Bovine papillomavirus type 1 DNA and E5 oncoprotein expression in water buffalo fibropapillomas.

Papillomas and fibropapillomas may occur in the skin and in different organs in animals. Ten different genotypes of bovine papillomavirus (BPV) have been identified. BPV-1 through BPV-10 are all strictly species-specific, but BPV-1/2 may also infect other species such as equids, inducing fibroblastic tumors. BPV-1 and BPV-2 are associated with fibropapillomas in cattle; these tumors are formed by excessive proliferation of virus-infected dermal fibroblasts and epidermal keratinocytes. Nine water buffalo (Bubalus bubalis) were examined for the presence of multiple cutaneous and perivulvar tumors. Cutaneous and perivulvar fibropapillomatosis were confirmed histologically. Negative-stain transmission electron microscopic examination revealed papillomavirus-like particles in the fibropapillomas, and papillomaviral DNA was also detected by the polymerase chain reaction. The amplified long control region (LCR) DNA sequence was identical to that of BPV-1. The BPV-1 E5 oncoprotein was strongly expressed in the tumor cells thus confirming a causal role of the virus. This article represents the first report of cutaneous, perivulvar, and vulvar fibropapilloma associated with BPV-1 infection in the water buffalo and describes another example of cross-species infection by BPV-1.

Transcriptional changes induced by bovine papillomavirus type 1 in equine fibroblasts.

Bovine papillomavirus type 1 (BPV-1) and, less commonly, BPV-2 are associated with the pathogenesis of common equine skin tumors termed sarcoids. In an attempt to understand the mechanisms by which BPV-1 induces sarcoids, we used gene expression profiling as a screening tool to identify candidate genes implicated in disease pathogenesis. Gene expression profiles of equine fibroblasts transformed by BPV-1 experimentally or from explanted tumors were compared with those of control equine fibroblasts to identify genes associated with expression of BPV-1. Analysis of the microarray data identified 81 probe sets that were significantly (P < 0.01) differentially expressed between the BPV-1-transformed and control cell lines. Expression of several deregulated genes, including MMP-1, CXCL5, FRA-1, NKG7, TLR4, and the gene encoding the major histocompatibility complex class I (MHC-I) protein, was confirmed using other BPV-1-transformed cell lines. Furthermore, expression of these genes was examined using a panel of 10 sarcoids. Increased expression of MMP-1, CXCL5, FRA-1, and NKG7 was detected in a subset of tumors, and TLR4 and MHC I showed robust down-regulation in all tumors. Deregulated expression was confirmed at the protein level for MMP-1 and MHC-I. The present report identifies genes modulated by BPV-1 transformation and will help identify the molecular mechanisms involved in disease pathogenesis.

Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: model systems for equine sarcoids.

It is now widely recognized that BPV-1 and less commonly BPV-2 are the causative agents of equine sarcoids. Here we present the generation of equine cell lines harboring BPV-1 genomes and expressing viral genes. These lines have been either explanted from sarcoid biopsies or generated in vitro by transfection of primary fibroblasts with BPV-1 DNA. Previously detected BPV-1 genome variations in equine sarcoids are also found in sarcoid cell lines, and only variant BPV-1 genomes can transform equine cells. These equine cell lines are morphologically transformed, proliferate faster than parental cells, have an extended life span and can grow independently of substrate. These characteristics are more marked the higher the level of viral E5, E6 and E7 gene expression. These findings confirm that the virus has an active role in the induction of sarcoids and the lines will be invaluable for further studies on the role of BPV-1 in sarcoid pathology.

Vaccination of sarcoid-bearing donkeys with chimeric virus-like particles of bovine papillomavirus type 1.

Equine sarcoids are fibroblastic skin tumours affecting equids worldwide. While the pathogenesis is not entirely understood, infection with bovine papillomavirus (BPV) type 1 (and less commonly type 2) has been implicated as a major factor in the disease process. Sarcoids very seldom regress and in fact often recrudesce following therapy. Nothing is known about the immune response of the equine host to BPV. Given that the viral genes are expressed in sarcoids, it is reasonable to assume that vaccination of animals against the expressed viral proteins would lead to the induction of an immune response against the antigens and possible tumour rejection. To this end we vaccinated sarcoid-bearing donkeys in a placebo-controlled trial using chimeric virus-like particles (CVLPs) comprising BPV-1 L1 and E7 proteins. The results show a tendency towards enhanced tumour regression and reduced progression in the vaccinated group compared to control animals. Although promising, further studies are required with larger animal groups to definitely conclude that vaccination with CVLPs is a potential therapy for the induction of sarcoid regression.

Preliminary studies of particle-mediated gene delivery to the joints of dogs.

This paper describes a preliminary evaluation of particle-mediated bombardment via the Helios gene gun for the delivery of therapeutic genes to synovial cells in culture. A reporter gene, enhanced green fluorescent protein, was delivered to rabbit synovial fibroblasts (HIG-82) using gold particle (1.0 microm) bombardment to evaluate transfection efficiency at helium pressures of 100 and 150 psi. Transfection of cells occurred at these pressures despite some cell death. The in vitro delivery of gold particles to samples of synovial membrane and articular cartilage from a freshly euthanased dog was also studied to examine depth of penetration of gold particles (1.0 microm) at helium pressures of 250 and 500 psi. Light microscopical examination of histological sections of the synovial membrane showed that particles of gold had penetrated the lining cells of the synovium. However, no gold particles had penetrated the articular cartilage even at 500 psi.

Inhibition of telomerase in canine cancer cells following telomestatin treatment.

Telomere shortening in normal somatic cells has been proposed as a major barrier to unlimited cellular proliferation. Telomerase is an enzyme capable of maintaining telomere length, and thus bypassing this barrier. In human beings, telomerase activity is restricted to cancer cells and cells of stem or germ cell lineages. Dogs represent a potentially useful clinical model for the development of telomerase-based therapies because telomerase activity is also restricted to cancer cells and stem cells in this species. We examined the ability of telomestatin to inhibit telomerase activity in telomerase-positive D17 and CMT7 canine cancer cell lines. At a concentration of 2 microM, telomestatin treatment resulted in a decrease in telomerase activity, telomere shortening, growth inhibition and apoptosis in telomerase-positive cancer cells. These effects were not seen in telomerase-negative skin fibroblasts or negative controls. These results confirm that telomestatin specifically inhibits telomerase activity in canine cancer cells and strengthens the usefulness of dogs as a model for testing telomerase-based therapies.

Telomerase activity and telomerase reverse transcriptase catalytic subunit expression in canine lymphoma: correlation with Ki67 immunoreactivity.

Increased telomerase activity (TA) has been found in human and canine solid tumours, stem cells and somatic tissues with enhanced proliferative potential. The relationship between TA in normal and malignant lymphoid tissues remains unclear. The TA and the expression of canine telomerase reverse transcriptase catalytic subunit (dogTERT) messenger RNA (mRNA) were analyzed in malignant lymph nodes from 30 dogs with lymphoma, from two dogs with non-neoplastic illness and from two clinically normal dogs, demonstrating a statistically significant difference between TA in lymphoma lymph nodes (n = 30) and normal nodes (n = 4) but no significant difference in dogTERT mRNA expression. In addition, the expression of telomerase reverse transcriptase catalytic subunit (TERT) protein and Ki67 was analyzed in malignant lymph nodes from 10 dogs with lymphoma and from two clinically normal dogs by immunohistochemistry. TERT expression was associated with Ki67 in all lymphoma nodes (n = 10), and differences were illustrated between TERT and Ki67 expression between lymphoma (n = 10) and non-lymphoma (n = 2) nodes. This data support further investigation of telomerase in canine haematopoietic neoplasia through large-scale prospective studies.

The canine telomerase catalytic subunit (dogTERT): characterisation of the gene promoter and identification of proximal core sequences necessary for specific transcriptional activity in canine telomerase positive cell lines.

Telomerase biology is complicated by studies that show that telomere expression and telomere biology differs between species, and that existing animal models do not closely resemble the human situation. We have previously reported a description of telomere/telomerase biology in the dog and have suggested this as an alternative model system. To further elucidate telomerase biology in this species we have cloned and characterised the canine reverse transcriptase (dogTERT) promoter. We demonstrate that core promoter activity is contained within a region extending approximately 300 bp upstream of the ATG codon. Transient transfections in telomerase-positive canine cell lines and telomerase negative fibroblasts showed that the promoter is only active in telomerase positive cell lines. Sequence analysis demonstrated that the 5' regulatory region is GC-rich and contains no TATA or CAAT box, similar to the human TERT promoter. Motif searches revealed the presence of multiple transcription factor binding sites common to both the human and canine TERT promoters, including a single E-box, Sp1, AP1, MZF-2 and ER/Sp1 binding sites. These findings suggest that the dogTERT gene shares similar transcriptional control to hTERT. Identification of the core promoter necessary for activity may allow the use of naturally occurring cancers in dogs as a preclinical testing ground for telomerase targeted therapies in human cancer patients.

Disease- and cell-type-specific transcriptional targeting of vectors for osteoarthritis gene therapy: further development of a clinical canine model.

OBJECTIVES: The potential for undesirable systemic effects related to constitutive expression of certain therapeutic transgenes may be limited through the development of transcriptionally targeted disease- and cell-type-specific vectors. The objective of this study was to analyse the canine matrix metalloproteinase-9 (MMP-9) promoter and deletion constructs for its ability to drive expression in response to pro-inflammatory cytokines (interleukin-1beta and tumour necrosis factor-alpha). METHODS: Initial analysis of MMP-9 deletion constructs was made using a luciferase reporter system. The promoter was subsequently engineered to incorporate multiple NF-kappaB sites. In parallel experiments we used the mouse collagen type XI promoter to study cell-type-specific promoter activity in chondrocyte-specific cells (SW1353) and undifferentiated chondroprogenitor cells (ATDC5). RESULTS: Incorporation of multiple NF-kappaB sites into the MMP-9 promoter enhanced activity while maintaining disease specificity. Further, manipulation of the mouse collagen type XI (mColXI) promoter by the incorporation of SOX9 enhancer sites downstream of a reporter gene, increased gene activity while maintaining cell type specificity. CONCLUSIONS: Manipulation of promoter and enhancer regions can improve transcriptionally targeted genes. A combination of these systems, in the context of the canine model, has the potential to improve the safety of osteoarthritis gene therapy vectors.

Feline epitheliotrophic T-cell lymphoma with paraneoplastic eosinophilia - immunochemotherapy with vinblastine and human recombinant interferon alpha(2b).

A cat with epitheliotrophic T-cell lymphoma with paraneoplastic eosinophilia is described. Initial attempts to control the disease with conventional therapies failed. The addition of recombinant human interferon alpha(2b) (rhINFalpha(2b)) resulted in a clinical, haematogenous and sonographic improvement for 49 days. The overall survival time from initial diagnosis was 100 days. Relapse was correlated with the development of serum antibodies directed against rhINFalpha(2b). To our knowledge, this is the first report describing the clinical use of IFNalpha in the treatment of neoplasia in the cat.

Equine telomeres and telomerase in cellular immortalisation and ageing.

To determine the role of telomeres in cellular ageing in equids, we analysed telomere lengths in peripheral blood derived DNA samples from a panel of donkeys (Equus asinus) ranging from 2 to 30 years of age. The average telomere lengths ranged from 7 to 21 kbp and a statistically significant inverse correlation between telomere lengths and donor age was demonstrated. Similarly, telomere lengths in primary fibroblasts isolated from a horse (Equus equus) demonstrated telomeric loss with in vitro ageing when cultured to senescence. We extended this study to evaluate activity of the enzyme telomerase in various equine cell cultures, normal equine tissues and equine benign tumour samples. Initially a panel of equine immortalised and primary cell cultures were evaluated for telomerase activity using a standard telomere repeat amplification protocol (TRAP) assay. High levels of telomerase activity were detected in equine immortalised cells with no activity evident in primary cell cultures. Similarly, no telomerase activity could be detected in normal equine tissues or equine benign tumour samples of the sarcoid or papilloma type. We conclude that telomere attrition may contribute to ageing in equids. However, it would appear that telomerase does not play a major role in the development of the most common benign tumours of the horse.

Cloning of canine IL-1ra, TNFR and TIMP-2.

This paper describes the cloning and sequence analysis of the cDNA's encoding the canine homologues of interleukin-1 receptor antagonist (IL-1ra), tumour necrosis factor receptor extra-cellular domain (TNFR/ECD) and tissue inhibitor of metalloproteinase-2 (TIMP-2). The coding sequences for canine IL-1ra and TNFR/ECD were obtained using reverse transcription polymerase chain reaction (RT-PCR) using RNA harvested from canine peripheral blood mononuclear cells (PBMC) and TIMP-2 was isolated in a similar fashion from the canine D17 osteosarcoma cell line. Sequence analysis of the canine genes demonstrated open reading frames of 531, 633 and 663 base pairs (bp), respectively. All three canine proteins IL-1ra, TNFR/ECD and TIMP-2 (177, 211 and 221 amino acids, respectively) showed considerable sequence similarity with the homologous sequences published for other species.

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.

Feline immunodeficiency virus (FIV)-associated lymphoma: a potential role for immune dysfunction in tumourigenesis.

To determine the potential role of immune dysfunction in feline immunodeficiency virus (FIV)-associated lymphomagenesis, we present the results of immunological monitoring during the chronic phase of experimental FIV infection in two cats which subsequently developed lymphoma. In one cat, C1, cell-mediated immunity was depressed throughout the monitoring period but particularly from 125-200 weeks post-infection (pi), when this cat demonstrated profoundly impaired lymphocyte blastogenesis and markedly increased interleukin-1 (IL-1) production compared to age-matched, uninfected control cats. Lymphocyte function in the other cat, C2, was preserved to a greater degree. Alterations in the levels of immunoglobulin isotypes M, A and G in CD4+-, CD8+- and CD21+-lymphocyte sub-sets were demonstrated in both cats. Southern blot analysis revealed the presence of integrated FIV-provirus in tumour DNA from C2 but not C1 indicating a possible direct role for the virus in the former case only. In this study we have characterised, for the first time, the FIV-induced immune dysfunction in cats which developed lymphoma, demonstrating potential indirect mechanisms of tumourigenesis.

Concentrations of isometamidium in the sera of cattle challenged with drug-resistant Trypanosoma congolense.

The relationship between serum concentrations of the prophylactic trypanocidal drug isometamidium chloride and protection against tsetse challenge with two populations of Trypanosoma congolense was investigated in Boran (Bos indicus) cattle, using an isometamidium-ELISA. Isometamidium chloride (Samorin) was administered to cattle at a dose rate of 1.0 mg/kg body weight by deep intramuscular injection. Thereafter, the animals were challenged at monthly intervals with either a drug-sensitive clone (T. congolense IL 1180) or a clone expressing a moderate level of resistance to isometamidium (T. congolense IL 3343). Untreated control cattle were used to confirm the infectivity of each challenge. Of ten drug-treated cattle that were challenged with T. congolense IL 3343, all were refractory to infection at the first challenge. 1 month after drug administration. However, all ten animals succumbed to infection at either the second (seven cattle) or third (three cattle) monthly challenges. By contrast, all five drug-treated cattle challenged with T. congolense IL 1180 resisted four monthly challenges. The mean isometamidium concentration at the time of the first, 1 month, challenge was 5.6 +/- 2.8 ng/ml. At the time of the second monthly challenge the mean concentration was 2.0 +/- 0.86 ng/ml: at this time, concentrations were not significantly different between those cattle refractory to challenge with T. congolense IL 3343 and those cattle that were not. Thus, differences in susceptibility to challenge at this time would appear to be due to differences in the drug sensitivity of the parasite challenge. Finally, the mean isometamidium concentration in uninfected cattle at the time of the fourth monthly challenge was 0.4 +/- 0.18 ng/ml. These results indicate that when T. congolense infection occurs in cattle under isometamidium prophylaxis, the parasites may be considered at least moderately drug resistant if the concentration of isometamidium in serum is 2.0 ng/ml. At concentrations between 0.4 and 2.0 ng/ml a low level of drug resistance may be inferred. Below 0.4 ng/ml, however, no inference regarding drug resistance should be made.

A longitudinal study of feline immunodeficiency virus-specific cytotoxic T lymphocytes in experimentally infected cats, using antigen-specific induction.

The evolution of the virus-specific cytotoxic T-lymphocyte response in two cats experimentally infected with feline immunodeficiency virus (FIV) was monitored. Effector cells were derived from peripheral blood lymphocytes during the acute and chronic phases of infection (0 to 21 and 62 to 127 weeks, respectively) and from the spleen and lymph nodes at 127 weeks after infection. Lymphocytes were restimulated in vitro with paraformaldehyde-fixed, autologous lymphoblasts which had been infected with recombinant vaccinia viruses expressing FIV GAG or ENV proteins. Unstimulated lymphocytes were also used as effectors in some assays. 51Cr-labelled autologous skin fibroblasts infected with recombinant vaccinia viruses were used as targets. FIV GAG-specific cytotoxic precursors were detected in restimulated circulating lymphocytes during acute infection in both cats. The onset of this activity was as early as 2 weeks postinfection (p.i.) in one cat. From 62 weeks p.i. neither FIV GAG- nor ENV-specific precursors could be detected in the peripheral blood. However, at 127 weeks p.i., GAG- and ENV-specific cytotoxic precursors were detected in lymphocytes isolated from lymph nodes. The FIV-specific cytotoxic cells were predominantly major histocompatibility complex class I restricted. No cytotoxic activity was detected from unstimulated lymphocytes. These studies demonstrate the use of an assay system for dissecting the FIV-specific cytotoxic cell response and show that precursor cells appear in the circulation very early after infection and prior to a detectable antibody response. Our results also suggest that the persistent high-level circulating antiviral cytotoxic T-lymphocyte responses seen in human immunodeficiency virus-infected humans may not be a feature of FIV infections in cats.

Generation of monoclonal antibodies to the anti-trypanosomal drug isometamidium.

Mice were immunized with either an isometamidium-human serum albumin (HSA) conjugate or an isometamidium-porcine thyroglobulin conjugate (PTG). Thereafter, monoclonal antibodies (MAbs) IL-A 1001, IL-A 1002, IL-A 1003, 5F7.B7, and 5F7.C9 were generated and selected on the basis that they recognized conjugated and unconjugated isometamidium, but lacked cross-reactivity with the carrier molecules. All five MAbs were of the IgG1 isotype. Each of the five MAbs was assessed in a competitive ELISA for isometamidium; in each case, the minimum level of detection was approximately 10 ng/ml. Each MAb exhibited approximately 0.1% cross-reactivity with the anti-trypanosomal compound diminazene. However, based on their cross-reactivity with the anti-trypanosomal compound homidium, the MAbs could be divided into two groups; IL-A 1001, IL-A 1002, and IL-A 1003, produced using an isometamidium-HSA conjugate as an immunogen, exhibited low levels of cross-reactivity (approximately 0.1%). In contrast, 5F7.B7 and 5F7.C9, produced using an isometamidium-PTG conjugate as an immunogen, exhibited high levels of cross-reactivity.

Isometamidium concentrations in the sera of Boran cattle: correlation with prophylaxis against tsetse-transmitted Trypanosoma congolense.

Fifteen Boran cattle from a trypanosomiasis-free area were injected intramuscularly with isometamidium chloride at a dose of 1 mg/kg body weight. Thereafter, the cattle were challenged at monthly intervals with Glossina morsitans centralis infected with one of three populations of Trypanosoma congolense (IL 3893, IL 3889 or IL 1180) until all animals became infected. Isometamidium concentrations in the sera of these cattle were measured using a competitive enzyme-linked immunosorbent assay over the first 105 days following treatment. All cattle challenged with IL 3893 or IL 3889 developed infection following the first challenge, at which time the mean serum drug concentration in all treated cattle was 6 ng/ml. Cattle challenged with IL 1180 became infected following 6 to 8 monthly challenges. The mean serum drug concentration in these cattle at the time of their third monthly challenge with IL 1180 was 0.75 ng/ml. Trypanosome populations IL 3893 and IL 3889 were considered to be highly resistant to isometamidium, while IL 1180, relatively sensitive. It was therefore concluded that T. congolense persisting at serum isometamidium concentrations greater than 0.75 ng/ml can be considered moderately resistant, while those persisting at concentrations greater than 6 ng/ml can be considered markedly resistant. These results will be most valuable in the investigation of isometamidium resistance of T. congolense in the field.

Evaluation and improvement of an enzyme-linked immunosorbent assay for the detection of isometamidium in bovine serum.

The control of bovine trypanosomiasis in Africa continues to rely heavily on the chemoprophylactic drug isometamidium (ISMM) chloride. However, despite many years of use, no methods are available that are sufficiently sensitive to measure drug levels in treated cattle. An enzyme-linked immunosorbent assay (ELISA) for the detection of ISMM in the serum of treated cattle has been developed and evaluated. Liquid-phase ISMM (sample) competes with solid-phase bound ISMM-protein conjugate for biotinylated sheep anti-ISMM IgG. The specific IgG is detected by streptavidin-peroxidase, using tetramethylbenzidine for colour development. Assay calibration is by four-parameter logistic curve-fitting. Factors contributing to absorbance variance were considered in assay optimization and improvement of precision and the lower limit of detection (approximately 0.1 ng/ml in serum). The ELISA was shown to detect serum ISMM for several months after treatment of cattle in a trypanosomiasis endemic country. The potential uses of this assay include the development of rational prophylactic drug regimens, and the indirect detection of drug-resistant trypanosomes.