Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization

International audienceUnconventional protein secretion represents an important process of the inflammatory response. The release of the pro-inflammatory cytokine interleukin (IL)-1β which burst during pyroptosis as a consequence of gasdermin D plasma membrane pore formation, can also occur through other unconventional secretion pathways dependent on caspase-1 activation. However, how caspase-1 mediates cytokine release independently of gasdermin D remains poorly understood. Here we show that following caspase-1 activation by different inflammasomes, caspase-1 cleaves early endosome autoantigen 1 (EEA1) protein at Asp127/132. Caspase-1 activation also results in the release of the endosomal EEA1 protein in a gasdermin D-independent manner. EEA1 knock-down results in adecreased release of caspase-1 and IL-1β, but the pyroptotic release of other inflammasome components and lactate dehydrogenase was not affected. This study shows how caspase-1 control the release of EEA1 and IL-1β in a pyroptotic-independent manner

ASC oligomer favors caspase-1CARD domain recruitment after intracellular potassium efflux

International audienceSignaling through the inflammasome is important for the inflammatory response. Low concentrations of intracellular K+ are associated with the specific oligomerization and activation of the NLRP3 inflammasome, a type of inflammasome involved in sterile inflammation. After NLRP3 oligomerization, ASC protein binds and forms oligomeric filaments that culminate in large protein complexes named ASC specks. ASC specks are also initiated from different inflammasome scaffolds, such as AIM2, NLRC4, or Pyrin. ASC oligomers recruit caspase-1 and then induce its activation through interactions between their respective caspase activation and recruitment domains (CARD). So far, ASC oligomerization and caspase-1 activation are K+-independent processes. Here, we found that when there is low intracellular K+, ASC oligomers change their structure independently of NLRP3 and make the ASCCARD domain more accessible for the recruitment of the pro-caspase-1CARD domain. Therefore, conditions that decrease intracellular K+ not only drive NLRP3 responses but also enhance the recruitment of the pro-caspase-1 CARD domain into the ASC specks

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton's tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1β release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation

Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation

The NLRP3 inflammasome is activated by a wide range of stimuli and drives diverse inflammatory diseases. The decrease of intracellular K+ concentration is a minimal upstream signal to most of the NLRP3 activation models. Here, we found that cellular K+ efflux induces a stable structural change in the inactive NLRP3, promoting an open conformation as a step preceding activation. This conformational change is facilitated by the specific NLRP3 FISNA domain and a unique flexible linker sequence between the PYD and FISNA domains. This linker also facilitates the ensemble of NLRP3PYD into a seed structure for ASC oligomerization. The introduction of the NLRP3 PYD-linker-FISNA sequence into NLRP6 resulted in a chimeric receptor able to be activated by K+ efflux–specific NLRP3 activators and promoted an in vivo inflammatory response to uric acid crystals. Our results establish that the amino-terminal sequence between PYD and NACHT domain of NLRP3 is key for inflammasome activation.I.H.-B. would like to acknowledge the funding by the Slovenian Research Agency (project grant J3-1746 and core funding P4-0176), and B.O. would like to acknowledge the funding by the Ministerio de Economía, Industria y Competitividad and ERDF (BIO2017-85329-R). This work was supported by grants to A.T.-A. from the internal support program of the Medical Faculty, University of Tübingen, Fortüne-Antrag Nr. 2615-0-0 and to P.P. from FEDER/Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación (grant SAF2017-88276-R), Fundación Séneca (grants 20859/PI/18, 21081/PDC/19, and 0003/COVI/20), and the European Research Council (ERC-2013-CoG grant 614578 and ERC-2019-PoC grant 899636)

Estudio de la expresión de VSTM1 en macrófagos humanos y su posible implicación en el desarrollo de cirrosis hepática

RESUMEN Uno de los objetivos de este trabajo fue el estudio bibliográfico del gen VSTM1, que codifica un receptor inhibidor recientemente descrito en fagocitos. Para ello se revisó la bibliografía publicada en revistas de investigación científica, así como las secuencias depositadas en las principales bases de datos biológicos disponibles en la red hasta el momento. Los resultados encontrados revelaron que la información depositada en las bases de datos por los diferentes autores era redundante y presentaban una nomenclatura diferente. Tras estudios de alineamiento con diferentes herramientas informáticas, pudimos establecer las equivalencias entre los datos publicados. De las 6 isoformas descritas, generadas por maduración alternativa del ARN, sólo algunas secuencias coincidieron en las bases de datos consultadas. Además, se detectó un error en la notación de la secuencia ACU00108, ya que se tradujo en glicina un codón que codifica para serina. El siguiente objetivo fue el estudio de la expresión del gen VSTM1 mediante qRT-PCR en macrófagos peritoneales obtenidos del líquido ascítico de pacientes con cirrosis hepática, y su comparación con los obtenidos para macrófagos de sangre periférica de donantes sanos, como población de referencia. Esta patología se asociada a un incremento de mediadores inflamatorios y podría estar relacionada con la desregulación de algunos de los mecanismos de control del sistema inmunitario. La qRT-PCR realizada con los cebadores diseñados para estudiar las isoformas VSTM1-v1 y v2, reveló que éstas se expresan en ambas poblaciones de macrófagos, siendo mayores los niveles de expresión del transcrito que codifica la isoforma proteíca de membrana (v1) que la secretada (v2). Además, la expresión génica de VSTM1-v1 aumenta con la diferenciación del macrófago. Sin embargo, aunque se aprecia una tendencia hacia una menor expresión en la población de macrófagos de pacientes con cirrosis, estas diferencias no fueron estadísticamente significativas.ABSTRACT One of the goals of this work was the bibliography study of the gene VSTM1, which encodes an inhibitory receptor recently described on phagocytes. To do this, we reviewed the bibliography from several scientific journals, as well as the sequences that so far now we can find in the main biological databases on the internet. Our results showed that the information available in the different databases was redundant and a different nomenclature was used for unique sequences. Using alignment studies with different bioinformatic tools we established equivalences between the published data. Taking into account the 6 isoforms described, generated by alternative splicing of the RNA, only some of them matched in the data bases revised. Furthermore, we detected a mistake in the sequence ACUU108, where a codon which encodes a serine was translated into glycine. Our next objective was to study the expression profile of the gene VSTM1, using qRT-PCR, on peritoneal macrophages obtained from ascitic fluid from patients with hepatic cirrhosis, and compared it with the expression on peripheral blood macrophages from healthy donors, which we used as a referential population. This pathology is related to an increase of inflammatory mediators that could be produced by a dysregulation of some control mechanisms involved in immune response. qRT-PCR performance using specific primers designed for VSTM1-v1 and v2, showed that these isoforms were expressed on both macrophage populations, and the level of expression was higher for the transcript which encodes the membrane protein (v1) than for the secreted one (v2). Moreover, we described that gene expression of VSTM1-v1 increases during macrophage differentiation. Nevertheless, although a tendency to a lower expression in cirrhotic macrophages was appreciated, this was not statistical significant

Estudio de la expresión del receptor LAIR-1 en macrófagos humanos. Posible implicación en el desarrollo de cirrosis hepática

RESUMEN El primer objetivo de este trabajo fue el estudio bibliográfico del gen LAIR-1, que codifica un receptor inhibidor descrito en fagocitos. Para ello se revisó la bibliografía publicada en revistas de investigación científica, así como las secuencias depositadas en las principales bases de datos biológicos disponibles en la red hasta el momento. Los resultados encontrados revelaron que la información depositada en las bases de datos por los diferentes autores era redundante y presentaba una nomenclatura diferente. Tras estudios de alineamiento con diferentes herramientas informáticas, pudimos establecer las equivalencias entre los datos publicados. De las 4 isoformas descritas, generadas por maduración alternativa del ARN, sólo LAIR-1a y LAIR-1b coincidieron completamente en las bases de datos consultadas. El siguiente objetivo fue el estudio de la expresión del gen LAIR-1 mediante qRT-PCR en macrófagos peritoneales obtenidos del líquido ascítico de pacientes con cirrosis hepática, y su comparación con los obtenidos para macrófagos de sangre periférica de donantes sanos, como población de referencia. Esta patología se asocia a un incremento de mediadores inflamatorios y podría estar relacionada con la desregulación de algunos de los mecanismos de control del sistema inmunitario. La qRT-PCR realizada con los cebadores diseñados para estudiar las isoformas LAIR-1a y -1b, reveló que éstas se expresan en ambas poblaciones de macrófagos. Además, los resultados mostraron que los niveles de expresión para los transcritos que codifican ambas isoformas son menores en la población de macrófagos de pacientes con cirrosis, en comparación con los macrófagos de donantes sanos. Se encontraron además diferencias significativas entre ambas poblaciones de macrófagos en la capacidad de respuesta a estímulos activadores como LPS y C. albicans. Estos resultados podrían contribuir a identificar nuevas dianas terapéuticas o diagnósticas para prevenir el daño hepático.ABSTRACT Our first goal in this work was the bibliographic study of the gene LAIR-1, which encodes an inhibitory receptor described on phagocytes. To do this, we reviewed the current bibliography from several scientific journals, as well as the sequences that so far now we can find in the main biological databases on the internet. Our results showed that the information available in the different databases was redundant and a different nomenclature was used for unique sequences. Using alignment studies with different bioinformatic tools, we established equivalences between the published data. Taking into account the 4 isoforms described, generated by alternative splicing of the RNA, only LAIR-1a and LAIR-1b matched in the databases reviewed. Our next objective was to study the expression profile of the gene LAIR-1, using qRT-PCR, on peritoneal macrophages obtained from ascitic fluid from patients with hepatic cirrhosis, and compared it with the expression on peripheral blood macrophages from healthy donors, which we used as a referential population. This pathology is related to an increase of inflammatory mediators that could be produced by a dysregulation of some control mechanisms involved in immune response. qRT-PCR performance using specific primers designed for LAIR-1a and -1b, showed that these isoforms were expressed on both macrophage populations. Results also showed significant differences between both macrophage populations in responsiveness to LPS and C. albicans activators. These results may help to identify new prognostic or therapeutic targets to prevent hepatic damage

Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production

Abstract Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory condition resulting from monoallelic NLRP3 variants that facilitate IL-1β production. Although these are gain-of-function variants characterized by hypersensitivity to cell priming, patients with CAPS and animal models of the disease may present inflammatory flares without identifiable external triggers. Here we find that CAPS-associated NLRP3 variants are forming constitutively active inflammasome, which induce increased basal cleavage of gasdermin D, IL-18 release and pyroptosis, with a concurrent basal pro-inflammatory gene expression signature, including the induction of nuclear receptors 4 A. The constitutively active NLRP3-inflammasome of CAPS is responsive to the selective NLRP3 inhibitor MCC950 and its activation is regulated by deubiquitination. Despite their preactivated state, the CAPS inflammasomes are responsive to activation of the NF-κB pathway. NLRP3-inflammasomes with CAPS-associated variants affect the immunometabolism of the myeloid compartment, leading to disruptions in lipids and amino acid pathways and impaired glycolysis, limiting IL-1β production. In summary, NLRP3 variants causing CAPS form a constitutively active inflammasome inducing pyroptosis and IL-18 release without cell priming, which enables the host’s innate defence against pathogens while also limiting IL-1β–dependent inflammatory episodes through immunometabolism modulation