BACKGROUND: Caveolin, a family of integral membrane proteins are a principal component of caveolae membranes. In this study, we investigated the effect of p38 kinase on differentiation and on inflammatory responses in sodium nitroprusside (SNP)- treated chondrocytes. METHODS: Rabbit articular chondrocytes were prepared from cartilage slices of 2-week-old New Zealand white rabbits by enzymatic digestion. SNP was used as a nitric oxide (NO) donor. In this experiments measuring SNP dose response, primary chondrocytes were treated with various concentrations of SNP for 24 h. The time course of the SNP response was determined by incubating cells with 1 mM SNP for the indicated time period (0~24 h). The cyclooxygenase-2 (COX-2) and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin E2 (PGE2) assay was used to measure the COX-2 activity. The tyrosine phosphorylation of caveolin-1 was determined by immunoblot analysis and immunostaining. RESULTS: SNP treatment stimulated tyrosine phosphorylation of caveolin-1 and activation of p38 kinase. SNP additionally caused dedifferentiation and inflammatory response. We showed previously that SNP treatment stimulated activation of p38 kinase and ERK-1/-2. Inhibition of p38 kinase with SB203580 reduced caveolin-1 tyrosine phosphorylation and COX-2 expression but enhanced dedifferentiation, whereas inhibition of ERK with PD98059 did not affect caveolin-1 tyrosine phosphorylation levels, suggesting that ERK at least is not related to dedifferentiation and COX-2 expression through caveolin-1 tyrosine phosphorylation. CONCLUSION: Our results indicate that SNP in articular chondrocytes stimulates dedifferentiation and inflammatory response via p38 kinase signaling in association with caveolin-1 phosphorylation.
Cartilage ; Caveolae ; Caveolin 1 ; Chondrocytes* ; Collagen Type II ; Cyclooxygenase 2* ; Digestion ; Dinoprostone ; Humans ; Membrane Proteins ; Membranes ; Nitric Oxide ; Nitroprusside ; Phosphorylation ; Phosphotransferases* ; Rabbits ; Tissue Donors ; Tyrosine

Current therapies for autoimmune diseases are not cures but merely palliatives, aimed at reducing symptoms. For the most part, these treatments provide nonspecific suppression of the immune system and thus do not distinguish between a pathogenic autoimmune response and a protective immune response. Recently emerging evidence not only has indicated the involvement of members of the TNF receptor/ligand superfamilies but also has revealed exciting innovative strategies for the treatment of autoimmune diseases and other chronic inflammatory diseases without depressing the immune response in general. In this review, we will discuss the regulatory mechanisms of TNF receptor/ligand family members, such as HVEM/ LIGHT, 4-1BB/4-1BBL, and GITR/GITRL that regulate T and B cell functions and participate in the process of inflammatory diseases. We will also discuss how intervening in the costimulatory pathways mediated by these molecules might have some potential as a therapeutic approach to immune disorders.
Animals ; Apoptosis ; Autoimmune Diseases/immunology/metabolism/pathology ; B-Lymphocytes/immunology/physiology ; Dendritic Cells/physiology ; Human ; Inflammation/*immunology ; Lymphocyte Activation/immunology ; Models, Biological ; Receptors, Tumor Necrosis Factor/*physiology ; T-Lymphocytes/immunology/physiology ; Tumor Necrosis Factor/immunology/*physiology

BACKGROUND: Chronic myelogenous leukemia is a chronic myeloproliferative disorder characterized by leukocytosis with myeloid elements at all stages of differentiation, t(9;22)(q34;q11) and bcr/abl rearrangement. We studied hydroxyurea induced apoptotic changes such as externalization of phosphatidylserine, caspase activities on human chronic myelogenous leukemic cell line, K562 cells. METHODS: K562 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and treated hydroxyurea. Viability was examined by MTT assay. Apoptosis were examined by annexin V stain, caspase (such as caspase-, caspase-, caspase-, caspase-, and caspase-) activities, and DNA fragmentation. RESULTS: The viability of K562 cells were markedly decreased in a dose dependent manner of hydroxyurea. Phosphatidylserine externalization was detected by annexin V stain after 3 hours in hydroxyurea treated K562 cells and the value of lactate dehydrogenase was not significantly changed in their culture media. The upstream effector of caspase- was slightly increased and had influenced on caspase-. And downstream acting caspase protease of caspase- was markedly increased in a time dependent manner at hydroxyurea treated K562 cells. In addition, however the activities of caspase- and caspase- were not increased. We also found DNA fragmentation at hydroxyurea treated K562 cells between 48 hours and 72 hours on agarose gel electrophoresis. CONCLUSIONS: Hydroxyurea induces apoptotic change in K562 cells via externalization of phosphatidylserine, activations of caspase-, caspase-, caspase- proteases, and DNA fragmentation.
Annexin A5 ; Apoptosis ; Cell Line* ; Culture Media ; DNA Fragmentation ; Electrophoresis, Agar Gel ; Humans ; Hydroxyurea* ; K562 Cells* ; L-Lactate Dehydrogenase ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukocytosis ; Myeloproliferative Disorders ; Peptide Hydrolases

BACKGROUND AND OBJECTIVES: Chronic sinusitis is a common disease in otolaryngology, but its pathologic mechanism has not been clearly known. Also, the evaluation method for the severity of chronic sinusitis is not established. The aim of this study was to analyze possible factors associated with the correlation between the radiological and pathological severity of chronic sinusitis. In addition, we assessed the profiles of inflammatory cells in the sinus mucosa and peripheral blood eosinophils in relation to the overall pathologic grades and OMU CT findings. SUBJECTS AND METHOD: Fifty specimens of pathologic sinus mucosa, obtained during endoscopic sinus surgery were stained with hematoxylin and eosin. Total inflammatory cells, plasma cells, neutrophils, lymphocytes and eosinophils were quantified. The preoperative OMU CT scans were scored by the staging system of Lund-Mackay. Also, the preoperative percentage of eosinophils in peripheral white blood cells were obtained with the complete blood count with differentiation. RESULTS: The count of total inflammatory cells, lymphocytes and eosinophils infiltrated in the diseased sinus mucosa correlated significantly with the severity of the pathologic grades and OMU CT scores. In addition, the CT scores assessed by Lund-Mackay system correlated significantly with the severity of the pathologic grades. CONCLUSION: The important indicators of the severity of the chronic inflammation in chronic sinusitis were OMU CT scores, overall pathologic grades, and total inflammatory cells, lymphocytes and eosinophils infiltrated in sinus mucosa.
Blood Cell Count ; Eosine Yellowish-(YS) ; Eosinophils ; Hematoxylin ; Inflammation* ; Leukocytes ; Lymphocytes ; Mucous Membrane ; Neutrophils ; Otolaryngology ; Plasma Cells ; Sinusitis* ; Tomography, X-Ray Computed

BACKGROUND: Diagnosis by direct microscopy and/or by culture of the Mycobacterium tuberculosis from body fluids or biopsy specimens is "Gold standard". However, the sensitivity of direct microscopy after Ziehl-Neelsen staining is relatively low and culture of mycobacteria is time consuming. Detection of mycobacterial DNA in clinical samples by the polymerase chain reaction is highly sensitive but laborious and expensive. Therefore, rapid, sensitive and readily applicable new tests need to be developed. So we had evaluated the clinical significance of serologic detection of antibody to 38 kDa antigen, which is known as the most specific to the M. tuberculosis complex, and culture filtrate antigen by ELISA in sputum AFB smear negative patients. METHOD: In this study, culture tests for acid fast bacilli with sputa or bronchial washing fluids of 183 consecutive patients who were negative of sputum AFB smear were performed. Simultaneously serum antibodies to 38 kDa antigen and unheated culture filtrate of M. tuberculosis were detected by an ELISA method. RESULTS: The optical densities of ELISA test with 38 kDa and culture filtrate antigen were significantly higher in active pulmonary tuberculosis cases than in non tuberculous pulmonary diseases (p<0.05), but in patients with active pulmonary tuberculosis, those of the sputum culture positive patients for M. tuberculosis were not significantly different from those of the sputum culture negative cases(p>O.05). In the smear-negative active pulmonary tuberculosis patients, the sensitivity of the ELISA using 38 kDa antigen and culture filtrate was 20.0% and 31.4%, respectively. The specificity was 95.3% and 93.9%, respectively. CONCLUSION: In active pulmonary tuberculosis but smear negative, the serologic detection of antibody to 38 kDa antigen and culture filtrate by ELISA cannot substitute traditional diagnostic tests and does not have clinically significant role to differenciate the patient with active pulmonary tuberculosis from other with non-tuberculous pulmonary diseases.
Antibodies ; Biopsy ; Body Fluids ; Diagnosis ; Diagnostic Tests, Routine ; DNA ; Enzyme-Linked Immunosorbent Assay* ; Humans ; Lung Diseases ; Microscopy ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymerase Chain Reaction ; Serologic Tests* ; Sputum ; Tuberculosis ; Tuberculosis, Pulmonary

PURPOSE: Numerous investigations have been conducted in order to determine the potential carcinogenic or chemopreventive activity of capsaicin. The aim of this study is to characterize the effects of capsaicin on colon cancer cells, and provide valuable information concerning the application of capsaicin in chemoprevention as well as for therapeutic purposes. METHODS: CoLo320DM and LoVo cells (human colon cancer cell line) were treated with capsaicin. In order to access cell viability and altered morphology, an MTT assay was performed and the cells were microscopically examined. Decreasing DNA staining was accessed by FACS. The cells were stained with FITC labeled annexin V and analyzed by FACS to detect cellular membrane alteration during apoptosis. The cells were stained with DiOC6(3) and Hydroethidine and analyzed by FACS in order to access ROS and dleta psi m. RESULTS: Capsaicin decreased cell viability in a dose-dependent manner. Capsaicin produced a cell morphology corresponding to the apoptotic features including cell shrinkage and chromatic condensation. Capsaicin treated cells induced a loss of nuclear DNA leading to hypoploidy in a dose-dependent manner. Cells were excluded by double staining with PI and FITC labeled annexin v and detected by FACS. We show that treatment of CoLo320DM, L0Vo cells with increasing concentrations of capsaicin parallel an increase in the percentage of red fluorescent cells (HE-->Eth) that reflect ROS hypergeneration and a decrease in the percentage of green fluorescent cells that reflect delta psi m disruption. CONCLUSION: These results clearly demonstrate that capsaicin-induced colon cancer cell death is apoptotic.
Annexin A5 ; Apoptosis* ; Capsaicin ; Cell Death ; Cell Line* ; Cell Survival ; Chemoprevention ; Colon* ; Colonic Neoplasms* ; DNA ; Fluorescein-5-isothiocyanate ; Humans* ; Membranes

Hepatocyte transplantation is a potential treatment modality for liver diseased patients. Purified hepatocytes stimulates allospecific cytotoxicity by expressing the MHC class I antigen. Also, during cold preservation, hepatocytes are damaged by lipid peroxidation with oxygen free radicals, which may induce apoptosis on cold preserved hepatocyte. For measuring the degree of antigenicity on cold- preserved mice hepatocytes with UW solution, we studied the expression of MHC class I antigen in various time period by FACS and RT-PCR. For analysis of apoptotic hepatocyte death, we studied morphological changes and DNA fragmentation. We used flow cytometry techniques with rhodamine 123,3,3'-dihexiloxadicarbocyanine (DiOC6 (3)) and propidium iodide (PI). DiOC6 (3) is mitochondrial probe to measure the mitochondrial transmembrane potential that drops early in apoptosis. The percentage of cells undergoing chromatinolysis (subdiploid cells) was determined by ethanol fixation followed by RNA digestion and PI staining. The cold preserved hepatocytes expressed MHC class I constitutively, but revealed no significant differences among various preservation period. However, apoptosis of hepatocytes occured progressively during cold preservation. These results provides that the cold preservation of mice hepatocyte induces apoptosis with involvement of an oxidative process, but does not stimulate over expression of MHC class I antigen.
Animals ; Apoptosis* ; Digestion ; DNA Fragmentation ; Ethanol ; Flow Cytometry ; Free Radicals ; Hepatocytes* ; Humans ; Lipid Peroxidation ; Liver ; Membrane Potentials ; Mice* ; Oxygen ; Propidium ; Rhodamines ; RNA

Herpes virus entry mediator (HVEM) is a newly discovered member of the tumor necrosis factor receptor (TNFR) superfamily that has a role in herpes simplex virus entry, in T cell activation and in tumor immunity. We generated mAb against HVEM and detected soluble HVEM (SHVEM) in the sera of patients with various autoimmune diseases. HVEM was constitutively expressed on CD4(+)and CD8(+)T cells, CD19(+)B cells, CD14(+)monocytes, neutrophils and dendritic cells. In three-way MLR, mAb 122 and 139 were agonists and mAb 108 had blocking activity. An ELISA was developed to detect sHVEM in patient sera. sHVEM levels were elevated in sera of patients with allergic asthma, atopic dermatitis and rheumatoid arthritis. The mAbs discussed here may be useful for studies of the role of HVEM in immune responses. Detection of soluble HVEM might have diagnostic and prognostic value in certain immunological disorders.
Animals ; Antibodies, Monoclonal/immunology ; Antibody Specificity ; Arthritis, Rheumatoid/blood/immunology ; Asthma/blood/immunology ; Autoimmune Diseases/*blood/immunology ; Cell Division ; Cell Line ; Dermatitis, Atopic/blood/immunology ; Female ; Flow Cytometry ; Humans ; Hypersensitivity/*blood/immunology ; Lymphocyte Culture Test, Mixed ; Mice ; Mice, Inbred BALB C ; Receptors, Tumor Necrosis Factor/*blood/immunology ; Receptors, Tumor Necrosis Factor, Member 14 ; Receptors, Virus/*blood/immunology ; Solubility