Introduction: The effects of Jin-Ying-Tang (JYT) on Toll-like Receptor 4 (TLR4) signalling transduction of lipopolysaccharide (LPS)-stimulated mouse mammary epithelial cells (MECs) in vitro were examined. Material and Methods: The cytotoxicity of JYT (0.06-62.50 mg/mL) on mouse MECs was determined by MTT assay. The MECs were co-cultured with LPS in the presence or absence of JYT (39.10 μg/mL, 391 μg/mL, 3910 μg/mL). The concentrations of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in the culture supernatants were detected by ELISA. The mRNA expression of TLR4 and downstream TLR4 signalling molecules such as myeloid differentiation factor 88 (MyD88), tumour necrosis factor receptor associated factor 6 (TRAF-6), inhibitor κB (IκB), and nuclear factor κB inducing kinase (NIK) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The results showed that the IC₅₀ of JYT on MECs was 12.25 mg/mL and JYT could significantly decrease the concentrations of IL-6 and TNF-α in LPS-stimulated MECs (P < 0.05). The mRNA expression of TLR4, MyD88, TRAF-6, IκB, and NIK was also significantly decreased when the LPS-stimulated MECs were cocultured at appropriate concentrations of JYT (P < 0.05, P < 0.01). Conclusion: These observations indicate a potential mechanism through which JYT attenuates the systemic inflammatory response to LPS-stimulated mouse mammary epithelial cells by inhibiting the activation of TLR4/MyD88/ TRAF-6/NIK pathway at the mRNA level.

Introduction: The cytotoxicity of anthelmintic agent, albendazole (ABZ) and its two major metabolites, sulfoxide (ABZSO) and sulfone (ABZ-SO₂), on non-hepatic Balb/c 3T3 line, two hepatoma cell lines (FaO, HepG2), and isolated rat hepatocytes was investigated. Material and Methods: Cell cultures were exposed for 24, 48, and 72 h to eight concentrations of the compounds ranging from 0.05 to 100 μg/mL (ABZ) and from 0.78 to 100 μg/mL (ABZ-SO and ABZ-SO₂). Three different assays were applied in which various biochemical endpoints were assessed: lysosomal activity - neutral red uptake (NRU) assay, proliferation - total protein contents (TPC) assay and lactate dehydrogenase (LDH) leakage assay. Results: The most toxic was albendazole whose EC₅₀ values calculated from the concentration effect curves ranged from 0.2 to 0.5 μg/mL (Balb/c 3T3) and from 0.4 to 73.3 μg/mL (HepG2). Rat hepatoma line and isolated rat hepatocytes were less sensitive to the impact of ABZ. Toxic action expressed as EC₅₀ was recorded after 72 h exposure only in LDH release assay at 0.8 μg/mL and 9.7 μg/mL respectively. The toxicity of metabolites was much lower. The most sensitive to ABZ-SO were fibroblasts and EC₅₀₋₇₂ₕ values were similar in all three assays used, i.e. NRU (14.1 μg/mL), TPC (15.8 μg/mL), and LDH (20.9 μg/mL). In the case of ABZ-SO₂ the mean effective concentrations were the highest, and could be reached only in one LDH assay. These values (μg/mL) were as follows: 65.3 (FaO), 65.4 (HepG2), 75.8 (hepatocytes), and 77.4 (Balb/c 3T3). Conclusion: The differences in in vitro toxicity of albendazole depend on metabolic ability of the cellular models. Primary cultured rat hepatocytes represent a valuable tool to study the impact of biotransformation on the cytotoxicity of drugs.

OBJECTIVE To compare gene expression patterns of T cells in porcine colostrum and peripheral blood. ANIMALS 10 multiparous sows. PROCEDURES Cytotoxic and CD4-CD8 double-positive T cells were separated from porcine colostrum and peripheral blood. Total RNA was extracted. The cDNA prepared from RNA was amplified, labeled, fragmented, and competitively hybridized to DNA microarray slides. The DNA microarray data were validated by use of a real-time reverse-transcription PCR assay, and expression of the genes FOS, NFKBI, IFNG, CXCR6, CCR5, ITGB2, CCR7, and SELL was assessed. Finally, DNA microarray data were validated at the protein level by use of flow cytometry via expression of c-Fos and integrin β-2. RESULTS Evaluation of gene expression profiles indicated that in contrast to results for peripheral blood, numerous cell-signaling pathways might be activated in colostrum. Profile analysis also revealed that FOS and NFKBI (genes of transcription factors) were involved in most cell-signaling pathways and that expression of these genes was significantly higher in colostral T cells than in peripheral blood T cells. Furthermore, CCR7 and SELL (genes of T-cell differentiation markers) in colostral T cells had expression patterns extremely similar to those found in effector or effector memory T cells. CONCLUSIONS AND CLINICAL RELEVANCE All or most of the T cells in colostrum had an effector-like phenotype and thus were more activated than those in peripheral blood. This gene expression profile would enable T cells to migrate to mammary glands, be secreted in colostrum, and likely contribute to passive immunity provided by sows to newborn pigs.