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The Characterization of Monoclonal Antibodies to Mouse TLT-1 Suggests That TLT-1 Plays a Role in Wound Healing.

ABSTRACT: Platelets play a vital role in hemostasis and inflammation. The membrane receptor TREM-like transcript-1 (TLT-1) is involved in platelet aggregation, bleeding, and inflammation, and it is localized in the α-granules of platelets. Upon platelet activation, TLT-1 is released from α-granules both in its transmembrane form and as a soluble fragment (sTLT-1). Higher levels of sTLT-1 have been detected in the plasma of patients with acute inflammation or sepsis, suggesting an important role for TLT-1 during inflammation. However, the roles of TLT-1 in hemostasis and inflammation are not well understood. We are developing the mouse model of TLT-1 to mechanistically test clinical associations of TLT-1 in health and disease. To facilitate our studies, monoclonal murine TLT-1 (mTLT-1) antibodies were produced by the immunization of a rabbit using the negatively charged region of the mTLT-1 extracellular domain 122PPVPGPREGEEAEDEK139. In the present study, we demonstrate that two selected clones, 4.6 and 4.8, are suitable for the detection of mTLT-1 by western blot, immunoprecipitation, immunofluorescent staining, flow cytometry and inhibit platelet aggregation in aggregometry assays. In addition, we found that the topical administration of clone 4.8 delayed the wound healing process in an experimental burn model. These results suggest that TLT-1 plays an important role in wound healing and because both clones specifically detect mTLT-1, they are suitable to further develop TLT-1 based models of inflammation and hemostasis in vivo.

SUBMITTER: Manfredi B

PROVIDER: S-EPMC5930940 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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The Characterization of Monoclonal Antibodies to Mouse TLT-1 Suggests That TLT-1 Plays a Role in Wound Healing.

Manfredi Barbara B Morales-Ortíz Jessica J Díaz-Díaz Lymarie M LM Hernandez-Matias Liz L Barreto-Vázquez Delmaliz D Menéndez-Pérez Javier J Rodríguez-Cordero J Alejandro JA Villalobos-Santos Juan C JC Santiago-Rivera Edgardo E Rivera-Dompenciel Adriana A Lozada-Delgado Eunice L EL Kuchibhotla Madhavi M Carrasquillo-Carrión Kelvin K Roche-Lima Abiel A Washington A Valance AV

Monoclonal antibodies in immunodiagnosis and immunotherapy 20180401 2

Platelets play a vital role in hemostasis and inflammation. The membrane receptor TREM-like transcript-1 (TLT-1) is involved in platelet aggregation, bleeding, and inflammation, and it is localized in the α-granules of platelets. Upon platelet activation, TLT-1 is released from α-granules both in its transmembrane form and as a soluble fragment (sTLT-1). Higher levels of sTLT-1 have been detected in the plasma of patients with acute inflammation or sepsis, suggesting an important role for TLT-1 ...[more]

PMID: 29708866

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Project description:BackgroundHevin is a matricellular protein involved in tissue repair and remodeling via interaction with the surrounding extracellular matrix (ECM) proteins. In this study, we examined the functional role of hevin using a corneal stromal wound healing model achieved by an excimer laser-induced irregular phototherapeutic keratectomy (IrrPTK) in hevin-null (hevin(-/-)) mice. We also investigated the effects of exogenous supplementation of recombinant human hevin (rhHevin) to rescue the stromal cellular components damaged by the excimer laser.Methodology/principal findingsWild type (WT) and hevin (-/-) mice were divided into three groups at 4 time points- 1, 2, 3 and 4 weeks. Group I served as naïve without any treatment. Group II received epithelial debridement and underwent IrrPTK using excimer laser. Group III received topical application of rhHevin after IrrPTK surgery for 3 days. Eyes were analyzed for corneal haze and matrix remodeling components using slit lamp biomicroscopy, in vivo confocal microscopy, light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) and western blotting (WB). IHC showed upregulation of hevin in IrrPTK-injured WT mice. Hevin (-/-) mice developed corneal haze as early as 1-2 weeks post IrrPTK-treatment compared to the WT group, which peaked at 3-4 weeks. They also exhibited accumulation of inflammatory cells, fibrotic components of ECM proteins and vascularized corneas as seen by IHC and WB. LM and TEM showed activated keratocytes (myofibroblasts), inflammatory debris and vascular tissues in the stroma. Exogenous application of rhHevin for 3 days reinstated inflammatory index of the corneal stroma similar to WT mice.Conclusions/significanceHevin is transiently expressed in the IrrPTK-injured corneas and loss of hevin predisposes them to aberrant wound healing. Hevin (-/-) mice develop early corneal haze characterized by severe chronic inflammation and stromal fibrosis that can be rescued with exogenous administration of rhHevin. Thus, hevin plays a pivotal role in the corneal wound healing.

Dataset Information

The Characterization of Monoclonal Antibodies to Mouse TLT-1 Suggests That TLT-1 Plays a Role in Wound Healing.

Publications

The Characterization of Monoclonal Antibodies to Mouse TLT-1 Suggests That TLT-1 Plays a Role in Wound Healing.

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