Mesenchymal stromal cells with chimaeric antigen receptors for enhanced immunosuppression.

Allogeneic mesenchymal stromal cells (MSCs) are a safe treatment option for many disorders of the immune system. However, clinical trials using MSCs have shown inconsistent therapeutic efficacy, mostly owing to MSCs providing insufficient immunosuppression in target tissues. Here we show that antigen-specific immunosuppression can be enhanced by genetically modifying MSCs with chimaeric antigen receptors (CARs), as we show for E-cadherin-targeted CAR-MSCs for the treatment of graft-versus-host disease in mice. CAR-MSCs led to superior T-cell suppression and localization to E-cadherin+ colonic cells, ameliorating the animals' symptoms and survival rates. On antigen-specific stimulation, CAR-MSCs upregulated the expression of immunosuppressive genes and receptors for T-cell inhibition as well as the production of immunosuppressive cytokines while maintaining their stem cell phenotype and safety profile in the animal models. CAR-MSCs may represent a widely applicable therapeutic technology for enhancing immunosuppression.

CD19 occupancy with tafasitamab increases therapeutic index of CART19 cell therapy and diminishes severity of CRS.

ABSTRACT: In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.

Desarrollo del dolor fetal y neonatal / Development of fetal and neonatal pain

Resumen: El dolor es el principal síntoma reportado a nivel mundial, es la principal causa de sufrimiento humano. Se considera que para que un individuo sienta dolor deben estar presentes: nociceptores, neurotransmisores del dolor, vías que llevan el estímulo al cerebro, el tálamo y conexión con la corteza. Por un tiempo se consideró que en esta etapa no se contaba con la madurez suficiente del sistema nervioso para sentir dolor; sin embargo, en la actualidad hay evidencia de que el feto presenta dolor; y su presencia altera el desarrollo del sistema nervioso. La presente revisión proporciona una visión actualizada de la ontogenia del dolor fetal y neonatal.

Abstract: Pain is the main symptom reported worldwide, it is the main cause of human suffering. It is considered that for an individual to feel pain, the following must be present: nociceptors, pain neurotransmitters, pathways that carry the stimulus to the brain, the thalamus and connection with the cortex. For a time it was considered that at this stage the nervous system was not mature enough to feel pain, however, there is currently evidence that the fetus has pain; and its presence alters the development of the nervous system. This review provides an updated view of the ontogeny of fetal and neonatal pain.

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia.

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells.

The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Two-Minute Step Test as a Complement to Six-Minute Walk Test in Subjects With Treated Coronary Artery Disease.

The 2-Minute Step Test (2MST) has been presented as an alternative to the 6-Minute Walk Test (6MWT) based on the association between the two tests in older adults; however, some authors propose that it should not be a substitute but rather a complement to the latter in the fitness evaluation. Specifically, in coronary disease, despite the potential and clinical utility of 2MST, the relationship of both tests in this population is unknown. This study aimed to determine the relationship between 6MWT and 2MST and to explore the relationship of biodemographic factors for both tests in subjects with treated coronary artery disease. For this, the 6MWT and the 2MST were applied to patients with coronary artery disease treated in 6 hospitals in Chile between May 2019 and February 2020. Additionally, lower limb strength was assessed by a chair-stand test, grip strength was assessed by a dynamometer, and physical measurements were applied. In total, 163 participants underwent both tests (average age = 58.7 ± 9.8 years; 73.6% men; 64.4% revascularized by angioplasty; 28.2% revascularized by surgery, and 7.4% treated by drugs or thrombolysis). Heart rate was higher at the end of the 6MWT, while the perception of effort was greater at the end of the 2MST. There was a weak positive correlation between the 6MWT and the 2MST in subjects with treated coronary disease (r = 0.28, p = 0.0003). While age (r = -0.27), weight (r = 0.25), height (r = 0.49), and strength of both lower limbs (r = 0.41) and grip strength (r = 0.53) correlated weakly or moderately to the covered distance in 6MWT, the number of steps by the 2MST correlated only weakly to height (r = 0.23), lower limb strength (r = 0.34), and grip strength (r = 0.34). Age, weight, height, lower limb strength, and grip strength would explain better the meters walked in the 6MWT than the steps achieved in the 2MST. With these findings, we can conclude that, in patients with treated coronary artery disease, it does not seem advisable to replace 6MWT with 2MST when it is possible to do so. Additionally, the 2MST may provide additional information in the fitness evaluation. However, the usefulness of 2MST in this population needs to be further studied.

GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity.

Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen receptor T (CART) cell therapy by contributing to the development of toxicities and resistance following infusion. We have previously shown that neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF) prevents these toxicities and enhances CART cell functions by inhibiting myeloid cell activation. In this report, we study the direct impact of GM-CSF disruption during the production of CD19-directed CART cells on their effector functions, independent of GM-CSF modulation of myeloid cells. In this study, we show that antigen-specific activation of GM-CSFKO CART19 cells consistently displayed reduced early activation, enhanced proliferation, and improved anti-tumor activity in a xenograft model for relapsed B cell malignancies. Activated CART19 cells significantly upregulate GM-CSF receptors. However, the interaction between GM-CSF and its upregulated receptors on CART cells was not the predominant mechanism of this activation phenotype. GM-CSFKO CART19 cell had reduced BH3 interacting-domain death agonist (Bid), suggesting an interaction between GM-CSF and intrinsic apoptosis pathways. In conclusion, our study demonstrates that CRISPR/Cas9-mediated GM-CSF knockout in CART cells directly ameliorates CART cell early activation and enhances anti-tumor activity in preclinical models.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography.

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Targeting cancer-associated fibroblasts in the bone marrow prevents resistance to CART-cell therapy in multiple myeloma.

Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.

Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity.

Although chimeric antigen receptor T (CART)-cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)-directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Eficacia terapéutica de la buprenorfina para tratamiento de dolor agudo postoperatorio en cirugía torácica por toracoscopía / Therapeutic efficacy of buprenorphine for treatment of acute postoperative pain in thoracic surgery by thoracoscopy

Resumen: Introducción: El dolor postoperatorio en toracoscopía tiene una incidencia del 80%. Estos procedimientos se realizan bajo anestesia general o sedoanalgesia. La buprenorfina en dosis de 4 μg/kg o más es un fármaco pilar para el tratamiento de dolor postoperatorio; sin embargo, dosis menores pueden ser suficientes. El objetivo de este estudio fue demostrar que dosis de 1-3 μg/kg de buprenorfina aporta una adecuada analgesia con menores efectos adversos durante el postoperatorio en pacientes sometidos a cirugía toracoscópica. Material y métodos: Se realizó el estudio prospectivo, transversal y doble ciego en 48 pacientes sometidos a toracoscopía, divididos en 3 grupos: A (1 μg/kg), B (2 μg/kg) y C (3 μg/kg) tratados con buprenorfina, se evaluaron efectos adversos, intensidad de dolor por escala verbal numérica (EVN) y sedación con escala de agitación-sedación de Richmond (RASS) de los 0 a los 120 minutos postanestesia. Resultados: Las dosis de 1 a 3 μg/kg de buprenorfina no ocasionaron efectos adversos. El mantenimiento de analgesia es mayor con 2 μg/kg (p = 0.019) y 3 μg/kg (p = 0.045). Las dosis de 1 y 2 μg/kg presentan un nivel de sedación menor. Conclusiones: La analgesia multimodal disminuyó un 200% el dolor postquirúrgico inmediato. Las dosis de 2-3 μg/kg de buprenorfina son efectivas para el control del dolor con menor incidencia de efectos adversos y un nivel de sedación superficial durante el postoperatorio.

Abstract: Introduction: Posterior pain in thoracoscopy has an 80% incidence. These procedures are done under general anesthesia or sedoanalgesia. The buprenorphin in dosage of 4 μg/kg it's the foundation for post-operatory pain treatment, nevertheless, smaller dosage can be sufficient. The objective of this study is to demonstrate that a dosage of buprenorphin from 1-3 μg/kg provides suitable analgesia with less side effects during post-operatory in patients who underwent thoracoscopic surgery. Material and methods: We conducted a double-blind, prospective, transversal study, in 48 patients which underwent a thoracoscopy divided in 3 groups: A (1 μg/kg), B (2 μg/kg), and C (3 μg/kg) treated with buprenorphin. We studied side effects, pain intensity (NVE), and sedative effect (RASS) from 0 to 120 minutes postanesthesia. Results: Dosage from 1 to 3 μg/kg did not generate side effects. The analgesia effect is higher with 2 μg/kg (p = 0.019) and 3 μg/kg (p = 0.045). Dosage of 1 and 2 μg/kg have lower sedative effect. Conclusion: The multimodal analgesia was diminished by 200% inmidiate postsurgical pain. Dosage from 2-3 μg/kg of buprenorphine are effective for pain control with less side effects and a superficial sedative effect during post-operatory.

CRISPR Takes the Front Seat in CART-Cell Development.

Chimeric antigen receptor T (CART)-cell immunotherapies have opened a door in the development of specialized gene therapies for hematological and solid cancers. Impressive response rates in pivotal trials led to the FDA approval of CART-cell therapy for certain hematological malignancies. However, autologous CART products are costly and time-intensive to manufacture, and most patients experience disease relapse within 1 year of CART administration. Additionally, CART-cell efficacy in solid tumors is extremely limited. CART-cell therapy is also associated with serious toxicities. Manufacturing difficulties, intrinsic T-cell defects, CART exhaustion, and treatment-associated toxicities are some of the current barriers to widespread adoption of CART-cell therapy. Genome editing tools such as CRISPR/Cas systems have demonstrated efficacy in further engineering CART cells to overcome these limitations. In this review, we will summarize the current approaches that use CRISPR to facilitate off-the-shelf CART products, increase CART-cell efficacy, and minimize CART-associated toxicities.

Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1+ CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.

Ejercicio: una herramienta clave en la prevención cardiovascular: Consenso de la Sociedad Chilena de Cardiología y Cirugía Cardiovascular y de la Sociedad Chilena de Kinesiología en Cardiología y Cirugía Cardiovascular / Excercise, a key intervention in Cardiovascular Prevention: Consensus of the Chilean Society of Cardiology and Cardiovascular Surgery and the Chilean Society of Kinesiology in Cardiology and Cardiovascular Surgery

This is a Document from de Chilean Society of Cardiology and Cardiovascular Surgery, along with the Chilean Society of Physical Therapy of Cardiology and Cardiovascular Surgery.

MALDI imaging: beyond classic diagnosis / Imagenología por MALDI: más allá del diagnóstico clásico

Abstract: Mass spectrometry has been the focus of technology development and application for imaging for several decades. Imaging mass spectrometry using matrix-assisted laser desorption ionization is a new and effective tool for molecular studies of complex biological samples such as tissue sections. As histological features remain intact throughout the analysis of a section, distribution maps of multiple analytes can be correlated with histological and clinical features. Spatial molecular arrangements can be assessed without the need for target-specific reagents, allowing the discovery of diagnostic and prognostic markers of different cancer types and enabling the determination of effective therapies.

Resumen: La espectrometría de masas ha sido el foco de atención del desarrollo y aplicación de tecnología para imagenología por varias décadas. La imagenología por espectrometría de masas utilizando ionización por desorción láser asistida por matriz es una herramienta novedosa y efectiva para el estudio molecular de muestras biológicas complejas como los cortes de tejidos. Ya que las características histológicas se mantienen intactas durante el análisis de una sección, pueden correlacionarse mapas de distribución de múltiples analitos con características clínicas e histológicas. Los arreglos moleculares espaciales pueden evaluarse sin la necesidad de reactivos específicos para el blanco, lo que permite el descubrimiento de marcadores pronósticos y diagnósticos de diferentes tipos de cáncer y permite la determinación de terapias efectivas.

MALDI imaging: beyond classic diagnosis.

Mass spectrometry has been the focus of technology development and application for imaging for several decades. Imaging mass spectrometry using matrix-assisted laser desorption ionization is a new and effective tool for molecular studies of complex biological samples such as tissue sections. As histological features remain intact throughout the analysis of a section, distribution maps of multiple analytes can be correlated with histological and clinical features. Spatial molecular arrangements can be assessed without the need for target-specific reagents, allowing the discovery of diagnostic and prognostic markers of different cancer types and enabling the determination of effective therapies.

Avaliação de qualidade no centro de material e esterilização / Quality evaluation in supply center

Esta pesquisa aborda um dos assuntos do padrão de qualidade, que é o Serviço de Atendimento ao Cliente (SAC), buscando revelar a percepção que os enfermeiros têm dos serviços que são prestados pelo Centro e Material e Esterilização (CME), além de desenvolver uma política entre os colaboradores que atuam nessa área, com o objetivo de instituir uma parceria na busca da qualidade. Utilizamos, para tanto, um instrumento com sete questões relacionadas com as atividades da equipe de esterilização, somado a um espaço destinado para comentários e sugestões que os participantes julgassem necessários registrar....