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Submitted to: Journal of Immunological Methods
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 4/25/2000 Publication Date: N/A Citation: N/A Interpretive Summary: The isolation of lymphocytes from young pigs is very critical for the understanding of the immune response of pigs at an early age. The characterization of porcine lymphocytes from mucosal tissues has been limited because of the difficulties in the processing and isolation of purified populations. The objective of this study was to standardize and describe a detailed protocol for the isolation of lymphocytes from the peripheral blood (PBMC), mesenteric lymph nodes (MLN), Peyer patches of jejunum (jejPP) and Ileum (ilPP), and the Intraepithelial (IE)and Lamina Propria (LP) compartments of the jejunum and ileum. The yield of lymphocytes from the IE and LP intestinal compartments was limited in very young animals (less than two weeks of age) due to the low numbers that are naturally found in these areas. Additionally, the presence of milk protein accumulation did limit the isolation procedure in the first week of age. Critical steps for the isolation of pure populations of cells were identified. The results of this study is an important reference for those scientific groups that use the pig as a model for the understanding of immune responses at the mucosal level. Knowledge of the intestinal immune system will help veterinarians to design new treatment strategies for disease control. Technical Abstract: The characterization of lymphoid subsets isolated from different anatomical sites is of great importance for understanding the mechanisms and interactions of normal and pathological immune reactions in the pig. The objective of this study was to standardize a protocol for the isolation of lymphocytes from mucosal tissues of neonatal pigs. Outbred pigs of ages between one to forty-five days from a high health commercial farm and thirteen miniature swine from an experimental farm were used as donors for the tissues examined. Specific protocols for the isolation of lymphocytes from the peripheral blood (PBMC), mesenteric lymph nodes (MLN), Peyer Patches of jejunum (jejPP) and ileum (ilPP), the Intraepithelial (IE) and Lamina Propria (LP) compartments of the jejunum and ileum are described in detail. The analysis of the cells isolated indicated a high viability (>90%). The histological sections from fragments collected from the intestine demonstrated that in nursing young pigs, the recovery of IE and LP lymphocytes may be limited because of the low numbers of cells present in early age. The presence of large intracytoplasmatic vacuoles and hyaline droplets between the columnar epithelial cells during the first week of age interferes with the isolation of pure lymphocytes from the IE and LP compartments. Optimal lymphocyte yields for all the samples analyzed was confirmed by immunostaining with the pan lymphocyte marker against CD45. The successful isolation and comparison of large numbers of pure populations from compartmentalized areas of the intestine and associated lymphoid tissues opens up a broad area for the investigation of immune responses of pigs at the mucosal level. |
