Epidemiological study using IgM and IgG antibody titers against SARS-CoV-2 in The University of Tokyo, Japan (UT-CATS).

INTRODUCTION: The worldwide pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to date. Given that some of the patients with coronavirus disease 2019 (COVID-19) are asymptomatic, antibody tests are useful to determine whether there is a previous infection with SARS-CoV-2. In this study, we measured IgM and IgG antibody titers against SARS-CoV-2 in the serum of asymptomatic healthy subjects in The University of Tokyo, Japan. METHODS: From June 2020, we recruited participants, who were students, staff, and faculty members of The University of Tokyo in the project named The University of Tokyo COVID-19 Antibody Titer Survey (UT-CATS). Following blood sample collection, participants were required to answer an online questionnaire about their social and health information. We measured IgG and IgM titers against SARS-CoV-2 using iFlash-SARS-CoV-2 IgM and IgG detection kit which applies a chemiluminescent immunoassay (CLIA) for the qualitative detection. RESULTS: There were 6609 volunteers in this study. After setting the cutoff value at 10 AU/mL, 32 (0.48%) were positive for IgG and 16 (0.24%) for IgM. Of six participants with a history of COVID-19, five were positive for IgG, whereas all were negative for IgM. The median titer of IgG was 0.40 AU/mL and 0.39 AU/mL for IgM. Both IgG and IgM titers were affected by gender, age, smoking status, and comorbidities. CONCLUSIONS: Positive rates of IgG and IgM titers were relatively low in our university. Serum levels of these antibodies were affected by several factors, which might affect the clinical course of COVID-19.

Genome-wide integration of microRNA and transcriptomic profiles of differentiating human alveolar epithelial cells.

The alveolar epithelium is comprised of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells, the latter being capable of self-renewal and transdifferentiation into AT1 cells for normal maintenance and restoration of epithelial integrity following injury. MicroRNAs (miRNAs) are critical regulators of several biological processes, including cell differentiation; however, their role in establishment/maintenance of cellular identity in adult alveolar epithelium is not well understood. To investigate this question, we performed genome-wide analysis of sequential changes in miRNA and gene expression profiles using a well-established model in which human AT2 (hAT2) cells transdifferentiate into AT1-like cells over time in culture that recapitulates many aspects of transdifferentiation in vivo. We defined three phases of miRNA expression during the transdifferentiation process as "early," "late," and "consistently" changed, which were further subclassified as up- or downregulated. miRNAs with altered expression at all time points during transdifferentiation were the largest subgroup, suggesting the need for consistent regulation of signaling pathways to mediate this process. Target prediction analysis and integration with previously published gene expression data identified glucocorticoid signaling as the top pathway regulated by miRNAs. Serum/glucocorticoid-regulated kinase 1 (SGK1) emerged as a central regulatory factor, whose downregulation correlated temporally with gain of hsa-miR-424 and hsa-miR-503 expression. Functional validation demonstrated specific targeting of these miRNAs to the 3'-untranslated region of SGK1. These data demonstrate the time-related contribution of miRNAs to the alveolar transdifferentiation process and suggest that inhibition of glucocorticoid signaling is necessary to achieve the AT1-like cell phenotype.

Claudin-18-mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis.

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Cross-Species Transcriptome Profiling Identifies New Alveolar Epithelial Type I Cell-Specific Genes.

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type-specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells' roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription-polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell-specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases.

Region-specific role for Pten in maintenance of epithelial phenotype and integrity.

Previous studies have demonstrated resistance to naphthalene-induced injury in proximal airways of mice with lung epithelial-specific deletion of the tumor-suppressor gene Pten, attributed to increased proliferation of airway progenitors. We tested effects of Pten loss following bleomycin injury, a model typically used to study distal lung epithelial injury, in conditional PtenSFTPC-cre knockout mice. Pten-deficient airway epithelium exhibited marked hyperplasia, particularly in small bronchioles and at bronchoalveolar duct junctions, with reduced E-cadherin and ß-catenin expression between cells toward the luminal aspect of the hyperplastic epithelium. Bronchiolar epithelial and alveolar epithelial type II (AT2) cells in PtenSFTPC-cre mice showed decreased expression of epithelial markers and increased expression of mesenchymal markers, suggesting at least partial epithelial-mesenchymal transition at baseline. Surprisingly, and in contrast to previous studies, mutant mice were exquisitely sensitive to bleomycin, manifesting rapid weight loss, respiratory distress, increased early mortality (by day 5), and reduced dynamic lung compliance. This was accompanied by sloughing of the hyperplastic airway epithelium with occlusion of small bronchioles by cellular debris, without evidence of increased parenchymal lung injury. Increased airway epithelial cell apoptosis due to loss of antioxidant defenses, reflected by decreased expression of superoxide dismutase 3, in combination with deficient intercellular adhesion, likely predisposed to airway sloughing in knockout mice. These findings demonstrate an important role for Pten in maintenance of airway epithelial phenotype integrity and indicate that responses to Pten deletion in respiratory epithelium following acute lung injury are highly context-dependent and region-specific.

The chimeric transcript RUNX1-GLRX5: a biomarker for good postoperative prognosis in Stage IA non-small-cell lung cancer.

Stage IA non-small-cell lung cancer cases have been recognized as having a low risk of relapse; however, occasionally, relapse may occur. To predict clinical outcome in Stage IA non-small-cell lung cancer patients, we searched for chimeric transcripts that can be used as biomarkers and identified a novel chimeric transcript, RUNX1-GLRX5, comprising RUNX1, a transcription factor, and GLRX5. This chimera was detected in approximately half of the investigated Stage IA non-small-cell lung cancer patients (44/104 cases, 42.3%). Although there was no significant difference in the overall survival rate between RUNX1-GLRX5-positive and -negative cases (P = 0.088), a significantly lower relapse rate was observed in the RUNX1-GLRX5-positive cases (P = 0.039), indicating that this chimera can be used as a biomarker for good prognosis in Stage IA patients. Detection of the RUNX1-GLRX5 chimeric transcript may therefore be useful for the determination of a postoperative treatment plan for Stage IA non-small-cell lung cancer patients.

Histone methylation-mediated silencing of miR-139 enhances invasion of non-small-cell lung cancer.

MicroRNA expression is frequently altered in human cancers, and some microRNAs act as oncogenes or tumor suppressors. MiR-139-5p (denoted thereafter as miR-139) has recently been reported to function as a tumor suppressor in several types of human cancer (hepatocellular carcinoma, colorectal cancer, breast cancer, and gastric cancer), but its function in non-small-cell lung cancer (NSCLC) and the mechanism of its suppression have not been studied in detail. MiR-139 was suppressed frequently in primary NSCLCs. MiR-139 is located within the intron of PDE2A and its expression was significantly correlated with the expression of PDE2A. A chromatin immunoprecipitation assay revealed that miR-139 was epigenetically silenced by histone H3 lysine 27 trimethylation (H3K27me3) of its host gene PDE2A and this process was independent of promoter DNA methylation. Pharmacological inhibition of both histone methylation and deacetylation-induced miR-139 with its host gene PDE2A. Ectopic expression of miR-139 in lung cancer cell lines did not affect the proliferation nor the migration but significantly suppressed the invasion through the extracellular matrix. In primary NSCLCs, decreased expression of miR-139 was significantly associated with distant lymph node metastasis and histological invasiveness (lymphatic invasion and vascular invasion) on both univariate and multivariate analyses. Collectively, these results suggest that H3K27me3-mediated silencing of miR-139 enhances an invasive and metastatic phenotype of NSCLC.

Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3.

Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation.

Clinical features of 280 hospitalized patients with lymphangioleiomyomatosis in Japan.

BACKGROUND AND OBJECTIVE: Lymphangioleiomyomatosis (LAM) is rare, but potentially life threatening owing to respiratory failure. However, knowledge is limited about the condition of hospitalized LAM patients. The objectives of this study were to investigate patient characteristics, comorbidities and causes of death among hospitalized LAM patients in Japan. METHODS: Using a national inpatient database in Japan, information on 280 LAM patients hospitalized between July 2010 and March 2013 was retrospectively collected. We divided the 280 patients into three groups according to their status regarding lung transplantation. RESULTS: For the study period, we identified 32 patients who had undergone lung transplantation ('after-transplantation' group), 12 patients admitted for lung transplantation ('for-transplantation') and 236 patients who had not undergone transplantation ('no-transplantation'). Although the clinical features of LAM patients in the 'no-transplantation' group were similar to previously reported findings, patients hospitalized in connection with transplantation showed the following: the activities of daily living score using the Barthel Index in the 'after-transplantation' group (89.4) was significantly higher than in the 'for-transplantation' group (64.6); the mortality rates in the after-transplantation group (3.1%) were significantly lower than in the for-transplantation group (25%). The most frequent comorbidity was pneumothorax, followed by respiratory failure and angiomyolipoma, although there was no significant difference in the prevalence among the three groups. CONCLUSIONS: We determined the clinical features, comorbidities and fatalities in hospitalized LAM patients. Patients with LAM after transplantation had higher activities of daily living scores than those before transplantation, which suggests that lung transplantation may improve activities of daily living.

Factors affecting mortality following emergency admission for chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide. Patients with COPD frequently have systemic comorbidities that often require unscheduled hospitalization for exacerbation and deterioration of physical conditions, and can have a poor prognosis. We verified factors affecting patients' short-term mortality, using a national inpatient database in Japan. METHODS: We retrospectively collected data for COPD patients (age: >40 years) with emergency admission between July 2010 and March 2013, using the Diagnosis Procedure Combination database. We performed multivariate logistic analyses fitted with a generalized estimating equation to assess factors associated with all-cause in-hospital mortality. RESULTS: A total of 177,207 patients (mean age: 77.5 years; males: 72.9%) were identified. All-cause in-hospital death occurred in 23,614 patients (13.7%). Higher mortality was associated with older age, male sex, lower body mass index, more severe dyspnea, lower level of consciousness, and worse activities of daily life. Higher mortality was also associated with comorbid conditions, including bacterial pneumonia, aspiration pneumonia, interstitial pneumonitis, pulmonary embolism, respiratory failure, lung cancer, heart failure, cerebral infarction, liver cirrhosis, and chronic renal failure. CONCLUSIONS: Our study demonstrated that all-cause in-hospital mortality in patients with COPD who required emergency hospitalization was associated with deteriorated general conditions and comorbidities at admission. Physicians should take into account these prognostic factors to choose better treatment options for COPD patients.

Detection of novel paraja ring finger 2-fer tyrosine kinase mRNA chimeras is associated with poor postoperative prognosis in non-small cell lung cancer.

Previously, we reported that the overexpression of fer tyrosine kinase (FER), a non-receptor tyrosine kinase, is correlated with poor postoperative prognosis and cancer-cell survival in non-small cell lung cancer (NSCLC). In the present study, we further analyzed FER-overexpressed NSCLC cases and identified various patterns of chimeric mRNAs, composed of paraja ring finger 2 (PJA2) and FER. We detected no genomic rearrangements between PJA2 and FER and attributed these chimeric mRNAs to alterations at the transcriptome level: i.e., trans-splicing. Several chimeric patterns were detected concurrently in each patient, and the pattern sets varied among patients, although the pattern in which PJA2 exon 1 was fused to FER exon 3 (designated as Pe1-Fe3 mRNA) was detected constantly. Therefore, in a wide screening for PJA2-FER mRNAs in NSCLC, we focused on this chimeric pattern as a representative chimera. In analyses of 167 NSCLC samples, Pe1-Fe3 mRNA was identified in about 10% of the patients, and the presence of chimeric mRNA was significantly correlated with a high expression level of parental FER mRNA. Furthermore, we found that the detection of Pe1-Fe3 mRNA was correlated with poor postoperative survival periods in NSCLC, consistent with a previous finding in which FER overexpression was correlated with poor postoperative prognosis in NSCLC. This report is the first to suggest a correlation between chimeric mRNA and the expression level of parental mRNA. Furthermore, our findings may be clinically beneficial, suggesting that PJA2-FER mRNAs might serve as a novel prognostic biomarker in NSCLC.

FER overexpression is associated with poor postoperative prognosis and cancer-cell survival in non-small cell lung cancer.

Here, we show that overexpression of fer tyrosine kinase (FER), a non-receptor tyrosine kinase, predicts poor postoperative outcome and might be involved in cancer-cell survival in non-small cell lung cancer (NSCLC). Systematic screening using in silico analyses and quantitative RT-PCR revealed that FER was overexpressed in about 10% of NSCLC patients. Evaluation of FER expression using immunohistochemistry (IHC) on tissue microarrays was consistent with the mRNA level detected using quantitative RT-PCR. In analyses of 135 NSCLC patients who had undergone potential curative resection, we found that FER overexpression detected using IHC had no association with clinicopathological features such as age, sex, smoking history, histological type, disease stage, T factor, N factor, adjuvant chemotherapy history, or EGFR mutation, but was correlated with poor postoperative survival periods. A multivariate Cox regression analysis showed that this prognostic impact was independent of other clinicopathological features. In functional analyses of FER in vitro, FER exhibited a transforming activity, suggesting that it possesses oncogenic functions. We also found that human lung cancer NCI-H661 cells, which exhibited FER-outlier expression, were led to apoptosis by the knockdown of FER using RNA interference. FER overexpression might serve as a prognostic biomarker and be involved in cancer-cell survival in NSCLC.

Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer.

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non-small-cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, whereas mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir-34b and mir-126 decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.

Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE).

Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.

[Case of sarcoidosis with squamous cell carcinoma which originated from solitary bronchial papilloma].

A 60-year-old man was given a clinical diagnosis of sarcoidosis, with enlarged mediastinal and hilar lymphadenopathy by chest CT, high levels of angiotensin-converting enzyme, and gallium scintigraphy findings. After 2 years follow-up, chest CT showed that only the right superior lobe bronchial lymph node had enlarged, occluding the right B1 bronchus, but other enlarged lymph nodes had not changed in size. We performed bronchoscopy to evaluate the occlusion of the right B1 bronchus, and recognized a polypoid lesion. Transbronchial tumor biopsy specimens revealed squamous cell lung carcinoma. A right upper lobectomy and drainage of the hilar and mediastinal lymph regions were performed. Histopathological examination revealed the coexistence of squamous cell carcinoma with many non-caseating epithelioid cell granulomas in all hilar and mediastinal drainage lymph nodes, but no metastasis. Non-caseating epithelioid cell granulomas were also seen in the lung interstitium. Histopathological examination suggested that the squamous cell carcinoma originated from a solitary bronchial papilloma. A diagnosis of lung cancer complicated with sarcoidosis was difficult by clinical imaging alone, including FDG-PET/CT. This suggests the importance of bronchoscopic examination, if a clinical course of the disease appears to be different from the usual course. This was a rare case of squamous cell carcinoma which originated from a solitary bronchial papilloma.

Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis.

DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 degrees C or 65 degrees C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

Mucosa associated lymphoid tissue (MALT) lymphoma of the thymus with trisomy 18.

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) of the thymus is rare and little is known about its karyotype abnormality. MALT lymphoma in general shows a good prognosis, but some reports suggest that the presence of trisomy 18 predicts recurrence. Here, we report a patient with MALT lymphoma of the thymus and the left parotid gland accompanied by Sjogren's syndrome. The karyotype analysis revealed that this is the first case of thymic MALT lymphoma with trisomy 18, which we believe is worth reporting. We also review cases with thymic MALT lymphoma previously reported in the literature.