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B W Calnek

Publications and source records attributed to B W Calnek.

At least 19 recordsLinked to original sources
PubMed2005

Classification of Marek's disease viruses according to pathotype: philosophy and methodology.

The concept of pathotype in Marek's disease (MD) probably dates from the recognition of a more virulent form of the disease in the late 1950s (Benton & Cover, 1957). Distinctions between MD virus strains were further expanded with the description of the vv pathotype in the early 1980s and of the vv+ pathotype in the 1990s. Pathotype designations reflect important biological properties that correlate with the break-through of vaccinal immunity in the field. However, pathotyping methods applied by various laboratories have not been uniform, preventing critical comparison of results. Better uniformity of pathotyping procedures is desirable.The Avian Disease and Oncology Laboratory (ADOL) method is based on induction of lymphoproliferative lesions in vaccinated chickens. This method has been used to pathotype more than 45 isolates and is the basis for the current pathotype classification of MD virus strains. Its limitations include requirements for a specific type of chickens (15x7 ab+), large numbers of animals, and a statistical method to compare lesion responses to those of JM/102W and Md5 control strains. Because of these limitations, it has not been and is not likely to be used in other laboratories. Comparability in pathotyping can be improved by the comparison of field isolates with standard prototype strains such as JM/102W, Md5 and 648A (American Type Culture Collection) or their equivalents. Data may be generated by different in vivo procedures that measure tumour induction, neurological disease (both neoplastic and non-neoplastic lesions), or solely non-neoplastic criteria (such as lymphoid organ weights or virus replication). Methods based on neoplastic criteria, especially when generated in MD-immunized chickens, will probably correlate most closely with that of the ADOL method and be most relevant to evolution of MD virus in the field. Based on data from several trials, a modification of the ADOL method that utilizes fewer chickens and can be conducted with commercial specific pathogen free strains is proposed. The modified method is based on "best fit" comparisons with prototype strains, and is expected to provide results generally comparable with the original method. A variety of other alternative criteria (see earlier) are also evaluated both for primary pathotyping and as adjuncts to other pathotyping methods. Advantages and disadvantages of alternative methods are presented.

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PubMed1995

Stages of Marek's disease virus latency defined by variable sensitivity to interferon modulation of viral antigen expression.

Cytokines in conditioned medium can suppress expression of viral internal antigens (VIA) in lymphocytes latently infected with Marek's disease virus. In the present study, conditioned media produced by spleen cells stimulated with concanavalin A or by mixed-lymphocyte reaction had significantly greater (P < 0.05) VIA-suppressive activity with lymphocytes harvested from birds at 14 days post infection than with those collected at 7 days. This finding defines two stages during the latent period in which sensitivity of lymphocytes to cytokine modulation of viral expression differs. Suppression involved proteins representing immediate-early, early and late viral antigens. Physico-chemical characterization of the suppressive factor in conditioned medium was consistent with that expected of interferon. Indeed, natural interferon prepared from avian reovirus-exposed chicken embryo cells, and recombinant chicken interferon, both mimicked the activity of conditioned medium and were more suppressive with lymphocytes from the later stage of latency.

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PubMed1992

Gordon Memorial Lecture. Chicken neoplasia--a model for cancer research.

1. The use of animal models has been immensely important for the advancement of our knowledge of the aetiology and pathogenesis of human diseases, including neoplasia. 2. Viruses, as oncogenic agents, were first described in the early 1900s when cell-free filtrates were used experimentally to transmit leukemias and sarcomas in chickens. In more recent years, studies with avian leukosis/sarcoma viruses have led the field in attempts to establish the genetic and molecular basis of viral oncogenesis. 3. Marek's disease of chickens was the first neoplasm proven to be caused by a herpesvirus and it remains the only neoplastic disease for which an effective vaccine has been developed and deployed. It serves as an elegant model as we seek an understanding of the pathogenesis of herpesvirus-induced lymphomas at both the cellular and molecular levels.

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PubMed1991

Two distinct alpha beta T-cell lineages can be distinguished by the differential usage of T-cell receptor V beta gene segments.

Avian T cells can be divided into three subpopulations based on their expression of distinct T-cell receptors (TCR1, TCR2, and TCR3), ontogeny, and tissue distribution. The TCR1 cells appear to be the equivalent of mammalian gamma delta cells, but the derivation of cells expressing TCR2 and TCR3 has been unclear. Here we report that chickens contain two families of TCR beta variable (V) gene segments, V beta 1 and V beta 2. Furthermore, TCR2 and TCR3 represent subsets of alpha beta cells that are defined by mutually exclusive usage of these two families of V beta gene segments. Sequence comparisons of V beta 1 and V beta 2 with mammalian TCR beta V segments reveal that V beta 1 gene segments encode the conserved amino acids used to define the mammalian V beta consensus subgroup I, while V beta 2 encodes the amino acids used to define the mammalian V beta subgroup II. Although the beta chains of TCR2 and TCR3 cells are encoded by the same diversity (D), joining (J), and constant (C) region segments, V beta 1 gene segments undergo rearrangement before V beta 2 gene segments during T-cell development. This may result from the fact that TCR2 cells undergo V-DJ joining by deletional rearrangement, whereas TCR3 cells undergo V-DJ joining by inversional rearrangement. These data suggest that the TCR alpha beta cells can be divided into two distinct and evolutionarily conserved lineages based on V beta gene segment usage. The clear-cut separation of these lineages in the chicken may help to define their immunologic role.

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PubMed1991

Transformation of T-lymphocyte subsets by Marek's disease herpesvirus.

Marek's disease herpesvirus (MDV)-transformed lymphoblastoid tumor cell lines were characterized for the presence of the surface markers. Monoclonal antibodies were used for CD3 (T-cell receptor [TCR] complex), TCR1, TCR2, and TCR3, CD4, CD8, and Ia antigen by indirect fluorescence staining followed by microscopic examination or flow cytometry. The lymphoblastoid cell lines were obtained from tumors from chickens infected with MDV (n = 44) or from local lesions induced by inoculation of allogeneic, MDV-infected chick kidney cells (n = 56). Lymphocytes were harvested from these lesions between 4 and 16 days postinoculation and cultured in vitro to establish cell lines. All cell lines expressed Ia antigen and CD3 and/or TCR and thus are activated T cells. Most of the cell lines developed from tumors were CD4+ CD8-; only one cell line was negative for both markers. Sixteen percent of the cell lines were TCR3+, while the remainder were TCR2+. The cell lines developed from local lesions were much more heterogeneous: 45% were CD4- CD8+, 34% were CD4- CD8-, and only 21% were CD4+ CD8-. The number of TCR3+ cell lines was larger than expected for the CD4- CD8+ and CD4- CD8- cell lines, as judged from the presence of these cells in the blood. These results indicate that several subsets of T lymphocytes can be transformed by MDV, depending on the pathogenesis of infection. Activation of T cells as a consequence of the normal pathogenesis or by allogeneic stimulation seem to be a first important step in the process of transformation.

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PubMed1989

Cytotoxic T lymphocytes in reticuloendotheliosis virus-infected chickens.

Cytotoxic T lymphocytes were functionally demonstrated in spleen cells from chickens 7 days post inoculation with reticuloendotheliosis virus using a Cr-release assay. Major histocompatibility complex (MHC)-restricted cytotoxicity was demonstrated using effector and target cells from two different strains of chickens of known avian MHC haplotype. Anti-viral specificity was shown and in vivo generation of MHC-restricted cytotoxicity was evaluated. Cytotoxic T cells were distinguished from macrophages and natural killer cells. Their cytotoxicity was not antibody dependent. Higher levels of cytolysis were found with cytotoxic T cells from embryonally bursectomized vs. intact chickens over a large range of effector to target cell ratios. Using monoclonal antibodies, cytotoxic T cells were further defined as Ia+ T cells by immunofluorescence, antibody plus complement-mediated lysis of effector cells and blocking of cytolysis in the Cr-release assay.

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PubMed1989

Immune response versus susceptibility to Marek's disease.

It was hypothesized that the generation of activated T cells through an efficient and rapid immune response during the early pathogenesis of Marek's disease virus (MDV) infection provides a large pool of target cells for transformation. Therefore, the correlation between genetic susceptibility to Marek's disease (MD) and in vitro mitogenic responses of lymphocytes as a measure of cell-mediated immune competence and efficiency was tested. In one series of trials, spleen cells from strains of chickens with differing levels of susceptibility to MD tumors were stimulated with graded doses of Concanavalin A (Con A) or phytohemagglutin (PHA). In a second series of trials, peripheral blood lymphocytes from individual chickens within genetic strains were tested at the same time chickens were challenged with MDV to determine susceptibility. Responsiveness was determined using one-way mixed lymphocyte reaction (MLR) tests as well as mitogen stimulation. Data from the tests comparing chicken strains supported the hypothesis in some but not all cases. The S13 chickens, which are more susceptible than P2a chickens to MD, were significantly more responsive, and highly resistant N2a chickens were significantly less responsive to Con A. In contrast, five other resistant strains were either more responsive (UCD-058, OS13) or equally responsive (UCD-140, OS5, C) to Con A when compared with P2a chickens. The PHA responses were even less predictive of MD susceptibility. No general correlation was observed between responsiveness to either mitogen or MLR tests and subsequent tumor development in trials comparing individuals within strains.

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PubMed1988

Maintenance of Marek's disease herpesvirus latency in vitro by a factor found in conditioned medium.

Chicken spleen cells latently infected with Marek's disease virus were cultured with and without conditioned medium (CM) obtained from concanavalin A-stimulated chicken spleen cell cultures. The expression of viral internal antigen(s) (VIA), which is usually associated with cultivation, was prevented or markedly reduced by the CM. This effect required the continued presence of CM, since its removal after 48 h resulted in the subsequent appearance of VIA. Although CM contains both gamma interferon (IFN-gamma) and interleukin 2, our studies suggest that the 'latency-maintaining activity' (LMF) may not be associated with either of these products of stimulated lymphocytes. However, IFN-gamma may also have had some suppressive effect. LMF appears to have an Mr greater than 10,000 and to be inactivated by heating to 90 degrees C for 5 min.

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PubMed1988

Effect of immunocompetence on the establishment and maintenance of latency with Marek's disease herpesvirus.

Marek's disease virus (MDV) infections normally have an early cytolytic phase in lymphoid organs at 3 to 6 days post-infection followed by a period of latent infection. Little is known about the mechanisms that govern latency with herpesvirus infections, including Marek's disease (MD). To investigate the importance of immunocompetence for either the establishment or the maintenance of latency in MD, immunosuppressive treatments were applied prior to infection with MDV or after latency was established. These included cyclosporin (Cs) or betamethasone (BM) treatments, neonatal thymectomy plus cyclophosphamide treatment (Tx/Cy), and infection at a young age before full competence. The effect of all the treatments was determined by examining tissues and spleen cells for evidence of virus replication before and after cultivation in vitro. Induced (Cs or Tx/Cy treatments) or natural (infection at a young age) incompetence resulted in prolonged and more widespread early cytolytic infection. Immunosuppression by Cs after latency had developed resulted in a reappearance of cytolytic infection in the spleen and it enhanced the cytolytic infection in the thymus and the bursa of Fabricius. After immunosuppression with Cs, cytolytic infection was found mostly in T cells, although many of the virus-positive cells did not have markers for either T cells or B cells. Immunosuppression by BM after latency had developed also resulted in the reappearance of cytolytic infection in the spleen but only at a very low level. These results suggest that immunocompetence is required for the establishment and maintenance of latency.

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PubMed1987

Expression of a putative tumor-associated surface antigen on normal versus Marek's disease virus-transformed lymphocytes.

Various avian tumor cell lines and normal spleen cells from 3 genetic strains of specific-pathogen-free (SPF) chickens were examined for expression of Marek's disease (MD) tumor-associated surface antigen (MATSA). Two anti-MATSA monoclonal antibodies (RPH 6 and EB 29) and a rabbit anti-MATSA antiserum were used in indirect fluorescent antibody tests, and cells were examined by fluorescence microscopy and with a fluorescence-activated cell sorter (FACS). Less than 5% MATSA-positive cells were observed in 2 non-MD tumor cell lines (LSCC-RP 9 and RECC-CU 60) with RPH 6, but 7-82% positive cells were observed with EB 29 or the rabbit antiserum. Five MD tumor cell lines (MDCC-CU 2, -CU 14, -CU 25, -CU 32, and -CU 41) had 12-72% positive cells detected with one or both monoclonals and 31-99% positive cells detected with the rabbit antiserum. Over 90% of cells in all MD lines were la and T3 positive, while values for the same parameters in LSCC-RP 9 were 100 and 3% and for RECC-CU 60, 48 and 51%, respectively. Evidence for cell-cycle-dependent expression of MATSA on MDCC-CU 2 was obtained from cell sorting experiments with the FACS and from examination of the MATSA-staining characteristics of 3 clones derived from the parent culture. Less than 5% MATSA-positive cells were observed in uncultured spleen cells from SPF chickens or in spleen cells stimulated for 48 hours with concanavalin A or phytohemagglutinin-M. However, with one exception, 10-53% of normal spleen cells were MATSA positive with RPH 6, after stimulation by mitogen for 24 or 48 hours followed by maintenance in conditioned medium (CM) for various times or after culture directly in CM for 3 days. More limited experiments with rabbit anti-MATSA antiserum yielded 55-85% MATSA-positive cells. From 60 to 97% of these MD virus-free, MATSA-positive cells were la-positive; and, in 2 cases, 89 and 90% were T3 positive.

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PubMed1986

Marek's disease--a model for herpesvirus oncology.

The article will review the salient features of pathogenesis of Marek's disease in terms of sequential events which occur from the time of virus entry to the development of frank lymphomas. A hypothesis will be presented which will offer a credible explanation for this specific sequence of changes. Then, various factors which influence the incidence of neoplasms will be identified and the possible mechanisms by which they are influential will be discussed. These include the variable oncogenic properties of different virus strains, the influence of host genotype, and immune responses.

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PubMed1985

Avian diseases.

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Animals↗
PubMed1984

Further characterization of Marek's disease virus-infected lymphocytes. I. In vivo infection.

Previous reports from this laboratory identified bursa-derived lymphocytes (B cells) and non-B cells as the predominant cell types respectively involved in the early cytolytic and subsequent latent infection of chickens with Marek's disease virus (MDV). It was not known whether these differences were qualitative or quantitative or if the method for detection of latent infection (viral antigen production after 48 h of in vitro cultivation) was sensitive enough. To further define the cells involved in the various phases of MDV infection, we used monoclonal antibodies which specifically react with B cells, or T cells, or la-antigen-bearing cells. Dual fluorescence tests to detect surface markers and viral internal antigen (VIA) were conducted with infected spleen cells freshly collected from MDV-infected chickens or after in vitro cultivation of those cells. The same antibodies were also used for a rosetting procedure to yield fractions enriched or depleted of T cells, B cells or la-bearing cells. These were examined directly for viral DNA by in situ hybridization or dot blot DNA hybridization and for VIA cultivation. We learned that infected T cells also comprise part of the early cytolytic phase of MDV infection but constitute a minority population (approximately 2-3%) compared to B cells (83-92%) at 3 or 4 days post infection. Latently infected cells were definitively identified as mostly la-bearing T cells, although a few (2-4%) were B cells. Prior to in vitro cultivation, latently infected cells apparently had insufficient viral DNA for detection by in situ hybridization, but the more sensitive dot blot procedure revealed viral DNA in fractions later found positive by VIA expression after in vitro cultivation. Viral DNA replication in latently infected cells apparently had occurred after 48 h cultivation because in situ hybridization detected infected cells at that time. Treatment of cell cultures with iodo-deoxyuridine, 12-O-tetradecanoyl phorbol-13-acetate or n-butyrate failed to increase the number of spleen cells which expressed VIA.

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PubMed1984

Further characterization of Marek's disease virus-infected lymphocytes. II. In vitro infection.

Lymphocyte cultures from chicken spleens had been shown to be susceptible to in vitro infection by Marek's disease virus (MDV)4 in an earlier report from this laboratory. In that study, virus infection was evidenced by virus isolation and detection of viral internal antigen (VIA) 2 days post inoculation (DPI), and serial passage was accomplished by adding fresh spleen cells at 2-day intervals. The susceptible cells were identified as bursa-derived lymphocytes (B cells). Using a dual fluorescence technique to identify surface markers for B cells, thymus-derived lymphocytes (T cells) or Ia antigen on VIA-positive cells, we have now shown that a small proportion (generally less than 10%) of VIA-positive cells observed 2 DPI are T cells, and that a low level of infection can be maintained by serial passage of MDV in cultures totally free of B cells. Most infected T cells in this study had Ia antigen. As the incubation period for infected cultures was extended from 2 to 4 or 5 days, the average number of viable cells decreased but the percentage of viable cells infected with MDV (VIA-positive) increased. Also, both the proportion and the actual number of infected T cells increased, significantly more so in cultures from genetically susceptible P-2 donors than from resistant N-2 donors. Spleen-cell cultures from resistant Line 6 chickens were markedly less susceptible than those from susceptible Line 7 chickens to in vitro MDV infection, as assessed by numbers of VIA-positive cells at 5 DPI.

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PubMed1984

Cell-specific antiviral activity of 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) against Marek's disease herpesvirus and turkey herpesvirus.

Three new fluoroarabinosylpyrimidine nucleosides (FIAC, FIAU and FMAU) were tested for in vitro activity against oncogenic and nononcogenic strains of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT). Marek's disease is a herpesvirus-induced lymphoma in chickens. Nononcogenic strains of MDV and HVT can protect against this disease. All viruses were inhibited by 1 microM of these drugs in chick kidney cell (CKC) cultures, but only FMAU and FIAU were active in chicken embryo fibroblast (CEF) and spleen cell cultures. It was determined that whereas CKC produced the enzyme 2'-deoxycytidine-deaminase which is needed to deaminate FIAC to FIAU, CEF were devoid of this enzyme activity. In addition, the deaminase inhibitor 3,4,5,6-tetrahydrouridine prevented the antiviral activity of FIAC in CKC. FMAU was not active against two Marek's disease-derived lymphoblastoid tumor cell lines.

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PubMed1983

Characterization of Marek's disease virus-infected lymphocytes: discrimination between cytolytically and latently infected cells.

Leukocyte suspensions derived from genetically Marek's disease (MD)-resistant N-line and MD-susceptible P-line chickens were fractionated at various times after exposure to the JM-10 clone of MD virus. At 3 and 5 days post exposure (DPE), during the productive-restrictive (cytolytic) phase, most infected spleen and thymus leukocytes were found to be low-density, nylon wool-adherent cells that possessed Fc receptors and surface Ia and IgM and were depleted by carbonyl iron treatment. This was true for leukocytes derived from N-line as well as those from P-line chickens. In contrast, most infected spleen cells derived from P-line chickens during the latent phase (i.e., after 7 DPE) were not found to have the above characteristics, with one exception: Ia antigen was demonstrated on the surface of latently infected cells. From these experiments it was concluded that the principal targets of the cytolytic JM-10 infection are B-cells, whereas the subsequent latent infection was found mostly in non-B-lymphocytes.

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