When less is more: Experimental Bishop-Koop technique for reduction in the use of laboratory animals for intestinal pathophysiological studies.

The use of animals to gain knowledge and understanding of diseases needs to be reduced and refined. In the field of intestinal research, because of the complexity of the gut immune system, living models testing is mandatory. Based on the 3Rs (replacement, reduction and refinement) principles, we aimed to developed and apply the derived-intestinal surgical procedure described by Bishop and Koop (BK) in rats to refine experimental gastrointestinal procedures and reduce the number of animals used for research employing two models of intestinal inflammation: intestinal ischemia-reperfusion injury and chemical-induced colitis. Our results show the feasibility of the application of the BK technique in rodents, with good success after surgical procedure in both small and large intestine (100% survival, clinical recovery and weight regain). A considerable reduction in the use of the number of rats in both intestinal inflammation models (80% in case of intestinal ischemia-reperfusion damage and 66.6% in chemical-induced colitis in our experimental design) was achieved. Compared with conventional experimental models described by various research groups, we report excellent reproducibility of intestinal damage and functionality, survival rate and clinical status of the animals when BK is applied.

Native Spleen Preservation During Visceral Transplantation Inhibits Graft-Versus-Host-Disease Development: Clinical and Experimental Study.

OBJECTIVE: We aimed to assess whether native spleen preservation during visceral transplantation (VT) affects graft-versus-host-disease (GVHD) incidence. SUMMARY BACKGROUND DATA: GVHD is one of the most severe and frequently lethal hematological complications after 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 3 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 2 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 <.001) and better survival ( P <.05) than those in the other groups. Skin and histological signs of GVHD were lower in the rats in the +SP group ( P <.05). The donor T-cell frequency in the bloodstream and skin was also significantly reduced when the native spleen was preserved ( P <.01 and P <.0001, respectively). CONCLUSIONS: The clinical and experimental data indicate that recipient spleen preservation protects against GVHD after VT, and donor cell clearance from the bloodstream by spleen macrophages could be the underlying mechanism. Therefore, spleen preservation should be considered in VT procedures, whenever possible.

Lidocaine reinforces the anti-inflammatory action of dexamethasone on myeloid and epithelial cells activated by inflammatory cytokines or SARS-CoV-2 infection.

BACKGROUND: Severe cases of Coronavirus Disease 2019 (COVID-19) that require admission to the Intensive Care Unit (ICU) and mechanical ventilation assistance show a high mortality rate with currently few therapeutic options available. Severe COVID-19 is characterized by a systemic inflammatory condition, also called "cytokine storm", which can lead to various multi-organ complications and ultimately death. Lidocaine, a safe local anesthetic that given intravenously is used to treat arrhythmias, has long been reported to have an anti-inflammatory and pro-homeostatic activity. METHODS: We studied the capacity of lidocaine to modulate cytokine secretion of mouse and human myeloid cell lines activated by different cytokines or Toll Like Receptor (TLR) ligands (flagellin (FliC), Lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (Poly I:C) and N-Palmitoyl-S- [2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine x 3HCl (Pam3Cys-SKKKK)) or by Severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) infection to epithelial cells. Reporter cell lines were used to study modulation of lidocaine of specific signaling pathways. RESULTS: Lidocaine used in combination with dexamethasone, had an additive effect in the modulation of cellular inflammatory response triggered by Tumoral Necrosis Factor alpha (TNFα), Interleukin 1 beta (IL-1ß) as well as different TLR ligands. We also found that lidocaine in combination with dexamethasone modulates the Nuclear factor kappa B (NF-κB) pathway, inflammasome activation as well as interferon gamma receptor (IFNγR) signaling without affecting the type I interferons (Type I IFNs) pathway. Furthermore, we showed that lidocaine and dexamethasone treatment of epithelial cells infected with SARS-CoV-2 modulated the expression of chemokines that contribute to pro-inflammatory effects in severe COVID. CONCLUSIONS: We reported for the first time in vitro anti-inflammatory capacity of lidocaine on SARS-CoV-2 triggered immune pathways. These results indicated the potential of lidocaine to treat COVID-19 patients and add tools to the therapeutic options available for these concerning cases.

Experimental study to assess the impact of vasopressors administered during maintenance of the brain-dead donation in the quality of the intestinal graft.

BACKGROUND: The hemodynamic maintenance of brain-dead donors will influence the quality of the organs procured for transplantation, including the intestine. Although norepinephrine (NE) and dopamine (DA) are commonly used to sustain mean arterial pressure in humans, there are no standardized protocols for their use during maintenance of brain-dead donors. Our aim was to compare the effects of each drug, in the intestinal graft quality using a rat brain-dead donation model. METHODS: Wistar rats (N = 17) underwent brain death (BD) for 2 hours with NE (NE group) or with DA (DA group) administration; the control group was mechanically ventilated for 2 hours without BD. Jejunum biopsies were obtained at the end of the maintenance period. Histological damage was evaluated using Park-Chiu scale. Villi/crypt ratio, mucosal thickness, Goblet cell count, and villi density were evaluated using ImageJ software (US National Institutes of Health, Bethesda, MD). Barrier damage was assessed by bacterial translocation culture counting on liver samples. The inflammatory status of the intestine was evaluated by CD3+ counting by immunohistochemistry and gene expression analysis of interleukin (IL)-6, IL-22, and CXCL10. RESULTS: Norepinephrine-treated donors had higher focal ischemic injury in the intestinal mucosa without a substantial modification of morphometrical parameters compared with DA-treated donors. CD3+ mucosal infiltration was greater in intestines procured from brain-dead donors, being highest in NE (p ˂ 0.001). Local inflammatory mediators were affected in BD: DA and NE groups showed a trend to lower expression of IL-22, whereas CXCL10 expression was higher in NE versus control group. Brain death promoted intestinal bacterial translocation, but the use of NE resulted in the highest bacterial counting in the liver (p ˂ 0.01). CONCLUSION: Our results favor the use of DA instead of NE as main vasoactive drug to manage BD-associated hemodynamic instability. Dopamine may contribute to improve the quality of the intestinal graft, by better preserving barrier function and lowering immune cell infiltration.

BACKGROUND: The hemodynamic maintenance of brain-dead donors will influence the quality of the organs procured for transplantation, including the intestine. Although norepinephrine (NE) and dopamine (DA) are commonly used to sustain mean arterial pressure in humans, there are no standardized protocols for their use during maintenance of brain-dead donors. Our aim was to compare the effects of each drug, in the intestinal graft quality using a rat brain-dead donation model. METHODS: Wistar rats (N = 17) underwent brain death (BD) for 2 hours with NE (NE group) or with DA (DA group) administration; the control group was mechanically ventilated for 2 hours without BD. Jejunum biopsies were obtained at the end of the maintenance period. Histological damage was evaluated using Park-Chiu scale. Villi/crypt ratio, mucosal thickness, Goblet cell count, and villi density were evaluated using ImageJ software (US National Institutes of Health, Bethesda, MD). Barrier damage was assessed by bacterial translocation culture counting on liver samples. The inflammatory status of the intestine was evaluated by CD3 + counting by immunohistochemistry and gene expression analysis of interleukin (IL)-6, IL-22, and CXCL10. RESULTS: Norepinephrine-treated donors had higher focal ischemic injury in the intestinal mucosa without a substantial modification of morphometrical parameters compared with DA-treated donors. CD3 + mucosal infiltration was greater in intestines procured from brain-dead donors, being highest in NE ( p ˂ 0.001). Local inflammatory mediators were affected in BD: DA and NE groups showed a trend to lower expression of IL-22, whereas CXCL10 expression was higher in NE versus control group. Brain death promoted intestinal bacterial translocation, but the use of NE resulted in the highest bacterial counting in the liver ( p ˂ 0.01). CONCLUSION: Our results favor the use of DA instead of NE as main vasoactive drug to manage BD-associated hemodynamic instability. Dopamine may contribute to improve the quality of the intestinal graft, by better preserving barrier function and lowering immune cell infiltration.

Induction Versus Maintenance Immunosuppression After Intestinal Transplant: Determining Which Treatment Most Impacts Long-Term Patient And Graft Survival.

OBJECTIVES: Immunosuppressive strategies for intestinal transplant have changed over time. However, specific intestinal transplant-oriented protocols and reports on long-term maintenance regimens are scarce. Our objective was to evaluate the impact of 2 different initial immunosuppressive protocols based on thymoglobulin (group A) and basiliximab (anti-interleukin 2 antibody) (group B) and of changes to maintenance immunosuppression over long-term follow-up in intestinal transplant recipients. MATERIALS AND METHODS: We performed a retrospective analysis of a prospectively established protocol for intestinal transplant immunosuppression, conducted between May 2006 and December 2020. We analyzed 51 intestinal transplant recipients, with 6 patients excluded because of early death or graft loss. Acute cellular rejection frequency and grade, number of acute cellular rejection episodes, time to the first acute cellular rejection episode, response to treatment, number of patients who progressed to chronic allograft rejection, kidney function, infections, incidence of posttransplant lymphoproliferative disorder and graft-versus-host disease, and patient and graft survival were analyzed. RESULTS: In the study groups, there were 87 acute cellular rejection episodes in 45 patients (33 in group A and 54 in group B). We found degree of acute cellular rejection to be mild in 45 patients, moderate in 18, and severe in 24 (not significant between groups). Our comparison of induction therapy (thymoglobulin [group A] vs interleukin 2 antibody [group B]) did not show any statistical difference during clinical followup. Long-term review showed that all patients were on tacrolimus. Five-year patient and graft survival rates were 62% and 45% for group A and 54% and 46% for group B, respectively (not significant). CONCLUSIONS: Long-term patient and graft outcomes reflected the use of an individualized follow-up with adjustments and changes in immunosuppressive medications according to the patient's clinical course and complications rather than based on the induction immunosuppressive protocol used.

Canonical and Non-canonical Inflammasome Activation by Outer Membrane Vesicles Derived From Bordetella pertussis.

Outer Membrane Vesicles (OMVs) derived from different Gram-negative bacteria have been proposed as an attractive vaccine platform because of their own immunogenic adjuvant properties. Pertussis or whooping cough is a highly contagious vaccine-preventable respiratory disease that resurged during the last decades in many countries. In response to the epidemiological situation, new boosters have been incorporated into vaccination schedules worldwide and new vaccine candidates have started to be designed. Particularly, our group designed a new pertussis vaccine candidate based on OMVs derived from Bordetella pertussis (BpOMVs). To continue with the characterization of the immune response induced by our OMV based vaccine candidate, this work aimed to investigate the ability of OMVs to activate the inflammasome pathway in macrophages. We observed that NLRP3, caspase-1/11, and gasdermin-D (GSDMD) are involved in inflammasome activation by BpOMVs. Moreover, we demonstrated that BpOMVs as well as transfected B. pertussis lipooligosaccharide (BpLOS) induce caspase-11 (Casp11) and guanylate-binding proteins (GBPs) dependent non-canonical inflammasome activation. Our results elucidate the mechanism by which BpOMVs trigger one central pathway of the innate response activation that is expected to skew the adaptive immune response elicited by BpOMVs vaccination.

Acute cellular rejection in small-bowel transplantation impairs NCR+ innate lymphoid cell subpopulation 3/interleukin 22 axis.

Acute cellular rejection (ACR) remains as one of the main causes of graft loss and death in intestinal transplant (ITx) patients. ACR promotes intestinal injury, disruption of the mucosal barrier, bacterial translocation, and organ dysfunction. As epithelial regeneration is critical in reversing these consequences, the functional axis between the innate lymphoid cell subpopulation 3 (ILC3) and interleukin 22 plays an essential role in that process. Natural-cytotoxic-receptor-positive (NCR+) ILC3 cells have been demonstrated to induce intestinal-stem-cell proliferation along with an IL-22-dependent expansion of that population in several intestinal pathologies, though thus far not after ITx. Therefore, we intended to determine the impact of chronic immunosuppression and ACR on ILC3 cells and interleukin-22 (IL-22) production in the lamina propria after that intervention. MATERIALS AND METHODS: We compared biopsies from healthy volunteers with biopsies from ITx recipients without or with mild-to-moderate ACR, using flow cytometry and the quantitative-PCR. RESULTS: NCR+ ILC3 cells were found to be unaffected by immunosuppression at different time points posttransplant when patients did not experience ACR, but were diminished upon the occurrence of ACR independently of the post-ITx time. Moreover, IL-22-expression levels were notably reduced in ACR. CONCLUSION: The NCR+-ILC3/IL-22 axis is impaired during ACR contributing to a delay in or lack of a complete and efficient epithelial regeneration. Thus, our findings reveal that IL-22 analogues could potentially be used as a new complementary therapeutic approach, in conjunction with immunosuppressant drugs, in order to promote mucosal regeneration upon ACR.

"Spexin improves adipose tissue inflammation and macrophage recruitment in obese mice".

Spexin (SPX) is a novel adipokine related to many metabolic effects, such as gastrointestinal movements, insulin and glucose homeostasis, lipid metabolism and energy balance. This study evaluates the role of SPX in the improvement of the metabolic and inflammatory profile in fructose-rich-diet obese mice. Adult Swiss mice were supplemented or not with fructose (20% in tap water, FRD and CTR, respectively) for 10 weeks. The last ten days, mice were treated or not with SPX (ip. 29 µg/Kg/day, FRD-SPX and CTR-SPX, respectively). A positive correlation was observed between body weight prior to treatment and weight loss after SPX challenge. Moreover, plasma and liver triglycerides and adipose tissue (AT) features (mass, adipocyte hypertrophy, mRNA of leptin) were improved. SPX also induced a reduction in epididymal AT (EAT) expression of TNFα, IL1ß and IL6 and an improvement in IL10 and CD206. M1 macrophages in EAT, principally the Ly6C- populations (M1a and M1b), were decreased. Adipocytes from FRD-SPX mice induced less macrophage activation (IL6, mRNA and secretion) than FRD after overnight co-culture with the monocyte cell line (RAW264.7) in stimulated conditions (M1 activation, LPS 100 ng/mL). Finally, in vitro, monocytes pre-incubated with SPX and stimulated with LPS showed decreased inflammatory mRNA markers compared to monocytes with LPS alone. In conclusion, SPX decreased body weight and improved the metabolic profile and adipocyte hypertrophy. Inflammatory Ly6C- macrophages decreased, together with inflammatory marker expression. In vitro studies demonstrate that SPX induced a decrease in M1 macrophage polarization directly or through mature adipocytes.

Modified Multivisceral Transplantation with Native Spleen Removal in Rats.

BACKGROUND: Modified multivisceral transplantation (MMVTx) refers to the use of a graft that includes all abdominal organs except the liver. The use of this type of transplant in children and adults expanded over the last years with good results. However, long-term survival in experimental models has not been reported. Our aim is to describe in detail some technical modifications of MMVTx to obtain long-term survival. MATERIALS AND METHODS: Syngeneic (Lewis-Lewis) heterotopic MMVTx was performed in 16 male rats (180-250 g). All procedures were performed under isoflurane anesthesia. The graft consisted of stomach, duodenopancreatic axis, spleen, and small bowel. The vascular pedicle consisted of a conduit of aorta, including the celiac trunk and the superior mesenteric artery (SMA), and the portal vein (PV). The engraftment was performed by end-to-side anastomosis to the infra-renal cava vein and aorta. After reperfusion, the graft was accommodated in the right side of the abdomen, and a terminal ileostomy performed. The native spleen was removed. RESULTS: Donor and recipient time was 39 ± 4.4 minutes and 69 ± 7 minutes, respectively; venous and arterial anastomosis time was 14 ± 1 minutes and 12.3 ± 1 minutes, respectively. Total ischemia time was 77.2 ± 7.9 minutes. Survival was 75% (12/16), six were sacrificed after 2 hours, and six were kept alive for long-term evaluation (more than 1 week). CONCLUSION: Long-term survival is reported after heterotopic MMVTx in rats. The heterotopic MMVTx with native spleen removal would potentially improve the existent models for transplant research. The usefulness of this model warrants further confirmation in allogeneic experiments.

M1 macrophage subtypes activation and adipocyte dysfunction worsen during prolonged consumption of a fructose-rich diet.

Fructose-rich diet (FRD) has been associated with obesity development, which is characterized by adipocytes hypertrophy and chronic low-grade inflammation. Interaction of adipocytes and immune cells plays a key role in adipose tissue (AT) alterations in obesity. We assessed the metabolic and immune impairments in AT in a murine obesity model induced by FRD at different periods. Adult Swiss mice were divided into groups of 6 and 10 weeks of fructose (FRD 6wk, FRD 10wk) or water intake (CTR 6wk, CTR 10wk). FRD induced increased in body weight, epidydimal AT mass, and plasmatic and liver Tg, and impaired insulin sensitivity. Also, hypertrophic adipocytes from FRD 6wk-10wk mice showed higher IL-6 when stimulated with LPS and leptin secretion. Several of these alterations worsened in FRD 10wk. Regarding AT inflammation, FRD mice have increased TNFα, IL-6 and IL1ß, and decrease in IL-10 and CD206 mRNA levels. Using CD11b, LY6C, CD11c and CD206 as macrophages markers, we identified for first time in AT M1 (M1a: Ly6C+/-CD11c+CD206- and M1b: Ly6C+/-CD11c+CD206+) and M2 subtypes (Ly6C+/-CD11c-CD206+). M1a phenotype increased from 6 weeks onward, while Ly6C+/- M1b phenotype increased only after 10 weeks. Finally, co-culture of RAW264.7 (monocytes cell line) and CTR or FRD adipocytes showed that FRD 10wk adipocytes increased IL-6 expression in non- or LPS-stimulated monocytes. Our results showed that AT dysfunction got worse as the period of fructose consumption was longer. Inflammatory macrophage subtypes increased depending on the period of FRD intake, and hypertrophic adipocytes were able to create an environment that favored M1 phenotype in vitro.

Compartmentalized Antimicrobial Defenses in Response to Flagellin.

Motility is often a pathogenicity determinant of bacteria targeting mucosal tissues. Flagella constitute the machinery that propels bacteria into appropriate niches. Besides motility, the structural component, flagellin, which forms the flagella, targets Toll-like receptor 5 (TLR5) to activate innate immunity. The compartmentalization of flagellin-mediated immunity and the contribution of epithelial cells and dendritic cells in detecting flagellin within luminal and basal sides are highlighted here, respectively. While a direct stimulation of the epithelium mainly results in recruitment of immune cells and production of antimicrobial molecules, TLR5 engagement on parenchymal dendritic cells can contribute to the stimulation of innate lymphocytes such as type 3 innate lymphoid cells, as well as T helper cells. This review, therefore, illustrates how the innate and adaptive immunity to flagellin are differentially regulated by the epithelium and the dendritic cells in response to pathogens that either colonize or invade mucosa.

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model.

BACKGROUND: Intestinal transplantation (ITx) faces many challenges due to the complexity of surgery and to the multiple immunological reactions that lead to the necessity of rigorous follow-up for early detection of acute cellular rejection (ACR). Our aim was to determine the kinetics of ACR using an experimental ITx model, with emphasis in the characterization of the process using different approaches, including the use of functional assays of absorptive and barrier function. METHODS: ITx in rats conducting serial sampling was performed. Clinical monitoring, graft histology, proinflammatory gene expression, and nitrosative stress determination were performed. Also, glucose absorption, barrier function using ovalbumin translocation, and contractile function were analyzed. RESULTS: The model used reproduced the different stages of ACR. Allogeneic ITx recipients showed signs of rejection from postoperative day (POD) 5, with increasing severity until 12 POD. Histological evaluation showed mild rejection in early sampling and severe rejection at late stages, with alterations in all graft layers. IL-6, CXCL 10, IFNg, and nitrite plasmas levels showed behavior coincident with histopathology. Remarkably, allogeneic grafts showed a marked alteration of glucose absorptive capacity from POD 5 that was sustained until endpoint. Coincidently, barrier function alteration was evidenced by luminal ovalbumin translocation to serum. Contractile function was progressively impaired along ACR. CONCLUSIONS: Glucose absorption and barrier function are altered at early stages of ACR when histological alterations or gene expression changes were much subtle. This observation may provide simple evaluation tools that could be eventually translated to the clinics to contribute to early ACR diagnosis.

Neutrophil Extracellular Traps Stimulate Proinflammatory Responses in Human Airway Epithelial Cells.

Tissue injury leads to the release of uric acid (UA). At high local concentrations, UA can form monosodium urate crystals (MSU). MSU and UA stimulate neutrophils to release extracellular traps (NET). Here, we investigated whether these NET could be involved in the development of inflammation by stimulating cytokine release by airway epithelial cells. We found that NET significantly increased the secretion of CXCL8/IL-8 and IL-6 by alveolar and bronchial epithelial cells. These effects were not observed when NETosis was inhibited by Diphenyleneiodonium, elastase inhibitor, or Cl-amidine. Similar findings were made with NET induced by cigarette smoke extract, suggesting that NET proinflammatory capacity is independent of the inducing stimulus. Furthermore, NET affected neither the viability and morphology of epithelial cells nor the barrier integrity of polarized cells. The epithelial stimulatory capacity of NET was not affected by degradation of DNA with micrococcal nuclease, treatment with heparin, or inhibition of the elastase immobilized to DNA, but it was significantly reduced by pretreatment with an anti-HMGB-1 blocking antibody. Altogether, our findings indicate that NET exert direct proinflammatory effects on airway epithelial cells that might contribute in vivo to the further recruitment of neutrophils and the perpetuation of inflammation upon lung tissue damage.

Lactate Inhibits the Pro-Inflammatory Response and Metabolic Reprogramming in Murine Macrophages in a GPR81-Independent Manner.

Lactate is an essential component of carbon metabolism in mammals. Recently, lactate was shown to signal through the G protein coupled receptor 81 (GPR81) and to thus modulate inflammatory processes. This study demonstrates that lactate inhibits pro-inflammatory signaling in a GPR81-independent fashion. While lipopolysaccharide (LPS) triggered expression of IL-6 and IL-12 p40, and CD40 in bone marrow-derived macrophages, lactate was able to abrogate these responses in a dose dependent manner in Gpr81-/- cells as well as in wild type cells. Macrophage activation was impaired when glycolysis was blocked by chemical inhibitors. Remarkably, lactate was found to inhibit LPS-induced glycolysis in wild type as well as in Gpr81-/- cells. In conclusion, our study suggests that lactate can induce GPR81-independent metabolic changes that modulate macrophage pro-inflammatory activation.

A galectin-specific signature in the gut delineates Crohn's disease and ulcerative colitis from other human inflammatory intestinal disorders.

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 ß-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.

Lactate and short chain fatty acids produced by microbial fermentation downregulate proinflammatory responses in intestinal epithelial cells and myeloid cells.

The use of short chain fatty acids to modulate gastrointestinal inflammatory conditions such as ulcerative colitis has produced encouraging results either in animal models or also in clinical trials. Identifying the key cellular and molecular targets of this activity will contribute to establish the appropriate combinations/targeting strategies to maximize the efficacy of anti-inflammatory interventions. In the present work, we evaluated in vitro the interaction of lactate, acetate, propionate and butyrate on cells relevant for innate immune response of the gastrointestinal tract. All molecules tested regulate the production of proinflammatory cytokines by TLR-4 and TLR-5 activated intestinal epithelial cells in a dose response manner. Furthermore SCFAs and lactate modulate cytokine secretion of TLR-activated bone marrow derived macrophages and also TLR-dependent CD40 upregulation in bone marrow derived dendritic in a dose-dependent manner. Butyrate and propionate have been effective at concentrations of 1 to 5mM whereas acetate and lactate produced modulatory effects at concentrations higher than 20-50mM in different assays. Our results indicate that in concentrations similar to found in large bowel lumen, all SCFAs tested and lactate can modulate activity of relevant sentinel cell types activated by TLR signals. Modulatory activity was not inhibited by pertussis toxin treatment indicating that the effects are not related to Gi signaling. The use of these molecules in combined or separately as intervention strategy in conditions where epithelial or myeloid cells are main triggers of the inflammatory situation seems appropriate.

Avances en Gastroenterología Trasplante intestinal desde la clínica a la investigación traslacional: contribuciones de una unidad integral de insuficiencia intestinal, rehabilitación y trasplante. Seguimiento biomédico del proceso de trasplante intestinal y su correlación con los distintos eventos fisiopatológicos que atraviesa el paciente.

One of the greatest achievements in gastroenterology and surgery of the last 50 years has been the capability to transplant different abdominal organs of the digestive system separately or as a whole. The complexity of the intestinal transplantation demands a multidisciplinary team engaged in the management of patients with intestinal failure responsible for defining the need for nutritional support, rehabilitation, or intestinal transplantation. This team should include a basic research area to provide answers to unresolved clinical problems. The aim of this work is to update the current status of intestinal transplantation, and to show the progress and results of our center; emphasizing our achievements in the clinical area, and the contributions of the translational research and mucosal immunology studies as part of the integral unit of intestinal failure, rehabilitation and transplantation. The data reported here demonstrate that the intestinal transplantation has been established as a therapeutic option in our country and Latin America, with long term results that have ranked our service at the level of the best centers in the world positioning us as referent in the specialty.

Current status of allograft tolerance in intestinal transplantation.

Solid organ transplantation has become a clinical practice after the development of different immunosuppressive drugs that allowed controlling rejection. The price to be paid for that is the permanent risk of infections and malignancies and a significant drug-associated toxicity. The establishment of transplant tolerance has been the "holy grail" for transplantation medicine since its beginnings. Different experimental approaches and clinical trials resulted in the accumulation of knowledge on mechanisms and strategies that favor the establishment of tolerance without achieving the objective of autonomous allograft tolerance in the clinical field. Development of tolerance in intestinal transplantation constitutes a challenging situation due to several particular features that contribute to the generation of a strong allogeneic response. In the present review, we summarize the different immune mechanisms that may contribute to allograft tolerance. The different barriers that should be bypassed in intestinal transplantation to tolerate the graft are discussed. Finally, we revise the strategies that were applied with different degrees of success in the clinical field including the most promising recent approaches and the forthcoming candidates in the field that might be translated into clinical trials in the near future.

Optimization of transgenesis conditions for the generation of CXCL2-luciferase reporter mice line

Background: Transgenesis by microinjection has been widely used for the generation of different mouse models. Different variables of the procedure may critically affect the efficiency of the process. A DNA construction that carries the CXCL2 promoter gene and firefly luciferase has been used to optimize aspects of the procedure. Three different concentrations (0.5, 1.0 and 4.0 ng/µl) of the DNA construction to microinject a total of 1981 zygotes has been tested. Intact/injected embryos, pregnancy and birth rate, survival of pups 7 days after birth, number of transgenic pups and overall transgenic efficiency was registered and analyzed by Z test of proportions for each group. Results: A total of seven transgenic founders were detected for the three DNA concentrations used, 1 in 46 alive pups in the 0.5 ng/µl group, 5 in 38 alive pups in the 1 ng/µl group and 1 in 21 alive pups in the 4 ng/µl group ( p < 0.1). The overall transgenic efficiency was higher for the 1 ng/µl concentration, with a transgenic rate of 13.2%. Conclusions: In conclusion, we have selected the best operative conditions to maximize the transgenesis efficiency. Furthermore, the transgenic lines developed could be used as a reporter model of innate immunity activation with many different applications in the fields of immunology, cancer and neurodegenerative diseases.