bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analytical Validation of Digital Spatial Profiling - a novel approach for multiplexed characterization of protein distribution and abundance in FFPE tissue sections
Authors: Merritt Chris , Barker Kristi , Metz Heather , Dennis Lucas , Webster Philippa , Beechem Joseph 
Published: January 2023

Abstract
Nanostring Technologies’ Digital Spatial Profiling (DSP) technology allows for the simultaneous analysis of 10’s to 100’s of proteins from discrete regions of interest (ROI), providing a morphological context to multiplexed protein analysis. The purpose of this study is to validate an antibody panel designed to characterize key immunology and tumor markers on the DSP platform.
IHC was performed on human FFPE tissues and human cell line pellets to evaluate binding specificity in both unconjugated and oligo-conjugated antibodies. The dynamic range of antibodies was tested using positive and negative FFPE cell pellets at different ratios. Interaction screens were performed to evaluate potential effects of multiplexing antibodies. A tissue microarray (TMA) containing normal and cancer tissues was employed to assess assay robustness.
IHC analysis of antibodies displayed indistinguishable staining patterns for both unconjugated and oligoconjugated antibodies. Mixed-proportion cell pellet assays revealed strong correlations between positive counts and positive cell numbers in an ROI. For example, CD3 displayed a LOD of 4% when assayed using cell pellet mixtures containing CD3+ CCRF-CEM cells and CD3− HEK293T cells. Interaction studies showed similar values for antibodies alone or in combination (R2 &gt; 0.8). TMA hierarchical clustering analysis demonstrated expected patterns for immune and tumor cells across different tissues.
These results demonstrate that indexing oligo conjugation does not interfere with antibody specificity and that these conjugated antibodies are robust reagents for quantification of protein abundance. Continued work on the DSP platform will expand the library of antibodies accessible for profiling.
Biomarker prediction to anti-PD-1 immunotherapy by using high dimensional single cell analysis
Authors: Krieg Carsten , Nowicka Malgorzata , Guglietta Silvia , Schindler Sabrina , Hartmann Felix J. , Weber Lukas M. , Dummer Reinhard , Robinson Mark D. , Levesque Mitchell P. , Becher Burkhard 
Published: January 2023

Abstract
Blockade of checkpoint inhibitors such as programmed cell death protein 1 (PD-1) set the pace for the immunotherapy of cancer. Following the initial success in metastatic melanoma, by showing a dramatic increase in progression-free survival, PD-1 immunotherapy was expanded to other cancers. As PD-1 monotherapy advances into first line treatment and combination of checkpoint inhibitors and other therapies start to show even increased response rates, still a large proportion of patients do not show durable responses. Therefore, predictive biomarkers of clinical response for stratification of patients most likely to respond to immunotherapy are urgently needed. In this study, we employed high-dimensional single cell mass cytometry and a generalized machine-learning bioinformatics workflow for the in-depth characterization of all immune cells in the peripheral blood of stage IV melanoma patients before and at 12 weeks of anti-PD-1 immunotherapy. In agreement with published literature a clear response in the T cell compartment of patients under immunotherapy was observed. To our surprise, prior to commencing PD-1 immunotherapy, a strong predictor of progression free and overall survival in responders to anti-PD-1 was a higher frequency of CD14+CD16−HLA-DRhi monocytes. We confirmed this finding by conventional flow cytometry in an independent, blinded validation cohort. Altogether, in this study, we identified for the first time a novel predictive biomarker for therapy decision in the treatment of metastatic melanoma and propose to extend this approach to select for positive responders in other cancers prior to immune checkpoint therapy.
Chemical screening based machine learning platform to identify MDSC-specific gene programs and immunomodulators to target solid tumor microenvironments
Authors: Chopra Gaurav , Kischuk Erin , Majumder Joydeb , Fine Jonathan , Lantz Travis Christopher , Ratliff Timothy L. 
Published: January 2023

Abstract
Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages can reprogram the pro-tumor microenvironment towards anti-tumor immunity. We hypothesized that MDSC suppressive function can be altered by targeting differentially expressed up-regulated genes of activated monocytic MDSCs in the tumor microenvironment compared to spleen. Using our proteome-scale target/antitarget network based interaction platform we identified and synthesized cell specific non-toxic chemical libraries with exclusive functions for anticancer potency and immunomodulation. MDSC were isolated from the tumor tissue of mice with bladder cancer, incubated with tumor explant supernantant under hypoxic condition to partially recreate the tumor microenvironment. The impact of synthetic compounds was assessed via changes in nitric oxide production and expression of function-associated genes, such as iNos, Arg1, CD274 (PD-L1). We used machine learning to iteratively predict activity that was verified experimentally. Feature selection based on proteome-compounds interactions simultaneously identified cell-specific gene programs and synthetic compound library targeting modular MDSC cell function ex vivo. We selected a compound that show no sensitivity to bladder cancer cells in vitro to assess if our cell-specific predictions translated in vivo. Significant reduction in tumor mass, lower frequency of MDSC expressing suppressive functional markers (iNOS, PD-L1) and increased IFNγ+T-cells was observed following treatment by oral gavage. We conclude that our platform can be used generally for desired immune cells to identify gene-program specific compounds to perturb the tumor microenvironment for combating solid tumors.
Stem cell, low-energy shock wave and phosphodiesterase type 5 inhibitor therapy to promote erectile function recovery in a rat pelvic neurovascular trauma model
Authors: Hertz Alexandria M. , George Evalyn I. , Reed-Maldonado Amanda B. , Brand Timothy C. , Salgar Shashikumar K. 
Published: January 2023

Abstract

Introduction
Battlefield pelvic injuries can have long-lasting debilitating effects, including erectile dysfunction (ED) in young soldiers. We investigated whether phosphodiesterase-5 inhibitor (PDE5i), low-energy shock wave (LESWT) and/or mesenchymal stem cell (MSC) therapy could improve erectile function (EF) after pelvic injury.

Methods
Lewis rats 10–12 weeks old were used. We had nine experimental groups (n=6/group): Sham, Injury alone, PDE5i, LESWT, MSC, LESWT+PDE5i, LESWT+MSC, MSC+PDE5i, and LESWT+PDE5i+MSC. Pelvic injury was created by performing bilateral cavernous nerve crush injury and internal pudendal bundle ligation. Bone marrow-derived MSCs (CD29+, CD90+, CD34−, CD31−) were injected (2×106 cells) into the corpora cavernosum. Rats received LESWT (1000 pulses at 0.06 mJ/mm2 and 3Hz) three times per week or sildenafil (PDE5i) in drinking water at ~10 mg/kg/day for four weeks. At 6thweek intracavernous pressure (ICP; mmHg) was recorded as EF outcome measure.

Results
Erectile function was significantly (P&lt;0.001) improved with PDE5i+LESWT therapy (max ICP 131±16, average ICP 116±11, change ICP 63±13) compared to untreated injury control (max ICP 88±24, average ICP 81±13, change ICP 16±23); ICPs became nearly normal, similar to uninjured sham group. LESWT alone improved (P&lt;0.05) EF (max ICP 105±14; change in ICP 39±15) compared to untreated injury control. Also, MSC alone improved (p&lt;0.05) EF compared to injury control. In both LESWT and MSC alone therapies EF recovery was partial. The other combination therapies and PDE5i alone did not show significant (P&gt;0.05) EF recovery.

Conclusion
LESWT, MSC and/or PDE5i combination therapy appears to be highly promising in EF recovery.
Fluorescent hypoxia probe for flow cytometry
Authors: Low Quentin , Calderon Veronica , Chen Aimei , Hu Yizhen , Mandavilli Bhaskar , Wang Xin , O’Grady Mike 
Published: January 2023

Abstract
Hypoxia is a condition where low levels of oxygen levels are present (1% to 2% O2). Hypoxia play a wide role in physiological and pathological conditions, from developmental angiogenesis to tumor progression and evasion. Hypoxia also modulates a number of immune functions from promoting inflammation to suppressing adaptive immunity. The current method for detecting hypoxic cells relies on an immunochemical approach. Pimonidazole react with peptide thiols in hypoxic cells and the resulting adduct is detected with an anti-pimonidazole antibody. To increase the availability of hypoxia detection reagents, we have developed a rhodamine-based hypoxia reagent (HR) for live cells. Using Jurkat leukemia cells incubated in hypoxic or normoxic conditions and stained with HR, we were able to clearly distinguish hypoxic cells from normoxic cells on a flow cytometer. We were also able to resolve cells incubated at differing levels of O2 (10%, 5% and 2.5% O2). To demonstrate that HR is compatible with other reagents, hypoxic cells were co-stained with a viability dye (SYTOX Red), a dye retained in intact mitochondria (TMRM) and with staining for a marker of apoptosis (annexin V). The results show that hypoxic cells excluded the dead cells dye while retaining TMRM, and were negative for annexin V staining at early time points. As expected, prolong incubation at hypoxia resulted in dead and apoptotic cells as evident in an increase in dead cells staining, a loss of mitochondrial integrity and an increase in annexin V+ cells. In summary, we present a sensitive reagent for detecting hypoxic cells without the need for cellular fixation and permeablization, while retaining it usage with other live cell flow cytometry detection reagents.
Identification of paired heavy and light chains from single B-cells from immunized Malian adults with rapid functional confirmation using iPair-BCR™, NGS, and iScreen™
Authors: Hou Xiaohong , Byrne-Steele Miranda L. , Pan Wenjing , Brown Brittany , Sanders Mollye , Eisenhower Mary , Coelho Camila , Hurtado Patricia Gonzales , Doritchamou Yai , Highsmith Kendrick , Taylor Justin J. , Schwartz Allison , Morrison Bob , Muratova Olga , Sagara Issaka , Doumbo Ogobara , Anderson Charles , Fried Michael , Duffy Patrick E. , Han Jian 
Published: January 2023

Abstract
Information about the cognate pairing of TCR alpha-beta chains and BCR IgH and IgKL chains encoded by individual T and B cells is key to understanding adaptive immune responses and developing therapeutic applications. We have previously reported the development of a sensitive technology that allows the amplification and identification of the paired human TCR alpha and beta chains from single T cells, termed iPair-TCR. Here, we report the extension of this technology to identify paired human BCR IgH and IgKL chains from antigen-specific single B cells. In this proof of concept study, we identified paired-VDJ-receptors from antigen-specific B-cells from nine Pfs230-EPA immunized Malian adults using the iPair-BCR method. Next, we developed a method to rapidly generate Fab fragments and demonstrate the binding of several of these single cell targets to the original Pfs230 antigen. Single cells of interest were identified based upon their repeated frequency on the plate, which indicates a clonal selection. The corresponding heavy and light chains were PCR amplified from selected wells. Using overlap extension PCR, all necessary elements for in vitro transcription and translation and either the CH1 or C-kappa-domain were added to both the 5′ and 3′ ends of the single cell VDJ. After in vitro transcription and translation, four out of five tested Fab fragments demonstrated binding through a colorimetric ELISA assay. The overall process after VDJ identification can be performed in under a week indicating the utility of our technology for rapid identification of antigen-specific BCRs and functional binding characteristics.
The ProQuantum immunoassay platform offers high performance in a simplified workflow for low-level measurement of cytokines in human and mouse sera/plasma
Authors: Bourdon David M. , Alohali Madhawi , Meng Lihao , Le Lucas , Freeman Alisha , Hu Jay 
Published: January 2023

Abstract
Immunoassays are not only a cornerstone of immunology but also biomedical research as a whole. A significant challenge that basic research scientists as well as translational investigators face is limitation of sample volumes obtained from mouse models and/or precious human samples. Researchers may need to compromise, make tradeoffs, and/or spend more time and resources to get enough samples for their experimental studies.
Thermo Fisher Scientific, the world leader in serving science, recently launched the ProQuantum High-Sensitivity Immunoassay platform (www.thermofisher.com/proquantum). These ready-to-use immunoassay kits combine the analyte specificity of high affinity antibody-antigen binding with the signal detection and amplification of real-time PCR.
The ProQuantum immunoassay platform features greater sensitivity and dynamic range than traditional methods using only 2 μL of sample. Assays are compatible with any real-time PCR instrument at 96- or 384-well configurations. Site-specific labeling chemistry preserves variable domain integrity of analyte-specific antibodies. The homogeneous (no wash) assay workflow provides sample-to-answer in 2 hours. An innovative web-browser based software application streamlines assay setup and provides robust data analytics.
Data presented here highlight human and mouse cytokine assay results for analytes important in the study of inflammation/autoimmunity. These performance data demonstrate the advantages of using ProQuantum immunoassays, especially when sample limitation is a concern.
Profiling of soluble immune checkpoint proteins as potential non-invasive biomarkers in colorectal cancer and sepsis
Authors: Pepin D , Godeny Michael , Russell Donna , Mehta Pramod , Lie Wen-Rong 
Published: January 2023

Abstract
In recent years, targeting immune checkpoint proteins has been shown to be an innovation for treatment of cancer and immune-related adverse events. Many checkpoint molecules have been identified on immune cells and/or tumor cells as activating receptors or inhibitory receptors. Many immune checkpoint proteins are also expressed as soluble forms in circulation, easily accessible as potential biomarker candidates. Here we report the development of a bead-based Luminex® multiplex assay for the quantitative profiling of a panel of co-inhibitory and co-stimulating immune checkpoint proteins: BTLA, CD27, CD28, CD40, CD80/B7-1, CD86/B7-2, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, TIM-3, and TLR-2. The aim of this study was to investigate a potential role for serum circulating immune-checkpoint proteins as putative biomarkers in colorectal cancer (CRC) and sepsis patients. We have used this novel 16-plex multiplex assay to measure multiple checkpoint protein expression levels in serum samples from CRC patients (n=20), sepsis patients (n=20), and a corresponding set of normal serum samples (n=20). Analysis of the soluble checkpoint protein signatures generated from this multiplex approach revealed differential expression of 9 soluble form of proteins, sBTLA, sCD27, sTIM-3, sHVEM, sCD40, sPD-1, sPD-L1, sCD86, and sTLR-2, in the CRC or sepsis serum samples, compared to the healthy serum controls. In summary, our results demonstrate that the multiplex immunoassay we developed is a useful research tool for the simultaneous quantitation of circulating immune checkpoint proteins, as well as its potential application in biomarker discovery and translational research.
Phosphoproteomic Analysis Reveals New SLP76 and PLCg1 Feedback Pathways That Regulate Lck in T Cells
Authors: Salomon Arthur R. , Belmont Judson , Gu Tao , Ji Qinqin 
Published: January 2023

Abstract
T cells play a central role in cell-mediated immunity against viruses, a variety of microbes, and cancer. Lck tyrosine kinase is the central regulator of T cell activation regulated through its phosphorylation state. The exact mechanisms that regulate the spatial equilibrium of Lck between the TCR and downstream regulators are not well defined. While some of the important regulatory proteins involved in Lck feedback loops in T cells are also beginning to be defined, the pathways leading to the activation and recruitment of these inhibitory molecules are much more obscure. Modern proteomic methods using quantitative mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways. Our recent data reveal a previously uncharacterized regulatory network where N-terminal tyrosine residues on SLP76 are involved in both positive and negative feedback loops, which regulate the phosphorylation of the activation loop on Lck. We also collected phosphoproteomic data that suggests that Lck targeting but not activity is regulated by PLCγ1. We examined the phosphoproteome of PLCγ1 deficient and reconstituted T cells through a timecourse of TCR stimulation. These data indicate that Lck activity was shifted from TCR to the SLP76 complex in the absence of PLCγ1. Altogether, wide-scale modern methods in quantitative mass spectrometry drive the molecular characterization of T cell signaling circuits and facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation and protein-protein interactions through a variety of activation states.
Immune cells avoid death from immunoproteasome inhibitors by switching to conventional proteasome
Authors: Ladi Ena , Everett Christine , Daniels Blake , Huestis Malcolm , Dere Ed , Gause Haley , Dirk Matthew , Rajapaksa Kathila , Staben Steve , Purkey Hans , Eidenschenk Celine , Siu Mike , Pappu Rajita 
Published: January 2023

Abstract
The pan-proteasome inhibitor bortezomib has demonstrated sustained clinical efficacy in off label trials in SLE patients by depleting pathogenic immune cells including highly secretory plasmablasts and plasmacytoid dendritic cells. But bortezomib also effects non-immune cells including neurons which raises significant safety concerns thus limiting the feasibility of pan-proteasome inhibitors as a treatment for autoimmune disease. As an alternative, targeting the immune cell specific immunoproteasome has been proposed as a promising therapy to deplete pathogenic immune cells while avoiding toxicity issues. Using highly specific immunoproteasome inhibitors, we show that this strategy does not lead to immune cell death as anticipated. We generated a large panel of small molecule inhibitors with varying specificity to the β5 subunit of the conventional and immuno- proteasome, and found that effects on viability of plasmablasts and pDCs correlate with the inhibition of the conventional proteasome, not the immunoproteasome. Our data indicates that the immune cells upregulate the conventional proteasomal subunits and prevent the accumulation of ubiquitinated protein. Widely used immunoproteasome inhibitors also block the conventional proteasome which, our data would indicate, is key to their ability to deplete immune cells.
The influence of berberine on co-stimulatory molecule expression and T cell activation
Authors: Vita Alexandra Adorno , Pullen Nicholas A. 
Published: January 2023

Abstract
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and deterioration within the joints, as well as extra-articular and systemic manifestations depending on severity. While there are many immunological factors that contribute to the pathology of RA, its progression is largely due to the activation of CD4+ T cells by antigen presenting cells (APCs), which then subsequently enhances the immune response of other cells within the area. Berberine (BBR), an isoquinoline alkaloid, has demonstrated the ability to attenuate mechanisms involved in RA in vitro and in vivo. Recent studies provide some evidence that BBR may interfere with the co-stimulatory mechanisms involved in a RA inflammatory response, however research specifically concerning BBR’s effect on co-stimulatory molecule expression and IL-12 production remains equivocal. In this study, we used flow cytometry to examine BBR’s effect on the cell surface expression of co-stimulatory molecules on bone marrow-derived dendritic cells (BMDCs) following stimulation with LPS, and on isolated splenic CD4+ T cells following activation with anti-CD3/anti-CD28. On BMDCs, the expression of CD40, CD80/86, and MHC class II was quantified, whereas CD40L, CD28, and IL-2R expression was examined on splenic CD4+ T cells. The amount of IL-12 secreted in BMDC supernatant was measured with an ELISA. The data collected on BBR’s effects will allow further insight into its potential as an alternative therapy for RA.
Disruption of self-tolerance in autoimmune diseases and therapeutic modulation to restore immune balance
Authors: Chakraborty Mousumi , Shashkova Elena , Cline-Smith Anna , Aurora Rajeev 
Published: January 2023

Abstract
Immune homeostasis is maintained by tolerogenic & immunogenic subsets of cells. They determine the intensity & type of response to infection & vaccination. Loss of tolerance results activation of self-reactive T-cells that attack self-tissues & develop pathologic conditions like arthritis. An important mechanism by which tolerance is maintained in periphery is by induction of regulatory T-cells (TREG).
Collagen induced arthritis (CIA) is a well-established model of rheumatoid arthritis. Immunization with collagen in presence of adjuvant in mice leads to production of proinflammatory cytokines, which leads to bone erosion by osteoclasts. Our laboratory has shown that osteoclasts function as antigen presenting cells (APCs), which induce FOXP3 in CD8+ T-cell (OC-iTcREG). Previous studies demonstrated that OC-iTcREG limit bone resorption in murine osteoporosis. We have also identified that, osteoclast express Notch ligand DLL4 & use Notch signaling to induce FOXP3 in CD8 T-cells. Expression of Notch ligand is also observed on other APCs, which we hypothesize are tolerogenic. Thus, we will characterize & examine whether these tolerogenic APCs can induce TREG & re-establish tolerance. We will present the results using adoptive transfer experiments, to measure immunomodulatory activity of DLL4 expressing APCs in CIA model. Efficacy of the treatment will be assessed by studying serum CTX, proinflammatory cytokines, immune-phenotypes of T-cell & APC subtypes from joint tissues of mice. Further, effect of these tolerogenic APCs will be compared with conventional APCs in CIA model to assess its therapeutic potential. This study will help to understand the complex interplay of tolerogenic cells in re-establishing immune homeostasis.