Skin Graft Donor Research Articles

Skin Bank Establishment in Treatment of Severe Burn Injuries: Overview and Experience with Skin Allografts at the Vienna Burn Center.

Depending on their extent, burn injuries require different treatment strategies. In cases of severe large-area trauma, the availability of vital skin for autografting is limited. Donor skin allografts are a well-established but rarely standardized option for temporary wound coverage. Ten patients were eligible for inclusion in this retrospective study. Overall, 202 donor skin grafts obtained from the in-house skin bank were applied in the Department of Plastic and Reconstructive and Aesthetic Surgery, Medical University of Vienna. Between 2017 and 2022, we analysed the results in patient treatment, the selection of skin donors, tissue procurement, tissue processing and storage of allografts, as well as the condition and morphology of the allografts before application. The average Abbreviated Burn Severity Index (ABSI) was 8.5 (range, 5-12), and the mean affected total body surface area (TBSA) was 46.1% (range, 20-80%). In total, allograft application was performed 14 times. In two cases, a total of eight allografts were removed due to local infection, accounting for 3.96% of skin grafts. Six patients survived the acute phase of treatment. Scanning electron microscope images and histology showed no signs of scaffold decomposition and intact tissue layers of the allografts. The skin banking program and the application of skin allografts at the Vienna Burn Center can be considered successful. In severe burn injuries, skin allografts provide time by serving as sufficient wound coverage after early necrosectomy. Having an in-house skin banking program at a dedicated burn centre is particularly advantageous since issues of availability and distribution can be minimized. Skin allografts provide a reliable treatment option in patients with extensive burn injuries.

The intragraft vascularized bone marrow induces secondary donor-specific mystacial pad allograft tolerance.

Vascularized bone marrow (VBM) is essential in tolerance induction through chimerism. We hypothesized that the inclusion of VBM contributes to the induction of mystacial pad allotransplantation tolerance. In this study, 19 VBM, nine mystacial pad, and six sequential VBM and mystacial pad allografts were transplanted from Brown Norway (BN) rats to Lewis (LEW) rats to test our hypothesis. The VBM recipients were divided into antilymphocyte serum (ALS) monotherapy group (two doses of ALS on day 3 pretransplantation and day 1 posttransplantation), immunosuppressant group [a week of 2 mg/kg/day tacrolimus (Tac) and 3 weeks of 3 mg/kg/day rapamycin (RPM)], and combined therapy group. The mystacial pad recipients were divided into VBM and non-VBM transplantation groups, and both groups were treated with an immunosuppression regimen that consists of ALS, Tac, and RPM. For the recipients of sequential VBM and mystacial pad allotransplantations, additional Tac was given 1 week after mystacial pad transplantation. Allograft survival, donor-specific tolerance, and chimerism level were evaluated. With the administration of ALS and short-term Tac and RPM treatments, VBM recipients demonstrated long-term graft survival (>120 days) with persistent chimerism for 30 days. CD3+ T cells from tolerant rats showed donor-specific hyporesponsiveness and tolerance to donor skin grafts but not to third-party counterparts. Furthermore, mystacial pad graft recipients with VBM transplantation exhibited a higher allograft survival rate than those without VBM transplantation [median survival time (MST) >90 days vs. 70 days, p < 0.05]. This study demonstrated that VBM transplantation is an efficient strategy to induce and maintain donor-specific tolerance for an osseous-free allograft.

Treatment of skin defects with PRP enriched with hyaluronic acid - histological aspects in rat model.

Tissue healing is a complex, dynamic process, characterized by the replacement of devitalized and absent cell and tissue structures. This can be obtained by different methods, these being found in the "reconstructive scale", which although it is very rich does not offer a universally valid solution for closing skin wounds. In plastic surgery, platelet-rich plasma (PRP) has proven effective in the treatment of skin graft donor areas, burn wounds, skin grafts, tendons, or varicose ulcers. Also, hyaluronic acid (HA) has found its utility in different areas of medicine, other than the esthetics field, with satisfactory results after its use in various lesions. The aim of our study was to find a method of healing wounds with skin defect that shortens the time of complete epithelialization compared to native healing, which is accessible to any patient both by its simplicity and by the lowest possible costs. So, we decided to test a preparation consisting of PRP and granular HA in this type of wounds on a group of 30 Wistar rats. Corroborating the macroscopic data with the microscopic ones, an important similarity can be observed between the healing of the adjuvant-treated lesion at 14 days postoperatively and the healing of the lesion left to natural healing at 21 days, thus shortening the healing period by seven days.

53 Use of Polylactic-acid-membrane in Split-thickness Skin Graft Donor Sites: A Prospective, Comparative, Randomized Study

IntroductionPolyurethane film (PU) dressings are commonly applied for coverage of split-thickness skin graft (SSG) donor sites, while previous studies have suggested reduced morbidity using a polylactic acid membrane (PLM). To further investigate the optimal treatment approach, the presented study compared outcome of donor sites in patients receiving either PLM or PU.MethodsThis randomized clinical trial allocated patients requiring SSG to receive either PLM or PU at the donor-site. Primary endpoint was difference in donor site scar appearance between groups 3 months postoperatively (Vancouver Scar Scale – VSS). Secondary endpoints included pain, the number of and time required for wound dressing changes, and costs related to the wound dressing.Results30 patients were allocated to each group. The median VSS scored lower for patients receiving PLM (PU: 3 (Q1: 2; Q3: 4) vs. PLM: 2 (Q1: 1; Q3: 3); p=0.049). Pain during change of wound dressing (PU: 2.0 ± 0.2 vs. PLM: 0.5 ± 0.2; p< 0.001) and mobilization (PU: 0.8 ± 0.2 vs. PLM: 0.3 ± 0.1; p=0.032) was reduced in the PLM group. Patients with PLM required less dressing changes per day of hospital stay (PU: 0.44 ± 0.06 vs. PLM: 0.28 ± 0.02; p=0.015). Mean time for wound dressing changes per patient was higher in the PU group (PU: 74.50 ± 5.72 vs. PLM: 21.43 ± 2.61 min; p< 0.001). Costs were higher in the PLM group (PU: 67.83 ± 5.56 vs. PLM: 162.79 ± 21.76 €; p< 0.001).ConclusionsPLM improves outcome of SSG donor sites, however, higher treatment costs must be taken into consideration.

Application of autologous platelet-rich plasma to graft donor sites to reduce pain and promote healing.

Platelet-rich plasma (PRP) is widely used for wound healing in medical care because of the numerous growth factors it contains. Traditionally, donor sites are left to heal with a primary dressing so wounds are not left open. However, a delay in healing accompanied by pain at a donor site is often seen. This study primarily throws light on the use of autologous PRP over split-thickness skin graft (STSG) donor sites to promote healing and reduce pain. The patients enrolled in this study in 2018-2019 were divided into two groups: the intervention group received autologous PRP applied topically at the donor site; in the control group, the wound was dressed traditionally. Pain scales were measured in the immediate postoperative period at six hours, 10 hours and 16 hours. The dressing was opened on the postoperative day 14 and observed for healing by an independent observer. A total of 100 patients were included in the study. Patients in the PRP group showed statistically significant faster healing at postoperative day 14 compared with the control group (p<0.05), who required dressings for 3-4 weeks postoperatively. Pain scale scores in the postoperative period were significantly less in the PRP group at six hours postoperatively compared with the control group (p<0.05). There was a reduced incidence of hypertrophic scar formation in the small number of patients in the PRP group who had developed hypertrophic scar previously. Application of PRP is a safe, cost-effective and easy method to achieve faster healing in graft donor site areas that are troublesome to both patients and doctors. It also reduces postoperative pain at donor sites. The authors recommend PRP is used more often in the management of donor sites for STSGs.

Effects of Administration Route of Adipose-Derived Stem Cells on the Survival of Allogeneic Skin Grafts in Mice

Composite tissue allotransplantation presents considerable potential for defective tissue reconstruction. However, the high immunogenicity of the allogeneic skin grafts can cause acute rejection. Adipose-derived stem cells (ADSCs) reportedly have an immunomodulation potential, which may improve the survival of allogeneic skin grafts. However, there is currently no consensus on administration route of ADSCs. This study compared the effectiveness of local injection vs intravenous (IV) administration of ADSCs in improving the survival of allogenic skin grafts in mice. BALB/c and C57BL/6 mice were used as skin graft donors and recipients, respectively. Mice were divided into 3 groups for IV injection of ADSCs (IV group) or phosphate-buffered saline (PBS; control), or for local injection in the fascial layer of the recipient bed (FL group). After allogeneic skin transplantation, 0.1 mL of PBS alone or with 1.5×105 ADSCs was immediately injected. The grafts were histologically evaluated on days 7 and 14 postoperation. The graft necrotic area was significantly smaller in the IV and FL groups than in the control group. Additionally, the grafts in these 2 groups exhibited decreased interleukin 17/6, tumor necrosis factor-α, and interferon-γ messenger mRNA levels; inflammatory changes; and cluster of differentiation 4 expression, and increased expression of vascular endothelial growth factor expression (P < .05). However, these 2 groups did not significantly differ (P > .05). ADSCs increased the survival of allogeneic skin grafts in mice regardless of IV or FL route of administration, and this effect is likely through anti-inflammatory and angiogenic effects of ADSCs.

Measuring the Impact of Targeting FcRn-Mediated IgG Recycling on Donor-Specific Alloantibodies in a Sensitized NHP Model.

BackgroundIn transplantation, plasmapheresis and IVIg provide the mainstay of treatment directed at reducing or removing circulating donor-specific antibody (DSA), yet both have limitations. We sought to test the efficacy of targeting the IgG recycling mechanism of the neonatal Fc receptor (FcRn) using anti-FcRn mAb therapy in a sensitized non-human primate (NHP) model, as a pharmacological means of lowering DSA.MethodsSix (6) rhesus macaque monkeys, previously sensitized by skin transplantation, received a single dose of 30mg/kg anti-RhFcRn IV, and effects on total IgG, as well as DSA IgG, were measured, in addition to IgM and protective immunity. Subsequently, 60mg/kg IV was given in the setting of kidney transplantation from skin graft donors. Kidney transplant recipients received RhATG, and tacrolimus, MMF, and steroid for maintenance immunosuppression.ResultsCirculating total IgG was reduced from a baseline 100% on D0 to 32.0% (mean, SD ± 10.6) on d4 post infusion (p<0.05), while using a DSA assay. T-cell flow cross match (TFXM) was reduced to 40.6±12.5% of baseline, and B-cell FXCM to 52.2±19.3%. Circulating total IgM and DSA IgM were unaffected by treatment. Pathogen-specific antibodies (anti-gB and anti-tetanus toxin IgG) were significantly reduced for 14d post infusion. Post-transplant, circulating IgG responded to anti-FcRn mAb treatment, but DSA increased rapidly.ConclusionTargeting the FcRn-mediated recycling of IgG is an effective means of lowering circulating donor-specific IgG in the sensitized recipient, although in the setting of organ transplantation mechanisms of rapid antibody rise post-transplant remains unaffected.

The cost-effectiveness of maintaining an in-house glycerol-preserved skin bank

IntroductionGlycerol-preserved skin allograft (GPA) plays a vital role, especially in the management of burns injury. Where it is utilized as temporary wound closure, the considerably cost-effective, simpler handling and storage of GPA makes it preferable in almost all clinical indications. The GPA was first introduced to the Hospital Universiti Sains Malaysia in 2001. The supply was imported from Euro Skin Bank, Beverwijk, The Netherlands. In the year 2013, our center had started maintaining an in-house glycerolized skin bank. MethodWe preserved donor skin grafts from patients who underwent plastic surgery-related procedures in 85% glycerol and stored them at +2 °C to +10 °C. Cost estimation of the GPA per cm2 was calculated to analyze the effectiveness of its preservation technique. ResultsThe cost of GPA from our skin bank is estimated to be almost 90% reduction in cost as compared to the supply from Euro Skin Bank. ConclusionThe selective and strategic use of the GPA in major burn patients assure effective advantages in the treatment of burns. The clinical significance of skin allograft usage is very high. The cost-effectiveness of maintaining an in-house skin bank made it possible for various centers for skin allograft usage.

The Effect of Medial Sural Artery Perforator Flap on Reconstruction of Soft Tissue Defects: A Meta-Analysis With Multiple Free Soft Flaps.

With the gradual popularity of relatively novel medial sural artery perforator flap (MSAPF), robust studies are needed to compare the surgical outcomes of MSAPF versus multiple free soft flaps (MFSFs) to verify the advantages and disadvantages of MSAPF. The authors searched PubMed, Web of Science, EMBASE, Cochrane Library, Chinese BioMedical Literature Database (CBM), and China National Knowledge Infrastructure (CNKI) until September, 2020, to identify studies that compared surgical outcomes of MSAPF and MFSFs. Two authors followed the PRISMA guidelines, individually extracted the data and performed the quality assessments. Survival rate of flaps, satisfaction degree of patients in recipient and donor site, skin grafting, and morbidity of recipient and donor site were evaluated. A total of 441 cases from 7 studies were included in our analysis. No significant differences were found regarding survival rate of flaps, recipient morbidity, and recipient satisfaction degree between the 2 groups. However, MSAPF group was significantly superior to MFSFs group in terms of skin grafting, morbidity, and satisfaction degree of donor site. Our meta-analysis showed that the MSPAF and MFSFs groups were similar in terms of survival rate of flaps, recipient morbidity, and recipient satisfaction degree. Medial sural artery perforator flap group was superior to MFSFs group in terms of morbidity and satisfaction degree of donor site. The results may prove that MSAPF is gaining popularity for a reason and is a good choice for repairing soft tissue defects.

Efficacy of a Natural Topical Skin Ointment for Managing Split-Thickness Skin Graft Donor Sites: A Pilot Double-blind Randomized Controlled Trial.

To evaluate the safety and efficacy of a new topical skin ointment with natural ingredients (aloe vera, honey, and peppermint) for dressing skin graft donor sites. A double-blind, placebo-controlled, randomized clinical trial. Researchers enrolled patients who were referred for split-thickness skin graft after burns or surgical wounds on the scalp or face area. For each patient, a thin layer of skin (depth, 0.04 mm; approximate size, less than 15 × 7 cm) was harvested from the thigh by a plastic surgeon with an electric or manual dermatome. The donor sites were divided and randomized to receive either natural ointment or petroleum jelly as a topical agent to dressing. Topical agents were applied on donor site wounds on days 0, 4, 7, and 14. Wound size, pain, erythema, pruritus, patient discomfort, complications, and physician satisfaction were evaluated at each visit. Among 28 patients, there was no significant difference between the two treatment agents regarding the rate of wound healing (P = .415), pain (P = .081), pruritus (P = .527), and patient discomfort (P = .616). The ointment was superior to petroleum jelly in reducing wound erythema (P = .001) and was associated with significantly better treatment satisfaction (P < .001). The natural topical ointment investigated in this study may be an acceptable alternative to petroleum jelly in caring for split-thickness donor skin graft donor site wounds to effectively promote wound healing, prevent infection and scarring, reduce pain, and comfort the patient.

Induction of immune tolerance towards allogeneic cells using fetal directed placental injection in a murine model

Objective: In utero exposure to foreign antigens prior to the development of the immune system induces immune tolerance. We aimed to induce immune tolerance towards allogeneic cells by early gestational transplacental injection under ultrasound guidance in a murine model. Methods: Bone marrow cells from C57BL/6-Green Fluorescence Protein transgenic mice were transplanted into the placenta of Balb/c fetal mice at 11-day of gestation under ultrasound guidance. Each fetus was injected with 2 × 105 cells/2.5 μl. After birth, we evaluated the immune response against allogeneic donor cells. Results: The birth survival rate was 21.2% for allogeneic mice. Survival of the donor skin graft was 75% and successful in mice injected with fetal transplacental cells, whereas the transplanted allogeneic skin was all rejected within 4 weeks in control naive mice (p=0.007). Cytotoxic immune reactivity against the allogeneic cells was suppressed according to the ELISPOT assay (p=0.002). Conclusion: We showed that early transplacental allogeneic cell injection can induce donor-specific tolerance sufficient to allow tissue graft.

Safety of Deep Sedation in Patients Undergoing Full-Thickness Skin Graft Harvesting and Skin Graft Reconstruction for Limb Salvage An Outcome Analysis.

Studies on obtaining donor skin graft using intravenous sedation for patients undergoing major foot surgeries in the same operating room visit have not previously been reported. The objective of this retrospective study is to demonstrate that intravenous sedation in this setting is both adequate and safe in patients undergoing skin graft reconstruction of the lower extremities in which donor skin graft is harvested from the same patient in one operating room visit. Medical records of 79 patients who underwent skin graft reconstruction of the lower extremities by one surgeon at the Yale New Haven Health System between November 1, 2008, and July 31, 2014, were reviewed. The patients' demographic characteristics, American Society of Anesthesiologists class, comorbid conditions, intraoperative analgesic administration, estimated blood loss, total operating room time, total postanesthesia care unit time, and postoperative complications within the first 72 hours were reviewed. This study found minimal blood loss and no postoperative complications, defined as any pulmonary or cardiac events, bleeding, admission to the intensive care unit, or requirement for invasive monitoring, in patients who underwent major foot surgery in conjunction with full-thickness skin graft. We propose that given the short duration and peripheral nature of the procedures, patients can safely undergo skin graft donor harvesting and skin graft reconstruction procedures with intravenous sedation regardless of American Society of Anesthesiologists class in one operating room visit.

Glycolytic Bias in mTORC2 Deficient Dendritic Cells Potentiates Antigen-Specific Immunity and Accelerates Graft Rejection

Background/Hypothesis The mechanistic target of rapamycin (mTOR) is known to function in two discrete complexes: mTOR complex 1 (mTORC1) and mTORC2. The function of mTORC1 in dendritic cells (DCs) has been studied extensively using rapamycin (RAPA) as an inhibitor. RAPA inhibition of mTORC1 prevents DC maturation, leading to decreased T effector cell proliferation and increased regulatory T cell (Treg) differentiation. Our group has recently demonstrated that mTORC2 deletion in DCs leads to both an enhanced pro-inflammatory DC phenotype and Th1/Th17 allogeneic T cell polarization and proliferation. However, the underlying mechanism has not been resolved. In addition, the role of mTORC2 in DCs in the context of transplantation has not been defined. We hypothesized that ablation of mTORC2 in DCs would alter metabolic activity, resulting in augmented antigen-specific T cell responses and accelerated graft rejection. Methods To assess the role of mTORC2 in DCs in transplantation, we used models of non-MHC mismatched skin transplantation in which either the graft donor or recipient was deficient in mTORC2 specifically in conventional CD11c+ DC (TORC2DC-/-). Graft survival was monitored; Banff rejection scoring was performed by a blinded pathologist. T cell infiltration and collagen degradation in the graft were determined by immunohistochemistry. To ascertain whether skin-resident TORC2DC-/- could augment inflammatory responses, we performed a cell-mediated delayed-type hypersensitivity assay. As mTORC2 has been implicated in cytoskeletal dynamics, we measured DC migration into secondary lymphoid tissue. To elucidate the role of mTORC2 in regulating DC metabolism we analyzed glycolytic capacity and mitochondrial respiratory activity of wild-type (WT) DC and TORC2DC-/- using a Seahorse XF Bioanalyzer. Mitochondrial mass and activity were determined via flow cytometric analysis of MitoTracker Green and TMRE uptake, respectively. ATP production was assessed using a luciferase-based luminescence assay. Results/Conclusions We demonstrate for the first time, that TORC2DC-/- deficiency in either skin graft donors or recipients accelerates immune-mediated rejection. TORC2DC-/- mice also exhibit enhanced T cell and inflammatory monocytic infiltration in the course of delayed-type hypersensitivity responses. These effects are not due to differential migration of TORC2DC-/- to secondary lymphoid tissue. TORC2DC-/- utilize an altered metabolic program, wherein glycolytic function is enhanced as compared to WT DCs. This metabolic phenotype corresponds with increased viability of TORC2DC-/- after stimulation, which may allow these TORC2DC-/- to persist in secondary lymphoid tissue longer than WT DCs. These findings reveal a novel role for mTORC2 in regulating DC immunometabolism, and may provide a basis for therapeutic targeting of DC metabolism to regulate immune responses in transplantation. NIH T32 AI74490.

Stability of Chimerism in Non-Obese Diabetic Mice Achieved By Rapid T Cell Depletion Is Associated With High Levels of Donor Cells Very Early After Transplant.

Stable mixed hematopoietic chimerism is a robust method for inducing donor-specific tolerance with the potential to prevent rejection of donor islets in recipients with autoimmune type-1 diabetes. However, with reduced intensity conditioning, fully allogeneic chimerism in a tolerance resistant autoimmune-prone non-obese diabetic (NOD) recipient has rarely been successful. In this setting, successful multilineage chimerism has required either partial major histocompatability complex matching, mega doses of bone marrow, or conditioning approaches that are not currently clinically feasible. Irradiation free protocols with moderate bone marrow doses have not generated full tolerance; donor skin grafts were rejected. We tested whether more efficient recipient T cell depletion would generate a more robust tolerance. We show that a combination of donor-specific transfusion-cyclophosphamide and multiple T cell depleting antibodies could induce stable high levels of fully allogeneic chimerism in NOD recipients. Less effective T cell depletion was associated with instability of chimerism. Stable chimeras appeared fully donor-specific tolerant, with clonal deletion of allospecific T cells and acceptance of donor skin grafts, while recovering substantial immunocompetence. The loss of chimerism months after transplant was significantly associated with a lower level of chimerism and donor T cells within the first 2 weeks after transplant. Thus, rapid and robust recipient T cell depletion allows for stable high levels of fully allogeneic chimerism and robust donor-specific tolerance in the stringent NOD model while using a clinically feasible protocol. In addition, these findings open the possibility of identifying recipients whose chimerism will later fail, stratifying patients for early intervention.

Skin grafting in the treatment of hard-to-heal leg ulcers

Some risk factors or comorbidities may make Chronic Leg Ulcers (CLU) very difficult to heal. These ulcers are usually defined refractory ulcers and may require an in-hospital intensive care to increase the healing rate. Aim of this retrospective study was to assess if our clinical routine in hospitalized patients, made up with surgical debridement followed by donor skin grafting (allografts), may favor the ulcer healing. The records of 120 patients (55 males and 65 females; mean age 73.9±11.3 years) with ulcers greater than 100 cm2 and lasting for more than 1 year were analyzed. The median ulcer size was 165 cm2 (IQR 130-250 cm2; range 100-1000 cm2). The median ulcer duration was 24 months (IQR 16-32 months; range 12-300 months). The ulcer pathophysiology was venous in 74 patients, arterial in 21, mixed in 12, vasculitis in 5 and post-traumatic in 8 patients. After debridement the patients were submitted to allograft procedures (single or multiple) up to the ulcer healing. When allograft was able to create an effective granulation tissue and reduce the ulcer size an autograft was performed to get the ulcer closure. 109 patients healed and 11 were lost at follow-up. 65 patients healed just with one allograft in 16 weeks (IQR 13-21 weeks). 42 patients healed with 2 procedures in 20 weeks (IQR 18-23 weeks). 31 of them received a final autograft while 11 healed with two allografts. 2 patients with an ulcer surface of 200 cm2, both affected by CLI, healed with 3 allografts procedures in 40 and 33 weeks, respectively. Pain and exudate amount were significantly decreased and even disappeared after the first allograft. Allografts alone or followed by an autograft are able to get the ulcer healing also in case of extensive and long lasting ulcers refractory to all previous treatments.

Anti-Interleukin-6 Promotes Allogeneic Bone Marrow Engraftment and Prolonged Graft Survival in an Irradiation-Free Murine Transplant Model.

Transfer of recipient regulatory T cells (Tregs) induces mixed chimerism and tolerance in an irradiation-free bone marrow (BM) transplantation (BMT) model involving short-course co-stimulation blockade and mTOR inhibition. Boosting endogenous Tregs pharmacologically in vivo would be an attractive alternative avoiding the current limitations of performing adoptive cell therapy in the routine clinical setting. Interleukin-6 (IL-6) potently inhibits Treg differentiation and its blockade was shown to increase Treg numbers in vivo. Therefore, we investigated whether IL-6 blockade can replace adoptive Treg transfer in irradiation-free allogeneic BMT. Treatment with anti-IL-6 instead of Treg transfer led to multi-lineage chimerism (persisting for ~12 weeks) in recipients of fully mismatched BM and significantly prolonged donor skin (MST 58 days) and heart (MST > 100 days) graft survival. Endogenous Foxp3+ Tregs expanded in anti-IL-6-treated BMT recipients, while dendritic cell (DC) activation and memory CD8+ T cell development were inhibited. Adding anti-IL-17 to anti-IL-6 treatment increased Treg frequencies, but did not further prolong donor skin graft survival significantly. These results demonstrate that IL-6 blockade promotes BM engraftment and donor graft survival in non-irradiated recipients and might provide an alternative to Treg cell therapy in the clinical setting.

A model of acute antibody mediated rejection in mouse renal transplantation

Objective To establish a model of acute antibody mediated rejection (AMR) in mouse kidney transplantation. Methods 6-8 weeks old male C3H (H2k) and BALB/c (H2d) mice were used as skin or kidney graft donors and recipients, respectively. All animals were provided by Beijing vital river experimental animal technical limited company. Recipient animals were divided into 2 groups. AMR group, recipient animals were presensitized by transplanting skin grafts before one week of kidney transplantation (n=10). Cell mediated rejection (CMR) group, recipient animals received kidney grafts without being presensitized by skin transplantation (n=10). Normal balb/c mice acted as control (n=3). Animal survival time was recorded and renal allograft function of creatinine (Cr) and blood urea nitrogen (BUN) was monitored. On the fifth day after kidney transplantation, recipient animals were sacrificed, analysis of renal graft histology based on Banff 2013 for rejection diagnosis, immunohistochemistry for mouse IgG, complement fragment C3d deposition detection intragraft, and flow cytometry for circulating donor specific antibody (DSA)-IgG, IgM detection were performed. Results The mean survival time of AMR group was (4.4±1.1) d and longer than 60 days for CMR goup. Graft function of serum Cr [ (415.6±25.6) μmol/L] and BUN [ (2 394.0±94.0) pmol/L] were remarkably increased on postoperative 2 d in AMR group. CMR control group showed normal serum levels of Cr [ (77.8±7.2) μmol/L] and BUN [ (1 343.0±233.9) pmol/L] on 60 days after kidney transplantation. On 5 d after kidney transplantation, the macroscopic morphology of renal grafts in AMR group was significantly swelling while grafts in CMR group were normal. The circulating IgG DSA level in AMR group was remarkably higher than CMR group with mean fluorescence intensity (MFI) values 223.0±29.0 vs. 134.5±39.6. The serum IgM DSA level did not change significantly with MFI values 9.2±0.1 vs. 10.0±0.7. Grafts in AMR group demonstrated dilation of peritubular capillaries (PTC), peritubular capillaritis, glomerulitis, glomerular/tubular necrosis and interstitial inflammation, hemorrhage, and edema. Extensively deposition of IgG and C3d intragraft were also observed in AMR group, while mild changes presented in CMR group which showed no significant difference from the normal control BALB/c mice. Conclusion We successfully established a model of acute AMR in C3H→BALB/c mouse kidney transplantation by presensitizing recipient mice using donor-strain derived skin transplantation at 1 week prior to kidney transplantation, which showed significantly different from non-presensitized recipient mice. Key words: Kidney transplantation; Antibody mediated rejection; Model, animal; Mice

Prevention of Donor Specific Antibody Production after Skin Allografting by Mobilization of Endogenous Stem Cells Using a Combination of AMD3100 and Low-dose FK506 in Rats

Background: We have developed a stem cell mobilizing strategy that enables long-term liver and kidney allograft survival without sustained immunosuppression in small and large animals using a safe combination of two FDA approved drugs AMD3100 and low-dose FK506 (AF). Furthermore, no increase in serum DSA levels was detected in animals displaying long-term survival even after donor skin grafts were rejected. Therefore, the purpose of this study is to determine if AF combination treatment prevents de novo DSA production in a rat model of skin allograft rejection. Methods: Split thickness skin allografts from Dark Agouti (DA, RT1a) rats were transplanted into adult Lewis (RT11) recipients. Recipient animals were randomly divided into AF treatment group (AMD3100 1 mg/kg and FK506 0.1 mg/kg, s.c., every other day for 14 days) and control group (same volume of saline, s.c.). Serum levels of DSA including both IgG and IgM were measured by flow cytometry at 14 days, 1 and 2 months after skin transplantation. The sensitization to donor antigens in Lewis rats at 2 months after skin allografting was further confirmed by transplanting donor DA kidneys. Full dose FK506 (1 mg/kg/day, s.c) was given to prevent acute cellular rejection after kidney transplantation. Results: Skin allografts were rejected in 8–10 days after transplantation in both groups. Serum levels of DSA-IgG increased 6 to 10 folds in control animals (n = 10), but only 2 to 3 folds in animals treated with AF combination (n = 11) at 1 month and remained at similar levels at 2 months after skin transplantation. All control animals (n = 5) died within 7 days after kidney transplantation. DSA deposition and AMR in kidney allografts were confirmed with signs of IgG deposition in and around glomeruli with C4d deposition on peritubular capillaries. In contrast, about 64% (7/11) animals with AF treatment after skin allografting survived over three weeks without antibody mediated rejection (AMR) and no increase in serum levels of DSA at 14 days after kidney transplantation. Conclusion: Mobilizing endogenous stem cells with AF combination treatment does not prolong skin allograft survival but prevents de novo DSA production after allograft rejection. AF treatment may inhibit B cell sensitization, prevent de novo DSA production and improve long-term allograft function after organ transplantation.

A Critical Role for TGF-β/Fc and Nonlytic IL-2/Fc Fusion Proteins in Promoting Chimerism and Donor-Specific Tolerance.

Immunoglobulin-cytokine fusion molecules have been shown to be the new generation of immunomodulating agents in transplantation tolerance induction. In the present study, we tested whether immunoregulatory cytokine fusion proteins of IL-10/Fc, TGF-β/Fc, or IL-2/Fc would enhance allogeneic bone marrow cell (BMC) engraftment and promote tolerance induction. B6 (H2) mice were conditioned with anti-CD154 (MR1) and rapamycin (Rapa) plus 100 cGy total body irradiation (MR1/Rapa/100 cGy) and transplanted with allogeneic B10.D2 (H2) BMC. Recipients were treated with lytic IL-2/Fc, nonlytic IL-2/Fc, TGF-β/Fc, or IL-10/Fc fusion proteins to promote chimerism to induce tolerance. Donor chimerism was achieved in 20% of recipients conditioned with MR1/Rapa/100 cGy. The addition of TGF-β/Fc (5- or 10-day treatment) or nonlytic IL-2/Fc (10-day treatment) fusion proteins to the conditioning resulted in engraftment in nearly 100% of recipients. In contrast, lytic IL-2/Fc or IL-10/Fc had no effect. The combination of nonlytic IL-2/Fc and TGF-β/Fc had a synergistic effect to promote engraftment and resulted in significantly higher donor chimerism compared with recipients conditioned with TGF-β/MR1/Rapa/100 cGy. Engraftment was durable in the majority of chimeras and increased over time. The chimeras accepted donor skin grafts and promptly rejected third-party skin grafts. Moreover, specific T cell receptor-Vβ5.½ and TCR-Vβ11 clonal deletion was detected in host T cells in chimeras, suggesting central tolerance to donor alloantigens. Allogeneic BMC engraftment is enhanced with TGF-β/Fc fusion protein treatment. TGF-β/Fc and nonlytic IL-2/Fc exert a synergistic effect in promotion of alloengraftment and donor-specific transplant tolerance, significantly decreasing the minimum total body irradiation dose required.

The modified antegrade digital artery island flap for severely flexion contracture of the burned finger

Objective To investigate the clinical outcomes of using modified antegrade digital artery island flap for the treatment of the severely flexion contracture of the burned finger. Methods Between August, 2013 to August, 2015, 21 patients (21 fingers) with severely flexion contracture of the burned finger were hospitalized for treatment. According to the Stren classification standard for the interphalangeal joint flexion contracture, all the patients were rated as type Ⅲ. The volar soft-tissue defect with exposed tendons, nerves, vessels or bone ranged from 1.0 cm × 2.0 cm to 2.5 cm × 4.0 cm after scar relaxation. The artery and the nerve defect were 1.5 to 4.5 cm and 2.0 to 4.2 cm, respectively. The wound were reconstructed with the modified antegrade digital artery island flap. The dorsal branches of the proper digital nerve of the flap were anastomosised with the proper digital nerve of the wound. The flap donor site was resurfaced with full-thickness skin grafting from inner aspect of the forearm. All the cases were called back for postoperative follow-up. Results All the reconstructed fingers and flaps survived completely without vascular problems. The donor skin graft survived and wound healed by first intention. All the patients were followed up with 11.5 months (range, 6-22 months). The finger appearance was satisfactory. The texture and color of flaps in all cases were good. There was no pigmentation and contraction relapse. The contracted fingers received no cold intolerance. At the final examination, the average values of static 2-point discrimination were 5.2 mm (range, 4.3-6.5 mm) of the flap. In the series, based on the Michigan Hand Outcome Questionnaire, 18 patients were strongly satisfied with the injured finger appearance and 3 patients satisfied with the appearance. Conclusion The modified antegrade digital artery island flap, which is easy to raise with large flap size and can result with the good finger appearance and function, is an ideal technique for reconstruction of the severe flexion contracture of the burned finger. Key words: Finger; Burn; Finger flexion contracture; Proper palmar digital artery; Island flap; Microsurgical operation