pyoverdin (PVD) could act as .OH catalysts. Purified PCH and PVD were iron loaded (Fe-PCH, Fe-PVD) and added to a hypoxanthine/xanthine oxidase superoxide- (.O2-) and hydrogen peroxide (H2O2)-generating system. Evidence for .OH generation was then sought using two different spin-trapping agents (5.5 dimethyl-pyrroline-1-oxide or N-t-butyl-alpha-phenylnitrone), as well as the deoxyribose oxidation assay. Regardless of methodology, .OH generation was detected in the presence of Fe-PCH but not Fe-PVD. Inhibition of the process by catalase and/or SOD suggested .OH formation with Fe-PCH occurred via the Haber-Weiss reaction. Similar results were obtained when stimulated neutrophils were used as the source of .O2- and H2O2. Addition of Fe-PCH but not Fe-PVD to stimulated neutrophils yielded .OH as detected by the above assay systems. Since PCH and PVD bind ferric (Fe3+) but not ferrous (Fe2+) iron, .OH catalysis with Fe-PCH would likely involve .O2(-)-mediated reduction of Fe3+ to Fe2+ with subsequent release of "free" Fe2+. This was confirmed by measuring formation of the Fe2(+)-ferrozine complex after exposure of Fe-PCH, but not Fe-PVD, to enzymatically generated .O2-. These data show that Fe-PCH, but not Fe-PVD, is capable of catalyzing generation of .OH. Such a process could represent as yet another mechanism of tissue injury at sites of infection with P. aeruginosa.

Authors

T J Coffman, C D Cox, B L Edeker, B E Britigan

Synergism between hepatic injuries and a nonhepatotropic reovirus in mice. Enhanced hepatic infection and death.

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Reovirus type 1, after intravenous inoculation in the adult mouse, is secreted via bile into the intestine in an infectious form. Although reovirus type 1 is rapidly removed from systemic circulation by the liver and the lung, very few hepatocytes express reovirus antigen during infection. In intestinal cells, reovirus replicates selectively in the crypts. This site preference may be due to active cell proliferation in the crypts. We hypothesized that the state of the cell may affect virus replication and tested this hypothesis by using chemical and surgical means to increase hepatic mitotic activity. Adult mice were treated with carbon tetrachloride or surgical trauma, inoculated with reovirus type 1 intravenously, and subsequently killed. Virus antigen was identified using a highly specific immunohistochemical technique. Liver sections were stained using immunoperoxidase with specific rabbit antireovirus antibody. Hepatotoxin and surgical trauma increase reovirus antigen detection in both Kupffer cells and hepatocytes. Only the sequential administration of CCl4 and virus caused mortality at doses sublethal for each alone. These data demonstrate a synergism between hepatic injury and reovirus which results in a significant increase in the magnitude of viral infection and contributes to mortality. Such synergism may be important in idiopathic liver disease.

Authors

D A Piccoli, C L Witzleben, C J Guico, A Morrison, D H Rubin

Relationship between the content of lysyl oxidase-dependent cross-links in skin collagen, nonenzymatic glycosylation, and long-term complications in type I diabetes mellitus.

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Many abnormalities in collagen have been reported in insulin-dependent diabetes mellitus, some or all of which have been attributed to increased cross-linking. Although recent work has focused on the role of glucose-derived collagen cross-links in the pathogenesis of diabetic complications, relatively few studies have investigated the role of lysyl oxidase-dependent (LOX) cross-links. In the present study, LOX cross-links and nonenzymatic glycosylation were quantified in skin collagen from diabetic subjects. There was an increase in the difunctional cross-link dihydroxylysinonorleucine (DHLNL) as well as in one of its trifunctional maturation products, hydroxypyridinium. All other LOX crosslinks were normal. Nonenzymatic glycosylation was increased in diabetic skin collagen, and this increase was correlated with increases in DHLNL (P less than 0.001). The biochemical results were examined for correlations with clinical data from the same subjects. Increases in DHLNL content were associated with duration of diabetes (P less than 0.003), glycohemoglobin levels (P less than 0.001), hand contractures (P less than 0.05), skin changes (P less than 0.005), and microalbuminuria (P less than 0.01). In nondiabetic subjects age was not correlated with collagen cross-link content with the exception that his-HLNL increased with age (r = 0.79, P less than 0.02). In diabetic subjects, PA levels decreased with age (r = 0.51, P less than 0.02). With increased duration of diabetes, DHLNL content was increased (r = 0.55, P less than 0.003) and OHP was increased (r = 0.59, P less than 0.01), whereas PA levels were decreased (r = -0.48, P less than 0.04). Nonenzymatic glycosylation of collagen was also increased with increased duration of diabetes (hex-lys, r = 0.47, P less than 0.02; hex-hyl, r = 0.39, P less than 0.05). We conclude that: (a) lysyl oxidase-dependent cross-linking is increased in skin collagen in diabetes and (b) that these changes in skin collagen are correlated with duration of diabetes, glycemic control, and long-term complications.

Authors

B Buckingham, K M Reiser

Platelet-derived growth factor in idiopathic pulmonary fibrosis.

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Fibrosis is a complex process involving an inflammatory reaction, fibroblast proliferation, and abnormal accumulation of interstitial collagens. Mononuclear cells are usually present in lung fibrosis. Activated monocytes and macrophages in culture have been shown to produce several growth factors including platelet-derived growth factor (PDGF). PDGF is a potent mitogen and chemoattractant for fibroblasts and smooth muscle cells and a stimulator of collagen synthesis. We have studied the expression of c-sis/PDGF-2 mRNA in lung tissues derived from five patients with idiopathic pulmonary fibrosis (IPF) and from four control individuals without IPF. Northern blot analysis of specimens obtained from four patients with IPF revealed the expression of the c-sis/PDGF-2 protooncogene. A control lung tissue without IPF did not express the c-sis protooncogene. In situ hybridization extended these studies demonstrating the expression of the c-sis mRNA in the five specimens with IPF but not in the four control specimens without IPF. The expression of c-sis mRNA was localized primarily in the epithelial cells. Invading alveolar macrophages also expressed c-sis mRNA. The expression of c-sis mRNA was accompanied by the expression of PDGF-like proteins in lung specimens with IPF but not in control lung specimens. These findings demonstrate the in vivo expression of the c-sis/PDGF-2 protooncogene and the production of PDGF-like proteins in the epithelial cells and macrophages of the fibrotic tissue. This localized and sustained production of PDGF-like mitogen may constitute an important contributing factor in the abnormal fibroblast proliferation and collagen production, events associated with pulmonary fibrosis.

Authors

H N Antoniades, M A Bravo, R E Avila, T Galanopoulos, J Neville-Golden, M Maxwell, M Selman

Fibronectin fragments containing the RGDS cell-binding domain mediate monocyte migration into the rabbit lung. A potential mechanism for C5 fragment-induced monocyte lung accumulation.

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Many inflammatory processes are characterized by an early phase of neutrophil migration and a later phase of monocyte migration into the inflammatory site. Mechanisms that govern the transition between phases are the subject of these investigations. Acute lung inflammation induced by C5 fragments in the rabbit leads to an initial neutrophil influx and plasma leakage into the alveolar space, followed by monocyte influx that we have previously shown to be dependent on prior emigration of neutrophils. Neutrophil enzymes are known to cleave intact fibronectin into fragments that are monocyte chemotaxins in vitro. Accordingly, generation of appropriate fibronectin fragments in situ by proteolytic enzymes from infiltrating neutrophils might represent a potential mechanism for attraction of monocytes into the lung. The studies reported herein demonstrate that a 120-kD fragment of fibronectin containing the RGDS fibroblast cell-binding domain induced monocyte migration into the rabbit lung in vivo. Intact fibronectin was inactive. A significant proportion of the monocyte migration was neutrophil independent. Intact fibronectin was present in bronchoalveolar lavage fluid from C5 fragment-treated animals rendered neutropenic, but absent in lavage from normal C5 fragment-treated animals. Fibronectin fragments were present in bronchoalveolar lavage fluid from both C5 fragment-treated and control rabbits. In addition, the amount of fibronectin was significantly increased in lavage of C5 fragment-treated normal but not neutropenic animals. Monoclonal antibodies directed against an epitope of fibronectin containing the RGDS cell-binding domain significantly inhibited the C5 fragment-induced monocyte migration, but not neutrophil migration. These studies suggest that chemotactic fibronectin fragments may in part be responsible for the recruitment of monocytes into areas of acute lung inflammation.

Authors

D E Doherty, P M Henson, R A Clark

Potassium depletion increases luminal Na+/H+ exchange and basolateral Na+:CO3=:HCO3- cotransport in rat renal cortex.

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Most HCO3- reabsorption in proximal tubules occurs via electroneutral Na+/H+ exchange in brush border membranes (BBMS) and electrogenic Na+:CO3=:HCO3- cotransport in basolateral membranes (BLMS). Since potassium depletion (KD) increases HCO3- reabsorption in proximal tubules, we evaluated these transport systems using BBM and BLM vesicles, respectively, from control (C) and KD rats. Feeding rats a potassium deficient diet for 3-4 wk resulted in lower plasma [K+] (2.94 mEq/liter, KD vs. 4.47 C), and higher arterial pH (7.51 KD vs. 7.39 C). KD rats gained less weight than C but had higher renal cortical weight. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0, 10% CO2, 90% N2) into BLM vesicles was 44% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was DIDS sensitive, suggesting that Na+:CO3=:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.2 mM in KD vs. 7.6 mM in C and Vmax of 278 nmol/min/mg protein in KD vs. 177 nmol/min/mg protein in C. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0) into BBM vesicles was 34% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 6.2 mM in KD vs. 7.1 mM in C and Vmax of 209 nmol/min/mg protein in KD vs. 144 nmol/min/mg protein in C. Uptakes of Na(+)-dependent [3H]glucose into BBM and [14C]succinate into BLM vesicles were not different in KD and C groups, suggesting that the Na+/H+ exchanger and Na+:CO3=:HCO3- cotransporter activities were specifically altered in KD. We conclude that adaptive increases in basolateral Na+:CO3=:HCO3- cotransport and luminal Na+H+ exchange are likely responsible for increased HCO3- reabsorption in proximal tubules of KD animals.

Authors

M Soleimani, J A Bergman, M A Hosford, T D McKinney

Mutation of the signal peptide-encoding region of the preproparathyroid hormone gene in familial isolated hypoparathyroidism.

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Preproparathyroid hormone (preproPTH) gene mutation has been proposed as a cause of familial isolated hypoparathyroidism (FIH). We cloned the preproPTH alleles of a patient with autosomal dominant FIH and sequenced the coding regions, 5' flanking regions, and splice junctions. The putatively abnormal (based on previous linkage studies) allele differed from the other allele's normal sequence at only one nucleotide. This T to C point mutation changes the codon for position 18 of the 31 amino acid prepro sequence from cysteine to arginine, disrupting the hydrophobic core of the signal sequence. Because the hydrophobic core is required by secreted proteins for efficient translocation across the endoplasmic reticulum, the mutant protein is likely to be inefficiently processed. Indeed, in vitro studies demonstrated dramatically impaired processing of the mutant preproPTH to proPTH. In summary, we observed a point mutation in the signal peptide-encoding region of a preproPTH gene in one FIH kindred and demonstrated a functional defect caused by the mutation. Mutation of the signal sequence constitutes a novel pathophysiologic mechanism in man, and further study may yield important insights both into this form of hormone deficiency and into the role of signal sequences in human physiology.

Authors

A Arnold, S A Horst, T J Gardella, H Baba, M A Levine, H M Kronenberg

Isolation of a human epidermal cDNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome.

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Autoantibodies present in the sera of patients with bullous pemphigoid (BP) bind to the basement membrane zone of normal human skin and commonly recognize two epidermal proteins, the BP240 and BP180 antigens. Two BP antigen cDNA clones from a lambda gt11 human keratinocyte library have been identified on the basis of reactivity with a BP serum. The fusion protein (FP) produced by one clone immunoadsorbed autoantibodies, which specifically recognized the BP180 by antigen, showing no cross-reactivity with BP240 by immunoblot analysis. The FP produced by the second clone immunoadsorbed autoantibodies which specifically reacted with the BP240 epidermal antigen. Northern blot analysis demonstrated that the BP180 and BP240 antigens are encoded by distinct RNA transcripts with lengths of 6.0 and 8.5 kb, respectively. Immunoblot analysis of the BP180 lysogen extract identified a 135-kD FP which was recognized by 7 of 16 BP sera and 7 of 8 herpes gestationis sera. A rabbit antiserum prepared against the lysogenic BP180 FP specifically recognized the BP180 antigen from human epidermal extracts by immunoblotting, labeled the BMZ by indirect immunofluorescence, and bound to human epidermal hemidesmosomes by immuno-electron microscopy. These results indicate that the BP180 antigen recognized by BP and herpes gestationis autoantibodies is a unique hemidesmosomal polypeptide, distinguishable from the BP240 antigen.

Authors

L A Diaz, H Ratrie 3rd, W S Saunders, S Futamura, H L Squiquera, G J Anhalt, G J Giudice

Endogenous prostaglandin endoperoxides and prostacyclin modulate the thrombolytic activity of tissue plasminogen activator. Effects of simultaneous inhibition of thromboxane A2 synthase and blockade of thromboxane A2/prostaglandin H2 receptors in a canine model of coronary thrombosis.

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We tested the hypothesis that simultaneous inhibition of TxA2 synthase and blockade of TxA2/PHG2 receptors is more effective in enhancing thrombolysis and preventing reocclusion after discontinuation of tissue plasminogen activator (t-PA) than either intervention alone. Coronary thrombosis was induced in 35 dogs by placing a copper coil into the left anterior descending coronary artery. Coronary flow was measured with a Doppler flow probe. 30 min after thrombus formation, the animals received saline (controls, n = 10); SQ 29548 (0.4 mg/kg bolus + 0.4 mg/kg per h infusion), a TxA2/PGH2 receptor antagonist (n = 8); dazoxiben (5 mg/kg bolus + 5 mg/kg per h infusion), a TxA2 synthase inhibitor (n = 9); or R 68070 (5 mg/kg bolus + 5 mg/kg per h infusion), a drug that blocks TxA2/PGH2 receptors and inhibits TxA2 synthase (n = 8). Then, all dogs received heparin (200 U/kg) and a bolus of t-PA (80 micrograms/kg) followed by a continuous infusion (8 micrograms/kg per min) for up to 90 min or until reperfusion was achieved. The time to thrombolysis did not change significantly in SQ 29548-treated dogs as compared with controls (42 +/- 5 vs. 56 +/- 7 min, respectively, P = NS), but it was significantly shortened by R 68070 and dazoxiben (11 +/- 2 and 25 +/- 6 min, respectively, P less than 0.001 vs. controls and SQ 29548-treated dogs). R 68070 administration resulted in a lysis time significantly shorter than that observed in the dazoxiben-treated group (P less than 0.01). Reocclusion was observed in eight of eight control dogs, five of seven SQ 29548-treated dogs, seven of nine dazoxiben-treated dogs, and zero of eight R 68070-treated animals (P less than 0.001). TxB2 and 6-keto-PGF1 alpha, measured in blood samples obtained from the coronary artery distal to the thrombus, were significantly increased at reperfusion and at reocclusion in control animals and in dogs receiving SQ 29548. R 68070 and dazoxiben prevented the increase in plasma TxB2 levels, whereas 6-keto-PGF1 alpha levels were significantly increased with respect to control and SQ 29548-treated dogs. Thus, simultaneous inhibition of TxA2 synthase and blockade of TxA2/PGH2 receptors is more effective than either intervention alone in this experimental model in enhancing thrombolysis and preventing reocclusion after t-PA administration.

Authors

P Golino, M Rosolowsky, S K Yao, J McNatt, F De Clerck, L M Buja, J T Willerson

Feedback inhibition of aldose reductase gene expression in rat renal medulla. Galactitol accumulation reduces enzyme mRNA levels and depletes cellular inositol content.

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Aldose reductase (AR) is an enzyme responsible for converting glucose into sorbitol and galactose into galactitol. In the renal inner medulla, where sorbitol production plays a role in cellular osmoregulation, AR gene expression has been shown to be osmotically regulated. The present study examined the effects of the accumulation of the AR end product, galactitol, induced by galactose feeding, on AR gene expression and on the balance of other cellular osmolytes, including inositol, in the renal medulla. To differentiate between the effects of excess substrate, product, and intervening osmotic factors, rats were fed either control, galactose, galactose and sorbinil (an AR inhibitor), or control plus sorbinil diets. Renal papillae were assayed for AR mRNA, sodium, urea, galactose, galactitol, sorbitol, inositol, and other organic osmolytes. Galactose feeding resulted in a great accumulation of galactitol and reduction in AR mRNA levels in renal papillae. Associated with these changes was a significant depletion of renal papillary sorbitol, inositol, and glycerolphosphocholine. These effects were largely attenuated by sorbinil. The present findings suggest that renal cellular accumulation of the enzyme's polyol product causes downregulation of AR gene expression. Furthermore, our findings suggest that the inositol depletion associated with sorbitol or galactitol accumulation in various cell types during hyperglycemia may be a function of cellular osmoregulation.

Authors

C Bondy, B D Cowley Jr, S L Lightman, P F Kador

Modulation of interleukin-1 beta RNA in monocytic cells infected with human immunodeficiency virus-1.

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The effect of HIV-1 infection on cytokine levels was studied in monocytic cells by using Northern blotting analysis. Monoblasts (THP-1, U937) did not express IL-1 beta RNA even if the cells were infected with HIV-1. After exposure to LPS (10 micrograms/ml) and 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) for 12 h, these HIV-1-infected monoblasts accumulated 8-15-fold greater levels of IL-1 beta RNA as compared with their HIV-1-uninfected counterparts that were similarly stimulated. In contrast, levels of RNAs coding for monocyte-colony-stimulating factor (M-CSF) and tumor necrosis factor-alpha (TNF alpha) were elevated less than twofold in the HIV-1-infected cells as compared with HIV-1-uninfected cells after their stimulation with LPS and TPA. Inhibition of new protein synthesis did not block the marked accumulation of IL-1 beta RNA produced by exposure to LPS and TPA in the HIV-1-infected cells. Time-course experiments showed that the maximal levels of IL-1 beta RNA occurred at 12 and 24 h after LPS and TPA stimulation of the HIV-1-infected and uninfected U937 cells, respectively. Studies of stability of RNA using actinomycin D showed that IL-1 beta RNA was equally stable in infected and uninfected U937 cells after their stimulation with TPA and LPS. Taken together, our data show that HIV-1 infection markedly augments IL-1 beta RNA accumulation in stimulated monocytic cells, probably through increasing rate of transcription of IL-1 beta.

Authors

K Yamato, Z el-Hajjaoui, K Simon, H P Koeffler

Essential fatty acid deficiency ameliorates acute renal dysfunction in the rat after the administration of the aminonucleoside of puromycin.

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The administration of the aminonucleoside of puromycin (PAN) to rats causes the nephrotic syndrome that is associated with an acute decline in renal function, and an interstitial infiltrate. We examined whether essential fatty acid deficiency (EFAD), which inhibits macrophage infiltration in glomerulonephritis, affects PAN-induced renal dysfunction. Both control and EFAD rats developed proteinuria that resolved over 28 d. After PAN administration, there was a prominent infiltration of macrophages in rats fed a normal diet. The infiltrate was prevented by the EFAD diet. The absence of a macrophage interstitial infiltrate was associated with a significantly higher Cin in the EFAD rats than in controls at 7 d (5.21 +/- 1.19 versus 0.39 +/- 0.08, P less than 0.002 ml/min/kg BW). In addition, CPAH fell to less than 10 ml/min/kg BW by day 7 in controls, but remained the same as normal in the EFAD. After administration of PAN to control rats, there was no increase in urinary thromboxane excretion or an increase in glomerular thromboxane production. Furthermore, the effect of EFAD could not be mimicked by the administration of a thromboxane synthase inhibitor. Irradiation-induced leukopenia in rats on a normal diet markedly improved glomerular filtration and renal blood flow in acutely nephrotic rats. EFAD prevents the interstitial cellular infiltrate and the renal ischemia associated with experimental nephrosis. The recruitment of mononuclear cells into the kidney following PAN directly contributes to the decline in renal function.

Authors

K P Harris, J B Lefkowith, S Klahr, G F Schreiner

Activation pathways of synovial T lymphocytes. Expression and function of the UM4D4/CDw60 antigen.

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Accumulating evidence implicates a central role for synovial T cells in the pathogenesis of rheumatoid arthritis, but the activation pathways that drive proliferation and effector function of these cells are not known. We have recently generated a novel monoclonal antibody against a rheumatoid synovial T cell line that recognizes an antigen termed UM4D4 (CDw60). This antigen is expressed on a minority of peripheral blood T cells, and represents the surface component of a distinct pathway of human T cell activation. The current studies were performed to examine the expression and function of UM4D4 on T cells obtained from synovial fluid and synovial membranes of patients with rheumatoid arthritis and other forms of inflammatory joint disease. The UM4D4 antigen is expressed at high surface density on about three-fourths of synovial fluid T cells and on a small subset of synovial fluid natural killer cells; in synovial tissue it is present on more than 90% of T cells in lymphoid aggregates, and on approximately 50% of T cells in stromal infiltrates In addition, UM4D4 is expressed in synovial tissue on a previously undescribed population of HLA-DR/DP-negative non-T cells with a dendritic morphology. Anti-UM4D4 was co-mitogenic for both RA and non-RA synovial fluid mononuclear cells, and induced IL-2 receptor expression. The UM4D4/CDw60 antigen may represent a functional activation pathway for synovial compartment T cells, which could play an important role in the pathogenesis of inflammatory arthritis.

Authors

D A Fox, J A Millard, L Kan, W S Zeldes, W Davis, J Higgs, F Emmrich, R W Kinne

High level transcription of the glucocerebrosidase pseudogene in normal subjects and patients with Gaucher disease.

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Gaucher disease is due to mutations involving the glucocerebrosidase gene. A closely homologous pseudogene is located approximately 16 kD downstream from the functional gene. Sequence analysis of clones from cDNA libraries made from skin fibroblast cultures showed several independent clones with the sequence of an aberrantly processed pseudogene message. Examination of cellular RNA from lymphoblasts or fibroblasts obtained from thirteen Gaucher disease patients, one Gaucher disease heterozygote, and four normal subjects showed that the pseudogene was consistently transcribed, and that in some cases the level of transcription seemed to be approximately equal to that of the functional gene. The transcription of the pseudogene must be taken into account when attempting to detect mutations of glucocerebrosidase by the study of cDNA libraries.

Authors

J Sorge, E Gross, C West, E Beutler

Apolipoprotein A-I and its amphipathic helix peptide analogues inhibit human immunodeficiency virus-induced syncytium formation.

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The envelope (membrane) glycoprotein of HIV is essential for virus attachment and entry into host cells. Additionally, when expressed on the plasma membrane of infected cells, the envelope protein is responsible for mediating cell-cell fusion which leads to the formation of multinucleated giant cells, one of the major cytopathic effects of HIV infections. The envelope glycoproteins of HIV contain regions that can fold into amphipathic alpha-helixes, and these regions have been suggested to play a role in subunit associations and in virus-induced cell fusion and cytopathic effects of HIV. We therefore tested the possibility that amphipathic helix-containing peptides and proteins may interfere with the HIV amphipathic peptides and inhibit those steps of HIV infection involving membrane fusion. Apolipoprotein A-I, the major protein component of high density lipoprotein, and its amphipathic peptide analogue were found to inhibit cell fusion, both in HIV-1-infected T cells and in recombinant vaccinia-virus-infected CD4+ HeLa cells expressing HIV envelope protein on their surfaces. The amphipathic peptides inhibited the infectivity of HIV-1. The inhibitory effects were manifest when the virus, but not cells, was pretreated with the peptides. Also, a reduction in HIV-induced cell killing was observed when virus-infected cell cultures were maintained in presence of amphipathic peptides. These results have potential implications for HIV biology and therapy.

Authors

B J Owens, G M Anantharamaiah, J B Kahlon, R V Srinivas, R W Compans, J P Segrest

Deletion of Alu sequences in the fifth c-sis intron in individuals with meningiomas.

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Abstract

An abnormality in the c-sis protooncogene was identified in leukocyte DNA from members of a family predisposed to the development of meningioma, and was found to be associated with the development of the tumor in those individuals. Molecular analysis of this abnormality demonstrated a deletion within the fifth intron of the c-sis gene. The normal c-sis gene has an Alu sequence in this region which includes two perfect 130 nucleotide repeated sequences separated by 5 bp. The deleted c-sis allele is missing precisely one copy of the 130 bp repeat and the intervening 5 bp. An identical deletion was also found in DNA from 1 of 13 sporadic meningiomas.

Authors

M Smidt, I Kirsch, L Ratner

Molecular basis of hereditary C3 deficiency.

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Hereditary deficiency of complement component C3 in a 10-yr-old boy was studied. C3 could not be detected by RIA of serum from the patient. Segregation of C3 S and C3 F allotypes within the family confirmed the presence of a null gene for C3, for which the patient was homozygous. 30 exons have been characterized, spanning the entire beta chain of C3 and the alpha chain as far as the C3d region. Sequence analysis of the exons derived from the C3 null gene showed no abnormalities in the coding sequences. A GT-AT mutation at the 5' donor splice site of the intervening sequence 18 was found in the C3 null gene. Exons 17-21 were amplified by the polymerase chain reaction (PCR) from first-strand cDNA synthesized from mRNA obtained from peripheral blood monocytes stimulated with LPS. This revealed a 61-bp deletion in exon 18, resulting from splicing of a cryptic 5' donor splice site in exon 18 with the normal 3' splice site in exon 19. This deletion leads to a disturbance of the reading frame of the mRNA with a stop codon 17 bp downstream from the abnormal splice in exon 18. His parents had both the normal and abnormal C3 mRNA and were shown to be heterozygous for this mutation by sequence analysis of genomic DNA amplified by PCR. Similar splice mutants have previously been reported in the beta-globin, phenylalanine hydroxylase, and porphobilinogen deaminase genes. This mutation is sufficient to cause the deficiency of C3 in the patient.

Authors

M Botto, K Y Fong, A K So, A Rudge, M J Walport

Endothelin enhances the contractile responsiveness of adult rat ventricular myocytes to calcium by a pertussis toxin-sensitive pathway.

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Abstract

It has long been assumed that the primary influences regulating cardiac contractility are the extent of mechanical loading of muscle fibers and the activity of the autonomic nervous system. However, the vasoactive peptide endothelin, initially found in vascular endothelium, is among the most potent positively inotropic agents yet described in mammalian myocardium. In isolated adult rat ventricular cells, endothelin's action was slow in onset but very long lasting with an EC50 of 50 pM that approximates the reported KD of the peptide for its receptor in rat heart. When the calcium activity of the buffer superfusing isolated single fura-2-loaded myocytes paced at 1.5 Hz was varied from 0.1 to 0.9 mM [Ca2+]o, 100 pM endothelin increased contractile amplitude with no significant change in diastolic or systolic [Ca2+]i, thus appearing to sensitize the myofilaments to intracellular calcium. Pertussis toxin, or prior exposure to a beta-adrenergic agonist, reduced or abolished the increase in myocyte contractility induced by endothelin. This novel and potent pharmacologic action of endothelin points to the potential importance of local, paracrine factors, perhaps derived from microvascular endothelium or endocardium, in the control of the contractile function of the heart.

Authors

R A Kelly, H Eid, B K Krämer, M O'Neill, B T Liang, M Reers, T W Smith

Apical membrane limits urea permeation across the rat inner medullary collecting duct.

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Abstract

Urea diffuses across the terminal inner medullary collecting duct (IMCD) via a facilitated transport pathway. To examine the mechanism of transcellular urea transport, membrane-apparent urea (Purea) and osmotic water (Pf) permeabilities of IMCD cells were measured by quantitative light microscopy in isolated IMCD-2 tubules perfused in the absence of vasopressin. Basolateral membrane Pf, determined by addition of raffinose to the bath, was 69 microns/s. Basolateral membrane Purea, determined by substituting urea for raffinose without change in osmolality, was 14 X 10(-5) cm/s. Bath phloretin inhibited basolateral Purea by 85% without a significant effect on Pf. The basolateral reflection coefficient for urea, determined by addition of urea in the presence of phloretin, was 1.0. These results indicate that urea crosses the basolateral membrane by diffusion, and not by solvent drag. In perfused tubules, the rate of cell swelling following substitution of urea for mannitol was significantly greater with bath than lumen changes. After correcting for membrane surface area, the basolateral membrane was twofold more permeable than the apical membrane. Conclusions: (a) in the absence of vasopressin, urea permeation across the IMCD cell is limited by the apical membrane; (b) the basolateral membrane contains a phloretin-sensitive urea transporter; (c) transepithelial urea transport occurs by movement of urea through the IMCD cell.

Authors

R A Star

Induction of renal growth and injury in the intact rat kidney by dietary deficiency of antioxidants.

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Abstract

We report induction of renal growth and injury in the intact rat kidney using a diet deficient in vitamin E and selenium. This diet was imposed in 3-wk-old male weanling rats, and after 9 wk, enhancement of growth, characterized by increased wet weight, dry weight, protein content, and DNA content appeared. Morphometric analyses revealed increased kidney volume, tubular epithelial volume, and mean glomerular volume. There were no differences in nephron number. The animals on the deficient diet displayed increased urinary protein excretion at 9 wk. Renal injury was also characterized by an interstitial cellular infiltrate and diminutions in glomerular filtration rate. Enhanced growth and injury were antedated by increased renal ammoniagenesis. The deficient diet did not induce metabolic acidosis, potassium depletion, glucose intolerance, or elevated plasma amino acid concentration. Enhancement of renal growth and ammoniagenesis by the deficient diet was not suppressible by chronic alkali therapy. Stimulation of renal growth could not be ascribed to increased intrarenal iron, induction of ornithine decarboxylase, or alterations in glomerular hemodynamics. Stimulation of renal ammoniagenesis by dietary deficiency of antioxidants is a novel finding, as is induction of growth and injury. We suggest that increased renal ammoniagenesis contributes to induction of renal growth and injury.

Authors

K A Nath, A K Salahudeen

Autoantibodies to the HLA-B27 sequence cross-react with the hypothetical peptide from the arthritis-associated Shigella plasmid.

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Abstract

We previously reported elevated serum antibody levels to a peptide representing the HLA-B27 polymorphic region (B27 peptide) in HLA-B27(+) ankylosing spondylitis (AS) patients. A plasmid (pHS-2) isolated from arthritogenic Shigella flexneri strains had been shown to encode an amino acid sequence homologous to HLA-B27. Rabbit antibody to this sequence (pHS-2 peptide) strongly cross-reacted with B27 peptide and, to a much lesser extent, with Klebsiella nitrogenase peptide. Serum antibody levels to pHS-2 peptide were studied in 160 spondylarthropathy patients. 12 of 115 (10.4%) AS patients, 2 of 45 (4.4%) patients with Reiter's syndrome or reactive arthritis as well as 6 of 147 (4.1%) normal controls were shown to have elevated anti-pHS-2 peptide antibodies. Antibody levels to B27 and pHS-2 peptides were significantly correlated in 134 HLA-B27(+) patients (r = 0.333, P less than 0.001). 13 of 15 affinity-purified anti-B27 peptide antibodies from patients strongly cross-reacted with pHS-2 peptide, whereas only 3 weakly cross-reacted to nitrogenase peptide. Leucine appeared to be a critical residue for this cross-reaction. AS patients' anti-B27 peptide antibodies reacted with HLA-B27 transfected L cells. These results may suggest that pHS-2 peptide more efficiently "mimics" B27 peptide than does nitrogenase peptide. Involvement of pHS-2 in pathogenesis of spondylarthropathy through molecular mimicry mechanisms requires further study.

Authors

N Tsuchiya, G Husby, R C Williams Jr, H Stieglitz, P E Lipsky, R D Inman

Immune modulation of metalloproteinase production in human macrophages. Selective pretranslational suppression of interstitial collagenase and stromelysin biosynthesis by interferon-gamma.

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Abstract

Interferon-gamma (IFN-gamma) is a lymphokine that activates mononuclear phagocytes. To test the hypothesis that IFN-gamma might have important effects upon the ability of human mononuclear phagocytes to degrade extracellular matrix, we have studied the action of this cytokine on the production of metalloproteinases and the counterregulatory tissue inhibitor of metalloproteinases (TIMP) by the human alveolar macrophage. We have found that IFN-gamma potently and selectively suppresses the lipopolysaccharide-induced production of two metalloproteinases--interstitial collagenase and stromelysin--by 50-90% at doses greater than or equal to 10 U/ml. The synthesis of TIMP and 92-kD type IV collagenase was also diminished by IFN-gamma, but these responses required 50- to 100-fold higher concentrations of the cytokine. All doses of IFN-gamma increased total and secreted protein synthesis slightly, indicating a highly specific effect on metalloenzyme biosynthesis. Inhibition of metalloproteinase expression occurred at a pretranslational level, as evidenced by parallel reductions in enzyme biosynthesis and collagenase-specific steady-state mRNA levels. Interestingly, the effect of IFN-gamma on metalloenzyme production was not readily reversible. Therefore, while IFN-gamma activates the macrophage and renders it tumoricidal, this enhanced function appears to be attained at the expense of the cell's capacity to degrade extracellular matrix.

Authors

S D Shapiro, E J Campbell, D K Kobayashi, H G Welgus

A direct comparison of biological response modulation and clinical side effects by interferon-beta ser, interferon-gamma, or the combination of interferons beta ser and gamma in humans.

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Abstract

To directly compare clinical side effects and biological response modification, IFN-beta ser, IFN-gamma, or the combination of IFN-beta ser plus IFN-gamma was administered to 21 cancer patients. Each IFN or the combination was given intravenously on days 1, 8, and 15 in varied order. Each IFN and the combination resulted in significant (P less than 0.05) modulation of IFN-induced proteins. IFN-beta ser was more effective than IFN-gamma in enhancing 2-5A synthetase activity (P = 0.001). IFN-gamma was more effective than IFN-beta ser in enhancing serum beta 2 microglobulin expression (P = 0.05) and indoleamine dioxygenase activity, as assessed by decreased serum tryptophan (P = 0.03). The combination enhanced tryptophan catabolism more effectively than IFN-beta ser in a dose-dependent manner (P less than 0.03). IFN-beta ser/IFN-gamma did not potentiate natural killer cells or antibody-dependent cellular toxicity (ADCC). IFN-beta ser/IFN-gamma enhanced monocyte guanylate cyclase activity, as assessed by serum neopterin, more effectively than IFN-gamma alone (P = 0.005). Both IFNs and the combination resulted in increases in HLA class II expression on monocytes. However, no significant difference in the level of induction of HLA DQ and HLA DR expression between IFN-beta ser/IFN-gamma and either IFN-beta ser or IFN-gamma was noted. Although frequency and servity of side effects of IFN-beta ser, IFN-gamma, or the combination were dose related, induction of induced proteins (with exception of influences on tryptophan catabolism) were not a function of dose administered over the 10-fold range. Continued treatment with the combination intravenously three times a week for 4 wk sustained but did not further potentiate, most of the changes in interferon-induced proteins. Thus, IFN-beta ser and IFN-gamma each resulted in effective and essentially equivalent patterns of induction of induced proteins. When combined, however, these IFNs did not result in potentiation of biological response modification in vivo.

Authors

J H Schiller, B Storer, D M Paulnock, R R Brown, S P Datta, P L Witt, E C Borden

Enhanced utilization of exogenous glucose improves cardiac function in hypoxic rabbit ventricle without increasing total glycolytic flux.

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Abstract

The effects of elevated glucose on cardiac function during hypoxia were investigated in isolated arterially perfused rabbit interventricular septa. Rest tension, developed tension, intracellular potential, 42K+ efflux, lactate production, exogenous glucose utilization, and tissue high-energy phosphate levels were measured during a 50-min period of hypoxia with 4, 5, or 50 mM glucose present (isosmotically balanced with sucrose) and during reoxygenation for 60 min with perfusate containing 5 mM glucose/45 mM sucrose. At physiologic (4 or 5 mM) and supraphysiologic glucose (50 mM), lactate production and high-energy phosphate levels during hypoxia were equally well maintained, yet cardiac dysfunction was markedly attenuated by 50 mM glucose. Despite identical rates of total glycolytic flux, exogenous glucose utilization was enhanced by 50 mM glucose so that tissue glycogen levels remained normal during hypoxia, whereas glycogen became depleted with 4 or 5 mM glucose present during hypoxia. Most of the beneficial effects of 50 mM glucose occurred during the first 25 min of hypoxia. Prior glycogen depletion had no deleterious effects during hypoxia with 50 mM glucose present, but exacerbated cardiac dysfunction during hypoxia with 5 mM glucose present. These findings indicate that enhanced utilization of exogenous glucose improved cardiac function during hypoxia without increasing total glycolytic flux or tissue high-energy phosphate levels, suggesting a novel cardioprotective mechanism.

Authors

E M Runnman, S T Lamp, J N Weiss

Interferon-gamma overcomes glucocorticoid suppression of cachectin/tumor necrosis factor biosynthesis by murine macrophages.

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Abstract

Glucocorticoids almost completely inhibit the synthesis by isolated macrophages of cachectin/tumor necrosis factor (TNF), a cytokine implicated as a major endogenous mediator of septic shock. Despite this in vitro effectiveness, the clinical use of glucocorticoids has failed to demonstrate any clear benefit in the treatment of septic shock. In an effort to understand what other mechanisms might play a role in the patient with sepsis, we examined the effect of interferon-gamma (IFN gamma) on the synthesis of cachectin/TNF. We show here that IFN gamma, although unable by itself to induce cachectin/TNF synthesis, enhanced the endotoxin-induced production of cachectin/TNF in vitro. Furthermore, IFN gamma overcame the inhibition of cachectin/TNF synthesis caused by the glucocorticoid, dexamethasone. These effects of IFN gamma were accounted for by increased levels of cachectin/TNF mRNA. The in vivo implications of these studies are discussed with emphasis on their relevance in human sepsis.

Authors

C E Luedke, A Cerami

Effect of inhibition of Na+/K(+)-adenosine triphosphatase on vascular action of vasopressin.

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Abstract

The present study was undertaken to examine the cellular interaction between a Na+/K(+)-ATPase inhibitor, ouabain, and arginine vasopressin (AVP) in rat vascular smooth muscle cells (VSMC) in culture. Preincubation with 10(-5) M ouabain for 60 min increased basal cytosolic free Ca2+ [( Ca2+]i) concentration and intracellular 45Ca2+ uptake. Ouabain, however, did not affect basal 45Ca2+ efflux or AVP-stimulated 45Ca2+ efflux. As assessed by cell shape change, preincubation with 10(-5) M ouabain for 60 min also enhanced the sustained cellular contractile effect of a submaximal (10(-8) M AVP, 21.5% vs. 30.5%, P less than 0.01) but not maximal dose of 10(-6) M AVP. Preincubation with 10(-5) M ouabain for 60 min did not change AVP-induced V1-specific surface receptor binding or AVP-induced inositol phosphate production but did however potentiate the mobilization of [Ca2+]i induced by a submaximal (10(-8) M AVP, 301 vs. 385 nM, P less than 0.01) but not a maximal dose of AVP. These effects of ouabain on the mobilization of [Ca2+]i were abolished by incubation in Ca2(+)-free buffer or 5 X 10(-5) M verapamil. Ouabain (10(-5) M) also enhanced the sustained cellular contractile effect of a direct protein kinase C activator, phorbol 12-myristate 13-acetate. The present results therefore indicate that the inhibition of Na+/K(+)-ATPase may enhance the vascular action of AVP, and perhaps other vasoconstrictors, by increasing the AVP-induced mobilization of [Ca2+]i and by potentiating the activity of protein kinase C stimulated by AVP through enhancing basal and AVP-stimulated cellular Ca2+ uptake.

Authors

K Okada, C Caramelo, P Tsai, R W Schrier

Polyclonal B cell activation in lupus-prone mice precedes and predicts the development of autoimmune disease.

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Abstract

Polyclonal B cell activation is an early feature of autoimmune disease in humans and mice with systemic lupus erythematosus. The contribution of polyclonal activation to the progression of autoimmunity is unclear, however, since it precedes the development of end-organ damage by months or years. To examine this issue, 109 autoimmune-prone (NZB X NZW)F1 X NZB backcross mice were hemi-splenectomized at 10 wk and the number and antigenic specificity of their Ig-secreting B cells quantitated by ELISA spot assay. Of the 61 mice that had polyclonally increased numbers of Ig-secreting cells/spleen, 31 died by 6 mo. In contrast, 0/48 backcross mice with normal numbers of Ig-secreting B cells at 10 wk died over the same period (P less than 0.001). Polyclonally activated mice also developed proteinuria earlier and more frequently than littermates with normal numbers of Ig-secreting cells (P less than 0.001). As adults, backcross mice with proteinuria expressed repertoires skewed towards the production of anti-DNA antibodies. At 10 wk these same mice expressed repertoires marked by polyclonal activation rather than preferential anti-DNA production. These findings indicate that autoimmune disease in SLE is accompanied by the autoantigen-driven production of autoantibodies but is preceded and predicted by polyclonal B cell activation.

Authors

D M Klinman

Lack of induction of suppressor T cells by intestinal epithelial cells from patients with inflammatory bowel disease.

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Abstract

The mechanisms underlying the chronic unrelenting inflammatory response seen in inflammatory bowel disease (IBD) are poorly understood. We have recently proposed a novel role for the normal intestinal enterocyte, that of antigen presenting cell. However, in contrast to conventional antigen presenting cells, normal enterocytes appear to selectively activate CD8+ antigen nonspecific suppressor T cells. To determine whether failure of this process may be occurring in inflammatory bowel disease, freshly isolated enterocytes from small and large bowel from normal patients, patients with Crohn's disease, ulcerative colitis, and inflammatory (diverticulitis, ischemic colitis, and gold induced colitis) controls were co-cultured with allogeneic T cells in a modified mixed lymphocyte reaction. In contrast to normal enterocytes, 42/42 Crohn's and 35/38 ulcerative colitis-derived epithelial cells stimulated CD4+ T cells, whereas 65/66 and 9/9 normal and inflammatory control enterocytes, respectively, stimulated CD8+ T cells (as previously described), suggesting that the results seen were not just a reflection of underlying inflammation. Furthermore, IBD enterocytes from both histologically involved and uninvolved tissue were similar in their ability to selectively activate CD4+ T cells, speaking for a more global defect in epithelial cells in IBD. Finally, activated T cells from IBD epithelial cell-stimulated mixed lymphocyte cultures displayed potent T helper activity in an antigen nonspecific fashion. Taken together, these data suggest that there may be an intrinsic defect in epithelial cells from patients with IBD, resulting in the inability to normally stimulate suppressor T cells in an antigen overloaded environment.

Authors

L Mayer, D Eisenhardt

Expression of ras oncogenes in cultured human cells alters the transcriptional and posttranscriptional regulation of cytokine genes.

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Abstract

Autonomous production of cytokines such as the hematopoietic colony-stimulating factors (CSFs), IL-1, or IL-6 has been demonstrated in numerous human and murine neoplasms, and may be involved in the pathogenesis of several paraneoplastic syndromes such as leukocytosis, fever, and hypercalcemia. Because of the high frequency with which mutations in ras protooncogenes have been detected in human tumors, as well as evidence linking ras gene products to activation of certain cellular functions, we investigated whether ras mutations might influence the regulation of cytokine genes. Normal human fibroblasts transfected with a mutant val12 H-ras oncogene expressed increased levels of mRNA transcripts encoding granulocyte-CSF (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and IL-1 beta compared with controls. Human mesothelioma cells transfected with a mutant asp12 N-ras oncogene exhibited similar alterations in cytokine gene expression. Estimates of transcriptional activity by nuclear run-on analysis revealed a selective increase in transcription only for the IL-1 gene. Analysis of mRNA half-life demonstrated a marked increase in the stability of numerous cytokine transcripts, including G-CSF, GM-CSF, IL-1, and IL-6. The addition of anti-IL-1 neutralizing antibody to cultures of cells expressing ras mutants did not block the expression of any of the cytokines examined, suggesting that the baseline expression of GM-CSF, G-CSF, and IL-6 was not a secondary event due to the increased transcription of IL-1. These results indicate that mutations in ras genes may alter expression of several cytokine genes through both transcriptional and posttranscriptional mechanisms.

Authors

G D Demetri, T J Ernst, E S Pratt 2nd, B W Zenzie, J G Rheinwald, J D Griffin

Role of lung fluid volume in growth and maturation of the fetal sheep lung.

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Abstract

We studied the effects of alterations in lung fluid volume on growth and maturation of the fetal lung. In a chronic fetal sheep preparation, right fetal lung volume was decreased by drainage of lung fluid while the volume of the left lung was expanded by mainstem bronchus ligation leading to lung fluid retention. After an experimental period of 25 d (from 105 to 129 d of gestation, term = 145 d), the right (deflated) lung was significantly hypoplastic and contained less DNA than the controls; 175.15 +/- 55.18 vs. 346.77 +/- 61.97 mg, respectively; P less than 0.001. In contrast, the left (expanded) lung was significantly hyperplastic and contained more DNA than the controls; 390.74 +/- 103.53 vs. 238.85 +/- 33.32 mg, respectively; P = 0.001. Biochemical indices of lung maturation, including total phospholipids, phosphatidylcholine, and disaturated phosphatidylcholine content expressed per unit of tissue DNA, were no different when comparing the hypoplastic, hyperplastic, and control lungs. These findings demonstrate that fetal lung cell multiplication is influenced by local distension with lung fluid, while the biochemical maturation of fetal lung surfactant is under systemic control.

Authors

A C Moessinger, R Harding, T M Adamson, M Singh, G T Kiu

Role of ventriculovascular coupling in cardiac response to increased contractility in closed-chest dogs.

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Abstract

While both dobutamine and pacing tachycardia augment left ventricular (LV) contractility, whether overall cardiovascular response to these stimuli is comparable is not known. To address this question we studied seven dogs previously instrumented with three LV diameter gauges and LV pressure manometers. After ganglionic blockade and sedation, caval occlusions were performed at heart rates of 120, 160, and 200 bpm before (C), and 160 and 200 bpm after administration of 10 micrograms/kg per min dobutamine, i.v. (D). The effective arterial elastance (Ea) went up from 14.2 +/- 4.5 mmHg/ml at C120 to 19.6 +/- 8.8 (P less than 0.025 vs C120) and 24.2 +/- 10.4 (P less than 0.001 vs C120) mmHg/ml at C160 and C200. Ees, the slope of the end-systolic pressure-volume relation, increased with pacing from 9.7 +/- 4.6 to 11.7 +/- 4.3 (P less than 0.02), and 13.2 +/- 5.7 (P less than 0.02) mmHg/ml at 160 and 200 bpm. With dobutamine infusion Ea went down, and Ees was further increased to 37.0 +/- 20.9 mmHg/ml at 160 bpm (P less than 0.002 vs C160), and 53.0 +/- 22.6 mmHg/ml at 200 bpm (P less than 0.002 vs C200). Comparison of stroke work and pressure-volume area from single beats with matched LV end-diastolic volumes showed that these were both increased by dobutamine, but not by pacing tachycardia. While increased heart rate after dobutamine markedly increased contractility, Ea was not changed, and neither stroke work nor pressure-volume was further increased. Thus, how well an increase in contractility is transmitted to the periphery is determined in part by arterial behavior. Assessment of both the arterial system and cardiac contractility is necessary to fully evaluate the overall impact of an inotropic stimulus.

Authors

G L Freeman, J T Colston

The immunoglobulin G subclass composition of immune complexes in cystic fibrosis. Implications for the pathogenesis of the Pseudomonas lung lesion.

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Abstract

It has been shown that pulmonary macrophage (PM) phagocytosis of Pseudomonas aeruginosa (PA) is inhibited in the presence of serum from cystic fibrosis (CF) patients colonized by Pseudomonas, and that these sera contain high concentrations of IgG2 antibodies. The goal of these studies was to investigate the role that IgG2-containing immune complexes (IC) play in this inhibition of both PM and neutrophil phagocytosis. We found that serum IgG2 concentrations were elevated significantly in CF patients with chronic PA colonization and that in selected sera from CF patients with chronic PA colonization (CF + IC, n = 10), the mean IC level was significantly elevated (2.90 +/- 0.22 mg/dl [SEM]). IgG2 comprised 74.5% of IgG precipitated in IC from CF + IC sera. An invitro phagocytic assay of [14C]PA uptake using CF + IC whole-sera opsonins confirmed that endocytosis by normal PM and neutrophils was significantly depressed. Removal of IC from CF + IC sera resulted in significantly decreased serum IgG2 concentrations without a significant change in the other subclass concentrations, and enhanced [14C]PA uptake by PM (26.6% uptake increased to 47.3%) and neutrophils (16.9% increased to 52.6%). Return of the soluble IgG2 IC to the original CF sera supernatants and the positive control sera resulted in return of the inhibitory capacity of the CF + IC sera. We conclude that immune sera from patients with chronic Pseudomonas infections characterized by elevated IgG2 subclass level functions poorly as an opsonin. In these individuals, IgG2 contributes significantly to circulating IC and removal of IC, matched by a simultaneous fall in IgG2, improves bacterial uptake by neutrophil and mononuclear phagocytes. IgG2 antibodies exert antiphagocytic effects by both direct inhibition and the formation of IC.

Authors

D B Hornick, R B Fick Jr

Myocardial localization and isoforms of neural cell adhesion molecule (N-CAM) in the developing and transplanted human heart.

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Abstract

Neural cell adhesion molecule (N-CAM) has been implicated in cellular interactions involved in cardiac morphogenesis and innervation. Immunohistochemical techniques and Western blot analysis were used to determine the localization and isoforms of N-CAM in the developing and extrinsically denervated human heart. Myocardial and conducting cells in the fetal heart (7-24 wk gestation) exhibited sarcolemmal immunoreactivity, the major desialo N-CAM isoforms being 150, 145, 120, 115, and 110 kD. N-CAM expression appeared to be downregulated in the myocardium during adult life, with relatively little sarcolemmal immunoreactivity being detected in normal donor tissues. In contrast to the temporal changes observed in the myocardium, both the developing and mature cardiac innervation displayed N-CAM immunofluorescence staining, localized to neuronal cell bodies, nerve fascicles and fibres. Extrinsically denervated cardiac allografts, obtained 2 d to 91 mo after transplantation, showed extensive sarcolemmal and intercalated disc immunostaining and expression of 125-, 120-, and 115-kD isoforms. Tissues from explanted recipient hearts and atrial appendage samples obtained during coronary bypass graft operations were also examined and displayed varying amounts of N-CAM immunoreactivity. We conclude that the expression of N-CAM immunoreactivity and isoforms in the human heart is developmentally regulated and may be modulated by factors such as cardiac innervation and myocardial hypertrophy.

Authors

L Gordon, J Wharton, S E Moore, F S Walsh, J G Moscoso, R Penketh, J Wallwork, K M Taylor, M H Yacoub, J M Polak

Cell surface expression of the 70-kD component of Ku, a DNA-binding nuclear autoantigen.

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Abstract

The Ku complex, a heterodimer of 86- and 70-kD proteins, is a nuclear DNA-binding autoantigen. However, hydrophobicity analysis of the deduced amino acid sequence of the 70-kD protein had strongly suggested that this might also be a membrane protein. In the present study, using antibodies to synthetic peptides and a polyclonal antiserum to the purified protein, we show that the 70-kD protein of the Ku complex is present in isolated plasma membranes of human cells. By indirect immunofluorescence microscopy and fluorescein-activated cell sorting, we demonstrate that this autoantigen is exposed on the cell surface. In addition, we have identified several domains of the protein that are exposed. Our study provides one of the first demonstrations of a eukaryotic, nuclear DNA-binding protein that is also on the cell membrane. Moreover, our results might help explain how autoantibodies to the Ku autoantigen could target cells for an autoimmune attack.

Authors

B S Prabhakar, G P Allaway, J Srinivasappa, A L Notkins

Chylomicron remnant clearance from the plasma is normal in familial hypercholesterolemic homozygotes with defined receptor defects.

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Abstract

The retinyl palmitate fat tolerance test was used to measure chylomicron remnant clearance in 10 normal subjects (apolipoprotein E [apo E] isotypes 3 or 4 only), 6 normolipidemic apo E2/2 homozygotes and 5 familial hypercholesterolemic homozygotes. Skin fibroblasts with fully upregulated LDL receptors from the latter subjects degraded rabbit 125I-beta VLDL in vitro at rates ranging from less than 10-48% of normal. Experiments in vivo revealed no significant differences between the normal and homozygous familial hypercholesterolemic (FHH) subjects in chylomicron remnant clearance assessed on the basis of "areas under the curves" for retinyl palmitate levels present in post-prandial serum, chylomicron remnants (Sf. less than 1,000), or chylomicrons (Sf. greater than 1,000). Remnant clearance was greatly decreased at all times in the apo E2/2 homozygotes, indicative of an important degree of flux control exerted by a receptor-mediated step involving apo E as ligand. The absence of any excess remnant accumulation in FHH subjects with varying "impairment" of LDL receptor-mediated degradation of apo E-containing lipoproteins, permits the conclusion that chylomicron remnants are initially cleared from the plasma by apo E-recognizing receptors which are genetically distinct from LDL receptors.

Authors

D C Rubinsztein, J C Cohen, G M Berger, D R van der Westhuyzen, G A Coetzee, W Gevers

Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat.

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Abstract

In vivo in the rat 1,25(OH)2D3 decreases and a low calcium increases PTH mRNA levels. We now report the effect of 3 and 8 wk of changes in dietary vitamin D and calcium on PTH mRNA levels. PTH mRNA levels were increased by 3 wk of calcium deficiency (five times), a vitamin D-deficient diet (two times), and combined deficiency (10 times), but not changed by high calcium. Vitamin D-deficient-diet rats' PTH mRNA did not decrease after a single large dose of 1,25(OH)2D3, but did decrease partially after repeated daily doses of 1,25(OH)2D3. Rats after a vitamin D-, calcium-deficient (-D-Ca) diet did not respond to changes in serum calcium at 1 h. Flow cytometry of isolated cells from parathyroid-thyroid tissue separated the smaller parathyroid from the larger thyroid cells and allowed an analysis of parathyroid cell number. In normal vitamin D/normal calcium (NDNCa) rats the parathyroid cells were 24.7 +/- 3.4% (n = 6) of the total cell number, whereas in -D-Ca rats they were 41.8 +/- 6.6% (n = 6) (P less than 0.05). That is, -D-Ca rats had 1.7 times the number of cells, whereas they had 10 times the amount of PTH mRNA, indicating the major contribution (6 times) of increased PTH gene expression per cell. Moreover, a calcium-deficient, more so than a vitamin D-deficient diet, amplifies the expression of the PTH gene, and vitamin D is necessary for an intact response of PTH mRNA to 1,25(OH)2D3 or calcium.

Authors

T Naveh-Many, J Silver

The complete nucleotide sequences of the heavy chain variable regions of six monospecific rheumatoid factors derived from Epstein-Barr virus-transformed B cells isolated from the synovial tissue of patients with rheumatoid arthritis. Further evidence that some autoantibodies are unmutated copies of germ line genes.

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Abstract

Structural studies of human monoclonal rheumatoid factors (RF) derived from patients with monoclonal gammapathies have revealed a restriction in the usage of heavy and light chain variable regions. These studies have suggested that germline genes with little if any somatic mutation can generate RF specificity. However, there is no information presently available regarding the primary structure and genetic origin of RF in rheumatoid arthritis. In this study, we have isolated and sequenced the VH regions of six monoclonal RF derived from the synovial membranes of two patients with rheumatoid arthritis and one with the juvenile polyarticular form of the disease. We found the same VH families as previously reported among monoclonal paraproteins with RF activity. However, our sample was diverse regarding the VH, DH, and JH gene segments used. Among VHI RF there was conservation in the length of CDRIII as well as restriction in the amino acid generated at the V-D junction, as opposed to VHIII RF and non-RF VHI molecules that are highly heterogeneous in these two aspects. We also found that different JH gene segments may contribute to RF specificity. The VH, DH, and JH elements of one RF in our study all had clearly identifiable germline counterparts. This RF displays a nearly germline configuration throughout its entire heavy chain and represents another example of an autoantibody encoded by one of the VH gene segments from the preimmune fetal repertoire.

Authors

V Pascual, I Randen, K Thompson, M Sioud, O Forre, J Natvig, J D Capra

Biogenesis of intestinal lactase-phlorizin hydrolase in adults with lactose intolerance. Evidence for reduced biosynthesis and slowed-down maturation in enterocytes.

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Abstract

Enzymatic activity, biosynthesis, and maturation of lactasephlorizin hydrolase (LPH) were investigated in adult volunteers with suspected lactose intolerance. Mean LPH activity in jejunal biopsy homogenates of these individuals was 31% compared to LPH-persistent individuals, and was accompanied by a reduced level of LPH-protein. Mean sucrase activity in individuals with low LPH was increased to 162% and was accompanied by an increase in sucrase-isomaltase (SI)-protein. Biosynthesis of LPH, SI, and aminopeptidase N (APN) was studied in organ culture of small intestinal biopsy specimens. In individuals with LPH restriction, the rate of synthesis of LPH was drastically decreased, reaching just 6% of the LPH-persistent group after 20 h of culture, while the rate of synthesis of SI appeared to be increased. In addition, maturation of pro-LPH to mature LPH occurred at a slower rate in LPH-restricted tissue. Immunoelectron microscopy revealed an accumulation of immunoreactive LPH in the Golgi region of enterocytes from LPH-restricted individuals and reduced labeling of microvillus membranes. Therefore, lactose intolerance in adults is mainly due to a decreased biosynthesis of LPH, either at the transcriptional or translational level. In addition, intracellular transport and maturation is retarded in some of the LPH-restricted individuals, and this leads to an accumulation of newly synthesized LPH in the Golgi and a failure of LPH to reach the microvillus membrane.

Authors

E E Sterchi, P R Mills, J A Fransen, H P Hauri, M J Lentze, H Y Naim, L Ginsel, J Bond

The biosynthetic basis of adult lactase deficiency.

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Abstract

The intestinal brush-border enzyme lactase splits lactose into its component monosaccharides, glucose and galactose. Relative deficiency of the enzyme during adulthood is a common condition worldwide and is frequently associated with symptoms of lactose intolerance. We studied the synthesis and processing of lactase in normal and adult hypolactasic subjects using human intestinal explants in organ culture. Metabolic labeling experiments in our control subjects with [35S]methionine followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, and fluorography demonstrated that newly synthesized lactase is initially recognized as a precursor molecule with a relative molecular weight (Mr) of 205,000. Over the course of several hours most of the labeled lactase was converted to a mature form of 150,000 Mr. Transiently appearing forms of 215,000 and 190,000 Mr were identified and were felt to represent intermediary species generated during intracellular processing. We identified two distinct alterations in lactase biosynthesis accounting for adult hypolactasia. Studies in three deficient subjects demonstrated markedly reduced synthesis of the precursor protein though posttranslational processing appeared identical to normal. Multiple studies in a fourth deficient subject demonstrated synthesis of ample amounts of precursor lactase but reduced conversion to the mature active form of the enzyme.

Authors

J Witte, M Lloyd, V Lorenzsonn, H Korsmo, W Olsen

An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels.

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Abstract

A polymorphism consisting of the presence or absence of a 250-bp DNA fragment was detected within the angiotensin I-converting enzyme gene (ACE) using the endothelial ACE cDNA probe. This polymorphism was used as a marker genotype in a study involving 80 healthy subjects, whose serum ACE levels were concomitantly measured. Allele frequencies were 0.6 for the shorter allele and 0.4 for the longer allele. A marked difference in serum ACE levels was observed between subjects in each of the three ACE genotype classes. Serum immunoreactive ACE concentrations were, respectively, 299.3 +/- 49, 392.6 +/- 66.8, and 494.1 +/- 88.3 micrograms/liter, for homozygotes with the longer allele (n = 14), and heterozygotes (n = 37) and homozygotes (n = 29) with the shorter allele. The insertion/deletion polymorphism accounted for 47% of the total phenotypic variance of serum ACE, showing that the ACE gene locus is the major locus that determines serum ACE concentration. Concomitant determination of the ACE genotype will improve discrimination between normal and abnormal serum ACE values by allowing comparison with a more appropriate reference interval.

Authors

B Rigat, C Hubert, F Alhenc-Gelas, F Cambien, P Corvol, F Soubrier

Failure of T cell receptor-anti-CD3 monoclonal antibody interaction in T cells from marrow recipients to induce increases in intracellular ionized calcium.

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Abstract

There are multiple immune defects in T cells from recipients after bone marrow transplantation (BMT). This study examines recipient T cells for increases in intracellular ionized calcium concentration [( Ca2+]i) after binding the T cell receptor-CD3 complex with anti-CD3 MAb. PBL from 10 of 23 short-term recipients (less than 1 yr after BMT) responded poorly (less than 35% of control) to anti-CD3 stimulation and PBL from 9 of 23 had blunted calcium flux responses (35-70% of control). Purified CD2+, CD56- cells from seven additional short-term recipients including three autologous marrow recipients were closely examined, and a sizable proportion of CD3+ cells from six of seven recipients did not increase [Ca2+]i after anti-CD3 stimulation. The decreased magnitude of the responses was due to decreased numbers of responding cells and not to a decrease in mean CD3 fluorescent intensity or in calcium flux responses on a single cell basis. Five of seven long-term recipients (greater than 1 yr after BMT) had PBL that responded normally and two of seven had PBL with blunted calcium flux responses. The data show that the signal transduction response mediated by the CD3-antigen receptor as measured by calcium flux is defective early after autologus or allogeneic BMT.

Authors

M Yamagami, P W McFadden, S M Koethe, V Ratanatharathorn, L G Lum

Endogenous angiotensin concentrations in specific intrarenal fluid compartments of the rat.

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Abstract

To examine angiotensin (ANG) concentrations in fluid compartments near known intrarenal ANG receptors, we measured ANG concentrations in glomerular filtrate (GF), star vessel plasma (SVP), and luminal fluid from the early, mid, and late proximal tubule (E, M, and L PT). Samples were collected from euvolemic Munich-Wistar rats by free-flow micropuncture; ANG concentrations were measured by RIA. In one group of rats, concentrations of total immunoreactive ANG (reflecting ANG II and lesser amounts of three fragments) in GF and E, M, and L PT fluid averaged 29-40 nM compared with 32 pM in systemic plasma. In a second group, immunoreactive ANG concentrations in SVP also exceeded systemic levels by a factor of 1,000. In a final group, samples of GF and LPT fluid were purified by HPLC before RIA to measure ANG II and III concentrations specifically: their respective concentrations were 6-8 nM and 14-25 nM. We interpret these results to indicate that substantial amounts of ANG peptides are released into or generated within intrarenal fluid compartments, in which local ANG is likely to effect regulation of renal function independently of systemic ANG.

Authors

M G Seikaly, B S Arant Jr, F D Seney Jr

Oligoclonal T cell receptor gene rearrangements in blood lymphocytes of patients with acute Epstein-Barr virus-induced infectious mononucleosis.

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Abstract

Gene rearrangement studies were performed on blood lymphocytes from eight patients with acute Epstein-Barr virus-induced infectious mononucleosis. The diagnosis in each case was based on characteristic clinical, hematologic, and serologic findings. The blood lymphocytes in each patient consisted predominantly of CD8+ T cells. EBV DNA was detected in seven patients by Southern blot analysis (EBV Bam HI W probe, Bam HI). A germline configuration was found for the immunoglobulin heavy and light chain genes (JH probe, Bam HI and Eco RI; C kappa probe, Bam HI; and C lambda probe, Eco RI). T cell receptor gene rearrangements were detected with J gamma and J beta 1 + 2 probes. Using a J gamma probe with two different restriction enzymes (Bgl II and Eco RI), the blood from each patient showed several bands corresponding to the polyclonal pattern previously described in the blood of normal individuals. Using J beta 1 + 2 probes with two different restriction enzymes (Bgl II and Bam HI), each case showed from 3 to about 12 extragermline bands of varying intensity and in different locations from case to case. In addition, each case showed relative deletion of the J beta 1 germline band. This oligoclonal pattern of T cell receptor gene rearrangements has not been previously reported in benign or malignant T cell populations.

Authors

J G Strickler, L A Movahed, K J Gajl-Peczalska, C A Horwitz, R D Brunning, L M Weiss

Platelet alpha-granule fibrinogen, albumin, and immunoglobulin G are not synthesized by rat and mouse megakaryocytes.

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Abstract

It has been assumed that endogenous synthesis by the platelet precursor cell, the bone marrow megakaryocyte, is the major source of platelet alpha-granule protein. To test this hypothesis, we used mRNA phenotyping to detect in megakaryocytes the presence of mRNA transcripts specific for various proteins. Our results indicate that megakaryocytes synthesize platelet factor 4, a protein relatively specific for platelets, but do not express mRNA transcripts for the fibrinogen, albumin, or IgG found in alpha-granules. We have previously shown that megakaryocytes endocytose circulating proteins, including fibrinogen, albumin, and IgG, and incorporate them into alpha-granules. Thus, platelets appear to contain a unique type of secretory granule whose contents originate by both endogenous synthesis and endocytosis from plasma. Under basal conditions, the source of alpha-granule fibrinogen is plasma.

Authors

P Handagama, D A Rappolee, Z Werb, J Levin, D F Bainton

In vivo activation by ultraviolet rays of the human immunodeficiency virus type 1 long terminal repeat.

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Abstract

It has been previously shown in vitro that the human immunodeficiency virus type 1 long terminal repeat (LTR) is activated by ultraviolet irradiation. In order to analyze if a similar effect could occur in vivo, transgenic mice carrying the lacZ gene under the control of the viral LTR were irradiated at 280-300 and 254 nm. These mice spontaneously expressed the transgene in the epidermis and the lens of both adults and embryos. Irradiations caused a significant increase in skin beta-galactosidase activity. This phenomenon might be involved in viral activation and could be of interest in regard to the skin pathology observed during an HIV infection.

Authors

C Cavard, A Zider, M Vernet, M Bennoun, S Saragosti, G Grimber, P Briand

Selective regulation of cellular cyclooxygenase by dexamethasone and endotoxin in mice.

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Abstract

We have studied the effect of glucocorticoids administered in vivo on the activity and synthesis of the cyclooxygenase enzyme (COX) in mice treated with or without concurrent intravenous administration of LPS. Mouse peritoneal macrophages from LPS-treated animals showed a two to three fold increase in COX activity determined by the production of PGE2 and PGI2 after stimulation of the cells with exogenous arachidonate. Dexamethasone injected simultaneously with LPS, 12 h before killing of the animal and removal of the macrophages, completely blocked the LPS-induced increase COX activity in peritoneal macrophages. The regulation observed in COX activity by LPS and dexamethasone are due primarily to changes in COX mass as determined by immunoprecipitation of [35S]methionine endogenously labeled enzyme. In contrast, the COX present in the nonadherent cells and in renal medullary microsomes obtained from the same animals, showed no significant changes between treatments. These results indicate that LPS in vivo stimulates COX synthesis in the peritoneal macrophages but not in the kidney. The effect of dexamethasone to inhibit COX synthesis is selective to the LPS-induced enzyme.

Authors

J L Masferrer, B S Zweifel, K Seibert, P Needleman