Depletion of Circular RNA circ_CORO1C Suppresses Gastric Cancer Development by Modulating miR-138-5p/KLF12 Axis.

BACKGROUND: Gastric cancer (GC) is a common and deadly malignancy in the world. CircRNAs have emerged as important regulators in human diseases, including GC. In this work, we intended to explore the role of circ_CORO1C in GC progression and potential mechanism. METHODS: Quantitative real-time PCR (qRT-PCR) or Western blot assay was performed to examine the expression of circRNA coronin-like actin-binding protein 1C (circ_CORO1C), microRNA (miR)-138-5p and Krueppel-like factor 12 (KLF12) in clinical samples and cells. Cell colony formation ability and viability were measured by colony formation assay and methyl thiazolyl tetrazolium (MTT) assay, respectively. Expression of cell proliferation and epithelia-mesenchymal transition (EMT) biomarker was detected by Western blot analysis. And cell metastasis, including migration and invasion, and apoptosis were analyzed via Transwell assay and flow cytometry, respectively. Target relationship among circ_CORO1C, miR-138-5p and KLF12 was validated by dual-luciferase reporter assay. The in vivo role of circ_CORO1C was investigated by tumor xenograft assay. RESULTS: Circ_CORO1C and KLF12 were upregulated, while miR-138-5p was downregulated in GC tissues and cells. Circ_CORO1C knockdown suppressed colony formation ability, viability, migration, invasion and EMT in GC cells, while promoted cell apoptosis in vitro. Circ_CORO1C targeted miR-138-5p, the inhibition of which could attenuate silenced circ_CORO1C-induced inhibitory effects on GC progression. MiR-138-5p repressed the aggressive malignant behaviors of GC cells by directly targeting KLF12. Circ_CORO1C deficiency inhibited GC tumor growth in vivo. CONCLUSION: Depletion of circ_CORO1C suppressed GC progression by regulating miR-138-5p/KLF12 axis, offering a potential molecular target for GC therapy.

Malondialdehyde Conjugated With Albumin Induces Pro-Inflammatory Activation of T Cells Isolated From Human Atherosclerotic Plaques Both Directly and Via Dendritic Cell-Mediated Mechanism.

Human dendritic cells were differentiated from blood monocytes and treated with malondialdehyde (MDA) conjugated with human serum albumin (HSA). Autologous T cells from human plaques or blood were co-cultured with the pre-treated dendritic cells or treated directly. MDA modifications were studied by mass spectrometry. MDA-HSA induced a pro-inflammatory DC-mediated T-cell activation and also a strong direct effect on T cells, inhibited by an inhibitor of oxidative stress and antibodies against MDA. Atherogenic heat shock protein-60 was strongly induced in T cells activated by MDA-HSA. Two peptide modifications in atherosclerotic patients' HSA were similar to those present in in vitro MDA-modified HSA.

IgM antibodies against phosphorylcholine promote polarization of T regulatory cells from patients with atherosclerotic plaques, systemic lupus erythematosus and healthy donors.

BACKGROUND AND AIMS: IgM antibodies against phosphorylcholine (anti-PC) are negatively associated with atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE), where the risk of CVD and atherosclerosis is high. We here study the effects of IgM anti-PC immune regulation. METHODS: Mononuclear leukocytes were isolated from peripheral blood (PBMC) obtained from healthy blood donors, six SLE patients with age- and sex-matched controls, and symptom-giving human atherosclerotic plaques. The proportion of Th17 (CD4+CCR6+) and Treg (CD4+CD25+CD127dim/-) cells was determined by flow cytometry in CD4+T cells after 6 days of culture with Th17 or Treg-polarizing cytokines, with PMA and Ionomycin stimulation. IgM anti-PC were extracted from total IgM, with flow-through IgM as controls. Dendritic cells (DC) were differentiated from PBMC. Antibody peptide/protein characterization was done by a proteomics de novo sequencing approach. RESULTS: IgM anti-PC increased significantly the proportion of Tregs from healthy donors, SLE patients and atherosclerotic plaque cells while control antibodies did not. T cells from SLE patients had a significantly lower proportion of Tregs and a higher proportion of Th17 cells as compared to matched controls. IgM anti-PC, but not control antibodies, significantly reduced the production of IL-17 and TNF-α in cell cultures from SLE patients and atherosclerotic plaque cells. IgM anti-PC interacted with CD40 and kept DCs in an immature stage, potentially being tolerogenic. We observed differences in the IgM peptide expression levels in anti-PC compared to control antibodies. CONCLUSIONS: IgM anti-PC promote polarization of Tregs, which could represent a novel protective mechanism in atherosclerosis and autoimmune conditions as SLE.

Human IgM Antibodies to Malondialdehyde Conjugated With Albumin Are Negatively Associated With Cardiovascular Disease Among 60-Year-Olds.

BACKGROUND: Malondialdehyde (MDA) is generated during lipid peroxidation as in oxidized low-density lipoprotein, but antibodies against oxidized low-density lipoprotein show variable results in clinical studies. We therefore studied the risk of cardiovascular disease (CVD) associated with IgM antibodies against MDA conjugated with human albumin (anti-MDA). METHODS AND RESULTS: In a 5- to 7-year follow-up of 60-year-old men and women from Stockholm County previously screened for cardiovascular risk factors (2039 men, 2193 women), 209 incident CVD cases (defined as new events of coronary heart disease, fatal and nonfatal myocardial infarction, ischemic stroke, and hospitalization for angina pectoris) and 620 age- and sex-matched controls were tested for IgM anti-MDA by ELISA. Antibody peptide/protein characterization was done using a proteomics de novo sequencing approach. After adjustment for smoking, body-mass index, type 2 diabetes mellitus, hyperlipidemia, and hypertension, an increased CVD risk was observed in the low IgM anti-MDA percentiles (below 10th and 25th) (odds ratio and 95% CI: 2.0; 1.19-3.36 and 1.67; 1.16-2.41, respectively). Anti-MDA above the 66th percentile was associated with a decreased CVD risk (odds ratio 0.68; CI: 0.48-0.98). After stratification by sex, associations were only present among men. IgM anti-MDA levels were lower among cases (median [interquartile range]: 141.0 [112.7-164.3] versus 147.4 [123.5-169.6]; P=0.0177), even more so among men (130.6 [107.7-155.3] versus 143.0 [120.1-165.2]; P=0.001). The IgM anti-MDA variable region profiles are distinctly different and also more homologous in their content (correlates strongly with fewer peptides) than control antibodies (not binding MDA). CONCLUSIONS: IgM anti-MDA is a protection marker for CVD. This finding could have diagnostic and therapeutic implications.

Oxidized Low-Density Lipoprotein (OxLDL)-Treated Dendritic Cells Promote Activation of T Cells in Human Atherosclerotic Plaque and Blood, Which Is Repressed by Statins: microRNA let-7c Is Integral to the Effect.

BACKGROUND: Activated T cells and dendritic cells (DCs) are colocalized in atherosclerotic plaques in association with plaque rupture. Oxidized low-density lipoprotein (oxLDL) promotes immune activation and inflammation. We studied the effects of statins (atorvastatin and simvastatin) on human DC maturation and T-cell activation. METHODS AND RESULTS: Human peripheral blood monocytes were differentiated to DCs and stimulated with oxLDL. T cells were isolated from carotid endarterectomy specimens from patients undergoing carotid endarterectomy or from healthy individuals. Naïve T cells were cocultured with pretreated DCs. The effects of statin were studied. OxLDL induced DC maturation and T-cell activation. OxLDL induced atherogenic heat shock proteins (HSP) 60 and 90 and decreased potentially atheroprotective heat shock protein 27, effects restored by atorvastatin. T cells exposed to oxLDL-treated DCs produced interferon-γ and interleukin (IL)-17. Atorvastatin and simvastatin suppressed the DC maturation showing lower expression of CD80, CD83, and CD86, and limited their production of tumor necrosis factor-α, IL-1ß and IL-6, and increased transforming growth factor-ß and IL-10 secretion. Statin-treated DCs inhibited Th1 and/or Th17 polarization by downregulation of transcriptional factors T-bet and RORγt expression, and induced T regulatory cells with IL-10 production. OxLDL-induced miRNA let7c and phosphorylation of Akt and ERK were repressed by statins. Let-7c had a pivotal role in mediating effect of oxLDL. Experiments on T cells derived from carotid atherosclerotic plaques or healthy individuals showed similar results. CONCLUSIONS: Statins repress human DC maturation induced by oxLDL, limit T-cell activation, and repress an atherogenic heat shock protein profile and promote induction of T regulatory cells. MicroRNA let-7c is integral to the effects.

Naturally occurring human phosphorylcholine antibodies are predominantly products of affinity-matured B cells in the adult.

Phosphorylcholine (PC) is a classic T-independent Ag that is exposed on apoptotic cells, oxidized phospholipids, and bacterial polysaccharides. Experimental as well as epidemiological studies have over the past decade implicated Abs against PC (anti-PC) as anti-inflammatory and a strong protective factor in cardiovascular disease. Although clinically important, little is known about the development of anti-PC in humans. This study was conceived to dissect the human anti-PC repertoire and generate human mAbs. We designed a PC-specific probe to identify, isolate, and characterize PC-reactive B cells from 10 healthy individuals. The donors had all mounted somatically mutated Abs toward PC using a broad variety of Ig genes. PC-reactive B cells were primarily found in the IgM(+) memory subset, although significant numbers also were detected among naive, IgG(+), and CD27(+)CD43(+) B cells. Abs from these subsets were clonally related, suggesting a common origin. mAbs derived from the same donors exhibited equivalent or higher affinity for PC than the well-characterized murine T-15 clone. These results provide novel insights into the cellular and molecular ontogeny of atheroprotective PC Abs, thereby offering new opportunities for Ab-based therapeutic interventions.

The transporter ABCB7 is a mediator of the phenotype of acquired refractory anemia with ring sideroblasts.

Refractory anemia with ring sideroblasts (RARS) is characterized by mitochondrial ferritin (FTMT) accumulation and markedly suppressed expression of the iron transporter ABCB7. To test the hypothesis that ABCB7 is a key mediator of ineffective erythropoiesis of RARS, we modulated its expression in hematopoietic cells. ABCB7 up and downregulation did not influence growth and survival of K562 cells. In normal bone marrow, ABCB7 downregulation reduced erythroid differentiation, growth and colony formation, and resulted in a gene expression pattern similar to that observed in intermediate RARS erythroblasts, and in the accumulation of FTMT. Importantly, forced ABCB7 expression restored erythroid colony growth and decreased FTMT expression level in RARS CD34+ marrow cells. Mutations in the SF3B1 gene, a core component of the RNA splicing machinery, were recently identified in a high proportion of patients with RARS and 11 of the 13 RARS patients in this study carried this mutation. Interestingly, ABCB7 exon usage differed between normal bone marrow and RARS, as well as within the RARS cohort. In addition, SF3B1 silencing resulted in downregulation of ABCB7 in K562 cells undergoing erythroid differentiation. Our findings support that ABCB7 is implicated in the phenotype of acquired RARS and suggest a relation between SF3B1 mutations and ABCB7 downregulation.

Exploration of the correlations between interferon-γ in patient serum and HEPACAM in bladder transitional cell carcinoma, and the interferon-γ mechanism inhibiting BIU-87 proliferation.

PURPOSE: Interferon-γ inhibits cancer cell proliferation and induces re-expression of different tumor suppressor genes. As a candidate, HEPACAM is almost lost in bladder transitional cell carcinoma. To our knowledge whether interferon-γ inhibits BIU-87 proliferation and re-expresses HEPACAM mRNA is still unknown. Thus, we probed the mechanism and examined the correlations between interferon-γ in patient serum and HEPACAM in bladder transitional cell carcinoma. MATERIALS AND METHODS: Using enzyme-linked immunosorbent assay we measured serum interferon-γ in 27 men and 6 women, and 15 volunteers. Disease was Ta-T1 in 12 patients, T2-T4 in 21, low grade in 25, high grade in 8, primary in 13 and recurrent in 20. A total of 33 cancer and 26 adjacent tissues were examined by immunohistochemistry to detect HEPACAM protein and ensure the position. Under interferon-γ stimulation we detected BIU-87 proliferation by MTT assay. Cell cycles were examined by flow cytometry. HEPACAM mRNA expression was determined by reverse transcription-polymerase chain reaction. Western blot was used to detect p21(WAF1). RESULTS: Interferon-γ was remarkably low in patients with bladder transitional cell carcinoma vs volunteers (p <0.01). HEPACAM protein was highly expressed in adjacent tissue, mainly at the cytomembrane, but it was almost absent in bladder transitional cell carcinoma (p <0.01). The interferon-γ decrease in the serum of patients with bladder transitional cell carcinoma and the low HEPACAM expression in tumors correlated linearly (r = 0.899, p <0.01). In vitro interferon-γ inhibited BIU-87 proliferation (p <0.01) and slightly re-expressed HEPACAM mRNA (p <0.05). The cell cycle was arrested at G(0)/G(1) and p21(WAF1) was concurrently increased in response to interferon-γ (p <0.01). CONCLUSIONS: Results suggest an important connection between HEPACAM and interferon-γ, which may inhibit BIU-87 proliferation through HEPACAM re-expression and p21(WAF1) up-regulation to arrest cells at the G(0)/G(1) phase.

Lymphoblastoid cell line with B1 cell characteristics established from a chronic lymphocytic leukemia clone by in vitro EBV infection.

Chronic lymphocytic leukemia (CLL) cells express the receptor for Epstein-Barr virus (EBV) and can be infected in vitro. Infected cells do not express the growth-promoting set of EBV-encoded genes and therefore they do not yield LCLs, in most experiments. With exceptional clones, lines were obtained however. We describe a new line, HG3, established by in vitro EBV-infection from an IGHV1-2 unmutated CLL patient clone. All cells expressed EBNA-2 and LMP-1, the EBV-encoded genes pivotal for transformation. The karyotype, FISH cytogenetics and SNP-array profile of the line and the patient's ex vivo clone showed biallelic 13q14 deletions with genomic loss of DLEU7, miR15a/miR16-1, the two micro-RNAs that are deleted in 50% of CLL cases. Further features of CLL cells were: expression of CD5/CD20/CD27/CD43 and release of IgM natural antibodies reacting with oxLDL-like epitopes on apoptotic cells (cf. stereotyped subset-1). Comparison with two LCLs established from normal B cells showed 32 genes expressed at higher levels (> 2-fold). Among these were LHX2 and LILRA. These genes may play a role in the development of the disease. LHX2 expression was shown in self-renewing multipotent hematopoietic stem cells, and LILRA4 codes for a receptor for bone marrow stromal cell antigen-2 that contributes to B cell development. Twenty-four genes were expressed at lower levels, among these PARD3 that is essential for asymmetric cell division. These genes may contribute to establish precursors of CLL clones by regulation of cellular phenotype in the hematopoietic compartment. Expression of CD5/CD20/CD27/CD43 and spontaneous production of natural antibodies may identify the CLL cell as a self-renewing B1 lymphocyte.

Activation of innate immunity by the leukotriene B 4 inhibits EBV induced B-cell transformation in cord-blood derived mononuclear cultures.

EBV specific cellular memory is not transferred from mother to child. By addition of the immunomodulators PSK and Trx80, that act on monocytes we could generate EBV-specific T cell response in cord-blood derived mononuclear cultures infected with the virus. In such cultures EBV infected B lymphocytes activated T and NK cells, and the immunostimulators activated the monocytes. Lymphokines produced by the monocytes induced the T and NK cells to progress into a functional activation state and they inhibited the EBV induced proliferation of B lymphocytes. Leukotrienes have been well studied in allergic and inflammatory responses, but less is known about their contribution to cellular immunity. In these experiments leukotriene LTB, produced by the activated monocytes was involved in the activation of effector cells. LTB4 was detected in the culture supernatants and addition of LTB4 biosynthesis inhibitors abolished the activation. These experiments showed thus that endogenously produced LTB4 can induce effector cell responses. Addition of LTB4 to the infected culture or readdition of LTB4 treated monocytes to the culture of infected monocyte depleted cell population induced also T cell activation and led to inhibition of B cell proliferation. These results demonstrated thus that LTB4 can contribute to the activation of innate immunity.

Leukotriene B4 activates T cells that inhibit B-cell proliferation in EBV-infected cord blood-derived mononuclear cell cultures.

Epstein-Barr virus (EBV)-specific cellular memory is not transferred from mother to child. Therefore, EBV-induced B-cell proliferation in in vitro-infected cord blood mononuclear cell cultures is not inhibited. However, by addition of immunomodulators, polysaccharide K (PSK) or truncated thioredoxin (Trx80) that activate monocytes, EBV-specific T-cell response could be generated in such cultures. Presently, we demonstrate that leukotriene B(4) (LTB(4)) is involved in the effect of the immunomodulators. LTB(4) was detected in the medium, and T-cell activation was compromised by addition of leukotriene biosynthesis inhibitors. Moreover, we found that LTB(4) added to infected cultures, which did not receive the immunomodulators, induced functional activation of the T cells. LTB(4) activated the monocytes and acted directly on the T cells. In consequence, addition of LTB(4) inhibited the EBV-induced proliferation of B lymphocytes. Specific cytotoxicity could be generated by restimulation of the T cells. The experiments showed successive stages of T-cell activation in acquisition of their immunologic effector function. This is orchestrated by complex cellular interactions, and autocrine loops mediated by soluble factors-here interferon (IFN)-gamma, interleukin (IL)-15, IL-12, and LTB(4). Importantly, the results indicate that endogenous LTB(4) can induce T-cell activation that inhibits the EBV-induced proliferation of B lymphocytes.

Concomitant increase of LMP1 and CD25 (IL-2-receptor alpha) expression induced by IL-10 in the EBV-positive NK lines SNK6 and KAI3.

Extranodal, nasal NK/T-cell lymphomas are regularly Epstein-Barr virus (EBV)-positive, with a type II latency pattern, expressing thus EBNA-1 and LMP1. The contribution of EBV to the tumor development is not known. Similarly to normal natural killer (NK) cells, cell lines derived from malignancies with a NK phenotype require IL-2 for in vitro proliferation. In our effort to explore the contribution of EBV, particularly the role of the LMP1 protein, to the pathogenesis of the NK lymphoma we found that its expression, studied in the NK-lines SNK6 and KAI3, depended on the supply of IL-2 or other cytokines. In the absence of IL-2 other cytokines, such as IL-10 and IFN-gamma, could maintain LMP1, but the cells did not proliferate. When grown in IL-2, the SNK6 cells produced IL-10 and IFN-gamma, and these cytokines mediated the expression of LMP1. IL-10 treatment enhanced, while IFN-gamma receptor blocking antibody reduced, the expression of CD25 and CD54 in the EBV-positive, but not in the EBV-negative lines. IL-10 treated cells required lower amount of IL-2 for proliferation compared to the untreated cells. This effect was seen only with the EBV-positive NK lines in which LMP1 and CD25 were concomitantly upregulated. By this mechanism EBV could have an important role in the development of NK lymphoma since the inflammatory component in the tumor tissue can provide these cytokines.

Carbazole is a naturally occurring inhibitor of angiogenesis and inflammation isolated from antipsoriatic coal tar.

Coal tar is one of the oldest and an effective treatment for psoriasis. Coal tar has been directly applied to the skin, or used in combination with UV light as part of the Goeckerman treatment. The use of coal tar has caused long-term remissions in psoriasis, but has fallen out of favor because the treatment requires hospitalization and coal tar is poorly acceptable aesthetically to patients. Thus, determining the active antipsoriatic component of coal tar is of considerable therapeutic interest. We fractionated coal tar into its components, and tested them using the SVR angiogenesis inhibitor assay. Treatment of SVR endothelial cells with coal tar fractions resulted in the isolation of a single fraction with antiangiogenic activity. The active antiangiogenic compound in coal tar is carbazole. In addition to antiangiogenic activity, carbazole inhibited the production of inflammatory IL-15 by human mononuclear cells. IL-15 is elevated in psoriasis and is thought to contribute to psoriatic inflammation. Carbazole treatment also reduced activity of inducible nitric oxide synthase (iNOS), which is proinflammatory and elevated in psoriasis. The effect of carbazole on upstream pathways in human psoriasis was determined, and carbazole was shown to inhibit signal transducer and activator of transcription (stat)3-mediated transcription, which has been shown to be relevant in human psoriasis. IL-15, iNOS, and stat3 activation require the activation of the small GTPase rac for optimal activity. Carbazole was found to inhibit rac activation as a mechanism for its inhibition of downstream inflammatory and angiogenic pathways. Given its antiangiogenic and anti-inflammatory activities, carbazole is likely a major component of the antipsoriatic activity of coal tar. Carbazole and derivatives may be useful in the therapy of human psoriasis.

EBV infection induces expression of the transcription factors ATF-2/c-Jun in B lymphocytes but not in B-CLL cells.

B cell type chronic lymphocytic leukaemia (B-CLL) cells carry the Epstein-Barr virus (EBV) receptor CD21 and can be infected in vitro with the virus. The infected cells exhibit an unusual EBV program, they express the nuclear proteins but not latent membrane protein 1 (LMP-1). Similar cells were encountered in lymphoid tissues of infectious mononucleosis (IM) patients and in lymphoproliferations of immunosuppressed patients. EBV infected B-CLL cells can be regarded as model for this viral program. In B cells the regulation of LMP-1 is executed mainly by EBV encoded nuclear antigen 2 (EBNA-2), interacting with several cellular proteins and these complexes bind to specific sequences in the LMP-1 promoter. ATF2 and c-Jun were shown to be among the interacting partners of EBNA-2. These molecules can be detected in experimentally infected B lymphocytes. We found c-Jun and/or phosphorylated ATF-2 (p-ATF-2) expression in some B-CLL ex vivo samples. They disappeared or their expression declined promptly in explanted cells, even if they were infected with EBV in vitro. Activation of the infected B-CLL cells by exposure to CD40L was accompanied by p-ATF-2 and c-Jun but not by LMP-1 expression. In one of three clones tested, subsequent treatment with histone deacetylase inhibitors (HDACi), TSA or n-butyrate, could induce LMP-1. Treatment with phorbol-12, 13-dibutyrate (PDB) induced LMP-1 expression in three of four clones. Neither the HDACi nor the PDB treated cells survived.

Leukotriene B4 plays a pivotal role in CD40-dependent activation of chronic B lymphocytic leukemia cells.

Biosynthesis of leukotrienes (LTs) occurs in human myeloid cells and B lymphocytes. However, the function of leukotrienes in B lymphocytes is unclear. Here, we report that B-cell chronic lymphocytic leukemia (B-CLL) cells produce leukotriene B(4), and that specific leukotriene biosynthesis inhibitors counteracted CD40-dependent activation of B-CLL cells. Studies on the expression of the high-affinity receptor for LTB(4) (BLT1) by flow cytometry analysis showed that the receptor was expressed, to a varying degree, in all investigated B-CLL clones. At a concentration of 100 nM, the drugs BWA4C (a specific 5-lipoxygenase inhibitor) and MK-886 (a specific 5-lipoxygenase activating protein inhibitor) markedly inhibited CD40-induced DNA synthesis (45% and 38%, respectively) and CD40-induced expression of CD23, CD54, and CD150. Addition of exogenous LTB(4) (150 nM) almost completely reversed the effect of the inhibitors on DNA synthesis and antigen expression. Taken together, the results of the present study suggest that leukotriene biosynthesis inhibitors may have a therapeutic role in B-CLL.

PSK and Trx80 inhibit B-cell growth in EBV-infected cord blood mononuclear cells through T cells activated by the monocyte products IL-15 and IL-12.

Epstein-Barr virus (EBV)-specific immunologic memory is not transferred from mother to child. In vitro infection of cord blood cells can therefore readily lead to the outgrowth of transformed B lymphocytes. We found that the immunomodulator polysaccharide K (PSK) or the mitogenic cytokine truncated thioredoxin (Trx80) inhibited the EBV-induced B-cell proliferation. Using signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) induction as a sign for T- and natural killer (NK) cell activation, we could follow it without any need for cell separation because neither macrophages nor B lymphocytes express SAP. The results suggest the following scenario: EBV infected and activated B lymphocytes. Upon interacting with these cells, T cells became posed for responding to cytokines produced by monocytes. Both PSK and Trx80, which is a secreted C-terminally truncated thioredoxin, activated the monocytes, which then produced cytokines in the presence of the primed T cells. PSK induced interleukin-15 (IL-15), while Trx80 induced IL-12 production. Both cytokines activated the T cells for function. Phosphatidylinositol 3-(PI 3)-kinase and reactive oxygen species (ROSs) were involved in the PSK-induced activation of monocytes. Restimulation of the cultures with EBV-transformed B cells generated specific cytotoxic activity.

Upregulation of LMP1 expression by histone deacetylase inhibitors in an EBV carrying NPC cell line.

OBJECTIVES: In about 60% of Epstein-Barr virus (EBV) carrying nasopharyngeal carcinomas (NPC) LMP1 expressing cells can be detected. The frequency of LMP1 positive cells and the expression level varies from cell to cell in the different tumors. Cell lines derived from EBV positive NPCs loose the virus during in vitro culture. The in vitro infected NPC cell line TWO3-EBV used in our study carries the neomycin-resistance gene containing EBV and expresses low level of LMP1. With this cell line it was thus possible to study the regulation of LMP1 expression by modification of chromatin acetylation state. STUDY DESIGN: The TWO-EBV cell line was treated with n -butyrate (NB) or trichostatin A (TSA). RESULTS: Shown by immunoblotting, the LMP1 level was elevated in the treated samples. Already 2 h after TSA exposure LMP1 expression was higher and it increased up to 24 h. Immunofluorescence staining showed that nearly all cells were LMP1 positive. Neither EBNA2 nor BZLF1 were induced. Tested first 2 h after the treatment, acetylated histone H3 and H4 were already detectable, and their level increased up to 8 h. Chromatin immunoprecipitation (ChIP) verified that the LMP1-promoter (LMP1p) (ED-L1) was acetylated after TSA treatment. CONCLUSION: EBV carrying epithelial cells do not express EBNA-2. We showed that LMP1 expression was upregulated by histone deacetylase inhibitors in an in vitro infected, EBV carrier NPC cell line.

The X-linked lymphoproliferative disease gene product SAP is expressed in activated T and NK cells.

The unique manifestation of the inherited immunodeficiency, X-linked lymphoproliferative disease (XLP), is the impaired control of EBV infection. The gene, which carries mutations or is deleted in the patients, has been identified (Xq25). The encoded protein (SAP, 128 aa) contains a single SH2 domain and binds to signaling lymphocytic activation molecule (SLAM) and to other related surface molecules that are expressed on activated T, B and NK cells. SAP modifies signal transduction through its association with these molecules. Initially it was assumed that SAP acts passively by interfering and blocking active interactions involving other SH2 carrying molecules. We demonstrated that SAP protein is expressed in activated T and NK, but not in activated B cells. This finding is in line with the fact that in vitro performance of effector cells derived from XLP patients is impaired. However, it is still not known why the severe symptoms (fatal mononucleosis or malignant lymphoproliferation in the survivors of the primary infection) are elicited by EBV. We studied SAP expression in several Burkitt lymphoma (BL) derived lines. In contrast to normal B cells, certain lines expressed SAP. These were all type I cells in the Burkitt line nomenclature: they expressed only one of the EBV encoded proteins (EBNA-1) and their phenotype corresponded to resting B cells. Lymphoblastoid cell lines and type III BLs, whose phenotype resembled activated B cells and expressed all nine EBV encoded proteins, were devoid of SAP. The relationship between cell activation and SAP expression is reciprocal in T and B cells i.e. BL lines, activated T and NK cells express SAP, while BL blasts do not express SAP. This opposite relationship may be exploited for studies about the function of SAP.

SH2D1A expression reflects activation of T and NK cells in cord blood lymphocytes infected with EBV and treated with the immunomodulator PSK.

We infected umbilical cord blood lymphocytes (CBL) with EBV in vitro. Analysis of the cell population in 3- and 6-day-old cultures showed a relative increase of B cells and outgrowth of B cells after prolonged culture period. The immunomodulator PSK was added to parallel cultures. In these cultures, B cell growth was inhibited and the activation of T and NK cells was potentiated. This was detected by assessment of SH2D1A (also named SAP or DSHP) expression (a molecule which participates in signal transduction and is mutated in X-linked lymphoproliferative disease, XLP). Upon further cultivation, irradiated autologous EBV infected B cells and IL-2 were added to the cultures. After 17 days, the B lymphocytes were removed from the PSK containing cultures. The remaining populations, containing mainly T and NK cells, exerted cytotoxic function which could act on EBV infected autologous B cells, allogeneic LCL and on K562. Since cellular immunity to EBV is not transmitted to the newborn, EBV specific memory is not involved in the activation of effector cells. Our finding of an in vitro response of T and NK cells to EBV infected B lymphocytes in the absence of EBV specific immunological memory is of particular interest, because it may also operate in vivo and participate in the scenario of primary infection. Its potentiation by immunomodulators may have practical significance.