Mucin is a glycoprotein found on the surface of cell membranes of adenocarcinomas. The purpose of these studies was to generate MUC1 multiple tandem repeat (VNTR)‐stimulated mononuclear cells (M1SMC). We first determined the optimal conditions to influence the immune response. In these studies, peripheral blood mononuclear cells (PBMC), from patients with adenocarcinomas, were stimulated by different numbers of M1SMC stimulations, various concentrations of MUC1 peptide, washing of PBMC prior to stimulation and days in culture, to determine the optimal conditions to influence the immune response. The results of this study indicate that the mononuclear cells (MC) stimulated twice 1 week apart with MUC1 VNTR1 produced a greater specific killing of the breast cancer cell line MCF‐7 than the 0, 1, 3 or 4 weekly stimulations. The optimal molarity for inducing cytotoxicity and cytokines (granulocyte macrophage colony‐stimulating factor, gamma‐interferon and interleukin‐10) was 45 × 10−8 m (1 μg/ml); except for tumour necrosis factor (TNF)‐alpha which was 22 × 10−8 m (0.5 μg/ml). The unwashed MC were superior to washing them with Ficoll–Hypaque. The optimal number of days in culture for cytotoxicity and cytokine production was after two stimulations (i.e. after day 7). Optimum conditions for generation of M1SMC identified in these studies were two stimulations with peptide, concentration of 45 × 10−8 m (1 μg/ml) peptide, unwashed cells, and after two stimulations or after 8 days in culture. M1SMC were generated from multiple patients with breast cancer which lysed adenocarcinoma cells.
Significance and Impact of the Study: Nanoparticle-assisted polymerase chain reaction (nanoPCR) assay is an improved PCR. NanoPCR is highly sensitive and specific because the nanofluids formed in the nanobuffer have high thermal conductivity, which reduces the time required to reach the target temperature. It is more sensitive than conventional PCR, and it could detect the cases earlier than conventional PCR. This report describes the first application of the highly efficient nanoPCR technology for the detection of porcine parvovirus (PPV). The PPV nanoPCR assay will be useful for the detection and study of PPV and will also be applicable to improve the detection of other viruses. AbstractA novel nanoparticle-assisted polymerase chain reaction (nanoPCR) assay to detect porcine parvovirus (PPV) is described here. Primers for this assay were designed based on the conserved region of the nonstructural protein 1 (NS1) gene of PPV, which encodes one of the nonstructural proteins. The sensitivity of the PPV nanoPCR assay was measured by using diluted recombinant plasmids in which the PPV NS1 gene had been inserted. The detection limit was 5Á6 9 10 1 copies ll À1 for the PPV nanoPCR assay vs 5Á6 9 10 3 copies ll À1 for conventional PCR assay. The results showed that the sensitivity of PPV nanoPCR assay was 100 times higher than that of conventional PCR assay. The PPV nanoPCR assay produced 142-bp product as expected when amplifying PPV DNA, while produced nothing when amplifying the DNA or cDNA of the following viruses: swine encephalomyocarditis virus, classical swine fever virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine teschovirus and porcine circovirus type II. PPV was detected in 108 of 109 clinical swine samples from Heilongjiang, Jilin and Henan provinces using the nanoPCR assay, and the results were confirmed by sequencing.
TIM‐3 is a member of the TIM (T‐cell immunoglobulin domain and mucin domain) family, which plays an important role in TH1 responses and autoimmune diseases. In this study, we cloned and characterized the porcine TIM‐3 gene. Real‐time PCR showed little expression of porcine TIM‐3 in muscle and stomach, low expression in kidney, brain, stomach and muscle, moderate expression in liver, small intestine and lymph, and high expression in spleen and lung. Transient transfection indicated that porcine TIM‐3 fusion protein was found to localize on the cell membranes or cytoplasm. Association analysis indicated that the SNP AccI in exon2 was significantly associated (P < 0.05) with red blood cell count mean corpuscular haemoglobin, packed cell volume, Lymphocyte percentage and Lymphocyte modulus. In conclusion, our results provide some information for conducting further studies on the functions of porcine TIM‐3 gene in type I diabetes and suggest that SNP AccI in exon2 may be utilized as a marker for molecular‐assisted selection in animal breeding.
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