Recurrence of hepatocellular carcinoma after liver transplantation: Prognostic and predictive factors of survival in a Latin American cohort.

BACKGROUND & AIM: Recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT) has a poor prognosis, and the adjusted effect of different treatments on post-recurrence survival (PRS) has not been well defined. This study aims to evaluate prognostic and predictive variables associated with PRS. METHODS: This Latin American multicenter retrospective cohort study included HCC patients who underwent LT between the years 2005-2018. We evaluated the effect of baseline characteristics at time of HCC recurrence diagnosis and PRS (Cox regression analysis). Early recurrences were those occurring within 12 months of LT. To evaluate the adjusted treatment effect for HCC recurrence, a propensity score matching analysis was performed to assess the probability of having received any specific treatment for recurrence. RESULTS: From a total of 1085 transplanted HCC patients, the cumulative incidence of recurrence was 16.6% (CI 13.5-20.3), with median time to recurrence of 13.0 months (IQR 6.0-26.0). Factors independently associated with PRS were early recurrence (47.6%), treatment with sorafenib and surgery/trans-arterial chemoembolization (TACE). Patients who underwent any treatment presented "early recurrences" less frequently, and more extrahepatic metastasis. This unbalanced distribution was included in the propensity score matching, with correct calibration and discrimination (receiving operator curve of 0.81 [CI 0.72;0.88]). After matching, the adjusted effect on PRS for any treatment was HR of 0.2 (0.10;0.33); P < .0001, for sorafenib therapy HR of 0.4 (0.27;0.77); P = .003, and for surgery/TACE HR of 0.4 (0.18;0.78); P = .009. CONCLUSION: Although early recurrence was associated with worse outcome, even in this population, systemic or locoregional treatments were associated with better PRS.

Tacrolimus-loaded Drug Delivery Systems in Vascularized Composite Allotransplantation: Lessons and Opportunities for Local Immunosuppression.

Long-term systemic immunosuppression is needed for vascularized composite allotransplantation (VCA). The high rate of acute rejection episodes in the first posttransplant year, the development of chronic rejection, and the adverse effects that come along with this treatment, currently prevent a wider clinical application of VCA. Opportunistic infections and metabolic disturbances are among the most observed side effects in VCA recipients. To overcome these challenges, local immunosuppression using biomaterial-based drug delivery systems (DDS) have been developed. The aim of these systems is to provide high local concentrations of immunosuppressive drugs while reducing their systemic load. This review provides a summary of recently investigated local DDS with different mechanisms of action such as on-demand, ultrasound-sensitive, or continuous drug delivery. In preclinical models, ranging from rodent to porcine and nonhuman primate models, this approach has been shown to reduce systemic tacrolimus (TAC) load and adverse effects, while prolonging graft survival. Localized immunosuppression using biomaterial-based DDS represents an encouraging approach to enhance graft survival and reduce toxic side effects of immunosuppressive drugs in VCA patients. Preclinical models using TAC-releasing DDS have demonstrated high local immunosuppressive effects with a low systemic burden. However, to reduce acute rejection events in translational animal models or in the clinical reality, the use of additional low-dose systemic TAC treatment may be envisaged. Patients may benefit through efficient graft immunosuppression and survival with negligible systemic adverse effects, resulting in better compliance and quality of life.

Immune Rejection of Cartilage in a Swine Vascularized Composite Allotransplantation Model.

BACKGROUND: Cartilage is a crucial tissue in vascularized composite allotransplantation (VCA) and plays a pivotal role in restoring motor function, especially in joint allotransplantation. Nevertheless, our understanding of immune rejection in cartilage remains limited and contentious. This study seeks to investigate the immune rejection of cartilage in a large animal model of VCA. METHODS: Cartilage, including articular cartilage and meniscus, as well as skin, muscle and lymph node, was retrieved from a swine heterotopic VCA graft when the skin of the graft suffered from grade III-IV rejection. Histologic examination, transmission electron microscopy and immunofluorescent staining were used to investigate immune rejection. RESULTS: Histologic examination revealed the infiltration of inflammatory cells and tissue destruction in cartilage. Transmission electron microscopy confirmed tissue damage and necrosis in cartilage. However, cartilage exhibited milder tissue damage when compared to rejected skin and muscle. Immunofluorescent staining revealed the activation of both the innate and adaptive immune systems, accompanied by an up-regulation of cell death biomarkers, including apoptosis and pyroptosis, in the rejected cartilage. CONCLUSION: Our study demonstrates that cartilage is not immunologically privileged and undergoes immune rejection concurrently with skin and muscle in the VCA graft, though with less severe inflammation and rejection.

Transcriptome profiling of immune rejection mechanisms in a porcine vascularized composite allotransplantation model.

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.

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.

Prolongation of the Time Window From Traumatic Limb Amputation to Replantation From 6 to 33 Hours Using Ex Vivo Limb Perfusion.

INTRODUCTION: Continuous extracorporeal perfusion (ECP), or machine perfusion, holds promise for prolonged skeletal muscle preservation in limb ischemia-reperfusion injury. This study aimed to extend the amputation-to-replantation time window from currently 6 hours to 33 hours using a 24-hour ECP approach. MATERIALS AND METHODS: Six large white pigs underwent surgical forelimb amputation under general anesthesia. After amputation, limbs were kept for 9 hours at room temperature and then perfused by 24-hour ECP with a modified histidine-tryptophan-ketoglutarate (HTK) solution. After ECP, limbs were orthotopically replanted and perfused in vivo for 12 hours. Clinical data, blood, and tissue samples were collected and analyzed. RESULTS: All 6 forelimbs could be successfully replanted and in vivo reperfused for 12 hours after 9 hours of room temperature ischemia followed by 24 hours ECP. Adequate limb perfusion was observed after replantation as shown by thermography and laser Doppler imaging. All pigs survived without severe organ failure, and no significant increase in inflammatory cytokines was found. Macroscopy and histology showed marked interstitial muscular edema of the limbs, whereas myofiber necrosis was not evident, implying the preservation of muscular integrity. CONCLUSIONS: The use of a 24-hour ECP has successfully extended limb preservation to 33 hours. The modified histidine-tryptophan-ketoglutarate perfusate demonstrated its ability for muscle protection. This innovative approach not only facilitates limb replantation after combat injuries, surmounting geographical barriers, but also broadens the prospects for well-matched limb allotransplants across countries and continents.