Biology subjects

de Jong, T. A.

Publications and source records attributed to de Jong, T. A..

3 recordsLinked to original sources

bioRxiv2024

Identification of stable reference genes for quantitative real-time PCR in human fibroblasts from lymph nodes and synovium

Real-time quantitative PCR (RT-qPCR) has emerged as an accurate and widely used technique for measuring gene expression levels. However, its reliability depends on the selection of appropriate reference genes to normalize for sample input. Accordingly, the identification of reference genes characterized by stable expression in cells and conditions of interest is essential for ensuring accurate expression values. To date, no study has specifically identified suitable reference genes for primary human cultured fibroblast-like synoviocytes (FLS) and lymph node stromal cells (LNSCs) within the context of rheumatoid arthritis (RA). These stromal cells play a critical role in the pathogenesis of disease. In this study, we evaluated the suitability of 15 candidate reference genes for normalizing transcript expression in FLS and LNSCs subjected to various in vitro stimuli. We included traditional reference genes often used for transcript normalization in fibroblasts as well as candidate genes identified as suitable reference genes via GeneVestigator analysis of publicly available transcriptomic data. RefFinder algorithms were used to identify the most stable reference genes for transcript normalization across the cell types and different experimental conditions. We determined that the optimal number of reference genes for every experimental condition tested was two; RPLP0 and POLR2G exhibited the greatest stability across different experimental conditions for LNSCs. However, for FLS, we observed greater variability in the most stable reference genes across different experimental conditions. Although POLR2G and TBP emerged as the most stable reference genes under unstimulated conditions, our findings indicated that FLS require distinct reference genes for transcript normalization depending on the specific experimental conditions. Validation of the selected reference genes for normalizing the expression levels of metabolic genes in unstimulated FLS emphasized the importance of prior evaluation of potential reference genes, as arbitrary selection of reference genes could lead to data misinterpretation. This study constitutes the first systematic analysis for selecting optimal reference genes for transcript normalization in different types of human fibroblasts. Our findings emphasize the importance of proper selection of reference genes for each experimental condition separately when applying standard quantitative PCR technology for assessing gene expression levels.

molecular biology↗

bioRxiv2023

Senescence phenotype of lymph node stromal cells from patients with rheumatoid arthritis is partly restored by dasatinib treatment

ObjectiveCellular senescence is a state of proliferation arrest of cells occurring during aging. The persistence and accumulation of senescent cells has been implicated in the pathogenesis of age-related diseases like rheumatoid arthritis (RA). RA is a chronic autoimmune disease in which loss of immune tolerance and systemic autoimmunity precedes clinical onset of disease. Lymph node stromal cells (LNSCs) are important regulators of immune tolerance. Accordingly, accumulating senescent LNSCs may potentially lead to defective immune tolerance and the development of systemic autoimmune disease. MethodsHuman LNSCs were isolated and cultured from inguinal lymph node needle biopsies from individuals at risk of developing RA (RA-risk individuals), RA patients and seronegative healthy volunteers. Senescence hallmarks and the effect of dasatinib treatment were assessed using quantitative PCR, flow cytometry, microscopy and live-cell imaging. ResultsCell size, granularity and autofluorescence were significantly higher in RA LNSCs compared with control LNSCs. Stainings indicate more senescence associated {beta}-galactosidase activity, more lipofuscin positive granules and increased DNA damage in RA-risk and RA LNSCs compared with control LNSCs. Moreover, we found altered gene expression levels of senescence associated genes in LNSCs from RA patients. Strikingly, the capacity to repair irradiation induced DNA damage was significantly lower in RA-risk and RA LNSCs compared with control LNSCs. Treating LNSCs with dasatinib significantly improved cell size and DNA repair capacity of cultured LNSCs. ConclusionWe observed multiple senescent hallmarks in RA LNSCs and to lesser extent already in RA-risk LNSCs, which could partly be restored by dasatinib treatment. KEY MESSAGES What is already known on this topic?- Synovial fibroblasts from RA patients display a senescent phenotype and accumulate in inflamed synovial tissue. What does this study add?- Lymph node stromal cells (LNSCs) from RA patients, and to a lesser extent from RA-risk, display key hallmarks of senescence. - Both ex vivo and in vitro LNSCs from RA patients have an increased cell size compared with control LNSCs. - RA and RA-risk LNSCs have an impaired ability to repair DNA damage - Treating LNSCs with dasatinib significantly improved cell size and DNA repair capacity of LNSCs. How might this study impact on clinical practice or future developments?- These hallmarks of senescence in LNSCs may indicate premature aging and loss of function of the immunomodulatory lymph node stromal compartment during RA development. Dasatinib treatment of LNSCs shows that senolytics may be an effective preclinical drug to restore cell function early in disease.

cell biology↗

bioRxiv2021

Synovial Gene Signatures Associated with the Development of Rheumatoid Arthritis in At Risk Individuals: a Prospective Study

BackgroundPrevious work has shown subtle infiltration of synovial T cells in the absence of overt synovial inflammation in individuals at risk of developing rheumatoid arthritis (RA). ObjectiveTo study the molecular changes in synovium preceding arthritis development in at risk individuals. Materials and methodsWe included sixty-seven individuals with arthralgia who were IgM rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) positive and without any evidence of arthritis. All individuals underwent mini-arthroscopic synovial tissue sampling of a knee joint at baseline and were followed prospectively. An explorative genome-wide transcriptional profiling study was performed on synovial tissue using Agilent arrays (discovery cohort). Survival analysis was used to identify transcripts associated with arthritis after follow up. Expression levels of differentially expressed genes were validated using quantitative real-time PCR (qPCR). Immunohistochemistry was used to study gene candidates at the protein level in situ. ResultsIn the discovery cohort, 6 of the 13 at risk individuals developed RA after a median follow-up time of 20 months (IQR 2 - 44; pre-RA). The 7 individuals who did not develop RA had a median follow-up time of 85 months (IQR 69 - 86). Using a False Discovery Rate of <5% we found increased expression of 3,151 transcripts correlating with a higher risk of arthritis development, whereas increased expression of 2,437 transcripts correlated with a lower risk. Gene set enrichment analysis revealed that synovial biopsies of pre-RA individuals display higher expression of genes involved in several immune response-related pathways compared with biopsies of individuals who did not develop RA. In contrast, lower expression was observed for genes involved in extracellular matrix receptor interaction, Wnt-mediated signal transduction and lipid metabolism. Two-way hierarchical cluster analysis of 27 genes measured by qPCR classified the synovial biopsies of 61 individuals into two groups, where pre-RA individuals (n=16) showed a preference to cluster together. Synovial tissue from pre-RA individuals were more likely to show podoplanin positive cells and lower lipid staining compared with synovial tissue from individuals who did not develop RA. ConclusionMolecular changes can be detected in synovial tissues before clinical onset of arthritis. Alterations in the immune response genes and lipid metabolism are associated with development of arthritis.

immunology↗