bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms by which Lactoferrin-derived chimeric peptide (LFch) enhances TGFβ1-induced Treg Differentiation
Authors: Kim Pyeung-Hyeun , Jang Young-Saeng , Park SunHee 
Published: November 2024

Abstract
We recently reported that LF substantially promoted Foxp3 expression by activated CD4+T cells and this activity was further enhanced by TGF-β1. We also found that the lactoferrin chimera (LFch, synthetic LF-derived chimeric peptides) dramatically enhanced TGFβ1-induced Foxp3 expression, although LFch itself did not stimulate Foxp3 expression. In the present study, we explored the mechanism by which LFch boosts TGFβ1-induced Treg differentiation. We applied biotinylated LFch (LFch-biotin) to elucidate this issue. Through the receptor ELISA and co-immunoprecipitation experiments, we observed that LFch-biotin directly binds to TβRIII and PI3K. Further, it also binds and diminishes phosphorylated-PI3K which is associated with CD28. As expected, LFch-biotin boosted TGFβ1-induced Foxp3 expression. The same was true when PI3K inhibitor was treated. Taken together, these results suggest that LFch upregulates TGFβ1-induced Treg differentiation by diminishing CD28 signal intensity through the complex of LFch/TβRIII/PI3K/CD28.
Targeting the SIRT1 pathway to modulate inflammation in a murine model of Kawasaki Disease vasculitis
Authors: Atici Asli , Jena Prasant , Gomez Angela , T. Carvalho Thacyana , Crother Timothy , Noval Rivas Magali 
Published: November 2024

Abstract
Kawasaki Disease (KD) is an acute febrile systemic vasculitis and the leading cause of acquired heart disease among children. Overactivation of the NLRP3 inflammasome and IL-1b production are critical for the development of cardiovascular lesions during KD. Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, is crucial for responding to cellular stress, and regulates NLRP3 activation as well as clearance of damaged mitochondria in cardiovascular diseases. However, its role in KD pathophysiology remains unknown. Here, we used the Lactobacillus casei cell wall extract (LCWE) murine model of KD to determine the role of SIRT1 on the development of cardiovascular lesions. We show that Sirt1 expression is downregulated in LCWE-injected mice with a significant reduction of nicotinamide, a product resulting from NAD+ degradation by SIRT1. In addition, supplementation of mice with NAD+ precursors significantly reduced the development of LCWE-induced cardiovascular lesions. Furthermore, KD development was less severe in SIRT1 overexpressing mice but was exacerbated with the specific deletion of Sirt1 in vascular smooth muscle cells. Treatment of LCWE-stimulated coronary smooth muscle cells with a SIRT1 activator favored mitophagy by inhibiting ribosomal S6 phosphorylation. These results indicate an impaired NAD+/SIRT1 axis during LCWE-induced KD vasculitis, which could potentially be targeted to reduce cardiovascular lesions in KD.
Homogeneous bioluminescent immunoassays detect profound synergistic regulation of IL-8 and MCP-1 release in cultured human monocytes
Authors: Lazar Dan , Ames Cassidy , Sequeira Paula , Kupcho Kevin , Mamott Daniel , O'Brien Martha , Mahan Sarah , Cali James 
Published: November 2024

Abstract
We have developed sensitive bioluminescent immunoassays for IL-8 and MCP-1 and applied these homogeneous assays to demonstrate synergistic regulation of their release in a culture model of human monocytes. IL-8 and MCP-1, key proinflammatory chemokines, play important roles in neutrophil and monocyte recruitment. Employing Lumit™ technology, we labeled candidate monoclonal antibodies for these chemokines with NanoBiT luciferase subunits (SmBiT or LgBiT), identifying pairs that produce strong luminescent signals upon target binding. These assays exhibited outstanding sensitivity (IL-8 at 0.4 pg/mL, MCP-1 at 0.8 pg/mL) and a broad linear range over three logs of analyte concentration, with high specificity against a panel of cytokines. Applying these assays to THP-1 monocytes in 96-well format, we observed lipopolysaccharide (LPS) induced a dose-dependent IL-8 release (max 1532 pg/mL), whereas IFN-γ triggered minimal release (169 pg/mL). However, LPS and IFN-γ together significantly increased IL-8 levels (8554 pg/mL). More profound synergy was observed for MCP-1 release; LPS or IFN-γ alone elicited modest responses (23 and 48 pg/mL, respectively), but their combination led to a dramatic increase (5902 pg/mL). This work establishes rapid, simple luminescent immunoassays for IL-8 and MCP-1 detection directly in cell culture and shows application of these assays to demonstrate synergistic regulation of chemokine release with implications for modulation of inflammatory responses.
Utilizing a surgical resection model for evaluating mRNA-based multi-antigen targeting CAR-T cell therapy against solid tumors
Authors: Dagher Oula , Posey Avery 
Published: November 2024

Abstract
Chimeric antigen receptor (CAR)-T cell therapies have demonstrated considerable remissions against hematological malignancies but only transient remission against glioblastoma. CAR-T cell engineering relies on viral transduction or nonviral delivery such as mRNA incorporation. However, current in vivo models are inadequate for mRNA-based CAR-T cell evaluation due to short-term CAR expression. Here, we investigated multi-antigen targeting mRNA-based CAR-T cells locally injected in a xenograft mouse model after surgical resection of primary tumor. NSG mice, subcutaneously inoculated with glioma cells, were divided into groups receiving either mock T or CAR-T cells. A resection cutoff of minimal residual disease was used to subdivide mice based on resection intensity (desired or undesired). Anti-tumor cytotoxicity was both resection- and dose-dependent. Notably, &gt;75% of mice undergoing undesired resection succumbed 49 days post-resection. However, CAR-T cell treated mice undergoing desired resection demonstrated complete or partial remissions along with significantly higher survival rates. Treatment of resection models with multivariant mRNA-based CAR-T cells demonstrates the translational utility of nonviral CAR-T cell approaches, building on previous reports targeting multiple antigens to offset solid tumor evasion. To sum up, our xenograft resection/CAR-T cell model is better tailored for evaluation of mRNA-based CAR-T cell therapies against solid tumors than existing models.
An image-based real time method to measure antibody-dependent cellular phagocytosis
Authors: Shi Yongchang , Wang Jianyong , Sun Yonglian , Koerber JT , Seki Akiko , Rutz Sascha 
Published: November 2024

Abstract
Antibody-dependent cellular phagocytosis (ADCP) is an important mechanism of action of therapeutic antibodies. In vitro quantification of ADCP has been technically challenging. Fluorescence microscopy and flow cytometry based assays were traditionally used to measure ADCP activity, these methods were static measurements at certain time point, thus provide no information about engulfment kinetics. Moreover, the flow cytometry assays require lifting the macrophage cells from the labware which may affect macrophage activities. We have developed a real time ADCP assay using a live cell analysis system (IncuCyte). The target cells labeled with a pH sensitive dye only fluoresce when they are engulfed in acidic phagosomes of macrophages in the presence of specific antibodies. Our assay is a plate-based with a simple mix and read protocol without disturbing the macrophages. The new method is quantitative and robust, can be readily used for high-throughput screening for functional antibodies. By studying the ADCP activities in different subtypes of macrophages, we found M2 microphages showed the strongest ADCP activity compare to M0 and M1 macrophages, due to their different expression levels of FcgRs. The assay is highly sensitive to differentiate the antibody variants with enhanced Fc function. It can also be used to study the kinetics and mechanisms of ADCP.
Developing a multiplex assay to monitor apoptosis and necrosis using the Cellaca™ PLX Image Cytometer
Authors: Chan Leo , Pierce Mackenzie , Huang Yongyang , Lin Allen , Lin Bo 
Published: November 2024

Abstract
Apoptosis is a critical pathway for programmed cell death that cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare cancer that can initiate the process of apoptosis, allowing for the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V-APC, Caspase 3/7, and Propidium Iodide (PI) using the Cellaca™ PLX Image Cytometer. First, apoptosis was induced in Jurkat and K562 with staurosporine and monitored over the course of 24 h. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early to late-stage apoptosis, and ultimately necrosis. A clear trend was observed during the first 2 h showing differences of up to 25% between the single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis amongst apoptotic populations, Annexin V+ only populations were higher than Caspase-3+ only populations by up to 30% between 0-2 hours. The proposed image-based detection method may provide an effective and efficient tool that allows researchers to better characterize and screen potential cancer therapeutic drug candidates in a high-throughput manner to determine their treatment efficacy.
Development of an ultra-sensitive multiplex cytokine assay utilizing a novel digital multi-omics detection technology
Authors: Lambert Jeremy , Proctor Angie , Seigfried Nate 
Published: November 2024

Abstract
Demand for rapid, highly sensitive measurement of proteins and nucleic acids has increased dramatically over the past several years to support biomarker research, drug development and diagnostics. In addition, the ability to interrogate both protein and nucleic acids (multi-omics) in a single sample allows critical information to be obtained relating the transcriptome to expressed protein levels. Despite this, current platforms for multiplex measurement of circulating biomarkers can lack sensitivity and specificity required for analytical and clinical validation of low abundance proteins and nucleic acids.
We report the development of an automated microfluidic-based technology capable of multiplexed measurement of protein and nucleic acid biomarkers utilizing a novel approach combining digital and analog detection modalities. Significant improvements in sensitivity and specificity are achieved through use of a bead-based microfluidic array resulting in a spatially-resolved series of single-plex signal amplification reactions. Critically, the isolation of reactions allows multiplex panels to be constructed in a modular manner while eliminating interference that often limits specificity of traditional multiplex biomarker detection technologies. A specific application of this technology we report here is a multiplexed assay measuring low abundance cytokine biomarkers from serum and plasma samples with utility in a broad range of research and clinical studies.
Genetic targeting of the mouse immunoglobulin locus to permit the expression of heavy-chain only antibodies against viral antigens
Authors: Borbet Timothy , Zaldana Kimberly , Morrison Tate , Boehringer Thomas , Gonzalez Sandra , Chan Kun-Wei , Luo Christina , Kirilov Milen , Kong Xiang-Peng , Wang Wei , Sheiffele Paul , Koralov Sergei 
Published: November 2024

Abstract
Conventional antibodies are composed of two identical sets of heavy and light chains, forming a dimer. The CH1 domain of the antibody heavy chain is essential for the pairing of heavy and light chains. However, certain animals, such as camels and sharks, evolved to produce heavy-chain-only antibodies (HCAbs) by losing this crucial CH1 domain in some of their antibody heavy chain constant regions. These HCAbs boast unique physicochemical properties that confer significant advantages: their smaller size allows for improved access to target antigens, increased hydrophilicity, enhanced tissue penetration, and greater structural stability compared to standard antibodies. Our research has successfully established a mouse model that intrinsically produces HCAbs from the mouse's own immunoglobulin heavy chain locus. Our findings demonstrate that heavy-chain-only B cell receptors, once introduced, facilitate normal B cell development, promote germinal center formation, and enhance plasma cell differentiation relative to B cells producing conventional antibodies. These genetically modified mice were vaccinated with viral antigens, resulting in the production of HCAbs that specifically target the SARS-CoV-2 spike protein and the HIV-1 Env trimer. Through single-cell sequencing, we have identified unique nucleotide sequences of clonotypes; the HCAbs are being synthesized to assess their viral neutralization potential and antigen-binding affinity to highlight the efficacy of our model.
Synthetic TCR/BCR RNA spike-in controls for assessing immune receptor profiling assay accuracy
Authors: Chenchik Alex , Liu Tianbing , Makhanov Mikhail , Hu Dongfang , Ghias Khadija , Diehl Paul 
Published: November 2024

Abstract
Synthetic TCR and BCR RNA spike-in controls can be used as universal standards to account for biases caused by PCR and NGS steps of adaptive immune receptor (AIR) repertoire profiling assays. We designed 48 BCR and 39 TCR mRNA synthetic constructs to cover nearly full-length V(D)JC structures, which represent all seven TCR (TRB, TRA, TRG, TRD) and BCR (IGH, IGK, and IGL) hypervariable chains. To identify cross-sample contamination, we spiked in 2 ul of BCR (16x3) and TCR (13x3) triplex isoform pools and detected the presence of the sequence in the sample. Furthermore, we integrated 48 BCR and 39 TCR premixed controls which are mixed in a 16:4:1 ratio into Cellecta’s DriverMap™ Adaptive Immune Receptor Profiling Assay, which employs a gene-specific, multiplex RT-PCR method with unique molecular indices (UMIs). The spike-in controls coupled with UMI-based PCR amplification can help distinguish between control and background sequences. We observed a consistent linear relationship between the concentration of spike-in controls and their molecular counts, with an average sequencing error rate between 0.4%-0.8% per base, which is in line with Illumina sequencing standards. These findings validate the effectiveness of our synthetic spike-in controls in correcting biases in AIR protocols, offering a reliable estimation of error and mutation rates in the DriverMap AIR assay or similar NGS-based immune receptor profiling methods.
The First Case of Autosomal Recessive CADINS-like Disease Caused by Novel Compound Heterozygous Hypomorphic Variants in CARD11.
Authors: Spratt Alison , Palterer Boaz , Bauman Bradly , Salvati Lorenzo , Mazzoni Alessio , Maggi Laura , Capone Manuela , Vanni Anna , Provenzano Aldesia , Giglio Sabrina , Cosmi Lorenzo , Liotta Francesco , Parronchi Paola , Annunziato Francesco , Snow Andrew 
Published: November 2024

Abstract
CARD11 is a lymphocyte-specific scaffold protein connecting antigen receptor ligation to signaling governing lymphocyte proliferation and differentiation. We and others have shown that heterozygous, dominant negative (DN) CARD11 mutations disrupting T cell receptor (TCR)-induced NF-κB signaling cause severe atopy in humans, often with recurrent infections and humoral immune deficits. Here we describe a patient with a history of recurrent upper respiratory infections, recalcitrant atopic dermatitis and common variable immunodeficiency, carrying novel compound heterozygous CARD11 variants (G689E, R920L) in regions of the protein (PDZ and L4, respectively) not normally associated with deleterious mutations. By transfecting variant expression plasmids into CARD11-deficient Jurkat T cells harboring an NF-κB-driven GFP reporter, we found that G689E was mildly hypomorphic, whereas R920L was a complete loss-of-function mutation with negligible DN activity against wild-type CARD11. However, TCR-induced NF-κB activity was markedly reduced upon variant co-expression, strongly suggesting this patient represents the first autosomal recessive case of CARD11-associated Atopy with Dominant Interference of NF-κB Signaling (CADINS) disease. Additional experiments examining different amino acid substitutions at these residues highlight key biochemical interactions between the PDZ and L4 domains, with important implications for future CARD11 structure-function and clinical diagnostic studies.
Deletion of MOF in CD4 T cells results in mitochondrial dysfunction, Th17 polarization, and colonic inflammation.
Authors: Teixeira Lima Felipe , Goddard Yana , Wheeler Matthew , Qu Ganlin , Diodati Nikolas , Sordo-Vieira Luis , V. Lazarte Arantxa , A. Schaller Matthew , Mehrad Borna 
Published: November 2024

Abstract
Introduction: The enzyme, MOF (males absent of the first), is a histone acetyltransferase that regulates the transcription of nuclear and mitochondrial genes. The role of MOF in acquired immunity has not been established.
Hypothesis: Expression of MOF controls CD4 T cell phenotype by regulating cellular metabolism.
Materials, Methods, and Results: We generated mice selectively deficient in MOF in CD4 T cells (Moff/f CD4cre). After ex vivo stimulation with CD3 and CD28, MOF-deficient splenic CD4 T cells displayed lower proliferation rate and viability, associated with altered mitochondrial morphology by electron microscopy, impaired oxidative phosphorylation, and higher glycolytic capacity, lactate production, and higher ATP production. In vivo, Moff/f CD4cre mice developed spontaneous rectal prolapse and colonic cryptitis. Intraepithelial and lamina propria CD4 T cell from these animals were disproportionately activated and displayed a Th17 phenotype. Treating these animals with a mitochondrion-targeted antioxidant, mitotempo, for a month resulted in normalization of T cell ATP production and lactate production.
Conclusion: The histone acetyltransferase MOF moderates T cell activation and polarization by controlling mitochondrial respiration, protecting against the spontaneous development of colitis.
Defining the role of PSTPIP2 in neutrophil inflammatory function
Authors: Momjian Claire , Ratitong Bridget , Fernandes Sheryl , Jorth Peter , Cassel Suzanne , Sutterwala Fayyaz 
Published: November 2024

Abstract
Our lab and others have found that neutrophils isolated from Pstpip2cmo mice, that are deficient in proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), display a hyperinflammatory phenotype with increased IL-1b and ROS secretion following stimulation. The hyperactive neutrophil phenotype is critical to the spontaneous development of an autoinflammatory disorder called chronic multifocal osteomyelitis. However, there are gaps in our understanding of the underlying mechanism that leads to the observed phenotype in Pstpip2cmo mice which could further our understanding of neutrophil biology. We first infected WT and Pstpip2cmo mice with P. aeruginosa to elucidate the importance of PSTPIP2 in vivo. 24-hours post infection, Pstpip2cmo mice had significantly lower bacterial burden compared to WT mice. We found increased neutrophils and monocytes in the bronchial alveolar fluid (BAL) of infected Pstpip2cmo mice, and these neutrophils are lower in side-scatter measured by flow cytometry. Under homeostatic conditions, we observed higher numbers of neutrophils in the bone marrow, blood, and spleen of Pstpip2cmo mice, and more granulocyte progenitors. In addition to elevated cell numbers, we found increased IL-1β secretion NETosis compared to WT mice. Together these data suggest that PSTPIP2 plays a critical role in restraining neutrophilic inflammatory response to bacterial infection and affects granulopoiesis in naïve mice.
Regulation of B1 cells response to Francisella tularensis LPS, a type-2 T-independent antigen
Authors: Barbosa Carlos Henrique D , Mello Guilherme Afonso , Re Fabio 
Published: November 2024

Abstract
Francisella tularensis (Ft), a Gram-negative bacterium, posesses atypical LPS that does not activate TLR4 and is considered a type 2 T-independent antigen (TI-2 Ag). Our group has recently shown that immunization with Ft-LPS elicits the production of protective Ft-LPS-specific IgM (IgMFt) by B1 cells independently of MyD88, in a mechanism that is dependent of the IL-25-ILC2-IL-5 axis. Immunization, but not infection, generates peritoneum-resident memory B1 cells that differentiates into IgMFt plasmacells upon secondary challenge but does not boost the production of IgMFt.
TI antigens were believed to be unable to generate memory response or long-lived plasma cells. This view is being challenged by several studies. This work aims to elucidate the mechanisms driving the class switch and memory response of B1 cells to Ft-LPS.
Here we show that peritoneal memory B1 cells transferred into Rag-/- mice produce higher amounts of IgM compared to naïve B1 cells. Moreover, the transferred naïve B1 cells did not class switch to IgG3/IgG2b upon immunization, suggesting involvement of FDCs and/or T cells. The presence of IgGFt inhibited the IgMFt response. Complement depletion in WT mice also inhibited class switch to IgGFt but did not affect IgMFt production and resulted in better boost effect. Our results might shed light on new strategies to improve vaccination against TI-2 Ags and on involvement of germinal center and complement proteins for the activation of B1 cells
Dietary and circadian cues regulate gut barrier integrity by synchronizing epithelial cell-macrophage crosstalk
Authors: Dende Chaitanya , Srinivasan Tarun , Propheter Daniel , Quinn Gabriella , Li Yun , Ruhn Kelly , Pendse Mihir , Brooks John , Maples Robert , Raj Prithvi , Pfeiffer Julie , Blander Julie , Lira Sergio , Hooper Lora 
Published: November 2024

Abstract
Small intestinal epithelial cells (sIECs) form a critical barrier essential for nutrient absorption and host defense. sIECs undergo continuous renewal to maintain their integrity. Phagocytes, such as macrophages, facilitate the clearance of apoptotic cells that enter subepithelial tissues. However, little is known about how sIEC-phagocyte crosstalk maintains barrier integrity. Our study reveals synchrony between sIEC death and the accumulation of a specific subset of macrophages engaged in engulfing dying cells. Notably, we observed a diurnal rhythmicity in the death of sIECs during homeostasis. A fraction of these dying cells enter the subepithelial space, where Tim4+ CD4+ macrophages rhythmically eliminate them. Our findings demonstrate that Tim4+ CD4+ macrophage maintenance depends on dietary vitamin A via macrophage-intrinsic retinoic acid receptor (RAR) signaling. We show that rhythmic Tim4+ CD4+ macrophage accumulation relies on light cues and the macrophage circadian clock. Impaired macrophage RAR signaling disrupts the clearance of dying sIECs, leading to compromised gut barrier integrity and increased bacterial translocation to distant organs. Our work reveals an intricate relationship between sIECs and phagocytes and identifies a crucial role for Tim4+ CD4+ macrophages in maintaining intestinal barrier integrity. Understanding these dynamics could suggest new strategies for maintaining intestinal barrier function during infection and inflammatory disease.