Characterization of N-glycolyneuraminic acid-containing glycosphingolipids from a Marek's disease lymphoma-derived chicken cell line, MSB1, as tumor-associated heterophile Hanganutziu-Deicher antigens

Heterophile, Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid-containing glycosphingolipids (GSLs) were detected as tumor-associated foreign antigens of a Marek's disease lymphoma-derived cell line, MSB1, by enzyme-immunoassay with chicken antibody against N-glycolylneuraminyl-lactosylceramide (anti-NeuGc-LacCer). At least three species of HD antigen-active GSLs were detected by two-dimensional thin-layer chromatography (TLC) combined with enzyme-immunoassay. The reactivities of the GSLs with anti-NeuGc-LacCer, their behaviors on two-dimensional TLC and the results of an endo-beta-galactosidase digestion study indicated that these three GSLs were NeuGc-LacCer (NeuGc alpha 2-2Gal beta 1-4Glc-Cer), NeuGc-nLcOse4Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and NeuGc-nLcOse6Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer).     

Cell-mediated cytotoxicity of lymphocytes from chickens inoculated with herpesvirus of turkey against a Marek's disease lymphoma cell line (MSB-1).

Using a new device which increases the sensitivity of detection of specific immune lysis of target cells by labeling them with [35S]-methionine, the in vitro cell-mediated cytotoxic response of spleen lymphocytes and peripheral blood lymphocytes from chickens vaccinated with herpesvirus of turkey (HVT), O1 strain, against MSB-1 line cells was clearly demonstrated. The cytotoxic activity was clearly inhibited by pretreatment of effector lymphocytes with anti-T lymphocyte serum and complement. The activity was greater using T cells purified from spleen lymphocytes and peripheral blood lymphocytes than with the unfractionated cells, indicating that T lymphocytes play the main role in effector activity. Using sera from HVG-vaccinated chickens, no significant cytotoxic effects were detected in the complement-dependent antibody cytotoxicity test against MSB-1 cells. These results suggest that cellular immunity against the surface antigen of Marek's disease (MD) lymphoma cells is mainly related to the preventive mechanism against MD incidence by HVT vaccination.     

Stable production of recombinant chicken antibody in CHO-K1 cell line.

When compared with mammalian IgG, chicken IgY is advantageous in terms of cross-reactivity. In this study, two plasmids were constructed for expression of recombinant chicken IgY derived from a chicken hybridoma. The first was for expression of the light (L) chain, and the other was for the heavy (H) chain with a histidine (His) tag at the carboxy-terminal. After transfection of recombinant chicken IgY gene into Chinese hamster ovary cells, a transfectant designated HF33 that secreted the specific antibody was selected. HF33 cells produced recombinant IgY with His tag at 10-15 microg/10(6) cells/24 h. On Western blotting analysis, the recombinant IgY was detected as one band for the H chain and two bands for the L chain. The recombinant IgY was successfully purified in a one-step procedure using a nickel-affinity resin. These results indicate that the present recombinant chicken IgY is useful for further applications.     

Persistence of genomes of both herpesvirus of turkeys and Marek's disease virus in a chicken T-lymphoblastoid cell line

A cell line tentatively designated as MDCC-BO1(T), was established from spleen cells of an apparently healthy chicken inoculated with herpesvirus of turkey (HVT). BO1(T) cells were T lymphoblastoid cells and the more than 95% of them had Marek's disease (MD) tumor-associated surface antigen (MATSA). However, no viral internal antigens or membrane antigens could be demonstrated in them by immunofluorescence tests using chicken anti-HVT and -MD virus (MDV) sera. The virus could be rescued from BO1(T) cells by co-cultivation with chick embryo fibroblasts (CEF). The DNA of the rescued virus was characterized as HVT DNA by its sedimentation profile in a neutral glycerol gradient and its endonuclease Hind III cleavage-pattern. Ultrastructural studies on CEF infected with the rescued virus revealed the presence of HVT-like virions. However, DNA-DNA reassociation kinetics showed that the BO1(T) cells contained a few copies of NVT and also MDV genomes.     

Propagation and infectivity titration of the Gifu-1 strain of chicken anemia agent in a cell line (MDCC-MSB1) derived from Marek's disease lymphoma

Chicken anemia agent (CAA) propagated in an established cell line derived from Marek's disease (MD) lymphoma (MDCC-MSB1). When passaged 19 times in MDCC-MSB1 cell cultures, it produced anemia of the same severity in chicks as it did before passage. Titration of the infectivity of CAA was performed successfully with subcultures of MDCC-MSB1 cell cultures which had been inoculated with serial tenfold dilutions of infected material. In it, no infected cultures could be subcultured. The propagation of CAA was also proved in the MD cell line, MDCC-JP2, and the avian lymphoid leukosis (LL) cell line, LSCC-1104B1, but not in the two MD cell lines, MDCC-RP1 and MDCC-BP1, or in the two LL cell lines, LSCC-1104X5 and LSCC-TLT. No CAA propagated in cell cultures prepared from skin and muscle, liver, or brain of chick embryos, or kidney, thymus, bursa of Fabricius, bone marrow, or white blood cells of chickens.     

In vitro cytotoxicity against Marek's disease lymphoblastoid cell lines after enzymatic removal of Marek's disease tumor-associated surface antigen.

Marek's disease tumor-associated surface antigen (MATSA) has been claimed to be the target of cytotoxic lymphocytes in in vitro tests for Marek's disease immunity. Treatment with papain, but not with trypsin or mixed glycosidases, removed MATSA from certain Marek's disease lymphoblastoid cell lines. Tumor cells with and without MATSA were used as target cells for in vitro studies on cell-mediated immune responses with sensitized spleen cells in a chromium release assay. The removal of MATSA did not influence the results of the chromium release assay. Attempts to block the cell-mediated cytotoxicity in vitro by coating tumor cells with an anti-MATSA serum failed. It was concluded that cell-mediated immune responses against Marek's disease tumor cells are directed against an as yet undefined antigen(s).     

Demonstration of a tumor-associated surface antigen in Marek's disease.

Surface antigenic markers were detected on three classes of Marek's disease (MD) tumor cells, i.e., MD lymphoma cells, cultured cells of the MSB-1 lymphoblastoid cell line, and JMV lymphoblastic leukemia cells, by indirect membrane immunofluorescent staining with serum from chickens immunized with JMV cells or from rabbits immunized with MSB-1 cells. This surface antigen was not detected on normal chicken lymphocytes, RPL-16 tumor cells (tranedormed by an avian RNA virus, or MD virus-infected fibroblasts that were positive for viral membrane antigen (MA). Furthermore, the surface antigen appeared unrelated to embryonic or histocompatibility antigens. This antigen is provisionally designated as a Marek's disease tumor-associated surface antigen (MATSA). The MATSA's on JMV, MSB-1 and MD lymphoma cells were related but not identical as demonstrated by antiserum titration, absorption and blocking tests with homologous and heterologous systems.     

Demonstration of the specificity of a monkey antiserum against human melanoma

Summary The reactivity spectrum of a monkey antiserum raised against in vitro-cultured human melanoma cells was compared by means of three different assays: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and mixed hemadsorption (MHA). After absorption with a pool of cells from T- and B-lymphoid cell lines the antiserum was specifically cytotoxic in CDC for cultured melanoma cells. The melanoma specificity of the antiserum was confirmed by quantitative absorption experiments with cultured melanoma and nonmelanoma cells. When the lymphoid cell-absorbed antiserum was assayed by MHA, however, a high reactivity against both melanoma and nonmelanoma cells was observed. Further absorption with nonmelanoma tumor cells removed the reactivity of the antiserum with different nonmelanoma cell lines, but did not abolish its reactivity with melanoma cells. After this second absorption, the antiserum remained cytolytic against melanoma cells in CDC. In ADCC experiments this antiserum was not able to induce any cytotoxicity even before absorption.Analysis of Sephadex G 200 fractions of the antiserum revealed that the melanoma-specific antibodies cytotoxic in CDC were localized exclusively in the IgM peak. This finding was confirmed in similar studies with four other nonhuman primate melanoma antisera. Abbreviations used in this paper: CDC, complement dependent cytotoxicity: ADCC, antibody dependent cell mediated cytotoxicity; MHA, mixed hemadsorption; FCS, fetal calf serum.     

Retroviral insertional activation of the c-myb proto-oncogene in a Marek's disease T-lymphoma cell line.

Marek's disease virus (MDV) is an avian herpesvirus that causes, in chickens, a lymphoproliferative disease characterized by malignant transformation of T lymphocytes. The rapid onset of polyclonal tumors indicates the existence of MDV-encoded oncogenic products. However, the molecular basis of MDV-induced lymphoproliferative disease and latency remains largely unclear. Several lines of evidence suggest that MDV and Rous-associated virus (RAV) might cooperate in the development of B-cell lymphomas induced by RAV. Our present results indicate for the first time that MDV and RAV might also act synergistically in the development of T-cell lymphomas. We report an example of an MDV-transformed T-lymphoblastoid cell line (T9) expressing high levels of a truncated C-MYB protein as a result of RAV integration within one c-myb allele. The chimeric RAV-c-myb mRNA species initiated in the 5' long terminal repeat of RAV are deprived of sequences corresponding to c-myb exons 1 to 3. The attenuation of MDV oncogenicity has been strongly related to structural changes in the MDV BamHI-D and BamHI-H DNA fragments. We have established that both DNA restriction fragments are rearranged in the T9 MDV-transformed cells. Our results suggest that retroviral insertional activation of the c-myb proto-oncogene is a critical factor involved in the maintenance of the transformed phenotype and the tumorigenic potential of this T-lymphoma cell line.     

Cytogenetic stability of chicken T-cell line transformed with Marek's disease virus: atomic force microscope, a new tool for investigation

The Marek's disease virus (MDV) integration may induce a novel organization of chromatin architecture with a modified genetic expression. In our opinion it is worthwhile trying to relate cytogenetic stability to functional modifications. Recently, atomic force microscopy technique was applied to study the structure of chromosomes at a nanoscale level. This high resolution allows to investigate the different structure of chromatin in order to study cytogenetic stability and chromosome aberrations due to MDV insertion. In this paper data are presented indicating a duplication [78,WZ,dup(1p)(p22-p23)] and a deletion [78,WZ cht del(3)(q2.10)] of Chromosomes 1 and 3 relatively. Relationships between GTG (G-bands by Trypsin using Giemsa) bands and the topography of chromosomes are also discussed, naming them Topographic Banding. The architecture of chromosomes observed by AFM can be related to the data obtained with classic banding techniques thus overcoming the optical resolution limits. The presence of chromatin bridges between sister chromatids at most of the heterochromatic regions is also evidenced. Besides, we present different studies of the longitudinal and transversal symmetry of the hetero and euchromatic regions to clearly demonstrate a different underlying architecture of these regions. It is indeed evident that the heterochromatic bands are more symmetrical than euchromatic bands.