Aim DNA vaccinating BALB/c mice with the constructed recombinant plasmid, pcDNA3, containing ROP1 gene from Toxoplasma gondii to observe the effect on the production of the cytokines, IFN- γ、 IL - 2 , and NO in the immunized mice. Methods Large-scale preparation of plasmid DNA by alkaline lysis,the DNA were injected through muscles of left leg in each mouse at the dosage of 100μg. A booster vaccine was given at the same dosage after two weeks. Control groups were injected with pcDNA3 blank plasmid and normal saline respectively. After 30,50 and 70 days of the booster injection, following tests were carried out 3 times separately :the serum IFN-γand IL-2 were detected by sandwich ELISA;the NO was detected by enzyme assay. Results The 3 times detected results of IFN-γγ、IL-2 and NO were significant higher in the vaccinated group than that of in control groups and the contents were increased with the vaccinated time prolonged. Conclusion The IFN - γγ、 IL - 2 and NO were produced by vaccinating BALB/c mice with the recombinant plasmid, pcROP1.

Objective To investigate the association between inflammatory biomarkers and survival in patients with stable chronic obstructive pulmonary disease(COPD).Methods A total of 1038 patients with stable COPD from January 2008 to December 2009 were included in a prospective cohort study.Clinical characteristics,pulmonary function tests,6 min walk test and a modified British Medical Research Council dyspnea scale (MMRC) were completed.Fasting blood was obtained to detect inflammatory biomarkers including neutrophils,C-reactive protein(CRP),fibrinogen,TNF-a,IL-6 and IL-8.Participants were evaluated every 3 months,all-cause mortality was used as the end event.Results 120 patients (9.2％) died in the period of follow-up.Kaplan-Meier analysis showed that 1 year,2-year-and 3-year survival rates were 94.4％,88.3％ and 84.0％,respectively.Compared with survivors,those who died had a higher level of inflammatory biomarkers.Cox multivariate regression analysis demonstrated that the independent risk factors for death were neutrophils (HR:1.262,95％CI:1.143-1.512,P=0.035),CRP (HR:1.234,95％CI:1.097-1.624,P=0.029),fibrinogen (HR:1.327,95％CI:1.141-1.619,P=0.026),TNF-α (HR:1.124,95％CI:1.043-1.659,P=0.045),IL-6 (HR:1,429,95％CI:1.237-1.816,P=0.014) and IL-8 (HR:1.188,95％CI:1.024-1.383,P=0.042).C statistical analysis showed that no single biomarker significantly improved the C statistic value on the base of clinical model,but it was further improved by the addition of all biomarkers (C =0.764,P =0.010).Conclusions The level of inflammatory biomarkers in the death with stable COPD is significantly increased.Age.BODE index,neutrophils,CRP,fibrinogen,TNF-α,IL-6 and IL-8 are independent risk factor for the prediction of mortality in patients with COPD.

Objective To establish and maintain the life cycle of Clonorchis sinensis in laboratory.Methods Adult worms and eggs of Clonorchis sinensis were collected from naturally infected cats.Eggs were ingested by freshwater snails in aquarium.When the cercariae were released from infected snails, they invaded into freshwater fishes.From the 30th day on after the release of cercariae, the infection rate and metacercariae density in freshwater fishes were determined.Results After 95 days the infected snails began shedding cercariae in a temperature range of 24.3-37.2 ℃, and no cercariae were found under 20 ℃.The infection rate in the snails Parafossarulus striatulus and Alocinma longicornis was 12.5% and 18.0%, respectively.Metacercariae were found in fish at 30 days after cercariae infection, and matured metacercariae were detected in 45 days.The number of metacercariae per gram of fish meat in Pseudorasbora parva, Ctenopharyngodon idellus, Rhodeus sinensis, Hypophthalmichthys nobilis, Cirrhinus molitorella, Carassius auratus, Cyprinus carpio and Oreochromis niloticus was 1 792, 16, 8, 6, 5, 4, 4, and 2, respectively.Rats and cats were fed with metacercariae from fish to receive adult worms.Conclusion Life cycle of Clonorchis sinensis has been established and maintained in the laboratory.

Objective　To construct the eukaryotic expression recombinant plasmid, pcIFN-γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3-rhoptry protein 1 （pc-ROP1） combined with pcIFN-γ against Toxoplasma gondii (T. gondii) infection in mice. Methods　A fragment of the IFN-γ gene was directly inserted into the pcDNA3 plasmid and identified by two restriction endonucleases digestion. pcIFN and pcROP1 DNA was injected into the left leg muscle of mice at a dosage of 100*!μg, and a booster vaccination was given at the same dosage after two weeks. Control groups were injected with pcDNA3 blank plasmid or normal saline. At 30, 50 and 70 days after booster injection, kinetic tests were carried out: MTT assay for the proliferation response of T lymphocyte cells and the activity of NK cells, sandwich ABC-ELISA for the determination of IFN-γ, IL-2 and IL-10; a serum enzymetic aassay for nitric oxide (NO) in sera and ELISA for the titer of IgG antibody in sera. Results　The recombinant plasmid, pcIFN-γ was constructed. The proliferation response of spleen T lymph cells, NK cell killing activity, and serum levels of IFN-γ, IL-2 and NO in mice injected with pcROP1 and pcIFN-γ were higher than in those injected with pcROP1 alone. There was no difference in IgG antibody levels between the two groups. Conclusion　The genetic adjuvant, pcIFN-γ, could enhance the cellular immune response induced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection.

Objective　To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods　Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed. Results　The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion　pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.

OBJECTIVETo characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis.

METHODSMice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 microg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-gamma, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally.

RESULTSSignificant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-gamma. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival.

CONCLUSIONHumoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation.

Animals ; Antibodies, Protozoan ; blood ; Antigens, Protozoan ; Cytokines ; biosynthesis ; Female ; Immunization ; Immunoglobulin G ; blood ; classification ; Interleukin-2 ; genetics ; Mice ; Protozoan Proteins ; genetics ; Protozoan Vaccines ; immunology ; Toxoplasma ; immunology ; Toxoplasmosis, Animal ; prevention & control ; Vaccines, DNA ; immunology

Objective To evaluate retrograde transpopliteal access for femoral-popliteal artery total occlusion with blind puncture.Methods Clinical data of 22 cases admitted from Sep 2014 to June 2016 undergoing endovascular treatment of the femoral-politeal artery occlusion with transpopliteal artery retrograde access by blind puncture were analyzed.Results A total of 22 patients underwent retrograde popliteal access with blind puncture after antegrade attempts failed.Puncture above the knee was performed in 18 cases and below the knee in 4 cases.The average increase of ABI was 0.57.Hematoma of puncture site was observed in 2 patients,other complications included pneumonia in 1 case and renal insufficiency in 2 cases.The mean follow-up time was (13 ± 5)months.Restenosis occurred in 8 patiens(36.4％)during the follow-up time.The primary patency was (86.4 ± 0.07) ％ at 6 months and (70.7-± 0.12) ％ at 12 months.There was no major amputation rate and mortality during the follow-up.Conclusions Retrograde transpopliteal access for femoral-popliteal artery occlusion with blind puncture is safe and effective.

Minimal hepatic encephalopathy (MHE) refers to a state of neuropsychological or neurophysiological abnormality and normal cognitive function in patients with liver cirrhosis, which is commonly seen in patients with liver cirrhosis. Early diagnosis and treatment of MHE can improve the quality of life of patients and reduce accidental deaths. At present, Psychometric Hepatic Encephalopathy Score is mainly used for the diagnosis of MHE, but its operation is complicated and time-consuming and is affected by age and educational level, with unsatisfactory reliability in clinical diagnosis. Serum biomarkers are objective reference indicators with simple and convenient measurement and can easily be promoted in clinical practice. Potential serum biomarkers such as S100β, 3-nitrotyrosine, and arterial blood ammonia have their own advantages and disadvantages in specificity, sensitivity, and diagnostic value. This article reviews the above-mentioned serum biomarkers.

OBJECTIVETo evaluate the immune responses induced by experimental DNA construct encoding Toxoplasma gondii (T. gondii) surface antigen1 (SAG1) and rhoptry protein 1 (ROP1) in mice as a hybrid gene.

METHODSTruncated SAG1 and ROP1 DNA fragments were amplified using polymerase chain reaction (PCR) and inserted into pEGFP-N3 vector to construct recombinant plasmid pSAG1-ROP1. NIH3T3 mammalian cells were transiently transfected with the DNA construct. Female BALB/c mice were given three intramuscular injections of 10 micro g plasmid DNA entrapped in liposome. Four weeks after the final booster injection, blood samples were collected and subjected to enzyme-linked immuno sorbent assay (ELISA) to investigate humoral and cell-mediated immune responses. Reversal transcript-polymerase chain reaction (RT-PCR) was used to evaluate the transcription of inoculated DNA-liposome complex in the injected site. Dot-blot hybridization was employed in order to detect whether or not the injected DNA was incorporated into the genomic DNA of the immunized mice.

RESULTSGreen fluorescence was observed in pSAG1-ROP1-transfected cells. Western blot analysis showed antibody recognition of the expressed SAG1-ROP1 was between 58 kDa and 75 kDa. No expression was observed in blank control plasmid-transfected cells. The sera of immunized mice exhibited antibodies to T. gondii tachyzoites and primarily interferon-gamma and interlukin-2. RT-PCR showed that the duration of transcribed inoculated liposome entrapped DNA in the injected muscular tissue was at least ten days post the first injection. Dot-blot hybridization revealed that the presence of foreign DNA in the splenocytes and peripheral blood leukocytes was transient and that no foreign DNA had inserted into the genomic DNA of mice immunized with pSAG1-ROP1.

CONCLUSIONSImmunization with a liposome-encapsulated DNA construct encoding the T. gondii SAG1 and ROP1 can induce humoral and cell-mediated immune responses.

Animals ; Antibody Formation ; Antigens, Protozoan ; genetics ; DNA ; Enzyme-Linked Immunosorbent Assay ; Female ; Genetic Vectors ; Immunity, Cellular ; Immunization ; Liposomes ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C ; Protozoan Proteins ; genetics ; Toxoplasma ; genetics ; immunology ; Transfection

Endovascular technology has become the first choice for the treatment of lower extremities arteriosclerotic obliterans. Bioresorbable vascular scaffolds have attracted more and more attention as a choice of endovascular technology. In the last decade, poly(L-lactic acid) bioresorbable scaffolds with or without drug coating have shown acceptable medium and long-term safety and efficacy in lower extremities arteriosclerotic obliterans, but the lesions of the subjects were relatively simple. Magnesium alloy bioresorbable scaffolds are safe but less effective in the treatment of lower extremities arteriosclerotic obliterans. Both iron and zinc alloy bioresorbable scaffolds have shown considerable results in animal experiments. In particular, the success of implantation of drug-coated iron alloy bioresorbable scaffolds in below-the-knee artery indicated that the iron alloy bioresorbable scaffolds have officially entered the clinical trial stage. Through the comprehensive summation of the previous clinical and experimental data of bioresorbable vascular scaffolds and the pathological characteristics of lower extremities arteriosclerotic obliterans, it is shown that the drug-coated poly(L-lactic acid) bioresorbable scaffolds and iron alloy bioresorbable scaffolds will have greater development potential in the treatment of lower extremities arteriosclerotic obliterans.