Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts DNA protection against hypoxia / 中国药理学通报

Aim To investigate the protective roles of sonic hedgehog( Shh) signaling pathway in hypoxia-in-duced DNA damage with the neonatal rat cardiomyo-cytes. Methods The hypoxia model on neonatal car-diomyocytes was established with one to two days old Sprague Dawley rats by deprivation of oxygen and glu-cose ( OGD) . After pretreated with Shh pathway ago-nist SAG1.3 or antagonist GANT61, the survival rates of cardiomyocytes were assayed by MTT after OGD 6 hours or 12 hours. The protein levels of Shh pathway, phosphorylated histone H2AX at serine 139 (γH2AX), phosphorylated ATM (p-ATM), phospho-rylated p53 ( p-p53 ) , cleaved-caspase-3, Bcl-2 and Bax were detected by Western blot. The γH2AX foci was detected by immunofluorescence. Results Com-pared to control group, the protein expression of γH2AX, p-ATM, cleaved-caspase-3, p-p53 in OGD cardiomyocytes significantly increased, and Bcl-2/Bax ratio proportionally decreased. Particularly, the ex-pression of γH2AX, p-ATM was highest at OGD 6 h, and then gradually declined after OGD 12 h. After SAG1.3 pretreatment, the expression of γH2AX, p-ATM, cleaved-caspase-3 and p-p53 dramatically de-creased and the Bcl2/Bax ratio increased in OGD 6 h or OGD 12 h cardiomyocytes. On the contrary, in GANT61 pretreatment group, the expression of γH2AX, p-ATM, cleaved-caspase-3 and p-p53 signifi-cantly increased and the Bcl-2/Bax ratio decreased compared to the OGD 6 h or OGD 12 h cardiomyo-cytes. Conclusion The activation of Shh pathway protects cardiomyocytes against hypoxia-induced apop-tosis through inhibition of DNA damage.

Immunogenicity of dominant epitopes of complex antigen rhoptry protein 2-sunface protein 1 derived from Toxoplasma gondii / 中华传染病杂志

Objective To analyze the immunogenicity of dominant epitope of complex antigen of rhoptry protein 2 (ROP2 ) and major surface protein 1 (SAG1 ) derived from Toxoplasma gondii (T .gondii) .Methods Dominant epitope of ROP2‐SAG1 containing both dominant T‐and B‐cell epitopes was predicted and selected from T . gondii with bioinformatics methods .The gene fragment cloned into pET32a expression vector was transformed into the competent cell (Escherichia coli strain Rosetta) and expressed under the induction .The protein purified by nitrilotriacetic acid (Ni‐NTA) agarose resin were finally identified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis . Japanese rabbits were immunized subcutaneously with purified epitope protein in contrast with control group immunized with pET32a protein or phosphate buffevred saline (PBS) .The sera from immunized rabbits were collected at week 0 , week 2 and week 4 for determination of epitope‐specific antibody IgG using enzyme‐linked immunosorbent assay ,and immunodot assay was used to further confirm the specificity of antibody .After BALB/c mice were immunized with purified epitope proteins ,the capacity of production of interferon‐γ(IFN‐γ) in splenocytes was detected by enzyme‐linked immunospot assay .Results Relative molecular weight 30 000 of dominant epitope was derived from prokaryotic system .Then the rabbits immunized with purified dominant epitope could produce corresponding epitope‐specific antibody IgG . And with the increased frequency of immunization ,the level of antibody gradually increased .At week 2 and 4 ,higher antibody response were observed in group of rabbit immunized with dominant epitope than those of control group(1.454±0.098vs0.616±0.084,F=0.000,P<0.05;2.299±0.224vs1.580±0.192,F=0 .112 ,P< 0 .05) .The antibody titer at week 4 was as high as 1∶40 960 .Immunodot assay further confirmed the antibody specificity against the dominant epitope .The level of IFN‐γ in splenocytes from mice immunized with dominant epitope after stimulation with three epitope specific CTL peptides (epitope peptides 1 [19 .333 ± 1 .528]/100 000 cells ,epitope peptides 2 ([40 .333 ± 1 .528]/100 000 cells) ,epitope peptides 3 ([70 .667 ± 1 .890]/100 000 cells) was significantly higher than that of PBS control group (epitope peptides 1 [1 .033 ± 0 .150]/100 000 cells ,epitope peptides 2 [1 .045 ± 0 .110]/100 000 cells , epitope peptides 3 [1 .041 ± 0 .120]/100 000 cells , F=0 .284 ,0 .000 and 0 .284 ,respectively ;all P<0 .05) . The level of IFN‐γ from splenocytes stimulated with combined peptide with cytotoxic T lymphocyte (CTL) epitope peptide and helper T cell epitope peptide (epitope peptides 3) was significantly higher than that with single CTL epitope peptides (epitope peptides 1 and epitope peptides 2 , F=5 .796 and 0 .000 ,respectively ;both P<0 .05) .Conclusion Screened dominant epitopes of ROP2‐SAG1 from T .gondii derived from prokaryotic expression system exhibit remarkable immunogenicity .

Evaluation of Recombinant SAG1, SAG2, and SAG3 Antigens for Serodiagnosis of Toxoplasmosis

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.

A Rapid Diagnostic Test for Toxoplasmosis using Recombinant Antigenic N-terminal Half of SAG1 Linked with Intrinsically Unstructured Domain of GRA2 Protein

Toxoplasma gondii is an apicomplexan parasite with a broad host range of most warm-blooded mammals including humans, of which one-thirds of the human population has been infected worldwide which can cause congenital defects, abortion, and neonatal complications. Here, we developed a rapid diagnostic test (RDT) for T. gondii infection. Antigenic N-terminal half of the major surface antigen (SAG1) was linked with intrinsically unstructured domain (IUD) of dense granule protein 2 (GRA2). The recombinant GST-GRA2-SAG1A protein was successfully expressed and purified as 51 kDa of molecular weight. Furthermore, antigenicity and solubility of the rGST-GRA2-SAG1A protein were significantly increased. The overall specificity and sensitivity of GST-GRA2-SAG1A loaded RDT (TgRDT) were estimated as 100% and 97.1% by comparing with ELISA result which uses T. gondii whole cell lysates as the antigen. The TgRDT tested with Uganda people sera for field trial and showed 31.9% of seroprevalence against T. gondii antibody. The TgRDT is proved to be a kit for rapid and easy to use with high accuracy, which would be a suitable serodiagnostic tool for toxoplasmosis.

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.

Development and Clinical Evaluation of a Rapid Serodiagnostic Test for Toxoplasmosis of Cats Using Recombinant SAG1 Antigen

Rapid serodiagnostic methods for Toxoplasma gondii infection in cats are urgently needed for effective control of transmission routes toward human infections. In this work, 4 recombinant T. gondii antigens (SAG1, SAG2, GRA3, and GRA6) were produced and tested for the development of rapid diagnostic test (RDT). The proteins were expressed in Escherichia coli, affinity-purified, and applied onto the nitrocellulose membrane of the test strip. The recombinant SAG1 (rSAG1) showed the strongest antigenic activity and highest specificity among them. We also performed clinical evaluation of the rSAG1-loaded RDT in 182 cat sera (55 household and 127 stray cats). The kit showed 0.88 of kappa value comparing with a commercialized ELISA kit, which indicated a significant correlation between rSAG1-loaded RDT and the ELISA kit. The overall sensitivity and specificity of the RDT were 100% (23/23) and 99.4% (158/159), respectively. The rSAG1-loaded RDT is rapid, easy to use, and highly accurate. Thus, it would be a suitable diagnostic tool for rapid detection of antibodies in T. gondii-infected cats under field conditions.

Primary structure of mature SAG1 gene of an Indonesian Toxoplasma gondii and comparison with other strains

Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1.

The genetic immunization in mice with three constructs of Toxoplasma gondii SAG1 gene / 热带医学杂志

Three forms of the major surface antigen (SAG1)of Toxoplasma gondii, that is the membrane form, the secrete form and the intracellular form, were constructed and used to immunize BALB/c mice. The humoral response in the mice immunized with the membrane form and the secrete form of SAG1 appeared earlier and stronger than those mice immunized with the intracellular form. Result from the challenging infection demonstrated that the protection in the mice immunized with the membrane and the secrete forms was also stronger than in the mice immunized with the intracellular form. We suggest that the immune efficiency of the three forms of SAG1 in the mouse model is different.