Human monocyte chemoattractant protein-1 (human MCP-1) mRNA accumulated in THP-1 cells 2 h after lipopolysaccharide (LPS) stimulation. DNase I footprinting revealed that LPS stimulation induced protein binding to the two closely located NF-B sites, A1 and A2. By electrophoretic gel mobility shift assay and supershift assay, the binding of (p65) 2 , c-Rel/p65, p50/p65, and p50/ c-Rel to the A2 oligonucleotide probe was detected after LPS stimulation. In contrast, 12-o-tetradecanoylphorbol 13-acetate did not induce a significant amount of MCP-1 mRNA in THP-1 cells 2 h after stimulation, and only p50/p65 bound to the A2 probe. trans-Activity of each NF-B/Rel dimer was investigated by transfecting P19 cells with p65, p50, and/or c-Rel expression vectors, and a luciferase construct containing the enhancer region of the human MCP-1 gene. Expression of recombinant p65 or p65 and c-Rel resulted in elevated luciferase activities, indicating that (p65) 2 and c-Rel/p65 had trans-activity. The binding of (p65) 2 and/or c-Rel/p65 to the A2 probe was also detected from 12-o-tetradecanoylphorbol 13-acetate-stimulated HeLa, HOS, and A172 cells in which expression of MCP-1 mRNA was elevated. Finally, the role of the A1 site was investigated. Both (p65) 2 and c-Rel/p65 bound to the A1 probe by electrophoretic mobility shift assay and a mutation in the A1 or A2 site resulted in a loss of the enhancer activity. These results suggest that the binding of (p65) 2 and c-Rel/p65 to the A1 and A2 sites of this gene is important for the tissue-and stimulus-specific transcription of the human MCP-1 gene.Blood monocytes infiltrate into the sites of inflammation and play major roles in host defense through their ability to present antigens and to produce various mediators. Although the mechanisms of monocyte infiltration have not been fully understood, locally produced monocyte chemoattractants seem to be responsible for the recruitment of blood monocytes into the sites of inflammatory reactions.Monocyte chemoattractant protein-1 (MCP-1) 1 is a member of the CC subfamily of the chemokine family and attracts blood monocytes both in vitro and in vivo (1-3). MCP-1 mRNA or protein was detected at high levels in the lesions of several diseases such as atherosclerosis (4, 5), arthritis (6), idiopathic pulmonary fibrosis (7,8), and various tumors (9 -11), strongly suggesting that MCP-1 plays a critical role in the recruitment of monocytes in these diseases. A wide variety of cells, including monocytes, fibroblasts, vascular endothelial cells, and smooth muscle cells, produces MCP-1 in vitro in response to various stimuli such as lipopolysaccharide (LPS), interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF␣), platelet-derived growth factor (PDGF), IFN-␥, or 12-o-tetradecanoylphorbol 13-acetate (TPA) (1-3). However, the mechanisms of MCP-1 production remain unknown.To understand the mechanisms involved in the expression of human MCP-1 mRNA in different types of cells at the molecular level, we previously investigated the transcription of human MCP-1 g...

Of 260 patients enrolled, 25 patients (9.6%) were associated with acquired von Willebrand syndrome (AvWS). We studied 25 patients with AvWS, retrospectively. AvWS was diagnosed by reduced levels of von Willebrand factor (vWF) (decrease of von Willebrand factor antigen [vWF:Ag] and von Willebrand ristocetin cofactor [vWF:RCoF]), a decrease of ristocetin-induced platelet agglutination (RIPA), sometimes decreased high-molecular-weight multimers, and prolonged bleeding time with neither prior nor family histories of bleeding problems and the evidence of normal vWF:RCoF in their families. The inhibitor of vWF was determined by mixing patient plasma with pooled normal plasma. Eight patients in this study had the inhibitors to vWF that were of the IgG class; the subclasses were IgG1 (7 cases) and IgG2 (1 case). Multimeric analysis of vWF showed selective loss of large multimers in most patients with AvWS similar to that of congenital type-2 von Willebrand disease (vWD). All inhibitors blocked ristocetin-mediated vWF binding to platelets. Five out of 6 IgGs evaluated here recognized the 39/34-kD fragment (residues 480/481-718) and Fragment III (residues 1-1365) that implied binding domain of glycoprotein Ib (GPIb), whereas 1 recognized Fragment I (residues 911-1365). A close relationship was found between the presence of the inhibitor and bleeding tendency. Of the 7 patients with inhibitors, 6 patients (86%) had a bleeding tendency, as well as 1 of the 15 patients without inhibitors (6%). The efficacy of treatment of underlying diseases and/or therapy with deamino D-arginine vasopressin (DDAVP) for the treatment of AvWS also depends on the presence of an inhibitor. Four of 8 patients with inhibitors (50%) had poor response to treatment of the underlying disease and/or therapy with DDAVP, as well as 1 of the 16 patients without inhibitors (6%). These results indicate that patients with AvWS developing inhibitors to vWF are likely to have bleeding problems and might be resistant to treatment of underlying diseases and/or therapy with DDAVP for bleeding to AvWS. We also showed evidence that intravenous immunoglobulin therapy (0.3 g/kg, 3 days) was effective to correct a hemostatic defect and manage severe bleeding in a patient with AvWS developing inhibitors. We might consider an additional treatment including expensive high-dose immunoglobulin therapy when uncontrollable bleeding is continued after the treatment of the underlying diseases and/or therapy with DDAVP.

We examined the hypothesis that superoxide mediates infiltration of neutrophils to the airways through nuclear factor (NF)-kappaB and interleukin-8 (IL-8) after acute exposure to cigarette smoke (CS) in vivo. Male Hartley strain guinea pigs were exposed to air or 20 puffs of CS and killed 5 h after the exposure. The differential cell count of bronchoalveolar lavage fluid and specific myeloperoxidase enzyme assay demonstrated that acute exposure to CS caused neutrophil accumulation to the airways and parenchyma, respectively. Acute exposure to CS increased DNA-binding activity of NF-kappaB in the lung. Acute exposure to CS also increased IL-8 messenger RNA (mRNA) expression in the lung. Pretreatment of guinea pigs with recombinant human superoxide dismutase (rhSOD) aerosols reduced the CS-induced neutrophil accumulation to the airways. Both activation of NF-kappaB and increased IL-8 mRNA expression were also inhibited by the pretreatment of rhSOD aerosols. Strong immunoreactivities for p65 and p50 were detected in the nuclei of alveolar macrophages after acute exposure to CS. The signal for IL-8 mRNA expression was demonstrated in the alveolar space after acute exposure to CS. Neither significant immunoreactivities for p65 and p50 nor IL-8 mRNA signals were observed in airway epithelium. These observations suggest that acute exposure to CS initiates superoxide-dependent mechanism that, through NF-kappaB activation and IL-8 mRNA expression, produces infiltration of neutrophils to the airways in vivo. It was also suggested that the alveolar macrophage is one potential source of NF-kappaB activation and IL-8 mRNA expression after acute exposure to CS.

Background-The sensation ofrespiratory effort may increase as expiratory muscles become fatigued during expiratory loading. A study was performed to determine whether expiratory muscle training (EMT) affects the sensation of respiratory effort during exercise in healthy subjects. Methods -Six subjects performed EMT for 15 minutes twice daily for four weeks using a pressure threshold device; another six subjects served as a control group. The expiratory threshold was set at 30% of the individual's maximum expiratory mouth pressure (PEmax). The sensation of respiratory effort was evaluated during a progressive exercise test using the Borg scale. Results -After EMT PEmax increased by 25% in the training group. The Borg score increased as exercise grade increased before and after EMT, but scores for each grade were lower after EMT. Minute ventilation during exercise decreased after EMT, as did the breathing frequency during exercise, while the expiratory time increased. Although there was no difference in the relationship between Borg score and minute ventilation before or after EMT, the curve shifted to a lower Borg score after EMT. There were no changes in PEmax, Borg score, minute ventilation, or breathing pattern after the four week study period in the control group. Conclusion -These findings suggest that EMT increases expiratory muscle strength and reduces the sensation of respiratory effort during exercise, presumably by reducing minute ventilation.

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