Yasuko Yoshioka

VEGF regulates the proliferation of acid-exposed alveolar lining epithelial cells

Background: Acid induced pneumonitis resulting in acute respiratory distress syndrome (ARDS) is characterised by increased alveolar permeability and accumulation of neutrophils. It is hypothesised that vascular endothelial growth factor (VEGF) is involved in the development of lung oedema. Furthermore, lower levels of VEGF are detected in bronchoalveolar lavage fluid from patients with ARDS than from non-ARDS patients. We hypothesised that VEGF acts cytoprotectively and have investigated this possibility in vitro with A549 cells. Methods: A549 cells were incubated in 24 well culture dishes 24 hours before exposure to acid, then incubated with serum free medium containing various concentrations of HCl for 30 minutes at 37°C in 5% CO2. The acidified medium was changed to normal complete medium; at specified incubation periods the supernatants were collected and the VEGF concentration measured and the number of adherent cells counted. Results: Proliferation of A549 cells and VEGF production were suppressed for at least 48 hours in HCl at a concentration of 50 mM. Restoration of cellular proliferation occurred following exogenous administration of VEGF (concentration of 1–250 ng/ml) and was inhibited by co-incubation with neutralising anti-VEGF antibody, indicating an interaction between VEGF molecules and A549 cells. Control cells were not influenced by administration of exogenous VEGF or anti-VEGF antibody. Treatment with neutralising anti-VEGF receptor (VEGFR) antibodies against VEGFR-1 and VEGFR-2 suppressed proliferation of acid exposed A549 cells but had no effect on control cells. Conclusions: Exogenous VEGF interacts with VEGFR-1 and VEGFR-2 on the surface and regulates the proliferation of injured alveolar lining epithelial cells in an autocrine or paracrine fashion.

Chronic intermittent hypoxia/reoxygenation facilitate amyloid-β generation in mice.

Previous studies have shown a high prevalence of obstructive sleep apnea (OSA) among patients with Alzheimers disease (AD). However, it is poorly assessed whether chronic intermittent hypoxia (CIH), which is a characteristic of OSA, affects the pathophysiology of AD. We aimed to investigate the direct effect of intermittent hypoxia (IH) in pathophysiology of AD in vivo and in vitro. In vivo, 15 male triple transgenic AD mice were exposed to either CIH or normoxia (5% O2 and 21% O2 every 10 min, 8 h/day for 4 weeks). Amyloid-β (Aβ) profile, cognitive brain function, and brain pathology were evaluated. In vitro, human neuroblastoma SH-SY5Y cells stably expressing wild-type amyloid-β protein precursor were exposed to either IH (8 cycles of 1% O2 for 10 min followed by 21% O2 for 20 min) or normoxia. The Aβ profile in the conditioned medium was analyzed. CIH significantly increased levels of Aβ42 but not Aβ40 in the brains of mice without the increase in hypoxia-inducible factor 1, alpha subunit (HIF-1α) expression. Furthermore, CIH significantly increased intracellular Aβ in the brain cortex. There were no significant changes in cognitive function. IH significantly increased levels of Aβ42 in the medium of SH-SY5Y cells without the increase in the HIF-1α expression. CIH directly and selectively increased levels of Aβ42 in the AD model. Our results suggest that OSA would aggravate AD. Early detection and intervention of OSA in AD may help to alleviate the progression of the disease.

Ultrasonographic evaluation of the diaphragm in patients with amyotrophic lateral sclerosis.

Abstract:  Real‐time diaphragmatic movement was evaluated with ultrasonography in three patients with amyotrophic lateral sclerosis (ALS). The initial complaint of two patients was weakness of the extremities followed by dyspnoea later in the disease course, while the third patient had dyspnoea as the initial symptom. Ultrasonographic analyses revealed that the contractile function of the diaphragm was not maintained during maximum inspiratory effort, with unsatisfactory diaphragmatic excursion and no change in diaphragmatic thickness during respiration, indicating diaphragmatic paralysis. Ultrasonography may be useful for the diagnosis and follow up of diaphragmatic involvement with amyotrophic lateral sclerosis and other motor‐neuron diseases.

Diagnostic significance of cerebrospinal fluid EGFR mutation analysis for leptomeningeal metastasis in non-small-cell lung cancer patients harboring an active EGFR mutation following gefitinib therapy failure

BACKGROUND Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been successfully used to treat patients with non-small cell lung cancer (NSCLC) harboring EGFR mutations. However, despite an initial excellent response, recurrence within one or two years is common. Diagnosis and treatment of leptomeningeal metastasis (LM), a form of NSCLC recurrence, remains particularly difficult. Here, we analyzed the EGFR mutation status of cerebrospinal fluid (CSF) directly using real-time polymerase chain reaction (PCR) and evaluated the efficacy of therapy with erlotinib, an EGFR TKI. PATIENTS AND METHODS Seven NSCLC patients harboring activating EGFR mutations who had developed LM during or after therapy with gefitinib, an EGFR TKI, were retrospectively analyzed. CSF was obtained and subjected to cytological examination and EGFR mutation analysis, including detection of the resistance-associated T790M mutation, using real-time PCR. RESULTS In all seven cases, the EGFR mutation detected in the CSF was the same as that detected in the primary tumor (sensitivity, 100%). Conversely, cytology results were positive in only two patients (sensitivity, 28.6%). No additional T790M mutations were detected. Erlotinib was efficacious in all cases, and improved performance status was achieved for five of the seven patients. The effect of erlotinib treatment was temporary, however, with time to treatment failure (TTF) ranging from 29 to 278 days (median, 65 days) and the interval between commencement of erlotinib treatment and death ranging from 45 to 347 days (median, 168 days). CONCLUSIONS Analysis of EGFR mutations in CSF using a highly sensitive real-time PCR assay is a potentially powerful diagnostic method for LM.

Inflammatory response and cathepsins in silica‐exposed Hermansky–Pudlak syndrome model pale ear mice

Hermansky–Pudlak syndrome (HPS) is a hereditary disorder involving the sorting processes of intracellular organelles such as lysosomes of reticuloendothelial cells. Pale ear (ep) mouse is known to have the HPS1 gene mutation, which is seen in patients with HPS and pulmonary fibrosis. As pulmonary fibrosis is not spontaneously observed in ep mice, we hypothesized that external stimuli are necessary for the genetic predisposition of its development. We used silica as the external stimulus to induce the alveolar macrophage‐mediated inflammatory response and evaluated the pathological changes of the lung and biochemical analysis of collagenolytic lysosomal enzymes cathepsins L and B in ep mice. Treatment with silica induced the following: persistent accumulation of activated macrophages; delayed clearance of silica from alveolar spaces; and increased collagen fibers in alveolar tissues, which were shown with trichrome staining in ep mice. The comparison of bronchoalveolar lavage cells between the naïve ep and control mice revealed: decreased enzymatic activities but increased antigenic levels of cathepsins L and B, resulting in significantly lower ratios of activity to antigen; increased ceroid deposits and cathepsin L antigens in lysosomes; and no abnormal forms of cathepsins were detected. After silica instillation, activities of cathepsin L in the ep mice increased but ratios of activity to antigen were still significantly low. These phenomena induced by silica suggest that external stimuli bring forth fibrogenesis in the animal models or humans that have HPS1 gene mutation.

High prevalence of gene abnormalities in young patients with lung cancer

BACKGROUND Recently, driver oncogenes in adenocarcinoma of the lung were identified, and several molecular target agents were introduced in the clinical setting. However, there are few reports on the frequency of gene abnormalities in young patients with lung cancer. MATERIALS AND METHODS Twelve patients with lung adenocarcinoma aged 40 or younger at Juntendo University Urayasu Hospital or Juntendo University Hospital from July 2004 to March 2010 were analyzed for driver oncogene status including EGFR activating mutation, EML4-ALK fusion gene, and K-ras mutation. RESULTS Four patients showed EGFR gene mutation. Five out of 7 EGFR mutation-negative patients showed positive results for EML4-ALK gene fusion. One case whose EGFR mutation was indeterminate. CONCLUSIONS Driver oncogene including EGFR mutation and EML4-ALK fusion gene was identified in 9 of 12 cases (75%). Examination of gene abnormalities is essential in young patients with non-small cell lung cancer to provide the best treatment.

Increased circulating CD16+ CD14dim monocytes in a patient with pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is characterized by filling of the alveoli with a periodic acid‐Schiff‐positive proteinaceous material. Although the pathogenesis of primary or idiopathic PAP remains unknown, it has been proposed that a deficiency or loss of responsiveness of the monocyte/macrophage lineage to granulocyte–macrophage colony stimulating factor (GM‐CSF) is involved in PAP. Secondary PAP is associated with haematological malignancies, especially in myeloid disorders. Herein, we report on an adult with PAP associated with myelodysplastic syndrome (MDS). The CD16+ CD14dim monocytes comprise 5–10% of circulating monocytes in healthy volunteers. Flow cytometric analysis of the patient in the present study revealed increased CD16+ CD14dim monocytes in the peripheral blood. It has been demonstrated that the expression of CD16 and CD14 is regulated by macrophage colony stimulating factor (M‐CSF) and GM‐CSF. Hence, serum cytokines were analysed in our patient and the concentration of serum GM‐CSF was found to be less than the lower limit of the assay. In addition, serum M‐CSF and granulocyte colony stimulating factor levels were only slightly increased above the normal range. These results suggest that the increase in the CD16+ CD14dim subpopulation in the circulation of our patient indicates another pathogenetic mechanism for secondary PAP, such as hyperresponsiveness of the monocyte/ macrophage lineage to these cytokines.

Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells.

Several recent studies have suggested that cancer stem cells (CSCs) are involved in resistance to gefitinib in non-small cell lung cancer (NSCLC). Oct4, a member of the POU-domain transcription factor family, has been shown to be involved in CSC properties of various cancers. We previously reported that Oct4 and the putative lung CSC marker CD133 were highly expressed in gefitinib-resistant persisters (GRPs) in NSCLC cells, and GRPs exhibited characteristic features of the CSCs phenotype. The aim of this study was to elucidate the role of Oct4 in the resistance to gefitinib in NSCLC cells with an activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9, which express the EGFR exon 19 deletion mutation, were transplanted into NOG mice, and were treated with gefitinib in vivo. After 14-17 days of gefitinib treatment, the tumors still remained; these tumors were referred to as gefitinib-resistant tumors (GRTs). PC9-GRTs showed higher expression of Oct4 and CD133. To investigate the role of Oct4 in the maintenance of gefitinib-resistant lung CSCs, we introduced the Oct4 gene into PC9 and HCC827 cells carrying an activating EGFR mutation by lentiviral infection. Transfection of Oct4 significantly increased CD133-positive GRPs and the number of sphere formation, reflecting the self-renewal activity, of PC9 and HCC827 cells under the high concentration of gefitinib in vitro. Furthermore, Oct4-overexpressing PC9 cells (PC9-Oct4) significantly formed tumors at 1 × 10 cells/injection in NOG mice as compared to control cells. In addition, PC9-Oct4 tumors were more resistant to gefitinib treatment as compared to control cells in vivo. Finally, immunohistochemical analysis revealed that Oct4 was highly expressed in tumor specimens of EGFR-mutant NSCLC patients with acquired resistance to gefitinib. Collectively, these findings suggest that Oct4 plays a pivotal role in the maintenance of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC.

Pancoast's syndrome in a patient with B‐cell lymphoma diagnosed and confirmed with immunoglobulin gene rearrangement

Abstract:  Pancoasts syndrome due to malignant lymphoma is extremely rare. A case of diffuse large B‐cell lymphoma presenting as Pancoasts syndrome is described. A 66‐year‐old man complained of pain and weakness of the right arm, and CXR revealed a right apical lung tumour. Histological findings were consistent with it being a diffuse large cell type lymphoma and Southern blot analysis revealed clonal rearrangement of the immunoglobulin heavy‐chain JH. Thus, the tumour in this patient was diagnosed to be diffuse large B‐cell lymphoma. Malignant lymphoma is an extremely rare cause of Pancoasts syndrome and only five cases have been described. This is the first reported case of Pancoasts syndrome caused by B‐cell lymphoma, which was accurately diagnosed by analysis of gene rearrangement.

Acid exposure potentiates intercellular adhesion molecule-1 and E-cadherin expression on A549 alveolar lining epithelial cells

Recruitment of neutrophils into the alveoli plays a major role in the pathogenesis of acid-induced pneumonitis. Preliminary data suggest that alteration in the expression of cellular adhesion molecules on the airway epithelial cells may play an important role in the recruitment of neutrophils following acid-induced lung injury. The aim of this study was to evaluate the change in the surface expression of intercellular adhesion molecule-1 (ICAM-1), E-cadherin, and vascular cell adhesion molecule -1 (VCAM-1) on acid-exposed A549 alveolar lining epithelial cells by flow cytometry and confocal laser microscopy. Acid exposure changed cell morphology, increased cell adhesion after trypsin-EDTA treatment, and up-regulated the expression of ICAM-1 and E-cadherin but not of VCAM-1. The up-regulation of ICAM-1 expression will induce the dysfunction of epithelial cells with or without accumulation of neutrophils in air spaces. Because the distribution of E-cadherin in acid-exposed A549 cells was at the sites where the cells attached to culture dish but not at the intercellular junctions between adjoining cells, up-regulated expression of E-cadherin will rather result in alterations of epithelial morphology and function of epithelial barrier. In addition, pentoxifylline suppressed the up-regulation of ICAM-1 and E-cadherin expression and may therefore attenuated the airway inflammation in acid-induced pneumonitis.