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Immunomodulatory potential of a bibenzyl-dihydrophenanthrene derivative isolated from Calanthe cardioglossa
Get PDFBibenzyls and dihydrophenanthrenes exhibit promising immunomodulatory effects in various human diseases. In this study, we isolated one new dihydrophenanthrene derivative (1), two new bibenzyl-dihydrophenanthrene derivatives (2, 3) along with 12 known compounds (4–15) from the methanol extract of Calanthe cardioglossa. These compounds were identified by using physicochemical analyses and spectroscopic methods. The three new compounds possess enantiomeric forms, and their configurations were determined by comparing the experimental electronic circular dichroism (ECD) spectra to data from the literature. The immunomodulatory activity of the isolated compounds was assessed in THP-1 monocytes and human peripheral blood mononuclear cells (PBMCs) derived from multiple sclerosis (MS) patients. Notably, five of the isolated compounds significantly reduced the TNF-α levels in LPS-stimulated THP-1 monocytes. Furthermore, calancardin B (2) exhibited a significant reduction in TNF-α levels in both THP-1 monocytes and CD14(+) monocytes from MS PBMCs
Dental and oral health assessments in the German National Cohort (NAKO)
Get PDFBACKGROUND: Despite considerable improvements in oral health in recent decades, caries and periodontitis are still widespread, ranking among the most prevalent diseases worldwide and requiring future research. The German National Cohort (NAKO Gesundheitsstudie, NAKO) is a large-scaled, multidisciplinary, nationwide, multi-centre, population-based, prospective cohort study with oral examinations that aims to provide a resource to study risk factors for major diseases. The aim of the present article is to provide the methodological background, to report on the data quality, and to present initial results of the oral examinations. METHODS: During baseline examinations (2014-2019), a total of 205,184 persons aged 19-74 years has been examined in 18 study centres, including, among others, a dental interview, stimulated saliva sampling, and recording of the numbers of present teeth and prostheses (standard Level 1 program). As part of the Level 2 program that was offered to 20% randomly selected participants, each study centre selected one of three modules, one of them being the Level 2 oral examination. This extended program was carried out in a subgroup of 20,828 participants, including collection of detailed information on the dental and prosthetic status as well as on periodontal, cariological and functional aspects. To ensure reliability and reproducibility, study nurses were trained and calibrated by dental experts. In addition, a reliability study was conducted among 794 Level 1 and 359 Level 2 participants, reporting intra class correlation and kappa coefficients. RESULTS: Intra class correlation and kappa coefficients for observer agreement and reliability were consistently above 0.7, indicating good to excellent reliability of all dental measurements. For example, intra class correlation was 0.937 for the number of present teeth (Level 1), 0.740 for mean probing depth (PD) and 0.797 for active mouth opening. An initial inspection of the data showed that the median number of present teeth was 27, of which on average 6.9 teeth were healthy and caries-free. Average mean PD was 1.92 mm. An orthodontic treatment was reported by 35.5% of participants. DISCUSSION: Overall, the dental study protocol was feasible and successfully integrated into the NAKO's overall assessment program. However, rigorous support of the study centres by dental professionals was required to ensure high quality data. In summary, high-quality data collection within the NAKO pave the way for future investigation of potential risk factors for oral diseases and links between oral and systemic diseases and conditions
Quality assurance in metabolomics and metabolic profiling
No full textMetabolic profiling (untargeted metabolomics) aims for a global unbiased analysis of metabolites in a cell or biological system. It remains a highly useful research tool used across various analytical platforms. Incremental improvements across multiple steps in the analytical process may have large consequences for the end quality of the data. Thus, this chapter concentrates on which aspects of quality assurance can be implemented by a lab in the (pre-)analytical stages of the analysis to improve the overall end quality of their data. The scope of this chapter is limited to liquid-chromatography-mass spectrometry (LC-MS)-based profiling, which is one of the most widely utilized platforms, although the general principles are applicable to all metabolomics experiments
Mucosal washes are useful for sampling intestinal mucus-associated microbiota despite low biomass
Get PDFUnderstanding the dynamic relationship between mucus-associated microbiota and host health is critical. However, studies predominantly using stool samples may not accurately represent these bacterial communities. Here, we investigated the mucus-associated microbiota in the gastrointestinal tract of mice and the terminal ileum of humans using different sample types: mucosal washes, brushes, scrapings, and intestinal contents in mice and biopsies, brushes and mucosal washes in humans. We used DNA quantification and 16S rRNA amplicon sequencing to evaluate the comparability of the information yielded from the different sample types under a controlled benchmark. In mice, mucosal washes and brushes had comparative bacterial DNA and host DNA contamination than scraping samples. Similarly, in humans, washes outperformed biopsies in bacterial DNA content. Read counts and microbiota alpha diversity remained remarkably similar in mice and between brushes and washes in humans. The composition of the microbiota varied based on the subsegment and sample type in mice and sample type in humans. We conclude that washes and brushes reduce host contamination without inducing substantial compositional bias when sampling mucosal microbiota. Our findings suggest that mucosal washes and brushes are a viable alternative to biopsies in humans and scrapings in mice, thereby improving the transferability of results across hosts. Our study highlights the importance of focusing on mucus-associated microbiota to better capture host–microbiome interactions at their closer interface
A kinetics-based model of hematopoiesis reveals extrinsic regulation of skewed lineage output from stem cells
Get PDFResiding at the top of the hematopoietic hierarchy, long-term hematopoietic stem cells (HSCs) are capable of self-renewal and sustained blood cell regeneration. Over the past decades, single-cell and clonal analyses have revealed substantial functional and molecular heterogeneity within this compartment, challenging the notion that self-renewal is inherently tied to balanced, multi-lineage blood production. However, a cohesive model that explains the relationships among these diverse HSC states remains elusive. Here, we combined single-cell transplantations of over 1,000 highly purified murine long-term HSCs with in-depth phenotyping of their clonal progeny to achieve a detailed, time-resolved understanding of heterogeneous reconstitution outcomes. We identified reconstitution kinetics as an overall unifying metric of HSC functional potency, with the most potent HSCs displaying the greatest delay in hematopoietic reconstitution. Importantly, a progressive acceleration in reconstitution kinetics was also associated with a gradual shift in mature cell production from platelet and erythro-myeloid bias to balanced, and eventually lymphoid bias. Serial single-cell transplantations of HSCs revealed a unidirectional acceleration in reconstitution kinetics accompanied by a gradual decline in functional potency of daughter HSCs, aligning diverse phenotypes along a linear hierarchical trajectory. Mathematical modeling, together with experimental modulation of lineage-biased blood production, demonstrated that apparent lineage biases actually arise from cell-extrinsic feedback regulation and clonal competition between slow- and fast-engrafting clones to occupy the limited compartment sizes of mature lineages. Our study reconciles multiple layers of HSC heterogeneity into a unifying framework, prompting a reevaluation of the meaning of lineage biases in both normal and diseased hematopoiesis, with broad implications for other regenerating tissues during development, homeostasis, and repair
Merkel cell polyomavirus infection and persistence modelled in skin organoids
No full textMerkel cell polyomavirus (MCPyV) causes most Merkel cell carcinomas (MCC). The virus is one of the few known human tumor viruses, and due to its direct role in this skin cancer development, it is a bona fide model for viral tumorigenesis and tumorigenesis in general. Chronic viruses in humans such as MCPyV are highly adapted to their host and current models to study infection, persistence and pathogenesis are highly limited. We here use an induced pluripotent stem cell (iPSC)-derived hair-bearing skin organoid (SkO) system to demonstrate efficient viral infection, progression and spread of MCPyV. Using bulk-, single cell - and spatial- transcriptomics, combined with immunostaining and nucleic acid hybridization technologies, we show that MCPyV ensures persistence due to a quasi-latency state within the majority of dermal fibroblasts carry the viral genome. Further, we identify the cell type of productive infection with papillary fibroblasts and dermal sheath fibroblasts supporting viral replication and progeny production. Our high-resolution methods demonstrate that the virus in these cells evades the innate immune response, as evidenced by the efficacy of interferon-beta treatment or ruxolitinib, a JAK/STAT inhibitor, in suppressing or stimulating viral replication. We show that iPSC-derived SkOs are able to support infection and long-term persistence of the virus under conditions very similar to those found in humans. Thus, this infection model provides a robust platform for understanding and characterizing the interaction of this virus with the immune system in infection, testing treatment strategies to control reactivation and map processes involved in tumor development
Identification of V0g propriospinal neurons and their role in locomotor control
Get PDFPropriospinal neurons relay sensory and motor information across the spinal cord and are critical components of the circuits coordinating body movements. Their diversity and roles in motor control are not clearly defined yet. In this study, by combining anatomical, molecular, and functional analyses in mice, we identified and characterized an ascending subtype of propriospinal neurons belonging to the Pitx2(+) V0 family of spinal neurons. We found that Pitx2(+) ascending neurons are integrated in spinal sensorimotor circuits and their function is important for the execution of precise limb movements required for effectively moving in challenging environments, like walking on a horizontal ladder or a balance beam. This work advances our understanding of the functional organization of propriospinal and V0 neurons, highlighting a previously unappreciated role in adjusting body movements to the more demanding needs of skilled locomotor tasks
Neurite orientation dispersion and density imaging in myelin oligodendrocyte glycoprotein antibody-associated disease and neuromyelitis optica spectrum disorders
Get PDFBACKGROUND: Aquaporin-4 antibody positive (AQP4+) neuromyelitis optica spectrum disorders (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) are two distinct antibody-mediated neuroinflammatory diseases. Diffusion Tensor Imaging (DTI) and Neurite Orientation Dispersion and Density Imaging (NODDI) are advanced diffusion-weighted MRI models providing quantitative metrics sensitive to cerebral microstructural changes. This study aims to differentiate brain tissue damage in NMOSD and MOGAD from controls and investigate its association with clinical disability, using NODDI and DTI-derived measures, including fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity. METHODS: This study included 31 AQP4+ NMOSD, 21 MOGAD patients and 45 healthy controls. Clinical information included disease duration, Expanded Disability Status Scale (EDSS), Timed 25 Foot Walk test (T25FW), Nine-Hole Peg Test (9HPT), Symbol Digit Modalities Test (SDMT) and monocular 100 % high contrast visual acuity (HCVA). All participants underwent MRI scanning with multi-shell diffusion-weighted imaging, T2w fluid-attenuated inversion recovery and T1w magnetization prepared-rapid acquisition gradient echo sequences to obtain manually segmented T2-hyperintense white matter lesions (WML) and normal-appearing brain tissue (NABT) masks, including white matter (NAWM), cortical and deep gray matter (NACGM, NADGM). DTI and NODDI metrics were compared between groups using region-of-interest (ROI) analysis and tract-based spatial statistics. Tissue-weighted means were obtained for the NODDI metrics (weighted neurite density index, wNDI; weighted orientation dispersion index, wODI). Group differences in ROI analyses were assessed using age and sex adjusted linear regression models, followed by post-hoc comparisons with estimated marginal means. Stepwise multivariable linear regression models were used to evaluate the association between MRI biomarkers and clinical outcomes. RESULTS: NMOSD patients had higher T2 lesion volume (1120.5 mm(3) vs. 374.6 mm(3), p<.001) and number (median 22 vs. 6, p<.001) than MOGAD patients. Both NMOSD and MOGAD lesions displayed lower wNDI and higher isotropic volume fraction (ISOVF) compared to microvascular lesions in controls (p<.05). In NACGM, NMOSD patients showed higher wODI but lower ISOVF compared to HC (p=.029). MOGAD patients had lower wNDI in NACGM compared to NMOSD (p=.012). Tract-based spatial statistics revealed damage to specific white matter abnormalities in NMOSD, with higher AD, ODI and ISOVF compared to controls, particularly in the corpus callosum and corticospinal tract. Clinical associations in NMOSD included higher EDSS with higher NAWM ISOVF (R(2)=0.46, p=.006), higher 9HPT with lower intralesional FA and higher NAWM MD (R(2)=0.54, p=.022), lower SDMT with lower intralesional FA and higher NACGM ISOVF (R(2)=0.54, p=.013), worse visual acuity with higher NAWM wODI. In MOGAD, higher EDSS was associated with lower NAWM FA (R(2)=0.29, p=.022), slower T25FW with higher NADGM ISOVF (R(2)=0.48, p<.001), lower SDMT with higher NAWM ISOVF (R(2)=0.62, p=.005) and worse visual acuity with higher NADGM MD. CONCLUSION: NODDI and DTI measures are sensitive to pathological alterations in myelin and axon integrity, as water diffusion is less restricted in demyelinated tissue. Compared to MOGAD, patients with NMOSD tend to exhibit more extensive chronic white matter damage, demyelination or axonal injury. NODDI demonstrates greater sensitivity and specificity to alterations in NACGM compared to DTI. Given their association with clinical disability, NODDI metrics appear to be valuable neuroimaging biomarkers for assessing microstructural damage in clinical research
High incidence and impact of suspected exocrine pancreatic insufficiency in patients post-hematopoietic stem cell transplantation: a single-center prospective observational study
Get PDFExocrine pancreatic insufficiency (EPI) is suspected but remains understudied in immunosuppressed conditions such as post-hematopoietic stem cell transplantation (HSCT). This prospective observational study aimed to investigate the incidence, impact, and risk factors of EPI in a cohort of 83 pediatric and young adult patients who underwent allogeneic HSCT at Charité - Universitätsmedizin Berlin between 2020 and 2023. Fecal pancreatic elastase (PE) measurements and transabdominal ultrasound were utilized to evaluate pancreatic function over a one-year period. Secondary analysis explored the association of EPI with clinical complications and included a multivariable regression analysis of potential risk factors. Low PE levels significantly correlated with pathological pancreatic imaging findings, independent of concurrent diarrhea. EPI was suspected in 45% (32/71) of patients (95%CI: [34.1%, 56.6%]), with 29% (13/45) (95%CI: [17.7%, 43.4%]) showing signs of prolonged EPI (pEPI) lasting at least 8 weeks. After excluding cases with confounding factors such as missing enteral nutrition and diarrhea, the cumulative incidence of prolonged EPI was 20% (8/41) (95%CI: 10.2%–34.0%) in the overall cohort. EPI was associated with weight loss, prolonged dependence on parenteral nutrition, and extended hospitalizations. Adenovirus (ADV) infection emerged as a significant risk factor for EPI (hazard ratio 3.22 [95%CI:1.26–8.2], p = 0.014), along with additional factors such as higher BMI pre-HSCT, pre-existing pancreatic damage and early post-HSCT fasting. The persistence of pancreatic atrophy and EPI beyond two years post-HSCT in individual cases suggests a potential for permanent pancreatic damage. This study underscores that EPI is a common complication following HSCT, with ADV infection being an important risk factor. The findings support routine PE measurements and early initiation of pancreatic enzyme replacement therapy (PERT), alongside aggressive treatment of ADV infections. Further research is necessary to evaluate the effects of PERT in this population and to explore the applicability of these findings in other immunosuppressed groups
Spatial-temporal diversity of extrachromosomal DNA shapes urothelial carcinoma evolution and tumor-immune microenvironment
Get PDFExtrachromosomal DNA (ecDNA) presents a promising target for cancer therapy; however, its spatial–temporal diversity and influence on tumor evolution and the immune microenvironment remain largely unclear. We apply computational methods to analyze ecDNA from whole-genome sequencing data of 595 patients with urothelial carcinoma. We demonstrate that ecDNA drives clonal evolution through structural rearrangements during malignant transformation and recurrence of urothelial carcinoma. This supports a model wherein tumors evolve via the selective expansion of ecDNA-bearing cells. Through multiregional sampling of tumors, we demonstrate that ecDNA contributes to the evolution of multifocality and increased intratumoral heterogeneity. ecDNA is present in 36% of urothelial carcinoma tumors and correlates with an immunosuppressive phenotype and poor prognosis. Single-cell RNA sequencing analyses reveal that ecDNA(+) malignant cells exhibit diminished expression of MHC class I molecules, enabling them to evade T-cell immunity. Finally, we show that sequencing of urinary sediment–derived DNA has excellent specificity in detecting ecDNA. SIGNIFICANCE: Our comprehensive analysis of ecDNA in urothelial carcinoma reveals its crucial role in driving the evolution and heterogeneity of multifocal cancer, as well as its early involvement in tumorigenesis. Moreover, this study sheds light on immune evasion mechanisms associated with ecDNA and offers valuable insights for developing targeted therapeutic strategies