Phenotypes of HeLa S3 variant cell lines resistant to growth inhibition by sodium butyrate.

HeLa cell variants capable of multiplying in the presence of sodium butyrate were used to study the relationship of cell cycle position to human chorionic gonadotropin (hCG) production and regulation of the genes encoding hCG alpha- and beta-subunits. The butyrate-resistant variants exhibit several different stable phenotypes. In wild-type HeLa cells, butyrate arrests cell division and modulates synthesis of alpha- and beta-subunits of glycoprotein hormones by coordinately regulating steady-state levels of their respective mRNAs. Because the variant cell lines replicate, in addition to producing hCG subunits in the presence of butyrate, cell cycle arrest does not seem to be a requirement for expression of glycoprotein hormone genes. Studies of histone modification suggest that neither hyperacetylation of histones H3 and H4 nor dephosphorylation of histones H1 and H2A mediates inhibition of cell replication. In the variants, alpha-subunit and hCG beta levels are independently regulated, as a consequence of independent regulation of alpha- and beta-hCG mRNA levels. Long-term effects of butyrate include derepression of some genes (hCG beta in the variant AO) and repression of others (hCG alpha in variant AO). Moreover, hormone production correlates with the steady-state levels of mRNA for each of the subunits, suggesting that regulation occurs before translation. These findings indicate that the butyrate-resistant variant cell lines are valuable for studies of the molecular mechanisms involved in regulation of expression of ectopic hormones.

Macrophage activation by Micropolyspora faeni does not suppress anamnestic pulmonary immunologic reactivity.

Determinants of lung immunologic response to antigens are not known, but could include alveolar macrophage (AM) activation. We tested the ability of AM activation to modify the anamnestic response by administering bovine serum albumin (BSA) intratracheally, activating AM (by intratracheal Micropolyspora faeni), and then exposing rabbits again to intratracheal BSA. We compared the results from 4 groups of animals: intratracheal administration of either 50 mg M. faeni or normal saline and later administration of either intratracheal or intramuscular BSA. M. faeni administered intratracheally increased the number of AM. These AM were activated (increased phagocytosis of IgG-coated particles). We found no difference in the amount of antibody in either lavage fluid or serum or in antigen-induced pulmonary parenchymal and hilar node lymphocyte proliferation among these 4 groups.

Terminal complement component deficiencies and rheumatic disease: development of a rheumatic syndrome and anticomplementary activity in a patient with complete C6 deficiency.

Hereditary deficiencies of early complement components have usually been associated with the development of rheumatic diseases like systemic lupus erythematosus (SLE), while terminal component deficiency is well known to predispose to recurrent neisserial infection. In contrast, only recently have patients been reported with rheumatic disease and hereditary deficiency of a terminal component. The clinical syndrome in these patients has been characterised as 'SLE-like'. We describe here a third patient with complete C6 deficiency and a systemic rheumatic illness characterised by fever, anaemia, lymphadenopathy, hepatosplenomegaly, episcleritis, and asymmetric arthritis. After blood transfusion her serum became anticomplementary; IgG antibody to human C6 was found to be the cause of anticomplement activity. Persistent absence of C6 in this patient and production of anti-C6 antibody after antigenic challenge indicate hereditary C6 deficiency. This case supports an association between hereditary deficiency of a terminal complement protein and the development of systemic rheumatic disease.

Fibronectin mediates chemotactic factor-stimulated neutrophil substrate adhesion.

Plasma fibronectin has been implicated as an important determinant of neutrophil adhesion to plastic surfaces. Using a monoclonal antifibronectin antibody, we examined the role of fibronectin (Fn) in chemotactic factor-mediated neutrophil attachment to various substrates. The chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (FMLP) significantly enhanced neutrophil adherence to multiple substrates including gelatin, gelatin coated with Fc fragments of human IgG or Fn, plastic alone, plastic coated with Fc fragments, or purified plasma Fn. An IgM monoclonal antibody to plasma Fn significantly inhibited FMLP-stimulated neutrophil attachment to gelatin, gelatin-Fc, gelatin-Fn, plastic, plastic-Fc, and plastic-Fn substrates when compared with the parent line myeloma supernatant or an irrelevant IgM monoclonal antibody. No reduction in FMLP-stimulated adherence to the gelatin-plasma or plastic-plasma substrates occurred in the presence of antibody. Anti-Fn antibody reduced FMLP-stimulated adhesion only when present during the entire assay; incubation of cells or substrates alone with antibody, followed by removal of excess antibody before addition of stimulus incubation, failed to alter adherence. These data suggest that neutrophil-derived Fn may play a role in chemotactic factor-induced neutrophil adherence to both collagenous and noncollagenous substrates. Further support for the hypothesis was suggested by the demonstration of release of immunoreactive Fn into incubation media from FMLP-stimulated neutrophils.

Aggravation by propranolol of hyperglycaemic effect of hydrochlorothiazide in type II diabetics without alteration of insulin secretion.

14 hypertensive men with type II diabetes sequentially received, in random order, hydrochlorothiazide 50 mg twice a day, propranolol 80 mg twice a day, and both drugs in combination. The 3-week treatment periods were separated by a 1-week washout period. Hydrochlorothiazide significantly increased fasting glucose by 31% (p less than 0.05) and glycosylated haemoglobin (HbA1c) by 6.0% (p less than 0.10). A similar treatment period of propranolol 80 mg twice a day caused no significant increases. However, when both drugs were taken in combination, fasting glucose rose by 56% and HbA1c by 14.7% (p less than 0.01). The hyperglycaemic effect of hydrochlorothiazide and its potentiation by propranolol were independent of serum potassium and of endogenous insulin secretion as measured by urine C-peptide excretion. The combination of hydrochlorothiazide and propranolol thus seems to cause serious disturbances in glycaemic control in type II diabetics by mechanisms independent of insulin secretion.

Pulmonary response to repeated exposure to Micropolyspora faeni.

Most human exposure to agents that cause hypersensitivity pneumonitis (HP) result in transient episodes of HP that resolve quickly. We repeatedly injected Micropolyspora faeni, which is responsible for farmer's lung disease, into rabbits in an attempt to elucidate mechanisms for this phenomenom (i.e., resolution of abnormalities). The character and the extent of lung disease, the amount of anti-M. faeni serum antibody, and skin reactivity to M. faeni were evaluated after 3 sensitizing and 2, 4, or 8 challenge injections. We also determined the fate of 125I labeled M. faeni injected intratracheally into both normal and previously exposed rabbits. Increased numbers of lymphocytes, macrophages, and few polymorphonuclear leukocytes were present in interstitial and intraalveolar regions and bronchial walls. Interstitial fibrosis was not observed. The extent of cellular abnormalities was maximal after 2 challenges and regressed thereafter, despite continuing intratracheal injections. Serum anti-M. faeni antibody peaked after 4 intratracheal challenges. Anti-M. faeni antibody level at the time of death appeared to be proportional to the extent of inflammatory reaction within the lung. Previous exposure of rabbits to M. faeni was associated with more rapid appearance of 125I in blood in the first 2 h after intratracheal injection of 125I M. faeni. However, 24 h after injection, there was less 125I in the lungs and more in the urine of immunized rabbits than in normal rabbits. Repeated intratracheal injections of M. faeni into rabbits produces transient interstitial, intraalveolar, and peribronchial inflammatory infiltration that regresses without fibrosis despite continued antigenic challenge. Immunization appears to markedly decrease pulmonary exposure to antigen that results from an intratracheal injection of M. faeni.

Inhibition of zymosan-induced alternative complement pathway activation by concanavalin A.

Zymosan, a polysaccharide composed primarily of glucan and mannan residues, activates the complement system through the alternative complement pathway. We showed that zymosan-induced complement activation is inhibited by zymosan-bound lectins with carbohydrate specificities for mannosyl and glycosyl residues. Lectins unable to bind mannosyl or glucosyl residues did not inhibit zymosan-induced complement activation.

Rapid fibrinolysis, augmented Hageman factor (factor XII) titers, and decreased C1 esterase inhibitor titers in women taking oral contraceptives.

The use of OCAs has been associated with multiple hemostatic abnormalities and an increased risk of thromboembolic disease. These changes have been attributed to increased synthesis of various clotting factors and decreased titers of antithrombin III. Paradoxically, enhanced in vitro fibrinolytic activity is also found in plasmas of women using OCAs. The present study demonstrates marked elevation of both procoagulant and antigenic HF titers in plasmas of women using OCAs, accompanied by a simultaneous decrease in plasma C-1-INH concentration. Titers of plasma prekallikrein, HMW kininogen, PTA, and alpha 2-Pl were unchanged. The rate of kaolin-assisted fibrinolysis was related directly to the titer of HF and inversely to C-1-INH concentration. Further, the addition of human HF to normal plasma enhanced fibrinolytic activity to a degree similar to that observed in plasmas of women taking OCAs. These data suggest that the increase in plasma HF concentration may participate in the phenomenon of enhanced in vitro fibrinolysis associated with OCA use, possibly augmented by diminished inhibitory control by C-1-INH. The relationship of these phenomena to the increased incidence of thrombosis is not known.

Quantitative measurement of properdin in normal human serum by electroimmunoassay and single radial immunodiffusion.

Properdin in normal serum was measured by electroimmunoassay (EIA) and single radial immunodiffusion (SRID). Fresh sera gave much lower properdin values in SRID in gels containing Mg2+ ions. Storage of sera at 4 degrees C resulted in a gradual increase of the properdin values measured by SRID but not of those of the EIA. With 10 mM of EDTA in the gels no difference between the properdin values obtained by the different methods was found. Evidence is presented that immunodiffusion values of properdin might be affected by precipitation of a C3-properdin complex in gels containing Mg2+ ions after the activation of the properdin system by agarose.

Radioimmunoassay of human Hageman factor (factor XII).

A secific, sensitive, and reproducible radioimmunoassay for human Hageman factor (HF, factory XII) has been developed with purified human HF and monospecific rabbit antibody. Precise measurements of HF antigen were possible for concentrations as low as 0.1% of that in normal pooled plasma. A good correlation (correlation co-efficient = 0.82) existed between the titers of HF measured by clot-promoting assays and radioimmunoassays among 42 normal adults. Confirming earlier studies, HF antigen was absent in Hageman trait plasma, but other congenital deficient plasmas, including those of individuals with Fletcher trait and Fitzgerald trait, contained normal amounts of HF antigen. HF antigen was reduced in the plasmas of patients with disseminated intravascular coagulation or advanced liver cirrhosis, but it was normal in those of patients with chronic renal failure or patients under treatment with warfarin. HF antigen was detected by this assay in plasmas of primates, but not detectable in plasmas of 11 nonprimate mammalian and one avian species.

Estrogen binding protein of rat liver.

An estrogen binding protein for estradiol-17beta is present in the liver cytosol of female intact and one day oophorectomized rats. The dissociation constant reveals high affinity binding (Kd: 0.69 +/- 0.14 times 10(-10) M). Quantitation of EBP using a dextran-coated charcoal method shows that this specific macromolecular binding is much less than in the rat uterus, but similar to that in DMBA-induced mammary tumors. Sucrose density gradient analysis shows sedimentation at 8-9 S and 4-5 S when compared to bovine serum albumin.

Measurement of circulating desialylated glycoproteins and correlation with hepatocellular damage.

Addition of increasing amounts of (125)I-labeled desialylated thyroxine-binding globulin (DTBG) to hepatic cell membranes resulted in a progressive increase in binding. Saturability of membrane sites was indicated by a concentration beyond which further increases in [(125)I]DTBG resulted in no further binding. The binding curve for [(125)I]DTBG was similar to binding curves of desialylated orosomucoid, fetuin, and ceruloplasmin. An inhibition assay system using hepatic cell membranes showed that desialylated orosomucoid had a greater affinity for membrane binding sites than did DTBG but desialylated fetuin and ceruloplasmin bound less avidly than DTBG. Serum from normal persons and patients with a variety of illnesses was tested for its ability to inhibit [(125)I]DTBG binding. The inhibitory activity of 1 ml of normal serum was equivalent to that of 0.2-2 mug DTBG. Patients with Laënnec's cirrhosis, biliary cirrhosis, and hepatic metastases had greatly increased inhibitory activity in their serum. Patients with jaundice due to extrahepatic obstruction had inhibitory activity not significantly different from that found in normal serum. Column chromatography of normal serum on Sephadex G-200 resulted in inhibitory activity throughout the range of protein molecular weight. Desialylation of normal serum with neuraminidase enhanced the inhibitory activity but did not change the distribution of the activity. Gel chromatography of cirrhotic serum showed markedly increased inhibitory activity associated with the macroglobulins and the 4.5S peak and a new peak of inhibitory activity in the low molecular weight area was also seen. Inhibition of desialylated glycoprotein binding to liver cell membranes by serum from patients with hepatocellular disease raises the possibility that desialylated serum glycoproteins accumulate in the circulation and that patients with compromised hepatocellular function may no longer be able to clear them from the circulation. Alternatively, accumulation of desialylated glycoproteins in the circulation could result from defective protein synthesis by the diseased liver.

Partial purification of plasma thromboplastin antecedent (factor XI) and its activation by trypsin.

A persistent puzzle in our understanding of hemostasis has been the absence of hemorrhagic symptoms in the majority of patients with Hageman trait, the hereditary deficiency of Hageman factor (factor XII). One proposed hypothesis is that alternative mechanisms exist in blood through which plasma thromboplastin antecedent (PTA, factor XI) can become active in the absence of Hageman factor. In order to test this hypothesis, the effect of several proteolytic enzymes, among them thrombin, plasma kallikrein, and trypsin, was tested upon unactivated PTA. PTA was prepared from normal human plasma by Ca(3)(PO(4))(2) adsorption, ammonium sulfate fractionation, and successive chromatography on QAE-Sephadex (twice). Sephadex-G150, and SP-Sephadex. The partially purified PTA was almost all in its native form, with a specific activity of 45-70 U/mg protein; the yield was about 10%. It contained no measurable amounts of other known clotting factors, plasmin, plasminogen, nor IgG. Incubation of PTA with trypsin generated potent clot-promoting activity that corrected the abnormally long clotting time of plasma deficient in Hageman factor or PTA but not in Christmas factor. This clot-promoting agent behaved like activated PTA on gel filtration (apparent molecular weight: 185,000) and was specifically inhibited by an antiserum directed against activated PTA. These data suggested that PTA can be converted into its active form by trypsin. PTA was not activated by thrombin, chymotrypsin, papain, ficin, plasmin, plasma kallikrein, tissue thromboplastin, or C. Trypsin converted PTA to its active form enzymatically. Whether trypsin serves to activate PTA in vivo is not yet clear.