bluuebunny/crossref_metadata_2025_split huggingface dataset:
Innate amplification of CD4 T cell responses during infection: NLRs and TLRs converge (P3342)
Authors: O'Donnell Hope , Monack Denise , McSorley Stephen 
Published: January 2023

Abstract
During infection T cells are activated in an antigen-specific manner and can then secrete effector cytokines in response to secondary interactions with antigen-presenting cells. Effector cytokines are critical for pathogen clearance, but are tightly controlled to limit host pathology. However, mounting evidence suggests alternative non-cognate pathways can induce these effector responses. Here, we examine the amplification of Th1 CD4 T cell production of IFNγ by innate stimuli to show that this non-specific response occurs in a broad variety of infectious and stimulatory contexts and requires cooperation of both Nod-like receptor and Toll-like receptor pathways. Using knock-out and mixed bone marrow chimeric mice we show a requirement for the inflammasome, T cell intrinsic IL18 receptor and extrinsic Toll-like receptors. This suggests a model wherein infected cells undergo inflammasome activation, and upon further innate stimulation, secrete cytokines to non-specifically amplify nearby activated T cell effector responses. The mechanisms of such a pathway could lead to a better understanding of immune-mediated clearance of pathogens, as well as immune-mediated host pathology.
STAT4-dependent and -independent T helper type-2 immunity mediates protection against Pneumocystis lung infection (P3304)
Authors: Myers Riley , Dunaway Chad , Nelson Michael , Trevor Jennifer , Steele Chad 
Published: January 2023

Abstract
Although it is clear that the loss of CD4+ T cells is a predisposing factor for the development of Pneumocystis pneumonia, the protective mechanisms mediated by CD4+ T cells are not well understood. Augmented production of Th1-type cytokines in the lungs of IL-10-/- mice correlated with enhanced P. murina clearance, whereas neutralization of IL-17 in normal mice compromised fungal clearance, suggesting that Th1 and Th17 cells play an important role during infection. STAT4 is critical for optimal Th1 and Th17 development, therefore we investigated its role in P. murina host defense. Our data shows that STAT4 was required for Th1 and, unexpectedly, Th2 responses in the lungs of C57BL/6 (BL/6) and Balb/c mice 14 days after infection, but only Balb/c STAT4-/- mice were susceptible to P. murina infection. Th2 responses in the lungs of BL/6 STAT4-/- mice, but not Balb/c STAT4-/- mice, were intact 28 days after infection and correlated with elevated M2 macrophage polarization. Additionally, anti-P. murina class-switched antibodies were increased in BL/6 STAT4-/- mice, but not Balb/c STAT4-/- mice, as a result of hyper-Th2 responses in the draining lymph nodes. Supporting our experimental observations, we found that lower IL-4, IL-5 and IL-13 levels in plasma from HIV+ patients correlated with Pneumocystis colonization. Collectively, our data suggests that robust local and systemic Th2-mediated responses are critical for immunity to Pneumocystis.
Dendritic cells are the major antigen presenting cells in inflammatory lesions of murine mycoplasma respiratory disease and contribute to disease severity (P3306)
Authors: Simecka Jerry , Sun Xiangle , Dobbs Nicole , Bodhankar Sheetal , Jones Harlan 
Published: January 2023

Abstract
Mycoplasmas cause chronic respiratory diseases in animals and humans, and vaccine development has been problematic. T cell responses were shown to confer protection as well as promote immunopathology in murine mycoplasma pneumonia. Because T cells play a critical role, the role of antigen presenting cells (APC) was examined as they likely influence either an increase in disease severity or promote protective immunity. The roles of APC, such as dendritic cells (DC) and macrophages, in mycoplasma disease are currently unknown. In this study, we examined the ability pulmonary APC populations and their contribution to T cell responses during disease pathogenesis. Both macrophages and DC increased in the lungs of mice after infection. These cell populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC from the lungs of infected mice were most capable of stimulating mycoplasma-specific T helper (Th) cell responses in vitro. In vivo, DC cells were co-localized with Th cells in inflammatory lesions in the lungs of mycoplasma-infected mice. Intratracheal inoculation of mycoplasma antigen-pulsed DC resulted in increased disease severity after subsequent infection. Thus, DC appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these DC likely contribute to responses impacting disease pathogenesis.
Laborsaving production of site-specific anti-phospho monoclonal antibodies by simultaneous screening for multiple phosphopeptides using multiplex bead assay (P3372)
Authors: Ise Tomoko , Abe Yasuhiro , Nagata Satoshi 
Published: January 2023

Abstract
Monoclonal antibodies (MAbs) to phosphotyrosines at specific sites of cellular proteins are critical tools to analyze the functional status of proteins. However, time consuming and laborious screening steps in current methods limits production. Here, we introduce a bead-based multiplex flow cytometry method for simultaneous evaluation of the generated MAbs for the cross-reactivity to different peptide sequences. In this proof of concept study, we chose four phosphorylation motifs from different receptors expressed on B cells (Ig-alpha, membrane IgG and IgE, and FCRL5). They were synthesized as a series of 11-mer peptides containing a tyrosine or a phosphotyrosine at the center and surrounded by unique amino acid sequences. They were conjugated with different color-coded beads which can be identified by red laser excitation in flow cytometry. Antibody binding to the peptides was measured as the level of blue laser excitable R-Phycoerythrin fluorescence. Mice immunized with phosphopeptide-KLH conjugates were used for making hybridomas. As expected, the simultaneous screening revealed unique specificities of the 10-30 MAbs generated in each fusion experiment. In particular, we obtained anti-phospho MAbs specific to Ig-alpha and membrane IgG as well as pan anti-phospho tyrosine Ab. Our results suggest that this method is suitable for production of anti-phospho MAbs that need to recognize a specific phosphopeptide among other phosphopeptides with structural similarity.
Use of antibody gene library for the isolation of specific single chain antibodies by ampicillinantigen conjugates (P3378)
Authors: Messerschmidt Katrin , Neumann-Schaal Meina , Heilmann Katja 
Published: January 2023

Abstract
Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cellsurface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (antifluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single-chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.
Model structure of a full-length anti-IgE complexed with IgE (P3384)
Authors: Wright Jon , Sargsyan Karen , Chu Hsing Mao , Lim Carmay , Chang Tse Wen 
Published: January 2023

Abstract
Modeling antibody/antigen (Ab/Ag) complexes remains a challenging problem and is important for understanding how proteins associate, which in turn can aid drug design. Recently, we have implemented a method (EMAP) for producing the conformations of Ab/Ag complexes in the widely used molecular dynamics program CHARMM. Here, we apply this method to model the structure of IgE in complex with omalizumab, a humanized IgG1 antibody that has been approved in many countries for treating severe, persistent allergic asthma. First, the EMAP method was calibrated by using it to reproduce 24 nonredundant Ab/Ag complexes whose bound and unbound structures have been solved. It could yield near-native conformations for 22 of the 24 complexes, whereas other docking methods such as ZDOCK yield less near-native conformations. Having validated the EMAP method, it was used to dock full-length models of omalizumab and IgE together. The resulting IgE/omalizumab complex structure provides insights into the relative importance of the residues involved in binding. It also provides a plausible explanation for why omalizumab and IgE do not form large immune complexes.
The RiboPuroMycylation method reveals surprising protein synthesis in bone marrow T cells within a day of vaccinia virus infection (P3385)
Authors: Seedhom Mina , David Alexandre , Bennink Jack , Yewdell Jonathan 
Published: January 2023

Abstract
Protein translation is an essential and intricately regulated cellular process. We recently described the RiboPuroMycylation Method (RPM) to measure protein translation in cultured cells. RPM is based on ribosome-catalyzed addition of puromycin to nascent chains followed by detection with a monoclonal antibody specific for puromycin in fixed and permeabilized cells. To apply RPM in vivo, we administered puromycin intravenously and measured puromycin staining via flow cytometry. We were able to detect a robust signal that was blocked by ribosome targeting antibiotics that prevent nascent chain puromycylation. After infection with either vaccinia virus (VACV) or influenza virus, we find profound increases in protein translation in activated T and B cells. In mice with adoptively transferred OT-1 T cells we find that translation of individual cells peaks 3 days after a foot pad infection, i.e. several days before T cell numbers peak. One day after an intra-peritoneal VACV infection, almost 40% of the CD4+ and CD8+ T cells in the bone marrow (BM) have increased protein translation as compared to only 3% of the T cells in the spleen. Using transgenic OT-1 T cells and VACV with or without SIINFEKL, we find that the increased protein translation in the spleen and BM is antigen-dependent. Taken together, these findings illustrate the ability of in vivo RPM to provide novel information regarding the participation of translational regulation in workings of the immune system.
A robust and sensitive flow cytometric method for multiplex RNA in-situ hybridization analysis of blood cells using oligo probes and branched DNA based signal amplification (P3356)
Authors: Lai Chunfai , Levinson Shauna , Lin Audrey , Pache Jared , Goltsev Yury , Nolan Garry , Bers George , Nguyen Quan 
Published: January 2023

Abstract
We present here a novel method for in-situ hybridization (ISH) detection of multiple RNA targets in single cells using standard flow cytometer. This method is based on the use of oligo pairs as probes and bDNA signal amplification for highly sensitive and specific ISH detection of RNA. The oligo pairs are designed to hybridize to specific target sequences and can be readily synthesized. The oligo probes/RNA target complex is detected after signal is generated by bDNA signal amplification. The method is compatible with immunostaining, so simultaneous staining of protein and RNA in single cells is feasible. We demonstrated the co-staining of RNA and surface marker proteins in PBMC, B cells, T cells and leukemic cell lines of U937, K562, Jurkat and M1. In addition, the flow cytometric analysis of cytokine gene induction by lipopolysaccharides/R848 in PBMC was shown by staining with antibody for CD14 protein and probes for RNAs encoding interleukin-1 beta, -6, -8 and tumor necrosis factor-alpha. Taken together, this novel RNA ISH flow cytometry assay offers an invaluable tool for studying immune response, stem cell biology and infectious diseases.
IL-17 mediates the protective actions of PPAR γ blockade during enteroaggregative Escherichia coli infection (P3238)
Authors: Philipson Casandra , Bassaganya-Riera Josep , Hontecillas Raquel 
Published: January 2023

Abstract
Enteroaggregative Escherichia coli (EAEC) is an etiologic agent of enterocolitis and persistent diarrhea worldwide, especially in malnourished children and immunosuppressed individuals. Expression of IL-17, which is produced by a number of immune and epithelial cells, increases during EAEC infection. We previously demonstrated that blocking peroxisome proliferator-activated receptor gamma (PPARγ) enhances mucosal Th17 responses that contribute to bacterial clearance in EAEC-infected mice. Here we investigate whether IL-17 mediates the effects of PPARγ blockade in the gut. We show that increasing doses of PPARγ antagonist GW9662 (0.5, 1, or 2µM) directly correlated to increased IL-17 expression in the colon. C57BL/6 wild type mice were challenged with EAEC strain JM221 on day 0 and administered anti IL-17 neutralizing antibody (50μg) i.p. on days 0, 2, and 4 of the study. Anti (α)-IL17 antibody-mediated neutralization of IL-17 abrogated the beneficial effects of PPARγ blockade on weight loss and fecal EAEC burden. In addition, infiltration of CD11c+ dendritic cells and CD4+ T cells into the colonic lamina propria, as well as colonic antimicrobial peptide expression were significantly reduced in mice treated with αIL-17 and GW9662 when compared to mice receiving GW9662 alone. These findings suggest that the beneficial effects from PPARγ blockade in disease activity and bacterial burden during EAEC infection are largely mediated through an IL-17-dependent mechanism.
Annexin 1 in microparticles promotes intestinal mucosal wound repair during inflammation. (P3264)
Authors: Nusrat Asma , Leoni Giovanna , Neumann Philipp-Alexander , Alam Ashfaqul , Lambeth David , Hilgarth Roland , Kusters Dennis , Reutelingsperger Chris , Perretti Mauro , Parkos Charles , Neish Andrew 
Published: January 2023

Abstract
Inflammation-induced epithelial injury results in compromised mucosal barrier function. In response to injury, epithelial cells migrate as a sheet to cover denuded surfaces. We have recently reported that a calcium dependent phospholipid binding protein Annexin A1 (AnxA1) is a pro-resolving mediator that signals via epithelial formyl peptide receptors (FPRs) to promote intestinal mucosal wound repair. Analysis of signaling pathways downstream of epithelial FPRs identified intestinal epithelial NADPH oxidase-1 (Nox1) and Rac1 as key elements responsible for generation of reactive oxygen species (ROS). We determined that ROS-induced oxidative modification of phosphatases, in turn, activate proteins involved in controlling focal cell matrix adhesions and epithelial cell migration. Exogenous AnxA1 administration was observed to promote intestinal mucosal wound closure in WT mice but not in mice lacking intestinal epithelial Nox1 (Nox1-/-IEC). Analysis of AnxA1 released from the epithelial cells revealed that it is shed in membranes that have properties of microparticles (&lt;1 µm) as an N-terminal cleaved product. Incubation of wounded epithelial monolayers with AnxA1containing microparticles promoted wound closure. These findings define a mechanism by which intracellular AnxA1 is cleaved and released in epithelial-derived microparticles during inflammation which can then signal in a paracrine fashion via FPR and Nox1 to promote intestinal mucosal wound repair.
Toll-like receptor signaling regulates mucosal barrier function and antigen acquisition in the small intestine (P3266)
Authors: Miller Mark , Xu Jack , McDole Jeremiah , McDonald Keely , Knoop Kathryn , Newberry Rodney 
Published: January 2023

Abstract
The mucosal immune system must efficiently recognize foreign antigen during intestinal infection and yet avoid triggering inappropriate inflammatory responses to commensal microbes and food antigens. In vivo two-photon imaging revealed that low-molecular weight material can cross the epithelium of the small intestine during goblet cell secretion, a phenomenon termed goblet cell-associated antigen passages (GAPs). GAPs are abundant in the steady-state and selectively deliver foreign antigen to CD103+ lamina propria dendritic cells with tolerogenic potential. However, under pathological conditions, goblet cell secretion and epithelial permeability are dramatically altered. The cellular mechanisms that regulate barrier function in response to luminal microbial products are poorly understood, but have important ramifications for intestinal pathogenesis and oral vaccine efficacy. We found that Toll-like receptor (TLR) ligands introduced acutely into the intestines of germ free mice modulated GAP formation in vivo. Furthermore, mice deficient in TLRs that signal through TRIF, lacked GAPs and had reduced epithelial permeability. We propose that a subset of villus epithelial cells expressing TLR3/TLR4 and containing high levels of serotonin (5HT) can induce GAP formation in the small intestine by secreting 5HT in response to TLR ligands.
Delineating residential macrophage heterogeneity in lung disease (P3282)
Authors: Duan Mubing , Li Waichu , Steinfort Daniel , Irving Louis , Anderson Gary , Hibbs Margaret 
Published: January 2023

Abstract
In healthy lungs, alveolar macrophages comprise &gt;95% of the immune cells in the alveolar airspaces where they regulate local lung surfactant production and act as the primary sentinels of respiratory pathogens. Interestingly, increased AMΦ numbers are observed in many animal models of chronic lung diseases and also clinically in patients with COPD. Combining flow cytometry and chimeric mice studies, we observed that residential AMΦs form two distinct subpopulations (by either remaining Mac-1neg/low or turning Mac-1pos) during acute inflammation and in chronic inflammatory lung disease subsequent to SHIP-1 deletion. SHIP-1-/- mice which spontaneously develop COPD-like lung disease maintain both Mac-1neg/low and Mac-1pos AMΦ subpopulations. This additional Mac-1pos subpopulation highly expresses MMP-12 and tracks with the induction of lung disease using SHIP-1-/- chimeric mice. Altogether, our results suggest that residential AMΦ heterogeneity is a component of acute lung inflammation and chronic inflammatory lung disease. Consequently, whether residential AMΦ heterogeneity may be a novel hallmark of inflammation-driven human lung diseases has been our final area of study. Overall, our studies of both animal models and patient samples may allow us to better understand the role of AMΦ heterogeneity in lung homeostasis and disease.
Oxidation resistance 1 attenuated lupus nephritis by suppressing oxidative stress and inflammation in kidney (P5119)
Authors: Li Quanzhen , Li Yajuan , Yan Mei , Raman Indu , Mohan Chandra 
Published: January 2023

Abstract
Oxidation resistance 1 (Oxr1) was identified to be up-regulated in mouse kidney upon anti-GBM antibody challenge and may play a role in anti-GBM disease and lupus nephritis. Since Oxr1 has been shown to confer resistance against oxidative stress by detoxify reactive oxygen species (ROS), we speculate that Oxr1 may have protective effect against oxidative stress induced renal damage. Thus, we utilized mesenchymal stem cells (MSCs) as vehicles to deliver Oxr1 into the injured kidneys of lupus nephritis model mice and investigated the influence of Oxr1 on glomerulonephritis. Human OXR1 gene was transfected into mouse bone marrow-derived MSCs via lentiviral vector pCDH to establish a stable hOXR1-MSC cell line. 106 hOXR1-MSCs were injected (i.v.) into B6.Sle1.Sle2.Sle3 lupus mice and nephritis phenotype was observed for 6 months. Compared with vector-MSCs treated mice, the hOXR1-MSCs administrated mice showed significantly decreased blood urea nitrogen (BUN), proteinuria and ameliorated renal pathology. hOXR1-MSCs transplantation significantly reduced macrophage and T lymphocyte infiltration by inhibiting the expression of CCL2, CCL7, IL-1β, IL-6 and NFκB. hOXR1-MSCs prevented hydrogen peroxide induced oxidative stress and its implantation reduced NO in mouse serum and urine and increased catalase and GPX1 to inhibit tubular cell apoptosis. In conclusion, OXR1 exerted protective effect on nephritis by suppressing inflammation and oxidative stress.
Oxidative damage and transmethylation micronutrient effects on the T cell epigenome in lupus (P5141)
Authors: Ray Donna , Richardson Bruce 
Published: January 2023

Abstract
The development of human lupus is attributed in part to T cell DNA demethylation. The mechanisms causing T cell DNA demethylation in lupus patients are poorly understood. DNA methylation is dependent on the methyl donor S-adenosylmethionine (SAM) and DNA methyltransferase (Dnmt) activity, and is inhibited by S-adenosylhomocysteine (SAH). Several dietary nutrients affect SAM levels including folate and methionine. Additionally, Dnmt levels are low in lupus patients due to defective ERK pathway signaling, caused by oxidative stress resulting in PKCδ nitration. We hypothesized that T cells subjected to oxidative stress will overexpress genes normally suppressed by DNA methylation due to low Dnmt levels, and the overexpression will increase in the presence of low folate and methionine levels. We treated PHA-stimulated human T cells with the oxidant peroxynitrite, cultured the cells in media containing high or low folate or methionine levels, and compared the effects on expression of the methylation sensitive genes CD70 and KIR. Peroxynitrite treated T cells cultured in lower nutrient concentrations overexpressed CD70 and KIR compared to those cultured in higher concentrations and/or no oxidant. These results suggest that low nutrient levels may increase T cell epigenetic changes caused by oxidation. Since several environmental insults resulting in oxidative stress are associated with lupus, dietary supplementation may mitigate the damage.