Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy.

Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin-deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 106) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

Establishing the Feasibility of Face Transplantation in Granulomatosis With Polyangiitis.

Granulomatosis with polyangiitis (GPA; formerly Wegener's granulomatosis) is a rare vasculitis that commonly starts in the craniofacial region. We report a case that was masked by prior facial trauma and associated with pyoderma gangrenosum (PG). Disease progression and aggressive debridements led to severe facial tissue loss. The decision to perform a face transplant was controversial because of the risk of disease relapse on the facial allograft. We reviewed renal transplant outcomes in GPA for possible relevance. A PubMed search retrieved 29 studies. Patient and graft survival, relapse, morbidity, mortality, rejection and immunosuppression were assessed. Ten-year patient survival and graft survival were 84.4% and 72.6%, respectively. GPA relapse occurred in 31.5%, and upper airway/ocular relapse occurred in 17.8% (resolved in 76.9%). Mortality was 12.3%. Acute and chronic rejection rates were 14.9% and 6.8%, respectively. Traditional posttransplant immunosuppression was effective. Our review suggests that GPA renal transplant outcomes are comparable to general renal transplant cohorts. Furthermore, transplanted GPA patients exhibit lower disease relapse secondary to lifelong immunosuppression. This supported our decision to perform a face transplant in this patient, which has been successful up to the present time (1-year posttransplantation). Untreated GPA and PG are potential causes of worse surgical outcomes in the craniofacial region.

A four-year pathology review of the near total face transplant.

In December of 2008, our institution performed a near total face transplant. The patient was monitored for signs of rejection assessed by paired skin and mucosa biopsies. The results of histological review of 120 biopsies collected during the first 4 years posttransplant are discussed. All biopsies were stained with hematoxylin and eosin, periodic acid-Schiff, immunohistochemical and TUNEL assays and graded using the Banff 2007 classification. Grade III rejection was diagnosed clinically at weeks 45 and 66, posttransplant; week 45 was determined as folliculitis while the erythema episode at week 66 confirmed an acute rejection (AR) that required hospitalization. The mucosa frequently showed interface inflammation without clinical signs of rejection and was not present in skin biopsies. In all, 34 of the 45 mucosal biopsies (75%) showed these interface changes. Clinical symptoms concurred with skin pathology in two grade III rejections. The mucosa showed histologic signs of rejection more frequently, which may indicate: increased mucosal sensitivity to rejection, a different type or subtype of AR that is specific to the mucosa, or a nonspecific process such as a drug effect. With more data and world experience, the diagnosis of face transplant rejection will be better defined and the Banff classification enhanced.

Face transplantation: a leading surgeon's perspective.

Twenty years of experience in the field of vascularized composite allografts (VCA) leading to the first US face transplantation is presented. Different experimental models and cadaveric studies in the VCA models are outlined. Development of face transplantation protocol and consent forms for Institutional Review Board approval is discussed. Different scientific, regulatory, and financial considerations that were required before approval of face transplantation are presented. The effort, importance, and role of multidisciplinary team approach are emphasized. Finally, the technical aspects of face transplantation and related immunologic and functional outcomes of the patients are discussed.

Cost analysis of conventional facial reconstruction procedures followed by face transplantation.

For the first time, this study analyzes the cost of multiple conventional reconstructions and face transplantation in a single patient. This patient is a 46-year-old female victim of a shotgun blast resulting in loss of multiple functional and aesthetic subunits. For over 5 years, she underwent multiple conventional reconstructions with suboptimal results. In December 2008, she became the recipient of the first U.S. face transplant. This has provided the unique opportunity to present the cost of 23 separate conventional reconstructive procedures and the first face transplant in the United States. The combined cost of conventional reconstructive procedures and the first U.S. face transplant was calculated to be $353 480 and $349 959, respectively. The combined cost posttransplant totaled $115 463. The direct cost pretransplant was $206 646, $232 893 peritransplant and $74 236 posttransplant. The two largest areas of cost utilization were surgical ($79 625; 38.5%) and nursing ($55 860; 27%), followed by anesthesia ($24 808; 12%) and pharmacy ($16 581; 8%). This study demonstrates that the cost of the first U.S. face transplant is similar to multiple conventional reconstructions. Although the cost of facial transplantation is considerable, the alleviation of psychological and physiological suffering, exceptional functional recovery and fulfillment of long-lasting hope for social reintegration may be priceless.

Overview of guidelines for establishing a face transplant program: a work in progress.

Since 2005, nine face transplants have been performed in four countries: France, the United States (US), China and Spain. These encouraging short-term outcomes, with the longest survivor approaching 5 years, have led to an increased interest in establishing face transplant programs worldwide. Therefore, the purpose of this article is to facilitate the dissemination of relevant details as per our experience in an effort to assist those medical centers interested in establishing a face transplant program. In this article, we address the logistical challenges involved with face transplantation; including essential program requirements, protocol details, face transplant team assembly, project funding, the organ procurement organization and the coroner. It must be emphasized that face transplantation is still experimental and its therapeutic value remains to be validated. All surgical teams pursuing this endeavor must dedicate an attention to detail and should accept a responsibility to publish their outcomes in a transparent manner in order to contribute to the international field. However, due to its inherent complexity, facial transplantation should only be performed by university-affiliated medical institutions capable of orchestrating a specialized multidisciplinary team with a long-term commitment to its success.

Composite tissue allotransplantation: a proposed classification system based on relative complexity.

Numerous achievements have been made encompassing a wide array of composite tissue allograft (CTA) subtypes. We sought to develop a simple, reproducible CTA classification system for the purpose of comparing clinical investigation. Each CTA subtype differs in relative complexity and can therefore be theoretically classified based on its unique combination of multiple factors. Eight complexity factors (CFs) are hypothesized: anatomic detail, psychological obstacles, rejection risk, required rehabilitation, relative antigenicity, functionality/cosmesis, skin ratio, and salvageability. A distribution of total complexity scores, ranging from 8 to 24, is classified into 3 ordered categories representing varying degrees of complexity. In conclusion, we have created a new classification system so that ongoing research and future data may be compared in a type-specific fashion.

Hematopoietic stem cell engraftment and seeding permits multi-lymphoid chimerism in vascularized bone marrow transplants.

Vascularized bone marrow transplantation (VBMT) across a MHC barrier under a 7-day alphabeta-TCR mAb and CsA protocol facilitated multiple hematolymphoid chimerism via trafficking of the immature (CD90) bone marrow cells (BMC) between donor and recipient compartments. Early engraftment of donor BMC [BN(RT1(n))] into the recipient BM compartment [LEW(RT1(l))] was achieved at 1 week posttransplant and this was associated with active hematopoiesis within allografted bone and correlated with high chimerism in the hematolymphoid organs. Two-way trafficking between donor and recipient BM compartments was confirmed by the presence of recipient MHC class I cells (RT1(l)) within the allografted bone up to 3 weeks posttransplant. At 10 weeks posttransplant, decline of BMC viability in allografted bone corresponded with bone fibrosis and lack of hematopoiesis. In contrast, active hematopoiesis was present in the recipient bone as evidenced by the presence of donor-specific immature (CD90/RT1(n)) cells, which correlated with chimerism maintenance. Clonogenic activity of donor-origin cells (RT1(n)) engrafted into the host BM compartment was confirmed by colony-forming units (CFU) assay. These results confirm that hematolymphoid chimerism is developed early post-VBMT by T-cell lineage and despite allografted bone fibrosis chimerism maintenance is supported by B-cell linage and active hematopoiesis of donor-origin cells in the host BM compartment.

Current status of composite tissue allotransplantation.

Composite tissue allotransplantation (CTA) is emerging as a potential treatment for complex tissue defects. A major drawback for CTA remains the requirement of lifelong immunosuppression. Up to date numerous experimental CTA models have been introduced to the transplantation literature. After the first hand transplantation in 1998, the clinical applicability of CTA has attracted a lot of attention. This article will outline the historical background of CTA and our experience in different CTA models.

Donor-origin cell engraftment after intraosseous or intravenous bone marrow transplantation in a rat model.

We compared the effects of intraosseous BMT with those of standard i.v. BMT on the efficacy on donor-cell engraftment into the BM and lymphoid organs across an MHC barrier in rats. Twenty-four intraosseous and 24 i.v. BMTs were performed from 48 ACI (RT1(a)) donors to 48 Lewis (RT1(l)) recipients. Each transplant group received either intraosseous or i.v. BMT. Groups I and II served as controls without immunosuppression (n=16); groups III and IV received cyclosporine monotherapy (n=16); and V and VI received alphabeta-TCR monoclonal antibody and cyclosporine A (alphabeta-TCR/CsA) for 7 days (n=16). In each group, four rats received 35 x 10(6) transplanted bone marrow cells (BMCs) and four received 70 x 10(6) cells. All animals survived without GVHD. Mean (+/-s.d.) donor-cell engraftment into BM of recipients after intraosseous BMT was 7.9% (+/-1.3%) in recipients receiving alphabeta-TCR-CsA and 70 x 10(6) BMCs, and 4.2% (+/-1.4%) in recipients after i.v. transplantation. The seeding efficacy of donor cells into lymphoid tissue was greater after intraosseous BMT and alphabeta-TCR-CsA than after standard i.v. transplantation. In our model, intraosseous BMT facilitated donor-cell engraftment under short-term immunodepletive alphabeta-TCR/CsA protocol, which resulted in a temporary state of immune unresponsiveness.

Anatomic characteristics of a fascia and its bands overlying the ulnar nerve in the proximal forearm: a cadaver study.

This study describes the characteristics of a fascia overlying the ulnar nerve for 10 cm distal to the midpoint of the retrocondylar groove. A total of 28 cadaver upper extremities were dissected. The ulnar nerve between the flexor carpi ulnaris and flexor digitorum profundus was traced distally underneath a thin fascia. The length of the fascia was measured and examined for the presence of segmental fascial thickenings, referred to as 'Bands'. Two types of fascia were found. In Type I, three Bands were identified within the fascia and the mean length of the fascia was 5.6 cm. In Type II, four Bands were identified and the mean length of the fascia was 7.7 cm. The presence of Bands within the fascia overlying the ulnar nerve in the proximal forearm may require release at the time of decompression, or anterior transposition, of the ulnar nerve at the cubital tunnel.

Trafficking of donor-derived bone marrow correlates with chimerism and extension of composite allograft survival across MHC barrier.

We proposed to evaluate differences between recipient's immune response to vascularized skin and combined vascularized skin/bone allografts, under a 7-day alphabeta-TCR plus cyclosporine (CsA) treatment protocol. Thirty-six transplantations were performed in six groups: group I (isograft control-vascularized skin graft; n=6); group II (isograft control-combined vascularized skin/bone graft; n=6); group III (allograft rejection control group-vascularized skin graft; n=6); group IV (allograft rejection control-combined vascularized skin/bone graft; n=6); group V (allograft treatment-vascularized skin graft; n=6); and group VI (allograft treatment-combined vascularized skin/bone graft; n=6). Isograft transplantations were performed between Lewis rats and allografts were transplanted across the MHC barrier from Brown Norway to Lewis rats. In the allograft treatment group, a combined alphabeta-TCR+CsA protocol was applied for 7 days. All groups were compared clinically, immunologically and histologically. Statistical significance was determined with two-tailed Student's t test. Indefinite graft survival was achieved in the isograft control group (>300 days). Allograft rejection controls rejected within 5 to 9 days posttransplant; chimerism levels were undetectable (<.5%). Allografts under the alphabeta-TCR+CsA protocol had significantly extended survival when skin was combined with bone (61-125 days) compared to vascularized skin allografts (43-61 days). Lymphoid macrochimerism was significantly higher in group VI than group V. Histology confirmed skin and bone viability. Combined vascularized skin/bone allografts had higher and sustained levels of donor-specific chimerism and extended allograft survival.

Laryngeal transplantation in 2005: a review.

There is no good surgical, medical or prosthetic solution to the problems faced by those with a larynx whose function is irreversibly damaged by tumor or trauma. Over the past 10 years, the pace of research designed to establish laryngeal transplantation as a therapeutic option for these persons has increased steadily. The biggest milestone in this field was the world's first true laryngeal transplant performed in Cleveland, Ohio in 1998. The recipient's graft continues to function well, in many respects, even after 7 years. However, it has also highlighted the remaining barriers to full-scale clinical trials. Stimulated by these observations, several groups have accumulated data which point to answers to some of the outstanding questions surrounding functional reinnervation and immunomodulation. This review seeks to outline the progress achieved in this field by 2005 and to point the way forward for laryngeal transplantation research in the 21st century.

The endoscopic management of cubital tunnel syndrome.

The overall success rate of surgical interventions for cubital tunnel syndrome is reported to be within 80% to 90% (Szabo, 1999). The discussion, however, whether to perform in situ nerve decompression or anterior transposition continues. In this paper, we present the results of our endoscopic approach to in situ cubital tunnel release, its rationale, clinical and anatomical indications and a detailed description of the technique.

Until they have faces: the ethics of facial allograft transplantation.

The ethical discussion of facial allograft transplantation (FAT) for severe facial deformity, popularly known as facial transplantation, has been one sided and sensationalistic. It is based on film and fiction rather than science and clinical experience. Based on our experience in developing the first IRB approved protocol for FAT, we critically discuss the problems with this discussion, which overlooks the plight of individuals with severe facial deformities. We discuss why FAT for facial deformity is ethically and surgically justified despite its negative portrayal in the media.

A new method of bone marrow transplantation leads to extension of skin allograft survival.

Tolerance induction through allogeneic bone marrow transplantation is an alternative method to chronic immunosuppression in maintaining long-term allograft survival. In this article, we introduce a new method of bone marrow allotransplantation, which preserves its natural microenvironment and does not require marrow processing or recipient conditioning. A total of 43 skin graft transplantations were performed in nine experimental groups between isogeneic [Lewis to Lewis (LEW, RT1(1))] and allogeneic [Lewis x Brown Norway (LBN --> F1, RT1(1+n)) to Lewis] rats under 35-day protocol of alphabeta T-cell receptor (TCR) monoclonal antibody (mAb) and cyclosporine (CsA) protocol. Monotherapies combined with "crude" bone marrow transplantation resulted in extended survival up to 21 days under CsA and up to 10 days under alphabeta-TCR mAb protocol. The use of combined protocol of alphabeta-TCRmAb/CsA with crude bone marrow transplantation resulted in the extension of skin allograft survival up to 65 days (P < .05). This new simple method of "crude" bone marrow allotransplantation without recipient conditioning is a promising, minimally invasive technique with a potential for direct clinical application.

Intraosseus transplantation of donor-derived hematopoietic stem and progenitor cells induces donor-specific chimerism and extends composite tissue allograft survival.

We investigated the effect of the intraosseous allotransplantation of the donor-derived hematopoietic stem cells (HSC) CD90+ on chimerism induction and survival of rat hind limb transplants. Eighteen rat hind limb transplantations were performed between Lewis-Brown-Norway and Lewis rats in three groups. Isograft and allograft rejection controls received no treatment. In the experimental group, 0.8 to 1.2 x 10(6) of separated and purified CD90+ HSC cells were transplanted intramedullary into the bone marrow cavity of the recipient's tibia during opposite hind limb transplantation, without immunosuppressive therapy. Transplants from isograft group survived indefinitely. Allograft controls rejected transplants on day 7 posttransplant. The injection of separated and purified CD90+ cells of the donor origin extended survival of the transplanted limbs up to 15 days in group III. We introduced a novel method of transplantation of the CD90+ cells of the donor origin into the recipient's bone marrow cavity. This technique resulted in extended allograft survival, without immunosuppressive therapy.

Different routes of donor-derived hematopoietic stem cell transplantation for donor-specific chimerism induction across MHC barrier.

Transplantation of donor-derived stem cells can improve organ allograft survival in animal models. This study was designed to investigate the effect of different routes of bone marrow cell (BMC) transplantation on donor-specific tolerance induction across MHC barrier under short-term CsA monotherapy and alphabetaTCR/CsA treatment protocols. Forty-eight BMC transplantations were performed between BN(RT1(n)) donors and LEW(RT1(1)) recipients. Intraosseous and intravenous BMC transplantation was studied in six groups of eight animals each receiving 35 x 10(6) (n = 4) and 70 x 10(6) (n = 4) bone marrow cells. Groups I and II (controls) received BMC transplantation but no treatment, groups III and IV CsA monotherapy, and groups V, VI alphabetaTCR/CsA protocol for 7 days. Flow cytometry monitored immunodepletion and donor-specific chimerism for MHC class I RT1(n)/CD4, RT1(n)/CD8 and RT1(n)/CD45RA antigens. All animals survived without graft-versus-host disease. At day 63 under CsA monotherapy a low level of chimerism for RT1(n)/CD4 was induced after intraosseous (1.9%) and intravenous (0.8%) transplantation of (70 x 10(6)) BMC. Under alphabetaTCR/CsA protocol chimerism for RT1(n)/CD4 revealed 6.5% and 0.9% in intraosseous and intravenous (70 x 10(6)) BMC transplantation, respectively. The total number of chimerism in intraosseous and intravenous (70 x 10(6)) BMC transplantation groups was 9.9% and 3.4%, respectively. Following intraosseous BMC transplantation under alphabetaTCR/CsA protocol chimerism was 50% higher in a group receiving 70 x 10(6) (9.9%) vs 35 x 10(6) (4.9%) BMC. Intraosseous transplantation of donor BMC under alphabetaTCR/CsA protocol was 75% more efficient in induction of donor-specific chimerism compared to intravenous transplantation.