A prospective survey of familial canine lymphosarcoma.

The incidence and case distribution of lymphosarcoma were determined prospectively in one breed of dogs, the bull mastiff. Fifty-nine dogs from three households were followed for 3 years. Two of the households had a previous history of lymphosarcoma cases, whereas the third had no history of such cases. During the survey period, 9 of the 59 dogs died from lymphosarcoma with multiple cases occurring in each household. On the basis of this survey, the annual incidence rate in the general bull mastiff population is likely to be on the order of 5,000 cases per 100,000 dogs, which constitutes the highest incidence rate recorded in this species. The distribution of the cases displayed a familial pattern. The possible involvement of genetic and infectious factors in the development of this disease pattern is discussed.

Synergy between a human c-myc transgene and p53 null genotype in murine thymic lymphomas: contrasting effects of homozygous and heterozygous p53 loss.

Activation of the c-myc oncogene and functional loss of the p53 tumour suppressor gene are among the most frequently recorded genetic lesions in neoplasia but their combined effect has not previously been investigated. By breeding together mice transgenic for human c-myc (CD2-myc) and mice carrying an inactive p53 allele (p53-/-) we found that these genetic lesions act synergistically in vivo. Offspring carrying the CD2-myc transgene and the homozygous p53 null mutation (p53-/-/CD2-myc) were viable but developed thymic lymphomas with dramatically increased frequency and reduced latency compared to both parental groups. The tumour phenotype was similar to that previously recorded for CD2-myc mice (predominantly CD3+, CD4+8+) but tumour clonal complexity and metastasis was significantly greater in the p53-/-/CD2-myc mice. In contrast, no significant increase in tumour incidence was seen in p53+/-/CD2-myc vs p53+/+/CD2-myc mice over a 6 month observation period. However, the loss of wild type p53 in a proportion of tumour cells in p53+/-/CD2-myc lymphomas suggests that wild type allele loss can occur as a late progression step rather than an initiating step in these tumours. We suggest that p53 loss of function may collaborate with the CD2-myc transgene at more than one stage in thymic lymphoma development.

Fas-independent apoptosis in T-cell tumours induced by the CD2-myc transgene.

Depending on the cellular context, the Myc oncoprotein is capable of promoting cell proliferation or death by apoptosis. These observations suggest that apoptosis in response to deregulated gene expression may represent a natural brake to tumour development. The pathways by which Myc induces apoptosis are as yet poorly characterised although recent observations on rat fibroblasts over-expressing Myc have demonstrated a requirement for the Fas pathway. To investigate the role of Fas in Myc-induced lymphomagenesis we backcrossed CD2-myc mice onto an lpr background. Rates of tumour development and phenotypic properties, including levels of apoptosis were indistinguishable from CD2-myc controls. Further, tumour cell lines derived from mice expressing a regulatable form of Myc showed inducible apoptosis at similar rates regardless of their lpr genotype. These results show that activation of c-myc and loss of Fas do not collaborate in T lymphoma development and that Myc-induced apoptosis in T-cells occurs by Fas-independent pathways.

Criteria for the definition of Epstein-Barr virus association in Hodgkin's disease.

There is a clear association between the Epstein-Barr virus (EBV) and Hodgkin's disease (HD). EBV is not, however, detectable within the affected tissues of all cases. The proportion of positive cases varies from 15-79% depending on the assay used to detect EBV. The techniques utilised vary not only in sensitivity but in their ability to detect viral DNA, RNA, or protein and in their ability to demonstrate the cellular localisation of the virus. Thus, the biological significance of a positive result will vary depending on the method of analysis. In the present study, four different methods of detecting EBV were compared. RNA in situ hybridization was found to be the most practical method of detecting EBV in tumour cells. Using this assay EBV was detected in the Reed-Sternberg cells of 33% and 45% of the two series of HD cases examined in this study. We believe that these cases should be considered EBV-associated.

Sequences of the ribonucleotide reductase-encoding genes of equine herpesvirus 4.

The equine herpesvirus 4 (EHV-4) genes encoding the two subunits of the enzyme ribonucleotide reductase (RR) were cloned and their nucleotide (nt) sequences determined. The large subunit (RR1) is predicted to comprise 789 amino acids (aa), which compares with lengths of 790, 775 and 1137 aa for the RR1 proteins encoded by equine herpesvirus 1 (EHV-1) gene 21, varicella zoster virus (VZV) gene 19 and herpes simplex virus type 1 (HSV-1) UL39, respectively. In common with VZV RR1, the EHV-4 RR1 protein lacks the N-terminal domain of HSV-1 RR1 which possesses protein kinase activity. EHV-4 RR1 demonstrates identities of 88, 52 and 29% with the RR1 proteins of EHV-1, VZV and HSV-1, respectively. The small subunit (RR2) is predicted to be 320 aa in length, which compares with lengths of 321, 306 and 340 aa for the RR2 proteins encoded by EHV-1 gene 20, VZV gene 18 and HSV-1 UL40, respectively. The EHV-4 RR2 protein exhibits identities of 90, 60 and 55% with the RR2 proteins of EHV-1, VZV and HSV-1, respectively.

An improved cosmid vector for the cloning of equine herpesvirus DNA.

We have modified the commercial cosmid vector, triple helix vector (THV), such that I-Sce-I restriction endonuclease sites flank the cloning site. I-Sce-I is a rare-cutting endonuclease which recognizes an 18-bp sequence. It does not restrict the genome of either of the equine herpesvirus 1 or 4 (EHV-1 and EHV-4) strains we have cosmid cloned. Thus, cosmid-cloned EHV fragments can be excised intact from the vector by I-Sce-I digestion, facilitating production of large overlapping EHV fragments for use in transfections to produce recombinant virus.

Case clustering, Epstein-Barr virus Reed-Sternberg cell status and herpes virus serology in Hodgkin's disease: results of a case-control study.

The Leukaemia Research Fund Data Collection Study (DCS) is a specialist registry of leukaemias and lymphomas. The present study involves 494 cases of Hodgkin's disease (HD) registered with the DCS between 1985 and 1989. This entire data set has been tested for localised spatial clustering using an established nearest neighbour method with 18% of all cases in young people classified as clustered (P < 0.05). No clustering was found in older cases. Subsamples were selected from the registered cases for a pilot study in which case clustering, herpes virus antibody titres and Epstein-Barr virus (EBV) presence within the Reed-Sternberg (RS) cells (EBV-RS status) were investigated together. Firstly, a case-control study of HD in young people or nodular sclerosing (NS) subtype (39 HD cases and 26 healthy controls) found significant elevation of antibody titres to EBV-viral capsid antigen (VCA), EBV-early antigen (EA) and human herpes virus 6 (HHV-6) in HD cases compared with controls. EBV viral genome was present in 5 cases and 4 of these were in clusters of HD in young people. Elevation of antibody titres to the EBV antigens was not associated with case clustering or EBV-RS status. Antibody titres to HHV-6 differed significantly between EBV-RS+ and EBV-RS- cases (P = 0.04). Geometric mean titres for HHV-6 for EBV-RS+ and EBV-RS- cases were 11.5 and 73.7, respectively, with the former lower than the control value of 20.5. Secondly, a cluster study included all other cases (n = 14) in clusters containing known EBV-RS+ cases. 3 further cases were EBV-RS+ positive but no cluster consisted entirely of positive cases. Overall, 5/16 clustered, 2/12 peripheral and 1/25 random cases in these studies were EBV-RS+ (P = 0.017). The interpretation of these results in terms of shared aetiological exposures of cases within clusters and the roles of EBV and HHV-6 is discussed, and hypotheses for testing in future studies proposed.

A model to study viral and cytokine involvement in Sjögren's syndrome.

To investigate mechanisms that may be important in the pathogenesis of Sjögren's syndrome (SS) we developed a protocol for the growth of salivary gland epithelial cells in culture. We examined the effect that viral infection has on the cellular location of the autoantigen La. Autoantibodies to La are common in SS and it has been proposed that viral infection may result in cell membrane expression of La. Co-expression of MHC class II molecules in infected cells could lead to the presentation of La peptides to the immune system. Advenovirus infection of salivary gland epithelial cells resulted in an altered nuclear staining of La. Treatment with interferon-gamma resulted in the expression of La in the cell cytoplasm and HLA-DR molecules at the cell surface. These findings suggest that a cytokine-driven mechanism may generate an autoimmune response to La in SS. Using the polymerase chain reaction (PCR) we tested salivary gland epithelial cell cultures for the presence of human herpesvirus-6 (HHV-6) and Epstein-Barr virus (EBV). Only HHV-6 was detected in 2 of 10 salivary gland epithelial cell cultures although the presence of HHV-6 was not associated with SS. Primary salivary gland cultures may prove useful as an in vitro model to study mechanisms of autoimmunity in SS.

Feline leukaemia viruses: molecular biology and pathogenesis.

The feline leukaemia virus (FeLV) group represents one of the most important viral pathogens of the domestic cat. In addition, this virus - host system is one of the major experimental models for retroviral pathogenesis. Under natural conditions, the virus is horizontally transmitted through the cat population. The outcome of infection depends on a variety of factors including the virus does encountered and the age and immune status of the host. FeLVs can establish persistent infection, either overt or latent. Degenerative diseases of the haemopoietic system are the most common result of persistent infection and immunosuppression with secondary infection accounts for more deaths than does neoplastic disease. However, more is known about the molecular mechanisms of oncogenesis in this system and there are now numerous examples of field case tumours where FeLV has transduced an oncogene or acted as an insertional mutagen. The factors affecting the relative frequency of these mechanisms are considered as is the possibility that recombinant env gene recombinants play a role in FeLV pathogenesis.

Detection of canine herpesvirus 1 in a wide range of tissues using the polymerase chain reaction.

Canine herpesvirus 1 (CHV-1), a member of the alphaherpesvirus sub-family, is known to cause fatal infections in litters of puppies and may also be involved in infertility, abortion, and stillbirths in adult dogs. The purpose of this study was to determine the presence of CHV-1 DNA using the polymerase chain reaction (PCR) in twelve key sites that have been associated with latency for the other herpesviruses. A 605 base pair portion of the viral glycoprotein B (gB) gene was amplified using degenerate primers, cloned, and sequenced. Conventional 20 mer primers were designed using this sequence information to amplify a 120 bp fragment of gB situated between the original degenerate primers. The specificity of amplification was confirmed by Southern Blot hybridisation using an internal oligonucleotide probe. DNA was extracted from tissue samples taken from twelve dogs at post mortem and from twenty-four blood samples. Nine out of twelve dogs showed evidence of infection with CHV-1; the tissues most commonly affected were lumbo-sacral ganglia (5/12 dogs), tonsil (5/12), parotid salivary gland (4/9), and liver (4/9). No positive results were detected within the twenty-four blood samples. These results indicate that exposure to CHV-1 may be much more common than previously suggested.

Expression of feline recombinant interferon-gamma in baculovirus and demonstration of biological activity.

We have previously reported the cloning of the coding sequence for feline-specific interferon-gamma. Here, we describe the expression of this sequence in a baculovirus system and demonstrate the biological activity of the recombinant protein. The coding sequence for feline interferon was directionally cloned into the baculovirus transfer vector pAcCL29-1. Transfer vector and linearized wild-type AcMNPV (BacPAK6) were used to co-transfect Sf9 cells by calcium phosphate coprecipitation. Subsequently, wild-type and recombinant viruses were separated by plaque assay. Recombinant plaques were expanded and a master stock of virus is produced. Production of biologically active interferon-gamma from infected Sf9 cells was demonstrated using a standard cytopathic effect reduction assay, utilising vesicular stomatitis virus (VSV), and an MHC class II induction assay.

Characterization of the feline thyroglobulin promoter.

The feline thyroglobulin promoter was identified by a combination of standard polymerase chain reaction (PCR) techniques, using primers designed according to regions of homology in published sequences from other species, then adaptor ligated PCR. A 310 bp fragment of the feline thyroglobulin promoter was generated, including 8 nucleotides of adaptor sequence at the 5' end and, based on the putative transcription start site, 36 nucleotides of the thyroglobulin mRNA (untranslated portion). The homology between the feline promoter sequence (from 193 bp upstream to the putative cap site) and canine, bovine and human sequences was 89%, 81% and 78%, respectively. Transient transfection studies, using reporter constructs in which the feline promoter controlled expression of chloramphenicol acetyl transferase, demonstrated promoter activity in thyroid cells, but no activity in non-thyroid cells. The data presented here demonstrate that the feline thyroglobulin promoter may provide a targeting mechanism for somatic gene therapy of feline thyroid disease.

Cultures of pig macrophages.

Freshly collected blood from pigs was defibrinated with glass beads. The white cells were collected on a Ficoll-Hypaque mixture and grown in RPMI 1640 medium containing 20 per cent fetal calf serum. Monocytes adhered to the plastic or glass flasks and the erythrocytes and lymphocytes remained in suspension. The adherent cells assumed the appearance of macrophages and the mean cell size at eight days was 40 mum. Phagocytosis was demonstrated and, in the first few days, incorporation of tritiated thymidine occurred. Cells remained viable in culture for more than eight weeks. Histo-chemical studies and the appearance of the cells under the electron microscope are described.

Diagnosis of equid herpesviruses -1 and -4 by polymerase chain reaction.

The polymerase chain reaction (PCR) is a sensitive technique used to detect DNA of viral pathogens. We have applied the technique to the detection of Equid herpesviruses-1 and -4 (EHV-1 and EHV-4) DNA within nasopharyngeal swab samples from horses. Ninety-eight samples from suspected field cases and in-contact horses were analysed. The assays were conducted blind and later decoded and compared with virus isolation data. Our results indicate that PCR is a sensitive and rapid technique for the diagnosis of EHV-1 and EHV-4 infection.

Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems.

Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 and p40 subunits or single chain IL-12 enhanced IFN-gamma production in cells derived from equine lymph nodes. Preincubation of IFN-gamma inducing preparations with anti-p40 monoclonal antibody resulted in a significant decrease in IFN-gamma induction capacity. Medium recovered from p35 and p40-expressing baculovirus infected cultures enhanced target cell IFN-gamma production and proliferation. Experimental studies in mice and other animals have revealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectious disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation of its potential application in equine species.

Xenotransplantation: an overview of microbiological risks and potentials for risk management.

Xenotransplantation is the use of animal organs, tissues or cells for transplantion into humans to treat a variety of medical conditions. If proven efficacious, the technique could be used as one means of alleviating the disparity between the growing demand for transplantable organs, tissues and cells, and the availability of human-origin transplants world-wide. Just as the practicality and efficacy of the technology need to be investigated, so too does the potential for associated infectious disease risk. While much remains to be learned about the microbiological risk associated with xenotransplantation, the elements to be incorporated into xenotransplantation risk management schemes can be considered, using what is currently known about the infectious agents potentially relevant to the xenotransplantation setting.

The cloning and sequencing of cDNAs encoding two isoforms of feline stem cell factor.

cDNA clones encoding two isoforms of feline stem cell factor (fSCF) have been isolated using RT-PCR and their sequences determined. The cDNAs encode a predicted full length fSCF protein of 274 amino-acids and a shorter isoform of 246 amino acids. Feline SCF shows a high degree of homology to the SCFs of other species at both the nucleic acid and protein level.

The isolation and sequence of canine interleukin-2.

A cDNA corresponding to canine IL-2 has been isolated and sequenced. The cDNA was synthesised using RT-PCR, with oligonucleotide primers designed from conserved regions of published IL-2 sequences. The cDNA encodes a predicted full length IL-2 protein of 155 amino-acids. At the nucleic acid level, the canine cDNA shows 92, 88, 88, 82 and 74% homology to published sequences of feline, human, equine, bovine and murine IL-2, respectively. The derived protein shows 90, 86, 85, 76 and 75% similarity to feline, human, equine, murine and bovine IL-2 homologs.

Cloning and sequencing of equine transforming growth factor-beta 1 (TGF beta-1) cDNA.

Transforming growth factor beta (TGF-beta) belongs to a family of peptide growth factors which control critical stages of cell proliferation and differentiation. We report the cloning and sequencing of the cDNA for TGF-beta type 1 isoform of the horse. The predicted mature equine TGF beta-1 peptide is 112 amino acids in length and exhibits 99% identity to mature human TGF beta-1.

Nucleotide sequence of equine caspase-1 cDNA.

Caspases are a family of cysteine proteases which have important roles in activation of cytokines and in apoptosis. Caspase-1, or interleukin-1 beta converting enzyme (ICE), promotes maturation of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) by proteolytic cleavage of precursor forms to generate biologically active peptides. We report the cloning and sequencing of equine caspase-1 cDNA. Equine caspase-1 is 405 amino acids in length and has 72% and 63% identity to human and mouse caspase-1, respectively, at the amino acid level. Sites of proteolytic cleavage and catalytic activity as identified in human caspase-1, are conserved.