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Background: Ischemia/reperfusion injury (IRI) is a complex pathological process, triggered by the restoration of blood flow following an interrupted blood supply. While restoring the blood flow is the only option to salvage the ischemic tissue, reperfusion after a prolonged period of ischemia initiates IRI, triggering a cascade of inflammatory responses ultimately leading to neutrophil recruitment to the inflamed tissue, where they release neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin and neutrophilic proteins, including peptidyl-arginine deiminase 2 and 4 (PAD2, PAD4), that, once outside, can citrullinate plasma proteins, irreversibly changing their conformation and potentially their function. While the involvement of NETs in IRI is known mainly from rodent models, we aimed to determine the effect of NET formation and especially PADs-mediated extracellular protein citrullination in a porcine model of limb IRI. Methods: We conducted our study on amputated pig forelimbs exposed to 1 h or 9 h of ischemia and then reperfused in vivo for 12 h. Limb weight, edema formation, compartmental pressure were measured, and skeletal muscle was analyzed by immunofluorescence (TUNEL assay and dystrophin staining) to evaluate tissue damage. Fibrin tissue deposition, complement deposition and NETs were investigated by immunofluorescence. Citrullinated plasma proteins were immunoprecipitated and citrullinated fibrinogen was identified in the plasma by Western blot and in the tissue by immunofluorescence and Western blot. Results: Our data consolidate the involvement of NETs in a porcine model of limb IRI, correlating their contribution to damage extension with the duration of the ischemic time. We found a massive infiltration of NETs in the group subjected to 9 h ischemia compared to the 1 h and citrullinated fibrinogen levels, in plasma and tissue, were higher in 9 h ischemia group. We propose fibrinogen citrullination as one of the mechanisms contributing to the worsening of IRI. NETs and protein citrullination represent a potential therapeutic target, but approaches are still a matter of debate. Here we introduce the idea of therapeutic approaches against citrullination to specifically inhibit PADs extracellularly, avoiding the downstream effects of hypercitrullination and keeping PADs' and NETs' intracellular regulatory functions. [ABSTRACT FROM AUTHOR]

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 Tcellular 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. [ABSTRACT FROM AUTHOR]

Background: Vascularized composite allotransplantation (VCA) offers the potential for a biological, functional reconstruction in individuals with limb loss or facial disfigurement. Yet, it faces substantial challenges due to heightened immune rejection rates compared to solid organ transplants. A deep understanding of the genetic and immunological drivers of VCA rejection is essential to improve VCA outcomes Methods: Heterotopic porcine hindlimb VCA models were established and followed until reaching the endpoint. Skin and muscle samples were obtained from VCA transplant recipient pigs for histological assessments and RNA sequencing analysis. The rejection groups included recipients with moderate pathological rejection, treated locally with tacrolimus encapsulated in triglycerol-monostearate gel (TGMS-TAC), as well as recipients with severe end-stage rejection presenting evident necrosis. Healthy donor tissue served as controls. Bioinformatics analysis, immunofluorescence, and electron microscopy were utilized to examine gene expression patterns and the expression of immune response markers. Results: Our comprehensive analyses encompassed differentially expressed genes, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathways, spanning various composite tissues including skin and muscle, in comparison to the healthy control group. The analysis revealed a consistency and reproducibility in alignment with the pathological rejection grading. Genes and pathways associated with innate immunity, notably pattern recognition receptors (PRRs), damage-associated molecular patterns (DAMPs), and antigen processing and presentation pathways, exhibited upregulation in the VCA rejection groups compared to the healthy controls. Our investigation identified significant shifts in gene expression related to cytokines, chemokines, complement pathways, and diverse immune cell types, with CD8 T cells and macrophages notably enriched in the VCA rejection tissues. Mechanisms of cell death, such as apoptosis, necroptosis and ferroptosis were observed and coexisted in rejected tissues. Conclusion: Our study provides insights into the genetic profile of tissue rejection in the porcine VCA model. We comprehensively analyze the molecular landscape of immune rejection mechanisms, from innate immunity activation to critical stages such as antigen recognition, cytotoxic rejection, and cell death. This research advances our understanding of graft rejection mechanisms and offers potential for improving diagnostic and therapeutic strategies to enhance the long-term success of VCA. [ABSTRACT FROM AUTHOR]

Osteomyelitis is probably the oldest known infection in the history of life. It can develop secondary to local tissue disruption, ischemia and associated chronic wounds or via hematogenous infection. Although it has been known to the medical community for a long time, treatment remains challenging. Detection of the microbial agent remains crucial for the associated antibiotic therapy. Also, tissue specimens for culture and histology must be obtained. Several factors such as biofilm formation, resistance development and special virulence factors can impede the efficiency of the antibiotic treatment. In the last two decades, developments of antibiotic agents with available data in the field of osteomyelitis primarily include brilacidin, ceftaroline, ceftobiprole, dalbavancin, daptomycin, tedizolid, telavancin, tigecycline. Many of them are not on the market, or under study, or only found in selected countries. However, they are expected to become more accessible in coming years. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Arenas Hoyos, Helmer, Yerly, Lese, Hirsiger, Zhang, Casoni, Garcia, Petrucci, Hammer, Duckova, Banz, Montani, Constantinescu, Vögelin, Bordon, Aleandri, Prost, Taddeo, Luciani, Rieben, Sorvillo and Olariu.)

Background: Surgical planning of corrective osteotomies is traditionally based on conventional radiographs and clinical findings. In the past 10 years, 3-dimensional (3D) preoperative planning approaches with patient-specific guides have been developed. However, the application of this technology to posttraumatic deformities of the metacarpals and phalangeal bones has not yet been investigated. Our goal was to evaluate the feasibility of the surgical application to the latter and to evaluate the extent and precision of correction., Methods: We present results of 6 patients (8 osteotomies) treated with phalangeal or metacarpal corrective osteotomy. Deformities were located in the third ray in 1, fourth ray in 3, and fifth ray in 4 cases. Six malunited metacarpal bones (1 intra-articular) and 2 deformed proximal phalanges were treated. Computer-based 3D preoperative planning using the contralateral hand as a template allowed the production of 3D-printed patient-specific guides that were used intraoperatively for navigation. The precision of the reduction was assessed using pre- and postoperative computed tomography by comparing the postoperative bone model with the preoperatively simulated osteotomy. Range of motion and grip strength were documented pre- and postoperatively., Results: The mean follow-up time was 6 months (range: 5-11 months). Rotational deformity was reduced from a mean of 10.0° (range: 7.2°-19.3°) preoperatively to 2.3° (range: 0.7°-3.7°) postoperatively, and translational incongruency decreased from a mean of 1.4 mm (range: 0.7-2.8 mm) to 0.4 mm (range: 0.1-0.9 mm)., Conclusion: Preliminary results indicate that a precise reduction for corrective osteotomies of metacarpal and phalangeal bones can be achieved by using 3D planning and patient-specific guides.