RESUMO
Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.
Assuntos
Heterogeneidade Genética , Hibridização in Situ Fluorescente , RNA/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunofenotipagem , Imunoterapia Adotiva , Hibridização in Situ Fluorescente/métodos , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Especificidade de Órgãos/genética , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To evaluate the effect of different treatment strategies on enterohaemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic syndrome. DESIGN: Multicentre retrospective case-control study. SETTING: 23 hospitals in northern Germany. PARTICIPANTS: 298 adults with enterohaemorrhagic E coli induced haemolytic uraemic syndrome. MAIN OUTCOME MEASURES: Dialysis, seizures, mechanical ventilation, abdominal surgery owing to perforation of the bowel or bowel necrosis, and death. RESULTS: 160 of the 298 patients (54%) temporarily required dialysis, with only three needing treatment long term. 37 patients (12%) had seizures, 54 (18%) required mechanical ventilation, and 12 (4%) died. No clear benefit was found from use of plasmapheresis or plasmapheresis with glucocorticoids. 67 of the patients were treated with eculizumab, a monoclonal antibody directed against the complement cascade. No short term benefit was detected that could be attributed to this treatment. 52 patients in one centre that used a strategy of aggressive treatment with combined antibiotics had fewer seizures (2% v 15%, P = 0.03), fewer deaths (0% v 5%, p = 0.029), required no abdominal surgery, and excreted E coli for a shorter duration. CONCLUSIONS: Enterohaemorrhagic E coli induced haemolytic uraemic syndrome is a severe self limiting acute condition. Our findings question the benefit of eculizumab and of plasmapheresis with or without glucocorticoids. Patients with established haemolytic uraemic syndrome seemed to benefit from antibiotic treatment and this should be investigated in a controlled trial.