Effects of induced stress from the live LaSota Newcastle disease vaccination on the growth performance and immune function in broiler chickens.

This study was conducted to assess the growth performance and immunological effects of vaccination-induced stress on broilers. The chickens were administered 0, 2, 4, 8, and 16 doses of live LaSota Newcastle disease (ND) vaccine and slaughtered on the 1st, 7th, 14th, and 21st day post vaccination. The results showed that the serum antibody titers after Newcastle disease virus (NDV) vaccination were elevated at day 7 post vaccination, peaked at day 14, then declined by day 21. Interestingly, the antibody titers peaked at 2 doses, and no further dose-dependent titer increases were observed. This study demonstrated that vaccination-induced stress increased serum adrenocorticotropic hormone and cortisol, affected growth performance (average daily gain, average daily feed intake, and feed conversion ratio), and triggered apoptosis in spleen lymphocytes by downregulating the ratio of Bcl-2 to BAX and upregulating the gene expressions of caspase-3 and -9, which was concordant with the activation of the enzymatic activities of caspase-3 and -9. This study suggests that NDV vaccine doses in broilers must be controlled judiciously because increasing the number of doses resulted in increased lymphocyte apoptosis while the peak of the antibody titer and optimal growth performance were achieved at a low number of doses (2 doses).

[Clinical study of S-1 single agent adjuvant chemotherapy in the radical resection of extrahepatic biliary carcinoma].

Objective: To explore the clinical efficacy of S-1 single agent adjuvant chemotherapy for the patients undergoing radical resection of extrahepatic biliary carcinoma. Methods: The clinical data of 108 patients with extrahepatic biliary carcinoma receiving radical resection who were admitted from January 2014 to June 2017 were retrospectively analyzed. There were 62 males(57.4%)and 46 females(42.6%),with a median age of 59 years (range:26 to 79 years),10 cases(9.3%) in stage Ⅱ,85 cases(78.7%) in stage Ⅲ, and 13 cases (12.0%) in stage Ⅳ, 40 cases(37.0%) of hilar cholangiocarcinoma, 8 cases(7.4%) of middle cholangiocarcinoma, 25 cases (23.2%) of distal cholangiocarcinoma, 35 cases(32.4%) of gallbladder carcinoma.After radical resection of extrahepatic biliary carcinoma, 49 patients receiving S-1 single agent chemotherapy and 59 patients receiving non-special treatment were divided into the chemotherapy group and the operation group,respectively. All the dates of the patients were followed up and collected with the overall survival time,tumor-free survival time,1,2 and 3-year survival rate after operation,and the rate of major toxic reaction during chemotherapy of the chemotherapy group. Survival curve was drawn by the Kaplan-Meier method, and survival analysis was done using the Log-rank test. Results: There were no significant differences in the general date of two groups(sex, age, tumor size, tumor site, TNM stages, degree of differentiation). The median overall survival time and the median tumor-free survival time in the chemotherapy group were 27 months and 21 months,respectively,and in the operation group were 21 months and 17 months,respectively. There were differences between the two groups in the overall survival rates(χ(2)=3.967,P<0.05) and the 2 and 3-year survival rate(63.3%,36.6%;41.6%,20.4%;χ(2)=4.510,P<0.05;χ(2)=6.143,P<0.05),but the 1-year overall survival rate (83.4%,79.7%)was not statistically significant(χ(2)=0.286,P>0.05). There were no significant differences in the tumor-free survival time,1,2 and 3-year tumor-free survival rate(77.6%,41.4%,33.1%;62.7%,30.9%,21.2%)between the two groups(χ(2)=0.876,P>0.05;χ(2)=0.252,P>0.05;χ(2)=1.571,P>0.05;χ(2)=3.323,P>0.05,respectively). The main toxic reaction during chemotherapy were dyspepsia(28.6%, 14/49), anemia(26.5%, 13/49), and leukopenia(22.5%, 11/49), all of which were mild. Conclusion: S-1 single agent chemotherapy after radical reseetion of extrahepatic biliary carcinoma could effectly improve the survival of patients and all of the main toxic reaction during chemotherapy were mild.

Molecular mechanism for the influence of gender dimorphism on alcoholic liver injury in mice.

It is known that women develop alcoholic liver injury more rapidly and have a lower alcohol toxic threshold than men. However, the detailed molecular mechanisms remain unclear. The precise mechanism responsible for the sex difference needs to be determined. Female and male mice were given ethanol by intragastric infusion every day for 4 weeks. The pathological changes were detected by hematoxylin-eosin, Sirius red, oil red O, periodic acid-Schiff, and Hochest33258 staining in the liver of female and male mice. The related gene and protein expression of hepatocytes stress, proliferation and apoptosis, glycogen synthesis, lipid metabolism, and hepatic fibrosis were also systematically analyzed in the female and male mice. Livers from ethanol-treated female mice had more serious hepatocyte necrosis, liver fibrosis ( P < 0.01), substantial micro/macrovesicular steatosis ( p < 0.01), glycogen consumption ( p < 0.05), and hepatocytes apoptosis ( p < 0.05) than ethanol-treated male mice. The expression of heat shock protein 27 (HSP27), HSP70, proliferating cell nuclear antigen, B-cell lymphoma/leukemia-2 (Bcl-2), and phosphorylated signal transducer and activators of transcription 3 (p-STAT3) was higher in ethanol-treated male mice than ethanol-treated female mice ( P < 0.05 or P < 0.01). But, the expression of Bax (Bcl-2-associated X protein), Caspase 3, CYP2E1 (cytochrome P4502E1), and transforming growth factor ßl had the contrary results. Our study suggested that ethanol treatment induced more expression of HSP27 and HSP70, faster hepatocyte proliferation, higher level of glycogen, and interleukin-6 signaling pathway activation, but less hepatocyte apoptosis and CYP2E1 expression in male mice than female mice, which could be helpful to understand the molecular mechanism for the influence of sex difference on alcoholic liver injury.

[Kindlin-2 promotes gallbladder cancer metastasis and invasion by inducing epithelial-mesenchymal transition].

Objective: To investigate the effects of Kindlin-2 on malignant phenotypes of human gallbladder cancer cells and discuss the mechanisms. Methods: The expression level of Kindlin-2 in 30 cases of gallbladder cancer tissues and adjacent non-tumoral tissues collected from the First Affiliated Hospital of Zhengzhou University between September 2012 and May 2013 was assessed by real-time PCR and immunohistochemistry.Lentivirus-mediated Kindlin-2 overexpression was used in gallbladder cancer cell lines GBC-SD and SGC-996.Transwell assay and adhesion assay were investigated to explore the functional role of Kindlin-2 on gallbladder cancer cells.Western Blot was used to test the protein change of epithelial-mesenchymal transition(EMT) characteristics. The t-test was used to analyzed results. Results: The RNA and protein levels of Kindlin-2 in gallbladder cancer tissues were higher than in the non-tumoral tissues (t=4.372, P=0.001; t=7.477, P=0.000). The expression level of Kindlin-2 in gallbladder cancer tissues was correlated with Nevin stage(χ(2)=5.932, P=0.035). Compared with control groups, the cell-matrix adhesion ability of GBC-SD and SGC-996 with Kindlin-2 overexpression was obviously promoted(1.66±0.03 vs. 1.07±0.22, t=2.710, P=0.041; 2.66±0.24 vs. 1.03±0.02, t=6.610, P=0.020). The number of GBC-SD and SGC-996 cells with Kindlin-2 overexpression passing through the Transwell chamber matrix increased significantly compared with the control groups(116.1±13.9 vs. 54.7±8.4, t=3.781, P=0.019; 136.3±7.5 vs. 64.3±6.4, t=7.302, P=0.002). The wound healing rate of GBC-SD with Kindlin-2 overexpression at 12-hour and 24-hour was higher than that of the group ((42.9±2.2)% vs. (29.7±1.7)%, t=4.690, P=0.009; (65.0±2.4)% vs.(40.4±2.0)%, t=7.945, P=0.001). The wound healing rate of SGC-996 with Kindlin-2 overexpression at 12-hour and 24-hour was also higher than that of the group ((32.9±1.3)% vs. (24.1±1.5)%, t=4.518, P=0.011; (51.3±1.1)% vs. (39.2±1.1)%, t=8.001, P=0.001). The characteristics of EMT were induced in gallbladder cancer cells with Kindlin-2 overexpression, including the up-regulation of N-cadherin, Vemintin and the down-regulation of E-cadherin. Conclusion: The expression of Kindlin-2 is up-regulated in gallbladder cancer tissues and Kindlin-2 promoted the malignant phenotypes of gallbladder cancer cells partially by epithelial-mesenchymal transition.

JAK/STAT5 signaling pathway inhibitor ruxolitinib reduces airway inflammation of neutrophilic asthma in mice model.

OBJECTIVE: The aim of this study was to explore the role of JAK/STAT signaling pathway inhibitor Ruxolitinib in neutrophilic airway inflammation and its possible immunological mechanism. MATERIALS AND METHODS: A total of 60 female C57BL/6 mice were randomly divided into neutrophilic asthma (NA) group, Ruxolitinib-treated (Ruxo) group and control (Con) group. Mice in NA and Ruxo groups were sensitized with ovalbumin (OVA) and excited to establish mice models of asthma. Bronchoalveolar lavage fluid (BALF) was collected at 24 h after the last atomization, and the total number of karyocyte and the percentages of sorted cells were detected. The activity of interleukin-17 (IL-17) in BALF was detected by enzyme-linked immunosorbent assay (ELISA). Lung tissue was separated and subjected to hematoxylin-eosin (HE) staining, and the pathological changes of lung tissue were observed under an optical microscope. The proportion of T helper 17 (Th17) cells in the lung was detected by flow cytometry (FCM). After successful modeling of NA mice, immunomagnetic bead purified mouse splenic cluster of differentiation 4+(CD4+) T was treated with IL-7 and Ruxolitinib, and the proportion of differentiated Th17 cells to CD4+ T cells and Ki-67, B-cell lymphoma 2 (Bcl-2) and activated Caspase3 expressions in Th17 cells were detected via FCM. RESULTS: Compared with those in NA group, the number of karyocytes and the percentages of neutrophils (NEU) and eosinophils (EOS) in BALF in Ruxo group were significantly reduced. The pathological changes of lung tissue in Ruxo group were overtly less than those in NA group. In comparison with NA group, Ruxo group had decreased IL-17A level in BALF and reduced proportion of Th17 cells in lung tissue. In in vitro experiment, compared with those in Con group, decreased percentages of Ki-67 and Bcl-2 proteins and increased percentage of Caspase3 in Th17 cells were found in Ruxo group. CONCLUSIONS: Ruxolitinib may suppress the survival of Th17 cells by inhibiting the JAK/STAT5 signaling pathway and regulate the anti-apoptosis proteins Bcl-2 and Caspase3, thus promoting the increase of Thl7 cells entering the apoptotic pathway and reducing airway inflammation in NA mice.

Decreased expression of humanized Fat-1 in porcine fetal fibroblasts following deletion of PGK-neomycin resistance.

The neomycin-resistance (neo(r)) gene is widely used as a selectable marker in eukaryotic expression vectors; however, its expression often affects that of target genes. Cre recombinase recognizes LoxP sites, leading to site-specific recombination and deletion of DNA and RNA between two LoxP sites. In the present study, a humanized Fat-1 gene (hFat-1) was generated by DNA Works and used to construct a pC-PGK-neo(r)-hfat-1 expression vector, in which PGK-neo(r) was flanked by two LoxP sites. The pC-PGK-neo(r)-hfat-1 plasmids were transfected into porcine fetal fibroblasts using liposomes, and three transgenic cell lines were obtained by culturing with 400 µg/mL G418 for 7 days. Next, these cell lines were transfected with a Cre recombinase expression plasmid, which contains a puromycin resistance gene, in order to delete neo(r), which was integrated into the genome. hFat-1-neo(r) negative cells were obtained following puromycin selection. Real-time quantitative polymerase chain reaction data indicated that neomycin-resistant cells had higher hFat-1 expression than neomycin-sensitive cells. High performance gas chromatography data suggested that the n-6/n-3 ratio was significantly lower in transfected cells than in wild-type cells. The n-6/n-3 ratio in Cre-treated hFat-1-transfected cells was higher than that in untreated cells, suggesting that deletion of PGK-neo(r) decreased hFat-1 expression.

A promoter trap vector for knocking out bovine myostatin gene with high targeting efficiency.

With the development of gene targeting approaches, genomic mutation technologies in livestock animals such as gene trapping, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats and their associated systems have been improved. Although ZFNs have been used for gene targeting in many species, the off-target sites are still present. Using gene trapping, the workload of screening of targeted clones was decreased by generating a smaller number of drug-resistant clones. Determining whether the efficiency of gene trapping is lower than that of ZFNs for a specific gene has been challenging. In this study, to knock out the bovine myostatin gene, we constructed a promoter trap vector and compared its efficiency with that of ZFNs. The promoter trap vector contained a green fluorescent protein sequence without the promoter and a neomycin phosphotransferase (neo(R)) cassette driven by the phosphoglycerate kinase promoter. When the trapping vector was inserted downstream of the endogenous promoter, the fluorescent protein gene was expressed. The targeted-positive cell clones were identified based on green fluorescence and G418 double selection, followed by polymerase chain reaction analysis and sequencing. The targeting efficiency reached 5%. Compared with the efficiency of ZFN pairs (5.17 and 2.86%), the promoter trap vector PIII-myostatin could knock out the bovine myostatin gene. Therefore, gene trapping may be an effective tool for genomic modification.

The important role of ADAM8 in the progression of hepatocellular carcinoma induced by diethylnitrosamine in mice.

This study focuses on investigating the concrete role of a disintegrin and metalloproteinase 8 (ADAM8) in the progression of hepatocellular carcinoma (HCC). Mice received anti-ADAM8 monoclonal antibody (mAb) of 100 µg/100 µl, 200 µg/100 µl or 300 µg/100 µl, respectively, in phosphate-buffered saline (PBS) or PBS intervention during the progression of HCC induced by diethylnitrosamine. The survival rate, body weight, and relative liver weight were determined in the mice. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and α-fetoprotein (AFP) level, hematoxylin-eosin staining, the expression level of vascular endothelial growth factor A (VEGF-A), proliferating cell nuclear antigen (PCNA), caspase 3 (Casp3), B cell leukemia 2 (Bcl2), B cell leukemia 2-associated X protein (Bax), protein p53 (P53), and ADAM8 were detected in the mice at the end of the 24th week. Our results showed that anti-ADAM8 mAb intervention effectively improved the survival rate, reduced the body weight loss and increased the relative liver weight in mice in a dose-dependent manner (p < 0.05 or p < 0.01). Anti-ADAM8 mAb intervention also significantly lowered serum AST, ALT, and AFP levels (p < 0.05 or p < 0.01), slowed the progression of HCC (p < 0.05 or p < 0.01), induced the expression of Casp3, Bax, and P53 (p < 0.05 or p < 0.01), and inhibited the expression of VEGF-A, PCNA, and Bcl2 in the liver of mice (p < 0.05 or p < 0.01) in a dose-dependent manner compared with the mice receiving PBS intervention. Our study suggested that ADAM8 might promote the progression of HCC by regulating the expression of these factors. Anti-ADAM8 mAb intervention might be suitable as a potential method for HCC therapy.

Neural respiratory drive in patients with COPD during exercise tests.

BACKGROUND: It is unknown whether neural drive is comparable in constant rate and incremental exercise tests. Few data have previously been available to address this question because of the lack of reliable methods to assess neural respiratory drive in patients with chronic obstructive pulmonary disease (COPD). OBJECTIVES: The aims of this study are to determine whether neural respiratory drive during constant rate exercise differs from that during incremental exercise and to determine whether neural respiratory drive was maximal at the end of exhaustive exercise tests. METHODS: We studied sixteen patients with moderate-severe COPD (mean ± SD FEV(1) 29 ± 10%). Both diaphragmatic electro-myogram (EMG) and transdiaphragmatic pressure were recorded with a combined multipair electrode balloon catheter during incremental and constant (80% of maximal oxygen consumption derived from a prior incremental exercise test) treadmill exercise. Minute ventilation and oxygen uptake were also measured. RESULTS: Root mean square (RMS) of the diaphragmatic EMG increased gradually without a plateau during incremental exercise, whereas the RMS increased initially and reached a plateau during constant work rate exercise. The RMS of the diaphragmatic EMG at the end of exercise was similar for both incremental and constant work rate exercise (176 ± 42 µV vs. 184 ± 39 µV); these values were 70 and 73% of maximal values recorded over the study. CONCLUSIONS: The pattern of increase in neural respiratory drive during incremental exercise is different to that observed during constant work rate exercise, but both exercise protocols are terminated when the patients achieve a similar but submaximal drive.

[Effect of xinhe granule on vascular endothelial damage and endothelial expressed endothelin and intercellular adhesion molecule-1 in rats fed with high lipid diet].

OBJECTIVE: To study the protective effect of Xinhe granule (XHG) on vascular endothelial damage in rats fed with high lipid diet. METHODS: Model of vascular endothelial damage was formed by feeding high lipid diet in rats. The model animal were divided into high dosage XHG group, low dosage XHG group, composite Salvia dripping pellet group and model control group, and a blank (normal) control group was also set up. The degree of endothelial damage and positive cell count of synthesizing and secreting endothelin (ET-1) and intercellular adhesion molecule-1 (ICAM-1) were determined by endothelial cell spreading technique and immunohistochemical technique quantitatively. RESULTS: Comparison of the degree on vascular endothelial damage and endothelial secreting ICAM-1 and ET-1 showed model control group > composite Salvia dripping pellet group > low dosage XHG group > high dosage XHG group > blank control group. XHG did not show obvious lowering action on blood lipid. CONCLUSION: XHG could inhibit the endothelial expressed ICAM-1 and ET-1 in hyperlipidemia rats, thus displaying the protective effect on vascular endothelium.

Uncv (uncovered): a new mutation causing hairloss on mouse chromosome 11.

A pair of mutant mice with a first sparse coat appeared spontaneously in the production stock of BALB/c mice with a normal coat. After being sib-mated, they produced three phenotypes in their progeny: mice with normal hair, mice with a first sparse coat and then a fuzzy coat, and uncovered mice. Genetic studies revealed the mutants had inherited an autosomal monogene that was semi-dominant. By using 11 biochemical loci--Idh, Car2, Mup1, Pgm1, Hbb, Es1, Es10, Gdc, Ce2, Mod1 and Es3--as genetic markers, two-point linkage tests were made. The results showed the gene was assigned to chromosome 11. The result of a three-point test with Es3 and D11Mit8 (microsatellite DNA) as markers showed that the mutation was linked to Es3 with the recombination fraction 7.89 +/- 2.19%, and linked to D11Mit8 with the recombination fraction 26.30 +/- 3.57%. The recombination fraction between Es3 and D11Mit8 was 32.90 +/- 3.81%. It is suggested that the mutation is a new genetic locus that affected the skin and hair structure of the mouse. The mutation was named uncovered, with the symbol Uncv. Further studies showed the mutation affected not only the histology of skin and hair but also the growth and reproductive performance of the mice. The molecular characterization of the Uncv locus needs to be further studied.