Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na⁺/H⁺ exchange

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Molecular basis of cardiac performance. Plasticity of the myocardium generated through protein isoform switches.

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B Nadal-Ginard, V Mahdavi

Regulation of interleukin 3 gene induction in normal human T cells.

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The regulation of IL-3 gene induction in human peripheral blood T cells was studied. IL-3 gene expression was inducible by crosslinking of the T cell receptor/CD3 complex using anti-CD3 MAb G19-4. Anti-CD3-induced IL-3 gene expression was found to be limited to the CD28+ T cell subset and could be augmented by costimulating T lymphocytes with antibodies directed against CD28. IL-3 expression could also be induced by costimulation of T cells with both phorbol ester and ionomycin, which are thought to mimic the intracellular effects of T cell receptor-antigen interaction. However, unlike other lymphokines such as IL-2 or granulocyte-macrophage colony-stimulating factor, IL-3 gene expression is not induced by stimulation of cells with phorbol myristate acetate and anti-CD28. We conclude that IL-3 gene regulation is under stringent control since IL-3 gene expression occurs only in the CD28+ subset of T cells, and since IL-3 induction obligately requires increased intracellular calcium.

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S C Guba, G Stella, L A Turka, C H June, C B Thompson, S G Emerson

Synthesis of a novel cytokine and its gene (LD78) expressions in hematopoietic fresh tumor cells and cell lines.

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The gene coding for the protein LD78 was isolated from stimulated human tonsillar lymphocytes by differential hybridization. The gene product consisted of 92 amino acids with characteristics of cytokines. LD78 gene transcripts were detected in eight of eight fresh samples of cells from patients with acute nonlymphocytic leukemia (ANLL) by Northern blot analysis. ANLL cells with monocytic features gave the strongest bands. RNA transcripts were found in two of three samples of cells from patients with adult T cell leukemia (ATL), eight of nine samples from patients with acute lymphocytic leukemia (ALL) of B cell lineage, and one of the three samples from patients with T cell ALL. KG-1, HL-60, HUT 102, MT-2, and MJ cell lines expressed the LD78 gene constitutively. The LD78 protein was detected in culture supernatants and cell lysates of HUT 102, MT-2, MJ, and fresh ATL cells by Western blot analysis. This protein was not found in culture supernatants or cell lysates of monocytic leukemia cells and HL-60 cells, although LD78 transcripts were found in those cells. The discrepancy between gene and protein expression might be explained by the stability of the mRNA. Thus, the protein may be involved in the neoplastic transformation of hematopoietic cells.

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Y Yamamura, T Hattori, K Obaru, K Sakai, N Asou, K Takatsuki, Y Ohmoto, H Nomiyama, K Shimada

The reactivity of desmosterol and other shellfish- and xanthomatosis-associated sterols in the macrophage sterol esterification reaction.

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The acyl-CoA: cholesterol acyl transferase (ACAT) reaction in macrophages is a critical step in atherosclerotic foam cell formation, but little is known about the reaction's sterol substrate specificity. In this report we examine the macrophage ACAT reactivity of the shellfish sterol, desmosterol, and other sterols found in man because of shellfish ingestion or in association with the foam cell diseases sitosterolemia and cerebrotendinous xanthomatosis (CTX). We first show that the J774 macrophage, a foam cell model with a hyperactive ACAT pathway, synthesizes desmosterol instead of cholesterol and that both endogenous and exogenous desmosterol are substrates and stimulators of the ACAT reaction in these cells. When exogenous desmosterol was added to human monocyte-derived macrophages, ACAT was stimulated 29- and 4-fold compared with control and cholesterol-treated cells, respectively. Steryl ester mass accumulation in desmosterol-treated human macrophages was 10-fold greater than in control cells and 3-fold greater than in cholesterol-treated cells. Another shellfish sterol, 24-methylene cholesterol, also stimulated ACAT in human macrophages, but most of the xanthomatosis-related sterols did not stimulate ACAT. These data suggest that: (a) the shellfish sterols desmosterol and 24-methylene cholesterol may be atherogenic; and (b) the excessive foam cell formation seen in sitosterolemia and CTX cannot be explained by ACAT hyperreactivity of their associated sterols.

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I Tabas, S J Feinmark, N Beatini

Apolipoprotein A-I variants. Naturally occurring substitutions of proline residues affect plasma concentration of apolipoprotein A-I.

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Six unrelated families with genetically determined structural variants of apo A-I were found in the course of an electrophoretic screening program for apo A-I variants in dried blood samples of newborns. The following structural variations were identified by the combined use of HPLC, time-of-flight secondary ion mass spectrometry (TOF-SIMS), and automated gas phase sequencing: Pro3----Arg (1x), Pro4----Arg (1x), and Pro165----Arg (4x). All variant carriers were heterozygous for their mutant of apo A-I. Subjects heterozygous for apo A-I(Pro165----Arg) (n = 12) were found to exhibit lower mean values for apo A-I (109 +/- 16 mg/dl) and HDL cholesterol (37 +/- 9 mg/dl) than unaffected family members (n = 9): 176 +/- 41 and 64 +/- 18 mg/dl, respectively (P less than 0.001). In 9 of 12 apo A-I(Pro165----Arg) variant carriers the concentrations of apo A-I were below the fifth percentile of sex-matched controls. By two-dimensional immunoelectrophoresis as well as by densitometry the relative concentration of the variant apo A-I in heterozygous carriers of apo A-I(Pro165----Arg) was determined to account for only 30% of the total plasma apo A-I mass instead of the expected 50%. Thus, the observed apo A-I deficiency may be largely a consequence of the decreased concentration of the variant apo A-I. In the case of the apo A-I(Pro3----Arg) mutant, densitometry of HDL apolipoproteins demonstrated a distinctly increased concentration of the variant proapo A-I relative to normal proapo A-I. This phenomenon was not observed in the apo A-I(Pro4----Arg) mutant or in other mutants. This suggests that the interspecies conserved proline residue in position 3 of mature apo A-I is functionally important for the regular enzymatic conversion of proapo A-I to mature apo A-I.

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A von Eckardstein, H Funke, A Henke, K Altland, A Benninghoven, G Assmann

Transin/stromelysin expression in the synovium of rats with experimental erosive arthritis. In situ localization and kinetics of expression of the transformation-associated metalloproteinase in euthymic and athymic Lewis rats.

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Transin is a neutral metalloproteinase initially isolated from malignantly transformed rat fibroblasts and subsequently shown to be homologous to human stromelysin. We performed Northern blot analysis on synovial tissue specimens from Lewis rats with proliferative and invasive streptococcal cell wall (SCW) arthritis. Transin mRNA was present in abundance, as was the mRNA of the c-myc oncogene, which is associated with cellular proliferation. Immunohistochemical staining of synovia from rats with chronic SCW arthritis showed high-level transin expression in the cells of the lining layer and underlying stroma, as well as in chondrocytes and osteoclasts in subchondral bone. Intense nuclear staining for the Myc oncoprotein was also detected with a cross-reactive antibody to v-Myc. Transin stained similarly in the early, rapid-onset, thymus-independent, acute phase of SCW arthritis. In the T cell-dependent adjuvant arthritis, transin expression was noted by day 4, 6 d before the influx of mononuclear cells and the onset of clinical disease. Athymic rats did not express transin. We concluded that transin is a marker of proliferative, invasive arthritis in rats and appears early in the course of disease development, but requires a competent immune system to sustain its expression in these model arthropathies.

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J P Case, H Sano, R Lafyatis, E F Remmers, G K Kumkumian, R L Wilder

Chronic norepinephrine elicits desensitization by uncoupling the beta-receptor.

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The goal of this study was to determine the mechanism of beta-adrenergic receptor desensitization after chronic elevation of circulating NE levels. Osmotic minipumps containing either NE or saline were implanted subcutaneously in dogs for 3-4 wk. Physiologic desensitization to isoproterenol was confirmed in conscious dogs, i.e., left ventricular dP/dt increased in response to isoproterenol (0.4 micrograms/kg per min) by 5,625 +/- 731 mmHg/s in control dogs with saline pumps, and significantly less, P less than 0.01, by 2,093 +/- 263 mmHg/s in dogs with NE pumps. Myocardial beta-adrenergic receptor density as determined with 125I-cyanopindolol binding was 49% higher (p less than 0.05) in the NE pump group. However, beta-adrenergic receptor agonist binding with isoproterenol demonstrated a significant shift into the low affinity state for the animals with NE pumps. Basal, GTP plus isoproterenol, 5'-guanylylimidodiphosphate, sodium fluoride, and forskolin-stimulated adenylate cyclase activity in the NE pump group were significantly depressed (P less than 0.05) by amounts ranging from 20 to 40%. The functional activity of the guanine nucleotide binding protein Gs was also reduced (P less than 0.05) in animals with NE pumps. Thus, the process of desensitization in response to chronic elevation of NE levels in intact, normal dogs does not involve a decrease in beta-adrenergic receptor density. Rather, it is characterized by reduced adenylate cyclase activation and uncoupling of the beta-adrenergic receptor in association with decreased activity of the GTP-coupling protein Gs.

Authors

D E Vatner, S F Vatner, J Nejima, N Uemura, E E Susanni, T H Hintze, C J Homcy

Genetic and environmental factors that regulate cytosolic epoxide hydrolase activity in normal human lymphocytes.

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To determine whether genetic mechanisms control large variations in cytosolic epoxide hydrolase (cEH) activity of unstimulated lymphocytes from normal human subjects, cEH activity was measured in (a) 6 sets of monozygotic (MZ) twins and 6 sets of dizygotic (DZ) twins; (b) 100 unrelated male subjects; and (c) 6 families. The twin study revealed predominantly genetic control (H2(1) = 0.95). Variability was markedly less within MZ (intrapair variance = 0.25) than DZ twins (intrapair variance = 6.33). In 100 unrelated male subjects the extent of interindividual variation was 11-fold. Unimodal distribution of values among 99 subjects encompassed a sixfold range. One outlier with very high activity clearly stood apart. Using the whole distribution curve we phenotyped members of six families. In the outlier's family, analysis of three generations suggested autosomal dominant transmission of high cEH activity. Analysis of the other 5 families and of 12 sets of twins, all from the large unimodal distribution, was consistent with either monogenic or polygenic control of variations within this mode. Several temporal host factors, including fever, the menstrual cycle, a 24-h fast, and diurnal variations, were investigated. Fever and fasting elevated cEH activity. Diurnal variations produced no observable alteration. During the menstrual cycle irregular fluctuations occurred.

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K K Norris, T M DeAngelo, E S Vesell

Epidermal growth factor enhances renal tubule cell regeneration and repair and accelerates the recovery of renal function in postischemic acute renal failure.

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To determine the timing and location of renal cell regeneration after ischemic injury to the kidney and to assess whether exogenous epidermal growth factor (EGF) enhances this regenerative repair process to accelerate recovery of renal function, experiments were undertaken in rats undergoing 30 min of bilateral renal artery clamp ischemia followed by reperfusion for varying time intervals. Renal cell regeneration, as reflected by incorporation of radiolabeled thymidine within the kidney, began between 24 to 48 h and reached a peak at 72 h after renal ischemia. As demonstrated by histoautoradiography, renal thymidine incorporation was essentially confined to tubule cells. Morphometric analysis of histoautoradiograph sections of renal tissue demonstrated that the majority of labeled cells were found in renal cortex, but some labeled cells were also located in the inner stripe of the outer medulla, suggesting that injury to medullary thick ascending limbs also occurs in this ischemic model. Exogenous EGF administration produced increases in renal thymidine incorporation compared with non-treated animals at 24, 48, and 72 h after ischemic injury. This accelerated DNA replicative process was associated with significantly lower peak blood urea nitrogen (BUN) and serum creatinine levels, averaging 63 +/- 20 and 3.1 +/- 0.4 mg/dl in EGF-treated ischemic rats compared with 149 +/- 20 and 5.1 +/- 0.1 mg/dl, respectively, in nontreated ischemic rats, and was also associated with a return to near normal BUN and serum creatinine levels in EGF-treated animals approximately 4 d earlier than that observed in nontreated animals. This report is the first demonstration that EGF accelerates the repair process of a visceral organ after an injurious insult.

Authors

H D Humes, D A Cieslinski, T M Coimbra, J M Messana, C Galvao

An arginine to glutamine mutation in residue 109 of human ornithine transcarbamylase completely abolishes enzymatic activity in Cos1 cells.

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Ornithine transcarbamylase (OTC) is an important enzyme in the detoxification of ammonia to urea, and its deficiency is the most common inborn error of ureagenesis in humans. Among 24 cases of OTC deficiency previously examined, three unrelated individuals all showed loss of a Taq I site in the OTC gene corresponding to codon 109, suggesting that this Taq I site may be prone to mutation. Two of these patients demonstrated the same C----T transition (in antisense strand) converting Arg109 to Gln. Although these studies implied a strong association between the missense mutation and OTC-deficient phenotype, a causal relationship could not be firmly established. We have investigated this relationship by reconstructing the mutation in vitro. A full-length human OTC cDNA was cloned into an SV40-based expression vector and has been reproducibly expressed at high levels in the cell line Cos1. By site-directed mutagenesis of this wild type sequence, we constructed a missense mutation which contains the C----T transition. Electroporation and transient assay in Cos1 indicated that the specific activity of mutant OTC was 100-fold lower than that of wild type. This result confirms that the Taq I alteration leading to the Gln missense is responsible for the OTC deficiency affecting the above patients.

Authors

J T Lee, R L Nussbaum

Induction of hypertrophy in cultured proximal tubule cells by extracellular NH4Cl.

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Ammonia production increases in several models of renal hypertrophy in vivo. The present study was designed to determine whether ammonia can directly modulate the growth of renal cells in the absence of a change in extracellular acidity. In serum-free media NH4Cl (0-20 mM) caused JTC cells and a primary culture of rabbit proximal tubule cells to hypertrophy (increase in cell protein content) in a dose-dependent fashion without a change in DNA synthesis. Studies in JTC cells revealed that the cell protein content increased as a result of both an increase in protein synthesis and a decrease in protein degradation. Total cell RNA content and ribosome number increased after NH4Cl exposure and the cell content of the lysosomal enzymes cathepsin B and L decreased. Inhibition of the Na+/H+ antiporter with amiloride did not prevent the hypertrophic response induced by NH4Cl. The results indicate that ammonia is an important modulator of renal cell growth and that hypertrophy can occur in the absence of functioning Na+/H+ antiport activity.

Authors

K Golchini, J Norman, R Bohman, I Kurtz

Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors.

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Hepatic lipocytes appear to be central to the pathogenesis of hepatic fibrosis, undergoing activation during inflammation to a matrix-producing, proliferative cell type. We have studied the activation process in culture by examining the response of lipocytes to conditioned medium from hepatic macrophages (Kupffer cells). Lipocytes exposed to Kupffer cell medium (KCM) exhibited cellular and nuclear enlargement associated with up to a threefold increase in collagen and total protein synthesis per cell. Cell proliferation was also stimulated as measured by [3H]thymidine incorporation and direct cell counting. The latter effect was serum dependent and inhibited by antibodies to platelet-derived growth factor (PDGF). Proliferation could be stimulated by recombinant PDGF, but only after preincubation of cells with KCM. These findings suggested that KCM was eliciting expression of the PDGF receptor in lipocytes, and this was confirmed by immunoblot analysis with antibodies to the PDGF receptor. DNA synthesis in lipocytes exposed to KCM occurred at 48 h, which reflected the time required for PDGF receptor expression (24 h) plus initiation of [3H]thymidine incorporation (24 h). These results indicate that KCM has multiple stimulatory effects on cultured lipocytes similar to activation of these cells observed in vivo.

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S L Friedman, M J Arthur

Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells.

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In vitro and in vivo studies suggest that liver fat-storing cells (FSC) may play an important role in the development of liver fibrosis. We explored the effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and TGF-beta, and basic fibroblast growth factor (bFGF) on DNA synthesis and growth of rat liver FSC. PDGF, EGF, TGF-alpha, and bFGF induced a dose-dependent increase in DNA synthesis with a peak effect at 24 h. PDGF produced the most striking effect with a maximum 18-fold increase over control. EGF, TGF-alpha, and bFGF elicited a maximum three- to fourfold increase in DNA synthesis. Analysis of growth curves revealed a similar pattern of potency of the growth factors. TGF-beta did not affect DNA synthesis of FSC; however, TGF-beta markedly potentiated the stimulatory effects of both EGF and PDGF. FSC showed high specific binding of 125I-PDGF and Scatchard analysis revealed high affinity receptors with an apparent Kd of 2.3 x 10(-10) M. Our data suggest that PDGF is a key mitogen for FSC and that the coordinate release of other growth factors together with PDGF by inflammatory cells represents a potent potential stimulus for FSC proliferation in conditions of chronic self-perpetuating liver inflammation.

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M Pinzani, L Gesualdo, G M Sabbah, H E Abboud

Nonopsonic antibodies in cystic fibrosis. Pseudomonas aeruginosa lipopolysaccharide-specific immunoglobulin G antibodies from infected patient sera inhibit neutrophil oxidative responses.

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Antibody opsonins from cystic fibrosis (CF) patients were investigated using nonmucoid and mucoid lipopolysaccharide (LPS) immunotype 1 Pseudomonas aeruginosa as bacterial ligands and PMN phagocytes. CF sera were compared to normal sera, polyvalent PA LPS hyperimmune globulin, and isotype switch variant monoclonal antibodies (MAbs) specific for type 1 PA LPS. Sera from PA-infected CF patients (CF PA+) had elevated levels of PA LPS and alginate IgG antibodies and promoted significantly greater antibody-dependent PMN chemiluminescence responses than sera from uninfected CF patients (CF PA-) or normal human sera (NHS). After adjustment for autologous IgG PA LPS antibody content, however, CF PA+ sera had less antibody-dependent opsonic activity than sera from CF PA- patients (P less than 0.025) or NHS (P less than 0.0025), suggesting qualitative opsonic defects of IgG PA LPS antibodies in CF PA+ sera. Antigen-specific immunoprecipitation of PA LPS antibodies enhanced opsonization by 40% of CF PA+ sera while uniformly reducing that from CF PA- sera (P less than 0.01), indicating LPS-specific nonopsonic antibodies in some CF PA+ sera. Alginate antibodies were not critical opsonins in most uninfected CF patient sera. PA LPS IgG antibodies isolated by immunoaffinity chromatography from NHS, hyperimmune globulin, and CF PA- sources were opsonic and had greater activity at equal antigen-binding concentration than identical antibodies isolated from infected CF patients (P less than 0.01-0.05); the majority of isolates from CF PA+ sera did not promote PMN oxidative responses above nonopsonic baseline. A potential isotypic basis for these findings was supported by differences in PMN responses to PA opsonized with MAbs of identical specificity but differing isotypes. PA LPS-specific IgG antibodies inhibiting PMN oxidative responses in infected patient sera demonstrate antigen-specific immunomodulation of host responses by chronic bacterial parasitism in CF, which may play a role in the pathophysiology of lung disease.

Authors

I Eichler, L Joris, Y P Hsu, J Van Wye, R Bram, R Moss

Cytochrome P450 metabolites of arachidonic acid are potent inhibitors of vasopressin action on rabbit cortical collecting duct.

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AA is metabolized by a cytochrome P450, NADPH-dependent epoxygenase to four regioisomeric epoxyeicosatrienoic acids (EETs). The EETs are further hydrated enzymatically to their respective diols, vic-dihydroxyeicosatrienoic acids (DHETs). We studied the effect of pretreatment with DHETs on 10 microU/cm2 arginine vasopressin (AVP)-stimulated hydraulic conductivity (Lp) (Lp x 10(-7) cm/atm/s, mean +/- SE) in rabbit cortical collecting ducts (CCDs) perfused in vitro at 37 degrees C. At 10(-6) M all four DHETs were potent inhibitors of the hydroosmotic effect of AVP. 14,15-DHET was the most potent isomer; it reduced AVP-induced Lp from a control value of 234.75 +/- 11.7, n = 17, to a value of 95.2 +/- 8.39, n = 5, P less than 0.0001, a reduction of AVP-mediated water flow of 60%. The inhibitory effect of 14,15-DHET was dose dependent and significant to nanomolar concentrations. 14,15-DHET at 10(-7) M was as potent an inhibitor of AVP's activity as was 10(-7) M PGE2. AVP's hydroosmotic effect is mediated through its intracellular second messenger, cAMP. 8-p-Chlorophenylthio-cAMP (CcAMP) at 10(-4) M induced a peak Lp of 189.6 +/- 11.0, n = 8; pretreatment with 10(-6) M 14,15-DHET reduced CcAMP-peak Lp to 132.0 +/- 13.4, n = 5, P less than 0.01, demonstrating a post-cAMP effect. Gas chromatography/mass spectroscopy suggests that EETs are present in extracts purified from CCDs. We conclude that cytochrome P450 epoxygenase eicosanoids are potent inhibitors of the hydroosmotic effect of vasopressin and are endogenous constituents of normal CCDs, the major target tissue for AVP.

Authors

D L Hirt, J Capdevila, J R Falck, M D Breyer, H R Jacobson

Macrophage/monocyte receptor for nonenzymatically glycosylated protein is upregulated by cachectin/tumor necrosis factor.

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Proteins of extracellular matrix undergo over time multiple reactions with glucose to form advanced glycosylation endproducts (AGEs) which are highly active in protein crosslinking, and have been implicated in tissue damage associated with aging and diabetes. A macrophage/monocyte receptor for AGE moieties mediates the uptake of AGE-modified proteins by a process that also induces cachectin/tumor necrosis factor (TNF) and IL-1 secretion. Reasoning that cytokines might regulate this AGE-receptor system, we have evaluated the effect of cachectin/TNF, IL-1, and IFN-gamma on AGE-protein processing. We report that cachectin/TNF induced a severalfold enhancement of binding, endocytosis, and degradation of AGE-BSA by both murine peritoneal macrophages and human blood monocytes in vitro, and that cachectin/TNF enhanced the rate of disappearance of AGE-modified red blood cells in vivo. IL-1 and IFN-gamma alone did not increase AGE processing, but IFN-gamma consistently enhanced cachectin/TNF-induced changes in AGE-receptor kinetics. Similar effects were induced by AGE-BSA and FFI-BSA, a chemically synthesized AGE, when used as macrophage stimulants, possibly via cachectin/TNF induction. All upregulatory responses were blocked by anticachectin/TNF monoclonal antibody. These data suggest that AGE-induced cachectin/TNF, in addition to influencing tissue regeneration and remodelling, may also normally regulate the disposal of tissue damaging AGE-proteins through an autocrine upregulation.

Authors

H Vlassara, L Moldawer, B Chan

Human mannose-binding protein activates the alternative complement pathway and enhances serum bactericidal activity on a mannose-rich isolate of Salmonella.

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The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains, a cysteine-rich NH2-terminal domain that stabilizes the collagen alpha helix of the second domain and a third COOH-terminal carbohydrate recognition domain. Previous studies have shown that both native and recombinant human MBP bind to wild-type virulent Salmonella montevideo that expresses a mannose-rich lipopolysaccharide. Interaction with MBP results in opsonization and killing by phagocytes. In this report we show that low concentration of MBP (less than 10 micrograms/ml) markedly enhance complement deposition via the alternative complement pathway on S. montevideo. Despite structural similarities between MBP and the C1q subcomponent of the first complement component, MBP did not restore classical pathway activity to C1q-deficient serum, nor did it activate C1s when added to a mixture of C1r and C1s. In the presence of MBP the C3 bound to S. montevideo during incubation in serum was in the form of C3b and iC3b at a ratio of 1:2. Presensitization of S. montevideo with MBP rendered this normally serum resistant organism susceptible to complement-mediated killing. These results emphasize that MBP and complement cooperate in first line defense of the nonimmune host.

Authors

J E Schweinle, R A Ezekowitz, A J Tenner, M Kuhlman, K A Joiner

Defective in vitro production of gamma-interferon and tumor necrosis factor-alpha by circulating T cells from patients with the hyper-immunoglobulin E syndrome.

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Circulating T cells from four patients with the hyper-IgE syndrome were found to produce significantly lower concentrations of interferon-gamma (IFN-gamma) in response to stimulation with phytohemagglutinin (PHA) than did T cells from eight age-matched healthy controls, three patients with atopic dermatitis and one patient with chronic granulomatous disease. A clonal analysis revealed that patients with hyper-IgE syndrome had markedly lower proportions of circulating T cells able to produce IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) in comparison with controls. In contrast, the proportions of peripheral blood T cells able to produce IL-4 or IL-2 were not significantly different in patients and controls. All the four patients with hyper-IgE syndrome showed high proportions of circulating CD4+ helper T cells able to induce IgE synthesis in allogeneic B cells, as well. Such an activity for IgE synthesis appeared to be positively correlated with IL-4 production by T cells and inversely related to the ability of the same T cells to produce IFN-gamma. Since IFN-gamma exerts an inhibitory effect on the synthesis of IgE and both IFN-gamma and TNF-alpha play an important role in inflammatory reactions, we suggest that the defective production of IFN-gamma may be responsible for hyperproduction of IgE and the combined defect of IFN-gamma and TNF-alpha may contribute to the undue susceptibility to infections seen in patients with hyper-IgE syndrome.

Authors

G Del Prete, A Tiri, E Maggi, M De Carli, D Macchia, P Parronchi, M E Rossi, M C Pietrogrande, M Ricci, S Romagnani

Coordinate regulation of transforming growth factor beta gene expression and cell proliferation in hamster lungs undergoing bleomycin-induced pulmonary fibrosis.

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The number of mesenchymal cells, as well as their ability to synthesize extracellular matrix (ECM) components, greatly increase in the interstitium of fibrotic lungs. We have previously shown that the transcription of type I procollagen and fibronectin genes in the lungs is preferentially elevated during the early stages of bleomycin-induced pulmonary fibrosis (Raghow, R., S. Lurie, J. M. Seyer, and A. H. Kang. 1985, J. Clin. Invest. 76:1734-1739. Since a cytokine-like transforming growth factor beta (TGF beta) that is capable of enhancing mesenchymal cell proliferation and ECM synthesis could be potentially involved in this process, we investigated the temporal relationship between the regulation of TGF beta gene transcription and cellular proliferation in the bleomycin-treated hamster lungs. We observed a transient 5-7-fold increase in the accumulation of TGF beta transcripts, a concomitant 3-4-fold elevation in the cellular proliferation, and 8-10-fold stimulation of DNA synthesis in these lungs; all three parameters peaked around day 10 after bleomycin administration. Based on these results, we conclude that regulation of TGF beta gene expression may contribute significantly to the early events that lead to bleomycin-induced pulmonary fibrosis.

Authors

B Raghow, P Irish, A H Kang

Developmental and hormonal regulation of glucocorticoid receptor messenger RNA in the rat.

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The biological action of glucocorticoids is dependent upon tissue-specific levels of the glucocorticoid receptor (GR). During stress, the hypothalamic-pituitary-adrenal axis is stimulated, and high levels of glucocorticoids circulate. This axis is modulated by negative feedback by glucocorticoids, which inhibit hypothalamic and pituitary hormone secretion and downregulate GR gene expression. To study the developmental tissue-specific regulation of the GR, we measured the relative concentration of GR mRNA in fetal, neonatal, adult, and aged rats and examined the effects of dexamethasone on GR gene expression. Three different tissue-specific developmental patterns of GR mRNA accumulation were found. In addition, there was an age-dependent tissue-specific pattern in the feedback regulation of GR mRNA by glucocorticoids. In the fetus and neonate, GR mRNA abundance was not regulated by circulating glucocorticoids. The adult pattern of glucocorticoid feedback inhibition of GR mRNA expression appeared between 2 and 7 d of life in liver, and after 7 but before 14 d of age in brain. The GR was biologically active in the 2-d-old neonate, however, since dexamethasone enhanced gene expression of angiotensinogen, which is another glucocorticoid responsive gene. These data demonstrate that the GR gene is regulated by both developmental and tissue-specific factors, and provide another molecular basis for ontogenic variations in the hypothalamic-pituitary-adrenal response to stress.

Authors

J E Kalinyak, C A Griffin, R W Hamilton, J G Bradshaw, A J Perlman, A R Hoffman

Defective dopamine-1 receptor adenylate cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat.

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The natriuretic effect of DA-1 agonists is less in the spontaneously hypertensive rat (SHR) than its normotensive control, the Wistar-Kyoto rat (WKY). To determine a mechanism of the decreased effect of DA-1 agonists on sodium transport, DA-1 receptors in renal proximal convoluted tubule (PCT) were studied by radioligand binding and by adenylate cyclase (AC) determinations. Specific binding of 125I-SCH 23982 (defined by 10 microM SCH 23390, a DA-1 antagonist) was concentration dependent, saturable, and stereoselective. The dissociation constant, maximum receptor density, and DA-1 antagonist inhibition constant were similar in SHR and WKY. The apparent molecular weight of the DA-1 receptor determined by the photoaffinity D1 probe 125I-MAB was also similar in WKY and SHR. However, DA-1 agonists competed more effectively for specific 125I-SCH 23982 binding sites in WKY than in SHR. Basal as well as forskolin, parathyroid hormone, GTP and Gpp(NH)p-stimulated-AC activities were similar. In contrast DA-1 agonists (fenoldopam, SKF 38393, SND 911C12) stimulated AC activity to a lesser extent in SHR. GTP and Gpp(NH)p enhanced the ability of DA-1 agonists to stimulate AC activity in WKY but not in SHR. These data suggest a defect in the DA-1 receptor-second messenger coupling mechanism in the PCT of the SHR.

Authors

S Kinoshita, A Sidhu, R A Felder

Ovarian failure and autoimmunity. Detection of autoantibodies directed against both the unoccupied luteinizing hormone/human chorionic gonadotropin receptor and the hormone-receptor complex of bovine corpus luteum.

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We developed an ELISA system for the detection of human anti-ovarian antibodies. Bovine corpora lutea were extracted in PBS (pH 7.2) and fractionated by ultracentrifugation. Both the soluble fraction obtained after 80,000 g (S80) and the Triton-extracted membrane fraction (ST288) were used as antigens. Additionally, the luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor was isolated by affinity chromatography (wheat germ agglutinin and LH-Sepharose) and also used as an antigen. In 7 of 14 patients with primary sterility and endometriosis a positive reaction was observed. Similarly, 6 of 16 patients with secondary sterility and endometriosis were also positive. Patients being stimulated for in vitro fertilization and presenting either primary or secondary sterility were positive in 5 of 22 and 6 of 16 cases, respectively. In the S80 test 41 of 60 sera presented IgG2 antibodies, whereas in the ST288 test 38 of 60 belonged to the IgG1 subclass. Kappa and lambda chains were equally distributed. Some patients could recognize the unoccupied LH/hCG receptor as an antigen, while others recognized only the complex formed by the hormone plus the hormone receptor. The S80 and ST288 antigens were isolated by affinity chromatography. Gel permeation of the purified antigens revealed in each case the presence of an antigen complex. The apparent molecular weight was between 2,000 and 36,000 D. Cross-reactivity studies using affinity-purified antibodies demonstrated an antigenic relationship of the membrane, soluble, and extractable fractions. NAc-(beta-1----4)-D-glucosaminide and -D-galactopyranoside were the main terminal glycosides.

Authors

H Moncayo, R Moncayo, R Benz, A Wolf, C Lauritzen

Ambient C1- ions modify rat mesangial cell contraction by modulating cell inositol trisphosphate and Ca2+ via enhanced prostaglandin E2.

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Abstract

Our recent observation showed that angiotensin II (AII) and arginine vasopressin (AVP) stimulate Ca2+-activated Cl- conductance in mesangial cells. These data raise the possibility that mesangial cell function may be modulated by extracellular chloride concentration [( Cl-]o). The present study was undertaken to test this possibility using cultured rat mesangial cells. When the [Cl-]o was reduced to zero, the percentage of mesangial cells showing contraction responding to AII and AVP was decreased from 72 +/- 9 to 33 +/- 10% and from 60 +/- 4 to 24 +/- 11%, respectively. Ca2+ transients induced by AII and AVP, measured in mesangial cells loaded with Ca2+-sensitive photoprotein aequorin, were attenuated as [Cl-]o decreased. Also, when [Cl-]o decreased, inositol trisphosphate (IP3) levels of mesangial cells were suppressed, both in the presence and absence of AII or AVP. PGE2 production by mesangial cells increased when [Cl-]o decreased and the effects of ambient Cl- deprivation could be restored by addition of indomethacin to the Cl- -free medium. Moreover, PGE2 decreased mesangial cell contractility, Ca2+ transients, and IP3 production in response to AII and AVP. These data suggest that the decrease in [Cl-]o attenuates mesangial cell contraction by suppressing IP3 production and thus Ca2+ transients in response to AII and AVP through enhanced PGE2 production.

Authors

T Okuda, I Kojima, E Ogata, K Kurokawa

Tumor necrosis factor participates in the pathogenesis of acute immune complex alveolitis in the rat.

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Abstract

We have examined the role of intrapulmonary TNF in a rat model of acute immune complex-triggered alveolitis. Intratracheal instillation of IgG anti-bovine serum albumin (anti-BSA) followed by intravenous infusion of BSA results in acute alveolitis. Over the 4-h course of evolving lung injury, a 10-fold increase in TNF activity occurred in bronchoalveolar lavage (BAL) fluid. Immunohistochemical analysis of lung sections and BAL cells revealed that alveolar macrophages are the chief source of TNF. Antibodies that specifically neutralize rat TNF activity were raised in rabbits immunized with recombinant mouse TNF alpha. When administered into the lungs with anti-BSA, anti-TNF resulted in a marked reduction (up to 61%) in lung injury. Intratracheal instillation of exogenous TNF alone, or in combination with anti-BSA, resulted in an increase in lung injury compared to controls. Morphometric analysis and measurements of myeloperoxidase activities in whole lung extracts from rats treated with anti-TNF revealed a marked reduction in neutrophils compared to positive controls. The anti-TNF antibody preparation did not inhibit in vitro complement activation or diminish neutrophil chemotactic activity present in activated rat serum. These data indicate that intrapulmonary TNF activity is required for the full development of acute immune complex-triggered alveolitis, that alveolar macrophages are the primary source of this cytokine, and that TNF participates in the pathogenesis of immune complex alveolitis through a mechanism involving neutrophil recruitment.

Authors

J S Warren, K R Yabroff, D G Remick, S L Kunkel, S W Chensue, R G Kunkel, K J Johnson, P A Ward

Distinct platelet thromboxane A2/prostaglandin H2 receptor subtypes. A radioligand binding study of human platelets.

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Abstract

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) may aggregate platelets via a common membrane receptor(s). To further characterize this receptor, binding of the radiolabeled TXA2/PGH2 mimetic [125I]BOP to washed human platelets (WP) was investigated. [125I]BOP was competitively displaced from its platelet binding site by stable TXA2/PGH2 analogues. Competition curves were shallow with Hill coefficients of -0.73 +/- 0.05 (P less than 0.001 different from unity) (90 +/- 1% specific binding). Scatchard plots were curvilinear and most consistent with two binding sites; a high-affinity site with Kd of 234 +/- 103 pM, Bmax of 0.7 +/- 0.3 pM/mg protein (180 +/- 87 sites/WP), and a lower affinity site with Kd of 2.31 +/- 0.86 nM, Bmax of 2.2 +/- 0.3 pM/mg protein (666 +/- 65 sites/WP). [125I]BOP association and dissociation kinetics gave a Kd of 157 pM without evidence of negative cooperativity. The EC50 for I-BOP-induced initial Ca2+ increase was 209 +/- 24 pM, shape change was 263 +/- 65 pM, and aggregation was 4.4 +/- 0.5 nM. Parallel binding studies using the TXA2/PGH2 receptor antagonist [125I]PTA-OH showed a single binding site. The rank order for TXA2/PGH2 analogues to displace [125I]PTA-OH was identical to that for [125I]BOP. These studies indicate that [125I]BOP binds to two distinct sites on human platelets that may represent platelet TXA2/PGH2 receptor subtypes. The close correlation of IC50 values for I-BOP-induced platelet shape change and aggregation with the two Kds for [125I]BOP binding suggests that these platelet responses may be independently mediated by the two putative receptors.

Authors

G W Dorn 2nd

Detection of three distinct patterns of T helper cell dysfunction in asymptomatic, human immunodeficiency virus-seropositive patients. Independence of CD4+ cell numbers and clinical staging.

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Abstract

We have tested the T helper cell (TH) potential of asymptomatic, HIV seropositive (HIV+) patients, using an in vitro assay for IL-2 production. Peripheral blood leukocytes (PBL) from 74 HIV+ patients and 70 HIV- control donors were tested for TH function when stimulated with influenza A virus (FLU), tetanus toxoid (TET), HLA alloantigens (ALLO), or PHA. Of the HIV+ patients, four different response patterns were observed: (a) patients who responded to all four stimuli (16%); (b) patients who were selectively unresponsive to FLU and TET, but responded to ALLO and PHA (54%); (c) patients who were unresponsive to FLU, TET, or ALLO, but responsive to PHA (16%); and (d) patients who failed to respond to any of these stimuli (14%). Our results indicate a time-dependent progression from a stage responsive to all four stimuli to a stage unresponsive to any of the stimuli tested, progressing in the order outlined above. The earliest TH defect is the loss of responses to FLU and TET, indicating a selective defect in CD4+ MHC self-restricted TH function. The later loss of ALLO and PHA IL-2 responses suggests more severe TH dysfunction involving both CD4+ and CD8+ T cells. None of these patterns of TH unresponsiveness in asymptomatic HIV+ individuals were correlated with CD4+ cell numbers nor with Walter Reed staging criteria. This study indicates that the in vitro TH assay used can detect multiple stages of immune dysregulation early in the course of HIV infection and raises the possibility that staging of HIV+ patients should include in vitro TH functional analyses of the type described here.

Authors

M Clerici, N I Stocks, R A Zajac, R N Boswell, D R Lucey, C S Via, G M Shearer

Quantitative contribution of systemic vascular autoregulation in acute hypertension in conscious dogs.

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Abstract

Experiments were performed in nine conscious dogs to quantitate the contribution of systemic vascular autoregulation to the increases in total peripheral resistance (TPR) and mean arterial pressure (MAP) produced by angiotensin II (ANG II), arginine vasopressin (AVP), and norepinephrine (NE). We hypothesized that if autoregulatory vasoconstriction is significant, then the increase in TPR produced by vasoconstrictor infusion will be greater when MAP is controlled at hypertensive values than when the increase in pressure is prevented by controlling MAP at the animal's normotensive value. Each drug was infused at a dose sufficient to increase MAP by 50%. Then, a constant rate of vasoconstrictor infusion was maintained while MAP was controlled at hypertensive or normotensive levels for 15-min periods using a gravity reservoir connected to the left common carotid artery. During AVP infusion, TPR was significantly greater when MAP was controlled at hypertensive than at normotensive values. This autoregulatory-mediated vasoconstriction accounted for approximately three-fourths of the increase in MAP produced by AVP. No significant autoregulatory component was identified for the increases in TPR and MAP produced by ANG II or NE. We conclude that systemic vascular autoregulation is a powerful physiological property that contributes to the hemodynamic response to pressor doses of AVP.

Authors

P J Metting, K A Kostrzewski, P M Stein, B A Stoos, S L Britton

High receptor binding affinity of lipoproteins in atypical dysbetalipoproteinemia (type III hyperlipoproteinemia).

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Abstract

Familial dysbetalipoproteinemia (or type III hyperlipoproteinemia) is characterized by the presence of abnormal, cholesteryl ester-rich beta-very low density lipoproteins (beta-VLDL) in the plasma. Subjects with typical dysbetalipoproteinemia are homozygous for an amino acid substitution in apolipoprotein (apo-) E at residue 158 and have defective apo-E-mediated binding of both pre-beta-VLDL and beta-VLDL to apo-B,E(LDL) (or LDL) receptors (1988. Chappell, D.A., J. Clin. Invest. 82:628-639). To understand the effect of substitutions in apo-E at sites other than residue 158, nine dysbetalipoproteinemic (dys-beta) subjects who were either homozygous or heterozygous for substitutions in apo-E at atypical sites were studied. These substitutions occurred at residue 142 (n = 6), 145 (n = 2), or 146 (n = 1) and are known to cause less defective binding than does the 158 substitution. The chemical composition and electrophoretic mobility of pre-beta-VLDL and beta-VLDL from atypical and typical dys-beta subjects were indistinguishable. However, lipoproteins from atypical and typical dys-beta subjects differed in their affinity for the apo-B,E(LDL) receptor on cultured human fibroblasts. The pre-beta-VLDL and beta-VLDL from atypical dys-beta subjects had 640- or 17-fold higher affinity, respectively, than did corresponding lipoproteins from typical dys-beta subjects. The higher binding affinity of lipoproteins from atypical dys-beta subjects was associated with a higher ratio of apo-E to total apo-C. Since higher binding affinity should cause more rapid receptor-mediated clearance of beta-VLDL in atypical than in typical dys-beta subjects in vivo, the mechanism of beta-VLDL accumulation may differ in these two groups.

Authors

D A Chappell

Localization of integrin receptors for fibronectin, collagen, and laminin in human skin. Variable expression in basal and squamous cell carcinomas.

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Abstract

VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin.

Authors

J Peltonen, H Larjava, S Jaakkola, H Gralnick, S K Akiyama, S S Yamada, K M Yamada, J Uitto

Regulation of active sodium and potassium transport in the distal colon of the rat. Role of the aldosterone and glucocorticoid receptors.

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Abstract

To determine whether mineralocorticosteroids and glucocorticosteroids have specific effects on colonic electrolyte transport, we compared the effect of aldosterone and RU 28362, a glucocorticoid receptor-specific agonist that does not bind to the aldosterone receptor, on unidirectional Na, Cl, and K fluxes across isolated mucosa of the rat distal colon. Continuous infusion of aldosterone for 7 d produced changes in four specific transport processes: induction of both active electrogenic, amiloride-sensitive sodium absorption and active electrogenic potassium secretion, enhancement of active electroneutral potassium absorption, and inhibition of electroneutral Na-Cl absorption, the predominant transport process in this epithelium. In contrast, continuous infusion of RU 28362 for 1-11 d produced a sustained increase in electroneutral Na-Cl absorption. This glucocorticoid receptor-specific agonist did not induce electrogenic sodium absorption nor affect either potassium absorption or secretion. These studies demonstrate that aldosterone (i.e., mineralocorticoid) and glucocorticoid receptors modulate separate and specific changes in active sodium and potassium transport. These results suggest that other glucocorticoids (e.g., dexamethasone, methylprednisolone) are not glucocorticoid receptor-specific and that their effects on electrogenic sodium absorption and potassium transport most likely represent the binding of these agonists to the aldosterone receptor.

Authors

S G Turnamian, H J Binder

Identification and characterization of a monocyte-derived neutrophil-activating factor in corticosteroid-resistant bronchial asthma.

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Abstract

Peripheral blood mononuclear cells (PBMC) were isolated from seven normal subjects, eight asthmatic subjects clinically sensitive to corticosteroids (CS), and eight asthmatic subjects clinically resistant to corticosteroids (CR). PBMC were cultured at 37 degrees C for 24 h in the absence or presence of 10(-16) to 10(-4) M hydrocortisone. Calcium ionophore (A23187)-activated neutrophils (PMN) primed by supernatants of PBMC from asthmatic subjects cultured in the absence of hydrocortisone generated approximately threefold more leukotriene B4 than PMN primed by supernatants of PBMC from normal subjects (P less than 0.05). Incubation of PBMC derived from CS subjects with 10(-8) M hydrocortisone completely inhibited the production of the enhancing activity (P less than 0.01), whereas in CR subjects hydrocortisone at concentrations up to 10(-4) M did not suppress the release of enhancing activity. The enhancing activity was produced by monocytes. Enhancing activity eluted with an Mr of 3,000 D and a pI of 7.1. It eluted at 10% acetonitrile after reverse-phase HPLC. The activity was destroyed by heating to 60 degrees C for 60 min and was sensitive to pronase treatment. The purified factor also enhanced superoxide generation by PMN which had been stimulated submaximally by phorbol myristate acetate.

Authors

J R Wilkinson, A E Crea, T J Clark, T H Lee

Identification of a satellite fatty acid ethyl ester synthase from human myocardium as a glutathione S-transferase.

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Abstract

Nonoxidative alcohol metabolism catalyzed by fatty acid ethyl ester (FAEE) synthases may contribute to extrahepatic injury resulting from alcohol abuse. Unlike rabbit myocardial FAEE synthase, that from human heart has a satellite minor synthase (I) eluting from DEAE cellulose at a conductivity of 5 mS. Synthase I was purified 1,118-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and Superose-12 fast-protein liquid chromatographies. SDS-PAGE showed a single polypeptide with a molecular mass of 26 kD and gel permeation chromatography indicated a molecular mass of 52 kD for the active enzyme. Homogeneous synthase I catalyzed ethyl ester synthesis at highest rates with unsaturated octadecanoic fatty acid substrates. The amino acid composition of synthase I was highly homologous to that of human myocardial major synthase, recently identified as an acidic glutathione (GSH) S-transferase. Antibody raised against homogeneous human heart major synthase cross-reacted with the 26-kD synthase I. FAEE synthase co-chromatographed with GSH S-transferase on DEAE cellulose, Sephadex G-100 and S-hexylglutathione agarose, and also displayed GSH S-transferase activity in catalyzing the conjugation of GSH with nitrobenzene-containing carcinogens. Thus, human myocardium contains a satellite peak of FAEE synthase activity and it is a neutral GSH S-transferase.

Authors

P S Bora, C A Spilburg, L G Lange

Intestinal graft-versus-host disease is initiated by donor T cells distinct from classic cytotoxic T lymphocytes.

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Abstract

In these studies, the role of T helper and T cytotoxic cells in generating intestinal graft-vs.-host disease (GVHD) was examined. Treatment of C57BL/6J (B6) splenocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes natural killer cells, cytotoxic T lymphocyte (CTL) precursors, and the capacity to cause lethal GVHD in irradiated B6xDBA/2 F1 (B6D2F1) mice while preserving T helper cell function. Neither control nor Leu-Leu-OMe-treated DBA/2 donor spleen and bone marrow cells were found to induce lethal GVHD in B6D2F1 recipients. However, extensive colonic GVHD developed in B6D2F1 recipients of DBA/2 bone marrow and spleen cells. Enteropathic GVHD in DBA/2----B6D2F1 mice was reduced in severity after anti-L3T4 + C treatment of donor cells, and was eliminated by anti-Thy1.2 + C or the combination of anti-L3T4 and anti-Lyt2 + C treatment of the donor cell inoculum. However, neither anti-Lyt2 + C, Leu-Leu-OMe, nor anti-Lyt2 + C and Leu-Leu-OMe treatment of donor cells significantly decreased severity of gut GVHD. Leu-Leu-OMe treatment of DBA/2 or B6 SpC was comparably effective in preventing in vitro or in vivo generation of B6D2F1-specific CTL. These findings, therefore, demonstrate that histologically severe enteropathic GVHD does not require participation of CTL and is not always associated with high mortality rates.

Authors

D L Thiele, M L Eigenbrodt, S E Bryde, E H Eigenbrodt, P E Lipsky

Complete and partial deficiencies of complement factor D in a Dutch family.

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Abstract

A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patient's serum of purified factor D to a final concentration of 1 microgram/ml resulted in full restoration of the activity of the alternative pathway. Using an enzyme-linked immunosorbent assay, it was shown that the patient's serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patient's serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patient's serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.

Authors

P S Hiemstra, E Langeler, B Compier, Y Keepers, P C Leijh, M T van den Barselaar, D Overbosch, M R Daha

Role of endogenous atrial natriuretic factor in acute congestive heart failure.

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Abstract

The current studies were designed to investigate the functional significance of elevated endogenous atrial natriuretic factor (ANF) in acute congestive heart failure (CHF). Integrated cardiorenal and endocrine function were measured in three models of acute low-output congestive heart failure with comparably reduced cardiac output (CO) and mean arterial pressure (MAP). Acute CHF was produced by rapid right ventricular pacing (group I, n = 5) which decreases CO and increases atrial pressures and plasma ANF. In group II, n = 5, thoracic inferior vena caval constriction (TIVCC) was produced to decrease venous return and CO but without increases in atrial pressure or plasma ANF. In group III, n = 5, TIVCC was performed and exogenous ANF infused to achieve plasma concentrations observed in acute CHF. In acute CHF with increases in endogenous ANF, sodium excretion (UNaV), renal blood flow (RBF), plasma renin activity (PRA), and plasma aldosterone (PA) were maintained despite decreases in CO and MAP. In contrast, TIVCC with similar reductions in CO and MAP but without increases in ANF resulted in decreases in UNaV and RBF and increases in PRA and PA. Exogenous administration of ANF in TIVCC to mimic levels in acute CHF prevented sodium retention, renal vasoconstriction, and activation of renin and aldosterone. These studies demonstrate that endogenous ANF serves as an important physiologic volume regulator in acute CHF to maintain sodium excretion and possibly participate in the suppression of activation of the renin-angiotensin-aldosterone system despite the stimulus of arterial hypotension.

Authors

M E Lee, W L Miller, B S Edwards, J C Burnett Jr

Glomerular hemodynamics in established glycerol-induced acute renal failure in the rat.

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Abstract

The glomerular dynamic correlates of failed filtration were studied in volume replete rats with established glycerol-induced acute renal failure (ARF). Over one-half of all nephrons formed virtually no filtrate, while the single nephron glomerular filtration rate (SNGFR) of fluid-filled nephrons, measured at the glomerulotubular junction to preclude the possibility of covert tubular leakage, averaged one-sixth of control (P less than 0.001). Even that low mean value was elevated by a few nephrons with a near normal SNGFR. Renal failure thus reflected both total filtration failure in the majority of nephrons and massively reduced filtration in most of the remainder. Glomerular capillary pressure (Pg) averaged some 14 mmHg below control (P less than 0.001), whereas the arterial colloid osmotic and Bowman's space pressures were not significantly altered. Renocortical and whole kidney blood flow were also unchanged. Marked internephron functional heterogeneity precluded estimates of the ultrafiltration coefficient. However, the fall in SNGFR correlated well with the markedly depressed Pg and afferent net filtration pressure (delta PnetA, P less than 0.001), which in turn were caused by increased preglomerular resistance and a reciprocal fall in efferent arteriolar resistance. This complex change in intrarenal resistances was largely, if not entirely, responsible for failed filtration in this ARF model.

Authors

A I Wolfert, D E Oken

Mechanism of therapeutic effect of high-dose intravenous immunoglobulin. Attenuation of acute, complement-dependent immune damage in a guinea pig model.

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Abstract

Studies were performed in in vitro and in vivo models to assess the effect of intravenous immunoglobulin (IVIG) on the development of acute complement-mediated tissue damage. IVIG significantly increased the duration of survival and frequently prevented the death of guinea pigs injected with anti-Forssman antiserum to cause lethal Forssman shock; no control animal treated with albumin and/or maltose vehicle survived. The most pronounced effect was achieve by delivering IVIG as one slow injection at 1,800 mg/kg 3 h before Forssman shock was elicited. Infusion of guinea pig IgG at the same dosage was similarly protective. A strong positive correlation was found between IgG plasma levels and survival time in guinea pigs treated with graded doses of IVIG. Therapy itself did not affect C3 and C4 levels nor the capacity to activate these components. In vitro studies showed almost complete inhibition of C3 uptake onto IgG-sensitized erythrocytes using serum from an IVIG-treated animal. We suggest that supraphysiologic levels of IVIG act in part by preventing active C3 fragments from binding to target cells. Infusion of high dose IVIG may be a rational approach to modulating acute, complement-dependent tissue damage in a variety of diseases in man.

Authors

M Basta, P Kirshbom, M M Frank, L F Fries

Complement component C1q enhances invasion of human mononuclear phagocytes and fibroblasts by Trypanosoma cruzi trypomastigotes.

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Abstract

Internalization and infectivity of Trypanosoma cruzi trypomastigotes by macrophages is enhanced by prior treatment of parasites with normal human serum. Heating serum or removing C1q from serum abrogates the enhancement, but augmentation of attachment and infectivity is restored by addition of purified C1q to either serum source. Although both noninfective epimastigotes (Epi) and vertebrate-stage tissue culture trypomastigotes (TCT) bind C1q in saturable fashion at 4 degrees C, internalization by monocytes and macrophages of TCT but not Epi-bearing C1q is enhanced in comparison to untreated parasites. Adherence of human monocytes and macrophages to surfaces coated with C1q also induces a marked enhancement of the internalization of native TCT. C1q enhances attachment of both Epi and TCT to human foreskin fibroblasts, but only when C1q is on the parasite and not when the fibroblasts are plated on C1q-coated surfaces. Only TCT coated with C1q show enhanced invasion into fibroblasts. Although trypomastigotes produce an inhibitor of the complement cascade which limits C3 deposition during incubation in normal human serum, C1q binds to the parasite and enhances entry of trypomastigotes into target cells.

Authors

M T Rimoldi, A J Tenner, D A Bobak, K A Joiner

Isolation of an acidic protein from cholesterol gallstones, which inhibits the precipitation of calcium carbonate in vitro.

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Abstract

In seeking to identify nucleating/antinucleating proteins involved in the pathogenesis of cholesterol gallstones, a major acidic protein was isolated from each of 13 samples of cholesterol gallstones. After the stones were extracted with methyl t-butyl ether to remove cholesterol, and methanol to remove bile salts and other lipids, they were demineralized with EDTA. The extracts were desalted with Sephadex-G25, and the proteins separated by PAGE. A protein was isolated, of molecular weight below 10 kD, which included firmly-bound diazo-positive yellow pigments and contained 24% acidic, but only 7% basic amino acid residues. The presence of N-acetyl glucosamine suggested that this was a glycoprotein. This protein at concentrations as low as 2 micrograms/ml, but neither human serum albumin nor its complex with bilirubin, inhibited calcium carbonate precipitation from a supersaturated solution in vitro. This protein could be precipitated from 0.15 M NaCl solution by the addition of 0.5 M calcium chloride. Considering that cholesterol gallstones contain calcium and pigment at their centers, and that small acidic proteins are important regulators in other biomineralization systems, this protein seems likely to play a role in the pathogenesis of cholesterol gallstones.

Authors

S Shimizu, B Sabsay, A Veis, J D Ostrow, R V Rege, L G Dawes

Placental corticotropin-releasing hormone may be a stimulator of maternal pituitary adrenocorticotropic hormone secretion in humans.

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Abstract

To clarify the physiological role of placental corticotropin-releasing hormone (CRH), we measured plasma CRH, ACTH, and cortisol throughout pregnancy. Cerebrospinal fluid (CSF) CRH levels and ACTH responsiveness to synthetic CRH were also quantified in pregnant and nonpregnant women. Maternal plasma CRH levels, which increased progressively during pregnancy, correlated well with both ACTH and cortisol in early labor, delivery, and postpartum samples, and also with cortisol levels in samples before labor. CSF CRH levels in term pregnant women did not differ from those of nonpregnant women. CRH infusion that attained similar plasma CRH levels to those found in late pregnancy elicited significant ACTH release in vivo and regular CRH test provoked normal ACTH response during early pregnancy but no response during late pregnancy. We concluded that: (a) maternal pituitary-adrenal axis correlates well with plasma CRH levels, which are high enough to provoke ACTH release from maternal pituitary; (b) hypothalamic CRH secretion in term pregnant women is not exaggerated; and (c) maternal pituitary is responsive to synthetic CRH in early but not late pregnancy, suggesting that maternal pituitary-adrenal axis is already activated by high circulating CRH. Placental CRH may be an important stimulator of the maternal pituitary-adrenal axis during pregnancy.

Authors

A Sasaki, O Shinkawa, K Yoshinaga

Activation of normal and cystic fibrosis Cl- channels by voltage, temperature, and trypsin.

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Abstract

In cystic fibrosis (CF) phosphorylation-dependent activation of outwardly rectifying apical membrane Cl- channels is defective. To further understand regulation of this channel we examined several other mechanisms of channel activation in normal and CF cells. Previous studies have shown that strong membrane depolarization can activate channels in excised cell-free membrane patches. Here we show that such activation is dependent on both the absolute membrane voltage and the duration of depolarization. Moreover, activation was reversible by membrane hyperpolarization. In some cases, excising patches of membrane from the cell caused channel activation, even in the absence of depolarization. However, the frequency of channel activation with patch excision increased when bath temperature was increased from 23 to 37 degrees C. Although the channel remained in the activated state when temperature was reduced to 23 degrees C, subsequent hyperpolarization inactivated the channel. In cell-attached patches, neither depolarization nor increasing bath temperature to 37 degrees C activated channels, suggesting that neither is physiologically important in regulation of the channel. Thus changes in membrane voltage and bath temperature appear to cause a nonenzymatic change in the channel's conformation; the interactions between voltage and temperature suggest that they may affect the same process. To determine if a proteolytic alteration of the channel could also cause activation, we added trypsin to the cytosolic surface of excised membrane patches. Trypsin activated channels, which could not then be inactivated by either hyperpolarization or phosphorylation with PKC, suggesting that trypsin removed or altered a region of the channel involved in inactivation. All of these interventions activated Cl- channels from both normal and CF cells. Thus many aspects of Cl- channel activation are normal in CF; only phosphorylation-dependent activation is defective.

Authors

M J Welsh, M Li, J D McCann

Serum levels of interleukin 6, a potent myeloma cell growth factor, as a reflect of disease severity in plasma cell dyscrasias.

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Abstract

Using a specific and very sensitive (1 pg = 1 U) bioassay, we investigated the presence of IL-6, a potent myeloma cell growth factor, in the sera of 131 subjects with plasma cell dyscrasias. 22 had monoclonal gammopathy of undetermined significance (MGUS), 13 had smoldering myeloma (SMM), 85 had overt multiple myeloma (MM), and 11 had plasma cell leukemia (PCL). Significant serum IL-6 levels were detected in only 3% of the MGUS/SMM group, but in 35% of the overt MM group and 100% of the PCL group. During overt MM, IL-6 was detected in 37% of the patients at diagnosis, 13% of those with stable MM, and 60% of those with fulminating disease. These data demonstrate that serum levels of IL-6, a potent myeloma cell growth factor in vitro, correlate with disease severity in plasma cell dyscrasias. Serial studies performed in 3 patients and correlative studies with labeling index in vivo in 25 patients have confirmed this concept. Taken together, this suggests that this cytokine is probably involved in vivo during the progressive phase of MM. Thus, anti-IL-6 or anti-IL-6 receptor antibodies could be useful as therapeutic agents at this stage of the disease.

Authors

R Bataille, M Jourdan, X G Zhang, B Klein

A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease.

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Abstract

A membrane-bound cytochrome b, a heterodimer formed by a 91-kD glycoprotein and a 22-kD polypeptide, is a critical component of the phagocyte NADPH-oxidase responsible for the generation of superoxide anion. Mutations in the gene for the 91-kD chain of this cytochrome result in the X-linked form of chronic granulomatous disease (CGD), in which phagocytes are unable to produce superoxide. Typically, there is a marked deficiency of the 91-kD subunit and the cytochrome spectrum is absent (X- CGD). In a variant form of CGD with X-linked inheritance, affected males have a normal visible absorbance spectrum of cytochrome b, yet fail to generate superoxide (X+ CGD). The size and abundance of the mRNA for the 91-kD subunit and its encoded protein were examined and appeared normal. To search for a putative mutation in the coding sequence of the 91-kD subunit gene, the corresponding RNA from an affected X+ male was amplified by the polymerase chain reaction and sequenced. A single nucleotide change, a C----A transversion, was identified that predicts a nonconservative Pro----His substitution at residue 415 of the encoded protein. Hybridization of amplified genomic DNA with allele-specific oligonucleotide probes demonstrated the mutation to be specific to affected X+ males and the carrier state. These results strengthen the concept that all X-linked CGD relates to mutations affecting the expression or structure of the 91-kD cytochrome b subunit. The mechanism by which the Pro 415----His mutation renders the oxidase nonfunctional is unknown, but may involve an impaired interaction with other components of the oxidase.

Authors

M C Dinauer, J T Curnutte, H Rosen, S H Orkin

Monocyte-chemotactic activity of defensins from human neutrophils.

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Abstract

We investigated the monocyte-chemotactic activity of fractionated extracts of human neutrophil granules. Monocyte-chemotactic activity was found predominantly in the defensin-containing fraction of the neutrophil granules. Purified preparations of each of the three human defensins (HNP-1, HNP-2, HNP-3) were then tested. HNP-1 demonstrated significant chemotactic activity for monocytes: Peak activity was seen at HNP-1 concentrations of 5 X 10(-9) M and was 49 +/- 20% (mean +/- SE, n = 9) of that elicited by 10(-8) M FMLP. HNP-2 (peak activity at 5 X 10(-9) M) was somewhat less active, yielding 19 +/- 10% (n = 11). HNP-3 failed to demonstrate chemotactic activity. Checkerboard analysis of monocyte response to HNP-1 and HNP-2 confirmed that their activity was chemotactic rather than chemokinetic. Neutrophils demonstrated a low level of response to defensins but this reaction was primarily chemokinetic. Defensins may play a role in the recruitment of monocytes by neutrophils into inflammatory sites.

Authors

M C Territo, T Ganz, M E Selsted, R Lehrer

Plasma Ip(a) concentration is inversely correlated with the ratio of Kringle IV/Kringle V encoding domains in the apo(a) gene.

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Abstract

Plasma Lp(a) levels correlate with atherosclerosis susceptibility. This lipoprotein consists of an LDL-like particle attached to a large glycoprotein called apo(a). Apo(a) is a complex glycoprotein containing multiple Kringle domains, found to be highly homologous to plasminogen Kringle IV, and a single Kringle domain homologous to plasminogen Kringle V. Lp(a) levels appear to be inversely correlated with apo(a) size in a given individual. In this study, we have used probes specific to the Kringles IV and V domains of apo(a) cDNA in quantitative Southern blotting analysis. By this method, we have determined the ratio of Kringle IV/Kringle V encoding domains in the apo(a) gene of 53 unrelated individuals with different plasma concentrations of Lp(a). This ratio was found to be inversely correlated with log Lp(a) levels (r = -0.90, P less than 0.0001) and directly correlated with apo(a) apparent molecular weight (Mr) (r = 0.79, P less than 0.0001). In summary, by showing that Lp(a) concentrations and apo(a) apparent size are highly correlated with the ratio of Kringle IV/Kringle V encoding domains in the apo(a) gene, we provide a DNA marker for this atherosclerosis risk factor as well as an important insight into the genetic mechanism regulating Lp(a) levels.

Authors

D Gavish, N Azrolan, J L Breslow