cobra-cardiotoxin-proteins and Inflammation

Lymphoma idiotype protein vaccines have shown therapeutic potential in previous clinical studies, and results from a completed pivotal, phase 3 controlled trial are promising. However, streamlined production of these patient-specific vaccines is required for eventual clinical application. Here, we show that second-generation, chemokine-fused idiotype DNA vaccines, when combined with myotoxins that induced sterile inflammation with recruitment of antigen-presenting cells at vaccination sites, were exceptional in their ability to provoke memory antitumor immunity in mice, compared with several TLR agonists. The combined vaccination strategy elicited both antigen-specific T-cell responses and humoral immunity. Unexpectedly, vaccine-induced tumor protection was intact in B cell-deficient mice but was abrogated completely by T-cell depletion in vivo, suggesting T-cell dependence. Furthermore, the optimal effect of myotoxins was observed with fusion vaccines that specifically targeted antigen delivery to antigen-presenting cells and not with vaccines lacking a targeting moiety, suggesting that the rational vaccine design will require combination strategies with novel, proinflammatory agents and highly optimized molecular vaccine constructs. These studies also challenge the paradigm that antibody responses are the primary of idiotype-specific antitumor effects and support the optimization of idiotype vaccines designed to induce primarily T-cell immunity.

Topics: Adjuvants, Immunologic; Animals; Antigen-Presenting Cells; B-Lymphocytes; Cancer Vaccines; Cell Line, Tumor; Cobra Cardiotoxin Proteins; Crotoxin; Drug Synergism; Immunity, Cellular; Immunoglobulin Idiotypes; Inflammation; Lymphoma; Mice; Mice, Inbred BALB C; T-Lymphocytes; Vaccines, DNA

The inflammatory response is thought to play important roles in tissue healing. The hypothesis of this study was that the inflammatory cytokine interferon (IFN)-gamma is produced endogenously following skeletal muscle injury and promotes efficient healing. We show that IFN-gamma is expressed at both mRNA and protein levels in skeletal muscle following injury, and that the time course of IFN-gamma expression correlated with the accumulation of macrophages, T-cells, and natural killer cells, as well as myoblasts, in damaged muscle. Cells of each type were isolated from injured muscle, and IFN-gamma expression was detected in each cell type. We also demonstrate that administration of an IFN-gamma receptor blocking antibody to wild-type mice impaired induction of interferon response factor-1, reduced cell proliferation, and decreased formation of regenerating fibers. IFN-gamma null mice showed similarly impaired muscle healing associated with impaired macrophage function and development of fibrosis. In vitro studies demonstrated that IFN-gamma and its receptor are expressed in the C2C12 muscle cell line, and that the IFN-gamma receptor blocking antibody reduced proliferation and fusion of these muscle cells. In summary, our results indicate that IFN-gamma promotes muscle healing, in part, by stimulating formation of new muscle fibers.

Topics: Animals; Cell Culture Techniques; Cell Division; Cobra Cardiotoxin Proteins; DNA Primers; Forelimb; Inflammation; Interferon gamma Receptor; Interferon-gamma; Intermediate Filament Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Myoblasts; Receptors, Interferon; Regeneration; Reverse Transcriptase Polymerase Chain Reaction

Severe tissue necrosis with a retarded wound healing process is a major symptom of a cobra snakebite. Cardiotoxins (CTXs) are major components of cobra venoms that belong to the Ly-6 protein family and are implicated in tissue damage. The interaction of the major CTX from Taiwan cobra, i.e. CTX A3, with sulfatides in the cell membrane has recently been shown to induce pore formation and cell internalization and to be responsible for cytotoxicity in cardiomyocytes (Wang, C.-H., Liu, J.-H., Lee, S.-C., Hsiao, C.-D., and Wu, W.-g. (2006) J. Biol. Chem. 281, 656-667). We show here that one of the non-cytotoxic CTXs, i.e. CTX A5 or cardiotoxin-like basic polypeptide, from Taiwan cobra specifically bound to alpha(v)beta3 integrin and inhibited bone resorption activity. We found that both membrane-bound and recombinant soluble alpha(v)beta3 integrins bound specifically to CTX A5 in a dose-dependent manner. Surface plasmon resonance analysis showed that human soluble alpha(v)beta3 bound to CTX A5 with an apparent affinity of approximately 0.3 microM. Calf pulmonary artery endothelial cells, which constitutively express alpha(v)beta3, showed a CTX A5 binding profile similar to that of membrane-bound and soluble alpha(v)beta3 integrins, suggesting that endothelial cells are a potential target for CTX action. We tested whether CTX A5 inhibits osteoclast differentiation and bone resorption, a process known to be involved in alpha(v)beta3 binding and inhibited by RGD-containing peptides. We demonstrate that CTX A5 inhibited both activities at a micromolar range by binding to murine alpha(v)beta3 integrin in osteoclasts and that CTX A5 co-localized with beta3 integrin. Finally, after comparing the integrin binding affinity among CTX homologs, we propose that the amino acid residues near the two loops of CTX A5 are involved in integrin binding. These results identify CTX A5 as a non-RGD integrin-binding protein with therapeutic potential as an integrin antagonist.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Bone Resorption; Cattle; Cell Adhesion; Cell Differentiation; Cell Line; Cell Proliferation; Cobra Cardiotoxin Proteins; Dose-Response Relationship, Drug; Elapidae; Endothelial Cells; Glycoproteins; Inflammation; Integrin alphaVbeta3; Integrins; Ligands; Mice; Microscopy, Electron, Scanning; Molecular Sequence Data; Necrosis; Neoplasms; Oligopeptides; Osteoclasts; Protein Binding; Protein Structure, Secondary; Pulmonary Artery; Recombinant Proteins; Sequence Homology, Amino Acid; Structure-Activity Relationship; Time Factors

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cobra Cardiotoxin Proteins; Cytokine TWEAK; Gene Expression Regulation; Humans; Inflammation; Mesenchymal Stem Cells; Mice; Models, Biological; Muscle Development; Muscle, Skeletal; Myoblasts; Receptors, Tumor Necrosis Factor; Regeneration; RNA, Messenger; Tumor Necrosis Factors; TWEAK Receptor