AMPK activation reduces vascular permeability and airway inflammation by regulating HIF/VEGFA pathway in a murine model of toluene diisocyanate-induced asthma

Background

Occupational asthma is characterized by airway inflammation and hyperresponsiveness associated with increased vascular permeability. AMP-activated protein kinase (AMPK) has been suggested to be a novel signaling molecule modulating inflammatory responses.

Objective

We sought to evaluate the involvement of AMPK in pathogenesis of occupational asthma and more specifically investigate the effect and molecular mechanisms of AMPK activation in regulating vascular permeability.

Methods

The mechanisms of action and therapeutic potential of an AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) were tested in a murine model of toluene diisocyanate (TDI)-induced asthma.

Results

AICAR attenuated airway inflammation and hyperresponsiveness increased by TDI inhalation. Moreover, TDI-induced increases in levels of hypoxia-inducible factor (HIF)-1α, HIF-2α, vascular endothelial growth factor A (VEGFA), and plasma exudation were substantially decreased by treatment with AICAR. Our results also showed that VEGFA expression was remarkably reduced by inhibition of HIF-1α and HIF-2α with 2-methoxyestradiol (2ME2) and that an inhibitor of VEGFA activity, CBO-P11 as well as 2ME2 significantly suppressed vascular permeability, airway infiltration of inflammatory cells, and airway hyperresponsiveness induced by TDI. In addition, AICAR reduced reactive oxygen species (ROS) generation and levels of malondialdehyde and T-helper type 2 cytokines (IL-4, IL-5, and IL-13), while this agent enhanced expression of an anti-inflammatory cytokine, IL-10.

Conclusions

These results suggest that AMPK activation ameliorates airway inflammatory responses by reducing vascular permeability via HIF/VEGFA pathway as well as by inhibiting ROS production and thus may be a possible therapeutic strategy for TDI-induced asthma and other airway inflammatory diseases.     

BRAFV600E Mutation Testing in Fine Needle Aspirates of Thyroid Nodules: Potential Value of Real-Time PCR

The BRAF(V600E) mutation is a valuable adjunctive diagnostic tool to ultrasound (US)-guided fine-needle aspiration (US-FNA). The objective of this study was to investigate the potential value of realtime PCR to detect BRAF(V600E) mutation. This study included 447 thyroid nodules in 420 patients who underwent US-FNA and BRAF(V600E) mutation analysis using dual priming oligonucleotide-based multiplex polymerase chain reaction (DPO-PCR) and real-time PCR. We calculated and compared the diagnostic performances of DPO-PCR and real-time PCR to detect BRAF(V600E) mutation in the thyroid nodules. Receiver operating characteristic (ROC) analysis was used to quantify the cut-off value of the Ct values of BRAF(V600E) mutation on real-time PCR. Optimal thresholds were determined (Youden index). We also compared the diagnostic performances between DPO-PCR and real-time PCR after applying the cut-off value on real-time PCR. Sensitivity, accuracy, and NPV were significantly higher in real-time PCR than DPO-PCR. When the optimal cut-off value of 32.4 at Ct values of BRAF(V600E) mutation was adjusted on real-time PCR, sensitivity was 66.2% and specificity was 100%. Sensitivity, accuracy, and NPV of real-time PCR were also significantly higher than DPO-PCR. In contrast, specificity and PPV were not significantly different between DPO-PCR and real-time PCR. Real time PCR can be a promising diagnostic method in detecting BRAF(V600E) mutation using optimal cut-off value.     

Involvement of endoplasmic reticulum stress in myofibroblastic differentiation of lung fibroblasts

Stress that impairs endoplasmic reticulum (ER) function leads to an accumulation of unfolded or misfolded proteins in the ER (ER stress) and triggers the unfolded protein response (UPR). Recent studies suggest that ER stress is involved in idiopathic pulmonary fibrosis (IPF). The present study was undertaken to determine the role of ER stress on myofibroblastic differentiation of fibroblasts. Fibroblasts in fibroblastic foci of IPF showed immunoreactivity for GRP78. To determine the role of ER stress on α-smooth muscle actin (α-SMA) and collagen type I expression in fibroblasts, mouse and human lung fibroblasts were treated with TGF-β1, and expression of ER stress-related proteins, α-SMA, and collagen type I was analyzed by Western blotting. TGF-β1 significantly increased expression of GRP78, XBP-1, and ATF6α, which was accompanied by increases in α-SMA and collagen type I expression in mouse and human fibroblasts. A chemical chaperone, 4-PBA, suppressed TGF-β1-induced UPR and α-SMA and collagen type I induction. We also showed that TGF-β1-induced UPR was mediated through the reactive oxygen species generation. Our study provides the first evidence implicating the UPR in myofibroblastic differentiation during fibrosis. These findings of the role of ER stress and chemical chaperones in pulmonary fibrosis may improve our understanding of the pathogenesis of IPF.     

Alpha modulation in parietal and retrosplenial cortex correlates with navigation performance

The present study investigated the brain dynamics accompanying spatial navigation based on distinct reference frames. Participants preferentially using an allocentric or an egocentric reference frame navigated through virtual tunnels and reported their homing direction at the end of each trial based on their spatial representation of the passage. Task-related electroencephalographic (EEG) dynamics were analyzed based on independent component analysis (ICA) and subsequent clustering of independent components. Parietal alpha desynchronization during encoding of spatial information predicted homing performance for participants using an egocentric reference frame. In contrast, retrosplenial and occipital alpha desynchronization during retrieval covaried with homing performance of participants using an allocentric reference frame. These results support the assumption of distinct neural networks underlying the computation of distinct reference frames and reveal a direct relationship of alpha modulation in parietal and retrosplenial areas with encoding and retrieval of spatial information for homing behavior.     

Effects of transcription factor activator protein-1 on interleukin-8 expression and enteritis in response to <Emphasis Type="Italic">Clostridium difficile</Emphasis> toxin A

Clostridium difficile toxin A causes acute colitis associated with intense infiltration of neutrophils. Although C. difficile toxin A is known to induce nuclear factor-kappaB-mediated chemokine expression in intestinal epithelial cells, little is known about its effect on the regulation of activator protein-1 (AP-1) by mitogen-activated protein kinase (MAPK). In the present study, we investigated whether the MAPK and AP-1 signaling pathway is involved in interleukin (IL)-8 expression and enteric inflammation in response to stimulation with toxin A. Toxin A activated MAPK and AP-1 composed of c-Jun/c-Fos heterodimers in primary intestinal epithelial cells and HT-29 cell lines. Transfection with mutant genes for Ras, c-Jun, p38, or c-Jun N-terminal kinase (JNK) significantly inhibited C. difficile toxin A-induced activation of AP-1 and expression of IL-8 in HT-29 cell lines. Furthermore, the p38 inhibitor SB203580 attenuated toxin A-induced inflammation in vivo in the mouse ileum, evidenced by a significant decrease in neutrophil infiltration, villous destruction, and mucosal congestion. Our results suggest that the Ras/MAPK cascade acts as the upstream signaling for AP-1 activation and IL-8 expression in toxin A-stimulated intestinal epithelial cells and may be involved in the development of enteritis after infection with toxin A-producing C. difficile.     

Outpatient-basis chemotherapy of oxaliplatin, 5-fluorouracil, and leucovorin as first-line treatment for patients with metastatic or recurrent colorectal cancer

The objectives of the present study were to evaluate the efficacy and safety of an outpatient-basis chemotherapy of oxaliplatin, 5-fluorouracil, and leucovorin as the first-line treatment for patients with advanced colorectal cancer. Forty-three histologically confirmed patients with metastatic or recurrent colorectal cancer were enrolled. The chemotherapy consisted of oxaliplatin 85 mg/m(2) as a 2-hr infusion on day 1, plus leucovorin 30 mg/m(2) over 10 min, followed by bolus 5-fluorouracil 400 mg/m(2) and an 8-hr infusion of 5-fluorouracil 600 mg/m(2) on days 1 and 2 (modified FOLFOX4), all of which were administered on an outpatient basis every 2 weeks. The median age was 58 yr (range 33-72 yr), and 25 (58.1%) patients had metastatic diseases. Eventually, 39 patients were assessable for efficacy and all assessable for toxicity. Four (9.3%) complete responses and 11 (25.6%) partial responses were confirmed, giving an overall response rate of 34.9% (95% CI; 20.0-49.7%). The median time to progression and median overall survival for all patients was 6.1 months and 17.4 months, respectively. Grade 3/4 neutropenia occurred in 2 patients (4.7%) and febrile neutropenia was observed in 1 patient (2.3%). Modified FOLFOX4, an outpatient-basis regimen, was found to be well-tolerated and effective as the first-line chemotherapy in patients with advanced colorectal cancer.     

Analysis of prognostic factors and treatment modality changes in breast cancer: a single institution study in Korea

PURPOSE: To determine the effects of new breast cancer treatments and to provide a baseline for monitoring the development of breast cancer in Korean women, we conducted an analysis at our institution to determine long-term clinicopathological features, survival rates, and prognostic factors. MATERIALS AND METHODS: This study retrospectively analyzed 2,403 patients between Sep 1994 and Dec 2002, who underwent breast cancer surgery at Samsung Medical Center in Korea. Demographic data, pathologic records and surgical records were collected. RESULTS: After a median follow-up duration of 121.9 (range: 2-158.1) months, the 5-year disease free survival (DFS) was 82.8% and the 10-year DFS was 74.7%. The 5-year and 10-year overall survival (OS) rates were 89.4% and 82.9%, respectively. Using multivariate analyses, we determined that the nodal status (p < 0.001), angioinvasion (p < 0.001), positive PR (p < 0.001), and C-erb-B2 (p < 0.001) were independent prognostic factors for OS. The frequency of breast conserving surgery was 33.9% before Dec 1999, and increased up to 44.1% by year Dec 2002. CONCLUSION: Most of the prognostic variables and clinical characteristics of the Korean breast cancer patients were similar to those reported for Western populations. However, the age distribution in Korean patients seemed to be different from that in patients from Western countries.     

Clinical features reflect exon sites of EGFR mutations in patients with resected non-small-cell lung cancer

The aim of the current study was to determine the clinical significance according to the subtypes of epidermal growth factor receptor (EGFR) mutations and presence of KRAS mutations in operable non-small-cell lung cancer (NSCLC). We sequenced exons 18-21 of the EGFR tyrosine kinase domain and examined mutations in codons 12 and 13 of KRAS in tissues of patients with NSCLC who had undergone surgical resection. EGFR mutations were more frequent in never-smokers than smokers (33% vs. 14%, respectively; p=0.009) and in females than in males (31% vs. 16%, respectively; p=0.036). Mutations in exon 18-19 and 20-21 were found in 10 and 22 patients, respectively. Never-smokers and broncho-alveolar cell carcinoma features were positively associated with a mutation in exon 18-19 (p=0.027 and 0.016, respectively). The five-year survival rate in patients with a mutation in exons 18-19 (100%) was higher than that in patients without such mutation (47%; p=0.021). KRAS mutations were found in 16 patients (12%) and were not related to the overall survival (p=0.742). Patients with an EGFR mutation in exons 18-19 had better survival than patients without such mutation. Subtypes of EGFR mutations may be prognostic factors in patients undergoing curative resection.     

Human placenta-derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: comparison with human bone marrow-derived feeders

Co-culture of human embryonic stem (ES) cells on mouse fibroblast feeders is the commonly used method for in vitro expansion of human ES cells in an undifferentiated state. However, it has potential risks of pathogen transmission from animals; thus, human cell-derived feeders have been employed to minimize this problem. In this study, we compared human placenta-derived feeders with bone marrow to demonstrate its effectiveness as feeders for in vitro long-term culture of human ES cells. We cultured a human ES cell line, SNUhES3, on human placenta-derived mesenchymal stem cell feeders and compared their culture efficiency with human bone marrow-derived feeders and control group (mouse fibroblast feeders, STO). The mean number of human ES cell colonies was 166 +/- 35 in the placenta feeders; this was significantly higher than bone marrow-derived feeders (87 +/- 16, p < 0.05). We could propagate the culture of SNUhES3 on the placenta feeders past the 50th week similar to control group. During the culture, the maintenance of undifferentiated state of SNUhES3 was demonstrated by the expression of SSEA-4, TRA-1-81, TRA-1-60, and Oct-4. However, we failed to propagate the culture of human ES cells on the human bone marrow-derived feeders past the 5th week. The efficiency of embryoid body formation was similar between placenta and control group, indicating the preservation of differentiation ability. Thus, placenta-derived feeders are more efficient for the long-term in vitro culture of human ES cells than bone marrow-derived feeders suggesting the possible role of placenta as a source for human cell-derived feeders.