RESUMEN
Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.
Asunto(s)
Inmunohistoquímica/métodos , Inmunofenotipificación/métodos , Receptores Quiméricos de Antígenos/análisis , Linfocitos T/inmunología , Animales , Biopsia , Ingeniería Celular , Técnicas de Cocultivo , Vectores Genéticos/genética , Virus de la Hepatitis B de la Marmota/genética , Humanos , Hibridación Fluorescente in Situ , Lentivirus/genética , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Transgénicos , Modelos Animales , Adhesión en Parafina , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Piel/citología , Piel/inmunología , Piel/patología , Linfocitos T/metabolismo , Linfocitos T/trasplante , Fijación del Tejido , Transducción Genética , Quimera por TrasplanteRESUMEN
To examine the effects of poor sanitation and hygiene on the prevalence of antimicrobial-resistant bacteria, we surveyed households in two rural and two urban communities in Guatemala (N = 196 randomly selected households). One adult (≥ 18-years old) and, when available, one child (≤ 5 years-old) provided a stool sample. Up to 48 presumptive Escherichia coli isolates were collected from each stool sample (n = 21,256 total) and were subjected to breakpoint assays for ten antibiotics. Mixed-effects logistic models were used to identify potential factors influencing the likelihood of harboring antibiotic-resistant bacteria. For nine out of ten antibiotics, the odds of detecting resistant bacteria decreased by ~ 32% (odds ratios, OR 0.53-0.8, P < 0.001) for every unit of improvement of a hygiene scale. Hygiene differences between households had a greater impact on prevalence compared to antibiotic use differences. The likelihood of detecting resistant isolates was lower for five antibiotics among households that boiled raw milk before consumption (OR 0.31-0.69), and higher for nine antibiotics in urban households (OR > 1.89-9.6). Poor hygiene conditions likely obscure effects of individual antibiotic use, presumably due to enhanced microbial transmission. Consequently, efforts to improve antibiotic stewardship should be coupled with improving hygiene conditions.