bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interferon Beta-1a Modulates the Innate Immune-response-mediated Regulation of Adaptive Immunity in Early Multiple Sclerosis (131.15)
Authors: Zhang Xin , Tang Y , Montes M , Jin J , Ramgolam V S , Markovic-Plese Silva 
Published: January 2023

Abstract
We performed Affymetrix gene arrays (HU133, ~38,500 genes) using RNA from the in vitro activated PBMCs from patients with clinically isolated syndrome suggestive of multiple sclerosis (CIS) (n=15) and matched healthy controls (HC) (n=8). Results were confirmed and further supported by RT-PCR and RayBio Cytokine Arrays. Affymetrix data revealed that IFNB-1a induced significant (p&lt;0.05) up-regulation of 711, and down-regulation of 587 genes in CIS patients, and 446 and 543 in HC, respectively. CXCL11 and CXCL10, the ligands of CXCR3 were significantly increased in CIS patients and HC, whereas proinflammatory chemokines CXCL2, CXCL3 and CXCL5 were decreased in CIS patients. CCL18 and CCL19 were increased in CIS, while CCL22 and CCL17 were decreased in CIS and HC. Those chemokine gene changes reflect the complex effects of IFNB-1a that likely affect differentiation/ maturation and chemotaxis of DCs and other cells bridging the innate and adaptive immune response. The anti-inflammatory cytokine IL-10 was increased in CIS, consistent with the previous findings in multiple sclerosis, while IL1A, IL1R1 and IL-16 were decreased in CIS and HC. IL-1 is an acute inflammatory phase cytokine whose inhibition may decrease differentiation of IL-17 secreting T-cells that is critical in the development of autoimmune response. SOCS7 and SCOS5 were decreased in CIS and HC that may result in impaired Th1 responses. TLR7 and its signal adapter MYD88 were increased in CIS and HC. IRAK4 and TRAF6, the components of MYD88-dependent pathway were also increased in CIS. IRF2 and IRF7 that may be involved in MYD88-independent TLR signaling pathway were increased in CIS and HC. Such effects on TLRs signaling pathways may inhibit DCs maturation and alter the innate immune response mediated regulation of adaptive immunity.
Genomic-based high throughput screening and identification of small molecule inhibitors targeting IFN α signaling pathways (131.17)
Authors: Chen Bo , Cibotti Ricardo , Zong Qin , Morris Chad , Castaneda Juana , Naiman Brian , Liu Derong , Glodek Anna , Soppet Dan , Horrigan Steve , Kiener Peter , Coyle Anthony , Audoly Laurent 
Published: January 2023

Abstract
Multiple lines of evidence suggest that inhibition of Type I Interferons (IFNs), including IFN(, may provide a therapeutic benefit in treating a number of autoimmune diseases such as Systemic Lupus Erythematosus (SLE). Herein, we describe the generation and use of AvalonRx-HITS®, a proprietary genomic based high-throughput screen (HTS), followed by immunological assays in primary human cells to identify inhibitors of the IFN( pathway. In the HTS step, THP1 cells were stimulated with IFN( and 268 defined small molecule inhibitors targeting 41 different intracellular signaling pathways were screened for their ability to block a specific IFN(-evoked gene signature. Four classes of compounds emerged from the HTS: NF-(B, JAK/STAT, Ubiquitin/Proteasome and HDAC inhibitors. In secondary assays, these compounds blocked both IFN(-induced human monocyte activation and differentiation into dendritic cells, and IFNs-associated gene signature induced by SLE serum in human monocytes. In addition to their dose-dependent anti-inflammatory effects, inhibitors targeting NF- (B or JAK/STAT signaling did not modulate IFN( anti-viral effects in an in vitro HSV-1 replication assay. In summary, we have developed a novel and robust platform using genomic, immunological and viral assays that may lead to the identification of small molecule inhibitors for the treatment of IFN(-associated autoimmune disorders.
Determination of affinity constants of adalimumab, infliximab and etanercept for soluble and membrane forms of TNF using kinetic exclusion assay (131.22)
Authors: Sakorafas Paul , Bose Sahana , Kaymakcalan Zehra 
Published: January 2023

Abstract
TNF neutralizing biologics adalimumab (Ada), infliximab (Inf), and etanercept (Eta) have distinct clinical efficacy profiles. It is not known whether their binding characteristics to TNF, especially their affinities for soluble (sTNF) vs transmembrane TNF (mTNF) plays an important role in different clinical outcomes. Previously, BIAcore and radioimmunoassay methods have been used to measure affinities for sTNF and mTNF, respectively. The affinity for mTNF as measured by RIA was much lower than the sTNF for all three biologics. In this study, we employed kinetic exclusion assay (KinExA) to measure overall affinities of unlabeled Ada, Inf and Eta for both sTNF and mTNF. A fixed amount of anti-TNFs were incubated with varying amounts of sTNF or cells transfected with a TNF deletion mutant that expressed a stable form of mTNF to reach to equilibrium. The overall affinities of Ada, Inf and Eta for sTNF were determined as 8.6, 4.2 and 1.2 pM; and for mTNF as 7.9, 7.0, and 11.2 pM. When measured by the same instrument, affinity for the membrane form of TNF was similar to soluble TNF for all three anti-TNFs. Eta had a higher affinity for sTNF, followed by Inf and Ada. Ada, Inf and Eta had similar affinities to mTNF as presented on the transfected cells.
Antagonizing HMGB1 inhibits proteinuria in a murine model of lupus-like disease (131.24)
Authors: Mao Su-Yau , Xu Yan , Zhang Jing , Schifferli Kevin , An Ling-Ling , Leininger Joel , Kiener Peter A , Wu Herren , Coyle Anthony J , Audoly Laurent P 
Published: January 2023

Abstract
High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts like an inflammatory cytokine when it is released by dying or stimulated cells. Elevated levels are detected at sites of inflammation in a number of autoimmune disorders including SLE. In addition, HMGB1 has been shown to contribute to the activation of plasmacytoid dendritic cells as well as B cells, cell types that are implicated in the pathogenesis of SLE. To address the role of HMGB1 in experimental lupus, we investigated the protective efficacy of a fully humanized mAb (IA-4, which recognizes both mouse and human HMGB1) in an IFNα-accelerated lupus model. In this model, adenovirus IFNα-treated NZB/W F1 mice exhibit 90–100% proteinuria incidence at 4–6 weeks post-treatment. The functionality of IA-4 has been demonstrated in vitro by its ability to inhibit rHMGB1-induced IL-6 secretion in human PBMC (IC50 = 0.9 nM). Prophylactic administration of the anti-HMGB1 mAb IA-4 at 10 mg/kg significantly inhibited the proteinuria severity score at seven weeks (65% inhibition; p&lt;0.05 compared to an isotype control). Consistent with the proteinuria data, histopathological assessment of the kidney also revealed protection with IA-4 treatment. Despite its protection in nephritis, IA-4 treatment showed no detectable effect on serum levels of IFN-α and autoantibodies against dsDNA or SSA/Ro. Taken together, these data suggest that HMGB1 is a critical effector molecule in lupus nephritis and that anti-HMGB1 treatment may provide a potential therapeutic benefit for the treatment of SLE.
Anti-BR3 antibodies – a new class of B cell immunotherapies combining cellular depletion and survival blockade (131.25)
Authors: Lin WeiYu , Gong Qian , Seshasayee Dhaya , Lin Zhoughua , Ou Qinglin , Ye Shiming , Suto Eric , Shu Jean , Lee Wyne Pun , Lee Vivian , Fuh Germaine , Yeh Sherry , Deforge Laura , Danilenko Dimitry , Thompson Jeffrey S. , Ambrose Christine , Balazs Mercedesz , Starovasnik Melissa , Martin Flavius 
Published: January 2023

Abstract
Pathogenic B lymphocytes removal through therapy with depleting monoclonal antibodies results in clinical benefit in both oncology and immunological indications. Incomplete rates of clinical response and the realization that B cell depletion following immunotherapy is incomplete fuels the need for better therapies. BAFF/BLyS signaling through its BAFFR/BR3 receptor have a critical role on B cell proliferation and survival in the mouse and to some extent in non-human primates and humans. In order to combine B cell depletion with the blockade of BAFF mediated B cell survival, we generated depleting, BAFF-blocking, non-agonistic anti-BR3 monoclonal antibodies. The results from this study indicate that in mice anti-BR3 monoclonal antibody reduces B cells to a higher degree when compared to anti-CD20 or BAFF-blockade through BR3-Fc. Anti-BR3 depletes B cells with a slower kinetics than anti-CD20 but showed high B cell depletion and a decrease in bone marrow plasma cells which was not seen with anti-CD20. Anti-BR3 treatment of NZB/W lupus mouse model results in significant survival benefit and reduction of established nephritic clinical symptoms indicating potential therapeutically value for human disease. In conclusion, in vivo comparison in mice of anti-BR3 antibodies with other B cell immunotherapies (anti-CD20 and BR3-Fc) shows characteristics in B cell subset reduction that can guide translation to treatment of human disease.
This work was supported by Genentech Inc.
Beneficial effects of Vitamin D receptor agonists on retinal autoimmunity (131.30)
Authors: Tang Jun , Zhu Wei , Uskokovic Milan , Sliver Phyllis , Su Shaobo , Chan Chi-Chao , Adorini Luciano , Caspi Rachel 
Published: January 2023

Abstract
1,25-Dihydroxyvitamin D3 (calcitriol) is a seco-steroid hormone of multiple bioactivities that regulates the differentiation, growth and function of a broad range of cells, including cells of the immune system, by binding to the nuclear Vitamin D receptor (VDR). BXL-35 is a synthetic analog of calcitriol with reduced effects on calcium metabolism. We examined their ability to inhibit experimental autoimmune uveoretinitis (EAU), an autoimmune disease mediated by Th1/Th17-type response, which serves as a model for human posterior uveitis. B10.RIII mice were immunized with an uveitogenic regimen of 8 μg IRBP in CFA and treated orally with calcitriol or with BXL-35, throughout (prevention) or starting 7 days after EAU induction (reversal). Calcitriol at 0.5 μg/kg and BXL-35 at 10 μg/kg prevented EAU and reduced Ag specific IFN-γ and IL-17 production by primed lymph node cells. In addition, BXL-35 but not calcitriol was able to reverse EAU at sub-toxic doses. Mechanistic studies revealed that dendritic cells from mice treated with calcitriol had reduced production of IL-1, TNF-α, IL-6 and
IL-12/23p40. Furthermore, calcitriol directly inhibited the ability of purified CD4+T cells to produce IL-17 in response to CD3/CD28 engagement under conditions favoring Th17 polarization. In contrast, calcitriol had no effect on the ability of T cells to adhere to endothelial cell monolayers. Our study suggests that the protective effects of VDR agonists on retinal autoimmunity involve direct and indirect inhibition of priming and function of autopathogenic T cells.
The protease inhibitor, Bowman-Birk inhibitor, suppresses experimental autoimmune encephalomyelitis (131.33)
Authors: Ciric Bogoljub , Touil Tarik , Gupta Anjali , Shindler Kenneth , Rostami Abdolmohamad 
Published: January 2023

Abstract
The Bowman-Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor. BBI concentrate (BBIC) is enriched with BBI, but is predominantly made up of other ingredients. In our previous study we found that oral administration of BBIC to Lewis rats with EAE resulted in significant clinical and histological suppression of disease. In the present study we investigated the effect of BBI using mouse EAE models and pure BBI. We found that both I.P. and oral treatment with BBI (typically 1 mg/day BBI) in SJL/PLP139–151, or C57BL/6/MOG35–55 models, significantly improved clinical and histological parameters of EAE (disease onset, severity, weight loss, inflammation and demyelination). This was true for different treatment regimens as regards the day of treatment initiation relative to immunization for EAE induction. In most experiments antigen-specific proliferation of immune cells derived from BBI-treated mice was significantly lower relative to control groups. In the SJL model of optic neuritis BBI significantly reduced the incidence of neuritis, inhibited inflammation and prevented loss of retinal ganglion cells. Using Boyden’s chamber assay we found that BBI inhibited invasiveness of activated splenocytes through the matrigel barrier. These results indicate that BBI is an excellent candidate for oral therapy in multiple sclerosis.
Prevention of Type 1 diabetes in NOD mice by helminth infection (131.36)
Authors: Liu Qian , Sundar Krishnan , Mousave Gity , Bleich David , Gause William 
Published: January 2023

Abstract
Parasitic helminth infection has been shown to modulate pathologic inflammation associated with allergy and autoimmune disease. The aim of this study was to examine whether infection with a helminth parasite could down-regulate the onset of type 1 diabetes in NOD mice and to examine the mechanisms involved in this protection. At 5 weeks of age, NOD mice were infected with Heligmosomoides polygyrus(Hp). At 36 weeks of age, these mice were compared with uninfected mice for the onset of type 1 diabetes (T1D). 70% of uninfected NOD mice became diabetic but only 10% of Hp infected mice developed T1D. Moreover, Hp infection also significantly reduced the development of the more aggressive cyclophosphamide-induced T1D in NOD mice with 5% developed in Hp-infected group compared to 78% in uninfected group. Further studies showed that infection with Hp in NOD mice:
markedly inhibited insulitis in the pancreas;elevated IL-4 and IL-13 levels in mesenteric and pancreatic lymph nodes as well as in spleens;increased the frequency of CD4+ CD25+ Foxp3+ Tregs in mesenteric and pancreatic lymph nodes;led to a consistent, massive accumulation of mononuclear cells in the regions of pancreatic fat.
These findings suggest that infection with Hp significantly inhibits the onset of T1D in NOD mice by influencing lymphocyte trafficking to the β-islet cells in the context of increased Th2 and T regulatory cell populations.
High Throughput Cell-Based Immunoassays Using Filtration Plates (132.1)
Authors: Park Jun , Kellard Libby , Lynch John , Sharma Ajay 
Published: January 2023

Abstract
Cell-based immunoassays require time consuming centrifugation/wash steps which can cause a substantial cell loss. We describe here automation compatible cell-based immunoassays using 96 well filtration plates. The method is rapid and robust, requiring no centrifugation based washing steps, and enables easy yet rigorous removal of unbound antibodies with little cell loss (&lt;20%) even after extensive filtration (&gt;10x).
We tested the method for detecting surface and intracellular proteins via a high throughput flow cytometer. In all cases, reproducible results with excellent signal to noise ratio were detected.
For surface protein detection, Jurkat cells were stained for CD45 and MFI (mean fluorescence intensity) of 504 +/-19 (CD45) and 12 +/-2 (isotype control) were obtained. For intracellular protein detection, anisomycin treated Jurkat cells and INF-γ treated U937 cells were stained for phospho-p38 (pT180/pY182) and phospho-Stat1 (pY701), respectively. MFI was 19.6 +/-1.8 (induced) and 5.2 +/-0.4 (not induced) for phosph-p38, and 44 +/-0.8 (induced) and 4.8+/-0.6 (not induced) for phospho-Stat1.
In conclusion, reported method allows for sample incubation/preparation in the same plate, while removing the rate limiting centrifugation/wash steps. While we demonstrate its use in flow cytometry, the method is suitable for other cell-based immunoassays such as chemiluminescent protein detection.
RNAi-mediated silencing in immune cells - From single gene analysis to screening applications (132.2)
Authors: Zumbansen Markus , Altrogge Ludger , Lenz Dietmar , Müller-Hartmann Herbert 
Published: January 2023

Abstract
RNAi-mediated gene knockdown is a powerful tool that is successfully applied to identify gene function and to elucidate biological pathways. RNAi experiments and large-scale siRNA screens require the efficient delivery of highly functional and specific nucleic acid substrates into the appropriate cell system. Cell types relevant for immunological research, such as primary T cells and several suspension cell lines, are poorly accessible using reagent based transfection approaches.
Nucleofection® is an established method for the effective, non-viral transfection of nucleic acids into primary and hard-to-transfect cell types. We expanded the method to the 96-well format making high throughput applications such as siRNA-library screenings possible in cell types highly relevant for immunological research.
We here present data showing the efficient RNAi-mediated gene knockdown in various cell lines and primary cells using 96-well nucleofection.
For example, data about the knockdown of Fas-mediated apoptosis in Jurkat cells will be presented. These studies were expanded to PLK1, which is a key regulator of mitotic progression in mammalian cells. Knockdown of PLK1 resulted in impaired proliferation and increased apoptosis. Starting from these single gene analyses RNAi-library screens are set up for the identification of anti-proliferative and pro-apoptotic genes.
Validation of an ELISPOT Based T cell Biomarker Assay for Monitoring Effects of Immune Modulating Agent (132.3)
Authors: Wullner Danika , Yue Erica , Swanson Steven , Jawa Vibha 
Published: January 2023

Abstract
Enzyme linked immunospot (ELISPOT) assays are widely used for detection of rare antigen specific cytokine producing T cells. These T cells are present in circulation during an infection or vaccination. A quantitative assessment of immunogen specific T cell responses may prove to be a meaningful surrogate marker of functional immunity in patients. To be an effective biomarker assay in a clinical trial evaluation, the assay needs to be evaluated in terms of its robustness and precision. This study describes the steps towards validating a T cell ELISPOT assay.
Validation experiments included assessment of variability during cell harvest and freezing procedures, and shipping and handling of cells from clinical site to the laboratory. A variability of below 5% (cell viability and cell count) and 9.3% was observed in ELISPOT counts throughout shipping experiments. A series of mitogens were screened to select the appropriate positive control for T cell activation. PHA and tetanus were selected as positive controls based on the consistently high spot counts in the ELISPOT. An increase in variability was observed with an increase in concentration of cells added to the ELISPOT wells. The optimal cell number to be plated was set at 300,000 cells/ well. Inter and intra assay variability was less than 20% for a PHA mitogen stimulated control. Analysis of variability results enabled the development of cut point for each stimulant. This was helpful in setting criterions for an actual response over false positives/negatives. Based on these validation results, a reliable ELISPOT assay was developed to quantify antigen specific T cell immunity and consequently used as a biomarker for monitoring the effects of immunomodulatory drugs.
Intracellular localization and trafficking using the ImageStream imaging flow cytometer (132.6)
Authors: Friend Sherree , Venkatachalam Vidya , George Thaddeus C , Hall Brian E , Morrissey Philip J. 
Published: January 2023

Abstract
Specific ligands or antibody-conjugated drugs can mediate their cellular effects by gaining entry into cells via receptor mediated endocytosis. Once internalized, molecules differ in their preferential endocytic pathway, and drug efficacy is highly dependant on the route of entry and interaction with cellular components. Evaluation of internalization and intracellular molecular trafficking events are traditionally performed using microscopy. These analyses are limited, because manual image acquistion and quantitative image analysis are time consuming processes. Here we describe a method for measuring internalized events using the ImageStream imaging flow cytometer, which automatically collects large numbers of images per data set and provides qunatitative image analysis tools. In addition, localization of internalized probes to endosomes and lysosomes is quantified in several model systems. Because the ImageStream collects multiple fluorescent images per cell, internalized marker colocalization to different cellular compartments can be done simultaneously in a quantitative manner.
The forkhead transcription factor Foxq1 enhances NF-kB activity and is critical for robust T cell activation and autoimmunity (35.22)
Authors: Bupp Melanie R. Gubbels , Peng Stanford L. 
Published: January 2023

Abstract
Essential immunoregulatory functions have recently been ascribed to several members of the forkhead transcription factor family. For example, Foxo3a and Foxj1 inhibit NF-kB activity and Foxp3 regulates lineage commitment and function of regulatory T cells. Here, we report that another family member, Foxq1, is critical for robust helper T cell activation. CD4+ T cells from mice bearing a mutant allele of Foxq1 displayed a significantly reduced proliferative response with impaired secretion of IL-2 and IFN-gamma after TCR ligation as compared to wild-type (WT) T cells. This defect is largely rescued by the addition of recombinant human IL-2, suggesting that Foxq1 is required for optimal production of IL-2 and consequently, robust T cell proliferation. Accordingly, in in vitro luciferase assays, Foxq1 augmented transcriptional activity of the IL-2 transactivator NF-kB while mutant Foxq1 did not retain this function. Finally, Foxq1-mutant mice exhibited reduced susceptibility to the progression of experimental autoimmune encephalomyelitis than their WT counterparts. Thus, Foxq1 is essential for helper T cell activation and autoaggressive T cell responses in vivo, likely via its ability to potentiate NF-kB activity and IL-2 production.
The epigenetic regulation of major histocompatibility complex class II (MHC-II) genes by the 19S proteasome (35.24)
Authors: Koues Olivia I , Greer Susanna F 
Published: January 2023

Abstract
Epigenetic modifications confer the accessibility of chromatin to transcription factors. Histones provide a scaffold for DNA to wind around and post-translational modifications to their N-terminal tails determine the open or closed state of chromatin. Essential to the adaptive immune response are MHC-II molecules which present antigenic peptides to CD4+T cells, thus initiating the immune system to protect against invading pathogens. Although much is known about the transcription factors necessary for MHC-II expression, there is little information as to the epigenetic histone modifications and the requisite enzymes needed to provide these transcription factors access to DNA. Yeast studies have shown the ATPases that comprise the19S component of the 26S proteasome target histone acetyltransferases (HATs) to active promoters. This targeting increases the level of acetylated histone H3 and opens the chromatin. To investigate the role of the 19S proteasome in the epigenetic regulation of MHC-II genes, we utilized siRNA to knockdown the 19S ATPase, S8. S8 knockdown decreased both HAT recruitment and levels of acetylated histone H3 at the MHC-II promoter. Our current study strongly implicates the 19S proteasome in modifying histones to initiate MHC-II transcription and provides novel insights into the regulation of mammalian transcription.
Research supported by NMSS, Georgia Cancer Coalition and GSU.