Goblet cell mucin distribution in the small intestine of newborn goat kids fed lyophilized bovine colostrum

The number of goblet cells containing neutral and acidic mucins, including sulphomucins and sialomucins, was investigated in the small intestine of goat kids fed with lyophilized bovine colostrum in the period of passive immunity acquisition. At 0, 7 and 14 h of life, 15 male newborns received 5% of body weight of lyophilized bovine colostrum (LBC) and 14 male newborns received goat colostrum (GC), both with 55 mg/mL of IgG. Three additional animals were sampled at birth, without colostrum intake. Duodenum, jejunum and ileum samples were collected at 18, 36 and 96 h of life. Histological stains, periodic acid-Schiff, 1% alcian blue pH 2.5 and 1% alcian blue pH 1.0 were used to identify neutral and acidic mucins and acidic sulphated mucins, respectively. The number of goblet cells containing neutral and acidic mucins, including sulphomucins and sialomucins, does not differ in the duodenum (P>0.05). In the jejunum, LBC showed a higher number of goblet cells containing sialomucins compared to GC (P<0.05). The highest number of goblet cells containing acidic and neutral mucins and total number of goblet cells were observed at 96 h (P<0.05). In this segment, vacuoles of colostrum were present at 18 and 36 h mainly in the upper region of the villi, while the goblet cells were located at the bottom. At 96 h, vacuoles of colostrum were not detected, only goblet cells distributed throughout the villi. In the ileum, the number of goblet cells containing sulphomucins was higher (P<0.05) at 96 h than at 18 h. The LBC group showed higher (P<0.05) number of goblet cells containing sulphomucins at 96 h and total number of goblet cells at 36 and 96 h than the 0-h group. The present work revealed that the greater the absorption of colostrum in the goat kids' jejunum epithelium, the smaller the number of goblet cells. Considering this segment, feeding newborns with heterologous colostrum caused alteration in the number of goblet cells containing sialomucin. This condition suggested a reaction of the intestinal epithelium with increasing secretion due to the presence of non-recognized substances from the lyophilized bovine colostrum.     

外源粪菌干预对受体猪肠道屏障功能的影响研究

为研究外源粪菌干预对受体猪肠道屏障功能的影响,试验选取初生重相近、胎次相同、同日出生的杜×长×大三元杂交哺乳仔猪12窝,随机分为2组,每组6窝(每窝10~12头)。试验组于1~10日龄隔天灌喂1 m L金华猪粪菌悬液进行外源粪菌干预;对照组灌喂等量无菌0.1 mol/L无菌磷酸盐缓冲液(PBS)。试验期间,仔猪自由哺乳采食和饮水,分别于12日龄和27日龄进行屠宰、采样。结果表明:外源粪菌干预可明显减少腹泻,提高受体猪的平均日增重和成活率(P0.05);外源粪菌干预能显著降低27日龄受体猪肠道隐窝深度(P0.05),显著提高肠道绒毛高度/隐窝深度(P0.05);外源粪菌干预能显著增加肠道杯状细胞数和12日龄肠道分泌型免疫球蛋白A阳性(SIgA~+)细胞数(P0.05),同时显著提高肠道黏蛋白Muc2、紧密连接蛋白ZO-1和Occludin表达水平(P0.05),而27日龄试验组的肠道SIgA~+细胞数与对照组无显著差异(P0.05)。综上可知,外源粪菌干预能通过增加肠道杯状细胞和SIgA~+细胞数、提高黏蛋白和紧密连接蛋白表达的方式调节受体猪肠道屏障功能,具有维持猪肠道功能稳态和促进肠道健康的作用。     

培植牛黄成因研究

对１３１头牛采用三种不同植黄方法，研究影响牛黄形成、增产的有关因素及牛黄形成机制。三种植黄方法牛胆汁中的胆汁酸、胆汁钙、胆红素、ｐＨ值均降低。牛胆囊内植黄期间胆汁粘蛋白、总蛋白含量升高（Ｐ〈０．０１），胆汁粘度增大（Ｐ〈０．０５），在牛腹腔模拟胆囊内（以下简称ＡＧ）和牛双胆囊（以下简称ＤＧ）内植黄期间胆汁粘蛋白、总蛋白含量降低，粘度变小。３种不同植黄方法牛胆囊内胆汁充盈度、胆汁郁滞量和胆道内压力不     

Mucin biosynthesis in the bovine goblet cell induced by Cooperia oncophora infection

Mucin hypersecretion is considered to be one of the most common components of the immune response to gastrointestinal nematode infection. However, investigations have not been conducted in the Cattle–Cooperia oncophora system to verify the findings largely derived from murine models. In this study, we examined the expression of seven mucins and seven enzymes in the mucin biosynthesis pathway involved in O-linked glycosylation in the bovine small intestine including goblet cells enriched using laser capture microdissection during a primary C. oncophora infection. At the mRNA level, MUC2 expression was significantly higher in both lamina propria and goblet cells at 28 days post-infection compared to the naïve control. MUC5B expression at the mRNA level was also higher in lamina propria at 28 dpi. Expression of MUC1, MUC4, MUC5AC, and MUC6 was extremely low or not detectable in goblet cells, columnar epithelial cells, and lamina propria from both naïve control and infected animals. Among the seven enzymes involved in post-translational O-linked glycosylation of mucins, GCNT3, which may represent one of the key rate-limiting steps in mucin biosynthesis, was up-regulated in goblet cells, columnar epithelial cells, lamina propria, and gross small intestine tissue during the course of infection. Western blot analysis revealed that MUC2 glycoprotein was strongly induced by infection in both gross small intestine tissue and its mucosal layer. In contrast, the higher MUC5B protein expression was observed only in the mucosal layer. Immunohistochemistry provided further evidence of the mucin glycoprotein production and localization. Our results provided insight into regulation of mucin biosynthesis in various cell types in the bovine small intestine during gastrointestinal nematode infection and will facilitate our understanding of mucins and their role in immune response against parasitic nematodes.     

In vivo immunogenic characteristics of cytotoxic T lymphocyte epitopes from pancreatic tumor antigen MUC4

AIM: Peptide vaccines have been conceived as promising therapies for tumor-inflicted patients due to their easy production and capability of inducing specific immune response required for defending the tumor. During our previous research, 4 HLA-A2-restricted peptides had been identified as immunogenic in vivo. In this study, we aimed to testify the in vivo immunogenicity of the 4 peptides. METHODS: BALB/c mice were vaccinated with HLA-A2 restricted peptides emulsified in incomplete Freund's adjuvant (IFA) subcutaneously in combination with the epitope at the adjacent location. After the 3rd peptide vaccination for 10 d, the peptide-specific immune response was evaluated by ELISPOT and ELISA. The ability to induce T cell response was investigated by using cytotoxicity assay in vivo and the presence of peptide-specific CD8⁺ T cells capable of recognizing the MHC-peptide was detected by flow cytometry. RESULTS: Among the 4 candidate HLA-A2 restricted peptides, the immune response elicited by P2004-1Y9V was superior to that of the other 3 peptides. The CTLs induced by P2004 and P2004-1Y9V lysed CAPAN-2 cells. P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell lines of CAPAN-2 than the native peptide-specific CTLs. Intracellular cytokine staining assay indicated the presence of P2004-1Y9V specific CD8⁺T cells in the P2004-1Y9V vaccinated mice. CONCLUSION: P2004-1Y9V is the most immunogenic peptide in vivo, and can be explored as potential tumor peptide vaccine in the future clinical study.     

Human dendritic cells transfected with MUC1 mRNA induce lethal effect of cytotoxic T-lymphocytes on non-small cell lung cancer in vitro

AIM：To investigate the specific anti-tumor effects of mature dendritic cells (DCs) transfected with amplified mucin 1 (MUC1) mRNA in vitro. METHODS：DCs separated and purified from the peripheral blood mononuclear cells were induced in vitro and then identified by flow cytometry. pcDNA3.1（+）-MUC1 plasmid was constructed and was able to transcribe MUC1 mRNA in vitro. The MUC1 mRNA was transfected into DCs by electroporation. MUC1-transfected DCs were used to induce T cells to be cytotoxic T-lymphocytes. Quantitative real-time PCR was performed to assess MUC1 mRNA expression in transfected DCs. The proliferation of T cells was examined by MTT assay. The proportion of CD8+ cells in the T cells was determined by flow cytometry and the specific cytotoxicity was measured by LDH assay. The secretion of IFN-γ was detected by ELISA. RESULTS：The marker gene expression in the DCs transfected with MUC1 mRNA was significantly increased compared with control group, peaking at 24 h. The transfection group showed the higher capacity to stimulate the proliferation of T cells compared with control group when the ratio of DCs to T cells was 1∶10. The proportion of CD8+ cells in transfection group was higher than that in control group. The lethal effect of special cytotoxic T-lymphocytes on target cells in transfection group was stronger than that in control group. The level of IFN-γ in the cell supernatant of transfection group was higher than that in control group. CONCLUSION：DCs plus MUC1 mRNA by electrical transfection induces specific anti-tumor effects, which provides an experiment evidence of using MUC1 as a target for immunotherapeutic strategy against non-small cell lung cancer.     

Refined methodology to purify mucins from pig colonic mucosa

Mucin, the main macromolecular component of mucus, contains a peptide backbone rich in proline, threonine and serine (PTS) and oligosaccharide side chains. Glycosylation increases the molecular size of mucins and restricts access of proteases to mucin. Based on these characteristics, Faure et al. [Faure, M., Moënnoz, D., Montigon, F., Fay, L.B., Breuille, D., Finot, P.A., Ballèvre, O. and J. Boza. 2002. Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats. Anal. Biochem 307: 244–251.] developed a method to purify mucins from intestinal mucosa. In this method, mucins are reduced (RD) and digested with protease (dig) prior to isolation using size-exclusion chromatography. Our objective was to refine this method to purify mucins from pig colonic mucosa. Mucosal scrapings were homogenized in 5 mM EDTA and were either treated with 7 μL of protease inhibitor cocktail (PIC; UndigUnRD) or incubated with 100 μL of Protease at 37 °C for 80 min and then subsequently treated with PIC (digUnRD). Homogenates were fractionated based on size using a Sepharose CL-4B column. Mucin-containing fractions were identified by a combination of SDS-PAGE and staining techniques and were pooled. Mucin purified using digUnRD method had higher proportions of PTS compared to crude mucin purified using UndigUnRD method (31.9 vs. 23.5 mol/100 mol AA; P < 0.005). The proportion of PTS in mucin purified using digUnRD was also higher than mucin purified using digRD (31.9 vs. 25.7 mol/100 mol AA; P < 0.05). The refined method employing protease digestion and no reduction can be successfully applied to purify mucins from pig colonic mucosa.