Construction and characterization of a hypervesiculation strain of Escherichia coli Nissle 1917.

Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in a host. OMVs secreted by probiotic probiotic strain Escherichia coli Nissle 1917 (EcN) have been reported to induce an immune response. In this study, we aimed to increase the OMV production of EcN. The double gene knockout of mlaE and nlpI was conducted in EcN because the ΔmlaEΔnlpI of experimental strain E. coli K12 showed the highest OMV production in our previous report. The ΔmlaEΔnlpI of EcN showed approximately 8 times higher OMV production compared with the parental (wild-type) strain. Quick-freeze, deep-etch replica electron microscopy revealed that plasmolysis occurred in the elongated ΔmlaEΔnlpI cells and the peptidoglycan (PG) had numerous holes. While these phenomena are similar to the findings for the ΔmlaEΔnlpI of K12, there were more PG holes in the ΔmlaEΔnlpI of EcN than the K12 strain, which were observed not only at the tip of the long axis but also in the whole PG structure. Further analysis clarified that the viability of ΔmlaEΔnlpI of EcN decreased compared with that of the wild-type. Although the amount of PG in ΔmlaEΔnlpI cells was about half of that in wild-type, the components of amino acids in PG did not change in ΔmlaEΔnlpI. Although the viability decreased compared to the wild-type, the ΔmlaEΔnlpI grew in normal culture conditions. The hypervesiculation strain constructed here is expected to be used as an enhanced probiotic strain.

Type A thymoma: a rare cause of neoplastic cardiac tamponade with long-term survival.

BACKGROUND: The prognosis of thymoma with cardiac tamponade is generally poor. Most of the reported thymomas with cardiac tamponade were type B or type AB (mixed thymoma), and cardiac tamponade due to type A thymoma, which has a better prognosis compared to type B thymoma, is extremely rare. CASE PRESENTATION: We encountered a case of cardiac tamponade in a 71-year-old male. He visited our emergency department due to exacerbation of fatigue and dyspnea on exertion that lasted for two weeks. Chest imaging revealed a large amount of pericardial fluid and a contrast-enhanced tumor with calcification in the anterior mediastinum. The patient underwent thoracoscopic tumor biopsy and pathological examinations revealed type A thymoma. In this case, long-term disease-free survival (7.5 years) was achieved by multidisciplinary treatment (preoperative chemotherapy, surgical excision, and postoperative radiation therapy), in accordance with the histological type. CONCLUSIONS: This case indicates that neoplastic cardiac tamponade, even in elderly patients, should not necessarily be regarded as a terminal cancer and requires a systematic investigation for underlying causes.

Diagnosis of visceral pleural invasion using confocal laser endomicroscopy during lung cancer surgery.

BACKGROUND: Visceral pleural invasion (VPI) in lung cancer is a significant prognostic factor; however, it is difficult to diagnose preoperatively or intraoperatively. In this study, we examined the possibility of intraoperative diagnosis of VPI using confocal laser endomicroscopy (CLE). METHODS: Among patients with primary lung cancer who underwent surgery between April 2018 and August 2019, those in whom the tumor was in contact with the pleura on chest computed tomography and whose pleural changes were intraoperatively confirmed were enrolled in this study. In the 35 patients who underwent lung resection (6 cases with visceral pleural infiltration), the area where pleural change was noted was observed and a short video was recorded using CLE. Based on the video images, three evaluators determined the defect ratio (0%, 25%, 50%, 75%, and 100%) of the autofluorescence-positive structure. The area under the receiver operating characteristic curve was used to evaluate the diagnostic performance for VPI. In 15 cases (3 cases with VPI), a validation study was performed for intraoperative VPI according to the cutoff value of the defect ratio of the autofluorescence-positive structure. RESULTS: The areas under the receiver operating characteristic curve for the defect ratio of the autofluorescence-positive structure were 0.86-0.91 for the three readers. Using defect ratio of autofluorescence-positive structure cutoff of ≥50% as predictor of VPI, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 83.3-100.0%, 57.7-73.1%, 35.3-41.7%, 95.0-100.0%, and 75.0-78.1%, respectively, for the three readers. In the validation study, the sensitivity was 100%, the specificity was 83.3%, and the diagnostic accuracy rate was 86.7%. CONCLUSIONS: The diagnosis of VPI through CLE is simple, non-invasive, and has high diagnostic accuracy rates. This method may be applicable for determining surgical procedures.

DNA methylation of GHSR, GNG4, HOXD9 and SALL3 is a common epigenetic alteration in thymic carcinoma.

Thymic epithelial tumors comprise thymoma, thymic carcinoma and neuroendocrine tumors of the thymus. Recent studies have revealed that the incidence of somatic non­synonymous mutations is significantly higher in thymic carcinoma than in thymoma. However, limited information is currently available on epigenetic alterations in these types of cancer. In this study, we thus performed genome­wide screening of aberrantly methylated CpG islands in thymoma and thymic carcinoma using Illumina HumanMethylation450 K BeadChip. We identified 92 CpG islands significantly hypermethylated in thymic carcinoma in relation to thymoma and selected G protein subunit gamma 4 (GNG4), growth hormone secretagogue receptor (GHSR), homeobox D9 (HOXD9) and spalt like transcription factor 3 (SALL3), which are related to cancer. We examined the promoter methylation of 4 genes in 46 thymic epithelial tumors and 20 paired thymus tissues using bisulfite pyrosequencing. Promoter methylation was significantly higher in thymic carcinoma than in thymoma and revealed a high discrimination between thymic carcinoma and thymoma in all 4 genes. Promoter methylation was higher in thymic carcinoma than in the thymus. No significant differences were observed in the promoter methylation of GNG4, HOXD9, or SALL3 between thymoma and the thymus. The promoter methylation of the 4 genes was not significantly higher in advanced­stage tumors than in early­stage tumors in all thymic epithelial tumors. Among the 4 genes, relapse­free survival was significantly worse in tumors with a higher DNA methylation than in those with a lower DNA methylation in all thymic epithelial tumors. Moreover, relapse­free survival was significantly worse in thymomas with a higher DNA methylation of HOXD9 and SALL3 than in those with a lower DNA methylation. On the whole, the findings of this study indicated that the promoter methylation of cancer­related genes was significantly higher in thymic carcinoma than in thymoma and the thymus. This is a common epigenetic alteration of high diagnostic value in thymic carcinoma and may be involved in the carcinogenesis of thymic carcinoma. However, epigenetic alterations in the 3 genes, apart from GHSR, are not involved in the tumorigenesis of thymoma.

Evaluation of the components of mediastinal cystic lesions using imaging techniques.

OBJECTIVE: To identify and differentiate patients with mediastinal cysts from those with cystic tumors requiring surgery. METHODS: A total of 36 patients with mediastinal cystic lesions were enrolled. The patients were separated into two groups based on pathological findings : those with mediastinal cysts (n=23) and those with mediastinal tumors (n=13). The cystic components were measured using imaging parameters including mean computed tomography (CT) value, apparent diffusion coefficient (ADC), T1 signal intensity ratio (T1SIratio), and T2 signal intensity ratio (T2SI-ratio), acquired from magnetic resonance imaging (MRI) ; and standardized maximum uptake value (SUVmax) from18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT). Both groups were statistically compared. RESULTS: Comparative parameters between the cysts and tumors revealed the following ratios : CT value, 40.9?21.2 versus (vs) 24.8?12.9 (p = 0.019) ;SUVmax, 1.18?0.50 vs 4.32?3.52 (p = 0.003) ; ADC, 3.46?0.96 vs 2.68?0.74 (p = 0.022) ; T1SI-ratio, 1.06?0.60 vs 1.35? 0.92 (p = 0.648) ; T2SI-ratio, 5.40?1.80 vs 4.33?1.58 (p = 0.194). However, there was no correlation between FDG uptake and ADC value. CONCLUSIONS: SUVmax from18F-FDG PET/CT and ADC derived from MRI were effective in facilitating preoperative diagnosis to differentiate mediastinal cysts from tumors. However, these examinations may be complementary to one another, not dominant. J. Med. Invest. 66 : 106-111, February, 2019.

Transbronchial Biopsy Using an Ultrathin Bronchoscope Guided by Cone-Beam Computed Tomography and Virtual Bronchoscopic Navigation in the Diagnosis of Pulmonary Nodules.

BACKGROUND: Transbronchial biopsy is a safe diagnostic approach for patients with peripheral pulmonary lesions; however, the diagnostic yield is low. OBJECTIVES: This study was conducted to evaluate the feasibility and diagnostic yield of transbronchial biopsy using the combination of an ultrathin bronchoscope, virtual bronchoscopic navigation (VBN), and cone-beam computed tomography (CBCT). METHODS: Patients with peripheral pulmonary lesions, no >30 mm, with the responsible bronchus, were prospectively included. An ultrathin bronchoscope and biopsy forceps were advanced to the target bronchus under VBN, 2D-fluoroscopy, and CBCT. We categorized the CBCT findings before biopsy into 3 types according to positions of the target lesion and forceps (CBCT target-forceps sign). In type A, the forceps reached the inside of the target lesion. In type C, the forceps could not reach the lesion. When the CBCT findings could not be categorized into either type A or C, the sign was categorized as type B. RESULTS: Although the target lesions were invisible by conventional C-arm fluoroscopy in 29 patients, CBCT visualized all 40 lesions. The overall diagnostic yield was 90.0%, and diagnostic yields for malignant and benign lesions were 92.0 and 86.7%, respectively. Diagnostic yields for CBCT target-forceps sign types A, B, and C were 100, 75.0, and 0%, respectively. Four undiagnosed patients proceeded to other diagnostic procedures based on the CBCT target-forceps sign (type B: n = 2, type C: n = 2) and were correctly diagnosed without delay. CONCLUSIONS: Transbronchial biopsy using an ultrathin bronchoscope guided by CBCT and VBN showed a very high yield in the diagnosis of pulmonary nodules.

Indocyanine green fluorescence imaging for resection of pulmonary metastasis of hepatocellular carcinoma.

BACKGROUND: Indocyanine green (ICG) accumulates in hepatocellular carcinoma (HCC), and tumor fluorescence can be observed under irradiation with near infrared light (NIR). This study investigated the clinical utility of ICG fluorescence imaging during resection of pulmonary metastases of HCC. METHODS: From April 2010 to June 2018, six patients with suspected pulmonary metastasis of HCC were enrolled prospectively. Prior to surgery, all patients underwent the ICG hepatic function test following intravenous administration of ICG (0.5 mg/kg body weight). During surgery, metastatic HCC was identified by observation of ICG fluorescence, allowing assessment of the surgical margin. Tumor fluorescence was also evaluated on cut sections. RESULTS: A total of 11 metastatic HCCs were resected in six patients at nine operations. Eight lesions were removed by wedge resection and 3 lesions were managed by lobectomy. During surgery, tumor fluorescence could be confirmed through the visceral pleura in 6 out of 7 lesions treated by wedge resection, while NIR irradiation was difficult for 1 lesion. For these 6 lesions, the median distance from the tumor to the visceral pleura and the median surgical margin were 0 mm (range, 0-2 mm) and 14 mm (range, 11-17 mm), respectively. When cut sections were examined, all tumors emitted fluorescence. All lesions were histologically confirmed to be metastatic HCC. CONCLUSIONS: In patients with pulmonary metastasis of HCC, ICG fluorescence imaging is useful for identifying the tumor and securing its margin when the lesion is peripheral and wedge resection is planned.

A feasibility study of postoperative adjuvant chemotherapy with fluoropyrimidine S-1 in patients with stage II-IIIA non-small cell lung cancer.

BACKGROUND: Adjuvant chemotherapy with uracil tegafur (UFT) improved survival among patients with completely resected stage I lung adenocarcinoma. S-1, an oral dihydropyrimidine dehydrogenase (DPD)-inhibitory 5-fluorouracil, is a more potent DPD inhibitor than UFT;therefore, we hypothesized that postoperative adjuvant chemotherapy with S-1 would be effective for advanced non-small cell lung cancer (NSCLC). We conducted a feasibility study of S-1 as postoperative adjuvant chemotherapy in patients with curatively resected pathological stage bold I back 10 bold I and bold I back 10 bold I back 20 bold I A NSCLC. METHODS: Adjuvant chemotherapy consisted of 9 courses (4-week administration, 2-week withdrawal) of S-1 at 80-120 mg/body per day. Twenty-four patients with completely resected NSCLC were enrolled in this study from November 2007 through December 2010. The primary endpoint was the rate of completion of the scheduled adjuvant chemotherapy. The secondary endpoints were safety, overall survival, and relapse-free survival. RESULTS: Five patients were censored because of disease recurrence. The planned 9 courses of S-1 were administered to completion in 8 patients. Twelve patients completed more than 70% of the planned courses. Grade 3 adverse reactions, such as elevated total bilirubin (4.2%) and pneumonitis (4.2%), were observed, but there were no Grade 4 adverse reactions. Patients who completed more than 70% of the 9 courses demonstrated better overall survival than those who completed less than 70%. CONCLUSION: Postoperative administration of S-1 may be possible with few severe adverse events as adjuvant chemotherapy for patients with curatively resected pathological stage bold I back 10 bold I -bold I back 10 bold I back 20 bold I A NSCLC. J. Med. Invest. 65:90-95, February, 2018.

Frequent silencing of RASSF1A by DNA methylation in thymic neuroendocrine tumours.

OBJECTIVES: Aberrant methylation of promoter CpG islands (CGIs) of tumour suppressor genes is a common epigenetic mechanism underlying cancer pathogenesis. The methylation patterns of thymic tumours have not been studied in detail since such tumours are rare. Herein, we sought to identify genes that could serve as epigenetic targets for thymic neuroendocrine tumour (NET) therapy. MATERIALS AND METHODS: Genome-wide screening for aberrantly methylated CGIs was performed in three NET samples, seven thymic carcinoma (TC) samples, and eight type-B3 thymoma samples. The methylation status of thymic epithelial tumours (TETs) samples was validated by pyrosequencing in a larger cohort. The expression status was analysed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. RESULTS: We identified a CGI on a novel gene, RASSF1A, which was strongly hypermethylated in NET, but not in thymic carcinoma or B3 thymoma. RASSF1A was identified as a candidate gene statistically and bibliographically, as it showed frequent CGI hypermethylation in NET by genome-wide screening. Pyrosequencing confirmed significant hypermethylation of a RASSF1A CGI in NET. Low-grade NET tissue was more strongly methylated than high-grade NET. Quantitative PCR and immunohistochemical staining revealed that RASSF1A mRNA and protein expression levels were negatively regulated by DNA methylation. CONCLUSIONS: RASSF1A is a tumour suppressor gene epigenetically dysregulated in NET. Aberrant methylation of RASSF1A has been reported in various tumours, but this is the first report of RASSF1A hypermethylation in TETs. RASSF1A may represent an epigenetic therapeutic target in thymic NET.

[Malignant Pleural Mesothelioma Presenting Refractory Pneumothorax Successfully Treated by Intrapleural Administration of Diluted Fibrin Glue;Report of a Case].

Malignant pleural mesothelioma sometimes accompanies intractable neumothorax due to the visceral pleural invasion of the tumor. A 68-years-old man was found to have massive pleural effusion and pleural mass combined with pneumothorax by computed tomography(CT). CT guided biopsy revealed the mass to be malignant pleural mesothelioma. Since continuous air leakage was observed by chest drainage, pleurodesis by OK-432 twice and bronchial occlusion by Endobronchial Watanabe Spigot (EWS)were performed. But air leakage continued, and the surgery was performed, however the treatment failed to stop the air leakage. Finally, the intrapleural administration of diluted fibrin glue was challenged and the air leakage stopped immediately after the treatment.

Disseminated tuberculosis after pregnancy progressed to paradoxical response to the treatment: report of two cases.

BACKGROUND: Early postpartum women are more likely to develop tuberculosis than nonpregnant women mainly due to immune reconstitution after delivery. Paradoxical response (PR) during antituberculosis treatment also arises via recovery from immunosuppression. However, no study focused on PR during antituberculosis treatment in a postpartum patient has been reported. CASE PRESENTATION: We present two sequential cases (Patient 1: 26-year-old; Patient 2: 29-year-old) of postpartum tuberculosis with pulmonary and extrapulmonary lesions (Patient 1: peritonitis; Patient 2: psoas abscess secondary to spondylitis). Both cases progressed to PR (worsening of pre-existing lung infiltrations (Patients 1, 2) and new contralateral effusion (Patient 2)) in a relatively short time after initiation of treatment (Patient 1: 1 week; Patient 2: 3 weeks), suggesting that immune modulations during pregnancy and delivery may contribute to the pathogenesis of both disseminated tuberculosis and its PR. The pulmonary lesions and effusion of both cases gradually improved without change of chemotherapy regimen. CONCLUSION: Physicians should recognize PR in tuberculosis patients with postpartum and then evaluate treatment efficacy.

A New Candidate Supporting Drug, Rikkunshito, for the QOL in Advanced Esophageal Cancer Patients with Chemotherapy Using Docetaxel/5-FU/CDDP.

Purpose. Docetaxel/5-FU/CDDP (DFP) therapy is a useful treatment for advanced esophageal cancer. However, adverse reactions such as chemotherapy-induced nausea and vomiting (CINV) interfere often with continuation of the chemotherapy. We investigated the efficacy of rikkunshito (TJ-43) on CINV. Methods. Nineteen patients who were going to undergo DFP therapy were enrolled. They were assigned to the following two groups: a TJ-43-treated group and -nontreated group. The following parameters were compared between the 2 groups: (1) the frequency of symptoms occurred, (2) vomiting, nausea, and anorexia score, and (3) QOL score. Results. The incidence of symptoms was lower in the TJ-43-treated group than that in the control group. The nausea score of the TJ-43-treated group was significantly lower than that of the control group. In the QOL score, the mood score and the ADL score decreased significantly in the control group. Conclusion. We recommend TJ-43 administration in patients undergoing DFP chemotherapy.

[Lung cancer in patient with a common trunk of left pulmonary veins; report of two cases].

A 69-year-old man and a 53-year-old man with lung cancer of left upper lobe underwent pulmonary resection. A preoperative chest computed tomography (CT) scan showed the left superior and inferior pulmonary veins forming a common trunk. In the former case, the common trunk was misidentified as the superior pulmonary vein. The latter was recognized preoperatively by using 3-dimensional CT, and successfully performed left superior segmentectomy. This variation type is surgically important because of a potential risk of intraoperative bleeding and damage to pulmonary circulation during pulmonary resection.

[Temporary effective treatment with L-asparaginase for a patient with refractory nasal NK/T-cell lymphoma].

A 48-year-old man was referred to Sakai Municipal Hospital with nasal discharge and right facial swelling. The pathological findings of a nasal cavity tumor revealed stage IIB NK/T-cell lymphoma. He was admitted to our hospital and received CHOP therapy, resulting in progressive disease. Irradiation therapy combined with DeVIC chemotherapy also could not shrink his lymphoma. Then, two courses of L-asparaginase(L-Asp) were administered, resulting in partial improvement of the nasal and pharynx lesions, resolution of the fever and improvement of his performance status. On the day before a third course of L-Asp, he again developed a lowgrade fever. Although L-Asp was administered for several days, marked elevation of serum LDH, AST, ALT level, and thrombocytopenia persisted, and he died. Post-mortem examinations revealed hemophagocytosis in the bone marrow and liver, and infiltration of lymphoma cells into multiple organs including left lower lung, liver, spleen and kidneys. Although L-Asp was effective against nasal NK/T-cell lymphoma resistant to combination chemotherapy and irradiation therapy, the effectiveness of the single agent with L-Asp was only transient. L-Asp based regimen should be used as first-line therapy if asparagine synthetase protein expression is low using an immunohistochemical method.

Laryngeal tuberculosis: a report of 15 cases.

Laryngeal tuberculosis is usually a complication of pulmonary tuberculosis. Recent studies have described a change in the clinical features of laryngeal tuberculosis. We present 15 cases of laryngeal tuberculosis treated at the Osaka Prefectural Habikino Hospital between 1993 and 2000. The results showed a mean age of 51 years, a male predominance by 2.75 to 1, and a 20% incidence (n = 3) of negative chest radiographic findings. The prominent presenting symptom was hoarseness (73.3%), and systemic symptoms were relatively rare. Seven patients showed ulcerative lesions, 5 showed granulomatous lesions, and the remaining 3 showed nonspecific inflammatory lesions in the larynx. Laryngeal lesions did not show any predilection for specific laryngeal sites in our series. In contrast to earlier studies, our study shows variations in clinical features of laryngeal tuberculosis. Physicians should consider tuberculosis in the differential diagnosis of laryngeal disease.