Helicobacter pylori stimulates inducible nitric oxide synthase expression and activity in a murine macrophage cell line

BACKGROUND & AIMS: Helicobacter pylori uniquely colonizes the human stomach and produces gastric mucosal inflammation. High-output nitric oxide production by inducible nitric oxide synthase (iNOS) is associated with immune activation and tissue injury. Because mononuclear cells comprise a major part of the cellular inflammatory response to H. pylori infection, the ability of H. pylori to induce iNOS in macrophages was assessed. METHODS: H. pylori preparations were added to RAW 264.7 murine macrophages, and iNOS expression was assessed by Northern blot analysis, enzyme activity assay, and NO2- release. RESULTS: Both whole H. pylori and French press lysates induced concentration-dependent NO2- production, with peak levels 20-fold above control. These findings were paralleled by marked increases in iNOS messenger RNA and enzyme activity levels. iNOS expression was synergistically increased with interferon gamma, indicating that the H. pylori effect can be amplified by other macrophage-activating factors. Studies of lipopolysaccharide (LPS) content and polymyxin B inhibition of LPS suggested that the H. pylori effect was attributable to both LPS- dependent and -independent mechanisms. CONCLUSIONS: iNOS expression in macrophages is activated by highly stable H. pylori products and may play an important role in the pathogenesis of H. pylori-associated gastric mucosal disease. (Gastroenterology 1996 Dec;111(6):1524-33)     

Acquired hypoprothrombinemia due to non-neutralizing antibodies to prothrombin: mechanism and management

A patient developed bleeding due to an acute acquired specific prothrombin deficiency. Unlike previously described patients, this patient had no evidence of an associated lupus anticoagulant. Prothrombin activity and antigen were decreased concordantly and the patient's plasma did not neutralize the activity of added prothrombin or interfere with its measurement by electroimmunoassay. Nevertheless, immunoelectrophoresis and experiments using 125I-prothrombin revealed a prothrombin-binding antibody. The residual prothrombin in the patient's plasma was in the form of a prothrombin-antibody complex. Administration of adrenal corticosteroids was associated with a rise in prothrombin activity and cessation of bleeding, but circulating prothrombin was still bound to the antibody. This suggests that non- neutralizing antibodies to prothrombin cause plasma prothrombin deficiency because of a rapid clearance of prothrombin-antibody complexes, which is slowed by adrenal corticosteroids. The antibody had a relatively low affinity for prothrombin (Kd 5 to 8 X 10(-7)) and was transient. It is possible, therefore, that the antibody arose not to prothrombin itself, but to an antigen sharing an epitope with prothrombin.     

An essential role for lysophosphatidylcholine in the inhibition of platelet aggregation by secretory phospholipase A2

The release of secretory phospholipase A2 (sPLA2) into the mammalian circulation may contribute to the development of hemorrhagic and inflammatory diseases. sPLA2 has previously been shown to alter the behavior of platelets, leukocytes, and endothelial cells, although the molecular basis for these cellular effects has not been established. Our studies indicate that the inhibition of platelet aggregation by snake, bee venom, and pancreatic sPLA2 is dependent on a plasma cofactor. This cofactor resides within the lipoprotein fraction of plasma, with 54%, 31%, and 11% of the activity present in the high- density lipoprotein (HDL), low-density lipoprotein (LDL), and very low density lipoprotein (VLDL) fractions, respectively. Delipidation of HDL and LDL was associated with the complete loss of platelet-inhibitory activity. Incubation of purified sPLA2 with the HDL fraction of plasma resulted in the time-dependent generation of lysophosphatidylcholine (lysoPC). The formation of lysoPC correlated with the inhibition of platelet aggregation. Purified lysoPC (10 to 100 micrograms/mL) inhibited platelet aggregation and dense granule release induced by thrombin (0.05 U/mL), collagen (1 micrograms/mL), ionophore A23187 (2 mumol/L), ADP (12.5 mumol/L), and adrenaline (3.2 mumol/L). The inhibition of platelet aggregation by lysoPC was dose-dependent and correlated with decreased fibrinogen binding to glycoprotein IIb-IIIa. Our studies indicate that the enzymatic generation of lysoPC from plasma lipoproteins is essential for the sPLA2-mediated inhibition of platelet activation in the presence of albumin. These results raise the possibility that the toxic effects of circulating sPLA2 may be due in part to the generation of the bioactive lysophospholipid, lysoPC.     

Age-related changes in deformability of human erythrocytes

The present study was designed to further the characterization of age- related changes in the deformability of human erythrocytes. The top (approximately young) and bottom (approximately old) 10% fractions of density-separated red cells from ten normal donors were subjected to graded levels of shear stress in a rheoscope. Measurements were made of steady-state elongation (cells tank treading in a state of dynamic equilibrium) and the time course of shape recovery following abrupt cessation of shear. In parallel with the rheologic experiments, several physical and chemical properties were assayed to determine correlates of mechanical properties. These included mean cell volume, mean corpuscular hemoglobin concentration, type A1 hemoglobin, glucosylation of membrane proteins, and membrane phospholipid and protein concentration. The microrheologic observations revealed that only about 90% of the old cells retained their capacity to tank tread. However, the tank-treading cells elongated less than their younger counterparts at corresponding levels of shear stress, thus demonstrating a reduced level of deformability. Further analysis of the data indicates that increases in membrane viscosity and elastic modulus along with a significant loss in excess surface area contribute to the limitation of the ability of the older cells to change shape.     

Inflammatory Renin-Angiotensin System Disruption Attenuates Sensory Hyperinnervation and Mechanical Hypersensitivity in a Rat Model of Provoked Vestibulodynia

Vestibulodynia is characterized by perivaginal mechanical hypersensitivity, hyperinnervation, and abundant inflammatory cells expressing renin-angiotensin system proteins. We developed a tractable rat model of vestibulodynia to further assess the contributions of the renin-angiotensin system. Complete Freund's adjuvant injected into the posterior vestibule induced marked vestibular hypersensitivity throughout a 7-day test period. Numbers of axons immunoreactive for PGP9.5, calcitonin gene-related peptide, and GFRα2 were increased. Numbers of macrophages and T cells were also increased whereas B cells were not. Renin-angiotensin-associated proteins were abundant, with T cells as well as macrophages contributing to increased renin and angiotensinogen. Media conditioned with inflamed vestibular tissue promoted neurite sprouting by rat dorsal root ganglion neurons in vitro, and this was blocked by the angiotensin II receptor type 2 receptor antagonist PD123319 or by an angiotensin II function blocking antibody. Sensory axon sprouting induced by inflamed tissue was dependent on activity of angiotensin-converting enzyme or chymase, but not cathepsin G. Thus, vestibular Complete Freund's adjuvant injection substantially recapitulates changes seen in patients with provoked vestibulodynia, and shows that manipulation of the local inflammatory renin-angiotensin system may be a useful therapeutic strategy.