Lipopolysaccharide potentiates endothelin-1-induced proliferation of pulmonary arterial smooth muscle cells by upregulating TRPC channels.

Lipopolysaccharide (LPS) and endothelin-1 (ET-1) are critical pathogenic factors in sepsis-induced pulmonary hypertension; however it is unknown whether they have a coordinated action in the pathogenesis of this disease. Here we found that although LPS did not change the contractility of rat pulmonary arterial smooth muscle cells (PASMCs) in response to ET-1, it significantly promoted ET-1-induced PASMC proliferation. Measurement of ET-1-evoked Ca(2+) transients in PASMCs showed that LPS dramatically enhanced Ca(2+) influx mediated by transient receptor potential canonical (TRPC) channels. LPS did not directly activate TRPC channels, instead it selectively upregulated the expression of TRPC3 and TRPC4 in pulmonary arteries. Small interfering RNA (siRNA) and chemical blockers against TRPC channels abolished LPS-induced PASMC proliferation. LPS-induced cell proliferation and TRPC expression was mediated by the Ca(2+)-dependent calcineurin/NFAT signaling pathway. We suggest that blocking TRPC channels could be an effective strategy in controlling pulmonary arterial remodeling after endotoxin exposure.

Involvement of TRPC channels in lung cancer cell differentiation and the correlation analysis in human non-small cell lung cancer.

The canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable cationic channels controlling the Ca(2+) influx evoked by G protein-coupled receptor activation and/or by Ca(2+) store depletion. Here we investigate the involvement of TRPCs in the cell differentiation of lung cancer. The expression of TRPCs and the correlation to cancer differentiation grade in non-small cell lung cancer (NSCLC) were analyzed by real-time PCR and immunostaining using tissue microarrays from 28 patient lung cancer samples. The association of TRPCs with cell differentiation was also investigated in the lung cancer cell line A549 by PCR and Western blotting. The channel activity was monitored by Ca(2+) imaging and patch recording after treatment with all-trans-retinoic acid (ATRA). The expression of TRPC1, 3, 4 and 6 was correlated to the differentiation grade of NSCLC in patients, but there was no correlation to age, sex, smoking history and lung cancer cell type. ATRA upregulated TRPC3, TRPC4 and TRPC6 expression and enhanced Ca(2+) influx in A549 cells, however, ATRA showed no direct effect on TRPC channels. Inhibition of TRPC channels by pore-blocking antibodies decreased the cell mitosis, which was counteracted by chronic treatment with ATRA. Blockade of TRPC channels inhibited A549 cell proliferation, while overexpression of TRPCs increased the proliferation. We conclude that TRPC expression correlates to lung cancer differentiation. TRPCs mediate the pharmacological effect of ATRA and play important roles in regulating lung cancer cell differentiation and proliferation, which gives a new understanding of lung cancer biology and potential anti-cancer therapy.

Pleural fluid soluble triggering receptor expressed on myeloid cells-1 as a marker of bacterial infection: a meta-analysis.

BACKGROUND: Pleural infection is a common clinical problem. Its successful treatment depends on rapid diagnosis and early initiation of antibiotics. The measurement of soluble triggering receptor expressed in myeloid cells-1 (sTREM-1) level in pleural effusions has proven to be a valuable diagnostic tool for differentiating bacterial effusions from effusions of other etiologies. Herein, we performed a meta-analysis to assess the accuracy of pleural fluid sTREM-1 in the diagnosis of bacterial infection. METHODS: We searched Web of Knowledge and Medline from 1990 through March 2011 for studies reporting diagnostic accuracy data regarding the use of sTREM-1 in the diagnosis of bacterial pleural effusions. Pooled sensitivity and specificity and summary measures of accuracy and Q* were calculated. RESULTS: Overall, the sensitivity of sTREM-1was 78% (95% CI: 72%-83%); the specificity was 84% (95% CI: 80%-87%); the positive likelihood ratio was 6.0 (95% CI: 3.3-10.7); and the negative likelihood ratio was 0.22 (95% CI: 0.12-0.40). The area under the SROC curve for sTREM-1 was 0.92. Statistical heterogeneity and inconsistency were found for sensitivity (p = 0.015, χ2 = 15.73, I2 = 61.9%), specificity (p = 0.000, χ2 = 29.90, I2 = 79.9%), positive likelihood ratio (p = 0.000, χ2 = 33.09, I2 = 81.9%), negative likelihood ratio (p = 0.008, χ2 = 17.25, I2 = 65.2%), and diagnostic odds ratio (p = 0.000, χ2 = 28.49, I2 = 78.9%). A meta-regression analysis performed showed that the Quality Assessment of Diagnostic Accuracy Studies score (p = 0.3245; RDOR, 4.34; 95% CI, 0.11 to 164.01), the Standards for Reporting of Diagnostic Accuracy score (p = 0.3331; RDOR, 1.70; 95% CI, 0.44 to 6.52), lack of blinding (p = 0.7439; RDOR, 0.60; 95% CI, 0.01 to 33.80), and whether the studies were prospective or retrospective studies (p = 0.2068; RDOR, 7.44; 95% CI, 0.18 to 301.17) did not affect the test accuracy. A funnel plot for publication bias suggested a remarkable trend of publication bias. CONCLUSIONS: Our findings suggest that sTREM-1 has a good diagnostic accuracy and may provide a useful adjunctive tool for the diagnosis of bacterial pleural effusions. However, further studies are needed in order to identify any differences in the diagnostic performance of sTREM-1 of parapneumonic effusions and empyemas.

Plasma orexin-a levels in COPD patients with hypercapnic respiratory failure.

Orexins have previously been shown to promote wakefulness, regulate lipid metabolism and participate in energy homeostasis. The aim of the study was to determine the relationship between plasma orexin-A and body composition in COPD in-patients with hypercapnic respiratory failure. 40 patients with hypercapnic respiratory failure and 22 healthy individuals were enrolled prospectively in this study. Plasma orexin-A levels, BMI, SaO(2), PaCO(2) and PaO(2) were noted for all the patients. Plasma orexin-A levels were higher in the underweight (UW) group, normal weight (NW) group and overweight (OW) group of COPD patients as compared with UW, NW and OW group of the control group (P < .05). Plasma orexin-A in COPD patients were higher in the OW group than in the NW group and the UW group. Plasma orexin-A levels showed significant correlation with body mass index (BMI), independent of PaO(2) (r = 0.576; P < .05) and %fat (r = 0.367; P < .05); a negative correlation was noted between plasma orexin-A levels and PaO(2) (r = -0.738; P < .05) and SaO(2) (r = -0.616; P < .05). Our results suggest that orexin-A levels are high in COPD patients with hypercapnic respiratory failure, and vary according to BMI and body composition. Orexin-A may be associated with the severity of hypoxemia in COPD patients with hypercapnic respiratory failure.

Novel interventional approaches for ALI/ARDS: cell-based gene therapy.

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), continue to be a major cause of morbidity and mortality in critically ill patients. The present therapeutic strategies for ALI/ARDS including supportive care, pharmacological treatments, and ventilator support are still controversial. More scientists are focusing on therapies involving stem cells, which have self-renewing capabilities and differentiate into multiple cell lineages, and, genomics therapy which has the potential to upregulate expression of anti-inflammatory mediators. Recently, the combination of cell and gene therapy which has been demonstrated to provide additive benefit has opened up a new chapter in therapeutic strategy and provides a basis for the development of an innovative approach for the prevention and treatment of ALI/ARDS.

LPS pretreatment ameliorates multiple organ injuries and improves survival in a murine model of polymicrobial sepsis.

OBJECTIVE: The endotoxin tolerance phenotype is characterized with decreased inflammation and increased phagocytosis. We hypothesized that endotoxin tolerance would provide protective effects on experimental sepsis with multiple organ injuries induced by cecal ligation and puncture (CLP). METHODS: Endotoxin tolerance was induced in male Sprague-Dawley rats with daily intraperitoneal injection of either 0.6 mg/kg of lipopolysaccharide (LPS) or vehicle for four consecutive days before subsequent CLP. Biochemical parameters, histological changes, inflammatory cytokine production, and lung tissue nuclear factor-κB (NF-κB) activation were assessed post-CLP. In a separate experiment, survival rate was monitored for 7 days after CLP. RESULTS: In vehicle-treated animals, CLP caused multiple organ injuries confirmed by the biochemical variables and histological examination. This was accompanied by an early activation of NF-κB in the lung and a substantial increase in plasma levels of tumor necrosis factor-α, interleukin-6, and interleukin-10. In contrast, pretreatment with LPS not only alleviated the development of multiple organ injuries after CLP, but also decreased sepsis-induced activation of pulmonary NF-κB and reduced plasma cytokines production. In addition, LPS pretreatment improved the survival in rats subjected to CLP. CONCLUSIONS: The beneficial effects of endotoxin tolerance indicate the potential of immunomodulatory strategies in the management of severe sepsis.

Protective effects of imipramine in murine endotoxin-induced acute lung injury.

The tricyclic antidepressant imipramine has recently emerged as a cytoprotective agent, exerting beneficial effects in inflammatory tissue injury. The present study aimed to investigate therapeutic effects of imipramine in murine model of endotoxin-induced acute lung injury. Mice were administrated intraperitoneally with LPS (lipopolysaccharide) from Escherichia coli or vehicle. Imipramine was administrated intraperitoneally 30 min before LPS challenge. Pretreatment of mice with imipramine reduced lethality. Impramine also significantly attenuated lung inflammation, lung edema, MPO (myeloperoxidase) activity, lung tissue pathological changes and nuclear factor-kappaB DNA binding activity. The results of this study suggest that imipramine can exert protective effects in endotoxin-induced acute lung injury by suppressing nuclear factor-kappaB-mediated expression of inflammatory genes. Thus, imipramine could be a potential novel therapeutic agent for the treatment for acute lung injury.

Up-regulation of growth factor independence 1 in endotoxin tolerant macrophages with low secretion of TNF-alpha and IL-6.

OBJECTIVE: Endotoxin tolerance refers to a low response to lipopolysaccharide (LPS). We hypothesized that growth factor independence 1 (Gfi1) involves in the endotoxin tolerance in macrophages. METHODS: Endotoxin tolerance was induced in the RAW264.7 cell line and thioglycolate-elicited murine peritoneal macrophages by incubation with 100 ng/ml LPS for 20 h. Macrophages without the pretreatment were set as control. Both endotoxin tolerant and control cells were then stimulated with 1,000 ng/ml LPS for indicated period of incubation. Gfi1 mRNA expression and protein production were investigated by real-time PCR and Western blotting, respectively. ELISA was performed to quantify the secretion of TNF-alpha and IL-6. RESULT: Compared with non-endotoxin tolerant macrophages, endotoxin tolerant cells secreted a lower amount of TNF-alpha and IL-6. The mRNA expression of Gfi1 in endotoxin tolerant macrophages was higher than that of control in both RAW264.7 cells and thioglycolate-elicited murine peritoneal macrophages. The protein production was accordingly up-regulated in endotoxin tolerant RAW264.7 cells. CONCLUSION: In in vitro endotoxin tolerant macrophages, the expression of Gfi1 mRNA and protein were up-regulated after high dose LPS stimulation, accompanied with a blunted TNF-alpha and IL-6 secretion. Gfi1 might participate in the mechanism of endotoxin tolerance.

[The characteristics of CT imaging and diagnosis of pulmonary cryptococcosis in 42 cases with non-acquired immune deficiency syndrome].

OBJECTIVE: To further elucidate the CT characteristics and diagnostic approaches to non-acquired immune deficiency syndrome patients with pulmonary cryptococcosis. METHODS: The histories of forty-two pulmonary cryptococcosis (PC) patients diagnosed in Zhongshan Hospital from 2003-2008 were collected and analyzed for demography data, underlying conditions, clinical symptoms, chest CT and diagnostic studies. RESULTS: None of the 42 PC patients had avian or its feces contacting history, and 71.4% (30/42) of them were immunocompetent. The most frequent CT lesions were multiple nodules (67.9%) with peripheral predominance (67.9%), and cavitations (50%) often presented within them. Masses/consolidation (31.4%) and patching lesions (2.9%) could exist occasionally. Positive detection rates of non-aggressive examinations including sputum, bronchoalveolar lavage fluid and bronchofibroscopy aspiration were 4.3%, 8.3% and 6.3% respectively, while those of aggressive approaches including transbronchial lung biopsy (TBLB), thin needle aspiration biopsy (TNAB) and pneumonectomy by surgery were 64.7%, 64.3% and 100% respectively. Non-aggressive serum cryptococcus antigen test was performed in 14 patients who had been diagnosed by histopathology or pathogen culture, and all of them were positive. CONCLUSION: Our study suggests that PC is common in immunocompetent population. Avian or its feces contacting is not so important as used opinion to PC differential diagnosis. CT characteristics of PC are diversiform and always change very slowly. Besides the most frequent multiple nodules with subpleural predominance, pulmonary lesions can present as masses, consolidation or patching. Aggressive techniques such as TBLB and TNAB are benefit to clinical diagnosis of PC, and non-aggressive serum cryptococcus antigen test may be promising for its early diagnosis as well as clinical course follow-up and therapeutic effect evaluation.

NAC is associated with additional alleviation of lung injury induced by invasive pulmonary aspergillosis in a neutropenic model.

AIM: Neutropenic individuals are at high risk for invasive pulmonary aspergillosis (IPA), a life-threatening infection. To evaluate the therapeutic potential of antioxidants, IPA was induced in neutropenic mice and the effect of N-acetyl-l-cysteine (NAC) on oxidative stress levels and lung injury was analyzed. METHODS: Mice were pretreated with three daily intraperitoneal injections of 150 mg/kg cyclophosphamide, followed by intratracheal inoculation with 4.5x10(6) conidia of Aspergillus fumigatus. The infected mice were then randomly assigned to an amphotericin B (AMB) group, an AMB plus NAC group, or an untreated control (C) group. In each group, the duration of treatment was 24, 48, or 72 h, and activities such as appearance, feeding, and dermal temperature were observed throughout the experiment. Sera and lung tissues were collected and analyzed by quantitative enzyme-linked immunosorbent assay (ELISA) for total protein, superoxide dismutase (SOD), malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-alpha), and interleukin-10 (IL-10) levels. The wet/dry weight ratio of the lung was also calculated and lung sections were stained with hematoxylin-eosin for pathological examination and with methenamine silver stain for fungus detection. RESULTS: Compared with the mice untreated with NAC, mice in the AMB plus NAC group had increased SOD and reduced MDA levels both systemically and locally at 24, 48, and 72 h after inoculation with conidia. NAC treatment also decreased the pulmonary protein content at 48 and 72 h and the lung wet/dry weight ratio at 24 and 48 h. Additionally, NAC enhanced pulmonary production of TNF-alpha and IL-10 at 24 h and 48 h. CONCLUSION: In combination with antifungal therapy, NAC treatment can alleviate oxidative stress and lung injury associated with IPA in neutropenic mice.Acta Pharmacologica Sinica (2009) 30: 980-986; doi: 10.1038/aps.2009.83.

Effects of N(omega)-nitro-L-arginine methyl ester and aminoguanidine on lipopolysaccharide-induced airway hyperresponsiveness in guinea pigs.

BACKGROUND: The down-regulation of constitutive nitric oxide synthase (cNOS) and up-regulation of inducible nitric oxide synthase (iNOS) are associated with the allergen-provocated airway hyperresponsiveness (AHR). This study aimed to determine whether their alteration also plays an important role in the AHR induced by lipopolysaccharide (LPS). METHODS: Hartley male guinea pigs, weighing between 250 g and 350 g, were injected with LPS at a dose of 1 mg/kg every 24 hours for three days. A non-selective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), or a selective inducible NOS inhibitor, aminoguanidine (AG), were used thirty minutes before each injection of LPS. Airway reactions, nitric oxide (NO) production and inflammatory changes were detected 24 hours after the last dose of LPS. RESULTS: AG significantly decreased the NO production in the bronchoalveolar lavage fluid (BALF) and sharply reduced the intensity of bronchoconstriction to histamine challenge. L-NAME also significantly decreased the NO production in the BALF, but had no effect on airway reactions or, perhaps, a tendency to enhance the intensity of AHR. CONCLUSIONS: The data suggest that inducible NOS contributes to the AHR induced by repetitive intraperitoneal LPS, and constitutive NOS was also involved.