Marek's Disease Lymphoblastoid Cell Research Articles

Gga-miR-130b-3p inhibits MSB1 cell proliferation, migration, invasion, and its downregulation in MD tumor is attributed to hypermethylation.

Marek's disease is an oncogenic and lymphoproliferative disease of chickens caused by Marek's disease virus. Hypermethylation or hypomethylation of CpG islands in gene promoter region are involved in the initiation and progression of carcinogenesis. In this study, we analyzed differential methylation levels of upstream region of gga-miR-130b-3p gene between Marek's disease virus-infected tumorous and non-infected spleens. Around the upstream 1 kb of gga-miR-130b-3p gene, two amplicons were designed that covered 616 bp. There were forty-eight CpG sites in this region. CpG sites in this region presented higher methylation level in tumorous spleens compared with that in non-infected ones. There were eight CpG sites significantly hypermethylated in tumorous spleens. The expression level of three DNA methyltransferases including DNMT1, DNMT3a and DNMT3b increased and the expression level of Tet ten-eleven translocation protein 2 remarkably decreased in tumorous spleens. Hypermethylation in the upstream region of gga-miR-130b-3p gene might be a direct reason for its downregulation in MD tumorous tissues. Moreover, cell proliferation of Marek's disease lymphoblastoid cell line MDCC-MSB1 was remarkably inhibited at 24, 36, 48, 60 and 72 h post-gga-miR-130b-3p-agomir transfection. The transwell migration assay indicated cell number of migration was significantly lower in miRNA agomir transfection group. Matrix metalloproteinases MMP2 and MMP9 are involved in tumor invasion, and their protein levels were significantly downregulated at 72 h post-miRNA-agomir transfection. Collectively, these results indicated that hypermethylation in upstream region of gga-miR-130b-3p gene contributed to its downregulation in tumorous tissues. Gga-miR-130b-3p plays an inhibitory role in lymphomatous cell transformation.

Characterization of Marek's Disease Virus BamHI-A-Specific cDNA Clones Obtained from a Marek's Disease Lymphoblastoid Cell Line

A cDNA library was constructed from poly(A) + RNA fractions obtained from a Marek's disease (MD) lymphoblastoid cell line, MDCC-CU41, in which viral gene expression is very limited. Three independent groups (1, 2, and 3) of MD virus (MDV)-specific clones were obtained, which were mapped in the inverted repeat region of the BamHI-A fragment of the MDV genome. Northern blot analysis showed that probes prepared from these cDNA clones hybridized with several transcripts of different sizes in poly(A) + RNA of MDCC-CU41, although the amounts of these transcripts were relatively small compared to those in MDV lytically infected cells. Moreover, a small open reading frame, which can encode a 94-amino-acid protein, was identified in the A41 cDNA clone (Group 3). By RNase protection assays, the 1.2-kb Group 3 transcriptional unit has been defined. In indirect immunofluorescent antibody assays, antiserum against the bacterially expressed fusion protein, glutathione S -transferase-A41, reacted specifically with the cytoplasmic regions of MDV (strain RB1B)-infected chick kidney cells. However, MDCC-CU41 did not contain a detectable lever of the protein determined by these methods.

Characterization of a Marek's disease virus BamHI-L-specific cDNA clone obtained from a Marek's disease lymphoblastoid cell line.

Two Marek's disease (MD) virus BamHI-L-specific cDNA clones were isolated from a cDNA library constructed from poly(A)+ RNA fractions of an MD lymphoblastoid cell line, MDCC-CU41 (CU41). These clones were mapped to the region corresponding to the BamHI-Q2 and L-regions. These clones hybridized with 2.5-, 0.8-, and 0.6-kb transcripts prepared from CU41. The transcriptional unit of the 0.6-kb transcript was determined by RNase protection assays. An open reading frame encoding a 107-amino-acid polypeptide was identified in the 0.6-kb transcript. Reverse transcriptase-PCR demonstrated the presence of this transcript in both CU41 and a reticuloendotheliosis virus-transformed cell line latently infected with MD virus.

Proliferation of Chicken Lymphoblastoid Cells after in vitro Infection with Marek's Disease Virus

Activated, thymus-derived (T) lymphoblasts were exposed to Marek's disease virus and cultivated in attempts to induce in vitro transformation. After 9 to 15 days, colonies or small clusters of proliferating lymphoblasts were observed in cultures from three of a total of 122 attempts. These developed into proliferating cell cultures that resembled conventional Marek's disease (MD) lymphoblastoid cell lines in terms of growth characteristics and morphology. All proliferative cultures were unusual in that 1) the expression of viral internal antigens consistently or periodically was very high (up to 30% of all cells) and 2) the cells deteriorated and/or proliferation ceased in all cases after culture periods of 45-176 days. The proliferative cultures were all characterized as CD2+ and CD3+, Ia-bearing T cells; one was CD4+/CD8- and TCR2+, the other two were CD4-/CD8- and TCR1+. The latter two are the only cultures of MD-infected cells known to be TCR1+.

Genetic Resistance to a Marek's Disease Transplantable Tumor Cell Line in Chicken Lines Selected for Different Immunological Characters

The tumor incidence and mortality of Marek's disease (MD) were determined for 471 progeny from four pairs of lines two-way selected for different immunological characters: graft versus host reaction (GVHR) competence, IgG levels, antibody response to rabbit serum albumin (RSA), and anaphylactic shock to BSA. All chicks were inoculated at 1 wk of age with 1 to 2 × 106 viable cells of a MD lymphoblastoid cell line, MDCC-MSB1-41C (41 C). Tumor incidence differed significantly between the high (H) and low (L) lines of each GVHR-, IgG-, RSA-, or BSA-selected group. There were also significant differences in mortality rates between H and L lines of all selected groups. The IgG-H, BSA-H, and RSA-H lines were more resistant than their respective L lines. The H line of the GVHR-selected lines was more susceptible than the L line. Tumor regression could be detected only in the IgG- and RSA-selected lines. No definite correlation could be found between genetic resistance to 41C and genetic resistance to MD virus.

Analysis of marek's disease tumor‐associated surface antigen on MDCC‐MSB1‐CLO.18 cells

The physico-chemical nature of Marek's disease tumor-associated surface antigen (MATSA) on Marek's disease (MD) lymphoblastoid tumor cell line (MDCC-MSB1-clo.18) was examined by the cellular enzyme-linked immunosorbent assay (CELISA) and the sandwich enzyme-linked immunosorbent assay (ELISA) using an anti-MATSA immune serum or a monoclonal antibody (MAb) 2B9 developed against MATSA. Our results indicate that MATSA is a glycoprotein and 2B9 recognizes an antigenic site in the protein moiety of MATSA. MATSA was solubilized from MSB1-clo.18 cells by treatment with 0.5% Nonidet P-40, and purified by affinity chromatography coupling with 2B9 and further by ion exchange chromatography on diethylaminoethylcellulose (IECD). MATSA was eluted with 0.2 to 0.3 M KCl in IECD and the purity of MATSA was increased about 2,500-fold. The purified MATSA was shown to have a molecular weight (Mr) of 70,000 by SDS-PAGE. The reactivity of purified MATSA with anti-thymus cell serum was examined. MATSA was detectable by anti-thymus cell serum, although 2B9, which was used to purify MATSA from MSB1-clo.18 cells, was not reactive to cells prepared from the thymus. However, MATSA was no longer detectable after the absorption of anti-thymus cell serum by chicken bursa cells. The absorption of anti-thymus cell serum by chicken red blood cells (RBC) had no effect on the reactivity against MATSA. These results suggest that MATSA may be a lymphocyte-specific antigen modified during leukemogenesis by Marek's disease virus (MDV).

Replicating Marek's disease virus (MDV) serotype 2 DNA with inserted MDV serotype 1 DNA sequences in a Marek's disease lymphoblastoid cell line MSB1-41C

We characterized the properties of herpes-type viruses which grew well in a Marek's disease lymphoblastoid cell line, MSB1-41C, inducing cytopathic effect characterized by the formation of syncytial giant cells. Examination of the infectious virus by field inversion gel electrophoresis revealed the presence of DNA of about 180 kbp in both the culture fluid and cell fractions of the infected MSB-41C cells. The DNA was found to consist of Marek's disease virus (MDV) serotype 2 (MDV2) and MDV serotype 1 (MDV1) DNA by Southern blot hybridization. The MDV1 DNA consisted of sequences mainly from the long inverted repeats including multiple copies of 132 bp direct tandem repeats. Molecular cloning of BamHI digests of the MDV2 DNA revealed a fragment of MDV1 DNA and MDV2 DNA fused together, indicating that the recombinant MDV2 DNA had been generated by genetic recombination with the latent MDV1 DNA.

In vivo growth inhibition of transplantable Marek's disease lymphomas by monoclonal antibodies against surface antigens on Marek's disease lymphoblastoid cell line.

In vivo growth inhibition of transplantable Marek's disease lymphomas by monoclonal antibodies against surface antigens on Marek's disease lymphoblastoid cell line.

Promoting effects of 105K glycoprotein on cell proliferation in chicken lymphoblastoid cell lines

The promoting effects on cell proliferation of the 105K glycoprotein (105Kgp), purified from sera of chickens to which a Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C (MSB1-41C), had been transplanted, were examined using culture cells from various sources. The MSB1-41C line as well as the other chicken lymphoblastoid cell lines examined were sensitive to the 105Kgp. The growth-promoting effects of 105Kgp showed a biphasic pattern depending upon the amount of 105Kgp added into the culture medium. These findings indicate that the 105Kgp may be a promoting factor for chicken growing cells, especially lymphoblastoid cell lines.

Promoting effects of 105K glycoprotein on cell proliferation in chicken lymphoblastoid cell lines.

The promoting effects on cell proliferation of the 105K glycoprotein (105Kgp), purified from sera of chickens to which a Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C (MSB1-41C), had been transplanted, were examined using culture cells from various sources. The MSB1-41C line as well as the other chicken lymphoblastoid cell lines examined were sensitive to the 105Kgp. The growth-promoting effects of 105Kgp showed a biphasic pattern depending upon the amount of 105Kgp added into the culture medium. These findings indicate that the 105Kgp may be a promoting factor for chicken growing cells, especially lymphoblastoid cell lines.

Suppression of NK activity of spleen cells by chicken fetal antigen present on Marek's disease lymphoblastoid cell line cells

Chicken fetal antigen (CFA) expressed on the cell surface of Marek's disease (MD) lymphoblastoid cell line (MDCC-MSBI) cells was purified by affinity chromatography using a monoclonal antibody (2H3). A 4-hr 51Cr release assay was performed using spleen cells as effector cells to investigate the role of the CFA in the immune response of chickens, especially in relation to natural killer (NK) activity. Spleen cells of specific-pathogen-free (SPF) chickens showed reduced NK activity in the presence of CFA in vitro, as did those cells treated with MSBI soluble antigen. NK activity of spleen cells from chickens treated with CFA was also suppressed when compared to the cells from untreated or ovalbumin-treated chickens. In addition, MSBI clo. 18 transplantable tumors grew progressively in some of the 14-day-old chickens treated with CFA, whereas the tumors regressed in age-matched chickens with or without treatment by ovalbumin.

Production of an immune suppressor factor by Marek's disease lymphoblastoid cell lines

Culture fluids from Marek's disease (MD) lymphoblastoid cell lines have suppressive activity against normal and mitogen-stimulated chicken spleen and bursal cells and also against the homologous cell lines. Suppressive activity was also present in supernatants from spleen cells infected in vitro with MD virus. The suppressor factor from MD cell lines was non-sedimentable, trypsin sensitive, heat resistant and partially dialysable. Preliminary studies suggest it has a molecular weight of 20,000 daltons. Studies were also conducted on the effect of the prostaglandin inhibitors indomethacin and aspirin on the production and action of the suppressor factor. At low concentrations they have a stimulatory effect on the cell lines suggesting that they inhibit the effects of suppressor factor; however only small amounts of prostaglandin E 2 were present in supernatants. Evidence was obtained that the suppressor factor may act indirectly by stimulating the production of prostaglandin by spleen cell cultures. The role of a suppressor factor in the immunosuppression observed in MD is discussed.

Cultivation and characterisation of avian lymphocytes with natural killer cell activity

Conditioned medium (CM), as a presumed source of lymphokines including interleukin-2, was prepared from chicken spleen cell cultures stimulated with concanavalin A (con A). When CM was used to cultivate spleen cells from 6- to 8-week-old P-2 chickens, eight of nine spleens yielded cell lines which grew continuously for at least 50 days. Six of the cultures were tested for natural killer (NK) cell activity against LSCC-RP9, a lymphoblastoid cell known to be susceptible to NK cells, and against several Marek's disease lymphoblastoid cell lines (MDCC-CU2, -CU36 and -MSB-1). All six cultures lysed the RP9 cells in a chromium release assay with high levels of specific release (30 to 50%) at effector cell to target cell ratios of 5:1 or 10:1. CU2 and CU36, which are NK-cell resistant, were not lysed, while there was a low level of specific activity against MSB-1. The cells were characterised for surface and internal antigens with monoclonal antibodies and were negative for thymocyte antigen, IgM, a T cell antigen also present on granulocytes and red blood cells, and two antigens found in macrophages. Two of the six lines examined had a low number of cells expressing la antigen, while the other four were negative. An antigen present on circulating T cells and a macrophage (sub)population was present on all lines. The majority of the cells had the morphological appearance of mammalian large granular lymphocytes (LGL) with a rather small kidney-shaped nucleus. Granules and vacuoles were present in the cytoplasm. However, there was a variable percentage of lymphoblastoid (LB) cells also present. Cell lines established with CM from con A-stimulated spleen lymphocytes in other studies were also shown to have high levels of NK activity regardless of the relative proportions of cells with the morphological appearance of LGLs or LBs, or the relative frequency of expression of the two T cell markers or la antigen, all of which varied markedly.

Identification of Marek's disease virus-specific antigens in Marek's disease lymphoblastoid cell lines using monoclonal antibody against virus-specific phosphorylated polypeptides.

For identification of the antigens specific to Marek's disease virus (MDV) in virus-non-producer lymphoblastoid cell lines established from a tumor of Marek's disease (MD), hybridomas producing monoclonal antibodies (MAbs) against the antigens were isolated. Immunogens for preparation of the hybridomas were purified from the lysate of an MD-lymphoblastoid cell line, MSB1, by affinity chromatography coupled with chicken anti-MDV serum immunoglobulin G. Three of the MAbs obtained, MB1, MB2 and MB3, were specific to MDV by immunofluorescence test. An immunofluorescence test using MB2 antibody showed that immunofluorescence-positive cells in non-producer MD-lymphoblastoid line cells became detectable when the culture temperature was shifted from 41 degrees C to 33 degrees C or when treatment with 5-iodo-2-deoxyuridine (IUdR) was performed, indicating that the antigen reactive with MB2 antibody is an MDV-specific early antigen. This temperature shift or IUdR-treatment did not induce other MDV-specific antigens, such as late gene products of MDV, detected with MAbs. MB1 and MB2 antibodies immunoprecipitated 4 MDV-specific phosphorylated polypeptides with molecular weights (MWs) of 43,000 (43kd), 39kd, 36kd and 24kd from chick embryo fibroblasts productively infected with virulent MDV. In the place of 43kd, phosphorylated 44kd polypeptide was precipitated from avirulent MDV-infected fibroblasts. However, MB3 antibody did not precipitate any MDV-specific polypeptides from infected fibroblasts. These results suggest that the phosphorylated polypeptides are MDV-specific polypeptides predominantly expressed in non-producer MD-lymphoblastoid cell lines.

Two serum glycoproteins in chickens inoculated with a transplantable Marek's disease cell line and preparation of monoclonal antibodies against the glycoproteins.

In the sera from chickens inoculated with a transplantable Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C, five peaks (I, II, III, IV and V) were separated by chromatofocusing in a gradient of pH 5-6. Peaks I, II, III and IV were not exactly detectable in normal chicken serum. The contents of two peaks (I and II) correlated to tumor size. Subsequently, Peaks I and II were each purified by Cellulofine gel filtration and six monoclonal antibodies to the purified materials were prepared by somatic cell hybridization. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) revealed that Peaks I and II were each a glycoprotein of the same molecular weight, approximately 105, 000 (105K) daltons, with different isoeltctric points. All monoclonal antibodies obtained cross-reacted equally with either Peak I and Peak II by enzyme-linked immunosorbent assay. By immunoblotting analysis using one (P5-13) of these antibodies, when the same samples from normal and MSB1-41C-transplanted chickens and the purified Peaks I and II were each subjected to SDS-PAGE and blotted on a sheet of nitrocellulose, a single band reacted with P5-13 was formed at a position corresponding with 105K dalton in the respective samples and the density of each band correlated to tumor size. These results suggest that the cross-reactive 105K molecules may increase when chickens develop tumors.

Transplantable Marek's disease lymphomas. II. Variable tumor immunity induced by different lymphoblastoid cells.

The effectiveness of cells from 6 different Marek's disease (MD) lymphoblastoid cell lines to induce immunity to syngeneic transplantable MD lymphomas was investigated in 2 related inbred lines of White Leghorn chickens (lines G-B1 and G-B2) that have different major histocompatibility complex (MHC) genotypes. Cells from 2 line G-B1 lymphomas (MDCT-NYM1 and MDCT-UG1) and 1 line G-B2 lymphoma (MDCT-UG2) were used for challenge. Three of the lymphoblastoid cell lines tested were developed from these lymphomas. Growth of palpable lymphomas was lowest among G-B2 chickens immunized with syngeneic lymphoblastoid cells. Protection against the early lethal effects of the highly virulent transplantable lymphomas was greatest in both lines of chickens when the lymphoblastoid cells were syngeneic with the hosts. Lymphoblastoid cells of unknown MHC type either failed to induce immunity to the lymphomas in both lines or protected some line G-B2 chickens challenged with syngeneic MDCT-UG2 lymphoma cells.

Separation of cytotoxic lymphocytes against Marek's disease lymphoblastoid cell lines on PercollRgradients1

A two-step method has been developed for the separation of lymphocytes cytotoxic for allogeneic Marek's disease lymphoblastoid cell lines. Ficoll-Paque-purified spleen cell suspensions were separated according to their densities on continuous gradients of Percoll. Pooled fractions were tested for specific release in a 4-hour chromium release assay. Cytotoxic activity was mainly detected in the fractions with a density of 1.060 to 1.070 g/ml. Levels of specific release were increased using those fractions compared to Ficoll-Paque-purified cells. A discontinuous Percoll gradient was developed based on the density range of cytotoxic cells. Continuous and discontinuous Percoll gradients were equally effective for the preparation of cytotoxic cells.

Immunization against Marek's Disease Transplantable Cell Lines

Continuous passage of MDCC-RP1, a highly tumorigenic Marek's disease (MD) lymphoblastoid cell line, in cell culture resulted in a gradual loss in ability of the cell line to cause progressive tumors in susceptible day-old chicks. Inoculation of day-old chicks with high-cell-culture-passaged (187th to 417th) nontumorigenic MDCC-RP1 cells gave excellent protection against challenge at 8 days with low-passaged tumorigenic MDCC-RP1 cells but failed to protect against primary tumors caused by inoculation with MD virus. Vaccination with the herpesvirus of turkeys, on the other hand, protected the chickens well against primary tumors caused by MD virus and against transplantable tumors caused by tumorigenic MDCC-RP1 cells, but it did not protect as well against another MD lymphoblastoid cell line, MDCC-RP4. It is unlikely, therefore, that vaccines prepared from passaged MDCC-RP1 cell lines will have value for protecting chickens against MD in the field.

In vivo Characteristics of a Transplantable Marek's Disease Lymphoblastoid Cell Line, MDCC-MSB1-41C

A Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C, was highly transplantable and lethal for chickens. Autopsies showed extensive metastasis in various organs. The transplantabilities of the parent cell line, MDCC-MSB1, and another derivative line, MDCC-MSB1-33C, were transient. MD virus (MDV) could be isolated from the kidneys but not from the peripheral blood leukocytes of chickens inoculated with the MSB1-41C cell line. In addition, anti-MDV antibodies were produced both in chickens inoculated with this cell line and in controls raised with inoculated chickens, but several attempts to isolate MDV from this cell line in vitro failed.

Characterization of an antigen associated with the Marek's disease lymphoblastoid cell line MSB-1.

A Marek's disease lymphoblastoid cell line (MSB-1) has been analysed by immunoprecipitation for expression of tumour-associated antigen, Marek's disease virus (MDV)-specific antigens and antigens specific to avian leukosis-sarcoma viruses. Rabbit antisera raised against two independently derived cell lines after extensive absorption with normal chick cells reacted with a polypeptide of mol. wt. 40 000 (40K) in extracts of MSB-1 cells. The 40K polypeptide was not present in myeloblasts or in chick embryo fibroblasts (CEF) infected with MDV and did not react with antiserum raised against normal chicken thymus antigens. The possibility that the 40K polypeptide is a tumour-associated antigen is discussed. Seven MDV-specific antigens were noted in infected CEF (mol. wt. 110K, 100K, 80K, 70K, 50K, 35K and 32K) but none of these was detected in MSB-1 cells. The avian leukosis-sarcoma group-specific antigen P27gag and its precursor Pr76gag were not found in MSB-1 cells, confirming that expression of mature gag protein is not required for transformation by MDV. However, two polypeptides of unknown origin and function (mol. wt. 180K and 110K) were precipitated from MSB-1 cells with a rabbit anti-Rous sarcoma (Schmidt-Rupin, subgroup D) antiserum.