ABSTRACT
Toxoplasma gondii can infect all the vertebrates including human, and leads to serious toxoplasmosis and considerable veterinary problems. T. gondii heat shock protein 60 (HSP60) is associated with the activation of antigen presenting cells by inducing initial immune responses and releasing inflammatory cytokines. It might be a potential DNA vaccine candidate for this parasite. A pVAX-HSP60 DNA vaccine was constructed and immune responses was evaluated in Kunming mice in this study. Our data indicated that the innate and adaptive immune responses was elicited by successive immunizations with pVAX-HSP60 DNA, showing apparent increases of CD3e+CD4+ and CD3e+CD8a+ T cells in spleen tissues of the HSP60 DNA-immunized mice (24.70±1.23% and 10.90±0.89%, P < 0.05) and higher levels of specific antibodies in sera. Furthermore, the survival period of the immunized mice (10.53±4.78 day) were significantly prolonged during the acute T. gondii infection. Decrease of brain cysts was significant in the experimental group during the chronic infection (P < 0.01). Taken together, TgHSP60 DNA can be as a vaccine candidate to prevent the acute and chronic T. gondii infections.
Subject(s)
Animals , Humans , Mice , Antibodies , Antigen-Presenting Cells , Brain , Chaperonin 60 , Cytokines , DNA , Immunization , Parasites , Spleen , T-Lymphocytes , Toxoplasma , Toxoplasmosis , VertebratesABSTRACT
Toxoplasma gondii is an opportunistic protozoan parasite that can infect almost all warm-blooded animals including humans with a worldwide distribution. Micronemes play an important role in invasion process of T. gondii, associated with the attachment, motility, and host cell recognition. In this research, sequence diversity in microneme protein 6 (MIC6) gene among 16 T. gondii isolates from different hosts and geographical regions and 1 reference strain was examined. The results showed that the sequence of all the examined T. gondii strains was 1,050 bp in length, and their A + T content was between 45.7% and 46.1%. Sequence analysis presented 33 nucleotide mutation positions (0-1.1%), resulting in 23 amino acid substitutions (0-2.3%) aligned with T. gondii RH strain. Moreover, T. gondii strains representing the 3 classical genotypes (Type I, II, and III) were separated into different clusters based on the locus of MIC6 using phylogenetic analyses by Bayesian inference (BI), maximum parsimony (MP), and maximum likelihood (ML), but T. gondii strains belonging to ToxoDB #9 were separated into different clusters. Our results suggested that MIC6 gene is not a suitable marker for T. gondii population genetic studies.
Subject(s)
Animals , Cats , Humans , Amino Acid Sequence , Base Sequence , Cell Adhesion Molecules/chemistry , Deer , Genetic Variation , Genotype , Goats , Molecular Sequence Data , Phylogeny , Protozoan Proteins/chemistry , Sequence Alignment , Sheep , Swine , Toxoplasma/classification , Toxoplasmosis/parasitology , Toxoplasmosis, Animal/parasitologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of phosphodiesterase type 5 (PDE5) small interfering RNA (siRNA) on cyclic guanosine monophosphatethe (cGMP) in the smooth muscle cells of human corpus cavernosum, and to provide laboratory evidence for the application of the RNA interference (RNAi) technique for the treatment of erectile dysfunction.</p><p><b>METHODS</b>The recombinant adenovirus rAd5-shRNA-PDE5A3 expressing three pairs of specific shRNA was constructed successfully. The smooth muscle cells of human corpus cavernosum were divided into an experimental, a negative control and a blank control group, and transfected respectively with rAd5-shRNA-PDE5A3, adenovirus rAd5-mock and phosphate buffered saline. The concentration of cGMP was measured by radioimmunoassay at 24, 48 and 72 hours after transfection, and the effect of rAd5-shRNA-PDE5A3 was detected on the cGMP in the smooth muscle cells of the corpus cavernosum.</p><p><b>RESULTS</b>The cGMP level in the smooth muscle cells of the corpus cavernosum was significantly higher in the rAd5-shRNA-PDE5A3 group than in the rAd5-mock control and blank control groups (P < 0.05), most significantly at 72 hours after transfection.</p><p><b>CONCLUSION</b>The rAd5-shRNA-PDE5A3 can obviously increase the cGMP level in the smooth muscle cells of human corpus cavernosum, and enhance the inhibition of the PDE5 gene.</p>
Subject(s)
Humans , Male , Adenoviridae , Cells, Cultured , Cyclic GMP , Metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5 , Genetics , Erectile Dysfunction , Genetics , Genetic Vectors , Myocytes, Smooth Muscle , Metabolism , Penis , Cell Biology , RNA, Small InterferingABSTRACT
<p><b>OBJECTIVE</b>To observe the influence of recombinant adenovirus-mediated PDE5-shRNAs on the free calcium level in rat penile smooth muscle cells and to explore the feasibility of gene therapy for erectile dysfunction (ED).</p><p><b>METHODS</b>Smooth muscle cells of the rat corpus cavernosum were transfected with constructed rAd-rPDE5-shRNAs and then dyed with the calcium fluorescent probe Fluo-3/AM at 24, 48 and 72 hours. The dynamic changes of the calcium fluorescence intensity were observed under the laser scanning confocal microscope (LSCM). The relative level of intracellular calcium was determined by fluorescence indexes.</p><p><b>RESULTS</b>The fluorescence indexes of calcium at 24, 48 and 72 hours were 829.3 +/- 7.8, 801.5 +/- 9.5 and 856.3 +/- 8.7 in the rAd-rPDES-shRNAs group, significantly lower than in the rAd-mock (1106.3 +/- 10.8, 1121.3 +/- 10.2 and 1058.5 +/- 12.1) and blank control group (1076.6 +/- 9.7, 1133.4 +/- 11.2 and 1104.3 +/- 10.5) (P < 0.05).</p><p><b>CONCLUSION</b>Adenovirus mediated shRNAs of the target PDE5 gene can significantly decrease the intracellular calcium level in the smooth muscle cells of the rat corpus cavernosum.</p>