The iNOS/Src/FAK axis is critical in Toll-like receptor-mediated cell motility in macrophages.

The Toll-like receptors (TLRs) play a pivotal role in innate immunity for the detection of highly conserved, pathogen-expressed molecules. Previously, we demonstrated that lipopolysaccharide (LPS, TLR4 ligand)-increased macrophage motility required the participation of Src and FAK, which was inducible nitric oxide synthase (iNOS)-dependent. To investigate whether this iNOS/Src/FAK pathway is a general mechanism for macrophages to mobilize in response to engagement of TLRs other than TLR4, peptidoglycan (PGN, TLR2 ligand), polyinosinic-polycytidylic acid (polyI:C, TLR3 ligand) and CpG-oligodeoxynucleotides (CpG, TLR9 ligand) were used to treat macrophages in this study. Like LPS stimulation, simultaneous increase of cell motility and Src (but not Fgr, Hck, and Lyn) was detected in RAW264.7, peritoneal macrophages, and bone marrow-derived macrophages exposed to PGN, polyI:C and CpG. Attenuation of Src suppressed PGN-, polyI:C-, and CpG-elicited movement and the level of FAK Pi-Tyr861, which could be reversed by the reintroduction of siRNA-resistant Src. Besides, knockdown of FAK reduced the mobility of macrophages stimulated with anyone of these TLR ligands. Remarkably, PGN-, polyI:C-, and CpG-induced Src expression, FAK Pi-Tyr861, and cell mobility were inhibited in macrophages devoid of iNOS, indicating the importance of iNOS. These findings corroborate that iNOS/Src/FAK axis occupies a central role in macrophage locomotion in response to engagement of TLRs.

Mechanical stress induces lung fibrosis by epithelial-mesenchymal transition.

OBJECTIVES: Many mechanically ventilated patients with acute respiratory distress syndrome develop pulmonary fibrosis. Stresses induced by mechanical ventilation may explain the development of fibrosis by a number of mechanisms (e.g., damage the alveolar epithelium, biotrauma). The objective of this study was t test the hypothesis that mechanical ventilation plays an important role in the pathogenesis of lung fibrosis. METHODS: C57BL/6 mice were randomized into four groups: healthy controls; hydrochloric acid aspiration alone; vehicle control solution followed 24 hrs later by mechanical ventilation (peak inspiratory pressure 22 cm H(2)O and positive end-expiratory pressure 2 cm H(2)O for 2 hrs); and acid aspiration followed 24 hrs later by mechanical ventilation. The animals were monitored for up to 15 days after acid aspiration. To explore the direct effects of mechanical stress on lung fibrotic formation, human lung epithelial cells (BEAS-2B) were exposed to mechanical stretch for up to 48 hrs. MEASUREMENT AND MAIN RESULTS: Impaired lung mechanics after mechanical ventilation was associated with increased lung hydroxyproline content, and increased expression of transforming growth factor-ß, ß-catenin, and mesenchymal markers (α-smooth muscle actin and vimentin) at both the gene and protein levels. Expression of epithelial markers including cytokeratin-8, E-cadherin, and prosurfactant protein B decreased. Lung histology demonstrated fibrosis formation and potential epithelia-mesenchymal transition. In vitro direct mechanical stretch of BEAS-2B cells resulted in similar fibrotic and epithelia-mesenchymal transition formation. CONCLUSIONS: Mechanical stress induces lung fibrosis, and epithelia-mesenchymal transition may play an important role in mediating the ventilator-induced lung fibrosis.

Human neutrophil peptides mediate endothelial-monocyte interaction, foam cell formation, and platelet activation.

OBJECTIVE: Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. METHODS AND RESULTS: We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor-related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. CONCLUSIONS: HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases.

Reliability and validity of the Taiwan (Mandarin Chinese) version of the chronic respiratory questionnaire.

PURPOSE: The chronic respiratory disease questionnaire (CRQ) has been validated and proved useful in assessing therapies for pulmonary diseases. We translated the CRQ into a Taiwan (Mandarin Chinese) version and surveyed its validity and reliability. METHODS: The CRQ includes 20 items divided into four domains: dyspnea, fatigue, emotional function, and mastery. We followed a forward-back translation procedure to create the Taiwan version. A cross-sectional survey was conducted among outpatients with chronic obstructive pulmonary disease. Participants underwent tests including the CRQ, the medical outcomes study short form (SF-36), the St. George respiratory questionnaire (SGRQ), lung function tests (LFTs), and a graded exercise test (GET). We used Cronbach's alpha to evaluate the internal consistency of the CRQ, intraclass coefficient for test-retest reliability, and Spearman's correlation for validity. RESULTS: Thirty-six men and 4 women (mean age 67.9 ± 9.9 years) were recruited. Evidence of good internal consistency, test-retest reliability, convergent, discriminant, concurrent, and construct validity of the CRQ was shown. Spearman's correlation showed moderate-to-strong correlation between the CRQ scores and scores of the SGRQ, subscales of the SF-36, and the results of LFTs and GET. CONCLUSIONS: The Taiwan version of the CRQ shows good validity, internal consistency, and test-retest reliability.

Association between spinal curvature disorders and injury: a nationwide population-based retrospective cohort study.

OBJECTIVES: Injury is an important issue in public health. Spinal curvature disorders are deformities characterised by excessive curves of the spine. The prevalence of spinal curvature disorders is not low, but its relationship with injury has not been studied. The aim of this study is to investigate whether spinal curvature disorders increase the risk of injury. DESIGN: Population-based retrospective cohort study. SETTING: Using data from the Taiwan National Health Insurance Research Database from 2000 to 2010. PARTICIPANTS AND EXPOSURE: Patients with spinal curvature disorders were selected using codes from the International Classification of Diseases, Ninth Revision, Clinical Modification. A cohort without spinal curvature was randomly frequency-matched to the spinal curvature disorders cohort at a ratio of 2:1 according to age, sex and index year. PRIMARY OUTCOME MEASURES: The risk of injury was analysed using Cox's proportional hazards regression models adjusting for age, sex, comorbidities, urbanisation level and socioeconomic status. RESULTS: A total of 20 566 patients with spinal curvature disorders and 41 132 controls were enrolled in this study. The risk of injury was 2.209 times higher (95% CI 2.118 to 2.303) in patients with spinal curvature disorders than in the control group. The spinal curvature disorders cohort exhibited higher risk of developing injury compared with the control group, regardless of age, sex, comorbidities, urbanisation level and subgroup of spinal curvature disorders. Based on the subgroup analysis, the spinal curvature disorders cohort had higher risks of unintentional injury and injury diagnoses such as fracture, dislocation, open wound, superficial injury/contusion, crushing and injury to nerves and spinal cord compared with the control cohort. CONCLUSIONS: Patients with spinal curvature disorders have a significantly higher risk of developing injury than patients without spinal curvature disorders. Aggressive detection and management of spinal curvature disorders may be beneficial for injury prevention.

Urinary biomarker of oxidative stress correlating with outcome in critically septic patients.

OBJECTIVE: To determine whether urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), an in vivo parameter of oxidative stress, is correlated with the outcome of critically septic patients. DESIGN AND SETTING: Clinical outcome study in an adult medical intensive care unit (ICU). PATIENTS: 85 consecutive septic patients (59 men, 26 women). MEASUREMENTS AND RESULTS: Patient characteristics and the clinical course were examined. Urinary 8-OHdG was analyzed using isotope-dilution liquid chromatography with tandem mass spectrometry (LC/MS/MS). ICU mortality was 25.9% (22/85) and hospital mortality 38.8% (33/85). Survivors' APACHEII scores on days 1 and 3 and the difference between them differed significantly from those of nonsurvivors (day 1, 21.0+/-7.1 vs. 25.9+/-8.0; day 3, 15.0+/-5.8 vs. 23.2+/-8.3; difference, 6.0+/-5.5 vs. 1.7+/-6.6). Urinary 8-OHdG was significantly lower in survivors than in nonsurvivors on day 1 (1.8+/-2.4 vs. 3.0+/-2.4). The area under receiver operating characteristic curve analysis for the association between day 1 urinary 8-OHdG and ICU mortality was 0.71. The comparison performed upon discharge from hospital revealed similar results. CONCLUSIONS: This is a preliminary study. Excretion of urinary 8-OHdG, as measured using isotope-dilution LC/MS/MS, and the APACHE II score were correlated with the outcome of critically septic patients in medical ICU.

Urinary biomarker of oxidative stress correlating with outcome in critically septic patients.

OBJECTIVE: To determine whether urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), an in vivo parameter of oxidative stress, is correlated with the outcome of critically septic patients. DESIGN AND SETTING: Clinical outcome study in an adult medical ICU. PATIENTS: Eighty-five consecutive septic patients: 59 men and 26 women. MEASUREMENTS AND RESULTS: Urinary 8-OHdG was analyzed using isotope-dilution liquid chromatography with tandem mass spectrometry (LC/MS/MS). ICU mortality in these 85 septic patients was 25.9% (n = 22) and hospital mortality 38.8% (n = 33). APACHE II scores of survivors on day 1, on day 3, and the difference between them differed significantly from those of nonsurvivors (day 1, 21.0 +/- 7.1 vs. 25.9 +/-8.0; day 3, 15.0 +/- 5.8 vs. 23.2 +/- 8.3; difference, 6.0 +/- 5.5 vs. 1.7 +/- 6.6). Urinary 8-OHdG was significantly lower in survivors than in nonsurvivors on day 1 (1.8 +/- 2.4 vs. 3.0 +/- 2.4). The area under receiver operating characteristic curve analysis for the association between day 1 urinary 8-OHdG and ICU mortality was 0.71. The comparison performed upon discharge from hospital revealed similar results. CONCLUSIONS: This is a preliminary study. The excretion of the urinary 8-OHdG, as measured using isotope-dilution LC/MS/MS, as the APACHE II score, were correlated with the outcome of critically septic patients in medical ICU.

Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement.

Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.

Economic Burden of Diabetes Mellitus on Patients with Respiratory Failure Requiring Mechanical Ventilation during Hospitalizations.

OBJECTIVES: To examine the economic burden of diabetes mellitus (DM) on medical expenditure among patients with respiratory failure (RF) requiring mechanical ventilation during hospitalization. METHODS: We extracted the data from Taiwan National Health Research Insurance Database for those adult patients on their first hospitalization for RF requiring mechanical ventilation between 2004 and 2010. We examined associations between medical expenditure and the presence of comorbid DM. We performed independent t tests, chi-square tests, and multivariate linear regression analysis to identify factors associated with excess medical expenditure. RESULTS: Of 347,961 patients hospitalized with first occurrence of RF requiring mechanical ventilation, 123,023 (35.36%) patients were documented to have a previous diagnosis of DM. Patients with RF and DM were sicker and consumed more health care resources than did patients with RF without DM. After adjusting for the specified covariates, mechanically ventilated patients with RF and DM consumed at least US $618 more of total inpatient medical expenditure than did patients with RF without DM. There were statistically significant interactions between age and DM on their total inpatient medical expenditure regardless of discharge status. CONCLUSIONS: DM was associated with more severe disease status and higher consumption of medical expenditure during hospitalizations among mechanically ventilated patients due to first occurrence of RF in Taiwan. These findings provide scientific evidence to facilitate appropriate resource allocation and formulate programs for higher quality of care in the future in Taiwan and other countries.

Impact of Taiwan's integrated prospective payment program on prolonged mechanical ventilation: a 6-year nationwide study.

OBJECTIVE: The integrated prospective payment program (IPP), which encourages the integrated care of mechanically ventilated patients in order to reduce the heavy utilization of high-cost ICUs, has been implemented by Taiwan's Bureau of National Health Insurance since July 2000. The aim of this study was to assess the impact of this program on weaning, hospital stay, mortality, and cost for patients requiring prolonged mechanical ventilation (PMV). METHODS: A data set of 1,000,000 randomly selected insurance holders from the National Health Research Insurance Database, Taiwan, was retrospectively analyzed. We enrolled 7,967 adult patients (age ≥ 17 y) who required PMV (duration ≥ 21 d) over a 6 year period. RESULTS: There were 3,275 patients on PMV before (1997-1999) and 4,692 patients on PMV after (2001-2003) the IPP implementation. After IPP implementation, PMV was found to be required in patients with a significantly higher age, lower urbanization level, higher income status, and a higher prevalence of neuromuscular disease (P < .001). In-hospital mortality was similar between the 2 periods (17.2% before vs 16.2% after, P = .26), but the weaning rate was significantly lower in the latter period (68.1% vs 64.2%, P < .001). Total hospital stay (75.3 d vs 95.1 d, P < .001) and duration of mechanical ventilation usage (55.8 d vs 71.6 d, P < .001) were both significantly higher after the IPP implementation. Total hospitalization cost in the PMV patients was significantly lower after IPP implementation. CONCLUSIONS: Implementation of the IPP program reduced the total hospitalization cost, increased the duration of mechanical ventilation usage and stay, and reduced the weaning rate in PMV patients.

The contribution of DNA apurinic/apyrimidinic endonuclease genotype and smoking habit to Taiwan lung cancer risk.

To evaluate the association and interaction of genotypic polymorphism the gene for DNA-apurinic/apyrimidinic endonuclease (APEX1) with personal smoking habit and lung cancer risk in Taiwan, the polymorphic variants of APEX1, Asp(148)Glu (rs1130409), were analyzed in association with lung cancer risk, and their joint effect with personal smoking habits on lung cancer susceptibility was discussed. In this hospital-based case-control study, 358 patients with lung cancer and 716 cancer-free controls, frequency-matched by age and sex, were recruited and genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The results showed that the percentages of TT, TG and GG APEX1 Asp(148)Glu genotypes were not significantly different at 43.0%, 41.1% and 15.9% in the lung cancer patient group and 39.9%, 46.1% and 14.0% in non-cancer control group, respectively. We further analyzed the genetic-lifestyle effects on lung cancer risk and found the contribution of APEX1 Asp(148)Glu genotypes to lung cancer susceptibility was neither enhanced in the cigarette smokers nor in the non-smokers (p=0.3550 and 0.8019, respectively). Our results provide evidence that the non-synonymous polymorphism of APEX1 Asp(148)Glu may not be directly associated with lung cancer risk, nor enhance the effects of smoking habit on lung cancer development.

Association of hypoxia inducible factor-1α polymorphisms with susceptibility to non-small-cell lung cancer.

Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of cellular response to hypoxia and has been suggested to play an important role in tumorigenesis and metastasis. The aim of this study was to investigate the role of HIF-1α-1772 C/T (P582S) and -1790 G/A (A588T) polymorphisms in the susceptibility to and severity of non-small-cell lung cancer (NSCLC). Using a case-control study design and polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis, the allele frequencies and genotype distributions of each single nucleotide polymorphism in 285 NSCLC cases and 300 gender-matched controls were compared. The distribution of the genotype frequencies of HIF-1α-1772 C/T and -1790 G/A were significantly different between the NSCLC and the controls. Logistic regression analysis revealed that higher odds ratios (ORs) for lung cancer were observed for individuals with HIF-1α-1772 T/T genotype against CC/CT genotypes (an OR of 4.04, 95% confidence interval [CI] = 2.02-8.08, P = 0.0001), and HIF-1α-1790 A/A genotype against GG/GA genotypes (an OR of 4.42, 95% CI 2.22-8.78, P < 0.0001). There were no relationship between HIF-1α-1772 C/T or -1790 G/A allele distribution and disease severity of NSCLC (P > 0.05). However, those patients carrying a HIF-1α-1772 T/T genotype or a HIF-1α-1790 A/A had a tendency toward inferior prognosis compared with other patients.

Association of polymorphisms in the genes of the urokinase plasminogen activation system with susceptibility to and severity of non-small cell lung cancer.

BACKGROUND: Urokinase plasminogen activating (uPA) system is implicated in neoplastic progression. High tissue levels of uPA system components correlate with a poor prognosis in lung cancer. The present study examined the single nucleotide polymorphisms (SNPs) of uPA and the corresponding receptor, uPAR, for exploring their roles in non-small cell lung cancer (NSCLC). METHODS: The allele frequencies and genotype distributions of uPA rs4065 C/T and uPAR rs344781 (-516 T/C) among 375 NSCLC cases and 380 healthy controls were examined using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis. Putative association between the above SNPs and clinicopathological characteristics of NSCLC were also analyzed. RESULTS: The genotype frequencies of the variant homozygotes of uPA and uPAR were significantly different between NSCLC and control subjects. Significant association was also observed between the examined genotypes and disease stage of NSCLC. Logistic regression analysis revealed that individuals with uPA rs4065 TT genotype have higher odds ratios (ORs) for lung cancer. Whereas, subjects with uPAR-344781 CC genotype have lower ORs for lung cancer. The patients carrying a homozygous TT genotype at uPA rs4065, or at least a T allele at uPAR-344781 (-516), had a tendency to develop advanced disease. CONCLUSIONS: Our results revealed that genetic polymorphisms of the uPA rs4065 C/T and uPAR rs344781 (-516 T/C) were associated with the susceptibility and severity of NSCLC.

Association of genetic polymorphisms of CXCL12/SDF1 gene and its receptor, CXCR4, to the susceptibility and prognosis of non-small cell lung cancer.

BACKGROUND: The aim of this study was to evaluate the relations of chemokine CXCL12, previously known as stromal cell-derived factor-1 (SDF1), and its receptor, CXCR4, gene variants on non-small cell lung cancer (NSCLC) risk and disease severity. METHODS: Through a case-control study design, genomic DNA samples of 247 NSCLC patients and 328 age and sex-matched controls were subjected to polymerase chain reaction-restriction fragment length polymorphism analysis. The validity of this technique was proven by direct sequencing of amplified products. Statistical analyses were conducted to explore the contribution of polymorphism of the CXCL12/SDF1 gene and CXCR4, in the susceptibility to and prognosis of NSCLC. RESULTS: Overall, the genotype frequencies of CXCL12/SDF1 gene and CXCR4, were significantly different between lung cancer patients and controls (p<0.0001), and also different between patients with lung cancers of various stages (p<0.0001). Logistic regression analysis revealed that higher odds ratios (ORs) for lung cancer were seen for individuals with CXCL12/SDF1 AA (an OR of 1.95, 95% CI 1.08-3.50, p=0.018), or CXCR4 TT (an OR of 4.71, 95% CI 1.99-11.2, p<0.0001), and for individuals with both CXCL12/SDF1 AA and CXCR4 TT genotypes (an OR of 12.4, 95% CI 1.56-98.3, p=0.002). The patients carrying a homologous AA genotype at CXCL12/SDF1, or a homologous TT genotype at CXCR4, had a tendency to advanced disease and toward poorer prognoses compared with other patients. CONCLUSION: A significant association between the polymorphisms of CXCL12/SDF1 and CXCR4, and the susceptibility to and prognosis of NSCLC was demonstrated.

Vascular endothelial growth factor C as a predictor of early recurrence and poor prognosis of resected stage I non-small cell lung cancer.

INTRODUCTION: Stage I non-small cell lung cancer (NSCLC) is potentially curable after completely resection, but early recurrence may infl uence prognosis. This study hypothesises that vascular endothelial growth factor C (VEGF-C) plays a key role in predicting early recurrence and poor survival of patients with stage I NSCLC. MATERIALS AND METHODS: The expression of VEGF-C was immuno-histochemically (IHC) analysed in tumour samples of primary stage I NSCLC and correlated to early recurrence (< 36 months), disease-free survival, and overall survival in all 49 patients. RESULTS: Early recurrence was identifi ed in 16 patients (33%), and the early recurrence rate in strong and weak VEGF-C activity was significantly different (P = 0.016). VEGF-C was also an independent risk factor in predicting early recurrence (HR = 3.98, P = 0.02). Patients with strong VEGF-C staining also had poor 3-year disease-free survival (P = 0.008) and overall survival (P = 0.007). CONCLUSION: Strong VEGF-C IHC staining could be a biomarker for predicting early recurrence and poor prognosis of resected stage I NSCLC, if the results of the present study are confirmed in a larger study. A more aggressive adjuvant therapy should be used in this group of patients.

Pigtail catheters vs large-bore chest tubes for management of secondary spontaneous pneumothoraces in adults.

It is still uncertain if large-bore chest tubes (20F-28F) is superior to pigtail catheter (10F-14F) in terms of the management of secondary spontaneous pneumothoraces (SSP). This study was designed to compare the efficacy and safety associated with placement of large-bore chest tubes vs pigtail catheters in adults experiencing the first episode of SSP. We conducted a retrospective chart review of 91 patients experiencing the first episode of SSP in a university hospital over a 3.5-year period who received treatment by either a large-bore chest tube or a pigtail catheter. Any patient who was younger than 18 years or experiencing mechanical ventilation-related barotraumas or pyopneumothorax was excluded from this study. Various parameters including demographical characteristics, size of pneumothorax, complications, time of pigtail or chest tube extubation, and length of hospital stay were collected and analyzed. Among the enrolled 91 patients, including 76 (83.5%) men with a mean age of 60 +/- 19 years, 69 were initially treated with a pigtail, and 22 patients received conventional chest tubes. Fifty patients (72.5%) undergoing the pigtail drainage and 16 (72.7%) undergoing large-bore chest tube treatment of SSP were successfully treated (P = .88). In addition, there was no significant difference in terms of length of hospital stay, extubation time, recurrence rate, and complication. Pigtail catheters offer a safe and effective alternative for large-bore chest tubes to adult patients experiencing the first episode of SSP, and we strongly suggested that pigtail tube drainage should be considered as the initial treatment of choice.