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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. 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 porcine and human peripheral neutrophils following incubation with tacrolimus. 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, NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus. 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.

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.

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.

Grafting of microencapsulated pancreatic islets has been proposed as an alternative to exogenous insulin for the treatment of type 1 diabetes mellitus. Microencapsulated islets are protected from direct contact with immune cells and larger immune‐active molecules such as immunoglobulins. Unfortunately, many islet cells in the microcapsules are lost in the immediate period after transplantation due to an early host immune response limiting long‐term function of the graft. Gusperimus has shown to reduce the inflammatory responses to grafted encapsulated islets, but it cannot be appropriately used because it is easily hydrolyzed leading to loss of activity. To temporarily modulate the inflammatory response directly after implantation and stabilize gusperimus, squalene‐gusperimus nanoparticles (Sq‐GusNPs) are developed and incorporated in human islets‐containing alginate‐based microcapsules. A prolonged and continuous release of gusperimus is achieved. This offers an anti‐inflammatory microenvironment in the vicinity of the microcapsules and a reduction of cytokine secretion by lipopolysaccharides‐activated human macrophages. Release of gusperimus from Sq‐GusNPs does not affect the in vitro viability or function of human pancreatic islets. The data illustrate that incorporation of Sq‐GusNPs in alginate microcapsules offers an opportunity to temporarily modulate the immediate immune response after the grafting procedure of encapsulated islets cells and reduce loss of islet cells. Incorporation of squalene‐gusperimus nanoparticles in alginate microcapsules allows a controlled and prolonged release of gusperimus. These nanoparticles proportionate an anti‐inflammatory microenvironment in the vicinity of the microcapsules and to the encapsulated islets without affecting their viability or function.

Objectives Bisphosphonates and denosumab are antiresorptive drugs used for the treatment of osteoporosis and oncological tumors. A severe side effect is osteonecrosis of the jaw. Monocyte/macrophage dysfunction is considered to play a distinct role in osteonecrosis. THP-1 monocytic cells were used in this study to elucidate the influence of zoledronate and denosumab on phorbol-12-myrisate-13-acetate (PMA)-induced macrophage differentiation and function in real-time. Materials and methods Macrophagic differentiation of the THP-1 suspension cells was measured by cell adherence in the presence or absence of different concentrations of zoledronate (0.5, 5, 50 μM) and denosumab (1, 10, 20, 40 μg/mL) using the real-time xCELLigence system. Additionally, a live/dead staining was performed by fluorescence microscopy. Results THP-1 cells demonstrated a regular initial PMA-induced differentiation to macrophages by live measurements of cell adherence and by an increase in CD68 surface expression as detected by flow cytometry. The addition of zoledronate led to cell detachment of the THP-1-derived macrophages in a dose-dependent manner in contrast to denosumab. Cell detachment was based on cell death as confirmed by live/dead staining, revealing elevated numbers of dead cells following addition of high zoledronate concentrations. However, denosumab did not deteriorate THP-1 cell viability. Conclusion Our results demonstrate that zoledronate but not denosumab suppresses monocytic THP-1 cell viability after macrophagic differentiation dose-dependently. Clinical relevance This is the first real-time study providing evidence for a dose-dependent immunosuppressive effect of zoledronate in contrast to denosumab on local macrophages.

Background Currently, successful transplantation of allografts requires the systemic use of immunosuppressive drugs. These can cause serious morbidity due to toxicity and increased susceptibility to cancer and infections. Local production of immunosuppressive molecules limited to the graft site would reduce the need for conventional, generalized immunosuppressive therapies and thus educe fewer side effects. This is particularly salient in a disease like type 1 diabetes, which is not immediately life‐threatening yet islet allografts can effect a cure. Methods We studied the efficacy of locally produced anti‐CD4 antibody, mediated by adenovirus (Adv‐anti‐CD4) transduction of islets, to enhance allograft survival. Adenovirus‐transduced islets were transplanted under the kidney capsule of diabetic recipients and graft rejection determined by monitoring blood glucose levels. Results Adv‐anti‐CD4 transduction of mouse islets afforded protection against allogeneic rejection after transplantation into fully mismatched recipients. In some recipients, the islet allograft survival was prolonged (persisting for at least 15 weeks), corresponding to the prolonged expression of the anti‐CD4 antibody. The effect was local, as absence of CD4+ T lymphocytes was observed primarily at the graft site. Conclusions Immunosuppressive effects can be restricted locally by our strategy. Local production of a single antibody against one subset of T lymphocytes can protect mouse islets from allograft rejection during transplantation to treat diabetes. Our findings foreshadow that this strategy may be even more effective when a combination of antibodies are used and that similar strategies may prevent xenograft rejection. Copyright © 2005 John Wiley & Sons, Ltd.

The purpose of this work was to study the local immunosuppressive effects of systemically administered methylprednisolone (MP) and its prodrug, dextran-methylprednisolone (DMP), in rat livers. Single 5 mg/kg (MP equivalent) doses of MP or DMP were injected intravenously to rats, and livers were isolated at different time points (0-72 h; n = 4/time point). Isolated livers were stimulated ex vivo with bacterial lipopolysaccharide, and outlet perfusate and bile samples were analyzed for their concentrations of tumor necrosis factor (TNF)-alpha by enzyme-linked immunosorbent assay. The area under the perfusate TNF-alpha concentration-time curve (AUC) was used as a measure of immune response. Hepatic concentrations of MP and DMP were also measured by high-performance liquid chromatography. Both MP and DMP resulted in a decrease in lipopolysaccharide-induced increase in TNF-alpha AUC. MP injection 8 h before liver isolation resulted in a maximum of 50% decrease in TNF-alpha AUC. Compared with MP, the maximum effect of the prodrug (DMP) was both more intense (approximately 80% reduction in TNF-alpha AUC) and delayed (maximum inhibition at 24 h). Overall, the area under the effect (% inhibition of TNF-alpha)-time (%inhibition-h) for DMP (3,680 +/- 406) was approximately four times more than that for the parent drug (846 +/- 114). Whereas the MP concentrations in the liver were not quantifiable after the injection of the parent drug, relatively large concentrations of DMP and regenerated MP were found in the liver of DMP-injected rats. After systemic administration to rats, both MP and DMP exhibit local immunosuppressive effects in the liver. The local effects of the prodrug (DMP), however, appear to be more intense and sustained than those of the parent drug (MP).

Early pregnancy factor (EPF), an extracellular chaperonin 10 homologue, has immunosuppressive and growth factor properties. In order to carry out more extensive studies on the in vivo characteristics of EPF, a recombinant form of the molecule has been prepared. Recombinant human EPF (rEPF) was expressed in Escherichia coli using the plasmid pGEX‐2T expression system. Potency of rEPF in vitro in the rosette inhibition test, the bioassay for EPF, was equivalent to that of native EPF (nEPF), purified from human platelets, and synthetic EPF (sEPF). However, the half‐life of activity (50% decrease in the log value) in serum, following i.p. injection, was significantly decreased (3.2 h, compared with nEPF 6.2 days, sEPF 5.8 days). This was thought to be due to modification of the N‐terminus of the recombinant molecule inhibiting binding to serum carrier proteins. Because EPF can modify Th1 responses, the ability of the recombinant molecule to suppress allogeneic graft rejection was investigated. Following skin grafts from Lewis rats to DA rats and vice versa, rEPF was delivered locally at the graft site and the effect on survival time of the allografts noted. Results demonstrated that rEPF treatment significantly prolonged skin graft survival time by as much as 55% in stringent models of transplantation across major histocompatibility barriers.

Dry eye disease is a very common multifactorial disease of the lacrimal functional unit that results in tear film instability, hyperosmolarity, chronic irritation and inflammation of the ocular surface. Diagnostic tools have been developing rapidly, however, both classic dry eye tests (Schirmer I test, fluorescein staining of the surface epithelium and tear film break-up time) and noninvasive imaging techniques are essential for an exact diagnosis. The management of dry eye syndrome can be either conservative or invasive based on the severity of the disease. The basic aim of treatment is to improve quality of life and reduce subjective complaints and objective ocular surface alterations in dry eye patients. The first line of treatment is tear substitution with artificial tear drops, gels and ointments. In moderate cases preservative-free tear supplementation, topical anti-inflammatory therapy and retinol treatment is recommended. Temporary or permanent punctal plug occlusion, therapeutic contact lenses or moisture chamber use constitute other options. In severe cases the application of topical autologous serum, systemic anti-inflammatory therapy, androgen substitution, secretagogues and surgical intervention can be effective. In the future, noninvasive diagnostic tools and instruments such as screening methods are likely to be developed. In addition, causal therapy of the dry eye will play a greater role, including cyclosporine therapy, secretion stimulation, growth factor-containing artificial tears, as well as secratogogues, immunomodulants and androgenic complexes for severe forms of the disease.