Estudio de las alteraciones inmunovirológicas de pacientes VIH "escasos repobladores": potenciales predictores y mecanismos implicados.

La infección por VIH provoca una depleción masiva de linfocitos T CD4 (CD4) que finalmente evoluciona a SIDA si no se inicia tratamiento. El tratamiento antirretroviral combinado (TARc) consigue suprimir la viremia VIH permitiendo un aumento del recuento de CD4. Sin embargo, existe un grupo de individuos, denominados “Escasos Repobladores” (ER), que no recuperan sus niveles de CD4 a pesar del TARc. Diversos factores hacen necesario el estudio de estos sujetos. En primer lugar, los ER constituyen un 25% de los sujetos que comienzan el TARc con bajos CD4. Por otro lado, los ER presentan un mayor riesgo de experimentar eventos clínicos no definitorios de SIDA (ENOS) y de muerte. Desafortunadamente, todas las intervenciones terapéuticas dirigidas a aumentar los recuentos de CD4 de los ER han fallado, dejando patente la necesidad de comprender mejor los mecanismos inmunológicos implicados y así diseñar nuevas estrategias terapéuticas. Se han propuestos diversos mecanismos inmunopatogénicos como potenciales desencadenantes de la escasa recuperación de CD4 tras el TARc. Sin embargo, todos los estudios previos se han llevado a cabo cuando los ER estaban bajo TARc, no pudiéndose descartar que las alteraciones descritas sean consecuencia de sus bajos recuentos CD4 en el momento de estudio. Para responder a dicha cuestión, nos propusimos estudiar los ER antes del inicio del TARc, cuando estos sujetos presentan recuentos de linfocitos T CD4 similares a los del grupo control. Este fue el primer objetivo de la presente Tesis Doctoral y fue abordado en el trabajo: “Higher levels of IL-6, CD4 turnover and Treg frequency are already present before cART in HIVinfected subjects with later low CD4 recovery”. El estudio de pacientes ER antes del inicio del TARc nos permitió determinar alteraciones inmunológicas que preceden a la escasa recuperación de linfocitos T CD4 y profundizar en potenciales factores asociados a dicha escasa reconstitución inmunológica. Aunque ya desde el principio de la epidemia de SIDA se conocía que el VIH provoca una inversión del ratio de linfocitos T CD4/CD8 (ratio CD4/CD8), no ha sido hasta hace poco cuando la inversión de dicho parámetro se ha asociado a un riesgo aumentado a experimentar ENOS y a muerte. Sin embargo, se desconocía si el ratio CD4/CD8 antes del inicio del TARc se asocia con la posterior recuperación de CD4 bajo el TARc. Esta cuestión ha sido abordada en el segundo artículo: “A lower baseline CD4/CD8 T-cell ratio is independently associated with immunodiscordant response to antiretroviral therapy in HIV-infected subjects”. Este estudio nos permitió asociar la escasa recuperación de CD4 con bajos niveles en el ratio CD4/CD8 antes del inicio del TARc. Por otro lado, los mecanismos involucrados en la inversión del ratio CD4/CD8 observada en el escenario VIH no están completamente dilucidados, y ningún trabajo previo había examinado el posible papel del timo, órgano responsable de la producción de novo de linfocitos CD4 y CD8 que se encuentra profundamente afectado en la infección por VIH, en dicho fenómeno. Esta cuestión fue abordada en el tercer artículo de la presente tesis: “Thymic funtion impacts the peripheral CD4/CD8 ratio of HIVinfected subjects”. Este estudio nos permitió dilucidar una asociación entre el ratio CD4/CD8 de sangre periférica y la función tímica antes del inicio del tratamiento antirretroviral, además de un papel del timo en la posterior normalización del ratio CD4/CD8 durante el TARc. La producción de nuevos linfocitos T por parte del timo tiene un papel fundamental en la recuperación de CD4. Sin embargo, hasta la fecha, no se había valorado el posible papel de los mecanismos de compensación periféricos o proliferación homeostática (PH) en dicho proceso. En primer lugar quisimos conocer más acerca del proceso de PH de linfocitos T CD4 humanos, ya que la mayoría de información existente había sido generada en modelos animales. Para ello desarrollamos un sistema de cultivos in vitro que nos permitió estudiar distintos tipos de PH e identificar marcadores celulares asociados a dicho fenómeno. Los resultados generados del estudio de la PH en linfocitos humanos, y la caracterización del modelo, fueron abordados en el quinto artículo de la presente tesis doctoral: “An in vitro system of autologous lymphocytes culture that allows studying the homeostatic proliferation mechanims of human naïve CD4 T-cells”. A continuación, para determinar la contribución de la PH sobre la escasa recuperación de linfocitos T CD4 de ER, determinamos la expresión de diversos marcadores celulares asociados al proceso de PH en ER antes del inicio del TARc y tras 24 meses de TARc supresor. Estos resultados fueron englobados en el quinto artículo de la presente tesis doctoral: “A high homeostatic proliferation precedes a poor CD4 T-cell recovery in response to HIV antiretroviral therapy”.Premio Extraordinario de Doctorado U

Orai2 Modulates Store-Operated Ca2+ Entry and Cell Cycle Progression in Breast Cancer Cells

Breast cancer is a heterogeneous disease from the histological and molecular expression point of view, and this heterogeneity determines cancer aggressiveness. Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is significantly remodeled in cancer cells and plays an important role in the development and support of different cancer hallmarks. The store-operated CRAC (Ca2+ release-activated Ca2+) channels are predominantly comprised of Orai1 but the participation of Orai2 and Orai3 subunits has been reported to modulate the magnitude of Ca2+ responses. Here we provide evidence for a heterogeneous expression of Orai2 among different breast cancer cell lines. In the HER2 and triple negative breast cancer cell lines SKBR3 and BT20, respectively, where the expression of Orai2 was greater, Orai2 modulates the magnitude of SOCE and sustain Ca2+ oscillations in response to carbachol. Interestingly, in these cells Orai2 modulates the activation of NFAT1 and NFAT4 in response to high and low agonist concentrations. Finally, we have found that, in cells with high Orai2 expression, Orai2 knockdown leads to cell cycle arrest at the G0-G1 phase and decreases apoptosis resistance upon cisplatin treatment. Altogether, these findings indicate that, in breast cancer cells with a high Orai2 expression, Orai2 plays a relevant functional role in agonist-evoked Ca2+ signals, cell proliferation and apoptosis resistance.Ministerio de Ciencia e Innovación: PID2019-104084GB-C21Ministerio de Ciencia e Innovación: PID2019-104084GB-C22Consejería de Educación y Empleo, Junta de Extremadura: IB20007Consejería de Educación y Empleo, Junta de Extremadura: GR18061Consejería de Educación y Empleo, Junta de Extremadura: PD16072Consejería de Educación y Empleo, Junta de Extremadura: GR2100

HIV-Infected Subjects With Poor CD4 T-Cell Recovery Despite Effective Therapy Express High Levels of OX40 and α4β7 on CD4 T-Cells Prior Therapy Initiation

Background HIV-infected subjects with suboptimal CD4 restoration despite suppressive combined antiretroviral treatment (cART) (immunodiscordant subjects) have been classically characterized after a variable period of time under cART. Recently, we have reported that an increased frequency of proliferating CD4 T-cells in these subjects is already present before the cART onset. The potential contribution of peripheral compensatory homeostatic proliferation (HP) is yet unknown. We aimed to analyze the expression of HP-related cellular markers on CD4 T-cells of immunodiscordant subjects before cART. Go to: Methods We analyzed the expression of OX40 and α4β7 on peripheral CD4 T-cells from immunodiscordant and control subjects (n = 21 each group) before cART initiation, and also on available follow-up samples (after 24 month of suppressive cART). Additionally, we tested the expression of these markers in an in vitro system for the study of human HP processes. Go to: Results Immunodiscordant subjects showed increased levels of OX40 and α4β7 on CD4 T-cells before cART initiation. While the cART tended to reduce these levels, immunodiscordant subjects still maintained comparatively higher levels of OX40 and α4β7 after 24 months under suppressive cART. These HP-related markers were upregulated in vitro during the human HP, especially during the fast HP. Go to: Conclusion Our results are compatible with exacerbated HP processes in immunodiscordant subjects, already before the cART onset.Fondo de Investigación Sanitaria FIS PI14/01693 PI13/0796 PI16/0503Fondos Europeos para el Desarrollo Regional (FEDER) CTS2593Junta de Andalucía. Consejería de Economía, Innovación, Ciencia y Empleo CTS2593AGAUR 2017SGR948GILEAD GLD14/293The Spanish AIDS Research Network of Excellence RD12/0017/0029 RD16/0025/0019 RD16/0025/0006Junta de Andalucía. Consejería de Salud y Bienestar Social C-0013-201

EFHB is a novel cytosolic Ca²+ sensor that modulates STIM1-SARAF interaction

FUNDAMENTOS/OBJETIVOS: STIM1 y Orai1 son los componentes clave de la entrada de Ca2+ en la tienda (SOCE). Entre las proteínas que participan en la regulación de la SOCE, la SARAF previene la activación espontánea de la SOCE y modula la función de STIM1. MÉTODOS: Se estimó la movilización de Ca2+ citosólico en células cargadas de fura-2 usando un microscopio invertido de epifluorescencia. La interacción de STIM1 con Orai1, EFHB (miembro de la familia B del dominio de la mano EF, también conocido como CFAP21) y SARAF se detectó mediante inmunoprecipitación seguida de Western blotting utilizando anticuerpos específicos. La participación de EFHB en la translocación de NFAT al núcleo se detectó mediante microscopía confocal. RESULTADOS: Aquí reportamos la identificación del EFHB como un nuevo regulador SOCE. El EFHB interactúa con el STIM1 al agotarse el almacenamiento y se disocia a través de un mecanismo dependiente de Ca2+-. El silenciamiento mediado por el ARNi así como los estudios de sobreexpresión revelaron que el EFHB juega un papel relevante en la interacción de STIM1 y Orai1 al agotarse las reservas, la activación de la translocación de SOCE y NFAT del citosol al núcleo. El silenciamiento de la expresión de la EFHB suprimió la disociación de la SARAF de la STIM1, lo que indica que la EFHB podría desempeñar un papel importante en la interacción dinámica entre ambas proteínas, lo que es pertinente para la activación de los canales de Orai1 al agotarse el almacenamiento de Ca2+ y su posterior modulación mediante la inactivación lenta dependiente del Ca2+. CONCLUSIÓN: Nuestros resultados indican que el EFHB es un nuevo regulador SOCE que modula la interacción STIM1-SARAF.BACKGROUND/AIMS: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Among the proteins involved in the regulation of SOCE, SARAF prevents spontaneous activation of SOCE and modulates STIM1 function. METHODS: Cytosolic Ca2+ mobilization was estimated in fura-2-loaded cells using an epifluorescence inverted microscope. STIM1 interaction with Orai1, EFHB (EF-hand domain family member B, also known as CFAP21) and SARAF was detected by immunoprecipitation followed by Western blotting using specific antibodies. The involvement of EFHB in the translocation of NFAT to the nucleus was detected by confocal microscopy. RESULTS: Here, we report the identification of EFHB as a new SOCE regulator. EFHB interacts with STIM1 upon store depletion and dissociates through a Ca2+- dependent mechanism. RNAi-mediated silencing as well as overexpression studies revealed that EFHB plays a relevant role in the interaction of STIM1 and Orai1 upon store depletion, the activation of SOCE and NFAT translocation from the cytosol to the nucleus. Silencing EFHB expression abolished the dissociation of SARAF from STIM1, which indicates that EFHB might play an important role in the dynamic interaction between both proteins, which is relevant for the activation of Orai1 channels upon Ca2+ store depletion and their subsequent modulation via slow Ca2+-dependent inactivation. CONCLUSION: Our results indicate that EFHB is a new SOCE regulator that modulates STIM1-SARAF interaction.• Ministerio de Economía y Competitividad. Contrato Juan de la Cierva IJCI-2015-25665, para Isaac Jardín Polo • Junta de Extremadura y Fondos FEDER. Contrato IB16046, para José Javier López Barba • Ministerio de Economía y Competitividad. Subvención BFU2016-74932-C2-1-P, para Letizia Albarrán Alonso • Ministerio de Economía y Competitividad. Subvenciones Subvenciones BFU2013-45564-C2-1-P/2-P y BFU2016-74932-C2-1-P/2-P • Junta de Extremadura y Fondos FEDER. Subvenciones IB16046 y GR18061peerReviewe

Immunological features beyond CD4/CD8 ratio values in older individuals

The CD4/CD8 T-cell ratio is emerging as a relevant marker of evolution for many pathologies and therapies. We aimed to explore immunological features beyond CD4/CD8 ratio values in older subjects (>65 years old) who were classified as having lower (2) ratio values. The lower group showed a lower thymic output (sj/β-TREC ratio) and frequency of naïve T-cells, concomitant with increased mature T-cells. In these subjects, the CD4 T-cell subset was enriched in CD95+ but depleted of CD98+ cells. The regulatory T-cell (Treg) compartment was enriched in CTLA-4+ cells. The CD8 T-cell pool exhibited increased frequencies of CD95+ cells but decreased frequencies of integrin-β7+ cells. Interestingly, in the intermediate group, the CD4 pool showed greater differences than the CD8 pool, mostly for cellular senescence. Regarding inflammation, only hsCRP was elevated in the lower group; however, negative correlations between the CD4/CD8 ratio and β2-microglobulin and sCD163 were detected. These subjects displayed trends of more comorbidities and less independence in daily activities. Altogether, our data reveal different thymic output and immune profiles for T-cells across CD4/CD8 ratio values that can define immune capabilities, affecting health status in older individuals. Thus, the CD4/CD8 ratio may be used as an integrative marker of biological age.This work was supported by grants from the Fondo de Investigación Sanitaria (FIS; PI18/01216), which is co-funded by Fondos Europeos para el Desarrollo Regional (FEDER) “Una manera de hacer Europa” and the Junta de Andalucía, Consejería de Economía, Innovación, Ciencia y Empleo (Proyecto de Investigación de Excelencia; CTS2593). The Spanish AIDS Research Network of Excellence also supported this study (RD16/0025/0019). V G-R, I O-M and A B-R were supported by Instituto de Salud Carlos III (FI19/00298, CM19/00051 and CD19/00143, respectively). YM. P was supported by the Consejería de Salud y Familias of Junta de Andalucía through the ‘‘Nicolás Monardes’’ program (C-0013-2017).Peer reviewe

Glutaminolysis and lipoproteins are key factors in late immune recovery in successfully treated HIV-infected patients

The immunological, biochemical and molecular mechanisms associated with poor immune recovery are far from known, and metabolomic profiling offers additional value to traditional soluble markers. Here, we present novel and relevant data that could contribute to better understanding of the molecular mechanisms preceding a discordant response and HIV progression under suppressive combined antiretroviral therapy (cART). Integrated data from nuclear magnetic resonance (NMR)-based lipoprotein profiles, mass spectrometry (MS)-based metabolomics and soluble plasma biomarkers help to build prognostic and immunological progression tools that enable the differentiation of HIV-infected subjects based on their immune recovery status after 96 weeks of suppressive cART. The metabolomic signature of ART-naïve HIV subjects with a subsequent late immune recovery is the expression of pro-inflammatory molecules and glutaminolysis, which is likely related to elevate T-cell turnover in these patients. The knowledge about how these metabolic pathways are interconnected and regulated provides new targets for future therapeutic interventions not only in HIV infection but also in other metabolic disorders such as human cancers where glutaminolysis is the alternative pathway for energy production in tumor cells to meet their requirement of rapid proliferation.Peer reviewe

A Lower Baseline CD4/CD8 T-Cell Ratio Is Independently Associated with Immunodiscordant Response to Antiretroviral Therapy in HIV-Infected Subjects

We explored if baseline CD4/CD8 T-cell ratio is associated with immunodiscordant response to antiretroviral therapy in HIV-infected subjects. Comparing immunodiscordant and immunoconcordant subjects matched by pretreatment CD4 counts, we observed a lower pretreatment CD4/CD8 T-cell ratio in immunodiscordant subjects. Furthermore, pretreatment CD4/CD8 T-cell ratio, but not CD4 counts, correlated with the main immunological alterations observed in immunodiscordants, including increased regulatory T-cell (Treg) frequency and T-cell turnover-related markers. Then, in a larger cohort, only baseline CD4/CD8 T-cell ratio was independently associated with immunodiscordance, after adjusting by the viral CXCR4-tropic HIV variants. Our results suggest that the CD4/CD8 T-cell ratio could be an accurate biomarker of the subjacent immunological damage triggering immunodiscordance.The HIV BioBank, integrated in the Spanish AIDS Research Network, is supported by Instituto de Salud Carlos III, Spanish Health Ministry (grants RD06/0006/0035 and RD12/0017/0037), as part of the Plan Nacional R + D + I and cofinanced by ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER) and Fundación para la investigación y prevención del SIDA en España (FIPSE). The RIS Cohort (CoRIS) is funded by the Instituto de Salud Carlos III through the Red Temática de Investigación Cooperativa en SIDA by the RD12/0017/0018 project, as part of the Plan Nacional R + D + I, and cofinanced by ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER). The project number is RIS_EPICLIN 20/2015. This work was supported by grants from the Fondo de Investigación Sanitaria, ISCIII (FIS; PI14/01693), and the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (Proyecto de Investigación de Excelencia; CTS2593), and cofunded by Fondos Europeos para el Desarrollo Regional (FEDER). I. Rosado-Sánchez was supported by the Spanish AIDS Research Network of Excellence (RIS; RD12/0017/0029). Y. M. Pacheco was supported by the Fondo de Investigación Sanitaria through the Miguel Servet program (CPII13/00037) and by the Servicio Andaluz de Salud through the Nicolás Monardes program (C-0010/13).Peer reviewe

HIV-Infected Subjects With Poor CD4 T-Cell Recovery Despite Effective Therapy Express High Levels of OX40 and α4β7 on CD4 T-Cells Prior Therapy Initiation

BackgroundHIV-infected subjects with suboptimal CD4 restoration despite suppressive combined antiretroviral treatment (cART) (immunodiscordant subjects) have been classically characterized after a variable period of time under cART. Recently, we have reported that an increased frequency of proliferating CD4 T-cells in these subjects is already present before the cART onset. The potential contribution of peripheral compensatory homeostatic proliferation (HP) is yet unknown. We aimed to analyze the expression of HP-related cellular markers on CD4 T-cells of immunodiscordant subjects before cART.MethodsWe analyzed the expression of OX40 and α4β7 on peripheral CD4 T-cells from immunodiscordant and control subjects (n = 21 each group) before cART initiation, and also on available follow-up samples (after 24 month of suppressive cART). Additionally, we tested the expression of these markers in an in vitro system for the study of human HP processes.ResultsImmunodiscordant subjects showed increased levels of OX40 and α4β7 on CD4 T-cells before cART initiation. While the cART tended to reduce these levels, immunodiscordant subjects still maintained comparatively higher levels of OX40 and α4β7 after 24 months under suppressive cART. These HP-related markers were upregulated in vitro during the human HP, especially during the fast HP.ConclusionOur results are compatible with exacerbated HP processes in immunodiscordant subjects, already before the cART onset

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat