RESUMEN
BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is a complication of cirrhosis characterized by multiple organ failure and high short-term mortality. The pathophysiology of ACLF involves elevated systemic inflammation leading to organ failure, along with immune dysfunction that heightens susceptibility to bacterial infections. However, it is unclear how these aspects are associated with recovery and nonrecovery in ACLF. APPROACH AND RESULTS: Here, we mapped the single-cell transcriptome of circulating immune cells from patients with ACLF and acute decompensated (AD) cirrhosis and healthy individuals. We further interrogate how these findings, as well as immunometabolic and functional profiles, associate with ACLF-recovery (ACLF-R) or nonrecovery (ACLF-NR). Our analysis unveiled 2 distinct states of classical monocytes (cMons). Hereto, ACLF-R cMons were characterized by transcripts associated with immune and stress tolerance, including anti-inflammatory genes such as RETN and LGALS1 . Additional metabolomic and functional validation experiments implicated an elevated oxidative phosphorylation metabolic program as well as an impaired ACLF-R cMon functionality. Interestingly, we observed a common stress-induced tolerant state, oxidative phosphorylation program, and blunted activation among lymphoid populations in patients with ACLF-R. Conversely, ACLF-NR cMon featured elevated expression of inflammatory and stress response genes such as VIM , LGALS2 , and TREM1 , along with blunted metabolic activity and increased functionality. CONCLUSIONS: This study identifies distinct immunometabolic cellular states that contribute to disease outcomes in patients with ACLF. Our findings provide valuable insights into the pathogenesis of ACLF, shedding light on factors driving either recovery or nonrecovery phenotypes, which may be harnessed as potential therapeutic targets in the future.
RESUMEN
BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), monocytes infiltrate visceral adipose tissue promoting local and hepatic inflammation. However, it remains unclear what drives inflammation and how the immune landscape in adipose tissue differs across the NAFLD severity spectrum. We aimed to assess adipose tissue macrophage (ATM) heterogeneity in a NAFLD cohort. METHODS: Visceral adipose tissue macrophages from lean and obese patients, stratified by NAFLD phenotypes, underwent single-cell RNA sequencing. Adipose tissue vascular integrity and breaching was assessed on a protein level via immunohistochemistry and immunofluorescence to determine targets of interest. RESULTS: We discovered multiple ATM populations, including resident vasculature-associated macrophages (ResVAMs) and distinct metabolically active macrophages (MMacs). Using trajectory analysis, we show that ResVAMs and MMacs are replenished by a common transitional macrophage (TransMac) subtype and that, during NASH, MMacs are not effectively replenished by TransMac precursors. We postulate an accessory role for MMacs and ResVAMs in protecting the adipose tissue vascular barrier, since they both interact with endothelial cells and localize around the vasculature. However, across the NAFLD severity spectrum, alterations occur in these subsets that parallel an adipose tissue vasculature breach characterized by albumin extravasation into the perivascular tissue. CONCLUSIONS: NAFLD-related macrophage dysfunction coincides with a loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. IMPACT AND IMPLICATIONS: Our study describes for the first time the myeloid cell landscape in human visceral adipose tissue at single-cell level within a cohort of well-characterized patients with non-alcoholic fatty liver disease. We report unique non-alcoholic steatohepatitis-specific transcriptional changes within metabolically active macrophages (MMacs) and resident vasculature-associated macrophages (ResVAMs) and we demonstrate their spatial location surrounding the vasculature. These dysfunctional transcriptional macrophage states coincided with the loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. Our study provides a theoretical basis for new therapeutic strategies to be directed towards reinstating the endogenous metabolic, homeostatic and cytoprotective functions of ResVAMs and MMacs, including their role in protecting vascular integrity.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Células Endoteliales/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Tejido Adiposo/metabolismo , Inflamación/metabolismoRESUMEN
Muscle-invasive bladder cancer (MIBC) remains a pressing health concern due to conventional treatment failure and significant molecular heterogeneity, hampering the development of novel targeted therapeutics. In our quest for novel targetable markers, recent glycoproteomics and bioinformatics data have pinpointed (glucose transporter 1) GLUT1 as a potential biomarker due to its increased expression in tumours compared to healthy tissues. This study explores this hypothesis in more detail, with emphasis on GLUT1 glycosylation patterns and cancer specificity. Immunohistochemistry analysis across a diverse set of human bladder tumours representing all disease stages revealed increasing GLUT1 expression with lesion severity, extending to metastasis, while remaining undetectable in healthy urothelium. In line with this, GLUT1 emerged as a marker of reduced overall survival. Revisiting nanoLC-EThcD-MS/MS data targeting immature O-glycosylation on muscle-invasive tumours identified GLUT1 as a carrier of short glycosylation associated with invasive disease. Precise glycosite mapping uncovered significant heterogeneity between patient samples, but also common glycopatterns that could provide the molecular basis for targeted solutions. Immature O-glycosylation conferred cancer specificity to GLUT1, laying the molecular groundwork for enhanced targeted therapeutics in bladder cancer. Future studies should focus on a comprehensive mapping of GLUT1 glycosites for highly specific cancer-targeted therapy development for bladder cancer.
Asunto(s)
Espectrometría de Masas en Tándem , Neoplasias de la Vejiga Urinaria , Humanos , Glicosilación , Transportador de Glucosa de Tipo 1/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Vejiga Urinaria/patologíaRESUMEN
Prion proteins constitute a major public health concern, which has partly overshadowed their physiological roles in several scenarios. Indeed, these proteins were implicated in male fertility but their role in female fertility is relatively less explored. This study was designed to evaluate the role of SPRN and PRNP prion family genes in bovine follicular steroidogenesis pathways. Post-transcriptional SPRN and PRNP silencing with siRNAs was established in bovine granulosa cell (GC) in vitro culture, and gene expression and progesterone and estradiol concentrations were evaluated. SPRN knockdown, led to a downregulation of CYP11A1 mRNA levels (2.1-fold), and PRNP knockdown led to an upregulation of SPRN mRNA levels (2.3-fold). CYP19A1 expression and estradiol synthesis was not detected in any experimental group. Finally, SPRN knockdown led to a mild reduction in progesterone production in GCs and this was the only experimental group that did not exhibit an increment in progesterone levels after 48 h of culture. As a conclusion, it was possible to detect the expression of the SPRN gene in bovine GCs, a potential interaction between SPRN and PRNP regulation, and the impact of SPRN expression on CYP11A1 and progesterone levels. These findings bring new insights into the role of these genes in ovarian steroidogenesis and female reproductive physiology.
Asunto(s)
Estradiol/metabolismo , Células de la Granulosa/fisiología , Proteínas Priónicas/genética , Progesterona/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Bovinos , Células Cultivadas , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Estradiol/genética , Femenino , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Priónicas/metabolismo , Progesterona/genética , Interferencia de ARNRESUMEN
Regulatory T cells (Treg) play a critical role in immune tolerance. The scarcity of Treg therapy clinical trials in humans has been largely due to the difficulty in obtaining sufficient Treg numbers. We performed a preclinical investigation on the potential of mesenchymal stromal cells (MSCs) to expand Treg in vitro to support future clinical trials. Human peripheral blood mononuclear cells from healthy donors were cocultured with allogeneic bone marrow-derived MSCs expanded under xenogeneic-free conditions. Our data show an increase in the counts and frequency of CD4+ CD25high Foxp3+ CD127low Treg cells (4- and 6-fold, respectively) after a 14-day coculture. However, natural Treg do not proliferate in coculture with MSCs. When purified conventional CD4 T cells (Tcon) are cocultured with MSCs, only cells that acquire a Treg-like phenotype proliferate. These MSC-induced Treg-like cells also resemble Treg functionally, since they suppress autologous Tcon proliferation. Importantly, the DNA methylation profile of MSC-induced Treg-like cells more closely resembles that of natural Treg than of Tcon, indicating that this population is stable. The expression of PD-1 is higher in Treg-like cells than in Tcon, whereas the frequency of PDL-1 increases in MSCs after coculture. TGF-ß levels are also significantly increased MSC cocultures. Overall, our data suggest that Treg enrichment by MSCs results from Tcon conversion into Treg-like cells, rather than to expansion of natural Treg, possibly through mechanisms involving TGF-ß and/or PD-1/PDL-1 expression. This MSC-induced Treg population closely resembles natural Treg in terms of phenotype, suppressive ability, and methylation profile.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Células Madre Mesenquimatosas/citología , Linfocitos T Reguladores/citología , Linfocitos T CD4-Positivos/metabolismo , Metilación de ADN , Humanos , Células Madre Mesenquimatosas/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
Esophageal cancer (EC) is a life-threatening disease, demanding the discovery of new biomarkers and molecular targets for precision oncology. Aberrantly glycosylated proteins hold tremendous potential towards this objective. In the current study, a series of esophageal squamous cell carcinomas (ESCC) and EC-derived circulating tumor cells (CTCs) were screened by immunoassays for the sialyl-Tn (STn) antigen, a glycan rarely expressed in healthy tissues and widely observed in aggressive gastrointestinal cancers. An ESCC cell model was glycoengineered to express STn and characterized in relation to cell proliferation and invasion in vitro. STn was found to be widely present in ESCC (70% of tumors) and in CTCs in 20% of patients, being associated with general recurrence and reduced survival. Furthermore, STn expression in ESCC cells increased invasion in vitro, while reducing cancer cells proliferation. In parallel, an ESCC mass spectrometry-based proteomics dataset, obtained from the PRIDE database, was comprehensively interrogated for abnormally glycosylated proteins. Data integration with the Target Score, an algorithm developed in-house, pinpointed the glucose transporter type 1 (GLUT1) as a biomarker of poor prognosis. GLUT1-STn glycoproteoforms were latter identified in tumor tissues in patients facing worst prognosis. Furthermore, healthy human tissues analysis suggested that STn glycosylation provided cancer specificity to GLUT1. In conclusion, STn is a biomarker of worst prognosis in EC and GLUT1-STn glycoforms may be used to increase its specificity on the stratification and targeting of aggressive ESCC forms.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , Transportador de Glucosa de Tipo 1/metabolismo , Proteoma/análisis , Programas Informáticos , Antígenos de Carbohidratos Asociados a Tumores/química , Apoptosis , Proliferación Celular , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/metabolismo , Regulación Neoplásica de la Expresión Génica , Transportador de Glucosa de Tipo 1/química , Glicosilación , Humanos , Masculino , Persona de Mediana Edad , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Pronóstico , Estudios Prospectivos , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Acute-on-chronic liver failure (ACLF) is associated with dysfunctional circulating monocytes whereby patients become highly susceptible to bacterial infections. Here, we identify the pathways underlying monocyte dysfunction in ACLF and we investigate whether metabolic rewiring reinstates their phagocytic and inflammatory capacity. DESIGN: Following phenotypic characterisation, we performed RNA sequencing on CD14+CD16- monocytes from patients with ACLF and decompensated alcoholic cirrhosis. Additionally, an in vitro model mimicking ACLF patient-derived features was implemented to investigate the efficacy of metabolic regulators on monocyte function. RESULTS: Monocytes from patients with ACLF featured elevated frequencies of interleukin (IL)-10-producing cells, reduced human leucocyte antigen DR isotype (HLA-DR) expression and impaired phagocytic and oxidative burst capacity. Transcriptional profiling of isolated CD14+CD16- monocytes in ACLF revealed upregulation of an array of immunosuppressive parameters and compromised antibacterial and antigen presentation machinery. In contrast, monocytes in decompensated cirrhosis showed intact capacity to respond to inflammatory triggers. Culturing healthy monocytes in ACLF plasma mimicked the immunosuppressive characteristics observed in patients, inducing a blunted phagocytic response and metabolic program associated with a tolerant state. Metabolic rewiring of the cells using a pharmacological inhibitor of glutamine synthetase, partially restored the phagocytic and inflammatory capacity of in vitro generated- as well as ACLF patient-derived monocytes. Highlighting its biological relevance, the glutamine synthetase/glutaminase ratio of ACLF patient-derived monocytes positively correlated with disease severity scores. CONCLUSION: In ACLF, monocytes feature a distinct transcriptional profile, polarised towards an immunotolerant state and altered metabolism. We demonstrated that metabolic rewiring of ACLF monocytes partially revives their function, opening up new options for therapeutic targeting in these patients.
Asunto(s)
Insuficiencia Hepática Crónica Agudizada/tratamiento farmacológico , Infecciones Bacterianas/tratamiento farmacológico , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Inmunosupresores/uso terapéutico , Monocitos/enzimología , Insuficiencia Hepática Crónica Agudizada/inmunología , Insuficiencia Hepática Crónica Agudizada/metabolismo , Adulto , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/patología , Células Cultivadas , Citocinas/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Fagocitosis , Estudios RetrospectivosRESUMEN
CD44 is a heavily glycosylated membrane receptor playing a key role in cell adhesion, signal transduction and cytoskeleton remodelling. It is also one of the most studied glycoproteins in cancer, frequently explored for stem cell identification, and associated with chemoresistance and metastasis. However, CD44 is a general designation for a large family of splicing variants exhibiting different degrees of glycosylation and, potentially, functionally distinct roles. Moreover, structural diversity associated with ambiguous nomenclature has delayed clinical developments. Herein, we attempt to comprehensively address these aspects and systematize CD44 nomenclature, setting milestones for biomarker discovery. In addition, we support that CD44 may be an important source of cancer neoantigens, most likely resulting from altered splicing and/or glycosylation. The discovery of potentially targetable CD44 (glyco)isoforms will require the combination of glycomics with proteogenomics approaches, exploring customized protein sequence databases generated using genomics and transcriptomics. Nevertheless, the necessary high-throughput analytical and bioinformatics tools are now available to address CD44 role in health and disease.
RESUMEN
Amphetamines are a class of psychotropic drugs with high abuse potential, as a result of their stimulant, euphoric, emphathogenic, entactogenic, and hallucinogenic properties. Although most amphetamines are synthetic drugs, of which methamphetamine, amphetamine, and 3,4-methylenedioxymethamphetamine ("ecstasy") represent well-recognized examples, the use of natural related compounds, namely cathinone and ephedrine, has been part of the history of humankind for thousands of years. Resulting from their amphiphilic nature, these drugs can easily cross the blood-brain barrier and elicit their well-known psychotropic effects. In the field of amphetamines' research, there is a general consensus that mitochondrial-dependent pathways can provide a major understanding concerning pathological processes underlying the neurotoxicity of these drugs. These events include alterations on tricarboxylic acid cycle's enzymes functioning, inhibition of mitochondrial electron transport chain's complexes, perturbations of mitochondrial clearance mechanisms, interference with mitochondrial dynamics, as well as oxidative modifications in mitochondrial macromolecules. Additionally, other studies indicate that amphetamines-induced neuronal toxicity is closely regulated by B cell lymphoma 2 superfamily of proteins with consequent activation of caspase-mediated downstream cell death pathway. Understanding the molecular mechanisms at mitochondrial level involved in amphetamines' neurotoxicity can help in defining target pathways or molecules mediating these effects, as well as in developing putative therapeutic approaches to prevent or treat the acute- or long-lasting neuropsychiatric complications seen in human abusers.
Asunto(s)
Trastornos Relacionados con Anfetaminas/complicaciones , Mitocondrias/efectos de los fármacos , Síndromes de Neurotoxicidad/etiología , Anfetaminas/administración & dosificación , Anfetaminas/farmacocinética , Anfetaminas/toxicidad , Animales , Barrera Hematoencefálica/metabolismo , Estimulantes del Sistema Nervioso Central/administración & dosificación , Estimulantes del Sistema Nervioso Central/farmacocinética , Estimulantes del Sistema Nervioso Central/toxicidad , Transporte de Electrón/efectos de los fármacos , Humanos , Síndromes de Neurotoxicidad/fisiopatologíaRESUMEN
Patient-centered smartphone applications have potential to support rheumatoid arthritis (RA) self-management but remain almost unexplored in literature. Therefore, this study evaluated the usefulness of a smartphone application to support RA self-management, the willingness of RA patients to use and pay for it and the features the application should have. In this cross-sectional study, a questionnaire was developed to collect information on population, device ownership, usefulness and willingness to use and pay for a RA self-management application and application features. Descriptive statistics, Chi-square, Fisher's exact test, t test or Mann-Whitney's test and multivariate analysis were used. One hundred RA patients answered the questionnaire. Patients' mean age was 57 ± 11.9 years, most were females (91 %), with multiple drug regimens and a 40 % treatment non-compliance rate. Most patients believed that could have a more active role in self-management (94 %) and reported it would be useful to develop a RA self-management application (86 %). Patients willing to use an application (83 %) were younger, with a possible more active role in self-management, with access to a smartphone, and using short message service, electronic mail and Internet. Multivariate analysis confirmed these results, except the associations regarding access to a smartphone and use of electronic mail and Internet. Fifty-eight patients (82 %) were willing to pay for a RA self-management application and the most requested feature for it was information in a simple format. This study suggested the usefulness and patients' willingness to use and pay for a RA self-management application and provided insight on patients' needs.
Asunto(s)
Artritis Reumatoide/terapia , Conductas Relacionadas con la Salud , Necesidades y Demandas de Servicios de Salud , Cooperación del Paciente , Autocuidado , Teléfono Inteligente , Anciano , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Estudios Transversales , Femenino , Humanos , Internet , Masculino , Persona de Mediana Edad , Relaciones Médico-Paciente , Encuestas y CuestionariosRESUMEN
BACKGROUND: Methotrexate (MTX) is widely used for rheumatoid arthritis (RA) treatment. Single nucleotide polymorphisms (SNPs) could be used as predictors of patients' therapeutic outcome variability. Therefore, this study aims to evaluate the influence of SNPs in genes encoding for MTX membrane transport proteins in order to predict clinical response to MTX. METHODS: Clinicopathological data from 233 RA patients treated with MTX were collected, clinical response defined, and patients genotyped for 23 SNPs. Genotype and haplotype analyses were performed using multivariate methods and a genetic risk index (GRI) for non-response was created. RESULTS: Increased risk for non-response was associated to SLC22A11 rs11231809 T carriers; ABCC1 rs246240 G carriers; ABCC1 rs3784864 G carriers; CGG haplotype for ABCC1 rs35592, rs2074087 and rs3784864; and CGG haplotype for ABCC1 rs35592, rs246240 and rs3784864. GRI demonstrated that patients with Index 3 were 16-fold more likely to be non-responders than those with Index 1. CONCLUSIONS: This study revealed that SLC22A11 and ABCC1 may be important to identify those patients who will not benefit from MTX treatment, highlighting the relevance in translating these results to clinical practice. However, further validation by independent studies is needed to develop the field of personalized medicine to predict clinical response to MTX treatment.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Metotrexato/uso terapéutico , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Transportadores de Anión Orgánico Sodio-Independiente/genética , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Antirreumáticos/farmacocinética , Artritis Reumatoide/genética , Femenino , Humanos , Masculino , Metotrexato/farmacocinética , Persona de Mediana Edad , FarmacogenéticaRESUMEN
Tubulin-associated unit (tau) has an important role in the pathogenesis and the diagnosis of Alzheimer's disease (AD) and other tauopathies. In view of the diversity of tau proteoforms, antibody-free methods represent a good approach for unbiased quantification. We adapted and evaluated the single-pot, solid-phase-enhanced sample-preparation (SP3) protocol for antibody-free extraction of the tau protein in cerebro-spinal fluid (CSF) mimic and in human brain. A total of 13 non-modified peptides were quantified by high-resolution mass spectrometry (HRMS) after digestion of tau by trypsin. We significantly improved the basic SP3 protocol by carefully optimizing the organic solvents and incubation time for tau binding, as well as the digestion step for the release directly from the SP3 beads of the 13 tau peptides. These optimizations proved to be primarily beneficial for the most hydrophilic tau peptides, increasing the sequence coverage of recombinant tau. Mean recovery in CSF mimic of the 13 non-modified peptides was of 53%, with LODs ranging from 0.75 to 10â ng/mL. Next, we tested the optimized SP3 protocol on pathological tau extracted from the soluble fraction from an AD brain sample (middle frontal gyrus). We could successfully identify and quantify biologically relevant tau peptides including representative peptides of two isoforms and two phospho-peptides (pTau217 and pTau181).
Asunto(s)
Enfermedad de Alzheimer , Tubulina (Proteína) , Humanos , Encéfalo , Anticuerpos , Espectrometría de Masas , PéptidosRESUMEN
Background and aims: A complete understanding of disease pathophysiology in advanced liver disease is hampered by the challenges posed by clinical specimen collection. Notably, in these patients, a transjugular liver biopsy (TJB) is the only safe way to obtain liver tissue. However, it remains unclear whether successful sequencing of this extremely small and fragile tissue can be achieved for downstream characterization of the hepatic landscape. Methods: Here we leveraged in-house available single-cell RNA-sequencing (scRNA-seq) and single-nucleus (snRNA-seq) technologies and accompanying tissue processing protocols and performed an in-patient comparison on TJB's from decompensated cirrhosis patients (n = 3). Results: We confirmed a high concordance between nuclear and whole cell transcriptomes and captured 31,410 single nuclei and 6,152 single cells, respectively. The two platforms revealed similar diversity since all 8 major cell types could be identified, albeit with different cellular proportions thereof. Most importantly, hepatocytes were most abundant in snRNA-seq, while lymphocyte frequencies were elevated in scRNA-seq. We next focused our attention on hepatic myeloid cells due to their key role in injury and repair during chronic liver disease. Comparison of their transcriptional signatures indicated that these were largely overlapping between the two platforms. However, the scRNA-seq platform failed to recover sufficient Kupffer cell numbers, and other monocytes/macrophages featured elevated expression of stress-related parameters. Conclusion: Our results indicate that single-nucleus transcriptome sequencing provides an effective means to overcome complications associated with clinical specimen collection and could sufficiently profile all major hepatic cell types including all myeloid cell subsets.
Asunto(s)
Perfilación de la Expresión Génica , Hepatopatías , Humanos , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Nuclear Pequeño , Cirrosis Hepática/genéticaRESUMEN
We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state of PR demonstrates the contribution of the ligand to increasing stability of the agonist conformation of helix-12 via a specific hydrogen-bond network including Glu(723). This interaction is absent when the full antagonist, RU486, binds to PR. Combined with a previously reported structure of Asoprisnil bound to the antagonist state of the receptor, this structure extends our understanding of the complex molecular interactions underlying the mixed agonist/antagonist profile of the compound. In addition, we present the structure of PR in its agonist conformation bound to the mixed profile compound Org3H whose reduced antagonistic activity and increased agonistic activity compared with reference antagonists is due to an induced fit around Trp(755), resulting in a decreased steric clash with Met(909) but inducing a new internal clash with Val(912) in helix-12. This structure also explains the previously published observation that 16α attachments to RU486 analogs induce mixed profiles by altering the binding of 11ß substituents. Together these structures further our understanding of the steric and electrostatic factors that contribute to the function of steroid receptor modulators, providing valuable insight for future compound design.
Asunto(s)
Estrenos/química , Mifepristona/química , Oximas/química , Receptores de Progesterona/agonistas , Receptores de Progesterona/química , Cristalografía por Rayos X , Humanos , Ligandos , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Haploidentical hematopoietic stem cell transplantation (HSCT) constitutes an important alternative for patients lacking a human leukocyte antigen (HLA)-matched donor. Although the use of haploidentical donors is increasingly common, the long-term impact of generating a donor-derived immune system in the context of an HLA-mismatched thymic environment remains poorly characterized. We performed an in-depth assessment of immune reconstitution in a group of haploidentical HSCT recipients 4 to 6 years posttransplantation, in parallel with the respective parental donors and age-matched healthy control subjects. Our data show that the proportion of naive and memory subsets in the recipients, both within CD8(+) and CD4(+) T cells, more closely resembled that observed in age-matched control subjects than in the donors. HSCT recipients displayed relatively high signal-joint T cell-receptor excision circle levels and a high frequency of the recent thymic emigrant-enriched CD31(+) subset within naive CD4(+) and naive regulatory T cells. Moreover, CD8(+), CD4(+), and regulatory T cells from HSCT recipients displayed a diverse T cell repertoire. These results support a key role for thymic output in T cell reconstitution. Nevertheless, HSCT recipients had significantly shorter telomeres within a naive-enriched CD4(+) T cell population than age-matched control subjects, despite the similar telomere length observed within the most differentiated CD8(+) and CD4(+) T cell subsets. Overall, our data suggest that long-term immune reconstitution was successfully achieved after haploidentical HSCT, a process that appears to have largely relied on de novo T cell production.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos HLA/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Subgrupos de Linfocitos T/inmunología , Adulto , Anemia Aplásica/inmunología , Anemia Aplásica/cirugía , Estudios Transversales , Femenino , Haploidia , Humanos , Memoria Inmunológica , Leucemia/inmunología , Leucemia/cirugía , Masculino , Persona de Mediana Edad , Donantes de Tejidos , Inmunología del Trasplante , Trasplante Homólogo , Adulto JovenRESUMEN
The synthesis and hit-to-lead SAR development from a pyrazolo[1,5-a]pyrimidine-derived hit 5 to the identification of a series of potent, pan-Pim inhibitors such as 11j are described.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Pirimidinas/química , Pirimidinas/farmacología , Humanos , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Proto-Oncogénicas c-pim-1/química , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacología , Pirimidinas/síntesis química , Relación Estructura-ActividadRESUMEN
Persistent viral infections and inflammatory syndromes induce the accumulation of T cells with characteristics of terminal differentiation or senescence. However, the mechanism that regulates the end-stage differentiation of these cells is unclear. Human CD4(+) effector memory (EM) T cells (CD27(-)CD45RA(-)) and also EM T cells that re-express CD45RA (CD27(-)CD45RA(+); EMRA) have many characteristics of end-stage differentiation. These include the expression of surface KLRG1 and CD57, reduced replicative capacity, decreased survival, and high expression of nuclear γH2AX after TCR activation. A paradoxical observation was that although CD4(+) EMRA T cells exhibit defective telomerase activity after activation, they have significantly longer telomeres than central memory (CM)-like (CD27(+)CD45RA(-)) and EM (CD27(-)CD45RA(-)) CD4(+) T cells. This suggested that telomerase activity was actively inhibited in this population. Because proinflammatory cytokines such as TNF-α inhibited telomerase activity in T cells via a p38 MAPK pathway, we investigated the involvement of p38 signaling in CD4(+) EMRA T cells. We found that the expression of both total and phosphorylated p38 was highest in the EM and EMRA compared with that of other CD4(+) T cell subsets. Furthermore, the inhibition of p38 signaling, especially in CD4(+) EMRA T cells, significantly enhanced their telomerase activity and survival after TCR activation. Thus, activation of the p38 MAPK pathway is directly involved in certain senescence characteristics of highly differentiated CD4(+) T cells. In particular, CD4(+) EMRA T cells have features of telomere-independent senescence that are regulated by active cell signaling pathways that are reversible.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Senescencia Celular/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Telomerasa/inmunología , Adulto , Western Blotting , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , Separación Celular , Citometría de Flujo , Humanos , Memoria Inmunológica/inmunología , Hibridación Fluorescente in Situ , Antígenos Comunes de Leucocito/biosíntesis , Antígenos Comunes de Leucocito/inmunología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/citología , Telomerasa/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/biosíntesis , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Pressure ulcers associated with the non-invasive ventilation mask can significantly reduce the quality of life of the patient who needs this therapy. This study aims to identify clinical interventions to prevent skin lesions associated with the use of non-invasive ventilation medical devices. METHODS: The Scoping Review followed the methodology of the Joanna Briggs Institute. For this study the research was carried out, during the month of January 2022, in several databases, such as PubMed, Web of Science, Scopus, EBSCOhost, RCAAP and OpenGrey, and studies published between 2010 and 2022 were included. RESULTS: Of the 33 articles identified, 11 articles were included in this review, in which we identified several interventions for the prevention of skin lesions associated with the use of medical devices for non-invasive ventilation. The interventions identified include skin assessment, optimal fixation of the device, and the use of interfaces, namely, hydrocolloid or foam dressing under the NIV mask, among others Conclusion: This scoping review demonstrates that there is some scientific evidence for prevention, however the methodological approaches are very different, which makes it difficult to clearly describe the referenced interventions. This study was not registered.
RESUMEN
The p38α mitogen-activated protein kinase regulates the synthesis of pro-inflammatory cytokines in response to stimulation by a diverse set of stress signals. Various different chemotypes and clinical candidates that inhibit p38α function have been reported over the years. In this publication, the novel structure of p38α cocrystallized with the clinical candidate TAK-715 is reported. Owing to the impact of crystallization conditions on the conformation of protein kinases (and in particular p38α), the structures of complexes of p38α with SB-203580, SCIO-469 and VX-745 have also been determined to enable in-depth comparison of ligand-induced protein conformations. The impact of experimental conditions on p38α-inhibitor complex structures, most importantly soaking versus cocrystallization, is discussed. Analysis of the structures and quantification of the protein-ligand interactions couples ligand-induced protein conformations to the number of interactions and to inhibitor selectivity against the human kinome. This shows that for the design of novel kinase inhibitors, selectivity is best obtained through maximization of the number of interactions throughout the ATP pocket and the exploitation of specific features in the active site.
Asunto(s)
Benzamidas/farmacología , Tiazoles/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/química , Adenosina Trifosfato/química , Dominio Catalítico , Clonación Molecular , Cristalización , Cristalografía por Rayos X/métodos , Humanos , Imidazoles/farmacología , Indoles/farmacología , Ligandos , Unión Proteica , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
OBJECTIVE: To compare the weight and insulin-like growth factor-1 in neonatal retinopathy (WINROP) to the growth and retinopathy of prematurity (G-ROP) model in a Portuguese cohort. DESIGN: Retrospective case series. METHODS: Clinical records of consecutive infants who underwent retinopathy of prematurity (ROP) screening from April 2012 to May 2019 were retrospectively reviewed. Both WINROP and G-ROP models were accessed for sensitivity and specificity for type 1 ROP. A separate analysis of both algorithms was performed in infants with gestational age (GA) <30 weeks. RESULTS: Of the 375 infants included in the study, 313 were eligible for G-ROP analysis and 311 for WINROP. In the G-ROP group, 22 infants developed type 1 ROP (sensitivity 90.91%, 95% confidence interval [CI] 70.84%-98.98%). In the WINROP group, 23 infants needed treatment (sensitivity of 86.96%, 95% CI 66.41%-97.22%). Both models reached 100% sensitivity for type 1 ROP if restricted to GA <30 weeks. CONCLUSIONS: Both models were easy to use and had similar sensitivities. If restricted to GA <30 weeks, both models detected all type 1 ROP.