bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimizing bacteriotherapy to prevent or treat food allergy
Authors: Hesser Lauren A. , Hubbell Jeffrey , Nagler Cathryn 
Published: January 2023

Abstract
Depletion of protective gut microbes strongly correlates with the increasing prevalence of non-communicable diseases, including food allergies. In previous work we transferred feces from healthy or cow’s milk allergic (CMA) infants to germ free mice and showed that healthy infants harbor intestinal bacteria that are causally protective against food allergy in this model. Monocolonization with a single bacterial species, Anaerostipes caccae, was sufficient to mimic the protective effect of the healthy microbiota. These studies have provided the conceptual basis for the use of bacteriotherapy to prevent food allergy. We are taking a synbiotic approach in which we co-deliver a new sub-strain (A. caccae_lahuc), isolated from the feces of infants in our study, with prebiotic substrates that enhance the ability of A. caccae_lahuc to grow in mice with a replete microbiota and produce butyrate, a short-chain fatty acid with immunoregulatory properties. In vitro studies of A. caccae_lahuc cultured with fecal bacteria from a CMA infant identified potato starch or the combination of lactate and acetate, the intermediate products of fiber breakdown, as prebiotics which maximize butyrate output. We are optimizing delivery of these prebiotics with A. caccae_lahuc to determine whether they stimulate butyrate production in vivo. We found that A. caccae_lahuc bacteriotherapy without prebiotics leads to its stable colonization in mice already colonized with the CMA microbiota without measurable increases in fecal or cecal butyrate. By decoupling growth from butyrate release, we are obtaining mechanistic insights into the components necessary to optimize a synbiotic formulation efficacious for the prevention or treatment of food allergy.
Physiologic expression of AhR regulates CX3CR1+ resident intestinal macrophages and promotes diabetes in NOD mice
Authors: Ehrlich Allison , Kahalehili Heather , Shan Rachel 
Published: January 2023

Abstract
Susceptibility to type 1 diabetes (T1D) is influenced by both genetic and environmental factors. The environmental sensor, the aryl hydrocarbon receptor (AhR), is expressed in mucosal immune cells and can be activated by metabolites derived from the diet and microbiome. We hypothesized that these metabolites may influence the autoimmune response and subsequent susceptibility to T1D. To test this hypothesis, we generated NOD AhR knockout mice and examined the intestinal immune response and development of T1D. Female AhR knockout mice had reduced insulitis at 12 weeks of age, and a decreased incidence and onset of hyperglycemia in comparison to their wild type littermates. To determine whether physiologic expression of AhR was altering the immune response, we analyzed CD4+ T cell subsets and identified an increase in IL-10R+Foxp3+ Tregs in the lamina propria (LP) of AhR knockout mice. We also discovered a population of resident-like macrophages (CD11b+CX3CR1+) in the small intestine LP that were negatively regulated by AhR. These cells have a very strong positive correlation to LP Tregs, and correlate with protection against insulitis in NOD mice. To identify the potential source of AhR ligands that regulate these resident-like macrophages, mice were fed an AhR ligand-deficient diet. These cells were not further regulated by dietary ligands, suggesting that AhR activation originates from the intestinal microbiome. Current studies are aimed at determining how AhR regulates the expression of LP resident-like macrophages and how these cells impose their immunoregulatory functions during T1D pathogenesis.
Roles for Leukemia-inhibitory factor receptor signaling in intestinal immunity
Authors: Kahn Laura Maria , Li Haiyan S. , Chrisikos Taylor T. , Zhou Yifan , Medik Yusra B. , Patel Bhakti , Babcock Rachel Lauren , Newton Kathryn , Watowich Stephanie S 
Published: January 2023

Abstract
Intestinal infections trigger immune responses that in some cases become dysregulated and lead to acute or chronic disease; understanding immune regulatory mechanisms in infection will help advance new therapies for intestinal disorders. Citrobacter rodentium infection in mice mimics enteropathogenic Escherichia coli infections in humans. We found that Leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, is upregulated in colon and serum following C. rodentium infection; however, the source, targets and effects of this cytokine during intestinal disease remain unclear. To investigate the role of LIF during infection, we generated mice with LIF receptor (LIFR) deficiency in endothelial and hematopoietic cells (Tie2cre Lifrf/f) or CD11c+ cells (CD11c cre Lifrf/f). Upon C. rodentium infection, Tie2cre Lifrf/fandCD11c cre Lifrf/f mice had reduced colon length, increased bacterial dissemination to spleen and liver, and greater colon pathology scores relative to infected controls. Comparison of cytokine and chemokine expression in the colon revealed upregulation of various factors in infected LIFR-deficient mice, with the pro-inflammatory cytokine IL-23 significantly increased in CD11c cre Lifrf/f mice. Our preliminary data from LIFR-deficient, bone marrow-derived myeloid cells suggests LIFR signaling inhibits inflammatory cytokine production in vitro. Collectively, our results suggest LIFR signaling in CD11c+ cells inhibits colonic IL-23 production and protects from immune dysregulation during C. rodentium infection. Our current studies are investigating LIF-producing and -responding populations in colon, to elucidate protective immune mechanisms during infection.
Curated, multi-omic, ML-driven single-cell atlas for characterizing the human immune system across disease states
Authors: Kiner Evgeny , Sharim Hila , Montalbano Antonino , Raveh-Sadka Tali , Yanover Uri , Wells Daniel K. , Satpathy Ansuman 
Published: January 2023

Abstract
The immune system plays a key role in various diseases such as cancer, autoimmune disorders and infections. It has been recently shown that cell surface protein markers on peripheral blood mononuclear cells (PBMCs) can be predictive of response to therapies1, but measurements of such markers is technically limited in the number of cells and markers that can be assessed. Single-cell RNA technology allows the measurement of the entire transcriptome in tens of thousands of cells, and therefore has the potential to survey the immune system in more depth and reveal rare cell states. However, no large single-cell datasets for immune cell states exist. Moreover, attempts to combine smaller distinct datasets are often hindered by batch effects stemming from differences in sample handling, single-cell technologies and computational analyses. We created a large curated multi-omic single-cell human PBMC atlas with clinical annotations from dozens of patients with several conditions across hundreds of thousands of cells. Our standardized end-to-end protocols and quality control processes provide a platform that allows production of large datasets with minimal batch effects. By combining single-cell RNA-seq (scRNA-seq) with surface marker identification by CITE-seq, we generate curated immune gene signatures and train a classifier to robustly identify many cell types and states across patients and different diseases, including rare cell populations. This curated, multi-omic clinically-annotated atlas is particularly suited for use by machine learning algorithms, and we are confident that as this data accumulates, it will be instrumental for inferring disease states and predict responses to various therapies.
CRISPR Cas9 ribonucleoprotein-mediated genomic editing in primary innate immune cells
Authors: Hildreth Andrew D , Riggan Luke , Rolot Marion , Wong Yung-Yu , Satyadi Will , Sun Ryan , Huerta Chris , O’Sullivan Timothy E. 
Published: January 2023

Abstract
CRISPR (clustered, regularly interspaced, short palindromic repeats) genome engineering has become a powerful tool to functionally investigate complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity for therapeutic purposes, current approaches are often inefficient or nonspecific, limiting their use in primary innate immune cells. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naïve NK cells and bone marrow-derived dendritic cell precursors. Using these methods, we demonstrate that XCR1+ conventional dendritic cells (cDC1) are sufficient and necessary for host protection to mouse cytomegalovirus (MCMV) in a MyD88-intrinsic manner. This scalable method will enhance target gene discovery and offer a specific and simplified approach to gene editing in the innate immune system.
Biomarker Trends: Luminex & Olink, Dried Blood Spots & Plasma
Authors: Rosenberg-Hasson Yael , Ho Tiffany C. , Simon Tamara D. , Liang Jing , Chang Serena , Herschmann Iris , Gotlib Ian H. , Maecker Holden T. 
Published: January 2023

Abstract
Biomarker discovery is at the forefront of understanding disease etiology, diagnosis and emerging treatments. One of the hurdles is to compare data generated by different platforms and technologies. Measurements of common biomarkers between vendors is another challenge which increases as we multiplex and generate more complex data. We address both these aspects concentrating on two major platforms: Luminex and Olink, in serum and cerebrospinal fluid samples. We find that among 30 overlapping cytokines and chemokines, about 20 display similar trends across these two platforms with comparable sensitivity. A few cytokines had differential and reduced sensitivity in one of these platforms emphasizing the need for improved biomarker detection. Our analysis offers a simple method to compare the data from the two platforms. In addition, we demonstrate the use of Dried Blood Spots as a reliable source for generating multiplex data. We compare plasma and DBS samples and observe similar trends for four out of the six cytokines analyzed, specifically: IL-1b, IFN-g, IL-6 and TNF-a. We discuss limitations of this approach and the need to analyze additional biomarkers in both matrices.
Global targeting of functional (phospho)tyrosines using sulfur-triazole exchange chemistry
Authors: Hsu Ku-Lung , Borne Adam L , Brulet Jeffrey W , Hahm Heung S , Toroitich Emmanuel K , Libby Adam H , Yuan Kun , Ware Timothy B , McCloud Rebecca L , Ciancone Anthony M 
Published: January 2023

Abstract
Chemoproteomic probes serve as valuable tools to study and perturb protein function on a global scale. Despite advances in methodologies for cysteine and lysine profiling, a large fraction of the human proteome remains inaccessible with current activity-based probes. Here, we describe development and application of sulfur-triazole exchange (SuTEx) chemistry for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can produce sulfonyl probes with high chemoselectivity for tyrosines over other nucleophilic amino acids to investigate thousands of tyrosine sites in lysates and live cells. We discover hyper-reactive tyrosines are enriched in enzymatic, protein–protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Our findings illustrate the broad potential for deploying SuTEx to globally investigate tyrosine reactivity, function and post-translational modification state in complex biological systems.
Single B cell isolation and cloning from rabbits to generate recombinant antibodies
Authors: Alves Bryce , Jelinek Mary Anne , Lu Yanan , Ritland Melissa , Velasco Patricia , Zhao Xi , Adams Eddie , Fernandez Joseph 
Published: January 2023

Abstract
For decades the method for generating antibodies with high avidity and affinity relied upon monoclonal antibody generation from immunized mice or polyclonal antibodies from rabbits. Both methods have their shortcomings. The murine immune system—specifically the high degree of immunological tolerance—prevents robust humoral responses to antigens that closely reasonable self-antigens in the mouse. Additionally, the cost and time necessary to generate a monoclonal antibody from mice can be prohibitively expensive, particularly if multiple attempts at hybridoma generation are required. A work around is to generate polyclonal antibodies in rabbits. Rabbits are further evolutionarily removed from humans than mice and mount a stronger immunological response to immunized human proteins or antigens. Unfortunately, the source of polyclonal antibodies from each rabbit is finite. To combat this problem, here we describe the development of a method to isolate single rabbit plasma B cells after immunization. Once isolated the VH and VL regions of the antibody are sequenced and the sequences are cloned into a recombinant rabbit IgG and IgK backbone that enables the recombinant expression of the single cell-derived, cloned rabbit antibodies. In doing so we have harnessed the immunological response of the rabbit host while eliminating the supply problem of finite reactive serum volumes and the cost of traditional murine monoclonal antibody generation.
Complete multi-chain adaptome amplification from whole blood and PBMC via novel dam-PCR technology
Authors: Depinet Mollye M , Pan Wenjing , Hou Xiaohong , Brown Brittany E , Eisenhower Mary , Weber Daniel , Byrne-Steele Miranda , Han Jian 
Published: January 2023

Abstract
Profiling the immune repertoire through NGS allows detailed, sequence-specific insight into the adaptive immune response. One of the key challenges during immune receptor amplification is the formation of dimers, which can compete with the immune amplicons of interest during library preparation. Therefore, we developed a novel PCR technique, dimer-avoided multiplex PCR (dam-PCR), that effectively avoids primer dimer formation and allows for the all-in-one qualitative amplification of all BCR and TCR receptors in a single, quantitative reaction. Here, we present this technology’s capabilities of bias-free immune repertoire amplification, even for low frequency CDR3s. First, we demonstrate the ability of this method to accurately detect a Jurkat cell line that was spiked into pre-screened sorted donor CD4+ cells at 1%, 0.1%, 0.01%, 0.001%, and 0.0001%. RNA from each spike-in percentage was extracted and amplified in duplicate using our standard amplification protocol to assess repeatability of CDR3 discovery between replicates, as well as establish the limits of detection and quantitation. Then, we demonstrate the method’s ability to amplify a synthetic repertoire, created by spiking RNA from two individuals of known repertoire into each other at 75%, 50%, and 25%. The mixed RNA, as well as pure RNA from each individual, was then amplified to assess correlation between the predicted repertoire and amplified repertoire at each spike-in percentage. For both the Jurkat cell level spike-in test, as well as the RNA level spike-in test, the amplified repertoire was successfully predicted at an R2 value of &gt; 0.99. This single reaction method will allow for a cost effective, all-inclusive, and quantitative analysis of immune repertoires.
Development of an in vivo model to assess immune and angiogenic responses to metallic implants using Humanized TLR-4 mice
Authors: Wood Steven C , Gotipatti Anuhya 
Published: January 2023

Abstract
Metal on metal(MOM) hips often fail due to inappropriate inflammation and angiogenesis which may be due to localized concentrations of metal ions and/or MOM wear particles in the joint. Critically, human, but not mouse TLR-4 receptors, are responsive to nickel, cobalt and MOM particles. Therefore, we are using humanized, hTLR-4 mice in these studies. MOM hips are predominately cobalt, so our initial experiments were focused upon this ion. Spleen cells of hTLR -4 mice were exposed to 3 concentrations of CoCl2 (2.5μM, 25μM, 250μM) for 3 and 6 days. The cultures were analyzed for endothelial cell markers, (MADCAM and CD31)and T cell markers, (CD4 and CD8). A dose dependent induction of endothelial markers was seen on day 3 with a dose dependent reduction on day 6, with no changes in CD4 or CD8 expression. Next, spleen cells of TLR-4 mice were exposed to 3 different ratios of cell to MOM particles (1:5, 1:50, 1:500 per cell) for 3 and 6 days. Endothelial markers were induced, but it was not dose dependent, however the endothelial cell numbers increased between 3 and 6 days. Again, T cell markers were not altered. To assess the in-vivo responses to MOM particles, nude mice were implanted with angioreactors containing MOM particles (2.5×105 and 2.5 × 106). On day 12, the angioreactors were removed and the was a dose dependent increase in endothelial cells. Currently, we are extending the MOM angiogenesis studies in hTLR-4 mice. In sum, both CoCl2 and MOM particles induced endothelial cells and angiogenesis. The hTLR-4 mouse model will greatly aid in the evaluating the angiogenic and immunotoxicological profile of ions and MOM wear debris, particularly during menopause. Funded by FDA Office of Women’s Health.
A robust and interpretable, end-to-end deep learning model for cytometry data
Authors: Hu Zicheng , Tang Alice , Singh Jaiveer , Bhattacharya Sanchita , Butte Atul 
Published: January 2023

Abstract
Cytometry technologies are essential tools for immunology research, providing high-throughput measurements of the immune cells at the single-cell level. Traditional approaches in interpreting and using cytometry measurements include manual or automated gating to identify cell subsets from the cytometry data, providing highly intuitive results but may lead to significant information loss, in that additional details in measured or correlated cell signals might be missed. In this study, we propose and test a deep convolutional neural network for analyzing cytometry data in an end-to-end fashion, allowing direct association between raw cytometry data and the clinical outcome of interest. Using nine large CyTOF studies from the open access ImmPort database, we demonstrated that the deep convolutional neural network model can accurately diagnose the latent cytomegalovirus (CMV) in healthy individuals, even in the presence of heavy batch effects between studies. In addition, we adopted three approaches for interpreting the deep convolutional neural network model and identified potentially novel immune cell subsets significantly associated with latent CMV infection.
HMGB1 A Box prevents neuronal injury from PD via restricting activation of microglia and inhibiting differentiation and infiltration of Th17 cells
Authors: Su Zhaoliang , Tian Yu , Xia Lin , Chen Rong , Xu Huaxi 
Published: January 2023

Abstract
Parkinson’s disease is a complexly neuronal injury and accompanied by many clinical challenges, for example, lacking of early diagnostic strategies and effective treatment. Here, we demonstrate that in the subacute Parkinson’s disease mice model, injection of HMGB1 competitive inhibitor protein HMGB1 A Box restored the number of dopaminergic neurons and TH+ fibers in the substantia nigra striatum. Our data also showed that A Box inhibited the activation of microglia mediated by neuronal injury and the production of TNF α and IL-1β by up-regulating microglia CD200-CD200R signal axis in vitro and in vivo. Microglia overexpressing CD200R produced fewer inflammatory mediators and restored TH+ neurons in vitro. Intravenous injection of A Box also reduced the levels of RANTES and CXCL12 in substantia nigra and significantly inhibited Th17 cells infiltration. The ability of activated microglia to promote naïve CD4+ T cells differentiation into Th17 in MPP+ model in vitro was also inhibited by A Box. Elevated levels of these two chemokines were also detected in the serum of the patients. In conclusion, our studies show that HMGB1 can induce neuronal damage by activating microglia to promote the production of inflammatory mediators. HMGB1 itself does not directly cause neuronal death. Moreover, the differentiation of T cells into Th17 and migration of T cells secondary to microglia activation induced by HMGB1 further aggravates neuronal damage. The significant effect of HMGB1 A Box indicates the possibility that this recombinant protein might become a potent inhibitor of neuroinflammation in Parkinson’s disease.
Immunosuppressive activity of cyclosporin A in vivo via NFAT-independent inhibition of TCR signaling
Authors: Otsuka Shizuka , Melis Nicolas , Dutta Debjani , Weigert Roberto , Ashwell Jonathan D 
Published: January 2023

Abstract
Calcineurin inhibitors (CNIs) such as cyclosporin A (CsA) and FK506 are widely used in treatment of autoimmune diseases and tissue transplant rejection. It is well known that CNIs inhibit NFAT-dependent gene expression, which is thought to be the major mechanism of their therapeutic effectiveness. We recently reported that CNIs inhibit TCR-proximal signaling and the upregulation of LFA-1/ICAM1 binding by preventing calcineurin’s dephosphorylation of Lck Ser-59 (LckS59), an inhibitory modification. This raises the possibility that CsA’s inhibition of TCR signaling may contribute to immunosuppression in clinical settings. Here we find that LckS59A (which cannot accept a phosphate at Ala59) knock-in T cells are resistant to CsA’s inhibition of TCR-proximal signaling. Correspondingly, whereas CsA prevented LckWT T cells from antigen-induced and LFA-1/ICAM-mediated binding to APCs in vitro, LckS59A T cells were resistant. Moreover, CsA was able to reverse existing LckWT T:APC interactions within minutes whereas LckS59A T:DC binding remained stable. To determine if disruption of T:DC binding occurs in vivo, we performed 2-photon imaging of lymph nodes from mice in which labeled antigen-specific T cells and antigen-pulsed DC had been injected. Antigen-specific T:DC formed as early as 3 hr after T cell injection. Notably, CsA markedly reduced the formation of antigen-specific LckWT T:DC but not LckS59A T:DC clusters in lymph nodes. Given that T:DC adhesion is an early and necessary step in the generation of T cell immunity, these data demonstrate a new and perhaps major immunosuppressive mechanism for CNIs immunosuppression.