A Nasal Inflammatory Cytokine Signature Is Associated with Early Graft-versus-Host Disease of the Lung after Allogeneic Hematopoietic Cell Transplantation: Proof of Concept.

Respiratory inflammation in bronchiolitis obliterans syndrome (BOS) after hematopoietic cell transplantation (HCT) is poorly understood. Clinical criteria for early-stage BOS (stage 0p) often capture HCT recipients without BOS. Measuring respiratory tract inflammation may help identify BOS, particularly early BOS. We conducted a prospective observational study in HCT recipients with new-onset BOS (n = 14), BOS stage 0p (n = 10), and recipients without lung impairment with (n = 3) or without (n = 8) chronic graft-versus-host disease and measured nasal inflammation using nasosorption at enrollment and then every 3 mo for 1 y. We divided BOS stage 0p into impairment that did not return to baseline values (preBOS, n = 6), or transient impairment (n = 4). We tested eluted nasal mucosal lining fluid from nasosorption matrices for inflammatory chemokines and cytokines using multiplex magnetic bead immunoassays. We analyzed between-group differences using the Kruskal-Wallis method, adjusting for multiple comparisons. We found increased nasal inflammation in preBOS and therefore directly compared patients with preBOS to those with transient impairment, as this would be of greatest diagnostic relevance. After adjusting for multiple corrections, we found significant increases in growth factors (FGF2, TGF-α, GM-CSF, VEGF), macrophage activation (CCL4, TNF-α, IL-6), neutrophil activation (CXCL2, IL-8), T cell activation (CD40 ligand, IL-2, IL-12p70, IL-15), type 2 inflammation (eotaxin, IL-4, IL-13), type 17 inflammation (IL-17A), dendritic maturation (FLT3 ligand, IL-7), and counterregulatory molecules (PD-L1, IL-1 receptor antagonist, IL-10) in preBOS patients compared to transient impairment. These differences waned over time. In conclusion, a transient multifaceted nasal inflammatory response is associated with preBOS. Our findings require validation in larger longitudinal cohorts.

Contributions of Circulating microRNAs for Early Detection of Lung Cancer.

There is unmet need to develop circulating biomarkers that would enable earlier interception of lung cancer when more effective treatment options are available. Here, a set of 30 miRNAs, selected from a review of the published literature were assessed for their predictive performance in identifying lung cancer cases in the pre-diagnostic setting. The 30 miRNAs were assayed using sera collected from 102 individuals diagnosed with lung cancer within one year following blood draw and 212 controls matched for age, sex, and smoking status. The additive performance of top-performing miRNA candidates in combination with a previously validated four-protein marker panel (4MP) consisting of the precursor form of surfactant protein B (Pro-SFTPB), cancer antigen 125 (CA125), carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA21-1) was additionally assessed. Of the 30 miRNAs evaluated, five (miR-320a-3p, miR-210-3p, miR-92a-3p, miR-21-5p, and miR-140-3p) were statistically significantly (Wilcoxon rank sum test p < 0.05) elevated in case sera compared to controls, with individual AUCs ranging from 0.57−0.62. Compared to the 4MP alone, the combination of 3-miRNAs + 4MP improved sensitivity at 95% specificity by 19.1% ((95% CI of difference 0.0−28.6); two-sided p: 0.006). Our findings demonstrate utility for miRNAs for early detection of lung cancer in combination with a four-protein marker panel.

A Comprehensive Search of Non-Canonical Proteins in Non-Small Cell Lung Cancer and Their Impact on the Immune Response.

There is substantial interest in mining neoantigens for cancer applications. Non-canonical proteins resulting from frameshift mutations have been identified as neoantigens in cancer. We investigated the landscape of non-canonical proteins in non-small cell lung cancer (NSCLC) and their induced immune response in the form of autoantibodies. A database of cryptoproteins was computationally constructed and comprised all alternate open reading frames (altORFs) and ORFs identified in pseudogenes, noncoding RNAs, and untranslated regions of mRNAs that did not align with known canonical proteins. Proteomic profiles of seventeen lung adenocarcinoma (LUAD) cell lines were searched to evaluate the occurrence of cryptoproteins. To assess the immunogenicity, immunoglobulin (Ig)-bound cryptoproteins in plasmas were profiled by mass spectrometry. The specimen set consisted of plasmas from 30 newly diagnosed NSCLC cases, pre-diagnostic plasmas from 51 NSCLC cases, and 102 control plasmas. An analysis of LUAD cell lines identified 420 cryptoproteins. Plasma Ig-bound analyses revealed 90 cryptoproteins uniquely found in cases and 14 cryptoproteins that had a fold-change >2 compared to controls. In pre-diagnostic samples, 17 Ig-bound cryptoproteins yielded an odds ratio ≥2. Eight Ig-bound cryptoproteins were elevated in both pre-diagnostic and newly diagnosed cases compared to controls. Cryptoproteins represent a class of neoantigens that induce an autoantibody response in NSCLC.

Blood-Based Biomarker Panel for Personalized Lung Cancer Risk Assessment.

PURPOSE: To investigate whether a panel of circulating protein biomarkers would improve risk assessment for lung cancer screening in combination with a risk model on the basis of participant characteristics. METHODS: A blinded validation study was performed using prostate lung colorectal ovarian (PLCO) Cancer Screening Trial data and biospecimens to evaluate the performance of a four-marker protein panel (4MP) consisting of the precursor form of surfactant protein B, cancer antigen 125, carcinoembryonic antigen, and cytokeratin-19 fragment in combination with a lung cancer risk prediction model (PLCOm2012) compared with current US Preventive Services Task Force (USPSTF) screening criteria. The 4MP was assayed in 1,299 sera collected preceding lung cancer diagnosis and 8,709 noncase sera. RESULTS: The 4MP alone yielded an area under the receiver operating characteristic curve of 0.79 (95% CI, 0.77 to 0.82) for case sera collected within 1-year preceding diagnosis and 0.74 (95% CI, 0.72 to 0.76) among the entire specimen set. The combined 4MP + PLCOm2012 model yielded an area under the receiver operating characteristic curve of 0.85 (95% CI, 0.82 to 0.88) for case sera collected within 1 year preceding diagnosis. The benefit of the 4MP in the combined model resulted from improvement in sensitivity at high specificity. Compared with the USPSTF2021 criteria, the combined 4MP + PLCOm2012 model exhibited statistically significant improvements in sensitivity and specificity. Among PLCO participants with ≥ 10 smoking pack-years, the 4MP + PLCOm2012 model would have identified for annual screening 9.2% more lung cancer cases and would have reduced referral by 13.7% among noncases compared with USPSTF2021 criteria. CONCLUSION: A blood-based biomarker panel in combination with PLCOm2012 significantly improves lung cancer risk assessment for lung cancer screening.

Protein citrullination as a source of cancer neoantigens.

BACKGROUND: Citrulline post-translational modification of proteins is mediated by protein arginine deiminase (PADI) family members and has been associated with autoimmune diseases. The role of PADI-citrullinome in immune response in cancer has not been evaluated. We hypothesized that PADI-mediated citrullinome is a source of neoantigens in cancer that induces immune response. METHODS: Protein expression of PADI family members was evaluated in 196 cancer cell lines by means of indepth proteomic profiling. Gene expression was assessed using messenger RNA data sets from The Cancer Genome Atlas. Immunohistochemical analysis of PADI2 and peptidyl-citrulline was performed using breast cancer tissue sections. Citrullinated 12-34-mer peptides in the putative Major Histocompatibility Complex-II (MHC-II) binding range were profiled in breast cancer cell lines to investigate the relationship between protein citrullination and antigen presentation. We further evaluated immunoglobulin-bound citrullinome by mass spectrometry using 156 patients with breast cancer and 113 cancer-free controls. RESULTS: Proteomic and gene expression analyses revealed PADI2 to be highly expressed in several cancer types including breast cancer. Immunohistochemical analysis of 422 breast tumor tissues revealed increased expression of PADI2 in ER- tumors (p<0.0001); PADI2 protein expression was positively correlated (p<0.0001) with peptidyl-citrulline staining. PADI2 expression exhibited strong positive correlations with a B cell immune signature and with MHC-II-bound citrullinated peptides. Increased circulating citrullinated antigen-antibody complexes occurred among newly diagnosed breast cancer cases relative to controls (p=0.0012). CONCLUSIONS: An immune response associated with citrullinome is a rich source of neoantigens in breast cancer with a potential for diagnostic and therapeutic applications.

Lead-Time Trajectory of CA19-9 as an Anchor Marker for Pancreatic Cancer Early Detection.

BACKGROUND & AIMS: There is substantial interest in liquid biopsy approaches for cancer early detection among subjects at risk, using multi-marker panels. CA19-9 is an established circulating biomarker for pancreatic cancer; however, its relevance for pancreatic cancer early detection or for monitoring subjects at risk has not been established. METHODS: CA19-9 levels were assessed in blinded sera from 175 subjects collected up to 5 years before diagnosis of pancreatic cancer and from 875 matched controls from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial. For comparison of performance, CA19-9 was assayed in blinded independent sets of samples collected at diagnosis from 129 subjects with resectable pancreatic cancer and 275 controls (100 healthy subjects; 50 with chronic pancreatitis; and 125 with noncancerous pancreatic cysts). The complementary value of 2 additional protein markers, TIMP1 and LRG1, was determined. RESULTS: In the PLCO cohort, levels of CA19-9 increased exponentially starting at 2 years before diagnosis with sensitivities reaching 60% at 99% specificity within 0 to 6 months before diagnosis for all cases and 50% at 99% specificity for cases diagnosed with early-stage disease. Performance was comparable for distinguishing newly diagnosed cases with resectable pancreatic cancer from healthy controls (64% sensitivity at 99% specificity). Comparison of resectable pancreatic cancer cases to subjects with chronic pancreatitis yielded 46% sensitivity at 99% specificity and for subjects with noncancerous cysts, 30% sensitivity at 99% specificity. For prediagnostic cases below cutoff value for CA19-9, the combination with LRG1 and TIMP1 yielded an increment of 13.2% in sensitivity at 99% specificity (P = .031) in identifying cases diagnosed within 1 year of blood collection. CONCLUSION: CA19-9 can serve as an anchor marker for pancreatic cancer early detection applications.

Contribution of a Blood-Based Protein Biomarker Panel to the Classification of Indeterminate Pulmonary Nodules.

RATIONALE: The workup and longitudinal monitoring for subjects presenting with pulmonary nodules is a pressing clinical problem. A blood-based biomarker panel potentially has utility for identifying subjects at higher risk for harboring a malignant nodule for whom additional workup would be indicated or subjects at reduced risk for whom imaging-based follow-up would be indicated. OBJECTIVES: To assess whether a previously described four-protein biomarker panel, reported to improve assessment of lung cancer risk compared with a smoking-based lung cancer risk model, can provide discrimination between benign and malignant indeterminate pulmonary nodules. METHODS: A previously validated multiplex enzyme-linked immunoassay was performed on matched case and control samples from each cohort. MEASUREMENTS: The biomarker panel was tested in two case-control cohorts of patients presenting with indeterminate pulmonary nodules at the University of Pittsburgh Medical Center and the University of Texas Southwestern. MAIN RESULTS: In both cohorts, the biomarker panel resulted in improved prediction of lung cancer risk over a model on the basis of nodule size alone. Of particular note, the addition of the marker panel to nodule size greatly improved sensitivity at a high specificity in both cohorts. CONCLUSIONS: A four-marker biomarker panel, previously validated to improve lung cancer risk prediction, was found to also have utility in distinguishing benign from malignant indeterminate pulmonary nodules. Its performance in improving sensitivity at a high specificity indicates potential utility of the marker panel in assessing likelihood of malignancy in otherwise indeterminate nodules.

Improving agreement of ASA physical status class between pre-anesthesia screening and day of surgery by adding institutional-specific and ASA-approved examples: a quality improvement project.

BACKGROUND: A successful anesthesia pre-assessment clinic needs to identify patients who need further testing, evaluation, and optimization prior to the day of surgery to avoid delays and cancelations. Although the ASA Physical Status Classification system (ASA PS) has been used widely for over 50 years, it has poor interrater agreement when only using the definitions. In 2014, ASA-approved examples for each ASA physical status class (ASA PS). In this quality improvement study, we developed and evaluated the effectiveness of institutional-specific examples on interrater reliability between anesthesia pre-anesthesia clinic (APAC) and the day of surgery evaluation (DOS). METHODS: A multi-step, multi-year quality improvement project was performed. Step 1, pre-intervention, was a retrospective review to determine the percentage agreement of ASA PS assignment between APAC and DOS for adult and pediatric patients. Step 2 was a retrospective review of the step 1 cases where the ASA PS assignment differed to determine which medical conditions were valued differently and then develop institutional-specific examples for medical conditions not addressed by ASA-approved examples. Step 3 was to educate clinicians about the newly implemented examples and how they should be used as a guide. Step 4, post-intervention, was a retrospective review to determine if the examples improved agreement between APAC and DOS ASA PS assignments. Weighted Kappa coefficient was used to measure of interrater agreement excluding chance agreement. RESULTS: Having only ASA PS definitions available, APAC and DOS agreement was only 74% for adults (n = 737) and 63% for pediatric patients (n = 216). For adults, 20 medical co-morbidity categories and, for pediatric patients, 9 medical co-morbidity categories accounted for > 90% the differences in ASA PS. After development and implementation of institutional-specific examples with ASA-approved examples, the percentage agreement increased for adult patients (n = 795) to 91% and for pediatric patients (n = 239) to 84%. Weighted Kappa coefficients increased significantly for all patients (from 0.62 to 0.85, p < .0001), adult patients (from 0.62 to 0.86, p < .0001), and pediatric patients (from 0.48 to 0.78, p < .0001). CONCLUSIONS: ASA-approved examples do not address all medical conditions that account for differences in the assignment of ASA PS between pre-anesthesia screening and day of anesthesia evaluation at our institution. The process of developing institutional-specific examples addressed the medical conditions that caused differences in assignment at one institution. The implementation of ASA PS examples improved consistency of assignment, and therefore communication of medical conditions of patients presenting for anesthesia care.

The neurological manifestations of COVID-19 - A case series.

Severe acute respiratory syndrome (SARS) is a fatal respiratory illness caused by the coronavirus (CoV). The first known case was reported in 2002, later coined as SARS-CoV. Over the last two decades, the CoV has periodically emerged in the general population, causing a varying degree of pneumonia. The most recent outbreak, now known as coronavirus disease of 2019 (COVID-19), has been on an exponential rise. Similar to its predecessors, COVID-19 causes a fatal form of pneumonia; however, in a small percentage of patients, COVID-19 has shown to cause neurological symptoms. Given that SARS-CoV and the new CoV strain share similar viral structures, COVID-19 may have the capability to invade the neurological system. We present a series of patients with COVID-19, the first of which presented with a seizure, whereas our second patient developed seizures during their hospital course. Neither patient had a previous history of epilepsy. RELEVANCE FOR PATIENTS: COVID-19 has rapidly evolved since it was first reported and has proven to be a fatal infective process. The last several months have been challenging for the medical community as we try to understand the complexities of this virus. Clinicians have attempted to assess the most common presenting symptoms based on reported cases. The purpose of this study was to help understand how COVID-19 presents itself when the neurological system is involved. This case series describes the common and uncommon neurological manifestations of COVID-19. By doing so, we hope to provide clinicians with additional information to help diagnose COVID-19 in this unprecedented time and to also be wary of the uncommon presenting features.

A Plasma-Derived Protein-Metabolite Multiplexed Panel for Early-Stage Pancreatic Cancer.

BACKGROUND: We applied a training and testing approach to develop and validate a plasma metabolite panel for the detection of early-stage pancreatic ductal adenocarcinoma (PDAC) alone and in combination with a previously validated protein panel for early-stage PDAC. METHODS: A comprehensive metabolomics platform was initially applied to plasmas collected from 20 PDAC cases and 80 controls. Candidate markers were filtered based on a second independent cohort that included nine invasive intraductal papillary mucinous neoplasm cases and 51 benign pancreatic cysts. Blinded validation of the resulting metabolite panel was performed in an independent test cohort consisting of 39 resectable PDAC cases and 82 matched healthy controls. The additive value of combining the metabolite panel with a previously validated protein panel was evaluated. RESULTS: Five metabolites (acetylspermidine, diacetylspermine, an indole-derivative, and two lysophosphatidylcholines) were selected as a panel based on filtering criteria. A combination rule was developed for distinguishing between PDAC and healthy controls using the Training Set. In the blinded validation study with early-stage PDAC samples and controls, the five metabolites yielded areas under the curve (AUCs) ranging from 0.726 to 0.842, and the combined metabolite model yielded an AUC of 0.892 (95% confidence interval [CI] = 0.828 to 0.956). Performance was further statistically significantly improved by combining the metabolite panel with a previously validated protein marker panel consisting of CA 19-9, LRG1, and TIMP1 (AUC = 0.924, 95% CI = 0.864 to 0.983, comparison DeLong test one-sided P= .02). CONCLUSIONS: A metabolite panel in combination with CA19-9, TIMP1, and LRG1 exhibited substantially improved performance in the detection of early-stage PDAC compared with a protein panel alone.

Assessment of Lung Cancer Risk on the Basis of a Biomarker Panel of Circulating Proteins.

Importance: There is an urgent need to improve lung cancer risk assessment because current screening criteria miss a large proportion of cases. Objective: To investigate whether a lung cancer risk prediction model based on a panel of selected circulating protein biomarkers can outperform a traditional risk prediction model and current US screening criteria. Design, Setting, and Participants: Prediagnostic samples from 108 ever-smoking patients with lung cancer diagnosed within 1 year after blood collection and samples from 216 smoking-matched controls from the Carotene and Retinol Efficacy Trial (CARET) cohort were used to develop a biomarker risk score based on 4 proteins (cancer antigen 125 [CA125], carcinoembryonic antigen [CEA], cytokeratin-19 fragment [CYFRA 21-1], and the precursor form of surfactant protein B [Pro-SFTPB]). The biomarker score was subsequently validated blindly using absolute risk estimates among 63 ever-smoking patients with lung cancer diagnosed within 1 year after blood collection and 90 matched controls from 2 large European population-based cohorts, the European Prospective Investigation into Cancer and Nutrition (EPIC) and the Northern Sweden Health and Disease Study (NSHDS). Main Outcomes and Measures: Model validity in discriminating between future lung cancer cases and controls. Discrimination estimates were weighted to reflect the background populations of EPIC and NSHDS validation studies (area under the receiver-operating characteristics curve [AUC], sensitivity, and specificity). Results: In the validation study of 63 ever-smoking patients with lung cancer and 90 matched controls (mean [SD] age, 57.7 [8.7] years; 68.6% men) from EPIC and NSHDS, an integrated risk prediction model that combined smoking exposure with the biomarker score yielded an AUC of 0.83 (95% CI, 0.76-0.90) compared with 0.73 (95% CI, 0.64-0.82) for a model based on smoking exposure alone (P = .003 for difference in AUC). At an overall specificity of 0.83, based on the US Preventive Services Task Force screening criteria, the sensitivity of the integrated risk prediction (biomarker) model was 0.63 compared with 0.43 for the smoking model. Conversely, at an overall sensitivity of 0.42, based on the US Preventive Services Task Force screening criteria, the integrated risk prediction model yielded a specificity of 0.95 compared with 0.86 for the smoking model. Conclusions and Relevance: This study provided a proof of principle in showing that a panel of circulating protein biomarkers may improve lung cancer risk assessment and may be used to define eligibility for computed tomography screening.

Proteinuria Predicts Resistance to Antiplatelet Therapy in Ischemic Stroke.

The occurrence of a stroke while on antiplatelet agents presents a therapeutic dilemma. One of the main causes for recurrent strokes is antiplatelet resistance more commonly known as high on treatment platelet reactivity (HTPR). Prior studies have established that proteinuria is associated with HTPR following myocardial infarction. Here, we investigated whether dipstick proteinuria correlates with HTPR in patients presenting with stroke. We performed a retrospective cohort analysis of 102 patients admitted for a recurrent ischemic stroke that had either a VerifyNow aspirin or VerifyNow clopidogrel laboratory test performed to assess platelet reactivity. Dipstick proteinuria was defined as > 30 mg/dl (2+ or more). HTPR was defined as an aspirin resistance unit > 550 for aspirin and a P2Y12 reactivity unit > 208 for clopidogrel. Patients with proteinuria on dipstick were significantly more likely to have HTPR to either aspirin (p value 0.017) or clopidogrel (p value 0.017). After controlling for age, smoking, diabetes, hypertension, CAD and GFR, proteinuria was an independent predictor of HTPR for patient taking aspirin (p = 0.025). Platelet resistance is an entity that undermines the activity of antiplatelet agents in reducing stroke risk. Here, we demonstrate an association with increased platelet reactivity and proteinuria. This highlights a potential new therapeutic target in reducing future stroke risk.

Sequential Validation of Blood-Based Protein Biomarker Candidates for Early-Stage Pancreatic Cancer.

Background: CA19-9, which is currently in clinical use as a pancreatic ductal adenocarcinoma (PDAC) biomarker, has limited performance in detecting early-stage disease. We and others have identified protein biomarker candidates that have the potential to complement CA19-9. We have carried out sequential validations starting with 17 protein biomarker candidates to determine which markers and marker combination would improve detection of early-stage disease compared with CA19-9 alone. Methods: Candidate biomarkers were subjected to enzyme-linked immunosorbent assay based sequential validation using independent multiple sample cohorts consisting of PDAC cases (n = 187), benign pancreatic disease (n = 93), and healthy controls (n = 169). A biomarker panel for early-stage PDAC was developed based on a logistic regression model. All statistical tests for the results presented below were one-sided. Results: Six out of the 17 biomarker candidates and CA19-9 were validated in a sample set consisting of 75 PDAC patients, 27 healthy subjects, and 19 chronic pancreatitis patients. A second independent set of 73 early-stage PDAC patients, 60 healthy subjects, and 74 benign pancreatic disease patients (combined validation set) yielded a model that consisted of TIMP1, LRG1, and CA19-9. Additional blinded testing of the model was done using an independent set of plasma samples from 39 resectable PDAC patients and 82 matched healthy subjects (test set). The model yielded areas under the curve (AUCs) of 0.949 (95% confidence interval [CI] = 0.917 to 0.981) and 0.887 (95% CI = 0.817 to 0.957) with sensitivities of 0.849 and 0.667 at 95% specificity in discriminating early-stage PDAC vs healthy subjects in the combined validation and test sets, respectively. The performance of the biomarker panel was statistically significantly improved compared with CA19-9 alone (P < .001, combined validation set; P = .008, test set). Conclusion: The addition of TIMP1 and LRG1 immunoassays to CA19-9 statistically significantly improves the detection of early-stage PDAC.

A single molecular probe for multi-analyte (Cr³âº, Al³âº and Fe³âº) detection in aqueous medium and its biological application.

An ESIPT based fluorescent sensor 1 was developed, which could selectively detect and differentiate trivalent metal ions Cr(3+), Al(3+) and Fe(3+) in aqueous medium. The cell imaging experiments confirmed that 1 can be used for monitoring intracellular Cr(3+) and Al(3+) levels in living cells.

Stacked stem cell sheets enhance cell-matrix interactions.

Cell sheet engineering has enabled the production of confluent cell sheets stacked together for use as a cardiac patch to increase cell survival rate and engraftment after transplantation, thereby providing a promising strategy for high density stem cell delivery for cardiac repair. One key challenge in using cell sheet technology is the difficulty of cell sheet handling due to its weak mechanical properties. A single-layer cell sheet is generally very fragile and tends to break or clump during harvest. Effective transfer and stacking methods are needed to move cell sheet technology into widespread clinical applications. In this study, we developed a simple and effective micropipette based method to aid cell sheet transfer and stacking. The cell viability after transfer was tested and multi-layer stem cell sheets were fabricated using the developed method. Furthermore, we examined the interactions between stacked stem cell sheets and fibrin matrix. Our results have shown that the preserved ECM associated with the detached cell sheet greatly facilitates its adherence to fibrin matrix and enhances the cell sheet-matrix interactions. Accelerated fibrin degradation caused by attached cell sheets was also observed.

Responsive systems for cell sheet detachment.

Cell sheet engineering has been progressing rapidly during the past few years and has emerged as a novel approach for cell based therapy. Cell sheet harvest technology enables fabrication of viable, transplantable cell sheets for various tissue engineering applications. Currently, the majority of cell sheet studies use thermo-responsive systems for cell sheet detachment. However, other responsive systems began showing their potentials for cell sheet harvest. This review provides an overview of current techniques in creating cell sheets using different types of responsive systems including thermo-responsive, electro-responsive, photo-responsive, pH-responsive and magnetic systems. Their mechanism, approach, as well as applications for cell detachment have been introduced. Further development of these responsive systems will allow efficient cell sheet harvesting and patterning of cells to reconstruct complex tissue for broad clinical applications.

DNA methylation and gene expression profiling of ewing sarcoma primary tumors reveal genes that are potential targets of epigenetic inactivation.

The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs) using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA)-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1,) and VCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.

Rapid cell sheet detachment using spin-coated pNIPAAm films retained on surfaces by an aminopropyltriethoxysilane network.

The ability to harvest cell sheets grown on thermoresponsive polymers, such as poly(N-isopropylacrylamide) (pNIPAAm), has been widely studied for use in tissue engineering applications. pNIPAAm is of special interest because of the phase change that it undergoes in a physiologically relevant temperature range. Two primary approaches have been adopted to graft pNIPAAm chains covalently onto tissue culture polystyrene dishes: electron beam irradiation and plasma polymerization. These approaches often involve non-easily accessible (e.g. e-beam) facilities and complicated procedures that have hindered most tissue culture laboratories in adopting this technology for their specific applications. In this study, we developed a simple and cost-effective approach to create thermoresponsive surfaces using commercially available pNIPAAm. Using a simple spin-coating technique, thermoresponsive thin films were deposited on glass slides or silicon wafers using pNIPAAm blended with a small amount of 3-aminopropyltriethoxysilane (APTES), which enhances the retention of pNIPAAm on the surface. We found that the thermoresponsive films created using our method support cell attachment and proliferation without additional adhesive proteins as well as cell sheet detachment within minutes.