UV-induced ligand exchange in MHC class I protein crystals

High-throughput structure determination of protein−ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, “conditional”, peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This “in crystallo” exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC β2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families

Class I major histocompatibility complexes loaded by a periodate trigger

Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide−MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide−MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide−MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands

Diagnóstico y tratamiento de la enfermedad inflamatoria intestinal: Primer Consenso Latinoamericano de la Pan American Crohn's and Colitis Organisation

La incidencia y la prevalencia de la enfermedad inflamatoria intestinal (EII) se han incrementado en los últimos a˜nos en varios países de Latinoamérica. Existe una necesidad de concientizar a gastroenterólogos y a la población en general para poder tener un diagnóstico y tratamiento oportunos en la colitis ulcerosa crónica idiopática (CUCI) y enfermedad de Crohn (EC). Es importante que todos los médicos tengan un criterio homogéneo acerca del diagnóstico y el tratamiento de la EII en América Latina. La Pan American Crohn’s and Colitis Organisation (PANCCO) es un organismo con el propósito de incluir a todos los países del continente americano pero se enfoca de manera específica a los países latinos. Este Consenso está dividido en 2 partes para su publicación: 1) diagnóstico y tratamiento, y 2) situaciones especiales. Este es el primer Consenso latinoamericano cuyo objetivo es promover una perspectiva adaptadaa nuestros países latinos para el diagnóstico, el tratamiento y la monitorización de pacientescon CUCI y EC.© 2016 Asociaci´on Mexicana de Gastroenterolog´ıa. Publicado por Masson Doyma M´exico S.A.Este es un art´ıculo Open Access bajo la licencia CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/). ABSTRACT The incidence and prevalence of inflammatory bowel disease (IBD) has increased inrecent years in several Latin American countries. There is a need to raise awareness in gastro-enterologists and the population in general, so that early diagnosis and treatment of ulcerativecolitis (UC) and Crohn’s Disease (CD) can be carried out. It is important for all physicians tohave homogeneous criteria regarding the diagnosis and treatment of IBD in Latin America. ThePan American Crohn’s and Colitis Organisation (PANCCO) is an organization that aims to includeall the countries of the Americas, but it specifically concentrates on Latin America. The presentConsensus was divided into two parts for publication: 1) Diagnosis and treatment and 2) Specialsituations.This is the first Latin American Consensus whose purpose is to promote a perspective adaptedto our Latin American countries for the diagnosis, treatment, and monitoring of patients withUC and CD.© 2016 Asociaci´on Mexicana de Gastroenterolog´ıa. Published by Masson Doyma M´exico S.A. Thisis an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Situaciones especiales en la enfermedad inflamatoria intestinal: primer consenso latinoamericano de la Pan American Crohn's and Colitis Organisation (PANCCO) (Segunda parte)

En los pacientes con enfermedad inflamatoria intestinal (EII) la fertilidad generalmente no se ve afectada (Nivel de evidencia: 3. Nivel de acuerdo: 100%) excepto en los pacientes con enfermedad de Crohn (EC) activa, mujeres con un historial de cirugía pélvica (Nivel de evidencia: 1. Nivel de acuerdo: 100%) u hombres que reciben tratamiento con sulfasalazina (Nivel de evidencia: 3. Nivel de acuerdo: 100%)

Reduction of Cross-Reactive Carbohydrate Determinants in Plant Foodstuff: Elucidation of Clinical Relevance and Implications for Allergy Diagnosis

Background: A longstanding debate in allergy is whether or not specific immunoglobulin-E antibodies (sIgE), recognizing cross-reactive carbohydrate determinants (CCD), are able to elicit clinical symptoms. In pollen and food allergy, $20% of patients display in-vitro CCD reactivity based on presence of a1,3-fucose and/or b1,2-xylose residues on N-glycans of plant (xylose/fucose) and insect (fucose) glycoproteins. Because the allergenicity of tomato glycoallergen Lyc e 2 was ascribed to N-glycan chains alone, this study aimed at evaluating clinical relevance of CCD-reduced foodstuff in patients with carbohydrate-specific IgE (CCD-sIgE). Methodology/Principal Findings: Tomato and/or potato plants with stable reduction of Lyc e 2 (tomato) or CCD formation in general were obtained via RNA interference, and gene-silencing was confirmed by immunoblot analyses. Two different CCD-positive patient groups were compared: one with tomato and/or potato food allergy and another with hymenopteravenom allergy (the latter to distinguish between CCD- and peptide-specific reactions in the food-allergic group). Nonallergic and CCD-negative food-allergic patients served as controls for immunoblot, basophil activation, and ImmunoCAP analyses. Basophil activation tests (BAT) revealed that Lyc e 2 is no key player among other tomato (glyco)allergens. CCDpositive patients showed decreased (re)activity with CCD-reduced foodstuff, most obvious in the hymenoptera venomallergic but less in the food-allergic group, suggesting that in-vivo reactivity is primarily based on peptide- and not CCDsIgE. Peptide epitopes remained unaffected in CCD-reduced plants, because CCD-negative patient sera showed reactivity similar to wild-type. In-house-made ImmunoCAPs, applied to investigate feasibility in routine diagnosis, confirmed BAT results at the sIgE level. Conclusions/Significance: CCD-positive hymenoptera venom-allergic patients (control group) showed basophil activation despite no allergic symptoms towards tomato and potato. Therefore, this proof-of-principle study demonstrates feasibility of CCD-reduced foodstuff to minimize ‘false-positive results’ in routine serum tests. Despite confirming low clinical relevance of CCD antibodies, we identified one patient with ambiguous in-vitro results, indicating need for further component-resolved diagnosis

Consenso mexicano sobre probióticos en gastroenterología

Introducción: El uso de los probióticos es común en la práctica clínica. Existe un número signi-ficativo de estudios que apoyan la eficacia de los probióticos en algunos trastornos digestivos.Sin embargo, el desconocimiento de la evidencia científica y las diferentes presentaciones ycomposiciones microbianas de los probióticos disponibles dificultan su prescripción.Objetivo: Proveer al clínico de una revisión consensuada sobre los probióticos y recomendacio-nes de su uso en gastroenterología.Material y métodos: Se seleccionaron los ensayos clínicos controlados, metaanálisis y revisio-nes sistemáticas publicados hasta 2015, usando los términos MESH: probiotics, gastrointestinaldiseases, humans, adults and children. Se utilizó la metodología Delphi. Diecisiete gastroente-rólogos de adultos y 12 de ni˜nos elaboraron enunciados los cuales fueron votados hasta obteneracuerdo > 70%. Para cada enunciado se evaluó el nivel de evidencia basado en el sistema GRADE.Resultados y conclusiones: Se generaron 11 enunciados sobre conceptos generales de probió-ticos y 27 enunciados sobre uso de probióticos en enfermedades gastrointestinales tanto enni˜nos como en adultos. El grupo de consenso recomienda el uso de probióticos en las siguientescondiciones clínicas: prevención de la diarrea asociada a antibióticos, tratamiento de la diarreaaguda infecciosa, prevención de infección por Clostridium difficile y enterocolitis necrosante,para disminuir los eventos adversos de la terapia de erradicación del Helicobacter pylori, elalivio de los síntomas del síndrome de intestino irritable, en el estre˜nimiento funcional deladulto, para inducir y mantener la remisión en pacientes con colitis ulcerosa crónica idiopáticay pouchitis, y en la encefalopatía hepática oculta y manifiesta.© 2016 Asociaci´on Mexicana de Gastroenterolog´ıa. Publicado por Masson Doyma M´exico S.A.Este es un art´ıculo Open Access bajo la licencia CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/). ASTRACT Introduction: Probiotics are frequently prescribed in clinical practice. Their efficacy in treatinggastrointestinal disorders is supported by a significant number of clinical trials. However, thecorrect prescription of these agents is hampered due to a lack of knowledge of the scientificevidence and to the different presentations and microbial compositions of the probiotics thatare currently available.Aim: To provide the clinician with a consensus review of probiotics and recommendations fortheir use in gastroenterology.Materials and methods: Controlled clinical trials, meta-analyses, and systematic reviewspublished up to 2015 were selected, using the MESH terms: probiotics, gastrointestinal diseases,humans, adults, AND children. The Delphi method was employed. Eighteen gastroenterologiststreating adult patients and 14 pediatric gastroenterologists formulated statements that werevoted on until agreement > 70% was reached. The level of evidence based on the GRADE systemwas evaluated for each statement.Results and conclusions: Eleven statements on the general concepts of probiotics and 27 sta-tements on the use of probiotics in gastrointestinal diseases in both adults and children wereformulated. The consensus group recommends the use of probiotics under the following clini-cal conditions: the prevention of diarrhea associated with antibiotics, the treatment of acuteinfectious diarrhea, the prevention of Clostridium difficile infection and necrotizing enteroco-litis, the reduction of adverse events from Helicobacter pylori eradication therapy, relief fromirritable bowel syndrome symptoms, the treatment of functional constipation in the adult, and the induction and maintenance of remission in patients with ulcerative colitis and pouchitis,and the treatment of covert and overt hepatic encephalopathy.© 2016 Asociaci´on Mexicana de Gastroenterolog´ıa. Published by Masson Doyma M´exico S.A. Thisis an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Update of the PANCCO clinical practice guidelines for the treatment of ulcerative colitis in the adult population

Ulcerative colitis (US) is a chronic disease of unknown etiology. It is incurable and its clinical course is intermittent, characterized by periods of remission and relapse. The prevalence and incidence of the disease has been increasing worldwide. The update presented herein includes the participation of healthcare professionals, decision-makers, and a representative of the patients, all of whom declared their conflicts of interest. Answerable clinical questions were formulated, and the outcomes were graded. The information search was conducted on the Medline/PubMed, Embase, Epistemonikos, and LILACS databases, and covered grey literature sources, as well. The search was updated on November 30, 2020, with no restrictions regarding date or language. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) classification system was implemented to establish the strength of the recommendation and quality of evidence. A formal consensus was developed, based on the RAND/UCLA methodology and the document was peer reviewed. The short version of the Clinical Practice Guidelines for the Treatment of Ulcerative Colitis in the Adult Population is presented herein, together with the supporting evidence and respective recommendations. In mild-to-moderate UC, budesonide MMX is an option when treatment with 5-ASA fails, and before using systemic steroids. In moderate-to-severe UC, infliximab, adalimumab, vedolizumab, ustekinumab, and tofacitinib can be used as first-line therapy. If there is anti-TNF therapy failure, ustekinumab and tofacitinib provide the best results. In patients with antibiotic-refractory pouchitis, anti-TNFs are the treatment of choice

Consenso Mexicano para el Tratamiento de la Hepatitis C

El objetivo del Consenso Mexicano para el Tratamiento de la Hepatitis C fue el de desarrollar un documento como guía en la práctica clínica con aplicabilidad en México. Se tomó en cuenta la opinión de expertos en el tema con especialidad en: gastroenterología, infectología y hepatología. Se realizó una revisión de la bibliografía en MEDLINE, EMBASE y CENTRAL mediante palabras claves referentes al tratamiento de la hepatitis C. Posteriormente se evaluó la calidad de la evidencia mediante el sistema GRADE y se redactaron enunciados, los cuales fueron sometidos a voto mediante un sistema modificado Delphi, y posteriormente se realizó revisión y corrección de los enunciados por un panel de 34 votantes. Finalmente se clasificó el nivel de acuerdo para cada oración. Esta guía busca dar recomendaciones con énfasis en los nuevos antivirales de acción directa y de esta manera facilitar su uso en la práctica clínica. Cada caso debe ser individualizado según sus comorbilidades y el manejo de estos pacientes siempre debe ser multidisciplinario. Abstract The aim of the Mexican Consensus on the Treatment of Hepatitis C was to develop clinical practice guidelines applicable to Mexico. The expert opinion of specialists in the following areas was taken into account: gastroenterology, infectious diseases, and hepatology. A search of the medical literature was carried out on the MEDLINE, EMBASE, and CENTRAL databases through keywords related to hepatitis C treatment. The quality of evidence was subsequently evaluated using the GRADE system and the consensus statements were formulated. The statements were then voted upon, using the modified Delphi system, and reviewed and corrected by a panel of 34 voting participants. Finally, the level of agreement was classified for each statement. The present guidelines provide recommendations with an emphasis on the new direct-acting antivirals, to facilitate their use in clinical practice. Each case must be individualized according to the comorbidities involved and patient management must always be multidisciplinary

El ácido acetilsalicílico inhibe la replicación del virus de la hepatitis a través de la vía de señalización de la ciclooxigena-2

Existen reportes de que el salicilato de sodio y la aspirina disminuyen la replicación de flavivirus. Por lo anterior, se consideró importante estudiar el efecto del AAS contra el VHC. Para ello se evaluó el efecto del AAS en la replicación y expresión de las proteínas virales, en el sistema de replicones subgenómicos del VHC. Las células fueron incubadas con 2-8 mM de AAS a diferentes tiempos y se determinaron los niveles del RNA y proteínas virales por PCR en tiempo real y western blot, respectivamente. Se observó que el AAS disminuyó los niveles del RNA y proteínas virales. También se encontró que el VHC indujo la expresión, síntesis y actividad de COX-2. Efectos que fueron regulados negativamente por AAS. Así mismo, la inhibición de p38 y MEK1/2 revirtió el efecto del AAS. Nuestros resultados sugieren que el efecto antiviral del AAS puede estar mediado por la vía COX-2/p38/MEK1/2; y la posibilidad de que el AAS podría ser un excelente adyuvante en el tratamiento de la infección crónica por VHC. It has been reported that sodium salicylate and aspirin inhibit the replication of flaviviruses. Therefore, we considered it important to test whether ASA had anti-HCV activity. To this end, we examined the effects of ASA on viral replication and protein expression, using an HCV subgenomic replicon cell culture system. We incubated these cells with 2-8 mM ASA at different times and measured HCV-RNA and protein levels by real time PCR and western blot analysis, respectively. We found that ASA has suppressive effect on HCVRNA and protein levels. We also observed that HCV-induced the expression, synthesis, and activity of COX-2; these effects were down-regulated by ASA. Inhibition of p38 and MEK1/2 MAPK prevented the antiviral effect of ASA. Our findings suggest that the anti-HCV effect of ASA can be mediated by COX-2/p38/MEK1/2 pathway; and the possibility that ASA could be an excellent adjuvant in the treatment of chronic HCV infection