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.

Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation.

Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients.

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype.

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.

Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2.

Immunization with vesicular stomatitis virus (VSV)-vectored COVID-19 vaccine candidates expressing the SARS-CoV-2 spike protein in place of the VSV glycoprotein relies implicitly on expression of the ACE2 receptor at the muscular injection site. Here, we report that such a viral vector vaccine did not induce protective immunity following intramuscular immunization of K18-hACE2 transgenic mice. However, when the viral vector was trans-complemented with the VSV glycoprotein, intramuscular immunization resulted in high titers of spike-specific neutralizing antibodies. The vaccinated animals were fully protected following infection with a lethal dose of SARS-CoV-2-SD614G via the nasal route, and partially protected if challenged with the SARS-CoV-2Delta variant. While dissemination of the challenge virus to the brain was completely inhibited, replication in the lung with consequent lung pathology was not entirely controlled. Thus, intramuscular immunization was clearly enhanced by trans-complementation of the VSV-vectored vaccines by the VSV glycoprotein and led to protection from COVID-19, although not achieving sterilizing immunity.

Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta.

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.

Presence of Donor Lymph Nodes Within Vascularized Composite Allotransplantation Ameliorates VEGF-C-mediated Lymphangiogenesis and Delays the Onset of Acute Rejection.

BACKGROUND: The lymphatic system plays an active role in modulating inflammation in autoimmune diseases and organ rejection. In this work, we hypothesized that the transfer of donor lymph node (LN) might be used to promote lymphangiogenesis and influence rejection in vascularized composite allotransplantation (VCA). METHODS: Hindlimb transplantations were performed in which (1) recipient rats received VCA containing donor LN (D:LN+), (2) recipient rats received VCA depleted of all donor LN (D:LN-), and (3) D:LN+ transplantations were followed by lymphangiogenesis inhibition using a vascular endothelial growth factor receptor-3 (VEGFR3) blocker. RESULTS: Our data show that graft rejection started significantly later in D:LN+ transplanted rats as compared to the D:LN- group. Moreover, we observed a higher level of VEGF-C and a quicker and more efficient lymphangiogenesis in the D:LN+ group as compared to the D:LN- group. The presence of donor LN within the graft was associated with reduced immunoactivation in the draining LN and increased frequency of circulating and skin-resident donor T regulatory cells. Blocking of the VEGF-C pathway using a VEGFR3 blocker disrupts the lymphangiogenesis process, accelerates rejection onset, and interferes with donor T-cell migration. CONCLUSIONS: This study demonstrates that VCA LNs play a pivotal role in the regulation of graft rejection and underlines the potential of specifically targeting the LN component of a VCA to control graft rejection.

Mixed Metal Oxide Nanoparticle Formulations for the Treatment of Seroma.

Seroma formation is a well-recognized postoperative complication for many plastic and general surgical procedures. Although various tissue adhesives and substances have been used in an effort to treat seroma formation, no therapies have been established clinically. Recently, the nano-bridging phenomenon has been introduced as a promising approach to achieve tissue adhesion and strong closure of deep skin wounds in rats. The present study seeks to assess the potential of nano-bridging beyond skin wounds in a rat model of seroma. Seromas were induced in 20 Lewis rats through bilateral axillary lymphadenectomy, excision of the latissimus dorsi and cutaneous maximus muscles, and disruption of dermal lymphatics. On postoperative day (POD) 7, the seroma was aspirated on both sides. A bioactive nanoparticle (NP) suspension based on zinc-doped strontium-substituted bioglass/ceria nanoparticles (NP group) or fibrin glue (fibrin group) was injected into the right seroma cavity, while the left side was left untreated. On POD 14, the NP group showed complete remission (no seromas at all), while the fibrin group recorded a reduction of only 63% in the seroma fluid volume. The NPs exerted local anti-inflammatory and neo-angiogenic effects, without any detectable systemic changes. Moreover, the ceria levels recorded in the organs did not surpass the background level, indicating that the nanoparticles stayed at the site of application. This study is a promising first example demonstrating the ability of inorganic nanoparticle formulations to reduce seroma formation in a rat model, without any detectable systemic adverse effects. These results emphasize the potential of nanotechnological solutions in the therapeutic management of seroma in the clinical setting.

The specific features of the developing T cell compartment of the neonatal lung are a determinant of respiratory syncytial virus immunopathogenesis.

The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the properties of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a translational model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4+ and CD8+ T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while γδ T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity.

SARS-CoV-2 spike D614G change enhances replication and transmission.

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.

Serpinb1a Is Dispensable for the Development and Cytokine Response of Invariant Natural Killer T Cell Subsets.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes. They quickly respond to antigenic stimulation by producing copious amounts of cytokines and chemokines. iNKT precursors differentiate into three subsets iNKT1, iNKT2, and iNKT17 with specific cytokine production signatures. While key transcription factors drive subset differentiation, factors that regulate iNKT subset homeostasis remain incompletely defined. Transcriptomic analyses of thymic iNKT subsets indicate that Serpinb1a is one of the most specific transcripts for iNKT17 cells suggesting that iNKT cell maintenance and function may be regulated by Serpinb1a. Serpinb1a is a major survival factor in neutrophils and prevents cell death in a cell-autonomous manner. It also controls inflammation in models of bacterial and viral infection as well as in LPS-driven inflammation. Here, we examined the iNKT subsets in neutropenic Serpinb1a-/- mice as well as in Serpinb1a-/- mice with normal neutrophil counts due to transgenic re-expression of SERPINB1 in neutrophils. In steady state, we found no significant effect of Serpinb1a-deficiency on the proliferation and numbers of iNKT subsets in thymus, lymph nodes, lung, liver and spleen. Following systemic activation with α-galactosylceramide, the prototypic glycolipid agonist of iNKT cells, we observed similar serum levels of IFN-γ and IL-4 between genotypes. Moreover, splenic dendritic cells showed normal upregulation of maturation markers following iNKT cell activation with α-galactosylceramide. Finally, lung instillation of α-galactosylceramide induced a similar recruitment of neutrophils and production of iNKT-derived cytokines IL-17, IFN-γ, and IL-4 in wild-type and Serpinb1a-/- mice. Taken together, our results indicate that Serpinb1a, while dominantly expressed in iNKT17 cells, is not essential for iNKT cell homeostasis, subset differentiation and cytokine release.

SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility.

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic 1 . However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro , it provides a real competitive advantage in vivo , particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

Development of vascularized nerve scaffold using perfusion-decellularization and recellularization.

INTRODUCTION: Vascularized nerve grafts (VNG) may offer an advantage in peripheral nerve regeneration by avoiding ischemic damage and central necrosis observed in non-VNG, particularly for the treatment of large and long nerve defects. However, surgical complexity, donor site morbidity and limited nerve availability remain important drawbacks for the clinical use of VNG. Here we explore the potential of perfusion-decellularization for bioengineering a VNG to be used in peripheral nerve reconstruction. METHODS: Porcine sciatic nerves were surgically procured along with their vascular pedicle attached. The specimens were decellularized via perfusion-decellularization and preservation of the extracellular matrix (ECM), vascular patency and tissue cytokine contents were examined. Scaffold reendothelialization was conducted with porcine aortic endothelial cells in a perfusion-bioreactor. RESULTS: Morphologic examination of decellularized VNG and analysis of the DNA content demonstrated cell clearance whereas ECM content and structures of the nerve fascicles were preserved. Using 3D micro-computed tomography imaging we observed optimal vasculature preservation in decellularized scaffolds, down to the capillary level. Cytokine quantification demonstrated measurable levels of growth factors after decellularization. Endothelial cell engraftment of the large caliber vessels was observed in reendothelialized scaffolds. CONCLUSIONS: In this study we provide evidence that perfusion-decellularization can be used to create vascularized nerve scaffolds in which the vasculature and the ECM component are well preserved. As compared to non-vascularized conduits, engineered vascularized nerve scaffolds may represent an ideal approach for promoting better nerve regeneration in larger nerve defect reconstructions.

Stromal Cell-Contact Dependent PI3K and APRIL Induced NF-κB Signaling Prevent Mitochondrial- and ER Stress Induced Death of Memory Plasma Cells.

The persistence of long-lived memory plasma cells in the bone marrow depends on survival factors available in the bone marrow, which are provided in niches organized by stromal cells. Using an ex vivo system in which we supply the known survival signals, direct cell contact to stromal cells, and the soluble cytokine a proliferation-inducing ligand (APRIL), we have elucidated the critical signaling pathways required for the survival of long-lived plasma cells. Integrin-mediated contact of bone marrow plasma cells with stromal cells activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway, leading to critical inactivation of Forkhead-Box-Protein O1/3 (FoxO1/3) and preventing the activation of mitochondrial stress-associated effector caspases 3 and 7. Accordingly, inhibition of PI3K signaling in vivo ablates bone marrow plasma cells. APRIL signaling, by the nuclear factor κB (NF-κB) pathway, blocks activation of the endoplasmic-reticulum-stress-associated initiator caspase 12. Thus, stromal-cell-contact-induced PI3K and APRIL-induced NF-κB signaling provide the necessary and complementary signals to maintain bone marrow memory plasma cells.

Regional Lymphatic Inclusion in Orthotopic Hindlimb Transplantation: Establishment and Assessment of Feasibility in a Rodent Model.

BACKGROUND: The lymphatic system may play an important role in local immunomodulation in vascularized composite allotransplantation (VCA). Currently, there is no standardized VCA model that includes the regional draining lymphatic tissue. The aim of this study was to develop a rapid and efficient orthotopic hindlimb transplantation model in rats that included the draining lymphatic basin to permit further evaluation of the lymphatic system's role in VCA. METHODS: Thirty transplantations from Brown Norway rats to Lewis rats were performed. To include the regional lymphatic tissue, the superficial epigastric vessels were preserved to allow retrieval of the corresponding inguinal lymph nodes, including the inguinal fat pad, with the hindlimb. A cuff technique was used for the vein, whereas the conventional microsurgical technique was used for the arterial anastomosis. Vascular patency was confirmed through laser Doppler analysis at postoperative day 1 and histological analysis after euthanasia. RESULTS: The presence and vascularization of the inguinal lymph nodes were verified with indocyanine green lymphoscintigraphy at the time of transplantation. Mean total ischemia time was 69 ± 24 minutes, and mean recipient operation time was 80 ± 19 minutes. Overall transplant survival rate was 93.3%. Laser Doppler analysis showed vascular (technical) success, indocyanine green lymphoscintigraphy confirmed the presence of lymph nodes and the histological analysis revealed patent anastomoses. CONCLUSIONS: We successfully developed an experimental orthotopic hindlimb transplantation model in rats that includes the draining inguinal lymphatic basin, which is an important asset in further research on lymphatic tissue and its role in VCA.

Point-of-Care Therapeutic Drug Monitoring for Precision Dosing of Immunosuppressive Drugs.

BACKGROUND: Immunosuppressive drugs (ISD) are an essential tool in the treatment of transplant rejection and immune-mediated diseases. Therapeutic drug monitoring (TDM) for determination of ISD concentrations in biological samples is an important instrument for dose personalization for improving efficacy while reducing side effects. While currently ISD concentration measurements are performed at specialized, centralized facilities, making the process complex and laborious for the patient, various innovative technical solutions have recently been proposed for bringing TDM to the point-of-care (POC). CONTENT: In this review, we evaluate current ISD-TDM and its value, limitations, and proposed implementations. Then, we discuss the potential of POC-TDM in the era of personalized medicine, and provide an updated review on the unmet needs and available technological solutions for the development of POC-TDM devices for ISD monitoring. Finally, we provide concrete suggestions for the generation of a meaningful and more patient-centric process for ISD monitoring. SUMMARY: POC-based ISD monitoring may improve clinical care by reducing turnaround time, by enabling more frequent measurements in order to obtain meaningful pharmacokinetic data (i.e., area under the curve) faster reaction in case of problems and by increasing patient convenience and compliance. The analysis of the ISD-TDM field prompts the evolution of POC testing toward the development of fully integrated platforms able to support clinical decision-making. We identify 4 major areas requiring careful combined implementation: patient usability, data meaningfulness, clinicians' acceptance, and cost-effectiveness.

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5 mg/kg) for 14 days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10 mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1 × 106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50 mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500 µL ip, POD 1, 5). Banff grade III or 120 days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28 days in CTRL (n = 7), 34 in ASC (n = 6), and 27.5 in ASC-CYP (n = 4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

Selective depletion of plasma cells in vivo based on the specificity of their secreted antibodies.

Antibody-mediated diseases affect more than 10% of the human population. For most, no cure is available, particularly when the pathogenic antibodies are secreted by long-lived plasma cells resistant to conventional immunosuppressive therapies. Current therapeutic approaches target not only the plasma cells that secrete pathogenic antibodies, but also those providing protective antibodies. Here, in a murine model bearing long-lived plasma cells secreting anti-OVA and -chicken gamma globulin (CGG) antibodies, we describe the first-time use of an antigen-antibody (OVA/anti-CD138 antibody) conjugate for in vivo labeling and selective ablation of plasma cells that secrete antibodies specific for the antigen OVA. The selective depletion also led to a stable reduction of the corresponding serum anti-OVA antibody levels. In contrast, CGG-specific plasma cells and circulating anti-CGG antibody levels remained unchanged. The method described here should enable the development of unique causative treatment strategies for established antibody-mediated diseases sparing humoral immunity.

Lymph node identification in skin malignancy using indocyanine green transcutaneously study: Study protocol for a diagnostic accuracy study.

INTRODUCTION: The incidence of malignant melanoma has been rising steadily over the past decades and Merkel cell carcinoma is a highly aggressive neuroendocrine skin tumor with a high mortality rate. Sentinel lymph node (SLN) biopsy is a recommended prognostic tool in primary cutaneous malignant melanomas of intermediate thickness and in all clinically node-negative Merkel cell carcinomas. The gold standard method for identification of SLNs is lymphoscintigraphy, which involves radioactive tracers. Indocyanine green-based near-infrared fluorescence imaging (NIRFI) has been also used for intraoperative SLN identification. We aim to evaluate the diagnostic sensitivity of the VisionSense VS3 NIRFI device for SNL identification. This device uses stereoscopic 3D high definition for both fluorescence and visible light imaging. Our hypothesis is that SLNs may be identified transcutaneously using fluorescent dye injections and NIRFI; therefore, obviating the need for lymphoscintigraphy in the future. METHODS AND ANALYSIS:: lymph node identification in skin malignancy using indocyanine green transcutaneously is a prospective diagnostic sensitivity study conducted at the Department of Plastic and Hand Surgery at the University Hospital Berne, Inselspital, Switzerland. The study aims at recruiting 93 patients (start date September 2017) to compare the accuracy of VisionSense VS3 camera at identifying SLNs transcutaneously with the current gold standard, lymphoscintigraphy. Moreover, a secondary objective is to determine if anatomical location of the SLN and patient factors (eg, body mass index, age) have an impact on the ability of VisionSense to detect SLNs when compared with the same gold standard. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03545334.

Delivery of Rapamycin Using In Situ Forming Implants Promotes Immunoregulation and Vascularized Composite Allograft Survival.

Vascularized composite allotransplantation (VCA), such as hand and face transplantation, is emerging as a potential solution in patients that suffered severe injuries. However, adverse effects of chronic high-dose immunosuppression regimens strongly limit the access to these procedures. In this study, we developed an in situ forming implant (ISFI) loaded with rapamycin to promote VCA acceptance. We hypothesized that the sustained delivery of low-dose rapamycin in proximity to the graft may promote graft survival and induce an immunoregulatory microenvironment, boosting the expansion of T regulatory cells (Treg). In vitro and in vivo analysis of rapamycin-loaded ISFI (Rapa-ISFI) showed sustained drug release with subtherapeutic systemic levels and persistent tissue levels. A single injection of Rapa-ISFI in the groin on the same side as a transplanted limb significantly prolonged VCA survival. Moreover, treatment with Rapa-ISFI increased the levels of multilineage mixed chimerism and the frequency of Treg both in the circulation and VCA-skin. Our study shows that Rapa-ISFI therapy represents a promising approach for minimizing immunosuppression, decreasing toxicity and increasing patient compliance. Importantly, the use of such a delivery system may favor the reprogramming of allogeneic responses towards a regulatory function in VCA and, potentially, in other transplants and inflammatory conditions.