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Systematic evaluation of neoepitope predictions challenges clinically observed T-cell responses and their impact on immune evasion
Full text linkPeptide presentation on human leukocyte antigens (HLAs) is essential for initiating T-cell responses and all consequences of this presentation including anticancer immunity or immune escape. Many studies have relied on in silico prediction tools rather than biological measurement of HLA presentation to study these effects. To better assess the frequency and consequences of neoantigen presentation, we overexpressed 125 combinations of full-length neoantigens and one HLA class I allele to experimentally validate presentation of mutated and non-mutated HLA ligands through HLA ligand isolation followed by tandem mass spectrometry. A successful presentation was observed only in 22% of predicted cases with strong implications on previously described downstream effects. For example, the association of HLA loss of heterozygosity with predicted neoepitopes was challenged for 58% (73/125) of combinations. Furthermore, when testing 51 sequences used for personalized messenger RNA neoepitope vaccines, we observed that clinical responses were independent of the presentation status of the neoepitopes. Even a presumably neoepitope-specific and strongly expanded T cell receptor clone from a neoantigen vaccination study could not be linked to a successfully presented neoepitope. Overall, these data highlight the importance of validating the presentation of neoepitopes to fully understand our interpretation of clinical mutation-specific responses and their related effects, including immune evasion
Brain atrophy staging in spinocerebellar ataxia type 3 for clinical prognosis and trial enrichment
Full text linkBACKGROUND: Spinocerebellar ataxia type 3 (SCA3) is characterised by progressive brain atrophy, with regional volume loss detectable via MRI prior to clinical manifestation. We aimed to identify the previously unknown sequence of brain atrophy in SCA3 and evaluate whether this sequence can be translated into an atrophy staging framework to enable accurate clinical prognosis and trial enrichment. METHODS: We included data from 322 SCA3 mutation carriers, enrolled in observational studies conducted across Europe, the Americas, and Asia. Participants underwent follow-up assessments up to five years after baseline. The Subtype and Stage Inference machine learning algorithm was applied to estimate the most likely atrophy sequence(s) from baseline anatomical MRI. The Scale for the Assessment and Rating of Ataxia (SARA) was used to capture ataxia severity. Atrophy stages were analysed in relation to SARA and time from disease onset. Interventional trials were simulated to estimate required sample sizes under different atrophy stage eligibility criteria. FINDINGS: We identified a uniform sequence of brain atrophy in SCA3, characterised by earliest volumetric decline in the caudal brainstem and substantial involvement of the white matter. Atrophy stage was associated with both SARA and time from disease onset. Atrophy staging outperformed single-region volumetrics in predicting SARA over time. Applying atrophy stage cut-offs substantially reduced the sample sizes needed to adequately power hypothetical clinical trials. INTERPRETATION: These findings yield mechanistic insights into the progression of neurodegeneration in SCA3 and possess immediate translational relevance, facilitating patient stratification and sample enrichment for interventional trials
Kidney disease reprograms microbiome-host signaling to promote heart failure
No full textGut microbiome-derived metabolites regulate host physiology, with systemic levels controlled by renal excretion. In kidney disease, impaired clearance leads to metabolite accumulation, inflammation, and secondary cardiovascular damage. We show that adverse cardiac remodeling following kidney disease critically depends on the microbiome. We identify microbiome-derived indoxyl sulfate-mediated activation of the aryl hydrocarbon receptor (AhR) as a key mechanism promoting IL-17A-producing T cells and cardiac fibrosis. AhR inhibition attenuates inflammation and cardiac remodeling. On population level, IL-17A is elevated in chronic kidney disease, particularly with coexisting heart failure. Mechanistically, AhR and IL-17A signaling induce a pro-fibrotic phenotype in cardiac fibroblasts, with conserved responses in human cells. This work identifies a microbiome-AhR-IL-17A axis as a mediator and druggable target of cardiovascular damage in kidney disease
Prognostic impact of TROP2 in adenocarcinoma of the esophageal junction and stomach
Full text linkINTRODUCTION
Adenocarcinoma of the esophageal junction and stomach (AEG/S) remains one of the deadliest cancers worldwide. New treatment options are urgently needed. A new target could be trophoblast cell surface protein 2 (TROP2), which is expressed in a variety of solid tumors and can be targeted, e.g., by sacituzumab govitecan, which has shown promising results in triple-negative breast cancer. This study investigates the expression of TROP2 in patients with AEG/S and correlates its expression with clinical and histopathological endpoints.
METHODS
TROP2 expression was assessed in a cohort of 250 patients who underwent primary surgery for AEG/S. Immunohistochemistry was performed on tissue microarrays constructed from primary tumors and lymph node metastases to quantify TROP2 expression intensity. Clinical variables, including overall survival and patient demographics, as well as tumor-specific characteristics such as stage and grade, were correlated with TROP2 expression to evaluate its potential prognostic relevance in AEG/S.
RESULTS
TROP2 was expressed in 86% of primary tumors and 81.3% of lymph node metastases. The intensity of TROP2 expression (low vs. medium vs. high) was correlated negatively with overall survival (p < 0.05, 70.9 months vs. 54.2 months vs. 39.5 months), lymphatic invasion (p = 0.05, V = 0.138), and higher grading (p = 0.037, V = 0.143). The intensity of TROP2 expression in lymph node metastases and primary tumors correlated significantly (p < 0.001, ρ = 0.444). There was a non-significant increase in positive lymphonodal status (p = 0.093, V = 0.138) in patients with higher TROP2 expression.
CONCLUSION
In Caucasian AEG/S patients, TROP2 is expressed in the majority of cases. TROP2 expression intensity itself has an impact on survival, which could be explained by a more aggressive phenotype, which leads to lymphatic invasion and lymph node metastasis
Confounders, diagnostic accuracy and reproducibility in CMR-feature tracking derived left atrial strain: a BER-CMR multisoftware, multi-site comparison
No full textAIMS: Cardiovascular Magnetic Resonance-feature tracking (CMR-FT) derived left atrial global longitudinal strain (LA-GLS) has prognostic relevance, even in the early stages of cardiovascular diseases. Identifying technical and subject-related confounders is essential for ensuring comparability across sites and for reliably distinguishing healthy from pathological conditions. This study aimed to evaluate the influence of post-processing software and subject-related factors on CMR-FT derived LA-GLS, diagnostic accuracy and to evaluate inter-site reproducibility. METHODS: This study included 149 healthy individuals and 40 patients with atrial fibrillation (AF; 19 persistent, 21 paroxysmal) from a single site. A subgroup of 18 traveling volunteers underwent CMR at four different sites. All participants underwent CMR in sinus rhythm. LA-GLS was assessed using three post-processing software packages (CVI42, TrufiStrain Research Prototype, Medis). Mixed models with repeated measures were applied to evaluate the effect of software, site and subject-related factors on LA-GLS components. ROC curve analysis was used to assess diagnostic accuracy across software in distinguishing healthy controls from AF patients. RESULTS: All GLS components differed across post-processing software (p<.001). Reservoir and contractile GLS were lowest in CVI42 (23.9% ± 3.3%, 9.9% ± 2.2%), followed by TrufiStrain (27.4% ± 6.3%, 15.0% ± 4.8%) and Medis (45.4% ± 9.7%, 20.3% ± 5.7%). Conduit GLS was lowest in TrufiStrain (12.4%±4.8%), followed by CVI42 (16.3% ± 4.5%) and Medis (25.1% ± 8.2%). Among traveling volunteers, LA-GLS values were consistent across sites when the same software was used. Across all software, reservoir GLS negatively correlated with age. Diagnostic accuracy was comparable across software packages (AUC for reservoir strain: CVI: 0.81 [0.69-0.90], TrufiStrain 0.76 [0.64-0.88], Medis: 0.84 [0.72-0.94]). CONCLUSION: Post-processing software is a significant confounder in CMR-FT based LA-GLS analysis and age substantially influences LA-GLS. LA-GLS demonstrates excellent inter-site reproducibility when analyzed with the same software and offers comparable diagnostic accuracy across platforms
CRISPR-MiX: a pooled single-stranded donor strategy to enhance HDR efficiency in human iPSCs
Full text linkCRISPR-Cas9 is widely used to model genetic disorders by introducing or correcting disease-associated mutations in induced pluripotent stem cells (iPSCs) through homology-directed repair (HDR). However, HDR efficiency in iPSCs remains low and is highly dependent on the target locus. Here, we developed CRISPR-MiX, an improved protocol to enhance HDR efficiency in human iPSCs. Using a GFP-to-BFP reporter system, we identified key single-stranded oligodeoxynucleotide (ssODN) donor design parameters, including homology arm symmetry, CRISPR/Cas-blocking mutations, and strand complementarity, which significantly influence HDR outcomes. We applied this approach to introduce pathogenic variants into five genes related to genetic cardiomyopathies. Quantitative analysis of HDR events showed that both the target locus and ssODN design strongly affect HDR efficiency. To address the locus- and design-specific limitations, we established CRISPR-MiX, a pooled ssODN-based method for scarless genome editing using ribonucleoproteins (RNPs) that does not require selection. CRISPR-MiX consistently improved HDR efficiency across multiple loci. This strategy offers a simple, robust, and versatile approach for precise genome engineering in iPSCs, supporting broad applications in disease modeling and functional genomics
Non-viral in situ gene editing effectively and safely rescues congenital ichthyosis-causing mutations in human skin
No full textAutosomal recessive congenital ichthyosis (ARCI) refers to a group of rare, highly debilitating skin disorders which significantly impair patients’ quality of life and lack any effective treatment options. Here, we report clinically-relevant in situ correction of the most common ARCI-causing mutation TGM1 c.877-2A>G, a splice-site aberration, in human disease models. Targeted skin barrier modulation followed by topical application of the cytosine base editor eTD packaged into lipid nanoparticles yielded functional restoration of ~30% of wild-type transglutaminase 1 activity in skin tissue. Toxicity studies and comprehensive off-target analysis demonstrated an excellent safety profile even after repeated application, without systemic distribution of the lipid nanoparticles or the genetic cargo as determined via highly-sensitive methods including DESI metabolic imaging. This study presents comprehensive preclinical data on the feasibility of in situ gene correction of genodermatoses-causing mutations showcasing its therapeutic potential and paving the way for curative next-generation treatments for severe genetic skin diseases
Associations of adiposity with gut microbiota composition among adults – results from a federated analysis of individual participant data from eight European observational studies
Full text linkGut microbiota may contribute to the adiposity-associated disease risk, but human studies reported inconsistent associations of adiposity with gut microbiota composition. We examined associations of body mass index (BMI) with alpha diversity and relative microbial abundance at the phylum and genus taxonomic levels (based on 16S rRNA amplicon sequencing or metagenomics) among 7,415 adults from eight European observational studies in a joint federated analysis of harmonized data using DataSHIELD. Higher BMI (per 5 kg/m2) was associated with lower alpha diversity (β: -0.05; 95%CI -0.07, -0.03), and on the phylum level positively associated with Proteobacteria, but neither with Firmicutes or Bacteroidetes nor their ratio, where high between-study heterogeneity was observed. On the genus level, BMI was inversely associated with the relative abundance of Faecalibacterium of the Firmicutes phylum (β: -0.11; 95%CI -0.14, -0.07), but positively with the odds of detection of Dorea, Streptococcus and Clostridium (all three Firmicutes) as well as Collinsella (Actinobacteria). This federated analysis of multiple studies found lower alpha diversity, alongside depleted Faecalibacterium, as well higher odds of detection of Dorea, Streptococcus, Clostridium and Collinsella with higher adiposity. By combining data from diverse study populations using harmonized data and statistical methods, our analysis partly overcomes sources of heterogeneity that may explain previously observed inconsistencies
CD11c(+) cells control platelet homeostasis in a murine bone marrow chimeric atherosclerosis model
Full text linkBACKGROUND/OBJECTIVES: Dendritic cells (DCs) are key regulators of immune responses in cardiovascular disease, yet their role in platelet homeostasis and thrombopoiesis remains incompletely understood. We previously demonstrated that chronic depletion of CD11c(+) cells accelerates atherosclerotic plaque development. The objective of this study was to determine whether sustained loss of CD11c(+) cells alters platelet production and systemic inflammatory signaling under atherogenic conditions. METHODS: CD11c-DTR bone marrow chimeric mice on ApoE(-/-) background were generated and fed a high-cholesterol diet. CD11c(+) cells were depleted by repeated diphtheria toxin administration over six weeks. Circulating platelet counts were quantified by automated hematology analysis. Systemic inflammatory changes were assessed using serum cytokine and chemokine profiling, and serum thrombopoietin (TPO) levels were measured by ELISA. RESULTS: Chronic CD11c(+) cell depletion resulted in a significant increase in circulating platelet counts in ApoE(-/-) mice. Serum cytokine profiling revealed broad inflammatory remodeling, including increased levels of cytokines associated with megakaryopoiesis and platelet activation, such as IL-4, MCP-1, CXCL9, IL-16, and IL-1α. In parallel, serum TPO levels were significantly elevated following CD11c(+) cell depletion. CONCLUSIONS: In the specific context of hyperlipidemic CD11c-DTR bone marrow chimeric mice, these findings demonstrate that loss of CD11c(+) cells is associated with a pro-thrombopoietic shift, elevated platelet counts, and systemic inflammatory changes. Our data identify a CD11c(+) cell-TPO-platelet axis linking immune regulation to platelet homeostasis and thrombo-inflammatory signaling under these specific atherogenic conditions
7-T potassium ((39)K) MRI to assess muscle K(+) depletion in primary aldosteronism
No full textBACKGROUND: Although 98% of potassium (K(+)) resides intracellularly, current clinical diagnostics assess only extracellular K(+) concentrations. Noninvasive imaging of tissue K(+) distribution could provide novel insights into pathophysiologic processes in diseases such as primary aldosteronism (PA). PA is characterized by excessive aldosterone production, which leads to electrolyte imbalances, hypertension, and increased cardiovascular risk. PURPOSE: To determine whether potassium 39 ((39)K) MRI combined with sodium 23 ((23)Na) MRI can help detect alterations in skeletal muscle K(+) and Na(+) distribution in participants with PA. MATERIALS AND METHODS: A prospective pre-post study in participants with PA and a cross-sectional case-control study in participants with PA and age- and sex-matched control participants were conducted between January 2019 and April 2024. Participants underwent 7-T (39)K and (23)Na MRI of the calf muscle before treatment and approximately 4 months after standard therapy (mineralocorticoid receptor antagonists or adrenalectomy). Control participants underwent a single scan. Apparent tissue K(+) concentrations (aTPCs) and apparent tissue Na(+) concentrations (aTSCs) were quantified; serum aldosterone and K(+) were measured and correlated with imaging. Fifty-five male mice underwent sham surgery or deoxycorticosterone acetate–pellet implantation for translational chemical muscle K(+) validation. Statistical tests included the Student t test or Mann-Whitney U test (between-group), Wilcoxon signed-rank test (within-participant), and Spearman correlation (two-sided α = .05). RESULTS: Forty-two participants were evaluated, including 21 participants with PA (mean age, 52 years ± 9 [SD]; 11 female participants) and 21 control participants (mean age, 53 years ± 9; 11 female participants). Participants with PA showed lower aTPC and higher aTSC than control participants (mean aTPC: 72.7 mmol/L ± 6.8 vs 79.1 mmol/L ± 10.0, P = .02; mean aTSC: 23.9 mmol/L ± 5.3 vs 19.0 mmol/L ± 3.0, P < .001). In within-participant comparisons, PA therapy increased aTPC (mean, 72.9 mmol/L ± 7.5 to 80.9 mmol/L ± 9.8; P = .001) but decreased aTSC (25.2 mmol/L ± 4.8 to 18.9 mmol/L ± 3.4; P < .001), with values approaching control participant values. Baseline aTPC did not correlate with serum K(+) (r(2) = .02, P = .54). Mouse muscle chemistry mirrored MRI-based K(+) depletion. CONCLUSION: Combined (39)K and (23)Na MRI enabled noninvasive detection of aldosterone-mediated tissue electrolyte shifts.
ClinicalTrials.gov Identifier: NCT0425178