Numerical research on the biomechanical behaviour of braided stents with different end shapes and stent-oesophagus interaction.

Quasi-static and dynamic numerical analyses are carried out by referring to computational models of commercial self-expandable braided stents with 3 commonly used end shapes, to evaluate the influence of different end shapes of stent on the biomechanical interaction between stent and oesophagus. The end shape has no influence on the equivalent stress, but has a great influence on the contact stress in the narrowest zone of the oesophagus-neoplasm system. However, the end shapes have significant effect on the equivalent stress and the contact stress in the healthy area of the oesophagus in contact with the stent ends. The results show that the maximum equivalent stress of the oesophagus occurs in the zone contact with the cup-shaped end and the maximum contact stress occurs in the zone contact with the edge of the trumpet-shaped stent end. Moreover, the stent apposition is almost not affected by the end shapes. Although small zones with an incomplete stent apposition appear in the transition zones of spherical-cup-shaped stent, such occurrence might not contribute to stent malapposition or stent migration. Therefore, these stents with 3 types of end shapes all have good stent apposition. Finally, the numerical simulation results can be used to assess the mechanical performance of stents with different end shapes, the effectiveness of stent expansion therapy, and the possibility of complications after stent implantation.

Numerical investigations of the mechanical properties of a braided non-vascular stent design using finite element method.

This paper discusses various issues relating to the mechanical properties of a braided non-vascular stent made of a Ni-Ti alloy. The design of the stent is a major factor which determines its reliability after implantation into a stenosed non-vascular cavity. This paper presents the effect of the main structural parameters on the mechanical properties of braided stents. A parametric analysis of a commercial stent model is developed using the commercial finite element code ANSYS. As a consequence of the analytical results that the pitch of wire has a greater effect than other structural parameters, a new design of a variable pitch stent is presented to improve mechanical properties of these braided stents. The effect of structural parameters on mechanical properties is compared for both stent models: constant and variable pitches. When the pitches of the left and right quarters of the stent are 50% larger and 100% larger than that of the central portion, respectively, the radial stiffness in the central portion increases by 10% and 38.8%, while the radial stiffness at the end portions decreases by 128% and 164.7%, the axial elongation by 25.6% and 56.6% and the bending deflection by 3.96% and 10.15%. It has been demonstrated by finite element analysis that the variable pitch stent can better meet the clinical requirements.

[Cloning and expression of Toxoplasma gondii autophagy-related protein 3 gene and preparation of its polyclonal antibody].

OBJECTIVE: To clone and express autophagy-related protein 3 (TgAtg3) gene of Toxoplasma gondii, and obtain the specific polyclonal antibody against TgAtg3. METHODS: TgAtg3 cDNA was inserted into prokaryotic expression vector pET28a. After identification, the constructed plasmid pET28a-TgAtg3 was transformed into E. coli Rosetta cells, and induced by special induction medium for expression of the protein. The recombinant protein was purified via Ni-NTA affinity chromatography. Western blotting assay was performed with anti-His tag mouse monoclonal antibody as the primary antibody. Rabbits were immunized with 125 µg purified TgAtg recombinant protein. Each rabbit received 4 immunizations at 2-week intervals with the same dose of antigen. The specific anti-TgAtg3 polyclonal antibody was obtained, and analyzed by Western blotting and indirect immunofluorescence assay (IFA). RESULTS: pET28a-TgAtg3 plasmid was identified by restriction enzyme digestion, PCR amplification and sequencing. SDS-PAGE and Western blotting analysis showed that the recombinant TgAtg3 protein (about Mr 44,000) was expressed in E. coli Rosetta cells. TgAtg3 protein from tachyzoite lysates was recognized by the specific anti-TgAtg3 polyclonal antibody. IFA assay determined that the specific polyclonal antibody bound to TgAtg3 protein from the cytoplasm of tachyzoites. CONCLUSION: The obtained soluble polyclonal antibody against TgAtg3 can specifically react to the endogenous TgAtg3 protein.

Development-specific differences in the proteomics of Angiostrongylus cantonensis.

Angiostrongyliasis is an emerging communicable disease. Several different hosts are required to complete the life cycle of Angiostrongylus cantonensis. However, we lack a complete understanding of variability of proteins across different developmental stages and their contribution to parasite survival and progression. In this study, we extracted soluble proteins from various stages of the A. cantonensis life cycle [female adults, male adults, the fifth-stage female larvae (FL5), the fifth-stage male larvae (ML5) and third-stage larvae (L3)], separated those proteins using two-dimensional difference gel electrophoresis (2D-DIGE) at pH 4-7, and analyzed the gel images using DeCyder 7.0 software. This proteomic analysis produced a total of 183 different dominant protein spots. Thirty-seven protein spots were found to have high confidence scores (>95%) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Comparative proteomic analyses revealed that 29 spots represented cytoskeleton-associated proteins and functional proteins. Eight spots were unnamed proteins. Twelve protein spots that were matched to the EST of different-stage larvae of A. cantonensis were identified. Two genes and the internal control 18s were chosen for quantitative real-time PCR (qPCR) and the qPCR results were consistent with those of the DIGE studies. These findings will provide a new basis for understanding the characteristics of growth and development of A. cantonensis and the host-parasite relationship. They may also assist searches for candidate proteins suitable for use in diagnostic assays and as drug targets for the control of eosinophilic meningitis caused by A. cantonensis.

Urine sample used for detection of toxoplasma gondii infection by loop-mediated isothermal amplification (LAMP).

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Toxoplasma gondii DNA in mice infected with T. gondii PRU strain. This LAMP assay was based on the sequence of highly repetitive B1 gene. The detection limit of T. gondii LAMP assay was 1 pg of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. The LAMP assay was also highly specific for T. gondii and able to detect T. gondii DNA in urine of mice treated with dexamethasone at 90 day post infection (p.i.), although this assay could not detect the DNA in mice urine 2-6 days p.i. These results demonstrated that LAMP is effective for evaluation of therapy effectiveness for T. gondii infection. The established LAMP assay may represent a useful and practical tool for the routine diagnosis and therapeutic evaluation of human toxoplasmosis.

In vitro study of the effects of Angiostrongylus cantonensis larvae extracts on apoptosis and dysfunction in the blood-brain barrier (BBB).

It has been hypothesized that blood-brain barrier (BBB) dysfunction in Angiostrongylus cantonensis infection might be due to the apoptosis of the hosts' BBB cells. Here, we evaluated this hypothesis through several methods, all based on an in vitro mouse BBB model consisting of primary culture brain microvascular endothelial cells (BMECs) and brain astrocytic cells (BACs). In the present study, a four-hour percolation and HRP permeability experiment showed that A. cantonensis larvae extracts can increase the permeability of the BBB. Apoptosis among BMECs and BACs after exposure to larvae extracts was monitored by TUNEL and annexin-V-FITC/PI double staining. A. cantonensis larvae extracts were found to induce apoptosis in both BMECs and BACs. For this reason, we concluded that the induction of apoptosis might participate in the BBB dysfunction observed during angiostrongyliasis. Improved fundamental understanding of how A. cantonensis induces apoptosis may lead to new approaches to the treatment or prevention of this parasitic disease.

Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II.

The Toxoplasma gondii nucleoside triphosphate hydrolase (TgNTPase) has apyrase activity, degrading ATP to the di- and mono-phosphate forms and may be used by the parasite to salvage purines from the host cell for survival and replication. To study the immune-protective value of TgNTPase-II, BALB/c mice were immunized with a recombinant form of the antigen rTgNTPase-II combined with alum. All immunized mice produced specific anti-rTgNTPase-II immunoglobulins, with high IgG antibody titers and a mixed IgG1/IgG2a response, with predominance of IgG2a production. The cellular immune response was associated with the production of IFN-γ and IL-2 cytokines and the increase of the percentage of CD8+ T cells. Vaccinated mice displayed significant protection against acute infection with the virulent RH strain (P<0.05 in survival rate) and also chronic infection with PRU cyst (62.9% and 57.6% reduction in brain parasite load for rTgNTPase-II+alum and rTgNTPase-II alone vaccinated groups) compared to the non-vaccinated control group. In conclusion, rTgNTPase-II elicits a strong specific Th1 immune response providing partial protection against both T. gondii acute and chronic infection.

[Microbial survey of bacterial contamination of shellfish products in Wenzhou].

OBJECTIVE: To investigate the status of bacterial contamination in the shellfish products in Wenzhou. METHODS: One hundred samples were collected and their bacterial populations including the total plate count were investigated. RESULTS: Of the 100 samples collected, 67 samples failed to not meet the national regulations due to bacterial contamination, accounting for 67% of the total samples. Among the contaminated samples, the most serious contamination was caused by coliforms (61.4% of the total plate count with contamination), followed by Salmonella (18.6%), Vibio parahaemolyticus (15.7%), Listeria spp. (4.3%) and others (6%). CONCLUSION: Microbial pollution has become a threat to the marine shellfish products in Wenzhou.

Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii.

Nucleoside triphosphate hydrolase (NTPase) is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii, which has a wide specificity toward NTP. In the present study, two monoclonal antibodies (mAbs, MNT1 and MNT2) against recombinant T. gondii NTPase-II (rTgNTPase-II) were developed. Western blot analysis displayed that these two mAbs can recognize specifically rTgNTPase-II as well as a 63kDa molecule in tachyzoites soluble antigens that corresponded to native NTPase-II. T. gondii tachyzoites pretreated with two mAbs were observed under Confocal Laser Microscope and a specific reaction was displayed on tachyzoites after indirect fluorescence antibody test (IFAT). When COS-7 cells were co-cultured with tachyzoites pretreated with two mAbs, the number of intracellular parasites per infected cell was significantly decreased compared with the control. Furthermore, incubation of T. gondii tachyzoites with two mAbs can inhibit NTPase activity in the presence of dithiothreitol, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the mAbs. The passive immunization test indicated that the transferred mAbs can significantly prolong the survival time of challenge infected mice. Taken together, we concluded that the mAbs against NTPase-II can reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.

[Application of double antibody sandwich ELISA for detection of nucleoside triphosphate hydrolase-II protein of Toxoplasma gondii].

OBJECTIVE: To establish a double antibody sandwich ELISA method for detection of nucleoside triphosphate hydrolase-II (NTPase-II) protein of Toxoplasma gondii. METHODS: BALB/c mice were immunized with recombinant NTPase-II (rTgNTPase-II) protein of T. gondii. The hybridomas that secreted high titer of monoclonal antibodies (mAbs) with high specificity were screened and used to establish the double antibody sandwich ELISA for the detection of rTgNTPase-II. In order to evaluate the sensitivity of the method, the concentration of whole-tachyzoite lysate and rTgNTPase-II was detected, respectively. Serum samples from patients with malaria (7 cases), schistosomiasis (12 cases), paragonimiasis (14 cases) and cysticercosis (10 cases) were examined by the same method. RESULTS: Two hybridoma cell lines, MNTI and MNT2, were developed for secreting mAbs against rTgNTPase-II. Western blotting analysis showed that the two mAbs specifically recognized rTgNTPase-II and whole-tachyzoite lysate. The MNT1 was used as coating antibody, and HRP-labeled MNT2 as secondary antibody. The double antibody sandwich ELISA detecting rTgNTPase-II was developed with a minimum concentration of 6 microg/ml for whole-tachyzoite lysate and 1.5 microg/ml for rTgNTPase-II. An overall specificity of 100% was determined. CONCLUSION: The double antibody sandwich ELISA based on MNT1 as coating antibody and MNT2 as secondary antibody has a high specificity.

[Preparation of monoclonal antibodies against the adhesion protein 33 of Trichomonas vaginalis].

OBJECTIVE: To prepare and characterize the monoclonal antibodies (McAbs) against recombinant adhesion protein 33 (AP33) of Trichomonas vaginalis. METHODS: The purified recombinant fusion protein AP33 was used as antigen to immunize BALB/c mice. Sp2/0 myeloma cells were fused with the splenocytes from immunized BALB/c mice. After ELISA screening and 4 times of limited dilution, 5 positive hybridoma cell lines were obtained, and the biological properties of the McAbs were identified by Western blotting. Indirect immunofluorescent antibody test (IFAT) was performed and the inhibition effect of McAbs on the cytoadherence of Trichomonas vaginalis to HeLa cell was assayed. RESULTS: Western blotting demonstrated that 5 McAbs, designated as 4A2, 4F11, 4F8, 4E7 and 4H11, specifically combined with the recombinant AP33 of T. vaginalis. The McAbs were IgG1 isotypes. Four of them (4F11, 4F8, 4E7 and 4H11) showed parasite recognition by IFAT. Parasite cytoadherence to a monolayer of HeLa cells was inhibited in vitro with a inhibition rate of 50.08%, 65.03%, 50.70% and 49.08% by the 4 McAbs under a concentration of 200, 200, 400 and 200 microg/ml, respectively. CONCLUSIONS: The prepared McAbs against the recombinant AP33 show a protective inhibition on cytoadherence of Trichomonas vaginalis in vitro.

[Expression and antigenicity analysis of NTPase gene of Toxoplasma gondii].

OBJECTIVE: To clone and express prokaryotic recombinant plasmid of nucleoside triphosphate hydrolase (NTPase) gene of Toxoplasma gondii, and analyze its antigenicity. METHOD: NTPase gene was amplified by PCR from RH strain of T. gondii and cloned into pGEM-T Easy vector. Positive clones were screened and identified by BglII, HindIII digestion and sequenced. The target gene was then subcloned into prokaryotic expression vector pBAD-HisB and transformed into E. coli BL21 (DE3). The expressed recombinant protein was purified with Ni-NTA agarose and further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS: NTPase-II gene was specifically amplified, and the homology of DNA sequence was 100% to that in the GenBank. SDS-PAGE showed that the recombinant NTPase protein with correct molecular weight was expressed highly in E.coli BL21 (DE3). Western blotting testified that the purified recombinant protein could be specifically recognized by mouse serum immunized with T. gondii and mouse anti-recombinant protein serum. CONCLUSION: The NTPase-II gene has been cloned and expressed in E.coli BL21 (DE3), and the purified protein of NTPase-II gene displays a specific antigenicity.

[Study on the infectivity of Angiostronglus cantonensis in the epidemic foci].

OBJECTIVE: To analyze the degrees on the epidemic foci of Angiostronglus cantonensis and to explore the measurement methods. METHODS: Snails (Pila gigas) were collected from the spots of Wenzhou, Cangnan, Yongjia, Yueqing in Zhejiang province and Minhou, Changle, Ningde in Fujian province. The snails were examined microscopically in order to calculate their infection rates and the average worm number in the positive snails, then taking the product of multiplication of both values as infestation index. RESULTS: The infection rates of the epidemic foci were 10.59% (9/85), 60.74% (181/298), 34.96% (79/226), 32.90% (76/231), 57.50% (184/320), 40.00% (82/205), 17.65% (12/68) and the rates of infectivity were 6.57, 183.54, 121.73, 93.45, 276.36, 76.08, 12.65, respectively. CONCLUSION: The epidemic foci were divided into five ranks (super, high, mid-range, low and non-epidemic foci) according to the value of infestation index which ranked from > 75, 30-75, 5-29, < 5 to 0.

[Preparation, characterization and preliminary application of recombinant protein AP33 of trichomonas vaginalis].

OBJECTIVE: To clone ap33 gene of Trichomonas vaginalis( T. v), construct prokaryotic expression system of the gene and identify its antigenicity and immunogenicity. METHODS: The total RNA was extracted from a clinical isolate Tv317 and the cDNA was synthesized by reverse transcription. The ap33 gene from cDNA of Tv317 was amplified by PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a (+) with inserted ap33 gene was constructed. Recombinant fusion protein AP33 was expressed in E. coli strain BL21DE3 induced by IPTG at different dosages. Western blotting was applied to determine immunoreactivity of the recombinant fusion protein AP33 with antibody against whole cell of T. v. Double agar diffusion was applied to determine immunogenicity of the recombinant fusion protein AP33 with rabbit antiserum immunized with the recombinant fusion protein AP33, and ELISA with antigen of T. v whole cell was applied to determine immunogenicity of the recombinant protein AP33. Positive human sera were tested by ELISA with the recombinant fusion protein AP33. RESULTS: High homology of nucleotide and amino acid sequences was revealed between the cloned ap33 and the corresponding gene. The recombinant protein showed a high expression level. The recombinant protein was recognized by anti-T. v polyclonal antibody from rabbit, and showed a high titer. The clinical T. v isolates showed high ap33 expression level and stimulated the production of specific antibody. Antibody against AP33 was detected in 78% of the 50 patients infected with T. v by ELISA. CONCLUSION: A prokaryotic expression system of T. v ap33 gene has been established. The expressed fusion protein AP33 shows satisfactory antigenicity and immunogenicity.

[Detection of Angiostrongylus cantonensis circulating antigen by monoclonal antibodies].

OBJECTIVE: To prepare monoclonal antibodies (McAbs) against soluble antigens of adult worms of Angiostrongylus cantonensis (A. cantonensis) on the purpose to detect CAg of A. cantonensis. METHODS: Female BALB/c mice were immunized with soluble antigens of adult worms of A. cantonensis and the spleen cells were fused with myeloma SP2/0 cells. The hybridoma cell strains were screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Two McAbs (3F1 and 4H2) were applied to detect the CAg in the sera of rats and mice infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA. RESULTS: Three McAbs against A. cantonensis adult were obtained. Two McAbs (3F1, 4H2) were identified as IgG1 and one McAb (2A2) was identified as IgM. The titers of culture fluid and ascites was 1:25,600, 1:25,600, 1:12,800 and 1:80,000, 1:80,000, 1:40,000 respectively. Western blotting results showed three McAb could be used to identify 15,000 protein of adult worms of A. cantonensis. The detection rates of the CAg in the sera of infected rats and mice were 84.2% (48/57) and 87.2% (41/47) respectively. The detection rate of the CAg in the sera of angiostrongyliasis patients was 86.4% (19/22), and no cross reactions with sera from patients with schistosomiasis, cysticercosis cellulose, paragonimiasis and trichinellosis were observed. The CAg in the sera from mice examined at different periods after infection revealed positive 2 week after inoculation and the titer of CAg peaked 4 week after inoculation. CONCLUSION: A new method of sandwich ELISA with high sensitivity and specificity to detect the serum A. cantonensis CAg has been obtained, it could be applicable to the diagnosis, observation of curative effect and epidemiology of angiostrongyliasis.

[Preparation and preliminary application of monoclonal antibodies against adult worm of Angiostrongylus cantonensis].

OBJECTIVE: To develop and identify monoclonal antibodies (McAbs) against adult worm of Angiostrongylus cantonensis and observe its applicability. METHODS: BALB/c mice were immunized with soluble antigen of adult worms of A. cantonensis. The spleen cells of immunized mice were fused with myeloma cell, and the hybridoma secreting high titer of McAbs with high specificity was screened. By using the McAbs, serum of angiostrongyliasis patient and sera of the rats infected with A. cantonensis were detected by Western blotting and double antibody sandwich ELISA respectively. RESULTS: Three McAbs were established (2A2, 3F1, 4H2), which all showed no cross reaction with antigens of Schistosoma japonicum, Paragonimus westermani, Cysticercus cellulosae and Trichinella spiralis. Western blotting analysis demonstrated that the three McAbs recognized a Mr 15,000 soluble antigen of adult worm of A. cantonensis and recognized the Mr 24,000 and Mr 15,000 circulating antigens from the serum of angiostrongyliasis patient. The double antibody sandwich ELISA detection showed a positive rate of 76.5%. CONCLUSION: Three hybridoma cell lines against adult worm of A. cantonensis have been established which secret high titer of McAbs with high specificity and seem promising in detecting the circulating antigen of the angiostrongyliasis patient.