CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging,... Show moreCRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina. Show less
T-cell dysregulation in chronic lymphocytic leukemia (CLL) associates with low response rates to autologous T cell-based therapies. How CLL affects antigen-specific T-cell responses remains largely... Show moreT-cell dysregulation in chronic lymphocytic leukemia (CLL) associates with low response rates to autologous T cell-based therapies. How CLL affects antigen-specific T-cell responses remains largely unknown. We investigated (epi)genetic and functional consequences of antigen-specific T-cell responses in presence of CLL in vitro and in an adoptive-transfer murine model. Already at steady-state, antigen-experienced patient-derived T cells were skewed towards short-lived effector cells (SLEC) at the expense of memory-precursor effector cells (MPEC). Stimulation of these T cells in vitro showed rapid induction of effector genes and suppression of key memory transcription factors only in presence of CLL cells, indicating epigenetic regulation. This was investigated in vivo by following antigen-specific responses of naive OT-I CD8(+) cells to mCMV-OVA in presence/absence of TCL1 B-cell leukemia. Presence of leukemia resulted in increased SLEC formation, with disturbed inflammatory cytokine production. Chromatin and transcriptome profiling revealed strong epigenetic modifications, leading to activation of an effector and silencing of a memory profile through presence of CLL cells. Secondary challenge in vivo confirmed dysfunctional memory responses by antigen-experienced OT-I cells generated in presence of CLL. Altogether, we show that presence of CLL induces a short-lived effector phenotype and impaired memory responses by epigenetic reprogramming during primary responses. Show less
Background: Plasma biomarkers may aid in the detection of anthracycline-related cardiomyopathy (ACMP). However, the currently available biomarkers have limited diagnostic value in long-term... Show moreBackground: Plasma biomarkers may aid in the detection of anthracycline-related cardiomyopathy (ACMP). However, the currently available biomarkers have limited diagnostic value in long-term childhood cancer survivors. This study sought to identify diagnostic plasma biomarkers for ACMP in childhood cancer survivors. Methods and Results: We measured 275 plasma proteins in 28 ACMP cases with left ventricular ejection fraction <45%, 29 anthracycline-treated controls with left ventricular ejection fraction >= 53% matched on sex, time after cancer, and anthracycline dose, and 29 patients with genetically determined dilated cardiomyopathy with left ventricular ejection fraction <45%. Multivariable linear regression was used to identify differentially expressed proteins. Elastic net model, including clinical characteristics, was used to assess discrimination of proteins diagnostic for ACMP. NT-proBNP (N-terminal pro-B-type natriuretic peptide) and the inflammatory markers CCL19 (C-C motif chemokine ligands 19) and CCL20, PSPD (pulmonary surfactant protein-D), and PTN (pleiotrophin) were significantly upregulated in ACMP compared with controls. An elastic net model selected 45 proteins, including NT-proBNP, CCL19, CCL20 and PSPD, but not PTN, that discriminated ACMP cases from controls with an area under the receiver operating characteristic curve (AUC) of 0.78. This model was not superior to a model including NT-proBNP and clinical characteristics (AUC=0.75; P=0.766). However, when excluding 8 ACMP cases with heart failure, the full model was superior to that including only NT-proBNP and clinical characteristics (AUC=0.75 versus AUC=0.50; P=0.022). The same 45 proteins also showed good discrimination between dilated cardiomyopathy and controls (AUC=0.89), underscoring their association with cardiomyopathy. Conclusions: We identified 3 specific inflammatory proteins as candidate plasma biomarkers for ACMP in long-term childhood cancer survivors and demonstrated protein overlap with dilated cardiomyopathy. Show less
Purpose: Late radiation toxicity is a major dose-limiting factor in curative cancer radiation therapy. Previous studies identified several risk factors for late radiation toxicity, including both... Show morePurpose: Late radiation toxicity is a major dose-limiting factor in curative cancer radiation therapy. Previous studies identified several risk factors for late radiation toxicity, including both dose-volume factors and genetic predisposition. Herein, we investigated the contribution of genetic predisposition, particularly compared with dose-volume factors, to the risk of late radiation toxicity in patients treated with highly conformal radiation therapy. Methods and Materials: We included 179 patients with prostate cancer who underwent treatment with curative external beam radiation therapy between 2009 and 2013. Toxicity was graded according to the Common Terminology Criteria for Adverse Events version 4.0. Transcriptional responsiveness of homologous recombination repair genes and g-H2AX foci decay ratios (FDRs) were determined in ex vivo irradiated lymphocytes in a previous analysis. Dose-volume parameters were retrieved by delineating the organs at risk (OARs) on CT planning images. Associations between risk factors and grade >= 2 urinary and were performed using the highest toxicity grade recorded during the follow-up per patient. Results: The median follow-up period was 31 months. One hundred and one patients (56%) developed grade >2 late radiation toxicity. Cumulative rates for urinary and bowel grade >2 late toxicities were 46% and 17%, respectively. In the multivariable analysis, factors significantly associated with grade >2 late toxicity were transurethral resection of the prostate (P = .013), y-H2AX FDR <3.41 (P = .008), and rectum V70 >11.52% (P = .017). Conclusions: Our results suggest that impaired DNA double-strand break repair in lymphocytes, as quantified by y-H2AX FDR, is the most critical determining factor of late radiation toxicity. The limited influence of dose-volume parameters could be due to the use of increasingly conformal techniques, leading to improved dose-volume parameters of the organs at risk. = 2021 Elsevier Inc. All rights reserved. Show less
Aims/hypothesisAnimal studies have indicated that disturbed diurnal rhythms of clock gene expression in adipose tissue can induce obesity and type 2 diabetes. The importance of the circadian timing... Show moreAims/hypothesisAnimal studies have indicated that disturbed diurnal rhythms of clock gene expression in adipose tissue can induce obesity and type 2 diabetes. The importance of the circadian timing system for energy metabolism is well established, but little is known about the diurnal regulation of (clock) gene expression in obese individuals with type 2 diabetes. In this study we aimed to identify key disturbances in the diurnal rhythms of the white adipose tissue transcriptome in obese individuals with type 2 diabetes.MethodsIn a case-control design, we included six obese individuals with type 2 diabetes and six healthy, lean control individuals. All participants were provided with three identical meals per day for 3days at zeitgeber time (ZT, with ZT 0:00 representing the time of lights on) 0:30, 6:00 and 11:30. Four sequential subcutaneous abdominal adipose tissue samples were obtained, on day 2 at ZT 15:30, and on day 3 at ZT 0:15, ZT 5:45 and ZT 11:15. Gene expression was measured using RNA sequencing.ResultsThe core clock genes showed reduced amplitude oscillations in the individuals with type 2 diabetes compared with the healthy control individuals. Moreover, in individuals with type 2 diabetes, only 1.8% (303 genes) of 16,818 expressed genes showed significant diurnal rhythmicity, compared with 8.4% (1421 genes) in healthy control individuals. Enrichment analysis revealed a loss of rhythm in individuals with type 2 diabetes of canonical metabolic pathways involved in the regulation of lipolysis. Enrichment analysis of genes with an altered mesor in individuals with type 2 diabetes showed decreased activity of the translation initiating pathway EIF2 signaling'. Individuals with type 2 diabetes showed a reduced diurnal rhythm in postprandial glucose concentrations.Conclusions/interpretationDiurnal clock and metabolic gene expression rhythms are decreased in subcutaneous adipose tissue of obese individuals with type 2 diabetes compared with lean control participants. Future investigation is needed to explore potential treatment targets as identified by our study, including clock enhancement and induction of EIF2 signalling.Data availabilityThe raw sequencing data and supplementary files for rhythmic expression analysis and Ingenuity Pathway Analysis have been deposited in NCBI Gene Expression Omnibus (GEO series accession number GSE104674). Show less
The efficacy of autologous (αβ) T-cell–based treatment strategies in chronic lymphocytic leukemia (CLL) has been modest. The Vγ9Vδ2-T cell subset consists of cytotoxic T lymphocytes with potent... Show moreThe efficacy of autologous (αβ) T-cell–based treatment strategies in chronic lymphocytic leukemia (CLL) has been modest. The Vγ9Vδ2-T cell subset consists of cytotoxic T lymphocytes with potent antilymphoma activity via a major histocompatibility complex–independent mechanism. We studied whether Vγ9Vδ2-T cells can be exploited as autologous effector lymphocytes in CLL. Healthy control Vγ9Vδ2-T cells were activated by and had potent cytolytic activity against CLL cells. However, CLL-derived Vγ9Vδ2-T cells proved dysfunctional with respect to effector cytokine production and degranulation, despite an increased frequency of the effector-type subset. Consequently, cytotoxicity against malignant B cells was hampered. A comparable dysfunctional phenotype was observed in healthy Vγ9Vδ2-T cells after coculture with CLL cells, indicating a leukemia-induced mechanism. Gene-expression profiling implicated alterations in synapse formation as a conceivable contributor to compromised Vγ9Vδ2-T–cell function in CLL patients. Dysfunction of Vγ9Vδ2-T cells was fully reversible upon activation with autologous monocyte-derived dendritic cells (moDCs). moDC activation resulted in efficient expansion and predominantly yielded Vγ9Vδ2-T cells with a memory phenotype. Furthermore, ibrutinib treatment promoted an antitumor T helper 1 (TH1) phenotype in Vγ9Vδ2-T cells, and we demonstrated binding of ibrutinib to IL-2-inducible kinase (ITK) in Vγ9Vδ2-T cells. Taken together, CLL-mediated dysfunction of autologous Vγ9Vδ2-T cells is fully reversible, resulting in potent cytotoxicity toward CLL cells. Our data support the potential use of Vγ9Vδ2-T cells as effector T cells in CLL immunotherapy and favor further exploration of combining Vγ9Vδ2-T-cell–based therapy with ibrutinib. Show less
BACKGROUND: Disrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the... Show moreBACKGROUND: Disrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity.OBJECTIVES: This study evaluates the potential of TRAF-STOP treatment in atherosclerosis.METHODS: The effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe-/-) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages.RESULTS: TRAF-STOP treatment of young Apoe-/- mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe-/- mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair "classical" immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and β2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe-/- mice.CONCLUSIONS: TRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.Published by Elsevier Inc.KEYWORDS: atherosclerosis; drug development; immunology; inflammation; nanotechnology Show less
