RESUMO
Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Neoplasias Pancreáticas/terapia , Receptores de Antígenos Quiméricos/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas Inibidoras de Diferenciação/imunologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Proteínas de Neoplasias/imunologia , Fatores de Transcrição SOXC/imunologiaRESUMO
The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.
Assuntos
Linfócitos T CD4-Positivos , Imunoterapia Adotiva , Leucemia , Receptores de Antígenos Quiméricos , Antígenos CD19/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Separação Celular , Humanos , Leucemia/imunologia , Leucemia/terapia , Receptores de Antígenos Quiméricos/imunologia , Fatores de TempoRESUMO
Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.
Assuntos
Cromotripsia , Evolução Molecular , Amplificação de Genes/genética , Neoplasias/genética , Oncogenes/genética , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Circular/química , DNA Circular/metabolismo , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , Proteína Quinase Ativada por DNA , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Células HeLa , Humanos , Micronúcleos com Defeito Cromossômico , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Seleção Genética , Sequenciamento Completo do GenomaRESUMO
The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes1-8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9-13 than in normal liver13-16, which enables positive selection to shape the genomic landscape9-13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17-19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.
Assuntos
Hepatopatias/genética , Hepatopatias/metabolismo , Fígado/metabolismo , Mutação/genética , Transporte Ativo do Núcleo Celular/genética , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Doença Crônica , Estudos de Coortes , Ácidos Graxos não Esterificados/metabolismo , Feminino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Resistência à Insulina , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/metabolismo , Masculino , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Triglicerídeos/metabolismoRESUMO
All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium1,2. Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.
Assuntos
Análise Mutacional de DNA , Endométrio/citologia , Endométrio/metabolismo , Epitélio/metabolismo , Saúde , Mutação , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Carcinogênese/genética , Células Clonais/citologia , Neoplasias do Endométrio/genética , Endométrio/patologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Paridade/genética , Fatores de Tempo , Adulto JovemRESUMO
Image data are universal in life sciences research. Their proper handling is not. A significant proportion of image data in research papers show signs of mishandling that undermine their interpretation. We propose that a precise description of the image processing and analysis applied is required to address this problem. A new norm for reporting reproducible image analyses will diminish mishandling, as it will alert co-authors, referees, and journals to aberrant image data processing or, if published nonetheless, it will document it to the reader. To promote this norm, we discuss the effectiveness of this approach and give some step-by-step instructions for publishing reproducible image data processing and analysis workflows.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador/normas , Editoração/normas , Confiabilidade dos Dados , Humanos , Reprodutibilidade dos Testes , Má Conduta Científica , Fluxo de TrabalhoRESUMO
The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.
Assuntos
Células Clonais/citologia , Células Clonais/patologia , Fibrose/genética , Fibrose/patologia , Fígado/citologia , Fígado/metabolismo , Mutação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células Clonais/metabolismo , Análise Mutacional de DNA , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Filogenia , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
BACKGROUND: Resistance to r-tPA (recombinant tissue-type plasminogen activator) is a well-known but poorly understood phenomenon that hampers successful recanalization in patients with acute ischemic stroke. Using clinically relevant thrombi from patients with acute ischemic stroke, we investigated if and how thrombus composition impacts r-tPA-mediated lysis. In addition, we explored strategies to overcome r-tPA resistance. METHODS: Thrombi were split into 2 parts, 1 of which was used for thrombolysis and the other for detailed histological analysis. Thrombolysis was performed in normal human plasma using r-tPA alone, using r-tPA in combination with DNase-1 or using r-tPA in combination with N,N'-diacetyl-l-cystine. Thrombus lysis was calculated as the percentage of residual thrombus weight compared with its initial weight and the degree of lysis was linked to thrombus composition determined via histology. RESULTS: Interestingly, we found that the efficacy of r-tPA-mediated thrombolysis was strongly correlated with the composition of the thrombi. Thrombi containing high amounts of red blood cells and low amounts of DNA and von Willebrand Factor were efficiently degraded by r-tPA, whereas thrombi containing low amounts of red blood cells and higher amounts of DNA and von Willebrand Factor were resistant to r-tPA. Importantly, combination of r-tPA with DNase-1 or N,N'-diacetyl-l-cystine significantly and specifically improved the lysis of these r-tPA-resistant thrombi. CONCLUSIONS: Using patient thrombus material, our results for the first time show that the composition of stroke thrombi largely determines their susceptibility to r-tPA-mediated thrombolysis. Red blood cell-poor thrombi have a specific resistance to r-tPA, which can be overcome by targeting nonfibrin components using DNase-1 or N,N'-diacetyl-l-cystine.
RESUMO
Thrombolytic therapies for acute ischemic stroke are widely available but only result in recanalization early enough, to be therapeutically useful, in 10% to 30% of cases. This large gap in treatment effectiveness could be filled by novel therapies that can increase the effectiveness of thrombus clearance without significantly increasing the risk of harm. This focused update will describe the current state of emerging adjuvant treatments for acute ischemic stroke reperfusion. We focus on new treatments that are designed to (1) target different components that make up a stroke thrombus, (2) enhance endogenous fibrinolytic systems, (3) reduce stagnant blood flow, and (4) improve recanalization of distal thrombi and postendovascular thrombectomy.
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CD19-directed chimeric antigen receptor-modified (CAR T) T cells achieve durable remissions in about 30% to 40% of relapsed/refractory large B-cell lymphomas. T-cell exhaustion and/or an immunosuppressive tumor microenvironment may contribute to CAR T-cell failure. Pembrolizumab, an anti-PD1 immune checkpoint inhibitor, may reverse T-cell exhaustion after CAR T-cell therapy. We treated 12 patients with B-cell lymphomas who were either refractory to (n = 9) or relapsed after (n = 3) CD19-directed CAR T-cell (4-1BB-costimulated) therapy with pembrolizumab 200 mg IV every 3 weeks. Median time from CAR T-cell infusion to first pembrolizumab dose was 3.3 months (range, 0.4-42.8 months). Pembrolizumab was well tolerated, and the only grade ≥3 adverse events related to pembrolizumab were neutropenia (n = 3; 25%). Best overall response rate after pembrolizumab was 25% (3 of 12 patients; 1 complete response; 2 partial responses). One (8%) patient had stable disease; thus, 4 of 12 (33%) patients had clinical benefit. After pembrolizumab, 4 patients with clinical benefit had an increase in percentage of CAR T cells by mass cytometry by time of flight (CyTOF); 3 of 4 of these patients also had increases in CAR19 transgene levels by quantitative polymerase chain reaction. Deep immune profiling using CyTOF revealed increased CAR T-cell activation and proliferation and less T-cell exhaustion in clinical responders. Together, PD1 blockade with pembrolizumab after CD19-directed CAR T-cell therapy appears safe and may achieve clinical responses in some patients with B-cell lymphomas refractory to or relapsed after CAR T-cell therapy. This trial was registered at www.clinicaltrials.gove as #NCT02650999.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD19/imunologia , Imunoterapia Adotiva/efeitos adversos , Linfoma de Células B/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Receptores de Antígenos Quiméricos/imunologia , Terapia de Salvação , Adulto , Idoso , Antineoplásicos Imunológicos/uso terapêutico , Feminino , Seguimentos , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Linfoma de Células B/terapia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Prognóstico , Estudos ProspectivosRESUMO
BACKGROUND: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. RESULTS: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. CONCLUSIONS: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.
Assuntos
Proteínas Nucleares/metabolismo , Splicing de RNA , Receptores de LDL/genética , Colesterol/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Lipoproteínas LDL/metabolismo , Fígado/metabolismo , Mutação , Proteínas Nucleares/genética , Receptores de LDL/metabolismo , Spliceossomos/metabolismoRESUMO
BACKGROUND: Topical corticosteroid phobia is associated with poor treatment adherence and is common among patients with skin disease. Knowledge about corticosteroid phobia and treatment adherence among patients with chronic hand eczema (CHE) is limited. OBJECTIVES: Investigate patient-reported outcomes regarding topical corticosteroids (TCS), and their impact on treatment adherence in CHE patients. METHODS: CHE patients from the Danish Skin Cohort answered a questionnaire including the Topical Corticosteroid Phobia (TOPICOP) scale and Medication Adherence Report Scale (MARS-5). Response rate was 69.2%. RESULTS: Of 927 with CHE, 75.5% totally or almost agreed that TCS damage the skin, 48.9% totally or almost agreed that TCS would affect their future health and 36.3% reported some degree of fear of TCS even though they were unaware of any TCS-associated risks. Most patients (77.9%) always or often stop treatment as soon as possible while 54.8% always or often wait as long as possible before starting treatment. Overall, 38.8% reported that they had taken less medicine than prescribed and 54.0% had stopped treatment throughout a period. Treatment adherence decreased with increasing corticosteroid phobia (p=0.004). LIMITATIONS: TOPICOP has not been validated in CHE patients. CONCLUSIONS: Corticosteroid phobia is common among CHE patients and negatively associated with treatment adherence.
RESUMO
BACKGROUND: Histopathologic overlap between cutaneous squamous cell carcinoma (cSCC) and its indolent mimics likely leads to the overdiagnosis of cSCC. OBJECTIVE: To perform a pilot study of the p53 immunohistochemical scoring system developed on vulvar squamous lesions in cSCC. METHODS: The consistency and reliability of p53 immunostaining using a scoring system developed on vulvar cases, as compared with TP53 genomic sequencing, was studied in an initial cohort of 28 cutaneous cases. p53 labeling was further assessed in an additional 63 cases of atypical squamous lesions, including 20 atypical squamous lesions classified by the authors as benign, 22 cases diagnosed as cSCC without high-risk features, and 21 cases of high-risk cSCC (cSCC-HR). RESULTS: The concordance of p53 labeling and TP53 sequencing was 82.1%. Four positive patterns of p53 mutation were identified: basal, parabasal/diffuse, null, and cytoplasmic. p53 positivity in atypical, benign squamous lesions (10%) was significantly lower than that of low-risk cSCC (63.6%, p = 0.0004) or cSCC-HR (90.5%, p < 0.0001). p53 positivity in low-risk cSCC versus cSCC-HR was not statistically significant (p = 0.07). CONCLUSION: p53 Labeling may be a helpful biomarker to support the diagnosis of cSCC and distinguish cSCC from atypical but benign mimics.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Cutâneas , Neoplasias Vulvares , Feminino , Humanos , Carcinoma de Células Escamosas/patologia , Proteína Supressora de Tumor p53/genética , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia , Projetos Piloto , Imuno-Histoquímica , Reprodutibilidade dos Testes , Neoplasias Vulvares/diagnóstico , Neoplasias Vulvares/patologiaRESUMO
Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.
Assuntos
5-Metilcitosina/metabolismo , Antígenos CD19/imunologia , Dioxigenases/genética , Imunoterapia/métodos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Linfócitos T/imunologia , Linfócitos T/transplante , Transferência Adotiva , Idoso , Alelos , Diferenciação Celular , Ensaios Clínicos como Assunto , Células Clonais/citologia , Células Clonais/imunologia , Dioxigenases/metabolismo , Epigênese Genética , Células HEK293 , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , TransgenesRESUMO
Epithelial monolayers are some of the best-studied models for collective cell migration due to their abundance in multicellular systems and their tractability. Experimentally, the collective migration of epithelial monolayers can be robustly steered e.g. using electric fields, via a process termed electrotaxis. Theoretically, however, the question of how to design an electric field to achieve a desired spatiotemporal movement pattern is underexplored. In this work, we construct and calibrate an ordinary differential equation model to predict the average velocity of the centre of mass of a cellular monolayer in response to stimulation with an electric field. We use this model, in conjunction with optimal control theory, to derive physically realistic optimal electric field designs to achieve a variety of aims, including maximising the total distance travelled by the monolayer, maximising the monolayer velocity, and keeping the monolayer velocity constant during stimulation. Together, this work is the first to present a unified framework for optimal control of collective monolayer electrotaxis and provides a blueprint to optimally steer collective migration using other external cues.
Assuntos
Movimento Celular , Células Epiteliais , Conceitos Matemáticos , Modelos Biológicos , Células Epiteliais/fisiologia , Células Epiteliais/citologia , Movimento Celular/fisiologia , Animais , Simulação por Computador , Resposta Táctica/fisiologia , Cães , Humanos , Células Madin Darby de Rim CaninoRESUMO
Several studies have shown the influence of commensal microbes on T cell function, specifically in the setting of checkpoint immunotherapy for cancer. In this study, we investigated how vancomycin-induced gut microbiota dysbiosis affects chimeric antigen receptor (CAR) T immunotherapy using multiple preclinical models as well as clinical correlates. In two murine tumor models, hematopoietic CD19+-A20 lymphoma and CD19+-B16 melanoma, mice receiving vancomycin in combination with CD19-directed CAR T cell (CART-19) therapy displayed increased tumor control and tumor-associated antigens (TAAs) cross-presentation compared with CART-19 alone. Fecal microbiota transplant from human healthy donors to pre-conditioned mice recapitulated the results obtained in naive gut microbiota mice. Last, B cell acute lymphoblastic leukemia patients treated with CART-19 and exposed to oral vancomycin showed higher CART-19 peak expansion compared with unexposed patients. These results substantiate the role of the gut microbiota on CAR T cell therapy and suggest that modulation of the gut microbiota using vancomycin may improve outcomes after CAR T cell therapy across tumor types.
Assuntos
Microbioma Gastrointestinal , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Receptores de Antígenos de Linfócitos T/genética , Apresentação Cruzada , Vancomicina/farmacologia , Imunoterapia , Linfócitos T , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/genética , Antígenos CD19RESUMO
Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.
Assuntos
Mesotelina , Neoplasias , Humanos , Proteínas Ligadas por GPI/genética , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Linfócitos TRESUMO
BACKGROUND: Optimal management of allergic rhinitis requires patient education with easy access to accurate information. However, previous online platforms have provided misleading information. The demand for online medical information continues to grow, especially with the introduction of advanced chatbots like ChatGPT. METHODS: This study aimed to evaluate the quality of information provided by ChatGPT regarding allergic rhinitis. A Likert scale was used to assess the accuracy of responses, ranging from 1 to 5. Four authors independently rated the responses from a healthcare professional's perspective. RESULTS: A total of 20 questions covering various aspects of allergic rhinitis were asked. Among the answers, eight received a score of 5 (no inaccuracies), five received a score of 4 (minor non-harmful inaccuracies), six received a score of 3 (potentially misinterpretable inaccuracies) and one answer had a score of 2 (minor potentially harmful inaccuracies). CONCLUSIONS: The variability in accuracy scores highlights the need for caution when relying solely on chatbots like ChatGPT for medical advice. Patients should consult qualified healthcare professionals and use online sources as a supplement. While ChatGPT has advantages in medical information delivery, its use should be approached with caution. ChatGPT can be useful for patient education but cannot replace healthcare professionals.
Assuntos
Inteligência Artificial , Rinite Alérgica , Humanos , Suplementos Nutricionais , Instalações de Saúde , Pessoal de SaúdeRESUMO
BACKGROUND: Flare patterns are not routinely considered in the severity classification or in clinical decision-making of atopic dermatitis (AD), but frequent or severe flares may contribute considerably to the disease burden. OBJECTIVES: To characterize patients with AD in relation to their flare pattern and compare flare patterns to disease severity, life quality and treatment satisfaction. METHODS: Patients with AD from the Danish Skin Cohort were included if they had active AD with and available data on number of flare-ups within the last 12 months. Categorical variables were presented as frequencies and percentages, whereas numerical variables were presented as median and interquartile ranges (IQR). Between-group differences were tested with chi-squared tests. RESULTS: A total of 1557 patients were included, with 57 reporting 0 flares, 698 (1-5 flares), 324 (6-10 flares) and 478 reporting >10 flares during the past 12 months. Both the severity measured by PO-SCORAD and the impairment of life quality measured by DLQI were higher among patients with more flares (median [IQR] PO-SCORAD: 13.0 [5.6-22.3], 29.7 [20.8-40.6], 36.3 [26.7-47.6]and 42.9 [30.7-55.6], respectively for the four flares strata, and median [IQR] DLQI: 1.0 [0.0-2.0], 3.0 [1.0-7.0], 4.0 [1.8-9.0] and 7.0 [3.0-11.0]). Satisfaction with the current treatment was generally higher among patients with no flares. However, 36.8%, 24.6% and 23.7% of patients with 1-5, 6-10 and >10 flares reported being extremely or very satisfied with their current treatment. CONCLUSIONS: Patients with many flares often report a higher severity and impairment of life quality compared to patients with fewer flares. Information on flaring could benefit treatment decisions, thereby decreasing undertreatment of patients with mild AD but severe flaring.
RESUMO
BACKGROUND: Several neurovascular procedures require temporary occlusion of cerebral arteries, leading to ischemia of unpredictable length, occasionally causing brain infarction. Experimental models of cerebral ischemia-reperfusion injury have established that platelet adhesion and coagulation play detrimental roles in reperfusion injury following transient cerebral ischemia. Therefore, in a model of cerebral ischemia-reperfusion injury (IRI), we investigated the therapeutic potential of a dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic which is able to bind to vascular injury sites. METHODS: Brain ischemia was induced in mice by transient occlusion of the right middle cerebral artery for 60 min. APAC, unfractionated heparin (UFH) (both at heparin equivalent doses of 0.5 mg/kg), or vehicle was intravenously administered 10 min before or 60 min after the start of ischemia. At 24 h later, mice were scored for their neurological and motor behavior, and brain damage was quantified. RESULTS: Both APAC and UFH administered before the onset of ischemia reduced brain injury. APAC and UFH pretreated mice had better neurological and motor functions (p < 0.05 and p < 0.01, respectively) and had significantly reduced cerebral infarct sizes (p < 0.01 and p < 0.001, respectively) at 24 h after transient occlusion compared with vehicle-treated mice. Importantly, no macroscopic bleeding complications were observed in either APAC- or UFH-treated animals. However, when APAC or UFH was administered 60 min after the start of ischemia, the therapeutic effect was lost, but without hemorrhaging either. CONCLUSIONS: Pretreatment with APAC or UFH was safe and effective in reducing brain injury in a model of cerebral ischemia induced by transient middle cerebral artery occlusion. Further studies on the use of APAC to limit ischemic injury during temporary occlusion in neurovascular procedures are indicated.