自体外周血干细胞移植治疗糖尿病足

目的：探讨外周血干细胞自体移植治疗糖尿病足的临床疗效.方法：患者共5例,9条肢体.移植前每日皮下注射rhG-CSF 300～600 μg进行外周血干细胞动员,第5 d行血细胞分离机采集干细胞,配成干细胞混悬液进行小腿肌肉局部按3 cm×3 cm距离进行注射移植治疗,观察移植后各项指标综合评估.结果：9条下肢均得以保存,症状好转.2例行数字减影下肢动脉造影显示侧支建立丰富,随访3月无移植相关并发症和不良反应.结论：动员后的外周血干细胞移植治疗糖尿病足是一种简单、安全、有效的方法.     

S‐I‐03 Differentiation of Mesenchymal Progenitor Cells Derived from Rat Bone Marrow

We showed mesenchymal progenitor cells(MPC) contain various cells, and the differentiational capacities and effects on wound healing were different each other. Bone marrow of the femur of adult F344 rat was suspended into culture medium and plated on a plastic dish.10–15 passaged cells were cloned in 100 types cells. For investigation of the differentiational capacity, the cells were treated by differentiational medium. The capacity of myogenesis was examined immunohistochemically with anti‐skeletal muscle myosin antibody. Osteochondrogenesis were investigated by alcian blue staining and adipogenesis by oil red O staining. The 53% of cells showed myogenesis, 78% osteochondrogenesis and 100% adipogenesis. The one type of cell showed amazing adipogenesis pattern, which we named “O” cell. Next we injected MPC and this “O” cell into the rats’ dorsal skin, and 1 cm full thickness incisional wounds were made immediately after. 2 weeks later, wounds were harvested and examined histologically. The wounds transplanted with high dose of MPC healed with very fine scar and collagen fibers were thick and aligned like normal dermis. But by transplanting the “O” cells, wounds healed with ordinary scar formation. By tansplantating mesenchymal progenitor cells, lesion healed with less scars. And there was a possibility that the MPC responded to the wound healing and regenerated dermal structure nearly normally. But the wounds healed not well by transplanting just “O” cell although it possesses the amazing differentiational capacity. The data demonstrate that plural types cells were necessary for wound healing.     

The Prostacyclin Analog Beraprost Sodium Augments the Efficacy of Therapeutic Angiogenesis Induced by Autologous Bone Marrow Cells

Implantation of autologous bone marrow (BM) mononuclear cells (MNCs) has been shown to augment neovascular formation in ischemic tissues in experimental animals and in humans. Prostaglandin derivatives improve the symptoms of patients with critical limb ischemia, possibly by their vasodilating and antiplatelet actions. We therefore examined whether therapeutic angiogenesis by implantation of autologous BM-MNCs would be enhanced by beraprost sodium (BPS), using a rabbit ischemic hindlimb model. Ischemia was induced by surgical resection of the left femoral artery. Twenty-five New Zealand white rabbits were divided into four groups. The first group (BM group, n = 4) received autologous BM-MNCs (2 × 10⁶/animal) implanted into the ischemic tissue 1 week after limb ischemia. The second group (BM+BPS group, n = 8) received BPS injected into the dorsal skin (300 μg/kg daily) starting 1 week before limb surgery. This group received BM-MNC implantation 1 week after surgery. Daily injection of BPS was continued until the end of the protocol. The third group (BPS group, n = 8) received BPS injected into the dorsal skin (600 μg/kg daily) starting 1 week before limb surgery. The fourth group received saline as a control (n = 4). The extent of angiogenesis in the ischemic hindlimb was assessed using the angiographic score (AS), ischemic/normal limb calf blood pressure ratio (CBPR), and tissue capillary density. Four weeks after limb ischemia, the ischemic/normal CBPR was highest in the BM+BPS group, followed by the BPS, BM, and control groups (0.56 ± 0.16, 0.51 ± 0.25, 0.44 ± 0.15, and 0.30 ± 0.10, respectively). The AS was also the greatest in the BM+BP group, followed by the BM, BP, and S group (1.63 ±0.21, 1.31 ± 0.25, 1.26 ± 0.21 and 0.80 ± 0.10, respectively). The TCD was greatest in the BM+BP group, followed in by the BM, BP, and S group? (46 ± 4.1, 34 ± 0.7, 33 ± 6.9, and 19 ± 1.8 per field, respectively). BP treatment is an effective means to enhance the efficacy of therapeutic angiogenesis induced by autologous BM-MNCs implantation in ischemic hindlimb tissues.     

P‐II‐04 Early Phase Regulation of Human Mesenchymal Stem Cells and the Unique Profile of the Proliferation by Bone Morphogenetic Protein‐2

Human mesenchymal stem cells (hMSCs) obtained from a single donor of the iliac crest, was further investigated for the cell proliferation, cell cycle profiles, gene expressions and the ultrastructures by electron microscopy. The hMSCs significantly increased the cell number by day 2 after by treatment of bone morphogenetic protein (BMP)‐2 alone, or basic fibroblast growth factor (bFGF) alone or combination of both in the serum‐free condition (P < 0.01). The hMSCs demonstrated the remarkable proliferation cell nuclear antigen notably at day 1 and pituitary tumor transforming gene all through the experiment, suggesting the cell cycle progression by BMP‐2 treatment as well as the strong cellular nuclear BrdU expression by immunocytochemistry. The fluorescence‐activated cell sorter also demonstrated the similar pattern of the cell cycle progression between BMP‐2 treatment in the serum‐free and 10 % fetal bovine serum treatment. The BMP‐2 treated hMSCs demonstrated the heterochromatin in the nucleus, suggesting the cell differentiation and the well‐developed granular endoplasmic reticulums, indicating the protein production. The hMSCs successfully proliferate, the cell cycle is progressed, and the cell ultrastructure morphology suggest the remarkable nuclear and of granular endoplasmic reticulum induction by BMP‐2 treatment in the serum‐free condition.     

032 Treatment of Chronic Ulcer with Allogeneic Cultured Dermal Substitute (CDS) composed of Collagen Sponge with Fibroblasts

We have been cultivating human epidermal cells for therapeutic purpose according to the original methods developed by Rheinwald and Green. Cultured epithelium (CE) was applied to patients with severe skin defects, burn wounds, chronic skin ulcers and cutaneous disorders like hypomelanosis. Autologous CE allows to restore massive skin surface in a short period compared with other conventional treatments. For grafts take, it is important to manage wound beds properly prior to CE grafting. The CDS was applied to prepare wound bed acceptable for CE grafting. The CDS was designed to secrete various types of cytokines, i.e., VEGF and KGF to stimulate wound healing. The successful management of deep wounds like chronic skin ulcer or burn ulcer requires granulation tissue formation and epithelialization to wound closure. This study aimed to evaluate the application of CDS in conjunction with CE for patients with chronic skin ulcer and burn ulcer. In some cases the wounds were cured by using CDS, and followed by CE grafting. All clinical trials achieved excellent or good results, showing no contracture and no hypertrophic scar after wound closure. The CDS was found to be useful to prepare wound beds and to facilitate wound management.     

Allospecific Rejection of MHC Class I‐Deficient Bone Marrow by CD8 T Cells

Avoidance of long‐term immunosuppression is a desired goal in organ transplantation. Mixed chimerism offers a promising approach to tolerance induction, and we have aimed to develop low‐toxicity, nonimmunodepleting approaches to achieve this outcome. In a mouse model achieving fully MHC‐mismatched allogeneic bone marrow engraftment with minimal conditioning (3 Gy total body irradiation followed by anti‐CD154 and T cell–depleted allogeneic bone marrow cells), CD4 T cells in the recipient are required to promote tolerance of preexisting alloreactive recipient CD8 T cells and thereby permit chimerism induction. We now demonstrate that mice devoid of CD4 T cells and NK cells reject MHC Class I‐deficient and Class I/Class II‐deficient marrow in a CD8 T cell–dependent manner. This rejection is specific for donor alloantigens, since recipient hematopoiesis is not affected by donor marrow rejection and MHC Class I‐deficient bone marrow that is syngeneic to the recipient is not rejected. Recipient CD8 T cells are activated and develop cytotoxicity against MHC Class I‐deficient donor cells in association with rejection. These data implicate a novel CD8 T cell–dependent bone marrow rejection pathway, wherein recipient CD8 T cells indirectly activated by donor alloantigens promote direct killing, in a T cell receptor–independent manner, of Class I‐deficient donor cells.     

自体骨髓干细胞移植治疗糖尿病足2例

对2例采用自体骨髓干细胞移植技术治疗糖尿病足患者进行观察护理,结果患者术后2周肢体疼痛、冷感觉 有显著好转,肢端皮温增高,溃疡基本愈合,未出现任何移植相关并发症和不良反应。提出术前注意与患者沟通, 讲明手术风险,做好各项准备,术后密切监测生命体征及穿刺部位、治疗区域情况,预防感染,加强护理。     

2例冠脉搭桥术后难愈性胸骨骨髓炎患者行自体骨髓干细胞移植治疗的护理

目的 对2例冠心病并存糖尿病行冠脉搭桥术后并发难愈性胸骨骨髓炎患者实施自体骨髓干细胞移植治疗和精心的护理,2例患者手术顺利,于术后14 d切口愈合出院,无并发症发生;随访12～24个月无复发.提出术前有效控制血糖,完善术前准备,加强健康教育指导,术后密切监护,积极抗感染和心理支持是保证手术成功的重要措施.     

1例骨髓移植术后并发重型肝炎患者行人工肝血浆置换术的护理

对1例骨髓移植术后在血型转换中并发重型肝炎的患者,通过人工肝支持系统进行4次血浆置换术,结果患者病情好转出院.提出做好充分的术前准备、加强血型抗体滴度的监测、选取合适血型的血浆,以及术中密切观察患者生命体征变化、预防并发症的发生,术后正确的饮食指导,是保证患者血浆置换术顺利完成的重要措施.     

Successful Bone Marrow Transplantation Reveals the Lack of Endothelial Progenitor Cells Mobilization in a Patient With Critical Limb Ischemia: A Case Report

Restoring blood flow to ischemic tissue is a prerequisite for treatment of ischemic diseases. Cell-based therapy based on bone marrow transplantation is a promising option for patients with critical limb ischemia (CLI). The efficacy of cell therapies to augment neovascularization seems to involve endothelial progenitor cells (EPCs); however, the mechanisms underlying the efficacy have not been fully elucidated. Herein we have described the case of a young patient with severe CLI, who experienced a 24-month beneficial clinical response to autologous bone marrow transplantation. The exceptional amelioration enabled him to perform standardized maximal treadmill exercise test that demonstrated lack of exercise-induced EPC mobilization, despite adequate stromal-derived factor 1 and vascular endothelial growth factor responses. Therefore, tissue ischemia is not sufficient to promote the recruitment of EPCs that have been demonstrated to be involved in the recovery from ischemia. The local implantation of marrow-derived elements may provide cells and/or trophic factors, which have the capacity to augment angiogenesis, opening new approaches to the etiopathogenesis of the disease.     

141HB107: A Pre‐Clinical Therapeutic Peptide

Peptides of the innate immune system play a vital role in the protection and repair of almost all biological systems. Such peptides have been implicated in a range of activities associated with prevention of disease and modulation of innate immunity. HB107 is a derivative of one such peptide, Cecropin B, that has demonstrated efficacy in enhancing wound healing in both burn and incision animal models. HB107 has been evaluated for efficacy in a mouse incision model and for safety and efficacy in a pig burn wound model. Topical application of the peptide gives RE⁵⁰(time needed for 50% re‐epithelialization) values of 10.28 days for 500 ug/mL and 12.72 days for 100 ug/mL compared to control 16.45 days. Additionally the peptide was well tolerated in terms of safety both topically and in an IV acute toxicity mouse model with no adverse effects observed. HB107 not only demonstrated efficacy and safety, but due to being a relatively short synthetic peptide, costs significantly less to manufacture than the current approved therapies.     

Mechanisms of Donor‐Specific Tolerance in Recipients of Haploidentical Combined Bone Marrow/Kidney Transplantation

We recently reported long‐term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor‐specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory‐type cell predominance and an increased proportion of CD4⁺CD25⁺CD127^?FOXP3⁺ Treg during the lymphopenic period. Complete donor‐specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL‐2‐producing and cytotoxic cells, developed and persisted for the 3‐year follow‐up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long‐term, systemic donor‐specific unresponsiveness in patients with HLA‐mismatched allograft tolerance. While regulatory cells may play an early role, long‐term tolerance appears to be maintained by a deletion or anergy mechanism.     

S‐I‐05 Increased Levels of Cell Division Cycle 25A Protein in Keloid Lesions

Aim: Keloid lesions develope as a result of abnormal growth of dermal fibroblasts after the injury. On the other hand, cell division cycle (Cdc) 25 is a family of phosphatases that activate the cell cycle regulating cyclin‐dependent kinases. The three members of this family, Cdc25A, Cdc25B, and Cdc25C act in different phases of the cell cycle. In this study, we examined the dgree of expression of these phosphatases in keloid fibroblasts. Methods: Primary cultures of keloid and adjacent normal dermal fibroblasts (n = 4) as well as frozen and paraffin‐embedded keloid and normal dermal tissues (n = 12 and n = 17 respectively) were examined by Western blot and immunohistochemical analyses for the expressions of Cdc25A, Cdc25B, Cdc25C and phosphorylated Cdk2. Results: Cdc25A protein levels were frequently increased in keloid fibroblasts as compared to the adjacent normal‐appearing fibroblasts or different normal dermal fibroblasts. In contrast, Cdc25B and Cdc25C were none or rarely expressed. The incrased levels of Cdc25A were associated with lower levels of its substrate, Cdk2, in a phosphorylated or inactive form. Conclusions: Taken together, our data show that Cdc25A protein levels increase in keloid fibroblasts and this increase may be sufficient to activate its substrate, Cdk2, and accelerate the cell cycling. These results may have implication for the development of strategies to silence Cdc25A activity after the wound healing as a therapeutic modality for the keloid lesions.     

Augmentation Mastopexy with Implant and Autologous Tissue for Correction of Moderate/Severe Ptosis

Purpose: Breast augmentation combined with mastopexy is associated with a significantly higher complication rate than augmentation alone. The combination of mastopexy and breast implants has revealed a moderate recurrence of breast ptosis in many patients particularly with use of medium to large implants. Ptosis is the “bottoming out” of the breast tissue with loss of the desired roundness, due to the ptosis of the breast implant and the mammary tissue. In this study, we hypothesize the need for careful planning and careful preoperative surgical execution to minimize this complication. Patients and Methods: Between January 2007 and July 2011, augmentation mastopexy with implant and autologous tissue (“double implant”) was performed for 25 patients with grade III mammary ptosis. All patients underwent inverted-T mastopexy with supramuscular moderately cohesive gel breast implant using an inferior-based flap of de-epitelialized dermoglandular tissue and a superior-based nipple-areola complex pedicle. Results: An inferior-based flap of deepithelialized dermoglandular tissue was used to stabilize the implant and is projection. Breast lifting was performed through a strong anchorage to fascia and to muscle of second intercostal space, improving the profile of the breast. Results were analyzed, no breast ptosis recurrence was noted at 30-month follow-up. Conclusions: Our technique presents the challenge of determining the amount of excess skin to be removed after implantation to create symmetry and provide for skin tightening without compromising tissue vascularization.     

Outcomes of Severe Pregnancy‐Related Liver Disease: Refining the Role of Transplantation

Severe liver disease in pregnancy is generally considered to have a favorable prognosis. The limited data available have not yielded disease‐specific prognostic criteria or guidance on who should undergo liver transplantation (LT). We retrospectively evaluated 54 admissions with pregnancy‐related liver disease to (1) evaluate if any admission parameters were associated with death and/or transplantation and (2) identify maternal complications. Eighteen had acute fatty liver of pregnancy and 32 had hypertension/eclampsia related disease. Seven patients (13%) died and four (7%) underwent LT. Survival rates were 43/48 if not listed for LT and 4/6 if listed. Of the four transplanted, three survived. Patients who died and/or underwent LT were more likely to have encephalopathy (p = 0.04) and hyperlactaemia (p = 0.03). Serum lactate was the best discriminant (ROC AUC 0.84). An admission lactate greater than 2.8mg/dL had 73% sensitivity and 75% specificity for predicting death or LT. The addition of encephalopathy to this parameter increased sensitivity and specificity to 90% and 86%, respectively. The King's College criteria were not effective in predicting outcome. This study confirms the overall favorable prognosis in pregnancy‐related liver failure but indicates that elevated lactate levels in the presence of encephalopathy best identify patients at greatest risk of death or LT.     

Study rationale,design, and pretransplantation alloantibody status: A first report of Clinical Trials in Organ Transplantation in Children‐04 (CTOTC‐04) in pediatric heart transplantation

Anti‐HLA donor‐specific antibodies are associated with worse outcomes after organ transplantation. Among sensitized pediatric heart candidates, requirement for negative donor‐specific cytotoxicity crossmatch increases wait times and mortality. However, transplantation with positive crossmatch may increase posttransplantation morbidity and mortality. We address this clinical challenge in a prospective, multicenter, observational cohort study of children listed for heart transplantation (Clinical Trials in Organ Transplantation in Children‐04 [CTOTC‐04]). Outcomes were compared among sensitized recipients who underwent transplantation with positive crossmatch, nonsensitized recipients, and sensitized recipients without positive crossmatch. Positive crossmatch recipients received antibody removal and augmented immunosuppression, while other recipients received standard immunosuppression with corticosteroid avoidance. This first CTOTC‐04 report summarizes study rationale and design and relates pretransplantation sensitization status using solid‐phase technology. Risk factors for sensitization were explored. Of 317 screened patients, 290 were enrolled and 240 underwent transplantation. Core laboratory evaluation demonstrated that more than half of patients were anti‐HLA sensitized. Greater than 80% of sensitized patients had class I (with or without class II) HLA antibodies, and one‐third of sensitized patients had at least 1 HLA antibody with median fluorescence intensity of ≥8000. Logistic regression models demonstrated male sex, weight, congenital heart disease history, prior allograft, and ventricular assist device are independent risk factors for sensitization.     

Novel RANK Antagonists for the Treatment of Bone‐Resorptive Disease: Theoretical Predictions and Experimental Validation

Receptor activator of nuclear factor‐κB (RANK) and RANK ligand (RANKL) play a pivotal role in bone metabolism, and selective targeting of RANK signaling has become a promising therapeutic strategy in the management of resorptive bone diseases. Existing antibody‐based therapies and novel inhibitors currently in development were designed to target the ligand, rather than the membrane receptor expressed on osteoclast precursors. We describe here an alternative approach to designing small peptides able to specifically bind to the hinge region of membrane RANK responsible for the conformational change upon RANKL association. A nonapeptide generated by this method was validated for its biological activity in vitro and in vivo and served as a lead compound for the generation of a series of peptide RANK antagonists derived from the original sequence. Our study presents a structure‐ and knowledge‐based strategy for the design of novel effective and affordable small peptide inhibitors specifically targeting the receptor RANK and opens a new therapeutic opportunity for the treatment of resorptive bone disease. © 2014 American Society for Bone and Mineral Research.