bluuebunny/crossref_metadata_2025_split huggingface dataset:
IFN-γ deficiency alters splenic compartment cellular composition and Brucella distribution in a murine model of brucellosis
Authors: Dadelahi Alexis S. , Lacey Carolyn A. , Skyberg Jerod A. 
Published: January 2023

Abstract
Despite more than a century of research, brucellosis remains a major zoonotic threat with an estimated 500,000 human cases annually. No licensed vaccine exists to counter Brucella infection, leaving many who contract brucellosis with a chronic and debilitating disease. Immunohistochemistry studies have identified targets of Brucella infection in vivo. However, due to the low detection limit of this technique, high infectious doses of Brucella or immunodeficient mice are employed which likely changes the composition of the infected tissue. We and others observed increased splenic bacterial burden and susceptibility to Brucella infection in mice deficient in IFN-γ. Therefore, to determine if IFN-γ changes splenic composition and cellular distribution of Brucella, we performed live cell sorting on spleens from Brucella- infected wild-type (WT) and IFN-γ−/− mice. Spleens from Brucella-infected IFN-γ−/− mice exhibited a lower proportion of B cells and an increased proportion of neutrophils and macrophages compared to WT mice. Interestingly, 90% of intracellular, viable Brucella from WT spleens during chronic infection was in B cells while the proportion of bacterial burden was similar amongst B cells, macrophages, and neutrophils within IFN-γ−/− spleens. In studies using B-cell deficient (μMT−/−) mice challenged with Brucella, we found significantly decreased bacterial burden in μMT−/− spleens compared to WT mice despite similar levels recovered from the joint between both groups. Collectively, these findings indicate that IFN-γ deficiency alters the distribution of cells within the splenic compartment and that B cells enhance Brucella burden in the spleen.
Peripheral Francisella Tularensis Infection Results In Neural Invasion and Pathologic Inflammation
Authors: Ramos Muniz Mireya G. , Spencer Charles T. , Contreras Ryan , Nunez Guillermo 
Published: January 2023

Abstract
Francisella tularensis infection triggers an overactive inflammatory response by stimulating production of host pro-inflammatory cytokines. Human tularemia, caused by F. tularensis, is a zoonotic disease transmitted by aerosol particles, direct contact, and via arthropod vectors, particularly ticks. With the known peripheral invasion of F. tularensis, and previously published clinical data indicating the presence of our bacterium in cerebral spinal fluid, many questions arise, which include the questions as to which specific neuronal cells are infected. In the periphery, macrophages provide innate immune defense against foreign material, including bacteria. Microglial cells are resident macrophages of the brain and spinal cord and previously have been reported to rapidly respond to pathological changes in the central nervous system, serving a similar function as macrophages. Their rapid activation is an important factor in guarding the neural parenchyma against infectious diseases, inflammation, and neurodegeneration while maintaining and facilitating the return to tissue homeostasis. Therefore, we hypothesized that, having invaded the brain by an unknown mechanism, F. tularensis may be capable of eliciting an immune response following infection of microglial cells. Since macrophage infection leads to elicitation of inflammation, infection of microglia similarly stimulate inflammation leading to neural pathology. Mice were inoculated on the rear flank with fluorescently labeled bacteria. Using fluorescent confocal microscopy, labeled F. tularensis was localized in the brains of infected mice. We analyzed brain homogenates for production of inflammatory cytokines at these sites through Milliplex and qPCR.
Mast Cells Play a Protective Role in Spontaneous Colitis
Authors: Lennon Elizabeth M. , Woodrow Jane S. , Thomason Courtney A. , Borst Luke B. , Edwards Laura E. , Moeser Adam James 
Published: January 2023

Abstract
Mast cells are modulators of inflammation and are activated during inflammatory bowel disease (IBD). Previous work has demonstrated that mast cells are proinflammatory in acute models of chemical colitis. However, the role of mast cells in chronic, spontaneous IBD models remains to be fully defined. We hypothesized that mast cells play a protective role in chronic, spontaneous colitis. We compared colitis and intestinal barrier function in IL10−/− mice to mast cell-deficient, colitis prone mice (double knockout (DKO): IL-10−/− x KitWsh/Wsh). Systemic engraftment of DKO mice with mast cells was performed. Mast cell-deficient DKO mice exhibited more severe colitis compared to mice that lacked IL-10 alone, indicated by increased colitis scores, mucosal hypertrophy, increased intestinal permeability, and spontaneous colonic cytokine production. Reconstitution of DKO mice with mast cells prevented colitis (reduced colitis scores, mucosal hypertrophy, TNF, and intestinal permeability). PCR array analyses demonstrated the influence of mast cells on cytokine expression. Analysis of the microbiota revealed sex-specific differences between IL10 and DKO mice, in particular, the abundance of Akkermansia muciniphila, a mucus degrader associated with colitis. Derivation of DKO mice into a germ-free environment revealed that the microbiota is required for disease development. RNA-seq on colon samples revealed candidate genes for the protective effects of mast cells. These results demonstrate that mast cell deficiency exacerbates disease in a chronic, spontaneous IBD model, indicating that mast cells may have protective functions in IBD, and potentially other chronic inflammatory disorders.
Lymphatic and uterine B cells contribute to maternal immunological tolerance during murine pregnancy
Authors: Genuino Ruth Marian Guzman , Eldi Preethi , Garcia-Valtanen Pablo , Hayball John D. , Diener Kerrilyn R. 
Published: January 2023

Abstract
B cells are best known for their ability to instigate positive immune responses by facilitating immune memory and producing antibodies. However, there are subpopulations of B cells that are capable of negative regulation. Collectively referred to as ‘regulatory B cells’ or ‘Bregs’, these B cell subpopulations have been shown to restore immune response to homeostatic levels in conditions requiring calibration of inflammation such as autoimmunity, cancer, and graft tolerance. Bregs carry out negative regulation by producing anti-inflammatory cytokines such as IL-10 and/or suppressing other immune cells by cognate interactions. In semi-allogeneic pregnancy where establishment of maternal immunological tolerance towards the fetus is the defining condition requisite to pregnancy success, the role of B cells in the local tissues has yet to be elucidated. In this study, kinetics of B cells during BALB/c x C57/BL6 murine pregnancy demonstrate transient increase in IL-10-producing B cells at insemination (0.5 days post-coitus), implantation (5.5 days pc), and delivery day in the para-aortic lymph nodes, as well as a significant increase in the uterus at 5.5 days pc and delivery day relative to the estrus virgin state. Moreover, flow cytometry revealed that these IL-10-producing B cells in the lymph nodes and uterus have elevated expression of PD-L1. In vitro suppression assays show that co-culturing lymphatic and uterine B cells from 5.5 days pc pregnant mice with CD4+ T cells significantly reduced the proliferation of T cells. These results suggest that B cells in the local pregnancy milieu function as regulatory B cells that contribute to the establishment of maternal immune tolerance during pregnancy.
ILC2 activation by leukotrienes: NFAT joins the team
Authors: von Moltke Jakob , O’Leary Claire E. , Barrett Nora , Kanaoka Yoshihide , Austen K. Frank , Locksley Richard M. 
Published: January 2023

Abstract
The initiation and maintenance of type 2 (allergic) immune responses in the lung requires production of the type 2 cytokines interleukin (IL)-4, IL-5, IL-13, and IL-9, which together drive eosinophilia and other hallmarks of type 2 inflammation. Group 2 innate lymphoid cells (ILC2s) and type 2 helper T cells (Th2) are the primary source of these cytokines. In Th2s, T cell receptor (TCR) signaling activates the transcription factors NFAT, AP-1, and NFκB, which cooperate to induce type 2 cytokines. ILC2s lack a TCR and respond instead to locally produced cytokines such as IL-33. While IL-33 induces AP-1 and NFκB, NFAT signaling has not been described in ILC2s and it remains unclear how maximal cytokine production is achieved in vivo. Here, we report the non-redundant NFAT-dependent role of lipid-derived leukotrienes in the activation of lung ILC2s. Using cytokine reporter and leukotriene-deficient mice in innate and adaptive models of type 2 airway inflammation, we find that while loss of leukotriene B4 or cysteinyl leukotriene signaling alone has modest effects, deletion of both pathways markedly diminishes ILC2 activation and eosinophilia. Type 2 responses are similarly attenuated in IL-33- and leukotriene-deficient mice, and optimal ILC2 activation reflects potent synergy between these pathways. We hypothesize that ILC2s integrate multiple signals as a substitute for TCR signaling. These findings therefore expand our understanding of ILC2 regulation and may have important implications for the treatment of airways disease.
Evaluation of activation and homing markers on regulatory T cells using a modular flow cytometry approach on the BD FACSLyric™ flow cytometer
Authors: Tyznik Aaron J. , Kara Nihan , Corselli Mirko , Warner Noel L. , Stall Alan M. , Trotter Joe , Saksena Suraj 
Published: January 2023

Abstract
Advancements in cell analysis capabilities have significantly expanded our understanding of the complexity of immunological systems. As the need for deeper analysis has increased, many laboratories have employed a set of lineage markers to define a cell population, followed by the addition of unique markers specific to their biological question. Utilizing regulatory T cells (Treg) as a model population, we describe the design of an 8-color backbone panel on a 12-Color BD FACSLyric™ flow cytometer that can be supplemented with 4 drop-in markers (colors) to characterize two critical facets of Treg biology: activation and homing. Intelligent panel design enabled identification of human naïve and activated Treg cells utilizing established Treg markers (CD3, CD4, CD25, CD127, FoxP3, CD45RA). CD15s and CD161 were included in the 8-color backbone panel to identify functionally suppressive effector and/or pro-inflammatory cytokine secreting Tregs. Importantly, addition of a 4-color drop-in activation panel (PI16, CD147, CD39 and HLA-DR) or a 4-color drop-in homing panel (CCR4, CCR6, CXCR3 and CD31) did not impact resolution of the backbone Treg population(s), while providing new and interesting insights into Treg biology. We highlight the utility of a modular panel design approach that provides an efficient, scalable, and standardized solution for complex analysis of essentially any cell population of interest.
B- and T-cell Immune Repertoire Characterization using Anchored Multiplex PCR and Next-Generation Sequencing
Authors: Haimes Josh D. , Harrison Thomas D. , Nair Namitha M. , Sims Jennifer , McKittrick Ian , Griffin Laura M. , Johnson Laura , Kudlow Brian A. 
Published: January 2023

Abstract

Introduction
NGS-based analysis of the immune repertoire (IR) is a powerful tool to monitor disease, adaptive immune responses to disease, vaccination and therapeutic interventions. IR characterization by NGS usually requires large primer panels, due to the extensive combinatorial diversity, and a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Anchored Multiplex PCR (AMP™) uses molecular barcoded (MBC) adapters and gene-specific primers (GSPs), enabling NGS-based immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation. Here, we describe AMP-based NGS assays for IR characterization, Immunoverse™ BCR and TCR, which utilize a minimal set of unidirectional GSPs and MBC adapters that reduce amplification bias.

Methods
The quantitative reproducibility and sensitivity of our assays was validated using mRNA isolated from PBMCs of healthy donors, B-cell chronic lymphocytic leukemia donors and formalin-fixed paraffin-embedded (FFPE) tissue.

Results
Both assays demonstrated high reproducibility between replicates with quantitative clone tracking down to 0.01%. The ability to determine isotype, clonotype and IGHV mutational status in a single assay was demonstrated. Preliminary TCR assay data indicates that CDR3 sequence capture is possible from FFPE tissue with clonotype calling being driven by input quantity, T-cell content, and, to a lesser degree, mRNA quality.

Conclusions
AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire.
Diagnostic Assessment of Immunological History by High-throughput TCR sequence Analyses.
Authors: Wolf Kyle , Maybruck Julie , DiPaolo Richard J. 
Published: January 2023

Abstract
T cell receptor (TCR) rearrangement results in germ-line editing of a T cell’s TCR loci. During infection, T cells with TCRs specific for the pathogen proliferate. Resulting pathogen-specific memory T cells retain the unique germ-line TCR DNA rearrangements, which we hypothesized may be used as molecular biomarkers of prior pathogen exposure. We performed high-throughput sequencing of the TCRβ repertoires of HLA-A2 transgenic mice pre- and post- inoculation with either the smallpox vaccine or highly-related monkeypox virus (MPXV). We generated a database of &gt;2×106 unique TCRβ clonotypes from samples pre- (naïve) and post-exposure to identify TCRs associated with vaccinated/infected samples vs naïve in 58 individual mice. We computationally identified 315 TCRβ sequences statistically associated with post-vaccinated samples, which we used to develop a diagnostic assay to classify samples as naïve or previously-exposed with 97% accuracy up to 9-months post-vaccination. We validated this assay in an independent cohort of MPXV infected mice. The diagnostic platform was adapted to analyze human TCR sequences to distinguish smallpox vaccinated or non-vaccinated individuals. We identified 239 vaccine-associated TCRβ sequences significantly expanded in 100 smallpox vaccinated individuals. A machine learning algorithm was developed to determine individuals’ vaccination status based on the presence of these 239 TCR sequences. Overall, the diagnostic assay correctly identified 74% of non-vaccinated samples and 85% of vaccinated samples. Thus, the analyses support that computational identification of pathogen-associated TCRs is a highly sensitive and specific method for determining prior exposure to an agent or pathogen.
New technology for development of host-targeted therapeutic vaccine against anthrax
Authors: Yang Huixiao , Yan Yongyong , Wang Haiyan , Chen Yanping , Zheng Zhichao , Kim Hyo , Wu Lihong , Franco Susana , Zeng Mingtao 
Published: January 2023

Abstract
Anthrax is a serious disease caused by Bacillus anthracis, a bacterium that can form spores for long-term survival and secrete anthrax toxins including protective antigen (PA), lethal factor (LF), and edema factor (EF). However, current anthrax postexposure treatments are inadequate at later stages of infection. Previously, our research have shown RNA inhibition (RNAi) on host cells silences anthrax toxin receptors (ATRs): tumor endothelial marker 8 (TEM8), capillary morphogenesis protein 2 (CMG2). This approach was protective against the cytotoxicity of anthrax toxins, indicating that ATR-targeted RNAi can be used as a therapy against anthrax. However, inefficient cytosolic delivery and toxicity of siRNA delivery vehicles limit the use siRNA as therapeutics. In this study, we have developed a detoxified anthrax edema toxin, which consists of PA and nontoxic N-terminal fragment of EF (EFn) conjugated with a peptide nona-D-arginine residues (EFn-9dR) to enable siRNA binding. The detoxified toxin was able to deliver specific siRNA to induce cmg2 gene silencing in different cell lines and C57BL/6 mice, and provide significant protection against anthrax lethal toxin challenge in vitro and in mice. Survived mice from toxin challenge were fully protected against lethal challenge with B. anthracis Sterne spores. The immune protective mechanism is mainly due to the significantly high serum neutralizing antibody response against anthrax toxins in these mice. These results suggest that detoxified anthrax toxin provides a tool for delivery of host-targeted siRNA into anthrax pathogenesis-associated host cells, and this new system could potentially be developed as a lifesaving postexposure therapeutic vaccine against anthrax.
Multi-parameter flow sorting with novel antibody fluorochrome conjugates that facilitate relabeling of targeted epitopes and reuse of fluorescence channels in downstream applications
Authors: Evaristo Cesar , Pankratz Jennifer , Schmachtenberg Sabine , Bergmann Anna , Jansen Nicole , Krauthäuser Susanne , Meiler Svenja , Yu Zhong , Schiminsky Esther , Völkel Andrea , Siewert Christiane , Dose Christian 
Published: January 2023

Abstract
Multi-parameter immunofluorescent labeling is the method of choice for flow sorting of cell populations out of heterogeneous mixtures. However, downstream applications of isolated cells are limited since fluorescence channels and epitopes are blocked by antibody fluorochrome conjugates utilized for the flow sorting experiment.
Here, we describe for the first time new types of antibody fluorochrome conjugates that enable a highly specific multi-parameter cell labeling. Unlike conventional fluorochrome-conjugated antibodies, the introduced conjugates rely on recombinantly engineered antibodies and a conjugation chemistry that facilitates the release of the whole conjugates from the cell surface after the flow sorting step. Thereby, previously blocked epitopes are free for relabeling, which provides maximal flexibility for renewed epitope targeting in downstream applications.
We demonstrate this flexibility in the context of a workflow for the isolation of highly pure human regulatory T cells (Treg). Tregs labeled with conventional CD4-VioBlue, CD25-PE and the new releasable CD127-APC were enriched by flow sorting. Final identification of the target cells was achieved by intranuclear labeling of the transcription factor FoxP3, a low expressed marker which requires a bright fluorochrome and fluorescence channel with high sensitivity for reliable detection. The selective removal of the CD127-APC conjugate facilitated the reuse of the APC-channel and enables maximum sensitivity for the cell sorting and analysis step.
In summary, this new generation of releasable antibody fluorochrome conjugates for multi-parameter cell labeling paves the way for so far impossible workflows in research and clinical applications.
IFN-γ regulates transcription of key molecules to prevent progressive experimental autoimmune encephalomyelitis
Authors: Robinson Rachel R. , Forsthuber Thomas 
Published: January 2023

Abstract
Multiple sclerosis (MS) is the most common disabling neurological condition of young adults. Most patients are initially diagnosed with relapsing-remitting MS. Problematically; 10 years after diagnosis approximately 60% of MS patients will have converted from the relapsing-remitting form of MS to secondary-progressive MS despite treatments. IFN-γ, a well-studied pro-inflammatory cytokine, has long been implicated in the pathogenesis of MS and its preclinical model, experimental autoimmune encephalomyelitis (EAE), and is often suppressed during treatments for MS. However, previous studies in our lab revealed that mice lacking IFN-γ signaling developed severe and progressive EAE due to increased presence of myelin debris and lipid peroxidation in the CNS, the cause of which is yet to be fully elucidated. We hypothesize that IFN-g prevents disease progression in this progressive EAE model by regulating the expression of molecules that promote the resolution of lipid peroxidation and clearance of myelin debris in the CNS during EAE. We identified a number of key molecules associated with lipid peroxidation which are temporally dysregulated in the absence of IFN-gR using qRT-PCR. To discover additional molecules involved in disease progression we have performed comprehensive RNA sequencing on the CNS of IFN-gR−/− vs WT mice through the course of disease. We will present the result of the analysis of these molecules and supporting studies. The results of our studies will contribute to a more thorough understanding of the underlying mechanisms and may lead to new treatments to prevent progression of MS.
RTA 1701 is an orally-bioavailable, potent, and selective RORγt inhibitor that suppresses Th17 differentiation in vitro and is efficacious in mouse models of autoimmune disease.
Authors: Dulubova Irina , Jiang Xin , Trevino Isaac , McCauley Lyndsey , Liu Liping , Hannigan Linda , Reisman Scott A. , Reisman Scott A. , Ferguson Deborah , Visnick Mike , Wigley Christian 
Published: January 2023

Abstract
Retinoic acid-related orphan receptor γt (RORγt) is a transcription factor that directs the differentiation of Th17 cells, a subset of CD4+ helper T cells involved in autoimmune disorders. Once differentiated, Th17 cells secrete interleukin-17A (IL-17A) and other key cytokines implicated in autoimmune and inflammatory disease. Antibodies that target Th17 cells are approved for several autoimmune disorders, validating the important role of Th17 cells in disease. RTA 1701 is a potent and selective RORγt inhibitor in preclinical development for autoimmune disorders. It inhibits the activity of RORγt, as assessed using ligand binding domain and full-length RORγt reporters. Moreover, RTA 1701 is selective for RORγt and does not inhibit the activity of two closely-related receptors, RORα and RORβ. By inhibiting RORγt, RTA 1701 blocks Th17 differentiation of naïve CD4+ T cells and IL-17A production by both Th17 polarized CD4+ T cells and activated peripheral blood mononuclear cells (PBMCs). The expression of other RORγt target genes and Th17 signature genes (eg, IL-17F, IL-21, CCL20) is also reduced following RTA 1701 treatment of Th17-polarized naïve CD4+ T cells, as assessed by qPCR. RTA 1701 is orally bioavailable with excellent pharmacokinetic properties in rodents and monkey. Furthermore, this compound is efficacious in animal models of autoimmune disease, including the collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis and the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Together, these results suggest that RORγt inhibition by RTA 1701 may have therapeutic potential in Th17-associated autoimmune disorders.