Experimental study on the effect and mechanism of adipose stem cell-derived exosomes combined with botulinum toxin A on skin trauma in rats.

BACKGROUND: Adipose stem cell-derived exosomes (ADSC-EXO) and botulinum toxin type A (BTX-A) individually showed a therapeutic effect on skin wound repair. AIMS: This study investigated their synergistic effect on promoting skin wound healing in vitro and in vivo and the underlying molecular events. METHODS: ADSCs were isolated from Sprague-Dawley (SD) rats to obtain ADSC-EXO by ultrafiltration and ultracentrifugation and were confirmed using nanoparticle tracking analysis and transmission electron microscopy. Human skin fibroblasts (HSF) were cultured and treated with or without ADSC-EXO, BTX-A, or their combination. Changes in cell phenotypes and protein expression were analyzed using different in vitro assays, and a rat skin wound model was used to assess their in vivo effects. RESULTS: The isolated ADSC-EXO from primarily cultured ADSCs had a circular vesicle shape with a 30-180 nm diameter. Treatment of HSF with ADSC-EXO and/or BTX-A significantly accelerated HSF migration in vitro and skin wound healing in a rat model. Moreover, ADSC-EXO plus BTX-A treatment dramatically induced VEGFA expression but reduced COL III and COL I levels in vivo. ADSC-EXO and/or BTX-A treatment significantly upregulated TGF-ß3 expression on Day 16 after surgery but downregulated TGF-ß1 expression, suggesting that ADSC-EXO plus BTX-A promoted skin wound healing and reduced inflammatory cell infiltration. CONCLUSIONS: The ADSC-EXO plus BTX-A treatment demonstrated a synergistic effect on skin wound healing through upregulation of VEGF expression and the TGF-ß3/TGF-ß1 and COL III/COL I ratio.

Construction of Exosomes that Overexpress CD47 and Evaluation of Their Immune Escape.

Our general purpose was to provide a theoretical and practical foundation for the use of exosomes (EXOs) that have high levels of CD47 as stable and efficient drug carriers. Thus, we prepared EXOs from adipose tissue-derived mesenchymal stromal cells (ADMSCs) that had high levels of CD47 (EXOsCD47) and control EXOs (without CD47), and then compared their immune escape in vivo and their resistance to phagocytosis in vitro. Nanoflow cytometry was used to determine the CD47 level in these EXOs, and the amount of EXOsCD47 that remained in rat plasma at 3 h after intraperitoneal injection. Phagocytosis of the EXOs was also determined using in vitro rat macrophage bone marrow (RMA-BM) experiments. Our in vitro results showed that macrophages ingested significantly more control EXOs than EXOsCD47 (p < 0.01), with confirmation by ultra-high-definition laser confocal microscopy. Consistently, our in vivo results showed that rats had 1.377-fold better retention of EXOsCD47 than control EXOs (p < 0.01). These results confirmed that these engineered EXOsCD47 had improved immune escape. Our results therefore verified that EXOsCD47 had increased immune evasion relative to control EXOs, and have potential for use as drug carriers.

Evidence for Dopamine Abnormalities Following Acute Methamphetamine Exposure Assessed by Neuromelanin-Sensitive Magnetic Resonance Imaging.

Background: Neuromelanin-sensitive magnetic resonance imaging (NM-MRI) is a newly developed MRI technique that provides a non-invasive way to indirectly measure of dopamine (DA) function. This study aimed to determine NM concentrations in brain regions following acute methamphetamine (MA) administration using NM-MRI and to explore whether NM-MRI can be used as a biomarker of DA function in non-neurodegenerative diseases. Methods: Baseline NM-MRI, T1-weighted and T2-weighted images were acquired from 27 rats before drug/placebo injection. The control group (n = 11) received acute placebo (Normal saline), while the experimental group (n = 16) received acute MA. NM-MRI scans were performed 5, 30, 60 and 90 min after injection. Regions of interest (ROIs), including the caudate putamen (CP), nucleus accumbens (NAc), hippocampus (HIP), substantia nigra (SN) and crus cerebri (CC), were manually drawn by an experienced radiologist. NM-MRI signal intensity in five brain regions at different time points (baseline and 5, 30, 60, and 90 min) were analyzed. Results: In both the control and experimental groups, at each time point (baseline and 5, 30, 60, and 90 min), the SN exhibited significantly higher NM-MRI signal intensity than the other brain regions (P < 0.05). In addition, acute MA administration resulted in a continuous upward trend in NM-MRI signal intensity in each brain region over time. However, there was no such trend over time in the control group. The NM-MRI signal intensity of SN in the experimental group was significantly higher at the 60 and 90 min compared with that in the control group (P values were 0.042 and 0.042 respectively). Within experimental group, the NM-MRI signal intensity of SN was significantly higher at the 60 and 90 min compared with that before MA administration (P values were 0.023 and 0.011 respectively). Increased amplitudes and rates of NM-MRI signal intensity were higher in the SN than in other brain regions after MA administration. Conclusion: Our results indicated that NM was mainly deposited in the SN, and the conversion of DA to NM was most significant in the SN after acute MA exposure. Increased DA release induced by acute MA exposure may lead to increased accumulation of NM in multiple brain regions that can be revealed by NM-MRI. NM-MRI may serve as a powerful imaging tool that could have diverse research and clinical applications for detecting pathological changes in drug addiction and related non-neurodegenerative diseases.

Efficacy of Retrobulbar Hyaluronidase Injection for Vision Loss Resulting from Hyaluronic Acid Filler Embolization.

BACKGROUND: Vision loss is a rare but serious complication of facial hyaluronic acid (HA) filler injection, for which there is no proven rescue therapy. Retrobulbar hyaluronidase injection is advocated by many plastic surgeons as an emergency treatment, but has not been carefully assessed for its efficacy. OBJECTIVES: To evaluate the efficacy of retrobulbar hyaluronidase injection as a rescue treatment for vision loss caused by HA filler embolization. METHODS: Patients with vision loss caused by HA filler embolization were treated with retrobulbar hyaluronidase injection. Their visual acuity and fundoscopic images before and after treatment were analyzed for efficacy assessment. RESULTS: One patient with branch retinal artery occlusion (BRAO), one patient with posterior ischemic optic neuropathy (PION), one patient with ophthalmic artery occlusion, and one patient with both BRAO and PION were treated with one or two retrobulbar injections of 1500 or 3000 units hyaluronidase. No patients demonstrated substantial retinal artery recanalization or vision acuity improvement after treatment. CONCLUSIONS: One or two retrobulbar injections of 1500 to 3000 IU hyaluronidase are unable to recanalize retinal artery occlusion or improve the visual outcome of patients who presented with vision loss caused by HA filler embolization at least four hours after onset. LEVEL OF EVIDENCE: 4.

Evolution and genetic characterization of hepatitis A virus isolates in China.

OBJECTIVES: Hepatitis A virus (HAV), transmitted mainly through the fecal-oral route, is one of the major causes of acute viral hepatitis worldwide. HAV is endemic in China. This study performed genetic and evolutionary analysis of HAV isolates circulated in the country. METHODS: Clinical samples were collected and HAV nucleotide and deduced amino acid sequences were analyzed. 70 representative sequences of HAV VP3-VP1-2A regions sampled from 1988 to 2014 were compared and characterized using the Bayesian Markov Chain Monte Carlo approach (BEAST software, Version1.7.5). RESULTS: All isolates from China in this study belonged to genotype I, with most of the samples clustering in subgenotype IA, while several unique amino acid variants were observed. The estimated mean substitution rate was 5.56×10(-4) substitutions / site / year, the time to the most recent common ancestor of genotype I isolates in China was calculated to be around 180 years ago. Skyline plots showed the incidence of HAV went down gradually from the mid-1990s. CONCLUSIONS: The evolution estimations were consistent with the laboratory and epidemiological results. Several isolates from China showed amino acid changes close to the immunodominant sites, which needs to be further analyzed. The study results have indicated the effectiveness of improving economic and sanitation levels together with HAV vaccination to control HAV-related infections in China.

[Establishment and application of TaqMan real-time RT-PCR for the detection of hepatitis A virus].

OBJECTIVE: To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis A virus in serum samples. METHODS: According to the references, primers-probe sets which were located in 5'-NCR, the most conservative part of HAV genome were designed and therefore we established a TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HAV RNA in serum from HAV patients. RESULTS: The HAV Real-time RT-PCR assay established in this study were able to detect HAV RNA and its detection limit ranged from 0.1CCID50/reaction to 0.01CCID50/reaction. When the detection of a same sample was repeated for three times, coefficients of varistion (CV) of intra- and inter-assay were calculated and they were all less than 2.0% and 2.6% respectively. Our data suggested that there were 5.18 x 10(2) - 4.93 x 10(7) RNA copies in 1 ml of the serum from acute HAV patients. CONCLUSION: The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HAV RNA. It was applied successfully in the pathogen detection of clinical samples.

[Hepatitis A virus mimotope mapping by phage display peptide library].

OBJECTIVE: A 12 mer phage display peptide library was used to identify hepatitis A virus mimotopes of antigenic determinants, to provide the feasibility of virus epitope mapping by using this approach. METHODS: Using purified anti-hepatitis A virus monoclonal antibody as affinity selective molecule, phage display peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing. RESULTS: 10 ELISA positive clones were chosen for DNA sequencing, and the displayed peptide sequences were deduced. 9 of them showed identical nucleotide sequence, and similarity in their amino acid sequence with VP1 of HAV HM175 was found, but no sequence homology was found between the other phage clone and the capsid proteins of HAV. Those peptides may behave as mimotopes of HAV. CONCLUSION: The mimotope of HAV was selected by using phage display peptide library screening. The results provide the potential of this method to search for the mimotopes of the virus.

[Characterization of capsid protein VP3-VP1 genes of hepatitis A virus prevalent strains circulated in China].

OBJECTIVE: To analyse the genetic characteristics of the capsid protein VP3-VP1 region of hepatitis A virus strains circulated in China. METHODS: The nucleotide sequences of VP3-VP1-2A region of 42 HAV IgM positive serum samples were sequenced and analysed for nucleotide and amino acid identities and genetic characteristics of the VP3-VP1 region. RESULTS: The nucleotide and amino acid identities in the VP1-2A junction region among the 42 strains were 89.1% - 100% and 97.3% - 100%; while in the complete VP3-VP1 region, the identities were 87.6%-100% and 98.8%-100%. Strains with identical nucleotide sequences in the VP1-2A junction region had 98.4%-100% nucleotide identity in the complete VP3-VP1 region and 0-2 amino acid differences in this region. There were no amino acid changes at neutralizing antigenetic sites of VP3-VP1 region within the 42 HAV strains. CONCLUSION: All the 42 HAV strains belonged to genotype I, with 40 strains clustering to sub-genotype IA and 2 to sub-genotype IB. Different HAV strains analysed in this paper differed in the nucleotide sequences of the VP3-VP1 region, but the amino acid sequences were highly conserved with no changes at neutralizing antigenetic sites. Both the nucleotide and amino acid sequences of the strains with the same VP1-2A junction region were identical or closely related when compared in the complete VP3-VP1 region.

The mechanism and kinetics of decomposition of 5-aminotetrazole.

The pathway and ab initio direct kinetics of the decomposition 5-aminotetrazole (5-ATZ) to HN(3) and NH(2)CN was investigated. Reactant, products and transition state were optimized with MP2 and B3LYP methods using 6-311G** and aug-cc-pVDZ basis sets. The intrinsic reaction coordinate curve of the reaction was calculated using the MP2 method with 6-311G** basis set. The energies were refined using CCSD(T)/6-311G**. Rate constants were evaluated by conventional transition-state theory (CVT) and canonical variational transition-state theory (TST), with tunneling effect over 300 to 2,500 K. The results indicated that the tunneling effect and the variational effect are small for the calculated rate constants. The fitted three-parameter expression calculated using the CVT and TST methods are k(T) = 4.07 x 10(11) x T(0.84) x e((-2.42 x 10(4))/T)s(-1) and k(T) = 2.09 x 10(11) x T(0.89) x e((-2.36 x 10(4))/T)s(-1), respectively.