Ammonia induces autophagy via circ-IFNLR1/miR-2188-5p/RNF182 axis in tracheas of chickens.

Ammonia (NH3 ), an air pollutant in the living environment, has many toxic effects on various tissues and organs. However, the underlying mechanisms of NH3 -induced tracheal cell autophagy remains poorly understood. In present study, chickens and LMH cells were used as NH3 exposure models to investigate toxic effects. The change of tracheal tissues ultrastructure showed that NH3 exposure induced autolysosomes. The differential expression of 12 circularRNAs (circRNAs) was induced by NH3 exposure using circRNAs transcriptome analysis in broiler tracheas. We further found that circ-IFNLR1 was down-regulated, and miR-2188-5p was up-regulated in tracheal tissues under NH3 exposure. Bioinformatics analysis and dual luciferase reporter system showed that circ-IFNLR1 bound directly to miR-2188-5p and regulated each other, and miR-2188-5p regulated RNF182. Overexpression of miR-2188-5p caused autophagy and its inhibition partially reversed autophagy in LMH cells which were caused by ammonia stimulation or knockdown of circ-IFNLR1. The expressions of three autophagy-related genes (LC3, Beclin 1, and BNIP3) were observably up-regulated. Our results indicated that NH3 exposure caused autophagy through circ-IFNLR1/miR-2188-5p/RNF182. These results provided new insights for the study of ammonia on environmental toxicology on ceRNA and circRNAs in vivo and vitro.

Recombinant pseudorabies virus expressing E2 of classical swine fever virus (CSFV) protects against both virulent pseudorabies virus and CSFV.

Both classical swine fever (CSF) and pseudorabies are highly contagious, economically significant diseases of swine in China. Although vaccination with the C-strain against classical swine fever virus (CSFV) is widely carried out and severe outbreaks of CSF seldom occur in China, CSF is sporadic in many pig herds and novel sub-subgenotypes of CSFV endlessly emerge. Thus, new measures are needed to eradicate CSFV from Chinese farms. The emergence of a pseudorabies virus (PRV) variant also posed a new challenge for the control of swine pseudorabies. Here, the recombinant PRV strain JS-2012-ΔgE/gI-E2 expressing E2 protein of CSFV was developed by inserting the E2 expression cassette into the intergenic region between the gG and gD genes of the gE/gI-deletion PRV variant strain JS-2012-ΔgE/gI. The recombinant virus was stable when passaged in vitro. A single vaccination of JS-2012-ΔgE/gI-E2 via intramuscular injection fully protected against lethal challenges of PRV and CSFV. Vaccination of piglets with the recombinant JS-2012-ΔgE/gI-E2 in the presence of high levels of maternally derived antibodies (Abs) to PRV can provide partial protection against lethal challenge of CSFV. Vaccination of the recombinant PRV JS-2012-ΔgE/gI-E2 strain did not induce the production of Abs to the gE protein of PRV or to the CSFV proteins other than E2. Thus, JS-2012-ΔgE/gI-E2 appears to be a promising recombinant marker vaccine candidate against PRV and CSFV for the control and eradication of the PRV variant and CSFV.

The correlation between HLA-DRB1 and HLA-DQB1 gene polymorphisms and cytokines in HPV16 infected women with advanced cervical cancer.

OBJECTIVE: To analyze the distribution of HLA-DRB1 and HLA-DQB1 alleles and its correlation with IFN-γ, IL-2, IL-6, IL-10 in HPV16 infected women with advanced cervical carcinoma. METHODS: We collected 137 blood samples of cervical carcinoma patients diagnosed by pathology as cervical cancer in stage IIb-IVb before the treatment, and we gathered 175 blood samples of healthy women living in the local. We determined the genetic subtypes of HLA-DRB1 and HLA-DQB1, and we measured the concentration of IFN-γ, IL-2, IL-6 and IL-10. We compared the difference of cytokines in patients with different clinical stages and the healthy in the control group. According to genetic subtypes of HLA-DRB1 and HLA-DQB1, we also compared the concentration of cytokine (CK) in different genetic subtypes. RESULTS: Eight HLA-DRB1 alleles and four HLA-DQB1 alleles were found. There were not significant differences between each allele in the concentration of IFN-γ, IL-2, IL-6, and IL-10. CONCLUSION: HLA-DRB1*07, HLA-DQB1*02 and HLA-DQB1*03 were the differentially expressed gene in HPV16 infected patients with advanced cervical cancer. There may be correlations between the occurrence, development of cervical cancer and IFN-γ, IL-2, IL-6, IL-10.

Protective effects of ginsenoside Rg1 against colistin sulfate-induced neurotoxicity in PC12 cells.

The present study aimed to examine the protective effect of ginsenoside Rg1 against colistin-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Ginsenoside Rg1 was shown to elevate cell viability, decrease levels of malondialdehyde and intracellular reactive oxygen species, enhance activity of superoxide dismutase and glutathione, and decrease the release of cytochrome-c, formation of DNA fragmentation in colistin-treated PC12 cells. Ginsenoside Rg1 also reversed the increased caspase-9 and -3 mRNA levels caused by colistin in PC12 cells. These results suggest that ginsenoside Rg1 exerts a neuroprotective effect on colistin-induced neurotoxicity in PC12 cells, at least in part, via the inhibition of oxidative stress, prevention of apoptosis mediated via mitochondria pathway. Co-administration of ginsenoside Rg1 highlights the potential to increase the therapeutic index of colistin.

Effects of Traditional Chinese Medicine Qinbai Qingfei Concentrated Pellet on Cellular Infectivity of Mycoplasma pneumoniae.

Aim. To study the effect and mechanism of traditional Chinese medicine Qinbai Qingfei concentrated pellet (QQCP) against Mycoplasma pneumoniae (MP). Methods. Rat airway smooth muscle (ASM) cells were used to examine the antimycoplasmal activity of QQCP via four drug-adding modes: pre- and postadding drugs, simultaneous-adding after drug and MP mixed, and simultaneous-adding drug and MP; taking roxithromycin dispersive tablets (RDT) as positive control, the cellular A 570 values were determined by MTT method. Results. All of A 570 values in QQCP group were significantly higher than those of the corresponding MP control group (P < 0.01) in four drug-adding modes; there was no significant difference in A 570 values between the QQCP group and that of the positive control group (P > 0.05), confirming that QQCP could significantly inhibit the infectivity of MP to ASM cells. Conclusion. QQCP had significant activity in preventing and treating MP infection, killing MP, and antiabsorption.

Pharmacokinetics of four different brands of colistimethate and formed colistin in rats.

OBJECTIVES: Very different labelling conventions are employed by different products of colistimethate (CMS), an inactive prodrug of colistin that is used as a last-line defence against Gram-negative 'superbugs'. This study examined the chemical composition and pharmacokinetics in rats of four commercial parenteral products of CMS. METHODS: Contents per vial of four brands of CMS from three different continents were weighed (n = 3). Elemental analysis and HPLC examination were conducted. The pharmacokinetics of CMS and formed colistin were investigated for each product after intravenous administration in rats (28.1 mg/kg CMS; n = 4). Blood was collected over 180 min, and concentrations of CMS and colistin were measured followed by pharmacokinetic analysis. RESULTS: X-GEN, Paddock and Atlantic products, labelled with 150 mg 'colistin base activity', contained 366.8 ± 0.80, 340.6 ± 0.08 and 380.0 ± 5.97 mg CMS (sodium) per vial, respectively; while the Forest product (labelled with 2 000 000 IU) contained 159.3 ± 1.75 mg CMS (sodium). The elemental compositions of the four products were similar; however, the HPLC profile of the Atlantic CMS was different from those of the other three products. The pharmacokinetics of CMS were generally comparable across brands; however, the molar ratios (%) of the AUC0-180min of colistin to CMS (1.68% ± 0.35% to 3.29% ± 0.43%) were significantly different (P = 0.0157). CONCLUSION: This is the first study to demonstrate that although different brands of CMS from various parts of the world have similar elemental compositions, they lead to different exposures to the microbiologically active formed colistin. The study has significant implications for the interpretation of pharmacological studies of CMS conducted in different parts of the world.

[Isolation and characterization of dendritic cells from normal human esophageal mucosa].

AIM: To investigate the characteristics of dendritic cells (DCs) from normal human esophageal mucosa. METHODS: We collected normal esophageal mucosa samples from healthy individuals using endoscope and isolated mononuclear cells from the samples using Ficoll-Hypaque density gradient centrifugation. DCs were further separated by magnetic-activated cell sorting (MACS) to analyze DCs subsets by flow cytometry. RESULTS: Three mononuclear cell populations were detected in normal human esophageal mucosa: HLA-DR(high)/CD13(low);, HLA-DR(med)/CD13(+); and HLA-DR(-)/CD13(+). HLA-DR(high)/CD13(low); cells expressed the DCs-associated molecules and were referred to as esophageal mucosal DCs. The DCs were immature since they expressed low levels of CD80, CD83 and CD86. CONCLUSION: HLA-DR(high)/CD13(low); cells isolated from the normal human esophageal mucosa were proved to be DCs. DCs can be successfully isolated from esophageal mucosa.

[Relation between gastric dendritic cells and H pylori-associated gastritis].

AIM: To investigate the effects of gastric dendritic cells(DCs) in H.pylori-associated gastritis. METHODS: Mononuclear cells were isolated from gastric samples of endoscoped healthy subjects or patients with gastritis by Ficoll-hypaquetechnique. HLA-DR(+);DCs were further isolated by MACS. Rapid urease test, warthin-starry dyer and C(14);-urea breath test were used to detect H.pylori. The relationship between the quantity of gastric DCs and H.pylori density or inflammatory background were analysed. RESULTS: The gastric mononuclear cells from H.pylori-infected normal gastric mucosa contained more HLA-DR(+);DCs (17.93 % ) than that from noninfected subjects (4.93%).The quantity of gastric DCs is positively correlated with H.pylori density. The quantity of gastric DCs were significantly higher in moderate and high inflammatory gastric mucosa than that in low inflammatory gastric mucosa. There was no difference in the quantity of gastric DCs between moderate and high inflammatory gastric mucosa. CONCLUSION: Successfully isolated gastric DCs. DCs may play a role in the early stage of H.pylori-associated gastritis.

[Association between HLA-Cw polymorphism and inflammatory bowel disease].

OBJECTIVE: To investigate the association of HLA-Cw alleles with inflammatory bowel disease(IBD), so as to identify IBD susceptibility gene. METHODS: The HLA-Cw genotype were analyzed in 100 patients with ulcerative colitis (UC), 73 patients with Crohn's disease (CD) and 106 randomly ethnically matched healthy controls by sequence specific primer polymerase chain (PCR-SSP). RESULTS: HLA-Cw*07 gene phenotype frequencies increased in patients with UC (0.430) compared with that in healthy controls (0.226), P = 0.002; while HLA-Cw*12 gene phenotype frequencies increased in patients with CD (0.356) compared with that in healthy controls (0.123), P = 0.000. CONCLUSION: HLA-Cw*07 allele and HLA-Cw*12 allele may be strongly associated with the susceptibility of UC and CD, respectively.

[Studies on the alkaloids from roots of Corydalis impatiens].

OBJECTIVE: To study the alkaloids from Corydalis impatiens. METHODS: The alkaloids were isolated and purified by chromatography and their structures were identified by spectral data and others methods. RESULTS: Seven alkaloids were isolated and identified as bicuculline(1), ochotensine(2), ochotensimine(3), ochrobirine(4), tetrahydrothalifendine(5), norochotensimine(6), N-methylactinodaphnine(7). CONCLUSION: All these compounds are isolated from this plant for the first time.

[ABVD chemotherapy scheme at day 1 and 8 for treatment of primary Hodgkin's lymphomas].

OBJECTIVE: To explore the effect of adriamycin, bleomycin, vincristine and dacarbazinum (ABVD) chemotherapy scheme executed at day 1 and day 8 for primary Hodgkin's lymphomas (HL). METHODS: 62 patients with primary HL in stages II - IV treated in our department from October 2005 to October 2006 were divided into group A and B at random with 31 patients in each group. The patients in group A received ABVD chemotherapy scheme executed at day 1 and day 8 for 6 - 8 cycles. The patients in group B received ABVD chemotherapy scheme executed at day 1 and day 15 for 6 - 8 cycles. The patients of the groups received radiotherapy by the same doctor after chemotherapy according to the patients condition and the radiotherapy regimens were not affected by the grouping. RESULTS: The complete remission rate (CR) in group A after chemotherapy was 90.3% (28/31); the one-year and two-year disease free survival (DFS) rates were 87.1% (27/31) and 80.0% (20/25) respectively. The CR rate in group B after chemotherapy was 83.9% (26/31); the one-year and two-year DFS rates were 80.6% (25/31) and 72.0% (18/25) respectively. The discrepancy of CR rates and the one-year and two-year DFS rates between the two groups was not significant (P > 0.05). The incidences of therapeutic side effects such as myocardial ischemia grade III - IV liver function impairment, pulmonary fibrosis and serious marrow inhibition between the two groups were not significant too (P > 0.05). Average chemotherapy period for the patients in group A was 159 days; it was 69 days shorter than that in group B. CONCLUSION: The CR rate, 1-year DFS rate and 2-year DFS rate of ABVD chemotherapy scheme executed at day 1 and 8 are similar to those of ABVD chemotherapy scheme executed at day 1 and 15 for primary HL in stages II - IV. The side-effects of chemotherapy between group A and B are similar too. The chemotherapy period in group A is shortened significantly.

[Screening and cloning gene of a hepatocyte protein interacting with HCV NS5ATP4A protein].

OBJECTIVE: To screen and clone hepatocyte protein interacting with hepatitis C virus NS5ATP4A protein for studying its biological functions. METHODS: Bait plasmids of hepatitis C virus NS5ATP4A were constructed. After verifying that hepatitis C virus NS5ATP4A protein could be steadily expressed in AH109 yeast strain, yeast-two hybrid assay was performed by mating AH109 with Y187 which pre-transformed with liver cDNA library plasmids pACT2, and the diploidy yeast cells were plated on quadruple dropout (QDO) medium and assayed for X-a-gal activity. Nineteen yeast colonies which grew on QDO and had a-gal activity were obtained, and then the library plasmids were extracted and sequenced. RESULTS: Seven genes were screened out and one of them was a formerly unknown gene. They were associated with RNA synthesis, protein translation, cell cycling and tumor immunity. CONCLUSION: NS5ATP4A binding proteins were successfully screened, which offers new clues for further studying the signal transduction pathway of NS5ATP4A and the pathogenic mechanism of HCV.

[Relationship between expression of inhibitory killer cell immunoglobulin-like receptor HLA-Cw ligand and susceptibility to inflammatory bowel disease].

OBJECTIVE: To investigate the distribution of different phenotypes of inhibitory killer cell immunoglobulin-like receptor (iKIR) and its ligand HLA-Cw in patients with inflammatory bowel disease (IBD), and to explore whether iKIR/HLA-Cw combinations are associated with IBD susceptibility. METHODS: Peripheral blood samples were collected from 100 patients with ulcerative colitis (UC), 73 patients with Crohn's disease, and 106 randomly selected ethnically matched healthy controls. Polymerase chain reaction with sequence-specific primers was used to analyze the iKIR phenotypes, and the HLA-Cw phenotypes was examined with HLA-C LOCUS SSP UNITRAY kit, and the combination of HLA-Cw and its corresponding iKIR in individual was analyzed subsequently. RESULTS: The KIR2DL1 and KIR2DL3 gene phenotype frequencies in UC patients were 0.710 and 0.620 respectively, both significantly lower than those in healthy controls (0.896, chi(2) = 11.405 P = 0.001, and 0.821, chi(2) = 10.362 P = 0.001 respectively). The KIR2DL1 gene phenotype frequency in CD patients was 0.740, significantly lower than that in healthy controls (0.896, chi(2) = 7.589, P = 0.006). The KIR2DL1 HLA-C2 combination (2DL1(+)-HLA-C2(+)) in UC patients and CD patients were 0.380 and 0.411 respectively, both significantly lower than that in healthy controls (0.575, chi(2) = 7.876 P = 0.005 and 0.575, chi(2) = 4.681 P = 0.030 respectively). CONCLUSION: The susceptibility to IBD is associated with decreased KIR2DL1-HLA-C2 combination.

[Construction and identification of matrix metalloproteinase inhibitor-1 siRNA eukaryotic expression vectors].

OBJECTIVE: To construct TIMP-1 siRNA eukaryotic expression vectors and evaluate their effect on TIMP-1 mRNA expression in hepatic stellate cells. METHODS: The combinant lone DNA with cutting sites of BamH I and Xho I enzyme according to the sequences of 447-465, 552-540 TIMP-1 of rats and nonspecific sequence were selected and cloned to pGEM-T vector and sub-cloned to pRNAT-U6.2. They were then identified by double enzyme digestion analysis and DNA sequencing. Three plasmids were transfected into T6 separately through an oligofectamine package. TIMP-1 mRNA expression was evaluated by RT-PCR. RESULTS: Targeting sequences of TIMP-1 siRNA eukaryotic expression vectors were correct. TIMP-1 mRNA expression was significantly reduced by transfecting them into the T6. CONCLUSION: We successfully constructed two TIMP-1 siRNA eukaryotic expression vectors and the transfected cells can significantly suppress the TIMP-1 expression.

US findings of bilateral primary breast cancer: retrospective study.

BACKGROUND: For women with breast cancer, the contralateral breast is at high risk. The bilateral cancers may be synchronous or metachronous. If the bilateral breast cancers have similar ultrasonography (US) appearances, the US findings of the first breast cancer (index cancer) might lead to early detection of the contralateral cancer. The purpose of this study was to identify the US characteristics of bilateral breast cancer and to determine whether bilateral breast cancers have similar US appearances and whether the US findings for one breast cancer might be predictive of the contralateral breast cancer. METHODS: We retrospectively reviewed the US manifestations of 58 patients with surgically proven bilateral primary breast cancer and compared the contralateral cancer with the index cancer by evaluation the margin, shape, inside echoes, posterior attenuation, calcification and color flow signals of 58 lesion pairs to investigate whether the bilateral breast cancers have similar US appearances. RESULTS: Bilateral primary breast cancers were more located in upper outer quadrant, frequently spiculation, taller than wide shape, with irregular margin, heterogeneous internal echo and acoustic shadowing, containing microcalcification and abundant color flow signals. The most common US appearances were taller than wide shape (75.0%, 87/116), irregular margins (79.3%, 92/116) and heterogeneous internal echo (86.2%, 100/116). Of the total 58 lesion pairs, 18 (31.0%) pairs had similar US characteristics, whereas 40 (69.0%) pairs had different US characteristics. CONCLUSIONS: US signs of the index cancer do not indicate the most likely appearance of the second cancer in the contralateral breast. Evaluation of the contralateral cancer should be performed without regard for the US findings for the index cancer.

[Expression of Crg-2 by a bacteria recombinant system of adenovirus].

AIM: To express an immunochemotactic and angiostatic anti-tumor molecule, CXC chemokine Crg-2, by E. coli recombinant system of adenovirus. METHODS: By E. coli recombination system of adenovirus, the shuttle vector pShuttle-cmv/crg-2 was co-transfected into BJ5183 E. coli with adenovirus backbone plasmid pAdEasy-1. The recombinants were transfected into 293 packaging cells and adenovirus was packaged and amplified. The molecular weight and bioactivity of recombinant protein were determined by Western blot and chemotaxis assay, respectively. RESULTS: Recombinant adenovirus genome backbone bearing crg-2 gene, pAd/crg-2, was successfully obtained as evidenced by endonucleases digestion and PCR. Adenovirus was packaged and amplified to a titre of 4x10(9) TCID50/L. The cultured supernatant of the infected 293 cells contented a protein of nearly 10,000 and presented a significantly chemotatic effect towards activated spleen lymphoblasts. CONCLUSION: Recombinant adenovirus Ad/crg-2 obtained by E. coli recombinant system of adenovirus can efficiently express bioactive Crg-2 chemokine.

[Study of huchang qingfei pellets on immune function in rats infected with Mycoplasma pneumoniae].

OBJECTIVE: To study the effects and mechanism of Huchang Qingfei pellets on immune function in rats infected with mycoplasma pneumoniae. METHOD: A rat model of mycoplasmal pneumonia (MP) was developed in repeated intranasal infectious routes and then the humoral and cellular immunocompetences were detected by radioimmunoassay, immune-turbidimetry and flow cytometry. RESULT: The levels of serum IgG,IgM and IL-2, IL-6 were enhinced obviously, the complement C3 and TNF-alpha were decreased and the ratio of CD4+ /CD8+ was improved significantly in the Huchang groups as compared with MP model group. CONCLUSION: Huchang Qingfei pellets can reinforce immune function via preventing both cellular and humoral immunity from depression in the rats with MP.

[Effect of Huchang Qingfei concentrated pellets on E-cadherin expression in lung tissue of mice infected with Mycoplasma pneumoniae].

OBJECTIVE: To investigate the effects of Huchang Qingfei concentrated pellets on the expression of E-cadherin (E-cd) in the lung tissue from mice infected with Mycoplasma pneumoniae (MP). METHOD: A mice model of Mycoplasmal pneumonia (MPP) was developed by repeatedly intranasal infectious route. Transmission electronic microscope (TEM) and immunohistochemistry stain were performed to observe the pathological changes and expression of E-cd in lung tissues. RESULT: Under TEM it was found that the cellular membrane was ruptured, mitochondria was denatured, crista was broken in the pulmonary cells of the model group; the all above parameters in Huchang medicated group were improved obviously. The immunohistochemistry test showed that strong positive brown stain of E-cd expression was found in the pulmonary epithelial cell membrane and bronchial periphery in the model group, however, in the medicated group, the E-cd expression level in the cellular membrane was decreased and the expression ratio was dropped significantly as compared with the model controls. CONCLUSION: Huchang Qingfei concentrated pellets can inhibit the overexpression of E-cd in the lung tissue of mice with MP-infection, which may be helpful for prevention and treatment of pulmonary injury caused by MPP.