bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adoptive transfer of Treg cells to B6 mice during Toxoplasma gondii infection induces down-modulation of exacerbated inflammatory immune response but does not generate protection (MPF1P.777)
Authors: Olguín Jonadab , Fernández Jacquelina , Castellanos Carlos , Campuzano Jaime , Terrazas Luis I. , Saavedra Rafael 
Published: January 2023

Abstract
Toxoplasmosis is an infection disease affecting one third of the world population and is caused by the protozoan parasite Toxoplasma gondii. TH1 immune response is necessary for protection, IFN-γ being the major mediator of resistance. An exacerbated TH1 immune response however, is detrimental. Regulatory T (Treg) are CD4+Foxp3+ cells that actively suppress pathological and physiological immune responses, thereby contributing to the maintenance of immune homeostasis. It has been reported that during acute toxoplasmosis in B6 mice, Treg cell number decreases. In order to analyze if Treg cells are involved in protection during T. gondii infection, we carried out adoptive transfer of Treg cells to infected mice. We observed a decreased mortality when Treg cells were adoptively transferred but an increased number of cysts in brain was detected. A decreased pathology in the small intestine, a decreased production of IFN-γ and TNF-α in serum, a decreased activation in CD4+ cells, and a decreased production of IFN-γ by CD4+ cells was also observed in the same mice. All these results suggest that adoptive transfer of Treg cells during T. gondii infection down-modulate the exacerbated immune response, but it does not generate protection in B6 mice. Thus Treg cells are not involved in protection during T. gondii infection.
Microbiota-derived GABA exacerbates Clostridium difficult-associated intestinal inflammation and suppresses development of protective immunity (MPF1P.768)
Authors: Dann Sara , Eaves-Pyles Tonyia , Peniche Alex , Choudhury Barun , Feng Hanping , Savidge Tor 
Published: January 2023

Abstract
Background: Clostridium difficile infection (CDI) is a major cause of nosocomial antibiotic-associated diarrhea and colitis. Previously we performed metagenomic and metabolomic analysis on stool samples collected from patients with and without CDI, and found a significant correlation between disease recurrence and intestinal lumen levels of the neurotransmitter GABA. Study objectives: To determine the role of GABA in CDI pathogenesis and the effects on host immune defenses. Methods & Results: C57BL/6 mice were treated with an antibiotic mixture +/- GABA in their drinking water and infected orally with C. difficile. Intestinal tissue was collected and examined for histopathology and gene expression. Administration of GABA exacerbated CDI- induced mucosal inflammation and damage, whereas inhibition of GABA with picrotoxin reduced disease severity. In addition, mice given daily doses of probiotic bacteria that secrete GABA had greater disease severity compared to controls. In vitro, GABA modulated epithelial cell cytokine responses to C. difficile toxins, LPS and flagellin. In vivo, GABA altered the cellular response to infection, and promoted the recruitment of CD11b+Gr1+ cells to the site of infection. The early immune changes were associated with decreased protective antibody production at week 2 post-infection. Conclusion: Microbial-derived GABA promotes CDI disease pathogenesis by modulating innate immune responses which leads to suppression of protective immunity.
Association of miRNA expression profile with Chlamydial-induced genital tract pathology (MPF2P.803)
Authors: Yeruva Venkat , Maurelli Anthony , Spencer Nicole , Praskievicz Nancy , McChesney Grant , Myers Garry , Rank Roger 
Published: January 2023

Abstract
Currently, with respect to chlamydial infections, one cannot predict the probability of an infected patient developing pelvic inflammatory disease. To determine if specific biomarkers may be associated with distinct chlamydial pathotypes, we utilized two Chlamydia muridarum variants (M4, M9) that showed differences in the development of upper tract pathology. M4 has a slower growth rate in vitro, induces pathology in 6 of 30 C57Bl/6 oviducts versus 25 of 30 oviducts in M9-infected mice. To determine if chemokine cytokine production within 24 h of infection will define the outcome of pathology, 15 chemokines and cytokines were measured. M4 infection induced significantly lower levels of CXCL 1, CXCL 2, TNF-α and CCL 2 in comparison to M9 infection with similar ribosomal RNA (rs16) levels for chlamydiae. To understand if chemokine cytokine responses would also be reflected in miRNA expression profile within 24 h of infection, 134 inflammation-related miRNAs were measured. Interestingly, 12 miRNAs (miR-135a-5p, miR155-5p, miR325-3p, miR338-5p, miR105, miR132-3p, miR142-3p, miR142-5p, miR147-3p, miR223-3p, miR298-5p and miR299a-3p) were expressed significantly higher during M4 infection than M9 infection, inversely correlating with the respective chemokine/cytokine responses. To our knowledge this is the first report demonstrating that early biomarkers elicited in the host could differentiate between two pathologic variants of chlamydiae and be predictive of upper tract disease.
Inhibitory role of β-arrestin2 in T cells protects against colitis. (MUC2P.823)
Authors: Sharma Deepika , Malik Ankit , Parameswaran Narayanan 
Published: January 2023

Abstract
β-arrestins have shown to be important regulators of inflammation. The aim of this study was to determine the role of β-arrestin2 in intestinal inflammation. In response to DSS-induced colitis, β-arrestin2 knockout (KO) mice exhibit greater body weight loss, higher disease activity index, shortened colon and loss of thymic weight as compared to wild type (WT) mice. Additionally, number of CD4+T cells and proportion of IL-17A+ (and RORγT+) and IFNγ+ CD4+ T cells were markedly higher in DSS-treated KO colonic lamina propria (cLP). Surprisingly, control KO mice too had higher number of CD4+ T cells and il17a mRNA expression in colon tissue compared to control WT mice. Furthermore, T-cell receptor (TCR) stimulation of MLN and splenic cells led to significantly higher production of IFNγ and IL-17 from KO compared to WT cells. Additionally, naïve CD4+ T cells from KO mice produced higher IFNγ and IL-17 and differentiated into Th1 and Th17 cells to a greater extent following TCR stimulation. These results suggest that the KO colon is likely predisposed to exacerbated mucosal inflammation by virtue of enhanced CD4+ T cell numbers and function. This overt response however, was independent of microbiota since, KO animals co-housed with WT mice also exhibited higher weight loss and colon shortening following DSS. Together, our results demonstrate that β-arrestin2 negatively regulates T cell activation and thereby modulates mucosal response under both basal and inflammatory conditions.
Development of non-overlapping multiplex antibody arrays for the quantitative measurement of 400 human and 200 mouse proteins in parallel (TECH1P.849)
Authors: Mao Yingqing , Yen Haw-han , Sun Yanni , Lv Zhiqiang , Huang Ruopan 
Published: January 2023

Abstract
Accurate detection of multiple cytokines in complex biological samples is essential to progress in immunological research. However, development of high-density multiplex sandwich-based ELISA panels is hampered by nonspecific cross-reactivity between and among target-specific reagents (i.e., capture antibodies, detection antibodies, and/or antigens); this is particularly challenging for bead-based assays, in which all three are free to interact in solution. To overcome this bottleneck, we tested for nonspecific cross-reactivity among reagents used on our quantitative antibody arrays (in which capture antibodies are affixed to a glass slide). Combinations of reagents exhibiting nonspecific interactions were separated on discrete arrays. Using this divide-and-conquer strategy, we developed and validated 10 human and 5 mouse non-overlapping 40-plex arrays for parallel, quantitative detection of 400 human and 200 mouse proteins respectively. These markers cover the most analyzed secretory proteins such as cytokines, chemokines, growth factors, soluble receptors, and other regulatory factors. To our best knowledge, this is the highest density multiplex antibody arrays developed to date. Such a system hold a great promise in determining key factors and mechanisms in immune cell physiology and pathology, in identifying novel drug targets, and in screening for putative biomarkers from patients' samples.
Real-time monitoring of MMP-9 secretion from monocytes using a nanoplasmonic-microfluidic platform (TECH1P.834)
Authors: Cheng Xuanhong , Wu Shu-han , Wei Pei-kun , Chiou Arthur 
Published: January 2023

Abstract
Leukocyte secretion is fundamental to the understanding of many immunological phenomena and is widely used in clinical diagnoses. Conventional methods, such as ELISA, ELISPOT and flow cytometry reveal significant information of cell secretion at fixed time points, but miss the secretion dynamics. To address this issue, we constructed an optofluidic platform for real-time monitoring of secretory activities of monocytes. The sensing element is a nanoslit array that uses the surface plasmon resonance mechanism to detect refractive index changes at the sensor/liquid interface. When functionalized with an antibody against matrix metalloproteinase 9 (MMP-9) and integrated with a cell-trapping microfluidic device, the sensor is able to monitor dynamic MMP-9 secretion from human acute monocytic leukemia cells in situ. Upon continuous lipopolysaccharide (LPS) stimulation, MMP-9 secretion is detected within two hours. This sensitivity is a result of ultralow surface detection limit, small culture compartments and close proximity of the sensor to the target cells. The average secretion measured using our system well matches previous reports while it requires orders of magnitude less cells. Furthermore, the cell secretion dynamics reveal an intermittent profile and the cause is still under investigation. Current effort is focused on imaging secretion from individual cells and multiplexing the sensors to study the secretion interplay of multiple molecules.
Auto-antibody profiling with over 9,500 human expressed proteins (TECH1P.853)
Authors: Lazar James , Amariei Flavia , Qin F. Xiao-Feng 
Published: January 2023

Abstract
Autoantibody profiling is a powerful method to probe the immune system for early signs of disease. However, progress has been limited by the availability of suitable antigens and high-throughput tools for screening large numbers of samples. Most antigens available have been produced in bacterial or insect cells and thus may not be optimal for autoantibody profiling. Although protein microarrays have been widely used to identify common autoantibodies in many diseases, creating smaller panels of antigens for larger screening studies has been difficult and costly. We have developed rapid methods for coupling proteins to Luminex beads allowing us to create and validate focused beads arrays in just a few days using native human proteins that have been expressed and purified in HEK293 cells. We have also developed a variable-stringency assay system that allows the user to optimize the detection specificity according to their particular application. Internal and external controls validate the presence of each protein and the IgG from the patient sample. Bead arrays for classic autoimmune antigens, as well as human breast, prostate, ovarian, pancreatic and lung cancer antigens have been prepared and custom arrays can be prepared from any of OriGene’s over 9500 purified proteins. Preliminary results with serum samples from traditional autoimmune patients, breast cancer, and Type II Diabetes support the utility of the approach.
A Versatile Simple Capture Assay for Assessing the Structural Integrity of MHC Multimer Reagents (TECH1P.859)
Authors: Reed Brendan , Chopp Laura , Girtman Megan , Van Keulen Virginia , Johnson Aaron , Schrum Adam , Pease Larry 
Published: January 2023

Abstract
MHC multimers including tetramers and pentamers provide a powerful way to visualize antigen specific T cell responses in both experimental and clinical immune assays. The multimers can be acquired commercially, from the NIH, or assembled in the laboratory for measuring immune responses to commonly-studied or novel-hypothetical antigens. The multimer reagents can be expensive and time consuming to acquire and in many cases are uncontrolled reagents as robust positive control T cells used to assess the reagents functionally may not be available. This is particularly problematic when using MHC multimers to investigate less well-defined T cell responses, such as those characterized by low T cell frequencies or by specificity to hypothetical antigens relevant to cancer, infection, or autoimmune disease. These problems are magnified when studying irreplaceable clinical trial samples where having confidence that reagents are biochemically intact is critical. To address this problem, we have devised a versatile, flow cytometry-based, capture assay to probe the structural integrity of MHC multimer reagents. In principle, the idea is to immobilize on a bead antibody specific for conformational determinants expressed on a properly folded MHC molecule. The particle becomes labeled and can be visualized and quantified by flow cytometry when a fluorescently tagged tetramer is adsorbed to the particulate upon binding to the antibody.
Big data approach towards the characterization of normal peripheral immune cells with data from ImmPort (TECH1P.860)
Authors: Andorf Sandra , Bollyky Jennifer , Bhattacharya Sanchita , Shankar Ravi , Dunn Patrick , Thomson Elizabeth , Wiser Jeffrey , Butte Atul 
Published: January 2023

Abstract
To date, our understanding of a normal immune system is far behind that of other healthy organ systems. One reason for this is the lack of a widespread adoption of standardization in the lab techniques, especially flow cytometry. Here we demonstrate the use of the Immunology Database and Analysis Portal (immport.niaid.nih.gov) as a source of publicly available flow cytometry files from hundreds of participants in several clinical trials, to study immune cells from the blood of healthy individuals. To characterize well-defined cells in a normal immune system, we used an unbiased, gate-free method to compare data from different cytometers and antibody staining panels, based on the distribution of fluorescence intensity of the markers. In order to perform the meta-analyses, the marker information from each file was obtained in an automated fashion and their nomenclature was made consistent. Various transformation and normalization strategies were applied and evaluated to make the flow cytometry data comparable across studies. Initial promising results were observed for the cell-surface markers used to define T and B cells in general, and some specific subsets including regulatory T cells as well as plasmablasts. We found high concordance in normal individuals from two studies performed at different times measured within the same facility. The extension of this approach to studies of healthy individuals across facilities promises to give insights towards the normal immune system.
Proteomic profiling of activated immune cells using quantitative multiplex bead-based assays to identify secretome and cell signaling protein signatures (TECH1P.869)
Authors: Lie Wen-Rong , Mistry Jehangir 
Published: January 2023

Abstract
Multiplex bead-based assays using the Luminex® xMAP® technology allows the simultaneous quantitation of soluble mediators involved in adaptive immune responses. Here we report on the quantitative profiling of the immune secretome and cell signaling target proteins to uncover the characteristics of a CD40L/IFN-γ-activated mature dendritic cell (DC)-based vaccine and the characteristics of T-cell responses generated upon vaccination. Over 200 secreted proteins were evaluated using the Human MILLIPLEX® MAP magnetic bead panels (Cytokine/Chemokine Panels 1-3, High Sensitivity T Cell Panel, CD8 Panel, Th17 Panel, Soluble Cytokine Receptor Panel, Angiogenesis/Growth Factor Panel , MMP & TIMP Panels, IGFBP Panel, Sepsis Panels, Cancer Panels 1-4, CVD Panels 1-3, and others). Additionally, the activation status of a DC-vaccine was profiled using MILLIPLEX® MAP intracellular MAPK/SAPK Pathway multiplex panel. Representative heat maps indicated differences in secreted protein concentrations, biomarker signatures and phosphorylation status in GM-CSF/IL-4-derived immature DCs and CD40L/IFN-γ-activated mature DCs. Furthermore, vaccine-induced antigen-specific CD8+ T cells presented 2 distinct phenotypes based on secretome profiling, and these profiles correlated with clinical outcomes. Consequently, these data demonstrate the utility of MILLIPLEX® panels using the Luminex® xMAP® technology for the characterization of adaptive immune responses in vaccine/immunotherapy setting.
Combinatorial approach to enumerate immunophenotypes in flow cytometry panels (TECH1P.872)
Authors: Bhattacharya Sanchita , Pouliot Yannick , Dunn Patrick , Thomson Elizabeth , Wiser Jeffrey , Butte Atul 
Published: January 2023

Abstract
The identification of cell populations from flow cytometry has rarely been mapped in a multi-dimensional space. Instead, the focus is typically limited to a small number of pre-specified cell types already described in the literature. We propose to re-conceptualize the flow cytometry panel as a discovery tool to disentangle complex flow data into the simplest, most clinically relevant cell types. We present a combinatorial method to enumerate all possible immunophenotypes within a flow panel from a clinical trial study. We applied this approach to 11,000 raw flow cytometry files from a multi-panel flow experiment in a ragweed allergy clinical trial (ImmPort:SDY1), available from the publicly accessible, NIAID-funded Immunology Database and Analysis portal (ImmPort; immport.niaid.nih.gov). Files were processed through an automated pipeline that applied ImmPort’s FLOCK autogating tool to segregate cell populations based on the expression of individual surface markers. This yielded roughly 59,000 unique cell populations. Unsupervised hierarchical clustering revealed cell populations that were highly correlated with treatment conditions, based on the Pearson similarity measure. From these results we identified a subset of monocytes with positive expression for CD23 that were correlated with clinical outcome. We believe this approach can help identify the minimal set of markers needed to characterize functionally distinct cell populations associated with clinical outcome.
Optimized antibody labeling efficiency through continuous centrifugal diafiltration (TECH1P.838)
Authors: Cappione Amedeo , Mabuchi Masaharu , Briggs David , Nadler Timothy 
Published: January 2023

Abstract
Immuno-detection encompasses a range of analytical methodologies. These assays, which quantify and/or localize expression of protein(s) in complex samples, are reliant upon tagged target-specific antibodies. While a range of labeling kits is available, the workflow is hampered by multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. Also, due to differences between labeling targets (and dyes), prior determination of labeling reaction parameters must occur. We present an alternative method for small-scale protein labeling based upon continuous flow diafiltration. By exploiting the rapid kinetics of labeling reactions, continuous diafiltration minimizes reaction time and exposure to excess dye, guaranteeing maximal target labeling and thus eliminating the need for up-front optimization of reaction conditions. All phases of the conjugation workflow can be performed in a single centrifugal device. When compared to other buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting, recovery, integrity). Originally designed for resin-based affinity purification, the device also provides a platform for initial antibody purification or albumin carrier removal. Overall, the method offers a simplified workflow with reduced processing time and fewer hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance.
Impact of mammalian target of rapamycin inhibition by everolimus on peripheral blood αβ and γδ T lymphocytes in renal transplant recipients (TRAN1P.875)
Authors: Gonzales Josephine , Dutt Shelley , Steinberg Steve , McKay Dianne , Jameson Julie 
Published: January 2023

Abstract
The kidney is an essential organ necessary for the human body to discharge harmful toxins and is imperative for filtering blood. According to the Centers for Disease Control and Prevention, kidney disease is the eighth leading cause of death in the United States. Diabetes and hypertension are two major factors that influence kidney function, which can consequently lead to end-stage renal disease. The primary method of treatment for end-stage renal disease is kidney transplantation, which requires a regimen of immunosuppressant drugs to prevent allograft rejection. Previously, our lab has shown that administration of the immunosuppressant drug, sirolimus a mammalian target of rapamycin inhibitor, also known as mTOR, impairs skin homeostasis and wound repair. To further understand the mechanism behind this adverse effect of mTOR suppression, we examined renal transplant recipients who were administered the sirolimus derivative everolimus. The number and activation of αβ and γδ T cells in the blood of patients administered everolimus were compared to a control group of patients not administered everolimus. Several of these T cell populations, including Vγ9Vδ2 T cells, have been previously displayed to home to sites of tissue damage to improve wound repair. This data explores which T lymphocyte populations require mTOR to maximize function in transplant recipients with implications in tissue repair and graft rejection.
A mutation in Transportin3 (Tnpo3) leads to generation of an individually distinct tumor-specific Kd-restricted epitope in the Meth A fibrosarcoma (TUM2P.899)
Authors: Duan Fei , Duitama Jorge , Al Seesi Sahar , Blanchard Tatiana , McMahon David , Sidney John , Sette Alessandro , Mandoiu Ion , Srivastava Pramod 
Published: January 2023

Abstract
The mutational repertoire of cancers creates the epitopes that make cancers immunogenic. Here, through a comprehensive genomic, bioinformatic and immunological analyses, we uncover over a hundred new neo-epitopes in the Meth A fibrosarcoma of BALB/c mice. Interestingly, the predicted affinities of neo-epitopes for MHC I have no bearing on protective anti-tumor immunogenicity; instead, the numerical difference of such affinities between the mutated and un-mutated sequences, named the Differential Agretopic Index (DAI), is a significant although imperfect predictor. The mutated Transportin 3 (Tnpo3)-derived epitope, the highest ranking (by DAI) epitope of Meth A is naturally presented by Meth A cells. Immunization with whole Meth A cells induces mutated Tnpo3 epitope-specific CD8+ T-cell responses. Immunization with mutated Tnpo3 peptide elicits CD8+ T-cell responses that recognize Meth A cells ex vivo, as well as significant protection from a tumor challenge. This tumor immunity is further enhanced by combination of immunization with mutant Tnpo3 with a Toll-like receptor 9 (TLR9) ligand or anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) blocking antibody.