Epidermolysis was never observed. in extremity transplantation. strong class=”kwd-title” Keywords: basic (laboratory) research/science, immunosuppressant, rejection: acute, rejection: T cell\mediated (TCMR), vascularized composite and reconstructive transplantation AbbreviationsALSanti\lymphocyte serumBNBrown Norway ratCTcomparative threshold cycleCTLA\4cytotoxic T lymphocyte\associated protein\4H&Ehematoxylin & eosinIFN\interferon\gammaipintraperitonealIHCimmunohistochemistryISimmunosuppressionLEWLewis ratPODpostoperative dayRTqPCRquantitative real\time polymerase chain reactionscsubcutaneousSEMstandard error of the meanTNF\tumor necrosis factor\alphaVCAvascularized composite tissue allograft/vascularized composite tissue allotransplantation 1.?INTRODUCTION With more than 150 performed cases of vascularized composite tissue INCA-6 allotransplantation (VCA) worldwide,1 the skin has come into focus, both as a site of immune reaction,2 and also as a target for therapeutic intervention. Exploring novel anti\skin rejection therapies would fulfill an important clinical need: reducing the?recipient’s exposure to chronic, systemic immunosuppression (IS). A recent study investigating cytokine expression in the skin of VCAs has shown highly upregulated IL\1 levels during allograft rejection.3, 4 IL\1 constitutes a proinflammatory signal inducing T cell infiltration, memory CD4?+? T cell activation, IL\6 expression, and Th\17 differentiation.5, 6 Blockers of IL\1 and IL\1 show significant effects in patients with autoinflammatory syndromes and have already been approved for treatment.7, 8, 9 Disrupting IL\1 function is therefore expected to decrease skin rejection in VCA. Here we tested the effect of an IL\1 blocking antibody on graft survival, rejection, cell infiltration, immune phenotype, and cytokine expression in an experimental rat hind\limb transplant model. 2.?MATERIALS AND METHODS 2.1. Animals Male Brown Norway rats Rabbit polyclonal to ZNF184 (BN) served as donors and Lewis rats (LEW) as recipients (Charles River, 200\250?g), representing a full MHC mismatch model in transplantation. Animals were housed under standard conditions with access to INCA-6 chow and water ad libitum. Experimental protocols were approved by the Austrian Federal Ministry of Science/Research. 2.2. Experimental design After orthotopic allogenic rat hind\limb transplantation, animals were treated as follows (Table?1): no immunosuppression (IS; group 1, n?=?5); baseline IS with anti\lymphocyte\serum (ALS, Accurate Chemical&Scientific Corporation; 0.5?mL days 0?+?3 intraperitoneal [ip]) and tacrolimus (Prograf, Astellas; 0.30?mg/kg/day until day 50 ip; group 2, n?=?5); baseline IS (see group 2) combined with a low\endotoxin, acid\free\purified anti\mouse/rat IL\1 monoclonal antibody (Clone B122, BioLegend; 1?mg/kg/week), administered subcutaneously (sc) into the transplanted limb (group 3, n?=?5); or contralateral limb (group 4, n?=?5). The immunosuppressive regimen was designed to overcome the immediate inflammation in response to ischemia/reperfusion and prevent an early and aggressive acute rejection. This IS regimen has been proven to be suitable in INCA-6 establishing the environment for testing the effect of intragraft targeted therapy in limb transplantation.10 Anti\IL\1 treatment was initiated on postoperative day (POD) 35, prior to weaning and cessation of tacrolimus therapy on POD 50 and continued once/week until POD 100. Tacrolimus blood trough levels have been shown to be below detection limits at 5?days after cessation.11 The antibody was delivered and equally dispensed in the subcutaneous compartment of the graft by 6\8 individual injections using a 27\gauge needle, distributed over the allograft/contralateral limb including the thigh, dorsum, and planta pedis. Table 1 Experimental groups thead valign=”top” th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Group /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Systemic treatment /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Local treatment /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Number /th /thead 1No treatmentNo52ALS 0.5?mL, days 0?+?3 and tacrolimus 0.3?mg/kg daily, ip, until day 50No53ALS 0.5?mL, days 0?+?3 and tacrolimus 0.3?mg/kg daily, ip, until day 50anti\IL\1 1?mg/kg weekly, sc, into transplanted limb, days 35\10054ALS 0.5?mL, days 0?+?3 and tacrolimus 0.3?mg/kg daily, ip, until day 50anti\IL\1 1?mg/kg weekly, sc, into contralateral, nontransplanted limb, days 35\1005 Open in a separate window ip, intraperitoneal; sc, subcutaneous. 2.3. Surgical procedure Anesthesia was performed with isoflurane inhalation anesthesia INCA-6 (3\4% for induction, 0.5\1.5% for maintenance). In addition, midazolam (Dormicum, 2.0?mg/kg), medetomidine (Domitor, INCA-6 0.15?mg/kg), and fentanyl (Fentanyl\Janssen, 0.005?mg/kg) were given. Surgical details have been described previously.12 Postoperative analgesia included buprenorphine (0.1?mg/kg) and carprofen (4.0?mg/kg) twice/day until POD 5 and 7, respectively. Grafts were monitored daily for macroscopic signs of rejection (Table?2). On POD 58, animals were anesthetized with isoflurane inhalation and a skin biopsy (5??5?mm) was collected from the allograft thigh for histopathologic examination. At the end\point (either grade III rejection or POD 100), animals were sacrificed and donor hind\limb tissues were collected..