Mechanism Of Transfer Factor As Enhancer For Canine Cellular Mediated Immunity

Transfer factor (TF) is produced by T lymphocytes, with a small molecular weight and chemically being complex of small peptide and oligonucleotide. TF can transfer donor's cell mediated immunity to receptor's cell mediated immunity. Particularly unspecific TF can enhance receptor's natural immunity and specific immunity, which makes TF being applied at large in treatment and prevention of animal and human diseases. So clarification of cellular and molecular mechanism of TF as enhancer for canine immune function will definitely contribute abundant theoretic evidences and instructions to control of canine diseases, especially canine infectious diseases. In this present study, the requisite swine TF and canine TF were prepared firstly. Afterwards, a range of hybrid shepherd dogs were selected to determine the canine lymphocytic proliferation effects in different time after the dogs were injected with swine TF and canine TF, respectively, the changes of subgroup T lymphocytes, the secretion of several important cytokines such as IL-2, IL-12, IL4 and IFN-γ, as well as the changes of neutrophilic granulocyte in canine peripheral blood of swine TF and canine TF, by the methods of lymphocytic transfer proliferation MTT, flow cytometry, ELISA and phagocytosis MTT. Then the therapeutic effects of combination formula containing swine TF against artificial and natural infected canine parvoviral disease were assessed. The results were as follows: 1. The prepared swine TF and canine TF showed resemble physical and chemical properties, and the trial products of TF met the same level as in the world and can be materials of this study. 2. In the lymphocytic transfer test, PHA-P at dosage of 12.5μg/mL performed facilitation effects for canine lymphocytic proliferation (p>0.05). ConA at 0.5～2.5μg/mL markedly proliferated T lymphocytes (p<0.05), with the best effect at 2.5μg/mL. LPS at 40μg/mL can significantly stimulate the proliferation of B lymphocytes. Homogeneous or heterogeneous TF can separately stimulate the proliferation of canine lymphocytes, but the proliferation effects of homogeneous TF were better than those of heterogeneous TF. When being combined with PHA-P, homogeneous TF can significantly proliferate canine lymphocytes in a larger dose range(0.097～12.5mg/mL), whereas heterogeneous TF only in a very limited dose range(0.097～3.125mg/mL)can significantly show the proliferation effects; But when being combined with ConA, homogeneous TF also in a very limited dose range(0.097～0.78mg/mL)can significantly show the proliferation effects, whereas heterogeneous TF only in a limited dose range 0.78～3.125mg/mL can significantly show the proliferation effects. Within 6～10 days after heterogeneous TF was injected, the proliferation ability of canine lymphocytes became stronger and stronger. Also within the 6～10 days, in the dogs injected with heterogeneous TF, the proliferation of lymphocytes, once again stimulated by TF itself or by TF and mitogen, showed a downtrend in the first 4～6 days and then was inhibited after 6 days. 3. With regard to enhancement of the number of lymphocyte in canine peripheral blood of homogeneous TF or heterogeneous TF, the increased were mainly CD4+ T-cells, according to the analysis of the ratio of CD4/CD8. CD4+ T-cells had increased for 20 days since the employed dogs were injected with homogeneous TF or heterogeneous TF, respectively. And the enhancive effect of homogeneous TF was better than that of heterogeneous TF. 4. Injecting dogs with homogeneous TF or heterogeneous TF separately can make the lymphocytes secret more IFN-γ. On the 10th day after the dogs were injected with canine TF, the canine lymphocytes showed the strongest ability of secreting IFN-γ; and on the 3rd day after the dogs were injected with swine TF, the canine lymphocytes showed the strongest ability of secreting IFN-γ. So it is very good that the homogeneous TF or heterogeneous TF stimulated canine lymphocytes to secret IFN-γin aspect of time. And the ability of secreting IL-2 of canine lymphocytes can be enhanced by injecting the dogs with homogeneous TF or heterogeneous TF, respectively. Injecting the dogs with TF can enhance lymphocytes to secret IL-12, with a "high-low-high"model, and there was no difference between homogeneous TF and heterogeneous TF. Either homogeneous TF or heterogeneous TF can strongly inhibit secreting of IL-4. 5. The better condition for MTT to be applied into determining the ability of phagocytosis of neutrophilic granulocyte against Escherichia coli in canine peripheral blood were explored. It can sensitively reflect the effects of phagocytosis of neutrophilic granulocyte against E. coli that 1.3×106 cells /mL neutrophilic granulocytes and 6×105 E. colies /mL had been mixed and incubated together for 2 hours followed by adding MTT and incubating for another 4 hours. The experiments in vitro showed that swine TF at 0.052～1.56 mg/mL markedly promoted the phagocytosis of neutrophilic granulocyte against E. coli, with the strongest effect to the phagocytosis of neutrophilic granulocyte against E. coli at 1.56mg/mL. The experiments in vivo indicated that the number of neutrophilic granulocytes in canine peripheral blood climbed to the summit and the effect of phagocytosis of neutrophilic granulocyte against E. coli was the strongest on the 2nd day after the dogs wereinjected with swine TF. 6. Combined with antibiotic etc, swine TF was used to treat artificial canine parvoviral disease and the recovery rate was 55.6% and apparent efficient rate was 77.8%, which was by far higher than that of serum treatment group in which the recovery rate was 25.0% and apparent efficient rate was 62.5% and that of medicinal treatment group in which the recovery rate was zero and apparent efficient rate was 37.5%. Combined with antibiotic etc, swine TF was used to treat natural canine parvoviral disease and the recovery rate was 56.3% and apparent efficient rate was 81.3%, which was by far higher than that of serum treatment group in which the recovery rate was 37.5% and apparent efficient rate was 68.8% and that of medicinal treatment group in which the recovery rate was 12.5% and apparent efficient rate was 56.3%. It was also found that the treatment time of the combination of swine TF and antibiotics etc was shorter than that of other groups. So it can be concluded here that either homogenous or heterogeneous TF can markedly enhance canine cellular mediated immunity, which put forward certain powerful experimental evidences and instructions for control of canine diseases especially canine infectious diseases and for enhancement of immune effects of canine vaccines.