RESUMEN
The clinical diagnosis of pathogen infectious diseases increasingly requires sensitive and rapid RNA detection technologies. The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system has shown immense potential in molecular diagnostics due to its trans-cleavage activity. However, most Cas13a-based detection methods require an amplicon transcription step, and the multi-step open-tube operations are prone to contamination, limiting their widespread application. Here, we propose an ultrasensitive (single-copy range, â¼aM) and rapid (within 40 min) isothermal one-pot RNA detection platform, termed SATCAS (Simultaneous Amplification and Testing platform based on Cas13a). This method effectively distinguishes viable bacteria (0%-100%) under constant total bacterial conditions, demonstrating its robustness and universality. SATCAS excels in identifying single nucleotide polymorphisms (SNPs), particularly detecting 0.5% drug-resistant mutations. We validated SATCAS by detecting infections in biological samples from 68 HBV, 23 EBV, and 48 SARS-CoV-2 patients, achieving 100% sensitivity, 92.86% specificity, and 97.06% accuracy in HBV infection testing. We anticipate that SATCAS has broad application potential in the early diagnosis, subtyping, drug resistance detection, and point-of-care monitoring of pathogen infectious diseases.
Asunto(s)
Técnicas Biosensibles , Sistemas CRISPR-Cas , Técnicas de Amplificación de Ácido Nucleico , Polimorfismo de Nucleótido Simple , SARS-CoV-2 , Humanos , Técnicas Biosensibles/métodos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , COVID-19/diagnóstico , COVID-19/virología , ARN Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificaciónRESUMEN
ABSTRACT: Graft-versus-host disease (GVHD) is a major life-threatening complication that occurs after allogeneic hematopoietic cell transplantation (HCT). Although adult tissue stem cells have been identified as targets of GVHD in the skin and gut, their role in hepatic GVHD is yet to be clarified. In the current study, we explored the fate of bile duct stem cells (BDSCs), capable of generating liver organoids in vitro, during hepatic GVHD after allogeneic HCT. We observed a significant expansion of biliary epithelial cells (BECs) on injury early after allogeneic HCT. Organoid-forming efficiency from the bile duct was also significantly increased early after allogeneic HCT. Subsequently, the organoid-forming efficiency from bile ducts was markedly decreased in association with the reduction of BECs and the elevation of plasma concentrations of bilirubin, suggesting that GVHD targets BDSCs and impairs the resilience of BECs. The growth of liver organoids in the presence of liver-infiltrating mononuclear cells from allogeneic recipients, but not from syngeneic recipients, was significantly reduced in a transforming growth factor-ß (TGF-ß)-dependent manner. Administration of SB-431542, an inhibitor of TGF-ß signaling, from day 14 to day 28, protected organoid-forming BDSCs against GVHD and mitigated biliary dysfunction after allogeneic HCT, suggesting that BDSCs are a promising therapeutic target for hepatic GVHD.
Asunto(s)
Conductos Biliares , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Organoides , Factor de Crecimiento Transformador beta , Animales , Enfermedad Injerto contra Huésped/patología , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/prevención & control , Conductos Biliares/patología , Factor de Crecimiento Transformador beta/metabolismo , Ratones , Células Madre/metabolismo , Células Madre/citología , Ratones Endogámicos C57BL , Masculino , Hígado/patología , Hígado/metabolismo , Dioxoles/farmacología , Benzamidas/farmacología , Células Epiteliales/metabolismo , Trasplante HomólogoRESUMEN
Calcineurin inhibitor-based graft-versus-host disease (GVHD) prophylaxis is standard in allogeneic hematopoietic stem cell transplantation (HCT) but fails to induce long-term tolerance without chronic GVHD (cGVHD) in a considerable number of patients. In this study, we addressed this long-standing question in mouse models of HCT. After HCT, alloreactive donor T cells rapidly differentiated into PD-1+ TIGIT+ terminally exhausted T cells (terminal Tex). GVHD prophylaxis with cyclosporine (CSP) suppressed donor T-cell expression of TOX, a master regulator to promote differentiation of transitory exhausted T cells (transitory Tex), expressing both inhibitory receptors and effector molecules, into terminal Tex, and inhibited tolerance induction. Adoptive transfer of transitory Tex, but not terminal Tex, into secondary recipients developed cGVHD. Transitory Tex maintained alloreactivity and thus PD-1 blockade restored graft-versus-leukemia (GVL) activity of transitory Tex and not terminal Tex. In conclusion, CSP inhibits tolerance induction by suppressing the terminal exhaustion of donor T cells, while maintaining GVL effects to suppress leukemia relapse.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Leucemia , Ratones , Animales , Inhibidores de la Calcineurina/farmacología , Linfocitos T , Enfermedad Injerto contra Huésped/prevención & control , Receptor de Muerte Celular Programada 1 , Ciclosporina/farmacología , Tolerancia InmunológicaRESUMEN
Granulopoiesis in the bone marrow adjusts cellular output as demand for neutrophils changes. Reactive granulopoiesis is induced by profound neutropenia, but its mechanism remains to be clarified. We herein explored its mechanisms using mouse models of syngeneic hematopoietic stem cell transplantation (SCT) and 5-fluorouracil-induced neutropenia. After SCT, T cell production of IL-17A was up-regulated. Neutrophil recovery was significantly delayed in IL-17A-deficient or T cell-deficient RAG1-/- mice, and adoptive transfer of wild-type (WT) T cells facilitated neutrophil engraftment. Gut decontamination with oral antibiotics suppressed T cell production of IL-17A and impaired neutrophil recovery. Transplantation of fecal microbiota collected from neutropenic, not naive, mice promoted neutrophil recovery in these mice, suggesting that neutropenia-associated microbiota had a potential to stimulate reactive granulopoiesis. Our study uncovered a cross talk between gut microbiota and neutropenia after SCT and chemotherapy.
Asunto(s)
Microbioma Gastrointestinal , Neutropenia , Ratones , Animales , Interleucina-17 , Linfocitos T , Ratones NoqueadosRESUMEN
Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a potentially curative therapy for FLT3 internal tandem duplication mutant (FLT3-ITD+) acute myeloid leukemia, but relapse rate is high. A recent study showed that sorafenib, a first generation FLT3 and multikinase inhibitor, enhanced graft-versus-leukemia (GVL) effects against FLT3-ITD+ leukemia via interleukin-15 (IL-15) production. However, it remains to be clarified whether this effect could be mediated by selective FLT3 inhibition. We investigated whether gilteritinib, a selective FLT3 inhibitor, could enhance GVL effects against FLT3-ITD transfected Ba/F3 leukemia (Ba/F3-FLT3-ITD) in mice. Oral administration of gilteritinib from day +5 to +14 after allo-SCT reduced expression of the co-inhibitory receptors PD-1 and TIGIT on donor CD8+ T cells and enhanced IL-15 expression in Ba/F3-FLT3-ITD. Bioluminescent imaging using luciferase-transfected Ba/F3-FLT3-ITD demonstrated that gilteritinib significantly suppressed leukemia expansion after allo-SCT, whereas it did not impact the morbidity or mortality of graft-versus-host disease (GVHD), resulting in significant improvement of overall survival. In conclusion, short-term administration of gilteritinib after allo-SCT enhanced GVL effects against FLT3-ITD+ leukemia without exacerbating GVHD.
Asunto(s)
Efecto Injerto vs Leucemia , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Compuestos de Anilina , Animales , Linfocitos T CD8-positivos , Interleucina-15 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Ratones , Mutación , Pirazinas , Trasplante Homólogo , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Posttransplant high-dose cyclophosphamide (PTCY) has been increasingly used as graft-versus-host disease (GVHD) prophylaxis after HLA-haploidentical or matched hematopoietic stem cell transplantation (SCT). However, PTCY alone is insufficient and requires additional immunosuppressants such as calcineurin inhibitors. In the current study, we evaluated effects of a novel GVHD prophylaxis with PTCY in combination with short-term KRP203, a selective agonist of sphingosine-1-phosphate receptor 1 that regulates egress of lymphocytes from the secondary lymphoid organs (SLOs) in mice. Short-term oral administration of KRP203 alone induced apoptosis of donor T cells in the SLOs and ameliorated GVHD. Administration of KRP203 significantly preserved graft-versus-leukemia effects compared to cyclosporin. A combination of KRP203 on days 0 to +4 and PTCY on day +3 synergistically suppressed donor T-cell migration into the intestine and skin, and ameliorated GVHD more potently than PTCY alone. A combination of short-term KRP203 and PTCY is a promising novel calcineurin-free GVHD prophylaxis in HLA-haploidentical SCT.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Animales , Ciclofosfamida , Ciclosporina/farmacología , Enfermedad Injerto contra Huésped/prevención & control , Inmunosupresores , RatonesRESUMEN
Myeloid differentiation factor 88 (MyD88) signaling has a crucial role in activation of both innate and adoptive immunity. MyD88 transduces signals via Toll-like receptor and interleukin-1 receptor superfamily to the NFκB pathway and inflammasome by forming a molecular complex with interleukin-1 receptor-associated kinase 4. The MyD88/interleukin-1 receptor-associated kinase 4 pathway plays an important role, not only in innate immunity, but also T-cell immunity; however, its role in donor T cells on the pathophysiology of graft-versus-host disease (GvHD) remains to be elucidated. We addressed this issue by using MyD88-deficient T cells in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-SCT). While MyD88-deficient and wild-type T cells proliferated equivalently after transplantation, MyD88-deficient T cells demonstrated impaired survival and differentiation toward Th1, Tc1, and Th17, and induced less severe GvHD compared to wild-type T cells. Administration of interleukin-1 receptor-associated kinase 4 inhibitor PF-06650833 significantly ameliorated GvHD after allo-SCT. These results thus demonstrate that donor T-cell MyD88/interleukin-1 receptor-associated kinase 4 pathway is a novel therapeutic target against GvHD after allo-SCT.