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关键词： Flow Cytometry   SARS-CoV-2   Immunophenotyping analysis   CD56bright   CD56dim  

摘要： Background: NK cells seem to be mainly involved in COVID-19 pneumonia. Little is known about NKT cells which represent a bridge between innate and adaptive immunity. Methods: We characterized peripheral blood T, NK and NKT cells in 45 patients with COVID-19 pneumonia (COVID-19 subjects) and 19 healthy donors (HDs). According to the severity of the disease, we stratified COVID-19 subjects into severe and non-severe groups. Results: Compared to HDs, COVID-19 subjects showed higher percentages of NK CD57+ and CD56dim NK cells and lower percentages of NKT and CD56bright cells. In the severe group we found a significantly lower percentage of NKT cells. In a multiple logistic regression analysis, NKT cell was independently associated with the severity of the disease. Conclusions: The low percentage of NKT cells in peripheral blood of COVID-19 subjects and the independent association with the severity of the disease suggests a potential role of this subset.

关键词： PCNS lymphoma   GCB-type   Hans algorithm   Survival   Age  

摘要： Primary CNS lymphomas (PCNSLs) are aggressive diffuse large B-cell lymphomas (DLBCLs) limited to the CNS that generally have a poor prognosis. Classification of DLBCL into germinal center B-cell (GCB) and activated B-cell (non-GCB) subtypes has prognostic value in systemic DLBCL, with GCB-type having a better prognosis. The aim of this study was to determine whether GCB versus non-GCB classification in PCNSLs has similar prognostic value. We analyzed clinical, radiological and histologic data from 24 patients with biopsy confirmed DLBCL of the CNS with classification into GCB versus non-GCB subtypes. We found that after a median follow-up of 15 months, only 39% of patients with non-GCB-type PCNS DLBCL were alive, whereas all patients with GCB-type were alive. Non-GCB-type had a median survival of 11 months, whereas all GCB-type patients were alive after a median follow-up of 22 months. As previously reported, we also found that patients younger than 70 years had longer survival (median 29 months) compared to older patients (median 8.8 months). There was no statistically significant difference between the ages, gender, focality, size or location of lesions, or treatment of non-GCB and GCB-type patients. Our findings suggest that classifying PCNSLs into GCB versus non-GCB subtype using the Hans algorithm may help stratify patients into two groups with different prognosis. (C) 2020 Elsevier Ltd. All rights reserved.

关键词： Adjuvant therapy   Clear cell ependymoma   Gross total resection   Overall survival   Progression-free survival  

摘要： BACKGROUND: The survival outcomes of clear cell ependymomas are poorly understood. This study clarifies the role of surgery and adjuvant therapy when this morphologically distinct tumor is encountered. METHODS: A systematic search for studies relating to clear cell ependymomas was conducted. Primary outcomes were progression-free survival and overall survival. Prognostic variables were age, sex, tumor consistency, extent of resection, and postoperative adjuvant therapy. Kaplan-Meier survival curves were generated and compared by the log-rank test. Multivariate Cox regression models were constructed, interrogated with Schoenfeld residuals, and used to identify independent prognostic factors. RESULTS: Of the 384 articles retrieved, 8 articles comprising 77 cases of clear cell ependymoma were included. Five-year overall survival and progression-free survival were 581% (95% confidence interval [CI], 46.3%-72.9%) and 46.3% (95% CI, 34.2%-62.8%), respectively. Kaplan-Meier analysis with the log-rank test showed that gross total resection was superior to subtotal resection in prolonging survival (P = 0.047) and delayed time to recurrence (P < 0.01). Multivariate analysis confirmed gross total resection as an independent protective factor against relapse (odds ratio, 0.39;95% CI, 017-019;P = 0.03). Age <50 years predicted longer overall survival (odds ratio, 0.16;95% CI, 0.05-0.49;P < 0.01). Postoperative adjuvant therapy after gross total resection did not affect overall survival (P = 0.98) or progression-free survival (P = 0.93). Adjuvant therapy after subtotal resection favored improved overall survival (P = 0.052). CONCLUSIONS: Clear cell ependymomas are particularly aggressive in those aged >50 years. Gross total resection remains the cornerstone of management. Postoperative adjuvant therapy is likely to be of survival benefit only after subtotal resection.

关键词： Non-small cell lung cancer   Brain metastases   Immunotherapy   Human leukocyte antigen (HLA) class 1  

摘要： Loss of human leukocyte antigen (HLA) class 1 expression is a mechanism of tumor immune escape and may contribute to resistance to immunotherapy. Patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors can have discordant responses between brain metastases and extracranial sites of disease. We sought to evaluate whether HLA class 1 expression was retained in metastatic NSCLC. Patients with paired primary NSCLC and brain metastases were identified from our institution's tissue registry. HLA class 1 cell membrane expression on tumor cells was determined by immunohistochemistry. Tumors with greater than the median of 10% HLA expression were considered positive. Agreement statistics (kappa) were used to assess the congruence of HLA expression. 51 patients with paired primary NSCLC and brain lesions were identified. The median HLA class 1 expression was 20% in the primary tumors (IQR 0-65%) and 10% in the brain metastases (IQR 5-40%). 27 primary tumors and 24 brain metastases were positive for HLA expression. There was disagreement in HLA positivity between paired lesions in 11 patients (22%, 95% CI 12-35%) (kappa = 0.57, 95% CI 0.35-0.79) (p = 0.0001). None of the patients received checkpoint inhibitors for treatment of these lesions. The results show that while there is moderate agreement in HLA class 1 expression between primary lung tumor and brain metastasis pairs, HLA expression is incongruent in nearly one quarter of patients. Loss of antigen presentation may represent one of the many potential mechanisms of discordant responses to checkpoint inhibitor therapy.

关键词： Epithelial kidney cells   SV40 T antigen   Transfection   Cell immortalization  

摘要： Sheep primary epithelial cells are short-lived in cell culture systems. For long-term in vitro studies, primary cells need to be immortalized. This study aims to establish and characterize T immortalized sheep embryo kidney cells (TISEKC). In this study, we used fetal lamb kidneys to derive primary cultures of epithelial cells. We subsequently immortalized these cells using the large T SV40 antigen to generate crude TISEKC and isolate TISEKC clones. Among numerous clones of immortalized cells, the selected TISEKC-5 maintained active division and cell growth over 20 passages but lacked expression of the oncogenic large T SV40 antigen. Morphologically, TISEKC-5 maintained their epithelial aspect similar to the parental primary epithelial cells. However, their growth properties showed quite different patterns. Crude TISEKC, as well as the clones of TISEKC proliferated highly in culture compared to the parental primary cells. In the early passages, immortalized cells showed heterogeneous polyploidy but in the late passages the karyotype of immortalized cells became progressively stable, identical to that of the primary cells, because the TISEKC-5 cell line has lost the large SV40 T antigen expression, this cell line is a valuable tool for veterinary sciences and biotechnological productions.

关键词： CRISPR interference   knockdown   inducible Tet-on system   multiplicity of infection   sgRNA  

摘要： To determine how nuclease deactivated Cas9 (dCas9) or single-guide RNA (sgRNA) expression levels affect the knockdown efficiency of CRISPRi, we created K562 cell clones expressing KRAB-dCas9 protein either with the inducible Tet-on system or with the constitutive SFFV promotor. Single clones were selected by fluorescence-activated cell sorting (FACS) for further study. Six genes with various expression levels were targeted using lentiviral sgRNA from two libraries in four cell clones with various KRAB-dCas9 expression levels. The expression level of dCas9 protein/sgRNA levels and the knockdown efficiency were determined by flow cytometry. The cell clone with the highest KRAB-dCas9 expression level achieved effective CRISPRi knockdown. The data describing this clone were statistically different from that on other clones, indicating the strong KRAB-dCas9 expression might be a prerequisite for CRISPRi. By adopting different multiplicity of infection (MOI) in lentiviral transduction of this clone, we modified the expression level of sgRNA and found that the knockdown efficiency was neither affected by the target gene expression level nor correlated with KRAB-dCas9 levels, which remained relatively constant across all knockdown experiments (coefficient of variation = 2.2%). As an example, the following levels of the knockdowns: 74.72, 72.28 and 39.08% for mmadhc, rpia and znf148 genes, respectively, were achieved. These knockdown efficiencies correlated well with the respective sgRNA expression levels. Linear regression models built using this data indicate that the knockdown efficiency may be significantly affected by the levels of both KRAB-dCas9 and sgRNA. Notably, the sgRNA levels have greater impact, being a major factor affecting CRISPRi efficiency.

关键词： cfDNA   Apoptosis   Necrosis   Active secretion   Liquid biopsy  

摘要： Cell-free DNA (cfDNA) as an emerging biomarker with huge potential for clinical application, especially in the field of liquid biopsy. The field is now in a critical transitional period in which cfDNA-based analysis is developing rapidly. No doubt learning more about the biological knowledge of cfDNA is beneficial to catalyze this transformation process. Therefore, in this review we have summarized the characteristics of cfDNA, including its structure and origin of tissues, in order to provide researchers with a more holistic insight of cfDNA. Subsequently, we focused on the pathways that cfDNA releases from cells, such as apoptosis, necrosis, and active secretion. Additionally, the clearance of cfDNA derived from both cellular death and active secretion in the physiological environment is also discussed. Finally, we have mentioned the link between cfDNA active secretion and tumor microenvironment.

关键词： Non-small cell lung cancer   PET/MRI   Prognostic markers  

摘要： Objectives: To investigate if the combined analysis of the apparent diffusion coefficient (ADC) and standardized uptake values (SUV) measured in F-18-fluoro-deoxy-glucose-positron emission tomography/magnetic resonance imaging (F-18-FDG PET/MRI) examinations correlates with overall survival in non-small cell lung cancer (NSCLC). Material and methods: A total of 92 patients with newly diagnosed, histopathologically proven NSCLC (44 women and 48 men, mean age 63.1 +/- 9.9y) underwent a dedicated thoracic F-18-FDG PET/MRI examination. A manually drawn polygonal region of interest (ROI), encompassing the entire primary tumor mass, was placed over the primary tumor on fused PET/MR images to determine the maximum and mean standardized uptake values (SUVmax;SDVmean) as well as on the ADC maps to quantify the mean and minimum ADC values (ADC(mean), ADC(min)). The impact of these parameters to predict patient's overall survival was tested using hazard ratios (HR). Pearson's correlation coefficients were calculated to assess dependencies between the different values. A p-value < 0.05 indicated statistical significance. Results: In all 92 patients (n = 59 dead at time of retrospective data collection, mean time till death: 19 +/- 16 month, n = 33 alive, mean time to last follow-up: 56 +/- 22 month) the Hazard ratios (HR) as independent predictors for overall survival (OS) of SUVmax were 2.37 (95 % CI: 1.23-4.59, p = 0.008) and for SUVmean 1.85 (95 % CI: 1.05-3.26, p = 0.03) while ADC(min) showed a HR of 0.95 (95 % CI: 0.57-1.59, p = 0.842) and ADC(mean) a HR of 2.01 (95 % CI: 1.2-3.38, p = 0.007). Furthermore, a combined analysis for SUVmax/ADC(mean), SUVmax/ ADC(min) and SUVmean/ADC(mean) revealed a HR of 2.01 (95 % CI: 1.10-3.67, p = 0.02), 1.75 (95 % CI: 0.97-3.15, p = 0.058) and 1.78 (95 % CI: 1.02-3.10, p = 0.04). Conclusion: SUVmax and SDVmean of the primary tumor are predictors for OS in therapy-naive NSCLC patients, whereas the combined analysis of SUV and ADC values d