ABSTRACT
We have recently identified a novel lymphocyte that is a dual expresser (DE) of TCRαß and BCR. DEs in T1D patients are predominated by a public BCR clonotype (clone-x) that encodes a potent autoantigen that cross-activates insulin-reactive T cells. Betts and colleagues were able to detect DEs but alleged to not detect high DE frequency, clone-x, or similar clones in T1D patients. Unfortunately, the authors did not follow our methods and when they did, their flow cytometric data at two sites were conflicting. Moreover, contrary to their claim, we identified clones similar to clone-x in their data along with clones bearing the core motif (DTAMVYYFDYW). Additionally, their report of no increased usage of clone-x VH/DH genes by bulk B cells confirms rather than challenges our results. Finally, the authors failed to provide data verifying purity of their sorted DEs, making it difficult to draw reliable conclusion of their repertoire analysis. This Matters Arising Response paper addresses the Japp et al. (2021) Matters Arising paper, published concurrently in Cell.
Subject(s)
Diabetes Mellitus, Type 1 , B-Lymphocytes , Clone Cells , Humans , Receptors, Antigen, T-Cell, alpha-beta , T-LymphocytesABSTRACT
T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.
Subject(s)
B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , Diabetes Mellitus, Type 1/immunology , Adolescent , Adult , Autoantigens/immunology , Child , Child, Preschool , Diabetes Mellitus, Type 1/metabolism , Epitopes/immunology , Female , HEK293 Cells , HLA-DQ Antigens/immunology , HLA-DQ Antigens/ultrastructure , Humans , Lymphocyte Activation/immunology , Lymphocytes/immunology , Lymphocytes/metabolism , Male , Middle Aged , Molecular Dynamics Simulation , Peptides , Protein Binding/immunologyABSTRACT
Increasing evidence shows pathophysiological significance of rare immune cells, necessitating the need for reliable and proper methods for their detection and analysis. We have recently identified a new lymphocyte that coexpresses lineage markers of T- and B-cells including T cell receptor and B cell receptor (called dual expressers, DEs). Because of the peculiar phenotype of DEs, we used multiple techniques to authenticate their identity (fluorescence-activated cell sorting [FACS], scRNAseq, EBV cell lines, and imaging flow cytometry). In an recent article published in this journal, Burel and colleagues successfully detected DEs using FACS and imaging microscopy. Yet they claimed, based on the profile of what they called naturally occurring CD3+ CD14+ T cell/monocyte complexes that the scRNAseq signature of DEs resembles that of cell-cell complexes. Serious flaws in their analysis, however, invalidate their conclusions. Unlike the CD3+ CD14+ complexes, DEs have a distinct identity due to expression of a unique set of signature genes. Without a clear explanation, Burel and colleagues excluded these genes from their analysis, thereby effectively stripped DEs from their identity. Inclusion of these genes as described in this communication restores the identity of DEs. Moreover, contrary to the claim of Burel and colleagues, B- and T-cell specific genes are similarly expressed in DE cells.
Subject(s)
B-Lymphocytes , Receptors, Antigen, T-Cell, alpha-beta , CD3 Complex/genetics , Flow Cytometry/methods , Lymphocytes/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-LymphocytesABSTRACT
Genetic deletion of Src associated in mitosis of 68kDa (Sam68), a pleiotropic adaptor protein prevents high-fat diet-induced weight gain and insulin resistance. To clarify the role of Sam68 in energy metabolism in the adult stage, we generated an inducible Sam68 knockout mice. Knockout of Sam68 was induced at the age of 7-10 weeks, and then we examined the metabolic profiles of the mice. Sam68 knockout mice gained less body weight over time and at 34 or 36 weeks old, had smaller fat mass without changes in food intake and absorption efficiency. Deletion of Sam68 in mice elevated thermogenesis, increased energy expenditure, and attenuated core-temperature drop during acute cold exposure. Furthermore, we examined younger Sam68 knockout mice at 11 weeks old before their body weights deviate, and confirmed increased energy expenditure and thermogenic gene program. Thus, Sam68 is essential for the control of adipose thermogenesis and energy homeostasis in the adult.
Subject(s)
Adaptor Proteins, Signal Transducing/deficiency , Energy Metabolism , Thermogenesis , Adaptor Proteins, Signal Transducing/metabolism , Animals , Male , Mice , Mice, Knockout , RNA-Binding Proteins/metabolismABSTRACT
Gastroesophageal junction (GEJ) cancer remains a clinically significant disease in Western countries due to its increasing incidence, which mirrors that of esophageal cancer, and poor prognosis. To develop novel and effective approaches for prevention, early detection, and treatment of patients with GEJ cancer, a better understanding of the mechanisms driving pathogenesis and malignant progression of this disease is required. These efforts have been limited by the small number of available cell lines and appropriate preclinical animal models for in vitro and in vivo studies. We have established and characterized a novel GEJ cancer cell line, GEAMP, derived from the malignant pleural effusion of a previously treated GEJ cancer patient. Comprehensive genetic analyses confirmed a clonal relationship between GEAMP cells and the primary tumor. Targeted next-generation sequencing identified 56 nonsynonymous alterations in 51 genes including TP53 and APC, which are commonly altered in GEJ cancer. In addition, multiple copy-number alterations were found including EGFR and K-RAS gene amplifications and loss of CDKN2A and CDKN2B. Histological examination of subcutaneous flank xenografts in nude and NOD-SCID mice showed a carcinoma with mixed squamous and glandular differentiation, suggesting GEAMP cells contain a subpopulation with multipotent potential. Finally, pharmacologic inhibition of the EGFR signaling pathway led to downregulation of key downstream kinases and inhibition of cell proliferation in vitro. Thus, GEAMP represents a valuable addition to the limited number of bona fide GEJ cancer cell lines.
Subject(s)
Adenocarcinoma/pathology , Cell Line, Tumor , Esophageal Neoplasms/pathology , Esophagogastric Junction/pathology , Pleural Effusion, Malignant/pathology , Adenocarcinoma/therapy , Animals , ErbB Receptors/antagonists & inhibitors , Esophageal Neoplasms/therapy , Fatal Outcome , Female , Humans , Male , Mice , Mice, Nude , Mice, SCID , Middle Aged , Pleural Effusion, Malignant/therapy , Xenograft Model Antitumor AssaysABSTRACT
While memory T-cells represent a hallmark of adaptive immunity, little is known about the genetic mechanisms regulating the longevity of memory CD4 T cells. Here, we studied the dynamics of gene expression in antigen specific CD4 T cells during infection, memory differentiation, and long-term survival up to nearly a year in mice. We observed that differentiation into long lived memory cells is associated with increased expression of genes inhibiting cell proliferation and apoptosis as well as genes promoting DNA repair response, lipid metabolism, and insulin resistance. We identified several transmembrane proteins in long-lived murine memory CD4 T cells, which co-localized exclusively within the responding antigen-specific memory CD4 T cells in human. The unique gene signatures of long-lived memory CD4 T cells, along with the new markers that we have defined, will enable a deeper understanding of memory CD4 T cell biology and allow for designing novel vaccines and therapeutics.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Immunologic Memory/immunology , Adult , Aging/genetics , Animals , CD4-Positive T-Lymphocytes/physiology , Cell Differentiation/immunology , Cell Proliferation/genetics , Cytokines/metabolism , Humans , Immunologic Memory/genetics , Interferon-gamma/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred BALB C , Mice, TransgenicABSTRACT
STUDY OBJECTIVE: To investigate the frequency of Beers Criteria (BC) medication and opioid use in patients age 65â¯years and older arriving in the Emergency Department. METHODS: We performed a retrospective observational study of a convenience sample of 400 patients, age 65â¯years and older, arriving to and discharged solely from the Emergency Department. We examined 400 sequential patient charts with visit dates April-July 2017, for the presence of a Beers Criteria medication or opioid prescription. We also examined each chart for nine specific chief complaints, including return visits and subsequent admissions. RESULTS: Of the 400 patients included in this study, 304 patients (76%; 95% confidence interval [CI] 72% to 80%) had at least 1 prescription at the index ED visit for an "avoid" or "use with caution" Beers Criteria medication. Of these patients, 194 (64%; 95% CI 58% to 69%) had ≥2 Beers medication prescriptions and 122 patients (40%; 95% CI 35% to 46%) had ≥3 Beers medication prescriptions. We found no difference in the number of patients with a chief complaint of interest between the BC medication list (28%) and lacking a BC medication (29%) (p-valueâ¯=â¯1). No patients returned in the next 7â¯days for a medication-related complaint. CONCLUSION: The results of this study call into question the routine application of lists without high-quality evidence to critique the prescribing of certain medications. Further patient-oriented study of the relevance of the Beers Criteria list, especially in light of the changed face of medication profiles and populations, is called for.
Subject(s)
Emergency Service, Hospital/statistics & numerical data , Potentially Inappropriate Medication List/statistics & numerical data , Practice Patterns, Physicians' , Analgesics, Opioid/administration & dosage , Female , Humans , Inappropriate Prescribing/statistics & numerical data , Male , Potentially Inappropriate Medication List/standards , Retrospective StudiesABSTRACT
The DNA entry and exit points on the nucleosome core regulate the initial invasion of the nucleosome by factors requiring access to the underlying DNA. Here we describe in vivo consequences of eliminating a single protein-DNA interaction at this position through mutagenesis of histone H3 Lys 42 to alanine. This substitution has a dramatic effect on the Saccharomyces cerevisiae transcriptome in both the transcriptional output and landscape of mRNA species produced. We attribute this in part to decreased histone H3 occupancy at transcriptionally active loci, leading to enhanced elongation. Additionally we show that this lysine is methylated in vivo, and genetic studies of methyl-lysine mimics suggest that this modification may be crucial in attenuating gene expression. Interestingly, this site of methylation is unique to Ascomycota, suggesting a recent evolutionary innovation that highlights the evolvability of post-translational modifications of chromatin.
Subject(s)
Evolution, Molecular , Gene Expression Regulation, Fungal , Histones/genetics , Histones/metabolism , Lysine/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Chromatin/chemistry , DNA Methylation , Gene Expression Profiling , Histones/chemistry , Lysine/chemistry , Models, Molecular , Mutation , Nuclear Proteins/metabolism , Peptide Elongation Factors/metabolism , Phenotype , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
BACKGROUND: Polycythemia vera is the ultimate phenotypic consequence of the V617F mutation in Janus kinase 2 (encoded by JAK2), but the extent to which this mutation influences the behavior of the involved CD34+ hematopoietic stem cells is unknown. METHODS: We analyzed gene expression in CD34+ peripheral-blood cells from 19 patients with polycythemia vera, using oligonucleotide microarray technology after correcting for potential confounding by sex, since the phenotypic features of the disease differ between men and women. RESULTS: Men with polycythemia vera had twice as many up-regulated or down-regulated genes as women with polycythemia vera, in a comparison of gene expression in the patients and in healthy persons of the same sex, but there were 102 genes with differential regulation that was concordant in men and women. When these genes were used for class discovery by means of unsupervised hierarchical clustering, the 19 patients could be divided into two groups that did not differ significantly with respect to age, neutrophil JAK2 V617F allele burden, white-cell count, platelet count, or clonal dominance. However, they did differ significantly with respect to disease duration; hemoglobin level; frequency of thromboembolic events, palpable splenomegaly, and splenectomy; chemotherapy exposure; leukemic transformation; and survival. The unsupervised clustering was confirmed by a supervised approach with the use of a top-scoring-pair classifier that segregated the 19 patients into the same two phenotypic groups with 100% accuracy. CONCLUSIONS: Removing sex as a potential confounder, we identified an accurate molecular method for classifying patients with polycythemia vera according to disease behavior, independently of their JAK2 V617F allele burden, and identified previously unrecognized molecular pathways in polycythemia vera outside the canonical JAK2 pathway that may be amenable to targeted therapy. (Funded by the Department of Defense and the National Institutes of Health.).
Subject(s)
Gene Expression , Janus Kinase 2/genetics , Phenotype , Polycythemia Vera/genetics , Aged , Aged, 80 and over , Antigens, CD34 , Blood Cell Count , Confounding Factors, Epidemiologic , Female , Gene Expression Regulation , Humans , Janus Kinase 2/metabolism , Male , Metabolic Networks and Pathways , Middle Aged , Oligonucleotide Array Sequence Analysis , Polycythemia Vera/classification , Polycythemia Vera/metabolism , Sex FactorsABSTRACT
Ischemia-reperfusion injury (IRI) is a major cause of AKI, and previous studies established important roles for conventional CD4(+) T cells, natural killer T cells, and CD4(+)CD25(+)FoxP3(+) Tregs in AKI pathogenesis. We recently identified CD4(-)CD8(-) (double-negative; DN) T cells as an important subset of αß T cell receptor-positive cells residing in mouse kidney. However, little is known about the pathophysiologic functions of kidney DN T cells. In this study, we phenotypically and functionally characterized murine kidney DN T cells in the steady state and in response to IRI. Unlike CD4(+) and CD8(+) T cells, DN T cells in the steady state expressed high levels of CD69, CD28, and CD40L; differentially expressed IL-27 and IL-10 anti-inflammatory cytokines; spontaneously proliferated at a very high rate; and suppressed in vitro proliferation of activated CD4(+) T cells. Within the first 3-24 hours after IRI, kidney DN T cells expanded significantly and upregulated expression of IL-10. In adoptive transfer experiments, DN T cells significantly protected recipients from AKI by an IL-10-dependent mechanism. DN T cells also made up a large fraction of the T cell compartment in human kidneys. Our results indicate that DN T cells are an important subset of the resident αß(+) T cell population in the mammalian kidney and are early responders to AKI that have anti-inflammatory properties.
Subject(s)
Acute Kidney Injury/immunology , Kidney/immunology , T-Lymphocytes/physiology , Animals , CD4 Antigens , CD8 Antigens , Humans , Male , Mice , Mice, Inbred C57BLABSTRACT
Reactivation of latent human cytomegalovirus is a significant infectious complication of organ transplantation and current therapies target viral replication once reactivation of latent virus has already occurred. The specific molecular pathways that activate viral gene expression in response to transplantation are not well understood. Our studies aim to identify these factors, with the goal of developing novel therapies that prevent transcriptional reactivation in transplant recipients. Murine cytomegalovirus (MCMV) is a valuable model for studying latency and reactivation of CMV in vivo. We previously demonstrated that transplantation of MCMV-latently infected kidneys into allogeneic recipients induces reactivation of immediate early (IE) gene expression and epigenetic reprogramming of the major immediate early promoter (MIEP) within 48âh. We hypothesize that these events are mediated by activation of signalling pathways that lead to binding of transcription factors to the MIEP, including AP-1 and NF-κB. Here we show that transplantation induces rapid activation of several members of the AP-1 and NF-κB transcription factor family and we demonstrate that canonical NF-κB (p65/p50), the junD component of AP-1, and nucleosome remodelling complexes are recruited to the MIEP following transplantation. Proteomic analysis of recipient plasma and transcriptome analysis of kidney RNA identified five extracellular ligands, including TNF, IL-1ß, IL-18, CD40L and IL-6, and three intracellular signalling pathways associated with reactivation of IE gene expression. Identification of the factors that mediate activation of these signalling pathways may eventually lead to new therapies to prevent reactivation of CMV and its sequelae.
Subject(s)
Herpesviridae Infections/genetics , Immediate-Early Proteins/genetics , Kidney Transplantation , Muromegalovirus/genetics , NF-kappa B/genetics , Transcription Factor AP-1/genetics , Virus Activation , Animals , CD40 Ligand/genetics , CD40 Ligand/immunology , Female , Gene Expression Regulation/immunology , Herpesviridae Infections/immunology , Herpesviridae Infections/pathology , Host-Pathogen Interactions , Immediate-Early Proteins/immunology , Interleukin-18/genetics , Interleukin-18/immunology , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Muromegalovirus/immunology , NF-kappa B/immunology , Nucleosomes/genetics , Nucleosomes/immunology , Promoter Regions, Genetic , Proteome/genetics , Proteome/immunology , Signal Transduction , Transcription Factor AP-1/immunology , Transplantation, Homologous , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Virus LatencyABSTRACT
Invariant natural killer T (iNKT) cells recognize glycolipids as antigens and diversify into NKT1 (IFN-γ), NKT2 (IL-4), and NKT17 (IL-17) functional subsets while developing in the thymus. Mechanisms that govern the balance between these functional subsets are poorly understood due, partly, to the lack of distinguishing surface markers. Here we identify the heparan sulfate proteoglycan syndecan-1 (sdc1) as a specific marker of naïve thymic NKT17 cells in mice and show that sdc1 deficiency significantly increases thymic NKT17 cells at the expense of NKT1 cells, leading to impaired iNKT cell-derived IFN-γ, both in vitro and in vivo. Using surface expression of sdc1 to identify NKT17 cells, we confirm differential tissue localization and interstrain variability of NKT17 cells, and reveal that NKT17 cells express high levels of TCR-ß, preferentially use Vß8, and are more highly sensitive to É-GalCer than to CD3/CD28 stimulation. These findings provide a novel, noninvasive, simple method for identification, and viable sorting of naïve NKT17 cells from unmanipulated mice, and suggest that sdc1 expression negatively regulates homeostasis in iNKT cells. In addition, these findings lay the groundwork for investigating the mechanisms by which sdc1 regulates NKT17 cells.
Subject(s)
Natural Killer T-Cells/immunology , Syndecan-1/immunology , T-Lymphocyte Subsets/immunology , Animals , Cell Separation/methods , Gene Expression Profiling , Interleukin-17/biosynthesis , Interleukin-17/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Natural Killer T-Cells/metabolism , Oligonucleotide Array Sequence Analysis , T-Lymphocyte Subsets/metabolismABSTRACT
The myeloid differentiation protein 88 (MyD88) adapter protein is an important mediator of kidney allograft rejection, yet the precise role of MyD88 signaling in directing the host immune response toward the development of kidney allograft rejection remains unclear. Using a stringent mouse model of allogeneic kidney transplantation, we demonstrated that acute allograft rejection occurred equally in MyD88-sufficient (wild-type [WT]) and MyD88(-/-) recipients. However, MyD88 deficiency resulted in spontaneous diminution of graft infiltrating effector cells, including CD11b(-)Gr-1(+) cells and activated CD8 T cells, as well as subsequent restoration of near-normal renal graft function, leading to long-term kidney allograft acceptance. Compared with T cells from WT recipients, T cells from MyD88(-/-) recipients failed to mount a robust recall response upon donor antigen restimulation in mixed lymphocyte cultures ex vivo. Notably, exogenous IL-6 restored the proliferation rate of T cells, particularly CD8 T cells, from MyD88(-/-) recipients to the proliferation rate of cells from WT recipients. Furthermore, MyD88(-/-) T cells exhibited diminished expression of chemokine receptors, specifically CCR4 and CXCR3, and the impaired ability to accumulate in the kidney allografts despite an otherwise MyD88-sufficient environment. These results provide a mechanism linking the lack of intrinsic MyD88 signaling in T cells to the effective control of the rejection response that results in spontaneous resolution of acute rejection and long-term graft protection.
Subject(s)
Graft Rejection , Immunologic Deficiency Syndromes/genetics , Kidney Transplantation , Kidney/immunology , Myeloid Differentiation Factor 88/genetics , Allografts , Animals , CD11b Antigen/metabolism , Cell Proliferation , Graft Survival , Interleukin-6/metabolism , Kidney/pathology , Lymphocyte Culture Test, Mixed , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Knockout , Primary Immunodeficiency Diseases , Receptors, CCR4/metabolism , Receptors, CXCR3/metabolism , Signal Transduction , Skin Transplantation , T-Lymphocytes/cytology , Transplantation, HomologousABSTRACT
UNLABELLED: Biomarkers predictive of recovery from acute kidney injury (AKI) after liver transplantation (LT) could enhance decision algorithms regarding the need for liver-kidney transplantation or renal sparing regimens. Multianalyte plasma/urine kidney injury protein panels were performed immediately before and 1 month post-LT in an initial test group divided by reversible pre-LT AKI (rAKI = post-LT renal recovery) versus no AKI (nAKI). This was followed by a larger validation set that included an additional group: irreversible pre-LT AKI (iAKI = no post-LT renal recovery). In the test group (n = 16), six pre-LT plasma (not urine) kidney injury proteins (osteopontin [OPN], neutrophil gelatinase-associated lipocalin, cystatin C, trefoil factor 3, tissue inhibitor of metalloproteinase [TIMP]-1, and ß-2-microglobulin) were higher in rAKI versus nAKI (P < 0.05) and returned to normal values with renal recovery post-LT. In the validation set (n = 46), a number of proteins were significantly higher in both rAKI and iAKI versus nAKI. However, only pre-LT plasma OPN (P = 0.009) and TIMP-1 (P = 0.019) levels were significantly higher in rAKI versus iAKI. Logistic regression modeling was used to correlate the probability of post-LT rAKI, factoring in both pre-LT protein markers and clinical variables. A combined model including elevated OPN and TIMP-1 levels, age <57, and absence of diabetes had the highest area under the curve of 0.82, compared to protein-only and clinical variable-only models. CONCLUSION: These data suggest that plasma protein profiles might improve the prediction of pre-LT kidney injury recovery after LT. However, multicenter, prospective studies are needed to validate these findings and ultimately test the value of such protein panels in perioperative management and decision making.
Subject(s)
Acute Kidney Injury/blood , Biomarkers/blood , Liver Diseases/blood , Liver Transplantation , Acute Kidney Injury/etiology , Acute-Phase Proteins , Aged , Cystatin C/blood , Female , Humans , Lipocalin-2 , Lipocalins/blood , Liver Diseases/complications , Liver Diseases/surgery , Male , Middle Aged , Osteopontin/blood , Peptides/blood , Predictive Value of Tests , Prospective Studies , Proto-Oncogene Proteins/blood , Recovery of Function , Tissue Inhibitor of Metalloproteinase-1/blood , Trefoil Factor-3 , beta 2-Microglobulin/bloodABSTRACT
BACKGROUND: Metastatic initiation has many phenotypic similarities to epithelial-to-mesenchymal transition, including loss of cell-cell adhesion, increased invasiveness, and increased cell mobility. We have previously demonstrated that drug resistance is associated with a metastatic phenotype in neuroblastoma (NB). The purpose of this project was to determine if the development of doxorubicin resistance is associated with characteristics of mesenchymal change in human NB cells. MATERIALS AND METHODS: Total RNA was isolated from wild type (WT) and doxorubicin-resistant (DoxR) human NB cell lines (SK-N-SH and SK-N-BE(2)C) and analyzed using the Illumina Human HT-12 version 4 Expression BeadChip. Differentially expressed genes (DEGs) were identified. Volcano plots and heat maps were generated. Genes of interest with a fold change in expression >1.5 and an adjusted P < 0.1 were analyzed. Immunofluorescence (IF) and Western blot analysis confirmed microarray results of interest. Matrigel invasion assay and migration wounding assays were performed. RESULTS: Volcano plots and heat maps visually demonstrated a similar pattern of DEGs in the SK-N-SH and SK-N-BE(2)C DoxR cell lines relative to their parental WT lines. Venn diagramming revealed 1594 DEGs common to both DoxR cell lines relative to their parental cell lines. Network analysis pointed to several significantly upregulated epithelial-to-mesenchymal transition pathways, through TGF-beta pathways via RhoA, PI3K, and ILK and via SMADs, as well as via notch signaling pathways. DoxR cell lines displayed a more invasive phenotype than respective WT cell lines. CONCLUSIONS: Human SK-N-SH and SK-N-BE(2)C NB cells display characteristics of mesenchymal change via multiple pathways in the transition to a drug-resistant state.
Subject(s)
Doxorubicin/pharmacology , Drug Resistance, Neoplasm/genetics , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Neuroblastoma , Antibiotics, Antineoplastic/pharmacology , Cell Adhesion , Cell Line, Tumor , Cell Movement , Female , Gene Expression Regulation, Neoplastic , Genome-Wide Association Study , Humans , Male , Neoplasm Invasiveness , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Neuroblastoma/secondary , Phenotype , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
Ectopic Myc expression plays a key role in human tumorigenesis, and Myc dose-dependent tumorigenesis has been well established in transgenic mice, but the Myc target genes that are dependent on Myc levels have not been well characterized. In this regard, we used the human P493-6 B cells, which have a preneoplastic state dependent on the Epstein-Barr viral EBNA2 protein and a neoplastic state with ectopic inducible Myc, to identify putative ectopic Myc target genes. Among the ectopic targets, JAG2 that encodes a Notch receptor ligand Jagged2, was directly induced by Myc. Inhibition of Notch signaling through RNAi targeting JAG2 or the gamma-secretase Notch inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) preferentially inhibited the neoplastic state in vitro. Furthermore, P493-6 tumorigenesis was inhibited by DAPT in vivo. Ectopic expression of JAG2 did not enhance aerobic cell proliferation, but increased proliferation of hypoxic cells in vitro and significantly increased in vivo tumorigenesis. Furthermore, the expression of Jagged2 in P493-6 tumors often overlapped with regions of hypoxia. These observations suggest that Notch signaling downstream of Myc enables cells to adapt in the tumor hypoxic microenvironment.
Subject(s)
Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Leukemic , Intercellular Signaling Peptides and Proteins/genetics , Lymphoma, B-Cell/genetics , Membrane Proteins/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Notch/metabolism , Animals , B-Lymphocytes/metabolism , Cell Hypoxia/genetics , Cell Proliferation , Dipeptides/pharmacology , Gene Expression Profiling , Humans , Jagged-2 Protein , Mice , Models, Biological , RNA Interference , Receptors, Notch/antagonists & inhibitors , Receptors, Notch/genetics , Transcriptional ActivationABSTRACT
Understanding the roles of different cell types in regulating T cell homeostasis in various tissues is critical for understanding adaptive immunity. Here, we show that RTECs (renal tubular epithelial cells) are intrinsically programmed to polyclonally stimulate proliferation of kidney αß T cells by a cell-cell contact mechanism that is major histocompatibility complex (MHC) independent and regulated by CD155, αVß3-integrin, and vitronectin. Peripheral CD4 and CD8 are resistant to RTEC-mediated stimulation, while the minor subset of double-negative (DN) T cells are responsive. This functional property of RTEC is discovered by using a coculture system that recapitulates spontaneous in vivo polyclonal proliferation of kidney T cells, which are mainly comprised of central memory T (TCM) and effector memory T (TEM) cells. This robust cell-intrinsic stimulatory role of RTECs could be underlying the steady-state spontaneous proliferation of kidney T cells. The results have conceptual implications for understanding roles of different cell types in regulating systemic and organ-specific T cell homeostasis.
Subject(s)
Acute Kidney Injury , T-Lymphocytes , Humans , Kidney , Epithelial Cells/metabolism , Acute Kidney Injury/metabolism , Coculture TechniquesABSTRACT
PURPOSE: Contact-based education, offering meaningful contact with individuals living in recovery with mental illness, reduces stigma. This study evaluated the effectiveness of the National Alliance on Mental Illness Provider Education Program (NAMI PEP) when implemented as a curricular requirement across two cohorts of third-year osteopathic medical students, comparing traditional, passive learning and active, online delivery formats. MATERIALS AND METHODS: Participants were two cohorts of third-year medical students (Cohort 1 n = 186; Cohort 2 n = 139; overall N = 325) who completed questionnaires measuring affect, beliefs, and behaviors toward patients with mental illness at pre-program, 1-week follow-up, and 6-month follow-up. For Cohort 1, the existing community-based NAMI PEP was implemented. For Cohort 2, the program was adapted to an online, active learning format tailored to medical students, and an additional 3-month follow-up assessment was added to better identify intermediate-term effects. RESULTS: The NAMI PEP was associated with longitudinal improvements in target outcomes, with enhanced effects with the adapted curriculum in Cohort 2. At 6-month follow-up, students reported less stigma, fewer stereotyping negative attitudes, and lower anxiety treating patients with mental illness. They also reported increased confidence integrating psychiatry into routine care and increased competence in principles of collaborative mental health treatment. CONCLUSIONS: This study demonstrates the longitudinal effectiveness of the NAMI PEP across two cohorts of medical students, with strengthened effects observed when the program is tailored to contemporary medical education.
Subject(s)
Mental Disorders , Psychiatry , Students, Medical , Humans , Problem-Based Learning , Students, Medical/psychology , Social Stigma , Mental Disorders/therapy , Mental Disorders/psychology , Psychiatry/educationABSTRACT
Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that the trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. A reduction in H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and the expression of genes associated with stem cell features, cell proliferation, the cell cycle, and DNA damage repair. A reduction in WDR82-mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas.
ABSTRACT
Human epidermal growth factor receptor 2 (HER2) overexpression leads to mammary tumorigenesis and its elevated levels lead to increase in cancer stem cells (CSCs), invasion, and metastasis. CSCs are resistant to radiation/chemotherapeutic drugs and are believed to be responsible for recurrence/relapse of cancer. CSCs are isolated using flow cytometry based sorting, although reliable, this technology hinders the convenient identification of molecular targets of CSCs. Therefore to understand the molecular players of increased CSC through HER2 overexpression and to develop meaningful targets for combination therapy, we isolated and characterized breast CSCs through convenient tumorsphere culture. We identified the altered protein expression in CSC as compared to non-CSC using LC-MS/MS and confirmed those results using qRT-PCR and Western blotting. Ferritin heavy chain 1 (FTH1) was identified as a candidate gene, which is involved in iron metabolism and iron depletion significantly decreased the self-renewal of CSCs. We further performed in silico analysis of altered genes in tumorsphere and identified a set of genes (PTMA, S100A4, S100A6, TNXRD1, COX-1, COX-2, KRT14, and FTH1), representing possible molecular targets, which in combination showed a promise to be used as prognostic markers for breast cancer.