PMID- 9802577 OWN - NLM STAT- MEDLINE DCOM- 19990112 LR - 20190831 IS - 0165-2427 (Print) IS - 0165-2427 (Linking) VI - 65 IP - 1 DP - 1998 Sep 16 TI - Construction of internal cDNA competitors for measuring IL-10 and IL-12 cytokine gene expression in swine. PG - 63-74 AB - A competitive PCR assay (cPCR) was used to quantify swine cytokine responses to parasite infection. Internal standards (deleted cDNA competitor molecules [DcDNA mimics]) were produced and tested for swine interleukin-12 (IL-12), interleukin-10 (IL-10) and hypoxanthine phosphoribosyltransferase (HPRT) from PCR generated cDNA cloned in plasmid vectors. Deletion clones for the cDNA competitor molecules (DcDNA mimics) were generated for IL-10, IL-12 and HPRT by PCR in a single step and verified by (1) amplification of the expected smaller PCR product with the original primers, (2) appropriate fragment size released by restriction digestion of the deleted clone, and (3) correct sequence of the new DcDNA insert. DcDNA mimics were used to quantitate cytokine gene mRNA production during experimental and natural infections of swine with the gastrointestinal nematode parasite Trichuris suis. Mesenteric lymph node cells were collected from control and infected pigs at the time of maximal pathogenicity (35 days after infection) and snap frozen. After RNA extraction, samples were reverse transcribed (RT) to cDNA. cPCR was performed using the housekeeping gene HPRT DcDNA mimic and HPRT specific primers to insure RNA integrity and concentration. Cytokine cDNA content in these samples was then quantitated using cytokine mimics and gene specific primers. IL-10 gene expression in MLN draining the colon of pigs experimentally infected with T. suis increased 10-20 fold at day 35 compared to control pigs. IL-12 gene expression was not detectable in MLN of these pigs, but was detectable in MLN of pigs exposed naturally to T. suis on a contaminated dirt lot that also exhibited signs of secondary bacterial invasion. Swine IL-10 and IL-12 gene expression can be quantitated in local mesenteric tissues. This cPCR assay will enable scientists to quantitate cytokine gene expression in swine and determine the nature of immune responses to important infectious diseases. FAU - Mansfield, L S AU - Mansfield LS AD - College of Veterinary Medicine, Michigan State University, East Lansing 48824, USA. mansfield@ahdlms.cvm.msu.edu FAU - Urban, J F AU - Urban JF FAU - Holley-Shanks, R R AU - Holley-Shanks RR FAU - Murtaugh, M P AU - Murtaugh MP FAU - Zarlenga, D S AU - Zarlenga DS FAU - Foss, D AU - Foss D FAU - Canals, A AU - Canals A FAU - Gause, W AU - Gause W FAU - Lunney, J K AU - Lunney JK LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. PL - Netherlands TA - Vet Immunol Immunopathol JT - Veterinary immunology and immunopathology JID - 8002006 RN - 0 (DNA Primers) RN - 0 (DNA, Complementary) RN - 0 (RNA, Messenger) RN - 130068-27-8 (Interleukin-10) RN - 187348-17-0 (Interleukin-12) RN - EC 2.4.2.8 (Hypoxanthine Phosphoribosyltransferase) SB - IM MH - Animals MH - Cloning, Molecular MH - Colon/immunology/parasitology MH - DNA Primers/chemistry MH - DNA, Complementary/*chemistry/genetics MH - Electrophoresis, Agar Gel/veterinary MH - Gene Expression Regulation MH - Hypoxanthine Phosphoribosyltransferase/chemistry/genetics MH - Interleukin-10/analysis/biosynthesis/*genetics MH - Interleukin-12/analysis/biosynthesis/*genetics MH - Lymph Nodes/immunology/parasitology MH - RNA, Messenger/chemistry MH - Reverse Transcriptase Polymerase Chain Reaction/methods/veterinary MH - Swine/genetics/*immunology/parasitology MH - Swine Diseases/immunology MH - Trichuriasis/immunology/veterinary MH - Trichuris/immunology EDAT- 1998/11/05 00:00 MHDA- 1998/11/05 00:01 CRDT- 1998/11/05 00:00 PHST- 1998/11/05 00:00 [pubmed] PHST- 1998/11/05 00:01 [medline] PHST- 1998/11/05 00:00 [entrez] AID - S0165-2427(98)00106-8 [pii] AID - 10.1016/s0165-2427(98)00106-8 [doi] PST - ppublish SO - Vet Immunol Immunopathol. 1998 Sep 16;65(1):63-74. doi: 10.1016/s0165-2427(98)00106-8.