Isolation, nucleotide sequence and expression of a cDNA encoding feline granulocyte colony-stimulating factor.

A cDNA encoding feline granulocyte colony stimulating factor (fG-CSF) was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction. The cDNA is 949 bp in length and encodes a predicted mature protein of 174 amino acids. Recombinant fG-CSF was expressed as a glutathione S-transferase fusion and purified by affinity chromatography. Biological activity of the recombinant protein was demonstrated using the murine myeloblastic cell line GNFS-60, which showed an ED50 for fG-CSF of approximately 2 ng/ml.

Feline leukemia virus DNA vaccine efficacy is enhanced by coadministration with interleukin-12 (IL-12) and IL-18 expression vectors.

The expectation that cell-mediated immunity is important in the control of feline leukemia virus (FeLV) infection led us to test a DNA vaccine administered alone or with cytokines that favored the development of a Th1 immune response. The vaccine consisted of two plasmids, one expressing the gag/pol genes and the other expressing the env gene of FeLV-A/Glasgow-1. The genetic adjuvants were plasmids encoding the feline cytokines interleukin-12 (IL-12), IL-18, or gamma interferon (IFN-gamma). Kittens were immunized by three intramuscular inoculations of the FeLV DNA vaccine alone or in combination with plasmids expressing IFN-gamma, IL-12, or both IL-12 and IL-18. Control kittens were inoculated with empty plasmid. Following immunization, anti-FeLV antibodies were not detected in any kitten. Three weeks after the final immunization, the kittens were challenged by the intraperitoneal inoculation of FeLV-A/Glasgow-1 and were then monitored for a further 15 weeks for the presence of virus in plasma and, at the end of the trial, for latent virus in bone marrow. The vaccine consisting of FeLV DNA with the IL-12 and IL-18 genes conferred significant immunity, protecting completely against transient and persistent viremia, and in five of six kittens protecting against latent infection. None of the other vaccines provided significant protection.

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.

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.

Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice.

Animal donors such as pigs could provide an alternative source of organs for transplantation. However, the promise of xenotransplantation is offset by the possible public health risk of a cross-species infection. All pigs contain several copies of porcine endogenous retroviruses (PERV), and at least three variants of PERV can infect human cell lines in vitro in co-culture, infectivity and pseudotyping experiments. Thus, if xenotransplantation of pig tissues results in PERV viral replication, there is a risk of spreading and adaptation of this retrovirus to the human host. C-type retroviruses related to PERV are associated with malignancies of haematopoietic lineage cells in their natural hosts. Here we show that pig pancreatic islets produce PERV and can infect human cells in culture. After transplantation into NOD/SCID (non-obese diabetic, severe combined immunodeficiency) mice, we detect ongoing viral expression and several tissue compartments become infected. This is the first evidence that PERV is transcriptionally active and infectious cross-species in vivo after transplantation of pig tissues. These results show that a concern for PERV infection risk associated with pig islet xenotransplantation in immunosuppressed human patients may be justified.

Cloning and sequencing of feline and canine ice-related cDNAs encoding hybrid caspase-1/caspase-13-like propeptides.

Caspases are cysteine proteases which have important roles in the activation of cytokines and in apoptosis. The ICE subfamily of caspases comprise peptides closely related to caspase-1, or interleukin-1beta (IL-1beta) converting enzyme (ICE), which promotes maturation of interleukin IL-1beta and interleukin-18 (IL-18) by proteolytic cleavage of precursor forms to generate biologically active peptides. Other members of this subfamily include caspase-4, -5, -13 and isoforms of these proteins. We report the cloning and sequencing of two feline and canine ICE-related cDNAs amplified by RT-PCR. The predicted proteins are 410 and 404 amino acids in length respectively and are most closely related to caspase-1 sequences across the N-terminal 115 amino acids and to human caspase-13 across the C-terminal sequence.

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.

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.

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.

Cloning, sequencing, and characterization of dog interleukin-18.

Interleukin-18, originally termed interferon-gamma inducing factor, is a recently described cytokine which is intimately involved in the generation of the immune response. The human and mouse sequences have been reported and studies have demonstrated the potent biological functions of this protein including the induction of interferon-gamma and the enhancement of NK cytotoxicity. This paper describes the cloning, sequencing, and characterization of the dog homologue of this gene. The coding sequence for dog IL-18 was obtained using reverse transcription-polymerase chain reaction from mRNA harvested from PMA-stimulated alveolar macrophages. Sequence analysis of the dog gene has demonstrated an open reading frame of 582 base pairs coding for a 193 amino acid precursor protein. The dog coding sequence shares 84% and 74% similarity to the human and mouse equivalents, respectively, at the nucleotide level. Based upon sequence analysis, we propose that this new gene is the dog equivalent of human IL-18.

DNA vaccination affords significant protection against feline immunodeficiency virus infection without inducing detectable antiviral antibodies.

To test the potential of a multigene DNA vaccine against lentivirus infection, we generated a defective mutant provirus of feline immunodeficiency virus (FIV) with an in-frame deletion in pol (FIVDeltaRT). In a first experiment, FIVDeltaRT DNA was administered intramuscularly to 10 animals, half of which also received feline gamma interferon (IFN-gamma) DNA. The DNA was administered in four 100-microg doses at 0, 10, and 23 weeks. Immunization with FIVDeltaRT elicited cytotoxic T-cell (CTL) responses to FIV Gag and Env in the absence of a serological response. After challenge with homologous virus at week 26, all 10 of the control animals became seropositive and viremic but 4 of the 10 vaccinates remained seronegative and virus free. Furthermore, quantitative virus isolation and quantitative PCR analysis of viral DNA in peripheral blood mononuclear cells revealed significantly lower virus loads in the FIVDeltaRT vaccinates than in the controls. Immunization with FIVDeltaRT in conjunction with IFN-gamma gave the highest proportion of protected cats, with only two of five vaccinates showing evidence of infection following challenge. In a second experiment involving two groups (FIVDeltaRT plus IFN-gamma and IFN-gamma alone), the immunization schedule was reduced to 0, 4, and 8 weeks. Once again, CTL responses were seen prior to challenge in the absence of detectable antibodies. Two of five cats receiving the proviral DNA vaccine were protected against infection, with an overall reduction in virus load compared to the five infected controls. These findings demonstrate that DNA vaccination can elicit protection against lentivirus infection in the absence of a serological response and suggest the need to reconsider efficacy criteria for lentivirus vaccines.

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.

Tumours derived from HTLV-I tax transgenic mice are characterized by enhanced levels of apoptosis and oncogene expression.

In order to investigate the role that the human T-lymphotropic virus type I (HTLV-I) tax oncogene plays in apoptosis and transformation in vivo, four lines of HTLV-I tax transgenic mice were generated under the regulatory control of the CD3-epsilon promoter-enhancer sequence. These mice develop a variety of phenotypes including mesenchymal tumours, which develop at wound sites, and salivary and mammary adenomas. In situ DNA fragment labelling and immunocytochemical analysis of these tumours reveals that they display enhanced levels of apoptosis, which is associated with elevated levels of Myc, Fos, Jun, and p53 protein expression. Furthermore, double immunofluorescent staining shows that Tax expression and apoptosis co-localize, indicating that Tax expression is closely associated with apoptosis in vivo.

Proviral insertions induce the expression of bone-specific isoforms of PEBP2alphaA (CBFA1): evidence for a new myc collaborating oncogene.

The til-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-myc transgenic mice. We now show that viral insertions at til-1 lead to transcriptional activation of PEBP2alphaA (CBFA1), a transcription factor related to the Drosophila segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNA-binding domain and share a novel N terminus different from the previously reported PEBP2alphaA products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated til-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2alphaA and indicate that the Myc and Runt family genes can cooperate in oncogenesis.

Complete DNA sequence of canine adenovirus type 1.

The complete DNA sequence of a field strain of canine adenovirus type 1 was determined by sequencing random fragments of viral DNA cloned into pBluescript. The virus has a genome of 30536 bp flanked by two identical 161 bp inverted terminal repeats. Thirty ORFs have been identified, based on genomic location or sequence identity with published adenoviruses. These are arranged into similar discrete regions found in the human adenoviruses. ORFs in the late region show greatest identity with published human adenovirus sequences, whereas the E3 and E4 ORFs show little or none.

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.

Skewed T-cell receptor Vbeta8.2 expression in transgenic CD2-myc induced thymic lymphoma: a role for antigen stimulation in tumour development?

Transgenic mice expressing the c-myc proto-oncogene under the control of the CD2-dominant control region show stochastic development of mainly clonal thymic lymphoma with long latency, indicating that cooperative events are needed for the development of the fully malignant phenotype. Previous studies have suggested that T-cell receptor-associated signals can contribute to tumour development. We have therefore used this transgenic model of T-cell transformation to determine whether antigen-specific responses could constitute an epigenetic event in lymphomagenesis. The T-cell receptor (TcR) repertoires of lymphoma clones were analysed with a panel of monoclonal antibodies (Abs) recognizing TcR Vbeta chains. The Vbeta repertoire of tumour clones arising in these mice was non-random with overrepresentation of Vbeta8.2 TcR species. The majority of Vbeta8.2+ clones were of a mature CD3+ CD8 single-positive (SP) phenotype. The biased TcR usage, together with a mature cell phenotype is consistent with the hypothesis that TcR-mediated signals cooperate with activated myc during T-cell transformation.

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.

Complete nucleotide sequence and transcriptional analysis of snakehead fish retrovirus.

The complete genome of the snakehead fish retrovirus has been cloned and sequenced, and its transcriptional profile in cell culture has been determined. The 11.2-kb provirus displays a complex expression pattern capable of encoding accessory proteins and is unique in the predicted location of the env initiation codon and signal peptide upstream of gag and the common splice donor site. The virus is distinguishable from all known retrovirus groups by the presence of an arginine tRNA primer binding site. The coding regions are highly divergent and show a number of unusual characteristics, including a large Gag coiled-coil region, a Pol domain of unknown function, and a long, lentiviral-like, Env cytoplasmic domain. Phylogenetic analysis of the Pol sequence emphasizes the divergent nature of the virus from the avian and mammalian retroviruses. The snakehead virus is also distinct from a previously characterized complex fish retrovirus, suggesting that discrete groups of these viruses have yet to be identified in the lower vertebrates.