BRAFV600E promotes anchorage-independent growth but inhibits anchorage-dependent growth in hTERT/Cdk4-Immortalized normal human bronchial epithelial cells.

Certain oncogenes, including mutant RAS and BRAF, induce a type of senescence known as oncogene-induced senescence (OIS) in normal cells in a cell-type-specific manner. OIS serves as a barrier to transformation by activated oncogenes. Our previous studies showed that mutant KRASV12 did not efficiently induce OIS in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line (HBEC3), but it did enhance both anchorage-dependent and anchorage-independent growth. In this study, we investigated whether mutant BRAF, a well-known inducer of OIS, could trigger OIS in HBEC3 cells. We also assessed the impact of mutant BRAF on the growth of HBEC3 cells, as no previous studies have examined this using a normal bronchial epithelial cell line model. We established an HBEC3 cell line, designated as HBEC3-BIN, that expresses mutant BRAFV600E in a doxycycline-regulated manner. Unlike our previous finding that KRASV12 upregulated both pERK and pAKT, mutant BRAFV600E upregulated pERK but not pAKT in HBEC3-BIN cells. Similar to KRASV12, BRAFV600E did not efficiently induce OIS. Interestingly, while BRAFV600E inhibited colony formation in anchorage-dependent conditions, it dramatically enhanced colony formation in anchorage-independent conditions in HBEC3-BIN. In HBEC3 cells without BRAFV600E or KRASV12 expression, p21 was only detected in the cytoplasm, and its localization was not altered by the expression of BRAFV600E or KRASV12. Next-generation sequencing analysis revealed an enrichment of gene sets known to be involved in carcinogenesis, including IL3/JAK/STAT3, IL2, STAT5, and the EMT pathway. Our results indicate that, unlike KRASV12, which promoted both, BRAFV600E enhances anchorage-independent growth but inhibits anchorage-dependent growth of HBEC3. This contrast may result from differences in activation signaling in the downstream pathways. Furthermore, HBEC3 cells appear to be inherently resistant to OIS, which may be partly due to the fact that p21 remains localized in the cytoplasm upon expression of BRAFV600E or KRASV12.

Neonatal lethality of mouse A/J-7SM consomic strain is caused by an insertion mutation in the Dchs1 gene.

Homosomic mice of the A/J-7SM consomic mouse strain that introduced the entire chromosome 7 (Chr 7) of SM/J into the A/J strain exhibited neonatal lethality. We tentatively maintained segregating inbred strains (A/J-7ASM and A/J-7DSM) in which the central portion of Chr 7 was heterozygous for the A/J and SM/J strains, and the centromeric and telomeric sides of Chr 7 were homozygous for the SM/J strain, instead of the A/J-7SM strain. Based on the chromosomal constitution of Chr 7 in A/J-7ASM and A/J-7DSM mice, the causative gene for neonatal lethality in homosomic mice was suggested to be located within an approximately 1.620 Mb region between D7Mit125 (104.879 Mb) and D7Mit355 (106.499 Mb) on Chr 7. RT-PCR analysis revealed that homosomic mice lacked dachsous cadherin-related 1 (Dchs1), which is located within the D7Mit125 to D7Mit355 region and functions in the regulation of planar cell polarity. Screening for mutations in Dchs1 indicated that homosomic mice possessed an early transposable (ETn)-like sequence in intron 1 of Dchs1. Moreover, an allelism test between Dchs1 ETn-like-insertion alleles detected in homosomic mice and CRISPR/Cas9-induced Dchs1 deletion alleles revealed that Dchs1 is a causative gene for neonatal lethality in homosomic mice. Based on these results, we concluded that in the A/J-7SM strain, ETn-like elements were inserted into intron 1 of SM/J-derived Dchs1 during strain development, which dramatically reduced Dchs1 expression, thus resulting in neonatal lethality in homosomic mice. Additionally, it was suggested that the timing of lethality in Dchs1 mutant mice is influenced by the genetic background.

Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line.

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called "oncogene-induced senescence (OIS)", prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called "HBEC3KT"), immortalized with hTERT ("T") and Cdk4 ("K"), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated ß-galactosidase staining (SA-ßG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.

Fibroblasts positive for meflin have anti-fibrotic properties in pulmonary fibrosis.

The prognosis of elderly individuals with idiopathic pulmonary fibrosis (IPF) remains poor. Fibroblastic foci, in which aggregates of proliferating fibroblasts and myofibroblasts are involved, are the pathological hallmark lesions in IPF to represent focal areas of active fibrogenesis. Fibroblast heterogeneity in fibrotic lesions hampers the discovery of the pathogenesis of pulmonary fibrosis. Therefore, to determine the pathogenesis of IPF, identification of functional fibroblasts is warranted. The aim of this study was to determine the role of fibroblasts positive for meflin, identified as a potential marker for mesenchymal stromal cells, during the development of pulmonary fibrosis.We characterised meflin-positive cells in a single-cell atlas established by single-cell RNA sequencing (scRNA-seq)-based profiling of 243 472 cells from 32 IPF lungs and 29 normal lung samples. We determined the role of fibroblasts positive for meflin using bleomycin (BLM)-induced pulmonary fibrosis.scRNA-seq combined with in situ RNA hybridisation identified proliferating fibroblasts positive for meflin in fibroblastic foci, not dense fibrosis, of fibrotic lungs in IPF patients. A BLM-induced lung fibrosis model for meflin-deficient mice showed that fibroblasts positive for meflin had anti-fibrotic properties to prevent pulmonary fibrosis. Although transforming growth factor-ß-induced fibrogenesis and cell senescence with the senescence-associated secretory phenotype were exacerbated in fibroblasts via the repression or lack of meflin, these were inhibited in meflin-deficient fibroblasts with meflin reconstitution.These findings provide evidence to show the biological importance of meflin expression on fibroblasts and myofibroblasts in the active fibrotic region of pulmonary fibrosis.

Miniaturization of Respiratory Measurement System in Artificial Ventilator for Small Animal Experiments to Reduce Dead Space and Its Application to Lung Elasticity Evaluation.

A respiratory measurement system composed of pressure and airflow sensors was introduced to precisely control the respiratory condition during animal experiments. The flow sensor was a hot-wire thermal airflow meter with a directional detection and airflow temperature change compensation function based on MEMS technology, and the pressure sensor was a commercially available one also produced by MEMS. The artificial dead space in the system was minimized to the value of 0.11 mL by integrating the two sensors on the same plate (26.0 mm × 15.0 mm). A balloon made of a silicone resin with a hardness of A30 was utilized as the simulated lung system and applied to the elasticity evaluation of the respiratory system in a living rat. The inside of the respiratory system was normally pressurized without damage, and we confirmed that the developed system was able to evaluate the elasticity of the lung tissue in the rat by using the pressure value obtained at the quasi-static conditions in the case of the ventilation in the animal experiments.

UHRF1, a Regulator of Methylation, as a Diagnostic and Prognostic Marker for Lung Cancer.

We evaluated the value of UHRF1, a regulator of methylation, as a biomarker for lung cancer. UHRF1 is expressed at higher levels in both lung adenocarcinoma (AD) and squamous cell carcinoma (SQ); however, a meta-analysis showed that UHRF1 expression is correlated with worse survival in patients with AD but not in those with SQ. UHRF1 knockdown suppressed the growth of lung cancer cell lines through G1 cell cycle arrest in some cell lines. These results suggest that UHRF1 may server as a diagnostic marker for AD and SQ and as a prognostic marker for AD in lung cancer.

Involvement of heme oxygenase-1 in suppression of T cell activation by quercetin.

AIM: Acute rejection is still a major problem in transplantation and one of the most important causes of late graft loss. Cyclosporine and tacrolimus are widely used for suppression of T cell function to avoid graft rejection, but long-term use of these compounds is associated with serious toxicities. Quercetin, a flavonoid found in fruits and vegetables, has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO) -1, an enzyme involved in heme catabolism. We hypothesized that quercetin induces HO-1 in T cells and suppresses T cell function via HO-1. In the present study, we showed that quercetin suppressed the A23187-mediated expression of interleukin (IL) -2 in T cells. METHODS: Mouse splenocytes, enriched T cells, and EL4 cells, a mouse T cell line, were treated with quercetin, and then stimulated with A23187, a calcium ionophore, concanavalin A, or anti-CD3ε and anti-CD28 antibodies. Cell proliferation, expression of IL-2, calcium mobilization, apoptosis, cell cycle, and phosphorylation of extracellular signal-regulated kinase (ERK) were investigated. RESULTS: Quercetin induced HO-1, and this induction of HO-1 was implicated in the suppression of IL-2 production. Furthermore, the induction of HO-1 by quercetin suppressed the influx of calcium ions, a known trigger of IL-2 production. Additionally, quercetin suppressed T cell proliferation through promotion of cell cycle arrest via HO-1 induction, but quercetin did not induce apoptosis. To investigate the role of the signal transduction pathway in quercetin's effect on cell proliferation, we evaluated the phosphorylation of ERK in T cells. Quercetin suppressed the A23187-mediated stimulation of ERK, an effect that was mediated through HO-1. These results suggested that HO-1 is involved in the suppressive effects of quercetin on T cell activation and proliferation. CONCLUSION: Our findings indicate that the quercetin may be a promising candidate for inducing HO-1 in T cells, thereby facilitating immunosuppressive effects.

Repressive role of stabilized hypoxia inducible factor 1α expression on transforming growth factor ß-induced extracellular matrix production in lung cancer cells.

Activation of transforming growth factor ß (TGF-ß) combined with persistent hypoxia often affects the tumor microenvironment. Disruption of cadherin/catenin complexes induced by these stimulations yields aberrant extracellular matrix (ECM) production, characteristics of epithelial-mesenchymal transition (EMT). Hypoxia-inducible factors (HIF), the hallmark of the response to hypoxia, play differential roles during development of diseases. Recent studies show that localization of cadherin/catenin complexes at the cell membrane might be tightly regulated by protein phosphatase activity. We aimed to investigate the role of stabilized HIF-1α expression by protein phosphatase activity on dissociation of the E-cadherin/ß-catenin complex and aberrant ECM expression in lung cancer cells under stimulation by TGF-ß. By using lung cancer cells treated with HIF-1α stabilizers or carrying doxycycline-dependent HIF-1α deletion or point mutants, we investigated the role of stabilized HIF-1α expression on TGF-ß-induced EMT in lung cancer cells. Furthermore, the underlying mechanisms were determined by inhibition of protein phosphatase activity. Persistent stimulation by TGF-ß and hypoxia induced EMT phenotypes in H358 cells in which stabilized HIF-1α expression was inhibited. Stabilized HIF-1α protein expression inhibited the TGF-ß-stimulated appearance of EMT phenotypes across cell types and species, independent of de novo vascular endothelial growth factor A (VEGFA) expression. Inhibition of protein phosphatase 2A activity abrogated the HIF-1α-induced repression of the TGF-ß-stimulated appearance of EMT phenotypes. This is the first study to show a direct role of stabilized HIF-1α expression on inhibition of TGF-ß-induced EMT phenotypes in lung cancer cells, in part, through protein phosphatase activity.

Exogenous induction of unphosphorylated PTEN reduces TGFß-induced extracellular matrix expressions in lung fibroblasts.

Transforming growth factor ß (TGFß) plays an important role in regulating aberrant extracellular matrix (ECM) production from alveolar/epithelial cells (AECs) and fibroblasts in pulmonary fibrosis. Although the tumor suppressor gene phosphatase and tensin homologue deleted from chromosome 10 (PTEN) can negatively control many TGFß-activated signaling pathways via the phosphatase activity, hyperactivation of the TGFß-related signaling pathways is often observed in fibrosis. Loss of PTEN expression might cause TGFß-induced ECM production. In addition, TGFß was recently shown to induce loss of PTEN enzymatic activity by phosphorylating the PTEN C-terminus. Therefore, we hypothesized that exogenous transfer of unphosphorylated PTEN (PTEN4A) might lead to reduce TGFß-induced ECM expression in not only epithelial cells but also fibroblasts. Adenovirus-based exogenous PTEN4A induction successfully reduced TGFß-induced fibronectin expression and retained ß-catenin at the cell membrane in human epithelial cells. Exogenous unphosphorylated PTEN also attenuated TGFß-induced ECM production and inhibited TGFß-induced ß-catenin translocation in a human fibroblast cell line and in mouse primary isolated lung fibroblasts. Conversely, TGFß-induced α-smooth muscle actin expression did not seem to be inhibited in these fibroblasts. Our data suggest that exogenous administration of unphosphorylated PTEN might be a promising strategy to restore TGFß-induced loss of PTEN activity and reduce aberrant TGFß-induced ECM production from epithelial cells and fibroblasts in lung fibrosis as compared with wild-type PTEN induction.

Hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers.

BACKGROUND: Persistent hypoxia stimulation, one of the most critical microenvironmental factors, accelerates the acquisition of epithelial-mesenchymal transition (EMT) phenotypes in lung cancer cells. Loss of phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression might accelerate the development of lung cancer in vivo. Recent studies suggest that tumor microenvironmental factors might modulate the PTEN activity though a decrease in total PTEN expression and an increase in phosphorylation of the PTEN C-terminus (p-PTEN), resulting in the acquisition of the EMT phenotypes. Nevertheless, it is not known whether persistent hypoxia can modulate PTEN phosphatase activity or whether hypoxia-induced EMT phenotypes are negatively regulated by the PTEN phosphatase activity. We aimed to investigate hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers. METHODS: Western blotting was performed in five lung cancer cell lines to evaluate total PTEN expression levels and the PTEN activation. In a xenograft model of lung cancer cells with endogenous PTEN expression, the PTEN expression was evaluated by immunohistochemistry. To examine the effect of hypoxia on phenotypic alterations in lung cancer cells in vitro, the cells were cultured under hypoxia. The effect of unphosphorylated PTEN (PTEN4A) induction on hypoxia-induced EMT phenotypes was evaluated, by using a Dox-dependent gene expression system. RESULTS: Lung cancer cells involving the EMT phenotypes showed a decrease in total PTEN expression and an increase in p-PTEN. In a xenograft model, loss of PTEN expression was observed in the tumor lesions showing tissue hypoxia. Persistent hypoxia yielded an approximately eight-fold increase in the p-PTEN/PTEN ratio in vitro. PTEN4A did not affect stabilization of hypoxia-inducible factor 1α. PTEN4A blunted hypoxia-induced EMT via inhibition of ß-catenin translocation into the cytoplasm and nucleus. CONCLUSION: Our study strengthens the therapeutic possibility that compensatory induction of unphosphorylated PTEN may inhibit the acquisition of EMT phenotypes in lung cancer cells under persistent hypoxia.

Direct regulation of transforming growth factor ß-induced epithelial-mesenchymal transition by the protein phosphatase activity of unphosphorylated PTEN in lung cancer cells.

Transforming growth factor ß (TGFß) causes the acquisition of epithelial-mesenchymal transition (EMT). Although the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) can negatively regulate many signaling pathways activated by TGFß, hyperactivation of these signaling pathways is observed in lung cancer cells. We recently showed that PTEN might be subject to TGFß-induced phosphorylation of its C-terminus, resulting in a loss of its enzyme activities; PTEN with an unphosphorylated C-terminus (PTEN4A), but not PTEN wild, inhibits TGFß-induced EMT. Nevertheless, whether or not the blockade of TGFß-induced EMT by the PTEN phosphatase activity might be attributed to the unphosphorylated PTEN C-terminus itself has not been fully determined. Furthermore, the lipid phosphatase activity of PTEN is well characterized, whereas the protein phosphatase activity has not been determined. By using lung cancer cells carrying PTEN domain deletions or point mutants, we investigated the role of PTEN protein phosphatase activities on TGFß-induced EMT in lung cancer cells. The unphosphorylated PTEN C-terminus might not directly retain the phosphatase activities and repress TGFß-induced EMT; the modification that keeps the PTEN C-terminus not phosphorylated might enable PTEN to retain the phosphatase activity. PTEN4A with G129E mutation, which lacks lipid phosphatase activity but retains protein phosphatase activity, repressed TGFß-induced EMT. Furthermore, the protein phosphatase activity of PTEN4A depended on an essential association between the C2 and phosphatase domains. These data suggest that the protein phosphatase activity of PTEN with an unphosphorylated C-terminus might be a therapeutic target to negatively regulate TGFß-induced EMT in lung cancer cells.

The Suppressive Effect of Quercetin on Toll-Like Receptor 7-Mediated Activation in Alveolar Macrophages.

Respiratory viral infections that cause chronic airway and lung disease can result in the activation of the innate immune response. Alveolar macrophages (AMs), one of the first lines of defense in the lung, are abundantly located in alveoli and the respiratory tract. Flavonoids found in fruits and vegetables exhibit cytoprotective effects on various cell types. In this study, we investigated the effect of quercetin on activation of AMs that had been exposed to imiquimod, a ligand of Toll-like receptor (TLR) 7. In both a mouse AM cell line (AMJ2-C11 cells) and mouse bronchoalveolar fluid cells, we demonstrated that quercetin attenuated TLR7-induced the expression of TNF-α and IL-6. In AMJ2-C11 cells, quercetin also attenuated the TLR7-induced CD40 expression; attenuated the translocation of p65; induced translocation of Nrf2 from cytosol to nucleus; and induced heme oxygenase (HO)-1 expression. Notably, tin protoporphyrin IX (SnPP), an inhibitor of HO-1, also attenuated TLR7-induced transcription of the TNF-α and IL-6 genes, suggesting that the effect of quercetin is mediated by HO-1. These results suggest that dietary supplementation with quercetin may have efficacy in the treatment of respiratory viral infection.

Protective effects of intratracheally administered quercetin on lipopolysaccharide-induced acute lung injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) can result in a life-threatening form of respiratory failure, and established, effective pharmacotherapies are therefore urgently required. Quercetin is one of the most common flavonoids found in fruits and vegetables, and has potent anti-inflammatory and anti-oxidant activities. Quercetin has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO)-1. Here, we investigated whether the intratracheal administration of quercetin could suppress lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as well as the involvement of HO-1 in quercetin's suppressive effects. METHODS: Mouse model of ALI were established by challenging intratracheally LPS. The wet lung-to-body weight ratio, matrix metalloproteinase (MMP)-9 activities, and pro-inflammatory cytokine productions, including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in bronchoalveolar lavage fluid (BALF) were examined in ALI mice with or without quercetin pretreatment. We also examined the effects of quercetin on LPS stimulation in the mouse alveolar macrophage cell line, AMJ2-C11 cells. RESULTS: Intratracheal administration of quercetin decreased the wet lung-to-body weight ratio. Moreover, quercetin decreased MMP-9 activity and the production of pro-inflammatory cytokines in BALF cells activated by LPS in advance. We determined the expression of quercetin-induced HO-1 in mouse lung, e.g., alveolar macrophages (AMs), alveolar and bronchial epithelial cells. When AMJ2-C11 cells were cultured with quercetin, a marked suppression of LPS-induced pro-inflammatory cytokine production was observed. The cytoprotective effects were attenuated by the addition of the HO-1 inhibitor SnPP. These results indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. CONCLUSIONS: Our findings indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. Intratracheal administration of quercetin will lead to new supportive strategies for cytoprotection in these serious lung conditions.

Degranulation and expression of cytokines were modulated by diazinon in activated mast cells.

Diazinon is an organophosphorus (OP) insecticides used in agriculture, home gardening and indoor pest control in Japan. It can activate macrophages and induce pro-inflammatory responses and has been reported to cause airway hyper-reactivity, suggesting the possibility of asthma exacerbation from exposure to OP insecticides. Despite the correlation between insecticide use and the pathogenesis of allergic diseases, there have been no reports on the effects of diazinon on mast cell function. Therefore, in this study, we investigated the effects of diazinon on mast cell function in rat basophilic leukemia (RBL)-2H3 cells. Surprisingly, we found that diazinon inhibited mast cell activation, although the degree of inhibition varied with concentration. Diazinon induced reactive oxygen species (ROS) generation and HO-1 expression at a concentration of 150 µM without affecting cell viability. Diazinon inhibited A23187-mediated degranulation and Tnf and Il4 expression in RBL-2H3 cells but did not affect calcium influx. Suppression of degranulation by diazinon was reversed when the culture supernatant was removed. As a signaling event downstream of calcium influx, diazinon inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) induced by A23187, whereas the phosphorylation of p38 had little effect. IgE cross-linking-mediated degranulation as well as the induction of Tnf and IL4 expression was significantly inhibited by diazinon, while diazinon had little effect on calcium influx. In conclusion, diazinon inhibited mast cell activation, including degranulation and cytokine expression. When evaluating the in vivo effects of diazinon, its potential to inhibit mast cell activation should be considered in the pathophysiology and development of allergic diseases in terms of basic and clinical aspects, respectively, although the effect of diazinon varies depending on the cell type.

Detection of loci for allergic asthma using SMXA recombinant inbred strains of mice.

Asthma is regarded as a multifactorial inflammatory disorder arising as a result of inappropriate immune responses in genetically susceptible individuals to common environmental antigens. However, the precise molecular basis is unknown. To identify genes for susceptibility to three asthma-related traits, airway hyperresponsiveness (AHR), eosinophil infiltration, and allergen-specific serum IgE levels, we conducted a genetic analysis using SMXA recombinant inbred (RI) strains of mice. Quantitative trait locus analysis detected a significant locus for AHR on chromosome 17. For eosinophil infiltration, significant loci were detected on chromosomes 9 and 16. Although we could not detect any significant loci for allergen-specific serum IgE, analysis of consomic strains showed that chromosomes 17 and 19 carried genes that affected this trait. We detected genetic susceptibility loci that separately regulated the three asthma-related phenotypes. Our results suggested that different genetic mechanisms regulate these asthma-related phenotypes. Genetic analyses using murine RI and consomic strains enhance understanding of the molecular mechanisms of asthma in human.

Aqueous fraction of Sauropus androgynus might be responsible for bronchiolitis obliterans.

BACKGROUND AND OBJECTIVE: Bronchiolitis obliterans (BO) has been reported to develop following ingestion of Sauropus androgynus (SA), a leafy shrub distributed in Southeast Asia. Little is known about direct effects of SA on airway resident cells or haematopoietic cells in vitro. Identification of the SA component responsible for the development of BO would be an important key to elucidate its mechanism. We sought to elucidate the direct effects of SA on airway resident cells or haematopoietic cells and identify the SA element responsible for the pathogenesis of BO. METHODS: SA dry powder was partitioned into fractions by solvent extraction. Human and murine monocytic cells, epithelial cells and endothelial cells were cultured with SA solution or fractions eluted from SA. We also investigated the effect of SA in vivo using a murine BO syndrome (BOS) model. RESULTS: The aqueous fraction of SA induced significant increases of inflammatory cytokine and chemokine production from monocytic lineage cells. This fraction also induced significant apoptosis of endothelial cells and enhanced intraluminal obstructive fibrosis in allogeneic trachea allograft in the murine BOS model. We found individual differences in tumour necrosis factor α (TNF-α) production from monocytes of healthy controls stimulated by this aqueous fraction of SA, whereas it induced high-level TNF-α production from monocytes of patients with SA-induced BO. CONCLUSIONS: These results suggest that an aqueous fraction of SA may be responsible for the pathogenesis of BO.

[Immunoreaction and blood transfusion--chairmen's introductory remarks].

Although blood transfusion is an extremely important therapeutic procedure that usually proceeds without complications, there are some risks associated with donated blood. Investigations into the causes of transfusion reactions and their prevention are important issues for transfusion therapy. In addition to nucleic acid amplification testing (NAT) for infectious diseases and the irradiation of blood to prevent post-transfusion GVHD, prestorage leukocyte reduction and diversion of the first part of the donation of blood were recently introduced into transfusion therapy. This symposium, entitled "Immunoreaction and blood transfusion", reviewed the immune responses associated with blood transfusion, which is probably the most frequent medical procedure performed in allogeneic organ transplantation, with four themes provided by the four featured invited speakers: transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO), high-dose intravenous immunoglobulin therapy for chronic inflammatory demyelinating polyradiculoneuropathy, transfusion-transmitted infectious disease surveillance, and transfusion-related immunomodulation.

Decrease in cholesterol in the cell membrane is essential for Nrf2 activation by quercetin.

Quercetin is a flavonoid with various cytoprotective effects. We previously reported that quercetin exerts anti-allergic, anti-oxidative, and anti-fibrotic activities via the induction of heme oxygenase (HO)-1. However, the mechanisms by which quercetin induces HO-1 to exhibit cytoprotective effects are poorly understood. We focused on its action on the cell membrane, which is the first part of the cell to interact with the extracellular environment. The cell membrane contains lipid rafts and caveolae, which play important roles in cellular signaling. A recent study showed that nuclear factor E2-related factor 2 (Nrf2), a transcription factor regulating anti-oxidative enzymes including HO-1, interacts with caveolin-1 (Cav-1), a component of caveolae, to regulate cellular anti-oxidative capacity. In this study, we investigated the changes in the cell membrane that leads to the induction of HO-1 by quercetin. Quercetin decreased the amount of cholesterol in the raft fractions, which in turn promoted the induction of HO-1. It also changed the composition of the lipid rafts and decreased and increased the expression of Cav-1 in the raft and non-raft fractions, respectively. Nrf2, which was localized in the cell membrane under resting conditions, was translocated along with Cav-1 to the nucleus after exposure to quercetin. These findings indicate for the first time that the HO-1-dependent cytoprotective effects of quercetin are mediated by the structural changes in lipid rafts brought about by decreasing the amount of cholesterol in the cell membrane, which thereby results in the translocation of the Cav-1-Nrf2 complex to the nucleus and induces the expression of HO-1.

Differential modulation of surfactant protein D under acute and persistent hypoxia in acute lung injury.

Hypoxia contributes to the development of fibrosis with epithelial-mesenchymal transition (EMT) via stimulation of hypoxia-inducible factor 1α (HIF-1α) and de novo twist expression. Although hypoxemia is associated with increasing levels of surfactant protein D (SP-D) in acute lung injury (ALI), the longitudinal effects of hypoxia on SP-D expression in lung tissue injury/fibrosis have not been fully evaluated. Here, the involvement of hypoxia and SP-D modulation was evaluated in a model of bleomycin-induced lung injury. We also investigated the molecular mechanisms by which hypoxia might modulate SP-D expression in alveolar cells, by using a doxycycline (Dox)-dependent HIF-1α expression system. Tissue hypoxia and altered SP-D levels were present in bleomycin-induced fibrotic lesions. Acute hypoxia induced SP-D expression, supported by the finding that Dox-induced expression of HIF-1α increased SP-D expression. In contrast, persistent hypoxia repressed SP-D expression coupled with an EMT phenotype and twist expression. Long-term expression of HIF-1α caused SP-D repression with twist expression. Ectopic twist expression repressed SP-D expression. The longitudinal observation of hypoxia and SP-D levels in ALI in vivo was supported by the finding that HIF-1α expression stabilized by acute hypoxia induced increasing SP-D expression in alveolar cells, whereas persistent hypoxia induced de novo twist expression in these cells, causing repression of SP-D and acquisition of an EMT phenotype. Thus this is the first study to demonstrate the molecular mechanisms, in which SP-D expression under acute and persistent hypoxia in acute lung injury might be differentially modulated by stabilized HIF-1α expression and de novo twist expression.

Quercetin protects against pulmonary oxidant stress via heme oxygenase-1 induction in lung epithelial cells.

The lung is a primary target for oxygen toxicity because of its constant exposure to high oxygen levels and environmental oxidants. Quercetin is one of the most commonly found dietary flavonoids, and it provides cytoprotective actions via activation of specific transcriptional factors and upregulation of endogenous defensive pathways. In the present study, we showed that quercetin increased the levels of heme oxygenase (HO)-1 expression and protected against hydrogen peroxide (H(2)O(2))-induced cytotoxicity in lung epithelial cell lines. Quercetin suppressed H(2)O(2)-induced apoptotic events, including hypodiploid cells, activation of caspase 3 enzyme activity and lactate dehydrogenase release. This cytoprotective effect was attenuated by the addition of the HO inhibitor, tin protoporphyrin IX. In addition, the end products of heme metabolites catalyzed by HO-1, carbon monoxide and bilirubin, protect against H(2)O(2)-induced cytotoxicity in LA-4 cells. Quercetin may well be one of the promising substances to attenuate oxidative epithelial cell injury in lung inflammation.