bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nedd4-family interacting proteins activate Nedd4-family E3 ubiquitin ligases in T cells to limit pathogenic inflammation. (P1382)
Authors: O'Leary Claire , Riling Chris , Oliver Paula 
Published: January 2023

Abstract
E3 ubiquitin ligases tune signaling pathways in activated T cells to regulate differentiation and cytokine production. Nedd4-family ligases have distinct roles in these processes. These ligases often function with adaptors, which have been shown to aid binding to substrates and/or E2 enzymes. We have found that Nedd4-family interacting protein 1 (Ndfip1) and Ndfip2 promote Nedd4-family E3 ligase catalytic activity by a new mechanism. Our in vitro studies also indicate that Ndfip1 and Ndfip2 have overlapping function. In vivo, Ndfip1 negatively regulates T cell activation and TH2 cytokine production. The in vivo function of Ndfip2 is unknown; therefore, we generated Ndfip2-/- mice. Our studies revealed that, unlike Ndfip1, Ndfip2 is not a prominent negative regulator of T cell activation or TH2 polarization. However, loss of Ndfip2 exacerbated the inflammatory Ndfip1-/- phenotype. This suggested that, like Ndfip1, Ndfip2 limits inflammation. Supporting this, using a T cell transfer model of colitis, we found that transfer of doubly deficient T cells caused more severe disease compared to transfer of Ndfip1-/- T cells. These data indicate that Ndfip2 negatively regulates effector cell function following activation. We have now tested the effects of Ndfip2 deficiency on distinct cellular subsets in models of both acute and chronic inflammatory bowel disease. These studies suggest that Ndfip1 and Ndfip2 are pleiotropic modulators of inflammation.
Replacement of Sϵ with Sμ reveals novel mechanistic insights into rearrangements of the IgH locus and cMyc chromosomal translocations (P1400)
Authors: Senger Kate , Misaghi Shahram , Qu Yan , Sai Tao , Hamidzadeh Kajal , Sun Yonglian , Nguyen Allen , Zhou Meijuan , Yan Donghong , Lin Zhonghua , Soukup Tim , Lorenzo Maria , Garris Christopher , Sebrell Andrew , Xu Min , Caplazi Patrick , Roose-Girma Merone , DeForge Laura , Warming Soren , Lee Wyne , Balazs Mercedesz , Dixit Vishva , Zarrin Ali 
Published: January 2023

Abstract
Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation induced cytidine deaminase (AID). We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest switch, Sϵ, is replaced with prominent recombination hotspot Sμ (SμKI). Inserted Sμ out-competed both endogenous Sγ1 and Sϵ to produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. Intra-switch recombination (ISR) within endogenous Sμ is also diminished in SμKI mice, although the insertion itself exhibits no ISR. Furthermore, reciprocal translocations between c-myc and endogenous Sμ are dramatically lower in SμKI mice, suggesting the knocked-in switch sequesters critical factors. We propose competition among S regions influences CSR, ISR, and chromosomal translocation.
Human blood-derived raw material: enabling controlled, consistent collection (P4415)
Authors: Ramos Thomas , Busby William , McPherson Gaytha , Burger Scott 
Published: January 2023

Abstract
Human cells are critical raw materials for manufacturing cell therapy products, but often introduce significant variability. Rigorous operational controls and quality systems, however, enable optimal collection of high-quality, consistent cellular material. HemaCare, a long-standing supplier of human-derived blood components, controls apheresis procedures and collection sites under a formal quality system, with GMP-compliant, validated procedures and equipment, and GTP-compliant donor screening and tracking. HemaCare performed 69,658 cellular apheresis collections in the last five years, including patient and normal-donor MNCs, G-CSF-mobilized PBPCs and plateletpheresis products, for use in research, clinical trials, and commercial products. Expanded capabilities include bone marrow, umbilical cord blood, and cord tissue collection, immunomagnetic selection, cryopreservation, and analysis by flow cytometry. HemaCare unmobilized apheresis products showed consistently high MNC purity, with 93.8% of products containing ≥75% MNC, and an average of 85.2% MNC ± 6.6% (mean ± 1 SD). Red blood cell contamination was low, with hematocrit averaging 1.8% ± 0.8%. Approximately 85% of HemaCare donors have donated apheresis products 5 or more times, and this repeat-donor pool also contributes to product consistency, as MNC content of individual donor apheresis products had an average coefficient of variation of 3.5%, compared to a CV of 7.7% for all apheresis products.
Evaluation of the adjuvant properties of chitosan-poly vinyl pyrrolidone nanoparticles (P4423)
Authors: Izaguirre Irma , Tenorio Aldo , Altuzar Victor , Mendoza Claudia , Vivanco Hector 
Published: January 2023

Abstract
Polymeric Nanoparticles (NPs) have recently been shown to possess significant potential as drug or antigen delivery systems. In addition to the carrier function, the adjuvant capacity is also an important and desirable property in the context of vaccination. Here, we evaluated the adjuvant properties for a new NPs formulation based on a two-polymer combination: CS-PVP. The NPs were synthesized by ionic gelation and characterized by TEM, DLS and FTIR. Moreover, biological properties of NPs were addressed in vitro by studying their effect on cell viability and Dendritic Cells (DCs) maturation. 5 x105 bone marrow DCs were stimulated with CS-PVP NPs at different concentrations for 24 hours. At the end of the time, we analyzed the surface expression for costimulatory molecules by flow cytometry. Our results showed an enhanced surface expression of CD40, CD80, CD86 and MHC II on NPs stimulated-DCs at two different concentrations: 250 y 500 μg/mL, confirming the maturation phenotype. Using Human Monocyte-derived Dendritic Cells, we evaluated by ELISA the production of TNF-α and IL-6. 2 x105 Human DCs were stimulated for 24 hours with 300 μg/mL of CS-PVP NPs. Intermediate levels of both TNF-α (260 pg/ml) and IL-6 (75 pg/mL) were detected comparing against LPS. These results clearly indicate that CS-PVP NPs formulation active both murine and human DCs, opening the possibility for using such as a potential adjuvant in vaccination.
In vivo PPAR-gamma activation inhibits MyD88 expression to protect mice from polymicrobial sepsis. (P4425)
Authors: Ferreira Ana , Sisti Flavia , Filgueiras Luciano , Wang Sue , Wang Julia , Cunha Fernando , Alves-Filho Jose , Serezani C. Henrique 
Published: January 2023

Abstract
Sepsis is a disorder characterized by systemic inflammatory response caused by an infectious agent and may progress to multiple organ dysfunction and death. Overwhelming inflammatory response is believed to underlie tissue damage and preceding multiple organ disfunction syndrome. Thus, therapeutic strategies to inhibit excessive inflammation in sepsis is needed. The nuclear receptor peroxisossome proliferating factor (PPAR)-g exhibit anti-inflammatory properties in different immune cells. We hypothesize that prolonged PPAR-g activation decreases systemic inflammatory responses, improving sepsis outcome. We employed a model of polymicrobial sepsis (cecum and ligation puncture-CLP) to evaluate whether prolonged PPAR-g activation inhibits inflammation and improves animal survival. PPAR-g mRNA expression is increased in both peritoneal and lung cells at 6 h after CLP. Our data show that the PPAR-g agonist pioglitazone treatment for 18 h enhances bacterial clearance in the peritoneal cavity; which was associated with enhanced neutrophil migration to the infectious focus. These data correlates with lower expression of the TLR adaptor MyD88 in pioglitazone in vivo and peritoneal macrophages in vitro. Together, our findings suggest that PPAR-g activation impairs TLR responses, by inhibiting expression of MyD88 and preventing overwhelming inflammatory response.
CD56negCD16+ NK cells are mature NK cells generated from CD56+CD16+ NK cells during HIV-1 infection (P4438)
Authors: York Vanessa , Milush Jeffrey , López-Vergès Sandra , Deeks Steven , Martin Jeffrey , Hecht Frederick , Lanier Lewis , Nixon Douglas 
Published: January 2023

Abstract
A subset of CD3negCD56negCD16+ Natural Killer (NK) cells is highly expanded during chronic HIV-1 infection, and the role of this subset in HIV-1 pathogenesis remains unclear. The lack of NK cell lineage-specific markers has complicated the study of minor NK cell subpopulations. Using CD7 as an additional NK cell marker, we found that CD3negCD56negCD16+ cells are a mixed population of CD7+ NK cells and CD7neg myeloid cells. CD7+CD56negCD16+ NK cells are significantly expanded in HIV-1 infection. CD7+CD56negCD16+ NK cells are mature and express KIRs, NKG2A, NKG2C, and natural cytotoxicity receptors similar to CD7+CD56+CD16+ cells. CD7+CD56neg NK cells in healthy donors produced minimal IFNg following K562 target cell or IL-12 plus IL-18 stimulation; however, they did degranulate in response to K562 cells similar to CD7+CD56+ NK cells. HIV-1 infection resulted in reduced IFNg secretion following K562 or cytokine stimulation by both NK cell subsets compared to healthy donors. Decreased granzyme B and perforin expression and increased expression of CD107a in the absence of stimulation, particularly in HIV-1-infected subjects, suggests that CD7+CD56negCD16+ NK cells may have recently engaged target cells. CD7+CD56negCD16+ NK cells have increased expression of the aging-related cyclin-dependent kinase inhibitors p16/INK4a and p21/Waf1. Taken together, CD7+CD56negCD16+ NK cells appear to be mature NK cells generated from chronic target engagement.
Effect of Clonorchis sinensis and N-nitrosodimethylamine on host cell proliferation and modulation of cancer related moleclues (P1356)
Authors: Kim Eun-min , Wi Hae Joo , Bae Young Mee 
Published: January 2023

Abstract
Clonorchis sinensis (C. sinensis) infection is known to cause cholangiocarcinomas in humans, but the mechanism is not clearly understood. In order to verify the role of excretory/secretory products (ESP) from C. sinensis in cell proliferation and the modulation of cancer related molecules, HEK293T cells were cultured with ESP and/or NDMA (N-nitrosodimethylamine), and then the mode of cell cycle and the expression of proteins were analyzed by using FACS, western blotting and confocal analysis. When the ESP of C. sinensis and NDMA were treated, the degree of cell proliferation was upregulated and the proportion of G2/M phase cells were increased by 25%-30% as compared with that of control. The expression pattern of the cell cycle proteins was also changed when both ESP and NDMA were added. Theses indicate that ESP from C. sinensis and NDMA synergistically affected the regulation of cell cycle proteins. In addition, the levels of Cox-2 and connexin 43 (Cx43) which is known as cancer related gap junction protein were upregulated by the addition of ESP and NDMA in vivo. In addition, the level of another gap junction protein, Cx32, which is also known as cancer inhibitor was down- regulated. Our results suggest that exposure to C. sinensis and a small amount of NDMA promote uncontrolled cellular proliferation of the bile duct epithelial cells and the upregulation of cancer related intracellular molecules.
GATA-3 regulation and stability in T helper type 2 cells (P1310)
Authors: Pellet Elsa , Peine Michael , Flossdorf Michael , Löhning Max , Höfer Thomas 
Published: January 2023

Abstract
GATA-3 and T-bet, the key transcription factors of T helper type 1 and type 2 cells, respectively, are specifically upregulated during differentiation of naive Th cells in response to antigen and cytokine signals. GATA-3 expression increases in response to IL-4/STAT6 signaling, whereas T-bet is induced by the IL-12/STAT4 and IFN-y/STAT1 pathways. GATA-3 and T-bet subsequently induce lineage-specific gene expression. Here, we study the regulation of GATA-3 in Th2 cells as well as the interactions between the Th1 and Th2 signaling pathways using time-resolved dose-response experiments and mathematical modeling. We measured the dose response of GATA-3, T-bet and pSTAT1/4/6 to IL-4 and IL-12 in vitro during differentiation and fitted mathematical models to the data. GATA-3 expression was graded and depended on the cytokines’ concentrations. The activation of STAT6 downstream of IL-4 and STAT4 downstream of IL-12 was stronger and longer-lasting with higher cytokine concentrations. We discovered several cross-regulatory mechanisms between the Th1 and Th2 pathways. Furthermore, the different amounts of GATA-3 reached in vitro were stably maintained in vivo for 30 days after transfer into naive mice, suggesting a cell-intrinsic mechanism allowing quantitative memory of GATA-3 expression in resting Th2 cells. Our results provide insight into the molecular mechanisms that underlie the induction and maintenance of Th2 cell differentiation.
Pulmonary hepcidin protects against acute lung injury (P3345)
Authors: Chen QiXing , Song ShengWen , Fang XiangMing 
Published: January 2023

Abstract
Hepcidin is a small cysteine-rich cationic peptide mainly produced by the liver, and is a principle iron regulatory hormone. Hepcidin could modulate acute inflammatory response via interaction with its receptor ferroportin. Recent studies showed that hepcidin was also expressed in tracheal epithelial cells. However, the role of this epithelia-derived hepcidin in lung inflammation remained unknown. Adenovirus-mediated hepcidin specific shRNA and a relative negative control were constructed and were administrated to mice intratracheally. Ten days later, the mice were subjected to cecal ligation and puncture to induce acute lung injury (ALI). The severity of lung injury was scored 24 hours after onset of ALI. The bacterial colony counts, white blood cell counts and total protein content in bronchoalveolar lavage fluid were also measured. Furthermore, the protein levels of ferroportin were analyzed using immunobloting. Adenovirus-mediated hepcidin specific shRNA significantly suppressed tracheal hepcidin levels ten days after administration. Interference of this epithelia-derived hepcidin increased bacterial proliferation, white blood cell counts and total protein content in bronchoalveolar lavage fluid, and thus exacerbated lung injury. However, the ferroportin levels were comparable between the two groups. Tracheal epithelia-derived hepcidin plays an important role in protecting against acute lung injury, which might function via a ferroportin-independent mechanism.
New fluorophore for multiplexing with violet laser excitation (P3289)
Authors: Anderson April , Low Quentin , Bone Bradley , Zhou Wenjun , Dubbels Bradley , Bradford Jolene , Gee Kyle 
Published: January 2023

Abstract
Flow cytometry instruments with solid state violet laser diodes are becoming more common due to their small size, low power requirements, cost, and reliability. The use of violet-excited fluorochromes in multiparametric flow cytometry has been essential in the expansion of multicolor flow cytometry, enabling greater numbers of markers to be detected in one sample. We have developed a novel violet-excited dye, Pacific Green™ dye, which has an excitation/emission maximum of 411 and 500 nm, respectively. This development addresses the need to transfer well-resolved markers off the Blue 488 nm and Red 635 nm excitation lines and onto the Violet 405 nm excitation line, thus enabling the detection of other markers with the 488 nm and 635 nm lasers. Pacific Green™ dye can be used for three color immunophenotyping using violet laser excitation with Pacific Blue™ and Pacific Orange ™ dyes with minimal compensation without 488 nm excitation. Data is shown for human lymphocytes and mouse splenocytes stained with Pacific Green™ direct conjugates. Six color immunophenotyping data is shown using direct conjugates of Pacific Green™, Alexa Fluor® 488, phycoerythrin, PE-Cy®7, Pacific Blue™ and Pacific Orange™ conjugates. Pacific Green™ dye can be used with the far red emitting QDot®605, 655, and 705 conjugates for higher multiplexing with violet laser excitation. Pacific Green™ dye can be paired with fixable blue or yellow-emitting viability stain with violet excitation to exclude dead cells.
Preservations in anhydrous sodium chloride reduces keratinocytes alloantigenicty. (P2223)
Authors: Domaszewska-Szostek Anna , Moscicka-Wesolowska Maria , Szymanska Beata , Olszewski Waldemar Lech 
Published: January 2023

Abstract
A large amount of human keratinocytes (KCs) is needed to cover burned skin areas. There is a need to develop an efficient method of long term preservation of these cells. We checked whether KCs isolated from skin preserved in anhydrous sodium chloride survive and can be successfully transplanted. Fragments of normal human skin were preserved in sodium chloride. After 7 or 30 days preservation they were rehydrated, the epidermis was separated and KCs were isolated. Subsequently, the cells were cultured for 7 or 21 days. Viability of cultured KCs was tested by the Viability/Cytotoxicity Test. KCs were transplanted to the dorsum of SCID mice. Histopathological evaluation revealed that the morphology of KCs was totally preserved. KCs from all epidermal layers could be identified. All grafts were taken by the recipients. Three weeks after transplantation, keratinocytes synthesized keratins 10 and 16, and expressed antigens specific for stem and transient amplifying cells, as well as markers of proliferation. Cells isolated from transplants and cultured in vitro remained viable and produced various enzymes. All graft cells remained HLA I+. Taken together, keratinocytes isolated from human skin and preserved in anhydric sodium chloride present very low alloantigenicity can be successfully transplanted to SCID mice.
Aberrant glycosylation of plasma proteins in HIV-infected patients (P6361)
Authors: Ruhaak L. Renee , Williams Cynthia , Fenton Anne , Nagy Lauren , Hong Qiuting , Dandekar Satya , Lebrilla Carlito 
Published: January 2023

Abstract
Background: Glycosylation is one of the most abundant post-translational modifications, and is involved in protein structure formation and protein-protein interactions. Aberrant glycosylation profiles are often observed on plasma proteins from patients with inflammatory diseases. Increased levels of non-galactosylated glycans have been reported on serum IgGs of HIV-infected patients. We investigated the effects of HIV infection on protein glycosylation by N-glycomic profiling of plasma and plasma IgG. Methods: We obtained plasma samples of 22 HIV infected patients (11 therapy-naïve, 11 receiving anti-retroviral therapy) and 11 HIV-negative controls. First, a nano-LC-TOF-MS strategy was employed for the evaluation of plasma N-glycan profiles in each of the samples. Then, a UPLC-QQQ-MS method was used to evaluate the IgG specific glycosylation patterns. N-glycan peak integrals were used for biostatistical analysis. Results and conclusion: Several neutral, fucosylated and sialylated glycan compositions as well as high mannose type glycans in plasma samples were significantly altered in therapy-naïve HIV infected patients compared to controls. Moreover, galactose-deficient glycans were increased on the IgG in these patients, independent of IgG subclass. Interestingly, these effects were largely reduced in HIV infected patients receiving therapy. These results suggest an important role for protein glycosylation in immune dysfunction that is driven by active HIV infection.
Comparative lipidomics reveals a global sampling of major cellular membrane lipids by human CD1 proteins (P5006)
Authors: Huang Shouxiong , Cheng Tan-Yun , Altman John , Moody D. Branch 
Published: January 2023

Abstract
Human CD1 proteins have differentially shaped grooves and present bacterial or self lipid antigens to T cells, but it is unknown whether the pool of endogenous lipids are differently sampled by each type of CD1 proteins. We used recently established lipidomic approach to analyze the molecules extracted from human CD1a, CD1b, CD1c, and CD1d proteins. In the study, we demonstrated that this highly sensitive and accurate lipidomic approach detected more than one thousand diverse molecules defined by accurate mass retention time values, which represented hundreds of lipid species associated with CD1 proteins. More than fifty percent of these molecules were shared by all four types of CD1 proteins and additional thirty percent shared by two or three CD1 isoforms. Further mass spectrometry analyses of collision-induced dissociation showed that the molecules eluted from all four types of CD1 proteins mainly are lipids from subcellular membranes, including phospholipids of conventional, ether-linked, and lyso forms and sphingolipids. Surprisingly, CD1 isoforms with bigger grooves preferred to lipids with shorter chains, which can be explained by small spacer or scaffold lipids that bind together with antigens. Whereas previous studies argued that one lipid molecule blocks CD1 grooves, analogous to MHC class II-associated CLIP peptide, we conclude that CD1 proteins broadly sample cellular lipids and two small lipids are able to be adapted into a big groove.
MHC II-peptide complex conformation constrained by interactions throughout the peptide binding groove determines HLA-DM susceptibility (P5014)
Authors: Yin Liusong , Trenh Peter , Stern Lawrence 
Published: January 2023

Abstract
HLA-DM (DM) mediates the exchange of peptides loaded onto MHC II. However, the determinants of DM-mediated peptide release remain unclear and controversial. In this study, we synthesized a series of peptides derived from HLA-A2104-117 and measured their kinetic stabilities when bound to HLA-DR1 (DR1) in the absence or presence of DM. As expected from previous work, we found that peptides with non-optimal pocket 1 residues were highly DM susceptible. Surprisingly we found that substitution of the pocket 9 residue can counteract the low binding affinity, low kinetic stability and high DM-susceptibility of peptides containing non-optimal pocket 1 residues. Surface Plasmon Resonance demonstrated that DM bound well to DR1 loaded with a peptide variant with pocket 1 alanine (A1), while no binding was observed for a rescue variant with leucine at pocket 9 (A1L9) or for the wild-type peptide (W1Q9). We determined the crystal structure of DR1-A1L9 to 2.3Å resolution, confirming the expected binding frame with alanine at pocket 1. DR1-A1 appeared to adopt a different conformation which can be better detected and edited by DM, as evidenced by the sodium dodecyl sulfate-resistance and recognition by a conformation-specific monoclonal antibody UL-5A1. Together with the results that multiple substitutions influence DM-susceptibility, our data suggest that conformation of MHC II-peptide complex constrained by interactions throughout the peptide binding site determines DM-susceptibility.