Il-Seob Shin

Hormonal change and cytokine mRNA expression in peripheral blood mononuclear cells during the development of canine autoimmune thyroiditis.

To elucidate the hormonal change and alteration in cytokine expression in peripheral blood mononuclear cells (PBMC) during the early stage of autoimmune thyroiditis, we have developed a canine model of this disease, in which normal dogs were immunized with bovine thyroglobulin (Tg) and/or canine thyroid extract. Serum samples were collected weekly, anti‐canine Tg antibody was measured by enzyme‐linked immunosorbent assay (ELISA) and thyroid stimulating hormone (TSH) and total T4 levels by radioimmunoassay. We also assayed T lymphocyte proliferation in response to Tg, as well as measuring cytokine mRNA by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR). All six dogs immunized with bovine Tg had both canine Tg autoantibody and anti‐T4 antibody. When the sample from the highest TgAA titre time‐point was compared with baseline the expression of mRNA encoding the Th1‐type cytokine such as interferon (IFN)‐γ, interleukin (IL)‐18 and IL‐15 was increased during the development of autoimmune thyroiditis. Expression of the Th2‐type cytokine, IL‐6 showed minimal change and IL‐4 expression was not detected in any of the samples. Expression of the T suppressive cytokine, IL‐10 and transforming growth factor (TGF)‐β was increased in the presence of antigen stimulation. These findings suggest that, although autoimmune thyroiditis is an organ‐specific autoimmune disease, systemic cytokine mRNA expression is also changed.

In vitro and in vivo gene therapy with CMV vector-mediated presumed dog β-nerve growth factor in pyridoxine-induced neuropathy dogs

Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog β-nerve growth factor (pdβ-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdβ-NGF protein reacted with a human β-NGF antibody and showed bioactivity in PC12 cells. The pdβ-NGF was shown to have similar bioactivity to the dog β-NGF. The recombinant pdβ-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L4 were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L4 and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.