bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tetracysteine tagged OVA (323–339) peptide as a novel tool for visualizing peptide-MHCII complexes in primary mouse cells
Authors: Al Souz Jafar A. , Akkaya Munir , Kamdar Rahul , Kamenyeva Olena , Shevach Ethan M. , Akkaya Billur 
Published: January 2023

Abstract
Professional APCs present peptides to CD4+ T cells in the context of MHCII. To understand the dynamics of peptide-MHCII complexes in T cell-APC interaction, real-time visualization of these complexes is crucial. However, current reagents such as peptide specific MHCII antibodies do not allow one to probe peptide-MHCII dynamics during live interactions in vivo, as they also block T cell-APC interactions. Fluorescent tags such as green fluorescent protein (GFP) consist of more than 200 amino acids and have the potential of disrupting the functionality of the antigenic peptides. Therefore, we constructed an OVA(323–339) peptide containing a pro-fluorescent tetracysteine tag (CCPGCC) at the C-terminus with aminocaproic acid linker (OVACACA). First, we tested whether the addition of tag had an impact on T cell stimulation and found that OVACACA pulsed APCs could activate OTII cells to same degree as OVA(323–339). Secondly, we treated the APCs with an organoarsenic compound to switch on the fluorescence and measured the signal by flow cytometry. We showed that the fluorescent signal specifically was derived from OVACACA-MHCII, as neither MHCII−/− APCs pulsed with OVACACA, nor MHCII+/+ APCs pulsed with OVA(323–339) produced the same signal. We also tracked the fluorescent signal in OTII-DC cultures in vitro and detected that it was concentrated at the synapse over time. Altogether our findings suggest that tetracysteine tagged peptides can be used for tracking peptide-MHCII complexes during the interactions of primary mouse T cells and APCs and have the potential to be an alternative to MHCII tetramers to detect antigen specific T cells.
Serum IGFBP2, IGFBP4, and sTNFR2 are predictive of concurrent clinical and histological nephritis in SLE
Authors: Haque Anam , Soliman Samar , Henderson Jared , Stanley Samantha , Saxena Ramesh , Mohan Chandra 
Published: January 2023

Abstract

Background
Systemic Lupus Erythematosus (SLE) is an autoimmune disorder that targets various body systems, including the kidneys, leading to Lupus Nephritis (LN). Present diagnostic methods for LN include the renal biopsy, which is invasive. This project takes advantage of a large cohort of LN samples where the serum samples were obtained at the time of renal biopsy. Our goal is to identify serum protein markers that are predictive of concurrent clinical and histological nephritis.

Methods
Serum samples from 10 healthy controls and 66 SLE patients with active renal disease obtained at the time of renal biopsy were ELISA-tested for levels of IGFBP2, IGFBP4, and sTNFR2, selected because they were elevated in LN patients in previous screening studies.

Results
Serum sTNRF2 (p &lt; 0.0001), IGFBP2 (p &lt; 0.0001) and IGFBP4 (p &lt; 0.05) were all elevated in LN patients compared to healthy controls. Concurrently measured Glomerular Filtration Rate was negatively correlated with serum IGFBP4 (p &lt; 0.0001), IGFBP2 (p &lt; 0.05) and sTNFR2 (p &lt; 0.001). Serum IGFBP4 correlated with SLEDAI (p &lt; 0.01), and Renal SLEDAI (p &lt; 0.01). All 3 serum proteins showed a positive correlation with Renal SLICC scores with IGFBP4 being the strongest (p &lt; 0.0001). There was also a significant correlation between various concurrent renal biopsy pathology lesions with the assayed serum proteins, notably IGFBP4 (p &lt; 0.0001).

Conclusion
Serum IGFBP2, IGFBP4, and sTNFR2 are predictive of concurrent clinical nephritis activity in LN as well as histological changes on renal biopsy. Whereas the biopsy is subject to sampling errors, the assayed proteins may represent more stable biomarkers of renal pathology changes in LN.
Tracking Plasmodium Invasion of Red Blood Cells by Simplified Flow Cytometry
Authors: Tran Kimvan Thi , Mulry James , Greco Beatrice , Tyagarajan Kamala 
Published: January 2023

Abstract
Malaria is an acute global problem with large number of deaths and infection on an annual basis. Study of malarial parasites, infection mechanism, action of antimalarial drugs and understanding disease dynamics requires culturing of red blood cells (RBC’s) with plasmodium strains followed by quantifying extent of invasion. The current “gold standard” methods for quantifying parasitemia utilizes microscopic examination of blood smears are laborious, time-consuming and extremely subjective in obtaining quantitative data and dependent on the skills of users. We have developed novel methods using simplified sample prep with analysis on the small footprint, touch-screen based Muse® Cell Analyzer that uses microcapillary cytometry to analyze thousands of RBC’s. The approaches utilize nucleic acid based staining to provide quantitation of infected red blood cells or parasitemia level or quantify parasite density in samples. The methods provide good sensitivity and high reproducibility across a wide parasitemia range and show valuable applicability to screening and monitoring of cultures. Approaches were applied to RBC’s infected with different P. falciparum strains followed by treatment with different doses and times with Chloroquine and Dihydroartemisinin. The studies demonstrate early and sensitive detection of parasitemia changes with treatment and showcase utility of the assays for studying action of anti-malarial drugs. The capability to obtain parasitemia results on infected RBC’s rapidly and easily on a small, accessible platform like Muse® Cell Analyzer can greatly increase ability of academic and clinical malarial researchers to obtain critical information relevant to status and progress of malarial disease
New Flow-Based Multiplex Assays for Simultaneous Quantification of 13 Human or Mouse Macrophage/Microglia Related Cytokines
Authors: Sun Binggang , Rughwani Priyanka , Chow Kay , Lehmann Jason , Ji Shaoquan 
Published: January 2023

Abstract
Immune responses are mediated by innate immune cells (e.g., macrophages) and subsequently, adaptive immune cells (e.g., T and B cells). Macrophages secrete an array of inflammatory mediators to affect their host defense, tissue repair, and immunoregulatory functions. Pro-inflammatory macrophages (M1) produce cytokines, such as IL-12p70, TNF-α, IL-6, IL-1β, IL-12p40, IL-18, IL-23, IFN-γ, and IP-10, while anti-inflammatory and tissue repairing macrophages (M2) release a different set of factors, such as IL-4, IL-10, IL-6, Arginase, TARC, MDC, IL-1RA and TGF-β1. We have developed new assay panels targeting human or mouse macrophage/microglia cells, using fluorescence–encoded beads that are suitable for use on commonly available flow cytometers. Each panel allows for the simultaneous quantification of 13 analytes related to macrophage function in humans and mice. These panels have been validated using biological samples to quantify analyte concentration changes in response to various stimuli. These high quality, low cost, easy-to-use panels provide an alternative multiplex solution to the biomedical research community.
Highly robust digital gene expression profiling of 96 gene targets for IO Lymphocyte and Myeloid activity in 96 Colorectal FFPE samples without any enzyme (RT or amplification) reactions
Authors: Merritt Christopher , Kharkia Anisha , Ong Giang 
Published: January 2023

Abstract
The NanoString nCounter Analysis platform uses a novel molecular barcoding technology to measure multiplexed gene expression of up to 800 targets. The assay counts fluorescent barcodes hybridized to targets to provide precise digital data. The standard nCounter gene expression assay can be used to process 12 samples per run. We recognize an additional need for a higher sample-throughput assay which would enable researchers to quickly evaluate tens of multiplexed targets in hundreds of samples. Here we present data from a new nCounter assay, nCounter PlexSet Reagents for gene expression, which is performed in a 96-sample format and is capable of digitally profiling up to 96 gene targets per sample. We used two preselected pathway panels for IO Lymphocyte Activity (96 gene targets) and IO Myeloid Activity (79 gene targets), chosen from our 400+ preselected pathway panels list, in order to evaluate the presence of immune signatures in 44 colorectal cancer (CRC) samples. Each CRC sample was ran in duplicates across the two panels in addition to a Reference Sample to calibrate for variability between PlexSet reagents. The results show that the PlexSet reagents for gene expression provides highly reproducible data with an average R2 &gt; 95% between duplicate samples.
Sample sparing platform for microscopy shows that CD45RB ligation increases Treg sensitivity to activation signals in vitro
Authors: Lee Joanne Haeun , Lee Jounghyun H. , Kam Lance C. 
Published: January 2023

Abstract
Small subpopulations of cells, e.g. regulatory T cells, often play key roles in the function of the entire immune system. While their smaller number makes them difficult to work with in conventional immunocytochemistry assays or flow cytometry, microscopy can provide functional and spatial information on a single cell basis. Combined with surface modification technology such as synthetic lipid bilayers and micropatterned antibodies, we are in a unique position to be able to study the spatial mobility of intracellular signals (e.g. show that mobility of Lck differs between mouse and human T cells). Even still, conventional microscopy platforms such as 96-well plates or flow cell systems require a minimum of 10k to 1 million cells and does not allow for an interchangeable surface. Conical wells with a 1mm bottom and 5mm top diameter (microwells), laid out in a 96-well configuration were mass manufactured out of polypropylene from Protolabs, and adhered to surface modified glass coverslips. 1k cells seeded and centrifuged at 100g for 10 mins resulted in 81% of the cells landing on an image-able area. Based on the report that anti-CD45RB mAb MB23G2 enhances graft-survival, we tested the hypothesis that anti-CD45RB ligation in vitro increases Treg sensitivity to activation signals. 3 different activation signal levels (1, 5, and 20 μg/mL), 2 cell types, and 2 treatment conditions conventionally required at least 150k cells per sample, or 1.8M cells total. On the microwell, we performed the experiment with 8k cells per sample, with quadruplicates for a total of 384k cells, to show that the ratio of phosphorylation of Lck at Tyr 394 to 505 sites, and alignment to micropatterned anti-CD3 dots are increased in Tregs treated with anti-CD45RB.
High throughput screening of IRAK4 small molecule inhibitors in TLR ligand stimulated whole blood
Authors: Sujatha-Bhaskar Swathi , Huang Zhiyu , Francis George , Bryan Callie , Zarrin Ali A. , Kiefer James , Ghilardi Nico , Brightbill Hans D. 
Published: January 2023

Abstract
The IRAK4 kinase is a key regulator of TLR and IL1R signaling. Small molecule inhibitors of IRAK4 represent an important advance in targeting these pathways. We developed a high throughput whole blood assay to identify potent IRAK4 inhibitors through the inhibition of TLR ligand (R848, LPS, and Gardiquimod) induced inflammatory cytokine production (IL6, TNF, IFNa). Extensive optimization was carried out to ensure rigorous measurement of small molecule potencies, percent maximum inhibition, and cytokine endpoint correlations to clearly distinguish compounds from one another in the context of human whole blood variance. Whole blood treated with IRAK4 inhibitors resulted in partial inhibition of TLR induced cytokines. Whole blood is a complex mixture of cell types and serum proteins. Our analysis shows various dependency of TLR signaling on IRAK4 kinase activity varies among various immune cells allowing for residual inflammatory outputs. Ex vivo whole blood stimulation assays have proven to be an important tool in drug discovery to identify potent IRAK4 small molecule clinical candidates and further define the role of IRAK4 signaling in immune responses.
Enforcing anergy of autoreactive B cells through inhibition of the PI3K pathway
Authors: Franks S. Elizabeth , Getahun Andrew , Cambier John C. 
Published: January 2023

Abstract
Variations in expression and/or function of a number of genes confer increased risk of development of autoimmunity. Among these variations is the expression of negative regulators of the PI3K pathway, whose activity is critical in maintaining B cell anergy. Anergy, in which autoreactive B cells are unresponsive to antigen, is maintained via upregulation of the inositol phosphatases PTEN and/or SHIP-1, which hydrolyze PI(3,4,5)P3, the product of PI3K, yielding PI(4,5)P2 and PI(3,4)P2, respectively. Levels of these negative regulators are reduced in SLE due to aberrant regulation by miRNA-7 and miRNA-155. Deletion of miRNA155, which raises SHIP-1 expression, has been shown to reduce autoantibody responses in the lpr autoimmune mouse model of Lupus. Acute deletion of SHIP-1 or PTEN in autoreactive B cells leads to autoimmunity. We report that it is possible to pharmacologically correct defects in PI3K pathway regulation which confer risk of autoimmunity, thus preventing autoimmunity while conferring minimal disruption of adaptive immune function. This intervention does not prevent autoimmunity caused by compromise of other risk allele emulating conditions, such as reduced SHP-1 expression. We hypothesize that PI3K inhibitors can be used therapeutically to compensate for decreased negative regulation of the PI3K pathway. The knowledge gained from these studies will aid in the development of precision treatments for autoimmunity that act by correcting effects of specific risk alleles.
Fingolimod ameliorates neuropsychiatric lupus without reducing circulating autoantibodies or brain cytokine expression.
Authors: Mike Elise V. , Putterman Chaim 
Published: January 2023

Abstract
Although the pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is still not well understood, autoantibodies, loss of brain barrier integrity, and pro-inflammatory cytokines have all been implicated in disease progression. Fingolimod (FL), an approved treatment for multiple sclerosis, improves neurologic disease through S1PR modulation and reducing passage of autoaggressive lymphocytes into the CNS. In initial studies, we found that FL treatment reduces lymphocytic infiltration of the choroid plexus in MRL/lpr lupus mice and attenuates memory impairment and depressive-like behavior. Here, we determined the effects of FL on systemic disease and pro-inflammatory cytokine expression in the CNS. Brain, spleen, lymph node, and serum samples were evaluated in MRL/lpr mice treated with FL three times weekly for 10 weeks starting at 10 weeks of age and from untreated controls (n=16/group).
FL-treated mice exhibited a significant reduction in splenomegaly and lymphadenopathy. However, there was no change in serum titers of anti-double stranded DNA antibodies. Moreover, immunofluorescence staining revealed no difference in IgG deposition between treated and control mice, suggesting that autoantibody reduction is not critical for FL-mediated disease improvement. Surprisingly, quantification of brain cortical cytokine levels revealed a significant increase in IL-1a, IL-1b, IFNg, IFNb and IL-12p70 in FL-treated mice, while IL-1a, IL-1b and MCP-1 were increased in the hippocampus.
These findings suggest that FL attenuates NPSLE, but the mechanism of improvement is not mediated by a reduction of autoantibodies or pro-inflammatory cytokine signaling.
A(1-7) reduces pathologies associated with SLE in MRL-lpr mice
Authors: Soto Maira , Gaffney Kevin J. , Jadhav Sachin S. , Rodgers Kathleen E. 
Published: January 2023

Abstract
Patients with Systemic Lupus Erythematosus (SLE) suffer from a chronic inflammatory autoimmune disease that causes damage to various organ systems. Even with the array of current treatments, patients with lupus are still in need of effective therapies to treat this complex, multi-system disease. Recent studies of the Renin Angiotensin System (RAS), widely known for its role in blood pressure regulation, have uncovered immunomodulatory effects. One of the more recently discovered components of RAS, angiotensin (1-7) [A(1-7)], is a bioactive peptides that affects various systems. In our study, MRL-lpr mice were treated daily with saline or A(1-7) for 6 weeks starting at 8 weeks of age. Proteinuria was monitored throughout the duration of the study. MRL-lpr mice treated with saline had higher levels of proteinuria compared to the MRL/MpJ- control mice, and A(1-7) treated MRL-lpr mice. A glomerulonephritis score revealed that the MRL-lpr mice had significant glomerular pathologies compared to MRL-MpJ mice of the same age. A(1-7) treatment significantly reduced the glomerular pathologies in the MRL-lpr mice. Treatment with A(1-7) reduced the size of both inguinal and axillary lymph nodes in MRl-lpr mice, and levels of anti-dsDNA IgG compared to the saline treated MRL-lpr mice. Treatment with A(1-7) also significantly reduced the severity of cutaneous pathologies resulting in only mild inflammation and hair loss compared to the saline treated MRL-lpr mice. Daily systemic treatment with A(1-7) significantly ameliorated disease in one of the most severe mouse models of SLE, making Mas agonists a potential target for future SLE therapies.
Sex hormones and gender influence the expression and function of regulatory T cells differentially in SLE patients
Authors: Singh Ram Pyare , Hahn Bevra H. 
Published: January 2023

Abstract
Regulatory T cells have been implicated in the regulation and maintenance of immune homeostasis. Whether gender and sex hormones differentially influence the expression and function of regulatory T cell phenotype and their influence on Foxp3 expression remain obscure. We provide evidence in this study that in humans the number and percent of regulatory T cells expressing CD4+ and CD8+ are significantly reduced in healthy females compared to healthy males. Both CD4+CD25+hiCD127low and CD8+CD25hi+CD127low subsets in healthy males have a 3–4-fold increase in Foxp3 mRNA expression compared to healthy females. Incubation of CD4+ regulatory T cells with 17b- estradiol at physiological levels either maintains or decreases FoxP3 expression in females with SLE, in contrast to inducing a significant increase in CD4+ Treg in healthy females. Of significance, testosterone significantly increases FoxP3 expression in CD4+CD25hiCD127low cells from females with SLE. Furthermore, plasma concentrations of testosterone positively correlate in those females with the expression of FoxP3 in their CD4+CD25+CD127lowT cells. These data demonstrate that females may be more susceptible than males to SLE and other autoimmune diseases in part because healthy females have fewer regulatory T cells and less FoxP3 expression within those cells. In addition, females with SLE, compared to healthy females, have less ability to generate CD4+Treg in response to physiologic concentrations of 17b-estradiol, whereas an active testosterone metabolite increases the generation of CD4+Tregs. These data clearly suggest that gender and sex hormones influence susceptibility to SLE via their effects on regulatory T cells and Foxp3 expression.
Apoptotic cell induced, TLR9-dependent AhR activity is a critical driver of tolerance induction and suppression of lupus
Authors: Shinde Rahul Suresh , Hezaveh Kebria , Halaby Marie Jo , Kloetgen Andreas , Lamorte Sara , Munn David , Tsirigos Aristotelis , Madaio Michael , Gabrielsson Sussane , Wither Joan , De Carvalho Daniel , McGaha Tracy 
Published: January 2023

Abstract
The Aryl hydrocarbon receptor (AhR) can potently modulate immunity at multiple levels, but its mechanistic role in immune regulation is still being elucidated. Here we show phagocytes exposed to apoptotic cells exhibit rapid activation of AhR driving IL-10 that antagonizes inflammatory cytokine production. AhR activation was dependent on apoptotic cell DNA-TLR9 interactions and reactive oxygen species (ROS) production in phagocytes that promoted nuclear accumulation and transcriptional activity. In vivo, apoptotic cell-induced AhR signals in myeloid cells were required for prevention of inflammatory innate and adaptive immunity against DNA and histones. Moreover, disease progression in lupus correlated with AhR signal strength, and disease course could be reduced or exacerbated by modulation of AhR activity. Finally, we observed myeloid specific deletion of AhR in mice resulted in systemic autoimmunity, and in SLE patients an increased AhR transcriptional signature correlated with disease. Thus, our findings suggest AhR activity influenced by apoptotic cell death is a key mechanism in maintenance of peripheral tolerance reducing inflammation and thereby retarding systemic autoimmune disease progression.
siRNA-mediated c-Rel knockdown ameliorates collagen-induced arthritis in mice
Authors: Ruan Qingguo , Fan Tingting , Zhong Fuhua , Liu Ruiling , Chen Youhai H. , Wang Ting 
Published: January 2023

Abstract
Previous studies have shown that inflammatory mediators involved in the development of rheumatoid arthritis (RA) are regulated by the Rel/nuclear factor-κB (Rel/NF-κB) transcription factor family. c-Rel, a member of the Rel/NF-κB family that is preferentially expressed by immune cells, is a risk factor for several inflammatory diseases including RA. In the current study, we investigated whether targeting c-Rel can be used to treat collagen-induced arthritis, an animal model for RA. c-Rel specific siRNA (siRel) delivered by nanoparticles was used to knockdown the expression of c-Rel. Our results showed that siRel treatment significantly ameliorated collagen-induced arthritis. Further study revealed that c-Rel expression in the dendritic cells and macrophages from mice treated with siRel was significantly down-regulated. Consistent with the phenotypical result, the expression of inflammatory cytokines TNF-α, IL-1β, IL-6, IL-12 and IL-23 by peritoneal macrophages and splenocytes were significantly decreased. In addition, attenuated systemic and collagen-specific Th1 and Th17 immune responses were observed. Furthermore, we found that the expression of inflammatory cytokines was significantly down-regulated and the infiltration of CD3+ T cells and F4/80+ macrophages was markedly reduced in hind paws of mice treated with siRel. Collectively, our study provides strong evidence that siRNA-mediated c-Rel knockdown can suppress the development of collagen-induced arthritis in mice. Therefore, blocking c-Rel may represent an attracting strategy for the treatment of human rheumatoid arthritis.