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Social engagement is critical to the personal and social development of children. Individuals with Autism Spectrum Disorder (ASD) often exhibit low levels of social engagement with peers and adults, with clearly fewer instances of eye contact, initiation, joint attention, and empathy. Given the pivotal role of social engagement, procedures that naturally evoke higher rates of social engagement may be critical. Prior research suggests that cartoons and other animated characters are more appealing to children with ASD than traditional face-to-face interactions with adults. However, this assertion lacks consistent empirical support. In this study, five elementary age students with ASD participated in a series of interactions with adults and live animation avatars. An alternating treatments design was used to evaluate participants’ social engagement comparing their interactions with avatars to those with humans. Data show all five participants were consistently more attentive to the avatar than to human interventionists. In addition, all participants and their parents indicated the procedures were socially valid (i.e., preferred, feasible, and effective).

Mucopolysaccharydosis IIIB is the second most frequent form of Sanfilippo syndrome, a degenerative, pediatric lysosomal storage disease (LSD) characterized by severe neurological disorders and death. We have generated two iPSCs lines derived from dermal fibroblast from a MPSIIIB homozygous (P358L) donor. Cells were reprogrammed with OriP/EBNA1-based episomal plasmids containing: OCT3/4, SOX2, KLF4, L-MYC, LIN28, BCL-xL and shp53. Both cell lines are homozygous for the P358L mutation of the α-N-acetylglucosaminidase (NAGLU) gene, have normal karyotype, are free of plasmid integration, express high levels of pluripotency-associated markers and can differentiate into the three germ layers. Resource table: Unlabelled TableUnique stem cell lines identifierIMEDEAi005-AIMEDEAi005-BAlternative names of stem cell linesiPS GM02931 CL2 (IMEDEAi005-A)iPS GM02931 CL3 (IMEDEAi005-B)InstitutionIMEDEA - Instituto Mediterráneo de Estudios AvanzadosContact information of distributorDaniel Bachiller; d.b@csic.esType of cell linesiPSCsOriginHumanCell SourceOriginal cell type: GM02931 Fibroblast from the Coriell InstituteClonalityClonal cell linesMethod of reprogrammingEpisomal Plasmids (Klf4, Sox2, Oct4, shp53, L-Myc, Lin28, Bcl-xL)Multiline rationaleSame disease, isogenic cell linesGene modificationNOType of modificationN/AAssociated diseaseMucopolysaccharidosis, Type IIIB; MPS3B(MPS IIIB; OMIM entry # 252920)Gene/locusNAGLU 17q21.2Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock date26/09/2018Cell line repository/bankRegistered in the Human Pluripotent Stem Cell Registry (https://hpscreg.eu).Ethical approvalOriginal line (GM02931 Fibroblast) has been obtained by Coriell Institute for Medical Research. Regarding policy of informed consent form, the Coriell Institute for Medical Research, which operates the NIGMS Human Genetic Cell Repository, has obtained a Certificate of Confidentiality from the National Institutes of Health to help ensure patient's privacy. Resource utility: Although the generation of iPSCs has been reported for some lysosomal storage diseases (LSD) in general, and from other mutations of the NAGLU gene in particular (Lemonnier et al., 2011), this is the first time that NAGLU Pro358Leu MPSIIIB-iPSCs lines have been generated and fully characterized demonstrating their quality as iPS cells. Resource details: Mucopolysaccharidosis IIIB (MPSIII, Sanfilippo syndrome type B) is a pediatric neurodegenerative disorder caused by a deficiency in NAGLU, an enzyme required for lysosomal degradation of heparin sulphate (HS). When the enzyme is absent or malfunctioning, HS accumulates in the cells of several tissues, with devastating effects in the brain and central nervous system. MPSIIIB is inherited in an autosomal recessive manner and presents an incidence between 0.03 and 0.78 cases per 1 × 105 live births (Fedele, 2015) depending on the country. Currently there is no therapy available.The NAGLU gene was identified in 1996, is located on chromosome 17q21.1 and contains 6 exons. More than 150 NAGLU mutations have been reported, being most of them missense (Valstar et al., 2010). All of them lead to MPSIIIB but, unlike MPSIIIA, none is predominant.The two iPSCs lines described in this report, IMEDEAi005-A and IMEDEAi005-B, (See Table 1) were generated from skin fibroblast obtained from a clinically affected homozygous donor. The mutant allele consists on a C > T transversion at nucleotide 1073 (1073 > T) resulting in a substitution of leucine for proline at codon 358 (Pro358Leu).Skin fibroblasts were reprogrammed to iPSCs by nucleofection with four OriP/EBNA1 (Epstein-Barr nuclear antigen-1) based episomal plasmids encoding 5 reprogramming genes (OCT3/4, SOX2, KLF4, L-Myc, LIN28 and BCL-xL), in addition to a short hairpin RNA against p53. The iPSCs lines showed morphology (Fig. 1A) and growth behaviour typical of human Embryonic Stem Cells (hESC), as well as normal female karyotype (46, XX) (Fig. 1B). After 12 passages, PCR analysis confirmed that both iPSCs lines had completely lost the episomal vectors (Fig. 1C). The identity of iPS cells and their parental fibroblasts was confirmed by STR analysis (Table 2, data not shown) in addition to the identification of the disease-associated mutation in the NAGLU gene by DNA sequencing (Fig. 1D). Regarding the iPSC phenotype, both lines expressed the pluripotency-associated markers: OCT3/4, NANOG, SOX2 and TRA-1-60 (Fig. 1E), and TRA-1-81 quantified by flow cytometry (Fig. 1G), resulting in 88.17% and 83.4% of TRA-1-81 positive cells in IMEDEAi005-A and IMEDEAi005-B respectively. Finally, the differentiation capacity of iPSCs lines was analyzed by embryoid body (EBs) formation. Expression of markers specific of the three germ layers was observed after at least 10 days of spontaneous differentiation (Fig. 1F). Mycoplasma analysis was negative for both iPSCs lines (Supplementary Fig. S1).Skin fibroblasts were reprogrammed to iPSCs by nucleofection with four OriP/EBNA1 (Epstein-Barr nuclear antigen-1) based episomal plasmids encoding 5 reprogramming genes (OCT3/4, SOX2, KLF4, L-Myc, LIN28 and BCL-xL), in addition to a short hairpin RNA against p53. The iPSCs lines showed morphology (Fig. 1A) and growth behaviour typical of human Embryonic Stem Cells (hESC), as well as normal female karyotype (46, XX) (Fig. 1B). After 12 passages, PCR analysis confirmed that both iPSCs lines had completely lost the episomal vectors (Fig. 1C). The identity of iPS cells and their parental fibroblasts was confirmed by STR analysis (Table 2, data not shown) in addition to the identification of the disease-associated mutation in the NAGLU gene by DNA sequencing (Fig. 1D). Regarding the iPSC phenotype, both lines expressed the pluripotency-associated markers: OCT3/4, NANOG, SOX2 and TRA-1-60 (Fig. 1E), and TRA-1-81 quantified by flow cytometry (Fig. 1G), resulting in 88.17% and 83.4% of TRA-1-81 positive cells in IMEDEAi005-A and IMEDEAi005-B respectively. Finally, the differentiation capacity of iPSCs lines was analyzed by embryoid body (EBs) formation. Expression of markers specific of the three germ layers was observed after at least 10 days of spontaneous differentiation (Fig. 1F). Mycoplasma analysis was negative for both iPSCs lines (Supplementary Fig. S1).In conclusion, we have successfully generated and characterized, for the first time to our knowledge, two human iPSCs lines from a MPSIIIB donor homozygous for the P358L NAGLU mutation. The new lines will complement the existing murine MPS IIIB model, with their potential to be used in a development of a purely human in vitro model of the disease.

A 32 base pair deletion in the C-C chemokine receptor type gene (CCR5-Delta 32), the main Human Immunodeficiency Virus (HIV) co-receptor results in a non-functional protein. Individuals homozygous for the CCR5-Delta 32 mutation are resistant to HIV infection. Here we report the generation, from pro-erythroblast enriched Peripheral Blood Mononuclear Cells (PBMCs) from a naturally occurring CCR5-Delta 32/Delta 32 individual, of the fully characterized iPSC line IMEDEAi008-A. The new line has normal karyotype, carry the Delta 32 mutation in homozygosity, is free of plasmid integrations, express high levels of pluripotency markers and can differentiate into all three germ layers., Funding was provided by the Spanish Ministry for Science and Innovation (RTC-2016-5324-1) and the Balearic Government (103/2019). JMMF was a postdoctoral Berrikertu fellow from the Basque government. AF was a recipient of Juan de la Cierva (JCI-2006-2675) and Torres Quevedo (PTQ-16-08496) postdoctoral fellowships from the Spanish Ministry for Science and Innovation.

Psoriasis is a chronic inflammatory skin disease that speeds up the life cycle of skin cells, forming scales and red patches that are itchy and sometimes painful. It is a complex disease of autoimmune origin and genetic predisposition with more than 10 different loci associated. Here we described the production of an iPSC line generated by Sendai Virus (Klf4, Oct3/4, Sox2 and c-Myc) reprogramming of Peripheral Blood Mononuclear Cells (PBMCs) from a Psoriasis patient. The iPSC line generated has normal 46XY karyotype, is free of SeV genome and transgenes insertions, express high levels of pluripotency markers and can differentiate into all three germ layers., Funding was provided by the Spanish Ministry for Science and Innovation (RTC-2016-5324-1). JMMF was a postdoctoral Berrikertu fellow from the Basque government. AF was a recipient of Juan de la Cierva (JCI-2006-2675) and Torres Quevedo (PTQ-16-08496) postdoctoral fellowships from the Spanish Ministry for Science and Innovation.

Easily accessible axially chiral substituted binols (95 to >99% ee) undergo an oxidative dearomatization process with the system Oxone/NaHCO3/acetone, under mild conditions, to afford pentacyclic h...

Cystic fibrosis (CF) is the main genetic cause of death among the Caucasian population. The disease is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth (meconium ileus), later extending to other organs, generally affecting the respiratory tract. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (cAMP)-dependent, phosphorylation-regulated chloride channel required for transport of chloride and other ions through cell membranes. There are more than 2,000 mutations described in the CFTR gene, but one of them, phenylalanine residue at amino acid position 508 (p.F508del), a recessive allele, is responsible for the vast majority of CF cases worldwide. Here, we present the results of the application of genome-editing techniques to the restoration of CFTR activity in p.F508del patient-derived induced pluripotent stem cells (iPSCs). Gene-edited iPSCs were subsequently used to produce intestinal organoids on which the physiological activity of the restored gene was tested in forskolin-induced swelling tests. The seamless restoration of the p.F508del mutation resulted in normal expression of the mature CFTR glycoprotein, full recovery of CFTR activity, and a normal response of the repaired organoids to treatment with two approved CF therapies: VX-770 and VX-809., Graphical Abstract, Cystic Fibrosis (CF) is the main genetic cause of death among the Caucasian population. Here, we present the results of the application of genome-editing techniques to repair the CF defect in patient-derived cells and organoids. This protocol could be the foundation for future treatments based on cell repair and transplantation.

This article also appears in: Lineage Tracer and Gene-editing in Stem Cells., Genome editing has become one of the most powerful tools in present‐day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator‐like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN‐F) or membrane receptors (TALEN‐M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence‐assisted cell sorting (FACS) or magnetic‐activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones. © 2020 by John Wiley & Sons, Inc., This work was supported by grants from the Spanish Ministry for Science and Innovation (PLE2009‐0091, RTC‐2014‐2207‐1 and IPT‐2011‐1402‐900000), ISCIII (PI14/01073 and PI18/00334), co‐funded by ERDF/ESF “Investing in your future”, the Balearic Government (16023/2008), the Spanish Cystic Fibrosis Federation (Pablo Motos grant), Federación ASEM‐Telemaratón “Todos somos raros, todos somos únicos”, Fundación Salud 2000, the European Commission (H2020, PHC‐667079), and an endowment from METROVACESA. J. M. M. F. was a postdoctoral Berrikertu fellow (granted by the Basque Government). A. F. was a recipient of Juan de la Cierva (JCI‐2006‐2675) and Torres Quevedo (PTQ‐16‐08496) postdoctoral fellowships from the Spanish Ministry for Science and Innovation.

The mutation E280A in PSEN1 (presenilin-1) is the most common cause of early-onset familial Alzheimer's Disease (fAD). It presents autosomal dominant inheritance and frequently leads to the manifestation of the disease in relatively young individuals. Here we report the generation of one PSEN1 E280A iPSC line derived from an early-onset patient. OriP/EBNA1-based episomal plasmids containing OCT3/4, SOX2, KLF4, L-MYC, LIN28, BCL-xL and shp53 were used to reprogram oral mucosa fibroblasts. The iPSC line generated has normal karyotype, carry the E280A mutation, is free of plasmid integration, express high levels of pluripotency markers and can differentiate into all three germ layers., Funding was provided by the Basque Government grant: Elkartek 20017 (DRUG4AD).

This paper evaluates the implementation of salinity gradient power (SGP) based on reverse electrodialysis (RED) in the Caribbean region of Colombia, specifically at the Magdalena River mouth (MRM). Our multidisciplinary approach comprises technical, technological, and technology diffusion assessments (market analysis). We estimate the theoretical energy potential in the MRM from spatial and temporal measurements of the physical and biochemical properties of the waters. Thereafter, we compare the power output of a tailored RED prototype using synthetic solutions and untreated natural waters from MRM. Finally, we propose a dynamic model for RED diffusion in Colombia. Our results show that the MRM exhibits suitable conditions for harnessing SGP given the prevailing thermohaline structure, tolerable water quality, and an existing physical barrier between the sea and the river. However, our experiments demonstrated that maximum power is reduced by half when feeding with MRM waters. This is attributed to the divalent ions and low concentration of the river. Finally, the dynamic model shows that the learning rate and the initial operational costs are key variables for technology diffusion. This research is a pioneer in integrating multidisciplinary perspectives of SGP in emerging economies. However, this baseline may be further complemented with socio-political, and economic analyses.

Background Individuals with autism often struggle to establish and maintain positive relationships with peers and adults due to communication and social difficulties. Reviews of the research literature on social skills curricula suggest these approaches may be less than effective and often fail to promote generalization of acquired skills. Advances in the availability and adaptability of live animation and digital avatars could provide interventionists a tool to maximize engagement during instruction and program for generalization. The purpose of this study was to examine the effects of an avatar-based social skills intervention on participants’ social initiations in a clinical setting and with same-age peers. Method The research team used a nonconcurrent multiple baseline design to evaluate the effects of instruction from an avatar on students' social skills. Systematic direct observation and the Social Skills Improvement System were used to evaluate the effects of an avatar delivered social skills lesson. Social validity was measured from the perspective of both participants and their parents. Results Following instruction from an avatar, participants’ percentage of independent steps completed in the skill “starting a conversation” increased to consistently above 80 %, or above typical mastery levels. Participants’ conversation skills generalized to interactions with same-age peers. Finally, parents reported small positive gains in social skills, and participants indicated the intervention was socially valid. Conclusions The findings from this study support the use of technology-aided interventions and instruction, specifically the use of live animation avatars. Future studies could extend use of this technology beyond the relatively simple, yet effective, application described here.

Resumen Se presenta y resuelve un modelo para diseño de pilas de Electrodiálisis inversa. Se propuso una nueva función de optimización para RED, que tienen en cuenta el consumo de agua de río, la densidad de potencia neta y la eficiencia termodinámica del proceso. Los parámetros de tiempos de residencia y espesor de compartimientos fueron optimizados correctamente usando la nueva función propuesta. Los resultados sugieren pequeños tiempos de residencia, compartimientos de espesores delgados, poca área transversal y relaciones bajas de Longitud/ Ancho del compartimiento para máxima generación de energía. Abstract A model for design of Reverse Electrodialysis stacks for energy generation is presented and solved. A new optimization function is proposed for RED, which accounts for river water consume, net power density and thermodynamic efficiency of the process. The parameters of residence time and thickness of the compartments are successfully optimized using the new proposed function. Results suggest small residence time, compartments thickness, and transversal area and Long/Width ratio for maximum energy generation.

Dendritic cells (DCs) control the type and location of immune responses. Ulcerative colitis (UC) is considered a Th2 disease mediated by IL-13 where up to one third of patients can develop extraintestinal manifestations. Colonic biopsies from inflamed and noninflamed areas of UC patients were cultured in vitro and their supernatants were used to condition human blood enriched DCs from healthy controls. Levels of IL-13 in the culture supernatants were below the detection limit in most cases and the cytokine profile suggested a mixed profile rather than a Th2 cytokine profile. IL-6 was the predominant cytokine found in inflamed areas from UC patients and its concentration correlated with the Mayo endoscopic score for severity of disease. DCs conditioned with noninflamed culture supernatants acquired a regulatory phenotype with decreased stimulatory capacity. However, DCs conditioned with inflamed culture supernatants acquired a proinflammatory phenotype with increased expression of the skin-homing chemokine CCR8. These DCs did not have decreased T-cell stimulatory capacity and primed T cells with the skin-homing CLA molecule in an IL-6-dependent mechanism. Our results highlight the role of IL-6 in UC and question the concept of UC as a Th2 disease and the relevance of IL-13 in its etiology.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., We studied whether celiac disease (CD) patients produce antibodies against a novel gliadin peptide specifically generated in the duodenum of CD patients by a previously described pattern of CD-specific duodenal proteases. Fingerprinting and ion-trap mass spectrometry of CD-specific duodenal gliadin-degrading protease pattern revealed a new 8-mer gliadin-derived peptide. An ELISA against synthetic deamidated 8-mer peptides (DGP 8-mer) was used to study the presence of IgA anti-DGP 8-mer antibodies in plasma samples from 81 children (31 active CD patients (aCD), 17 CD patients on a gluten-free diet (GFD), 10 healthy controls (C) and 23 patients with other gastrointestinal pathology (GP)) and 101 adults (16 aCD, 12 GFD, 27 C and 46 GP-patients). Deamidation of the 8-mer peptide significantly increased the reactivity of the IgA antibodies from CD patients against the peptide. Significant IgA anti-DGP 8-mer antibodies levels were detected in 93.5% of aCD-, 11.8% of GFD- and 4.3% of GP-patients in children. In adults, antibodies were detected in 81.3% of aCD-patients and 8.3% of GFD-patients while were absent in 100% of C- and GP-patients. Duodenal CD-specific gliadin degrading proteases release an 8-mer gliadin peptide that once deamidated is an antigen for specific IgA antibodies in CD patients which may provide a new accurate diagnostic tool in CD. © 2013 Vallejo-Diez et al., This work has been supported by grants (to EA) from the Instituto de Salud Carlos III – FEDER (PI070244, PI10/01647), Junta de Castilla y León (SAN126/VA31/09), AECID (A/019196/08), and Phadia Spain S.L (now ThermoFisher Scientific).

La Enfermedad Celiaca (EC) es una enteropatía sensible al gluten que se desarrolla en individuos genéticamente predispuestos tras la ingesta de trigo, cebada, centeno y algunas variedades de avena. Durante la digestión, las proteínas de gluten son hidrolizadas en pequeños péptidos, los cuales activan una respuesta inmune específica de los pacientes celiacos. En ensayos previos de zimografía realizados en nuestro laboratorio, se describió un patrón de degradación de la gliadina específico de los pacientes celiacos, y compuesto por 7 proteasas. El análisis de una de las bandas de degradación , dio como resultado la identificación del péptido 8-mer, como un péptido derivado de la omega gliadina específico de los pacientes celiacos. En esta Tesis, nos planteamos tres objetivos generales. El primero de ellos fue estudiar la capacidad de degradación de prolaminas por la mucosa duodenal de los pacientes celiacos. Hemos observado como la degradación de la gliadina es mucho más intensa y definida en la capa de mucus que recubre el epitelio intestinal, que en el propio tejido y que se mantiene de una forma más dispersa en el jugo duodenal de los pacientes celiacos. Además se realizó un estudio in silico del péptido 8-mer que demostró que este péptido se encuentra únicamente en los cereales tóxicos para los pacientes celiacos, y que su secuencia se solapa con uno de los epítopos de gluten de células T específicos de los pacientes celiacos. El segundo de los objetivos fue estudiar el efecto del péptido 8-mer sobre el epitelio intestinal, utilizando un modelo in vitro de células Caco-2. El incremento en la permeabilidad del epitelio en pacientes con EC, es indiscutible, sin embargo, no está muy claro si es una causa o una consecuencia de la propia enfermedad. Por ello quisimos además, estudiar si el contacto de las células Caco -2 con las muestras de jugo o mucus duodenal, condicionaría de alguna manera el efecto del péptido 8-mer sobre el epitelio. Tras realizar varios ensayos de PCR cuantitativa, analizamos los niveles de expresión de diferentes moléculas, por parte de las células Caco, implicadas en la patogenia de la enfermedad (la citocina proinflamatoria IL-15, la molécula de estrés de los enterocitos MICA, las proteínas de las uniones estrechas de los enterocitos Ocludina y Zo-1 y el factor de crecimiento transformante TGF beta). Los resultados, a pesar de ser preliminares, sugieren que el péptido 8-mer podría tener un efecto tóxico directo sobre el epitelio intestinal de los pacientes celiacos, el cual podría verse favorecido por el contacto directo del epitelio con la capa de mucus que lo recubre en los pacientes celiacos. Finalmente, nos planteamos la posibilidad de que el péptido 8-mer fuera capaz de activar una respuesta inmune en los pacientes celiacos. Se diseñó y estandarizó un ensayo ELISA indirecto para estudiar la presencia de anticuerpos IgA específicos frente al péptido 8-mer. En primer lugar observamos la existencia de estos anticuerpos en muestras de plasma de los pacientes celiacos, y que el reconocimiento del péptido se veía potenciado con la desaminación del mismo. Esto sugiere que el péptido 8-mer es generado in vivo en los pacientes celiacos, y posteriormente desaminado por la enzima transglutaminasa tisular. Estos anticuerpos se detectaron únicamente en los pacientes celiacos, estando ausentes en los pacientes control no celiacos, lo que confirma la capacidad del péptido de generar una respuesta inmune humoral. El ensayo ELISA diseñado constituye una nueva herramienta con múltiples utilidades tanto en el apoyo al diagnóstico de la enfermedad celiaca como en el seguimiento de la dieta sin gluten tanto en niños como en adultos, además de permitir discriminar entre la enfermedad celiaca y otras patologías gastrointestinales., Departamento de Pediatría e Inmunología, Obstetricia y Ginecología, Nutrición y Bromatología, Psiquiatría e Historia de la Ciencia

Dendritic cells (DCs) control the type and location of immune responses. Ulcerative colitis (UC) is considered a Th2 disease mediated by IL-13 where up to one third of patients can develop extraintestinal manifestations. Colonic biopsies from inflamed and noninflamed areas of UC patients were cultured in vitro and their supernatants were used to condition human blood enriched DCs from healthy controls. Levels of IL-13 in the culture supernatants were below the detection limit in most cases and the cytokine profile suggested a mixed profile rather than a Th2 cytokine profile. IL-6 was the predominant cytokine found in inflamed areas from UC patients and its concentration correlated with the Mayo endoscopic score for severity of disease. DCs conditioned with noninflamed culture supernatants acquired a regulatory phenotype with decreased stimulatory capacity. However, DCs conditioned with inflamed culture supernatants acquired a proinflammatory phenotype with increased expression of the skin-homing chemokine CCR8. These DCs did not have decreased T-cell stimulatory capacity and primed T cells with the skin-homing CLA molecule in an IL-6-dependent mechanism. Our results highlight the role of IL-6 in UC and question the concept of UC as a Th2 disease and the relevance of IL-13 in its etiology.

[Background]: The IL-15/NF-κB axis has an important role in coeliac disease (CD) and may represent a molecular target for immunomodulation. Ascorbate (vitamin C) is known to show inhibitory effects on NF-κB. Therefore, we studied if ascorbate supplementation to gliadin gliadin-stimulated biopsy culture could down-regulate the mucosal immune response to gliadin in CD. [Methods]: Duodenal biopsy explants from treated CD patients were gliadin challenged in vitro (100 οg/ml) with and without 20. mM ascorbate. An extra tissue explant in basal culture was used as internal control. Secretion levels of nitrites (3. h), and IFNγ, TNFα, IFNα, IL-17, IL-13, and IL-6 (24. h) were measured on the supernatants. IL-15 was assayed by western-blot on whole protein duodenal explants. [Results]: The addition of ascorbate to in vitro culture gliadin-challenged biopsies blocked the secretion of nitrites (p= 0.013), IFNγ (p= 0.0207), TNFα (p= 0.0099), IFNα (p= 0.0375), and IL-6 (p= 0.0036) compared to samples from non-ascorbate supplemented culture. Cytokine secretion was downregulated by ascorbate even to lower values than those observed in basal cultures (IFNγ: p= 0.0312; TNFα: p= 0.0312; IFNα: p= 0.0312; and IL-6: p= 0.0078). Gliadin-challenge induced IL-15 production in biopsies from treated CD patients, while the addition of ascorbate to culture medium completely inhibited IL-15 production. Moreover, the inhibition of IL-15 by ascorbate took place even in the only treated CD-patient who had basal IL-15 production. [Conclusions]: Ascorbate decreases the mucosal inflammatory response to gluten in an intestinal biopsy culture model, so it might have a role in future supplementary therapy in CD. © 2010 SEICAP., Spanish Ministry of Education (FPU, AP2002-2696), Junta de Castilla y León (FPI fellowship), Universidad de Valladolid (FPI fellowship), Instituto de Salud Carlos III, Spanish Ministry of Health (PI 070244 and CES08/016), Fundación IECSCYL, Junta de Castilla y León (SAN-GRS232/B/08), and Phadia Spain.

Methods: C57BL/6 mice received 4% dextran sodium sulfate (DSS) in their drinking water for 7 days to induce colitis. Mice were matched for body weight and treated IP daily with 500mg/kg TUDCA or PBS. Weight loss and mortality were monitored. On day 0, 3, 7 and 10, eight mice per group were sacrificed and colon length was assessed. Colonocytes were isolated and lysed for real-time PCR (qPCR) analysis of BIP, XBP1s, XBP1u, ATF4 and PDIA4. HT29 cells were treated with 10mM TUDCA for 24 hours and lysed for qPCR analysis. Results: Administration of DSS did not result in substantial activation of the UPR in colonic epithelial cells. A transient activation of IRE1 was seen, measured by the increased ratio of spliced to unspliced XBP1 levels at day 7 and 10. Activation of the PERK and ATF6 pathways was not observed, as ATF4 and PDIA4 expression remained unchanged. The expression of the key chaperone BIP diminished at day 3 and 7 and returned to baseline at day 10. Administration of TUDCA resulted in 100% survival as compared to 60% in vehicle treated mice (p < 0.05). Weight loss and colon shortening was significantly less in TUDCA treated mice (p < 0.01 and p = 0.02 respectively). Colonocytes from these mice showed 2.6-fold XBP1 splicing increase at day 3, while BIP levels were comparable to those in non-DSS treated mice. These findings were confirmed in vitro by the induction of XBP1 splicing and BIP expression in HT29 cells cultured in the presence of TUDCA (p < 0.001). Conclusions: Acute colitis is accompanied by a transient activation of the IRE1 pathway. TUDCA alters this response by forcing IRE1 activation and inducing its downstream target gene BIP. BIP protects cells from stress to the endoplasmic reticulum and could be beneficial to epithelial cells subjected to colonic damage. Chemical chaperones such as TUDCA merit further clinical investigation for the treatment of IBD.