Inactivated Sendai virus suppresses murine melanoma growth by inducing host immune responses and down-regulating ß-catenin expression.

OBJECTIVE: This paper aims to investigate the anti-tumor mechanism of inactivated Sendai virus (Hemagglutinating virus of Japan envelope, HVJ-E) for murine melanoma (B16F10). METHODS: The murine dendritic cells (DCs) were treated with HVJ-E, and then the cytokines secreted from DCs and costimulation-related molecules on DCs were measured. Meanwhile, the expression of ß-catenin in HVJ-E treated murine melanoma cells was detected. In addition, HVJ-E was intratumorally injected into the melanoma on C57BL/6 mice, and the immune cells, CTL response and tumor volume were analyzed. RESULTS: HVJ-E injected into B16F10 melanoma obviously inhibited the growth of the tumor and prolonged the survival time of the tumor-bearing mice. Profiles of cytokines secreted by dendritic cells (DCs) after HVJ-E stimulation showed that the number of cytokines released was significantly higher than that elicited by PBS (1P<0.05). The co-stimulation-related molecules on DCs were comparable to those stimulated by LPS. Immunohistochemical examinations demonstrated the repression of ß-catenin in B16F10 melanoma cells after HVJ-E treatment. Meanwhile, real-time reverse transcription PCR revealed that HVJ-E induced a remarkable infiltration of CD11c positive cells, chemokine ligand 10 (CXCL10) molecules, interleukin-2 (IL-2) molecule, CD4(+) and CD8(+) T cells into HVJ-E injected tumors. Furthermore, the mRNA expression level of ß-catenin in the HVJ-E injected tumors was also down-regulated. In addition, B16F10-specific CTLs were induced significantly after HVJ-E was injected into the tumor-bearing mice. CONCLUSION: This is the first report to show the effective inhibition of melanoma tumors by HVJ-E alone and the mechanism through which it induces antitumor immune responses and regulates important signal pathways for melanoma invasion. Therefore, HVJ-E shows its prospect as a novel therapeutic for melanoma therapy.

[Isolation of avian Escherichia coli and PCR detection of their F1 and HPI genes].

To further investigate into the prevalence of F1 fimbriae and high pathogenicity island (HPI) in avian Escherichia coli, a total of 69 bacteria isolates (29 from geese and 40 from chickens) were obtained from deceased poultry and characterized to be Escherichia coli by gram staining, culture characterizing and bio-chemical testing. Two sets of primers were designed or analyzed with DNAStar software based on the F1 and HPI sequences that deposited in GenBank and synthesized by Sangon Biological Engineering Technology and Service Co. Ltd. (Shanghai, P. R. China). All the primers were tested for their specificities by using reference E. coli strains, and it is confirmed that the PCR using primers F1-F and F1-R could identify F1+ E. coli, as well as the PCR using primers irp-F and irp-R could identify HPI+ E. coli. All the isolates were submitted to PCR detection, and the data shows 46 isolates (66.7%) were F1-positive, 10 isolates (14.5%) were F1+ HPI-positive and 2 isolates (20.0%) were HPI-positive. Furthermore analysis indicated that the prevalence of F1 and HPI have no different between the isolates from geese and chickens, and no different among the isolates from different tissue, such as liver, lung and duodenum. In addition, all the 69 E. coli isolates serological typed, and the results show that the isolates from geese were belonged to O26 (25.0%), O78 (12.5%), O18 (12.5%) and O117 (12.5%), while E coli from chickens were fell into O109 (37.5%), O24 (18.75%), O18 (12.5%), O139 (12.5%) and O78 (6.25%). Seven kinds of antibiotics were tested on 11 isolates, and it revealed that most isolates were sensitive to cefazolin, nitrofurantoin and gentamycin, while resistant to lincomycin, tetracycline and polymycin B.

Molecular structure, phylogenetic analysis, tissue distribution, and function characterization of interferon-γ-inducible lysosomal thiol reductase (GILT) gene in sheep (Ovis aries).

Interferon-γ-inducible-lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, a sheep cDNA, sGILT, encoding GILT protein was isolated from the spleen cDNA library of Ovis aries. It codes for a deduced protein of 244 amino acids with a putative molecular weight of 27.6 kDa, which has all the typical structural features of GILT protein including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 5 conserved cysteines. Sequence comparison indicated the amino acid sequence of sGILT showed high identity to cow GILT (93.03%). Phylogenetic analysis showed that sGILT and cow GILT shared the greatest homology. The result of real-time PCR suggested that sGILT mRNA was expressed in a tissue-specific manner and obviously up-regulated in splenocytes and PBMCs after induction with lipopolysaccharide (LPS). Recombinant sGILT fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Its expression was confirmed by SDS-PAGE and Western blotting analysis. Thiol reductase activity was assessed using antibody as the substrate. These results suggest that sGILT has the activity of disulfide bond reduction and indicate that sheep also express a protein that has been found to maintain first line of innate immune defense at basal level.