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BackgroundPorcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), is a serious economic problem for the swine industry in China. In this study, we investigated the genetic variation of PCV2 in China using strains isolated from 2004-2008. Viruses were isolated from samples collected from pigs with multi-systemic lesions and clinical signs of PMWS from different regions of China, and the genomes of these viruses were sequenced. The assembled sequences were used to define the genotypes of these strains; PCR-RFLP methodology was used to distinguish isolates and capture ELISA was used to demonstrate the antigenic changes resulted from ORF2 gene mutation of the isolates.ResultsWe identified 19 PCV2 isolates, including four newly emerging PCV2 mutant strains. The 19 isolates were designated into three genotypes (PCV2a, PCV2b and PCV2d). PCV2d represented a novel genotype and a shift from PCV2a to PCV2b as the predominant genotype in China was identified. This is the first report of 1766 nt PCV2 harboring a base deletion at other new different positions. Amino acid sequence analysis identified two novel ORF2 mutations (resulting in ORF2 sequences 705 and 708 nt in length) in three deletion strains (1766 nt) and one strain with a genome 1767 nt in length. Finding of two amino acids elongation of the ORF2-encoded Cap protein is firstly observed among PCV2 strains all over the world. The isolates were distinguished into different genotypes by PCR-RFLP methodology and antigenic changes were present in Cap protein of mutation isolates by capture ELISA.ConclusionsThe results of this study provide evidence that PCV2 is undergoing constant genetic variation and that the predominant strain in China as well as the antigenic situation has changed in recent years. Furthermore, the PCR-RFLP method presented here may be useful for the differential identification of PCV2 strains in future studies.

Hypoxia preconditioning enhances the therapeutic effect of mesenchymal stem cells (MSCs). However, the mechanism underlying hypoxia-induced augmentation of the protective effect of MSCs on myocardial infarction (MI) is poorly understood. We show that hypoxia-enhanced survival, mobility, and protection of cocultured cardiomyocytes were paralleled by increased expression of leptin and cell surface receptor CXCR4. The enhanced activities were abolished by either knockdown of leptin with a selective shRNA or by genetic deficiency of leptin or its receptor in MSCs derived, respectively, from ob/ob or db/db mice. To characterize the role of leptin in the regulation of MSC functions by hypoxia and its possible contribution to enhanced therapeutic efficacy, cell therapy using MSCs derived from wild-type, ob/ob, or db/db mice was implemented in mouse models of acute MI. Augmented protection by hypoxia pretreatment was only seen with MSCs from wild-type mice. Parameters that were differentially affected by hypoxia pretreatment included MSC engraftment, c-Kit+ cell recruitment to the infarct, vascular density, infarct size, and long-term contractile function. These data show that leptin signaling is an early and essential step for the enhanced survival, chemotaxis, and therapeutic properties of MSCs conferred by preculture under hypoxia. Leptin may play a physiological role in priming MSCs resident in the bone marrow endosteum for optimal response to systemic signaling molecules and subsequent tissue repair.

BackgroundPorcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS) in pigs. Currently, there is considerable interest in the immunology of PCV2; in particular, the immunological properties of the capsid protein. This protein is involved in PCV2 immunogenicity and is a potential target for vaccine development. In this study, we identified one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of PCV2.ResultsOne monoclonal antibody (mAb; 8E4), against the capsid protein of PCV2, was generated and characterized in this study. 8E4 reacted with the genotype PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains by an immunoperoxidase monolayer assay (IPMA) and a capture ELISA. Furthermore, the mAb had the capacity to neutralize PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains. One critical amino acid that determined a conformational neutralizing epitope was identified using mAb 8E4 and PCV2 infectious clone technique. Amino acid residues 47-72 in the capsid protein of PCV2a/CL were replaced with the corresponding region of PCV2b/YJ, and the reactivity of mAb 8E4 was lost. Further experiments demonstrated that one amino acid substitution, the alanine for arginine at position 59 (A59R) in the capsid protein of PCV2a (CL, LG and JF2) strains, inhibited completely the immunoreactivity of three PCV2a strains with mAb 8E4.ConclusionsIt is concluded that the alanine at position 59 in the capsid protein of PCV2a (CL, LG and JF2) strains is a critical amino acid, which determines one neutralizing epitope of PCV2a (CL, LG and JF2) strains. This study provides valuable information for further in-depth mapping of the conformational neutralizing epitope, understanding antigenic difference among PCV2 strains, and development of a useful vaccine for control of PCV2-associated disease.

Diabetic encephalopathy is clinically characterized by acquired behavior and cognitive dysfunction but its pathogenesis is not clear. This study aimed to explore the pathogenesis of diabetic encephalopathy and the mechanisms of ghrelin to ameliorate cognitive dysfunction in diabetic rats. Thirty-six streptozotocin diabetic rat models were established; 12 weeks later, all the rats were randomly divided into 3 groups [diabetic model group (D), ghrelin treatment group (T1), and ghrelin and D-lys-3-GHRP-6 treatment group (T2)] of 12 each. Twelve normoglycemic rats were classified in the normal group (N). Learning and memory behaviors were measured using a spatial version of the Morris water maze test. The brain-derived neurotrophic factor (BDNF), cAMP responsive element binding protein (CREB), phosphorylated CREB (p-CREB), phosphorylated ERK1/2 (p-ERK1/2), caspase-3, and Bcl-xl protein expressions in the hippocampi of all the rats were detected using immunohistochemistry. The mRNA expressions of BDNF, CREB, and caspase-3 were examined using reverse transcription-polymerase chain reaction. The hippocampus neuronal apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling method. We found that learning and memory level in the ghrelin treatment group improved significantly, expression of Bcl-xl, BDNF, CREB, p-CREB, and p-ERK1/2 in the hippocampus was increased in the ghrelin treatment group, and the number of apoptotic neurons in the hippocampus decreased remarkably. Our results showed that the changes of BDNF, CREB, and hippocampus neuronal apoptosis might be involved in the pathogenesis of diabetic encephalopathy. We suggested that ghrelin improved cognitive ability in streptozotocin-induced diabetic rats by improving the expressions of BDNF and CREB and by attenuating hippocampus neuronal apoptosis. The effects of ghrelin depend on the receptor of ghrelin, GHSR-1a, and ERK1/2 pathway.

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BackgroundPorcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), is a serious economic problem in the swine industry. Different genotypes (PCV2a, PCV2b and PCV2d) of the virus are present in the clinical cases in China, and it is necessary to elucidate the pathogenic difference among different genotypes of PCV2. In this study, four strains of different genotypes were isolated, two were ordinary strains and another two were mutation strains, which there are one and two amino acids elongation in the capsid protein (Cap) of PCV2, respectively. Representative strains of different genotypes of the virus were constructed by infectious molecular clone and biological characterization of the rescued viruses were identified in vitro.ResultsFour PCV2 isolates (PCV2a/CL, PCV2b/YJ, PCV2b/JF and PCV2d/BDH) of different genotypes were isolated from the clinical cases of PMWS in China. Four infectious clones of PCV2 were constructed and the rescued viruses were harvested after transfection into PK15 cells. The rescued viruses were verified by nucleotide sequence analysis, morphology of the viruses and immunoperoxidase monolayer assay (IPMA). The rescued viruses propagated stably after consecutive incubation for more than ten passages, and virus propagation reached its peak 72h post infection (PI), and the virus titers were up to 105.7 TCID50/ml. By using neutralizing 1D2 monoclonal antibody (mAb) of PCV2, the antigen capture ELISA showed that only the PCV2a/rCL and PCV2b/rJF strains has immunoreactivity with the 1D2 mAb, however, another two rescued strains (PCV2b/rYJ and PCV2d/rBDH) do not, which indicated the antigenic difference among the rescued viruses of different genotypes. In addition, here is the first report of obtaining the newly emerging PCV2 with mutation in vitro by infectious molecular clone technology.ConclusionsConclusions drawn from this study show that PCV2 has prevailing differences in genomic and ORF2 gene length and antigen in swine herds in China. Four representative clones for different genotypes were constructed and rescued, which will facilitate further studies on the pathogenic differences resulting from different subtypes of PCV2.