Transplantation of a whole ear allograft by immunological induction of donor-specific tolerance by bone marrow transplantation: An experimental study in rabbits

Previous studies in our laboratory have shown that giving bone marrow cells through the portal vein or intraosseous route is likely to be beneficial to tolerance of induction of allografts in rabbits. Using this model, we tested whether a less severe regimen for conditioning of the host can prevent rejection of allografts. Rabbits were given a single intraosseous injection of donor bone marrow cells and total body irradiation (7 Gy) and transplantation of skin and ear allografts. Mean skin allograft survival for this treatment was 88 days, which was similar to the results of our earlier study. A donor ear was accepted for more than a year with no signs of rejection. These results suggest that a single intraosseous injection of donor bone marrow cells is sufficient for induction of donor-specific tolerance in rabbits and that immunosuppressive agents may not be needed.     

骨髓移植诱导免疫耐受家兔全耳同种移植

目的　为了减轻免疫诱导过程中对受体的侵袭 ,进一步探讨以家兔为动物模型 ,通过异体骨髓移植诱导同种皮肤移植免疫耐受。方法　通过骨髓腔内直接注射异体骨髓细胞 ,进行骨髓移植。结果　皮片的平均存活时间是 (88.0± 6 .0 )天 (P <0 .0 0 1 )。为了探讨其普遍性 ,我们用此诱导方法 ,还进行了同种全耳移植。平均存活时间是 (1 4 6 .0± 1 5 .1 )天 ,并有 1只移植耳存活时间已超过 1年 ,未见任何排斥反应。结论　本研究证明了以家兔为动物模型 ,在不使用免疫抑制剂的情况下 ,通过骨髓腔内直接注射异体骨髓细胞 ,进行骨髓移植的方法 ,不仅在皮肤移植上可以诱导长期稳定的特异性免疫耐受 ,而且在实质性器官耳的同种移植上取得了良好的效果     

Requirement of a higher degree of chimerism for skin allograft tolerance in cyclophosphamide-induced tolerance

By using a cyclophosphamide (CP)-induced tolerance system, we previously raised the possibility that the degree of chimerism might determine the induction of heart and skin allograft tolerance. When C3H (H-2^k; Thy1.2, Mls-1^b) mice were intravenously primed with 1×10⁸ spleen cells (SCs) from H-2 matched AKR (H-2^k; Thy1.1, Mls-1^a) mice and then treated intraperitoneally with 200 mg/kg CP, the survival of AKR skin grafts was permanently prolonged in a tolerogen-specific fashion. After this treatment, a minimal degree of mixed chimerism and the clonal destruction of Mls-1^a-reactive CD4⁺V6⁺ T cells in the periphery were observed. When AKR SCs and 100 mg/kg CP were used for conditioning, the survival of the AKR skin grafts was mildly prolonged. The clonal destruction of CD4⁺V6⁺ T cells in the periphery was induced and a minimal degree of mixed chimerism was detectable. The degree of mixed chimerism induced with AKR SCs and 200 mg/kg CP was significantly higher than that with AKR SCs and 100 mg/kg CP during the observation. On the other hand, neither skin allograft prolongation nor permanent mixed chimerism could be induced when C3H mice were treated with AKR SCs and 50 mg/kg CP. In order to increase the degree of mixed chimerism, we injected 1×10⁸ tolerant AKR SCs on day 3 into the recipient C3H mice that had been treated with AKR SCs on day 0 and with 100 mg/kg CP on day 2. The reason that we used tolerant SCs was that untreated AKR SCs caused graft-versus-host disease in most of the recipients. Tolerant AKR SCs were harvested from AKR mice that had been treated with C3H SCs and 200 mg/kg CP 2 weeks earlier, and did not contain regulatory cells. By adoptive transfer, the degree of chimerism was stably and significantly increased in all recipients, and AKR skin graft tolerance was induced in half of the recipients. T-cell-depleted bone marrow cells (BMCs) from untreated AKR mice induced skin allograft tolerance in 83% of recipients. Thus, the present study strongly confirmed the hypothesis that a higher degree of chimerism is required for the induction of skin allograft tolerance in CP-induced tolerance.     

Requirement of a higher degree of chimerism for skin allograft tolerance in cyclophosphamide-induced tolerance

Abstract By using a cyclophosphamide (CP)‐induced tolerance system, we previously raised the possibility that the degree of chimerism might determine the induction of heart and skin allograft tolerance. When C3H (H‐2k; Thy 1.2, Mls‐lb) mice were intravenously primed with 1x108 spleen cells (SCs) from H‐2 matched AKR (H‐2k; Thy 1.1, Mls‐la) mice and then treated intraperitoneally with 200 mg/kg CP, the survival of AKR skin grafts was permanently prolonged in a tolerogen‐specific fashion. After this treatment, a minimal degree of mixed chimerism and the clonal destruction of Mls‐la‐reactive CD4+Vβ6+ T cells in the periphery were observed. When AKR SCs and 100 mg/kg CP were used for conditioning, the survival of the AKR skin grafts was mildly prolonged. The clonal destruction of CD4 + Vβ6+ T cells in the periphery was induced and a minimal degree of mixed chimerism was detectable. The degree of mixed chimerism induced with AKR SCs and 200 mg/ kg CP was significantly higher than that with AKR SCs and 100 mg/kg CP during the observation. On the other hand, neither skin allograft prolongation nor permanent mixed chimerism could be induced when C3H mice were treated with AKR SCs and 50 mg/kg CP. In order to increase the degree of mixed chimerism, we injected 1x108 tolerant AKR SCs on day 3 into the recipient C3H mice that had been treated with AKR SCs on day 0 and with 100 mg/kg CP on day 2. The reason that we used tolerant SCs was that untreated AKR SCs caused graft‐versus‐host disease in most of the recipients. Tolerant AKR SCs were harvested from AKR mice that had been treated with C3H SCs and 200 mg/kg CP 2 weeks earlier, and did not contain regulatory cells. By adoptive transfer, the degree of chimerism was stably and significantly increased in all recipients, and AKR skin graft tolerance was induced in half of the recipients. T‐cell‐depleted bone marrow cells (BMCs) from untreated AKR mice induced skin allograft tolerance in 83% of recipients. Thus, the present study strongly confirmed the hypothesis that a higher degree of chimerism is required for the induction of skin allograft tolerance in CP‐induced tolerance.     

Banking and use of glycerol preserved full-thickness skin allograft harvested from body contouring procedures

Introduction

The use of glycerol preserved skin allograft (GPA) became a main stay in burn treatment. However, harvesting of cadaveric skin is not yet legalized in many countries including Egypt.

Objectives

To estimate the feasibility of using skin harvested from body contouring procedures as a source of GPA and its clinical efficacy.

Patients and methods

Skin harvested from body contouring procedures done in Al-Azhar university hospitals was preserved by glycerolization and used in management of burn and complicated wounds.

Results

In the period between February 2012 and February 2013 skin was harvested from 24 abdomenoplasty cases, 6 bilateral breast reduction cases, and 1 case of thigh lift done in Al-Azhar university hospitals. This yielded about 22,000 cm² of skin preserved by glycerolization. This GPA was used in 15 excised burn wounds, in 9 cases of chronic burn wounds, and in 6 complicated wounds. Partial graft loss occurred in 3 cases and total graft loss occurred in 1 case.

Conclusion

The glycerolized full-thickness skin harvested from body contouring procedures is clinically effective in burn and wound management. In the presence of regional coordination, it can serve as an abundant source for skin banking in where cadaveric skin use is not legalized.     

骨髓移植诱导特异性免疫耐受同种皮肤移植的动物实验研究

目的　证实通过动物实验模型的骨髓移植可以诱导同种皮肤移植的免疫耐受。方法　将 114只日本白色家兔和Dutch家兔分为对照组和实验组 ,日本白色家兔作为供体 ,Dutch家兔作为受体。对照组 ,在不使用免疫抑制剂的情况下 ,将 12只日本白色家兔与 12只Dutch家兔行相同面积的背部全厚皮肤互换移植 ,观察其成活时间。实验组 ,将 4 5只日本白色家兔和 4 5只Dutch家兔行全厚皮肤移植的同时行骨髓移植 ,然后将作为受体的Dutch家兔分为A ,B ,C ,D四组 ,分别行非致死量的γ射线全身照射的骨髓细胞移植及同种皮肤移植 ,观察移植皮肤的成活时间。结果　对照组 ,供体与受体移植皮肤的平均成活时间分别为 (12 .0± 1.7)天和 (10 .3± 1.3)天。实验组 ,A ,B ,C ,D四组移植皮肤的平均成活时间分别为 (6 1.0± 7.2 )、(80 .7± 10 .4 )、(78.8± 12 .7)、(88.0± 6 .0 )天。结论　通过骨髓移植导特异性免疫耐受同种皮肤移植的动物实验 ,旨在为临床应用提供了理论基础及可靠依据 ,为同种组织重建提供一个新方法     

Total full-thickness skin grafting for treating patients with extensive facial burn injury: A 10-year experience

IntroductionFacial burns are not only a severe burn injury, but result in psychological disturbance. The improvement of the methods of treating facial burns remains topical. The aim of the study was to evaluate the effectiveness of approach based on full-thickness skin autografting for facial burn injuries.MethodsDuring 2000–2019, ninety seven patients with the facial burn were treated in Burn Center. All patient were divided into two groups. The comparative analysis between groups was done.ResultsGroup A was treated with full-thickness skin grafts (42 patients – 43.3%). Since 2010, total full-thickness skin graft was used in 11 patients from Group A. In group B, 55 patients (56.7%) were treated with split-thickness skin grafts, including 9 patients (16.4%) with total split-thickness skin graft transplantation. Total full-thickness skin graft was performed in case of a deep and extensive facial burn and cicatricial deformities. During the long-term period, a positive cosmetic result and the absence of indications for reconstructive operations were noted.ConclusionThe approach of facial burn treatment based on total full-thickness skin graft allows conditions for engraftment and adaptation of autograft, reduces the risk of scar developing and achieves maximum cosmetic results of treatment.     

The application of glycerol-preserved skin allograft in the treatment of burn injuries: An analysis based on indications

Introduction

Glycerol-preserved skin allograft (GPA) plays a crucial role in the management of burns. Its indications include wound-bed preparation, definitive dressing and sandwich grafting technique.

Objective

We analysed the experience of using GPA and its efficacy in burn treatment in our burn centre.

Methods

All burns managed with GPA in our burn centre from October 2001 to May 2008 were analysed.

Results

Mean total body surface area (TBSA) of 43 consecutive cases was 28.7%. GPA adhered to the wound for an average of 8.4 days before rejection. The length of hospital stay of the survivors was 42.5 days. The autograft take after wound-bed preparation with GPA was 88.4%. For sandwich grafting technique, the autograft take was 74.4%. When GPA was applied for partial-thickness burn as definitive dressing, all patients achieved complete healing within an average of 19 days without further surgical intervention. Despite colonisation of burn wounds after application of skin allograft, the outcomes of autograft take and wound healing were not significantly different.

Conclusion

The selective and strategic use of the GPA in major burn patients ensures optimal benefits in the management of burns. It is versatile in various categories of burn wounds with minimal morbidity.     

异基因小鼠髓腔内骨髓移植诱导免疫耐受

目的探讨异基因小鼠髓腔内骨髓移植(IBM-BMT)诱导免疫耐受的效果。方法雄性BALB/c小鼠和雌性C57BL/6小鼠分别作为骨髓移植的供、受者。受者预处理后进行IBM-BMT,建立异基因小鼠骨髓移植模型。通过皮肤移植和混合淋巴细胞反应(MLR)对受者的耐受状态进行检测。结果接受过IBM-BMT的受者进行供者来源的皮肤移植,移植物的存活时间>300d,较对照组的(12.7±1.63)d明显延长(P<0.01),而受者接受来自无关供者(KM小鼠)的皮肤移植物存活时间未见延长。接受过IBM-BMT的受者脾细胞对供者脾细胞的MLR增殖率均明显降低,与对照组比较,P<0.01,而对无关供者的脾细胞仍表现强烈的增殖反应。结论应用IBM-BMT可以诱导受者获得供者抗原的特异性免疫耐受,使移植物存活时间延长。     

Transfer of tolerance to heart and kidney allografts in the rat model.

Long-term allograft acceptance can be induced in the rat using a variety of maneuvers. One of the cardinal features of some models of tolerance is that once the tolerance state has been established, it can be perpetuated to naive recipients by the adoptive transfer of donor-specific regulatory cells. Such adoptive transfer studies have also addressed the capacity of T-cell subpopulations and non-T cells to transfer tolerance. However, tolerance cannot be transferred in all models. The underlying reasons for this are unclear with some studies pointing towards dose-dependent aspects and timing of expansion of T regulatory cells following tolerance transfer. Further exploration of this phenomenon will help us to understand better the mechanisms upon which allograft tolerance is based, and will provide new perspectives for further experimental studies.     

维生素D衍生物联合供者骨髓细胞输注诱导异基因大鼠心脏移植免疫耐受

目的 探讨同种异基因心脏移植后免疫耐受的诱导。方法 建立大鼠颈部异位心脏移植模型，按分组分别给予门静脉输注供者骨髓细胞(DBMC)(B组)、骨化三醇灌胃(C组)、输注DBMC及骨化三醇灌胃(D组)以及环孢素A(CsA)灌胃(E组)。观察移植心脏的存活时间及心肌组织病理改变，测定心肌组织中肿瘤坏死因子及细胞间粘附分子-1 mRNA的表达以及血清钙、磷浓度，进行受者与供者及无关第三品系大鼠脾细胞混合淋巴细胞培养(MLR)。结果 D组移植心脏的存活时间较其它各组显著延长(P＜0．05)；术后7d，C组、D组、E组受者脾细胞均能显著抑制供者及第三方无关供者脾细胞作为刺激细胞引起的MLR；D组手术前后血磷、血钙浓度的差异无显著性(P＞0．05)；各组急性排斥反应的程度，D组最轻；D组肿瘤坏死因子及细胞间粘附分子-1 mRNA的表达受到显著抑制，与对照组(A组)、B、C组比较，差异有显著性(P＜0．05)。结论 骨化三醇灌胃联合DBMC输注可显著延长移植心脏的存活时间，二者具有协同作用。     

Donor skin allograft survival after bone marrow transplantation: Case report and systematic review of the literature☆

Background

We present a case of skin allograft survival in a patient who previously received a bone marrow transplant from the same HLA-matched donor. DNA fingerprinting of skin biopsies showed mixed cellularity originating from the donor and recipient (68% and 32% donor DNA in the allograft skin and the native recipient's skin, respectively). Histologic sections demonstrated both grade 3/4 rejection and graft-versus-host-disease. We have conducted a systematic review in search for other cases of donor skin allograft survival after a bone marrow or hematopoietic stem cell transplantation.

The versatility of a glycerol-preserved skin allograft as an adjunctive treatment to free flap reconstruction

Skin allografts have been used in medical practice for over a century owing to their unique composition as a biological dressing. Skin allografts can be obtained in several preparations such as cryopreserved, glycerol-preserved, and fresh allograft. A glycerol-preserved allograft (GPA) was introduced in the early 1980s. It has several advantages compared with other dressings such as ease of processing, storage and transport, lower cost, less antigenicity, antimicrobial properties, and neo-vascularisation promoting properties. Skin allografts are mainly used in the management of severe burn injuries, chronic ulcers, and complex, traumatic wounds. Published reports of the use of skin allografts in association with free flap surgery are few or non existent. We would like to share our experience of several cases of free tissue transfer that utilised GPA as a temporary wound dressing in multiple scenarios. On the basis of this case series, we would like to recommend that a GPA be used as a temporary dressing in conjunction with free flap surgery when required to protect the flap pedicle, allowing time for the edema to subside and the wound can then be closed for a better aesthetic outcome.     

Abortive alloantigen presentation by donor dendritic cells leads to donor-specific tolerance: a study with a preoperative CTLA4Ig inoculation

Donor dendritic cells (DCs) within allografts initiate the induction of an allospecific T cell response, while an abortive alloantigen presentation by DCs may induce allospecific unresponsiveness. We thus investigated the tolerogenic effect of donor DCs that were made incompetent in alloantigen presentation by treatment of CTLA4Ig. When we treated rats with donor DCs (2 × 10⁶/rat i.v.) on the preoperative day, nine rejected allografts in an accelerated manner (5.0 ± 2.2 vs. 8.2 ± 1.6 days in the control group). Preoperative inoculation of DCs pulsed with CTLA4Ig, a procedure which suppresses an allogeneic mixed lymphocyte reaction (MLR), also provoked an accelerated rejection (5.6 ± 1.7 days). When DCs and CTLA4Ig (500 μg/rat i.p. on days −9, −7 and −5) were concomitantly inoculated, allograft survival was significantly prolonged (>38.7 ± 40.0 days); a preoperative CTLA4Ig inoculation alone failed to do so (7.5 ± 1.2 days). Long-term graft survivors tolerated skin grafts from the donor but not from those from a third party. These results indicate that abortive alloantigen presentation by donor DCs, upon which an accessory signal pathway is suppressed by CTLA4Ig, leads to prolonged graft survival and donor-specific tolerance. Received: 12 June 1999 / Accepted: 1 October 1999     

高压氧预处理促进皮瓣成活的研究

目的探讨高压氧（hyperbaric oxygen，HBO）预处理对提高皮肤缺血耐受的作用。方法18只雄性SD大鼠分为HB0预处理组和对照组。HBO预处理组大鼠在手术前2d行HBO治疗，对照组大鼠不做任何处理。麻醉后在大鼠腹部形成以右侧腹壁下浅动脉为蒂的扩大腹部皮瓣，大小约6cm×9cm。先用显微血管夹阻断皮瓣血供3h，然后恢复皮瓣血供。术后第5天观察皮瓣成活情况。用硫酸纸按设计皮瓣和成活皮瓣形状剪成相应的纸模并用扫描仪将纸模转换为相应的皮瓣图像。用Acrobat软件计算分析皮瓣面积。数据用SPSS软件进行统计学分析。结果对照组和预处理组的平均皮瓣设计面积分别为（51．59±6．62）cm^2和（52．71±2．05）cm^2。平均成活面积分别为（7．38±2．49）cm^2和（15．82±5．95）cm^2。两组在皮瓣成活面积上差异有统计学意义（t=4．14，P〈0．01）。结论HBO预处理能提高皮肤缺血耐受，提高皮瓣成活率。     

未成熟树突状细胞联合雷帕霉素诱导小鼠肤移植免疫耐受

状细胞组、联合组的皮肤移植物存活时间分别为(6.9±1.9)、(10.3±3.0)、(17.0±3.4)、(20.8±3.6)d.方差分析提示组间差异有统计学意义(P＜0.05);S-N-K检验提示各组之间的差异均有统计学意义(P＜0.05).结论 供者骨髓来源未成熟树突状细胞可诱导小鼠皮肤移植免疫耐受;联合使用雷帕霉素可延长免疫耐受的持续时间.     

早期削痂全厚皮片移植治疗小儿手部深度烧伤

自1990年以来,我们对18例21只小儿手部深度烧伤行早期削痂、全厚皮片移植,术后外形及功能均较满意,取得良好效果。该手术适用于生命体征较平稳、供皮区充裕的中小面积烧伤的患儿,且手术方法简便,可有效地预防小儿手部深度烧伤后的畸形。     

大剂量免疫抑制剂预处理后骨髓移植诱导小鼠皮肤移植耐受的实验研究

目的用大剂量免疫抑制剂作为骨髓移植前受者的预处理，诱导其对供者特异性器官移植免疫耐受，并探讨宏嵌合的形成与移植耐受的关系。方法雄性C57BL/6和雌性BALB／c小鼠作为皮肤移植的供者和受者。实验分为11组：第1组为对照组，仅作皮肤移植，不做其他处理；第2组为单纯供者骨髓细胞移植(BMT)后进行皮肤移植；第3～5组：分别使用免疫抑制剂他克莫司(FK506)、环孢素A(CsA)、环磷酰胺(CTX)后进行皮肤移植；第6～8组：分别应用FK506、CsA、CTX预处理后作骨髓移植，再进行皮肤移植。前8组皮肤及骨髓供者均为雄性C57BL／6小鼠。第9～11组：处理同第6～8组，但移植皮肤来自无关供者雄性ICR小鼠，以验证耐受的特异性。每组受鼠6只。观察皮肤移植存活时间、皮肤的排斥反应，并用多聚酶链式反应(PCR)检测嵌合体的形成。结果单独应用常规剂量的供者骨髓细胞输注或短期的免疫抑制治疗并不能延长移植物的存活，也没有宏嵌合形成。而6～8组的皮肤移植物存活时间较各对照组明显延长；宏嵌合检查呈阳性。来自无关供者的皮片存活时间无延长。结论采用短期应用大剂量免疫抑制剂这一温和的非照射预处理方法，再输注供者骨髓细胞，可获得一定程度的免疫耐受，达到延长皮肤移植物存活的目的。移植前输注供者骨髓细胞形成的宏嵌合与移植物耐受有关。     

体外光化学疗法对皮肤移植小鼠产生调节性T细胞的影响

目的  探讨输注体外光化学法处理的脾淋巴细胞对皮肤移植受体小鼠产生调节性T细胞(Treg)及移植物存活时间的影响。方法  以C57BL/6小鼠为供体,BALB/c小鼠为受体,建立小鼠皮肤移植模型。分离C57BL/6和BALB/c小鼠脾淋巴细胞(CSP、BSP),制备经8-甲氧基补骨脂素联合长波紫外线(PUVA)处理的小鼠脾淋巴细胞(PUVA-SP)。根据受者静脉输注的成分将实验动物随机分为5组(每组12只)：PUVA-BSP组、PUVA-CSP组、BSP组、CSP组及磷酸盐缓冲液(PBS)对照组,每组受体分别于术前7 d、手术当日和术后7 d按组别从尾静脉注入PUVA-BSP、PUVA-CSP、BSP、CSP或PBS。观察受体移植物的存活时间,检测受体外周血中CD4⁺CD25⁺Foxp3⁺Treg表达情况。结果  皮肤移植术后,PUVA-BSP组和PUVA-CSP组的受体小鼠外周细胞CD4⁺CD25⁺Foxp3⁺Treg的比例明显高于输注BSP组、CSP组和PBS对照组;PUVA-CSP组高于PUVA-BSP,BSP组和CSP组低于PBS对照组。PUVA-BSP组和PUVA-CSP组的受者小鼠移植皮片存活时间明显长于BSP组、CSP组和PBS对照组(均为P＜0.05)。结论  输注足够数量的PUVA-SP可诱导受体体内产生较多的CD4⁺CD25⁺Foxp3⁺Treg,可以显著延长移植物的存活时间。