Objective To investigate the serotypes of human enterovirus B ( HEV-B) species cau-sing hand, foot and mouth disease ( HFMD) and to analyze the genetic characteristics of VP1 region in cox-sackievirus B2 ( CVB2 ) and coxsackievirus B5 ( CVB5 ) strains circulating in Anyang area during 2011 to 2015. Methods Real-time RT-PCR and semi-nested RT-PCR were performed to identify coxsackievirus A16 (CVA16), enterovirus 71 (EV71) and other serotypes of enterovirus in order to obtain the complete etiologic composition of HFMD. The numbers of HEV-B serotypes and the percentages of specimens positive for every serotype in all enterovirus-positive specimens were calculated. As CVB2 and CVB5 were the pre-dominant serotypes of HEV-B species, five pairs of primers targeting the VP1 regions of CVB2 and CVB5 were designed to obtain the complete nucleotide sequences of CVB2 and CVB5 VP1 regions. The phylogenet-ic trees were constructed based on the VP1 sequences obtained in this study and those submitted to GenBank by using MEGA7. 0 and BioEdit7. 2. The selection pressures on VP1 regions of CVB2 and CVB5 strains cir-culating in China in recent years were evaluated with the online program of DataMonkey. Results A total of 57 specimens that belonged to 14 serotypes of HEV-B species were detected in Anyang area from 2011 to 2015. The 14 serotypes of HEV-B species accounted for 56% of all serotypes of enterovirus and the speci-mens positive for HEV-B species accounted for 3. 06% of all enterovirus-positive specimens. The HFMD ca-ses caused by most of the HEV-B serotypes were sporadic cases. Small outbreaks of HFMD could also be caused by some serotypes of HEV-B such as CVB2 and CVB5. The complete sequences of VP1 region were obtained from 8 CVB2 strains and 9 CVB5 strains. The phylogenetic trees based on the VP1 sequences dem-onstrated that the CVB2 strains were classified into four genotypes ( A-D) . The mean evolutionary distances between different genotypes ranged from 0. 191 to 0. 208 and the similarities in nucleotide sequences ranged from 79. 7% to 85. 8%. The CVB5 strains were classified into 6 genotypes (A-F). The mean evolutionary distances and the similarities in nucleotide sequences between different genotypes of CVB5 strains ranged from 0. 170 to 0. 285 and 76. 0% to 86. 8%, respectively. Strains of different genotypes varied significantly in the residues on positons 157 and 263 in the VP1 region of CVB2 strains and on positions 75, 90 and 95 in the VP1 region of CVB5 strains. All of the CVB2 strains isolated in Anyang area belonged to D genotype and located intensively in one lineage. The CVB5 strains circulated in Anyang area belonged to F genotype and located in two lineages. The selection pressures on CVB2 strains of D genotype and CVB5 strains of F geno-type circulating in China in recent years were 0. 037 and 0. 036, respectively. Six positively selected amino acid sites were found in the VP1 region of CVB5 strains, but no positively selected amino acid site was found in the VP1 region of CVB2 strains. Conclusion HEV-B species was an essential component of the etiologic spectrum of HFMD in Anyang area during 2011 to 2015, of which CVB5 and CVB2 were the predominant se-rotypes. The VP1 region of CVB5 was more complex and active than that of CVB2 over the course of evolution.

Objective To investigate the essential role of Toll-like receptor (TLR) signaling in dendritic cells in rejection responses of skin transplantation in mice.Method Immature wild-type BALB/c bone marrow dentritic cells (BMDCs) were stimulated by a TLR agonist,CpG,in the presence of ST2825,an inhibitor of key molecule myeloid differentiation factor 88 (MyD88) in TLR signal transduction,or in the absence for 24 h in vitro.Co-stimulatory molecules CD80 and CD86 were analyzed by flow cytometry.Wild-type C57BL/6 na ve T cells stained with CFSE,a fluorescent dye,were co-cultured with BALB/c DCs for 3 days in the presence or absence of ST2825.CD3 +/CFSE+cells were analyzed by flow cytometry.Minor antigen-mismatched (HY-mismatched) skin allograft model was employed in this study in which the donors were all males and the recipients were all females.The experimental groups were divided as follows:the control group,the donors and the recipients were all wide type BALB/c mice; the MyD88 group,the donors and the recipients were all MyD88-konckout BALB/c mice; the DCs group,the donors and the recipients were all MyD88-knockout BALB/c mice and the recipients were transfused with donor-sourced DCs after transplantation; the experimental group,the donors and the recipients were all MyD88-knockout BALB/c mice and the recipients were transfused with donor-sourced DCs pre-treated with ST2825after transplantation.The survival time of skin allografts was observed.Result ST2825 could dose dependently inhibit the high level expression of co-stimulatory molecules CD80 and CD86 induced with CpG.Similarly,and it could also dosedependently inhibit the allo-reactive T cell proliferation upon the stimulation with DCs.The survival time of every group was as follows:the control group (22.8 ±2.8) days vs.the MyD88 group (＞100 days) vs.the DCs group (9.7± 2) days vs.the experimental group (＞100 days,P＜0.05,vs.the DC group).Conclusion DCs may play an essential role in rejection responses of transplantation via TLR signaling.The inhibitor of TLR signaling,ST2825,could inhibit the activation of DCs and the biological functions,which might be contributed to the inhibitory effects of ST2825 on DCs maturation and indirect suppression on the proliferation of allo reactive T cells.

Objective To investigate the expression of COX-2,MMP-2 and TIMP-2 ,the pathological fea-tures ,and their relationship in breast cancer. Methods The expressions of COX-2 ,MMP-2 and TIMP-2 were deter-mined by S-P immunohistochemical method on tissue chips,which containing 127 cases of breast carcinoma. Results The positive rates of COX-2,MMP-2 and TIMP-2 protein were 81.1 (103/127)% ,96.9(123/127)% and 60.6 (77/127) % respectively;The expression of COX-2 was positively related to auxiliary lymph node metastasis and TNM staging (P<0.01 and P<0.05, respectively), and inversely related to PR expression (P<0.05). Further-more,the expression of COX-2 was positively correlated with MMP-2 (r=0. 290 ,P<0.01). Conclusions The ex-pression of COX-2 might be closely related to the invasion and metastasis of breast cancer and has a close relation-ship with MMP-2. The levels of MMP-2 might be partly regulated by COX-2.

Objective To observe the inhibitory effect of tumstatin related peptide T3 mediated by short peptide to osteosarcoma vascular. Methods Through MTS assay, wound healing assay, the inhibitory effect of targeting-T3 peptide and T3 peptide on the human umbilical veil endothelial cell was studied in vitro. After the preparation of 50 nude mice model bearing osteosarcoma, the nude mice bearing too large or too small tumors were eliminated and the left ones were divided into 4 groups (6 animals for each group: T3 peptide, targeting-T3 peptide, CTX, PBS) randomly. Through weight of tumor, histopathologicol slice and immunohistochemical methods. The inhibitory action of targeting-T3 peptide and T3 peptide on the neoge-netic vascular of osteosarcoma implanted in nude mouse was studied. Results In vitro, both T3 peptide and targeting-T3 peptide effectively inhibited the proliferation of human umbilical veil endothelial cell. In the experiment of vivo, the average weight of tumor of targeting-T3 peptide group was (1.104?.247) g, the average weight of the T3 peptide group was (1.484?.369) g. There was the statistical difference in tumor inhibition on the osteosarcoma betweent the targeting-T3 group and T3 group (F=16.353, P=0.000). The positive rate of vascular endothelial growth factor and metastasis in the lung in the targeting-T3 peptide group all descended than the T3 peptide group. Conclusion Because of the short peptide to osteosarcoma vascular, targeting-T3 peptide could significantly restrain the development of osteosarcoma. Coupling short peptide to T3 peptide increase the selective binding of T3 peptide to osteosarcoma vascular.

PURPOSE: This study aimed to investigate the effect of adipose-derived stem cell (ADSC) transplantation on atherosclerosis (AS) and its underlying mechanisms. MATERIALS AND METHODS: In our study, rat AS model was established, and ADSCs were isolated and cultured. Atherosclerotic plaque and pathological symptoms of thoracic aorta were measured by Oil Red O staining and Hematoxylin-Eosin staining, respectively. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured by an automatic biochemical analyzer. Expressions of vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), aortic endothelin-1 (ET-1), interleukin-6 (IL-6), c-reactive protein (CRP), and tumor necrosis factor α (TNF-α) were measured by enzyme linked immunosorbent assay, VEGF, VCAM-1, ICAM-1, ET-1, respectively, and NF-κB p65 mRNA expressions were detected by quantitative real-time polymerase chain reaction. Protein expressions of VEGF, VCAM-1, ICAM-1, ET-1, NF-κB p65, p-NF-κB p65, and IκBα were measured by western blot. Moreover, NF-κB p65 expression was measured by immunofluorescence staining. RESULTS: ADSC transplantation alleviated the pathological symptoms of aortic AS. ADSC transplantation decreased the levels of TC, TG, and LDL-C and increased serum HDL-C level. Meanwhile, ADSC transplantation decreased the levels of IL-6, CRP, and TNF-α in AS rats. Moreover, the expressions of VEGF, ET-1, VCAM-1, and ICAM-1 were decreased by ADSC transplantation. ADSC transplantation inhibited phosphorylation of NF-κB p65 and promoted IκBα expression in AS rats. CONCLUSION: Our study demonstrated that ADSC transplantation could inhibit vascular inflammatory responses and endothelial dysfunction by suppressing NF-κB pathway in AS rats.
Animals ; Aorta, Thoracic ; Atherosclerosis ; Blotting, Western ; C-Reactive Protein ; Cholesterol ; Endothelin-1 ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Intercellular Adhesion Molecule-1 ; Interleukin-6 ; Lipoproteins ; Phosphorylation ; Plaque, Atherosclerotic ; Rats ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Stem Cell Transplantation ; Stem Cells ; Triglycerides ; Tumor Necrosis Factor-alpha ; Vascular Cell Adhesion Molecule-1 ; Vascular Endothelial Growth Factor A

Objective To fulfill high-efficient expression of rTMP-GH,a recombinant fusion protein of thrombopoietin mimetic peptide and human growth hormone,in Pichia pastoris.Methods cDNA fragment coding for rTMP-GH was amplified by PCR,and subsequently cloned into the expression vector pPIC9K.The linearlized plasmid pPIC9K-rTMP-GH was transformed into Pichia pastoris GS115 cells and screened in MD/MM plates under pressure of G418.The recombinant fusion protein rTMP-GH was identified by Western blotting,and its biological activity was analyzed by its ability on colony formation of megakaryoblasts.Results The pPIC9K-rTMP-GH expression plasmid was successfully constructed.GS115 cells with high expression of rTMP-GH was screened out,and the rough purified rTMP-GH showed promotive effect on megakaryoblast colony formation.Conclusion High-efficient expression of rTMP-GH in Pichia pastoris is achieved.

Objective To establish a rabbit model of restenosis and analyze the expressions of VEGFmRNA and TGF-?_1mRNA during the intimal proliferation.We also explored the relationship between VEGFmRNA,TGF-?_1mRNA and restenosis.Methods 40 healthy male New Zealand white rabbits were evenly divided into three injury groups and one control group.Right carotid arteries were injured with PCI balloon in the injury groups.10 rabbits of each injury group were sacrificed on weeks 1,2 and 4 after the injury.VEGFmRNA and TGF-?_1mRNA were examined by in situ hybridization.All the samples were analyzed using a computerized imaging analysis system.Results In the injury groups,neointimal areas were significantly larger than those in control group(P

Objective To investigate the effects of rTMP-GH, a recombinant fusion protein of thrombopoietin mimetic peptide (TMP) and human growth hormone (GH), on the proliferation and thrombocytopoiesis of cultured megakaryocytes. Methods After being treated with 100 ng/ml of rTMP-GH for 7 d, Dami cells, a kind of megakaryocyte cell line, were analyzed by observing the numbers of colony forming unit. Meanwhile, Western blotting and RT-PCR were applied to detect the expression changes of globin transcription factor-1 (GATA-1), which is a main regulator of thrombocytopoiesis in megakaryocytes. Results The numbers of colony forming unit were markedly increased in cultured Dami cells incubated with rTMP-GH. Compared with normal control and GH treatment groups, both the mRNA and protein levels of GATA-1 were up-regulated significantly in Dami cells treated with rTMP-GH. Conclusion rTMP-GH has a strong ability to promote the proliferation and thrombocytopoiesis of megakaryocytes.

OBJECTIVE: To observe the micromorphological characteristic of bacterial biofilm on mucosa of chronic rhinosinusitis (CRS). METHOD: Mucosa samples of middle turbinate were obtained from 4 patients of CRS during ESS. The size of each sample was about 4 mm x 4 mm. The samples were fixed in 4% paraformaldehyde for 24 hours, then fixed in 1% osmium tetroxide for 2 hours, graded dehydration with ethanol, dried with carbon dioxide and sputter coated with gold. The ultrastructure of these samples was observed by scanning electron microscope. RESULT: Bacterial biofilms were found on samples in all 4 patients. The biofilms were mainly formed on the surface of cilia. The bacterial flagella and cilia were intertwined. The biofilms could be found in a lot kinds of bacterial infections or mixed infections which were caused by multiple bacteria and fungi. CONCLUSION Bacterial biofilm could be formed on ciliated epithelia.
Bacterial Infections ; microbiology ; Biofilms ; Cilia ; microbiology ; ultrastructure ; Epithelium ; microbiology ; ultrastructure ; Humans ; Microscopy, Electron, Scanning ; Sinusitis ; microbiology

Objective To establish a magnetic nanoparticles separation-based quantitative real-time PCR(RT-PCR)assay for fast and accurate detection of Plasmodium falciparum and providing a technical support for improving the control and preven-tion of imported malaria. Methods According to the conserved sequences of the P. falciparum genome 18SrRNA,the species-specific primers and probe were designed and synthetized. The RT-PCR was established by constructing the plasmid standard , fitting the standard curve and using magnetic nanoparticles separation. The sensitivity and specificity of the assay were evaluat-ed. Results The relationship between the threshold cycle(Ct)and logarithm of initial templates copies was linear over a range of 2.5×101 to 2.5×108 copies/μl(R2=0.999). Among 13 subjects of entry frontier,a P. falciparum carrier with low load was de-tected by using the assay and none was detected with the conventional examinations(microscopic examinations and rapid tests). Conclusion This assay shows a high sensitivity in detection of P. falciparum,with rapid and accurate characteristics,and is especially useful in diagnosis of P. falciparum infectors with low parasitaemia at entry-exit frontier ports.