bluuebunny/crossref_metadata_2025_split huggingface dataset:
Memory B cell recruitment to the vaginal mucosa is required for luminal antibody secretion
Authors: Oh Ji Eun , Iijima Norifumi , Iwasaki Akiko 
Published: January 2023

Abstract
Antibodies secreted into the mucosal barriers serve to protect the host from a variety of pathogens, and are the basis for successful vaccines. In type I mucosa such as the intestinal tract, dimeric IgA secreted by local plasma cells is transported through polymeric Ig receptors (pIgR). However, due to the paucity of pIgR and plasma cells, how and whether antibodies are delivered to the type II mucosa represented by the lower female reproductive tract (FRT) lumen remains unclear. Here, using genital herpes infection in mice, we show that primary infection does not establish plasma cells in the lamina propria of FRT. Instead, upon secondary challenge with herpes simplex virus 2 (HSV-2), circulating memory B cells that enter the FRT serve as the source of rapid and robust antibody secretion into the FRT lumen. CD4 tissue-resident memory T cells (TRM) secrete interferon gamma (IFN-γ), which induces expression of chemokines including CXCL9 and CXCL10. Circulating memory B cells are recruited to the vaginal mucosa in CXCR3-dependent manner, and secrete virus-specific IgG2b, IgG2c and IgA into the FRT lumen. These results reveal circulating memory B cells as a rapidly inducible source of mucosal antibodies for the FRT.
Single-cell RNA-seq of young and aged alveolar macrophages
Authors: Wu Yue , Zhu Bibo , Goplen Nick G. , Zhang Ruixuan , Li Ying , Cardani Amber N , Braciale Thomas J , Sun Jie 
Published: January 2023

Abstract
Alveolar Macrophages (AMs) are a unique population of innate immune cells that reside in the alveolar space and contribute to accommodate the ever-changing need of the lungs against the external environmental challenges. AMs derive from the fetal origins and are capable of maintaining themselves through self-renewal without the input of hematopoiesis during the adulthood. However, little is known about how aging is influencing the molecular characteristics and heterogeneity of the AMs during homeostasis. Using single-cell RNA sequencing (scRNA-seq) and Seurat clustering, we discovered AMs can be subdivided into different “subsets” with unique molecular characteristics during homeostasis. Interestingly, when we further investigate the AM population with the combined data from the young(~8w) and old(~2y) mice, the data suggested that the proliferating cell fall in the same cluster with reduced number in the old-mice group, while the poised inflammatory cells showed distinct molecular patterns between the group with different age. We hypothesize that the inflammatory and the self-renewal AM subpopulations may be maintained by distinct extracellular signals. With the progress of aging, the self-renewal ability of AMs would reduce because of the number of proliferating cells but not the change of molecular patterns. At the same time, the inflammatory population would change its characteristics with age, and potentially lead to functional change upon activation. Further validation of the AM subset identity as well as the developmental and functional interrelationship of these subsets are required. In addition, the extrinsic and intrinsic signals responsible for the maintenance of these AM subsets are to be explored.
Multiparametric analysis of cell health status using flow cytometry
Authors: Low Quentin , Calderon Veronica , Montoya Leticia , O'Grady Mike 
Published: January 2023

Abstract
Cellular responses to drug treatment can be varied and heterogeneous. Often, a multiparametric approach is necessary to identify cellular pathways that are responding or affected. To maximize the number of parameters analysed per sample, we aimed to combine several functional sensors into one assay. In addition, we set out to streamline the workflow focusing on live cell responses. To this end, we designed a flow cytometry-based detection system centered on a single incubation condition for sensors that have minimal spectral overlap. Analysing Jurkat cells treated with camptothecin, a topoisomerase inhibitor that triggers cellular apoptosis, we were able to simultaneously detect the loss of cells in G2/M phase using the stoichiometric DNA dye, Hoechst 33342; an increase in apoptotic cells with a sensor for activated caspase 3/7 (CellEvent Caspase-3/7 Green); the loss of mitochondrial membrane potential with tetramethylrhodamine, methyl ester (TMRM); and an increase in dead cells as well as cells in late apoptotic stage, using a SYTOX viability dye. We also tested another four-sensor combination that focused on detecting cellular stress. Upon prolonged camptothecin induction, we were able to detect an increase in mitochondrial stress with MitoSox, a sensor that detects mitochondrial oxidative stress; an increase in cellular stress with a CellROX reagent specific for reactive oxygen species. There was also a loss in mitochondrial membrane potential as well as a greater loss of cellular viability. In summary, using a model of cellular response to drug treatment, we performed multiparametric cell status analysis with a combination of cellular detection reagents on a flow cytometer.
A Robust and Scalable Method to Crosslink Antibodies and Proteins
Authors: Zhao Qin , Patel Deven , Wu Haiyan , Liao Jinfang , Diwu Zhenjun 
Published: January 2023

Abstract
The conjugations of biopolymers are critical tasks in life science field. A traditional method is to use SMCC, but SMCC-modified protein is unstable and easy to cause self-crosslink if the protein contains thiol group. Another known method is based on S-HyNic and Sulfo-S-4FB chemistry, but catalyst is required and the procedure is tedious and long.
We developed a Buccutite method to crosslink antibodies to biopolymers (e.g. enzymes and ligonucleotides) or nano particles (such as Qdots and latex beads). This new technique is based on a pair of crosslinkers: Buccutite FOL and MTA. One protein is labeled with MTA, and the other is pre-activated with FOL; when two parts are mixed, the conjugation occurs promptly without any catalyst required.
The Buccutite method was validated with the conjugation of antibody with HRP or phycobiliproteins (PE, APC or their tandems). ELISA assays showed the Buccutite antibody-HRP has the same sensitivity as traditional antibody-HRP conjugate. Flow cytometry result also showed the Buccutite conjugates performed as well as the ones prepared with traditional SMCC method when evaluated with human lymphocytes CD antibodies conjugates. Pre-activated proteins (e.g., PE-FOL) were stable for 12 months without any decreasing in performance. Buccutite conjugation was very efficient: there was less than ~10% of the free antibody left, while there was still more than 40% free antibody remaining for the traditional SMCC method.
In conclusion, Buccutite method has demonstrated a way to prepare conjugate at high efficiency in short reaction time and mild reaction conditions with the same performance. It is a superior and effective alternative to the existing methods for crosslinking biopolymers to their targets.
Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function
Authors: Shifrut Eric , Carnevale Julia , Ashworth Alan , Marson Alexander 
Published: January 2023

Abstract
Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. Critical biology of human immune cells, including key signaling pathways and effector functions, may not be recapitulated in immortalized cell lines. We developed a new method, sgRNA lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA-Seq revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. In summary, we have developed a novel pooled CRISPR screening technology with the potential to explore unmapped genetic circuits in primary human cells and to guide the design of engineered cell therapies.
Novel plate-based detection method for T cell activation/proliferation, migration, and cytotoxicity assay using image cytometry
Authors: Chan Leo 
Published: January 2023

Abstract
The field of cancer immunotherapy has grew significantly due to numerous successful trials in cancer vaccines, antibody-based treatments, CAR T cell therapy, and other immunotherapies. Specifically for T cell-based assays, they have been traditionally analyzed using ELISA or ELISpot to detect specific cytokines concentration for measuring T cell responses. Other methods such as T cell activation/proliferation, migration, and cytotoxicity assays have employed flow cytometry or microscopy to understand the functionality of T cells. In the recent years, image cytometry has been used to develop robust assays to monitor T cell immune response, which is highly critical for immuno-oncology research.
In this work, we demonstrate the use of Celigo Image Cytometer to perform high-throughput in vitro T cell-based functional assays. Three direct cell counting methods are developed for label-free T cell activation and proliferation measurement, T cell migration assay, as well as T cell-mediated cytotoxicity. First, primary T cells are seeded into a 96-well plate and stimulated with antibodies or co-culture with cancer cells. The cells are directly counted in the well over 48 hours in bright field images. Next, T cells are seeded in a transwell with chemokines in the bottom wells. As the T cells migrate through and drop to the bottom, the cells are directly counted using bright field or fluorescence with Hoechst staining. Finally, T cell-mediated cytotoxicity assay is performed by co-culturing CD8 T cells with calcein- or fluorescent protein-labeled cancer cells. As target cancer cells are killed, the fluorescence dissipates, and by counting the changes in the number of “live” target cells, the cytotoxicity percentages are measured.
The “Crisis of Reproducibility” of Animal Studies: Causes and Solutions
Authors: Shollenberger Lisa M , Hentze Hannes , Ibsen Eric 
Published: January 2023

Abstract
A majority of post-phase III clinical trial therapies have historically failed to receive regulatory approval due to a lack of demonstrated efficacy. According to a 2012 study published by Amgen researchers, 90% of the 50+ in vivo oncology studies were irreproducible. Over the last two years, questions and debates concerning the validity of the preclinical science data have resulted in multiple published reviews.
Suboptimal disease models, inadequate or inappropriate controls, and poor documentation practices (including electronic spreadsheets) will be discussed as primary causes of irreproducibility in animal research. To address these concerns, several best-practice resources and initiatives have been made available to the research community. The ARRIVE guidelines, PHISPS protocols, and the N3R Design Assistant will be described as practically-applicable concepts and publicly-available resources to address issues contributing to the “Crisis of Reproducibility”.
Researchers have increasingly adopted animal study workflow software applications and databases, which were developed to address this crisis. By applying such automated and time-saving technologies, researchers can now more easily standardize the highly variable but detailed processes of study design, procedure adherence and descriptions, animal welfare parameter monitoring, graphing, data analyses, report creation, task and measurement scheduling, and data preservation. Using some real-life examples, the advantages and disadvantages of preclinical study software applications and the use of electronic spreadsheets will be discussed in the context of helping to resolve the “Crisis of Reproducibility” in animal studies.
Adarza’s Ziva technology platform enables highly multiplexed label-free detection of serum analytes
Authors: Schmuke Jon J , Mueller Ernie , Darby Amanda , Wente-Roth Traci , Vohra Naila , Haley Jeaneen , Hodge Tracey , Pillers Michelle 
Published: January 2023

Abstract
New multiplex, label-free methods for the sensitive and quantitative detection of serum proteins are highly desirable, as they have the potential to dramatically accelerate biomedical research in the age of multi-omic, big data driven personalized medicine. We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array for the detection of human serum cytokines and inflammatory markers. The chip is able to provide quantitative information about protein concentrations, with limits of detection in picograms per mL. Provided in a standard 96-well microplate format, the label-free nature of the array will permit rapid expansion in plex size and enable simple, flexible creation of custom panels as there are no secondary antibodies or labels to cross-react. Here we demonstrate validation parameters of sensitivity, precision, and accuracy for inflammatory markers (&gt;20) in a new multiplex technology suitable for analyzing trends across cytokine and inflammatory marker profiles comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the Ziva instrument, the technology has been designed for the ultimate user-experience.
Optimum processing conditions for single cell RNA sequencing on frozen human PBMCs
Authors: Hanamsagar Richa , Marcus Robert , Chamberlain Mathew , de Rinaldis Emanuele , Savova Virginia 
Published: January 2023

Abstract
The field of single cell RNA sequencing (sc-SEQ) has exploded in the past few years. From picking up single cells manually under a microscope, to droplet-based encapsulation of cells using microfluidics – this technology has improved in leaps and bounds. Common droplet-based technologies include inDrop, Drop-seq and 10X Genomics Chromium. All three technologies utilize microfluidics for encapsulating single cells & uniquely barcoded beads within an oil droplet. They differ in their bead material/manufacturing, barcode design and the range to which their operation can be customized by the end user. However, the performance of each sc-SEQ each technology is dependent on factors such as ability to obtain pure, viable single-cell suspension, and ability to accurately quantify the number of cells before running them through the machine. Here, we compare and contrast different conditions for cell processing that can affect single-cell sequencing results – including cell counting and purifying methods, as well as cell subtype enrichment kits; followed by single cell encapsulation, library preparation and analysis using 10X Genomics Chromium workflow.
The contribution of tumor type, stage and antigen in the Antibody-Antigen kinetics by ELISA in adult and pediatric cancer patients
Authors: Marquez-Manriquez Juan P , MATUTE-BRISENO JOSE ANTONIO , CAMACHO-HERNANDEZ ALEJANDRO , ICEDO-ZAMORA SALVADOR , Alejandro Lucero-Diaz PEDRO ALEJANDRO 
Published: January 2023

Abstract

Background
Most of the ELISA studies in cancer patients use different source of antigens, which is mainly limited to recombinant proteins produced in E. coli and this may impact the global results between the studies using even the same antigen because most of the researchers assume that all the tumor types and all the antigens would behave equally, which we believe is not well established immunologically and clinically.

Methods
The repository bank was used (n=75 adults) and (n=35 pediatric) and (n=90) presumably healthy patients. The serum was analyzed by ELISA at different times starting at 1, 2, 4,6,12,24, 48 and 72 hours where the serum was placed at 1:200 dilution with a panel of 8 recombinant, purified proteins and predicted immunogenic peptides from all the 8 proteins. We correlate the kinetics of antibody-antigen binding with several clinical parameters such as stage, PFS, ECOG and the level of some acute phase proteins.

Results
We demonstrated that the optical density (OD) at 650 nm of the cancer patients is different depending of the tumor stage (p=0.0001) and age as in in pediatric tumors we found statistical significance in the antibody-binding with respect to the adult tumors (p=0.0005) in 2 hours versus 72 hours. Importantly, we found that the patients with early binding at 2 hours had better prognosis (mOS=19 months) than those at 72 hours (mOS=7 months). Also the best antigen to demonstrate the antigen specific immune response in cancer patients was the purified proteins (p=0.0001) and predicted peptides (p=0.001).

Discussion
This data demonstrate that the nature of the antigen, the kinetic of exposure of the antibody-antigen impacts the results and the clinic correlation with several parameters including tumor stage.
Optimizing the pharmacokinetic properties of polymeric IgA in mice
Authors: Rajan Sharmila , Lombaba Noelle Twyla , Mandikian Danielle , Boswell Andy , Yip Victor , Bravo-Perez Daniel , Chen Eugene , Wang Jianyong , Phung Wilson , Lee Sophia , Farahi Farzam , Estevez Alberto , Gill Avinash , Sandoval Wendy , Viajar Sharon , Ciferri Claudio , Matsumoto Masrissa , Spiess Christoph 
Published: January 2023

Abstract
IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance. Here, we present the biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA, which may enable the IgA platform the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure. This strategy may thus allow for targeting antigens previously untenable with conventional IgG1, while allowing for lower drug exposure in non-targeted regions.
T cell receptor repertoire detection from dry blood spots
Authors: Li Song , Pan Wenjing , Wu Shang-Gin , Liu Hongna , Byrne-Steele Miranda , Brown Brittany E , Depinet Mollye , Hou Xiaohong , Han Jian 
Published: January 2023

Abstract
Immune repertoire amplification of T cell receptors (TCRs) coupled with high throughput sequencing provides detailed insight into the immune system. A common starting sample type is total RNA extracted from whole blood by venipuncture. However, the costs, participant burden, regulatory constraints, and logistics associated with venipuncture and RNA handling are major barriers to clinical application or community-based research on various diseases. This study aims to identify TCR repertoires from dry blood spots (DBS), a method that could help collect real-world data for biomarker applications. Finger-prick blood was collected onto a Whatman filter card, and RNA was extracted from DBS. Fully automated multiplex PCR was performed to generate a TCRβ chain library for next generation sequencing (NGS) analysis of unique CDR3s (uCDR3). According to statistical analysis and laboratory confirmation, forty 2-mm punch disks from the filter cards were enough to detect the shared top clones and have a strong correlation in uCDR3 discovery with whole blood. uCDR3 discovery was not affected by storage temperatures (room temperature or −20°C) or storage durations (1, 14, and 28 days) when compared to whole blood. About 74 – 90% of top 50 uCDR3 clones of whole blood could also be detected from DBS. The DBS-based TCR repertoire profiling method is minimally invasive, provides convenient sampling, and incorporates fully automated library preparation. The system is sensitive to low RNA input, and the results are highly correlated with whole blood uCDR3 discovery allowing study scale-up to better understand the relationship and mutual influences between the immune repertoire and various disease states.
Complete all-in-one immune repertoire profiling of newborn babies via novel dimer-avoided multiplex PCR (dam-PCR)
Authors: Hou Xiaohong , Pan Wenjing , Depinet Mollye , Brown Brittany E , Eisenhower Mary , Byrne-Steele Miranda , Weber Michele , Dworsky Meyer , Callison Sharon , Han Jian 
Published: January 2023

Abstract
There is an increased demand for immune repertoire profiling in both basic research as well as in various clinical settings, such as during vaccine development, lymphocyte tracking in minimal residual disease (MRD), hematopoietic stem cell transplant recovery monitoring, immunotherapy development, and immunological monitoring for prognosis purposes during treatment. Although next generation sequencing of the immune repertoire allows detailed, sequence-specific insight into the adaptive immune response, it usually focuses on only one or two receptor chains. 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. We therefore developed a novel PCR technique, dimer-avoided multiplex PCR, that allows amplification of both BCRs and TCRs in a single, quantitative multiplex reaction. Our method generates next generation sequencing libraries for all TCR and BCR loci including TCR-beta, TCR-alpha, TCR-delta, TCR-gamma, BCR-IgH, and BCR-IgK and IgL. Here, we report the application of this method to investigate the immune repertoire of cord blood from newborn babies. Our study revealed the vast diversity and abundance of babies’ immune repertoire, the composition of TCR and BCR loci, and the sharing status of unique CDR3s among newborns. This single reaction, cost effective, all-inclusive, and quantitative immune-profiling analysis method shows promise for future applications in both basic research and clinical settings.