ABSTRACT
Increasing evidence supports a role for small extracellular vesicles (sEV, including exosomes) in Diffuse Large B-cell lymphoma (DLBCL) progression and resistance to treatment. CD20 and PD-L1 are found on DLBCL-derived sEV, but little is known about their patient-level heterogeneity. Moreover, the capacity of PD-L1+ sEV to modulate T cells needs to be clarified. Herein we analyzed sEV produced by human DLBCL cell lines and EBV-transformed B cell-lymphoblastoid cell lines (LCLs), a model allowing autologous T cell co-cultures. We determined CD20 and PD-L1 levels on plasma sEV from patient samples vs healthy volunteers (HV). sEV functional relevance was also investigated on CD4+ and CD8+ T cells. sEV derived from all cell lines showed an enrichment of CD20 and a high glycosylated PD-L1 expression when compared to cell lysates. High PD-L1 expression on LCL-derived sEV was associated with higher CD4+ and CD8+ T cell apoptosis. In patients, plasma sEV concentration was higher vs HV. Compared to sEV-CD20 level that seemed higher in patients, PD-L1 level in sEV was not different from those of HV. A high glycosylated PD-L1 level was shown in sEV from both patients and HV plasma samples, that was associated with the same inhibiting effect on activated T cells. We conclude that sEV derived from EBV-transformed B cells realize an immunosuppressive role that involved cell-cell interaction and probably at least PD-L1. Furthermore, our findings suggest the potential of circulating sEV as a source of biomarkers in DLBCL, notably to have information on immunotherapeutic target levels of parental tumor cells.
ABSTRACT
Early and sensitive biomarkers of liver dysfunction and drug-induced liver injury (DILI) are still needed, both for patient care and drug development. We developed the Serum Enhanced Binding (SEB) test to reveal post-transcriptional modifications (PTMs) of human serum albumin resulting from hepatocyte dysfunctions and further evaluated its performance in an animal model. The SEB test consists in spiking serum ex-vivo with ligands having specific binding sites related to the most relevant albumin PTMs and measuring their unbound fraction. To explore the hypothesis that albumin PTMs occur early during liver injury and can also be detected by the SEB test, we induced hepatotoxicity in male albino Wistar rats by administering high daily doses of ethanol and CCl4 over several days. Blood was collected for characterization and quantification of albumin isoforms by high-resolution mass spectrometry, for classical biochemical analyses as well as to apply the SEB test. In the exposed rats, the appearance of albumin isoforms paralleled the positivity of the SEB test ligands and histological injuries. These were observed as early as D3 in the Ethanol and CCl4 groups, whereas the classical liver tests (ALT, AST, PAL) significantly increased only at D7. The behavior of several ligands was supported by structural and molecular simulation analysis. The SEB test and albumin isoforms revealed hepatocyte damage early, before the current biochemical biomarkers. The SEB test should be easier to implement in the clinics than albumin isoform profiling.
Subject(s)
Chemical and Drug Induced Liver Injury , Liver , Rats , Male , Humans , Animals , Liver/metabolism , Rats, Wistar , Chemical and Drug Induced Liver Injury/pathology , Albumins/metabolism , Ethanol/metabolism , Biomarkers/metabolism , Protein Isoforms/metabolism , Carbon Tetrachloride/toxicityABSTRACT
BACKGROUND AND AIMS: The muscle retracting sign (MRS) can be present during endoscopic submucosal dissection (ESD) of macronodular colorectal lesions. The prevalence of MRS and its pathologic and clinical implications is unclear. This study evaluated the effect of MRS on the technical and clinical outcomes of ESD. METHODS: All patients referred for ESD of protruding lesions or granular mixed lesions with >10 mm macronodule granular mixed laterally spreading tumors (LST-GMs) in 2 academic centers from January 2017 to October 2022 were prospectively included. Size of the macronodule was analyzed retrospectively. The primary outcome was the curative resection rate according to MRS status. Secondary outcomes were R0 resection, perforation, secondary surgery rate, and risk factors for MRS. RESULTS: Of 694 lesions, 84 (12%) had MRS (MRS+). The curative resection rate was decreased by MRS (MRS+ 41.6% vs lesions without MRS [MRS-] 81.3%), whereas the perforation (MRS+ 22.6% vs MRS- 9.2%), submucosal cancer (MRS+ 34.9% vs MRS- 9.2%), and surgery (MRS+ 45.2% vs MRS- 6%) rates were increased. The R0 resection rate of MRS+ colonic lesions was lower than that of rectal lesions (53% vs 74.3%). In multivariate analysis, protruding lesions (odds ratio, 2.47; 95% confidence interval, 1.27-4.80) and macronodules >4 cm (odds ratio, 4.24; 95% confidence interval, 2.23-8.05) were risk factors for MRS. CONCLUSIONS: MRS reduces oncologic outcomes and increases the perforation rate. Consequently, procedures in the colon should be stopped if MRS is detected, and those in the rectum should be continued due to the morbidity of alternative therapy.
Subject(s)
Colorectal Neoplasms , Endoscopic Mucosal Resection , Humans , Prevalence , Retrospective Studies , Clinical Relevance , Dissection/methods , Muscles/pathology , Colorectal Neoplasms/pathology , Treatment Outcome , Intestinal Mucosa/surgery , Intestinal Mucosa/pathologyABSTRACT
Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3ß, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3ß, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.
Subject(s)
Intellectual Disability , Mental Retardation, X-Linked , Humans , Intellectual Disability/genetics , Creatine/genetics , Creatine/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Mental Retardation, X-Linked/genetics , Mental Retardation, X-Linked/metabolism , Brain/metabolism , Organoids/metabolismABSTRACT
Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8-/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8-/y mice and wild type and DCE-treated Slc6a8-/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD.
ABSTRACT
Background: In the context of personalized medicine, screening patients to identify targetable molecular alterations is essential for therapeutic decisions such as inclusion in clinical trials, early access to therapies, or compassionate treatment. The objective of this study was to determine the real-world impact of routine incorporation of FoundationOne analysis in cancers with a poor prognosis and limited treatment options, or in those progressing after at least one course of standard therapy. Methods: A FoundationOneCDx panel for solid tumor or liquid biopsy samples was offered to 204 eligible patients. Results: Samples from 150 patients were processed for genomic testing, with a data acquisition success rate of 93%. The analysis identified 2419 gene alterations, with a median of 11 alterations per tumor (range, 0-86). The most common or likely pathogenic variants were on TP53, TERT, PI3KCA, CDKN2A/B, KRAS, CCDN1, FGF19, FGF3, and SMAD4. The median tumor mutation burden was three mutations/Mb (range, 0-117) in 143 patients with available data. Of 150 patients with known or likely pathogenic actionable alterations, 13 (8.6%) received matched targeted therapy. Sixty-nine patients underwent Molecular Tumor Board, which resulted in recommendations in 60 cases. Treatment with genotype-directed therapy had no impact on overall survival (13 months vs. 14 months; p = 0.95; hazard ratio = 1.04 (95% confidence interval, 0.48-2.26)]. Conclusions: This study highlights that an organized center with a Multidisciplinary Molecular Tumor Board and an NGS screening system can obtain satisfactory results comparable with those of large centers for including patients in clinical trials.
ABSTRACT
We report the case of a 36-year-old patient who was initially managed for gynecomastia. The first biological analyses showed a moderately elevated alpha-fetoprotein (AFP) level. After an endocrine etiology was excluded, an abdominal computed tomography scan showed typical focal nodular hyperplasia (FNH) proven by biopsy and showing expression of AFP in FNH cells. After follow-up for 24 months, the serum AFP and liver radiology remained unchanged. The association between an elevated AFP and FNH is rarely described in the medical literature.
ABSTRACT
INTRODUCTION: In Western countries, debates between ESD vs piece-meal EMR as the best treatment for large colorectal adenomas persist regarding the difficulty of ESD the colon, and the safety and relatively good results of piece-meal endoscopic mucosal resection (EMR). Pocket-creation method (PCM) and double-clip countertraction (DCT) are two strategies recently published to facilitate ESD in this challenging situation. METHOD: This is a randomized animal study to compare PCM and DCT strategies for colonic ESD on ex vivo models (bovine colon) performed by 3 operators novice in ESD. Hybridknife type T was used to inject normal saline tinted with a small amount of blue dye in all procedures. Randomization was stratified according to the use of gravity assist. Primary endpoint was the difference in resection speed between PCM and DCT strategies. RESULTS: Resection speed was significantly higher in the DCT group than in the PCM group (56.3 vs. 31.6 mm2/min, p = 0.01). Technical success rate, defined as en bloc resection in under 60 min, was significantly better in the DCT group than in the PCM group (100% vs. 84.4%, p = 0.024), perforation rate was lower (0% vs. 18.8%, p = 0.012), and difficulty score was better (2.4 vs. 6.2, p < 0.0001) as was procedure duration (24.2 vs. 40.2 min, p < 0.0001). CONCLUSION: DCT was superior to PCM for ESD in our validated bovine colon model. This strategy is inexpensive, easy to use and adaptive. It might facilitate the widespread use of colonic ESD in Western countries and change Western ideas regarding the use of colonic ESD compared with piece-meal EMR for large benign lesions.
Subject(s)
Endoscopic Mucosal Resection/methods , Surgical Instruments , Traction , Animals , Cattle , Colorectal Neoplasms/surgery , Gravitation , Humans , Treatment OutcomeABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Subject(s)
Adenoma , Endoscopic Mucosal Resection , Adenoma/surgery , Humans , Surgical Instruments , TractionABSTRACT
Neurodegenerative diseases, including Alzheimer's and Parkinson's disease, are characterized by increased protein aggregation in the brain, progressive neuronal loss, increased inflammation, and neurogenesis impairment. We analyzed the effects of a new purine derivative drug, PDD005, in attenuating mechanisms involved in the pathogenesis of neurodegenerative diseases, using both in vivo and in vitro models. We show that PDD005 is distributed to the brain and can rescue cognitive deficits associated with aging in mice. Treatment with PDD005 prevents impairment of neurogenesis by increasing sex-determining region Y-box 2, nestin, and also enhances synaptic function through upregulation of synaptophysin and postsynaptic density protein 95. PDD005 treatment also reduced neuro-inflammation by decreasing interleukin-1ß expression, activation of astrocytes, and microglia. We identified prohibitin as a potential target in mediating the therapeutic effects of PDD005 for the treatment of cognitive deficit in aging mice. Additionally, in the current study, glycogen synthase kinase appears to attenuate tau pathology.
Subject(s)
Cognition Disorders/prevention & control , Hippocampus/drug effects , Molecular Targeted Therapy , Nerve Tissue Proteins/antagonists & inhibitors , Neuroprotective Agents/pharmacology , Repressor Proteins/antagonists & inhibitors , Tauopathies/prevention & control , Aging/psychology , Animals , Blood-Brain Barrier , Brain/metabolism , Cells, Cultured , Cognition Disorders/drug therapy , Donepezil/pharmacology , Drug Evaluation, Preclinical , Endothelial Cells/drug effects , Gene Expression Regulation/drug effects , Glycogen Synthase Kinase 3 beta/biosynthesis , Glycogen Synthase Kinase 3 beta/genetics , Interleukin-1beta/biosynthesis , Interleukin-1beta/genetics , Male , Maze Learning/drug effects , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/drug effects , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Neurogenesis/drug effects , Neuroglia/drug effects , Neuronal Plasticity/drug effects , Neuroprotective Agents/pharmacokinetics , Phosphorylation/drug effects , Prohibitins , Protein Processing, Post-Translational/drug effects , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Tauopathies/drug therapy , tau Proteins/metabolismSubject(s)
Abdominal Pain/etiology , Ehlers-Danlos Syndrome/diagnosis , Gastrointestinal Hemorrhage/diagnosis , Hematoma/diagnosis , Pelvic Pain/etiology , Abdominal Pain/surgery , Adult , Colectomy , Colon/diagnostic imaging , Colon/pathology , Ehlers-Danlos Syndrome/complications , Ehlers-Danlos Syndrome/therapy , Gastrointestinal Hemorrhage/etiology , Gastrointestinal Hemorrhage/surgery , Hematoma/etiology , Hematoma/surgery , Hospitalization , Humans , Intestinal Mucosa/diagnostic imaging , Intestinal Mucosa/pathology , Male , Pelvic Pain/surgery , Tomography, X-Ray ComputedABSTRACT
The development of effective central nervous system (CNS) drugs has been hampered by the lack of robust strategies to mimic the blood-brain barrier (BBB) and cerebrovascular impairments in vitro. Recent technological advancements in BBB modeling using induced pluripotent stem cells (iPSCs) allowed to overcome some of these obstacles, nonetheless the pertinence for their use in drug permeation study remains to be established. This mandatory information requires a cross comparison of in vitro and in vivo pharmacokinetic data in the same species to avoid failure in late clinical drug development. Here, we measured the BBB permeabilities of 8 clinical positron emission tomography (PET) radioligands with known pharmacokinetic parameters in human brain in vivo with a newly developed in vitro iPSC-based human BBB (iPSC-hBBB) model. Our findings showed a good correlation between in vitro and in vivo drug brain permeability (R2 = 0.83; P = 0.008) which contrasted with the limited correlation between in vitro apparent permeability for a set of 18 CNS/non-CNS compounds using the in vitro iPSCs-hBBB model and drug physicochemical properties. Our data suggest that the iPSC-hBBB model can be integrated in a flow scheme of CNS drug screening and potentially used to study species differences in BBB permeation.
Subject(s)
Blood-Brain Barrier/chemistry , Brain/diagnostic imaging , Induced Pluripotent Stem Cells/cytology , Neuroglia/cytology , Animals , Blood-Brain Barrier/diagnostic imaging , Brain/metabolism , Cell Differentiation , Cells, Cultured , Drug Evaluation, Preclinical , Humans , Induced Pluripotent Stem Cells/metabolism , Mice , Models, Biological , Neuroglia/metabolism , Permeability , Positron-Emission Tomography , Proof of Concept Study , RatsABSTRACT
PURPOSE: Assessment of the risk of recurrence is essential to determine the therapeutic strategy of meningioma treatment. Many relapsing or aggressive meningiomas show elevated mitotic and/or Ki67 indices, reflecting cell cycle deregulation. As CDKN2A is a key tumor suppressor gene involved in cell cycle control, we investigated whether CDKN2A alterations may be involved in tumor recurrence. METHODS: We carried out a comparative analysis of 17 recurrent and 13 non-recurrent meningiomas. CDKN2A single nucleotide variations (SNVs), deletions, methylation status of the promotor, and p16 expression were investigated. Results were correlated with the recurrent or non-recurrent status and clinicopathological data. RESULTS: We identified a CDKN2A SNV (NM_000077, exon2, c.G442A, p.Ala148Thr) in five meningiomas that was significantly associated with recurrence (p = 0.03). This mutation, confirmed by Sanger sequencing and referenced in the COSMIC database in various cancers, has not been reported in meningioma. The presence of one of the three following CDKN2A alterations-p.(Ala148Thr) mutation, whole homozygous or heterozygous gene loss, or promotor methylation > 8%-was observed in 13 of the 17 relapsing meningiomas and was strongly associated with recurrence (p < 0.0001) and a Ki67 labeling index > 7% (p = 0.004). CONCLUSION: We report an undescribed p.(Ala148Thr) CDKN2A mutation in meningioma that was only present in relapsing tumors. In our series, CDKN2A gene alterations were only found in recurrent meningiomas. However, our results need to be evaluated on a larger series to ensure that these CDKN2A alterations can be used as biomarkers of recurrence in meningioma.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p16/genetics , Meningeal Neoplasms/genetics , Meningioma/genetics , Neoplasm Recurrence, Local/genetics , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Meningeal Neoplasms/pathology , Meningioma/pathology , Middle Aged , Mutation , Neoplasm Recurrence, Local/pathology , Retrospective StudiesABSTRACT
Background and study aims ESD in the colon is more challenging technically than in other locations. Here, we report the first comparative case series of colon ESD using a systematic countertraction strategy using two clips and a rubber band. Patients and methods Retrospective comparative study of classic versus countertraction colon ESD performed in colon ESD cases collected prospectively at Lyon Edouard Herriot Hospital and Limoges University Hospital from January 2016 until December 2017. Results The study included 192 cases (controlâ=â76, countertractionâ=â116). Countertraction using the double clip and rubber band technique versus the control group resulted in a significant decrease in the procedure time (94.7 vs . 117 min; P â=â0.004) and significant increases in procedure speed (28.2 vs . 16.7âmm 2 /min; P â<â0.0001), en bloc resection rate (95.7â% vs . 76.3â%, P â<â0.0001), and R0 resection rate (78.5â% vs . 64.5â%, P â=â0.04). At an individual operator point of view, results varied between operators but the double clip countertraction strategy significantly increased the en bloc resection rate, R0 resection rate, and speed of dissection for each of the 4 operators. Conclusion Systematic countertraction using a double clip and rubber band facilitates colon ESD. This strategy should become the standard for colon ESD.
Subject(s)
Endoscopic Mucosal Resection , Esophageal Neoplasms , Lymphangioma, Cystic , Deglutition Disorders/diagnosis , Deglutition Disorders/etiology , Endoscopic Mucosal Resection/instrumentation , Endoscopic Mucosal Resection/methods , Esophageal Neoplasms/pathology , Esophageal Neoplasms/surgery , Female , Humans , Lymphangioma, Cystic/pathology , Lymphangioma, Cystic/surgery , Middle Aged , Treatment Outcome , Tumor BurdenSubject(s)
Carcinoma , Colon, Ascending , Colonic Neoplasms , Colonoscopy , Endoscopic Mucosal Resection , Gastroscopes , Adult , Carcinoma/pathology , Carcinoma/surgery , Colon, Ascending/pathology , Colon, Ascending/surgery , Colonic Neoplasms/pathology , Colonic Neoplasms/surgery , Colonoscopy/instrumentation , Colonoscopy/methods , Endoscopic Mucosal Resection/instrumentation , Endoscopic Mucosal Resection/methods , Humans , Male , Treatment OutcomeABSTRACT
Human mini-brains (MB) are cerebral organoids that recapitulate in part the complexity of the human brain in a unique three-dimensional in vitro model, yielding discrete brain regions reminiscent of the cerebral cortex. Specific proteins linked to neurodegenerative disorders are physiologically expressed in MBs, such as APP-derived amyloids (Aß), whose physiological and pathological roles and interactions with other proteins are not well established in humans. Here, we demonstrate that neuroectodermal organoids can be used to study the Aß accumulation implicated in Alzheimer's disease (AD). To enhance the process of protein secretion and accumulation, we adopted a chemical strategy of induction to modulate post-translational pathways of APP using an Amyloid-ß Forty-Two Inducer named Aftin-5. Secreted, soluble Aß fragment concentrations were analyzed in MB-conditioned media. An increase in the Aß42 fragment secretion was observed as was an increased Aß42/Aß40 ratio after drug treatment, which is consistent with the pathological-like phenotypes described in vivo in transgenic animal models and in vitro in induced pluripotent stem cell-derived neural cultures obtained from AD patients. Notably in this context we observe time-dependent Aß accumulation, which differs from protein accumulation occurring after treatment. We show that mini-brains obtained from a non-AD control cell line are responsive to chemical compound induction, producing a shift of physiological Aß concentrations, suggesting that this model can be used to identify environmental agents that may initiate the cascade of events ultimately leading to sporadic AD. Increases in both Aß oligomers and their target, the cellular prion protein (PrPC), support the possibility of using MBs to further understand the pathophysiological role that underlies their interaction in a human model. Finally, the potential application of MBs for modeling age-associated phenotypes and the study of neurological disorders is confirmed.