Identification of differential inflammation factors in nephroblastoma tissue and clinical significance / 中华泌尿外科杂志

Objective To identify the differential inflammation factors in nephroblastoma tissue using proteomics technology and analyze its relationship with clinical stage,pathological phenotype,lymph node metastasis,vascular invasion.Methods From Jan 2010 to Dec 2014,nephroblastoma tumor tissues from 40 patients were obtained.Meanwhile,the 35 tissue near proximal kidney and 25 tissues distal kidney were also obtained.The classification of clinical stage included Ⅰ stage in 6 cases,Ⅱ stage in 12 cases,Ⅲ stage in 13 cases and Ⅳ stage in 9 cases.Other characters contained good prognosis type in 37 case,poor prognosis type in 3 cases,lymphatic metastasis in 17 cases,no sign of lymphatic metastasis in 23 cases,vascular invasion in 9 cases and non-vascular invasion in 31 cases.The SELDI-TOF-MS was used for screening differential protein peaks among three groups.Then,SPE and TRICINE-SDS-PAGE were used to separate and purificate the protein,which showed high peaks expression in tumor tissue,respectively.After in-gel digestion,we received the identification of targeted proteins according to sequence information through Nano-LC-MS/MS.Finally we compared differential expression of inflammatory peaks in different groups of clinical stage,pathological type,lymph node metastasis and vascular invasion.Results All the peaks high expression in tumor tissue,m/z12138 and m/z 13462 are identified as MIF and NAP-2.Expression of two protein peaks in tumor tissue(1437.8 + 997.3,1730.4 + 1147.8) is higher than those in proximal tissue (952.6 + 591.2,1031.1 + 1120.8) and in distal tissue(315.4 + 296.5,114.7 + 118.9),which showed the significant difference (P ＜ 0.001).According to the clinic stage classification,the expression of those protein were 678.8 + 189.0,746.2 + 238.7 in stage Ⅰ,664.0 + 202.0,1180.7 + 404.9 in stage Ⅱ,1524.7+407.9,2160.4 + 1252.3 in stage Ⅲ and 2850.2 + 861.2,2498.4 + 1290.5 in stage Ⅳ.Based on the other characters,expression of those protein were the 1271.7 + 809.2,1553.3 + 991.4 in good prognosis type,3487.2 + 166.2,3915.1 +507.3 in poor prognosis type,2207.1 +961.7,2569.5 + 1285.2 in lymph node metastasis,869.2 + 474.6,1110.2 + 433.6 in non-lymph node metastasis,2850.2 + 861.2,2498.4 +1290.5 in vascular invasion and 1027.8 + 521.3,1507.5 + 1019.9 in non-vascular invasion.All the comparison results have significant statistical difference (P ＜ 0.001).Conclusion MIF and NAP-2significantly increase in nephroblastoma tumor tissue.Meanwhile,there was obvious relationship between those protein with clinical stage,pathological type,lymph node metastasis and vascular invasion.

In silico experiment with an-antigen-toll like receptor-5 agonist fusion construct for immunogenic application to Helicobacter pylori.

BACKGROUNDS: Helicobacter pylori colonize the gastric mucosa of half of the world's population. Although it is classified as a definitive type I carcinogen by World Health Organization, there is no effective vaccine against this bacterium. H. pylori evade the host immune response by avoiding toll-like detection, such as detection via toll-like receptor-5 (TLR-5). Thus, a chimeric construct consisting of selected epitopes from virulence factors that is incorporated into a TLR-5 ligand (Pseudomonas flagellin) could result in more potent innate and adaptive immune responses. MATERIALS AND METHODS: Based on the histocompatibility antigens of BALB/c mice, in silico techniques were used to select several fragments from H. pylori virulence factors with a high density of B- and T-cell epitopes. RESULTS: These segments consist of cytotoxin-associated geneA (residue 162-283), neutrophil activating protein (residue 30-135) and outer inflammatory protein A (residue 155-268). The secondary and tertiary structure of the chimeric constructs and other bioinformatics analyses such as stability, solubility, and antigenicity were performed. The chimeric construct containing antigenic segments of H. pylori proteins was fused with the D3 domain of Pseudomonas flagellin. This recombinant chimeric gene was optimized for expression in Escherichia coli. The in silico results showed that the conserved C- and N-terminal domains of flagellin and the antigenicity of selected fragments were retained. DISCUSSION: In silico analysis showed that Pseudomonas flagellin is a suitable platform for incorporation of an antigenic construct from H. pylori. This strategy may be an effective tool for the control of H. pylori and other persistent infections.

Screening of antigenic epitope of neutrophil-activating protein of Helicobacter pylori by phage display peptide Hbrary / 中华微生物学和免疫学杂志

Objective To identify epitope of neutrophil-activating protein(NAP) of Helicobacter pylori(Hp).Methods Using the mouse monoclonal antibodies against NAP as selective molecular and immunoscrcening phage-display random 7-peptides library.The positive clones were sequenced and analyzed.Phage clones were chosen to immunize mice and to evaluate the potential of phagotopes as effective vaccines.Results One mimotope(FAHLATQ)showed a good match with the NAP at 140-143 AA(AHLA)and the serum of mice induced by the phage clone clearly recognized NAP.Conclusion This study suggests thatthe antigenic epitope could be mapped through screening the phage-display peptide library with monoclonalantibody and a mimotopo of NAP providing an ahernative approach for the diagnosis and development of avaccine for Hp.

Preliminary study on immunotherapy of an oral recombinant DNA vaccine of Helic obacter pylori neutrophil activating protein / 中华消化杂志

Objective To construct an oral recombinant DNA vaccine of Helicobacter pylori(H. pylori) neutrophil activating protein (Hp-NAP), and to evaluate its immunotherapeutic effects. Methods The napA gene (encoding Hp-NAP) was amplified by poly mera se chain reaction(PCR) and cloned into TA cloning vector pBT. After nucleotide s equencing and sequence analysis, the target sequence was subcloned into an eukar yot ic expression vector pIRES. Then the identified recombinant plasmid, pIRES-napA , was transformed into a live attenuated Salmonella typhimurium(S. typhimurium ) strain SL7207, and lavaged into a long-term(30 weeks) model of BALB/c mice infected by Sydney strain(SS1) of H. pylori. Results A 435 bp target gene of napA was amplified by PCR. Seq uenci ng and BLAST analysis showed that most of the cloned napA sequence was homologou s with that of SS1 strain of H. pylori. provided by GenBank, and the homolog y of neucleotide and protein was over 98%, respectively. PCR and restriction enzyme digestion id entification indicated that a recombinant live attenuated S. typhimurium DNA vaccine strain carrying Hp-napA gene was successfully constructed. After 4 wee ks of oral immunization, 75% of mice treated with DNA vaccine were rapid urease test negative, while those with vacant plasmid or normal saline alone were all p ositive (P= 0.0476). The titer of serum Hp-NAP antibody was signific antly elevated in treatment group. Conclusions The successful construction of an effective oral recom binant DNA vaccine of Hp-NAP may be helpful for the further development of polyvalent DNA vaccine against H. pylori infection.