A Case Report of a Bleeding Duodenal Gastro-Intestinal Stromal Tumor and its Emergent Management

A gastrointestinal stromal tumor (GIST) arising in the duodenum is a rare subtype of mesenchymal tumor. GISTs have a reported incidence of 11.9–19.6 per million population and duodenal GISTs make up just 5% of these tumors. Common presentation of duodenal GISTs is through an upper gastrointestinal bleed, of which, they are responsible for less than 1% of all gastrointestinal bleeding. In an elective setting, surgical management remains the mainstay of treatment. In this Case Report, the emergent management of a localized duodenal resection was performed by an acute care surgeon, in an unstable patient. The risk factors for malignancy include tumor size and a high mitotic cell index. Tumor recurrence is determined by tumor size, tumor rupture, high mitotic cell index, a non-gastric location and gastrointestinal bleeding.

P041 CDKN2B-AS1 (ANRIL) expression is decreased in Inflammatory Bowel Disease epithelia and in Celiac, and its reduction is linked with induced cells proliferation

Background Long non-coding RNAs (lncRNAs) have attenuated expression in several immune-mediated disorders. Using mRNAseq of intestinal biopsies, we identified a widespread dysregulation of 459 lncRNAs in the ileum of treatment-naïve pediatric Crohn Disease (CD) patients. We noted that large fraction of downregulated lncRNAs were correlated with epithelial functions. CDKN2B-AS1 (ANRIL) is one of the top most down-regulated lncRNA in CD, and showed decreased expression in Ulcerative Colitis (UC) colon tissues in recent studies. Methods Transcriptomics data is used to evaluate CDKN2B-AS1 expression in mucosal biopsies datasets and in gut epithelia. siRNA oligonucleotides targeting most CDKN2B-AS1 transcripts using Lipofectamine RNAiMAX is used to modulate CDKN2B-AS1 function, and RT-PCR to evaluate mRNA expression. xCELLigence system with gold microelectrodes in plate base is used to measure the impedance as an indicator for cell index. Results CDKN2B-AS1 is down-regulated in bulk mucosal biopsies obtained from CD ileum [fold change (FC) -8, corrected p=1.6E-5], in UC rectum (FC -9.4, corrected p=3.9E-18), and in celiac duodenum (FC -2.3, corrected p=0.03) in comparison to controls. CDKN2B-AS1 is detected under basal conditions in HT-29 cells. Two sets of CDKN2B-AS1 siRNA oligonucleotides (targeting most transcripts) achieved up to 60% reduction in CDKN2B-AS1 expression in HT-29 cells (p=1E-04) as confirmed by RT-PCR using two sets of primers. CDKN2B-AS1 reduction significantly increased cell index as measured by xCELLigence (doubled the index, p=1.7E-03). mRNAseq of the CDKN2B-AS1 reduced HT-29, showed reduction of the tumor suppressor gene APC in CDKN2B-AS1 siRNA treated cells (FC=-1.75, p=0.04) and of TGFBR2 (FC=-1.75, p=0.04). In contrast, reduction of CDKN2B-AS1 resulted in increase in WNT11 (FC=2.1, p=0.02) and TGFB1 (FC=1.9, p=0.04), and induction of the replication associated topoisomerase TOP1MT (FC=2.7, p=0.001). We confirmed these mRNAseq results using RT-PCR. Finally, mimicking inflammation by treating HT-29 with IL1β and LPS, reduced CDKN2B-AS1 expression (by 70%, p=6.3E-04) and doubled the cell index (p=1.8E-03). Conclusion We detected reduced CDKN2B-AS1 expression in three inflammatory disease affecting the gut in different location along the GI tract. Using HT-29 model system we were able to show, as was previously shown, an increase in cell index in CDKN2B-AS1 siRNA treated cells. We supplement those showing effect on down-stream genes that may be relevant in controlling cell proliferation. We further show that upon inflammatory triggering of HT-29 cells, CDKN2B-AS1 expression is reduced and cell index is increased, which may suggest a potential role in epithelial renewal in inflammation.

Application of xCELLigence real-time cell analysis to the microplate assay for pertussis toxin induced clustering in CHO cells

The microplate assay with Chinese Hamster Ovary (CHO) cells is currently used as a safety test to monitor the residual pertussis toxin (PT) amount in acellular pertussis antigens prior to vaccine formulation. The assay is based on the findings that the exposure of CHO cells to PT results in a concentration-dependent clustering response which can be used to estimate the amount of PT in a sample preparation. A major challenge with the current CHO cell assay methodology is that scoring of PT-induced clustering is dependent on subjective operator visual assessment using light microscopy. In this work, we have explored the feasibility of replacing the microscopy readout for the CHO cell assay with the xCELLigence Real-Time Cell Analysis system (ACEA BioSciences, a part of Agilent). The xCELLigence equipment is designed to monitor cell adhesion and growth. The electrical impedance generated from cell attachment and proliferation is quantified via gold electrodes at the bottom of the cell culture plate wells, which is then translated into a unitless readout called cell index. Results showed significant decrease in the cell index readouts of CHO cells exposed to PT compared to the cell index of unexposed CHO cells. Similar endpoint concentrations were obtained when the PT reference standard was titrated with either xCELLigence or microscopy. Testing genetically detoxified pertussis samples unspiked or spiked with PT further supported the sensitivity and reproducibility of the xCELLigence assay in comparison with the conventional microscopy assay. In conclusion, the xCELLigence RTCA system offers an alternative automated and higher throughput method for evaluating PT-induced clustering in CHO cells.

A Comparative Analysis of Methods for Titering Reovirus

Background: A key challenge in the process of virus amplification is the need for a simple and convenient method for measuring virus titers. Objective: Real-time unlabeled cell analysis (RTCA) was used to establish a standard curve of correlation between half-cell index time (CIT50) and virus titer. At the same time, the virus titer from tunable resistance pulse detection (TRPS) technology was compared with the traditional median tissue culture infectious dose (TCID50) method to evaluate the feasibility and application value of the RTCA technique and TRPS technology. Methods: : Cell index (CI) values for L929 cells under different culture conditions were detected, and the appropriate initial cell inoculation density was screened. The half-cell index (CI50) values of reovirus infected L929 cells with TCID50 titers were analyzed by RTCA, the CI50-TCID50 standard curve was created, and a regression equation was developed. RTCA, TCID50, and TRPS methods were used to detect the reovirus titer obtained by the amplification, and the sensitivity and feasibility of the CIT50-TCID50 standard curve method were analyzed. The virus titer was detected by TRPS technology and the TCID50 method. Results: L929 cells were best propagated at an initial density of 6 × 103 cells/well. After infecting L929 cells with different titers of reference reovirus, the linear correlation of CIT50 and TCID50 was y = -2.1806x + 71.023 (R2 = 0.9742). The titer resulting from the RTCA assay was 7×109.6821 pfu/mL, from the TRPS assay was 4.52×1010 pfu/mL, and from the TCID50 assay was 7×109.467 pfu/mL. Conclusion: The CIT50-TCID50 standard curve method established by the RTCA technique can be used to quantitatively detect reovirus titer with L929 cells. Compared with the TCID50 method, it takes a relatively short time and has high sensitivity and accuracy. The TRPS technology requires even less time to quantify the virus, but its precision is lower than that of the TCID50 method and RTCA technology. This study provides new technical methods for assessing the virulence of infectious live reovirus particles. Lay Summary: After amplification of the virus, we need to detect the virus titers (the virulence of the virus). The traditional method is to use the virus to infect cells, and then the virus titers can be calculated by 50% of the cells infected. However, this traditional method is time consuming. The ways of RTCA (a real-time cell analysis technique) and TRPS (a nano-bioparticle analysis technique) help us to detect viral titers. The consistency of these three methods determines their feasibility and accuracy. If they are feasible, then these two simple technologies will provide new ideas for detecting viral titers.

Relationship of Red Cell Index with the Severity of COPD

Background: Chronic obstructive pulmonary disease (COPD) has always attracted attention due to its high prevalence and high mortality. How to predict and diagnose COPD and assess the severity of the disease is our top priority. We aimed to evaluate the association between red cell index (RCI) and the severity of COPD, and compare predictive value among RCI, neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) of indicating the severity of COPD.Methods: A total of 207 participants were recruited (100 COPD patients and 107 healthy controls). COPD patients were divided into two groups according to receiver operating characteristics (ROC) curves cut-off value of RCI (RCI < 1.75, n = 54; RCI > 1.75, n = 46). Pearson’s correlation test, logistic regression analysis and other tests were performed.Results: Compared with low RCI group, the forced expiration volume in 1 second (FEV1) and FEV1 in percent of the predicted value (FEV1%) of high RCI group decreased (p = 0.016, p = 0.001). There is a negative correlation between RCI and FEV1% (r = -0.302, p = 0.004), while no correlation between FEV1% and NLR and PLR. RCI’s ability to predict Global Initiative for Chronic Obstructive Lung Disease classification, is better than NLR and PLR, with a cut of 1.75, specificity of 85.2%, sensitivity of 57.6% and area under the curve (AUC) of 0.729 (p = 0.001). Multivariate logistic regression analysis proved RCI was an independent factor affecting lung function in COPD patients (odds ratio [OR] = 4.27, 95%CI: 1.57 - 11.63, p = 0.004). Conclusion: RCI is a novel biomarker that can better assess respiratory function and severity of COPD than NLR and PLR. Higher RCI is independently related to deterioration of respiratory function.

Estimating Dynamic Cellular Morphological Properties via the Combination of the RTCA System and a Hough-Transform-Based Algorithm

The xCELLigence real-time cell analysis (RTCA) system has the potential to detect cellular proliferation, migration, cytotoxicity, adherence, and remodeling. Although the RTCA system is widely recognized as a noninvasive and efficient tool for real-time monitoring of cellular fate, it cannot describe detailed cell morphological parameters, such as length and intensity. Transforming growth factor beta(TGF-β) induced the epithelial–mesenchymal transition (EMT), which produces significant changes in cellular morphology, so we used TGF-β to treat A549 epithelial cells in this study. We compared it with lipopolysaccharide (LPS) and cigarette smoke extract (CSE) as stimulators. We developed an efficient algorithm to quantify the morphological cell changes. This algorithm is comprised of three major parts: image preprocessing, Hough transform (HT), and post-processing. We used the RTCA system to record the A549 cell index. Western blot was used to confirm the EMT. The RTCA system showed that different stimulators produce different cell index curves. The algorithm determined the lengths of the detected lines of cells, and the results were similar to the RTCA system in the TGF-β group. The Western blot results show that TGF-β changed the EMT markers, but the other stimulator remained unchanged. Optics-based computer vision techniques can supply the requisite information for the RTCA system based on good correspondence between the results.