bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissecting the cellular responses of immune cells and Lgr5+ stem cells in the inflamed mouse colon with the RNAscope in situ hybridization technology
Authors: Anderson Courtney M , Li Na M , Todorov Courtney , Dikshit Anushka , Ma Xiao-Jun 
Published: January 2023

Abstract
Due to its exposure to a harsh luminal environment, the intestinal epithelium has a remarkably fast turnover rate that is facilitated by a resident intestinal stem cell (ISC) population present at the base of the intestinal crypt. These ISCs, marked by the GPCR Lgr5, allow the intestinal epithelium to adapt to different types of damage, such as inflammation. Chronic intestinal inflammation is a hallmark of the inflammatory bowel diseases (IBD) yet the interplay between inflammatory immune cells and ISCs remains to be elucidated. Therefore, we utilized the single-molecule RNA in situ hybridization (ISH) technology RNAscope to visualize the expression of multiple immune and ISC markers within the inflamed intestinal tissue environment. To interrogate the expression pattern of inflammatory immune cell and ISC markers within the intestinal crypt, we performed the assay on colons from either control or TNBS-treated mice. We visualized the location of each intestinal cell population, including the resident Lgr5+ ISC population, within the crypt. The impact of inflammation on the Lgr5+ ISC population, as well as the Wnt/β-catenin pathway, was also examined. Using a multiplex assay, we assessed infiltration of regulatory T cells and Th17 cells in the inflamed region. Lastly, we examined the expression of several receptor-ligand pairs for cytokines and ISC markers. Taken together, these results demonstrate the ability of the RNAscope assay to visualize the ISCs within the morphological context of the intestinal crypt and in relationship to inflammatory immune cells. This work may help understand mechanisms behind the pathogenesis of IBD and other inflammatory diseases and also aid in the development of potential therapies.
Immune Activation of the Blood Brain Barrier and Implications for Neuroinflammation in Schizophrenia
Authors: Crockett Alexis Mary , Ryan Sean K. , Vasquez Adriana Hernandez , Canning Caroline , Kanyuch Nickole , Ceja Guadalupe , Gesualdi James , Kebir Hania , Kapoor Richa , Benallegue Nail , Anderson Stewart A. , Alvarez Jorge I. 
Published: January 2023

Abstract
Neuroimmune dysregulation has been implicated in various neuropsychiatric disorders, including schizophrenia (SZ). Multiple studies indicate peripheral inflammation, including increased serum IL-6 and circulating Th17 cells, and neuroinflammation in the context of psychosis. Reports that the blood brain barrier (BBB), the highly specialized CNS endothelial cells, is disrupted in patients suggest that vascular alterations contribute to neuroimmune dysfunction in SZ. Therefore, we investigated whether the BBB is intrinsically compromised in the most common genetic risk factor for SZ, the hemizygous deletion of chromosome 22q11.2 (22qDS). We employed an innovative approach based on the differentiation of human induced pluripotent stem cells (iPSCs) from 22qDS patients with SZ into BBB-like endothelium and further substantiated our findings in vivo in a mouse model of 22qDS. We demonstrate that barrier function and immune privilege properties of the BBB are compromised in 22qDS, as we found compromised expression of the tight junction protein claudin-5 and upregulation of the cell adhesion molecule ICAM-1 (CD54). To assess the immunological consequences of such imbalances, we determined the impact of the BBB on transmigrating immune cells and found that the 22qDS BBB supported leukocyte migration and activation. Furthermore, we observed increased astrocyte activation and IL-6 expression in 22qDS mice and post-mortem 22qDS patients, suggesting that compromised BBB may promote astrocyte-mediated neuroinflammation in 22qDS. These results indicate that 22qDS is associated with changes in the BBB, and suggest that the BBB may play a role in the increased risk for neuropsychiatric disease in 22qDS patients.
CCR9+ dendritic cells promote the differentiation of Foxp3+ regulatory CD4 T cells
Authors: Lal Girdhari , Pathak Manisha , Halder Namrita , Padghan Priyanka 
Published: January 2023

Abstract
Chemokine receptor CCR9 is known to play an important role in the migration of immune cells to the gut. Intestinal epithelial cells under gut inflammation or in inflammatory bowel disease (IBD) produce several folds higher CCL25, the only known ligand for CCR9, and drive the recruitment of CCR9+ immune cells. Due to its gut tropism activity, CCR9 and CCL25 are suggested as a potential therapeutic target. Still, many of the clinical trials targeting CCR9/CCL25 did not give a positive outcome; instead, they increase the severity of the disease. The non-chemotactic function of CCR9 in the gut inflammation and immunity is not known.
Using dextran sodium sulfate (DSS)-induced gut inflammation model in C57BL/6 mice, we showed that CCR9+ DCs, specifically CD11b−CD103+ DCs recruited in high frequency to the gut and gut-associated lymphoid tissues (GALT) in DSS treated mice as compared to control group. These CCR9+ DCs showed lower MHC II and CD86 molecules and had higher regulatory surface markers (FasL and Latency-associated peptide, LAP) in the GALT. Further, we demonstrate that thymic stromal lymphopoietin (TSLP) produced by CCR9+ DCs but not IL-10 or TGF-β, promotes the differentiation of Foxp3+ Treg. Furthermore, adoptive transfer of CCR9+ DCs in C57BL/6 mice promoted Tregs but reduced the Th17 cells in the GALT, and also suppressed the ovalbumin-specific gut allergic immune response. Together, these results suggest that CCR9+ DCs have a regulatory function and can be explored as an adoptive cellular therapy to control the gut inflammation and allergic immune response.
GPR120 regulates neutrophil functions and intestinal homeostasis
Authors: Zhou Zheng , Yang Wenjing , Yu Yanbo , Zhao Xiaojing , Yu Yu , Bilotta Anthony J , Yao Suxia , Cong Yingzi 
Published: January 2023

Abstract
GPR120 (also known as free fatty acid receptor 4, FFAR4) has been identified recently as a bona fide receptor for long-chain fatty acids (LCFA), and has a critical role in various physiological homeostasis mechanisms such as adipogenesis. However, if and how GPR120 regulates intestinal homeostasis is still largely unknown. In this study, we found that GPR120−/− mice showed more severe colitis induced by DSS compared to WT mice, which was evident as increased weight loss and higher histopathology scores. Depletion of neutrophils with anti-Ly6G increased the colitis severity, indicating protective roles of GPR120 and neutrophils in the intestines from inflammation. GPR120 agonist, CpdA, promoted neutrophil production of IL-17 and IL-22, but not TNF-α and TGF-β expression. Consistently, GPR120−/− neutrophils showed impaired IL-17 and IL-22 production compared with WT neutrophils. Additionally, treatment with CpdA and LCFA Omega-3, DHA, enhanced neutrophil production of reactive oxygen species (ROS). Furthermore, CpdA increased antimicrobial peptide S100A9, but not S100A8, production in neutrophils. Mechanically, inhibition of mTOR pathway by mTOR inhibitors, rapamycin and AZD8055, suppressed neutrophil IL-22 and IL-17 production induced by CpdA, while it did not affect the GPR120 agonist induction of ROS. Additionally, inhibition of glycolysis by glycolysis inhibitor 2-DG suppressed GPR120 agonist-induced IL-17 but not IL-22 and ROS production in neutrophils, indicating mTOR and glycolysis differentially regulate GPR120 induction of IL-17, IL-22 and ROS. Taken together, our results highlight a critical role of GPR120 in regulating neutrophil functions, thus contributing to the maintenance of intestinal homeostasis.
Quantitative immune profiling of human tumor tissues with multiplexed ChipCytometry
Authors: Northcutt Adam J , Christians Arne , Forys Jason T , Campbell Thomas D , Winkeler Crystal L 
Published: January 2023

Abstract

Objective
Many immune cell profiling methods have demonstrated limits on the number of biomarkers that can be reliably assayed from the same cell; to remedy this, we present here ChipCytometry, an imaging technology for quantitative immune profiling of cells and sectioned tissues.

Methods
ChipCytometry is a fluorescence-based imaging system that utilizes multiplexed immuno-fluorescence staining in combination with high-dynamic-range (HDR) imaging to facilitate quantitative phenotyping of individual cells within tissue samples. Employing this technology to profile metastatic and primary tumors, multiple biomarkers were stained in iterative multiplex assays to profile single cells with an AI-powered cell segmentation algorithm. This immune profiling was completed on 6 different cryosectioned human tumor tissues (head & neck, liver, lung, breast, colon, and pancreas).

Results
ChipCytometry allowed for the quantification of both tumor and immune cells in all 6 of the human tumor tissues that were analyzed, revealing the diversity of T cells (helper, cytotoxic, regulatory, naïve/effector/memory), B-cells, granulocytes, monocytes, dendritic cells, and natural killer cells present in the tumor microenvironment. The resolution, dynamic range, and AI-powered cell segmentation offered by ChipCytometry allowed for quantification of 15 immune cell types at the single-cell level. The data show exact counts of each specific cell type, relative percentages within each tissue, and the precise spatial distribution of each cell.

Conclusion
These results demonstrate the potential of ChipCytometry to broadly probe a diverse set of human tumor tissue types and acquire a deep immunological cell type profile.
ImmuneSpace enables multicohort immune correlates analysis
Authors: Kim Ju Yeong , Henrich Evan , Miller Helen , Jiang Mike , Wagner Jake , Finak Greg , Gottardo Raphael 
Published: January 2023

Abstract
ImmuneSpace is a data repository and analysis platform of the Human Immunology Project Consortium (HIPC), a multi-center collaborative effort to characterize the status of the immune system in different populations under diverse stimulations and disease states. This ongoing effort has generated large amounts of varied high-throughput, high-dimensional immunological data. ImmuneSpace directly extracts data from ImmPort -- the central NIAID database, and further standardizes it for meta-analysis and reporting, making it ready to be explored using state-of-art visualization and analysis tools. ImmuneSpace will act as a central immunological hub, allowing experimentalists, statisticians, and bioinformaticians to freely retrieve, explore and compare data across assays and across studies generated within and outside of HIPC. We present a selection of multi-cohort analyses using standardized gene expression and flow cytometry data sets across multiple studies identifying novel baseline predictors of vaccine efficacy across a variety of pathogens.
Organ-on-Chip Meets Immunology: Let’s Start with the Lungs
Authors: Benam Kambez Hajipouran 
Published: January 2023

Abstract
Organs-on-chips are biomimetic, microfluidic, cell culture devices created with microchip manufacturing methods that contain continuously perfused hollow microchannels inhabited by living tissue cells arranged to simulate organ-level physiology. By recapitulating the multicellular architectures, tissue-tissue interfaces, chemical gradients, mechanical cues, and vascular perfusion of the body, these devices produce levels of tissue and organ functionality not possible with conventional 2D or 3D culture systems. They also enable high-resolution, real-time imaging and in vitro analysis of biochemical, genetic and metabolic activities of living human cells in a functional human tissue and organ context. Here, we discuss development of ‘Human Lung Small Airway-on-a-Chip’ – an engineered cell-by-cell reconstituted microfluidic device with living human tissues to model airway inflammation in the context of debilitating lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). In addition, we will present our preliminary data on recreating a hematopoietically active bone marrow niche in vitro (‘BM-on-a-Chip’) for microphysiological integration with the Small Airway-on-a-Chip to emulate innate immune cell egress into circulation (from the BM) and their recruitment to the lung airway.
Expanding the capabilities of the Maxpar Direct Immune Profiling Assay with additional markers, and customization of the analysis model in Maxpar Pathsetter
Authors: Li Stephen K.H. , Mujib Shariq , Cohen Michael , Yao Huihui , Majonis Daniel , Loh Christina 
Published: January 2023

Abstract
Mass cytometry, which utilizes CyTOF® technology, is a single-cell analysis platform that uses metal-tagged antibodies. CyTOF can resolve more than 50 parameters in a single panel without the need for compensation, making it an ideal solution for routine enumeration of immune cells. The Maxpar® Direct™ Immune Profiling System is a sample-to-answer solution for human immune profiling using mass cytometry. The Maxpar®Direct™ Immune Profiling Assay™ is an optimized 30-marker panel contained in a dry single-tube format for human whole blood or PBMC staining, and samples are acquired on the Helios™ system. Maxpar Pathsetter™ is an automated software that reports cell counts, percentage calculations, and staining intensity. It also produces graphical elements such as dot plots and a Cen-se′™ graph for 37 immune cell populations. The panel can be tailored by adding markers to open channels, the Maxpar Pathsetter model can then be customized to measure expression markers on existing classified populations or identify additional immune cell subsets.
We present data where the Maxpar Direct Immune Profiling Assay is used as a core immunophenotyping panel and additional markers are added to create a nearly 50 marker panel. Added markers are used to identify MDSCs, further classify existing cell populations, and measure immuno-oncology related markers including OX40, TIM-3, Fas, PD-1, PD-L1, ICOS, and TIGIT. We demonstrate how the Maxpar Pathsetter model is modified to incorporate the added markers.
The ability to customize the Maxpar Pathsetter model and expand the Maxpar Direct Immune Profiling Assay allows for flexibility of the system. It allows researchers to have a streamlined solution for broad immune profiling using mass cytometry.
Immune monitoring for immuno-oncology applications
Authors: Chattopadhyay Pratip K , Lomas Woodrow E , Gao Guo-Jian , Li Na , Saksena Suraj 
Published: January 2023

Abstract
The immunotherapy revolution has spurred the development of many new drugs and drug regimens for patient treatment. A key challenge is to identify the factors that drive patient toxicities and responses to treatment, with a particularly acute need for predictive biomarkers that can discriminate patients destined to respond and fail treatment. Technologies to interrogate immune cells are now readily available, but important gaps remain in their application, which limit the full realization of the promise of precision oncology.
High parameter flow cytometry is a gold-standard but is limited by difficulty of panel design and lack of standardization. We present Color Wheel, a panel design tool, which builds optimized antibody panels based on the user’s instrument. These optimized panels have been manufactured in a dried, ready to use format to drive workflow and assay standardization. Initial results demonstrate excellent concordance between the dried and liquid versions of the high parameter multicolor panel(s).
Molecular cytometry represents an exciting new approach to high dimensional immune analysis because it can measure at least 102 proteins and 400 mRNA targets simultaneously per cell. An important application gap for this technology is lack of data indicating the sequencing depth needed for adequate resolution. Here, we present results from a molecular cytometry experiment sequenced deeply, and then bioinformatically sub-sampled at different levels to identify the minimum level of sequencing needed for clear identification of cells, which can serve as a reference guide for users to save time and cost in their experiments. We also present preliminary data generated using dried version of molecular cytometry panel(s).
High throughput subpopulation signaling analysis: protocol considerations for multiplexed intracellular flow cytometry
Authors: MacDonald Robert J , Manning Christopher , Alonzo Emily 
Published: January 2023

Abstract
While much of our knowledge of signaling pathways has come from bulk analytical methods such as western blotting, more granular techniques enable us to deepen our understanding of how signaling profiles may vary in heterogeneous cell populations. Flow cytometry offers a high-throughput avenue for subpopulation signaling analysis. Combining antibodies against extracellular CD markers with those against intracellular signaling proteins or post-translational modifications enables researchers to explore variations in signaling profiles by cell type.
Performing a staining protocol that allows for accurate performance of antibodies against both extracellular and intracellular epitopes is key. Manufacturers commonly validate antibodies using specific protocols; straying from these may lead to drastically different experimental results and conclusions. Antibodies against CD markers, for example, are often validated for use on live cells, and may perform poorly on fixed and permeabilized cells. Compromised CD marker detection may lead to inaccurate gating and thus inappropriate subpopulation analyses. In turn, this can lead to anomalous conclusions about the signaling profiles of subpopulations of interest.
In order to mitigate the risk of such occurrences, we present a general guide to protocol considerations when performing multiplexed intracellular flow cytometry through the lens of immune cell stimulation. Highlighting subpopulations of primary mouse immune cells treated with 2′3′-cGAMP, we demonstrate that CD marker staining protocol variation can lead to erroneous subpopulation characterization, which can then impact interpretation of innate immune signaling profiles within these populations.
Development of a novel model: Using songbirds to elucidate the pro-neurogenic role of microglia in healthy and natural neurodegenerative states
Authors: Larson Tracy A , Wolkoff Matthew E , Tokareva Kate , Cole Marianne Marritt , Brenowitz Eliot A 
Published: January 2023

Abstract
Inflammatory cells like microglia exert beneficial effects on neuronal survival, synaptic maintenance, and circuit plasticity. Most research has focused on neural injury or insult; roles in normal development and homeostasis remain less clear. To characterize the behavior of microglia under non-pathological conditions, we exploit seasonal death and replacement of neurons in a sensorimotor brain region responsible for singing behavior in white-crowned sparrow, Zonotrichia leucophrys. The song control nucleus HVC incorporates 50,000 new projection neurons into an existing pool of 100,000 neurons within one week of transitioning to breeding conditions. Within four days of transitioning back to nonbreeding conditions, an equal number of HVC neurons dies. Seasonal neuronal death in HVC increases neural stem cell proliferation in the adjacent ventral ventricular zone. This growth and regression correlates to respective increases and decreases in song production rate and quality. We found that microglia activation mediates pro-neurogenic responses in each of two paradigms: (i) inflammation induced locally with lipopolysaccharide microinjection in HVC was sufficient to induce neural stem cell proliferation; (ii) inflammation, including activation of microglia, was necessary to induce neural stem cell proliferation following seasonal neuronal loss. Having established that activation of microglia drives pro-neurogenic responses under non-pathological conditions, we are enabling future mechanistic studies with essential genetic and molecular tools, including a high quality annotated genome and transcriptome, cell-type specific molecular markers, in vivo imaging, and CRISPR/Cas9 mutagenesis.
CD34 count using Image based-immunofluorescence cell counter with filterpaper imbeded microchip
Authors: Lim Chae Seung , Jang Woong Sik , Nam Jeonghun 
Published: January 2023

Abstract

Background
The accurate measurment of CD34 positive cells is importance for successful hematopoietic stem cell transplantation. Until now, flowcytometric assay is the standard method for enumerating CD34+ stem cells it has some limitations. Microscanner C3(Biozentech, Korea) is noble image based cell counter for blood components adopt microscopy based cell counter with disposable plastic microchips. This system is composed of the staining solutions, the microscopic instrument and the image analysis software. We developed 0.5 um sized filter paper imbeded plastic microchip for enrichment of rare cells. and applied it to image based-immunofluorescence cell counter for CD34+ cells.

Methods
Leukocyte and CD34-positive cells were stained by CD45-BB515 and CD34-PE (Becton Dickinson, USA). We compared the reproducibility and linearity between the standard flowcytometric method (Beckman Coulter Cytomics FC500 ) and image based-immunofluorescence cell counter with filter paper imbeded microchip by using CD34 positive leukocyte quality control materials (300, 100, 50, 25,10 cells/μl).

Results
CD34 measurement by image based-immunofluorescence cell counter with filter paper imbeded microchip was performed within 5 min. The repeatability was within the range of 10–300 cells/μ, and the coefficient of variation was 8.2–20.3%. The correlation with the CD34 measured by flow cytometry was (R2&gt; 0.9746, P &lt;0.0001).

Conclusions
CD34 measurement by image based-immunofluorescence cell counter with filter paper imbeded plastic microchips is fast, simple and reliable, it could be used as simple measuring tools for rare blood cells in the blood.
Treatment with Multiple-linked Myelin Peptides Encapsulated within Nanoparticles Induces Antigen-specific Tolerance in SJL/J Relapsing-remitting Experimental Autoimmune Encephalomyelitis
Authors: Beddow Sara A , Neef Tobias , Ifergan Igal , Podojil Joseph R , Getts Daniel , Miller Stephen D 
Published: January 2023

Abstract
Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a demyelinating disease of the central nervous system (CNS), and serves as a fit-for-purpose pre-clinical model of Multiple Sclerosis (MS). Data show that tolerogenic immune-modifying nanoparticles (TIMPs) encapsulating peptides/proteins is an effective therapeutic that induces antigen-specific tolerance to the encapsulated peptide/protein. The effectiveness of this therapeutic platform has been be demonstrated in multiple mouse models, as well as in a recently completed phase 2 double-blinded placebo-controlled clinical trial for the treatment of celiac disease. While previous EAE studies have utilized single peptides, the present study utilized a polypeptide containing the SJL/J mouse dominant encephalitogenic peptides (PLP139–151, PLP178–191, MBP84–104, and MOG92-10) linked together with intervening capsaicin S cleavage sites. The use of the multiple-linked myelin peptides was produced to achieve broader coverage of myelin-derived epitopes, which will be required for the treatment of MS. The present data show that this multiple-linked myelin peptide emulsified in CFA induced both CD4+ T cell responses and EAE in SJL/J mice similar to PLP139–151/CFA. Our data go on to show that treatment of SJL/J mice with multiple-linked myelin peptide TIMP inhibited both PLP139–151/CFA-induced R-EAE, as well as multiple-linked myelin peptide/CFA-induced EAE. Furthermore, treatment of SJL/J mice with multiple-linked myelin peptide TIMP significantly decreased TH17 cell responses and increased Tr1 cell responses. The present findings suggest that utilizing multiple-linked peptides may be clinically translatable for the treatment of autoimmune disease.