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D Sargan

Publications and source records attributed to D Sargan.

14 recordsLinked to original sources
PubMed2006

Understanding the genetics of autoimmune disease: two loci that regulate late onset Addison's disease in Portuguese Water Dogs.

Addison's disease, an immune-mediated disorder caused by destruction of the adrenal glands, is a rare disorder of Western European populations. Studies indicate that the disorder is polygenic in nature, involving specific alleles of the CTLA-4, DRB1*04 and DQ, Cyp27B1, VDR and MIC-A and -B loci. A similar immune form of Addison's disease occurs in several breeds of domestic dog, with frequencies ranging from 1.5 to 9.0%. The high frequency of the disease in domestic dog breeds likely reflects the small number of founders associated with many breeds, subsequent inbreeding, and the frequent use of popular sires. The Portuguese Water Dog (PWD) is a significantly affected breed. An analysis of 11,384 PWDs surveyed between 1985 and 1996 suggests a breed-specific disease incidence of 1.5%. As with humans, the disease is typically of late onset. This study involves a genetic comparison of Addison's disease in the PWD to the analogous disease in humans. The study is facilitated by the existence of complete pedigrees and a relatively high degree of inbreeding among PWDs. The breed originated from 31 founders, with 10 animals responsible for 90% of the current gene pool. We describe, specifically, the identification of two disease-associated loci, on Canis familiaris (CFA) chromosomes CFA12 and 37, which are syntenic with the human DRB1 histocompatibility locus alleles HLA-DRB1*04 and DRB1*0301, and to a locus for immunosuppression syntenic with CTLA-4. Strong similarities exist therefore in the complex genetic background of Addison's disease in humans and in the PWD. With the completion of the canine and human genome sequence, the purebred dog is set to become an important comparative model for Addison's as well as other human immune disorders.

Addison Disease↗
PubMed2003

Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina.

Recombinant adeno-associated virus (rAAV) vectors are attractive candidates for the treatment of inherited and acquired retinal disease. Although rAAV vectors are well characterized in rodent models, a prerequisite to their clinical application in human patients is the thorough evaluation of their efficacy and safety in intermediate animal models. In this study, we describe rAAV-2-mediated expression of GFP reporter gene in retinal cells following local vector delivery in dogs. Subretinal delivery of rAAV.CMV.GFP was performed unilaterally in eight normal dogs from 6 weeks of age. The area of retinal transduction was maximized by the optimization of surgical techniques for subretinal vector delivery by pars-plana vitrectomy and the use of fine-gauge subretinal cannulae to create multiple retinotomies. rAAV-2 vectors mediated efficient stable reporter gene expression in photoreceptors and retinal pigment epithelial cells. We found efficient transduction of cone photoreceptors in addition to rods in both the canine retina and after subretinal vector delivery in another intermediate animal model, the feline retina. GFP expression in dogs was confined to the area of the retinal bleb and was sustained in cells at this site for at least 18 months. Electroretinography demonstrated a modest reduction in global rod-mediated retinal function following subretinal delivery of rAAV.CMV.GFP. Three of the eight animals developed delayed-onset intraocular inflammation, in two cases associated with a serum antibody response to GFP protein. We conclude that rAAV-2 vectors mediate efficient sustained transgene expression in rod and cone photoreceptors following subretinal delivery in this intermediate animal model. The possibility of adverse effects including intraocular immune responses and reduced retinal function requires further investigation prior to clinical applications in patients.

Animals↗
PubMed1999

A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps.

Cross-species reciprocal chromosome painting was used to delineate homologous chromosomal segments between domestic dog, red fox, and human. Whole sets of chromosome-specific painting probes for the red fox and dog were made by PCR amplification of flow-sorted chromosomes from established cell cultures. Based on their hybridization patterns, a complete comparative chromosome map of the three species has been built. Thirty-nine of the 44 synteny groups from the published radiation hybrid map and 33 of the 40 linkage groups in the linkage map of the dog have been assigned to specific chromosomes by fluorescence in situ hybridization and PCR-based genotyping. Each canine chromosome has at least one DNA marker assigned to it. The human-canid map shows that the canid karyotypes are among the most extensively rearranged karyotypes in mammals. Twenty-two human autosomal paints delineated 73 homologous regions on 38 canine autosomes, while paints from 38 dog autosomes detected 90 homologous segments in the human genome. Of the 22 human autosomes, only the syntenies of three chromosomes (14, 20, and 21) have been maintained intact in the canid genome. The dog-fox map and DAPI banding comparison demonstrate that the remarkable karyotype differences between fox (2n = 34 + 0-8 Bs) and dog (2n = 78) are due to 26 chromosomal fusion events and 4 fission events. It is proposed that the more easily karyotyped fox chromosomes can be used as a common reference and control system for future gene mapping in the DogMap project and CGH analysis of canine tumor DNA.

Animals↗
PubMed1996

Rod opsin cDNAs.

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Animals↗
PubMed1995

Molecular cloning and expression of DNA encoding ovine interleukin 2.

We have generated DNA encoding the mature form of ovine interleukin 2 (IL-2) by polymerase chain reaction (PCR) using primers complementary to sequences at the 5' and 3' ends of human, murine and bovine IL-2 cDNA. The predicted PCR product of 400 bp was ligated into the yeast Ty-P1 galactose-inducible expression vector pOGS40 which was used to transform yeast spheroplasts. The fusion protein, with a Factor Xa proteolytic cleavage site between ovine IL-2 and the P1 fusion partner, was expressed from galactose-induced transformed yeast. P1:IL-2 fusion protein, which self-assembles into virus-like particles (VLPs) due to the interaction of the P1 protein, was purified from lysates of mechanically disrupted yeast by centrifugation on a discontinuous sucrose gradient. Fusion protein was detected in Western blot analysis with polyclonal antisera raised to recombinant bovine IL-2. Soluble recombinant ovine IL-2 was released from the P1 fusion protein by cleavage with Factor Xa enzyme. After purification recombinant ovine IL-2 was functionally active as shown by its ability to support the proliferation of Con A-activated T cells and was capable of generating maedi visna virus-specific cytotoxic T cells from primed precursor cells. The availability of recombinant ovine IL-2 will greatly help the analysis of the specificity of pathogen-specific cells in the sheep.

Amino Acid Sequence↗
PubMed1995

The survival and growth of ovine afferent lymph dendritic cells in culture depends on tumour necrosis factor-alpha and is enhanced by granulocyte-macrophage colony-stimulating factor but inhibited by interferon-gamma.

An in vitro culture system is described which allows an analysis of the signals responsible for the survival, growth and functional maturation of afferent lymph dendritic cells (ALDC), a subpopulation of migrating dermal dendritic cells involved in antigen carriage and presentation to T-cells. Purified ALDC survived and grew for up to 30 days in lymph node conditioned medium and survived 14 days in recombinant ovine (rov) TNF-alpha whereas none were detected after 24 h in rov GM-CSF, rov IFN-gamma or rh M-CSF. However, when rov GM-CSF was added to cultures along with rov TNF-alpha, increased numbers of ALDC compared with input numbers (growth) were recorded on Days 14 and 21. In contrast, when 50-200 units ml-1 of rov IFN-gamma were added to cultures of ALDC along with TNF-alpha or rov TNF-alpha plus rov GM-CSF, cell survival and growth was inhibited. Antibody blocking studies confirmed the cytokine specificity of these effects. ALDC cultured in rov TNF-alpha or rov TNF-alpha plus rov GM-CSF retained MHC Class-II and ov CD-1 antigen expression and accessory function for autologous ov CD-4 T-cell proliferation, although at reduced levels compared with freshly isolated cells. Neither fresh nor cultured ALDC expressed coagulation factor XIIIa.

Animals↗
PubMed1995

The restricted IgG1 antibody response to maedi visna virus is seen following infection but not following immunization with recombinant gag protein.

Maedi-visna (MVV) is a retrovirus of the subfamily lentivirinae which includes HIV, simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Infection of its natural host, the sheep, does not cause overt immunodeficiency, but rather a chronic inflammatory disease. However, subtle immunological changes following infection have been reported including a sheep IgG1 subclass-restricted MVV-neutralizing antibody. Here we demonstrate by Western blotting that there is no IgG2 serum antibody response to any MVV antigen after MVV infection, in contrast to infection with the parapox virus Orf, when serum IgG2 anti-Orf antibody is readily detected. By ELISA, the IgG1 antibody titres to Orf are higher than to MVV, but the minimum MVV serum antibody IgG1/IgG2 ratio is significantly raised compared with that for Orf virus antibody in the same sheep, indicating that the IgG2 defect in MVV infection cannot be accounted for by differences in the sensitivity of the Orf and MVV ELISA. Serum IgG2 anti-MVV gag p. 25 can be detected in both normal and MVV-infected sheep following immunization with purified recombinant MVV gag p 25 protein in Freund's complete adjuvant. The failure to make an IgG2 MVV-specific antibody indicates that immunological dysfunction can arise with macrophage tropic lentiviruses, and it may aid viral persistence.

Animals↗
PubMed1993

Early events in immune evasion by the lentivirus maedi-visna occurring within infected lymphoid tissue.

Infections caused by lentiviruses, including human immunodeficiency virus, are characterized by slowly progressive disease in the presence of a virus-specific immune response. The earliest events in the virus-host interaction are likely to be important in determining disease establishment and progression, and the kinetics of these early events following lentiviral infection are described here. Lymphatic cannulation in the sheep has been used to monitor both the virus and the immune response in efferent lymph after infection of the node with maedi-visna virus (MVV). Viral replication and dissemination could be detected and consisted of a wave of MVV-infected cells leaving the node around 9 to 18 days postinfection. No cell-free virus was recovered despite the fact that soluble MVV p25 was detected in lymph plasma. The maximum frequency of MVV-infected cells was only 11 in 10(6) but over the first 20 days of infection amounted to greater than 10(4) virus-infected cells leaving the node. There was a profound increase in the output of activated lymphoblast from the lymph nodes of infected sheep, characterized by an increased percentage of CD8+ lymphoblasts. All of the CD8+ lymphoblasts at the peak of the response expressed both major histocompatibility complex class II DR and DQ molecules but not interleukin-2 receptor (CD25). The in vitro proliferative response of efferent lymph cells existing the node after challenge with MVV to both recombinant human interleukin-2 and the mitogen concanavalin A was decreased between days 8 and 16 postinfection, and a specific proliferative response to MVV was not detected until after day 15. Despite the high level of CD8+ lymphoblasts in efferent lymph, direct MVV-specific cytotoxic activity was demonstrated in only one of the five MVV-challenged sheep. MVV-specific antibody responses, including neutralization and MVV p25 immune complexes in efferent lymph, were detectable during the major period of virus dissemination. The relationship of these findings to the evasion of the host's acute immune response by MVV is discussed.

Animals↗
PubMed1992

Clinicopathological investigation of primary, uncomplicated maedi-visna virus infection.

Maedi-visna virus infection in a flock of sheep in Scotland was associated with respiratory disease, neurological disease, mastitis and lameness. The major clinical signs were dyspnoea (particularly on exercise), progressive fore- and hindlimb ataxia and balance defects, mammary induration and multilimb lameness, occasionally with enlarged carpal joints. Pathological examinations revealed lesions in the lungs, central nervous system, mammary glands and joints which were consistent with those induced by maedi-visna virus. The was no clinical or pathological evidence of concurrent sheep pulmonary adenomatosis, and pulmonary bacterial infections, when they occurred, were superimposed on the lesions due to maedi-visna virus.

Animals↗
PubMed1992

Cloning of a cDNA encoding the ovine interleukin-2 receptor 55-kDa protein, CD25.

A 1.3-kb cDNA that encodes the entire 825-bp coding region of ovine CD25, the interleukin-2 receptor 55-kDa protein, has been isolated. Comparison of the deduced amino acid sequence with CD25 proteins from other species shows the ovine sequence to have the greatest homology with that of the bovine species.

Amino Acid Sequence↗
PubMed1992

Phenotypic analysis of lymphocyte populations in the lungs and regional lymphoid tissue of sheep naturally infected with maedi visna virus.

We have analysed the phenotype of lymphocytes in lung and regional lymph node of symptomatic and asymptomatic sheep infected with the ovine lentivirus, maedi visna virus (MVV). Compared to equivalent tissues from age-matched, non-infected controls, MVV-infected sheep show increased numbers of lymphocytes in the lung, both in the bronchus-associated lymphoid tissue (BALT) and in the alveolar septae. Both CD8+ and CD4+ T lymphocyte numbers in alveolar septae were increased, particularly in animals with clinical respiratory disease. The ratio of CD8+ to CD4+ lymphocytes was similar to that in normal lung. In both MVV-infected and uninfected animals a high proportion of pulmonary lymphocytes, particularly in the alveolar septae, did not express the CD5 antigen, suggesting that they were activated. The number of activated cells was higher in infected sheep. Variable numbers of alveolar macrophages containing MVV-core protein were present in alveolar lumina, the majority of positive cells showing morphological evidence of activation. In regional lymphoid tissue there were increased numbers of CD8+ and gamma delta expressing T cells in lymphoid follicles and germinal centres of infected animals. The specificity of these cells is unknown and we could find no evidence for the presence of cells productively infected with the virus in these structures. This study shows that activated T lymphocytes, particularly of the CD8 subset, play a major part in the pathogenesis of MVV-induced pulmonary and regional lymph node lesions.

Animals↗