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Pitcher, Carol, Stefan Höning, Anja Fingerhut, Katherine Bowers, and Mark Marsh. "Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation." Molecular Biology of the Cell 10, no. 3 (March 1999): 677–91. http://dx.doi.org/10.1091/mbc.10.3.677.
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Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56 lck , undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56 lck , we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.
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La Russa, Raffaele, Aniello Maiese, Rocco Valerio Viola, Alessandra De Matteis, Enrica Pinchi, Paola Frati, and Vittorio Fineschi. "Searching for highly sensitive and specific biomarkers for sepsis: State-of-the-art in post-mortem diagnosis of sepsis through immunohistochemical analysis." International Journal of Immunopathology and Pharmacology 33 (January 2019): 205873841985522. http://dx.doi.org/10.1177/2058738419855226.
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The autoptical observations commonly ascribed to sepsis deal with unspecific general and local signs of inflammation or ischemia, such as myocardial inflammation, pulmonary edema and infiltration, cerebral swallowing, and tubular necrosis in the kidney. In the two last decades, some studies have been carried out to implement immunohistochemical markers for post-mortem diagnosis. All of these target molecules are specifically up-regulated or down-regulated during systemic inflammatory responses, especially for infective causes. Among these, we found some antigens expressed on leukocyte surfaces (very late antigen-4 (VLA-4), cluster differentiation-15 (CD15)), enzyme contained in neutrophils granules (lysozyme (LZ), lactoferrin (LF)), endothelial markers and junctions (E-selectin, vascular endothelial cadherin (VE-cadherin)), and soluble factors (vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNFα), procalcitonin (PCT), soluble triggering receptor expressed on myeloid cells-1 (s-TREM-1)). All of these showed potential reliability in differentiating sepsis cases from controls. Further studies are needed to provide a concrete validation for a combination of markers on specific organ samples in order to reach a post-mortem diagnosis of sepsis also in the absence of clinical records.
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Rudqvist, Nils, Claire Lhuillier, Maud Charpentier, Erik Wennerberg, Sheila Spada, Caroline Sheridan, Xi Kathy Zhou, et al. "465 Radiotherapy and CTLA-4 blockade expand anti-tumor T cells differentiation states and cooperate with CD40 agonist to induce tumor rejection." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A494—A495. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0465.
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BackgroundRadiotherapy (RT) in combination with CTLA-4 inhibition (CTLA4i) can expand and activate T-cells to reject tumors in both mice and some patients with tumors unresponsive to CTLA4i alone.1 2 However, only a subset of patients achieves long-term control of metastatic disease. Similar responses to RT+CTLA4i are seen in the 4T1 mouse model of triple negative breast cancer (TNBC), making it an ideal model to interrogate the interaction between RT and CTLA4i, and identify barriers to its effectiveness.MethodsMice were inoculated in one or both flanks with 4T1 cells. In some experiments one tumor was removed for analysis before start of treatment with RT (3 × 8 Gy) and/or anti-CTLA-4 antibody (9H10, 3 × 200 ?g i.p.). The intratumoral T cell response was assessed using bulk and single cell RNA/TCR sequencing. The METABRIC dataset3 was used to associate gene expression signatures with patient survival. In some experiments, RT+CTLA4i was combined with PD-1, LAG-3, or CD40 Abs.ResultsRT, alone and with CTLA4i, increased the TCR repertoire clonality and activated T cells density in the tumors (figure 1A-G). In untreated tumors, Gzmb+Prf1+Lag3+Pd1+Cd8+ T cells (cluster 0) were most common. CTLA4i ‘unlocked’ Ifng+Cd40lg+ Cd4+ T cells (cluster 2) while RT favored expansion/persistence of Cd8+ T cell clusters. In tumors of mice treated with RT+CTLA4i activated Treg cells (cluster 1) were decreased and Ifng+Cd40lg+Cd4+ T cells (cluster 2) increased. Relatively among CD8+ T cells, Ifng+Tnf+Cd8+ (cluster 4) was expanded at the expense of cluster 0 (figure 2A-F). Gene signatures defining clusters 0, 2, and 4 were associated with improved survival in the METABRIC TNBC patient cohort using a multivariate model (figure 2G-H). In mice, AH1-tumor antigen-specific CD8+ T cells occupied different transcriptional states, with a shift to cluster 4 in mice treated with RT+CTLA4i (figure 2I), suggesting that multiple functional T cell states are required for tumor rejection. Based on the T cell phenotypes expanded by RT+CTLA4i, antibodies to PD-1, LAG-3, and CD40 were tested for the ability to enhance RT+CTLA4i therapy. Only CD40-agonist improved significantly tumor control (figure 3A-B).Abstract 465 Figure 1RT, alone and with CTLA4i, increased the TCR repertoire clonality and density of activated T cells in the tumors. (A) Design of the experiment enabling collection of pre- and post-treatment (pre-tx and post-tx) 4T1 tumor tissue that was analyzed using RNA- and TCR-sequencing. (B) Tumor growth curves. Statistical significance in tumor volume growth between groups was determined using 2-way repeated measures ANOVA between day 15–21 and t-test at day 21. (C) Shannon clonality of paired pre- and post-tx TCR repertoires. Pairwise and paired t-tests were used to evaluate statistical significance of differences between and within groups, respectively. (D) RNA-seq based gene expression heatmap of selected canonical T cell markers in post-tx tumors. (E) Linear regression between Cd3e and Cd4 or Cd8 gene expression in post-tx tumors. R2 and p indicate R-square and p-value for the models, respectively (F) Ingenuity Pathway Analysis Canonical Pathway and (G) Upstream Regulation analysis. Z-scores indicate predicted activation (> 2) or inhibition (< -2) of pathways and upstream regulators. (all panels) *, **, and ***, and #, ## and ### indicate p-values of pairwise and paired statistical tests, respectively. Tukey’s and Holm’s method for adjusting p-values corrected for multiple comparison was used for the ANOVA and t-tests, respectively. (Abbreviations) tx, treatment; RT, radiation therapy; CTLA4, CTLA-4 Ab therapy; TCR, T cell receptorAbstract 465 Figure 2RT+CTLA-4i increased tumor infiltration by Gzmb+Prf1+Lag3+Pd1+Cd8+, Ifng+Cd40lg+Cd4+, and Ifng+Tnf+Cd8+ T cells in 4T1 tumors. (A) Design of experiment enabling single cell analysis of T cells infiltrating 4T1 tumors. (B) Based on gene expression levels, the T cells were divided into 17 clusters (indicated by colors) and visualized in 2D using UMAP dimensionality reduction algorithm. (C) Gene expression levels of selected high-level T cell markers. (D) Table with main phenotype, key genes representative for each cluster, and the distribution of T cells from each condition falling into the different clusters. (E) Proportion of Cd4+ and Cd8+ T cells for the different treatment groups. (F) The expression of cluster-specific gene signatures in bulk 4T1 tumors for clusters 0, 2, and 4. (G) Survival curves and (H) multivariate analysis of the association between survival and enrichment of the gene signatures of clusters 0, 2, and 4. (I) The positioning of the all AH1-dextramer+ Cd8+T cell clones within the UMAP plot. Color annotate cluster. (Abbreviations) tx, treatment; RT, radiation therapy; Untr., untreated; CTLA4, CTLA-4 Ab; TCR CDR3, T cell receptor complementary determining region 3; UMAP, Uniform Manifold Approximation and Projection for dimension reduction; AH1, tumor antigen in 4T1 tumors; SPSYVYHQF peptide derived from gp70 and restricted to H2-LdAbstract 465 Figure 3Agonistic CD40 treatment improves RT+CTLA-4 therapy. Individual tumor growth curves for untreated, RT+CTLA-4, or RT+CTLA-4+CD40 treated mice. Color annotate group. *, **, ***, and **** indicate p-values < 0.05, 0.01, 0.001, and 0.0001, respectively, calculated using a linear mixed-effects model. (A) and (B) represent two individual experiments. (Abbreviations) RT, radiation therapy; CTLA4, CTLA-4 Ab; CD40, anti-CD40 Ab; mm3, cubic millimeter; d, daysConclusionsAltogether, these results revealed that RT and CTLA4i have complementary effects and besides driving T cells into tumors shape CD4 and CD8 T cell functional differentiation towards subsets that are associated with improved survival in patients. Unexpectedly, inhibition of checkpoint receptors expressed by a large CD8 T cells cluster did not further improve responses to RT+CTLA4i, whereas agonistic CD40 therapy did, suggesting new therapeutic strategies.AcknowledgementsGrant support: R01CA198533ReferencesK, Ferrari de Andrade L, Wucherpfennig KW, Heguy A, Imai N, Gnjatic S, Emerson RO, Zhou XK, Zhang T, Chachoua A, Demaria S. Radiotherapy induces responses of lung cancer to CTLA-4 blockade. Nat Med 2018;24(12):1845–51.Rudqvist NP, Pilones KA, Lhuillier C, Wennerberg E, Sidhom JW, Emerson RO, Robins HS, Schneck J, Formenti SC, Demaria S. Radiotherapy and CTLA-4 Blockade Shape the TCR Repertoire of Tumor-Infiltrating T Cells. Cancer Immunol Res 2018;6(2):139–50.Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, Speed D, Lynch AG, Samarajiwa S, Yuan Y, Graf S, Ha G, Haffari G, Bashashati A, Russell R, McKinney S, Group M, Langerod A, Green A, Provenzano E, Wishart G, Pinder S, Watson P, Markowetz F, Murphy L, Ellis I, Purushotham A, Borresen-Dale AL, Brenton JD, Tavare S, Caldas C, Aparicio S. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 2012;486(7403):346–52.
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Peri, Francesco, Valentina Calabrese, Matteo Piazza, and Roberto Cighetti. "Synthetic molecules and functionalized nanoparticles targeting the LPS-TLR4 signaling: A new generation of immunotherapeutics." Pure and Applied Chemistry 84, no. 1 (December 8, 2011): 97–106. http://dx.doi.org/10.1351/pac-con-11-10-35.
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Toll-like receptor 4 (TLR4), the receptor of bacterial endotoxins in mammalians, plays a pivotal role in the induction of innate immunity and inflammation. TLR4 activation by bacterial lipopolysaccharide (LPS) is achieved by the coordinate and sequential action of three other proteins, the lipopolysaccharide binding protein (LBP), the cluster differentiation antigen CD14, and the myeloid differentiation protein (MD-2) receptors, that bind LPS and present it in a monomeric form to TLR4 by forming the activated [TLR4·MD-2·LPS]2 complex. Small molecules and nanoparticles active in modulating the TLR4 signal by targeting directly the MD-2·TLR4 complex or by interfering in other points of the TLR4 signaling are presented in this paper. These compounds have great pharmacological interest as vaccine adjuvants, immunotherapeutics, anti-sepsis, and anti-inflammatory agents.
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Muhammad Rashad, Jaffar Muhammad Baqir, and Ahmed Abdul jabbar Jaloob Aljanaby. "ROLE OF INTERLEUKIN-2, INTERLEUKIN-4 AND CLUSTER OF DIFFERENTIATION-22 AS AN IMMUNE MARKERS IN INDIVIDUALS INFECTED WITH Helicobacter pylori." Journal of Experimental Biology and Agricultural Sciences 9, no. 3 (June 25, 2021): 388–93. http://dx.doi.org/10.18006/2021.9(3).388.393.
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Helicobacter pylori is a gram-negative, intracellular, microaerophilic bacteria which causing Peptic ulcer. This bacterium can change its shape which helps the bacteria to survive in the host gastric microenvironment. The Peptic ulcer caused by this bacterium stimulates the humoral and cellular immune response in individuals. The current study was carried out to access the role of interleukin-2, interleukin-4, and cluster differentiation-22 as immune markers in the identification of H. pylori infection. The presence of H. pylori has been diagnosed by feces test (antigen rapid test). In this study, the presence of three immunological markers viz., IL-2, IL-4, and CD22 were measured in the serum of 60 individuals infected with H. pylori and 30 healthy individuals by the Enzyme-Linked Immune-sorbent Assay method. Results of this study indicated a significant increase (P-value=0.0307*) in the concentration of IL-2 (294.27ng/ml), IL-4(151.28ng/ml), and CD22 (492.73ng/ml) in the serum of individuals infected with H. pylori while these concentrations were reported 235.98ng/ml, 116.14ng/ml and 369.33ng/ml respectively in the healthy individuals. Results of the study can be concluded that H.pylori infection stimulates the Cellular and humoral immune response which resulted in the increased production of IL-2, IL-4, and CD22.
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Weber, E., D. Schmitter, H. Resch, J. A. Zarn, R. Waibel, M. Mabry, P. Huguenin, and R. A. Stahel. "Radiation Studies on B Cell Differentiation Marker CD24/SCLC Cluster-4 Antigen Expressing and Non-expressing Lung Cancer Cell Lines and Mouse Fibroblasts." International Journal of Radiation Biology 68, no. 2 (January 1995): 205–13. http://dx.doi.org/10.1080/09553009514551111.
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Lal, Sean, Rodney Lui, Lisa Nguyen, Peter Macdonald, Gareth Denyer, and Cristobal dos Remedios. "Increases in leukocyte cluster of differentiation antigen expression during cardiopulmonary bypass in patients undergoing heart transplantation (vol. 4, Issue 7, pp. 1918-1926)." PROTEOMICS 4, no. 11 (November 2004): 3660. http://dx.doi.org/10.1002/pmic.200490072.
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Solinas, Cinzia, Chunyan Gu-Trantien, and Karen Willard-Gallo. "The rationale behind targeting the ICOS-ICOS ligand costimulatory pathway in cancer immunotherapy." ESMO Open 5, no. 1 (January 2020): e000544. http://dx.doi.org/10.1136/esmoopen-2019-000544.
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Inducible T cell costimulator (ICOS, cluster of differentiation (CD278)) is an activating costimulatory immune checkpoint expressed on activated T cells. Its ligand, ICOSL is expressed on antigen-presenting cells and somatic cells, including tumour cells in the tumour microenvironment. ICOS and ICOSL expression is linked to the release of soluble factors (cytokines), induced by activation of the immune response. ICOS and ICOSL binding generates various activities among the diversity of T cell subpopulations, including T cell activation and effector functions and when sustained also suppressive activities mediated by regulatory T cells. This dual role in both antitumour and protumour activities makes targeting the ICOS/ICOSL pathway attractive for enhancement of antitumour immune responses. This review summarises the biological background and rationale for targeting ICOS/ICOSL in cancer together with an overview of the principal ongoing clinical trials that are testing it in combination with anti-cytotoxic T lymphocyte antigen-4 and anti-programmed cell death-1 or anti-programmed cell death ligand-1 based immune checkpoint blockade.
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Colden-Stanfield, Margaret. "Clustering of very late antigen-4 integrins modulates K+ currents to alter Ca2+-mediated monocyte function." American Journal of Physiology-Cell Physiology 283, no. 3 (September 1, 2002): C990—C1000. http://dx.doi.org/10.1152/ajpcell.00481.2001.
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Endothelial cell vascular cell adhesion molecule-1 (VCAM-1) activates adherent monocytes by clustering their very late antigen-4 (VLA-4) receptors, resulting in the modulation of the inwardly rectifying ( I ir) and delayed rectifying ( I dr) K+ currents, hyperpolarization of the cells, and enhanced Ca2+ influx (Colden-Stanfield M and Gallin EK. Am J Physiol Cell Physiol 275: C267–C277, 1998; Colden-Stanfield M and Scanlon M. Am J Physiol Cell Physiol 279: C488–C494, 2000). The present study was undertaken to test the hypothesis that monoclonal antibodies (MAbs) against VLA-4 (MAbVLA-4) mimic VCAM-1 to cluster VLA-4 integrins, which play a key role in signaling an increase in the secretion of the proinflammatory cytokine interleukin-8 (IL-8). Whole cell ionic currents and IL-8 secretion from THP-1 monocytes that were incubated on polystyrene, VCAM-1-immobilized MAbVLA-4 or an isotype-matched MAb against CD45 (MAbCD45) were measured. Clustering of VLA-4 integrins with a cross-linked MAbVLA-4, but not a monovalent MAbVLA-4, modulated the K+ currents in an identical manner to incubation of cells on VCAM-1. Similarly, cross-linked MAbVLA-4 or VCAM-1 augmented Ca2+-mediated IL-8 secretion from THP-1 monocytes and was completely abolished by exposure to CsCl, an I ir blocker. Thus VLA-4 integrin clustering by cross-linked MAbVLA-4 mimics VCAM-1/VLA-4 interactions sufficiently to be associated with events leading to monocyte differentiation, enhanced Ca2+-mediated macrophage function, and possibly atherosclerotic plaque formation.
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Harada, Satoshi, Takafumi Segawa, Shigeru Ehara, and Takahiro Sato. "Treatment of primary and metastatic tumors through cancer immunotherapy and abscopal effect by targeted antigen-capturing nanoparticles with programmed death-1 blockade." International Journal of PIXE 28, no. 03n04 (January 2018): 69–76. http://dx.doi.org/10.1142/s0129083518500158.
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Microcapsules that release antigen-capturing nanoparticles (AC-NPs) with macrophage inflammatory protein-3 alpha (MIP-3[Formula: see text]) and anti-programmed death-1 (PD-1) antibody are developed, and these microcapsules have the ability to enhance immunoresponses through cross-priming of cluster of differentiation 8+ (CD8+) T cells by dendritic cells (DCs) in vivo in BALB/c mice. Lipid protamine hyaluronic acid nanoparticles containing AC-NPs generated via nanoprecipitation of 4 mg/mL of polylactic-co-glycolic acid (PLGA), 1,000 ng/mL of MIP-3[Formula: see text] and 400 [Formula: see text]g of anti-PD-1 were mixed with 1 mL of 4.0% alginate and 3.0% of hyaluronate and then sprayed with 0.5 mM of ferrous chloride. These capsules were injected subcutaneously around LM17 tumor in the left hind legs of BALB/c mice. The tumors were exposed to a radiation dose of 10 or 20 Gy from 100 keV soft X-ray radiation. PLGA AC-NPs and MIP-3[Formula: see text] were released in response to the radiation dose. PLGA AC-NPs captured tumor-derived protein antigens are released by exposure to radiation, and these antigens were transported to DCs that were recruited and activated by MIP-3[Formula: see text], intensifying the DC-associated cross-priming of CD8+ T cells. These treatments resulted in increased antitumor effect and reduced metastasis by abscopal effect. Our targeted immunotherapy may lead to better tumor therapy.
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Oehler, Leopold, Otto Majdic, Winfried F. Pickl, Johannes Stöckl, Elisabeth Riedl, Johannes Drach, Klemens Rappersberger, Klaus Geissler, and Walter Knapp. "Neutrophil Granulocyte–committed Cells Can Be Driven to Acquire Dendritic Cell Characteristics." Journal of Experimental Medicine 187, no. 7 (April 6, 1998): 1019–28. http://dx.doi.org/10.1084/jem.187.7.1019.
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Polymorphonuclear granulocytes (PMNs) are thought to fulfill their role in host defense primarily via phagocytosis and release of cytotoxic compounds and to be inefficient in antigen presentation and stimulation of specific T cells. Dendritic cells (DCs), in contrast, are potent antigen-presenting cells with the unique capacity to initiate primary immune responses. We demonstrate here that highly purified lactoferrin-positive immediate precursors of end-stage neutrophilic PMN (PMNp) can be reverted in their functional maturation program and driven to acquire characteristic DC features. Upon culture with the cytokine combination granulocyte/macrophage colony-stimulating factor plus interleukin 4 plus tumor necrosis factor α, they develop DC morphology and acquire molecular features characteristic for DCs. These molecular changes include neo-expression of the DC-associated surface molecules cluster of differentiation (CD)1a, CD1b, CD1c, human leukocyte antigen (HLA)-DR, HLA-DQ, CD80, CD86, CD40, CD54, and CD5, and downregulation of CD15 and CD65s. Additional stimulation with CD40 ligand induces also expression of CD83 and upregulates CD80, CD86, and HLA-DR. The neutrophil-derived DCs are potent T cell stimulators in allogeneic, as well as autologous, mixed lymphocyte reactions (MLRs), whereas freshly isolated neutrophils are completely unable to do so. In addition, neutrophil-derived DCs are at least 10,000 times more efficient in presenting soluble antigen to autologous T cells when compared to freshly isolated monocytes. Also, in functional terms, these neutrophil-derived DCs thus closely resemble “classical” DC populations.
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Ashouri, Judith F., Lih-Yun Hsu, Steven Yu, Dmitry Rychkov, Yiling Chen, Debra A. Cheng, Marina Sirota, et al. "Reporters of TCR signaling identify arthritogenic T cells in murine and human autoimmune arthritis." Proceedings of the National Academy of Sciences 116, no. 37 (August 27, 2019): 18517–27. http://dx.doi.org/10.1073/pnas.1904271116.
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How pathogenic cluster of differentiation 4 (CD4) T cells in rheumatoid arthritis (RA) develop remains poorly understood. We used Nur77—a marker of T cell antigen receptor (TCR) signaling—to identify antigen-activated CD4 T cells in the SKG mouse model of autoimmune arthritis and in patients with RA. Using a fluorescent reporter of Nur77 expression in SKG mice, we found that higher levels of Nur77-eGFP in SKG CD4 T cells marked their autoreactivity, arthritogenic potential, and ability to more readily differentiate into interleukin-17 (IL-17)–producing cells. The T cells with increased autoreactivity, nonetheless had diminished ex vivo inducible TCR signaling, perhaps reflective of adaptive inhibitory mechanisms induced by chronic autoantigen exposure in vivo. The enhanced autoreactivity was associated with up-regulation of IL-6 cytokine signaling machinery, which might be attributable, in part, to a reduced amount of expression of suppressor of cytokine signaling 3 (SOCS3)—a key negative regulator of IL-6 signaling. As a result, the more autoreactive GFPhiCD4 T cells from SKGNur mice were hyperresponsive to IL-6 receptor signaling. Consistent with findings from SKGNur mice,SOCS3expression was similarly down-regulated in RA synovium. This suggests that despite impaired TCR signaling, autoreactive T cells exposed to chronic antigen stimulation exhibit heightened sensitivity to IL-6, which contributes to the arthritogenicity in SKG mice, and perhaps in patients with RA.
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Mayerhofer, Matthias, Karl J. Aichberger, Stefan Florian, Maria-Theresa Krauth, Martin Bilban, Harald Esterbauer, Oswald Wagner, et al. "c-kit D816V Provides a Strong Signal for Myelomastocytic Differentiation and Cluster Formation in Murine Ba/F3 Cells." Blood 104, no. 11 (November 16, 2004): 485. http://dx.doi.org/10.1182/blood.v104.11.485.485.
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Abstract The pathologic hallmark of systemic mastocytosis (SM) is differentiation and cluster formation of mast cells (MC) in hematopoietic tissues. The somatic c-kit mutation D816V is detectable in a majority of all SM patients independent of the proliferation-status of MC or subtype (indolent or aggressive) of disease. To investigate the role of c-kit D816V in the pathogenesis of SM, we established a Ba/F3 cell line with doxycycline-inducible expression of c-kit D816V. We found that c-kit D816V provides a strong signal for mast cell differentiation and cluster formation in Ba/F3 hematopoietic progenitor cells without enhancing their growth thereby resembling the clinical presentation of indolent SM (ISM). As assessed by gene chip analysis, induction of c-kit D816V resulted in expression of various differentiation antigens including mouse mast cell protease 5, mi transcription factor, histidine decarboxylase (HDC), secretory granule proteoglycan, IL-4 receptor, ICAM-1, and CD63 consistent with an early phase of mastopoiesis. By contrast, c-kit D816V did neither induce expression of granulo-monocytic antigens such as myeloperoxidase, IL-3 receptor, or GM-CSF receptor, nor expression of ‘late stage’ mast cell antigens such as FcεRI. The c-kit D816V-induced synthesis of histamine in Ba/F3 cells was confirmed by RIA. To examine the role of c-kit D816V in the pathogenesis of mastocytosis, we extended our analysis to bone marrow biopsy sections obtained from patients with ISM. In these experiments, the D816V-mutated form of c-kit was detected more frequently in micro-dissected tryptase-positive MC obtained from dense compact infiltrates (44.2%) than in diffusely spread MC in these patients (22.6%) (p<0.05). In summary, our data establish a role for c-kit D816V in differentiation and cluster formation of neoplastic (mast) cells. Additional genetic hits, apart from c-kit D816V, may be responsible for aggressive growth of MC in advanced MC neoplasms.
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Vijtiuk, N., K. Trutin-Ostovič, T. Balenovič, M. Popovič, and I. Valpotič. "Functional and phenotypic analyses of porcine gut immune cells immunized by oral administration of F4ac+nonenterotoxigenic Escherichia coli strains." Veterinární Medicína 47, No. 12 (March 30, 2012): 333–41. http://dx.doi.org/10.17221/5844-vetmed.
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The aim of this study was to determine the priming effect of experimentally inoculated non-ETEC strains (2407, 1466) on gastrointestinal mucosal lymphocytes. Five 4-week-old pigs per group were orally inoculated with either F4ac<sup>+</sup>(1466 or 2407) or F4– (1467) non-ETEC strains. The control pigs were given broth containing 1.2% sodium bicarbonate. At postinoculation Day 6 the pigs were killed, their gut lymphocytes were isolated, and their responsiveness was tested in vitro with F4 antigen, peptidoglycan monomer (PGM), pokeweed mitogen (PWM), phytohemagglutinin (PHA) and lipopolysaccharide (LPS). Additionally, the patterns of cluster of differentiation (CD) antigen expression on T and B cells in the single cell suspensions from JLP, IPP, and MLN were determined by flow cytometry using anti-swine CD-specific monoclonal antibodies. F4ac+ non-ETEC strain 2407 and, to a lesser extent 1466, activated lymphocytes from PP and MLN to respond better to common mitogens (PHA, PWM, LPS), purified fimbrial (F4ac) antigen or immunologic response modifier (PGM). An increase of CD2a<sup>+</sup>and CD8a<sup>+</sup>T cells in JLP, and species-specific SWC1<sup>+</sup>T cells in MLN (P < 0.05) was detected in 2407-treated pigs. In conclusion, inoculation with non-ETEC strain 2407 exhibited stimulatory properties to porcine gut immune cells, and thus, could be used in the vaccination programs to control the postweaning colibacillosis in pigs.
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Gupta, Sakshee, Bharti Malhotra, Jitendra Kumar Tiwari, Prabhu Dayal Khandelwal, and Rakesh Kumar Maheshwari. "Cluster of differentiation 4+ T-cell counts and human immunodeficiency virus-1 viral load in patients coinfected with hepatitis B virus and hepatitis C virus." Journal of Laboratory Physicians 10, no. 02 (April 2018): 162–67. http://dx.doi.org/10.4103/jlp.jlp_37_17.
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ABSTRACT BACKGROUND: Coinfections of human immunodeficiency virus (HIV) with hepatitis viruses may affect the progress of disease and response to therapy. OBJECTIVES: To study the incidence of hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfections in HIV–positive patients and their influence on HIV–1 viral load and cluster of differentiation 4+ (CD4+) T–cell counts. MATERIALS AND METHODS: This pilot study was done on 179 HIV–positive patients attending antiretroviral therapy (ART) centre. Their blood samples were tested for HIV-1 viral load, CD4+ T–cell counts, hepatitis B surface antigen, anti–HCV antibodies, HBV DNA and HCV RNA polymerase chain reaction. RESULTS: Among the 179 patients, 7.82% (14/179) were coinfected with HBV and 4.46% (8/179) with HCV. Median CD4+ T–cell count of HIV monoinfected patients was 200 cells/µl and viral load was 1.67 log10 copies/µl. Median CD4+ T–cell counts of 193 cells/µl for HBV (P = 0.230) and 197 cells/µl for HCV (P = 0.610) coinfected patients were similar to that of HIV monoinfected patients. Viral load was higher in both HBV and HCV infected patients but statistically significant only for HCV (P = 0.017). Increase in CD4+ T–cell counts and decrease in HIV–1 viral load in coinfected patients on 2 years of ART were lower than that in HIV monoinfected patients. CONCLUSION: HBV/HCV coinfected HIV patients had similar CD4+ T–cell counts as in HIV monoinfected patients, higher HIV viral load both in chemo–naive patients and in those on ART as compared to HIV monoinfected patients. However, this study needs to be done on a large scale to assess the impact of coinfection on CD4 count and HIV viral load with proper follow–up of patients every 6 months till at least 2 years.
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Ragab, Ehab, Usama Shaheen, Ammar Bader, Khaled Elokely, and Mohammed Ghoneim. "Computational Study and Biological Evaluation of Isolated Saponins from the Fruits of Gleditsia aquatica and Gleditsia caspica." Records of Natural Products 15, no. 2 (November 28, 2020): 142–47. http://dx.doi.org/10.25135/rnp.202.20.08.1764.
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Phytochemical study of the ethanolic extract of the fruits of Gleditsia aquatica and Gleditsia caspica resulted in the isolation of the triterpene saponins; aquaticasaponin A (1), aquaticasaponin B (2), caspicaoside L (3) and caspicaoside M (4). Compound (1) showed good activity against methicillin resistant Staphylococcus aureus (MRSA) (IC50 =16.3 μg/mL) and Staphylococcus aureus (non-MRSA), (IC50 =12.2 μg/mL) and it expressed considerable cytotoxic activity against BT-549 (Human Ductal Carcinoma, Breast), KB (Human Epidermal Carcinoma, Oral) and SK-MEL (Human Malignant Melanoma) with IC50 values of 8.3, 10.0 and 3.3 μg/mL, respectively. Compounds (2) and (3) showed potent cytotoxicity against BT-549 with IC50 values of 5.3 and 7.3 μg/mL, and against SK-MEL with IC50 values of 4.3 and 3.1 μg/mL, respectively. Compound (4) showed good cytotoxicity against KB with IC50 value of 7.3 μg/mL. In consistent, the study of molecular determinates of cytotoxic activity of these new scaffolds showed close high docking scores to CD81 (Cluster of Differentiation 81) human antigen which could be of great importance for the development of new cytotoxic candidates. The structure identification of isolated metabolites was carried out using 1D and 2D NMR and mass spectra.
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Yeku, Oladapo O., and Renier J. Brentjens. "Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy." Biochemical Society Transactions 44, no. 2 (April 11, 2016): 412–18. http://dx.doi.org/10.1042/bst20150291.
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Chimaeric antigen receptor (CAR) T-cells are T-cells that have been genetically modified to express an artificial construct consisting of a synthetic T-cell receptor (TCR) targeted to a predetermined antigen expressed on a tumour. Coupling the T-cell receptor to a CD3ζ signalling domain paved the way for first generation CAR T-cells that were efficacious against cluster of differentiation (CD)19-expressing B-cell malignancies. Optimization with additional signalling domains such as CD28 or 4-1BB in addition to CD3ζ provided T-cell activation signal 2 and further improved the efficacy and persistence of these second generation CAR T-cells. Third generation CAR T-cells which utilize two tandem costimulatory domains have also been reported. In this review, we discuss a different approach to optimization of CAR T-cells. Through additional genetic modifications, these resultant armored CAR T-cells are typically modified second generation CAR T-cells that have been further optimized to inducibly or constitutively secrete active cytokines or express ligands that further armor CAR T-cells to improve efficacy and persistence. The choice of the ‘armor’ agent is based on knowledge of the tumour microenvironment and the roles of other elements of the innate and adaptive immune system. Although there are several variants of armored CAR T-cells under investigation, here we focus on three unique approaches using interleukin-12 (IL-12), CD40L and 4-1BBL. These agents have been shown to further enhance CAR T-cell efficacy and persistence in the face of a hostile tumour microenvironment via different mechanisms.
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Ramzi, Mani, Nargess Arandi, Mahdiyar Iravani Saadi, Ramin Yaghobi, and Bita Geramizadeh. "Genetic Variation of Costimulatory Molecules, Including Cytotoxic T-Lymphocyte Antigen 4, Inducible T-Cell Costimulator, Cluster Differentiation 28, and Programmed Cell Death 1 Genes, in Iranian Patients With Leukemia." Experimental and Clinical Transplantation 18, no. 6 (November 2020): 719–24. http://dx.doi.org/10.6002/ect.2017.0176.
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Shaheen, Sameerah, Maha M. Arafah, Aliah R. Alshanwani, Laila Mohammed Fadda, Ahlam M. Alhusaini, Hanaa M. Ali, Iman H. Hasan, Hanan Hagar, Fatima MB Alharbi, and Alaa AlHarthii. "Chitosan nanoparticles as a promising candidate for liver injury induced by 2-nitropropane: Implications of P53, iNOS, VEGF, PCNA, and CD68 pathways." Science Progress 104, no. 2 (April 2021): 003685042110118. http://dx.doi.org/10.1177/00368504211011839.
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The current article was designed to assess the role of chitosan nanoparticles (CNPs) in the management of hepatic injury induced by the hepatocarcinogen 2-nitropropane (2-NP). Rats were divided into three groups. The first group served as a control, the second group was injected with 2-NP, while the third group was treated with CNPs 1 h before 2-NP injection every other day for 4 weeks. The 2-NP injection upregulated serum AST and ALT activities, as well as hepatic TNF- α, IL-6, and MDA levels and the expression of vascular endothelial growth factor (VEGF) and caspase-3, whereas GSH contents and SOD activity were decreased. Immunohistochemistry investigations revealed that the hepatic protein expression of collagen I, inducible nitric oxide synthetase, proliferating cell nuclear antigen, cluster of differentiation, and p53 were upregulated. hematoxylin and eosin (H&E) and Masson’s trichrome stains supported the previous parameters, and CNPs ameliorated most of the previous biochemical parameters. CNPs achieved promising results in the limitation of 2-NP hepatotoxicity.
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Aries, James, Sarah Charrot, Jennifer Ball, Matthew Mee, Symeon Theocharidis, Sofie Van Gassen, Sameena Iqbal, John G. Gribben, and Jeff Davies. "Integrated Immune Signature Analyses Identifies Evolution of Distinct Immunoregulatory Cell Populations Which Control Alloreactivity after Allogeneic HSCT." Blood 134, Supplement_1 (November 13, 2019): 595. http://dx.doi.org/10.1182/blood-2019-124875.
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Background. Treatment failure after allogeneic haematopoietic stem-cell transplantation (AHST) using reduced-intensity conditioning (RIC) results from too much alloreactivity and harmful acute Graft-versus-Host Disease (aGVHD). Studies have identified many reconstituting immune cell subsets associated with development of clinical alloreactivity but the functionally dominant parameters at different time-points remain unknown. We therefore used mass cytometry (MS) to simultaneously assess multiple alloreactive and immunoregulatory cell populations to identify dominant immune reconstitution signatures associated with subsequent development of aGvHD after AHST. Methods. Phenotypic markers identifying more than 30 immune cell subsets known to influence alloreactivity were combined in a single MS panel. Peripheral blood from 58 patients with haematological cancers was analysed after T-replete HLA-matched RIC-AHST using uniform conditioning. Normalization of individual test samples spiked with CD45-barcoded healthy control cells was used to reduce batch effects. Complementary high-dimensional analytic tools were used to generate cellular profiles across the whole cohort and identify differences between patients grouped by subsequent development of aGVHD. Results. Unsupervised clustering analysis identified 40 phenotypically distinct T, B and NK cell clusters post-transplant. Significant batch effects were effectively reduced with a novel R-based algorithm normalising data to control cells. Cluster diversity analysis early post-transplant demonstrated lower cluster diversity in patients who subsequently developed aGvHD consistent with perturbation of phenotypic clusters in these patients. Two specific clusters were significantly different in abundance at D+30 in patients who went on to develop aGvHD and those who remained aGVHD-free. A cluster with a CD56brightCD16negCD27+/- regulatory NK cell (NKreg) phenotype was reduced in patients going on to develop aGvHD using both Phenograph and FlowSOM algorithms (p=0.001). These findings were validated by forward analysis using the CITRUS algorithm, revealing a similar differentiating cell population. CD56bright NKreg reconstitution was independent of CMV reactivation and did not impede reconstitution of WT1 and PR1 tumor-associated antigen-specific T cells. The reduction in NKreg in patients who subsequently developed aGvHD was accompanied by a significant increase in alloreactive CCR5+CD45RA-CCR7- CD4 effector memory T cells (Tem). We next used correlation analysis of cluster abundance across the whole cohort to identify all clusters contributing to the immune 'regulome' (those inversely correlated with alloreactive CD4 EM and/or CD8 EM T cell clusters). Notably, at D+30 the regulome consisted of 4 phenotypically distinct CD56bright NKreg clusters and a CD4+CD8+ double positive (DP) cluster, but not FOXP3+ CD4 regulatory T cells (Treg), Figure 1A Both the identity of differentiating clusters between patients subsequently developing aGVHD and those who remained aGvHD-free, and the dominant constituents of the regulome changed over time. By D+60 a CD56bright NKreg cluster (with a distinct phenotype to the differentiating cluster identified at D+30) and a DP T cell cluster were significantly reduced in patients subsequently developing aGvHD. The D+60 regulome consisted of multiple distinct CD56bright clusters, a DP T cell cluster and CD4 Treg, Figure 1B. Importantly by D+90 the immune regulome consisted of a reduced number of CD56bright NKreg clusters and increasing dominance of CD4Treg, Figure 1C. Conclusion. We show proof-of-concept that a novel acquisition and analysis pipeline can be applied to MS data to identify multiple immunoregulatory cells after AHST that contribute to the control of reconstituting alloreactive T cells. This approach identified a loss of NK cell-mediated control of alloreactive CD4 Tem cells as the dominant immune process preceding the development of aGvHD early post-transplant. Importantly, we show that specific immunoregulatory subsets are dominant at different time-points, with increasing influence of DP T cells and CD4 Treg at later time points. Our data provide mechanistic insight into the dynamic pattern of control of alloreactivity over time and show that strategies to expand or potentiate immunoregulatory cells to prevent aGvHD should be time-dependent. Disclosures Gribben: Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding.
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Cirulli, V., L. Crisa, G. M. Beattie, M. I. Mally, A. D. Lopez, A. Fannon, A. Ptasznik, et al. "KSA Antigen Ep-CAM Mediates Cell–Cell Adhesion of Pancreatic Epithelial Cells: Morphoregulatory Roles in Pancreatic Islet Development." Journal of Cell Biology 140, no. 6 (March 23, 1998): 1519–34. http://dx.doi.org/10.1083/jcb.140.6.1519.
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Cell adhesion molecules (CAMs) are important mediators of cell–cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell–cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.
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Wei, Spencer C., Nana-Ama A. S. Anang, Roshan Sharma, Miles C. Andrews, Alexandre Reuben, Jacob H. Levine, Alexandria P. Cogdill, et al. "Combination anti–CTLA-4 plus anti–PD-1 checkpoint blockade utilizes cellular mechanisms partially distinct from monotherapies." Proceedings of the National Academy of Sciences 116, no. 45 (October 21, 2019): 22699–709. http://dx.doi.org/10.1073/pnas.1821218116.
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Immune checkpoint blockade therapy targets T cell-negative costimulatory molecules such as cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1). Combination anti–CTLA-4 and anti–PD-1 blockade therapy has enhanced efficacy, but it remains unclear through what mechanisms such effects are mediated. A critical question is whether combination therapy targets and modulates the same T cell populations as monotherapies. Using a mass cytometry-based systems approach, we comprehensively profiled the response of T cell populations to monotherapy and combination anti–CTLA-4 plus anti–PD-1 therapy in syngeneic murine tumors and clinical samples. Most effects of monotherapies were additive in the context of combination therapy; however, multiple combination therapy-specific effects were observed. Highly phenotypically exhausted cluster of differentiation 8 (CD8) T cells expand in frequency following anti–PD-1 monotherapy but not combination therapy, while activated terminally differentiated effector CD8 T cells expand only following combination therapy. Combination therapy also led to further increased frequency of T helper type 1 (Th1)-like CD4 effector T cells even though anti–PD-1 monotherapy is not sufficient to do so. Mass cytometry analyses of peripheral blood from melanoma patients treated with immune checkpoint blockade therapies similarly revealed mostly additive effects on the frequencies of T cell subsets along with unique modulation of terminally differentiated effector CD8 T cells by combination ipilimumab plus nivolumab therapy. Together, these findings indicate that dual blockade of CTLA-4 and PD-1 therapy is sufficient to induce unique cellular responses compared with either monotherapy.
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Ey, P. L., R. H. Andrews, and G. Mayrhofer. "Differentiation of major genotypes ofGiardia intestinalisby polymerase chain reaction analysis of a gene encoding a trophozoite surface antigen." Parasitology 106, no. 4 (May 1993): 347–56. http://dx.doi.org/10.1017/s0031182000067081.
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SUMMARYThe polymerase chain reaction (PCR) has been used to amplifyin vitroa semi-conserved region of a gene encoding anMr68–72000 surface antigen ofGiardia intestinalistrophozoites. Using primers specific for conserved nucleotide sequences identified within the promoter-distal portion of two homologous genes (tsp11 andtsa417) cloned previously from theG. intestinalisisolates Ad-1 (from Australia) and WB (from Afghanistan), a single PCR-amplified DNA fragment of the expected size (0·52 kilobases) was obtained in high yield from either purified DNA or whole trophozoites of the Ad-1 isolate and from every 1 of 9 other axenicG. intestinalisisolates belonging to genetic groups I and II (defined previously on the basis of allozyme electrophoresis data—Andrewset al.1989). Discernible product was recovered from as few as 2–4 trophozoites. In contrast, 6G. intestinalisisolates that were assigned by allozymic analysis to genetic groups III/IV yielded small amounts of a 0·37-kilobase (kb) amplification product (with evidence in some samples of an additional 0·4 or 0·18 kb fragment) but no 0·52 kb product. Two animal-derived isolates ofG. duodenalis(one from an Australian native rodent,Notomys alexis, the other from a domestic cat) also yielded a single 0·37 kb PCR-amplified fragment, whereas an isolate from another cat produced a 0·34 kb fragment. No product was recovered fromG. muris, a morphologically distinct species ofGiardia. The results demonstrate that different genotypes ofG. duodenaliscan be distinguished using this assay and that it is diagnostic for isolates belonging to two major clusters (groups I/II and III/IV) ofG. intestinalis. The amplified DNA segment appears to be relatively conserved among group I and group II isolates ofG. intestinalis. A related but clearly distinct sequence seems to be conserved among group III/IV isolates ofG. intestinalisand some isolates ofG. duodenalis.
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Wu, Yongxia, David Bastian, Jessica Lauren Heinrichs, Jianing Fu, Hung Nguyen, Qi-Jing Li, Changqing Xia, and Xue-Zhong Yu. "Microrna-17-92 Cluster: Novel Target for Controlling Gvhd While Preserving GVL Effect." Blood 124, no. 21 (December 6, 2014): 845. http://dx.doi.org/10.1182/blood.v124.21.845.845.
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Abstract Graft-versus-host disease (GVHD) remains a life threatening complication after allogeneic hematopoietic stem cell transplantation (HCT). Donor T cells are the key pathogenic effectors in the induction of GVHD. MicroRNAs (miRs) have been shown to play an important role in orchestrating immune response, among which miR-17-92 cluster is one of the best characterized miR clusters that encodes 6 miRs including 17, 18a, 19a, 20a, 19b-1 and 92-1. Although regulatory functions of miR-17-92 cluster have been elaborated in a variety of immune responses including anti-infection, anti-tumor, and autoimmunity, the role of this miR cluster in the modulation of T-cell response to alloantigens and the development of GVHD has not been explored previously. Based on the previous report that miR-17-92 promotes Th1 responses and inhibits induced regulatory T-cell (iTreg) differentiation in vitro, we hypothesized that blockade of miR-17-92 would constrain T-cell alloresponse and attenuate GVHD. To evaluate the function of miR-17-92 on T-cell alloresponse, we utilized the mice with miR-17-92 conditional knock-out (KO) on T cells as donors, and compared the alloresponse of WT and KO T cells after allogeneic bone marrow transplantation (allo-BMT). We observed that KO T cells had substantially reduced ability to proliferate and produce IFNγ as compared to WT counterparts 4 days after cell transfer. Interestingly, CD4 but not CD8 KO T cells had increased cell death in the population of fast-dividing T cells. Thus, miR-17-92 cluster promotes activation and expansion of both CD4 and CD8 T cells, and inhibits activation-induced cell death of CD4 but not CD8 T cells at the early stage of alloresponse in vivo. We further evaluated the role of miR-17-92 on T cells in the development of acute GVHD in a fully MHC-mismatched BMT model. In sharp contrast to WT T cells that caused severe GVHD and resulted in 100% mortality of the recipients, KO T cells were impaired in causing severe GVHD reflected by mild clinical manifestations and no mortality. These observations were extended to MHC-matched but minor antigen-mismatched as well as haploidentical BMT models that are more clinically relevant. We next addressed the critical question whether T cells deficient for miR-17-92 are still capable of mediating graft-versus-leukemia (GVL) effect. Using A20 lymphoma and P815 mastocytoma cell lines, we demonstrated that the KO T cells essentially retained the GVL activity in MHC-mismatched and haploidentical BMT model, respectively. Mechanistic studies revealed that miR-17-92 promoted CD4 T-cell proliferation, survival, migration to target organs, and Th1-differentiation, but reduced Th2-differentiation and iTreg generation. However, miR-17-92 had less impact on CD8 T-cell proliferation, survival, IFNγ production, and cytolytic activity reflected by granzyme B and CD107a expression. Moreover, miR-17-92 negatively regulated TNFα production by both CD4 and CD8 T cells. We therefore conclude that miR-17-92 cluster is required for T cells to induce severe GVHD, but it is dispensable for T cells to mediate the GVL effect. To increase translational potential of our findings, we designed the locked nucleic acid (LNA) antagomirs specific for miR-17 or miR-19, which have been reported to be the key members in this cluster. We observed that the treatment with anti-miR-17 significantly inhibited T-cell expansion and IFNγ production in response to alloantigen in vivo, and anti-miR-19 was more effective. Furthermore, our ongoing experiment showed the treatment with anti-miR-17 or anti-miR-19 was able to considerably attenuate the severity of GVHD as compared to scrambled antagomir in a MHC-mismatched BMT model. Taken together, the current work reveals that miR-17-92 cluster is essential for T-cell alloresponse and GVHD development, and validates miR-17-92 cluster as promising therapeutic target for the control of GVHD while preserving GVL activity in allogeneic HCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Migliorini, Denis, Valérie Dutoit, Mathilde Allard, Nicole Grandjean Hallez, Eliana Marinari, Valérie Widmer, Géraldine Philippin, et al. "Phase I/II trial testing safety and immunogenicity of the multipeptide IMA950/poly-ICLC vaccine in newly diagnosed adult malignant astrocytoma patients." Neuro-Oncology 21, no. 7 (May 3, 2019): 923–33. http://dx.doi.org/10.1093/neuonc/noz040.
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Abstract Background Peptide vaccines offer the opportunity to elicit glioma-specific T cells with tumor killing ability. Using antigens eluted from the surface of glioblastoma samples, we designed a phase I/II study to test safety and immunogenicity of the IMA950 multipeptide vaccine adjuvanted with poly-ICLC (polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose) in human leukocyte antigen A2+ glioma patients. Methods Adult patients with newly diagnosed glioblastoma (n = 16) and grade III astrocytoma (n = 3) were treated with radiochemotherapy followed by IMA950/poly-ICLC vaccination. The first 6 patients received IMA950 (9 major histocompatibility complex [MHC] class I and 2 MHC class II peptides) intradermally and poly-ICLC intramuscularly (i.m.). After protocol amendment, IMA950 and poly-ICLC were mixed and injected subcutaneously (n = 7) or i.m. (n = 6). Primary endpoints were safety and immunogenicity. Secondary endpoints were overall survival, progression-free survival at 6 and 9 months, and vaccine-specific peripheral cluster of differentiation (CD)4 and CD8 T-cell responses. Results The IMA950/poly-ICLC vaccine was safe and well tolerated. Four patients presented cerebral edema with rapid recovery. For the first 6 patients, vaccine-induced CD8 T-cell responses were restricted to a single peptide and CD4 responses were absent. After optimization of vaccine formulation, we observed multipeptide CD8 and sustained T helper 1 CD4 T-cell responses. For the entire cohort, CD8 T-cell responses to a single or multiple peptides were observed in 63.2% and 36.8% of patients, respectively. Median overall survival was 19 months for glioblastoma patients. Conclusion We provide, in a clinical trial, using cell surface-presented antigens, insights into optimization of vaccines generating effector T cells for glioma patients. Trial registration Clinicaltrials.gov NCT01920191.
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Brown, D., and G. L. Waneck. "Glycosyl-phosphatidylinositol-anchored membrane proteins." Journal of the American Society of Nephrology 3, no. 4 (October 1992): 895–906. http://dx.doi.org/10.1681/asn.v34895.
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Many proteins of eukaryotic cells are anchored to membranes by covalent linkage to glycosyl-phosphatidylinositol (GPI). These proteins lack a transmembrane domain, have no cytoplasmic tail, and are, therefore, located exclusively on the extracellular side of the plasma membrane. GPI-anchored proteins form a diverse family of molecules that includes membrane-associated enzymes, adhesion molecules, activation antigens, differentiation markers, protozoan coat components, and other miscellaneous glycoproteins. In the kidney, several GPI-anchored proteins have been identified, including uromodulin (Tamm-Horsfall glycoprotein), carbonic anhydrase type IV, alkaline phosphatase, Thy-1, BP-3, aminopeptidase P, and dipeptidylpeptidase. GPI-anchored proteins can be released from membranes with specific phospholipases and can be recovered from the detergent-insoluble pellet after Triton X-114 treatment of membranes. All GPI-anchored proteins are initially synthesized with a transmembrane anchor, but after translocation across the membrane of the endoplasmic reticulum, the ecto-domain of the protein is cleaved and covalently linked to a preformed GPI anchor by a specific transamidase enzyme. Although it remains obscure why so many proteins are endowed with a GPI anchor, the presence of a GPI anchor does confer some functional characteristics to proteins: (1) it is a strong apical targeting signal in polarized epithelial cells; (2) GPI-anchored proteins do not cluster into clathrin-coated pits but instead are concentrated into specialized lipid domains in the membrane, including so-called smooth pinocytotic vesicles, or caveoli; (3) GPI-anchored proteins can act as activation antigens in the immune system; (4) when the GPI anchor is cleaved by PI-phospholipase C or PI-phospholipase D, second messengers for signal transduction may be generated; (5) the GPI anchor can modulate antigen presentation by major histocompatibility complex molecules. Finally, at least one human disease, paroxysmal nocturnal hemoglobinuria, is a result of defective GPI anchor addition to plasma membrane proteins.
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Sun, Shu, Chen Jin, Yueying Li, Jia Si, Yueli Cui, Matthew T. Rondina, Fuchou Tang, and Qian-fei Wang. "Transcriptional and Spatial Heterogeneity of Mouse Megakaryocytes at Single-Cell Resolution." Blood 134, Supplement_1 (November 13, 2019): 275. http://dx.doi.org/10.1182/blood-2019-129471.
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Megakaryocytes (MKs) have long been described solely as platelet progenitors. However, recent studies show that MKs also act as an essential component of the bone marrow niche to maintain hematopoietic stem cell function, and combat infection by engulfing and presenting antigens. However, it is not known whether these diverse programs are executed by a single cell population or distinct subsets of cells. We performed single-cell RNA sequencing (scRNA-seq) to dissect the heterogeneity of MKs. To overcome the difficulty in obtaining the rare (0.1% in BM) and oversized (up to 65μm) highly polyploid, fragile MKs, we developed an efficient isolation strategy by combining fluorescence-activated cell sorting (FACS) sorting, manual selection of highly viable cells, and FISH verification of ploidy. We obtained 920 CD41+ highly-purified, bone-marrow derived, murine MKs spanning each ploidy stages (2N-32N) for scRNA-seq with modified Smart-seq2 protocol. On average, we detected over 6800 expressed genes and 250,000 transcripts in each MK. All cells expressed classical markers such as Pf4 and Itga2b (CD41). Four cell clusters were identified using an unsupervised clustering method. Cells in Cluster 1 expressed mature MK markers CD42 and CD61, were enriched for hemostasis and platelet activation expression signatures, and consist of ≥8N cells, suggesting these MKs may represent platelet generating MKs. Cells in cluster 2 had lower CD42 and CD61 expression, were low ploidy (≤8N), and had higher expression of inflammation-related genes, including Ctss and Itgam ("inflammatory response-associated MKs"). Cells in Cluster 3 were enriched for DNA replication and DNA strand elongation (GO terms) and were in all ploidy stages ("MKs in polyploidization stage"). Cluster 4, most of which were high-ploidy, expressed high levels of CD42, CD61, TGF-β, and IGF1: factors regulating HSC behavior ("HSC niche cells"). Furthermore, we identified cell population-specific surface markers and transcription factors (TFs) for each of the 4 clusters. Then, immunostaining using antibodies against corresponding markers were performed to confirm the presence of respective MK subpopulations in the bone marrow. Our analyses suggest that defining MK stages by ploidy and traditional markers CD42 and CD61 alone may result in a genetically and developmentally heterogenous population of MK. Rather, MKs at various stages may be more specifically identified by these gene signatures. MKs with different functions are known to have specific spatial distribution in the bone marrow (BM) niche. To test whether MKs with distinct expression signatures are uniquely localized within BM, we performed immunofluorescence staining on BM sections using antibodies against cell population-specific marker genes. Remarkably, we observed that Cluster 1 cells directly contacted blood vessels while most of Cluster 4 cells resided within one cell diameter of HSCs. The unique spatial distribution of cluster 1 and 4 population are in consistency with their respective transcriptomic signatures, and support that platelet generation and HSC maintenance are carried out by two distinct MK subpopulations. We further investigated the potential intrinsic relationship of these four Clusters during megakaryopoiesis. Developmental time courses were reconstructed using Monocle analysis, demonstrating that polyploidization (Cluster 3) occurs at the early stage of MK development with subsequent differentiation toward three orientations. While MKs with low ploidy appear to have two distinct cell fates (immuno-modulation or polyploidization), MKs with high ploidy (≥8N) differentiate towards populations associated with platelet production or stem cell regulation. In summary, our study provides the first in vivo transcriptomic profile of megakaryopoiesis and a potential map of megakaryocyte heterogeneity at the single-cell resolution. MKs may be classified into different functional subpopulations irrespective to their developmental stage and degree of ploidy. These observations suggest that megakaryopoiesis does not occur merely in a stepwise process, but is dynamic and adaptive to locations in the BM and biological needs. Disclosures No relevant conflicts of interest to declare.
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Darma, Sidrah, Herry D. Nawing, Ninny Meutia Pelupessy, and Husein Albar. "A Child with HIV (Human Immunodeficiency Virus) Infection Accompanied by Severe Acute Malnutrition: A Case Report." Green Medical Journal 2, no. 3 (December 30, 2020): 112–20. http://dx.doi.org/10.33096/gmj.v2i3.66.
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Joint United Nations Programme in HIV/AIDS (UNAIDS) reported that 1.8 million children under 15 years old had HIV with 150,000 new pediatric cases in 2015, and only 49% had an antiretroviral (ARV) therapy. Mortality in HIV-infected children with severe acute malnutrition was 30.4% in Africa. A 1-year and 8-months-old girl was hospitalized due to diarrhea, vomiting, oral thrush, and recurrent fever before admission. She has been hospitalized for HIV infection one month ago and treated with ARV. Her mother was treated with ARV before. Physical examination showed a severely ill, poorly nourished, stunting, and conscious child with normal vital signs. There was oral thrush. The evidence of nutritional marasmus was old man face, piano sign, wasting, and baggy pants. Laboratory findings revealed anemia, positive antigen and antibody of HIV infection, and low Cluster of Differentiation 4 (CD4). She was treated with ARV, Cotrimoxazole, and management of malnutrition and diarrhea. The prognosis of the patient was poor. A 1-year and 8-months-old girl with HIV infection complicated with severe acute malnutrition, acute diarrhea, oral thrush, and anemia of chronic disease were reported. The diagnosis was based on clinical and laboratory findings. Management focused on the therapy of HIV and accompanying illness. The prognosis was poor.
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Rantam, Fedik Abdul, Viol Dhea Kharisma, Christrijogo Sumartono, Jusak Nugraha, Andi Yasmin Wijaya, Helen Susilowati, Suryo Kuncorojakti, and Alexander Patera Nugraha. "Molecular docking and dynamic simulation of conserved B cell epitope of SARS-CoV-2 glycoprotein Indonesian isolates: an immunoinformatic approach." F1000Research 10 (August 16, 2021): 813. http://dx.doi.org/10.12688/f1000research.54258.1.
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Background: An immunoinformatic approach may be useful to investigate the conserved region in the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Indonesia isolates. The aim of this study was to investigate Indonesian SARS-CoV-2 isolates based on B cell epitopes by targeting the conserved regions in the spike glycoprotein to trigger increased multi-variant virus neutralization and memory response for the development of vaccine seed candidates. Methods: SARS-CoV-2 spike glycoprotein gene sequences originating from Indonesia were compared with Wuhan (China), the United Kingdom, South Africa, India, the United States, and Brazil isolates obtained from the NCBI and GISAID databases. The recognition of antigens was carried out directly using B cells through the B cell receptor (BCR). An indirect B cell activation by Cluster of Differentiation (CD)4+ T cells and major histocompatibility complex (MHC)-II was predicted through the binding with human leukocyte antigen (HLA) based on IC50 value. In addition, vaccine allergenicity and toxicity were investigated. During the molecular complex examination, the 3D peptide structure was investigated and the lowest amount of energy formed when the vaccine candidate peptide bound to BCR and MHC-II was calculated. Results: As a result, the spike glycoprotein sequences of Indonesian SARS-CoV-2 isolates had conserved regions which were very similar to reference countries such as China, the United Kingdom, South Africa, India, the United States, and Brazil. Conclusion: It was predicted that the conserved regions could be identified as the epitope of B and T CD4+ cells that produced the peptides for vaccine candidate with antigenic, non-allergen, and non-toxic properties.
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Yin, Xiaoyan, Subha Subramanian, Shih-Jen Hwang, Christopher J. O’Donnell, Caroline S. Fox, Paul Courchesne, Pieter Muntendam, et al. "Protein Biomarkers of New-Onset Cardiovascular Disease." Arteriosclerosis, Thrombosis, and Vascular Biology 34, no. 4 (April 2014): 939–45. http://dx.doi.org/10.1161/atvbaha.113.302918.
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Objective— Incorporation of novel plasma protein biomarkers may improve current models for prediction of atherosclerotic cardiovascular disease (ASCVD) risk. Approach and Results— We used discovery mass spectrometry (MS) to determine plasma concentrations of 861 proteins in 135 myocardial infarction (MI) cases and 135 matched controls. Then, we measured 59 markers by targeted MS in 336 ASCVD case–control pairs. Associations with MI or ASCVD were tested in single-marker and multiple-marker analyses adjusted for established ASCVD risk factors. Twelve single markers from discovery MS were associated with MI incidence (at P <0.01), adjusting for clinical risk factors. Seven proteins in aggregate (cyclophilin A, cluster of differentiation 5 molecule [CD5] antigen-like, cell-surface glycoprotein mucin cell surface associated protein 18 [MUC-18], collagen-α 1 [XVIII] chain, salivary α-amylase 1, C-reactive protein, and multimerin-2) were highly associated with MI ( P <0.0001) and significantly improved its prediction compared with a model with clinical risk factors alone (C-statistic of 0.71 versus 0.84). Through targeted MS, 12 single proteins were predictors of ASCVD (at P <0.05) after adjusting for established risk factors. In multiple-marker analyses, 4 proteins in combination (α-1–acid glycoprotein 1, paraoxonase 1, tetranectin, and CD5 antigen-like) predicted incident ASCVD ( P <0.0001) and moderately improved the C-statistic from the model with clinical covariates alone (C-statistic of 0.69 versus 0.73). Conclusions— Proteomics profiling identified single- and multiple-marker protein panels that are associated with new-onset ASCVD and may lead to a better understanding of underlying disease mechanisms. Our findings include many novel protein biomarkers that, if externally validated, may improve risk assessment for MI and ASCVD.
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Odagiri, Naoshi, Hoang Hai, Le Thi Thanh Thuy, Minh Phuong Dong, Maito Suoh, Kohei Kotani, Atsushi Hagihara, et al. "Early Change in the Plasma Levels of Circulating Soluble Immune Checkpoint Proteins in Patients with Unresectable Hepatocellular Carcinoma Treated by Lenvatinib or Transcatheter Arterial Chemoembolization." Cancers 12, no. 8 (July 24, 2020): 2045. http://dx.doi.org/10.3390/cancers12082045.
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Immune checkpoint inhibitors, combined with anti-angiogenic agents or locoregional treatments (e.g., transarterial chemoembolization (TACE)), are expected to become standard-of-care for unresectable hepatocellular carcinoma (HCC). We measured the plasma levels of 16 soluble checkpoint proteins using multiplexed fluorescent bead-based immunoassays in patients with HCC who underwent lenvatinib (n = 24) or TACE (n = 22) treatment. In lenvatinib-treated patients, plasma levels of sCD27 (soluble cluster of differentiation 27) decreased (p = 0.040) and levels of sCD40 (p = 0.014) and sTIM-3 (p < 0.001) were increased at Week 1, while levels of sCD27 (p < 0.001) were increased significantly at Weeks 2 through 4. At Week 1 of TACE, in addition to sCD27 (p = 0.028), sCD40 (p < 0.001), and sTIM-3 (soluble T-cell immunoglobulin and mucin domain–3) (p < 0.001), levels of sHVEM (soluble herpesvirus entry mediator) (p = 0.003), sTLR-2 (soluble Toll-like receptor 2) (p = 0.009), sCD80 (p = 0.036), sCTLA-4 (soluble cytotoxic T-lymphocyte antigen 4) (p = 0.005), sGITR (soluble glucocorticoid-induced tumor necrosis factor receptor) (p = 0.030), sGITRL (soluble glucocorticoid-induced TNFR-related ligand) (p = 0.090), and sPD-L1 (soluble programmed death-ligand 1) (p = 0.070) also increased. The fold-changes in soluble checkpoint receptors and their ligands, including sCTLA-4 with sCD80/sCD86 and sPD-1 (soluble programmed cell death domain–1) with sPD-L1 were positively correlated in both the lenvatinib and TACE treatment groups. Our results suggest that there are some limited differences in immunomodulatory effects between anti-angiogenic agents and TACE. Further studies from multicenters may help to identify an effective combination therapy.
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Li, X., M. Li, S. Huang, S. Qiao, C. Kang, and D. Shi. "165 THE EFFECT OF shRNA TARGETING CLUSTER OF DIFFERENTIATION ANTIGEN 14 ON GENE EXPRESSION OF TNF-α, TLR4, AND IL-6 IN LIPOPOLYSACCHARIDE-INDUCED BUFFALO PERIPHERAL BLOOD MONOCYTE/MACROPHAGE." Reproduction, Fertility and Development 25, no. 1 (2013): 231. http://dx.doi.org/10.1071/rdv25n1ab165.
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Cluster of differentiation antigen 14 (CD14) plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, leading to inflammation. Several studies have proved that upstream inhibition of bacterial LPS/toll-like receptor 4 (TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. In this study, to explore the effect of CD14 down-regulation on TLR4 signal conductive-related genes expression after stimulation by LPS, five CD14 shRNA (319/421/755/970/1041) sequences and a negative control sequence (NC-1864) were synthesised and used to construct lentiviral recombinant plasmid pSicoR-GFP-shRNA. Lentiviral recombinant plasmids of pSicoR-GFP-shRNA and fusion expression vector of pDsRed-N1-buffalo CD14 were co-transfected into HEK293 using liposome. At 72 h after transfection, the expression of exogenous buffalo CD14 mRNA was reduced at different level for all shRNA plasmids, in which shRNA-1041 had the highest interfering efficiency by RT-qPCR and fluorescence-activated cell sorting analysis. Then, buffalo peripheral blood monocyte/macrophage was purified and infected by the CD14 shRNA lentivirus. After 7 days of infection, the cells were stimulated by 1 µg mL–1 LPS for 3 h, then the mRNA expression level of CD14, TLR4, IL-6, and TNF-α transcripts in the cells were detected by the RT-qPCR method. After stimulation by LPS, the expression of endogenous CD14 was significantly reduced by CD14 shRNA-1041, the mRNA expression level of TLR4, IL-6, and TNF-α genes was also significantly down-regulated in comparison with control group (P ≤ 0.01). In conclusion, the selected CD14 shRNA-1041 cannot only inhibit the expression of endogenous CD14 mRNA in buffalo peripheral blood monocyte/macrophage, but also downregulate the mRNA expression of CD14, TLR4, IL-6, and TNF-α. The above results demonstrate that knockdown of endogenous CD14 has obvious coordination effects on the signal conductive function of TLR4 after stimulating by LPS, and shRNA technology will provide a new way to prevent endotoxin-related diseases in livestock. This work was supported by the National Transgenic Project (2009ZX08007-009B), Guangxi natural science funding (2012GXNSFCB053002), and funding of State Key Laboratory of Subtropical Bioresource Conservation and Utilisation (KSL-CUSAb-2012-02).
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Liu, C., B. Sun, B. Xu, X. Meng, L. Li, Q. Song, S. Wu, and J. Yu. "A Panel Containing PD-1, IL-2Rα, IL-10, and CA15-3 as a Biomarker to Discriminate Breast Cancer from Benign Breast Disease." Journal of Global Oncology 4, Supplement 2 (October 1, 2018): 204s. http://dx.doi.org/10.1200/jgo.18.82400.
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Background: Programmed cell death protein 1 (PD-1), an immune checkpoint molecule, has recently been recognized as a predictive and prognostic biomarker in several malignant tumors, but its diagnostic value remains largely unknown. Aim: We aimed to investigate the differential diagnostic efficiency of PD-1 and other immune molecules, and propose a panel of immune molecules combined with cancer antigen 15-3 (CA15-3) to distinguish breast cancer (BC) from benign breast disease (BBD). Methods: Ninety-one eligible BC patients and 31 BBD patients were enrolled. Pretreatment peripheral blood was collected and tested for mRNA expression of PD-1, cytotoxic T lymphocyte antigen 4 (CTLA-4), forkhead box P3 (FOXP3), transforming growth factor beta (TGF-β), interleukin-10 (IL-10), IL-2 receptor alpha (IL-2Rα), and cluster of differentiation 28 (CD28) by quantitative real-time PCR. Results: The diagnostic areas under curve (AUCs) of PD-1, IL-2Rα, and IL-10 for BC-BBD discrimination were 0.764, 0.758, and 0.743, respectively. The diagnostic efficiencies of these 2 parameters in distinguishing early-stage or advanced BC from BBD were consistent with a role in BC-BBD discrimination. A panel of PD-1 + IL-10 + IL-2Rα + CA15-3 showed the highest AUC (0.862), with sensitivity of 0.933 and specificity of 0.724, for BC-BBD discrimination. In addition, for early-stage BC discrimination, this panel also had the highest AUC (0.811), with a sensitivity of 0.933 and specificity of 0.614, while for advanced BC discrimination, a panel of PD-1 + IL-10 + CA15-3 exhibited the highest AUC (0.896), with a sensitivity of 0.933 and specificity of 0.783. Conclusion: These data indicate that the panel containing PD-1, IL-2Rα, IL-10, and CA15-3 can effectively discriminate BC from BBD with a high efficiency. After further confirmation, it could be used to complement conventional imaging modalities, especially in discriminating early-stage BC from BBD.
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Vackova, Julie, Ingrid Polakova, Shweta Dilip Johari, and Michal Smahel. "CD80 Expression on Tumor Cells Alters Tumor Microenvironment and Efficacy of Cancer Immunotherapy by CTLA-4 Blockade." Cancers 13, no. 8 (April 16, 2021): 1935. http://dx.doi.org/10.3390/cancers13081935.
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Cluster of differentiation (CD) 80 is mainly expressed in immune cells but can also be found in several types of cancer cells. This molecule may either activate or inhibit immune reactions. Here, we determined the immunosuppressive role of CD80 in the tumor microenvironment by CRISPR/Cas9-mediated deactivation of the corresponding gene in the mouse oncogenic TC-1 cell line. The tumor cells with deactivated CD80 (TC-1/dCD80-1) were more immunogenic than parental cells and induced tumors that gained sensitivity to cytotoxic T-lymphocyte antigen 4 (CTLA-4) blockade, as compared with the TC-1 cells. In vivo depletion experiments showed that the deactivation of CD80 switched the pro-tumorigenic effect of macrophages observed in TC-1-induced tumors into an anti-tumorigenic effect in TC-1/dCD80-1 tumors and induced the pro-tumorigenic activity of CD4+ cells. Moreover, the frequency of lymphoid and myeloid cells and the CTLA-4 expression by T helper (Th)17 cells were increased in TC-1/dCD80-1- compared with that in the TC-1-induced tumors. CTLA-4 blockade downregulated the frequencies of most immune cell types and upregulated the frequency of M2 macrophages in the TC-1 tumors, while it increased the frequency of lymphoid cells in TC-1/dCD80-1-induced tumors. Furthermore, the anti-CTLA-4 therapy enhanced the frequency of CD8+ T cells as well as CD4+ T cells, especially for a Th1 subset. Regulatory T cells (Treg) formed the most abundant CD4+ T cell subset in untreated tumors. The anti-CTLA-4 treatment downregulated the frequency of Treg cells with limited immunosuppressive potential in the TC-1 tumors, whereas it enriched this type of Treg cells and decreased the Treg cells with high immunosuppressive potential in TC-1/dCD80-1-induced tumors. The immunosuppressive role of tumor-cell-expressed CD80 should be considered in research into biomarkers for the prediction of cancer patients’ sensitivity to immune checkpoint inhibitors and for the development of a tumor-cell-specific CD80 blockade.
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Mandic, Robert, Oliver T. Fackler, Matthias Geyer, Thomas Linnemann, Yong-Hui Zheng, and B. Matija Peterlin. "Negative Factor from SIV Binds to the Catalytic Subunit of the V-ATPase to Internalize CD4 and to Increase Viral Infectivity." Molecular Biology of the Cell 12, no. 2 (February 2001): 463–73. http://dx.doi.org/10.1091/mbc.12.2.463.
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The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.
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Chouw, Angliana, Bayu Winata Putera, Cynthia Retna Sartika, and Ajeng Diantini. "Positive Correlation between Very Small Embryonic Stem Cell, Hematopoietic Stem Cell, and Endothelial Progenitor Cell in Umbilical Cord Blood Unit." Indonesian Biomedical Journal 10, no. 3 (December 28, 2018): 231–5. http://dx.doi.org/10.18585/inabj.v10i3.455.
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BACKGROUND: Umbilical Cord Blood (UCB) has been widely use as regenerative medicine due to the content of undifferentiated cell which have capability to do self-renewal and differentiation into various type of cell called stem cells. Recent studies show that UCB contains not only hematopoietic stem cell (HSC) but also others stem cell and progenitor cell such as endothelial progenitor cell (EPC) and very small embryonic-like stem cell (VSEL). It is beliefs that HSC and EPC shared the same progenitor. In this study, correlation between the cell number of HSC, EPC and VSEL is analyzed in umbilical cord blood as the source of stem cell for clinical application.METHODS: The cell number of HSC, EPC and VSEL is counted from cryopreserved UCB collected from 22 women delivered via cesarean section which already stored for more than 2 years in this study. Sample were incubated with antibodies such as cluster of differentiation (CD)34-phycoerythrin (PE)/CD45-fluorescein isothiocyanate (FITC), CD133/1 (anti-CD (AC)133)-antigen-presenting cell (APC) and, CD184 (C-X-C chemokine receptor (CXCR)4)-PE.Vio770 to detect the present of HSC, EPC and VSEL in UCB. Sample were analyze using flowcytometer BD FACS Canto II.RESULTS: The cell population of HSC and late-EPC is 0.009% and 0.01% of total cell in UCB. VSEL only represented 0.001% from total cell in UCB, showing the lowest number of cell population in UCB. The correlation between the cell number of HSC and EPC is r=0,483*, p=0.023) and between HSC and VSEL is r=0.510*, p=0.015.CONCLUSION: In this study, both EPC and VSEL have a significant positive correlation with HSC.KEYWORDS: stem cell, umbilical cord blood, endothelial progenitor, flowcytometry
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Chu, Hsueh-Yao, Long-Sheng Lu, Wanying Cho, Shin-Yao Wu, Yu-Cheng Chang, Chien-Ping Lin, Chih-Yung Yang, Chi-Hung Lin, Jeng-Kai Jiang, and Fan-Gang Tseng. "Enumerating Circulating Tumor Cells with a Self-Assembled Cell Array (SACA) Chip: A Feasibility Study in Patients with Colorectal Cancer." Cancers 11, no. 1 (January 8, 2019): 56. http://dx.doi.org/10.3390/cancers11010056.
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Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide. Detecting and enumerating circulating tumor cells (CTCs) in patients with colorectal cancer emerged as an important prognostic tool which provides a direct estimate of metastatic potential. Improving the turnaround time and decreasing sample volume is critical for incorporating this liquid biopsy tool into routine practice. The objective of the current study was to validate the clinical feasibility of a self-assembled cell array (SACA) chip, a CTC counting platform with less than 4 h turnaround time, in patients with newly diagnosed colorectal cancers. In total, 179 patients with newly diagnosed colorectal cancers from a single institute were enrolled. Epithelial cell adhesion molecule positive (EpCAM(+)), cluster of differentiation 45 negative (CD45(−)) cells were isolated and enumerated from 2 mL of peripheral vein blood (PB) and inferior mesenteric vein blood (IMV) samples obtained during surgery. We found that the CTC count in PB but not IMV correlates with disease stages. Neoadjuvant chemotherapy did not lead to decreased CTC count in both types of blood samples. With cutoffs of four CTCs per 2 mL of blood, and serum carcinoembryonic antigen (CEA) level of 5 ng/mL, patients with non-metastatic disease were more likely to experience recurrence if they had high PB CTC count and high serum CEA concentration (odds ratio, 8.9). Our study demonstrates the feasibility of enumerating CTCs with a SACA chip in patients with colorectal cancer.
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Su, Zhaoqian, Kalyani Dhusia, and Yinghao Wu. "A computational study of co-inhibitory immune complex assembly at the interface between T cells and antigen presenting cells." PLOS Computational Biology 17, no. 3 (March 8, 2021): e1008825. http://dx.doi.org/10.1371/journal.pcbi.1008825.
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The activation and differentiation of T-cells are mainly directly by their co-regulatory receptors. T lymphocyte-associated protein-4 (CTLA-4) and programed cell death-1 (PD-1) are two of the most important co-regulatory receptors. Binding of PD-1 and CTLA-4 with their corresponding ligands programed cell death-ligand 1 (PD-L1) and B7 on the antigen presenting cells (APC) activates two central co-inhibitory signaling pathways to suppress T cell functions. Interestingly, recent experiments have identified a new cis-interaction between PD-L1 and B7, suggesting that a crosstalk exists between two co-inhibitory receptors and the two pairs of ligand-receptor complexes can undergo dynamic oligomerization. Inspired by these experimental evidences, we developed a coarse-grained model to characterize the assembling of an immune complex consisting of CLTA-4, B7, PD-L1 and PD-1. These four proteins and their interactions form a small network motif. The temporal dynamics and spatial pattern formation of this network was simulated by a diffusion-reaction algorithm. Our simulation method incorporates the membrane confinement of cell surface proteins and geometric arrangement of different binding interfaces between these proteins. A wide range of binding constants was tested for the interactions involved in the network. Interestingly, we show that the CTLA-4/B7 ligand-receptor complexes can first form linear oligomers, while these oligomers further align together into two-dimensional clusters. Similar phenomenon has also been observed in other systems of cell surface proteins. Our test results further indicate that both co-inhibitory signaling pathways activated by B7 and PD-L1 can be down-regulated by the new cis-interaction between these two ligands, consistent with previous experimental evidences. Finally, the simulations also suggest that the dynamic and the spatial properties of the immune complex assembly are highly determined by the energetics of molecular interactions in the network. Our study, therefore, brings new insights to the co-regulatory mechanisms of T cell activation.
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Poe, Jonathan C., Dadong Zhang, Jichun Xie, Rachel A. DiCioccio, Xiaodi Qin, Jiyuan Fang, Vincent T. Ho, et al. "Single-Cell RNA-Seq Identifies Potentially Pathogenic B Cell Populations That Uniquely Circulate in Patients with Chronic Gvhd." Blood 134, Supplement_1 (November 13, 2019): 874. http://dx.doi.org/10.1182/blood-2019-130928.
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While B cells are known to contribute to the pathogenesis of chronic graft-versus-host disease (cGVHD) in mice, it has been challenging to elucidate intrinsic mechanisms of tolerance loss in patients. To identify distinct and potentially targetable B-cell subsets in cGVHD, we employed single-cell RNA-Seq along with an unsupervised hierarchical clustering analysis, targeting 10,000 single B cells from each of eight patients who were >12 months post-allogeneic hematopoietic stem cell transplantation (HCT) and either had active cGVHD manifestations (n=4) or never developed cGVHD (n=4). Bioinformatics analysis of pooled cell data (using R with Seurat extension package) identified 6 major B cell clusters common to all patients (Figure 1A). "Intra-cluster" gene comparison (using R package DESeq2, false-discovery rate 0.05) revealed numerous differentially expressed genes between patient groups. The greatest number of differentially-expressed genes occurred in a cluster referred to herein as 'Cluster 6' (Figure 1A, in yellow with asterisk). Within Cluster 6, B cells from active cGVHD patients expressed significantly increased ITGAX (CD11c, Padj =0.007), TNFRSF13B (TACI, a receptor for BAFF, Padj =0.003), IGHG1 (IgG1, Padj =9.3e-06) and IGHG3 (IgG3, Padj =1.7e-12), along with 44 additional genes (to be discussed). Thus, Cluster 6 in cGVHD patients may represent a CD11cpos, BAFF-responsive B cell subset primed to undergo isotype switching in response to alloantigen. Flow cytometry analysis on PBMCs from an independent HCT patient cohort (n=10) confirmed that CD11cpos B cells were indeed significantly expanded in cGVHD (P < 0.01, Figure 1B), and revealed these B cells were also TACIpos, CD19high, forward scatter high (FSChigh) blast-like cells (Figure 1C). We found that these CD11cpos B cells had mixed expression of CD21, CD27, IgD and CD24 (Figure 1C). Remarkably, other recent studies on bulk patient B cells have suggested that similar CD11cposCD21negCD19highT-BETpos cells are critical drivers of humoral autoimmunity in diseases including systemic lupus erythematosus (SLE; Scharer et al. 2019; Rubtsova et al. 2017; Rubtsov et al. 2011). This subset now identified by single-cell RNA-Seq is consistent with a population of TACIhigh B cells that produced IgG in response to BAFF treatment ex vivo (Sarantopoulos 2009). Data suggest we have identified functionally distinct and potentially targetable B cell subpopulations. We are employing functional assays to determine whether the additional molecular pathways now elucidated account for our previous work showing greater ex vivo B cell survival rates and hyper-responsiveness to surrogate antigen (Allen et al. 2012, 2014), certain TLR agonists (Suthers et al. 2017), and NOTCH ligand (Poe et al. 2017). In addition to more deeply characterizing B-cell subsets in cGVHD, our single-cell RNA-Seq analyses identified several genes significantly altered across multiple B cell clusters in the cGVHD group, implicating more broad alterations of some genes in this disease. Among these is CKS2, a critical cell cycle regulator, which was significantly increased in cGVHD B cells (Padj 1.0e-10 to 0.018, depending on the cluster evaluated). Increased CKS2 expression was validated by qPCR analysis on B cells from a separate HCT patient cohort with or without cGVHD (P < 0.001, Figure 1D), suggesting that the majority of cGVHD B cells are primed to enter the cell cycle at multiple stages of differentiation when exposed to the proper stimuli. In summary, we used an unbiased approach to identify and further characterize an extrafollicular CD11cposTACIposCD19high B cell population in cGVHD patients that appears to be activated and undergoing active IgG isotype switching. This plasmablast-like B cell population is potentially amenable to therapeutic intervention to prevent pathogenic antibody production. Importantly, we also identify gene alterations across the cGVHD peripheral B cell compartment that potentially underpin promotion of hyperactivated B cells in this disease. Therapeutic strategies to target these pathways will also be discussed. This work was supported by a National Institutes of Health grant, R01HL129061. Disclosures Ho: Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Horwitz:Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Rizzieri:Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Stemline: Research Funding.
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Dias, V. L., J. C. Young, and K. L. Loveland. "247. BMP signalling in the induction of germline precursors from mouse embryonic stem cells in vitro." Reproduction, Fertility and Development 20, no. 9 (2008): 47. http://dx.doi.org/10.1071/srb08abs247.
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BMP signalling is critical for germline lineage establishment during mouse embryogenesis. To assess its importance in the induction of germline precursors in vitro, a mouse embryonic stem (ES) cell line harbouring an Oct4 promoter-EGFP reporter construct was used to derive embryoid body (EB) aggregates cultured in the absence or presence of combinations of BMP2, BMP4 and BMP8b for 3 to 10 days. At both day 5 and 10 of culture, clearly defined clusters of Oct4-EGFP were observed in EBs cultured with BMPs, while these clusters were minimal to absent in untreated EBs. Quantitative mRNA analysis of day 3 to day 10 EBs revealed a significant elevation in the expression of genes associated with primordial germ cell specification in EBs grown in the presence of BMPs. Moreover, a transient elevation of early germ cell markers Blimp1, Fragilis and Stella was detected in day 3–4 EBs cultured with BMPs, followed their decline by day 5. In contrast, levels of the pluripotency markers, Oct3/4 and Nanog, and the later germ cell markers, Dazl and Vasa, progressively increased from day 3 to day 5. Levels of TGFβ superfamily signalling components ALK2, Smad1 and Smad5 remained relatively constant during this period. Wholemount immunofluorescence of day 5 Oct4-EGFP EBs exposed to BMP4 demonstrated co-localisation of primordial germ cell markers Oct3/4, Stella, and the cell surface antigen SSEA-1 with EGFP+ clusters. These results demonstrate that signalling by BMP2 and 4 enhances germ cell marker expression within EBs and identifies the day 3 to 5 timeframe in EB differentiation as a critical window when putative germ cells are first specified in vitro.
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Shponka, V., S. Kroft, A. M. Harrington, K. Monahan, and E. Atallah. "Non-Acute Myeloid Neoplasms With CD34(-) Blasts: Immunophenotypic And Clinical Analysis Of 5 Cases." American Journal of Clinical Pathology 154, Supplement_1 (October 2020): S105—S106. http://dx.doi.org/10.1093/ajcp/aqaa161.231.
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Abstract Introduction/Objective Non-acute myeloid neoplasms (NAMNs) comprise clonal hematopoietic disorders, characterized by ≤20% myeloblasts in the peripheral blood (PB) and/or bone marrow (BM). The blasts of NAMN are almost exclusively CD34(+), contrasting with blasts in some acute myeloid leukemias (AML). We detail the clinicopathologic features of NAMN with CD34(-) blasts. Methods We searched 981 NAMN pathology reports for CD34(-) blasts by flow cytometry (FC). 8-color FC data was analyzed using cluster analysis to identify all relevant cell populations with a comprehensive antibody panel. CD34(-) blast populations were defined as reproducible clusters with similar light scatter properties across antibody tubes, after exclusion of other cell populations (e.g. basophils, neutrophils, mast cells). Blast aberrancies were defined as differences from established normal antigen expression patterns. PB and BM morphology was reviewed and chart reviews were performed. Results We identified 5 NAMN patients with CD34(-) blasts (2M, 3F; 57-84y/o) including 3 myelodysplastic syndrome (MDSs) with excess blasts, 1 therapy-related MDS, and 1 chronic myelomonocytic leukemia-1 (CMML). Blasts ranged from 0-1.5% in PB and 2.8-8% in BM. CD34(-) blasts accounted for 0.85-8.2% of BM events by FC, with all showing other aberrancies. The CMML patient had CD34(-) blasts with monocytic differentiation; all other CD34(-) blast populations showed expression of CD13, CD33, and CD117 and aberrant under-expression of CD38. Aberrancies were also present in the CD34(+) blasts in all patients (1-4/patient), accounting for 0.01-0.55% of events; 3/5 pts had under-expression of CD33, CD38, CD45, and/or HLA-DR. No cases had adverse cytogenetics. Patient outcomes included: indolent disease (1); died from disease 3 years post-diagnosis and chemotherapy (1); transformed to AML (2; 1-alive in complete remission; 1-died); and alive following allogeneic stem cell transplant (1). Conclusion Our data confirm that CD34(-) blasts are rarely observed in NAMN, and these patients have variable outcomes. Interestingly, all cases showed a concomitant aberrant CD34(+) blast population.
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Ogata, Atsushi, Tomihiro Wakamiya, Masashi Nishihara, Tatsuya Tanaka, Taichiro Mizokami, Jun Masuoka, Nobuaki Momozaki, Shuji Sakata, Hiroyuki Irie, and Tatsuya Abe. "Association between Pericytes in Intraplaque Neovessels and Magnetic Resonance Angiography Findings." International Journal of Molecular Sciences 21, no. 6 (March 13, 2020): 1980. http://dx.doi.org/10.3390/ijms21061980.
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(1) Background: Pericytes are involved in intraplaque neovascularization of advanced and complicated atherosclerotic lesions. However, the role of pericytes in human carotid plaques is unclear. An unstable carotid plaque that shows high-intensity signals on time-of-flight (TOF) magnetic resonance angiography (MRA) is often a cause of ischemic stroke. The aim of the present study is to examine the relationship between the pericytes in intraplaque neovessels and MRA findings. (2) Methods: A total of 46 patients with 49 carotid artery stenoses who underwent carotid endarterectomy at our hospitals were enrolled. The patients with carotid plaques that were histopathologically evaluated were retrospectively analyzed. Intraplaque hemorrhage was evaluated using glycophorin A staining, and intraplaque neovessels were evaluated using CD34 (Cluster of differentiation) stain as an endothelial cell marker or NG2 (Neuron-glial antigen 2) and CD146 stains as pericyte markers. Additionally, the relationships between the TOF-MRA findings and the carotid plaque pathologies were evaluated. (3) Results: Of the 49 stenoses, 28 had high-intensity signals (TOF-HIS group) and 21 had iso-intensity signals (TOF-IIS group) on TOF-MRA. The density of the CD34-positive neovessels was equivalent in both groups. However, the NG2- and CD146-positive neovessels had significantly higher densities in the TOF-HIS group than in the TOF-IIS group. (4) Conclusion: The presence of a high-intensity signal on TOF-MRA in carotid plaques was associated with intraplaque hemorrhage and few pericytes in intraplaque neovessels. These findings may contribute to the development of new therapeutic strategies focusing on pericytes.
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Imran, Jakir Hossain, and Jung Kyung Kim. "A Nut-and-Bolt Microfluidic Mixing System for the Rapid Labeling of Immune Cells with Antibodies." Micromachines 11, no. 3 (March 9, 2020): 280. http://dx.doi.org/10.3390/mi11030280.
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A nut-and-bolt microfluidic system was previously developed for a point-of-care (POC) human immunodeficiency virus (HIV) test and was able to acquire images of CD4 (cluster of differentiation 4) + T-lymphocytes in a sample drop of blood followed by image analysis. However, as the system was not fully integrated with a sample reaction module, the mixing of the sample with the antibody reagent was carried out manually. To achieve a rapid reaction with a reduced amount of costly reagent in a POC diagnostic system, an efficient sample mixing function must be implemented. Here, we propose a novel method to drastically accelerate the process of sample mixing and increase the reaction rate in the nut-and-bolt microfluidic system, where the sample is mixed with the reagent in a reaction chamber formed by connecting a nut with a bolt-like sample cartridge. The mixing is facilitated by rotating the sample cartridge bidirectionally using a DC motor, which agitates the sample in a chaotic manner. A microbead complex formed by the avidin–biotin interaction was used as a model reaction system to examine the feasibility of our mixing module. We found that the reaction time for the avidin–biotin binding by mixing was 7.5 times shorter than in the incubation method, achieving a reaction efficiency of over 95%. The performance of our mixing system was further demonstrated by measuring the concentration of CD4 cells labeled with a fluorescent antibody in the blood sample. The antigen–antibody reaction mixing was faster by a factor of 20, reaching a reaction efficiency comparable to the conventional incubation method.
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Islam, Md Faridul, Md Abdul Alim, Abdul Ali Mia, Md Abdur Razzak, and AFM Shamsul Haque. "Biological therapy for Rheumatoid Arthritis an update." Journal of Armed Forces Medical College, Bangladesh 10, no. 1 (April 8, 2015): 99–102. http://dx.doi.org/10.3329/jafmc.v10i1.22933.
Full textAbstract:
Disease-Modifying Antirheumatic Drugs (DMARDs) play a vital role in the management of Rheumatoid Arthritis (RA). This update aims at focusing some important and novel aspects of biological DMARDs. Recent advances in biological therapy have opened a new window of opportunity for this potentially crippling disorder particularly patients refractory to conventional DMARDs. Close association of cytokine network in the pathophysiology of rheumatoid arthritis has facilitated the development of new biological agents and revolutionized the treatment. Novel drugs such as anti-tumor necrosis factor-alpha (anti-TNF-?) (eg, certolizumab), anti-interleukin-1 (anti-IL-1) (eg, anakinra), anti-interleukin-6 (antiIL-6) (eg, tocilizumab), T-cell depletor (eg, abatacept), anti-cluster differentiation 20 (anti-CD-20) (eg, rituximab) have recently joined with the existing biological therapy in the arena of RA. Emerging agents like adhesion molecule inhibitors, anti-interleukin-15 (anti-IL-15), fusion protein-cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin G1 (CTLA4Ig) are also under investigations. Higher potency, quicker onset of action, less frequency of administration are the main advantages as opposed to potential serious side effects such as infection susceptibility, injection site reaction, Systemic Lupus Erythematosus-like (SLE-like) and Multiple Sclerosis-like (MS-like) symptoms and higher price. Combination of biologicals is not recommended because of higher rate of adverse events and lack of additive effects. But biological DMARDs in combination of Methotrexate (MTX) are now a preferred choice of many rheumatologists. The last but not the least option for aggressive and refractory patients of RA is biological DMARDs. DOI: http://dx.doi.org/10.3329/jafmc.v10i1.22933 Journal of Armed Forces Medical College Bangladesh Vol.10(1) 2014
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Foti, Michelangelo, Aram Mangasarian, Vincent Piguet, Daniel P. Lew, Karl-Heinz Krause, Didier Trono, and Jean-Louis Carpentier. "Nef-mediated Clathrin-coated Pit Formation." Journal of Cell Biology 139, no. 1 (October 6, 1997): 37–47. http://dx.doi.org/10.1083/jcb.139.1.37.
Full textAbstract:
The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.
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Ohshima, Shino, Tatsuya Matsubara, Asuka Miyamoto, Atsuko Shigenari, Noriaki Imaeda, Masaki Takasu, Masafumi Tanaka, et al. "Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs." PLOS ONE 15, no. 11 (November 25, 2020): e0242572. http://dx.doi.org/10.1371/journal.pone.0242572.
Full textAbstract:
Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.
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Burks, Stephen Shelby, Ross C. Puffer, Iahn Cajigas, David Valdivia, Andrew E. Rosenberg, Robert J. Spinner, and Allan D. Levi. "Synovial Sarcoma of the Nerve—Clinical and Pathological Features: Case Series and Systematic Review." Neurosurgery 85, no. 6 (August 21, 2019): E975—E991. http://dx.doi.org/10.1093/neuros/nyz321.
Full textAbstract:
Abstract BACKGROUND Synovial sarcoma of the nerve is a rare entity with several cases and case series reported in the literature. Despite an improved understanding of the biology, the clinical course is difficult to predict. OBJECTIVE To compile a series of patients with synovial sarcoma of the peripheral nerve (SSPN) and assess clinical and pathological factors and their contribution to survival and recurrence. METHODS Cases from 2 institutions collected in patients undergoing surgical intervention for SSPN. Systematic review including PubMed and Scopus databases were searched for related articles published from 1970 to December 2018. Eligibility criteria: (1) case reports or case series reporting on SSPN, (2) clinical course and/or pathological features of the tumor reported, and (3) articles published in English. RESULTS From patients treated at our institutions (13) the average follow-up period was 3.2 yr. Tumor recurrence was seen in 4 cases and death in 3. Systematic review of the literature yielded 44 additional cases with an average follow-up period of 3.6 yr. From pooled data, there were 10 recurrences and 7 deaths (20% and 14%, respectively). Adjuvant treatment used in 62.5% of cases. Immunohistochemical markers used in diagnosis varied widely; the most common are the following: Epithelial membrane antigen (EMA), cytokeratin, vimentin, cluster of differentiation (CD34), and transducin-like enhancer of split 1 (TLE1). Statistical analysis illustrated tumor size and use of chemotherapy to be negative predictors of survival. No other factors, clinically or from pathologist review, were correlated with recurrence or survival. CONCLUSION By combining cases from our institution with historical data and performing statistical analysis we show correlation between tumor size and death.
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Wang, Meng, Iulian Pruteanu, Adam D. Cohen, Alfred L. Garfall, Michael C. Milone, Lifeng Tian, Vanessa E. Gonzalez, et al. "Identification and Validation of Predictive Biomarkers to CD19- and BCMA-Specific CAR T-Cell Responses in CAR T-Cell Precursors." Blood 134, Supplement_1 (November 13, 2019): 622. http://dx.doi.org/10.1182/blood-2019-122513.
Full textAbstract:
CD19-specific chimeric antigen receptor (CAR) T cell therapies have been highly effective against B cell malignancies. We previously demonstrated that differential responses to anti-CD19 CAR T cell therapy in chronic lymphocytic leukemia (CLL) are associated with early memory T cell signature in apheresed, pre-manufacturing T-cells (CAR T-cell precursors). We tested the hypothesis that the composition of CAR-T precursor cells determines clinical efficacy in adult and pediatric Acute Lymphoblastic Leukemia (ALL), Non-Hodgkin's Lymphoma (NHL), Multiple Myeloma (MM), and CLL. Apheresed T cells were engineered to express 4-1BB plus CD3-zeta-signaling CARs targeting CD19, or B cell maturation antigen (BCMA). The same 9-day manufacturing process was used for all trials. CAR T cell kinetics were monitored using a CAR gene-specific quantitative PCR assay and standard clinical response assessments were performed. Apheresed T cells from 36 CLL, 30 adult ALL, 58 pediatric ALL, 33 NHL, and 25 MM patients were immunophenotyped by flow cytometry. The CLL cohort was used to discover phenotypically distinct subpopulations associated with the two main response groups; these associations were validated in the remaining patient cohorts. Eight CD8+ T cell populations or clusters were identified using the shared-nearest-neighbor clustering method (PMID: 31178118) in the CLL cohort. T cell subsets exhibiting naive (cluster 6) or early memory (cluster 4) features were significantly enriched in responding patients, whereas an effector memory CD8 subpopulation (cluster 2) marked the non-responding patients. Mapping these clusters onto apheresed CD8+ T cells from the other four diseases showed that cluster 4 predicted response to CAR T cell therapy in NHL and myeloma but not in adult and pediatric ALL. We also examined the expression of activation-regulated molecules including HLA-DR, Ki67, and exhaustion-related molecules PD1, CTLA4, TIM3, and LAG3. A CD27+ CD8+ population expressing low level CTLA4 but none of the activation or negative regulatory molecules was significantly enriched in responding CLL patients; this cluster validated in NHL and myeloma. A similar analysis on apheresed CD4+ T cells identified an early memory population (cluster 6) enriched in CLL responders, which expresses CCR7 and CD27 but not CD45RO, CD127, CD28, or other late memory/effector molecules. However, this population did not validate in any of the other diseases. Though not statistically significant, the CD4+ clusters with the largest effect size for enrichment in responders from NHL and myeloma trials exhibited early memory T cell features and lack of HLA-DR expression, suggesting that quiescent early memory state in CD4 may also be associated with clinical responses. A separate analysis of checkpoint inhibitory receptors and activation markers in memory CD4 T cell subsets confirmed the early memory, non-activated state of this population in CLL and was validated in myeloma but none of the other diseases. In vivo activation was a shared theme in CD4+ T cells for non-responding patients as well, though these CLL-defined CD4+ apheresed T cells clusters did not significantly validate in other diseases. In summary, our data confirm and extend our predictive biomarker profile in CLL to mature B cell and plasma cell malignancies by showing that a non-cycling, non-activated early memory CD8+ T cell population in pre-manufacturing cells was validated as a biomarker in myeloma, and NHL. We also showed that responder-associated apheresed CD4+ T cells with early memory features identified in CLL after CD19 CAR T infusions are validated in myeloma after BCMA CAR T. Thus, differentiation state and in vivo activation, and potentially exhaustion, separate response groups. Our findings inform next-generation CAR T-cell manufacturing using the populations identified herein as a starting population. Disclosures Pruteanu: Novartis: Employment. Cohen:Poseida Therapeutics, Inc.: Research Funding. Garfall:Surface Oncology: Consultancy; Novartis: Research Funding; Janssen: Research Funding; Amgen: Research Funding; Tmunity: Research Funding. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Gill:Novartis: Research Funding; Tmunity: Research Funding; Carisma: Equity Ownership, Research Funding; Sensei: Consultancy; Aro: Consultancy; Fate: Consultancy. Frey:Novartis: Research Funding. Ruella:Nanostring: Consultancy, Speakers Bureau; Novartis: Patents & Royalties: CART for cancer; AbClon: Membership on an entity's Board of Directors or advisory committees. Lacey:Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding; Novartis: Research Funding. Svoboda:Merck: Research Funding; BMS: Consultancy, Research Funding; Incyte: Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Kite: Consultancy; Seattle Genetics: Consultancy, Research Funding; Kyowa: Consultancy; AstraZeneca: Consultancy. Chong:Tessa: Consultancy; Novartis: Consultancy; Merck: Research Funding. Fraietta:LEK Consulting: Consultancy; Cabaletta: Research Funding; Tmunity: Research Funding. Davis:Cabaletta: Research Funding; Tmunity: Research Funding. Nasta:Rafael: Research Funding; Aileron: Research Funding; Takeda/Millennium: Research Funding; Incyte: Research Funding; Roche/Genentech: Research Funding; Merck: Consultancy; Atara: Research Funding; Debiopharm: Research Funding. Levine:CRC Oncology: Consultancy; Vycellix: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership; Novartis: Consultancy, Patents & Royalties, Research Funding; Cure Genetics: Consultancy; Avectas: Membership on an entity's Board of Directors or advisory committees; Brammer Bio: Membership on an entity's Board of Directors or advisory committees; Incysus: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Maude:Kite: Consultancy; Novartis: Consultancy. Schuster:Nordic Nanovector: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Pharmacyclics: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Loxo Oncology: Honoraria; Merck: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Novartis: Honoraria, Patents & Royalties: Combination Therapies of CAR and PD-1 Inhibitors with royalties paid to Novartis, Research Funding; AbbVie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Stadtmauer:Celgene: Consultancy; Tmunity: Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Research Funding. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Cure Genetics: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards. Porter:Incyte: Membership on an entity's Board of Directors or advisory committees; American Board of Internal Medicine: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Glenmark Pharm: Membership on an entity's Board of Directors or advisory committees; Immunovative: Membership on an entity's Board of Directors or advisory committees; Genentech: Employment; Wiley and Sons: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:Novartis: Research Funding, Speakers Bureau; Parker Institute for Cancer Immunotherapy: Research Funding; Stand Up to Cancer: Research Funding; Incyte: Research Funding; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; National Institutes of Health: Research Funding.
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Fedyanina, O. S., A. E. Zadorozhnaya, A. N. Khvastunova, E. M. Koltsova, E. N. Balashova, L. A. Timofeeva, A. L. Karavaeva, et al. "Leukocyte subgroup distribution and morphology in blood of premature and full-term newborn babies studied by the cell microarray." Pediatric Hematology/Oncology and Immunopathology 17, no. 4 (January 13, 2019): 11–16. http://dx.doi.org/10.24287/1726-1708-2018-17-4-11-16.
Full textAbstract:
Both the ratio of different leukocyte subgroup content and the leukocyte morphology in peripheral blood of newborns are important in diagnosis of several diseases including combined immunodeficiency and neonatal septicemia. There is a need for development of screening methods for parallel study of the leukocyte morphology and population structure in the newborn peripheral blood. We aimed to determine the relative abundance of different leukocyte subsets and to study their morphology in full-term and premature newborn babies and healthy adult volunteers using the cell-binding microarray – a transparent support with immobilized antibodies against leukocyte cluster-of-differentiation antigens. The work was supported by the Scientific council and approved by the ethical committee of the Centre. We have studied the peripheral blood of 12 full-term newborns (38–40 weeks gestation), 9 premature newborns (22–32 weeks gestation) and 18 healthy adults. The relative abundance of the leukocyte and their morphology were determined using the cell-binding microarray including antibodies against CD2, СD3, СD4, CD5, СD7, CD8, CD10, СD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD33, CD38, CD41a, CD45, CD45RA, CD45RO, CD61, CD64, CD117, CD123, HLA-DR. The percentage of leukocytes positive for every of the studied surface CD antigens among the peripheral blood mononuclear cells of full-term and preterm newborn babies and healthy adults determined on the cell–binding microarray are in good agreement with published flow cytometry data. CD11b+ leukocytes both in premature and full-term newborns included up to 21% myelocytes and 27% metamyelocytes. The reported data can be used as reference values in cell-binding microarray application in diagnosis of combined immunodeficiency or neonatal septicemia.
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Hastings, R. H., D. Summers-Torres, B. Yaszay, J. LeSueur, D. W. Burton, and L. J. Deftos. "Parathyroid hormone-related protein, an autocrine growth inhibitor of alveolar type II cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 272, no. 3 (March 1, 1997): L394—L399. http://dx.doi.org/10.1152/ajplung.1997.272.3.l394.
Full textAbstract:
Parathyroid hormone-related protein (PTHRP) is an autocrine regulator of differentiation for type II pneumocytes [R. H. Hastings, D. Summers-Torres, T. C. Cheung, L. S. Ditmer, D. W. Burton, E. M. Petrin, R. G. Spragg, J. Li, and L. J. Deftos. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L353-L361, 1996]. We investigated autocrine effects on growth by decreasing endogenous PTHRP in rat type II cells. Cultured cells were incubated with antibodies against PTHRP-(1-34) (8B12) or PTHRP-(109-141) (9H7) or an irrelevant antibody (1 microg/ml) for 3 days. Conditioned media from the irrelevant antibody group contained 143 +/- 8 fg PTHRP/ 100,000 cells. The 8B12 and 9H7 reduced levels to 45 +/- 8 and 88 +/- 16 fg PTHRP/100,000 cells, respectively (n = 4 cell isolations, P < 0.05). Cells treated with the PTHRP antibodies nearly tripled in number. The irrelevant antibody had no effect on growth. Exogenous PTHRP-(1-34) (2.5 nM) blocked the growth-stimulating effect of 9H7. Instilled intratracheal 8B12 and 9H7 induced expression of proliferating cell nuclear antigen in clusters of alveolar cells in rats. Clustered cells expressed surfactant apoproteins and cytokeratin 19. These data suggest that endogenous PTHRP-(1-34) inhibits proliferation of type II cells in vivo and in vitro.