👤 Klaudia Walter

Chronic neuroinflammation is associated with comorbidities in people with HIV (PWH) on antiretroviral therapy (ART). While cannabis use is associated with reduced neuroinflammation and neurocognitive impairment (NCI) in PWH, the underlying mechanisms are unknown. To address this gap in knowledge, we analyzed monocyte-derived macrophages (MDMs) from a cohort of 50 PWH and 33 people without HIV (mean age: 61.9 years), categorized by frequency of cannabis use (naïve/low, moderate, daily). We performed immunocytochemistry, RNA sequencing, and qPCR on MDMs and quantified related biomarkers in donor plasma. In this cohort study, daily cannabis use in PWH was associated with less global neurocognitive deficits, and with an anti-inflammatory immunometabolic-phenotype in MDMs characterized by (1) a metabolic shift from glycolysis to oxidative phosphorylation, (2) higher mitochondrial numbers, (3) altered cytokine profiles (pro-inflammatory downregulation, anti-inflammatory upregulation), and (4) higher brain-derived neurotrophic factor (BDNF) expression. These cellular changes were corroborated by a plasma biomarker profile in PWH including (1) lower levels of growth differentiation factor 15 and soluble triggering receptor expressed on myeloid cells 2, and (2) higher mature BDNF/precursor BDNF ratios that correlated with better cognition. Thus, cannabis use may mitigate NCI in PWH by immunometabolically reprogramming MDM function towards an anti-inflammatory and neuroprotective state. Show less

Epidemiological analyses suggest that the Ɛ4 allele of apolipoprotein E (ApoE) genes may influence the effects of alcohol on cognitive and executive function and dementia risk compared to the Ɛ3 allele. Here, we investigated this question in female rats given that women are more vulnerable than men to the Ɛ4 genotype effects on various diseases. Experiment 1 examined the effects of alcohol drinking on performance in a Barnes maze and an operant strategy set-shifting (OSS) task during abstinence in wildtype (WT) and homozygous ApoE4 knock-in (E4) rats. Experiment 2 repeated the behavioral assessments to assess the effects of heavy alcohol exposure and explored seizure susceptibility in E4 and homozygous ApoE3 knock-in (E3) rats. The experiments revealed that E4 rats drank significantly higher doses of alcohol than did the WT and E3 rats. However, there was no genotype or alcohol effect on performance in the Barnes maze and the OSS task. Notably, E4 rats had a shorter latency to kainate-induced seizures and maintained worse seizures compared to age-matched E3 rats. These findings suggest that the Ɛ4 allele may confer a higher risk for increased alcohol drinking without significantly exacerbating alcohol-associated decline in cognitive and executive function in females. Given the scarcity and discrepant reports regarding the role of ApoE polymorphism on seizure disorders among human and rodent studies, results of this study also underscore the need for more rigorous clinical and preclinical studies to determine the role of ApoE in sporadic and alcohol withdrawal seizures. Show less

To characterize the clinical, radiological, and molecular characteristics of CNS tumors associated with Noonan syndrome (NS) and other non-Neurofibromatosis type 1 RASopathies. Twenty-four patients with concern for NS underwent clinical and central radiological review in this multi-institutional study. Whole-exome sequencing, RNA sequencing, and methylation analyses of peripheral blood and/or tumor specimens were performed. Nineteen (79%) of 24 participants had NS, 17/19 (89%) of which had a germline The online version contains supplementary material available at 10.1007/s11060-026-05478-7. Show less

To characterize the clinical, radiological, and molecular characteristics of CNS tumors associated with Noonan syndrome (NS) and other non-Neurofibromatosis type 1 RASopathies. Twenty-four patients with concern for NS underwent clinical and central radiological review in this multi-institutional study. Whole-exome sequencing, RNA sequencing, and methylation analyses of peripheral blood and/or tumor specimens were performed. Nineteen (79%) of 24 participants had NS, 17/19 (89%) of which had a germline Show less

Gene fusions are common primary drivers of pediatric leukemias and are the result of underlying structural variants (SVs). Current clinical workflows to detect such alterations rely on a multimodal approach, which often increases analysis time and overall cost of testing. In this study, we used long-read sequencing (lrSeq) as a proof-of-concept to determine whether clinically relevant (cr) SVs could be detected within a small (n = 17) pediatric leukemia cohort. We show that this methodology successfully determined all known crSVs (n = 5/5) detected through routine clinical testing. This approach also identified crSVs that resulted in the classification of a leukemia genetic subtype for four additional patients (n = 4/12), such as an ins(11;10)(q23.3;p12p12) forming a KMT2A::MLLT10 fusion, that were missed by routine clinical approaches. This study demonstrates the diagnostic potential of lrSeq as an assay for SV detection in pediatric leukemia and supports lrSeq as a valuable tool for the accurate detection of crSVs. Show less

Pediatric AML with KMT2A::MLLT10 accounts for 10%-15% of KMT2A-rearranged AML and is associated with poor prognosis. Lately, the assessment of measurable residual disease (MRD) by reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become an important tool for disease management; however, in the pediatric setting, it lacks standardized protocols. Therefore, we investigated the prognostic relevance of MRD monitoring by RT-qPCR during high-dose polychemotherapy in pediatric patients with AML expressing KMT2A::MLLT10. Using RNA sequencing, we determined the fusion breakpoints and designed RT-qPCR assays for MRD monitoring. Bone marrow samples collected from 41 patients, who were treated in the AML-BFM or AIEOP study, were analyzed for MRD by RT-qPCR. MRD positivity after the second treatment course resulted in a significantly worse probability of overall survival (pOS) compared to MRD negative patients (33.3% ± 19.2% vs. 80.6% ± 7.8%, p = 0.032). Moreover, the probability of event-free survival (pEFS) (16.7% ± 15.2% vs. 76.9% ± 8.3%, p = 0.003) and cumulative incidence of relapse (CIR) (83.3% ± 40.8% vs. 19.2% ± 40.2%, p = 0.001) were significantly worse for patients in complete morphologic remission who remained MRD positive after the second treatment course. Thus, MRD monitoring enables the identification of a subgroup of pediatric patients with AML carrying KMT2A::MLLT10 in complete morphologic remission with a dismal prognosis despite the current intensive therapy regimen. AML-BFM study 2004: ClinicalTrials.gov Identifier: NCT00111345; AML-BFM registry 2012 and AML-BFM study 2012: EudraCT 2013-000018-39; AML-BFM registry 2017: DRKS number: DRKS00013030. Show less

Gene fusions are common primary drivers of pediatric leukemias and are the result of underlying structural variant (SVs). Current clinical workflows to detect such alterations rely on a multimodal approach, which often increases analysis time and overall cost of testing. In this study, we used long-read sequencing (lrSeq) as a proof-of-concept to determine whether clinically relevant (cr) SVs could be detected within a small (n = 17) pediatric leukemia cohort. We show that this methodology successfully determined all known crSVs detected through routine clinical testing. We also identified crSVs, such as an ins(11;10)(q23.3;p12p12) forming a KMT2A::MLLT10 fusion, missed by routine clinical approaches, resulting in the classification of leukemia genetic subtypes for four additional patients. This study demonstrates the diagnostic potential of lrSeq as an assay for SV detection in pediatric leukemia and supports lrSeq as a valuable tool for the accurate detection of crSVs. Show less

New methods like panel-based RNA fusion sequencing (RNA-FS) promise improved diagnostics in various malignancies. We here analyzed the impact of RNA-FS on the initial diagnostics of 241 cases with pediatric acute myeloid leukemia (AML). We show that, compared to classical cytogenetics (CCG), RNA-FS reliably detected risk-relevant fusion genes in pediatric AML. In addition, RNA-FS strongly improved the detection of cryptic fusion genes like NUP98::NSD1, KMT2A::MLLT10 and CBFA2T3::GLIS2 and thereby resulted in an improved risk stratification in 25 patients (10.4%). Validation of additionally detected non-risk-relevant high confidence fusion calls identified PIM3::BRD1, C22orf34::BRD1, PSPC1::ZMYM2 and ARHGAP26::NR3C1 as common genetic variants and MYB::GATA1 as recurrent aberration, which we here describe in AML subtypes M0 and M7 for the first time. However, it failed to detect rare cytogenetically confirmed fusion events like MNX1::ETV6 and other chromosome 12p-abnormalities. As add-on benefit, the proportion of patients for whom measurable residual disease (MRD) monitoring became possible was increased by RNA-FS from 44.4 to 75.5% as the information on the fusion transcripts' sequence allowed the design of new MRD assays. Show less

We evaluated the suitability of 2% human platelet lysate medium (2%HPL) as a replacement for 2% fetal bovine serum medium (2%FBS) for the xeno-free organ culture of human donor corneas. A total of 32 corneas from 16 human donors were cultured in 2%FBS for 3 days (TP1), then evaluated using phase contrast microscopy (endothelial cell density (ECD) and cell morphology). Following an additional 25-day culture period (TP2) in either 2%FBS or 2%HPL, the pairs were again compared using microscopy; then stroma and Descemet membrane/endothelium (DmE) were processed for next generation sequencing (NGS). At TP2 the ECD was higher in the 2%HPL group (2179 ± 288 cells/mm Show less

Application of next-generation sequencing may lead to the detection of secondary findings (SF) not related to the initially analyzed disease but to other severe medically actionable diseases. However, the analysis of SFs is not yet routinely performed. We mined whole-exome sequencing data of 231 pediatric cancer patients and their parents who had been treated in our center for the presence of SFs. By this approach, we identified in 6 children (2.6%) pathogenic germline variants in 5 of the noncancer-related genes on the American College of Medical Genetics and Genomics (ACMG) SF v3.0 list, of which the majority were related to cardiovascular diseases ( RYR2 , MYBPC3 , KCNQ1 ). Interestingly, only the patient harboring the KCNQ1 variant showed at the time point of the analysis signs of the related Long QT syndrome. Moreover, we report 3 variants of unknown significance which, although not classified as pathogenic, have been reported in the literature to occur in individuals with the respective disease. While the frequency of patients with SFs is low, the impact of such findings on the patients' life is enormous, with regard to the potential prevention of life-threatening diseases. Hence, we are convinced that such actionable SF should be routinely analyzed. Show less

Lipodystrophy syndromes cause hypertriglyceridemia that improves with leptin treatment using metreleptin. Mechanisms causing hypertriglyceridemia and improvements after metreleptin are incompletely understood. Determine relationship of circulating lipoprotein lipase (LPL) modulators with hypertriglyceridemia in healthy controls and in patients with lipodystrophy before and after metreleptin. Cross-sectional comparison of patients with lipodystrophy (generalized lipodystrophy n = 3; partial lipodystrophy n = 11) vs age/sex-matched healthy controls (n = 28), and longitudinal analyses in patients before and after 2 weeks and 6 months of metreleptin. The study was carried out at the National Institutes of Health, Bethesda, Maryland. Outcomes were LPL stimulators apolipoprotein (apo) C-II and apoA-V and inhibitors apoC-III and angiopoietin-like proteins (ANGPTLs) 3, 4, and 8; ex vivo activation of LPL by plasma. Patients with lipodystrophy were hypertriglyceridemic and had higher levels of all LPL stimulators and inhibitors vs controls except for ANGPTL4, with >300-fold higher ANGPTL8, 4-fold higher apoC-III, 3.5-fold higher apoC-II, 1.9-fold higher apoA-V, 1.6-fold higher ANGPTL3 ( Elevations in LPL inhibitors apoC-III and ANGPTL8 may contribute to hypertriglyceridemia in lipodystrophy, and may mediate reductions in circulating and hepatic triglycerides after metreleptin. These therefore are strong candidates for therapies to lower triglycerides in these patients. Show less

Smoking is the leading cause of preventable disease. Although smoking results in an acute effect of relaxation and positive mood through dopamine release, smoking is thought to increase stress symptoms such as heart rate and blood pressure from nicotine-induced effects on the HPA axis and increased cortisol. Despite the importance in understanding the mechanisms in smoking maintenance, little is known about the overall protein and physiological response to smoking. There may be multiple functions involved that if identified might help in improving methods for behavioral and pharmacological interventions. Therefore, our goal for this pilot study was to identify proteins in the saliva that change in response to an acute smoking event versus acute sham smoking event in smokers and non-smokers, respectively. We employed the iTRAQ technique followed by Mass Spectrometry to identify differentially expressed proteins in saliva of smokers and non-smokers after smoking cigarettes and sham smoking, respectively. We also validated some of the salivary proteins by ELISA or western blotting. In addition, salivary cortisol and salivary amylase (sAA) activity were measured. In all, 484 salivary proteins were identified. Several proteins were elevated as well as decreased in smokers compared to non-smokers. Among these were proteins associated with stress response including fibrinogen alpha, cystatin A and sAA. Our investigation also highlights methodological considerations in study design, sampling and iTRAQ analysis. We suggest further investigation of other differentially expressed proteins in this study including ACBP, A2ML1, APOA4, BPIB1, BPIA2, CAH1, CAH6, CYTA, DSG1, EST1, GRP78, GSTO1, sAA, SAP, STAT, TCO1, and TGM3 that might assist in improving methods for behavioral and pharmacological interventions for smokers. Show less

Circulating metabolite levels are biomarkers for cardiovascular disease (CVD). Here we studied, association of rare variants and 226 serum lipoproteins, lipids and amino acids in 7,142 (discovery plus follow-up) healthy participants. We leveraged the information from multiple metabolite measurements on the same participants to improve discovery in rare variant association analyses for gene-based and gene-set tests by incorporating correlated metabolites as covariates in the validation stage. Gene-based analysis corrected for the effective number of tests performed, confirmed established associations at APOB, APOC3, PAH, HAL and PCSK (p<1.32x10-7) and identified novel gene-trait associations at a lower stringency threshold with ACSL1, MYCN, FBXO36 and B4GALNT3 (p<2.5x10-6). Regulation of the pyruvate dehydrogenase (PDH) complex was associated for the first time, in gene-set analyses also corrected for effective number of tests, with IDL and LDL parameters, as well as circulating cholesterol (pMETASKAT<2.41x10-6). In conclusion, using an approach that leverages metabolite measurements obtained in the same participants, we identified novel loci and pathways involved in the regulation of these important metabolic biomarkers. As large-scale biobanks continue to amass sequencing and phenotypic information, analytical approaches such as ours will be useful to fully exploit the copious amounts of biological data generated in these efforts. Show less

The aim of the present study was to characterise key enzymes involved in polyunsaturated fatty acid (PUFA) synthesis in the testis and epididymis collected from 2-year-old healthy warmblood stallions (n=10). The mRNA expression of fatty acid synthase, the Δ9-, Δ6-, Δ5- and Δ4-desaturases and elongases 6, 5 and 2 (encoded by the fatty acid synthase (FASN), the stearoyl-CoA desaturase (SCD), the fatty acid desaturase 2 (FADS2), the fatty acid desaturase 1 (FADS1), the delta 4-desaturase, sphingolipid 1 (DEGS1), ELOVL fatty acid elongase 6(ELOVL6), ELOVL fatty acid elongase 5 (ELOVL5), ELOVL fatty acid elongase 2 (ELOVL2) genes respectively) was determined in equine testis and epididymis. All enzymes were present in testicular tissue and along the epididymis, but mRNA expression differed among localisations. The protein localisation of FADS1, FADS2 and ELOVL5 was determined by immunohistochemistry. In the testes, FADS1 was expressed in the germinal cells and ELOVL5 was expressed in germinal and Leydig cells; FADS2 was not detected. In the epididymis, FADS1 and FADS2 were expressed in the principal and basal cells, whereas ELOVL5 was found only in the principal cells of the caput. All three enzymes were present in epididymal vesicles secreted by an apocrine mechanism. These results suggest active PUFA metabolism during spermatogenesis and epididymal sperm maturation in stallions. Show less

The role of rare variants in complex traits remains uncharted. Here, we conduct deep whole genome sequencing of 1457 individuals from an isolated population, and test for rare variant burdens across six cardiometabolic traits. We identify a role for rare regulatory variation, which has hitherto been missed. We find evidence of rare variant burdens that are independent of established common variant signals (ADIPOQ and adiponectin, P = 4.2 × 10 Show less

Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0.33, discovery + replication p = 6.38 × 10(-10)) and a rare synonymous variant in GFI1B (rs150813342, MAF = 0.009, discovery + replication p = 1.79 × 10(-27)). By performing CRISPR/Cas9 genome editing in hematopoietic cell lines and follow-up targeted knockdown experiments in primary human hematopoietic stem and progenitor cells, we demonstrate an alternative splicing mechanism by which the GFI1B rs150813342 variant suppresses formation of a GFI1B isoform that preferentially promotes megakaryocyte differentiation and platelet production. These results demonstrate how unbiased studies of natural variation in blood cell traits can provide insight into the regulation of human hematopoiesis. Show less

The analysis of rich catalogues of genetic variation from population-based sequencing provides an opportunity to screen for functional effects. Here we report a rare variant in APOC3 (rs138326449-A, minor allele frequency ~0.25% (UK)) associated with plasma triglyceride (TG) levels (-1.43 s.d. (s.e.=0.27 per minor allele (P-value=8.0 × 10(-8))) discovered in 3,202 individuals with low read-depth, whole-genome sequence. We replicate this in 12,831 participants from five additional samples of Northern and Southern European origin (-1.0 s.d. (s.e.=0.173), P-value=7.32 × 10(-9)). This is consistent with an effect between 0.5 and 1.5 mmol l(-1) dependent on population. We show that a single predicted splice donor variant is responsible for association signals and is independent of known common variants. Analyses suggest an independent relationship between rs138326449 and high-density lipoprotein (HDL) levels. This represents one of the first examples of a rare, large effect variant identified from whole-genome sequencing at a population scale. Show less

Recent studies have underlined the role of nuclear receptors in the involvement of prostate cancer signalling pathways. A total of 84 benign prostate hyperplasia (BPH), 84 low risk prostate cancer (LPC) and 64 advanced disease (APC) cases were sampled on a tissue microarray (TMA) and stained for retinoic acid receptor (RAR)-α, retionoid X receptor (RXR)-α, liver X receptor (LXR)-α, farnesoid X receptor (FXR) and proliferate-activated receptor gamma (PPAR)-γ and the (pro)-inflammatory molecules cyclooxygenase 2 (COX2), tumor necrosis factor (TNF)-α and inducible Nitric oxide synthase (iNOS) immunohistochemically. PPAR-γ expression in APC tissues was found to be significantly higher than that in LPC and BPH specimens (p<0.001). In contrast, RXR-a expression was significantly lower (p<0.001). COX2 staining demonstrated a trend towards overexpression in APC (p=0.025). No significant differences were found for RAR-α, iNOS and TNF-α expression. Staining of FXR and LXR was seen diffusely in the cytoplasm as well as in the nucleus, preventing sufficient evaluation by definition. This study provides the basis for applying PPAR-γ ligands clinically in treatment of APC. Show less

This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future. Show less