Suppression of Ventilation-Induced Diaphragm Fibrosis through the Phosphoinositide 3-Kinase-γ in a Murine Bleomycin-Induced Acute Lung Injury Model.

Mechanical ventilation (MV), used in patients with acute lung injury (ALI), induces diaphragmatic myofiber atrophy and contractile inactivity, termed ventilator-induced diaphragm dysfunction. Phosphoinositide 3-kinase-γ (PI3K-γ) is crucial in modulating fibrogenesis during the reparative phase of ALI; however, the mechanisms regulating the interactions among MV, myofiber fibrosis, and PI3K-γ remain unclear. We hypothesized that MV with or without bleomycin treatment would increase diaphragm muscle fibrosis through the PI3K-γ pathway. Five days after receiving a single bolus of 0.075 units of bleomycin intratracheally, C57BL/6 mice were exposed to 6 or 10 mL/kg of MV for 8 h after receiving 5 mg/kg of AS605240 intraperitoneally. In wild-type mice, bleomycin exposure followed by MV 10 mL/kg prompted significant increases in disruptions of diaphragmatic myofibrillar organization, transforming growth factor-ß1, oxidative loads, Masson's trichrome staining, extracellular collagen levels, positive staining of α-smooth muscle actin, PI3K-γ expression, and myonuclear apoptosis (p < 0.05). Decreased diaphragm contractility and peroxisome proliferator-activated receptor-γ coactivator-1α levels were also observed (p < 0.05). MV-augmented bleomycin-induced diaphragm fibrosis and myonuclear apoptosis were attenuated in PI3K-γ-deficient mice and through AS605240-induced inhibition of PI3K-γ activity (p < 0.05). MV-augmented diaphragm fibrosis after bleomycin-induced ALI is partially mediated by PI3K-γ. Therapy targeting PI3K-γ may ameliorate MV-associated diaphragm fibrosis.

Increased Production of Interleukin-10 and Tumor Necrosis Factor-Alpha in Stimulated Peripheral Blood Mononuclear Cells after Inhibition of S100A12.

Sepsis may induce immunosuppression and result in death. S100A12 can bind to the receptor for advanced glycation end-products (RAGE) and Toll-like receptor (TLR)4 following induction of various inflammatory responses. It is unclear whether S100A12 significantly influences the immune system, which may be associated with sepsis-related mortality. We measured plasma S100A12 levels and cytokine responses (mean ± standard error mean) of lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) after S100A12 inhibition in healthy controls and patients with sepsis on days one and seven. Day one plasma soluble RAGE (sRAGE) and S100A12 levels in patients with sepsis were significantly higher than those in controls (2481.3 ± 295.0 vs. 1273.0 ± 108.2 pg/mL, p < 0.001; 530.3 ± 18.2 vs. 310.1 ± 28.1 pg/mL, p < 0.001, respectively). Day seven plasma S100A12 levels in non-survivors were significantly higher than those in survivors (593.1 ± 12.7 vs. 499.3 ± 23.8 pg/mL, p = 0.002, respectively). In survivors, plasma sRAGE levels were significantly decreased after 6 days (2297.3 ± 320.3 vs. 1530.1 ± 219.1 pg/mL, p = 0.009, respectively), but not in non-survivors. Inhibiting S100A12 increased the production of tumor necrosis factor (TNF)-α and interleukin (IL)-10 in stimulated PBMCs for both controls and patients. Therefore, S100A12 plays an important role in sepsis pathogenesis. S100A12 may competitively bind to TLR4 and RAGE, resulting in decreased IL-10 and TNF-α production.

Effects of Curcuma longa L., Eucommia ulmoides Oliv. and Gynostemma pentaphyllum (Thunb.) Makino on Cytokine Production in Stimulated Peripheral Blood Mononuclear Cells in Patients with Tuberculosis.

BACKGROUND: Tuberculosis (TB) infection triggers the innate and adaptive immune responses. Eucommia ulmoides Oliv., Gynostemma pentaphyllum (Thunb.) Makino, and Curcuma longa L. extracts exhibit various immunomodulatory effects. OBJECTIVE: This study aimed to determine the effects of 3 extracts used in Traditional Chinese Medicine (TCM) on cytokine production in peripheral blood mononuclear cells (PBMCs) obtained from patients with TB. DESIGN: The research team performed an in vitro study with self controls. SETTING: The study took place at the Department of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Taiwan. PARTICIPANTS: 18 patients diagnosed with pulmonary TB were enrolled in the study. INTERVENTION: Purified protein derivative (PPD)-stimulated PBMCs were cultured for 48 h in the presence and absence of 0.05 or 0.1 mg/mL of herbal extracts. OUTCOME MEASURES: Cytokine levels of interferon (IFN)-γ, interleukin (IL)-10, IL-12, tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-ß1 in the culture supernatant were measured. RESULTS: C longa L., E ulmoides Oliv. and G pentaphyllum (Thunb.) Makino extracts decreased IFN-γ production in PPD-stimulated PBMCs. C longa L. extract did not exhibit a marked and consistent effect on the production of IL-10, IL-12, TNF-α and TGF-ß1. E ulmoides Oliv. extract increased the production of IL-10, TNF-α and TGF-ß1. G pentaphyllum (Thunb.) Makino extract increased the production of IL-10, IL-12, TNF-α and TGF-ß1. CONCLUSION: These results show that G pentaphyllum (Thunb.) Makino might enhance cell immunity since it increased the production of IL-12 and TNF-α with dose effect.

Increased Death of Peripheral Blood Mononuclear Cells after TLR4 Inhibition in Sepsis Is Not via TNF/TNF Receptor-Mediated Apoptotic Pathway.

BACKGROUND: Apoptosis is one of the causes of immune depression in sepsis. Pyroptosis also occurs in sepsis. The toll-like receptor (TLR) 4 and receptor for advanced glycation end products (RAGE) have been shown to play important roles in apoptosis and pyroptosis. However, it is still unknown whether TLR4 inhibition decreases apoptosis in sepsis. METHODS: Stimulated peripheral blood mononuclear cells (PBMCs) with or without lipopolysaccharides (LPS) and high-mobility group box 1 (HMGB1) were cultured with or without TLR4 inhibition using monoclonal antibodies from 20 patients with sepsis. Caspase-3, caspase-8, and caspase-9 activities were measured. The expression of B cell lymphoma 2 (Bcl2) and Bcl2-associated X (Bax) was measured. The cell death of PBMCs was detected using a flow cytofluorimeter. RESULTS: After TLR4 inhibition, Bcl2 to Bax ratio elevated both in LPS and HMGB1-stimulated PBMCs. The activities of caspase-3, caspase-8, and caspase-9 did not change in LPS or HMGB1-stimulated PBMCs. The cell death of LPS and HMGB1-stimulated CD8 lymphocytes and monocytes increased after TLR4 inhibition. The cell death of CD4 lymphocytes was unchanged. CONCLUSION: The apoptosis did not decrease, while TLR4 was inhibited. After TLR4 inhibition, there was an unknown mechanism to keep cell death in stimulated PBMCs in patients with sepsis.

Effect of interleukin-17 on in vitro cytokine production in healthy controls and patients with severe sepsis.

BACKGROUND/PURPOSE: Interleukin (IL)-17 family members (IL-17A to IL-17F) are appearing to play key roles in host defense and inflammatory disease. Recently, several cytokines, such as IL-6, IL-10, IL-12, and transforming growth factor (TGF)-ß1, were shown to have vital roles in severe sepsis. However, the influence of IL-17 on these cytokine responses from peripheral blood mononuclear cells (PBMCs) is unclear. METHODS: Fifty-two patients who were admitted to our intensive care unit (ICU) because of severe sepsis were enrolled into this study. To validate experimental findings, 25 healthy controls were enrolled. Lipopolysaccharide-stimulated PBMCs with IL-17 or anti-IL-17 treatments were cultured for 24 hours. IL-6, IL-10, IL-12, and TGF-ß1 levels in supernatants were measured. RESULTS: The IL-12 production from stimulated PBMCs was increased after IL-17 treatment in both control and patient groups. Additional treatment of anti-IL-17 enhanced IL-10 production but decreased IL-12 production from stimulated PBMCs of healthy controls and patients with severe sepsis. CONCLUSION: IL-17 was helpful for inflammation in severe sepsis. Lack of IL-17 decreased IL-12 and enhanced IL-10 production from PBMCs, which resulted in immune imbalance.

Differential mRNA expression of Toll-like receptors and their adaptors in pleural effusions.

BACKGROUND AND OBJECTIVE: Binding of Toll-like receptors (TLRs) to microbial or endogenous ligands activates and triggers the associated signalling pathway, which leads to the production of inflammatory cytokines and type I interferons. The extent of TLR pathway activation may vary with the ligands present in different pleural diseases. METHODS: The relative mRNA expression levels of TLRs and their adaptors in pleural fluid were determined by PCR and gel electrophoresis in 36 transudative, 25 infectious and 39 malignant pleural effusions. RESULTS: The relative mRNA expression levels of TLR8 and myeloid differentiation factor 88 were low in infectious effusions and that of ras-related C3 botulinum toxin substrate 1 was low in malignant pleural effusions. Different cellular components correlated significantly with the relative mRNA expression of TLRs or their adaptors in pleural effusions with different aetiologies. CONCLUSIONS: The relative mRNA expression profiles of TLRs and their adaptors in pleural fluid differ among transudative, infectious and malignant pleural effusions.

Pleural fluid viscosity may help identifying malignant pleural effusions.

BACKGROUND AND OBJECTIVE: Cancer cells are larger in size and more rigid than blood cells. As the size and rigidity of cells contribute to blood viscosity, an association may exist between high pleural fluid viscosity and cancer cells in pleural effusions. The aim of this study was to determine the correlation between pleural fluid viscosity and cell constituents or laboratory data in pleural diseases with different aetiologies. METHODS: Fluid viscosities were determined in pleural effusions obtained via thoracocentesis. Pleural fluid viscosities were correlated with the laboratory data and with the percentages of different cellular constituents as assessed by cytological examination. RESULTS: Pleural fluid viscosity was highest in malignant pleural effusions with positive results on cytological examination, and was correlated with the percentages of tumour cells (Spearman's rho = 0.24, P = 0.037) and mitotic figures (rho = 0.23, P = 0.041) in the exudates. Multivariate logistic regression analysis showed that pleural fluid viscosity was a significant determinant of positive results on cytological examination (odds ratio (OR) 6.26, 95% confidence interval (CI) 1.32-29.8), as were the levels of protein (OR 1.48, 95% CI 1.01-2.16) and LDH (OR 1.001, 95% CI 1-1.002). CONCLUSION: High pleural fluid viscosity may suggest a potential diagnosis of malignant pleural effusion.

Fumagillin treatment of hepatocellular carcinoma in rats: an in vivo study of antiangiogenesis.

AIM: To investigate the effect and possible mechanisms of antiangiogenesis therapy for HCC in rats. METHODS: Adult male LEW/SsN rats were divided into 3 groups, 25 animals each. Group A was the control group. Groups B and C were given diethylnitrosamine, 5 mg/kg/d. In addition, group C rats received an intraperitoneal injection of fumagillin, 30 mg/(kg x d). Five animals in each group were killed at 6th, 12th, 18th, 20th and 24th wk to evaluate the development of HCC and metastasis. Weight of the rats, liver tumors, and number of organs involved by HCC were measured at each stage. We compared methionine aminopeptidase-2 (MetAP-2) mRNA, Bcl-2 mRNA, telomerase mRNA, and telomerase activity at 24th wk in the liver tissue of group A rats and tumor tissue of HCC from group B and C rats. RESULTS: No HCC developed in group A, but tumors were present in group B and C rats by the 18th wk. At wk 20 and 24, the median liver weight in group B was 0.64 g (range: 0.58-0.70 g) and 0.79 g (range: 0.70-0.90 g) (P = 0.04), and that in group C was 0.37 g (range: 0.35-0.42 g) and 0.39 g (range: 0.35-0.47 g) (P = 0.67). The liver weight in group C rats was significantly lower than that in group B rats (P = 0.009). At the same time, the median metastasis score (number of organ systems involved) was 3 (range2-3) in group B, and 1 (range 1-2) in group C, a significant difference between the groups (P = 0.007, 0.004). The levels of MetAP-2 mRNA were significantly higher in groups B and C than in group A (P = 0.025), and significantly higher in group C than in group B (P = 0.047). The level of Bcl-2 mRNA was significantly higher in group B than in group A (P = 0.024), but lower in group C than in group B, although not significantly (P = 0.072). Telomerase mRNA was significantly higher in group B than in group A (P = 0.025), but significantly lower in group C than in group B (P = 0.016). The same inter-group relationship was also true for telomerase activity (P = 0.025 and 0.046). CONCLUSION: Fumagillin effectively inhibits both liver tumor growth and metastasis in rats in vivo. A possible mechanism is fumagillin-induced inhibition of MetAP-2, which plays an essential role in endothelial cell proliferation. Inhibition of MetAP-2 also results in inhibition of Bcl-2 and telomerase activity.

Prognostic significance of preoperative circulating vascular endothelial growth factor messenger RNA expression in resectable hepatocellular carcinoma: a prospective study.

AIM: To investigate the prognostic value of vascular endothelial growth factor messenger RNA (VEGF mRNA) in the peripheral blood (PB) of patients with hepatocellular carcinoma (HCC) undergoing curative resection. METHODS: Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA in the PB was determined prospectively in 50 controls and in 50 consecutive patients undergoing curative resection for HCC. RESULTS: Among the isoforms of VEGF mRNA, VEGF(165) and VEGF(121) were expressed. By multivariate analysis, a higher level of VEGF(165) in preoperative PB correlated with a risk of HCC recurrence with borderline significance (P=0.050) and significantly with recurrence-related mortality (P=0.048); while VEGF(121) did not. Other significant predictors of HCC recurrence included cellular dedifferentiation (P=0.033), an absent or incomplete capsule (P=0.020), vascular permeation (P=0.018), and daughter nodules (P=0.006). The other significant parameter of recurrence related mortality was cellular dedifferentiation (P=0.053). The level of circulating VEGF mRNA, however, did not significantly correlate with tumor size, cellular differentiation, capsule, daughter nodules, vascular permeation, necrosis and hemorrhage of tumors. CONCLUSION: The preoperative level of circulating VEGF mRNA, especially isoform VEGF(165), plays a significant role in the prediction of postoperative recurrence of HCC.

Is the vascular endothelial growth factor messenger RNA expression in resectable hepatocellular carcinoma of prognostic value after resection?

AIM: To study whether vascular endothelial growth factor messenger RNA (VEGF mRNA) in the hepatocellular carcinoma (HCC) tissues obtained after curative resection has a prognostic value. METHODS: Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA was determined prospectively in liver tissues of 50 controls and in HCC tissues of 50 consecutive patients undergoing curative resection for HCC. RESULTS: Among the isoforms of VEGF mRNA, VEGF(165) and VEGF(121) were expressed. By multivariate analysis, a higher level of VEGF(165) in HCC tissue correlated with a significant risk of HCC recurrence (P=0.038) and significantly with recurrence-related mortality (P=0.045); while VEGF(121) did not. Other significant predictors of HCC recurrence included cellular dedifferentiation (P=0.033), an absent or incomplete capsule (P=0.020), vascular permeation (P=0.018), and daughter nodules (P=0.006). The other significant variables of recurrence related mortality consisted of vascular permeation (P=0.045), and cellular dedifferentiation (P=0.053). The level of VEGF mRNA in HCC tissues, however, did not significantly correlate with tumor size, cellular differentiation, capsule, daughter nodules, vascular permeation, necrosis and hemorrhage of tumors. CONCLUSION: The expression of VEGF mRNA, especially isoform VEGF(165), in HCC tissues, may play a significant and independent role in the prediction of postoperative recurrence of HCC.