Cbl Ubiquitin Ligases Control B Cell Exit from the Germinal-Center Reaction.

Selective expansion of high-affinity antigen-specific B cells in germinal centers (GCs) is a key event in antibody affinity maturation. GC B cells with improved affinity can either continue affinity-driven selection or exit the GC to differentiate into plasma cells (PCs) or memory B cells. Here we found that deleting E3 ubiquitin ligases Cbl and Cbl-b (Cbls) in GC B cells resulted in the early exit of high-affinity antigen-specific B cells from the GC reaction and thus impaired clonal expansion. Cbls were highly expressed in GC light zone (LZ) B cells, where they promoted the ubiquitination and degradation of Irf4, a transcription factor facilitating PC fate choice. Strong CD40 and BCR stimulation triggered the Cbl degradation, resulting in increased Irf4 expression and exit from GC affinity selection. Thus, a regulatory cascade that is centered on the Cbl ubiquitin ligases ensures affinity-driven clonal expansion by connecting BCR affinity signals with differentiation programs.

[Altered expression of endogenous transforming growth factor ß1 and early calcification related genes in rat endplate].

OBJECTIVE: To explore the relationship between endogenous transforming growth factor (TGF)-ß1 and calcification-related genes through an in vitro degeneration model by propagating rat endplate chondrocytes during a natural degeneration process. METHODS: Endplate chondrocytes were extracted from rat lumbar vertebrae, isolated by enzyme digestion and P2 and P4 generations selected for a 6-day in vitro culture. The specimens were photographed microscopically to observe the cellular differences by alizarin red staining. Type II collagen marker gene, transcription factor SOX-9 gene and metabolism-related genes proteoglycan. matrix metalloproteinase (MMP)-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 were detected by RT-PCR to verify the degeneration model. Based on this model, the changes of growth factor TGF-ß1 and calcification-related genes ankyrin (ANK), ectonucleotide pyrophosphatase (ENPP), tissue-nonspecific alkaline phosphatase (TNAP) were continuously tested. RESULTS: Compared with P2 cells, P4 cells tended to assume a spindle-shaped morphology. And alizarin red staining showed no change between them. The level of transcription factor SOX-9 of P4 cells (P4/P2 = 0.0690, P = 0.0489) was significantly lower than that of P2 cells. Type II collagen (P4/P2 = 0.0535, P = 0.009) and proteoglycan (P4/P2 = 0.2672, P = 0.0343) were also significantly lower than those of P2 cells. No significant changes were observed in other metabolism-related genes. TGF-ß1 (P4/P2 = 0.5934, P = 0.0482) was significantly lower. The expressions of TNAP (P4/P2 = 0.0385, P = 0.0139) and ANK (P4/P2 = 0.2121, P = 0.0009) were significantly lower. But ENPP showed no significant change. CONCLUSION: P4 endplate chondrocytes undergo natural degeneration in vitro with the rising passage number. Type II collagen, SOX-9 and proteoglycan are significantly reduced. Endogenous TGF-ß1 gene and calcification-related genes are down-regulated. The decrease of ANK gene may be caused by the down-regulation of endogenous TGF-ß1. Modulating the expression of endogenous TGF-ß1 gene in endplate chondrocytes may become a new therapeutic approach for the degeneration of intervertebral disc.

Ubiquitin Ligases CBL and CBL-B Maintain the Homeostasis and Immune Quiescence of Dendritic Cells.

Dendritic cells (DCs) are composed of multiple lineages of hematopoietic cells and orchestrate immune responses upon detecting the danger and inflammatory signals associated with pathogen and damaged tissues. Under steady-state, DCs are maintained at limited numbers and the functionally quiescent status. While it is known that a fine balance in the DC homeostasis and activation status is also important to prevent autoimmune diseases and hyperinflammation, mechanisms that control DC development and activation under stead-state remain not fully understood. Here we show that DC-specific ablation of CBL and CBL-B (CBL-/-CBL-B-/-) leads to spontaneous liver inflammation and fibrosis and early death of the mice. The mutant mice have a marked expansion of classic CD8α+/CD103+ DCs (cDC1s) in peripheral lymphoid organs and the liver. These DCs exhibit atypical activation phenotypes characterized by an increased production of inflammatory cytokines and chemokines but not the cell surface MHC-II and costimulatory ligands. While the mutant mice also have massive T cell activation, lymphocytes are not required for the disease development. The CBL-/-CBL-B-/- mutation enhances FLT3-mTOR signaling, due to defective FLT3 ubiquitination and degradation. Blockade of FLT3-mTOR signaling normalizes the homeostasis of cDC1s and attenuates liver inflammation. Our result thus reveals a critical role of CBLs in the maintenance of DC homeostasis and immune quiescence. This regulation could be relevant to liver inflammatory diseases and fibrosis in humans.

Polyethylenimine600-ß-cyclodextrin: a promising nanopolymer for nonviral gene delivery of primary mesenchymal stem cells.

Genetically modified mesenchymal stem cells (MSCs) have great potential in the application of regenerative medicine and molecular therapy. In the present manuscript, we introduce a nanopolymer, polyethylenimine600-ß-cyclodextrin (PEI600-ß-CyD), as an efficient polyplex-forming plasmid delivery agent with low toxicity and ideal transfection efficiency on primary MSCs. PEI600-ß-CyD causes significantly less cytotoxicity and apoptosis on MSCs than 25 kDa high-molecular-weight PEI (PEI25kDa). PEI600-ß-CyD also exhibits similar transfection efficiency as PEI25kDa on MSCs, which is higher than that of PEI600Da. Quantum dot-labeled plasmids show that PEI600-ß-CyD or PEI25kDa delivers the plasmids in a more scattered manner than PEI600Da does. Further study shows that PEI600-ß-CyD and PEI25kDa are more capable of delivering plasmids into the cell lysosome and nucleus than PEI600Da, which correlates well with the results of their transfection-efficiency assay. Moreover, among the three vectors, PEI600-ß-CyD has the most capacity of enhancing the alkaline phosphatase activity of MSCs by transfecting bone morphogenetic protein 2, 7, or special AT-rich sequence-binding protein 2. These results clearly indicate that PEI600-ß-CyD is a safe and effective candidate for the nonviral gene delivery of MSCs because of its ideal inclusion ability and proton sponge effect, and the application of this nanopolymer warrants further investigation.

Uptake mechanisms of non-viral gene delivery.

Non-viral gene delivery is currently a hot subject for its relative safety and simplicity of use; however, it is still far from being ideal enough to be clinically used for its comparatively lower efficiency than viral gene delivery. To improve the efficiency of non-viral gene delivery needs a comprehensive understanding of the uptake mechanisms. Macromolecules are internalized into cells by a variety of mechanisms, and their intracellular fates are usually relevant with the uptake pathways. The uptake pathways of non-viral gene complexes are usually determined by not only the gene/carrier interaction but also by the interaction between complexes and target cells. The best-characterized uptake pathway is the so-called clathrin-mediated endocytic pathway. However, there are numerous updates of knowledge about endocytic pathways and even non-endocytic pathways in recent years with the development of novel technologies for tracking and inhibiting. In this review, we will try to sort out our current understanding of the uptake mechanisms of non-viral gene delivery. In addition, factors for pathway selection are summarized in the third section. Finally, the useful inhibitors or tools for the study of these pathways will also be concluded in the last section.

Biscarbamate cross-linked low molecular weight PEI for delivering IL-1 receptor antagonist gene to synoviocytes for arthritis therapy.

Cytoxicity is an essential concern for polyethyleneimine 25 kDa (PEI 25 kDa), a widely reported, highly effective transfection agent used in gene delivery. In our recent experiments, Small molecular weight cross-linked poly(ethylene imine) by biscarbamate linkage (PEI-Bu) (Mn: 3278, Mw: 4289) can reduce target cell apoptosis induced by polycationic transfection, and has almost the same DNA condensation capability as PEI 25 kDa. PEI-Bu showed significantly higher activity and lower cytotoxicity than PEI 25 kDa in transfecting the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra) gene to rat synoviocytes, an optimal target for arthritis treatment. The expression of IL-1Ra in synoviocytes then suppresses the expression of metalloproteases 13 (MMP13) gene, which is responsible for cartilage destruction regulated by IL-1ß in arthritis. In conclusion, PEI-Bu is a promising tool for delivering IL-1Ra gene to synoviocytes for arthritis therapy.

Progress and prospects of chitosan and its derivatives as non-viral gene vectors in gene therapy.

Although cell transfection by viral vectors is highly efficient, undesirable side effects including immunogenicity, toxicity and carcinogenesis have to be taken into consideration before their clinical applications. In contrast, most non-viral vectors, such as chitosan, are advantageous due to their biocompatibility, biodegradability, low toxicity and immunogenicity. However, the tranfection efficiency of chitosan as gene vector is rather low because of its low stability and low buffering capacity. Recent technological progress in chemical modification of chitosan has led to improvements of its transfection efficiency without disturbing its biocompatibility and biodegradability. These advances have led to a better understanding of the relationship between the physicochemical characteristics of a non-viral vector and its transfection efficiency. In this review, we summarize the obstacles encountered during the transfection process of chitosan and its derivatives, and then focus on strategies to overcome these obstacles. An accurate method for determining the rate-limiting step and intracellular unpacking kinetics of chitosan and its derivatives is also presented. Lastly, gene-silencing chitosan/small interfering RNA (chitosan/siRNA) complexes and prospects of feasible methods for enhancing the transfection efficiency of chitosan and its derivatives are discussed.