枸杞多糖对免疫抑制小鼠血清中IL-6、IL-10和IL-12/IL23 p40分泌水平的影响

通过研究枸杞多糖(LBP)对免疫抑制小鼠血清中细胞因子IL-6、IL-12/IL-23p40和IL-10分泌的影响,探讨LBP的免疫调节机制,为LBP的开发利用提供理论依据。60只ICR雌性小鼠,随机分为5组,对各组小鼠进行连续4d每日腹腔注射环磷酰胺(40mg/kg),间隔3d后,再连续4d每日腹腔注射环磷酰胺(40mg/kg);同时,用高(40mg/1kg)、中(20mg/1kg)、低剂量(10mg/1kg)的LBP给小鼠连续灌胃2周,阳性对照和阴性对照分别用LPS(1mg/kg)和PBS,连续2周。小鼠眼眶取血,分离血清,ELISA检测血清中IL-6、IL-12/IL-23p40和IL-10的分泌水平。结果显示,与阳性对照和阴性对照组相比,LBP能提高免疫抑制小鼠血清中IL-6、IL-12/IL-23p40和IL-10的分泌水平,提示LBP能增强小鼠细胞免疫与体液免疫应答,为枸杞多糖作为免疫增强剂的开发应用奠定了一定的理论基础。     

Cytokine and catabolic enzyme expression in synovium, synovial fluid and articular cartilage of naturally osteoarthritic equine carpi

Reasons for performing study: Understanding the expression of catabolic and anabolic genes during osteoarthritis progression should help to identify the major mediators of the disease. Objective: To compare the cytokine and anabolic marker concentrations in synovium, synovial fluid and cartilage between normal and osteoarthritic joints. Methods: Carpi from horses age 2–11 years were used. Tissues were harvested at the time of surgery or euthanasia, and RNA was isolated for RT‐PCR analysis. Tumour necrosis factor alpha (TNFα), interleukin‐1beta (IL‐1β), aggrecanase 1 (ADAMTS‐4), aggrecanase 2 (ADAMTS‐5), matrix metalloproteinase‐13 (MMP‐13), interleukin 17 (IL‐17) and collagen type I alpha 1(Col‐1) expression were determined in synovium. TNFα, IL‐1β, ADAMTS‐4, ADAMTS‐5, MMP‐13, IL‐17, collagen type IIB (Col‐2B), and aggrecan expression were determined in cartilage. TNFα concentration in the synovial fluid was determined by enzyme‐linked immunosorbent assay (ELISA). Results: Expression of TNFα, ADAMTS‐5 and MMP‐13 was significantly increased in synovial tissue from OA joints. Synovial membrane IL‐1β abundance showed only moderate elevations in OA, without reaching significant levels. Cytokine expression was increased significantly in OA cartilage samples, particularly TNFα, IL‐1β, ADAMTS‐4 and MMP‐13; and collagen type I expression was significantly increased in synovial tissues from OA groups. Collagen type II message was diminished in mild and moderate stages of OA, but rebounded to significant elevations in severely degenerate joints. Conversely, aggrecan levels significantly declined in cartilage from all OA groups. Synovial fluid TNFα peptide concentration was significantly increased in severe OA cases. Conclusion: TNFα was increased in all degrees of equine OA, and was abundantly expressed in synovial membrane and cartilage. IL‐1β was overexpressed in OA cartilage, but not to a significant extent in synovium. Potential relevance: Control of TNFα should be considered further as a target in the treatment of OA. ADAMTS‐4 may be the primary aggrecanase causing cartilage breakdown in OA.     

PRRSV和PCV2共感染对猪肺泡巨噬细胞细胞因子IL-10、IL-12p40和IFN-γ mRNA转录的影响

用猪繁殖与呼吸综合征病毒(PRRSV)和猪圆环病毒2型(PCV2)共感染40日龄健康仔猪,利用实时荧光定量PCR技术对共感染仔猪肺泡巨噬细胞(PAM)细胞因子IL-10、IL-12p40和IFN-γ的mRNA转录水平的变化进行了定量分析。结果表明,IL-12p40 mRNA转录从攻毒后3 d开始显著上调(P<0.05),7 d达高峰,14 d开始下降,但仍显著高于对照组(P<0.05),28 d和42 d低于对照组;IL-10 mRNA在3 d显著上调(P<0.05),14 d达到转录高峰(P<0.01),之后逐渐下降,42 d接近对照组水平;IFN-γmRNA转录水平尽管在3 d和28 d时出现2次转录低峰1、4 d和42 d时出现2次高峰,但只在3 d和7 d差异显著(P<0.05)。由此表明,PRRSV和PCV2共感染可导致PAM细胞因子IL-12p40和IL-10 mRNA的转录在感染早期被激活,而IFN-γ mRNA转录则呈较复杂的动态变化,提示PRRSV和PCV2共感染猪PAM的免疫应答、免疫调节和抗感染功能均受到不同程度的影响。     

IL‐12p40 gene expression in lung and hilar lymph nodes of MPS‐resistant pigs

Mycoplasma pneumonia of swine (MPS) is caused by Mycoplasma hyopneumoniae (M.hp) and is a common chronic respiratory disease of pigs. Recently, a genetically selected variant of the Landrace pig (Miyagino L2) has a lower incidence of pulmonary MPS lesions. We investigated the pathological and immunological characteristics of MPS resistance in these pigs (n = 24) by comparing with the normal landrace pig (control: n = 24). The pathological MPS lung lesion score in MPS‐selected landrace pigs was significantly lower than in the control. The gene expression of interleukin (IL)‐12p40, which acts as a chemoattractant and a component of the bioactive cytokines IL‐12 and IL‐23, was significantly higher at the hilar lymph nodes, lung, and spleen in MPS‐selected landrace pigs than in control landrace pigs, and these were negatively correlated with the macroscopic MPS lung lesion score. In summary, we demonstrate that resistance against MPS in Miyagino L2 pigs is associated with IL‐12p40 up‐regulation, in comparison with normal landrace pigs without the MPS vaccine. In addition, a comparative study of macroscopic MPS lung lesions and IL‐12p40 gene expression in lung and hilar lymph nodes may lead to beneficial selection traits for the genetic selection for MPS resistance in pigs.     

Molecular cloning, expression and characterization of an interleukin 27 p28 homolog in pig (Sus scrofa)

IL-27 is the newest member of the IL-12 cytokine family and plays an important role in the immune regulation. It is composed of two subunits, p28 and EBV-induced gene 3(EBI3). Although human and mouse IL-27 p28 genes have been cloned, pig IL-27 p28 gene has not ever been reported. In the present study, we have cloned and characterized the full-length cDNA of IL-27 p28 from pig. The open reading frame of pig IL-27 p28 gene is 720 bp, which encodes a protein of 239 amino acids with a predicted molecular mass of 26.6 kDa. The deduced amino acid sequence of pig IL-27 p28 showed a high degree of homology to human (63%) and mouse (58%). It was a 4-helix cytokine and belonged to 4-helix cytokine superfamily. Pig IL-27 p28 has one transmembrane region, one signal peptide, and one N-glycosylation site, two Protein kinase C phosphorylation sites, three Casein kinase II phosphorylation sites and one N-myristoylation site. For the expression of pig IL-27 p28 protein in a eukaryotic expression system the recombinant plasmid was constructed. The expression of pig IL-27 p28 in mammalian cells were confirmed by flow cytometry analysis, immunofluorescence and Western blot. The analysis also confirmed a cross reactivity with anti-mouse IL-27 p28 antibody. This is the first report of cloning and characterization of IL-27 p28 in pig.     

A long‐term study of the effects of SLC12A1 homozygous mutation (g.62382825G>A,p.Pro372Leu) in Japanese Black cattle

Recessive missense mutation in the solute carrier family 12, member 1 (SLC12A1) gene (g.62382825G>A) is associated with hydrallantois, which is the accumulation of fluid in the allantoic cavity of a pregnant animal, and usually causes fetal death in Japanese Black cattle. However, the symptoms of a homozygote with this mutation that do not result in fetal death have not previously been tracked and evaluated. In the present study, we observed a homozygote with the SLC12A1 risk allele over a long‐term period. The calf did not show any obvious clinical symptoms, although it did exhibit a slight growth retardation that accompanied mild calciuria. At 28 months of age, the homozygote showed renal dysfunction, which in turn resulted in hydronephrosis. The time course of the symptoms was consistent with the phenotype of Bartter syndrome in humans. Additionally, the risk heterozygous genotype did not any effects on carcass traits, which indicates that eliminating the risk allele would not have any unfavorable effects. Therefore, we emphasize that both the fetal‐ and late‐stage symptoms associated with the SLC12A1 risk allele compromise animal welfare, and consequently may result in severe economic losses for individual farmers if the SLC12A1 risk allele is not eliminated from the population.     

Furosemide loading test in a case of homozygous solute carrier family 12, member 1 (SLC12A1) mutation (g.62382825G>A,p.Pro372Leu) in Japanese Black cattle

Hydrallantois is the excessive accumulation of fluid in the allantoic cavity in a pregnant animal and is associated with fetal death. We recently identified a recessive missense mutation in the solute carrier family 12, member 1 (SLC 12A1 ) gene (g.62382825G>A, p.Pro372Leu) that is associated with hydrallantois in Japanese Black cattle. Unexpectedly, we found a case of the homozygous risk‐allele for SLC 12A1 in a calf, using a PCR ‐based direct DNA sequencing test. The homozygote was outwardly healthy up to 3 months of age and the mother did not exhibit any clinical symptoms of hydrallantois. In order to validate these observations, we performed confirmation tests for the genotype and a diuretic loading test using furosemide, which inhibits the transporter activity of the SLC 12A1 protein. The results showed that the calf was really homozygous for the risk‐allele. In the homozygous calf, administration of furosemide did not alter urinary Na⁺ or Cl^? levels, in contrast to the heterozygote and wild‐type calves in which these were significantly increased. These results demonstrate that the SLC 12A1 (g.62382825G>A, p.Pro372Leu) is a hypomorphic or loss‐of‐function mutation and the hydrallantois with this mutation shows incomplete penetrance in Japanese Black cattle.