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There is an urgent need to address the shortage of potential multivisceral grafts in order to reduce the average time in waiting list. Since donation after circulatory death (DCD) has been successfully employed for other solid organs, a thorough evaluation of the use of intestinal grafts from DCD is warranted. Here, we have generated a model of Maastricht III DCD in rodents, focusing on the viability of intestinal and multivisceral grafts at five (DCD5) and twenty (DCD20) minutes of cardiac arrest compared to living and brain death donors. DCD groups exhibited time-dependent damage. DCD20 generated substantial intestinal mucosal injury and decreased number of Goblet cells whereas grafts from DCD5 closely resemble those of brain death and living donors groups in terms intestinal morphology, expression of tight junction proteins and number of Paneth and Globet cells. Upon transplantation, intestines from DCD5 showed increased ischemia/reperfusion damage compared to living donor grafts, however mucosal integrity was recovered 48 h after transplantation. No differences in terms of graft rejection, gene expression and absorptive function between DCD5 and living donor were observed at 7 post-transplant days. Collectively, our results highlight DCD as a possible strategy to increase multivisceral donation and transplantation procedures.

Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by chronic, relapsing intestinal inflammation. Galectin-1 (Gal-1) is an endogenous lectin with key pro-resolving roles, including induction of T-cell apoptosis and secretion of immunosuppressive cytokines. Despite considerable progress, the relevance of Gal-1-induced T-cell death in inflamed tissue from human IBD patients has not been ascertained. Intestinal biopsies and surgical specimens from control patients (n = 52) and patients with active or inactive IBD (n = 97) were studied. Gal-1 expression was studied by RT-qPCR, immunoblotting, ELISA and immunohistochemistry. Gal-1-specific ligands and Gal-1-induced apoptosis of lamina propria (LP) T-cells were determined by TUNEL and flow cytometry. We found a transient expression of asialo core 1-O-glycans in LP T-cells from inflamed areas (p

Intestinal passenger T leukocytes are responsible for graft-versus-host disease (GvHD) in intestinal transplantation (ITx). We hypothesized that ex vivo fludarabine treatment of the bowel graft would diminish the risk of GvHD and improve overall survival post-transplant. We performed isolated heterotopic small bowel transplantations from Lewis (LEW) to Brown Norway (BN) rat strains, which generated GvHD signs from the fourth day post-transplant. These symptoms included rash, weight loss, piloerection, and diarrhea. The grafts of one of the experimental groups were immersed and sealed in cold Celsior preservation solution with 1000 µm fludarabine for 1 h, prior to its implantation into recipient animals. No histological signs of intestinal tissue alterations were observed after fludarabine treatment. Fludarabine-treated bowel recipients showed significantly later and milder clinical signs of GvHD and reduced total donor cell chimerism, as determined by flow cytometry using strain-specific anti-HLA antibodies. Additionally, fludarabine treatment prolonged recipients' overall survival (13.5 days ± 0.3 days vs. 9.2 days ± 0.5). We conclude that active modification of the intestinal leukocyte composition is advantageous in our ITx animal model. Immunosuppression with fludarabine during the surgical procedure, which could be translated directly to the clinic, protects bowel recipients from GvHD and improves overall post-transplant survival.

OBJECTIVE We aimed to assess whether native spleen preservation during VT affects GVHD incidence. SUMMARY BACKGROUND DATA Graft-versus-host-disease (GVHD) is one of the most severe and frequently lethal hematological complications after visceral transplantation (VT) procedures. Because there is no specific treatment for GVHD, it is imperative to develop a strategy to reduce donor lymphocyte engraftment and proliferation. METHODS Our study included both clinical and experimental data. A total of 108 patients were divided into three groups: a native spleen preservation group, a native spleen removal with no donor spleen group, and a donor spleen included (allogeneic spleen) group. We also used an allogeneic VT rat model, in which recipients were divided into two groups: a native spleen preservation (+SP) group and a native spleen removal (-S) group. Skin rash appearance, histopathological changes, chimerism, and spleen effects on circulating allogeneic T-cells were assessed. RESULTS The patients with native spleen preservation showed a lower rate of GVHD (p

Coronavirus disease 2019 (COVID-19) pandemic has become one of the most challenging episodes in the history of modern public health, with particular emphasis in high-risk population. However, the evidence regarding their response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent responsible for COVID-19 is scant. Herein, we present the clinical and therapeutic course of a SARS-CoV-2 infection in a patient with multivisceral transplant and a recent tuberculosis infection., Instituto de Estudios Inmunológicos y Fisiopatológicos

Background Two different mitochondrial fractions (MFs) have been characterized in the human placenta: the "light" and "heavy" fractions (LMF and HMF). Although these organelles are the main source of reactive oxygen species, an imbalance between their production and the rate of detoxification represents a serious threat to mitochondrial homeostasis and, in the case of the placenta, also to the fetus. The aim of this study was to evaluate the antioxidant capacity and susceptibility to oxidative stress in both types of MFs. Methods Human MFs were isolated from healthy donors (n = 11) and either incubated or not with H2 O2 . Catalase (CAT) activity, and reduced glutathione (GSH), lipid peroxidation (LP), and protein carbonylation (PC) levels were determined. Results H2 O2 treatment increased LP and PC levels and decreased CAT activity. GSH levels were similar in control and treated MFs. Conclusion H2 O2 caused oxidative damage in both LMF and HMF and the antioxidant system measured in these two MFs responded similarly. To the best of our knowledge, this is the first partial description of the antioxidant defense in placental HMF and LMF performed in a cell-free assay. The small number of antioxidant system parameters measured did not allow detecting differences between HMF and LMF.

Background: Galactomannan (GAL), a polysaccharide present on the cell wall of several fungi, has shown an ability to modulate inflammatory responses through the dectin-1 receptor in human macrophages. However, studies evaluating the modulatory properties of this polysaccharide in in vivo inflammatory scenarios are scarce. We hypothesized that GAL pretreatment would modulate local and remote damage related to intestinal reperfusion after an ischemic insult. Materials and methods: Adult male Balb/c mice were subjected to intestinal ischemia–reperfusion injury by reversible occlusion of the superior mesenteric artery, consisting of 45 min of ischemia followed by 3 or 24 h of reperfusion. Intragastric GAL (70 mg/kg) was administered 12 h before ischemia, and saline solution was used in the control animals. Jejunum, lung, and blood samples were taken for the analysis of histology, gene expression, plasma cytokine levels, and nitrosative stress. Results: Intestinal and lung histologic alterations were attenuated by GAL pretreatment, showing significant differences compared with nontreated animals. Interleukin 1β, monocyte chemoattractant protein 1, and IL-6 messenger RNA expression were considerably downregulated in the small intestine of the GAL group. In addition, GAL treatment significantly prevented plasma interleukin 6 and monocyte chemoattractant protein 1 upregulation and diminished nitrate and nitrite levels after 3 h of intestinal reperfusion. Conclusions: GAL pretreatment constitutes a novel and promising therapy to reduce local and remote damage triggered by intestinal ischemia–reperfusion injury. Further in vivo and in vitro studies to understand GAL's modulatory effects are warranted., Facultad de Ciencias Veterinarias, Instituto de Estudios Inmunológicos y Fisiopatológicos

Galectins play key roles in the inflammatory cascade. In this study, we aimed to analyze the effect of galectin-1 (Gal-1) in the function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro-inflammatory cytokines, and Gal-1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal-1 binding and modulation of IECs. We found an enhanced binding of Gal-1 to: (a) murine IECs exposed to IL-1β, TNF, and IL-13; (b) IECs from inflamed areas in intestinal tissue from IBD patients; (c) small bowel of allergic mice; and (d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal-1 favored a tolerogenic micro-environment, whereas high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase-9 and modulation of Bcl-2 protein family members. Our results showed that, when added in the presence of diverse pro-inflammatory cytokines such as tumor necrosis factor (TNF), IL-13 and IL-5, Gal-1 differentially promoted the secretion of growth factors including thymic stromal lymphopoietin (TSLP), epidermal growth factor (EGF), IL-10, IL-25, and transforming growth factor (TGF-β1 ). In conclusion, we found an augmented binding of Gal-1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal-1 concentration. These findings highlight the importance of the inflammatory micro-environment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal-1 and its role in epithelial cell homeostasis.

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions of the gastrointestinal tract including Crohn's disease (CD) and ulcerative colitis (UC). Galectins, defined by shared consensus amino acid sequence and affinity for β-galactosides, are critical modulators of the inflammatory response. However, the relevance of the galectin network in the pathogenesis of human IBD has not yet been explored. Here, we analyzed the expression of relevant members of the galectin family in intestinal biopsies, and identified their contribution as novel mucosal markers in IBD. Colonic biopsies were obtained from 59 IBD patients (22 CD and 37 UC), 9 patients with gut rejection after transplantation, 8 adult celiac patients, and 32 non-IBD donors. Galectin mRNA expression was analyzed by RT-PCR and qPCR using specific primers for individual galectins. A linear discriminant analysis (LDA) was used to analyze galectin expression in individual intestinal samples. Expression of common mucosal-associated galectins (Gal-1, -3, -4, -9) is dysregulated in inflamed tissues of IBD patients compared with non-inflamed IBD or control samples. LDA discriminated between different inflammation grades in active IBD and showed that remission IBD samples were clusterized with control samples. Galectin profiling could not distinguish CD and UC. Furthermore, inflamed IBD was discriminated from inflamed tissue of rejected gut in transplanted patients and duodenum of celiac patients, which could not be distinguished from control duodenum samples. The integrative analysis of galectins discriminated IBD from other intestinal inflammatory conditions and could be used as potential mucosal biomarker.

Galectins play key roles in the inflammatory cascade. In this study, we aimed to analyze the effect of galectin-1 (Gal-1) in the function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro-inflammatory cytokines, and Gal-1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal-1 binding and modulation of IECs. We found an enhanced binding of Gal-1 to: (a) murine IECs exposed to IL-1β, TNF, and IL-13; (b) IECs from inflamed areas in intestinal tissue from IBD patients; (c) small bowel of allergic mice; and (d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal-1 favored a tolerogenic micro-environment, whereas high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase-9 and modulation of Bcl-2 protein family members. Our results showed that, when added in the presence of diverse pro-inflammatory cytokines such as tumor necrosis factor (TNF), IL-13 and IL-5, Gal-1 differentially promoted the secretion of growth factors including thymic stromal lymphopoietin (TSLP), epidermal growth factor (EGF), IL-10, IL-25, and transforming growth factor (TGF-β1 ). In conclusion, we found an augmented binding of Gal-1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal-1 concentration. These findings highlight the importance of the inflammatory micro-environment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal-1 and its role in epithelial cell homeostasis.

Las Enfermedades Inflamatorias Intestinales (EII) son un conjunto de patologías crónicas multifactoriales en las cuales se produce una respuesta inmune aberrante contra componentes de la microbiota intestinal. Dentro de las EII, la colitis ulcerosa (CU) y la enfermedad de Crohn (EC) son las dos formas más estudiadas. Si bien existen diferencias en la presentación clínica de estas patologías, en ambos casos los linfocitos T (LT) de la mucosa sobre-expresan citoquinas pro-inflamatorias del perfil Th1. La cronicidad de este proceso desencadena complicaciones tales como la formación de abscesos, fístulas, estenosis e inclusive la necesidad de resección quirúrgica de diversos segmentos del intestino debido a lo extenso del daño. Galectina-1 (Gal-1) es una lectina endógena con propiedades anti-infamatorias ampliamente documentadas. Entre los distintos efectos descriptos para esta proteína podríamos mencionar el control de la sobrevida de los LT Th1 y la modulación en la producción de citoquinas. En el presente trabajo de tesis se propuso estudiar la expresión de Gal-1 en la mucosa colónica humana y murina en situaciones fisiológicas y patológicas; analizar la correlación entre la producción de esta lectina y la actividad inflamatoria del tejido y su capacidad de modular la sobrevida de los LT y de las células epiteliales intestinales (CEI) en dichos entornos. Pudimos demostrar que el análisis conjunto de la expresión intestinal de diferentes galectinas permite distinguir entre pacientes con EII activa y pacientes control, EII inactiva o en remisión. Además, concluimos que este análisis es una herramienta eficiente para determinar el grado de inflamación de dicha mucosa y distinguirla de otras patologías inflamatorias intestinales agudas y crónicas. Por lo tanto, podría constituir un marcador de la historia de la patología en el paciente. En cuanto a su rol en la fisiología intestinal, en homeostasis Gal-1 ejerce un efecto pro-apoptótico sobre los LT de la lámina propia colónica, efecto que permite controlar la activación celular frente al constante desafío antigénico por parte de la microbiota. Además, comprobamos que la respuesta fisiológica del intestino frente a un estímulo inflamatorio consiste en inducir un aumento en los niveles de expresión de esta lectina. Sin embargo, este mecanismo compensatorio resulta inefectivo dado que los LT de la lámina propia alteran su maquinaria de glicosilacióin, inhibiendo la expresión de los ligandos específicos para Gal-1. Asimismo, las proteasas presentes en el entorno inflamatorio tisular degradan a esta proteína. Ambos efectos en la lámina propia favorecen la persistencia del foco inflamatorio y la no resolución del daño tisular. Por otra parte, la inflamación sostenida incrementa el porcentaje de CEI que expresan los ligandos para esta lectina. Este fenómeno intensifica el efecto pro-apoptótico que Gal-1 ejerce sobre dichas células. Este mecanismo contribuye a la perdida de la integridad epitelial, la alteración de la permeabilidad intestinal y propicia la traslocación de los antígenos del lumen a la lámina propia. Por lo tanto, en condiciones de homeostasis, Gal-1 contribuiría a la normal fisiología del intestino. Sin embargo, en procesos inflamatorios crónicos, dicho rol se vería alterado, hecho que propiciaría la continuidad de la enfermedad. Una mejor comprensión de los mecanismos que gobiernan la expresión de los ligandos específicos de esta lectina, y el rol que ésta ejerce sobre otras células del sistema inmune, permitirá plantear nuevas terapias o procedimientos para restablecer el glicofenotipo de los LT y de las CEI y, de esta manera, controlar la inflamación intestinal, Facultad de Ciencias Exactas