bluuebunny/crossref_metadata_2025_split huggingface dataset:
An efficient human whole blood workflow using CyTOF technology: a lyophilized 30-plex antibody panel coupled with automated data analysis
Authors: Li Stephen K.H. , Majonis Daniel , Bagwell C. Bruce , Hunsberger Benjamin C. , Baranov Vladimir , Ornatsky Olga 
Published: January 2023

Abstract
CyTOF® mass cytometry is a highly multiplexed, single-cell analysis platform that uses metal-tagged antibodies. A significant advantage of this technology is its ability to resolve more than 30 markers in a single panel without the need for compensation. Nonetheless, a highly multiplexed panel requires optimization of antibody titers, full panel verification, sample preparation, data acquisition, and analysis. Preparation of a staining cocktail from 30 individual tubes may be prone to errors. Not only is data analysis time-consuming for complex panels, it is difficult to interpret without expertise in immunology.
We have developed a lyophilized 30-plex immunphenotyping panel contained in a single tube, an efficient workflow, and an automated software solution for human whole blood analysis using mass cytometry. The panel focuses on T cell lineage while also capturing other relevant immune populations. Blood is added directly to the lyophilized antibody tube, followed by RBC lysis, wash, and fixation steps and finally data acquisition of stained samples on the Helios™ system. The dedicated software tool accepts FCS3.0 files and in under 10 minutes automatically generates reports on the number of live cells, percentage of specific cell populations, staining intensities, histograms, 2D dot plots, and tSNE graphs. The auto-calculated frequencies of populations are comparable to manual gating, with a validated correlation coefficient over 0.9. The panel and software tool enable researchers to streamline immunophenotyping of whole blood while accurately and reproducibly monitoring changes in immune cell subsets in patient samples.
Exploring the applicability of ctDNA technology as a diagnostic tool for improved Cancer care and management within the Pacific
Authors: Sika-Paotonu Dianne , Guilford Parry 
Published: January 2023

Abstract
For Pacific peoples living in New Zealand and in the Pacific region, the burden of cancer remains a public health concern of significance. The medical situation for cancer patients in New Zealand and also in the Pacific is of relevance given the health inequalities that exist for Pacific peoples and the global burden of cancer. This work sought to obtain clarity around the cancer diagnostic, management practices and treatments that exist for cancer patients in the Cook Islands. Circulating tumour DNA (ctDNA) is a novel cancer detection method and refers to small fragments of tumour DNA found in the blood circulation. These DNA fragments can be isolated and quantified to obtain data about the cancer’s size and progression. The possibility of utilizing a simple blood sample to allow detection and monitoring of cancer growth is highly desirable. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer diagnostics and surveillance and also lead to improved cancer health outcomes for Pacific peoples in the Cook Islands. This work sought to investigate the potential utilization of ctDNA to support and improve the diagnosis, care and health outcomes of Pacific cancer patients in the Cook Islands. A high degree of interest was expressed by the medical, nursing, health and community research staff and lay persons in the development of ctDNA as a potential diagnostic tool within the Cook Islands. Of particular interest was the potential to monitor patients remotely using the technology rather than be inconvenienced by travelling far distances with their families to the Main Hospital. ctDNA remains a feasible tool with potential to support cancer patients in the Cook Islands.
High-throughput single-cell linking of antigen specificities with T cell receptor sequences using de novo generated DNA-linked MHC tetramers
Authors: Jiang Ning 
Published: January 2023

Abstract
Identification of correlated antigens recognized by T cell and its T cell receptor (TCR) sequences at single cell level is extremely useful to the understanding and treating immune-related diseases. Although CyTOF has pushed the number of antigenic peptides that can be interrogated at once to around 100, the destructive nature of CyTOF prohibits linking pMHCs recognized by a T cell with its TCR receptor sequences.
Here we describe a new technology, TetATCR-Seq for Tetramer Associated TCR sequencing, that is capable of de novo generating a large amount of peptides and mapping antigen recognition with single cell TCR sequences by DNA barcoded tetramers. It evolves three major steps: 1) de novo peptide generation, 2) DNA barcoded tetramer generation, and 3) single cell sequencing of tetramer DNA barcode and TCR sequences.
Using a published set of cancer neo-antigen sequences, we showed that these peptides and their wildtype counterparts can be quickly generated. Resulted tetramers can be used to clearly identify a population of neo-antigen only or wild type antigen only or double positive T cells. Subsequent single cell TCR sequencing showed that TCR paired a and b chain sequences can be obtained in more than 80% of single T cells. Simultaneous tetramer DNA barcode sequencing showed that many T cells are found to cross-reactive to both neo-antigen and its wild type counterpart, suggesting the necessity of strict screening for neo-antigen only TCR in adoptive cell transfer therapy.
In summary, TetaTCR-seq enables one to quickly survey antigen repertoire again TCR repertoire at single cell level for a large number of peptides. It is a useful technology to screen for T cell cross-reactivity and select neo-antigen specific TCR for cancer immunotherapy.
Multiplex immunoassay detection of autoimmune disease autoantibodies in serum and plasma
Authors: gilliam brooke e. , Pepin D , Xiao Qiang 
Published: January 2023

Abstract
Autoantibody formation is principal to the pathogenesis of a variety of autoimmune diseases. Dysregulated apoptosis and the subsequent defective clearance of cellular debris leads to the exposure of autoantigens and the generation of autoantibodies. The presence of autoantibodies may indicate disease activity, prognosis, and clinical associations related to a variety of autoimmune diseases, including systemic lupus erythematosus (SLE), Sjögren’s Syndrome, Scleroderma, Polymyositis/Dermatomyositis, and various overlap syndromes of these diseases. Here we report the development of a multiplex immunoassay to monitor 20 autoantibodies present in blood that are involved in a variety of autoimmune diseases: SSA/Ro60, SSA/Ro52, SSB/La, RNP, RNP/Sm, Sm, Ribosomal P, Proteinase 3, Myeloperoxidase, PCNA, β 2-Glycoprotein, CENP-A, CENP-B, Scl-70, Jo-1, C1q, PM/Scl-100, Ku, Mi-2, and PL-12. Using the 20-plex immunoassay, we measured expression of autoantibodies in serum and plasma from SLE patients with various disease manifestations and overlap syndromes, healthy controls, and CDC reference samples available for 11 autoantibodies. Additionally, control beads were included in the immunoassay. Levels of SSA/Ro60, SSA/Ro52, SSB/La, RNP, RNP/Sm, and Sm matched clinical laboratory features noted at the time of SLE sample collection. Additionally, anti-C1q antibody levels were associated with lupus nephritis. The CDC controls exhibited elevated MFI for the autoantibodies for which they were expected to react strongly. This study demonstrates the value of using multiplex technology to evaluate multiple autoantibodies, allowing for an extensive autoantibody profile to evaluate patients with various autoimmune diseases.
Evaluation of a B-Cell Biosensor Immunoassay for Rapid and Sensitive Detection of Ricin, a Harmful Plant Protein Toxin
Authors: Venkateswaran Kodumudi , Selvan Senthamil R. , Sarwar Jawad , Parameswaran Nishanth , Babu Prasanti , Koval Sophia , Venkateswaran Roma , Krishnan Krish VV. , Simpson Jeanette , Hodge David R. 
Published: January 2023

Abstract
Ricin is a carbohydrate binding protein produced in the castor bean seeds and is very toxic to humans. Ricin is one of the most frequently used toxin as a biothreat agent in many individual incidents. PathSensors has licensed the Cellular Analysis and Notification of Antigen Risks and Yields (CANARY) technology from Massachusetts Institute of Technology and developed Zephyr biosensor sandwich immunoassay for rapid detection of Ricin. In this assay, CANARY technology utilizes engineered B Cells that express ricin specific monoclonal antibody on the surface as biosensors. Luminescence, emitted from the B cells upon binding of the toxin, is measured as photons in a Luminometer to detect ricin in a suspicious sample. In this study, a detailed performance evaluation of this rapid ricin detection test was done in multiple phases using well characterized materials. The dynamic range of this assay was determined by testing wide ranging concentrations (0 to 100 ng/test) of ricin and limit of detection (LOD) was calculated by PROBIT regression analysis. Further validation of assay was performed by testing inclusivity, informational, near neighbor, lectin, and white powder panels, and also environmental aerosol filter sample testing. LOD for ricin was found to be 1 ng/test. Repeatability testing showed more than 99% sensitivity and 100% specificity. There was no false positive detection of environmental background panel and other potentially interfering materials except Ricinus communis agglutinin also known as RCA120. The validation indicates that the ricin biosensor test is user-friendly laboratory detection method which has great potential for use in national and international public health and environmental test laboratories.
The Benzathine Penicillin G (BPG) reformulation preferences study-edging closer towards a new Penicillin for Rheumatic Fever and Rheumatic Heart Disease
Authors: Sika-Paotonu Dianne , Tiatia Ramona , Sung Yun K. , Thornley Craig , Betty Bryan , Wineera-Parai Ranei , Eddie Barbara , Baker Michael , Puni Ronald , Dalton Estere , Taueetia-Suá Tua , Maloney Margaret , Sanchez-Felix Manuel , Spector Jonathan , Carapetis Jonathan 
Published: January 2023

Abstract
Acute Rheumatic Fever (ARF) is the autoimmune condition triggered by untreated Group A Streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Severe and recurrent untreated attacks of ARF can cause cardiac damage, known as Rheumatic Heart Disease (RHD). RHD remains a significant cause of morbidity and mortality globally and in Australia, New Zealand (NZ) and the Pacific Region, the disease burden of ARF and RHD amongst Indigenous and Pacific communities is one of the highest in the world, usually affecting children and young adults. The most effective recommended preventative measure for ARF requires painful monthly intramuscular injections of Benzathine Penicillin G (BPG) for 10 years or more. Known as secondary prophylaxis, these injections aim to prevent GAS infections that may lead to recurrence of ARF and either cause or worsen RHD. Adherence to secondary prophylaxis remains a challenge in many settings and improved understanding of barriers and a BPG reformulation that is more appropriate are urgently needed. This work sought to explore the BPG reformulation preferences and perspectives of predominantly Māori and Pacific children/teens in New Zealand currently receiving monthly BPG intramuscular injections, their families and healthcare providers using three software applications developed for this purpose. A total of 82 participants comprised of children/teens, family members and health professionals have thus far participated in this research project. This is the first time software applications have been successfully developed to collect qualitative and quantitative data on individual preferences for BPG formulations and dosing regimens in New Zealand.
Efficient isolation of particle-free human ILC2s from peripheral blood mononuclear cells
Authors: Poon Grace F.T. , Kyei Stephen K. , Antignano Frann , McQueen Karina L. , Peters Carrie E. , Takei Fumio , Valdez Yanet , Kokaji Andy I. , Woodside Steven M. , Eaves Allen C. , Thomas Terry E. 
Published: January 2023

Abstract
Group 2 innate lymphoid cells (ILC2s) have important roles in type-2 immune responses and are implicated in allergies, asthma, helminth infections and other metabolic diseases. To better understand their role in immunity, highly purified ILC2s are required. ILC2s are phenotypically defined as CD45+ lineage-negative CRTH2+ CD127+ CD161+, and comprise approximately 0.05% of peripheral blood mononuclear cells (PBMCs). Fluorescence activated cell sorting is the most common method to isolate ILC2s but it is both time-consuming and expensive because the cells are so rare. To address this issue, we have developed an immunomagnetic isolation method to obtain highly pure, particle-free ILC2s from human PBMCs in 2.5 hours. First, CRTH2 positive cells are labelled with a CRTH2-PE monoclonal antibody. These cells are then positively selected using an anti-PE antibody complex, EasySep™ Releasable RapidSpheres™ and an EasySep™ magnet. After separation, the magnetic particles are released from the positively selected cells and a second cocktail of antibody complexes and EasySep™ Dextran RapidSpheres™ is added to label non-ILC2s within the CRTH2 positive population. After a second separation, the released particles and unwanted cells are retained in the tube within the magnet, whereas the ILC2s are simply poured off and ready for use. With this method, the purity of ILC2s isolated from PBMCs was 84–95% (median 91%, n=18) with a recovery of 0.4–14.3×103 ILC2s per 108 starting PBMCs (median 2.46×103, n=18). The isolated ILC2s are functional, producing IL-13 upon in vitro stimulation with IL-33 and IL-2. Overall, EasySep™ Human ILC2 Isolation Kit allows researchers to easily and efficiently isolate highly pure and functional ILC2s.
Induction of neuroantigen-specific CD8+ T cell responses as a treatment for relapsing autoimmune demyelinating disease
Authors: Brate Ashley A. , Sinha Sushmita , Itani Farah R. , Pewe Lecia L. , Harty John T. , Karandikar Nitin 
Published: January 2023

Abstract
Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). We have shown that CNS-specific CD8+ T cells possess a disease suppressive function in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In humans, this suppressive function of CD8+ T cells is deficient during MS relapses and is restored during quiescence, suggesting that lack of CD8+ T cell mediated regulatory function may contribute to relapses in MS. We thus evaluated the role of CD8+ T cells in the context of the relapsing-remitting model (RR-EAE), using SJL mice. We demonstrate here that PLP178–191- and MBP84–104-specific CD8+ T cells ameliorated disease severity in an antigen-specific manner. Moreover, PLP178–191 CD8+ T cells reduced the number of relapses in PLP178–191-induced disease, even when transferred as a treatment during ongoing disease. We also probed the role of endogenously generated CNS-specific CD8+ T cell responses by using Listeria monocytogenes (LM) encoding CNS antigens to preferentially prime suppressive CD8+ T cells in vivo. Using this system, we show infection with LM expressing PLP175–194 endogenously induced disease suppressive CD8+ T cells that protected and treated PLP178–191 disease. Importantly, LM-PLP175–194 infection during disease remission of PLP139–151-induced EAE could effectively prevent relapses. These studies support a potential immunotherapeutic strategy using LM vaccination to endogenously prime disease regulatory CD8+ T cells.
Differential expression of CNS-specific proteins in progressive EAE point to potential biomarkers for progressive MS
Authors: Chase Carol Ann , Raphael Itay , Forsthuber Thomas 
Published: January 2023

Abstract
The lack of reliable markers for assessing the progression of multiple sclerosis (MS) from the relapsing-remitting to the secondary progressive form hinders the ability for therapeutic intervention and results in continued physiological deterioration. To begin to address the urgent need for biomarkers of progressive MS we investigated proteome changes over the disease course of progressive experimental autoimmune encephalomyelitis (EAE) in NOD mice as a preclinical model of the disease using a high-throughput quantitative proteomic technique pioneered in our lab. In this study, we utilized this established proteomic technique as well as bioinformatics tools to prioritize key proteins whose expression level in the CNS correlated specifically with the progressive phase of disease in the NOD EAE model. Hierarchical clustering of timepoints indicate that samples cluster based on progression of disease. Specifically, late time points cluster separately from naïve and peak time points. Importantly, statistical testing identified proteins with differential expression in the CNS of mice across the time course of NOD EAE, several of which are CNS specific. We are seeking to detect corollary changes in these CNS-specific proteins in the serum, pointing to a minimally invasive means of monitoring disease progress and measuring drug efficacy. Our studies will provide proof-of-concept for identifying homologous human biomarkers to guide treatment in individual patients. Furthermore, our results may provide insights into mechanisms that contribute to disease pathology and offer additional therapeutic targets for slowing the progression of MS.
The mechanism of preventing EAE via gene therapy
Authors: Keeler Geoffrey D. , Cote Isabelle , Gaddie Cristina D. , Hoffman Brad E. 
Published: January 2023

Abstract
Similar to Multiple Sclerosis, EAE is a complex disease in which immune tolerance is lost and antigen specific (Ag-sp) Tregs are known to play a role. We have developed a gene immunotherapy that has been shown to induce MOG specific Tregs, restore tolerance, and effectively prevent, and/or reverse, significant disease. However, the spatiotemporal profile of the induced Tregs is unclear. To identify the tissue specific populations of AAV8.MOG-induced specific Tregs, we used a transgenic Foxp3 reporter mouse with a MOG35–55/I-Ab tetramer. Two weeks prior to EAE induction, mice were tolerized via a single injection of vector. At the peak of disease, tissues were harvested and fluorescently labeled for phenotypic identification. FACS analysis revealed a higher proportion of Ag-sp Tregs in the cervical and inguinal lymph nodes (LN) and spleen, as compared to controls. In contrast, a higher proportion of Ag-sp effector cells were seen in control mice as compared to treated. Furthermore, a significant number of infiltrating effector T cells were isolated from the spinal cords of control mice, while very few T cells were recovered from treated mice. This aligns with cytokine data that was collected showing there to be higher production of IFN-γ in the spinal cord and spleen of control mice. These results suggest that AAV.MOG induced Ag-sp Treg populations accumulate in the peripheral organs, and may prevent effector cells from migrating to the spinal cord. This work provides new insights on tolerance induction mechanisms. These insights will be useful for the development of effective cell based therapies capable of treating autoimmune diseases.
Small molecule modulators of prion protein (PrpC) in Dendritic cells regulate inflammatory responses in a model of Multiple Sclerosis
Authors: Fallarino Francesca , Barreca Maria Letizia , Manni Giorgia , Scalisi Giulia , Biasini Emiliano , Gargaro Marco , Manfroni Giuseppe 
Published: January 2023

Abstract
Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for human multiple sclerosis, an autoimmune disease driven by differentiated Th1 and Th17 cells, and inflammatory dendritic cells (DCs). Recent data show that EAE is worsened in mice lacking PrPC protein and disease exacerbation has been attributed to T cells, which would differentiate into more aggressive effectors, when deprived of PrPC.
Novel compounds, modulating PrPC activity, have been recently developed, although not tested in EAE models yet. Interestingly, by specific gene array studies, we found that PrPC was highly expressed in DCs and differentially expressed among selected DC subsets. Moreover, by using a series of computational, and biochemical assays we identified novel small molecules, able to modulate PrPC function in specific cell lines overexpressing PrPC. By employing such tools, we studied the impact of PrPC modulation in promoting regulatory DC subsets. To this aim, DCs were treated either with novel PrPC modulators or a reference PrPC binding molecule Fe (III)-TMPyP. Then, they were co-cultured with sorted naïve CD4+ T cells in vitro. Notably, we found a significant expansion of FOXP3+CD4+ T regulatory (Treg) cells in cultures containing DC subsets, which were pre-treated with the specific PrPC modulators. Moreover, in a EAE model, we showed that systemic administration of such PrPC regulators, resulted in significant reduction of disease severity, compared to untreated controls. Overall, our results suggest that PrPC modulation in selected DC subsets may represent a novel means to restrain inflammatory T cell effectors, resulting in reduced inflammation, demyelination, and axonal injury in a model of EAE.
Tumor necrosis factor receptor 2 promotes neuroprotection during chronic autoimmune neuroinflammation
Authors: Gomez-Rivera Francisco , Raphael Itay , Raphael Rebecca , Robinson Rachel R. , Nalawade Saisha A. , Forsthuber Thomas 
Published: January 2023

Abstract
Multiple sclerosis (MS) is an inflammatory autoimmune disorder affecting the central nervous system (CNS) which affects over 400,000 Americans and over 2.5 million people worldwide. Although most patients are initially diagnosed with relapsing-remitting MS, the majority of these patients later develop a chronic-progressive form of MS, for which there is no well-established mouse model. The most common genetic factor associated with MS susceptibility is the Human Leukocyte Antigen (HLA)-DR2b haplotype. Additionally, studies in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, showed that lack of TNF signaling through its receptor TNFR2 leads to disease exacerbation and severe demyelination. Here, we developed a mouse model which expresses HLA-DR2b and lacks TNFR2, designated DR2bΔR2. Strikingly, DR2bΔR2 mice develop progressive EAE with pathology and clinical features observed in progressive MS patients. Adoptive transfer studies revealed that the clinical phenotype of EAE in DR2bΔR2 mice are largely dependent on TNFR2 expression in the CNS. Subsequently, we showed that DR2bΔR2 mice have a significant increase of lesions in the cerebellum, associated with reduced of oligodendrocyte progenitor cells (OPC) recruitment or function. Moreover, we showed that DR2bΔR2 mice are presented with chronic astrogliosis in demyelinating lesions. Our studies provide key insights into CNS repair and regulatory mechanisms controlled by TNFR2 during neuroinflammation and provide novel therapeutic strategies for treating progressive MS.