OBJECTIVETo explore the protective effects of mitochondrial ATP-sensitive potassium channel opener diazoxide on hyperoxia-induced apoptosis of type II alveolar epithelial cells (A549 cells) and possible mechanisms.

METHODSA549 cells were cultured in vitro and divided randomly into control, hyperoxia and diazoxide group. The hyperoxia group was exposed to a mixture of O2 (900 mL/L) and CO2 (50 mL/L) for 10 minutes, then cultured in a closed environment. The diazoxide group was pretreated with diazoxide of 100 μmol/L for 24 hrs before hyperxia induction. The cells were collected 12, 24 and 48 hrs after culture. The morphologic changes of A549 cells were observed under an inverted microscope. A549 cell apoptosis was detected by flow cytometry. The expression of Omi/HtrA2 in the endochylema of A549 cells was determined by immunohistochemistry.

RESULTSA549 cells were damaged and the changes in morphology of the cells were serious in the hyperoxia group. The apoptosis rate of A549 cells and the expression of Omi/HtrA2 in the endochylema increased in the hyperoxia group compared with the control group (P<0.05). The growth and the morphology of A549 cells were greatly improved and the cell injuries were obviously alleviated in the diazoxide group. The expression of Omi/HtrA2 in the endochylema and the apoptosis rate of A549 cells were significantly reduced in the diazoxide group compared with the hyperoxia group (P<0.05).

CONCLUSIONSDiazoxide as an opener of mitoKATP channel can reduce the expression of Omi/HtrA2 and the apoptosis rate of A549 cells, thus relieves the injury of A549 cells induced by hyperoxia.

Apoptosis ; Cells, Cultured ; Cytoprotection ; Diazoxide ; pharmacology ; High-Temperature Requirement A Serine Peptidase 2 ; Humans ; Hyperoxia ; complications ; Lung ; pathology ; Mitochondrial Proteins ; analysis ; Potassium Channels ; physiology ; Serine Endopeptidases ; analysis

Dendritic Cells ; immunology ; Granzymes ; Humans ; Immune Tolerance ; Killer Cells, Natural ; immunology ; Multivariate Analysis ; S100 Proteins ; analysis ; Serine Endopeptidases ; analysis ; Stomach Neoplasms ; immunology ; pathology ; T-Lymphocytes, Cytotoxic ; immunology

Although active inflammation may be deleterious and indicate immunologic activation in chronically rejected grafts, the underlying mechanism of tissue destruction has been little studied. Twenty-four cases of chronic rejection (CR) with or without acute rejection (AR) were stained with antibodies against CD3, CD8, CD68, granzyme B and TIA-1, and the number of positive cells were counted. Eleven cases of AR served as controls. The number of CD3 and CD8 positive cells increased in the acute on CR group compared to the CR group. About a half of CD3 positive T cells were CD8 positive in both groups, however, the proportion of TIA-1 or granzyme B positive cells was higher in the acute on CR group. The numbers of CD3, CD68, granzyme B and TIA-1 positive cells were higher in the AR group than the acute on CR group, however, no significant difference was found between the two groups. Serum creatinine level and proteinuria at the time of biopsy and the percentages of late onset AR and graft failure rate were higher in the acute on CR group than the CR group. Summarizing, these results suggest that infiltration of activated T cells containing cytotoxic granules plays a role in graft destruction in acute on CR.
Adult ; Antigens, CD3/analysis ; Antigens, CD8/analysis ; Female ; Follow-Up Studies ; *Graft Rejection ; Human ; Immunohistochemistry ; *Kidney Transplantation ; Male ; Membrane Proteins/*analysis ; RNA-Binding Proteins/*analysis ; Serine Endopeptidases/*metabolism ; Transplantation, Homologous

OBJECTIVETo study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression.

METHODSPersistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins.

RESULTSIn the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed.

CONCLUSIONSThe virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.

Carcinoma, Hepatocellular ; virology ; Defective Viruses ; physiology ; Encephalitis Virus, Japanese ; chemistry ; genetics ; physiology ; Humans ; Membrane Glycoproteins ; analysis ; Mutation ; RNA Helicases ; Serine Endopeptidases ; Tumor Cells, Cultured ; Viral Envelope Proteins ; analysis ; Viral Nonstructural Proteins ; analysis

BACKGROUNDProtease activated receptor-2 is cleaved and activated by trypsin or mast cell tryptase and may play an important role in inflammation. However, it is unknown whether PAR-2 can mediate tryptase-induced inflammatory reaction. This study was conduct to investigate whether PAR-2 could be the activated by mast cell tryptase and medicated the tryptase induced interleukin-8 expression in endothelial cells.

METHODSProtease activated receptor-2 expression was found in endothelial cell lines ECV304 cell by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Interleukin-8 stimulated by purified human mast cell tryptase was determined by RT-PCR and enzyme linked immunosorbent assay (ELISA). Data were analysed by the S-N-K one-way ANOVA test.

RESULTSThe present study shows that mRNA and protein of protease activated receptor-2 could be expressed in ECV304 cells, and tryptase upregulated the expression levels of both interleukin-8 mRNA and protein. The increased expression of interleukin-8 was inhibited by an antiprotease activated receptor-2 monoclonal antibody, SAM11. An additional band was observed by Western blotting after the incubation of ECV304 cells with tryptase for 2 hours, which suggested that protease activated receptor-2 was activated.

CONCLUSIONProtease activated receptor-2 can mediate the mast cell tryptase stimulated expression of interleukin-8 in ECV304 cell.

Antibodies, Monoclonal ; immunology ; Cell Line ; Endothelial Cells ; metabolism ; Gene Expression Regulation ; Humans ; Interleukin-8 ; genetics ; RNA, Messenger ; analysis ; Receptor, PAR-2 ; analysis ; genetics ; physiology ; Serine Endopeptidases ; physiology ; Tryptases ; Up-Regulation

BACKGROUNDLittle is known about the role of dual angiotensin II forming pathways during heart failure. In the present study, the changes of chymase and angiotensin converting enzyme (ACE) expressions in the failing hearts of hamsters were analysed.

METHODSHeart failure was induced by ligation of left anterior descending branch of the coronary artery. Chymase, ACE and angiotensin II type 1 receptor (AT1R) mRNA levels were analysed by reverse transcription polymerase chain reaction (RT-PCR). The activities of chymase and ACE were determined by radioimmunoassay (RIA). Myocardial collagen fibre analysis was performed under optical microscope.

RESULTSLeft ventricular systolic pressure (LVSP) and maximum left ventricular developed pressure increase rate (dp/dtmax, mmHg/s) gradually moved lower at 2, 3, 4 and 8 weeks after operation. On the other hand, left ventricular end-diastolic pressure (LVEDP) increased gradually after operation. Compared with the control group (3.55 +/- 0.06, 4.79 +/- 0.70), the heart weight/body weight ratio in operation group had increased significantly at 4 weeks and 8 weeks (4.28 +/- 0.43, 6.17 +/- 0.73) (P < 0.01). Collagen staining showed that the quantity of myocardial collagen fibre increased significantly in the operation group. RT-PCR showed that the chymase mRNA level in the operation group was consistently greater than that in the control group. AT1R mRNA level was also increased significantly at 3 weeks and 4 weeks, both being 1.3 times that of the control group (P < 0.01), whereas ACE mRNA level was not changed. Higher activity of chymase was detected in operation group, being 4, 8, 13 and 19 times that of the control group at 2, 3, 4 and 8 weeks (P < 0.01), respectively. ACE activity was also significantly higher at the same time, being 7, 10, 10 and 3.5 times that of the control (P < 0.01). Angiotensin II (Ang II) level in operation group increased significantly, being 2.5, 2.7, 3.5 and 2 times that of the control group at 2, 3, 4 and 8 weeks, respectively (P < 0.01).

CONCLUSIONSA dual Ang II forming pathway from both ACE and chymase in the hamster hearts plays an important role during the development of heart failure. At the decompensatory stage, the reduction of AngII level may be associated with the decrease of ACE activity.

Angiotensin II ; analysis ; Animals ; Body Weight ; Chymases ; Cricetinae ; Heart Failure ; metabolism ; Male ; Myocardium ; metabolism ; Peptidyl-Dipeptidase A ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptor, Angiotensin, Type 1 ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Serine Endopeptidases ; genetics ; physiology ; Ventricular Function, Left

OBJECTIVETo compare the clinical and pathological manifestations of patients with antineutrophil cytoplasmic autoantibodies (ANCA) directed against proteinase 3 (anti-PR3) or myeloperoxidase (anti-MPO).

METHODSOne hundred and forty patients with ANCA were detected for anti-PR3 and anti-MPO by ELISA. The clinical features at presentation, histopathological characteristics and outcome of all patients who were tested positive for anti-PR3 or anti-MPO were analysed.

RESULTSIn anti-PR3 group (n = 21), 16 cases (76.2%) had systemic vasculitis, in which Wegener's granulomatosis prevailed (13 cases, 61.9%). In anti-MPO group (n = 31), 19 cases (61.3%) were diagnosed as systemic vasculitis and 12 cases (38.7%) as microscopic angiitis. For vasculitic patients with anti-PR3 and anti-MPO, the disease duration at diagnosis was 9.6 +/- 2.0 m and 4.4 +/- 0.9 m respectively, P < 0.05; vasculitis activity index (BVAS) and mean number of affected organ were 22.5 +/- 2.1, 5.0 +/- 0.4 and 25.1 +/- 1.7, 4.8 +/- 0.4 respectively, P > 0.05; upper respiratory tract, eye and joint involvements were 11(68.8%), 7(43.8%), 11(68.8%) and 7(36.8%), 2(10.5%), 5(26.3%) respectively, P < 0.05. Although there was no statistical difference in renal involvement between these two groups, patients with serum creatine > 500 micromol/L were more commonly seen in anti-MPO group than in anti-PR3 group, which were 8(42.1%) and 2(12.5%) respectively, P < 0.05. Ten relapses were seen in anti-PR3 group and only 2 in anti-MPO group, but the acute mortality rate in anti-MPO group (5/19, 27.4%) was much higher than that in anti-PR3 group (1/16, 6.3%).

CONCLUSIONSAnti-PR3 and anti-MPO occurred mainly in systemic vasculitis. A large divergence was seen in the disease spectrum between patients with anti-PR3 and those with anti-MPO. In particular, upper respiratory tract, eye and joint involvements, granuloma formation and relapse were more prominent in anti-PR3 patients. By contrast, the anti-MPO patients had a more acute disease onset, more rapid progressive renal involvement and a higher acute mortality rate.

Antibodies, Antineutrophil Cytoplasmic ; analysis ; Autoantibodies ; analysis ; Follow-Up Studies ; Granulomatosis with Polyangiitis ; drug therapy ; immunology ; pathology ; Humans ; Immunosuppressive Agents ; therapeutic use ; Kidney ; pathology ; Myeloblastin ; Peroxidase ; immunology ; Respiratory System ; pathology ; Serine Endopeptidases ; immunology ; Vasculitis ; drug therapy ; immunology ; pathology

Acute Kidney Injury ; etiology ; Betacoronavirus ; Coronavirus Infections ; complications ; Humans ; Kidney ; enzymology ; Pandemics ; Peptidyl-Dipeptidase A ; physiology ; Pneumonia, Viral ; complications ; Sequence Analysis, RNA ; methods ; Serine Endopeptidases ; physiology ; Single-Cell Analysis ; methods

BACKGROUNDPrevious studies have indicated that thrombin (TM) may play a major role in brain edema after intracerebral hemorrhages (ICHs). However, the mechanism of TM-induced brain edema is poorly understood. In this study, we explored the effect of TM on the permeability of the blood brain barrier (BBB) and investigated its possible mechanism, aiming at providing a potential target for brain edema therapy after ICHs.

METHODSTM or TM + cathepsin G (CATG) was stereotaxically injected into the right caudate nucleus of Sprague-Dawley rats in vivo. BBB permeability was measured by Evans-Blue extravasation. Brain water content was determined by the dry-wet weight method. Brain microvascular endothelial cells were then cultured in vitro. After TM or TM + CATG was added to the endothelial cell medium, changes in the morphology of cells were dynamically observed by phase-contrast light microscopy, and the expression of matrix metalloproteinase-2 (MMP-2) protein was measured by immunohistochemical method.

RESULTSBBB permeability increased at 6 hours after a TM injection into the ipsilateral caudate nucleus (P < 0.05), peaked between 24 hours (P < 0.01) and 48 hours (P < 0.05) after the injection, and then declined. Brain water content changed in parallel with the changes in BBB permeability. However, at all time points, BBB permeability and brain water content after a TM + CATG injection were not significantly different from the respective parameters in the control group (P > 0.05). TM induced endothelial cell contraction in vitro in a time-dependent manner and enhanced the expression of MMP-2 protein. After incubation with TM + CATG, cell morphology and MMP-2 expression did not change significantly as compared to the control group (P > 0.05).

CONCLUSIONSIncreased BBB permeability may be one of the mechanisms behind TM-induced cerebral edema. TM induces endothelial cell contraction and promotes MMP-2 expression by activating protease activated receptor-1 (PAR-1), possibly leading to the opening of the BBB.

Animals ; Blood-Brain Barrier ; drug effects ; Body Water ; metabolism ; Brain Edema ; etiology ; Cathepsin G ; Cathepsins ; pharmacology ; Cerebral Hemorrhage ; complications ; Matrix Metalloproteinase 2 ; analysis ; Permeability ; Rats ; Rats, Sprague-Dawley ; Receptor, PAR-1 ; physiology ; Serine Endopeptidases ; Thrombin ; toxicity

OBJECTIVETo analyze the gene expression level of fibroblast activation protein in HBV related hepatocellular carcinoma patients and discuss its clinical significance.

METHODSFAP gene expression in 33 hepatocellular carcinoma patients cancer tissues, peficancerous tissues, distant relative normal liver tissues and 13 normal liver tissues were examined by reverse transcription PCR; and real-time fluorescent quantitative PCR (qRT-PCR) was used to quantify their expression.

RESULTSFAP were expressed in all the tissues,the relative expression values in cancer tissues, peficancerous tissues and distant relative normal liver tissues were 5.14 +/- 6.69, 1.58 +/- 0.96, 1.63 +/- 0.94, respectively, the differences were statistically significant (F = 4.401, P < 0.05); and in TNM stage I, II, IIII, they were 2.89 +/- 3.35, 4.15 +/- 4.69, 10.09 +/- 9.51 respectively; in well-differentiated, differentiated and poorly differentiated hepatocellular carcinoma were 1.62 +/- 1.74, 3.84 +/- 3.79, 1.26 +/- 13.34 respectively. The differences were all statistically significant (P < 0.05).

CONCLUSIONFAP may play an important role in the occurrence and development of HBV related hepatocellular carcinoma.

Adult ; Aged ; Carcinoma, Hepatocellular ; etiology ; metabolism ; Female ; Gelatinases ; genetics ; physiology ; Gene Expression Regulation, Neoplastic ; Hepatitis B ; complications ; Humans ; Liver Neoplasms ; etiology ; metabolism ; Male ; Membrane Proteins ; genetics ; physiology ; Middle Aged ; RNA, Messenger ; analysis ; Serine Endopeptidases ; genetics ; physiology