Antibodies against type II collagen(CII)are essential for development of collagen-induced arthritis(CIA),but how and where the B-cell response to CII is initiated is not fully known.We show here that naive DBA/1 mice ...Antibodies against type II collagen(CII)are essential for development of collagen-induced arthritis(CIA),but how and where the B-cell response to CII is initiated is not fully known.We show here that naive DBA/1 mice display naturally reactive IgM and IgG anti-CII producing B cells prior to immunization.The CII-reactive B cells were observed in the spleen and recognized as marginal zone(MZ)B cells.After CII immunization,CII-specific B cells expanded rapidly in the spleen,in contrast to the lymph nodes,with the initial response derived from MZ B cells and later by follicular(FO)B cells.This was evident despite that the MZ B cells were subject to stringent tolerance mechanisms by having a greater Fc gamma receptor IIb expression than the FO B cells.Further,the MZ B cells migrated to the FO areas upon immunization,possibly providing antigen and activating FO T cells and subsequently FO B cells.Thus,around CIA onset increased numbers of IgG anti-CII producing FO B cells was seen in the spleen,which was dominated by IgG2a-and IgG2b-positive cells.These data demonstrate that CII-reactive MZ B cells are present before and expand after CII immunization,suggesting an initiating role of MZ B cells in the development of CIA.展开更多
The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were...The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.展开更多
Effective treatment of rheumatoid arthritis can be mediated by native chicken type II collagen(n CCII), recombinant peptide containing n CCII tolerogenic epitopes(CTEs), or a therapeutic DNA vaccine encoding the full-...Effective treatment of rheumatoid arthritis can be mediated by native chicken type II collagen(n CCII), recombinant peptide containing n CCII tolerogenic epitopes(CTEs), or a therapeutic DNA vaccine encoding the full-length CCOL2 A1 c DNA. As recombinant CCII(r CCII) might avoid potential pathogenic virus contamination during n CCII preparation or chromosomal integration and oncogene activation associated with DNA vaccines, here we evaluated the importance of propeptide and telopeptide domains on r CCII triple helix molecular assembly. We constructed p C-and p N-procollagen(without N-or Cpropeptides, respectively) as well as CTEs located in the triple helical domain lacking both propeptides and telopeptides, and expressed these in yeast Pichia pastoris host strain GS115(his4, Mut+) simultaneously with recombinant chicken prolyl-4-hydroxylase α and β subunits. Both p C-and p N-procollagen monomers accumulated inside P. pastoris cells, whereas CTE was assembled into homotrimers with stable conformation and secreted into the supernatants, suggesting that the large molecular weight p C-or p N-procollagens were retained within the endoplasmic reticulum whereas the smaller CTEs proceeded through the secretory pathway. Furthermore, resulting recombinant chicken type II collagen p Cα1(II) can induce collagen-induced arthritis(CIA) rat model, which seems to be as effective as the current standard n CCII. Notably, protease digestion assays showed that r CCII could assemble in the absence of C-and N-propeptides or telopeptides. These findings provide new insights into the minimal structural requirements for r CCII expression and folding.展开更多
基金This research project was supported by the Swedish Research Council and The King Gustav V’s 80 years Foundation
文摘Antibodies against type II collagen(CII)are essential for development of collagen-induced arthritis(CIA),but how and where the B-cell response to CII is initiated is not fully known.We show here that naive DBA/1 mice display naturally reactive IgM and IgG anti-CII producing B cells prior to immunization.The CII-reactive B cells were observed in the spleen and recognized as marginal zone(MZ)B cells.After CII immunization,CII-specific B cells expanded rapidly in the spleen,in contrast to the lymph nodes,with the initial response derived from MZ B cells and later by follicular(FO)B cells.This was evident despite that the MZ B cells were subject to stringent tolerance mechanisms by having a greater Fc gamma receptor IIb expression than the FO B cells.Further,the MZ B cells migrated to the FO areas upon immunization,possibly providing antigen and activating FO T cells and subsequently FO B cells.Thus,around CIA onset increased numbers of IgG anti-CII producing FO B cells was seen in the spleen,which was dominated by IgG2a-and IgG2b-positive cells.These data demonstrate that CII-reactive MZ B cells are present before and expand after CII immunization,suggesting an initiating role of MZ B cells in the development of CIA.
基金Supported by the National Natural Science Foundation of China (Grant No. 30271318)
文摘The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.
文摘Effective treatment of rheumatoid arthritis can be mediated by native chicken type II collagen(n CCII), recombinant peptide containing n CCII tolerogenic epitopes(CTEs), or a therapeutic DNA vaccine encoding the full-length CCOL2 A1 c DNA. As recombinant CCII(r CCII) might avoid potential pathogenic virus contamination during n CCII preparation or chromosomal integration and oncogene activation associated with DNA vaccines, here we evaluated the importance of propeptide and telopeptide domains on r CCII triple helix molecular assembly. We constructed p C-and p N-procollagen(without N-or Cpropeptides, respectively) as well as CTEs located in the triple helical domain lacking both propeptides and telopeptides, and expressed these in yeast Pichia pastoris host strain GS115(his4, Mut+) simultaneously with recombinant chicken prolyl-4-hydroxylase α and β subunits. Both p C-and p N-procollagen monomers accumulated inside P. pastoris cells, whereas CTE was assembled into homotrimers with stable conformation and secreted into the supernatants, suggesting that the large molecular weight p C-or p N-procollagens were retained within the endoplasmic reticulum whereas the smaller CTEs proceeded through the secretory pathway. Furthermore, resulting recombinant chicken type II collagen p Cα1(II) can induce collagen-induced arthritis(CIA) rat model, which seems to be as effective as the current standard n CCII. Notably, protease digestion assays showed that r CCII could assemble in the absence of C-and N-propeptides or telopeptides. These findings provide new insights into the minimal structural requirements for r CCII expression and folding.
