Clinical and radiographic features of a cohort of adult and pediatric subjects in the Pacific Northwest with myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD).

BACKGROUND: Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is a newly described clinical entity comprised of isolated or recurrent attacks of optic neuritis, transverse myelitis, acute disseminated encephalomyelitis (ADEM), encephalitis, or seronegative NMOSD. Prior studies report that 30-80 % of children and adults with MOGAD go on to have relapses though there are no reliable predictors. The objectives of this study were to (1) describe the demographic, clinical, and radiographic patterns of MOGAD at our center and (2) identify possible predictors of relapsing disease. METHODS: Single-center retrospective cohort study of pediatric and adult subjects with MOGAD evaluated at least once at our center between January 1, 2017 and September 30, 2022. Eligible subjects had a history of positive MOG-IgG and consistent clinical syndrome comprised of an initial attack of optic neuritis (ON), transverse myelitis (TM), ADEM, cerebral cortical encephalitis, seronegative neuromyelitis optica (simultaneous ON and TM), isolated brainstem or cerebellar syndrome, or other (not fitting into another group). Relapsing subjects or those remaining monophasic at 12 months were included in the analyses of predictors of relapsing disease. Covariates included age, sex, race/ethnicity, and index event phenotype. Unadjusted and adjusted risk ratios were calculated for pediatric and adult subjects. RESULTS: We describe the demographic, clinical, and radiographic characteristics of 58 subjects with MOGAD. Covariates from 48 subjects were analyzed for predictors of relapsing disease. In adults, Hispanics and non-White non-Hispanics were at increased risk of relapsing disease compared to non-Hispanic Whites [Adjusted RR 1.52 (95 % CI: 1.01, 2.30)]. There were no significant associations in the pediatric group. CONCLUSION: This study is the first to describe a cohort of MOGAD in the Pacific Northwest. Our findings highlight racial and ethnic differences in risk of relapsing MOGAD in adults. Further studies on racial and ethnic differences in MOGAD are needed to confirm these findings.

Vascular disease risk factors in multiple sclerosis: Effect on metabolism and brain volumes.

BACKGROUND: Vascular disease risk factors (VDRF) such as hypertension, hyperlipidemia, obesity, diabetes and heart disease likely play a role in disease progression in people with multiple sclerosis (PwMS) (Marrie, Rudick et al. 2010). Studies exploring the mechanistic connection between vascular disease and MS disease progression are scant. We hypothesized that phosphate energy metabolism impairment in PwMS with VDRFs (VDRF+) will be greater compared to PwMS without VDRFs (VDRF-) and is related to increased brain atrophy in VDRF+. To test this hypothesis, we planned to study the differences in the high energy phosphate (HEP) metabolites in cerebral gray matter as assessed by 31P magnetic resonance spectroscopic imaging (MRSI) and MRI brain volumetric in the VDRF+ and VDRF- PwMS at four different timepoints over a 3 yearlong period using a 7T MR system. We present here the results from the cross-sectional evaluation of HEP metabolites and brain volumes. We also evaluated the differences in clinical impairment, blood metabolic biomarkers and quality of life in VDRF+ and VDRF- PwMS in this cohort. METHODS: Group differences in high energy phosphate metabolites were assessed from a volume of interest in the occipital region using linear mixed models. Brain parenchymal and white matter lesion volumes were determined from MR anatomic images. We present here the cross-sectional analysis of the baseline data collected as part of a longitudinal 3 yearlong study where we obtained baseline and subsequent 6-monthly clinical and laboratory data and annual 7T MRI volumetric and 31P MR spectroscopic imaging (MRSI) data on 52 PwMS with and without VDRF. Key clinical and laboratory outcomes included: body mass index (BMI), waist and thigh circumferences and disability [Expanded Disability Status Scale (EDSS)], safety (complete blood count with differential, complete metabolic), lipid panel including total cholesterol and HbA1C. We analyzed clinical and laboratory data for the group differences using student's t or χ2 test. We investigated relationship between phosphate metabolites and VDRF using mixed effect linear regression. RESULTS: Complete MRI data were available for 29 VDRF+, age 56.3 (6.8) years [mean (SD)] (83% female), and 23 VDRF-, age 52.5 (7.5) years (57% female) individuals with MS. The mean value of normalized adenosine triphosphate (ATP) (calculated as the ratio of ATP to total phosphate signal in a voxel) was decreased by 4.5% (p < .05) in VDRF+ compared to VDRF- MS group. White matter lesion (WML) volume fraction in VDRF+ individuals {0.007 (0.007)} was more than doubled compared to VDRF- participants {0.003 (0.006), p= .02}. CONCLUSIONS: We found significantly lower brain ATP and higher inorganic phosphate (Pi) in those PwMS with VDRFs compared to those without. ATP depletion may reflect mitochondrial dysfunction. Ongoing longitudinal data analysis from this study, not presented here, will evaluate the relationship of phosphate metabolites, brain atrophy and disease progression in PwMS with and without vascular disease.

Epigenetic Plasticity Enables CNS-Trafficking of EBV-infected B Lymphocytes.

Subpopulations of B-lymphocytes traffic to different sites and organs to provide diverse and tissue-specific functions. Here, we provide evidence that epigenetic differences confer a neuroinvasive phenotype. An EBV+ B cell lymphoma cell line (M14) with low frequency trafficking to the CNS was neuroadapted to generate a highly neuroinvasive B-cell population (MUN14). MUN14 B cells efficiently infiltrated the CNS within one week and produced neurological pathologies. We compared the gene expression profiles of viral and cellular genes using RNA-Seq and identified one viral (EBNA1) and several cellular gene candidates, including secreted phosphoprotein 1/osteopontin (SPP1/OPN), neuron navigator 3 (NAV3), CXCR4, and germinal center-associated signaling and motility protein (GCSAM) that were selectively upregulated in MUN14. ATAC-Seq and ChIP-qPCR revealed that these gene expression changes correlated with epigenetic changes at gene regulatory elements. The neuroinvasive phenotype could be attenuated with a neutralizing antibody to OPN, confirming the functional role of this protein in trafficking EBV+ B cells to the CNS. These studies indicate that B-cell trafficking to the CNS can be acquired by epigenetic adaptations and provide a new model to study B-cell neuroinvasion associated CNS lymphoma and autoimmune disease of the CNS, including multiple sclerosis (MS).

Defective Epstein-Barr Virus Genomes and Atypical Viral Gene Expression in B-Cell Lines Derived from Multiple Myeloma Patients.

Epstein-Barr virus (EBV) is a human gammaherpesvirus that is causally associated with various lymphomas and carcinomas. Although EBV is not typically associated with multiple myeloma (MM), it can be found in some B-cell lines derived from MM patients. Here, we analyzed two EBV-positive MM-patient-derived cell lines, IM9 and ARH77, and found defective viral genomes and atypical viral gene expression patterns. We performed transcriptome sequencing to characterize the viral and cellular properties of the two EBV-positive cell lines, compared to the canonical MM cell line 8226. Principal-component analyses indicated that IM9 and ARH77 clustered together and distinct from 8226. Immunological Genome Project analysis designated these cells as stem cell and bone marrow derived. IM9 and ARH77 displayed atypical viral gene expression, including leaky lytic cycle gene expression with an absence of lytic DNA amplification. Genome sequencing revealed that the EBV genomes in ARH77 contain large deletions, while IM9 has copy number losses in multiple EBV loci. Both IM9 and ARH77 showed EBV genome heterogeneity, suggesting cells harboring multiple and variant viral genomes. We identified atypical high-level expression of lytic genes BLRF1 and BLRF2. We demonstrated that short hairpin RNA (shRNA) depletion of BLRF2 altered viral and host gene expression, including a reduction in lytic gene activation and DNA amplification. These findings demonstrate that aberrant viral genomes and lytic gene expression persist in rare B cells derived from MM tumors, and they suggest that EBV may contribute to the etiology of MM. IMPORTANCE EBV is an oncogenic herpesvirus, but its mechanisms of oncogenesis are not fully understood. A role for EBV in MM has not yet been established. We analyzed EBV-positive B-cell lines derived from MM patients and found that the cells harbored defective viral genomes with aberrant viral gene expression patterns and cell gene signatures for bone marrow-derived lymphoid stem cells. These findings suggest that aberrant EBV latent infection may contribute to the etiology of MM.

PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

Epstein Barr virus (EBV) is a potentially oncogenic gammaherpesvirus that establishes a chronic, latent infection in memory B cells. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type. CTCF is posttranslationally modified by the host enzyme PARP1. PARP1, or poly(ADP-ribose) polymerase 1, catalyzes the transfer of a poly(ADP-ribose) (PAR) moiety from NAD+ onto acceptor proteins, including itself, histone proteins, and CTCF. PARylation of CTCF by PARP1 can affect CTCF's insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Thus, we predicted that pharmacological inhibition with PARP1 inhibitors would affect EBV latency type through a chromatin-specific mechanism. Here, we show that PARP1 and CTCF colocalize at specific sites throughout the EBV genome and provide evidence to suggest that PARP1 acts to stabilize CTCF binding and maintain the open chromatin landscape at the active Cp promoter during type III latency. Further, PARP1 activity is important in maintaining latency type-specific viral gene expression. The data presented here provide a rationale for the use of PARP inhibitors in the treatment of EBV-associated cancers exhibiting type III latency and ultimately could contribute to an EBV-specific treatment strategy for AIDS-related or posttransplant lymphomas.IMPORTANCE EBV is a human gammaherpesvirus that infects more than 95% of individuals worldwide. Upon infection, EBV circularizes as an episome and establishes a chronic, latent infection in B cells. In doing so, the virus utilizes host cell machinery to regulate and maintain the viral genome. In otherwise healthy individuals, EBV infection is typically nonpathological; however, latent infection is potentially oncogenic and is responsible for 1% of human cancers. During latent infection, EBV expresses specific sets of proteins according to the given latency type, each of which is associated with specific types of cancers. For example, type III latency, in which the virus expresses its full repertoire of latent proteins, is characteristic of AIDS-associated and posttransplant lymphomas associated with EBV infection. Understanding how viral latency type is regulated at the chromatin level may reveal potential targets for EBV-specific pharmacological intervention in EBV-associated cancers.

Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

The enzyme Poly(ADP-ribose) polymerase 1 (PARP1) plays a very important role in the DNA damage response, but its role in numerous aspects is not fully understood. We recently showed that in the absence of DNA damage, PARP1 regulates the expression of the chromatin-modifying enzyme EZH2. Work from other groups has shown that EZH2 participates in the DNA damage response. These combined data suggest that EZH2 could be a target of PARP1 in both untreated and genotoxic agent-treated conditions. In this work we tested the hypothesis that, in response to DNA damage, PARP1 regulates EZH2 activity. Here we report that PARP1 regulates EZH2 activity after DNA damage. In particular, we find that EZH2 is a direct target of PARP1 upon induction of alkylating and UV-induced DNA damage in cells and in vitro. PARylation of EZH2 inhibits EZH2 histone methyltransferase (H3K27me) enzymatic activity. We observed in cells that the induction of PARP1 activity by DNA alkylating agents decreases the association of EZH2 with chromatin, and PARylation of histone H3 reduces EZH2 affinity for its target histone H3. Our findings establish that PARP1 and PARylation are important regulators of EZH2 function and link EZH2-mediated heterochromatin formation, DNA damage and PARylation. These findings may also have clinical implications, as they suggest that inhibitors of EZH2 can improve anti-tumor effects of PARP1 inhibitors in BRCA1/2-deficient cancers.

Interferon-γ Signaling in Melanocytes and Melanoma Cells Regulates Expression of CTLA-4.

CTLA4 is a cell surface receptor on T cells that functions as an immune checkpoint molecule to enforce tolerance to cognate antigens. Anti-CTLA4 immunotherapy is highly effective at reactivating T-cell responses against melanoma, which is postulated to be due to targeting CTLA4 on T cells. Here, we report that CTLA4 is also highly expressed by most human melanoma cell lines, as well as in normal human melanocytes. Interferon-γ (IFNG) signaling activated the expression of the human CTLA4 gene in a melanocyte and melanoma cell-specific manner. Mechanistically, IFNG activated CTLA4 expression through JAK1/2-dependent phosphorylation of STAT1, which bound a specific gamma-activated sequence site on the CTLA4 promoter, thereby licensing CBP/p300-mediated histone acetylation and local chromatin opening. In melanoma cell lines, elevated baseline expression relied upon constitutive activation of the MAPK pathway. Notably, RNA-seq analyses of melanoma specimens obtained from patients who had received anti-CTLA4 immunotherapy (ipilimumab) showed upregulation of an IFNG-response gene expression signature, including CTLA4 itself, which correlated significantly with durable response. Taken together, our results raise the possibility that CTLA4 targeting on melanoma cells may contribute to the clinical immunobiology of anti-CTLA4 responses.Significance: These findings show that human melanoma cells express high levels of the immune checkpoint molecule CTLA4, with important possible implications for understanding how anti-CTLA4 immunotherapy mediates its therapeutic effects. Cancer Res; 78(2); 436-50. ©2017 AACR.

Coordinate Regulation of TET2 and EBNA2 Controls the DNA Methylation State of Latent Epstein-Barr Virus.

Epstein-Barr virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the ten-eleven translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that short hairpin RNA (shRNA) depletion of TET2 results in a decrease in latency gene expression but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine and a corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-jκ binding and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene itself is regulated in a fashion similar to that of the EBV genome. Chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) revealed that the TET2 gene contains EBNA2-dependent RBP-jκ and EBF1 binding sites and is subject to DNA methylation-associated transcriptional silencing similar to what is seen in EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-jκ binding sites and can interact with EBNA2 by coimmunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes and that TET2 protein is a cofactor of EBNA2 and coregulator of the EBV type III latency program and DNA methylation state.IMPORTANCE Epstein-Barr virus (EBV) latency and carcinogenesis involve the selective epigenetic modification of viral and cellular genes. Here, we show that TET2, a cellular tumor suppressor involved in active DNA demethylation, plays a central role in regulating the DNA methylation state during EBV latency. TET2 is coordinately regulated and functionally interacts with the viral oncogene EBNA2. TET2 and EBNA2 function cooperatively to demethylate genes important for EBV-driven B-cell growth transformation.

Small molecule perturbation of the CAND1-Cullin1-ubiquitin cycle stabilizes p53 and triggers Epstein-Barr virus reactivation.

The chemical probe C60 efficiently triggers Epstein-Barr Virus (EBV) reactivation from latency through an unknown mechanism. Here, we identify the Cullin exchange factor CAND1 as a biochemical target of C60. We also identified CAND1 in an shRNA library screen for EBV lytic reactivation. Gene expression profiling revealed that C60 activates the p53 pathway and protein analysis revealed a strong stabilization and S15 phosphorylation of p53. C60 reduced Cullin1 association with CAND1 and led to a global accumulation of ubiquitylated substrates. C60 also stabilized the EBV immediate early protein ZTA through a Cullin-CAND1-interaction motif in the ZTA transcription activation domain. We propose that C60 perturbs the normal interaction and function of CAND1 with Cullins to promote the stabilization of substrates like ZTA and p53, leading to EBV reactivation from latency. Understanding the mechanism of action of C60 may provide new approaches for treatment of EBV associated tumors, as well as new tools to stabilize p53.

IGH/MYC Translocation Associates with BRCA2 Deficiency and Synthetic Lethality to PARP1 Inhibitors.

Burkitt lymphoma/leukemia cells carry t(8;14)(q24;q32) chromosomal translocation encoding IGH/MYC, which results in the constitutive expression of the MYC oncogene. Here, it is demonstrated that untreated and cytarabine (AraC)-treated IGH/MYC-positive Burkitt lymphoma cells accumulate a high number of potentially lethal DNA double-strand breaks (DSB) and display low levels of the BRCA2 tumor suppressor protein, which is a key element of homologous recombination (HR)-mediated DSB repair. BRCA2 deficiency in IGH/MYC-positive cells was associated with diminished HR activity and hypersensitivity to PARP1 inhibitors (olaparib, talazoparib) used alone or in combination with cytarabine in vitro Moreover, talazoparib exerted a therapeutic effect in NGS mice bearing primary Burkitt lymphoma xenografts. In conclusion, IGH/MYC-positive Burkitt lymphoma/leukemia cells have decreased BRCA2 and are sensitive to PARP1 inhibition alone or in combination with other chemotherapies.Implications: This study postulates that IGH/MYC-induced BRCA2 deficiency may predispose Burkitt lymphoma cells to synthetic lethality triggered by PARP1 inhibitors.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/15/8/967/F1.large.jpgMol Cancer Res; 15(8); 967-72. ©2017 AACR.

PARP1 restricts Epstein Barr Virus lytic reactivation by binding the BZLF1 promoter.

The Epstein Barr virus (EBV) genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type, and in other herpesviruses, loss of CTCF binding at specific regions correlates with viral reactivation. Here, we demonstrate that binding of PARP1, an important cofactor of CTCF, at the BZLF1 lytic switch promoter restricts EBV reactivation. Knockdown of PARP1 in the Akata-EBV cell line significantly increases viral copy number and lytic protein expression. Interestingly, CTCF knockdown has no effect on viral reactivation, and CTCF binding across the EBV genome is largely unchanged following reactivation. Moreover, EBV reactivation attenuates PARP activity, and Zta expression alone is sufficient to decrease PARP activity. Here we demonstrate a restrictive function of PARP1 in EBV lytic reactivation.

HIV-1 Vpr disrupts mitochondria axonal transport and accelerates neuronal aging.

Disruption of mitochondria axonal transport, essential for the maintenance of synaptic and neuronal integrity and function, has been identified in neurodegenerative diseases. Whether HIV-1 viral proteins affect mitochondria axonal transport is unknown, albeit HIV-associated neurocognitive disorders occur in around half of the patients living with HIV. Therefore, we sought to examine the effect of HIV-1 viral protein R (Vpr) on mitochondria axonal transport. Using mice primary neuronal cultures, we demonstrated that 4-day Vpr treatment reduced the ratio of moving mitochondria associated with (i) less energy (ATP) supply, (ii) reduction in Miro-1 and (iii) increase of α-synuclein which led to loss of microtubule stability as demonstrated by inconsecutive distribution of acetylated α-tubulin along the axons. Interestingly, the effect of Vpr on mitochondria axonal transport was partially restored in the presence of bongkrekic acid, a compound that negatively affected the Vpr-adenine nucleotide translocator (ANT) interaction and totally restored the ATP level in neurons. This indicated Vpr impaired mitochondria axonal transport partially related to its interaction with ANT. The above effect of Vpr was similar to the data obtained from hippocampal tissues isolated from 18-month-old aging mice compared to 5-month-old mice. In accord with previous clinical findings that HIV infection prematurely ages the brain and increases the susceptibility to HAND, we found that Vpr induced aging markers in neurons. Thus, we concluded that instead of causing cell death, low concentration of HIV-1 Vpr altered neuronal function related with inhibition of mitochondria axonal transport which might contribute to the accelerated neuronal aging.

Novel STIM1-dependent control of Ca2+ clearance regulates NFAT activity during T-cell activation.

Antigen presentation to the T-cell receptor leads to sustained cytosolic Ca2+ elevation, which is critical for T-cell activation. We previously showed that in activated T cells, Ca2+ clearance is inhibited by the endoplasmic reticulum Ca2+ sensor stromal interacting molecule 1 (STIM1) via association with the plasma membrane Ca2+/ATPase 4 (PMCA4) Ca2+ pump. Having further observed that expression of both proteins is increased in activated T cells, the current study focused on mechanisms regulating both up-regulation of STIM1 and PMCA4 and assessing how this up-regulation contributes to control of Ca2+ clearance. Using a STIM1 promoter luciferase vector, we found that the zinc finger transcription factors early growth response (EGR) 1 and EGR4, but not EGR2 or EGR3, drive luciferase activity. We further found that neither STIM1 nor PMCA4 is up-regulated when both EGR1 and EGR4 are knocked down using RNA interference. Further, under these conditions, activation-induced Ca2+ clearance inhibition was eliminated with little effect on Ca2+ entry. Finally, we found that nuclear factor of activated T-cell (NFAT) activity is profoundly attenuated if Ca2+ clearance is not inhibited by STIM1. These findings reveal a critical role for STIM1-mediated control of Ca2+ clearance in NFAT induction during T-cell activation.-Samakai, E., Hooper, R., Martin, K. A., Shmurak, M., Zhang, Y., Kappes, D. J., Tempera, I., Soboloff, J. Novel STIM1-dependent control of Ca2+ clearance regulates NFAT activity during T-cell activation.

Epstein-Barr Virus Oncoprotein LMP1 Mediates Epigenetic Changes in Host Gene Expression through PARP1.

UNLABELLED: The latent infection of Epstein-Barr virus (EBV) is associated with 1% of human cancer incidence. Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) that mediate EBV replication during latency. In this study, we detail the mechanisms that drive cellular PARylation during latent EBV infection and the effects of PARylation on host gene expression and cellular function. EBV-infected B cells had higher PAR levels than EBV-negative B cells. Moreover, cellular PAR levels were up to 2-fold greater in type III than type I latently infected EBV B cells. We identified a positive association between expression of the EBV genome-encoded latency membrane protein 1 (LMP1) and PAR levels that was dependent upon PARP1. PARP1 regulates gene expression by numerous mechanisms, including modifying chromatin structure and altering the function of chromatin-modifying enzymes. Since LMP1 is essential in establishing EBV latency and promoting tumorigenesis, we explored the model that disruption in cellular PARylation, driven by LMP1 expression, subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP1 inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes. Inhibition of PARP1, or abrogation of PARP1 expression, also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP1 activity in LMP1-induced gene expression and cellular transformation associated with LMP1. In summary, we identified a novel mechanism by which LMP1 drives expression of host tumor-promoting genes by blocking generation of the inhibitory histone modification H3K27me3 through PARP1 activation. IMPORTANCE: EBV is causally linked to several malignancies and is responsible for 1% of cancer incidence worldwide. The EBV-encoded protein LMP1 is essential for promoting viral tumorigenesis by aberrant activation of several well-known intracellular signaling pathways. We have identified and defined an additional novel molecular mechanism by which LMP1 regulates the expression of tumor-promoting host genes. We found that LMP1 activates the cellular protein PARP1, leading to a decrease in a repressive histone modification, accompanied by induction in expression of multiple cancer-related genes. PARP1 inhibition or depletion led to a decrease in LMP1-induced cellular transformation. Therefore, targeting PARP1 activity may be an effective treatment for EBV-associated malignancies.

Genomic alterations in abnormal neutrophils isolated from adult patients with systemic lupus erythematosus.

INTRODUCTION: Patients with systemic lupus erythematosus (SLE) have an abnormal population of neutrophils, called low-density granulocytes (LDGs), that express the surface markers of mature neutrophils, yet their nuclear morphology resembles an immature cell. Because a similar discrepancy in maturation status is observed in myelodysplasias, and disruption of neutrophil development is frequently associated with genomic alterations, genomic DNA isolated from autologous pairs of LDGs and normal-density neutrophils was compared for genomic changes. METHODS: Alterations in copy number and losses of heterozygosity (LOH) were detected by cytogenetic microarray analysis. Microsatellite instability (MSI) was detected by capillary gel electrophoresis of fluorescently labeled PCR products. RESULTS: Control neutrophils and normal-density SLE neutrophils had similar levels of copy number variations, while the autologous SLE LDGs had an over twofold greater number of copy number alterations per genome. The additional copy number alterations found in LDGs were prevalent in six of the thirteen SLE patients, and occurred preferentially on chromosome 19, 17, 8, and X. These same SLE patients also displayed an increase in LOH. Several SLE patients had a common LOH on chromosome 5q that includes several cytokine genes and a DNA repair enzyme. In addition, three SLE patients displayed MSI. Two patients displayed MSI in greater than one marker, and one patient had MSI and increased copy number alterations. No correlations between genomic instability and immunosuppressive drugs, disease activity or disease manifestations were apparent. CONCLUSIONS: The increased level of copy number alterations and LOH in the LDG samples relative to autologous normal-density SLE neutrophils suggests somatic alterations that are consistent with DNA strand break repair, while MSI suggests a replication error-prone status. Thus, the LDGs isolated have elevated levels of somatic alterations that are consistent with genetic damage or genomic instability. This suggests that the LDGs in adult SLE patients are derived from cell progenitors that are distinct from the autologous normal-density neutrophils, and may reflect a role for genomic instability in the disease.

Epigenetic deregulation of the LMP1/LMP2 locus of Epstein-Barr virus by mutation of a single CTCF-cohesin binding site.

The chromatin regulatory factors CTCF and cohesin have been implicated in the coordinated control of multiple gene loci in Epstein-Barr virus (EBV) latency. We have found that CTCF and cohesin are highly enriched at the convergent and partially overlapping transcripts for the LMP1 and LMP2A genes, but it is not yet known how CTCF and cohesin may coordinately regulate these transcripts. We now show that genetic disruption of this CTCF binding site (EBVΔCTCF166) leads to a deregulation of LMP1, LMP2A, and LMP2B transcription in EBV-immortalized B lymphocytes. EBVΔCTCF166 virus-immortalized primary B lymphocytes showed a decrease in LMP1 and LMP2A mRNA and a corresponding increase in LMP2B mRNA. The reduction of LMP1 and LMP2A correlated with a loss of euchromatic histone modification H3K9ac and a corresponding increase in heterochromatic histone modification H3K9me3 at the LMP2A promoter region in EBVΔCTCF166. Chromosome conformation capture (3C) revealed that DNA loop formation with the origin of plasmid replication (OriP) enhancer was eliminated in EBVΔCTCF166. We also observed that the EBV episome copy number was elevated in EBVΔCTCF166 and that this was not due to increased lytic cycle activity. These findings suggest that a single CTCF binding site controls LMP2A and LMP1 promoter selection, chromatin boundary function, DNA loop formation, and episome copy number control during EBV latency.

Human fetal testis xenografts are resistant to phthalate-induced endocrine disruption.

BACKGROUND: In utero exposure to endocrine-disrupting chemicals may contribute to testicular dysgenesis syndrome (TDS), a proposed constellation of increasingly common male reproductive tract abnormalities (including hypospadias, cryptorchidism, hypospermatogenesis, and testicular cancer). Male rats exposed in utero to certain phthalate plasticizers exhibit multinucleated germ cell (MNG) induction and suppressed steroidogenic gene expression and testosterone production in the fetal testis, causing TDS-consistent effects of hypospadias and cryptorchidism. Mice exposed to phthalates in utero exhibit MNG induction only. This disparity in response demonstrates a species-specific sensitivity to phthalate-induced suppression of fetal Leydig cell steroidogenesis. Importantly, ex vivo phthalate exposure of the fetal testis does not recapitulate the species-specific endocrine disruption, demonstrating the need for a new bioassay to assess the human response to phthalates. OBJECTIVES: In this study, we aimed to develop and validate a rat and mouse testis xenograft bioassay of phthalate exposure and examine the human fetal testis response. METHODS: Fetal rat, mouse, and human testes were xenografted into immunodeficient rodent hosts, and hosts were gavaged with a range of phthalate doses over multiple days. Xenografts were harvested and assessed for histopathology and steroidogenic end points. RESULTS: Consistent with the in utero response, phthalate exposure induced MNG formation in rat and mouse xenografts, but only rats exhibited suppressed steroidogenesis. Across a range of doses, human fetal testis xenografts exhibited MNG induction but were resistant to suppression of steroidogenic gene expression. CONCLUSIONS: Phthalate exposure of grafted human fetal testis altered fetal germ cells but did not reduce expression of genes that regulate fetal testosterone biosynthesis.