Search

Norfloxacin (NOR) in milk may influence mammalian cell replication and bring about a decrease in the efficiency for treating infection in humans. However, current techniques for detecting NOR usually require expensive instruments and trained personnel. In this work, we have developed a low-cost and simple method via paper-based fluorescent immunoassay for highly sensitive and selective detection of NOR in milk at picogram level. The NOR monoclonal antibody labeled with quantum dots is used as a detection probe to recognize the corresponding NOR, which can quantitatively detect NOR on paper-based devices. The detection limits in aqueous solution and milk are 1 pg/mL and 10 pg/mL, respectively. The developed paper-based method provides a cheap, sensitive, eco-friendly, and rapid approach for quantitative detection of trace NOR in milk, which may find wide applications in food safety inspection. Noteworthy, the method is especially suitable for applications at resource-limited and on-site settings.

Identification of specific antibodies in patient plasma is an essential part of many diagnostic procedures and is critical for safe blood transfusion. Current techniques require laboratory infrastructure and long turnaround times which limits access to those nearby tertiary healthcare providers. Addressing this challenge, a novel and rapid paper-based antibody test is reported. We validate antibody detection with reverse blood typing using IgM antibodies and then generalise the validity by adapting to detect SARS CoV-2 (COVID-19) antibodies in patient serum samples. Reagent red blood cells (RBC) are first combined with the patient plasma containing the screened antibody and a droplet of the mixture is then deposited onto paper. The light intensity profile is analyzed to identify test results, which can be detected by eye and/or with image processing to allow full automation. The efficacy of this test to perform reverse blood typing is demonstrated and the performance and sensitivity of this test using different paper types and RBC reagents was investigated using clinical samples. As an example of the flexibility of this approach, we labeled the RBC reagent with an antibody-peptide conjugate to detect SARS CoV-2 (COVID-19) antibodies in patient serum samples. This concept could be generalized to any agglutination-based antibody diagnostics with blood plasma.

We developed a multiplexed point-of-care immunodiagnostic assay for antibody detection in human sera made through the vertical stacking of functional paper layers. In this multiplexed vertical flow immunodiagnostic assay (xVFA), a colorimetric signal is generated by gold nanoparticles captured on a spatially-multiplexed sensing membrane containing specific antigens. The assay is completed in 20 minutes, following which the sensing membrane is imaged by a cost-effective mobile-phone reader. The images are sent to a server, where the results are rapidly analyzed and relayed back to the user. The performance of the assay was evaluated by measuring Lyme-specific antibodies in human sera as model target antibodies. The presented platform is rapid, simple, inexpensive, and allows for simultaneous and quantitative measurement of multiple antibodies and/or antigens making it a suitable point-of-care platform for disease diagnostics.

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9–13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event – a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies’ input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.

This paper presents a wireless, self-powered, affinity-based biosensor based on the integration of paper-based microfluidics with our previously reported method for self-assembling radio-frequency (RF) antennas. At the core of the proposed approach is a silver-enhancement technique that grows portions of a RF antenna in regions where target antigens hybridize with target specific affinity probes. The hybridization regions are defined by a network of nitrocellulose based microfluidic channels which implement a self-powered approach to sample the reagent and control its flow and mixing. The integration substrate for the biosensor has been constructed using polyethylene and the patterning of the antenna on the substrate has been achieved using a low-cost ink-jet printing technique. The substrate has been integrated with passive radio-frequency identification (RFID) tags to demonstrate that the resulting sensor-tag can be used for continuous monitoring in a food supply-chain where direct measurement of analytes is typically considered to be impractical. We validate the proof-of-concept operation of the proposed sensor-tag using IgG as a model analyte and using a 915 MHz Ultra-high-frequency (UHF) RFID tagging technology.

Microfluidic paper-based analytical devices (μPADs) are emerging as effective analytical platforms for point-of-care assays in resource-limited areas. Simple and cost-effective fabrication method still remains challenging on μPADs. A simple and cost-effective method for fabricating paper-based devices was presented in this work by using of dipping strategy with the recycled polystyrene in chloroform as the hydrophobic reagent. Adhesive tape was employed as mask to transfer the hydrophilic channel pattern to the paper substrate. With the single-sided adhesive tape stuck on the hydrophilic parts of the paper surface, the paper-based device was immersed in chloroform solution with dissolving recycling polystyrene for several seconds. Then the hydrophilic pattern can be achieved and all the other parts on the paper surface were hydrophobic. The adhesive tape was torn off from the hydrophilic parts. The highest contact angle value of 114° of the hydrophobic part was acquired with this simple fabrication method. By using of the sandwich-type immunoreactions and luminol-H2O2 p-iodophenol (PIP) chemiluminescence(CL) system, three cancer biomarkers were simultaneously detected in human serum samples on μPADs with the linear range of 0.05–80.0 ng·mL−1 for carcinoembryonic antigen (CEA), 5.0–80.0 ng·mL−1 for alpha-fetal protein (AFP) and 1.0–50.0 ng·mL−1 for prostate-specific antigen (PSA). The fabricating strategy with recycling polystyrene and adhesive tape provides a versatile platform for prototyping of μPADs in both developed and resource constrained region.

Brentuximab vedotin (BV) is approved for the treatment of patients with relapsed or refractory CD30-positive Hodgkin lymphoma, and relapsed or refractory systemic anaplastic large-cell lymphoma. Several uncertainties remain regarding the optimal use of the drug in its approved indications as well as outside them. This article reports recommendations on the use of BV issued during a consensus project, sponsored by the Italian Society of Hematology (SIE) and its affiliate societies, Società Italiana di Ematologia Sperimentale (SIES) and Gruppo Italiano Trapianto di Midollo Osseo (GITMO). Scientific evidence on BV was evaluated by a panel of experts, and consensus was developed by group discussion for key questions selected according to the clinical relevance. The following key issues were addressed: testing CD30 positivity to assess eligibility to BV; assessing practice indications of BV in Hodgkin lymphoma and systemic anaplastic large-cell lymphoma; providing pretreatment evaluation of patients candidates to BV; monitoring the response to BV; managing patients treated with BV; and assessing the role of BV in other CD30-positive lymphomas.

Both the ABO and Rhesus (Rh) blood groups play crucial roles in blood transfusion medicine. Herein, we report a simple and low-cost paper-based analytical device (PAD) for phenotyping red blood cell (RBC) antigens. Using this Rh typing format, 5 Rh antigens on RBCs can be simultaneously detected and macroscopically visualized within 12 min. The proposed Rh phenotyping relies on the presence or absence of hemagglutination in the sample zones after immobilizing the antibodies targeting each Rh antigen. The PAD was optimized in terms of filter paper type, antibodies, and distance of the visualization zone. In this study, the optimal conditions were Whatman filter paper Grade 4; anti-D, -C, -E, -c, and -e antibodies; RBC suspension of 30%; and a visualization zone of 1 cm above the sample zone. The accuracy of simultaneously phenotyping the five Rh RBC antigens in the blood samples (n = 4692) was 99.19%, comparable with the accuracy of the gold-standard tube method used by blood bank laboratories in several regions of Thailand. Furthermore, decision making based on this method can be assisted by deep learning. After implementing a two-stage objective detection algorithm (YOLO v4-tiny) and classification model (DenseNet-201), the ambiguous images (n = 48) were interpreted with 100% accuracy. The PAD integrated with customized-region convolutional neural networks can reduce the interpretation discrepancies in RBC antigen phenotyping in any laboratory.

A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow μPADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model μPAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 μg mL−1.

A novel ultra‐low‐cost biochemical analysis platform to quantify protein dissociation binding constants and kinetics using paper microfluidics is reported. This approach marries video imaging with one of humankind's oldest materials: paper, requiring no large, expensive laboratory equipment, complex microfluidics or external power. Temporal measurements of nanoparticle–antibody conjugates binding on paper is found to follow the Langmuir Adsorption Model. This is exploited to measure a series of antibody–antigen dissociation constants on paper, showing excellent agreement with a gold‐standard benchtop interferometer. The concept is demonstrated with a camera and low‐end smartphone, 500‐fold cheaper than the reference method, and can be multiplexed to measure ten reactions in parallel. These findings will help to widen access to quantitative analytical biochemistry, for diverse applications spanning disease diagnostics, drug discovery, and environmental analysis in resource‐limited settings.

Branched zinc oxide nanorods (BZR) arrays, an array with high charge carries collection efficiency and specific surface area, are grown on the reduced graphene oxide-paper working electrode for the first time to construct a paper-based electrochemical (EC) immunosensor. Typically, the BZR are fabricated via a simple hydrothermal process, which can provide abundant sites for antibodies loading. By combining the large surface area of porous zinc oxide (PZS) and good biocompatibility of gold nanoparticles (AuNPs), PZS–AuNPs (PZS@Au) nanocomposites are designed to label horseradish peroxide (HRP) and antigens. After a competitive reaction between antigens and PZS@Au nanocomposites labeled antigens, the signal labels are introduced into the immunosensor, in which, HRP participate in biocatalytic precipitation process. The produced precipitate reduces the electrode surface area and hinders the electron transfer. With the increase of concentration of antigens, the signal labels introduced into the sensor decrease, thus, a signal-on immunoassay for α-fetoprotein detection is constructed. The proposed paper-based EC immunosensor combines enzymatic biocatalytic precipitation reaction and competitive immunoassay mode for the first time, and possesses a wide linear range from 0.2 pg mL −1 to 500 ng mL −1 with a detection limit of 0.08 pg mL −1 . In addition, the proposed method is simple, sensitive and specific and can be a promising platform for other protein detection.

“Immune normalization” has emerged as a new paradigm in immunotherapy, which is proposed in cancer patients instead of conventional “immune-enhancement” therapy. Immune normalization may also be implemented in cancer prevention of “sub-healthy” individuals. We established in vitro cultured mixed-natural killer (NKM) cells to achieve immune normalization. The in vitro cytotoxicity of NKM cells was tenfold higher than that of peripheral blood mononuclear cells (PBMCs). The cytotoxicity of NKM cells was negatively correlated with the proportion of T-helper cells (cluster of differentiation: CD3+CD4+ T), and positively correlated with the proportion of NK cells (especially CD56brightCD16bright NK cells). Then, we defined “sub-healthy individuals” after measuring Programmed cell death protein-1 (PD-1) expression in PBMCs from 95 donors aged > 50 years. Furthermore, we evaluated the potential clinical application of NKM-cell therapy in 11 patients with malignant lymphoma, one patient with pancreatic cancer, and four sub-healthy individuals. NKM-cell therapy elicited good tolerance and side-effects were not found. In sub-healthy individuals, the proportion of CD3+PD-1+ T cells and CD3+CD8+PD-1+ T cells was reduced significantly after NKM-cell treatment. We demonstrated that a new method using NKM cells was safe and efficacious as adjuvant treatment for cancer patients as well as therapy for sub-healthy individuals. Normalization of the peripheral immune system through NKM-cell therapy could expand its scope of application in different disorders.

Supernumerary centrioles are frequently observed in diverse types of cancer cells. In this study, we investigated the mechanism underlying the generation of supernumerary centrioles during the M phase. We generated the TP53;PCNT;CEP215 triple knockout (KO) cells and determined the configurations of the centriole during the cell cycle. The triple KO cells exhibited a precocious separation of centrioles and unscheduled centriole assembly in the M phase. Supernumerary centrioles in the triple KO cells were present throughout the cell cycle; however, among all the centrioles, only two maintained an intact composition, including CEP135, CEP192, CEP295 and CEP152. Intact centrioles were formed during the S phase and the rest of the centrioles may be generated during the M phase. M-phase-assembled centrioles lacked the ability to organize microtubules in the interphase; however, a fraction of them may acquire pericentriolar material to organize microtubules during the M phase. Taken together, our work reveals the heterogeneity of the supernumerary centrioles in the triple KO cells. .

Rationale: Therapeutic antibody conjugates allow for the specific delivery of cytotoxic agents or immune cells to tumors, thus enhancing the antitumor activity of these agents and minimizing adverse systemic effects. Most current antibody conjugates are prepared by nonspecific modification of antibody cysteine or lysine residues, inevitably resulting in the generation of heterogeneous conjugates with limited therapeutic efficacies. Traditional strategies to prepare homogeneous antibody conjugates require antibody engineering or chemical/enzymatic treatments, processes that often affect antibody folding and stability, as well as yield and cost. Developing a simple and cost-effective way to precisely couple functional payloads to native antibodies is of great importance. Methods: We describe a simple proximity-induced antibody conjugation method (pClick) that enables the synthesis of homogeneous antibody conjugates from native antibodies without requiring additional antibody engineering or post-synthesis treatments. A proximity-activated crosslinker is introduced into a chemically synthesized affinity peptide modified with a bioorthogonal handle. Upon binding to a specific antibody site, the affinity peptide covalently attaches to the antibody via spontaneous crosslinking, yielding an antibody molecule ready for bioorthogonal conjugation with payloads. Results: We have prepared well-defined antibody-drug conjugates and bispecific small molecule-antibody conjugates using pClick technology. The resulting conjugates exhibit excellent in vitro cytotoxic activity against cancer cells and, in the case of bispecific conjugates, superb antitumor activity in mouse xenograft models. Conclusions: Our pClick technology enables efficient, simple, and site-specific conjugation of various moieties to the existing native antibodies. This technology does not require antibody engineering or additional UV/chemical/enzymatic treatments, therefore providing a general, convenient strategy for developing novel antibody conjugates.

Background: Multiple myeloma (MM) is incurable in spite of recent treatment improvements, highlighting the development of new therapies. Chimeric antigen receptor (CAR) T-cell therapy has dramatically changed the therapeutic effectiveness in high-risk B-cell malignancies. For relapsed/refractory multiple myeloma (RRMM), preclinical evaluations of CAR-T therapy have shown promising efficacy, thus various active clinical trials are under way. Herein, we conducted this review to summarize efficacy and safety of CAR-T therapy and provide more evidence to guide clinical treatments. Method: We systematically searched literature based on databases (PubMed, EMBASE, Cochrane Central Register of Controlled Trials), and conference abstracts reported from American Society of Hematology (ASH), European Hematology Association (EHA) and American Society of Clinical Oncology (ASCO), in addition to other sources (www.clinicaltrials.gov, article citations). Data assessed efficacy and safety of CAR-T therapy in patients with RRMM were extracted and evaluated, and then systematically analyzed by Comprehensive Meta-analysis 3.0 (CMA 3.0). Results: A total of 23 studies including 350 participants from different countries, diagnosed as RRMM and treated with CAR-T therapy (containing 7 antigens targeted by CARs) were combined. In summary, we discovered the pooled overall response rate (77%), complete response rate (37%) and minimal residual disease (MRD) negativity rate within responders (78%). Furthermore, the pooled relapse rate of responders was 38% and median progression-free survival was 8 months. The pooled survival rate was 87% at last follow-up (median, 12 months). In addition, the pooled grade 3-4 rates of cytokine release syndrome (CRS) and neurologic toxicities (NT) were 14% and 13%, respectively. Conclusion: Our study suggests that CAR-T therapy has demonstrated efficacy and safety in RRMM patients. BCMA-targeted CAR-T and anti-BCMA contained regimen have shown better efficacy.

In vivo tracking of dendritic cell (DC) migration to the lymphatic system is essential for evaluating the outcome of DC-based immunotherapies. Novel multimodal imaging strategies with high analytical performance are urgently needed to supply complementary information about the migration and colonization of DCs. In this study, we designed a bimodal imaging agent, namely Au@Prussian blue-Gd@ovalbumin nanoparticles (APG@OVA NPs), for activating DCs and real-time tracking of DC migration process by magnetic resonance imaging (MRI). Moreover, the distribution of the colonized DCs in the lymphatic system was profiled at the single-cell levels based on surface-enhanced Raman scattering (SERS) technique. Methods: In this strategy, PBs as cyanide (CN)-bridged coordination blocks were assembled onto the gold nanoparticles core to provide SERS signal in the Raman-silent region (1800 and 2800 cm-1), which could avoid background signal interference. The doping Gd3+ located in the lattice of PB enables the MRI ability with high relaxivity of the probe. Ovalbumin, an egg allergen, was used as an antigen to activate DCs due to its immunological properties. The prepared APG@OVA NP agents were used to activate DCs with high efficacy and to track their migration and distribution in vivo through SERS/MR bimodal imaging. Results: The APG@OVA NP agents could not only enable DC activating and labeling, but also achieve real-time monitoring of DC migration in vivo and accurate profiling of DC distribution in the lymphatic system. MR imaging indicated the time-dependent migration of the APG@OVA NP-labeled DCs from the footpad to the sentinel lymph node. The background-free Raman mapping of the lymph node tissue slice demonstrated that the activated DCs have successfully colonized to the sentinel lymph node. Conclusion: Concerning the high activating efficacy, dual complementary imaging readouts, and low biological toxicity, the APG@OVA NPs act as high-performance tracking agents for DC-based immunotherapies.

In the ongoing COVID-19 pandemic, simple, rapid, point-of-care tests not requiring trained personnel for primary care testing are essential. Saliva-based antigen rapid tests (ARTs) can fulfil this need, but these tests require overnight-fasted samples; without which independent studies have demonstrated sensitivities of only 11.7 to 23.1%. Herein, we report an Amplified Parallel ART (AP-ART) with sensitivity above 90%, even with non-fasted samples. The virus was captured multimodally, using both anti-spike protein antibodies and Angiotensin Converting Enzyme 2 (ACE2) protein. It also featured two parallel flow channels. The first contained spike protein binding gold nanoparticles which produced a visible red line upon encountering the virus. The second contained signal amplifying nanoparticles that complex with the former and amplify the signal without any linker. Compared to existing dual gold amplification techniques, a limit of detection of one order of magnitude lower was achieved (0.0064 ng·mL–1). AP-ART performance in detecting SARS-CoV-2 in saliva of COVID-19 patients was investigated using a case–control study (139 participants enrolled and 162 saliva samples tested). Unlike commercially available ARTs, the sensitivity of AP-ART was maintained even when non-fasting saliva was used. Compared to the gold standard reverse transcription-polymerase chain reaction testing on nasopharyngeal samples, non-fasting saliva tested on AP-ART showed a sensitivity of 97.0% (95% CI: 84.7–99.8); without amplification, the sensitivity was 72.7% (95% CI: 83.7–94.8). Thus, AP-ART has the potential to be developed for point-of-care testing, which may be particularly important in resource-limited settings, and for early diagnosis to initiate newly approved therapies to reduce COVID-19 severity. Graphical abstract

India is in the grip of a devastating second wave of Covid-19. Many experts believe new variants of concern (VOC) are behind this unprecedented surge. Some media reports are hinting toward higher risk of Covid infection following Covid vaccinations. 'Original antigenic sin,' in which a prior exposure to an antigen leads to an ineffective response to a related antigen, may offer one immunological explanation for this unusual association. There is an urgent need of undertaking a detailed study to prove/disprove this association.

Air pollution might affect atherosclerosis through DNA methylation changes in cells crucial to atherosclerosis, such as monocytes. We conducted an epigenome-wide study of DNA methylation in CD14+ monocytes and long-term ambient air pollution exposure in adults participating in the Multi-Ethnic Study of Atherosclerosis (MESA). We also assessed the association between differentially methylated signals and cis-gene expression. Using spatiotemporal models, one-year average concentrations of outdoor fine particulate matter (PM2.5) and oxides of nitrogen (NOX) were estimated at participants��� homes. We assessed DNA methylation and gene expression using Illumina 450k and HumanHT-12 v4 Expression BeadChips, respectively (n = 1,207). We used bump hunting and site-specific approaches to identify differentially methylated signals (false discovery rate of 0.05) and used linear models to assess associations between differentially methylated signals and cis-gene expression. Four differentially methylated regions (DMRs) located on chromosomes 5, 6, 7, and 16 (within or near SDHAP3, ZFP57, HOXA5, and PRM1, respectively) were associated with PM2.5. The DMRs on chromosomes 5 and 6 also associated with NOX. The DMR on chromosome 5 had the smallest p-value for both PM2.5 (p = 1.4��10���6) and NOX (p = 7.7��10���6). Three differentially methylated CpGs were identified for PM2.5, and cg05926640 (near TOMM20) had the smallest p-value (p = 5.6��10���8). NOX significantly associated with cg11756214 within ZNF347 (p = 5.6��10���8). Several differentially methylated signals were also associated with cis-gene expression. The DMR located on chromosome 7 was associated with the expression of HOXA5, HOXA9, and HOXA10. The DMRs located on chromosomes 5 and 16 were associated with expression of MRPL36 and DEXI, respectively. The CpG cg05926640 was associated with expression of ARID4B, IRF2BP2, and TOMM20. We identified differential DNA methylation in monocytes associated with long-term air pollution exposure. Methylation signals associated with gene expression might help explain how air pollution contributes to cardiovascular disease.

Streptococcus suis bacteria are one of the most serious health problems for pigs and an emerging zoonotic agent in humans working in the swine industry. S. suis bacteria express capsular polysaccharides (CPS) a major bacterial virulence factor that define the serotypes. Oligosaccharides resembling the CPS of S. suis serotypes 2, 3, 9, and 14 have been synthesized, glycans related to serotypes 2 and 9 were placed on glycan array surfaces to screen blood from infected pigs. Lead antigens for the development of semi‐synthetic S. suis serotypes 2 and 9 glycoconjugate veterinary vaccines were identified in this way., 30 novel oligosaccharides resembling the capsular polysaccharides related to four major serotypes (2, 3, 9, 14) of Streptococcus suis (S. suis) were synthesized. The conjugation‐ready glycans were used to screen the sera of pigs and rabbits. With a glycan array study, glycan epitopes of lead antigens for the development of semi‐synthetic glycoconjugate vaccines to protect from S. suis serotypes 2 and 9 were identified.

The effect of Toxocara canis antigens on cytokine production by human THP-1 macrophages was studied in vitro. Toxocara Excretory-Secretory products (TES) and recombinant mucins (Tc-MUC-2, Tc-MUC-3, Tc-MUC-4, and Tc-MUC-5) as well as deglycosylated forms of these antigens were used in the study. TES products stimulated macrophages to produce the innate proinflammatory IL-1β, IL-6, and TNF-α cytokines regardless of the presence of glycans. Recombinant mucins induced glycan-dependent cytokine production. Sugar moieties led to at least 3-fold higher production of regulatory IL-10 as well as proinflammatory cytokines. The presence of glycans on mucins also affected the downstream signalling pathways in stimulated cells. The most prominent difference was noted in AKT and AMPK kinase activation. AKT phosphorylation was observed in cells stimulated with glycosylated mucins, whereas treatment with deglycosylated antigens led to AMPK phosphorylation. MAP kinase family members such as JNK and p38 and c-Jun transcription factor were phosphorylated in both cases what suggests that toll-like receptor signalling may be involved in mucin-treated macrophages. This pathway is however modified by other signalling molecules as only mucins containing intact sugars significantly induced the production of cytokines.

This longitudinal comparative study investigated the effect of preventive chemotherapy (PC) on covert tissue changes associated with lymphatic filariasis (LF) among young people living in an LF-endemic area in Myanmar.Tissue compressibility and extracellular free fluid in the lower limbs of people aged 10-21 years were measured using indurometry and bioimpedance spectroscopy (BIS). Baseline measures were taken in October 2014, annual mass drug administration (MDA) of PC was delivered in December, and in March 2015 further PC was offered to LF-positive cases who had missed MDA. Follow-up measures were taken in February and June 2015.A total of 50 antigen-positive cases and 46 antigen-negative controls were included. Self-reported PC consumption was 60.1% during 2014 MDA and 66.2% overall. At second follow-up, 24 of 34 cases and 27 of 43 controls had consumed PC. Significant and clinically relevant between-group differences at baseline were not found post-PC. Bayesian linear mixed models showed a significant change in indurometer scores at both calves for antigen-positive cases who consumed any PC (dominant calf: -0.30 [95% CI -0.52, -0.07], P 0.05 and non-dominant calf: -0.35 [95% CI -0.58, -0.12], P 0.01). Changes in antigen-negative participants or those not consuming PC were not significant.This study is the first attempt to use simple field-friendly tools to track fluid and tissue changes after treatment of asymptomatic people infected with LF. Results suggested that PC alone is sufficient to reverse covert lymphatic disturbance. Longer follow-up of larger cohorts is required to confirm these improvements and whether they persist over time. These findings should prompt increased efforts to overcome low PC coverage, which misses many infected young people, particularly males, who are unaware of their infection status, unmotivated to take PC and at risk of developing lymphoedema. Indurometry and BIS should be considered in assessment of lymphatic filariasis-related lymphedema.Cette étude comparative longitudinale a investigué l'effet de la chimiothérapie préventive (CP) sur les modifications tissulaires cachées associées à la filariose lymphatique (FL) chez les jeunes vivant dans une zone d'endémie pour la FL au Myanmar. MÉTHODES: La compressibilité des tissus et le liquide libre extracellulaire dans les membres inférieurs des personnes âgées de 10 à 21 ans ont été mesurés par indurométrie et spectroscopie de bioimpédance (BIS). Les mesures de base ont été prises en octobre 2014, la distribution en masse de médicament (DMM) annuelle a été administrée en décembre et en mars 2015, et une CP additionnelle a été offerte aux cas positifs pour la FL qui avaient manqué la DMM. Des mesures de suivi ont été prises en février et juin 2015. RÉSULTATS: 50 cas positifs pour l'antigène et 46 témoins négatifs ont été inclus. L'administration de CP auto-déclarée était de 60,1% durant la DMM de 2014 et de 66,2% au total. Au deuxième suivi, 24 des 34 cas et 27 des 43 témoins avaient pris la CP. Des différences significatives et cliniquement pertinentes entre les groupes au départ n'ont pas été trouvées après la CP. Les modèles mixtes linéaires bayésiens ont montré un changement significatif des scores d'indurometrie aux deux mollets pour les cas positifs pour l'antigène qui prenaient une CP (mollet dominant: -0,30 [IC95%: -0,52, -0,07], p0,05, mollet non dominant: - 0,35 [IC95%: -0,58, -0,12], p0,01). Les changements chez les participants négatifs pour l'antigène ou ceux qui ne prenaient pas de CP n’étaient pas significatifs.Cette étude est la première tentative d'utilisation d'outils simples, conviviaux sur le terrain, pour suivre les modifications du tissu conjonctif après le traitement de personnes asymptomatiques infectées par la FL. Les résultats suggèrent que la CP seule est suffisante pour inverser les modifications lymphatiques cachées. Un suivi plus long de plus grandes cohortes est nécessaire pour confirmer ces améliorations et déterminer si elles persistent ou non. Ces résultats devraient inciter à redoubler d'efforts pour surmonter la faible couverture en CP, qui rate beaucoup de jeunes infectés, en particulier les hommes, qui ne sont pas au courant de leur statut d'infection, qui ne sont pas motivés pour prendre une CP et risquent de développer un lymphœdème. L'indurométrie et la BIS devraient être considérées dans l’évaluation du lymphoedème associé à la filariose lymphatique.

Background Sleep Disordered Breathing (SDB) is associated with a wide range of pathophysiological changes due, in part, to hypoxemia during sleep. We sought to identify gene transcription associations with measures of SDB and hypoxemia during sleep, and study their response to treatment. Methods In two discovery cohorts, Framingham Offspring Study (FOS; N = 571) and the Multi-Ethnic Study of Atherosclerosis (MESA; N = 580), we studied gene expression in peripheral blood mononuclear cells in association with three measures of SDB: Apnea Hypopnea Index (AHI); average oxyhemoglobin saturation (avgO2) during sleep; and minimum oxyhemoglobin saturation (minO2) during sleep. Associated genes were used for analysis of gene expression in the blood of 15 participants with moderate or severe obstructive sleep apnea (OSA) from the Heart Biomarkers In Apnea Treatment (HeartBEAT) trial. These genes were studied pre- and post-treatment (three months) with continuous positive airway pressure (CPAP). We also performed Gene Set Enrichment Analysis (GSEA) on all traits and cohort analyses. Findings Twenty-two genes were associated with SDB traits in both MESA and FOS. Of these, lower expression of CD1D and RAB20 was associated with lower avgO2 in MESA and FOS. CPAP treatment increased the expression of these genes in HeartBEAT participants. Immunity and inflammation pathways were up-regulated in subjects with lower avgO2; i.e., in those with a more severe SDB phenotype (MESA), whereas immuno-inflammatory processes were down-regulated following CPAP treatment (HeartBEAT). Interpretation Low oxygen saturation during sleep is associated with alterations in gene expression and transcriptional programs that are partially reversed by CPAP treatment.

[Background and Purpose]: The humoral immune response in cancer patients can be used for early detection of the disease. Autoantibodies raised against tumor-associated antigens (TAAs) are promising clinical biomarkers for reliable cancer diagnosis, prognosis, and therapy monitoring. In this study, an electrochemical disposable multiplexed immunosensing platform able to integrate difficult- and easy-to-express colorectal cancer (CRC) TAAs is reported for the sensitive determination of eight CRC-specific autoantibodies., [Methods]: The electrochemical immunosensing approach involves the use of magnetic microcarriers (MBs) as solid supports modified with covalently immobilized HaloTag fusion proteins for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric responses measured using the hydroquinone (HQ)/H2O2 system were related to the levels of autoantibodies in plasma., [Results]: The biosensing platform was applied to the analysis of autoantibodies against 8 TAAs described for the first time in this work in plasma samples from healthy asymptomatic individuals (n=3), and patients with high-risk of developing CRC (n=3), and from patients already diagnosed with colorectal (n=3), lung (n=2) or breast (n=2) cancer. The developed bioplatform demonstrated an improved discrimination between CRC patients and controls (asymptomatic healthy individuals and breast and lung cancer patients) compared to an ELISA-like luminescence test., [Conclusions]: The proposed methodology uses a just-in-time produced protein in a simpler protocol, with low sample volume, and involves cost-effective instrumentation, which could be used in a high-throughput manner for reliable population screening to facilitate the detection of early CRC patients at affordable cost., This work was supported by the financial support of the PI17CIII/00045 grant from the AES-ISCIII program to R.B., cofounded by FEDER funds. The financial support of the CTQ2015-64402-C2-1-R (Spanish Ministerio de Economía y Competitividad) and RTI2018-096135-B-I00 (Ministerio de Ciencia, Innovación y Universidades) Research Projects and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) to P.Y-S., S.C. and JM.P are gratefully acknowledged. G.D. acknowledges the financial support of PI15/00246 grant of the FIS and Cátedra UAM-Roche en Medicina de Innovación. M.G-A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. A.M-C. is a recipient of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte. G.S-F. is recipient of a predoctoral contract (grant number 1193818N) supported by The Flanders Research Foundation (FWO). Predoctoral contracts from the Spanish Ministerio de Economía y Competitividad (E.P.) and Universidad Complutense de Madrid (V.R.V-M.) are also gratefully acknowledged.

The fetal isoform A of the insulin receptor (IR-A) is frequently overexpressed in a variety of malignancies including breast cancer. IR overexpression has a recognized role in cancer progression and resistance to anticancer therapies. In particular, IR-A has a peculiar mitogenic potential and is activated not only by insulin but also by IGF-2. Previously, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis. We now evaluated whether DDR1 is able to exert a role in breast cancer biology by functionally cross-talking with IR. In MCF-7 human breast cancer cells, IR and DDR1 co-immunoprecipitated and co-localized after insulin or IGF-2 stimulation. In a panel of breast cancer cells, DDR1 knockdown by specific siRNAs markedly inhibited IR downstream signaling as well as proliferation, migration and colony formation in response to insulin and IGF-2. These effects were accompanied by reduction of IR protein and mRNA expression, which involved both transcriptional and post-transcriptional effects. DDR1 overexpression elicited opposite effects. Bioinformatics analysis of public domain databases showed that IR and DDR1 co-expression significantly correlates with several clinically relevant histopathological and molecular features of human breast carcinomas. These findings demonstrate that, in human breast cancer cells, DDR1 regulates IR expression and ligand dependent biological actions. This novel functional crosstalk is likely clinically relevant and may become a new molecular target in breast cancer.

// Yaomei Tian 1, * , Meng Li 2, * , Chaoheng Yu 1 , Rui Zhang 1 , Xueyan Zhang 1 , Rong Huang 1 , Lian Lu 1 , Fengjiao Yuan 1 , Yingzi Fan 1 , Bailing Zhou 1 , Ke Men 1 , Heng Xu 1 and Li Yang 1 1 State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, Sichuan, China 2 Chengdu Blood Center, Chengdu, Sichuan, China * These authors contributed equally to this work Correspondence to: Li Yang, email: yl.tracy73@gmail.com Keywords: combinatorial adjuvant, aluminum salts, CpG oligodeoxynucleotide, innate defense regulator peptide HH2, anti-tumor immune responses Received: December 07, 2016 Accepted: April 02, 2017 Published: April 28, 2017 ABSTRACT Single-component adjuvant is prone to eliciting a specific type of Th1 or Th2 response. So, the development of combinatorial adjuvants inducing a robust mixed Th1/Th2 response is a promising vaccination strategy against cancer. Here, we describe a novel combination of aluminum salts (alum), CpG oligodeoxynucleotide (CpG) and innate defense regulator peptide HH2 for improving anti-tumor immune responses. The CpG-HH2 complex significantly enhanced the production of IFN-γ, TNF-α and IL-1β, promoted the uptake of antigen and strengthened the activation of p38, Erk1/2 and NF-κB in vitro , compared to CpG or HH2 alone. Immunization with NY-ESO-1 antigen plus alum-CpG-HH2 combinatorial adjuvant effectively inhibited tumor growth and reduced tumor burden in prophylactic and therapeutic tumor models and even in passive serum or cellular therapy. In addition, co-administration of NY-ESO-1 with alum-CpG-HH2 combinatorial adjuvant markedly activated NK cell cytotoxicity, induced antibody-dependent cellular cytotoxicity (ADCC), dramatically elicited cytotoxic T lymphocytes (CTLs) response, and increased infiltrating lymphocytes in tumors. Moreover, in vivo depletion of CD8 + T cells completely and depletion of NK cells partially blocked the anti-tumor activity of NY-ESO-1-alum-CpG-HH2 immunization. Overall, our results demonstrate a novel adjuvant combination for cancer vaccine with efficient immunomodulation by stimulating innate immunity and mediating adaptive immunity.

Summary Global eradication of the guinea worm (Dracunculus medinensis) is near, although perhaps delayed a little by the discovery of a transmission cycle in dogs. It is therefore an appropriate time to reflect on the severe impact of this infection on the life of the communities where it was endemic prior to the start of the global eradication programme in 1981. From 1971 to 1974, we conducted a series of unpublished studies on guinea worm in a group of villages in Katsina State, northern Nigeria, where the infection was highly endemic. These studies demonstrated the high rate of infection in affected communities, the frequent recurrence of the infection in some subjects and the long‐standing disability that remained in some infected individuals. Immunological studies showed a high level of immediate hypersensitivity to adult worm and larval antigens but a downregulation of Th1‐type T‐cell responses to worm antigens. Freeing communities such as those described in this article from the scourge of guinea worm infection for good will be an important public health triumph.

Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II-containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4+ T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4+ T cell stimulation.

Poor distribution of antigen/adjuvant to target sites and inadequate induction of T cell responses remain major challenges in cancer immunotherapy because of the lack of appropriate delivery systems. Nanocarrier-based antigen delivery systems have emerged as an innovative strategy to improve vaccine efficacy. Here we present polymeric hybrid micelles (PHMs) as a simple and potent antigen/adjuvant co-delivery system with highly tunable properties. PHMs consist of two amphiphilic diblock copolymers, polycaprolactone-polyethylenimine (PCL-PEI) and polycaprolactone-polyethyleneglycol (PCL-PEG). PHMs with different proportions of cationic PCL-PEI were prepared and loaded with tyrosinase-related protein 2 (Trp2) peptide and adjuvant CpG oligodeoxynucleotide to generate the Trp2/PHM/CpG co-delivery system. Lymphatic and intracellular antigen delivery as a function of PCL-PEI proportion was evaluated in vitro and in vivo. PHMs containing 10% (w/w) PCL-PEI (Trp2/PHM10/CpG) showed the optimal balance of good distribution to lymph nodes, strong immunization effect after subcutaneous administration, and low toxicity to dendritic cells. In a mouse model of B16F10 melanoma, Trp2/PHM10/CpG showed significantly higher antigen-specific cytotoxic T lymphocyte activity and greater anticancer efficacy than Trp2/PHM0/CpG without PCL-PEI or a mixture of free Trp2 and CpG. These results provide new insights into how cationic segments affect the efficiency of antigen delivery by cationic nanocarriers. They also suggest that PHMs can serve as a structurally simple and highly tunable platform for co-delivery of antigen and adjuvant in cancer immunotherapy.

Dendritic cells (DCs) are the most potent specialized antigen-presenting cells as now known, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses. Immunologically, the motilities and T cell activation capabilities of DCs are closely related to the resulting immune responses. However, due to the complexity of the immune system, the dynamic changes in the number of cells during the peripheral tissue (e.g. skin and mucosa) immune response induced by DCs are still poorly understood. Therefore, this study simulated dynamic number changes of DCs and T cells in this process by constructing several ordinary differential equations and setting the initial conditions of the functions and parameters. The results showed that these equations could simulate dynamic numerical changes of DCs and T cells in peripheral tissue and lymph node, which was in accordance with the physiological conditions such as the duration of immune response, the proliferation rates and the motilities of DCs and T cells. This model provided a theoretical reference for studying the immunologic functions of DCs and practical guidance for the clinical DCs-based therapy against immune-related diseases.

Purpose Tryptophan’s (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. Methods Selective Trp oxidation was induced by 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. Results Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. Conclusions Trp oxidation in CDRs can adversely impact mAbs’ conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product. Electronic supplementary material The online version of this article (10.1007/s11095-018-2545-8) contains supplementary material, which is available to authorized users.

// Hui Li 1, * , Dongsheng Yue 1, 2, * , Joy Q. Jin 1 , Gavitt A. Woodard 1 , Bhairavi Tolani 1 , Thomas M. Luh 1 , Etienne Giroux-Leprieur 1 , Minli Mo 3 , Zhao Chen 3 , Juanjuan Che 4 , Zhenfa Zhang 2 , Yong Zhou 1 , Lei Wang 1, 5 , Xishan Hao 2 , David Jablons 1 , Changli Wang 2 , Biao He 1 1 Thoracic Oncology Program, Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94115, USA 2 Department of Lung Cancer, Lung Cancer Center, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China 3 Beijing ACCB Biotech Ltd., Beijing 100084, China 4 Department of Oncology, Beijing Friendship Hospital of Capital Medical University, Beijing 100050, China 5 Department of Thoracic Surgery, Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, China * These authors have contributed equally to this work Correspondence to: Biao He, email: biao.he@ucsfmedctr.org Changli Wang, email: yuedongsheng_cg@163.com Keywords: sonic hedgehog, gli, epithelial-mesenchymal transition, lung cancer, adenocarcinoma Received: May 09, 2016 Accepted: July 28, 2016 Published: August 12, 2016 ABSTRACT Adenocarcinoma is the most common type of lung cancer. Epithelial-mesenchymal transition (EMT) is required for tumor invasion/metastasis and the components that control this process are potential therapeutic targets. This study we examined the role of Gli in lung adenocarcinoma and whether its activation regulates metastasis through EMT in lung adenocarcinoma. We found that tumors with high Gli expression had significantly lower E-Cadherin expression in two independent cohorts of patients with lung adenocarcinoma that we studied. In vitro up-regulation of SHh resulted in increased cell migration while small molecule inhibitors of Smo or Gli significantly reduced cell mobility both in a wound healing assay and in a 3D cell invasion assay. Inhibition of Gli in vivo decreased tumor growth and induced an increase in E-Cadherin expression. Our results indicate that Gli may be critical for lung adenocarcinoma metastasis and that a novel Gli inhibitor shows promise as a therapeutic agent by preventing cell migration and invasion in vitro and significantly reducing tumor growth and increasing E-Cadherin expression in vivo .

// Nan Meng 1 , Min Xia 2 , Ya-Qi Lu 1 , Mi Wang 1 , Krishna M. Boini 2 , Pin-Lan Li 2 and Wang-Xian Tang 1 1 Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2 Department of Pharmacology and Toxicology, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA Correspondence to: Wang-Xian Tang, email: // Keywords : NLRP3 inflammasome, hepatic stellate cell, cathepsin B, Schistosoma J., Pathology Section Received : February 23, 2016 Accepted : June 01, 2016 Published : June 14, 2016 Abstract The major pathological changes during Schistosoma J. infection are characterized by granulomatous inflammation in the liver, a cellular immune response to schistosomal egg antigens. The molecular mechanisms initiating or promoting this schistosomal granulomatous inflammation remain poorly understood. In the present study, we first demonstrated that in mice infected with Schistosoma J . for 6 weeks exhibited increased levels of IL-1β in liver, a major product of NLRP3 inflammasomes and collagen deposition around the eosinophilic granuloma with Schistosoma J. eggs, which was substantially attenuated by caspase-1 inhibitor, YVAD. This activation of the NLRP3 inflammasome occurred in hepatic stellate cells (HSCs), as shown by a marked increase in co-localization of IL-1β with HSCs marker, desmin. Using isolated, cultured mouse HSCs, we further explored the mechanisms by which soluble egg antigen (SEA) from Schistosoma J. activates NLRP3 inflammasomes. SEA induced the formation and activation of NLRP3 inflammasomes, which was associated with both redox regulation and lysosomal dysfunction, but not with potassium channel activation. These results suggest that NLRP3 inflammasome activation in HSCs may serve as an early mechanism to turn on the inflammatory response and thereby instigate liver fibrosis during Schistosoma J. infection.

Aged subjects display increased susceptibility to mucosal diseases. Retinoic Acid (RA) plays a major role in inducing tolerance in the mucosa. RA acts on Dendritic cells (DCs) to induce mucosal tolerance. Here we compared the response of DCs from aged and young individuals to RA with a view to understand the role of DCs in age-associated increased susceptibility to mucosal diseases. Our investigations revealed that compared to young DCs, RA stimulated DCs from aged subjects are defective in inducing IL-10 and T regulatory cells. Examinations of the underlying mechanisms indicated that RA exposure led to the upregulation of CD141 and GARP on DCs which rendered the DCs tolerogenic. CD141(hi), GARP(+) DCs displayed enhanced capacity to induce T regulatory cells compared to CD141(lo) and GARP(-) DCs. Unlike RA stimulated DCs from young, DCs from aged subjects exhibited diminished upregulation of both CD141 and GARP. The percentage of DCs expressing CD141 and GARP on RA treatment was significantly reduced in DCs from aged individuals. Furthermore, the remaining CD141(hi), GARP(+) DCs from aged individuals were also deficient in inducing T regs. In summary, reduced response of aged DCs to RA enhances mucosal inflammation in the elderly, increasing their susceptibility to mucosal diseases.

Distribution of the isoelectric point (pI) was calculated for the hypervariable regions of Fab fragments of the antibody molecules, which structure is annotated in the structural antibody database SabDab. The distribution is consistent with the universal for all organisms dividing the proteome into two sets of acidic and basic proteins. It shows the additional fine structure in a form of the narrow-sized peaks of pI values. This is an explanation why a small change of the environmental pH can have a strong effect on the antibody-antigen affinity. To show this, a typical enzyme-linked immunospecific assay experiment for testing the reaction of goat anti-human IgA antibodies with human IgA immunoglobulins of saliva as antigens was modified in such a way that Fe3O4 magnetic nanoparticles were added to PBS buffer. The magnetic nanoparticles were remotely heated by the radio frequency magnetic field providing the local change of temperature and pH. It was observed that short times of the heating were significantly increasing the antibody-antigen binding strength while it was not the case for a longer time. The finding discussed in the study can be useful for biopharmaceuticals using antibodies, the immunoassay techniques as well as for control over the use of hyperthermia.

Targeting specifically primary prostate cancer (PCa) cells for immune therapy, gene therapy or molecular imaging is of high importance. The PCA3 long non-coding RNA is a unique PCa biomarker and oncogene that has been widely studied. This gene has been mainly exploited as an accurate diagnostic urine biomarker for PCa detection. In this study, the PCA3 promoter was introduced into a new transcriptional amplification system named the 3-Step Transcriptional Amplification System (PCA3-3STA) and cloned into type 5 adenovirus. PCA3-3STA activity was highly specific for PCa cells, ranging between 98.7- and 108.0-fold higher than that for benign primary prostate epithelial or non-PCa cells, respectively. In human PCa xenografts, PCA3-3STA displayed robust bioluminescent signals at levels that are sufficient to translate to positron emission tomography (PET)-based reporter imaging. Remarkably, when freshly isolated benign or cancerous prostate biopsies were infected with PCA3-3STA, the optical signal produced from primary PCa biopsies was significantly higher than from benign prostate biopsies (4.4-fold, p < 0.0001). PCA3-3STA therefore represents a PCa-specific expression system with the potential to target, with high accuracy, primary or metastatic PCa epithelial cells for imaging, vaccines, or gene therapy.

Epidermal growth factor receptor (EGFR)-mutation-positive non-small cell lung cancer (NSCLC) is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs). We investigated receptor tyrosine kinases (RTKs), Src family kinases and focal adhesion kinase (FAK) as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2) of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2) inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1) was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p = 0.0407) and overall survival (hazard ratio of 2.23, p = 0.0192). A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing., Graphical Abstract Unlabelled Image, Highlights • AXL and CDCP1 are co-expressed in treatment-naïve EGFR-mutation-positive NSCLC patients. • AXL and CDCP1 are related to shorter progression-free survival with EGFR inhibitors and shorter overall survival. • Src family kinases and YAP1 are regulatory nodes for AXL and CDCP1 expression. • The combination of EGFR TKI with TPX-0005 is synergistic in EGFR-mutation-positive lung tumors in culture and in vivo. We explore the molecular changes that occur after the application of an EGFR inhibitor in EGFR-mutation positive tumors. The tumors do not acquire secondary drivers to overcome a primary driver but, counter-regulatory nodes observable before treatment, are immediately made apparent by pathway-specific intervention. The expression of the receptor tyrosine kinase AXL and the transmembrane protein CDCP1 in baseline samples of EGFR-mutation positive NSCLC patients can provide us with information on the treatment outcome. The upfront combination of an EGFR inhibitor with a multikinase inhibitor, that controls the regulatory nodes for RTKs activation, is a therapeutic approach that deserves to be further explored.

A single donor-specific transfusion induces alloantibodies and Treg cells, Donor-specific blood transfusion is known to induce alloresponses and lead to immunosuppression. We examined their underlying mechanisms by employing fully allogeneic rat combinations. Transfused recipients efficiently produced alloantibodies of the IgM and IgG subclasses directed against donor class I MHC. The recipients exhibited active expansion of CD4+ T cells and CD4+FOXP3+ regulatory T cells (Treg cells), followed by CD45R+ B cells and IgM+ or IgG subclass+ antibody-forming cells mainly in the spleen. From 1.5 days, the resident MHCII+CD103+ dendritic cells (DCs) in the splenic T-cell area, periarterial lymphocyte sheath, formed clusters with recipient BrdU+ or 5-ethynyl-2′-deoxyuridine+ cells, from which the proliferative response of CD4+ T cells originated peaking at 3–4 days. Transfusion-induced antibodies had donor passenger cell-depleting activity in vitro and in vivo and could suppress acute GvH disease caused by donor T cells. Furthermore, Treg cells significantly suppressed mixed leukocyte reactions in a donor-specific manner. In conclusion, single blood transfusion efficiently induced a helper T-cell-dependent anti-donor class I MHC antibody-forming cell response with immunoglobulin class switching, and a donor-specific Treg cell response mainly in the spleen, probably by way of the indirect allorecognition via resident DCs. These antibodies and Treg cells may be involved, at least partly, in the donor-specific transfusion-induced suppression of allograft rejection.

Computational vaccine design, also known as computational vaccinology, encompasses epitope mapping, antigen selection and immunogen design using computational tools. The iVAX toolkit is an integrated set of tools that has been in development since 1998 by De Groot and Martin. It comprises a suite of immunoinformatics algorithms for triaging candidate antigens, selecting immunogenic and conserved T cell epitopes, eliminating regulatory T cell epitopes, and optimizing antigens for immunogenicity and protection against disease. iVAX has been applied to vaccine development programs for emerging infectious diseases, cancer antigens and biodefense targets. Several iVAX vaccine design projects have had success in pre-clinical studies in animal models and are progressing toward clinical studies. The toolkit now incorporates a range of immunoinformatics tools for infectious disease and cancer immunotherapy vaccine design. This article will provide a guide to the iVAX approach to computational vaccinology.

With increasing age, the kidney undergoes characteristic changes in the glomerular and tubulo-interstitial compartments, which are ultimately accompanied by reduced kidney function. Studies have shown age-related loss of peritubular vessels. Normal peritubular vessel tone, function and survival depend on neighboring pericytes. Pericyte detachment leads to vascular damage, which can be accompanied by their differentiation to fibroblasts and myofibroblasts, a state that favors matrix production. To better understand the fate of pericytes in the aged kidney, 27 month-old mice were studied. Compared to 3 month-old young adult mice, aged kidneys showed a substantial decrease in capillaries, identified by CD31 staining, in both cortex and medulla. This was accompanied by a marked decrease in surrounding NG2+ / PDGFRβ+ pericytes. This decrease was more pronounced in the medulla. Capillaries devoid of pericytes were typically dilated in aged mice. Aged kidneys were also characterized by interstitial fibrosis due to increased collagen-I and -III staining. This was accompanied by an increase in the number of pericytes that acquired a pro-fibrotic phenotype, identified by increased PDGFRβ+ / αSMA+ staining. These findings are consistent with the decline in kidney interstitial pericytes as a critical step in the development of changes to the peritubular vasculature with aging, and accompanying fibrosis.

// Sridevi Yadavilli 1 , Joseph Scafidi 2 , Oren J. Becher 3 , Amanda M. Saratsis 4 , Rebecca L. Hiner 5 , Madhuri Kambhampati 1 , Santi Mariarita 6 , Tobey J. MacDonald 7 , Kari-Elise Codispoti 8 , Suresh N. Magge 9 , Jyoti K. Jaiswal 1,10 , Roger J. Packer 11 and Javad Nazarian 1,10 1 Research Center for Genetic Medicine, Children’s National Health System, Washington, DC, USA 2 Department of Neurology and Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA 3 Department of Pediatrics and Pathology, Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA 4 Division of Neurosurgery, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA 5 Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, NY, USA 6 Department of Pathology and Lab Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 7 Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA 8 Department of Pathology, Children’s National Health System, Washington, DC, USA 9 Division of Neurosurgery, Children’s National Health System, Washington, DC, USA 10 Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, USA 11 Brain Tumor Institute, Center for Neuroscience and Behavioral Medicine, Children’s National Health System, Washington, DC, USA Correspondence to: Javad Nazarian, email: // Keywords : DIPG, NG2, PDGF, histone 3, glioma Received : March 04, 2015 Accepted : March 11, 2015 Published : March 30, 2015 Abstract Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to induce gliomagenesis. Here, we investigated NG2 expression in a cohort of DIPG specimens (n= 50) . We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2. Treatment with 5-Azacytidine, a methyltransferase inhibitor, results in NG2 downregulation in DIPG primary tumor cells in vitro . NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein, GFAP) markers, indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.

Atopic, obese asthmatics exhibit airway obstruction with variable degrees of eosinophilic airway inflammation. We previously reported that mice obese as a result of a genetic deficiency in either leptin ( ob/ ob mice) or the long isoform of the leptin receptor ( db/ db mice) exhibit enhanced airway obstruction in the presence of decreased numbers of bronchoalveolar lavage fluid (BALF) eosinophils compared with lean, wild-type mice following antigen (ovalbumin; OVA) sensitization and challenge. To determine whether the genetic modality of obesity induction influences the development of OVA-induced airway obstruction and OVA-induced pulmonary inflammation, we examined indices of these sequelae in mice obese as a result of a genetic deficiency in carboxypeptidase E, an enzyme that processes prohormones and proneuropeptides involved in satiety and energy expenditure ( Cpefatmice). Accordingly, Cpefatand lean, wild-type (C57BL/6) mice were sensitized to OVA and then challenged with either aerosolized PBS or OVA. Compared with genotype-matched, OVA-sensitized and PBS-challenged mice, OVA sensitization and challenge elicited airway obstruction and increased BALF eosinophils, macrophages, neutrophils, IL-4, IL-13, IL-18, and chemerin. However, OVA challenge enhanced airway obstruction and pulmonary inflammation in Cpefatcompared with wild-type mice. These results demonstrate that OVA sensitization and challenge enhance airway obstruction in obese mice regardless of the genetic basis of obesity, whereas the degree of OVA-induced pulmonary inflammation is dependent on the genetic modality of obesity induction. These results have important implications for animal models of asthma, as modeling the pulmonary phenotypes for subpopulations of atopic, obese asthmatics critically depends on selecting the appropriate mouse model.

Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry-based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.

Tick-borne infectious diseases and allergies are a growing problem worldwide. Tick bite allergy has been associated with the direct effect of immunoglobulin E (IgE) response to tick salivary antigens, or secondary to the induction of allergy to red meat consumption through IgE antibodies against the carbohydrate α-Gal (Gal α 1-3Gal β 1-(3)4GlcNAc-R). However, despite the growing burden of this pathology, the proteins associated with anaphylaxis to tick bite have not been characterized. To address this question, a comparative proteomics approach was used to characterize tick proteins producing an IgE antibody response in a healthy individual with record of tick bites, which had not resulted in any allergic reactions, and two patients with anaphylactic reactions to Rhipicephalus bursa or Hyalomma marginatum tick bites. Both patients and the healthy individual were red meat tolerant. The results supported a patientspecific IgE antibody response to tick species responsible for the anaphylaxis to tick bite. Both patients and the healthy individual serologically recognized tick proteins with and without a-Gal modifications, with proteins differentially recognized by patients but not control sera. These proteins could be used as potential antigens for diagnostics, treatment and prevention of tick bite-induced allergies., Part of this research was supported by the European Union FP7 ANTIGONE project number 278976. L.M-H was supported by the University of Castilla La Mancha (UCLM, Spain) fellowship. M.V and I.G.FM were supported by the Research Plan of UCLM. F.R-F was supported by the ‘Ramón y Cajal’ program of the Spanish Ministry of Economy and Competitiveness.

// Weibo Yu 1 , Xiaotian Qin 2 , Yusheng Jin 1 , Yawei Li 2 , Chintda Santiskulvong 3 , Victor Vu 1 , Gang Zeng 4, 5 , Zuofeng Zhang 6 , Michelle Chow 1 , Jianyu Rao 1, 5 1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 2 Beijing Boyuantaihe Biological Technology Co., Ltd., Beijing, China 3 Genomics Core, Cedars-Sinai Medical Center, Los Angeles, CA, USA 4 Department of Urology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 5 Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, CA, USA 6 Department of Epidemiology, School of Public Health, University of California at Los Angeles, Los Angeles, CA, USA Correspondence to: Jianyu Rao, email: jrao@mednet.ucla.edu Keywords: TSY-1, hematopoietic cells, Telomerase, TERT, methylation Received: September 08, 2016 Accepted: November 24, 2016 Published: December 15, 2016 ABSTRACT Telomere and Telomerase have recently been explored as anti-aging and anti-cancer drug targets with only limited success. Previously we showed that the Chinese herbal medicine Tianshengyuan-1 (TSY-1), an agent used to treat bone marrow deficiency, has a profound effect on stimulating Telomerase activity in hematopoietic cells. Here, the mechanism of TSY-1 on cellular Telomerase activity was further investigated using HL60, a promyelocytic leukemia cell line, normal peripheral blood mononuclear cells, and CD34+ hematopoietic stem cells derived from umbilical cord blood. TSY-1 increases Telomerase activity in normal peripheral blood mononuclear cells and CD34+ hematopoietic stem cells with innately low Telomerase activity but decreases Telomerase activity in HL60 cells with high intrinsic Telomerase activity, both in a dose-response manner. Gene profiling analysis identified Telomerase reverse transcriptase (TERT) as the potential target gene associated with the TSY-1 effect, which was verified by both RT-PCR and western blot analysis. The β-galactosidase reporter staining assay showed that the effect of TSY-1 on Telomerase activity correlates with cell senescence. TSY-1 induced hypomethylation within TERT core promoter in HL60 cells but induced hypermethylation within TERT core promoter in normal peripheral blood mononuclear cells and CD34+ hematopoietic stem cells. TSY-1 appears to affect the Telomerase activity in different cell lines differently and the effect is associated with TERT expression, possibly via the methylation of TERT promoter.

// Elizabeth Shurell 1, * , Maria E. Vergara-Lluri 2, * , Yunfeng Li 3 , Joseph G. Crompton 1 , Arun Singh 4 , Nicholas Bernthal 5 , Hong Wu 3 , Fritz C. Eilber 1 , Sarah M. Dry 2 1 Division of Surgical Oncology, University of California, Los Angeles, CA 90095, USA 2 Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA 90095, USA 3 Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095, USA 4 Department of Hematology/Oncology, University of California, Los Angeles, CA 90095, USA 5 Department of Orthopaedic Surgery, University of California, Los Angeles, CA 90095, USA * Co-first author Correspondence to: Fritz C. Eilber, email: fceilber@mednet.ucla.edu Keywords: NY-ESO-1, sarcoma, MPNST, liposarcoma, immunotherapy Received: July 28, 2016 Accepted: September 09, 2016 Published: September 17, 2016 ABSTRACT Background: Immunotherapy targeting cancer-testis antigen NY-ESO-1 shows promise for tumors with poor response to chemoradiation. Malignant peripheral nerve sheath tumors (MPNSTs) and liposarcomas (LPS) are chemoresistant and have few effective treatment options. Materials Methods: Using a comprehensive tissue microarray (TMA) of both benign and malignant tumors in primary, recurrent, and metastatic samples, we examined NY-ESO-1 expression in peripheral nerve sheath tumor (PNST) and adipocytic tumors. The PNST TMA included 42 MPNSTs (spontaneous n = 26, NF1-associated n = 16), 35 neurofibromas (spontaneous n = 22, NF-1 associated n = 13), 11 schwannomas, and 18 normal nerves. The LPS TMA included 48 well-differentiated/dedifferentiated (WD/DD) LPS, 13 myxoid/round cell LPS, 3 pleomorphic LPS, 8 lipomas, 1 myelolipoma, and 3 normal adipocytic tissue samples. Stained in triplicate, NY-ESO-1 intensity and density were scored. Results: NY-ESO-1 expression was exclusive to malignant tumors. 100% of myxoid/round cell LPS demonstrated NY-ESO-1 expression, while only 6% of WD/DD LPS showed protein expression, one of which was WD LPS. Of MPNST, 4/26 (15%) spontaneous and 2/16 (12%) NF1-associated MPNSTs demonstrated NY-ESO-1 expression. Strong NY-ESO-1 expression was observed in myxoid/round cell and dedifferentiated LPS, and MPNST in primary, neoadjuvant, and metastatic settings. Conclusions: We found higher prevalence of NY-ESO-1 expression in MPNSTs than previously reported, highlighting a subset of MPNST patients who may benefit from immunotherapy. This study expands our understanding of NY-ESO-1 in WD/DD LPS and is the first demonstration of staining in a WD LPS and metastatic/recurrent myxoid/round cell LPS. These results suggest immunotherapy targeting NY-ESO-1 may benefit patients with aggressive tumors resistant to conventional therapy.

// Ibrahim Omar 1 , Antonio Lapenna 1 , Leonor Cohen-Daniel 1 , Boaz Tirosh 2 and Michael Berger 1 1 The Lautenberg Center for Immunology and Cancer Research, The Biomedical Research Institute Israel-Canada of the Faculty of Medicine, The Hebrew University Hadassah Medical School, Jerusalem, Israel 2 Institute for Drug Research, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel Correspondence to: Michael Berger, email: // Keywords : ER stress, UPR, Slfn2, quiescence, XBP1, Immunology and Microbiology Section, Immune response, Immunity Received : May 08, 2016 Accepted : May 22, 2016 Published : June 03, 2016 Abstract Immunologically naive lymphocytes are kept in a quiescent state until antigen engagement. These quiescent immune cells are characterized by small cell size, lack of spontaneous proliferation and low metabolic rate. Lymphocyte quiescence is actively enforced condition which ensures the preservation of proper differentiation and proliferation capabilities of naive and memory lymphocytes. Previously we described a chemically induced mutation in Schlafen2 (Slfn2), termed elektra , which breaks quiescence and compromises immunity. However, the mechanism by which Slfn2 maintains quiescence remains unknown. Here we demonstrate that elektra T cells display chronic ER stress under steady state conditions. Modulation of ER stress response by depletion of either UPR mediators XBP1 or CHOP, improved viability and partially corrected the developmental abnormalities and proliferation capabilities of elektra T cells. Altogether, our results demonstrate a functional connection between Slfn2 induced quiescence in T cells and ER homeostasis, clarifying a novel mechanism by which immune cell quiescence is maintained.

Loss of polarity and quiescence along with increased cellular invasiveness are associated with breast tumor progression. ROCK plays a central role in actin-cytoskeletal rearrangement. We used physiologically relevant 3D cultures of nonmalignant and cancer cells in gels made of laminin-rich extracellular matrix, to investigate ROCK function. Whereas expression levels of ROCK1 and ROCK2 were elevated in cancer cells compared to nonmalignant cells, this was not observed in 2D cultures. Malignant cells showed increased phosphorylation of MLC, corresponding to disorganized F-actin. Inhibition of ROCK signaling restored polarity, decreased disorganization of F-actin, and led to reduction of proliferation. Inhibition of ROCK also decreased EGFR and Integrinβ1 levels, and consequently suppressed activation of Akt, MAPK and FAK as well as GLUT3 and LDHA levels. Again, ROCK inhibition did not inhibit these molecules in 2D. A triple negative breast cancer cell line, which lacks E-cadherin, had high levels of ROCK but was less sensitive to ROCK inhibitors. Exogenous overexpression of E-cadherin, however, rendered these cells strikingly sensitive to ROCK inhibition. Our results add to the growing literature that demonstrate the importance of context and tissue architecture in determining not only regulation of normal and malignant phenotypes but also drug response.

// Alexander Ring 1, 2 , Neal Mineyev 3 , Weizhu Zhu 1, 2 , Emily Park 4 , Chip Lomas 5 , Vasu Punj 2, 6 , Min Yu 2, 7 , Dany Barrak 1, 2 , Victoria Forte 2 , Tania Porras 1, 2 , Debu Tripathy 8 , Julie E. Lang 1, 2 1 Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 2 Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA 3 Department of Surgery, Lenox Hospital New York, New York, NY 10065, USA 4 Advanced Cell Diagnostics, Research and Development, Hayward, CA 94545, USA 5 BD Biosciences, Research and Development, San Jose, CA 95131, USA 6 Division of Hematology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 7 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 8 Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Correspondence to: Julie E. Lang, e-mail: Julie.Lang@med.usc.edu Keywords: breast cancer, circulating tumor cells, IE/FACS, EpCAM Received: July 10, 2015 Accepted: October 09, 2015 Published: October 19, 2015 ABSTRACT Purpose: The potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes. Results: Cancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups ( p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB. Materials and Methods: Ten cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB. Conclusions: EpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations.