Issue published May 1, 1991

A utoantibodies to phospholipids (APA) occur frequently in systemic lupus erythematosus (SLE) and other autoimmune disorders and predispose to intravascular thromboses. Major histocompatibility complex (MHC) class II alleles (HLA-DR and DQ) were determined by restriction fragment length polymorphisms (RFLP) in 20 patients with APA (lupus anticoagulant). HLA-DQw7 (DQB1*0301), linked to HLA-DR5 and -DR4 haplotypes, occurred in 70% and was significantly increased compared to 139 race-matched normal controls (P = 0.002, P corrected [pc] = 0.05, odds ratio [OR] = 5.1). Moreover, the frequency of HLA-DQw7 was significantly higher in SLE patients with APA as compared with patients without APA but with other autoantibodies, including anti-Ro and La (P = 0.0001, pc = 0.002, OR = 10.7), anti-Ro alone (P = 0.001, pc = 0.02, OR = 11.2), anti-dsDNA (P = 0.001, pc = 0.02, OR = 7.1), and possibly anti-Sm (P = 0.04, pc = NS, OR = 6.8) and anti-nRNP (U1-RNP) (P = 0.01, pc = NS, OR = 7.8). The DQB1*0301 allele of DQw7 showed the strongest association, while the frequencies of the DQA1*0301 (45%) and DQA1*0501 (50%) alleles did not differ from the controls. Among the HLA-DQB1*0301 (DQw7) negative patients, all possessed HLA-DQw8 (DQB1*0302) and/or HLA-DQw6 (DQB1*0602 or DQB1*0603) alleles. The HLA-DQB1*0301 chain shares an identical seven amino acid sequence with DQB1*0302, *0602, and *0603 chains in the third hypervariable region of the HLA-DQ molecule. This candidate "epitope" may play a role in mediating an autoimmune response to APA.

P revious studies in cultured rat hepatocytes revealed that initial uptake of sulfobromophthalein (BSP) was markedly reduced upon removal of Cl- from the medium. In the present study, unidirectional Cl- gradients were established in short-term cultured rat hepatocytes and their effect on BSP uptake was determined. These investigations revealed that BSP uptake requires external Cl- and is not stimulated by unidirectional Cl- gradients, suggesting that BSP transport is not coupled to Cl- transport. In contrast, BSP transport is stimulated by an inside-to-outside OH- gradient, consistent with OH- exchange or H+ cotransport. As the presence of Cl- is essential for but not directly coupled to BSP transport, binding of 35S-BSP to hepatocytes was determined at 4 degrees C. This revealed an approximately 10-fold higher affinity of cells for BSP in the presence as compared to the absence of Cl- (Ka = 3.2 +/- 0.8 vs. 0.42 +/- 0.09 microM-1; P less than 0.02). Affinity of BSP for albumin was Cl(-)-independent, and was approximately 10% of its affinity for cells in the presence of Cl-. These results indicate that extracellular Cl- modulates the affinity of BSP for its hepatocyte transporter.

T o evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport.

T o examine in vivo the separate effects on distal tubule JtCO2, of dietary chloride restriction, bicarbonate loading, and changes in luminal chloride concentration, we microperfused distal tubules at a physiologic flow rate (8 nl/min) with solutions containing either 45 or 0 mM chloride (after gluconate substitution). Rats were fed a diet containing zero, minimal, or normal amounts of chloride, while drinking either water or a solution of 0.15 M sodium bicarbonate. Neither extracellular fluid volume contraction nor negative chloride balance ensued. Analysis of covariance with repeated measures demonstrated that dietary chloride, drinking sodium bicarbonate, and perfusion with either 45 mM or zero chloride, each have separate and significant modulating effects on distal tubule bicarbonate secretion. During mild alkalemia, there is modest bicarbonate secretion which is significantly different from zero (-9.9 +/- 3.2 pmol.min-1.mm-1, P less than 0.01), and which is suppressed after perfusion with zero chloride. In contrast, during more pronounced metabolic alkalosis after supplemental bicarbonate drinking, the bicarbonate secretory flux is brisk (-26 +/- 3 pmol.min-1.mm-1) and significantly different from zero and persists (-11 +/- 3 pmol.min-1.mm-1) even during perfusion with zero luminal chloride. Accordingly, in this two-day model of alkalosis induced by dietary chloride restriction, there is regulatory secretion of bicarbonate by distal tubules in vivo which is modulated by luminal chloride concentration.

T his study describes a methodology for generating stable, cloned, EBV-transformed IgG- and IgM-producing human B cell lines. Using these lines we have characterized immunoglobulin V gene utilization in an anti-DNA-associated idiotypic system. The 31 anti-DNA-associated idiotype is encoded preferentially by the VK1 gene family, and, in all probability, reflects a germ line gene-encoded framework determinant. Analysis of these lines indicates that the DNA-binding antibodies produced by B cell lines from SLE patients may differ from DNA binding myeloma proteins and from natural autoantibodies.

T he protective effect of stable nitroxide radicals against oxidative damage was studied using cardiomyocyte cultures obtained from newborn rats. Monolayered cardiomyocytes were exposed to H2O2 and the effect on spontaneous beating and leakage of LDH was determined. Hydrogen peroxide irreversibly blocked rhythmic beating and resulted in a significant membrane injury as shown by release of LDH. The injury was prevented by catalase which removes H2O2 and by cell-permeable, metal-chelating agents such as desferrioxamine or bipyridine. In contrast, reagents which are excluded from the cell such as superoxide dismutase or DTPA did not protect the cells against H2O2. Five- and six-membered ring, stable nitroxide radicals which have previously been shown to chemically act as low-molecular weight, membrane-permeable, SOD-mimetic compounds provided full protection. The nitroxides prevented leakage of LDH and preserved normal cardiomyocyte contractility, presumably by intercepting intracellular O2-radicals. Alternatively, protection may result through nitroxides reacting with reduced transition metal ions or by detoxifying secondary organic radicals.

W e recently observed that specific antibodies to type II collagen do not bind in appreciable amounts to the intact surface of articular cartilage, whereas antibodies to the minor collagen types V, VI, and IX do. These results suggest that the outermost cartilage surface layer prevented interaction of the antibodies with the major collagen type in articular cartilage. The present studies were designed to investigate the pathogenic mechanisms involved in the disruption of the cartilage surface layer in inflammatory arthritis. Articular cartilage obtained from rabbits undergoing acute antigen-induced arthritis of 72 h duration showed a significant increase in binding of anti-type II antibody to cartilage surfaces compared with normal control cartilage (P less than 0.01). Augmentation of anti-type II binding was also observed upon in vitro incubation of bovine articular slices or intact rabbit patellar cartilage for 1 h with human polymorphonuclear neutrophils (PMN), PMN lysates, or purified human PMN elastase. This increase was not inhibited by sodium azide, nor was it enhanced by incubation of cartilage with the strong oxidant hypochlorous acid. Chondrocyte-mediated matrix proteoglycan degradation in cartilage explants cultured in the presence of cytokines failed to increase antibody binding appreciably. The augmentation in antibody binding seen with PMN lysates was inhibited by the nonspecific serine-esterase inhibitor PMSF, but not by the divalent metal chelator EDTA. The elastase-specific inhibitor AAPVCMK also inhibited most of the PMN-induced increase in antibody binding, whereas the cathepsin G-specific inhibitor GLPCMK was much less effective. Incubation of intact cartilage with purified human PMN elastase indicated that this serine esterase could account for the increase in anti-type II collagen antibody binding to intact cartilage surfaces. These studies suggest that in an inflammatory response, PMN-derived elastase degrades the outer layer of articular cartilage, exposing epitopes on type II collagen. They also help clarify the pathogenic mechanisms involved in early articular cartilage damage in inflammatory joint diseases.

T he acute effects of TNF on the microcirculation were studied by in vivo microscopy in rat cremaster muscle. The changes in arteriolar diameter after topical administration of recombinant TNF (rTNF; 10(-4)-10(4) ng/ml) were studied in second-, third-, and fourth-order arterioles (A2-A4) whose mean diameters under control conditions were 64.3, 30.7, and 14.8 microns respectively. rTNF induced a concentration-dependent vasodilation whose amplitude was largest for the smallest arterioles. At the highest concentration tested, arteriolar diameter increased by 21, 29, and 41% of control diameter for the A2, A3, and A4 arterioles, respectively. Indomethacin or mefenamic acid, two structurally different prostaglandin synthesis inhibitors, markedly inhibited the degree of vasodilation induced by rTNF in the three arteriolar orders. As regards the effect of rTNF on vasoconstriction in response to norepinephrine, vasoconstriction was greatest for the smallest arterioles, and did not change 10 min after rTNF administration for any of the three arteriolar orders. We conclude that (a) rTNF has a direct vasodilatory effect which is greatest in the smallest arterioles, (b) this vasodilation is at least partly mediated by prostaglandins, and (c) administration of rTNF in itself does not acutely alter the response of the arterioles to vasopressive drugs.

W e have attempted to identify mRNA for IL-5 in endobronchial mucosal biopsies from asthmatics and controls, using the technique of in situ hybridization. Bronchial biopsies were obtained from 10 asthmatics and 9 nonatopic normal controls. A radio-labeled cRNA probe was prepared from an IL-5 cDNA and hybridized to permeabilized sections. These were washed extensively before processing for autoradiography. An IL-5-producing T cell clone derived from a patient with the hyper-IgE syndrome was used as a positive control. As a negative control, sections were also treated with a "sense" IL-5 probe. Specific hybridization signals for IL-5 mRNA were demonstrated within the bronchial mucosa in 6 out of the 10 asthmatic subjects. Cells exhibiting hybridization signals were located beneath the epithelial basement membrane. In contrast, there was no hybridization in the control group. No hybridization was observed with the sense probe. The six IL-5 mRNA-positive asthmatics tended to have more severe disease than the negative asthmatics, as assessed by symptoms and lung function, and showed a significant increase in the degree of infiltration of the bronchial mucosa by secreting (EG2+) eosinophils and activated (CD25+) T lymphocytes. Within the subjects who showed positive IL-5 mRNA, there was a correlation between IL-5 mRNA expression and the number of CD25+ and EG2+ cells and total eosinophil count. This study provides evidence for the cellular localization of IL-5 mRNA in the bronchial mucosa of asthmatics and supports the concept that this cytokine regulates eosinophil function in bronchial asthma.

T he effects of guanosine 5'-triphosphate (GTP) and GTP-gamma-S, known activators of GTP binding proteins, on proton transport were investigated in endosome-enriched vesicles (endosomes). Endosomes were prepared from rabbit renal cortex following the intravenous injection of FITC-dextran. The rate of intravesicular acidification was determined by measuring changes in fluorescence of FITC-dextran. Both GTP and GTP-gamma-S stimulated significantly the initial rate of proton transport. In contrast, GDP-beta-S, which does not activate GTP binding proteins, inhibited proton transport. The rank order of stimulation was GTP-gamma-S greater than GTP greater than control greater than GDP-beta-S. GTP-gamma-S stimulation of proton transport was also observed under conditions in which chloride entry was eliminated, i.e., 0 mM external chloride concentration in the presence of potassium/valinomycin voltage clamping. GTP-gamma-S did not affect proton leak in endosomes as determined by collapse of H+ ATPase-generated pH gradients. ADP ribosylation by treatment of endosomal membranes with pertussis toxin revealed two substrates corresponding to the 39-41 kD region and comigrating with alpha i subunits. Pretreatment of the membranes with pertussis toxin had no effect on proton transport in the absence of GTP or GTP-gamma-S. However, pretreatment with pertussis toxin blocked the stimulation of proton transport by GTP. In contrast, as reported in other membranes by others previously, pertussis toxin did not prevent the stimulation of proton transport by GTP-gamma-S. These findings, taken together, indicate that GTP binding proteins are present in endosomal membranes derived from renal cortex and that activation of G protein by GTP and GTP-gamma-S stimulates proton transport in a rank order identical to that reported for other transport pathways modulated by Gi proteins. Therefore, these studies suggest that G proteins are capable of stimulating the vacuolar H ATPase of endosomes directly.

C l-/HCO3- exchange is present in all three cell types of the rabbit cortical collecting tubule, yet may mediate a different function in each cell type. The purpose of this study was to characterize further the location, function, and regulation of Cl-/HCO3- exchange in two cell types using measurements of intracellular pH (pHi). In the principal cell there was no evidence for apical Cl-/HCO3- exchange, including no change in pHi with increases in luminal HCO3-. The principal cell possesses a basolateral Cl-/HCO3- exchanger that is inactive normally but stimulated by intracellular alkalosis. Decreased PCO2 results in increased pHi associated with activation of Cl-/HCO3- exchange and partial recovery of pHi. In contrast, the beta-intercalated cell possesses an apical Cl-/HCO3- exchanger and alkalinizes with increases in luminal HCO3-. Also in contrast to the principal cell, the beta-intercalated cell apical Cl-/HCO3- exchanger does not appear to be involved in pHi regulation and may be specifically modified for transcellular HCO3- transport. In conclusion, the separate Cl-/HCO3- exchangers in the principal cell and the beta-intercalated cell not only have opposite polarity but are regulated differently.

T his study was undertaken to determine potential tissue sources of plasma cholesteryl ester transfer protein (CETP), and to assess the influence of CETP on lipoprotein concentrations and atherosclerosis. In a group of 28 cynomolgus monkeys fed high fat, high cholesterol diets, plasma CETP concentration was strongly correlated with the abundance of CETP mRNA in liver and in adipose tissue, and with the output of CETP in liver perfusates. Plasma CETP concentration showed a strong inverse correlation with HDL cholesterol concentrations (r = -0.62, P less than 0.001) and a positive correlation with LDL cholesterol concentration (r = 0.54, P less than 0.005) and molecular weight (r = 0.57, P less than 0.001). The extent of coronary artery atherosclerosis was positively correlated with LDL cholesterol concentration and molecular weight, and with plasma CETP concentration. Thus, in monkeys fed an atherogenic diet, individual variation in CETP mRNA abundance in liver and adipose tissue probably plays a major role in the determination of plasma CETP levels. In plasma, CETP influences the distribution of cholesteryl esters between LDL and HDL, and CETP concentration appears to be a key determinant of the relative atherogenicity of the plasma lipoproteins.

T cell clones were established from peripheral blood of a patient with severe aplastic anemia. 8 of 18 individual clonal T cell populations stably coexpressed CD4 and CD8 molecules, a phenotype characteristic for thymocytes and a minor subpopulation of circulating T lymphocytes. Analysis of T cell receptor genes revealed identical rearrangements of T cell receptor beta chain genes, suggesting clonality of these T cells. CD4+/CD8+ T cells clones were found to be efficiently cytotoxic towards autologous lymphoblasts. Autocytotoxicity could be blocked by a CD3 MAb, a MAb specific for monomorphic MHC class II determinants, and particularly, by an MHC-DP-specific MAb, suggesting specificity for autologous DP molecules. Perhaps more important, CD4+/CD8+ T cell clones inhibited differentiation of autologous progenitor enriched bone marrow cells in vitro by a direct cell-mediated mechanism. These data suggest that circulating cytotoxic CD4+/CD8+ T cell clones specific for autologous MHC-DP determinants may be involved in hematopoietic failure in some cases of aplastic anemia.

T he endothelial cytoskeleton is believed to play an important role in the regulation of endothelial permeability. We used botulinum C2 toxin to perturb cellular actin and determined its effect on the permeability of endothelial cell monolayers derived from porcine pulmonary arteries. The substrate for botulinum C2 toxin is nonmuscle monomeric actin which becomes ADP-ribosylated. This modified actin cannot participate in actin polymerization and, in addition, acts as a capping protein. Exposure of endothelial cell monolayers to botulinum C2 toxin resulted in a dose- (3-100 ng/ml) and time-dependent (30-120 min) increase in the hydraulic conductivity and decrease in the selectivity of the cell monolayers. The effects of C2 toxin were accompanied by a time- and dose-dependent increase in ADP-ribosylatin of G-actin. G-Actin content increased and F-actin content decreased time- and dose-dependently in C2 toxin-treated endothelial cells. Phalloidin which stabilizes filamentous actin prevented the effects of botulinum C2 toxin on endothelial permeability. Botulinum C2 toxin induced interendothelial gaps. The effects occurred in the absence of overt cell damage and were not reversible within 2 h. The data suggest that the endothelial microfilament system is important for the regulation of endothelial permeability.

T he Capnocytophaga are inhabitants of the hypoxic human gingival crevice that are normally prevented by neutrophils from causing periodontal and systemic infection. To identify potential nonoxidative bactericidal mechanisms against Capnocytophaga within human neutrophils, gel filtration chromatography was used to fractionate neutrophil granule extracts. Seven granule fractions, designated A through G, were obtained. The Capnocytophaga were most sensitive to killing by fraction D. Fraction D exhibited substantial bactericidal activity under aerobic and anaerobic conditions. The bactericidal activity associated with ion-exchange subfractions D8-D11, which contained primarily cathepsin G as assessed by enzymatic activity, amino acid composition, and NH2-terminal sequence. Heat-inactivation, diisopropylfluorophosphate, PMSF, and N-benzyloxycarbonylglycylleucylphenylalanyl-chloromethyl ketone inhibited bactericidal activity against Capnocytophaga sputigena but not Escherichia coli. We conclude that (a) human neutrophil cathepsin G is an important antimicrobial system against the Capnocytophaga, (b) the bactericidal activity of cathepsin G against Capnocytophaga is oxygen independent, and (c) an intact enzyme active site is involved in the killing of C. sputigena but not E. coli. We suggest that human neutrophil cathepsin G is an important antimicrobial system against certain oral bacteria and that cathepsin G kills bacteria by two distinct mechanisms.

R emodeling of myocyte interconnections may be an important determinant of ventricular tachycardia in regions bordering healed infarcts. We used quantitative electron microscopy to characterize the distribution of gap junctions in 10 canine left ventricles 3-10 wk after coronary occlusion. In three normal canine left ventricles analyzed ultrastructurally, myocardial gap junctions were distributed anisotropically; gap junction profile length was significantly greater in the transverse than in longitudinal planes of section. In infarct border zone tissues, the normal anisotropic distribution was completely abolished and fewer gap junctions per unit intercalated disk length were observed. Analysis of individual gap junction profile length distributions revealed selective disruption of the largest gap junctions that collectively comprised only 9.6% of total junction profiles, but encompassed nearly 40% of aggregate gap junction length in the transverse plane of section. Three-dimensional reconstructions of myocyte interconnections by high resolution quantitative light microscopy of serial sections demonstrated a reduction in the number of cells connected by intercalated disks to a single myocyte from 11.2 +/- 1.0 in normal myocardium to 6.5 +/- 1.3 in border zone tissues (P less than 0.001). Connections of cells in primarily side-to-side apposition were reduced by 75%, whereas primarily end-to-end connections were reduced by only 22% (P less than 0.05). These alterations would disproportionately enhance axial resistivity in the transverse direction, potentially contributing to development of reentrant arrhythmias.

T o better understand the structural basis for rheumatoid factor activity, the nucleotide sequence of the light chain variable regions of nine human monospecific IgM rheumatoid factors were analyzed. Rheumatoid factors were isolated from three patients with rheumatoid arthritis, a patient with systemic lupus erythematosus, and a normal individual. The VL gene segments used by these rheumatoid factors are not as restricted as previous work on mixed cryoglobulin rheumatoid factors had suggested. Each of the different VK families is represented and there are two examples where a V lambda gene segment is used. Molecules with structures similar to those of the Wa and Po CRI, characteristic of mixed cryoglobulin rheumatoid factors, are not common among these rheumatoid factors isolated from patients with rheumatoid arthritis. While there are clear examples of rheumatoid factors that are direct copies of germline genes, most of the sequence data suggest that the processes of antigenic selection and somatic mutation contribute significantly to the generation of monospecific rheumatoid factors in patients with autoimmune disease.

P atients with hereditary angioneurotic edema (HANE) have serum levels of functionally active inhibitor of the first component of complement (C1 INH) between 5 and 30% of normal, instead of the 50% expected from the single normal allele. Increases in rates of catabolism have been documented in patients with HANE and certainly account for some of decrease in C1 INH level. A possible role for a decrease in synthesis of C1 INH in producing serum levels of C1 INH below the expected 50% of normal has not been well studied. We studied the synthesis of C1 INH in skin fibroblast lines, which produce easily detectable amounts of C1 INH. In type I HANE cells, C1 INH synthesis was 19.6 +/- 4.0% (mean +/- SD) of normal, much less than the 50% predicted. In type II HANE cells, the total amount of C1 INH synthesis (functional and dysfunctional) was 98.9 +/- 17% of normal; the functional protein comprised 43% of the total. Thus, type II HANE cells synthesized functional C1 INH at a much greater rate than for the type I cells. In both type I and II HANE cells, amounts of steady-state C1 INH mRNA levels paralleled rates of C1 INH synthesis, indicating that control of C1 INH synthesis occurred at pretranslational levels. Both type I and type II fibroblasts synthesized normal amounts of C1r and C1s. These data suggest that the lower than expected amounts of functionally active C1 INH in type I HANE may be due, in part, to a decrease in rate of synthesis of the protein, and that the expressions of the normal C1 INH allele in HANE is influenced by the type of abnormal allele present.

W e examined the hypothesis that respiratory sinus arrhythmia (RSA) is primarily a central phenomenon and thus that RSA is directly correlated with respiratory controller output. RSA was measured in nine anesthetized dogs, first during spontaneous breathing (SB) and then during constant flow ventilation (CFV), a technique whereby phasic chest wall movements and thoracic pressure swings are eliminated. Measurements of the heart rate and of the moving time averaged (MTA) phrenic neurogram during these two ventilatory modes were made during progressive hypercapnia and progressive hypoxia. RSA divided by the MTA phrenic amplitude (RSAa) showed a power-law relationship with both arterial carbon dioxide partial pressure (PaCO2) and oxygen saturation (SaO2), but with different exponents for different conditions. However, the power-law relation between RSAa and respiratory frequency had an exponent indistinguishable from -2 whether hypoxia or hypercapnia was the stimulus for increased respiratory drive, and during both CFV and spontaneous breathing (-1.9 +/- 0.4, hypoxia, SB; -1.8 +/- 0.7, hypoxia, CFV; -2.1 +/- 0.8, hypercapnia, SB; -1.9 +/- 0.7, hypercapnia, CFV). We conclude that respiratory sinus arrhythmia is centrally mediated and directly related to respiratory drive, and that changes in blood gases and phasic afferent signals affect RSA primarily by influencing respiratory drive.

V iral growth in specific tissue is usually required in order to lead to pathology. Two reovirus isolates (type 1 Lang and type 3 Dearing) differ in their capacity to grow in cultured mouse heart cells. The mammalian reoviruses contain a genome of 10 double-stranded RNA gene segments. By the use of 37 reassortant viruses (consisting of viruses with different combinations of genes derived from the two parents), difference in capacity of different strains to grow in heart cells was mapped to three different genes, all of which encode viral core proteins: the M1 gene (P less than 0.000044); the L1 gene (P = 0.00094); and the L3 gene (P = 0.019). Using the same set of reassortant viruses, the L1 (P = 0.00015) and L3 (P = 0.0065) genes were involved in differences of the ability of viral strains to grow in mouse L cells (fibroblasts), but the M1 gene (P = 0.12) was not. These findings suggest that the M1 gene plays an important and specific role in determining the relative capacity of certain viral strains to grow in the heart. Thus, we have identified viral genes responsible for differing growth capacity in heart muscle cells in culture. These findings provide a novel system for studies of viral myocarditis at a molecular genetic level.

T he effects of thyroid-stimulating antibodies (TSAb) and of thyrotropin (TSH) were compared, on the generation of cyclic AMP and inositol phosphates (InsP), in human thyroid slices incubated in vitro, and on the Rapoport cyclic AMP bioassay. The TSAb positive sera were obtained from 19 patients with Graves' disease. In 14 experiments with the slices system, TSH significantly increased cyclic AMP accumulation (TSH, 0.03-10 mU/ml) as well as the cyclic AMP-independent inositol trisphosphate (InsP3) generation (TSH, 1-10 mU/ml). In the same 14 experiments, TSAb (0.10-28 mg/ml) enhanced cyclic AMP intracellular levels as expected while they did not induce any InsP accumulation. Even when TSAb increased cyclic AMP levels to the same or higher values as those obtained with TSH concentrations allowing InsP3 generation. TSAb were still unable to activate the phosphatidylinositol-Ca2+ cascade. The patterns of the response curves of TSAb and TSH on cyclic AMP accumulation were different, suggesting that different mechanisms may be involved. In addition, unlike TSH, TSAb were not able to stimulate H2O2 generation, which in human tissue mainly depends on the activation of the phosphatidylinositol-Ca2+ cascade. Immunoglobulins from six additional Graves' patients lacking measurable cyclic AMP-stimulating activity in both slices and cells systems did not activate phospholipase C either. In conclusion, our results show that TSAb do not share all the metabolic actions of TSH on human thyroid tissue. The data provide support for the concept that the pathogenesis of Graves' disease can be fully accounted for by the ability of TSAb to stimulate adenylate cyclase. This work also confirms that TSH activates the cyclic AMP and the phosphatidylinositol cascade by independent pathways in the human thyroid.

A possible relationship between protein kinase C activation and impaired receptor-mediated endothelium-dependent relaxation in diabetes mellitus was examined in isolated aorta from normal rabbit exposed to elevated glucose. Aorta treated for 10 min with 4-phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, showed decreased relaxations to the endothelium-dependent vasodilator, acetylcholine, similar to normal aorta exposed to elevated glucose (22 and 44 mM) for 6 h. Relaxations to the receptor-independent endothelium-dependent vasodilator, A23187, and those caused by the direct smooth muscle vasodilator, sodium nitroprusside, were unaffected by treatment with PMA or exposure to elevated glucose. Indomethacin increased relaxations to acetylcholine of aorta treated with PMA indicating a role for vasoconstrictor prostanoids. PMA caused a significant increase in basal and acetylcholine-stimulated release of vasoconstrictor prostanoids including thromboxane A2 from aortic segments with, but not without endothelium. Protein kinase C inhibitors, H-7 or sphingosine, restored the abnormal acetylcholine-induced relaxations as well as suppressed the abnormal release of prostanoids in aorta exposed to elevated glucose. These findings suggest that the dysfunction of receptor-mediated endothelium-dependent relaxation associated with exposure to elevated glucose is due to increased production of vasoconstrictor prostanoids by the endothelium as a consequence of protein kinase C activation.

A trial natriuretic factor (ANF) is a peptide hormone of cardiac origin elevated in acute congestive heart failure (CHF), which is degraded by the enzyme neutral endopeptidase 24.11 (NEP). This study was designed to investigate the pulmonary and urinary clearance of ANF before and after the initiation of acute experimental CHF in dogs, and to assess the contribution of enzymatic degradation to these clearances in CHF. This study demonstrated a significant clearance of plasma ANF across the pulmonary circulation at baseline, and a tendency for pulmonary clearance to decrease in CHF (1115 +/- 268 to 498 +/- 173 ml/min, NS). The pulmonary extraction of ANF present at baseline was not altered with acute CHF (36.0 +/- 7.8 to 34.9 +/- 12.1%, NS). NEP inhibition (NEPI) abolished both the clearance and extraction of plasma ANF across the lung in CHF. Similarly, significant urinary clearance of ANF was present at baseline, and in acute CHF the urinary clearance of ANF decreased (0.14 +/- 0.02 to 0.02 +/- 0.01 ml/min, P less than 0.05). NEPI prevented the decrease in the urinary clearance of ANF, and enhanced the renal response to endogenous ANF, independent of further increases in plasma ANF during CHF. This study supports an important role for NEP in the pulmonary and urinary metabolism of endogenous ANF during acute CHF.

A lthough endothelium-derived relaxing factor (EDRF) has been studied extensively in large vessels, little is known about its role in the preglomerular afferent arteriole (Af-Art). We tested the hypothesis that EDRF, which is produced locally in the Af-Art, modulates arteriolar responses to angiotensin II (AII). A single rabbit Af-Art with its glomerulus intact was microperfused in vitro at 60 mmHg. When 0.1 microM AII was first applied, luminal diameter decreased by 49 +/- 7.0% (n = 9; P less than 0.0001); however, constriction waned, with the decrease becoming 15 +/- 3.5% at 1 min. After washing the Af-Art, repeated AII caused less constriction (13 +/- 4.0%; P less than 0.0002 vs. first application), showing tachyphylaxis. Pretreatment with Nw-nitro-L-arginine (N-Arg), which inhibits synthesis of nitric oxide (an EDRF), decreased basal diameter by 18 +/- 3.0% (n = 14; P less than 0.0001). N-Arg also augmented AII-induced constriction (86 +/- 6.8%; P less than 0.02 vs. nontreated Af-Art) and rendered it persistent (82 +/- 6.9% at 1 min). Even after pretreatment with N-Arg, repeated AII caused a weaker response, which was restored by washing with kidney homogenate rich in angiotensinase. In conclusion, this study provides evidence that local production of EDRF is an important determinant of the tone of the Af-Art. Our results suggest that the transient nature of AII-induced constriction of the Af-Art may be due to production of EDRF, while tachyphylaxis may be the result of long lasting receptor occupancy.

S teroid 11 beta-hydroxylase (P450c11) deficiency (failure to convert 11-deoxycortisol to cortisol) causes less than 10% of cases of congenital adrenal hyperplasia in most populations, but it is relatively frequent in Jews of Moroccan origin. P450c11 is encoded by the CYP11B1 gene which is located on chromosome 8q22 along with a homologous gene of unknown function, CYP11B2. To identify mutations in CYP11B1 associated with 11 beta-hydroxylase deficiency in Moroccan Jews, oligonucleotides were used that selectively amplified portions of CYP11B1 in polymerase chain reactions without amplifying CYP11B2. Sequence analysis of amplified fragments from one patient revealed a single base substitution in exon 8, codon 448 from CGC (arginine) to CAC (histidine). This residue is within the "heme binding" peptide that contains a cysteine that is a ligand to the heme group. The equivalent of Arg-448 is found in every known eukaryotic P450, and therefore it seems likely that a mutation of this residue would adversely affect enzymatic activity. 11 of 12 affected alleles from six Moroccan Jewish families carried the mutation in codon 448. This mutation is not normally present in CYP11B2 and thus appears to have arisen in CYP11B1 as a true point mutation rather than a gene conversion.

W e examined the possibility that the cutaneous permeability barrier regulates epidermal DNA synthesis in two acute and two chronic models of barrier perturbation. In animals treated topically with acetone, DNA synthesis is increased 102%, in tape-stripped animals 127%, in essential fatty acid deficient animals 50%, and in animals chronically treated with topical lovastatin 64%. This linkage between disturbances in barrier function and increased DNA synthesis is further supported by specific and correlative observations: (a) in these disparate models, artificial replacement of the barrier with a water-impermeable membrane inhibits the expected increase in DNA synthesis; (b) the extent of the burst in DNA synthesis is proportional to the degree of barrier abrogation; (c) the inhibition of DNA synthesis by membranes is directly related to the degree of permeability of these occlusive membranes, i.e., the more impermeable the greater the degree of inhibition; (d) topical treatment with lipids that restore barrier function corrects the increase in DNA synthesis; and (e) barrier abrogation with acetone produces an increase in epidermal DNA synthesis without altering bulk protein synthetic rates in contrast to events known to follow injury or cell replacement. Autoradiographic studies show that the increase in DNA synthesis after acetone treatment is limited to the epidermal basal layer. This constellation of findings strongly suggests that cutaneous barrier function is one factor that regulates epidermal DNA synthesis.

I n bacterial meningitis, LPS induces production in cerebrospinal fluid of the cytokines IL-1 beta and tumor necrosis factor alpha (TNF alpha), which are the principle mediators of meningeal inflammation. IL-1 beta and TNF alpha induce fever, and elevated temperature may affect cytokine expression. Dexamethasone treatment improves outcome in bacterial meningitis possibly by inhibiting IL-1 beta and TNF alpha. In this report, the effects of elevated temperature and dexamethasone on LPS-stimulated IL-1 beta and TNF alpha mRNA gene expression and protein synthesis were studied in human astrocytoma cell lines and primary cultures of human fetal astrocytes. Cells cultured at 40 degrees C exhibited smaller peaks of IL-1 beta and TNF alpha transcription and protein synthesis compared with cells cultured at 37 degrees C. The addition of dexamethasone before, during, or after exposure of the cells to LPS resulted in temperature-dependent inhibition of IL-1 beta transcription and protein synthesis. The most extensive inhibition occurred in pretreated cells cultured at 37 degrees C. Cotreatment with LPS and dexamethasone also inhibited TNF alpha mRNA transcription at both temperatures. The effects of another antiinflammatory agent, indomethacin, on LPS induction of IL-1 beta and TNF alpha mRNA were temperature and cell line dependent. These findings provide a possible explanation for the efficacy of dexamethasone treatment of bacterial meningitis and support the proposal that fever may be beneficial to the host in this disease.

P ositron emission tomography in combination with the newly introduced catecholamine analogue [11C]hydroxyephedrine ([11C]HED) enables the noninvasive delineation of sympathetic nerve terminals of the heart. To address the ongoing controversy over possible reinnervation of the human transplant, 5 healthy control subjects and 11 patients were studied after cardiac transplant by this imaging approach. Regional [11C]HED retention was compared to regional blood flow as assessed by rubidium-82. Transplant patients were divided into two groups. Group I had recent (less than 1 yr, 4.4 +/- 2.3 mo) surgery, while group II patients underwent cardiac transplantation more than 2 yr before imaging (3.5 +/- 1.3 yr). [11C]HED retention paralleled blood flow in normals, but was homogeneously reduced in group I. In contrast, group II patients revealed heterogeneous [11C]HED retention, with increased uptake in the proximal anterior and septal wall. Quantitative evaluation of [11C]HED retention revealed a 70% reduction in group I and 59% reduction in group II patients (P less than 0.001). In group II patients, [11C]HED retention reached 60% of normal in the proximal anterior wall. These data suggest the presence of neuronal tissue in the transplanted human heart, which may reflect regional sympathetic reinnervation.

L oss of heterozygosity (LOH) at specific loci may help localize tumor suppressor genes involved in the formation of various familial and sporadic tumors. In addition, the genetic loci for a number of familial tumor syndromes have been mapped by linkage analysis. To explore the possible role of tumor suppressor genes in endocrine tumors, we tested 41 pheochromocytomas (34 sporadic and 7 familial) and 11 medullary thyroid cancers (MTC) (10 sporadic and 1 familial) for LOH near a variety of potentially important genetic loci: (a) the multiple endocrine neoplasia type 2A (MEN 2A) locus on chromosome 10; (b) the von Hippel-Lindau locus on 3p; and (c) the p53 and neurofibromatosis 1 loci on 17. We also examined chromosomes 1p and 22q because previous studies in a small number of pheochromocytomas and MTCs suggested LOH in these regions. Background rates for LOH were assessed using several "random" probes. Finally, we examined a number of clinical and histologic characteristics of these tumors for possible correlations with specific genetic alterations. LOH in the region of the MEN 2A locus was uncommon (0% for MTCs, 5% for pheochromocytomas). However, we found significant allelic losses in pheochromocytomas on chromosomes 1p (42%), 3p (16%), 17p (24%), and 22q (31%). We also noted a correlation between LOH on 1p and urinary excretion of metanephrine by these patients (P = 0.02). LOH on 1p, 3p, and 17p also appeared to be associated with increased tumor volume. Analysis of the smaller number of MTCs demonstrated allelic losses on chromosomes 1p and 22q. Our results suggest that tumor formation and/or progression in pheochromocytomas and MTCs involves multiple genes, analogous with the model proposed for colon carcinoma.

W e postulated that vascular phosphoinositide metabolism is attenuated during pregnancy, and thereby could contribute to maternal vasodilation and reduced vascular reactivity. The basal rate of incorporation of [3H]myo-inositol and [3H]glycerol into phosphoinositides of aortae from pregnant rats in vitro was significantly reduced, when compared with vessels from virgin animals. After injection of [3H]myo-inositol intravenously into chronically instrumented conscious pregnant and virgin rats, the incorporation of the label by phosphatidylinositol was 66 +/- 4% less in aortae of gravid versus virgin animals (P less than 0.001), despite comparable plasma concentrations of radioactivity. Fold stimulation of total [3H]inositol phosphates by arginine vasopressin, norepinephrine, and angiotensin II over a 15-min period was not different between aortic segments from virgin and gravid rats, although both absolute basal and stimulated levels were significantly less in vessels from pregnant animals. After 45 s of incubation with 10(-7) M arginine vasopressin, however, the fold-stimulation of [3H]inositol trisplus tetrakisphosphate was reduced in aortae from gravid rats, when compared with vessels from virgin animals (P less than 0.005). By HPLC, greater than 90% of the radioactivity in the [3H]inositol trisplus tetrakisphosphate column fraction after 30 and 60 s of agonist stimulation was [3H]inositol-1,4,5-trisphosphate. We further observed that the rate of uptake of [3H]myo-inositol by aortic vasculature obtained from gravid rats was significantly (24%) less than uptake by vessels from virgin animals. Plasma myo-inositol concentrations were not significantly different, but presumably as a consequence of reduced uptake, aortic segments freshly isolated from pregnant rats contained 22 +/- 6% less myo-inositol than vessels from virgin controls as measured by gas chromatography-mass spectrometry (P less than 0.03). We conclude that myo-inositol uptake and content, phosphoinositide turnover, and inositol phosphate production are reduced in aortic vasculature of gravid rats.

T he mechanisms by which HIV-1 infection kills T lymphocytes are not clearly established. Apoptosis is an internally programmed cell death pathway that may regulate both T cell development and senescence, and that is characterized by cleavage of DNA at internucleosomal regions. The present experiments show that acute HIV-1 infection of MT2 lymphoblasts and activated normal peripheral blood mononuclear cells induces apoptosis. The addition of anti-gp120 neutralizing antibody, after HIV-1 infection of MT2 cells, permitted sustained high levels of viral replication, but blocked apoptosis and cell death. Apoptosis may account for the direct cytopathologic effects of HIV-1 in T cells.

W e report methods allowing the culture of rapidly dividing gastric epithelial cells to investigate the regulation of mucosal cell replication. Cells from canine fundic mucosa were dispersed by enzyme treatment, enriched by filtration and elutriation, and cultured on collagen gel in DMEM/F12 medium. After 48 h, greater than 95% of the cells displayed immunoreactivity with antibody to cytokeratin, an epithelial marker. The cells formed confluent monolayers by 72 h with a transmembrane resistance of 1,600 ohm.cm2 when mounted in a Ussing chamber indicating retention of epithelial cell characteristics. Calf serum (0.1-2%) produced a dose-dependent mitogenic effect evident by increases in [3H]-thymidine incorporation into acid-precipitated material and in cell number. After an 18-24-h incubation with [3H]-thymidine, approximately 55% of the cells cultured in 2% serum showed evidence of DNA synthesis by autoradiography and all of the replicating cells were cytokeratin positive. Using comparable culture conditions, a similar proportion of cells incubated for 18-24 h with bromodeoxyuridine displayed nuclear anti-bromodeoxyuridine immunoreactivity, thus indicating that over half of the cells in these cultures synthesized DNA during this period. As with serum, epidermal growth factor and transforming growth factor alpha (TGF alpha) (10 pM to 1 nM), insulin (10 nM to 1 microM) and insulinlike growth factor-I (IGF-I, 1-100 nM) increased [3H]-thymidine uptake. The greater potency of IGF-I, compared to insulin, suggests the presence of IGF-I receptors. We conclude that this culture preparation is composed of fundic mucosal epithelial cells and contains a predominance of dividing epithelial cells. EGF/TGF alpha and IGF-I are potential factors directly regulating proliferation of fundic mucosal cells.

A polipoprotein C-III is a major protein constituent of triglyceride rich lipoproteins and HDL. It occurs in plasma in three isoforms differing by their sialic acid content. Apo C-III putatively inhibits lipolysis and the apo E mediated hepatic uptake of remnants from triglyceride rich particles. We identified a heterozygous carrier of an apolipoprotein C-III variant by the presence of additional bands after isoelectric focusing (IEF) of VLDL. Structural analysis of the variant protein by HPLC, time-of-flight secondary ion mass spectrometry, and automated gas phase sequencing revealed a lysine to glutamic acid replacement in position 58. The underlying A to G exchange was verified by direct sequencing subsequent to amplification by polymerase chain reaction of exon 4 of the apo C-III gene. Family studies revealed vertical transmission of this defect. The two variant carriers exhibited plasma concentrations of HDL cholesterol and apo A-I above the 95th percentiles of sex matched controls whereas the unaffected father and sister showed normal values. The plasma concentrations of apo C-III in the two variant carriers were decreased by 30-40% compared with those of the two unaffected family members and to random controls. Using two-dimensional immunoelectrophoresis as well as IEF and subsequent scanning densitometry, we found that the low serum concentration of apo C-III was a consequence of diminished concentrations of the variant apo C-III isoproteins in both VLDL (15% of normal) and HDL (25% of normal). Apo C-III(Lys58----Glu) heterozygotes possessed unusual HDL as demonstrated by nondenaturing gradient gel electrophoresis. They consisted mainly of HDL2b and contained a proportion of atypically large particles, enriched in apo E, with a Stokes diameter of 13-18 nm and resembling HDLc. In conclusion, heterozygosity for a structural apo C-III variant--apo C-III(Lys58----Glu)--was identified in two hyperalphalipoproteinemic subjects characterized by the presence of low plasma apo C-III concentrations and atypically large HDL.

I n cultured intact LLC-PK1 renal epithelial cells, a nonhydrolyzable ATP analogue, ATP gamma S, inhibits AVP-stimulated cAMP formation. In LLC-PK1 membranes, several ATP analogues inhibit basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in a dose-dependent manner. The rank order potency of inhibition by ATP analogues suggests that a P2y type of ATP receptor is involved in this inhibition. The compound ATP gamma S inhibits agonist-stimulated adenylate cyclase activity in solubilized and in isobutylmethylxanthine (IBMX) and quinacrine pretreated membranes, suggesting that ATP gamma S inhibition occurs independent of AVP and A1 adenosine receptors and of phospholipase A2 activity. The ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity is not affected by pertussis toxin but is attenuated by GDP beta S, suggesting a possible role for a pertussis toxin insensitive G protein in the inhibition. Exposure of intact LLC-PK cells to ATP gamma S results in a significant increase in protein kinase C activity. However, neither of two protein kinase C inhibitors (staurosporine and H-7) prevents ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity, suggesting that this inhibition occurs by a protein kinase C independent mechanism. These findings suggest the presence of functional P2y purinoceptors coupled to two signal transduction pathways in cultured renal epithelial cells. The effect of P2y purinoceptors to inhibit AVP-stimulated adenylate cyclase activity may be mediated, at least in part, by a pertussis toxin insensitive G protein.

G lucocorticosteroids have an inhibitory effect on the expression of interleukin 2 (IL-2) and interleukin 2 receptor (IL-2R) genes. To determine the mechanisms of this inhibition, human T lymphocytes were stimulated with mitogens in the presence of dexamethasone. Nuclear transcription run-off assays showed that high doses of dexamethasone inhibited the transcription of the IL-2 gene but not that of the IL-2R gene. Post-transcriptionally, high doses of dexamethasone (10(-4) M) were required to inhibit IL-2R mRNA levels by 50%, whereas lower doses (10(-6) M) inhibited by greater than 70% the accumulation of IL-2 mRNA. IL-2 mRNA half-life decreased in the presence of dexamethasone (10(-6) M) by approximately 50%. At the protein product level, dexamethasone inhibited both IL-2 production, as well as cell surface and soluble forms of IL-2R. IL-2R gene expression was inhibited for at least 72 h after exposure of cells to dexamethasone. In the presence of exogenous IL-2, dexamethasone failed to exert a significant effect on the production of IL-2R protein. These data indicate that dexamethasone has a greater effect on the expression of the IL-2 gene than on the IL-2R gene. Dexamethasone both inhibits transcription of the IL-2 gene and decreases the stability of IL-2 mRNA. The effect of dexamethasone on the IL-2R gene is post-transcriptional and may result indirectly from decreased IL-2 production.

W e describe a kindred in which the proband and 6 of his 12 children have hypobetalipoproteinemia. The plasma lipoproteins of the affected subjects contained a unique species of apolipoprotein (apo) B, apo B67, in addition to the normal species, apo B100 and apo B48. The size of apo B67 and immunochemical studies with a panel of apo B-specific antibodies indicated that apo B67 was a truncated species of apo B that contained approximately the amino-terminal 3,000-3,100 amino acids of apo B100. Sequencing of genomic apo B clones revealed that affected family members were heterozygous for a mutant apo B allele containing a single nucleotide deletion in exon 26 (cDNA nucleotide 9327). This frameshift mutation is predicted to result in the synthesis of a truncated apo B containing 3,040 amino acids. Apo B67 is present in low levels in the plasma but is easily detectable within the very low density lipoprotein and low density lipoprotein fractions. Examination of the proband's immediate family revealed seven normolipidemic subjects and seven subjects with hypobetalipoproteinemia. In the affected subjects, the mean total and low density lipoprotein cholesterol levels were 120 and 42 mg/dl, respectively. A significantly higher mean high density lipoprotein cholesterol level was found in the affected subjects (75 vs. 55 mg/dl). We hypothesize that the elevated high density lipoprotein cholesterol levels in subjects heterozygous for the apo B67 mutation may be metabolically linked to the low levels of apo B-containing lipoproteins in their plasma.

D iabetes was induced in rats by administration of a single i.p. injection of streptozotocin (50 mg/kg body wt). After 7 d, diabetic rats were further treated with insulin or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] for an additional 5-7 d. Control, diabetic, diabetic + insulin, and diabetic + 1,25(OH)2D3 rats were then killed, their proximal small intestines were removed, and villus-tip epithelial cells were isolated and used to prepare brush-border membrane vesicles. Preparations from each of these groups were then analyzed and compared with respect to their amiloride-sensitive, electroneutral Na(+)-H+ exchange activity, using 22Na uptake as well as acridine orange techniques. The results of these experiments demonstrated that (a) H+ gradient-dependent 22Na uptake as well as Na+ gradient-dependent transmembrane H+ fluxes were significantly increased in diabetic vesicles compared to their control counterparts, (b) kinetic studies demonstrated that this enhanced 22Na uptake in diabetes was a result of increased maximal velocity (Vmax) of this exchanger with no change in apparent affinity (Km) for Na+, (c) serum levels of 1,25(OH)2D3 were significantly lower in diabetic animals compared with their control counterparts; and (d) insulin or 1,25(OH)2D3 treatment restored the Vmax alterations to control values, without any significant changes in Km, concomitant with significantly increasing the serum levels of 1,25(OH)2D3 in diabetic animals. These results indicate that Na(+)-H+ activity is significantly increased in proximal small intestinal luminal membranes of streptozotocin-induced diabetic rats. Moreover, alterations in the serum levels of 1,25(OH)2D3 may, at least in part, explain this enhanced antiporter activity and its correction by insulin.

M edium from cocultures of human aortic endothelial cells (HAEC) and smooth muscle cells (HASMC) taken from the same donor contained approximately two- to fourfold more macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, and up to 5.1-fold more transforming growth factor beta than could be accounted for by the sum of the activities of media from equivalent numbers of HAEC and HASMC cultured separately. After pulse labeling, immunoprecipitated [35S]fibronectin and [14C]collagen were also found to be substantially increased in the coculture compared to the sum of HAEC and HASMC cultured separately. The cocultivation of HAEC and HASMC resulted in a 2.7-fold increase in connexin43 messenger RNA. When direct physical contact between HAEC and HASMC was prevented by a membrane that was permeable to medium, the levels of [35S]fibronectin and [14C]collagen in the coculture were significantly reduced. Monocytes cultured alone contained low levels of [35S]fibronectin and [14C]collagen but when added to the coculture there was up to a 22-fold increase in [35S]fibronectin and a 1.9-fold increase in [14C]collagen compared to the coculture alone. The increase in fibronectin was prevented in the presence of neutralizing antibody to interleukin 1 and antibody to interleukin 6 by 45% and 67%, respectively. Addition of monocytes to cocultures also induced the levels of mRNA for connexin43 by 2.8-fold. We conclude that the interaction of HAEC, HASMC, and monocytes in coculture can result in marked increases in the levels of several biologically important molecules and that increased gap junction formation between the cells and interleukins 1 and 6 may be partially responsible for these changes.

M onocytes in the circulation of normal individuals express two receptors for the constant region of immunoglobulin, Fc gamma RI and Fc gamma RII. In contrast, we have observed that AIDS monocytes express significant levels of a third Fc gamma R, Fc gamma RIII (CD16), which is normally associated with activation or maturation of the monocyte population. By dual-fluorescence analysis using a monoclonal antibody specific for Fc gamma RIII (MAb 3G8), 38.5 +/- 3.2% of the LeuM3 (CD14)-positive monocytes in AIDS patients were CD16 positive as compared to 10.4 +/- 1.0% for healthy individuals (n = 29; P less than 0.005). Furthermore, AIDS monocytes expressed Fc gamma RIII-specific mRNA which is expressed minimally or not at all in control monocytes. As a recently identified inducer of Fc gamma RIII expression on blood monocytes, transforming growth factor-beta (TGF-beta) was found to be elevated in the serum and/or plasma of AIDS patients. Moreover, incubation of normal monocytes with AIDS serum or plasma induced CD16 expression which correlated with serum TGF-beta levels (r = 0.74, P less than 0.001) and was inhibited with a neutralizing antibody to TGF-beta. Thus, the increased CD16 expression on peripheral blood monocytes in AIDS patients may be the consequence of elevated circulating levels of the polypeptide hormone TGF-beta.

E scherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT.

T he intravenous administration of heparin to patients before open heart surgery reduced ristocetin cofactor activity by 58% (P less than 0.01, t test), and this impairment of von Willebrand factor-dependent platelet function was closely related to plasma heparin levels (r2 = 0.9), but not to plasma von Willebrand factor (vWF) levels. We hypothesized that heparin may inhibit vWF-dependent platelet hemostatic functions by directly binding vWF in solution and interfering with vWF-GpIb binding. Using the in vitro techniques of ristocetin-induced platelet agglutination, fluorescent flow cytometric measurement of vWF-platelet binding, and conventional radioligand binding assays we observed that heparin inhibited both vWF-dependent platelet function and vWF-platelet binding in a parallel and dose-dependent manner. Heparin also inhibited platelet agglutination induced by bovine vWF and inhibited the binding of human asialo-vWF to platelets in ristocetin-free systems. The inhibitory potency of heparin was not dependent upon its affinity for antithrombin III, but was molecular weight dependent: homogeneous preparations of lower molecular weight were less inhibitory. Heparin impairment of vWF function may explain why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.

P revious studies have demonstrated that ionizing radiation induces the expression of certain cytokines, such as TNF alpha/cachectin. However, there is presently no available information regarding the molecular mechanisms responsible for the regulation of cytokine gene expression by ionizing radiation. In this report, we describe the regulation of the TNF gene by ionizing radiation in human myeloid leukemia cells. The increase in TNF transcripts by x rays was both time- and dose-dependent as determined by Northern blot analysis. Similar findings were obtained in human peripheral blood monocytes. Transcriptional run-on analyses have demonstrated that ionizing radiation stimulates the rate of TNF gene transcription. Furthermore, induction of TNF mRNA was increased in the absence of protein synthesis. In contrast, ionizing radiation had little effect on the half-life of TNF transcripts. These findings indicate that the increase in TNF mRNA observed after irradiation is regulated by transcriptional mechanisms and suggest that production of this cytokine by myeloid cells may play a role in the pathophysiologic effects of ionizing radiation.

I n vivo microscopy was used to assess the relationships among shear rate (and shear stress), leukocyte rolling velocity, and leukocyte adherence in a cat mesentery preparation. Shear rate in individual venules and arterioles of 25-35 microns diameter were varied over a wide range by graded occlusion of an arterial loop. There was a linear decline in leukocyte rolling velocity (Vwbc) as red cell velocity (Vrbc) was reduced. The ratio Vwbc/Vrbc remained constant despite variations in shear stress from 5-25 dyn/cm2. A reduction in shear stress was associated with an increased leukocyte adherence, particularly when Vwbc was reduced below 50 microns/s. Reduction in wall shear rate below 500 s-1 in arterioles allowed 1-3 leukocytes to adhere per 100 microns length of vessel, while venules exposed to the same shear rates had 5-16 adherent leukocytes. In arterioles, leukocyte rolling was only observed at low shear rates. At shear rates less than 250 s-1 leukocyte rolling velocity was faster in arterioles than venules, and the ratio Vwbc/Vrbc for arterioles was 0.08 +/- 0.02, which was fourfold higher than the ratio obtained in venules at similar shear rates. Pretreatment with the CD18-specific antibody (mAb) IB4 increased leukocyte rolling velocity in venules by approximately 20 microns/s at red cell velocities below 2,000 microns/s. mAb IB4 largely prevented the leukocyte adherence to arterioles and venules, and increased the ratio Vwbc/Vrbc observed in venules at low shear elicit a CD18-dependent adhesive interaction between leukocytes and microvascular endothelium, and that differences in shear rates cannot explain the greater propensity for leukocyte rolling and adhesion in venules than arterioles.

T o better understand the events involved in the local migration of inflammatory cells into sites of allergic reactions, we studied expression of the cytokine inducible endothelial cell (EC) neutrophil adhesion molecule, endothelial-leukocyte adhesion molecule (ELAM-1), in sequential skin biopsies from patients with respiratory allergy during the late phase reaction (LPR) between 20 min and until 24 h after intradermal allergen (ragweed or dust mites) injection. In 7 of 7 atopic patients but in only 1 of 4 apparently normal controls, allergen induced appearance of ELAM-1 on EC. ELAM-1 expression occurred concurrently with the development of inflammatory cell infiltrates by 3-4 h after intradermal injection. Saline injected sites in all subjects were negative. Skin organ cultures demonstrated that allergen could produce the same EC changes in vitro whether allergen was injected in vivo 20 min before culture or added during skin culture. These EC changes in organ culture were inhibited by the presence of combined anti-sera to both TNF-alpha and IL-1, but not by antisera to either cytokine alone. We conclude that EC activation occurs in elicited LPR and suggest that cytokine-induced EC activation may play a role in the migration of inflammatory cells into allergic skin reactions. Furthermore, resident cells in the skin rather than infiltrating leukocytes appear to be the source of the cytokines that mediate endothelial activation.

P hospholipase A2 (PLA2) activities in cytosolic, mitochondrial, and microsomal fractions of rat kidneys were characterized under control conditions, after ischemia, and subsequent to ischemia and reperfusion. Two forms of PLA2 activity were present in the cytosolic fraction: a high molecular weight form, active against phosphatidylcholine (PC), and phosphatidylethanolamine (PE), which upon purification has a molecular mass of 110 kD; and smaller form (Mr approximately 14 kD), active against PE. In mitochondrial and microsomal fractions a single form (Mr approximately 14 kD), active against both PC and PE, was dominant. Activities in each fraction were optimal at pH 8.5-9.5. Cytosolic PLA2 activity was enhanced when Ca2+ concentration [( Ca2+]) was increased over the range of 10(-7) to 10(-6) M. Mitochondrial PLA2 activity required higher [Ca2+] for activation (greater than 10(-6) M). After 45 min of ischemia cytosolic PLA2 activity was decreased, whereas mitochondrial and microsomal activities were increased. When ischemia was followed by 1 h of reperfusion, cytosolic, mitochondrial, and microsomal activities were enhanced. Ischemia alone did not change the gel filtration chromatography patterns of PLA2 activity, but ischemia and reperfusion resulted in the appearance of a new peak of activity in cytosolic and mitochondrial fractions (Mr approximately 2-3 kD). Thus, the rat kidney has multiple forms of PLA2 activity, likely representing distinct enzymes, with Ca2+ dependencies suggesting regulation by Ca2+ in vivo. Ischemia and reperfusion result in stable increases of PLA2 activity in each subcellular fraction, perhaps related to covalent modifications of PLA2's, which likely account for membrane phospholipid degradation, and increased tissue levels of unsaturated free fatty acids.

W e sought to determine whether systemic administration of proteases ameliorates membranous nephritis induced in rats by immunization and challenge with cationic bovine gamma globulin, and whether targeting of protease to glomerular capillaries increases efficacy. Proteases substituted with biotin were targeted via the cationic protein avidin A, which by virtue of its charge has affinity for the glomerular basement membrane. Despite identical pretreatment proteinuria, rats given untargeted protease (biotin-conjugated without avidin, or unconjugated plus avidin) had significantly less proteinuria than saline-treated controls and nephrotic rats given avidin plus biotin-conjugated (targeted) protease had even less proteinuria and reduced glomerular rat IgG and C3. Among more severely nephrotic rats, targeted protease was again more effective than untargeted protease at reducing proteinuria, and also decreased the size of electron-dense glomerular deposits, hypercholesterolemia, and creatininemia. Inactivated targeted proteases had no effect on proteinuria, hypercholesterolemia, or azotemia. Finally, active targeted protease did not affect proteinuria in the nonimmune mediated nephrosis induced by puromycin aminonucleoside. We conclude that systemic protease can specifically diminish glomerular immune deposits, proteinuria, hyperlipidemia, and creatininemia associated with experimental immune complex glomerulonephritis but not toxic nephrosis, and that targeted protease is more effective than untargeted protease.

N ormal structure and function of the lung parenchyma depend upon elastic fibers. Amorphous elastin is biochemically stable in vitro, and may provide a metabolically stable structural framework for the lung parenchyma. To test the metabolic stability of elastin in the normal human lung parenchyma, we have (a) estimated the time elapsed since the synthesis of the protein through measurement of aspartic acid racemization and (b) modeled the elastin turnover through measurement of the prevalence of nuclear weapons-related 14C. Elastin purified by a new technique from normal lung parenchyma was hydrolyzed; then the prevalences of D-aspartate and 14C were measured by gas chromatography and accelerator-mass spectrometry, respectively. D-aspartate increased linearly with age; Kasp (1.76 x 10(-3) yr(-1) was similar to that previously found for extraordinarily stable human tissues, indicating that the age of lung parenchymal elastin corresponded with the age of the subject. Radiocarbon prevalence data also were consistent with extraordinary metabolic stability of elastin; the calculated mean carbon residence time in elastin was 74 yr (95% confidence limits, 40-174 yr). These results indicate that airspace enlargement characteristic of "aging lung" is not associated with appreciable new synthesis of lung parenchymal elastin. The present study provides the first tissue-specific evaluation of turnover of an extracellular matrix component in humans and underscores the potential importance of elastin for maintenance of normal lung structure. Most importantly, the present work provides a foundation for strategies to directly evaluate extracellular matrix injury and repair in diseases of lung (especially pulmonary emphysema), vascular tissue, and skin.

L ymphocyte homing receptors (HRs) defined by Hermes antibodies (anti-CD44) and lymphocyte function associated antigen-1 (LFA-1, CD11a/CD18) are involved in lymphocyte binding to endothelial cells of high endothelial venules (HEVs) at sites where lymphocytes exit the blood. Their expression was correlated to the clinical behavior of 245 non-Hodgkin's lymphomas followed up for the median of 87 mo after the diagnosis. Lymphomas that showed no or weak staining intensity for HRs were more often of stage I (P = 0.005), disseminated less frequently hematogenously (P = 0.003), and had more favorable prognosis than lymphomas with intensive staining for HRs (P less than 0.0001) despite that they were more often histologically of high grade malignancy (P = 0.002). Expression of LFA-1 beta chain (CD18) did not correlate significantly with stage or survival, but had prognostic value in a subgroup of HR expression negative lymphomas (P = 0.03). HR staining intensity was an independent prognostic factor in a multivariate analysis. These findings indicate that Hermes/CD44 molecule is associated to the determination of the metastatic potential and prognosis of non-Hodgkin's lymphomas. They also reveal a new entity among non-Hodgkin's lymphomas, because lymphomas that express low levels of HR have favorable prognosis despite their often highly malignant histological appearance.

D irect measurement of de novo lipogenesis has not previously been possible in humans. We measured de novo hepatic lipogenesis in normal men by means of stable isotopes and by combining the acetylated-xenobiotic probe technique with mass isotopomer analysis of secreted very low density lipoprotein-fatty acids (VLDL-FA). Sulfamethoxazole (SMX) was administered with [13C]acetate during an overnight fast followed by refeeding with intravenous glucose (7-10 mg/kg of weight per min), oral Ensure (7-10 mg of carbohydrate/kg of weight per min), or a high-carbohydrate mixed-meal breakfast (3.5 g of carbohydrate/kg of weight). Respiratory quotients remained less than 1.0. High-performance liquid chromatography/mass spectrometry-determined enrichments in SMX-acetate attained stable plateau values, and hepatic acetyl-coenzyme A (CoA) dilution rate did not increase with refeeding (approximately 0.024 mmol/kg per min). The fraction of VLDL-palmitate derived from de novo lipogenesis was only 0.91 +/- 0.27% (fasted) and 1.64-1.97% (fed). For stearate, this was 0.37 +/- 0.08% and 0.47-0.64%. Precursor enrichments predicted from isotopomer ratios were close to measured SMX-acetate enrichments, indicating that SMX-acetate samples the true lipogenic acetyl-CoA pool. Stearate synthesis was less than palmitate and the two did not move in parallel. Estimated total VLDL-FA synthesis is less than 500 mg/day. Thus, de novo hepatic lipogenesis is a quantitatively minor pathway, consistent with gas exchange estimates; fatty acid futile cycling (oxidation/resynthesis) is not thermogenically significant; and synthesis rates of different nonessential fatty acids by human liver are not identical in nonoverfed normal men. The contribution and regulation of de novo lipogenesis in other settings can be studied using this technique.

H uman hepatocyte growth factor (hHGF) has recently been expressed as a recombinant polypeptide from Chinese hampster ovary cell transfectants. Using a primary rat hepatocyte bioassay, we have tested the biological activity of recombinant hHGF and compared it with native hHGF. Dose-response curves were almost identical, with half-maximal stimulation of DNA synthesis at 1-2 ng/ml (equivalent to approximately 10 pM). S-phase labeling index was similarly enhanced and numerous mitotic cells were observed. Recombinant and native hHGF also stimulated DNA synthesis and S-phase labeling index in primary adult human hepatocytes. Human cells were more responsive than rat hepatocytes, with recombinant hHGF slightly more potent than native hHGF (half-maximal stimulation 0.3 and 0.6 ng/ml, respectively). Since HGF levels rise in patients with fulminant hepatic failure and in animals after partial hepatectomy or administration of hepatotoxins, situations where liver regeneration occurs, HGF is suggested to play a key role in regulation of hepatic growth. The high potency of the factor on human hepatocytes reinforces its candidacy as a critical mitogen in human liver growth. The availability of a recombinant hHGF opens the way for in vivo experimental studies and to the possibility of using hHGF as a clinical therapeutic agent, either alone or in combination with other factors.

L ong-Evans Cinnamon (LEC) rats, an inbred strain of a mutant rat isolated from Long-Evans rats, develop hereditary hepatitis. To elucidate the role of copper metabolism in the development of the hepatitis in LEC rats, we examined the copper concentration in the tissues and serum levels of copper and ceruloplasmin. Copper concentration in the liver of LEC rats was over 40 times that of normal Long-Evans Agouti (LEA) rats, while the serum ceruloplasmin and copper concentrations in LEC rats decreased significantly. The hepatocytes of LEC rats show steatosis in cytoplasm and pleomorphism of mitochondria, resembling the histologic features of the liver in Wilson's disease. These findings suggest that the hereditary hepatitis in LEC rats is closely associated with copper toxicity, and may be dealing with a rat form of Wilson's disease. Thus the LEC rats will provide a unique and useful animal model for clarifying the mechanism and for developing treatment strategies for Wilson's disease and other abnormal copper metabolism in humans.

B ranched chain alpha-ketoacid dehydrogenase (BCKDH) deficiency results in maple syrup urine disease (MSUD). We examined the molecular basis of familial cases of MSUD by analyzing the activity, subunit structure, mRNA sequence, and genome structure of the affected enzyme. The BCKDH activity in the proband with MSUD was approximately 6% of the normal control level. Immunoblot analysis revealed that the E1 beta subunit of BCKDH was absent and that the E1 alpha subunit of BCKDH was markedly reduced. We amplified the cDNAs of the E1 alpha subunit and the E1 beta subunit of the BCKDH complex obtained from cells of the patient, using the polymerase chain reaction method, then sequenced the amplified cDNAs. The deduced amino acid sequence for the E1 alpha subunit of the patient's cell was normal. An 11-bp deletion was identified in the region that encoded the mitochondrial targeting leader peptide in the E1 beta cDNA. This 11-bp sequence is found in the first exon of the BCKDH-E1 beta gene, as a direct tandem repeat. Amplification of genomic DNA revealed that the consanguineous parents were heterozygous for this mutant allele, and sister and brother of the patient with the disease were homozygous for this mutant allele. This 11-bp deletion mutation caused a change in the reading frame and the mature E1 beta protein was defective. These observations show the biological importance of the E1 beta subunit of BCKDH to maintain normal function of the enzyme activity. The absence of the E1 beta subunit results in instability of the E1 alpha subunit.

W e studied whether a novel vasoconstrictor peptide, endothelin-1 (ET-1), is synthesized by and released from human carcinoma cell lines, and whether ET-1 stimulates proliferation of these tumor cells. ET-1-like immunoreactivity was released from both HeLa and HEp-2 cells as a function of time. Reverse-phase HPLC of the conditioned media from HeLa cells revealed a major peak coeluting with standard ET-1. Northern blot analysis demonstrated the expression of mRNA for ET-1 precursor in both tumor cell lines. Both cell lines contained a single class of specific binding sites for ET-1. ET-1 dose-dependently induced increases in cytosolic free Ca2+ concentration in fura-2-loaded tumor cells, whose effect was completely abolished by chelating extracellular Ca2+ or by Ca(2+)-channel blocker. ET-1 stimulated proliferation of the quiescent cell lines in a dose-dependent manner, whose effect was inhibited by Ca(2+)-channel blocker. Polyclonal antibody for ET-1 inhibited proliferation of these cell lines, whereas nonimmune serum had no effect. These results demonstrate that ET-1 is synthesized by and released from human epithelial carcinoma cell lines, and that exogenous and endogenous ET-1 stimulates proliferation of the cells possibly through Ca2+ influx, suggesting its role as an autocrine/paracrine growth factor for certain tumor cells.