Neutrophil extracellular traps and citrullinated fibrinogen contribute to injury in a porcine model of limb ischemia and reperfusion.

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.

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.

Infección por Sporothrix schenckii en agricultores del caserío "Peñón de Gabante": Municipio Tovar. Estado Aragua. Venezuela / Infection caused by Sporothrix schenckii in farmers from the village "Peñón de Gabante": Tovar municipality. Aragua state. Venezuela

In Aragua state (Venezuela) there are farming communities such as the Tovar municipality that meet geographical and climatic conditions favourable for the development of the fungus Sporothrix schenckii, in which farmer workers would be particularly exposed and at risk of infection and development of sporotrichosis. Accordingly, it was determined the prevalence of this infection in 148 farmers of both sexes from the village "Peñón de Gabante" through the intradermoreaction to esporotrichin. The overall prevalence was 24% and the frequency of infection increased with the age of the farmers, the time working as farmers and it was higher in men (27.6%) than in women (20.8%), reaching values of 39.1% in persons of 50 years and older and 28% in those with more than 10 years occupied in agriculture, although it was independent of these variables. Also, it was found a lower percentage of intradermal positivity (21.8%) in farmers that reported regular use of protective equipment in compare to those whom used it irregularly (27.9%) even though the differences weren't statistically significant. Statistically significant association (p< 0.005) was found between the clinical signs referred by farmers and the intradermopositivity to the esporotrichin, direct contact with plants with thorny branches and/or fruits of prickly shell (p< 0.001) and direct contact with the soil during the process of preparation, fertilization and sowing the crops (p<0.005). These findings suggest that the studied area is endemic to Sporothrix schenckii and farmers found in occupational risk of acquiring this infection.

En el estado Aragua, Venezuela, existen comunidades agrícolas como las del municipio Tovar, que reúnen condiciones geográficas y climáticas favorables para el desarrollo del hongo Sporothrix schenckii, en las que los agricultores estarían particularmente expuestos y a riesgo de adquirir la infección y desarrollar esporotricosis. En conse cuencia, se determinó la prevalencia de esta infección en 148 agricultores de ambos sexos del caserío "Peñón de Gabante", mediante la intradermorreacción a la esporotriquina. La prevalencia fue 24% y la frecuencia de infección incrementó con la edad de los agricultores, el tiempo desarrollando esta labor y fue mayor en hombres (27,6%) que en mujeres (20,8%), alcanzando valores de 39,1% en las personas con edades de 50 años o más y de 28% en aquellos con más de 10 años laborando en la agricultura, aunque resultó independiente de estas variables. Se halló un menor porcentaje de positividad (21,8%) en los agricultores que utilizan en forma regular equipos de protección en comparación con aquellos que lo hacen irregularmente (27,9%), aunque las diferencias no fueron estadísticamente significativas. Se encontró asociación estadísticamente significativa entre la intradermopositividad a la esporotriquina y los signos clínicos relacionados a la esporotricosis (p< 0,005), el contacto directo tanto con plantas de ramas espinosas y/o frutos de cáscara espinosa (p< 0,001) y con el contacto directo con la tierra durante el proceso de preparación, abono y/o siembra de sus cultivos (p< 0,005). Estos hallazgos sugieren que la zona estudiada es endémica para Sporothrix schenckii y los agricultores se encuentran en riesgo ocupacional de contraer esta infección.