We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.
Adenoviridae ; genetics ; metabolism ; Adiponectin ; biosynthesis ; genetics ; Animals ; Cell Line ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the effects of total flavonoids from astragalus complanatus (FAC) on paraquat poisoning-induced pulmonary fibrosis in rats.

METHODSThe rats were divided into six groups randomly: control group, paraquat group, prednisolone group and FAC low-dose, middle-dose, high-dose group. Pulmonary fibrosis model was replicated by intratracheal injection of paraquat. In the mext day,the rats were treated by intragastric administration once a day. After 28 days, the rats were sacrificed. The lung index and the levels of HYP and T-AOC were measured, and the pathologic changes of the lung tissue were obtained by HE staining. The levels of TGF-β, Smad2, α-SMA protein were analyzed by Western blot.

RESULTSFAC improved the activity of T-AOC in serum and reduced pulmonary index and the content of HYP as well (P<0.05 or P<0.01), the alveolitis and fibrosis extent were attenuated. The expression of Smad2 significantly decreased in groups of FAC low-dose, middle-dose and high-dose (0.31±0.11, 0.45±0.12 and 0.30±0.05) as compared with that of the PQ group (0.85±0.34) (P<0.05). The expression of α-SMA significantly decreased in groups of FAC low-dose, middle-dose and high-dose (0.31±0.11, 0.35±0.07 and 0.32±0.10) as compared with that of the PQ group (0.45±0.08) (P<0.05). The expression of TGF-β significantly decreased in groups of FAC low-dose, middle-dose and high-dose (0.35±0.04, 0.27±0.05 and 0.18±0.04)as compared with that of the PQ group (0.63±0.11) (P<0.05).

CONCLUSIONFAC can alleviate PQ-induced pulmonary fibrosis in rats through inhibiting TGF-β/Smad signaling pathway.

Actins ; metabolism ; Animals ; Astragalus Plant ; chemistry ; Flavonoids ; pharmacology ; Lung ; pathology ; Paraquat ; poisoning ; Phytochemicals ; pharmacology ; Pulmonary Fibrosis ; chemically induced ; drug therapy ; Rats ; Smad2 Protein ; metabolism ; Transforming Growth Factor beta ; metabolism

Chimeric antigen receptor (CAR)-T cell therapy has achieved remarkable success in the treatment of hematological malignancies. Based on the immunomodulatory capability of CAR-T cells, efforts have turned toward exploring their potential in treating autoimmune diseases. Bibliometric analysis of 210 records from 128 academic journals published by 372 institutions in 40 countries/regions indicates a growing number of publications on CAR-T therapy for autoimmune diseases, covering a range of subtypes such as systemic lupus erythematosus, multiple sclerosis, among others. CAR-T therapy holds promise in mitigating several shortcomings, including the indiscriminate suppression of the immune system by traditional immunosuppressants, and non-sustaining therapeutic levels of monoclonal antibodies due to inherent pharmacokinetic constraints. By persisting and proliferating in vivo, CAR-T cells can offer a tailored and precise therapeutics. This paper reviewed preclinical experiments and clinical trials involving CAR-T and CAR-related therapies in various autoimmune diseases, incorporating innovations well-studied in the field of hematological tumors, aiming to explore a safe and effective therapeutic option for relapsed/refractory autoimmune diseases.

The endovascular exclusion is an effective treatment of aortic aneurysm diseases in frail and elderly patients who cannot suffer the open surgery. However, as the key treatment device of this technique, traditional stent-grafts are not suitable to treat complex aortic aneurysm diseases in emergency. The emergence of the fenestrated stent-graft and in-situ fenestration has brought new dawn to the treatment of these patients. This study reviews the advances in complex aortic aneurysms treated by the fenestrated stent-graft and the in-situ fenestration. In addition, the novel concept of the fabric structure designed for "in-situ fenestrated stent-graft" is proposed for the in-situ fenestration technique. It is expected to break through the bottleneck of the present fenestrated stent-grafts. It would be beneficial to the bailout of complex aortic aneurysm diseases and thereby benefitting more patients.

Objective:To determine the content of the released nickel ion through the 7 extraction media to extract the Ni-Ti wires and to plot the curve of the released nickel ion so as to identify a leaching medium that can be substituted for blood for in vitro Ni release evaluation. Methods:The release of Ni through microwave digestion/inductively coupled plasma mass spectrometry (ICP-MS) in the goat serum was determined. Because of the high content of Ni release, it could be determined by diluting the extraction medium, and other extraction media could be determined directly. Ni release standard curves were plotted by the release amount and different time point variables. Though the different extraction media Ni release curves confirm the specificity of extraction media instead of blood.Results:By analyzing the Ni release curves of seven leaching media, it was found that none of these seven extraction media was suitable for the evaluation of Ni release in in vitro leaching media. Considering the safety of the leaching medium and the simplicity of preparation, hydrochloric acid solution was chosen as the leaching medium, but the concentration needed to be diluted accordingly. Finally, a hydrochloric acid solution was created as an alternative to blood for the in vitro study of Ni release from Ni-Ti alloy cardiovascular products, with a volume fraction of 0.005%. Conclusions:The in vitro leaching medium that can replace blood was found to be hydrochloric acid for the time being, but its concentration was too high, resulting in too much Ni release as well, which deviated from the actual situation. Therefore, the hydrochloric acid solution was diluted step by step, and the Ni release curve was examined until it was close to the clinical release level, and the actual concentration was determined, thus laying a solid foundation for the subsequent evaluation of the safety and risk.