1 6697 167 VARICELLA-ZOSTER VIRUS HUMAN GANGLIONIC LATENCY: A CURRENT SUMMARY. VARICELLA-ZOSTER VIRUS (VZV) IS A UBIQUITOUS HUMAN HERPES VIRUS TYPICALLY ACQUIRED IN CHILDHOOD WHEN IT CAUSES VARICELLA (CHICKENPOX), FOLLOWING WHICH THE VIRUS ESTABLISHES A LATENT INFECTION IN TRIGEMINAL AND DORSAL ROOT GANGLIA THAT LASTS FOR THE LIFE OF THE INDIVIDUAL. VZV SUBSEQUENTLY REACTIVATES, SPONTANEOUSLY OR AFTER SPECIFIC TRIGGERING FACTORS, TO CAUSE HERPES ZOSTER (SHINGLES), WHICH MAY BE COMPLICATED BY POSTHERPETIC NEURALGIA AND SEVERAL OTHER NEUROLOGICAL COMPLICATIONS INCLUDING VASCULOPATHY. OUR UNDERSTANDING OF VZV LATENCY LAGS BEHIND OUR KNOWLEDGE OF HERPES SIMPLEX VIRUS TYPE 1 (HSV-1) LATENCY PRIMARILY DUE TO THE DIFFICULTY IN PROPAGATING THE VIRUS TO HIGH TITERS IN A CELL-FREE STATE, AND THE LACK OF A SUITABLE SMALL-ANIMAL MODEL FOR STUDYING VIRUS LATENCY AND REACTIVATION. IT IS NOW ESTABLISHED BEYOND DOUBT THAT LATENT VZV IS PREDOMINANTLY LOCATED IN HUMAN GANGLIONIC NEURONS. VIRUS GENE TRANSCRIPTION DURING LATENCY IS EPIGENETICALLY REGULATED, AND APPEARS TO BE RESTRICTED TO EXPRESSION OF AT LEAST SIX GENES, WITH EXPRESSION OF GENE 63 BEING THE HALLMARK OF LATENCY. HOWEVER, VIRAL GENE TRANSCRIPTION MAY BE MORE EXTENSIVE THAN PREVIOUSLY THOUGHT. THERE IS ALSO EVIDENCE FOR SEVERAL VZV GENES BEING EXPRESSED AT THE PROTEIN LEVEL, INCLUDING VZV GENE 63-ENCODED PROTEIN, BUT RECENT EVIDENCE SUGGESTS THAT THIS MAY NOT BE A COMMON EVENT. THE NATURE AND EXTENT OF THE CHRONIC INFLAMMATORY RESPONSE IN LATENTLY INFECTED GANGLIA IS ALSO OF CURRENT INTEREST. THERE REMAIN SEVERAL QUESTIONS CONCERNING THE VZV LATENCY PROCESS THAT STILL NEED TO BE RESOLVED UNAMBIGUOUSLY AND IT IS LIKELY THAT THIS WILL REQUIRE THE USE OF NEWLY DEVELOPED MOLECULAR TECHNOLOGIES, SUCH AS GEXPS MULTIPLEX POLYMERASE CHAIN REACTION (PCR) FOR VIRUS TRANSCRIPTIONAL ANALYSIS AND CHIP-SEQ TO STUDY THE EPIGENETIC OF LATENT VIRUS GENOME ( LIU ET AL, 2010 , BMC BIOL 8: 56). 2010 2 2787 33 EZH2-MEDIATED EPIGENETIC MODIFICATION IS REQUIRED FOR ALLOGENEIC T CELL-INDUCED LUPUS DISEASE. BACKGROUND: THE MECHANISMS INVOLVED IN THE PATHOGENESIS OF AUTOIMMUNE DISORDERS, INCLUDING SYSTEMIC LUPUS ERYTHEMATOSUS (SLE), HAVE NOT BEEN FULLY ELUCIDATED. SOME OF THESE MECHANISMS INVOLVE EPIGENETIC REGULATION OF GENE EXPRESSION. THE HISTONE METHYLTRANSFERASE EZH2 CONTRIBUTES TO EPIGENETIC REGULATION OF GENE EXPRESSION, IS HIGHLY EXPRESSED IN GERMINAL CENTER (GC) B CELLS AND FOLLICULAR T HELPER (T(FH)) CELLS, AND MAY BE INVOLVED IN LUPUS PATHOGENESIS. METHODS: THE MURINE BM12 MODEL OF LUPUS-LIKE CHRONIC GRAFT VERSUS HOST DISEASE (CGVHD) WAS INDUCED BY INTRA-PERITONEAL INJECTION OF NEGATIVELY ISOLATED ALLOGENEIC CD4(+) T CELLS. LUPUS-LIKE DISEASE DEVELOPMENT WAS MONITORED BY ELISA DETERMINATION OF SERUM ANTI-DSDNA AND ANTI-CHROMATIN ANTIBODY TITERS. IMMUNE CELL ACTIVATION AND EZH2 EXPRESSION WERE EVALUATED BY FLOW CYTOMETRY AND WESTERN BLOTTING. RESULTS: DECREASED AUTOANTIBODY PRODUCTION AND GC FORMATION ARE OBSERVED WHEN EZH2-DEFICIENT CD4(+) T CELLS ARE USED INSTEAD OF WILD-TYPE (WT) TO INDUCE CGVHD AND WHEN MICE THAT RECEIVE ALLOGENEIC WT DONOR T CELLS TO INDUCE CGVHD ARE TREATED WITH GSK503, AN EZH2-SPECIFIC INHIBITOR. IN THE BM12 CGVHD MODEL, WT DONOR T CELLS ARE NORMALLY FULLY ACTIVATED 1 WEEK AFTER INFUSION INTO AN ALLOGENEIC HOST, EXHIBIT A T(FH) CELL (PD-1(HI)/CXCR5(HI)) PHENOTYPE WITH UPREGULATED EZH2, AND ACTIVATE B CELLS TO FORM GERMINAL CENTERS (GCS). IN CONTRAST, EZH2-DEFICIENT DONOR T CELLS GENERATE FEWER T(FH) CELLS THAT FAIL TO ACTIVATE B CELLS OR PROMOTE GC FORMATION. DESPITE SIMILAR T-INDEPENDENT, LPS-INDUCED B CELL RESPONSES, OVA-IMMUNIZED CD4.EZH2-KO MICE HAD A SKEWED LOW-AFFINITY IGM PHENOTYPE IN COMPARISON TO SIMILARLY TREATED WT MICE. IN ADDITION, EARLY AFTER OVA IMMUNIZATION, MORE CD4(+) T CELLS FROM B6.CD4.EZH2-KO MICE HAD A CD44(LO)/CD62L(LO) PHENOTYPE, WHICH SUGGESTS ARRESTED OR DELAYED ACTIVATION, THAN CD4(+) T CELLS FROM OVALBUMIN-IMMUNIZED B6.WT MICE. CONCLUSION: EZH2 GENE DELETION OR PHARMACOLOGICAL EZH2 INHIBITION SUPPRESSES AUTOANTIBODY PRODUCTION AND GC FORMATION IN BM12 LUPUS-LIKE CGVHD AND DECREASES AFFINITY MATURATION AND ISOTYPE SWITCHING IN RESPONSE TO IMMUNIZATION WITH A T CELL-DEPENDENT ANTIGEN. EZH2 INHIBITION MAY BE USEFUL FOR THE TREATMENT OF LUPUS AND OTHER AUTOIMMUNE DISORDERS. 2020 3 40 67 A COMPARISON OF HERPES SIMPLEX VIRUS TYPE 1 AND VARICELLA-ZOSTER VIRUS LATENCY AND REACTIVATION. HERPES SIMPLEX VIRUS TYPE 1 (HSV-1; HUMAN HERPESVIRUS 1) AND VARICELLA-ZOSTER VIRUS (VZV; HUMAN HERPESVIRUS 3) ARE HUMAN NEUROTROPIC ALPHAHERPESVIRUSES THAT CAUSE LIFELONG INFECTIONS IN GANGLIA. FOLLOWING PRIMARY INFECTION AND ESTABLISHMENT OF LATENCY, HSV-1 REACTIVATION TYPICALLY RESULTS IN HERPES LABIALIS (COLD SORES), BUT CAN OCCUR FREQUENTLY ELSEWHERE ON THE BODY AT THE SITE OF PRIMARY INFECTION (E.G. WHITLOW), PARTICULARLY AT THE GENITALS. RARELY, HSV-1 REACTIVATION CAN CAUSE ENCEPHALITIS; HOWEVER, A THIRD OF THE CASES OF HSV-1 ENCEPHALITIS ARE ASSOCIATED WITH HSV-1 PRIMARY INFECTION. PRIMARY VZV INFECTION CAUSES VARICELLA (CHICKENPOX) FOLLOWING WHICH LATENT VIRUS MAY REACTIVATE DECADES LATER TO PRODUCE HERPES ZOSTER (SHINGLES), AS WELL AS AN INCREASINGLY RECOGNIZED NUMBER OF SUBACUTE, ACUTE AND CHRONIC NEUROLOGICAL CONDITIONS. FOLLOWING PRIMARY INFECTION, BOTH VIRUSES ESTABLISH A LATENT INFECTION IN NEURONAL CELLS IN HUMAN PERIPHERAL GANGLIA. HOWEVER, THE DETAILED MECHANISMS OF VIRAL LATENCY AND REACTIVATION HAVE YET TO BE UNRAVELLED. IN BOTH CASES LATENT VIRAL DNA EXISTS IN AN 'END-LESS' STATE WHERE THE ENDS OF THE VIRUS GENOME ARE JOINED TO FORM STRUCTURES CONSISTENT WITH UNIT LENGTH EPISOMES AND CONCATEMERS, FROM WHICH VIRAL GENE TRANSCRIPTION IS RESTRICTED. IN LATENTLY INFECTED GANGLIA, THE MOST ABUNDANTLY DETECTED HSV-1 RNAS ARE THE SPLICED PRODUCTS ORIGINATING FROM THE PRIMARY LATENCY ASSOCIATED TRANSCRIPT (LAT). THIS PRIMARY LAT IS AN 8.3 KB UNSTABLE TRANSCRIPT FROM WHICH TWO STABLE (1.5 AND 2.0 KB) INTRONS ARE SPLICED. TRANSCRIPTS MAPPING TO 12 VZV GENES HAVE BEEN DETECTED IN HUMAN GANGLIA REMOVED AT AUTOPSY; HOWEVER, IT IS DIFFICULT TO ASCRIBE THESE AS TRANSCRIPTS PRESENT DURING LATENT INFECTION AS EARLY-STAGE VIRUS REACTIVATION MAY HAVE TRANSPIRED IN THE POST-MORTEM TIME PERIOD IN THE GANGLIA. NONETHELESS, LOW-LEVEL TRANSCRIPTION OF VZV ORF63 HAS BEEN REPEATEDLY DETECTED IN MULTIPLE GANGLIA REMOVED AS CLOSE TO DEATH AS POSSIBLE. THERE IS INCREASING EVIDENCE THAT HSV-1 AND VZV LATENCY IS EPIGENETICALLY REGULATED. IN VITRO MODELS THAT PERMIT PATHWAY ANALYSIS AND IDENTIFICATION OF BOTH EPIGENETIC MODULATIONS AND GLOBAL TRANSCRIPTIONAL MECHANISMS OF HSV-1 AND VZV LATENCY HOLD MUCH PROMISE FOR OUR FUTURE UNDERSTANDING IN THIS COMPLEX AREA. THIS REVIEW SUMMARIZES THE MOLECULAR BIOLOGY OF HSV-1 AND VZV LATENCY AND REACTIVATION, AND ALSO PRESENTS FUTURE DIRECTIONS FOR STUDY. 2015 4 4751 33 NOVEL RISK MARKERS FOR GASTRIC CANCER SCREENING: PRESENT STATUS AND FUTURE PROSPECTS. INITIAL IDENTIFICATION OF POPULATIONS AT HIGH RISK OF GASTRIC CANCER (GC) IS IMPORTANT FOR ENDOSCOPIC SCREENING OF GC. AS SERUM PEPSINOGEN (PG) TEST-POSITIVE SUBJECTS WITH PROGRESSION OF CHRONIC ATROPHIC GASTRITIS (CAG) SHOW A HIGH LIKELIHOOD OF FUTURE CANCER DEVELOPMENT, THIS POPULATION WARRANTS CAREFUL FOLLOW-UP OBSERVATION AS A HIGH-RISK GC GROUP. BY COMBINING THE PG TEST WITH HELICOBACTER PYLORI (HP) ANTIBODY TITERS, THE HP-RELATED CHRONIC GASTRITIS STAGE CAN BE CLASSIFIED, THUS IDENTIFYING NOT ONLY A GC HIGH-RISK GROUP BUT ALSO A LOW-RISK GROUP. AMONG PG TEST-NEGATIVE PATIENTS WITHOUT CAG, THOSE WITH HIGH SERUM PG II LEVELS AND HP ANTIBODY TITERS ARE THOUGHT TO HAVE SEVERE GASTRIC MUCOSAL INFLAMMATION AND THE RISK OF DIFFUSE-TYPE GC IS ALSO HIGH. MEANWHILE, IN GASTRIC MUCOSAE OBTAINED BY ENDOSCOPIC BIOPSY, HP INFECTION INDUCES ABERRANT DNA METHYLATION IN CPG ISLANDS IN MULTIPLE GENE REGIONS AND THE EXTENT OF METHYLATION CLEARLY CORRELATES WITH GC RISK. BY QUANTIFYING ABERRANT DNA METHYLATION IN SUITABLE GENE MARKERS, WE CAN DETERMINE THE EXTENT OF THE EPIGENETIC FIELD FOR CANCERIZATION. THESE NOVEL CONCEPTS AND RISK MARKERS WILL HAVE MANY CLINICAL APPLICATIONS IN GASTROINTESTINAL ENDOSCOPY, INCLUDING MORE EFFICIENT ENDOSCOPIC GC SCREENING AND A STRATEGIC APPROACH TO METACHRONOUS MULTIPLE GCS AFTER ENDOSCOPIC TREATMENT. 2010 5 6071 21 THE DNA METHYLATION INHIBITOR 5-AZACYTIDINE INCREASES REGULATORY T CELLS AND ALLEVIATES AIRWAY INFLAMMATION IN OVALBUMIN-SENSITIZED MICE. BACKGROUND: ASTHMA IS CHARACTERIZED AS A CHRONIC INFLAMMATORY DISORDER OF THE AIRWAYS ASSOCIATED WITH AN ENHANCED TH2 RESPONSE TO INHALED ALLERGENS. CD4+ T REGULATORY (TREG) CELLS ARE CONTROLLED BY THE MASTER TRANSCRIPTION FACTOR FOXP3 AND STRICTLY MAINTAIN PERIPHERAL IMMUNOTOLERANCE. EPIGENETIC REGULATION OF FOXP3 BY DNA METHYLTRANSFERASE INHIBITORS, SUCH AS 5-AZACYTIDINE (AZA), CAN GENERATE A STEADY SUPPLY OF FUNCTIONAL TREG CELLS. THEREFORE, WE PROPOSE THAT AZA CAN AUGMENT TREG CELLS IN VIVO TO PREVENT THE PATHOGENESIS OF ASTHMA. METHODS: BALB/C MICE WERE SENSITIZED WITH CHICKEN OVALBUMIN (OVA) AND TREATED WITH DIFFERENT DOSES OF AZA. AIRWAY HYPERRESPONSIVENESS TO METHACHOLINE, EOSINOPHILIA IN BRONCHOALVEOLAR LAVAGE FLUID, CIRCULATING TITERS OF OVA-SPECIFIC IGG1 AND IGE, AND STIMULATING LEVELS OF TH2 CYTOKINES FROM SPLENOCYTES WERE THEN DETERMINED. CELLULAR POPULATIONS WERE EXAMINED BY FLOW CYTOMETRY. PC61 ANTIBODY, WHICH DEPLETES CD25+ CELLS, WAS USED TO VERIFY THE ROLE OF CD25+ CELLS IN AZA-INDUCED TOLERANCE. RESULTS: ADMINISTRATION OF AZA TO OVA-SENSITIZED MICE DIMINISHED AIRWAY HYPERREACTIVITY, PULMONARY EOSINOPHILIA, LEVELS OF OVA-SPECIFIC IGG1 AND IGE IN SERUM, AND SECRETION OF TH2 CYTOKINES FROM OVA-STIMULATED SPLENOCYTES IN A DOSE-DEPENDENT MANNER. PERCENTAGES OF CD25+ AND FOXP3+ CELLS IN THE CD4+ CELL POPULATION WERE NOTABLY INCREASED IN AZA-TREATED MICE COMPARED TO SENSITIZED CONTROL MICE. FURTHERMORE, THE MAJOR SYMPTOMS OF ASTHMA WERE EXACERBATED BY DEPLETING CD25+ CELLS IN AZA-TREATED MICE. CONCLUSIONS: AZA MAY HAVE APPLICATIONS AS A NOVEL CLINICAL STRATEGY TO INCREASE THE PRODUCTION OF TREG CELLS IN ORDER TO MODULATE THE AIRWAY INFLAMMATION ASSOCIATED WITH ASTHMA. 2013 6 1007 24 CHRONIC VIRUS INFECTION ENFORCES DEMETHYLATION OF THE LOCUS THAT ENCODES PD-1 IN ANTIGEN-SPECIFIC CD8(+) T CELLS. FUNCTIONALLY EXHAUSTED T CELLS HAVE HIGH EXPRESSION OF THE PD-1 INHIBITORY RECEPTOR, AND THERAPIES THAT BLOCK PD-1 SIGNALING SHOW PROMISE FOR RESOLVING CHRONIC VIRAL INFECTIONS AND CANCER. BY USING HUMAN AND MURINE SYSTEMS OF ACUTE AND CHRONIC VIRAL INFECTIONS, WE ANALYZED EPIGENETIC REGULATION OF PD-1 EXPRESSION DURING CD8(+) T CELL DIFFERENTIATION. DURING ACUTE INFECTION, NAIVE TO EFFECTOR CD8(+) T CELL DIFFERENTIATION WAS ACCOMPANIED BY A TRANSIENT LOSS OF DNA METHYLATION OF THE PDCD1 LOCUS THAT WAS DIRECTLY COUPLED TO THE DURATION AND STRENGTH OF T CELL RECEPTOR SIGNALING. FURTHER DIFFERENTIATION INTO FUNCTIONAL MEMORY CELLS COINCIDED WITH PDCD1 REMETHYLATION, PROVIDING AN ADAPTED PROGRAM FOR REGULATION OF PD-1 EXPRESSION. IN CONTRAST, THE PDCD1 REGULATORY REGION WAS COMPLETELY DEMETHYLATED IN EXHAUSTED CD8(+) T CELLS AND REMAINED UNMETHYLATED EVEN WHEN VIRUS TITERS DECREASED. THIS LACK OF DNA REMETHYLATION LEAVES THE PDCD1 LOCUS POISED FOR RAPID EXPRESSION, POTENTIALLY PROVIDING A SIGNAL FOR PREMATURE TERMINATION OF ANTIVIRAL FUNCTIONS. 2011 7 1001 30 CHRONIC TCDD EXPOSURE RESULTS IN THE DYSREGULATION OF GENE EXPRESSION IN SPLENIC B-LYMPHOCYTES AND IN THE IMPAIRMENTS IN T-CELL AND B-CELL DIFFERENTIATION IN MOUSE MODEL. 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) EXPOSURE IN HUMANS IS ASSOCIATED WITH MARKED IMMUNE SUPPRESSIONS AND INCREASED INCIDENCE OF LYMPHOBLASTIC DISEASES. TO ELUCIDATE MECHANISMS OF IMPAIRMENTS IN HUMORAL IMMUNE RESPONSES, WE USED A MURINE MODEL. FOLLOWING A 20-WEEK ADMINISTRATION OF LOW DOSES OF TCDD, WE OBSERVED SEVERELY REDUCED ANTIBODY TITERS, DRAMATICALLY DECREASED NUMBER OF SPLENIC TH1 AND TH2 CELLS AND AN INCREASE IN CD19(+) B CELLS. TRANSCRIPTIONAL PROFILING OF CD19(+) B CELLS SHOWED THAT MARKERS OF PRE-B CELLS WERE SIGNIFICANTLY ELEVATED, INDICATING DELAYED B CELL MATURATION. THESE CHANGES IN B CELLS WERE ACCOMPANIED BY DECREASES OF T HELPER CELL NUMBERS AND REDUCED IGM AND IGG TITERS. A TRANSCRIPTOME ANALYSIS OF SPLENIC B CELLS FOLLOWED BY INGENUITY PATHWAY ANALYSIS (IPA) REVEALED A SET OF DIFFERENTIALLY EXPRESSED GENES KNOWN TO PLAY ROLES IN TUMORIGENESIS, CELL-PROLIFERATION AND CELL-MIGRATION. THE MOST UP-REGULATED TRANSCRIPT GENE WAS EPH RECEPTOR A2 (EPHA2), A KNOWN ONCOGENE, AND THE MOST DOWN-REGULATED TRANSCRIPT WAS ZBTB16 THAT CODES FOR A NEGATIVE TRANSCRIPTIONAL REGULATOR IMPORTANT IN EPIGENETIC CHROMATIN REMODELING. IPA IDENTIFIED CAMP-RESPONSIVE ELEMENT MODULATOR (CREM) AND CAMP-RESPONSIVE ELEMENT BINDING PROTEIN 1 (CREB1) AS TOP UPSTREAM REGULATORS. CONSISTENTLY, A MAPPER PROMOTER DATABASE ANALYSIS SHOWED THAT ALL TOP DYSREGULATED GENES HAD CREM AND/OR CREB1 BINDING SITES IN THEIR PROMOTER REGIONS. IN SUMMARY, OUR DATA SHOWED THAT CHRONIC TCDD EXPOSURE IN MICE CAUSED SUPPRESSED HUMORAL IMMUNITY ACCOMPANIED WITH PROFOUND DYSREGULATION OF GENE EXPRESSION IN SPLENIC B-LYMPHOCYTES, LIKELY THROUGH CAMP-DEPENDENT PATHWAYS. THIS DYSREGULATION RESULTED IN IMPAIRMENTS IN T-CELL AND B-CELL DIFFERENTIATION AND ACTIVATION OF THE TUMORIGENIC TRANSCRIPTION PROGRAM. 2016 8 1061 29 CLINICAL REMISSION OF SIGHT-THREATENING NON-INFECTIOUS UVEITIS IS CHARACTERIZED BY AN UPREGULATION OF PERIPHERAL T-REGULATORY CELL POLARIZED TOWARDS T-BET AND TIGIT. BACKGROUND: NON-INFECTIOUS UVEITIS CAN CAUSE CHRONIC RELAPSING AND REMITTING OCULAR INFLAMMATION, WHICH MAY REQUIRE HIGH DOSE SYSTEMIC IMMUNOSUPPRESSION TO PREVENT SEVERE SIGHT LOSS. IT HAS BEEN CLASSICALLY DESCRIBED AS AN AUTOIMMUNE DISEASE, MEDIATED BY PRO-INFLAMMATORY TH1 AND TH17 T-CELL SUBSETS. STUDIES SUGGEST THAT NATURAL IMMUNOSUPPRESSIVE CD4(+)CD25(+)FOXP3(+) T-REGULATORY CELLS (TREGS) ARE INVOLVED IN RESOLUTION OF INFLAMMATION AND MAY BE INVOLVED IN THE MAINTENANCE OF CLINICAL REMISSION. OBJECTIVE: TO INVESTIGATE WHETHER THERE IS A PERIPHERAL BLOOD IMMUNOREGULATORY PHENOTYPE ASSOCIATED WITH CLINICAL REMISSION OF SIGHT-THREATENING NON-INFECTIOUS UVEITIS BY COMPARING PERIPHERAL BLOOD LEVELS OF TREG, TH1, AND TH17, AND ASSOCIATED DNA METHYLATION AND CYTOKINE LEVELS IN PATIENTS WITH ACTIVE UVEITIC DISEASE, CONTROL SUBJECTS AND PATIENTS (WITH PREVIOUSLY ACTIVE DISEASE) IN CLINICAL REMISSION INDUCED BY IMMUNOSUPPRESSIVE DRUGS. METHODS: ISOLATED PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM PERIPHERAL BLOOD SAMPLES FROM PROSPECTIVELY RECRUITED SUBJECTS WERE ANALYZED BY FLOW CYTOMETRY FOR CD3, CD4, FOXP3, TIGIT, T-BET, AND RELATED ORPHAN RECEPTOR GAMMAT. EPIGENETIC DNA METHYLATION LEVELS OF FOXP3 TREG-SPECIFIC DEMETHYLATED REGION (TSDR), FOXP3 PROMOTER, TBX21, RORC2, AND TIGIT LOCI WERE DETERMINED IN CRYOPRESERVED PBMC USING A NEXT-GENERATION SEQUENCING APPROACH. RELATED CYTOKINES WERE MEASURED IN BLOOD SERA. FUNCTIONAL SUPPRESSIVE CAPACITY OF TREG WAS ASSESSED USING T-CELL PROLIFERATION ASSAYS. RESULTS: FIFTY PATIENTS WITH UVEITIS (INTERMEDIATE, POSTERIOR, AND PANUVEITIS) AND 10 CONTROL SUBJECTS WERE RECRUITED. THE FREQUENCY OF CD4(+)CD25(+)FOXP3(+) TREG, TIGIT(+) TREG, AND T-BET(+) TREG AND THE RATIO OF TREG TO TH1 WERE SIGNIFICANTLY HIGHER IN REMISSION PATIENTS COMPARED WITH PATIENTS WITH ACTIVE UVEITIC DISEASE; AND TIGIT(+) TREGS WERE A SIGNIFICANT PREDICTOR OF CLINICAL REMISSION. TREG FROM PATIENTS IN CLINICAL REMISSION DEMONSTRATED A HIGH LEVEL OF IN VITRO SUPPRESSIVE FUNCTION COMPARED WITH TREG FROM CONTROL SUBJECTS AND FROM PATIENTS WITH UNTREATED ACTIVE DISEASE. PBMC FROM PATIENTS IN CLINICAL REMISSION HAD SIGNIFICANTLY LOWER METHYLATION LEVELS AT THE FOXP3 TSDR, FOXP3 PROMOTER, AND TIGIT LOCI AND HIGHER LEVELS AT RORC LOCI THAN THOSE WITH ACTIVE DISEASE. CLINICAL REMISSION WAS ALSO ASSOCIATED WITH SIGNIFICANTLY HIGHER SERUM LEVELS OF TRANSFORMING GROWTH FACTOR BETA AND IL-10, WHICH POSITIVELY CORRELATED WITH TREG LEVELS, AND LOWER SERUM LEVELS OF IFNGAMMA, IL-17A, AND IL-22 COMPARED WITH PATIENTS WITH ACTIVE DISEASE. CONCLUSION: CLINICAL REMISSION OF SIGHT-THREATENING NON-INFECTIOUS UVEITIS HAS AN IMMUNOREGULATORY PHENOTYPE CHARACTERIZED BY UPREGULATION OF PERIPHERAL TREG, POLARIZED TOWARD T-BET AND TIGIT. THESE FINDINGS MAY ASSIST WITH INDIVIDUALIZED THERAPY OF UVEITIS, BY INFORMING WHETHER DRUG THERAPY HAS INDUCED PHENOTYPICALLY STABLE TREG ASSOCIATED WITH LONG-TERM CLINICAL REMISSION. 2018 9 3373 23 HISTONE MODULATION BLOCKS TREG-INDUCED FOXP3 BINDING TO THE IL-2 PROMOTER OF VIRUS-SPECIFIC CD8(+) T CELLS FROM FELINE IMMUNODEFICIENCY VIRUS-INFECTED CATS. CD8(+) T CELLS ARE CRITICAL FOR CONTROLLING HIV INFECTION. DURING THE CHRONIC PHASE OF LENTIVIRAL INFECTION, CD8(+) T CELLS LOSE THEIR PROLIFERATIVE CAPACITY AND EXHIBIT IMPAIRED ANTIVIRAL FUNCTION. THIS LOSS OF CD8(+) T CELL FUNCTION IS DUE, IN PART, TO CD4(+)CD25(+) T REGULATORY (TREG) CELL-MEDIATED SUPPRESSION. OUR RESEARCH GROUP HAS DEMONSTRATED THAT LENTIVIRUS-ACTIVATED CD4(+)CD25(+) TREG CELLS INDUCE THE REPRESSIVE TRANSCRIPTION FACTOR FORKHEAD BOX P3 (FOXP3) IN AUTOLOGOUS CD8(+) T CELLS FOLLOWING CO-CULTURE. WE HAVE RECENTLY REPORTED THAT TREG-INDUCED FOXP3 BINDS THE INTERLEUKIN-2 (IL-2), INTERFERON-GAMMA (IFN- GAMMA), AND TUMOR NECROSIS FACTOR-ALPHA (TNF-ALPHA) PROMOTERS IN VIRUS-SPECIFIC CD8(+) T CELLS. THESE DATA SUGGEST AN IMPORTANT ROLE OF FOXP3-MEDIATED CD8(+) T CELL DYSFUNCTION IN LENTIVIRAL INFECTION. TO ELUCIDATE THE MECHANISM OF THIS SUPPRESSION, WE PREVIOUSLY REPORTED THAT DECREASED METHYLATION FACILITATES FOXP3 BINDING IN MITOGEN-ACTIVATED CD8(+) T CELLS FROM FELINE IMMUNODEFICIENCY VIRUS (FIV)-INFECTED CATS. WE DEMONSTRATED THE REDUCED BINDING OF FOXP3 TO THE IL-2 PROMOTER BY INCREASING METHYLATION OF CD8(+) T CELLS. IN THE STUDIES PRESENTED HERE, WE ASK IF ANOTHER FORM OF EPIGENETIC MODULATION MIGHT ALLEVIATE FOXP3-MEDIATED SUPPRESSION IN CD8(+) T CELLS. WE HYPOTHESIZED THAT DECREASING HISTONE ACETYLATION IN VIRUS-SPECIFIC CD8(+) T CELLS WOULD DECREASE TREG-INDUCED FOXP3 BINDING TO THE IL-2 PROMOTER. INDEED, USING ANACARDIC ACID (AA), A KNOWN HISTONE ACETYL TRANSFERASE (HAT) INHIBITOR, WE DEMONSTRATE A REDUCTION IN FOXP3 BINDING TO THE IL-2 PROMOTER IN VIRUS-SPECIFIC CD8(+) T CELLS CO-CULTURED WITH AUTOLOGOUS TREG CELLS. THESE DATA IDENTIFY A NOVEL MECHANISM OF FOXP3-MEDIATED CD8(+) T CELL DYSFUNCTION DURING LENTIVIRAL INFECTION. 2018 10 5908 25 TARGETED DE-METHYLATION OF THE FOXP3-TSDR IS SUFFICIENT TO INDUCE PHYSIOLOGICAL FOXP3 EXPRESSION BUT NOT A FUNCTIONAL TREG PHENOTYPE. CD4+ REGULATORY T CELLS (TREGS) ARE KEY MEDIATORS OF IMMUNOLOGICAL TOLERANCE AND PROMISING EFFECTOR CELLS FOR IMMUNO-SUPPRESSIVE ADOPTIVE CELLULAR THERAPY TO FIGHT AUTOIMMUNITY AND CHRONIC INFLAMMATION. THEIR FUNCTIONAL STABILITY IS CRITICAL FOR THEIR CLINICAL UTILITY AND HAS BEEN CORRELATED TO THE DEMETHYLATED STATE OF THE TSDR/CNS2 ENHANCER ELEMENT IN THE TREG LINEAGE TRANSCRIPTION FACTOR FOXP3. HOWEVER, PROOF FOR A CAUSAL CONTRIBUTION OF THE TSDR DE-METHYLATION TO FOXP3 STABILITY AND TREG INDUCTION IS SO FAR LACKING. WE HERE ESTABLISHED A POWERFUL TRANSIENT-TRANSFECTION CRISPR-CAS9-BASED EPIGENETIC EDITING METHOD FOR THE SELECTIVE DE-METHYLATION OF THE TSDR WITHIN THE ENDOGENOUS CHROMATIN ENVIRONMENT OF A LIVING CELL. THE INDUCED DE-METHYLATED STATE WAS STABLE OVER WEEKS IN CLONAL T CELL PROLIFERATION CULTURES EVEN AFTER EXPRESSION OF THE EDITING COMPLEX HAD CEASED. EPIGENETIC EDITING OF THE TSDR RESULTED IN FOXP3 EXPRESSION, EVEN IN ITS PHYSIOLOGICAL ISOFORM DISTRIBUTION, PROVING A CAUSAL ROLE FOR THE DE-METHYLATED TSDR IN FOXP3 REGULATION. HOWEVER, SUCCESSFUL FOXP3 INDUCTION WAS NOT ASSOCIATED WITH A SWITCH TOWARDS A FUNCTIONAL TREG PHENOTYPE, IN CONTRAST TO WHAT HAS BEEN REPORTED FROM FOXP3 OVEREXPRESSION APPROACHES. THUS, TSDR DE-METHYLATION IS REQUIRED, BUT NOT SUFFICIENT FOR A STABLE TREG PHENOTYPE INDUCTION. THEREFORE, TARGETED DEMETHYLATION OF THE TSDR MAY BE A CRITICAL ADDITION TO PUBLISHED IN VITRO TREG INDUCTION PROTOCOLS WHICH SO FAR LACK FOXP3 STABILITY. 2020 11 3454 30 HYPOMETHYLATION AT THE REGULATORY T CELL-SPECIFIC DEMETHYLATED REGION IN CD25HI T CELLS IS DECOUPLED FROM FOXP3 EXPRESSION AT THE INFLAMED SITE IN CHILDHOOD ARTHRITIS. THE MAINTENANCE OF FOXP3 EXPRESSION IN CD25(HI) REGULATORY T CELLS (TREGS) IS CRUCIAL TO THE CONTROL OF INFLAMMATION AND ESSENTIAL FOR SUCCESSFUL TREG TRANSFER THERAPIES. COEXPRESSION OF CD25 AND FOXP3 IN COMBINATION WITH A HYPOMETHYLATED REGION WITHIN THE FOXP3 GENE, CALLED THE TREG-SPECIFIC DEMETHYLATED REGION (TSDR), IS CONSIDERED THE HALLMARK OF STABLE TREGS. THE TSDR IS AN EPIGENETIC MOTIF THAT IS IMPORTANT FOR STABLE FOXP3 EXPRESSION AND IS USED AS A BIOMARKER TO MEASURE TREG LINEAGE COMMITMENT. IN THIS STUDY, WE REPORT THAT, UNLIKE IN PERIPHERAL BLOOD, CD4(+) T CELL EXPRESSION OF CD25 AND FOXP3 IS FREQUENTLY DISSOCIATED AT THE INFLAMED SITE IN PATIENTS WITH JUVENILE IDIOPATHIC ARTHRITIS, WHICH LED US TO QUESTION THE STABILITY OF HUMAN TREGS IN CHRONIC INFLAMMATORY ENVIRONMENTS. WE DESCRIBE A NOVEL CD4(+)CD127(LO)CD25(HI) HUMAN T CELL POPULATION THAT EXHIBITS EXTENSIVE TSDR AND PROMOTER DEMETHYLATION IN THE ABSENCE OF STABLE FOXP3 EXPRESSION. THIS POPULATION EXPRESSES HIGH LEVELS OF CTLA-4 AND CAN SUPPRESS T CONVENTIONAL CELL PROLIFERATION IN VITRO. THESE DATA COLLECTIVELY SUGGEST THAT THIS POPULATION MAY REPRESENT A CHRONICALLY ACTIVATED FOXP3(LO) TREG POPULATION. WE SHOW THAT THESE CELLS HAVE DEFECTS IN IL-2 SIGNALING AND REDUCED EXPRESSION OF A DEUBIQUITINASE IMPORTANT FOR FOXP3 STABILITY. CLINICALLY, THE PROPORTIONS OF THESE CELLS WITHIN THE CD25(HI) T CELL SUBSET ARE INCREASED IN PATIENTS WITH THE MORE SEVERE COURSES OF DISEASE. OUR STUDY DEMONSTRATES, THEREFORE, THAT HYPOMETHYLATION AT THE TSDR CAN BE DECOUPLED FROM FOXP3 EXPRESSION IN HUMAN T CELLS AND THAT ENVIRONMENT-SPECIFIC BREAKDOWN IN FOXP3 STABILITY MAY COMPROMISE THE RESOLUTION OF INFLAMMATION IN JUVENILE IDIOPATHIC ARTHRITIS. 2014 12 295 36 AGING INDUCES B CELL DEFECTS AND DECREASED ANTIBODY RESPONSES TO INFLUENZA INFECTION AND VACCINATION. BACKGROUND: AGING IS CHARACTERIZED BY A PROGRESSIVE DECLINE IN THE CAPACITY OF THE IMMUNE SYSTEM TO FIGHT INFLUENZA VIRUS INFECTION AND TO RESPOND TO VACCINATION. AMONG THE SEVERAL FACTORS INVOLVED, IN ADDITION TO INCREASED FRAILTY AND HIGH-RISK CONDITIONS, THE AGE-ASSOCIATED DECREASE IN CELLULAR AND HUMORAL IMMUNE RESPONSES PLAYS A RELEVANT ROLE. THIS IS IN LARGE PART DUE TO INFLAMMAGING, THE CHRONIC LOW-GRADE INFLAMMATORY STATUS OF THE ELDERLY, ASSOCIATED WITH INTRINSIC INFLAMMATION OF THE IMMUNE CELLS AND DECREASED IMMUNE FUNCTION. RESULTS: AGING IS USUALLY ASSOCIATED WITH REDUCED INFLUENZA VIRUS-SPECIFIC AND INFLUENZA VACCINE-SPECIFIC ANTIBODY RESPONSES BUT SOME ELDERLY INDIVIDUALS WITH HIGHER PRE-EXPOSURE ANTIBODY TITERS, DUE TO A PREVIOUS INFECTION OR VACCINATION, HAVE LESS PROBABILITY TO GET INFECTED. EXAMPLES OF THIS EXCEPTION ARE THE ELDERLY INDIVIDUALS INFECTED DURING THE 2009 PANDEMIC SEASON WHO MADE ANTIBODIES WITH BROADER EPITOPE RECOGNITION AND HIGHER AVIDITY THAN THOSE MADE BY YOUNGER INDIVIDUALS. SEVERAL STUDIES HAVE ALLOWED THE IDENTIFICATION OF B CELL INTRINSIC DEFECTS ACCOUNTING FOR SUB-OPTIMAL ANTIBODY RESPONSES OF ELDERLY INDIVIDUALS. THESE DEFECTS INCLUDE 1) REDUCED CLASS SWITCH RECOMBINATION, RESPONSIBLE FOR THE GENERATION OF A SECONDARY RESPONSE OF CLASS SWITCHED ANTIBODIES, 2) REDUCED DE NOVO SOMATIC HYPERMUTATION OF THE ANTIBODY VARIABLE REGION, 3) REDUCED BINDING AND NEUTRALIZATION CAPACITY, AS WELL AS BINDING SPECIFICITY, OF THE SECRETED ANTIBODIES, 4) INCREASED EPIGENETIC MODIFICATIONS THAT ARE ASSOCIATED WITH LOWER ANTIBODY RESPONSES, 5) INCREASED FREQUENCIES OF INFLAMMATORY B CELL SUBSETS, AND 6) SHORTER TELOMERES. CONCLUSIONS: ALTHOUGH INFLUENZA VACCINATION REPRESENTS THE MOST EFFECTIVE WAY TO PREVENT INFLUENZA INFECTION, VACCINES WITH GREATER IMMUNOGENICITY ARE NEEDED TO IMPROVE THE RESPONSE OF ELDERLY INDIVIDUALS. RECENT ADVANCES IN TECHNOLOGY HAVE MADE POSSIBLE A BROAD APPROACH TO BETTER UNDERSTAND THE AGE-ASSOCIATED CHANGES IN IMMUNE CELLS, NEEDED TO DESIGN TAILORED VACCINES AND EFFECTIVE THERAPEUTIC STRATEGIES THAT WILL BE ABLE TO IMPROVE THE IMMUNE RESPONSE OF VULNERABLE INDIVIDUALS. 2020 13 2765 28 EXPRESSION, EPIGENETIC REGULATION, AND HUMORAL IMMUNOGENICITY OF CANCER-TESTIS ANTIGENS IN CHRONIC MYELOID LEUKEMIA. OBJECTIVE: CANCER-TESTIS (CT) ANTIGENS REPRESENT ATTRACTIVE TARGETS FOR TUMOR IMMUNOTHERAPY BASED ON THEIR TUMOR-RESTRICTED EXPRESSION AND IMMUNOGENICITY. HOWEVER, A BROAD PICTURE OF THE EXPRESSION OF CT ANTIGENS AND ASSOCIATED HUMORAL IMMUNE RESPONSES IN CHRONIC MYELOID LEUKEMIA (CML) IS STILL MISSING. METHODS: WE SCREENED CML CELL LINES AND BONE MARROW (BM) SAMPLES FROM HEALTHY DONORS BY RT-PCR FOR THE EXPRESSION OF 31 CT ANTIGENS BEFORE AND AFTER TREATMENT WITH EPIGENETIC AGENTS. EXPRESSION OF TUMOR-RESTRICTED ANTIGENS WAS FURTHER EXAMINED IN 60 CML PATIENTS AND HUMORAL IMMUNE RESPONSES AGAINST 15 CT ANTIGENS WERE SCREENED BY ELISA. RESULTS: IN UNTREATED CELL LINES WE DETECTED THE EXPRESSION OF 17 CT ANTIGENS THAT WERE ABSENT FROM NORMAL BM. EXPRESSION OF MOST ANTIGENS INCREASED FOLLOWING DEMETHYLATING TREATMENT WITH 5'-AZA-2'-DEOXYCYTIDINE. IN THESE SAMPLES, ONLY PRAME WAS REPEATEDLY DETECTED AND EXPRESSION CORRELATED WITH SEVERAL CLINICOPATHOLOGICAL PARAMETERS AND DECREASED OVERALL SURVIVAL. WE FURTHER SHOW THAT A LOWER FREQUENCY OF PRAME-POSITIVE SAMPLES DURING IMATINIB TREATMENT WAS NOT CAUSED BY GENE-SPECIFIC DOWNREGULATION. ANALYZING THE PATIENTS' ANTIBODY RESPONSES WE FOUND THAT THE VAST MAJORITY OF PATIENTS LACKED SPONTANEOUS IMMUNITY AGAINST CT ANTIGENS INCLUDING PRAME. CONCLUSIONS: CT ANTIGEN EXPRESSION CAN BE INCREASED BY THE APPLICATION OF EPIGENETIC AGENTS AND THE EXPRESSION OF PRAME CORRELATES WITH CLINICOPATHOLOGICAL PARAMETERS AND OVERALL SURVIVAL IN PATIENTS WITH CML, BUT DOES NOT LEAD TO HUMORAL IMMUNE RESPONSES. PRAME-SPECIFIC IMMUNOTHERAPY MIGHT REPRESENT A PROMISING APPROACH FOR THE ERADICATION OF RESIDUAL THERAPY-RESISTANT LEUKEMIC CELLS DUE TO ITS FREQUENT EXPRESSION AND STABILITY UNDER IMATINIB TREATMENT. 2010 14 2392 23 EPIGENETIC REPRESSION OF INTERLEUKIN 2 EXPRESSION IN SENESCENT CD4+ T CELLS DURING CHRONIC HIV TYPE 1 INFECTION. THE MOLECULAR MECHANISMS FOR IL2 GENE-SPECIFIC DYSREGULATION DURING CHRONIC HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) INFECTION ARE UNKNOWN. HERE, WE INVESTIGATED THE ROLE OF DNA METHYLATION IN SUPPRESSING INTERLEUKIN 2 (IL-2) EXPRESSION IN MEMORY CD4(+) T CELLS DURING CHRONIC HIV-1 INFECTION. WE OBSERVED THAT CPG SITES IN THE IL2 PROMOTER OF CD4(+) T CELLS WERE FULLY METHYLATED IN NAIVE CD4(+) T CELLS AND SIGNIFICANTLY DEMETHYLATED IN THE MEMORY POPULATIONS. INTERESTINGLY, WE FOUND THAT THE MEMORY CELLS THAT HAD A TERMINALLY DIFFERENTIATED PHENOTYPE AND EXPRESSED CD57 HAD INCREASED IL2 PROMOTER METHYLATION RELATIVE TO LESS DIFFERENTIATED MEMORY CELLS IN HEALTHY INDIVIDUALS. IMPORTANTLY, EARLY EFFECTOR MEMORY SUBSETS FROM HIV-1-INFECTED SUBJECTS EXPRESSED HIGH LEVELS OF CD57 AND WERE HIGHLY METHYLATED AT THE IL2 LOCUS. FURTHERMORE, THE INCREASED CD57 EXPRESSION ON MEMORY CD4(+) T CELLS WAS INVERSELY CORRELATED WITH IL-2 PRODUCTION. THESE DATA SUGGEST THAT DNA METHYLATION AT THE IL2 LOCUS IN CD4(+) T CELLS IS COUPLED TO IMMUNOSENESCENCE AND PLAYS A CRITICAL ROLE IN THE BROAD DYSFUNCTION THAT OCCURS IN POLYCLONAL T CELLS DURING HIV-1 INFECTION. 2015 15 2326 28 EPIGENETIC REGULATION OF HOTAIR IN ADVANCED CHRONIC MYELOID LEUKEMIA. PURPOSE: CHRONIC MYELOID LEUKEMIA (CML) ACCOUNTS FOR ~10% OF LEUKEMIA CASES, AND ITS PROGRESSION INVOLVES EPIGENETIC GENE REGULATION. THIS STUDY INVESTIGATED EPIGENETIC REGULATION OF HOTAIR AND ITS TARGET MICRORNA, MIR-143, IN ADVANCED CML. PATIENTS AND METHODS: WE FIRST ISOLATED BONE MARROW MONONUCLEAR CELLS FROM 70 PATIENTS WITH DIFFERENT PHASES OF CML AND FROM HEALTHY DONORS AS NORMAL CONTROL; WE ALSO CULTURED K562 AND KCL22 CELLS, TREATED WITH DEMETHYLATION DRUG; MTT ASSAY, FLOW CYTOMETRY, QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), WESTERN BLOT, LUCIFERASE ASSAY, RNA PULL-DOWN ASSAY AND RNA-BINDING PROTEIN IMMUNOPRECIPITATION (RIP) ASSAY WERE PERFORMED. RESULT: AS MEASURED BY QPCR, HOTAIR EXPRESSION IN K562 CELLS, KCL22 CELLS, AND SAMPLES FROM CASES OF ADVANCED-STAGE CML INCREASED WITH LEVELS OF SEVERAL DNA METHYLTRANSFERASES AND HISTONE DEACETYLATES, INCLUDING DNMT1, DNMT3A, HDAC1, EZH2, AND LSD1, AND MIR-143 LEVELS WERE DECREASED AND HOTAIR LEVELS WERE INCREASED. TREATMENT WITH 5-AZACYTIDINE, A DNA METHYLATION INHIBITOR, DECREASED DNMT1, DNMT3A, HDAC1, EZH2, LSD1 MRNA, PROTEIN LEVELS, AND HOTAIR MRNA LEVELS BUT INCREASED MIR-143 LEVELS. HOTAIR KNOCKDOWN AND MIR-143 OVEREXPRESSION BOTH INHIBITED PROLIFERATION AND PROMOTED APOPTOSIS IN KCL22 AND K562 CELLS THROUGH THE PI3K/AKT PATHWAY. RNA PULL-DOWN, MASS SPECTROMETRY, AND RIP ASSAYS SHOWED THAT HOTAIR INTERACTED WITH EZH2 AND LSD1. A DUAL-LUCIFERASE ASSAY DEMONSTRATED THAT HOTAIR INTERACTED WITH MIR-143. CONCLUSION: OUR FINDINGS DEMONSTRATE THE KEY EPIGENETIC INTERACTIONS OF HOTAIR RELATED TO CML PROGRESSION AND SUGGEST HOTAIR AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AS A CML TREATMENT STRATEGY. 2018 16 556 33 AZACYTIDINE TREATMENT INHIBITS THE PROGRESSION OF HERPES STROMAL KERATITIS BY ENHANCING REGULATORY T CELL FUNCTION. OCULAR INFECTION WITH HERPES SIMPLEX VIRUS 1 (HSV-1) SETS OFF AN INFLAMMATORY REACTION IN THE CORNEA WHICH LEADS TO BOTH VIRUS CLEARANCE AND CHRONIC LESIONS THAT ARE ORCHESTRATED BY CD4 T CELLS. APPROACHES THAT ENHANCE THE FUNCTION OF REGULATORY T CELLS (TREG) AND DAMPEN EFFECTOR T CELLS CAN BE EFFECTIVE TO LIMIT STROMAL KERATITIS (SK) LESION SEVERITY. IN THIS REPORT, WE EXPLORE THE NOVEL APPROACH OF INHIBITING DNA METHYLTRANSFERASE ACTIVITY USING 5-AZACYTIDINE (AZA; A CYTOSINE ANALOG) TO LIMIT HSV-1-INDUCED OCULAR LESIONS. WE SHOW THAT THERAPY BEGUN AFTER INFECTION WHEN VIRUS WAS NO LONGER ACTIVELY REPLICATING RESULTED IN A PRONOUNCED REDUCTION IN LESION SEVERITY, WITH MARKEDLY DIMINISHED NUMBERS OF T CELLS AND NONLYMPHOID INFLAMMATORY CELLS, ALONG WITH REDUCED CYTOKINE MEDIATORS. THE REMAINING INFLAMMATORY REACTIONS HAD A CHANGE IN THE RATIO OF CD4 FOXP3(+) TREG TO EFFECTOR TH1 CD4 T CELLS IN OCULAR LESIONS AND LYMPHOID TISSUES, WITH TREG BECOMING PREDOMINANT OVER THE EFFECTORS. IN ADDITION, COMPARED TO THOSE FROM CONTROL MICE, TREG FROM AZA-TREATED MICE SHOWED MORE SUPPRESSOR ACTIVITY IN VITRO AND EXPRESSED HIGHER LEVELS OF ACTIVATION MOLECULES. ADDITIONALLY, CELLS INDUCED IN VITRO IN THE PRESENCE OF AZA SHOWED EPIGENETIC DIFFERENCES IN THE TREG-SPECIFIC DEMETHYLATED REGION (TSDR) OF FOXP3 AND WERE MORE STABLE WHEN EXPOSED TO INFLAMMATORY CYTOKINES. OUR RESULTS SHOW THAT THERAPY WITH AZA IS AN EFFECTIVE MEANS OF CONTROLLING A VIRUS-INDUCED INFLAMMATORY REACTION AND MAY ACT MAINLY BY THE EFFECTS ON TREG.IMPORTANCE HSV-1 INFECTION HAS BEEN SHOWN TO INITIATE AN INFLAMMATORY REACTION IN THE CORNEA THAT LEADS TO TISSUE DAMAGE AND LOSS OF VISION. THE INFLAMMATORY REACTION IS ORCHESTRATED BY GAMMA INTERFERON (IFN-GAMMA)-SECRETING TH1 CELLS, AND REGULATORY T CELLS PLAY A PROTECTIVE ROLE. HENCE, NOVEL THERAPEUTICS THAT CAN REBALANCE THE RATIO OF REGULATORY T CELLS TO EFFECTORS ARE A RELEVANT ISSUE. THIS STUDY OPENS UP A NEW AVENUE IN TREATING HSV-INDUCED SK LESIONS BY INCREASING THE STABILITY AND FUNCTION OF REGULATORY T CELLS USING THE DNA METHYLTRANSFERASE INHIBITOR 5-AZACYTIDINE (AZA). AZA INCREASED THE FUNCTION OF REGULATORY T CELLS, LEADING TO ENHANCED SUPPRESSIVE ACTIVITY AND DIMINISHED LESIONS. HENCE, THERAPY WITH AZA, WHICH ACTS MAINLY BY ITS EFFECTS ON TREG, CAN BE AN EFFECTIVE MEANS TO CONTROL VIRUS-INDUCED INFLAMMATORY LESIONS. 2017 17 3037 34 GENOME AND METHYLOME VARIATION IN HELICOBACTER PYLORI WITH A CAG PATHOGENICITY ISLAND DURING EARLY STAGES OF HUMAN INFECTION. BACKGROUND & AIMS: HELICOBACTER PYLORI IS REMARKABLE FOR ITS GENETIC VARIATION; YET, LITTLE IS KNOWN ABOUT ITS GENETIC CHANGES DURING EARLY STAGES OF HUMAN INFECTION, AS THE BACTERIA ADAPT TO THEIR NEW ENVIRONMENT. WE ANALYZED GENOME AND METHYLOME VARIATIONS IN A FULLY VIRULENT STRAIN OF H PYLORI DURING EXPERIMENTAL INFECTION. METHODS: WE PERFORMED A RANDOMIZED PHASE I/II, OBSERVER-BLIND, PLACEBO-CONTROLLED STUDY OF 12 HEALTHY, H PYLORI-NEGATIVE ADULTS IN GERMANY FROM OCTOBER 2008 THROUGH MARCH 2010. THE VOLUNTEERS WERE GIVEN A PROPHYLACTIC VACCINE CANDIDATE (N = 7) OR PLACEBO (N = 5) AND THEN CHALLENGED WITH H PYLORI STRAIN BCM-300. BIOPSY SAMPLES WERE COLLECTED AND H PYLORI WERE ISOLATED. GENOMES OF THE CHALLENGE STRAIN AND 12 REISOLATES, OBTAINED 12 WEEKS AFTER (OR IN 1 CASE, 62 WEEKS AFTER) INFECTION WERE SEQUENCED BY SINGLE-MOLECULE, REAL-TIME TECHNOLOGY, WHICH, IN PARALLEL, PERMITTED DETERMINATION OF GENOME-WIDE METHYLATION PATTERNS FOR ALL STRAINS. FUNCTIONAL EFFECTS OF GENETIC CHANGES OBSERVED IN H PYLORI STRAINS DURING HUMAN INFECTION WERE ASSESSED BY MEASURING RELEASE OF INTERLEUKIN 8 FROM AGS CELLS (TO DETECT CAG PATHOGENICITY ISLAND FUNCTION), NEUTRAL RED UPTAKE (TO DETECT VACUOLATING CYTOTOXIN ACTIVITY), AND ADHESION ASSAYS. RESULTS: THE OBSERVED MUTATION RATE WAS IN AGREEMENT WITH RATES PREVIOUSLY DETERMINED FROM PATIENTS WITH CHRONIC H PYLORI INFECTIONS, WITHOUT EVIDENCE OF A MUTATION BURST. A LOSS OF CAG PATHOGENICITY ISLAND FUNCTION WAS OBSERVED IN 3 REISOLATES. IN ADDITION, 3 REISOLATES FROM THE VACCINE GROUP ACQUIRED MUTATIONS IN THE VACUOLATING CYTOTOXIN GENE VACA, RESULTING IN LOSS OF VACUOLIZATION ACTIVITY. WE OBSERVED INTERSTRAIN VARIATION IN METHYLOMES DUE TO PHASE VARIATION IN GENES ENCODING METHYLTRANSFERASES. CONCLUSIONS: WE ANALYZED ADAPTATION OF A FULLY VIRULENT STRAIN OF H PYLORI TO 12 DIFFERENT VOLUNTEERS TO OBTAIN A ROBUST ESTIMATE OF THE FREQUENCY OF GENETIC AND EPIGENETIC CHANGES IN THE ABSENCE OF INTERSTRAIN RECOMBINATION. OUR FINDINGS INDICATE THAT THE LARGE AMOUNT OF GENETIC VARIATION IN H PYLORI POSES A CHALLENGE TO VACCINE DEVELOPMENT. CLINICALTRIALS.GOV NO: NCT00736476. 2018 18 5058 31 PHENOTYPIC ALTERATION OF CD8+ T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA IS ASSOCIATED WITH EPIGENETIC REPROGRAMMING. IMMUNOSUPPRESSION IS A PREVALENT CLINICAL FEATURE IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) PATIENTS, WITH MANY PATIENTS DEMONSTRATING INCREASED SUSCEPTIBILITY TO INFECTIONS AS WELL AS INCREASED FAILURE OF AN ANTITUMOR IMMUNE RESPONSE. HOWEVER, MUCH IS CURRENTLY NOT UNDERSTOOD REGARDING THE PRECISE MECHANISMS THAT ATTRIBUTE TO THIS IMMUNOSUPPRESSIVE PHENOTYPE IN CLL. TO PROVIDE FURTHER CLARITY TO THIS PARTICULAR PHENOMENON, WE ANALYZED THE T-CELL PROFILE OF CLL PATIENT SAMPLES WITHIN A LARGE COHORT AND OBSERVED THAT PATIENTS WITH AN INVERTED CD4/CD8 RATIO HAD A SHORTER TIME TO FIRST TREATMENT AS WELL AS OVERALL SURVIVAL. THESE OBSERVATIONS COINCIDED WITH HIGHER EXPRESSION OF THE IMMUNE CHECKPOINT RECEPTOR PD-1 IN CLL PATIENT CD8+ T CELLS WHEN COMPARED TO AGE-MATCHED HEALTHY DONORS. INTERESTINGLY, WE DISCOVERED THAT INCREASED PD-1 EXPRESSION IN CD8+ T CELLS CORRESPONDS WITH DECREASED DNA METHYLATION LEVELS IN A DISTAL UPSTREAM LOCUS OF THE PD-1 GENE PDCD1. FURTHER ANALYSIS USING LUCIFERASE REPORTER ASSAYS SUGGESTS THAT THE IDENTIFIED PDCD1 DISTAL UPSTREAM REGION ACTS AS AN ENHANCER FOR PDCD1 TRANSCRIPTION AND THIS REGION BECOMES DEMETHYLATED DURING ACTIVATION OF NAIVE CD8+ T CELLS BY ANTI-CD3/ANTI-CD28 ANTIBODIES AND IL2. FINALLY, WE CONDUCTED A GENOME-WIDE DNA METHYLATION ANALYSIS COMPARING CD8+ T CELLS FROM CLL PATIENTS AGAINST HEALTHY DONORS AND IDENTIFIED ADDITIONAL DIFFERENTIALLY METHYLATED GENES WITH KNOWN IMMUNE REGULATORY FUNCTIONS INCLUDING CCR6 AND KLRG1. TAKEN TOGETHER, OUR FINDINGS REVEAL THE OCCURRENCE OF EPIGENETIC REPROGRAMMING TAKING PLACE WITHIN CLL PATIENT CD8+ T CELLS AND HIGHLIGHT THE POTENTIAL MECHANISM OF HOW IMMUNOSUPPRESSION IS ACCOMPLISHED IN CLL. 2016 19 912 17 CHRONIC EXPOSURE TO WATER POLLUTANT TRICHLOROETHYLENE INCREASED EPIGENETIC DRIFT IN CD4(+) T CELLS. AIM: AUTOIMMUNE DISEASE AND CD4(+) T-CELL ALTERATIONS ARE INDUCED IN MICE EXPOSED TO THE WATER POLLUTANT TRICHLOROETHYLENE (TCE). WE EXAMINED HERE WHETHER TCE ALTERED GENE-SPECIFIC DNA METHYLATION IN CD4(+) T CELLS AS A POSSIBLE MECHANISM OF IMMUNOTOXICITY. MATERIALS & METHODS: NAIVE AND EFFECTOR/MEMORY CD4(+) T CELLS FROM MICE EXPOSED TO TCE (0.5 MG/ML IN DRINKING WATER) FOR 40 WEEKS WERE EXAMINED BY BISULFITE NEXT-GENERATION DNA SEQUENCING. RESULTS: A PROBABILISTIC MODEL CALCULATED FROM MULTIPLE GENES SHOWED THAT TCE DECREASED METHYLATION CONTROL IN CD4(+) T CELLS. DATA FROM INDIVIDUAL GENES FITTED TO A QUADRATIC REGRESSION MODEL SHOWED THAT TCE INCREASED GENE-SPECIFIC METHYLATION VARIANCE IN BOTH CD4 SUBSETS. CONCLUSION: TCE INCREASED EPIGENETIC DRIFT OF SPECIFIC CPG SITES IN CD4(+) T CELLS. 2016 20 1158 32 CONTEXT-DEPENDENT EPIGENETIC REGULATION OF NUCLEAR FACTOR OF ACTIVATED T CELLS 1 IN PANCREATIC PLASTICITY. BACKGROUND & AIMS: THE ABILITY OF EXOCRINE PANCREATIC CELLS TO CHANGE THE CELLULAR PHENOTYPE IS REQUIRED FOR TISSUE REGENERATION UPON INJURY, BUT ALSO CONTRIBUTES TO THEIR MALIGNANT TRANSFORMATION AND TUMOR PROGRESSION. WE INVESTIGATED CONTEXT-DEPENDENT SIGNALING AND TRANSCRIPTION MECHANISMS THAT DETERMINE PANCREATIC CELL FATE DECISIONS TOWARD REGENERATION AND MALIGNANCY. IN PARTICULAR, WE STUDIED THE FUNCTION AND REGULATION OF THE INFLAMMATORY TRANSCRIPTION FACTOR NUCLEAR FACTOR OF ACTIVATED T CELLS 1 (NFATC1) IN PANCREATIC CELL PLASTICITY AND TISSUE ADAPTATION. METHODS: WE ANALYZED CELL PLASTICITY DURING PANCREATIC REGENERATION AND TRANSFORMATION IN MICE WITH PANCREAS-SPECIFIC EXPRESSION OF A CONSTITUTIVELY ACTIVE FORM OF NFATC1, OR DEPLETION OF ENHANCER OF ZESTE 2 HOMOLOGUE 2 (EZH2), IN THE CONTEXT OF WILD-TYPE OR CONSTITUTIVELY ACTIVATE KRAS, RESPECTIVELY. ACUTE AND CHRONIC PANCREATITIS WERE INDUCED BY INTRAPERITONEAL INJECTION OF CAERULEIN. EZH2-DEPENDENT REGULATION OF NFATC1 EXPRESSION WAS STUDIED IN MOUSE IN HUMAN PANCREATIC TISSUE AND CELLS BY IMMUNOHISTOCHEMISTRY, IMMUNOBLOTTING, AND QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION. WE USED GENETIC AND PHARMACOLOGIC APPROACHES OF EZH2 AND NFATC1 INHIBITION TO STUDY THE CONSEQUENCES OF PATHWAY DISRUPTION ON PANCREATIC MORPHOLOGY AND FUNCTION. EPIGENETIC MODIFICATIONS ON THE NFATC1 GENE WERE INVESTIGATED BY CHROMATIN IMMUNOPRECIPITATION ASSAYS. RESULTS: NFATC1 WAS RAPIDLY AND TRANSIENTLY INDUCED IN EARLY ADAPTATION TO ACINAR CELL INJURY IN HUMAN SAMPLES AND IN MICE, WHERE IT PROMOTED ACINAR CELL TRANSDIFFERENTIATION AND BLOCKED PROLIFERATION OF METAPLASTIC PANCREATIC CELLS. HOWEVER, IN LATE STAGES OF REGENERATION, NFATC1 WAS EPIGENETICALLY SILENCED BY EZH2-DEPENDENT HISTONE METHYLATION, TO ENABLE ACINAR CELL REDIFFERENTIATION AND PREVENT ORGAN ATROPHY AND EXOCRINE INSUFFICIENCY. IN CONTRAST, ONCOGENIC ACTIVATION OF KRAS SIGNALING IN PANCREATIC DUCTAL ADENOCARCINOMA CELLS REVERSED THE EZH2-DEPENDENT EFFECTS ON THE NFATC1 GENE AND WAS REQUIRED FOR EZH2-MEDIATED TRANSCRIPTIONAL ACTIVATION OF NFATC1. CONCLUSIONS: IN STUDIES OF HUMAN AND MOUSE PANCREATIC CELLS AND TISSUE, WE IDENTIFIED CONTEXT-SPECIFIC EPIGENETIC REGULATION OF NFATC1 ACTIVITY AS AN IMPORTANT MECHANISM OF PANCREATIC CELL PLASTICITY. INHIBITORS OF EZH2 MIGHT THEREFORE INTERFERE WITH ONCOGENIC ACTIVITY OF NFATC1 AND BE USED IN TREATMENT OF PANCREATIC DUCTAL ADENOCARCINOMA. 2017