Anti-neutrophil cytoplasmic antibody-negative microscopic polyangiitis: A case report and literature review.

The aim of this study was to investigate the clinical characteristics, diagnosis and treatment of anti-neutrophil cytoplasmic antibody (ANCA)-negative microscopic polyangiitis (MPA). We described the case of a patient with ANCA-negative MPA and conducted analyses and a review of the relevant literature. Based on the collected data, the epidemiology, diagnosis and treatment of ANCA-negative MPA were discussed. The patient, a 69-year-old male, was initially diagnosed with pneumonia and interstitial lung disease (ILD) based on his clinical symptoms. The patient was ANCA-negative. The follow-up and consultations with the relevant departments after the ANCA testing led to a diagnosis of MPA being considered. The administration of glucocorticoids and immunosuppressant drugs was found to improve the symptoms of the patient. The clinical symptoms of MPA are unspecific. The majority of MPA cases are ANCA-positive, but misdiagnosis should be considered as a possibility in ANCA-negative cases. When patients are suspected to have MPA, therefore, ANCA tests should be immediately performed. Test results should be analyzed for the early diagnosis of MPA in order to enable the provision of immediate treatment, improve patient prognosis and reduce mortality rate.

Expression variations of connective tissue growth factor in pulmonary arteries from smokers with and without chronic obstructive pulmonary disease.

Cigarette smoking contributes to the development of pulmonary hypertension (PH) complicated with chronic obstructive pulmonary disease (COPD), and the pulmonary vascular remodeling, the structural basis of PH, could be attributed to abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs).In this study, morphometrical analysis showed that the pulmonary vessel wall thickness in smoker group and COPD group was significantly greater than in nonsmokers. In addition, we determined the expression patterns of connective tissue growth factor (CTGF) and cyclin D1 in PASMCs harvested from smokers with normal lung function or mild to moderate COPD, finding that the expression levels of CTGF and cyclin D1 were significantly increased in smoker group and COPD group. In vitro experiment showed that the expression of CTGF, cyclin D1 and E2F were significantly increased in human PASMCs (HPASMCs) treated with 2% cigarette smoke extract (CSE), and two CTGF siRNAs with different mRNA hits successfully attenuated the upregulated cyclin D1 and E2F, and significantly restored the CSE-induced proliferation of HPASMCs by causing cell cycle arrest in G0. These findings suggest that CTGF may contribute to the pathogenesis of abnormal proliferation of HPASMCs by promoting the expression of its downstream effectors in smokers with or without COPD.

Expression of IL-17A and IL-17F in lipopolysaccharide-induced acute lung injury and the counteraction of anisodamine or methylprednisolone.

Th17 cytokines IL-17A and IL-17F as pro-inflammatory cytokines played an important role in triggering inflammatory responses. However, little was known about the expression of IL-17A and IL-17F in acute lung injury (ALI). Therefore, the present study investigated the expression of IL-17A and IL-17F in lipopolysaccharide (LPS)-induced ALI in rats and rat pulmonary microvascular endothelial cells (PMVEC) by enzyme-linked immunosorbant assay or reverse transcription-polymerase chains reaction. Anisodamine and methylprednisolone were also investigated as anti-inflammatory strategy in the process of LPS-induced ALI. Lung injury was evaluated by histological changes, right lung wet weight:body weight (LW/BW) ratios, and protein education and total leukocyte count of bronchoalveolar lavage fluid (BALF). Our findings showed that LPS exposure elevated the levels of leukocyte number, protein education in BALF and the ratios of LW/BW, increased the expression of IL-17A and IL-17F in the lung tissues homogenate, BALF and serum of ALI rats. Up-regulation of IL-17F expression was also observed after LPS challenge in rat PMVEC. Treatment with anisodamine or methylprednisolone significantly inhibited the increases of parameters of ALI induced by LPS, and markedly reduced the expression of IL-17A and IL-17F in rats and the IL-17F expression in PMVEC. These data suggested that IL-17A and IL-17F maybe play an important role in LPS-induced ALI via autocrine and paracrine mechanisms, and anisodamine is similar in extent to methylprednisolone that contributes to relieve LPS-induced ALI.

[Effect of tumor necrosis factor-α on expression of interleukin-17 receptor C in pulmonary microvascular endothelial cells in rats].

OBJECTIVE: To explore the effects of tumor necrosis factor-alpha (TNF-α) or methylprednisolone on the expression of interleukin-17 receptor C (IL-17RC) in rat pulmonary microvascular endothelial cells (RPMVEC). METHODS: Culture RPMVEC were randomly divided into dose-dependent and time-dependent groups. In dose-dependent group, cells were cultured with TNF-α (0, 0.1, 1, 10 µg/L TNF-α) for 3 h. In time-dependent group, cells were cultured with TNF-α (10 µg/L) for 0, 1, 3, 6, 12 h. In the drug intervention group, cells were cultured with TNF-α (10 µg/L) or methylprednisolone (200 µg/L) +TNF-α (10 µg/L) for 3 h respectively. The expression of IL-17RC in isolated and cultured RPMVEC was identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry. The changes of IL-17RC mRNA were detected by RT-PCR after the stimulation of RPMVEC by TNF-α or methylprednisolone. RESULTS: RT-PCR and Western blot revealed that IL-17RC mRNA and protein were present in RPMVEC. The product of IL-17RC immunocytochemical reaction was predominantly located in cytoplasm and cytomembrane. In RPMVEC TNF-α significantly up-regulated IL-17RC mRNA in a dose-dependent manner (0 µg/L TNF-α group: 0.241 ± 0.010, 0.1 µg/L TNF-α group: 0.372 ± 0.017, 1 µg/L TNF-α group: 0.452 ± 0.017, 10 µg/L TNF-α group: 0.643 ± 0.042, F = 33.774, P < 0.05). In time-dependent group, the expression of IL-17RC mRNA rose at 1 h (0.417 ± 0.038), peaked at 3 h (0.674 ± 0.018), then decreased gradually at 6 h (0.378 ± 0.035), but stayed higher at 12 h (0.318 ± 0.032). When compared with 0 h group (0.197 ± 0.008), there were significant differences (F = 37.903, P < 0.05). Methylprednisolone caused a marked attenuation of TNF-α-induced IL-17RC expression (0.333 ± 0.031 vs 0.660 ± 0.026, F = 89.637, P < 0.05). CONCLUSIONS: IL-17RC is predominantly present in cytomembrane and cytoplasm of RPMVEC. TNF-α up-regulates the expression of IL-17RC mRNA. Methylprednisolone inhibits the elevated expression of IL-17RC mRNA induced by TNF-α so as to relieve the inflammatory response of PMVEC.

Caveolin-1 regulates Rac1 activation and rat pulmonary microvascular endothelial hyperpermeability induced by TNF-α.

A multiplicity of vital cellular and tissue level functions are controlled by caveolin-1 and it is considered to be an important candidate for targeted therapeutics. Rac1-cortactin signaling plays an important role in maintaining the functions of the endothelial barrier in microvascular endothelial cells. The activity of Rac1 has been shown to be regulated by caveolin-1. Therefore, the present study investigated the consequences of down-regulating caveolin-1 and the subsequent changes in activity of Rac1 and the endothelial barrier functions in primary rat pulmonary microvascular endothelial cells (RPMVECs). RPMVECs were transfected with a small hairpin RNA duplex to down-regulate caveolin-1 expression. This procedure significantly increased the activity of Rac1. Moreover, down-regulation of caveolin-1 attenuated TNF-α-induced decrease in TER, increase in the flux of FITC-BSA and the disappearance of cortactin from the cell periphery in RPMVEC. Rac1 inhibitors significantly abolished this barrier-protective effect induced by down-regulation of caveolin-1 in response to TNF-α in RPMVECs. In conclusion, our data suggest a mechanism for the regulation of Rac1 activity by caveolin-1, with consequences for activation of endothelial cells in response to TNF-α.

[Role of caveolin-1 in pulmonary microvascular endothelial cells injury induced by lipopolysaccharide in rat].

OBJECTIVE: To investigate the role of caveolin-1 (Cav-1) in the modulation of rat pulmonary microvascular endothelial cells (RPMVEC) injury induced by lipopolysaccharide (LPS). METHODS: Cultured RPMVEC were randomly divided into time-dependent injury group induced by LPS and intervention group in which cells were pretreated by protein kinase A inhibitor (PKI). In the time-dependent injury group, monolayers of cells were constructed to determine permeability changes after 10 µg/mL LPS challenge for 0, 1, 3, 6, 12 and 24 hours with the method of Evans blue-labeled albumin flux across the monolayer (Pd). Western blotting was used to determine the Cav-1 expression after LPS stimulation and the phosphorylation-Cav-1 (p-Cav-1) expression after LPS challenge for 0, 10, 30, 60, 90, 120 minutes. In the intervention group, after pre-treatment with 10 µmol/L PKI for 30 minutes, RPMVECs were challenged with 10 µg/mL LPS, and the expression of p-Cav-1 was determined 30 minutes after LPS challenge, the permeability and the Cav-1 protein expression were assessed by Pd and Western blotting, respectively. Non-stimulation group and single PKI simulation group served as controls. RESULTS: Western blotting revealed that the expression of Cav-1 protein was elevated at 1 hour (2.97 ± 0.07), peaking at 3 hours (3.77 ± 0.37), then it lowered gradually, but it was still higher at 24 hours (1.45 ± 0.18) when compared with 0 hour group (1.12±0.08) with significant differences (F=178.047, P=0.000). After RPMVEC monolayers were challenged by LPS for different periods (0, 1, 3, 6, 12 and 24 hours), there were significant increases in a time-dependent manner in Cav-1 expression in the permeability as measured by Pd [(99.67 ± 4.32)%, (118.17 ± 2.32)%, (159.00 ± 2.61)%, (141.17 ± 2.64)%, (120.17 ± 2.79)% and (108.83 ± 2.04)%, F=345.869, P=0.000] which was similar to the changes in Cav-1 expression. LPS also increased Cav-1 phosphorylation in a time-dependent manner occurring at 10 minutes (2.41 ± 0.11), peaking at 30 minutes (2.83 ± 0.10), and then it decreased gradually, finally returned to basal levels (1.03±0.04) at 120 minutes (1.04 ± 0.04) after LPS treatment with significant difference (F=519.417, P=0.000). When PKI was pre-treated with RPMVEC the expression of Cav-1 was significantly increased (5.07 ± 0.22 vs. 3.81 ± 0.23, P<0.01), and p-Cav-1 (3.93 ± 0.23 vs. 2.77±0.10, P<0.01), and RPMVEC monolayers permeability was enhanced [(184.17 ± 5.49)% vs. (151.50 ± 3.08)%, P<0.01] after being challenged. CONCLUSIONS: Up-regulated expression of Cav-1 and phosphorylation-Cav-1 that may be modulated by protein kinase A signal pathway plays an important role in RPMVEC permeability injury as induced by LPS.

[Methods for measurement of permeability coefficient of pulmonary microvascular endothelial cells in rat].

OBJECTIVE: To explore an optimal method for the measurement of pulmonary microvascular endothelial cell (PMVEC) permeability coefficient. METHODS: A monolayer of rat PMVEC model was constructed by culturing a cell suspension on transwell filter or polycarbonate filter membrane. After the state of confluence of cells was affirmed with epithelial volt-ohm meter or inverted microscope, the permeability coefficient was determined by means of transendothelial electrical resistance (TER), fluoresceinisothiocyanate-dextran (Pd), and permeation of Hanks solution (Lp) across monolayers. Meanwhile,changes in PMVEC permeability expressed by the ratio of the observed value and the original value were observed after lipopolysaccharide (LPS) challenge for 0. 5 hour or 2 hours. RESULTS: The cells reached the state of confluence as observed under inverted microscope on the third day post-seeding, and the TER and Pdat this time-point were C(39. 45 ± 3. 96) ( 2 cm2] and [(8. 52 + 0. 50) X 10-6cm/s], respectively. After PMVEC were seeded on transwell filters, the TER increased steadily in a time-dependent manner after seeding of PMVEC, reaching the summit at the fourth day post-seeding C(49. 84 ± 3. 93)f " cm2].Under the natural state, the TER, Pd and Lp of confluent PMVEC monolayers were (49.84 ±3.93) ·.* cm2, (6.15±0.63) X 106 cm/s and (6.80 + 0.62) X10< cm * s-' * cm HZO-', respectively.After PMVEC monolayers were challenged with 10 mg/L LPS for both 0. 5 hour and 2 hours, there was significant decrease in the permeability coefficient as measured by TER (0. 87+ 0. 03, 0.45 0. 04 vs. 1.00+0.08, respectively, both P< 0.05), and an increase in the permeability coefficient measured by Pd (1.33±0. 11, 2.43±0. 14 vs. 1.00+0.10, respectively, both P<0. 05) and the permeability coefficient measured by Lp (1.30± 0.07, 2.38 0.15 vs. 1.00 + 0.11, respectively, both P< 0.05) when compared with the normal group. CONCLUSION: Three methods, namely TER, Pd and Lp are available to use to assess PMVEC permeability coefficient. The combination of an inverted microscope, TER and Pd enhances the accuracy in determining PMVEC permeability coefficient, and it provides an experimental technique for studying the pathogenesis of acute lung injury in vitro.

Role of src-suppressed C kinase substrate in rat pulmonary microvascular endothelial hyperpermeability stimulated by inflammatory cytokines.

OBJECTIVE: The aim of the study was to investigate the role of src-suppressed C kinase substrate (SSeCKS) in the modulation of rat pulmonary microvascular endothelial cells (RPMVEC) permeability elicited by interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. METHODS: The gene expression of SSeCKS was analyzed by reverse transcription-polymerase chain reaction. Immunoblotting was used to determine the SSeCKS protein expression and the activation of the protein kinase C (PKC) signaling pathway. A RPMVEC monolayer was constructed to determine changes of transendothelial electrical resistance (TER) and FITC-dextran flux (P (d)) across the monolayer. SSeCKS-specific small interfering RNA was transfected into RPMVEC. RESULTS: IL-1ß and TNF-α activated the PKC signaling pathway in RPMVEC, and up-regulated the gene and protein expression of SSeCKS. Depletion of endogenous SSeCKS in RPMVEC significantly attenuated cytokine-induced decrease in TER and increase in P (d), but not to the basal levels. PKC inhibitors also significantly decreased cytokine-induced hyperpermeability and SSeCKS expression. CONCLUSIONS: SSeCKS is involved in the endothelial hyperpermeability induced by IL-1ß and TNF-α in inflammatory process.

[Induction effect of platelet-activating factor on Src-suppressed C kinase substrate gene expression in rat pulmonary microvascular endothelial cells].

OBJECTIVE: To investigate the effect of platelet-activating factor (PAF) on the production of Src-suppressed C kinase substrate (SSeCKS) mRNA in rat pulmonary microvascular endothelial cell (RPMVEC) and its signal transduction pathways. METHODS: Cellular in situ hybridization was performed to reveal changes in SSeCKS mRNA expression in the cultured RPMVECs after giving PAF stimulation. RESULTS: Normal RPMVECs expressed SSeCKS mRNA at a low level, which appeared throughout the cytoplasm with specific hybridization signals. 1.5 hours of 10(-10), 10(-9), 10(-8), 10(-7) mol/L PAF incubation induced a progressive increase in SSeCKS mRNA expression. When compared to the normal control group each difference had statistical significance (0.054 9 + or - 0.000 7, 0.059 9 + or - 0.001 8, 0.069 0 + or - 0.001 8, 0.075 4 + or - 0.001 9 vs. 0.036 8 + or - 0.003 7, respectively, all P<0.05). Within 0.5, 1.5, 3, 6, 12, 24 hours of 10(-7) mol/L PAF challenge, the level of SSeCKS mRNA expression raised at 0.5 hour markedly (0.071 0 + or - 0.001 5), peaked at 1.5 hours (0.075 6 + or - 0.001 7), then began to decline gradually, and still persisted at a higher level than the normal control group until 24 hours (0.043 9 + or - 0.001 0 vs. 0.038 2 + or - 0.004 1, all P<0.05). Pre-incubation of 10 micromol/L pyrrolidine dithiocarbamate (PDTC) that inhibits activity of nuclear factor-KappaB (NF-KappaB) in RPMVECs caused a conspicuous attenuation of PAF-induced SSeCKS mRNA expression (0.049 7 + or - 0.003 2 vs. 0.071 9 + or - 0.001 9, P<0.05), whereas no change of PAF-induced effect was found by pretreatment of protein kinase C (PKC) inhibitor bis-indolylmaleimide (BIM, 0.069 7 + or - 0.002 1 vs. 0.071 9 + or - 0.001 9, P>0.05). CONCLUSION: PAF can up regulate the expression of SSeCKS mRNA in a dose- and time-dependent manner in RPMVECs. It is NF-KappaB rather than PKC signal pathway that is involved in modulation of the intracellular signaling process.

Interleukin-17F-induced pulmonary microvascular endothelial monolayer hyperpermeability via the protein kinase C pathway.

BACKGROUND: Interleukin (IL)-17F is involved in lung inflammation, but the effect of IL-17F on endothelial permeability and its signaling pathway remain ill-defined. The current study sought to investigate the effect of IL-17F on endothelium and assess the role of protein kinase C (PKC) and src-suppressed C kinase substrate (SSeCKS) in this process. METHODS: Rat pulmonary microvascular endothelial monolayers were constructed to determine changes of permeability as measured by means of FITC-dextran and Hank's solution flux across monolayers and transendothelial electrical resistance with or without IL-17F and PKC inhibitors. Additional monolayers were stained using FITC-phalloidin for filamentous actin (F-actin). The gene expression of SSeCKS was analyzed by the reverse transcription-polymerase chains. Alterations of SSeCKS protein were investigated by immunoblotting and immunoprecipitation. RESULTS: IL-17F increased endothelial monolayer permeability in a dose- and time-dependent manner. F-actin staining revealed that permeability changes were accompanied by reorganization of cytoskeleton. In the presence of PKC inhibitors, the IL-17F-induced hyperpermeability and reorganization of F-actin were attenuated. The gene and protein expression of SSeCKS were conspicuously elevated after IL-17F challenge. The process of SSeCKS phosphorylation followed a time course that mirrored the time course of hyperpermeability induced by IL-17F. IL-17F-induced SSeCKS phosphorylation was abrogated after PKC inhibitors pretreatment. The translocation of SSeCKS from the cytosol to the membrane and a significant increase in the SSeCKS association with the cytoskeleton were found after IL-17F treatment. CONCLUSIONS: IL-17F is an important mediator of increased endothelial permeability. PKC and SSeCKS are integral signaling components essential for IL-17F-induced hyperpermeability.

[The effect of intrapleural pingyangmycin administration on activity of fibrinolytic system and transforming growth factor-beta1 in malignant pleural effusion].

OBJECTIVE: To observe the changes of the concentrations of plasminogen activator inhibitor-1(PAI-1), tissue-type plasminogen activator(t-PA), transforming growth factor-beta(1)(TGF-beta(1)) in peripheral blood and pleural effusion before and after intrapleural pingyangmycin administration, and therefore to investigate the mechanism by which pingyangmycin produces pleurodesis. METHODS: Since February to September 2008, a total of 26 patients with malignant pleural effusion (MPE) underwent intrapleural pingyangmycin administration. The concentrations of PAI-1, t-PA, TGF-beta(1) and the number of leucocytes in peripheral blood and pleural effusion before and after treatment were detected. The pleurodesis efficacy according to WHO standard was evaluated 1 month later. Patients who showed complete disappearance of pleural effusion lasting more than 1 month and reduction of pleural effusion more than 50% were classified as the effective group, while the others were classified as the failure group. RESULTS: One month after intrapleural pingyangmycin administration, the total response rate of MPE control was 57.7% (effective group = 15 cases). The number of leucocytes and neutrophils in peripheral blood were significantly higher after intrapleural pingyangmycin administration [the number of leucocytes: effective group (9.2 +/- 2.0) x 10(9)/L, failure group (9.4 +/- 3.8) x 10(9)/L; neutrophil count: effective group (7.9 +/- 2.1) x 10(9)/L, failure group (8.1 +/- 3.3) x 10(9)/L] than in those before[the number of leucocytes: effective group (6.6 +/- 1.4) x 10(9)/L, failure group (5.6 +/- 0.9) x 10(9)/L; neutrophil count: effective group (4.5 +/- 1.3) x 10(9)/L, failure group (4.2 +/- 1.0) x 10(9)/L. F = 30.80, 46.08 respectively, all P < 0.01]. However, the concentrations of PAI-1, t-PA and TGF-beta(1) in the peripheral blood showed no significant difference before and after treatment(P > 0.05). The concentrations of PAI-1 were significantly lower in the pleural effusion before treatment [effective group (1054 +/- 1039) microg/L, failure group (1027 +/- 955) microg/L] than after treatment [24 h after intrapleural pingyangmycin administration: effective group (2195 +/- 861) microg/L, failure group (1099 +/- 568) microg/L]; 72 h after treatment: effective group (1688 +/- 703) microg/L, failure group (1383 +/- 797) microg/L(F = 6.29, P = 0.01). The levels of t-PA were significantly higher in the pleural effusion before treatment [the effective group (42 +/- 33) microg/L, failure group (54 +/- 25) microg/L] than after treatment[24 h after intrapleural pingyangmycin administration: the effective group (49 +/- 49) microg/L, failure group (53 +/- 40) microg/L; 72 h after treatment: the effective group (17 +/- 20) microg/L, failure group (28 +/- 22) microg/L (F = 19.85, P < 0.01). The levels of TGF-beta(1) in the pleural effusion showed no significant difference before and after treatment (P > 0.05). The number of leucocytes in pleural effusion was significantly higher after intrapleural pingyangmycin administration [the effective group (4.7 +/- 4.7) x 10(9)/L, failure group (2.1 +/- 1.4) x 10(9)/L] than before [the effective group (2.3 +/- 1.9) x 10(9)/L, failure group (1.0 +/- 0.9) x 10(9)/L. F = 8.05, P < 0.01]. The number of leucocytes, neutrophils and the concentrations of PAI-1, t-PA, TGF-beta(1) in the peripheral blood showed no significant difference between the effective group and the failure group before and after treatment (P > 0.05). However, the concentrations of PAI-1 in pleural effusion after treatment were higher in the effective group than in the failure group (F = 8.51, P < 0.01). CONCLUSION: Intrapleural pingyangmycin administration is a safe and effective treatment for MPE. The mechanism of pleurodesis is related to inhibiting the activity of plasminogen in the pleural cavity.

[Effect of endotoxin on expression of angiotensin converting enzyme-2 in cultured rat pulmonary microvascular endothelial cells].

OBJECTIVE: To observe the effects of endotoxin on angiotensin converting enzyme-2 (ACE2) expression in rat pulmonary microvascular endothelial cells (RPMVECs) in vitro, and discuss the role of ACE2 in endotoxin-induced acute lung injury (ALI). METHODS: In vitro, cultured RPMVECs were incubated with endotoxin (10 mg/L), the expression of ACE2 mRNA and protein were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting techniques at time points of 3, 6, 12, and 24 hours respectively after incubation with endotoxin. RESULTS: After the treatment of RPMVECs with endotoxin (10 mg/L), the levels of ACE2 mRNA and protein decreased significantly, and then they increased slightly at 6 hours, followed by a continuous decrease until 24 hours. The statistic analysis demonstrated that, except at the 6-hour time point, the levels of ACE2 mRNA and protein expression at other time points were lower than those of control group significantly (P<0.05 or P<0.01). CONCLUSION: The downregulation of ACE2 mRNA and protein expression in RPMVECs by endotoxin may play an important role in pathogenesis of ALI.

[Studies on the expression of Src-suppressed C kinase substrate mRNA in pulmonary microvascular endothelial cells induced by lipopolysaccharide].

OBJECTIVE: To study the expression of Src-suppressed C kinase substrate (SSeCKS) mRNA in pulmonary microvascular endothelial cells(PMVEC) induced by lipopolysaccharide, and the interfering effect of methylprednisolone sodium succinate. METHODS: Rat PMVEC (RPMVEC) were isolated and cultured in vitro, then grouped randomly according to different culture time and different dosage of LPS, and the expression of SSeCKS mRNA in RPMVEC of different groups were determined by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Normally, the expression of the SSeCKS mRNA in RPMVEC was maintained on a low level, and it could be elevated by stimulation of LPS, and the changes in the expression exhibited in dosage and time dependent manner. After being stimulated with LPS for 1 hour, the extent of increase in SSeCKS mRNA in RPMVEC coincided with the increased dosage of LPS (control: 0.263+/-0.033; LPS of 0.1 mg/L: 0.529+/-0.066; LPS of 1 mg/L: 1.391+/-0.048; LPS of 10 mg/L: 2.339+/-0.055; LPS of 100 mg/L: 2.861+/-0.069, F=639.096, P<0.05). When RPMVEC were stimulated with 10 mg/L of LPS, the expression of SSeCKS mRNA began to increase at 0.5 hour, peaked at 1 hour, then decreased gradually, and it remained high even at 12 hours (control: 0.301+/-0.022; LPS 0.5 hour: 1.617+/-0.018; 1 hour: 2.378+/-0.031; 3 hours: 2.148+/-0.056; 6 hours: 1.322+/-0.042; 12 hours: 0.772+/-0.044, F=726.346, P<0.05); After methylprednisolone sodium succinate had been given, the increase of SSeCKS mRNA induced by LPS is inhibited (2.664+/-0.104 vs. 1.759+/-0.151, F=156.000, P<0.05). CONCLUSION: (1) LPS could induce up-regulation of SSeCKS mRNA, the elevation is in time and dosage dependent manner. This indicate SSeCKS is related with LPS induced injury of RPMVEC. (2) Methylprednisolone sodium succinate inhibit the increase of SSeCKS mRNA induced by LPS.

[Effect of sodium hyaluronate on pleural adhesions and fibrosis in a rabbit model of empyema].

OBJECTIVE: To study the effect of sodium hyaluronate (SH) on pleural adhesions and fibrosis in experimental empyema. METHODS: Twenty rabbits were randomly divided into two groups: a treatment group and a control group, 10 rabbits in each group. Chest tubes were placed into the right pleural cavity of the rabbits. Empyema was induced via intrapleural injection of turpentine followed by instillation of 10(8) CFU Staphylococcus aureus into the pleural space 3 h later. After an empyema was verified by pleural fluid analysis at 24 h, SH 3 ml (30 mg) was introduced into the right pleural cavity in the treatment group, but normal saline 3 ml was used in the control group. The rabbits received procaine penicillin G, 200 000 U intramuscularly daily after 24 h. Pleural effusion was analyzed at 24 h and 96 h. All animals were sacrificed on Day 8. RESULTS: Eighteen rabbits completed the experiment, 9 rabbits in each group. Pleural fluid analysis at 96 h in the treatment group and the control group showed that, leukocyte count was (24.7 +/- 13.0) x 10(9)/L and (36.9 +/- 10.1) x 10(9)/L, respectively; neutrophil percentage was (27.1 +/- 11.2)% and (49.6 +/- 10.9)%, respectively; protein was (30.1 +/- 3.6) g/L and (35.6 +/- 4.3) g/L, respectively. The three parameters of the treatment group were significantly lower than those of the control group (P < 0.05). The level of glucose was (2.44 +/- 0.52) mmol/L at 96 h in the treatment group and significantly higher than the control group (3.78 +/- 1.28) mmol/L (P < 0.05). In the treatment group and the control group the pleurodesis score was 0.7 +/- 0.5 and 3.2 +/- 0.7, respectively; visceral pleural thickness score was (28 +/- 10) microm and (156 +/- 42) microm, respectively; and fibroblast score was (37 +/- 15)/mm(2) and (163 +/- 58)/mm(2), respectively. The above parameters of the treatment group were significantly lower than those of the control group. CONCLUSION: Early intrapleural injection of a high molecular weight SH was safe and effective in decreasing pleural adhesions and fibrosis in a rabbit model of empyema.

Diagnostic accuracy of human telomerase reverse transcriptase mRNA in malignant pleural effusions: A preliminary report for in situ hybridization detection.

BACKGROUND: Detection of human telomerase reverse transcriptase (hTERT) mRNA by in situ hybridization (ISH) may be valuable in the diagnosis of cancer. We assessed the diagnostic performance of hTERT mRNA in cells from pleural fluid in malignant pleural effusions (PEs). METHODS: We used a 2-step ISH with digoxin-labelled oligonucleotide probes to detect hTERT mRNA in a blinded prospective study of cells from 103 unselected pleural fluid specimens. The reference standard for malignant PEs was clinical evaluation and pleural fluid cytology, combined with pleural biopsy, other examination and follow-up as needed. RESULTS: According to the final diagnoses, there were 41 malignant PEs, 55 benign PEs and 7 cases with uncertain etiology. When the 7 cryptogenic cases were excluded, the sensitivity and specificity of detectable hTERT mRNA for malignancy were 80% and 95%, respectively. When detection of hTERT mRNA was combined with clinical repeated pleural fluid cytology, the sensitivity and specificity were 90% and 95%, respectively. CONCLUSIONS: Detection of hTERT mRNA in cells from pleural fluid by ISH could potentially be used in diagnosing malignant PEs as an aid. Further investigations with stricter controls and cross-validation tests will be warranted.

Expression and regulation of AT1 receptor in rat lung microvascular endothelial cell.

BACKGROUND: The renin-angiotensin system is thought to be involved in the development and progression of vascular endothelium inflammation, thereby contributing to vascular endothelium injury. To clarify the role of angiotensin II (Ang II) in rat pulmonary microvascular endothelial cells (RPMVECs), we examined the expression and functional significance of angiotensin II (Ang II) receptors in normal and lipopolysacchride (LPS) treated RPMVECs. METHODS: The expressions of Ang II type 1(AT(1)) and Ang II type 2 (AT(2)) receptors in cultured RPMVECs were identified by the reverse transcription-polymerase chain reaction (RT-PCR) technique, Western blot and (125)I-labeled [Sar(1),Ile(8)] Ang II binding assays. The RPMVECs were treated with LPS (0.1-100 microg/ml) and Ang II (10(-8)-10(-5) M) for 24 h, respectively. Next, RPMVECs were treated with 10 microg/ml LPS or 10(-7) M Ang II for various times (3, 6, 12, and 24 h). The mRNA and protein levels of, AT(1) and AT(2) receptors, were evaluated at 3, 6, 12, and 24 h, respectively. RESULTS: The presence of specific Ang II binding sites in RPMVECs was found by Ang II saturated assays. RT-PCR revealed that only the AT(1) receptor mRNA is presented in RPMVECs. Western blot analysis of the RPMVECs protein extracts showed only one prominent band of the protein at approximately 41 KDa when probed with anti-AT(1) antibody and anti-AT(2) antibody. No AT(2) receptor mRNA and protein was detected. LPS treated cells resulted in an increase in the mRNA and protein levels of AT(1) receptor, whereas, Ang II treated cells showed a decrease in the mRNA and protein levels of AT(1) receptor. CONCLUSIONS: We found that primary cultured RPMVECs expressed only AT(1) receptor, but not AT(2) receptor. LPS up-regulated the transcriptional and post-transcriptional expression of AT(1) receptor in RPMVECS; in contrast, Ang II treatment caused a reduction in the mRNA and protein of AT(1) receptor in a time-dependent manner.

LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor.

Lipopolysaccharide (LPS) is known to stimulate the circulation and local production of angiotensin II (Ang II). To assess whether Ang II plays a role in LPS-induced acute lung injury, rats were injected with LPS, the microvascular endothelial permeability injury was evaluated by histological changes, increased pulmonary wet/dry weight ratio, and pulmonary microvascular protein leak. Besides, increased rat pulmonary microvascular endothelial cell monolayer permeability coefficient (K(f)) was measured after treatment with LPS and/or Ang II, respectively. LPS/Ang II, treatment resulted in a significant increase in K(f). Ang II cooperates with LPS to further increase K(f). Hence, LPS increases pulmonary microvascular endothelial permeability both in vitro and in vivo. Local lung Ang II was increased in response to LPS challenge, and elevated Ang II ulteriorly exacerbates LPS-induced endothelium injury. [Sar(1),Ile(8)]Ang II, a selective block of Ang II type 1 (AT(1)) receptors, eliminated these changes significantly. Our conclusion is that the LPS-induced lung injury may be mediated by the AT(1) receptor.

[Inflammatory injurious effect of angiotensin II on pulmonary microvascular endothelium in rat].

OBJECTIVE: To investigate the effects of angiotensin II (AngII) and its receptors on monolayer permeability of pulmonary microvascular endothelial cells in rat. METHODS: The following examinations were done on cultured rat pulmonary microvascular endothelial cells (RPMVECs). 1. Micro-filter was used to assay the variations of lipopolysaccharide (LPS)-induced increased RPMVECs monolayer permeability coefficient (Kf) in different periods. 2. Effect of angiotensin II on LPS-induced permeability injury to the endothelium, and preventive effects of angiotensin II type 1 receptor antagonist (Sar1, Ile8)-Ang II. RESULTS: LPS increased RPMVECs monolayer permeability compared with normal control, and AngII exacerbated LPS-induced RPMVECs monolayer permeability significantly. This synergistic effect was significantly prevented by the addition of (Sar1, Ile8 )-Ang II. CONCLUSION: AngII and LPS have synergistic injurious effects to the pulmonary microvascular endothelium in rat, and AngII exacerbates increase in LPS-induced RPMVECs monolayer permeability. This synergistic effect is significantly prevented by the addition of (Sar1, Ile8)-Ang II.