Enhancement of T Follicular Helper Cell-Mediated Humoral Immunity Reponses During Development of Experimental Autoimmune Myasthenia Gravis / 神经科学通报·英文版

Myasthenia gravis (MG) is a prototypical antibody-mediated neurological autoimmune disease with the involvement of humoral immune responses in its pathogenesis. T follicular helper (Tfh) cells have been implicated in many autoimmune diseases. However, whether and how Tfh cells are involved in MG remain unclear. Here, we established and studied a widely-used and approved animal model of human MG, the rat model with acetylcholine receptor alpha (AChRα) subunit (R-AChR)-induced experimental autoimmune myasthenia gravis (EAMG). This model presented mild body-weight loss 10 days after the first immunization (representing the early stage of disease) and more obvious clinical manifestations and body-weight loss 7 days after the second immunization (representing the late stage of disease). AChR-specific pre-Tfh cells and mature Tfh cells were detected in these two stages, respectively. In co-cultures of Tfh cells and B cells, the number of IgG2b-secreting B cells and the level of anti-AChR antibodies in the supernatant were higher in the cultures containing EAMG-derived Tfh cells. In immunohistochemistry and immunofluorescence assays, a substantial number of CD4/Bcl-6 T cells and a greater number of larger germinal centers were observed in lymph node tissues resected from EAMG rats. Based on these results, we hypothesize that an AChR-specific Tfh cell-mediated humoral immune response contributes to the development of EAMG.

Role of interelenkin-17, -6 and endothelin-1 in statins attenuated no-reflow phenomenon of rat acute myocardial infarction and reperfusion / 中国地方病学杂志

ObjectiveTo study the relevant effect of proinflammatory cytokines interelenkin-17(IL-17), -6 and endothelin-1 (ET-1) on statins attenuating no-reflow phenomenon after myocardial ischemia-reperfusion in rats.MethodsEighteen healthy male Wistar rats were randomly divided into 3 groups according to body weight: sham operation, injury, preconditioning groups. The preconditioning group was given atorvastatin 2 mg·kg-1 ·d-1 and the other two groups were given the same volume of saline once. After 7 days, the rats were anesthetized with an intraperitoneal injection of chloral hydrate, and then the thoracic cavity was opened. The coronary artery of injury group and preconditioning group were ligated for 60 minutes, and then opened for 15 minutes, to establish the rat acute myocardial ischemia-reperfusion model. The sham operation group was was treated with a seam through the coronary artery without ligation. Eleetrocardiogram was checked before ligation, and ligation was carried out for 15, 30, 45 minutes and then reperfusion for 15 minutes. After reperfusion for 15 minutes, the thioflavine S and Even's were injected from femoral venous, then the heart and blood were obtained(keeping left ventricular only). Hearts were flushed with saline and sliced transversely into five to seven sections. Finally, observed at 365 nm wave length the existence of non-fluorescent areas, which was no-reflow zone. The level of serum IL-17, IL-6 and ET-1 was detected by ELISA. Results The electrocardiogram confirmed that the sham operation group had no ischemic damage and the model of myocardial ischemia- reperfusion was established in preconditioning group and injury group. The noreflow phenomenon could be observed under 365 nm wave length in preconditioning group and injury group. The ligated area[LA％, (57.34 ± 11.49)％, (53.08 ± 8.66)％] of injury group and preconditioning group was higher than that of sham operation group(0, all P ＜ 0.05); the area of no-reflow[ANF％, (48.96 ± 6.94)％, (21.37 ±3.35)％] of injury group and preconditioning group was higher than that of sham operation group(0, all P ＜ 0.05),and the ANF％ of preconditioning group was lower than that of injury group(P ＜ 0.05) ; the level of serum IL-17,IL-6 and ET-1[(151.67 ± 11.19) × 10-9, (167.89 ± 5.13) × 10-9, (322.37 ± 19.08) × 10-9 g/L] of injury group was higher than those of sham group and preconditioning operation group[(49.75 ± 14.06) × 10-9, (59.32 ± 5.26) ×10-9, (109.9 ± 12.12) × 10-9, (90.45 ± 11.63) × 10-9, (112.47 ± 10.40) × 10-9 and(198.91 ± 27.88) × 10-9 g/L,P ＜ 0.05], the level of serum IL-17, IL-6 and ET-1 of preconditioning group was higher than those of sham operation group(P＜ 0.05). Conclusionsno-reflow phenomenon is related with IL-17 and ET-1 which can promote the expression of IL-6, statins decreases the expression of IL-17 and ET-1, and then decreases the on-reflow phenomenon.

Effects of human DAF and CD59 co-expression in vascular endothelia in transgenic mice on heart work during human plasma peffasion / 中华器官移植杂志

Objective To construct transgenic mice tissue-specifically co-expressing human DAF/CD59 in the vascular endothelia and to investigate the ability to protect against human comple- ment-mediated attack.Methods Transgenic mice were generated by co-microinjection of hDAF/CD59 expression constructs driven by the human intercellular adhesion molecule-2(ICAM-2)promoter.PCR and Southern blot of genomic DNA were used to assess the presence of hDAF/CD59 in the genome of the founders,and the expression at protein level was measured by flow cytometry.Immunohistochemis- try was used to detect the distribution of hDAF/CD59.An ex vivo perfusion model was used to com- pare hearts from these hDAF/CD59 transgenic mice with hDAF hearts.Results After microinjection of genes,11 of 135 mice born were shown to be double-transgenic,and human DAF/CD59 were ex- pressed on the surface of leucocytes in 6 of the 11 DAF/CD59-integrated mice.Expression levels of 6 founders ranged from 80% to 95% of that in human leucocytes.Human DAF/CD59 were strongly expressed in the vascular endothelia of heart,kidney,with little or no positive staining observed in non- endothelial cells.Compared to hDAF hearts that maintained approximately 20% maximum work during perfusion with 20% human plasma,these endothelial-specific hDAF/CD59 hearts were further protected with work maintained at 40% of the maximum level during 60 min.Conclusion The intro- duced hDAF/CD59 genes have been integrated and specifically expressed in the vascular endothelia. The endothelial co-expression of hDAF/CD59 can provide greater protection against human complement-mediated attack than the expression of hDAF alone.

Construction of recombinant human CD59 using ICAM-2 promoter for endothelial-specific expression in xenotransplantation / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct a recombinant human CD59 gene containing intercellular adhesion molecule-2 promoter for high level endothelial-specific expression in xenotransplantation.</p><p><b>METHODS</b>ICAM-2 promotor fragment and CD59-intron 1 fragment were produced by PCR from the human blood genome, and then clone these fragments into a pcDNA3-CD59 eukaryotic expression vector which was followed by digestion with the specific restricted endonuclease (for example: EcoRI, Hind III). The ICAM-2 promoter and CD59-intron 1 fragments were identified by PCR, and sequencing. The recombinant was then transfected into pig aorta endothelial cells with Lipofection, and the expression was measured by flow cytometer.</p><p><b>RESULTS</b>Products of the sequences measured were in accord with the frames of the gene bank. The expression of the protein of this recombinant was positive.</p><p><b>CONCLUSION</b>The CD59 recombinant gene is constructed successfully, providing a basis for transgenic research.</p>