Jignesh V. Unadkat

Marginal mass islet transplantation with autologous mesenchymal stem cells promotes long-term islet allograft survival and sustained normoglycemia.

Allogeneic islet transplantation is an option to treat diabetes however there are obstacles that are limiting its clinical use. We have examined whether mesenchymal stem cells (MSC) improve islet graft survival and whether such therapy allows for better graft acceptance with reduced requirement for immunosuppression. In vitro-expanded syngeneic bone marrow-derived MSC were co-transplanted with islets into omental pouch in a rat model of streptozotocin-induced diabetes. Marginal mass syngeneic islet transplantation into the omentum with MSC promoted sustained normoglycemia. Interestingly, allogeneic islets +MSC, but not islets alone, with short-term use of immunosuppression enhanced long-term islet graft survival, insulin expression in the grafts and induced normal serum insulin levels and normoglycemia. T cells from recipients transplanted with allogeneic islets +MSC produced low levels of IFN-gamma and TNF-alpha upon ex-vivo activation, and this transplantation protocol promoted the generation of IL-10-secreting CD4(+) T cells. These data encourage further preclinical and eventually, clinical MSC-based islet transplantation to improve the outcome of allogeneic islet transplantation in the treatment of diabetes.

Co-infusion of donor bone marrow with host mesenchymal stem cells treats GVHD and promotes vascularized skin allograft survival in rats.

We investigated the effect of autologous mesenchymal stem cells (MSC) on multiple unmodified donor bone marrow (BM) infusions and vascularized skin graft outcome. BM-derived rat MSC were examined for phenotype and function. MSC/MSC-conditioned-medium suppressed IFN-gamma production by T cells and modified DC function. Infusions of MSC with one-time BM improved vascularized skin graft survival, while with one-two-times BM reversed graft versus host disease (GVHD). Mixed chimerism was enhanced in recipients given two-four-times BM with MSC infusions. Interestingly, four-times BM infusions with MSC delayed GVHD onset, reduced host tissue damage and enhanced vascularized skin allograft survival compared to four-times BM alone. These data demonstrate that, the co-infusion of MSC with unmodified BM limit the toxicity of allogeneic BM transplantation, enhance mixed chimerism and improve vascularized skin graft survival. These findings provide insights for the development of autologous MSC-based BM transplantation and prevention of graft rejection or treatment of autoimmunity.

Composite tissue vasculopathy and degeneration following multiple episodes of acute rejection in reconstructive transplantation.

Transplant vasculopathy has not been systematically investigated in composite tissue allotransplantation (CTA). The impact of multiple acute rejections (ARs) on long‐term graft outcomes in reconstructive transplantation remains unknown. This study in a rat hind‐limb allotransplantation model systematically analyzes vasculopathy and tissue‐specific pathological changes secondary to multiple AR episodes. LEW rats were transplanted with BN rat hind limbs and treated as follows: Group 1 (Iso): isografts. Group 2 (CsA): Cyclosporine (CsA) qd; Group 3 (mult AR): CsA and dexamethasone only when AR was observed. No AR was observed in Groups 1 and 2. Multiple AR were observed in Group 3, and each episode was completely reversed (clinically) with pulsed CsA + dexamethasone treatment. Group 3 animals demonstrated significant vascular lesions along with skin and muscle atrophy, upregulation of profibrotic gene expression and fibrosis when compared to Groups 1 and 2. In addition, allograft bone was sclerotic, weak and prone to malunion and nonunion. Interestingly, vasculopathy was a late finding, whereas muscle atrophy with macrophage infiltration was seen early, after only a few AR episodes. Taken together, multiple AR episodes lead to vasculopathy and tissue‐specific pathology in CTA. This is the first evidence of ‘composite tissue vasculopathy and degeneration (CTVD)’ in CTA.

Long-Term Survival of Limb Allografts Induced by Pharmacologically Conditioned, Donor Alloantigen-Pulsed Dendritic Cells Without Maintenance Immunosuppression

Background. We showed recently that limb allograft survival could be enhanced by administration of alloantigen (Ag)-pulsed immature dendritic cells (DC) after transplantation. Since indefinite graft survival was not achieved, we have further modified the DC by pharmacologic (rapamycin; Rapa) conditioning and ascertained their influence on graft survival, without continued immunosuppressive therapy. Methods. We compared the ability of donor Ag-pulsed, Rapa-conditioned rat myeloid DC (Rapa DC) and control DC (CTR DC) to inhibit alloreactive T-cell responses after limb transplantation in antilymphocyte serum (ALS)-treated recipients given a short postoperative course of cyclosporine (CsA). Results. Both DC populations expressed similar levels of major histocompatibility complex (MHC) II, CD40 and CD54, but Rapa DC expressed lower CD86. After toll-like receptor activation, both populations produced minimal interleukin (IL)-12p70, but Rapa DC secreted lower levels of IL-6 and IL-10. The capacity of DCs to stimulate T-cell proliferation in mixed leukocyte reactions was very low. Pulsing of the DC with donor Ag did not alter their phenotype or function. Interestingly, posttransplant administration of donor Ag-pulsed Rapa DC to rats given perioperative ALS and 21 days CsA significantly delayed graft rejection and promoted long-term (>125 days) graft survival. AlloAg-pulsed Rapa DC induced T-cell hyporesponsiveness and promoted the generation of IL-10-secreting CD4+ T cells upon ex vivo challenge. Conclusions. Infusion of donor Ag-pulsed, Rapa-conditioned DC after composite tissue transplantation can prevent rejection of the grafts, including skin, across a full MHC mismatch and in the absence of continued immunosuppressive therapy.

Daily topical tacrolimus therapy prevents skin rejection in a rodent hind limb allograft model.

Background: Skin is the most immunogenic component of a composite tissue allograft. Topical immunotherapy is an attractive therapeutic modality with which to provide local immunosuppression, with minimal systemic toxicity. The present study was performed to investigate the potential of topical tacrolimus to prolong survival of the skin component of a composite tissue allograft. Methods: Wistar Furth–to-Lewis rat orthotopic hind limb transplants were performed. Group I consisted of rats treated with topical tacrolimus; group II, antilymphocyte serum plus 21 days cyclosporine; and group III, antilymphocyte serum plus 21 days of cyclosporine plus topical tacrolimus. In group IV, tacrolimus levels in blood, skin, and muscle were measured in an autograft control group. Results: All animals in group I (n = 8) developed grade III clinical rejection by postoperative day 9. In group II (n = 9), the median onset of grade III rejection was postoperative day 40 (range, postoperative days 34 to 44). In group III (n = 6), two animals developed focal grade III rejection on postoperative days 35 and 56. The remaining four animals reached the 100-day endpoint without grade III rejection. In group IV, tacrolimus levels were low or undetectable in blood, whereas skin levels were 100-fold higher than underlying muscle. Conclusions: Topical tacrolimus therapy has the potential to prevent skin rejection in a composite tissue allograft. Preoperative depletion of T cells with antilymphocyte serum, along with a short course of systemic immunosuppression, prevents acute rejection, whereas topical tacrolimus inhibits immune cell function in the skin. Concentrations of tacrolimus are substantially higher in skin compared with underlying muscle and peripheral blood. Topical immunotherapy could reduce the morbidity associated with systemic immunosuppression in clinical composite tissue allografts.

A Clinical Review of Total Body Lift Surgery

BACKGROUND Until 2001, body contouring surgery after massive weight loss was uncoordinated, with few patients achieving compete rehabilitation. OBJECTIVE The authors report a 5-year, retrospective, 75-case clinical review to determine the effectiveness, reliability, and safety of single and multistage total body lift (TBL) surgery. METHODS Between January 2001 and June 2006, 59 single-stage, 15 two-stage, and 1 three-stage TBL surgeries were performed, involving a total of 605 separate procedures. Outcomes and complications were compared among all TBL patients and a contemporaneous published series. RESULTS Three representative cases are described. Overall, in patients under 55 years of age with a body mass index of less than 30, there was no significant difference in the choice of procedure (ie, single-stage TBL [95% confidence interval, 1.236-2.302] or multiple-stage TBL [95% confidence interval, 1.687-4.892]; P = .1882). Although there was no significant association between major complications and the number of procedures performed in this cohort of patients, there were increased wound healing problems following multiple-stage TBL (P > .5). Single-stage TBL surgery averaged 8.4 hours. Two-stage surgery took 7.4 hours for the first stage and 4.6 hours for the second stage, for a total of 11 hours. Banked blood transfusions for single-stage surgery were 1.5 per single-stage case and 0.78 per multi-stage case. Seventy-six percent of the patients experienced complications, mostly related to wound healing. All preoperative and postoperative Pittsburgh rating grades improved. CONCLUSIONS TBL is customized for individuals who desire a comprehensive approach to improvement of their loose skin. The rate of complications was high and comparable to other published series. There was no difference between the complications of the single-and two-stage patients. While there was an observable reduction in deformity and a high rate of satisfactory aesthetic outcomes, this high number of complications indicates a need to improve clinical performance.

Investigation of Antibody-Mediated Rejection in Composite Tissue Allotransplantation in a Rat Limb Transplant Model

BACKGROUND Despite the widely accepted implication of antidonor antibodies and complement in solid organ transplantation, their role in reconstructive allotransplantation is not clear. The aim of this study was to analyze the humoral immune response using a rat orthotopic limb transplantation model. METHODS We used the Brown Norway to Lewis rat orthotopic hind-limb transplant model: Group 1, isografts; group 2, allografts with daily continuous cyclosporine treatment to prevent acute rejection; and group 3, allografts undergoing multiple episodes of acute rejection. Samples were taken at 30, 60, and 90 days. Serum was analyzed by FACS for antidonor antibodies. Tissue deposition of antibodies and complement was investigated by immunofluorescence. RESULTS By day 90, animals in group 3 had undergone 19 (+/-3.2) acute rejection episodes. There was no difference in the occurrence of serum antidonor antibodies between the three groups at any time point. However, at 90 days, anti-third-party antibodies were significantly greater among group 3. There was no difference in antibody or complement deposition in muscles between the 3 groups. CONCLUSION Despite the increased antibody against a third party after multiple rejection episodes in this animal model, there was no clear evidence of an antibody-mediated alloresponse in limb transplantation.

Biologics and Donor Bone Marrow Cells for Targeted Immunomodulation in Vascularized Composite Allotransplantation: A Translational Trial in Swine

INTRODUCTION Bone marrow (BM) infusion following organ transplantation is a prerequisite for potential donor-antigen-specific tolerance induction. We developed a preclinical swine model to determine the optimal dose of BM cells to achieve microchimerism. Furthermore, induction therapy was optimized by augmenting the BM infusion with biologics in the form of costimulatory blockade: cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4-Ig). MATERIALS AND METHODS Yucatan miniature swine (n = 12) underwent total body and thymic irradiation for cytodepletion. Animal groups received 15, 30, or 60 million cells per kilogram of whole unmodified BM. The optimal dose of BM cell infusion (BMT) was then applied to subsequent experiments evaluating the addition of CTLA4lg. Group 1 (control) received no treatment. Group 2 received FK506 only; group 3 received irradiation, BMT, and FK506; group 4 received FK506 and CTLA4-lg. RESULTS Microchimerism was established in all animals after BM cell infusion; at postoperative day 9, it was significantly increased for 60 million cells per kilogram (P = .0001). Transplanted animals in group 1 rejected the allograft 5 to 8 days after transplantation. Group 2 rejected the allograft (skin and muscle) 30 to 32 days after transplantation (2 days after cessation of immunosuppression). Group 3 rejected the skin portion of the allograft at 50, 52, and 53 days posttransplant. Remaining allograft components (muscle, bone, nerve, vessel) survived indefinitely. Group 4 animals demonstrated significantly prolonged muscle survival beyond 150 days posttransplant; the skin component survived past 150 days in two of three animals. Skin and muscle histology in all long-term surviving animals were normal. CONCLUSIONS BM cell infusion with 60 million cells per kilogram results in stable levels of microchimerism. The addition of costimulatory blockade (CTLA4lg) prolonged allograft skin survival and overall graft survival. Such targeted immunomodulatory protocols might facilitate immune tolerance and eliminate the need for multidrug immunosuppression to maintain graft survival after vascularized composite allotransplantation.

A Model for Functional Recovery and Cortical Reintegration after Hemifacial Composite Tissue Allotransplantation

Background: The ability to achieve optimal functional recovery is important in both face and hand transplantation. The purpose of this study was to develop a functional rat hemifacial transplant model optimal for studying both functional outcome and cortical reintegration in composite tissue allotransplantation. Methods: Five syngeneic transplants with motor and sensory nerve appositions (group 1) and five syngeneic transplants without nerve appositions (group 2) were performed. Five allogeneic transplants were performed with motor and sensory nerve appositions (group 3). Lewis (RT1l) rats were used for syngeneic transplants and Brown-Norway (RT1n) donors and Lewis (RT1l) recipients were used for allogeneic transplants. Allografts received cyclosporine A monotherapy. Functional recovery was assessed by recordings of nerve conduction velocity and cortical neural activity evoked by facial nerve and sensory (tactile) stimuli, respectively. Results: All animals in groups 1 and 3 showed evidence of motor function return on nerve conduction testing, whereas animals in group 2, which did not have nerve appositions, did not show electrical activity on electromyographic analysis (p < 0.001). All animals in groups 1 and 3 showed evidence of reafferentation on recording from the somatosensory cortex after whisker stimulation. Animals in group 2 did not show a cortical response on stimulation of the whiskers (p < 0.001). Conclusion: The authors have established a hemiface transplant model in the rat that has several modalities for the comprehensive study of motor and sensory recovery and cortical reintegration after composite tissue allotransplantation.

The Importance of Early Operative Treatment in Open Fractures of the Fingers

IntroductionCurrent guidelines suggest early surgical treatment of open fractures. This rule in open hand fractures is not well supported and may be practically difficult to observe. Furthermore, desirable washout can be obtained in the emergency department (ED). The purpose of this study was to determine the importance of early surgery in our institution. MethodsSeventy patients with open fractures of the hand were retrospectively reviewed for demographics, fracture characteristics, and complications. Statistical analysis included univariate analysis, Fisher exact test, and Akaike information criterion. ResultsIntravenous antibiotics were administered early in 53 (75.7%) patients. Mean (SD) time to surgery was 2.3 (134.9) hours. The infection rate was 11.4%. No significant relationship was found between fracture type, finger involved, hand dominance, comorbidities, and infection. Antibiotic administration was significantly related to infection (P = 0.007), whereas time to surgery was not (P = 0.33). Age was weakly related to infection (P = 0.08). DiscussionAdministration of intravenous antibiotics in the ED was the most significant factor in preventing infection, whereas the time to operation was not significant. Because a thorough washout and debridement can be performed on open hand fractures in the ED due to the ability to provide adequate anesthesia, the actual time to surgery may possibly be delayed without increasing the risk of infection. Future prospective studies may allow for better guidelines for the treatment of open hand fractures.