Competitive and noncompetitive fluorescence polarization immunoassays for the detection of benzothiostrobin using FITC-labeled dendrimer-like peptides.

Peptides obtained from phage display libraries are valuable reagents for small-molecule immunoassays. However, their application in fluorescence polarization immunoassays (FPIAs) is limited by phage particles. Here, monomer, dendrimer-like dimer, tetramer peptidomimetic and anti-immunocomplex tracers were designed and synthesized using lysine as special scaffolds and spacers to develop competitive and noncompetitive FPIAs for benzothiostrobin. The affinity between tracers and monoclonal antibodies or immunocomplexes increased with the tracer valence. A higher signal-to-noise ratio and sensitivity could be generated in the FPIAs based on tetramer tracers. The sensitivities of competitive (50% inhibitory concentration) and noncompetitive (50% saturation concentration) FPIAs were 19.71 ± 4.65 and 40.43 ± 2.73 ng mL-1, respectively. The spiked recoveries were 78.3%-105.2% with relative standard deviations (RSDs) of 0.7%-15.4% for the competitive FPIA, while 78.7%-115.3% with RSDs of 0.7%-12.5% for the noncompetitive FPIA. The amounts of benzothiostrobin in rice detected by the FPIAs were consistent with those detected by high performance liquid chromatography.

Angiogenesis and nerve regeneration induced by local administration of plasmid pBud-coVEGF165-coFGF2 into the intact rat sciatic nerve.

Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) are well-known growth factors involved in the regeneration of various tissues and organs, including peripheral nerve system. In the present study, we elucidated the local and systemic effects of plasmid construct рBud-coVEGF165-coFGF2 injected into the epineurium of intact rat sciatic nerve. Results of histological examination of sciatic nerve and multiplex immunoassays of serum showed the absence of immunogenicity and biosafety of plasmid рBud-coVEGF165-coFGF2. Moreover, local administration of plasmid DNA construct resulted in significantly decreased levels of pro-inflammatory cytokines in the peripheral blood, including tumor necrosis factor α (TNFα) and interleukin-12, and significantly increased levels of cytokines and chemokines including Regulated upon Activation, Normal T Cell Expressed and Presumably Secrete (RANTES), epidermal growth factor, interleukin-2, and monocyte chemoattractant protein 1. These changes in the peripheral blood on day 7 after injection of plasmid construct рBud-coVEGF165-coFGF2 show that the plasmid construct has systemic effects and may modulate immune response. At the same time, reverse transcription-polymerase chain reaction revealed transient expression of coFGF2, coVEGF165, ratFGF2 and ratVEGFA with direct transport of transcripts from distal part to proximal part of the sciatic nerve. Immunohistochemical staining revealed prolonged presence of VEGFA in sciatic nerve till 14 days post-injection. These findings suggest that local administration of plasmid construct рBud-coVEGF165-coFGF2 at a concentration of 30 ng/µL results in the formation of pro-angiogenic stimuli and, and the plasmid construct, used as a drug for gene therapy, might potentially facilitate regeneration of the sciatic nerve. The study was approved by the Animal Ethics Committee of Kazan Federal University, procedures were approved by the Local Ethics Committee (approval No. 5) on May 27, 2014.

Allogenic Adipose Derived Stem Cells Transplantation Improved Sciatic Nerve Regeneration in Rats: Autologous Nerve Graft Model.

We examined the effect of transplantation of allogenic adipose-derived stem cells (ADSCs) with properties of mesenchymal stem cells (MSCs) on posttraumatic sciatic nerve regeneration in rats. We suggested an approach to rat sciatic nerve reconstruction using the nerve from the other leg as a graft. The comparison was that of a critical 10 mm nerve defect repaired by means of autologous nerve grafting versus an identical lesion on the contralateral side. In this experimental model, the same animal acts simultaneously as a test model, and control. Regeneration of the left nerve was enhanced by the use of ADSCs, whereas the right nerve healed under natural conditions. Thus the effects of individual differences were excluded and a result closer to clinical practice obtained. We observed significant destructive changes in the sciatic nerve tissue after surgery which resulted in the formation of combined contractures in knee and ankle joints of both limbs and neurotrophic ulcers only on the right limb. The stimulation of regeneration by ADSCs increased the survival of spinal L5 ganglia neurons by 26.4%, improved sciatic nerve vascularization by 35.68% and increased the number of myelin fibers in the distal nerve by 41.87%. Moreover, we have demonstrated that S100, PMP2, and PMP22 gene expression levels are suppressed in response to trauma as compared to intact animals. We have shown that ADSC-based therapy contributes to significant improvement in the regeneration.