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INTRODUCTION: Since the first report of a novel coronavirus infection caused by SARS-CoV-2 in late 2019, the infection has spread rapidly and had a significant impact on our lives. In the early stages of the COVID-19 pandemic, there was no adequate testing system in place, despite an urgent need for infection control measures in student dormitories. METHODS: We have been monitoring SARS-CoV-2 in wastewater as part of our infection control efforts in the university facilities since fall 2020. In the four dormitories, absorbent cotton was placed in the drains that the facility wastewater passed through, and samples were collected twice a week and processed by RT-PCR for SARS-CoV-2. The dormitory residents were informed of the monitoring results the next morning. RESULTS: The positivity of residents in the dormitories was highly consistent with the positivity of wastewater. Wastewater was positive in 89 % of cases before any residents were tested and found positive. Facility wastewater monitoring showed sensitivities of 80.4 % and specificities of 87.6 %. No traceable resident-to-resident transmission of infection within the facility was confirmed during the study period. CONCLUSION: Sampling a single wastewater outlet in a building for SARS-CoV-2 PCR can effectively indicate the presence or absence of COVID-19 cases and be very useful for infection control of a facility. This simple and effective monitoring is applicable to future outbreaks of both emerging and re-emerging infectious diseases.
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INTRODUCTION: The usefulness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests in asymptomatic individuals has not been well validated, although they have satisfied sensitivity and specificity in symptomatic patients. In this study, we investigated the significance of IgM and IgG antibody titers against SARS-CoV-2 in the serum of asymptomatic healthy subjects. METHODS: From June 2020, we recruited 10,039 participants to the project named the University of Tokyo COVID-19 Antibody Titer Survey (UT-CATS), and measured iFlash-SARS-CoV-2 IgM and IgG (YHLO IgM and IgG) titers in the collected serum. For the samples with increased IgM or IgG titers, we performed additional measurements using Elecsys Anti-SARS-CoV-2 Ig (Roche total Ig) and Architect SARS-CoV-2 IgG (Abbott IgG) and investigated the reactivity to N, S1, and receptor binding domain (RBD) proteins. RESULTS: After setting the cutoff value at 5 AU/mL, 61 (0.61%) were positive for YHLO IgM and 104 (1.04%) for YHLO IgG. Few samples with elevated YHLO IgM showed reactivity to S1 or RBD proteins, and IgG titers did not increase during the follow-up in any samples. The samples with elevated YHLO IgG consisted of two groups: one reacted to S1 or RBD proteins and the other did not, which was reflected in the results of Roche total Ig. CONCLUSIONS: In SARS-CoV-2 seroepidemiological studies of asymptomatic participants, sufficient attention should be given to the interpretation of the results of YHLO IgM and IgG, and the combined use of YHLO IgG and Roche total Ig might be more reliable.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Voluntários Saudáveis , Humanos , Imunoglobulina G , Imunoglobulina M , Estudos SoroepidemiológicosRESUMO
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.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Estudos Epidemiológicos , Humanos , Imunoglobulina G , Imunoglobulina M , Japão/epidemiologiaRESUMO
A 57-year-old woman was referred to our hospital for investigation of multiple tiny nodules in the lung fields bilaterally on computed tomography (CT). Video-assisted thoracoscopic lung biopsy was performed to diagnose the pulmonary lesions. Histological analysis showed nodular lesions with interstitial proliferation of uniform, round to oval cells with variable widening of the alveolar septa. Immunohistochemically, the cells were positive for EMA, CD56 and the progesterone receptor, but negative for chromogranin and synaptophysin. The diagnosis was "diffuse pulmonary meningotheliomatosis", with multiple diffuse "minute pulmonary meningothelial-like nodules". Diffuse pulmonary meningotheliomatosis should be kept in mind when we encounter small nodular shadows on a CT scan.
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Neoplasias Pulmonares , Biópsia , Diagnóstico Diferencial , Feminino , Humanos , Pulmão , Neoplasias Pulmonares/diagnóstico por imagem , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios XRESUMO
A 50-year-old man was admitted with respiratory failure. Chest X-ray and computed tomography revealed massive left pleural effusion and mediastinal shift. Pleural effusion showed abnormally high amylase levels of 42,600 IU/l and a high protein level of 3.2 g/dl. The serum amylase level was also 42,100 IU/l, and the proportion of pancreatic-type amylase was 88%. We diagnosed the patient with pancreatic effusion. Chest and abdominal enhanced computed tomography and magnetic resonance cholangiopancreatography revealed no pancreaticopleural fistula. He underwent a thoracoscopic examination that revealed brown pleural effusion as well as fibrin clots and thickness of the pleura. Histologically, there was no malignancy and the cause of pleural effusion was considered to be chronic pancreatitis.
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Doenças Pleurais , Derrame Pleural , Humanos , Masculino , Doenças do Mediastino , Pessoa de Meia-Idade , Pâncreas , Pancreatopatias , Tomografia Computadorizada por Raios XRESUMO
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.
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Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , MicroRNAs/fisiologia , Adulto , Idoso , Proteínas de Ligação ao Cálcio , Carcinoma Pulmonar de Células não Pequenas/patologia , Imunoprecipitação da Cromatina , Metilação de DNA , Família de Proteínas EGF , Fatores de Crescimento Endotelial/genética , Epigênese Genética , Feminino , Inativação Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Invasividade NeoplásicaRESUMO
We investigated whether the CpG island methylation of certain microRNAs was associated with the clinicopathological features and the prognosis of non-small-cell lung cancer. The methylation of mir-152, -9-3, -124-1, -124-2, and -124-3 was analyzed in 96 non-small-cell lung cancer specimens using a combined bisulfite restriction analysis. The median observation period was 49.5 months. The methylation of mir-9-3, -124-2, and -124-3 was individually associated with an advanced T factor independent of age, sex, and smoking habit. Moreover, the methylation of multiple microRNA loci was associated with a poorer progression-free survival in a univariate analysis. Our result enlightens the accumulation of aberrant DNA methylation which occurs in concordance with the tumor progression.
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Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Ilhas de CpG , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Idoso , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Metilação de DNA , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Recidiva Local de NeoplasiaRESUMO
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.
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DNA Complementar/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Biologia Molecular/métodos , DNA Ligases/metabolismo , Primers do DNA/genética , DNA Complementar/genética , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
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.
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Fosfatase Alcalina/química , Ilhas de CpG , Metilação de DNA , DNA/análise , Exodesoxirribonucleases/química , Reação em Cadeia da Polimerase , Animais , Linhagem Celular , DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Temperatura Alta , Humanos , Sulfitos/químicaRESUMO
Epigenetic changes can lead to abnormal expression of genes in cancer, and several genes have been reported to have aberrant promoter DNA methylation in non-small-cell lung cancer (NSCLC). We identified aberrantly methylated genes in NSCLC by combination of in silico and experimental approaches. We first applied bioinformatics, and from microarray datasets, we selected genes with low expression and having functions related to cancer. Next, combined bisulfite restriction analysis was carried out in 10 pooled resected lung cancer tissues to screen for genes that were aberrantly methylated, and the methylation ratio (the fraction of methylated DNA in extracted DNA from a cancer tissue sample) was quantified using quantitative analysis of methylated alleles. We identified 8 methylated genes (ARPC1B, DNAH9, FLRT2, G0S2, IRS2, PKP1, SPOCK1 and UCHL1) previously unreported in NSCLC. Analyses of methylation profiles of 101 resected lung cancer tissue samples revealed quantitatively low methylation in whole, methylation ratios were almost less than 30% even in the methylated samples, and no significant correlation to prognosis after 2 years of follow-up using hierarchical clustering. DNA methylation of G0S2 gene was significantly more frequent in squamous lung cancer (n = 18, mean of methylation ratios: 15%) compared with nonsquamous lung cancer (n = 83, mean of methylation ratios: 2.6%) (Mann-Whitney U test, p < 0.001). DNA methylation of G0S2 can be an important biomarker for squamous lung cancer.
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Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ciclo Celular/genética , Metilação de DNA/genética , Neoplasias Pulmonares/genética , Análise por Conglomerados , Epigênese Genética , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
We recently identified that DNA methylation of the G0S2 gene was significantly more frequent in squamous lung cancer than in non-squamous lung cancer. However, the significance of G0S2 methylation levels on cancer cells is not yet known. We investigated the effect of G0S2 methylation levels on cell growth, mRNA expression, and chromatin structure using squamous lung cancer cell lines and normal human bronchial epithelial cells. DNA methylation and mRNA expression of G0S2 were inversely correlated, and in one of the squamous lung cancer cell lines, LC-1 sq, G0S2 was completely methylated and suppressed. Overexpression of G0S2 in LC-1 sq did not show growth arrest or apoptosis. The G0S2 gene has been reported to be a target gene of all-trans retinoic acid and peroxisome proliferator-activated receptor agonists. We treated LC-1 sq with 5-Aza-2'-deoxycytidine, Trichostatin A, all-trans retinoic acid, Wy 14643, or Pioglitazone either alone or in combination. Only 5-Aza-2'-deoxycytidine restored mRNA expression of G0S2. Chromatin immunoprecipitation revealed that histone H3 lysine 9 was methylated regardless of DNA methylation or mRNA expression. In summary, mRNA expression of G0S2 was regulated mainly by DNA methylation in squamous lung cancer cell lines. When the G0S2 gene was methylated, nuclear receptor agonists could not restore mRNA expression of G0S2 and did not show any additive effect on mRNA expression of G0S2 even after the treatment with 5-Aza-2'-deoxycytidine.
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Proteínas de Ciclo Celular/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias de Células Escamosas/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Humanos , Ácidos Hidroxâmicos/farmacologia , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Pioglitazona , Tiazolidinedionas/farmacologia , Transcrição Gênica , Tretinoína/farmacologiaRESUMO
Infliximab, an anti-TNF-alpha agent, is highly effective against rheumatoid arthritis and Crohn's disease. However, respiratory infection can occur as a complication. We report two cases complicated by respiratory infection following administration of infliximab. The first case, a 67-year-old woman with rheumatoid arthritis, developed pneumocystis pneumonia after three courses of infliximab therapy. The second case, a 31-year-old man with Crohn's disease, developed pulmonary tuberculosis after four courses of infliximab therapy. Respiratory complications associated with anti-TNF therapy include infectious diseases such as pneumocystis pneumonia, tuberculosis, and bacterial pneumonia. They often lead a fulminant course, and early diagnosis is essential. The final report of a survey of the initial 5000 cases with rheumatoid arthritis treated with infliximab in Japan was released in April 2006; pulmonary infectious complications included 22 cases of pneumocystis pneumonia, 14 cases of tuberculosis, and 108 cases of bacterial pneumonia. The growing use of anti-TNF therapy might lead to increasing pulmonary complications. Accumulation of similar cases is expected to elucidate the mechanism of the complications and methods for effective prophylaxis.