ABSTRACT
Background: The identification of fragment sequences, or motifs, within a therapeutic protein that may elicit an immune response when processed by T-cells can be provided by computer-aided approaches. Immunogenicity is a significant problem associated with protein therapeutics and should be investigated in the early stage of protein-based drug development to avoid treatment resistance and potentially life-threatening immune responses. Purpose: To provide a combined computer-aided protocol for investigating the immunogenic profile of a recombinant Kunitz-type inhibitor, which has been reported as promising antitumor agent by our research group. Methods: The combination of databases searching (IEDB and SYFPEITHI) and molecular docking simulations was exploited, herein. This combined protocol has allowed the identification of potential epitopes before in vitro/in vivo evaluation. Predictors of human proteasome cleavage transport and major histocompatibility complex (MHC) binding were considered as overall score assigning the corresponding intrinsic potential of being a T cell epitope to each fragment sequence. The peptides or motifs better classified in the two databases were docked into the three-dimensional (3D) structure of MHC (class I and II) complex to verify the calculated binding affinity. The binding interactions regarding the molecular recognition process by T-cells were also exploited through the MHC:ligand:T-cell complexes. Results: Regarding the Kunitz-type sequence, four motifs were identified as potentially epitopes for MHC-I and three motifs were found for MHC-II. But, those motifs were classified as moderately immunogenic. Final remarks: The combined computer-aided protocol has significantly reduced the number of potential epitopes to be considered for further analysis and could be useful to identify immunogenic fragments (high, moderate and low) in protein pharmaceutics before in vitro/in vivo experimentation.
ABSTRACT
Background: The identification of fragment sequences, or motifs, within a therapeutic protein that may elicit an immune response when processed by T-cells can be provided by computer-aided approaches. Immunogenicity is a significant problem associated with protein therapeutics and should be investigated in the early stage of protein-based drug development to avoid treatment resistance and potentially life-threatening immune responses. Purpose: To provide a combined computer-aided protocol for investigating the immunogenic profile of a recombinant Kunitz-type inhibitor, which has been reported as promising antitumor agent by our research group. Methods: The combination of databases searching (IEDB and SYFPEITHI) and molecular docking simulations was exploited, herein. This combined protocol has allowed the identification of potential epitopes before in vitro/in vivo evaluation. Predictors of human proteasome cleavage transport and major histocompatibility complex (MHC) binding were considered as overall score assigning the corresponding intrinsic potential of being a T cell epitope to each fragment sequence. The peptides or motifs better classified in the two databases were docked into the three-dimensional (3D) structure of MHC (class I and II) complex to verify the calculated binding affinity. The binding interactions regarding the molecular recognition process by T-cells were also exploited through the MHC:ligand:T-cell complexes. Results: Regarding the Kunitz-type sequence, four motifs were identified as potentially epitopes for MHC-I and three motifs were found for MHC-II. But, those motifs were classified as moderately immunogenic. Final remarks: The combined computer-aided protocol has significantly reduced the number of potential epitopes to be considered for further analysis and could be useful to identify immunogenic fragments (high, moderate and low) in protein pharmaceutics before in vitro/in vivo experimentation.
ABSTRACT
Background Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolin's roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosac's in vivo effects using a skin wound healing model in rats. Methods Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. Results The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. Conclusion The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations.(AU)
Subject(s)
Skin/injuries , Wound Healing , Wounds and Injuries , Proteins , Cell Proliferation , Epithelial Cells , LepidopteraABSTRACT
Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolins roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosacs in vivo effects using a skin wound healing model in rats. Methods Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. Results The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. Conclusion The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations.
Subject(s)
Wound Healing , Apoptosis Regulatory Proteins/analysis , Apoptosis Regulatory Proteins/adverse effects , Lepidoptera/chemistryABSTRACT
Background: Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolin's roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosac's in vivo effects using a skin wound healing model in rats. Methods: Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. Results: The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. Conclusion: The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations
