Orthotopic Rat Forelimb Transplantation

Vascularized composite allotransplantation (VCA) involves the transplantation of multiple tissue types -- including skin, muscle, bone, and nerves -- ...

Johns Hopkins University School of Medicine

Costimulation blockade-based regimens are a promising strategy for management of transplant recipients. However, maintenance immunosuppression via CTLA4-Ig monotherapy is characterized by high frequency of rejection episodes. Recent evidence suggests that inflammatory cytokines contribute to alloreactive T cell activation in a CD28-independent manner, a reasonable contributor to the limited efficacy of CTLA4-Ig. In this study, we investigated the possible synergism of a combined short-term inhibition of cytokine signaling and CD28 engagement on the modulation of rejection. Our results demonstrate that the JAK/STAT inhibitor tofacitinib restored the immunomodulatory effect of CTLA4-Ig on mouse alloreactive T cells in the presence of inflammatory cytokines. Tofacitinib exposure conferred dendritic cells with a tolerogenic phenotype reducing their cytokine secretion and costimulatory molecules expression. JAK inhibition also directly affected T cell activation. In vivo, the combination of CTLA4-Ig and tofacitinib induced long-term survival of heart allografts and, importantly, it was equally effective when using grafts subjected to prolonged ischemia. Transplant survival correlated with a reduction in effector T cells and intragraft accumulation of regulatory T cells. Collectively, our studies demonstrate a powerful synergism between CTLA4-Ig and tofacitinib and suggest their combined use is a promising strategy for improved management of transplanted patients.

A short course of tofacitinib sustains the immunoregulatory effect of CTLA4-Ig in the presence of inflammatory cytokines and promotes long-term survival of murine cardiac allografts.

Static cold storage is the cheapest and easiest method and current gold standard to store and preserve donor organs. This study aimed to compare the preservative capacity of gluconate-lactobionate-dextran (Unisol) solutions to histidine-tryptophan-ketoglutarate (HTK) solution. Murine syngeneic heterotopic heart transplantations (Balb/c-Balb/c) were carried out after 18 h of static cold storage. Cardiac grafts were either flushed and stored with Unisol-based solutions with high-(UHK) and low-potassium (ULK) ± glutathione, or HTK. Cardiac grafts were assessed for rebeating and functionality, histomorphologic alterations, and cytokine expression. Unisol-based solutions demonstrated a faster rebeating time (UHK 56 s, UHK + Glut 44 s, ULK 45 s, ULK + Glut 47 s) compared to HTK (119.5 s) along with a better contractility early after reperfusion and at the endpoint on POD 3. Ischemic injury led to a significantly increased leukocyte recruitment, with similar degrees of tissue damage and inflammatory infiltrate in all groups, yet the number of apoptotic cells tended to be lower in ULK compared to HTK. In UHK- and ULK-treated animals, a trend toward decreased expression of proinflammatory markers was seen when compared to HTK. Unisol-based solutions showed an improved preservative capacity compared with the gold standard HTK early after cardiac transplantation. Supplemented glutathione did not further improve tissue-protective properties.

Gluconate-Lactobionate-Dextran Perfusion Solutions Attenuate Ischemic Injury and Improve Function in a Murine Cardiac Transplant Model.

The use of therapeutic proteins and peptides is of great interest for the treatment of many diseases, and advances in nanotechnology offer a path toward their stable delivery via preferred routes of administration. In this study, we sought to design and formulate a nanostructured lipid carrier (NLC) containing a nominal antigen (insulin peptide) for oral delivery. We utilized the design of experiments (DOE) statistical method to determine the dependencies of formulation variables on physicochemical particle characteristics including particle size, polydispersity (PDI), melting point, and latent heat of melting. The particles were determined to be non-toxic in vitro, readily taken up by primary immune cells, and found to accumulate in regional lymph nodes following oral administration. We believe that this platform technology could be broadly useful for the treatment of autoimmune diseases by supporting the development of oral delivery-based antigen specific immunotherapies.

Design and characterization of lipid nanocarriers for oral delivery of immunotherapeutic peptides.

Static cold storage (SCS), the current clinical gold standard for organ preservation, provides surgeons with a limited window of time between procurement and transplantation. In vascularized composite allotransplantation (VCA), this time limitation prevents many viable allografts from being designated to the best-matched recipients. Machine perfusion (MP) systems hold significant promise for extending and improving organ preservation. Most of the prior MP systems for VCA have been built and tested for large animal models. However, small animal models are beneficial for high-throughput biomolecular investigations. This study describes the design and development of a multiparametric bioreactor with a circuit customized to perfuse rat abdominal wall VCAs. To demonstrate its concept and functionality, this bioreactor system was employed in a small-scale demonstrative study in which biomolecular metrics pertaining to graft viability were evaluated non-invasively and in real time. We additionally report a low incidence of cell death from ischemic necrosis as well as minimal interstitial edema in machine perfused grafts. After up to 12 h of continuous perfusion, grafts were shown to survive transplantation and reperfusion, successfully integrating with recipient tissues and vasculature. Our multiparametric bioreactor system for rat abdominal wall VCA provides an advanced framework to test novel techniques to enhance normothermic and sub-normothermic VCA preservations in small animal models.

Design of a Multiparametric Perfusion Bioreactor System for Evaluating Sub-Normothermic Preservation of Rat Abdominal Wall Vascularized Composite Allografts.

Ex vivo machine perfusion (EVMP) is a versatile platform utilized in vascularized composite allotransplantation (VCA) to prolong preservation, salvage tissue, and evaluate graft viability. However, there is no consensus on best practices for VCA. This article discusses the common components, modifications, and considerations necessary for a successful VCA perfusion.

A Guide to the Implementation and Design of Ex Vivo Perfusion Machines for Vascularized Composite Allotransplantation.

Vascularized composite allotransplantation (VCA) has revolutionized restorative surgery of devastating injuries. Unfortunately, these grafts undergo significant injury during prolonged cold ischemia and subsequent reperfusion.  machine perfusion (EVMP) is a technique that has shown significant promise in solid organ transplant, but study of its utility in VCA has been limited. A systematic review was conducted to identify preclinical publications investigating perfusion in limb VCAs. Articles published through June 2023 were screened. 29 articles met inclusion criteria, comprising 370 VCA limbs from swine, rats, canines, and humans. EVMP was conducted under normothermic (n = 6), near-normothermic (n = 11), sub-normothermic (n = 3), or hypothermic (n = 13) conditions. While each study used a unique perfusate recipe, most were based on a premade medium. Many incorporated additives, including antibiotics and red blood cells. The duration varied from 3 to over 24 h. Multiple studies showed improved or equivalent biomarkers, histology, and outcomes for normothermic or near-normothermic EVMP (n = 4) and hypothermic EVMP (n = 8) compared to static cold storage, suggesting that EVMP may be a superior storage method to SCS. While there is no definitive evidence regarding the optimal temperature, perfusate composition, or perfusion time for VCAs, each perfusion factor should be chosen and adapted based on the individual goals of the study. This review offers a summary of the current literature to serve as an accessible reference for the design of future protocols in this field.

Article	Year
A short course of tofacitinib sustains the immunoregulatory effect of CTLA4-Ig in the presence of inflammatory cytokines and promotes long-term survival of murine cardiac allografts. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons\| PubMed ID: 33331121	2021
Gluconate-Lactobionate-Dextran Perfusion Solutions Attenuate Ischemic Injury and Improve Function in a Murine Cardiac Transplant Model. Cells\| PubMed ID: 35626690	2022
Design and characterization of lipid nanocarriers for oral delivery of immunotherapeutic peptides. Journal of biomedical materials research. Part A\| PubMed ID: 36585800	2023
Design of a Multiparametric Perfusion Bioreactor System for Evaluating Sub-Normothermic Preservation of Rat Abdominal Wall Vascularized Composite Allografts. Bioengineering (Basel, Switzerland)\| PubMed ID: 38671729	2024
A Guide to the Implementation and Design of Ex Vivo Perfusion Machines for Vascularized Composite Allotransplantation. Plastic and reconstructive surgery. Global open\| PubMed ID: 39534373	2024
Perfusion of Limb Vascularized Composite Allotransplants: A Systematic Review of Published Protocols. Transplant international : official journal of the European Society for Organ Transplantation\| PubMed ID: 40458543	2025

Yichuan Zhang

Biography

JoVE Journal Publications

Other Publications