A local drug delivery system prolongs graft survival by dampening T cell infiltration and neutrophil extracellular trap formation in vascularized composite allografts.

Introduction: The standard treatment for preventing rejection in vascularized composite allotransplantation (VCA) currently relies on systemic immunosuppression, which exposes the host to well-known side effects. Locally administered immunosuppression strategies have shown promising results to bypass this hurdle. Nevertheless, their progress has been slow, partially attributed to a limited understanding of the essential mechanisms underlying graft rejection. Recent discoveries highlight the crucial involvement of innate immune components, such as neutrophil extracellular traps (NETs), in organ transplantation. Here we aimed to prolong graft survival through a tacrolimus-based drug delivery system and to understand the role of NETs in VCA graft rejection. Methods: To prevent off-target toxicity and promote graft survival, we tested a locally administered tacrolimus-loaded on-demand drug delivery system (TGMS-TAC) in a multiple MHC-mismatched porcine VCA model. Off-target toxicity was assessed in tissue and blood. Graft rejection was evaluated macroscopically while the complement system, T cells, neutrophils and NETs were analyzed in graft tissues by immunofluorescence and/or western blot. Plasmatic levels of inflammatory cytokines were measured using a Luminex magnetic-bead porcine panel, and NETs were measured in plasma and tissue using DNA-MPO ELISA. Lastly, to evaluate the effect of tacrolimus on NET formation, NETs were induced in-vitro in porcine and human peripheral neutrophils following incubation with tacrolimus. Results: Repeated intra-graft administrations of TGMS-TAC minimized systemic toxicity and prolonged graft survival. Nevertheless, signs of rejection were observed at endpoint. Systemically, there were no increases in cytokine levels, complement anaphylatoxins, T-cell subpopulations, or neutrophils during rejection. Yet, tissue analysis showed local infiltration of T cells and neutrophils, together with neutrophil extracellular traps (NETs) in rejected grafts. Interestingly, intra-graft administration of tacrolimus contributed to a reduction in both T-cellular infiltration and NETs. In fact, in-vitro NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus. Conclusion: Our data indicate that the proposed local delivery of immunosuppression avoids off-target toxicity while prolonging graft survival in a multiple MHC-mismatch VCA model. Furthermore, NETs are found to play a role in graft rejection and could therefore be a potential innovative therapeutic target.

Nontuberculous mycobacterial infection following cat scratch in the setting of topical steroid use.

Infections due to nontuberculous mycobacteria (NTM) are caused by mycobacterial species other than Mycobacterium tuberculosis, M. leprae, and M. bovis. Patients who are immunocompromised have increased susceptibility to pulmonary, lymphatic, and skin infections by these pathogens. We present a case of a 78-year-old male who presented to dermatology with a left dorsolateral hand infection after sustaining cat scratches in the setting of topical steroid therapy for suspected pyoderma gangrenosum. A shave biopsy of the lesion showed granulomatous dermatitis and associated acid-fast bacilli, while tissue culture grew Mycobacterium chelonae. This case demonstrates cat scratches as an uncommon risk factor for cutaneous NTM disease. Although an association between cat scratches and human NTM infections has only been reported in two previous cases, it must be considered in cases of unusual and persistent cutaneous lesions, especially in immunocompromised patients, even those with only local immunosuppression from topical agents.

Local Dexamethasone Administration Delays Allogeneic Islet Graft Rejection in the Anterior Chamber of the Eye of Non-Human Primates.

Pancreatic islet transplantation into the anterior chamber of the eye (ACE) has been shown to improve glycemic control and metabolic parameters of diabetes in both murine and primate models. This novel transplantation site also allows the delivery of therapeutic agents, such as immunosuppressive drugs, locally to prevent islet graft rejection and circumvent unwanted systemic side effects. Local intravitreal administration of micronized dexamethasone implant was performed prior to allogeneic islet transplantation into the ACEs of non-human primates. Two study groups were observed namely allogeneic graft without immunosuppression (n = 4 eyes) and allogeneic graft with local immunosuppression (n = 8 eyes). Survival of islet grafts and dexamethasone concentration in the ACE were assessed in parallel for 24 weeks. Allogeneic islet grafts with local dexamethasone treatment showed significantly better survival than those with no immunosuppression (median survival time- 15 weeks vs 3 weeks, log-rank test p<0.0001). Around 73% of the grafts still survived at week 10 with a single local dexamethasone implant, where the control group showed no graft survival. Dexamethasone treated islet grafts revealed a good functional response to high glucose stimulation despite there was a transient suppression of insulin secretion from week 8 to 12. Our findings show a significant improvement of allografts survival in the ACE with local dexamethasone treatment. These results highlight the feasibility of local administration of pharmacological compounds in the ACE to improve islet graft survival and function. By eliminating the need for systemic immunosuppression, these findings may impact clinical islet transplantation in the treatment of diabetes, and the ACE may serve as a novel therapeutic islet transplantation site with high potential for local pharmacological intervention.

Emerging local immunomodulatory strategies to circumvent systemic immunosuppression in cell transplantation.

INTRODUCTION: Cell transplantation is a promising curative therapeutic strategy whereby impaired organ function can be restored without the need for whole-organ transplantation. A key challenge in allotransplantation is the requirement for life-long systemic immunosuppression to prevent rejection, which is associated with serious adverse effects such as increased risk of opportunistic infections and the development of neoplasms. This challenge underscores the urgent need for novel strategies to prevent graft rejection while abrogating toxicity-associated adverse events. AREAS COVERED: We review recent advances in immunoengineering strategies for localized immunomodulation that aim to support allograft function and provide immune tolerance in a safe and effective manner. EXPERT OPINION: Immunoengineering strategies are tailored approaches for achieving immunomodulation of the transplant microenvironment. Biomaterials can be adapted for localized and controlled release of immunomodulatory agents, decreasing the effective dose threshold and frequency of administration. The future of transplant rejection management lies in the shift from systemic to local immunomodulation with suppression of effector and activation of regulatory T cells, to promote immune tolerance.

The impact of locally-delivered tacrolimus-releasing microspheres and polyethylene glycol-based islet surface modification on xenogeneic islet survival.

Pancreatic islet replacement therapy is an advanced choice for severe cases of type I diabetes. Nevertheless, extensive host immune response toward islet grafts remains a huge challenge for long-term graft function, and a lack of islet donors further increases the difficulties associated with upscaling this therapy. Mounting evidence suggests local delivery of immunosuppressive agents provides a feasible means of enhancing graft-protection. Among many immunosuppressants, tacrolimus (FK506) is one of the most potent interleukin-2 (IL-2)-mediated T-cell proliferation blockers. Here, we reported the effect of locally-delivered FK506-releasing PLGA microspheres (FK506-M) combined with polyethylene glycol (PEG)-based islet surface modification on xenogeneic islet survival in C57BL/6 mouse model. FK506-M was prepared using an emulsion method to a particle size of 10-40 µm and released FK506 over 40 days in vitro. Around 80% of the initial dose of FK506-M stably localized near transplanted islets, as observed under a bioimaging instrument and by immunofluorescence staining of islet grafts. Interestingly, FK506-M at very low-doses (equivalent to 150 to 2400 ng FK506 per recipient) was found to inhibit the infiltration of immune cells into grafts and reduce serum IL-1ß levels, thereby improving graft survival times dose-dependently. The PEGylation of islets alone was not enough to protect islets from early rejection. However, combined treatment with FK506-M additively prolonged xenograft survival. In conclusion, this study describes a safe, effective approach for translating a systemic exposure-free local drug delivery into clinical trials of islet transplantation.

Neovascularized implantable cell homing encapsulation platform with tunable local immunosuppressant delivery for allogeneic cell transplantation.

Cell encapsulation is an attractive transplantation strategy to treat endocrine disorders. Transplanted cells offer a dynamic and stimulus-responsive system that secretes therapeutics based on patient need. Despite significant advancements, a challenge in allogeneic cell encapsulation is maintaining sufficient oxygen and nutrient exchange, while providing protection from the host immune system. To this end, we developed a subcutaneously implantable dual-reservoir encapsulation system integrating in situ prevascularization and local immunosuppressant delivery, termed NICHE. NICHE structure is 3D-printed in biocompatible polyamide 2200 and comprises of independent cell and drug reservoirs separated by a nanoporous membrane for sustained local release of immunosuppressant. Here we present the development and characterization of NICHE, as well as efficacy validation for allogeneic cell transplantation in an immunocompetent rat model. We established biocompatibility and mechanical stability of NICHE. Further, NICHE vascularization was achieved with the aid of mesenchymal stem cells. Our study demonstrated sustained local elution of immunosuppressant (CTLA4Ig) into the cell reservoir protected transcutaneously-transplanted allogeneic Leydig cells from host immune destruction during a 31-day study, and reduced systemic drug exposure by 12-fold. In summary, NICHE is the first encapsulation platform achieving both in situ vascularization and immunosuppressant delivery, presenting a viable strategy for allogeneic cell transplantation.

A local application of mesenchymal stem cells and cyclosporine A attenuates immune response by a switch in macrophage phenotype.

The immunosuppressive effects of systemically administered mesenchymal stem cells (MSCs) and immunosuppressive drugs have been well documented. We analysed the mechanisms underlying the therapeutic effect of MSCs applied locally in combination with non-specific immunosuppression in a mouse model of allogeneic skin transplantation. The MSC-seeded and cyclosporine A (CsA)-loaded nanofibre scaffolds were applied topically to skin allografts in a mouse model and the local immune response was assessed and characterized. MSCs migrated from the scaffold into the side of injury and were detected in the graft region and draining lymph nodes (DLNs). The numbers of graft-infiltrating macrophages and the production of nitric oxide (NO) were significantly decreased in recipients treated with MSCs and CsA, and this reduction correlated with impaired production of IFNγ in the graft and DLNs. In contrast, the proportion of alternatively activated macrophages (F4/80+ CD206+ cells) and the production of IL-10 by intragraft macrophages were significantly upregulated. The ability of MSCs to alter the phenotype of macrophages from the M1 type into an M2 population was confirmed in a co-culture system in vitro. We suggest that the topical application of MSCs in combination with CsA induces a switch in macrophages to a population with an alternatively activated 'healing' phenotype and producing elevated levels of IL-10. These alterations in macrophage phenotype and function could represent one of the mechanisms of immunosuppressive action of MSCs applied in combination with CsA. Copyright © 2015 John Wiley & Sons, Ltd.

Altered macrophagic THP-1 cell phagocytosis and migration in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Local immune dysfunction via macrophages is a proposed aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study aimed to clarify the effects of various bisphosphonates on macrophage function using a THP-1 monocytic model to examine migration, phagocytosis, and fibrin structure. MATERIALS AND METHODS: THP-1 cell migration was measured in the presence and absence of zoledronate, ibandronate, risedronate, alendronate, pamidronate (0.5, 5 and 50 µM) and clodronate (125, 250 and 500 µM) using the real-time xCELLigence system. Phagocytosis and actin fibre assays were performed after 72 h with zoledronate, ibandronate, alendronate and clodronate. RESULTS: Time to maximum migration for THP-1 cells was significantly reduced (p < 0.05) for high dosages of zoledronate, ibandronate and alendronate compared to controls. All dosages of clodronate and a low dose of zoledronate exhibited prolonged migrations. Phagocytic capacity was significantly reduced in high dosages of all bisphosphonates and for 5 µM zoledronate and ibandronate (p < 0.05). Low bisphosphonate exposure was accompanied by overcharged phagosoms. Altered appearance in F-actin fibrin structure was observed in bisphosphonate-exposed cells. CONCLUSIONS: All bisphosphonates altered the migration of THP-1 cells dose-dependently. Low doses also prolonged migration and altered cell morphology. These findings support the idea of a disturbed local immune function of macrophages even in jaw bone exposed to low concentrations of bisphosphonate. CLINICAL RELEVANCE: These are the first real-time results for disrupted migration and function of macrophagic THP-1 cells in high doses. Low dosages also demonstrated altered macrophage phagocytosis and cell morphology, suggesting a disturbed local immune function in BRONJ pathogenesis.

Cytomegalovirus-associated gastric ulcer: a side effect of steroid injections for pyloric stenosis.

The local injection of triamcinolone acetonide (TA) is effective in preventing pyloric stenosis and deformity following large endoscopic submucosal dissection (ESD). However, because of its long-acting nature, TA can induce long-term local immunosuppression and subsequent adverse events. We report a case of a cytomegalovirus (CMV) ulcer that formed only at the TA local injection site. A 68-year-old man underwent ESD to treat early gastric cancer that formed over the pylorus. The lesion extended to the duodenum, and an artificial ulcer covered more than two-thirds of the circumference of the pylorus. To prevent pyloric stenosis, TA was locally injected into the ulcer floor. On day 12, a deeper ulcer 10 mm in diameter was discovered in the center of the post-ESD ulcer. Biopsies revealed large cells with intranuclear inclusion bodies, which stained positive for the anti-CMV antibody. Local TA injections are useful, however, CMV ulcer might occur as adverse events.