T follicular helper (Tfh) cells are essential for the development of germinal center B cells and high-affinity antibody-producing B cells in humans and mice. Here, we identify the guanine nucleotide ...exchange factor (GEF) Rin-like (Rinl) as a negative regulator of Tfh generation. Loss of Rinl leads to an increase of Tfh in aging, upon in vivo immunization and acute LCMV Armstrong infection in mice, and in human CD4+ T cell in vitro cultures. Mechanistically, adoptive transfer experiments using WT and Rinl-KO naïve CD4+ T cells unraveled T cell-intrinsic GEF-dependent functions of Rinl. Further, Rinl regulates CD28 internalization and signaling, thereby shaping CD4+ T cell activation and differentiation. Thus, our results identify the GEF Rinl as a negative regulator of global Tfh differentiation in an immunological context and species-independent manner, and furthermore, connect Rinl with CD28 internalization and signaling pathways in CD4+ T cells, demonstrating for the first time the importance of endocytic processes for Tfh differentiation.
Energy metabolism, involving the ATP-dependent AMPK-PgC-Ppar pathway impacts metabolic health immensely, in that its impairment can lead to obesity, giving rise to disease. Based on observations that ...individuals with Gilbert's syndrome (GS; UGT1A1(*)28 promoter mutation) are generally lighter, leaner and healthier than controls, specific inter-group differences in the AMPK pathway regulation were explored. Therefore, a case-control study involving 120 fasted, healthy, age- and gender matched subjects with/without GS, was conducted. By utilising intra-cellular flow cytometry (next to assessing AMPKα1 gene expression), levels of functioning proteins (phospho-AMPK α1/α2, PgC 1 α, Ppar α and γ) were measured in PBMCs (peripheral blood mononucleated cells). In GS individuals, rates of phospho-AMPK α1/α2, -Ppar α/γ and of PgC 1α were significantly higher, attesting to a boosted fasting response in this condition. In line with this finding, AMPKα1 gene expression was equal between the groups, possibly stressing the post-translational importance of boosted fasting effects in GS. In reflection of an apparently improved health status, GS individuals had significantly lower BMI, glucose, insulin, C-peptide and triglyceride levels. Herewith, we propose a new theory to explain why individuals having GS are leaner and healthier, and are therefore less likely to contract metabolic diseases or die prematurely thereof.
Progesterone therapy is a viable treatment for complex atypical hyperplasia (CAH) and endometrial adenocarcinoma, though reliable molecular determinants of response are not available. To explore if ...analysis of pre-therapy endometrial biopsies could yield biomarkers of response to progesterone, patients with CAH or adenocarcinoma undergoing treatment with progestins were included in this cross-sectional study. Immunohistochemistry for progesterone receptor (PR) was performed. Manual PR expression scores (PRES) were first calculated for biopsies by counting PR-positive nuclei in 12 sensitive vs 9 resistant samples. Significant differences in manual PRES were detected in the stroma (
p
< 0.01) and total endometrium (
p
< 0.01) for sensitive vs resistant patients. Manual PRES in the stroma had the highest accuracy in segregating sensitive vs resistant patients (96%). Differences in epithelial PRES were not significant. To validate these findings, a correlation between manual PRES and visual PRES was performed in the 21 patients. An additional 11 patients were analyzed to test if visual PRES would be predictive of response to progesterone. Visual PRES in epithelia and stroma in the 32 specimens was calculated. Significant differences in visual PRES were detected in the stroma for sensitive vs resistant samples (
p
< 0.01), while differences in epithelial and total endometrium were not significant. Whole genome bisulfite sequencing was performed on DNA isolated using pre-therapy biopsies from 6 sensitive and 6 resistant patients in this cohort. Differentially methylated regions were identified in the stroma and epithelium when evaluating sensitive vs resistant samples. Pathways involved in cell adhesion demonstrated the greatest difference in methylation in these samples.
The protective role of mildly elevated bilirubin against CVD and diabetes mellitus type 2 (DMT2) is associated with a favorable lipid phenotype. As the mechanistic understanding of this protection in ...humans remains elusive, we aimed to assess the metabolomics profile of mildly hyperbilirubinemic (Gilbert's syndrome; GS) individuals especially targeting lipid catabolism.
Using NMR serum metabolomics of 56 GS individuals and 56 age and gender-matched healthy controls, GS individuals demonstrated significantly greater concentrations of acetylcarnitine (+20%, p < 0.001) and the ketone bodies, 3-hydroxybutyric acid (+132%, p < 0.001), acetoacetic acid (+95%, p < 0.001) and acetone (+46%, p < 0.001). Metabolites associated with an increased mitochondrial lipid metabolism such as citrate (+15%, p < 0.001), anaplerotic amino acid intermediates and creatinine were significantly greater and creatine significantly reduced in GS individuals. Stimulators of lipid catabolism including AMPK (+59%, p < 0.001), pPPARα (+24%, p < 0.001) and T3 (+9%, p = 0.009) supported the metabolomics data while concomitantly blood glucose and insulin (−33%, p = 0.002) levels were significantly reduced. We further showed that the increased lipid catabolism partially mediates the favorable lipid phenotype (lower triglycerides) of GS individuals. Increased trimethylamine (+35%, p < 0.001) indicated changes in trimethylamine metabolism, an emerging predictor of metabolic health.
We showed an enhanced lipid catabolism in mildly hyperbilirubinemic individuals, novel evidence as to why these individuals are leaner and protected against chronic metabolic diseases emphasizing bilirubin to be a promising future target in obese and dyslipidemia patients.
•Mildly hyperbilirubinemic individuals have substantially increased ketone bodies and acetylcarnitine.•Increased AMPK, PPARα and T3 levels are consistent with increased lipid catabolites suggesting enhanced lipid catabolism.•Lipid catabolism partially mediates the favorable blood triglyceride levels of mildly hyperbilirubinemic individuals.•This mechanism seems to be the key strategy in the protective role of bilirubin against chronic metabolic diseases.
Telomere length (TL) in blood cells is widely used in human studies as a molecular marker of ageing. Circulating cell-free DNA (cfDNA) as well as unconjugated bilirubin (UCB) are dynamic blood ...constituents whose involvement in age-associated diseases is largely unexplored. To our knowledge, there are no published studies integrating all three parameters, especially in individuals of advanced age. Here we present a secondary analysis from the Vienna Active Aging Study (VAAS), a randomized controlled intervention trial in institutionalized elderly individuals (n = 101). Using an exploratory approach we combine three blood-based molecular markers (TL, UCB and cfDNA) with a range of primary and secondary outcomes from the intervention. We further look at the changes occurring in these parameters after 6-month resistance exercise training with or without supplementation. A correlation between UCB and TL was evident at baseline (p < 0.05), and both were associated with increased chromosomal anomalies such as nucleoplasmatic bridges and nuclear buds (p < 0.05). Of the three main markers explored in this paper, only cfDNA decreased significantly (p < 0.05) after 6-month training and dietary intervention. No clear relationship could be established between cfDNA and either UCB or TL. The trial was registered at ClinicalTrials.gov (NCT01775111).
Increased DNA and chromosomal damage are linked to aging and age-related diseases like cardiovascular diseases, diabetes or cancer. Physical activity and an optimal status of micro- and ...macronutrients are known to reduce the incidence of MN, a marker for chromosomal instability and mutagenicity. Once older people reach a certain age they change from a home-living situation to an institutionalized situation, which is often accompanied by malnutrition, depression and inactivity. We conducted the current study to investigate the effect of a six month progressive resistance training (RT), with or without protein and vitamin supplementation (RTS) or cognitive training (CT) only, on chromosomal damage measured by the cytokinesis block micronucleus cytome assay in 97 Austrian institutionalized women and men (65–98years). All three intervention groups demonstrated a tendency of a reduced frequency of cells with MN (−15%) as well as for the total number of MN (−20%), however no significant time-effect was observed. Besides a significant increase in plasma B12 and red blood cell folate status, the six month change of B12 was negatively correlated with the six month change of the MN frequency in the RTS group (r=−0.584, p=0.009). Our results suggest that in this age group either physical or cognitive training may result in similar biochemical changes and therefore enhance resistance against genomic instability. Supplementation with the vitamins B12 and folic acid could contribute to reduced chromosomal damage in institutionalized elderly.
•In the elderly, social activities seem to have an impact on genome stability.•Strength training tends to lower chromosomal damage in institutionalized elderly.•Increasing the vitamin B12 plasma level correlates negatively to chromosomal damage.•Study compliance seems to be influenced by baseline physical conditions.
Bilirubin (BR) is a natural endogenous compound with a potent bioactivity. Gilbert's Syndrome (GS) is a benign hereditary condition of increased unconjugated bilirubin (UCB) in serum and serves as a ...convenient model for studying the effects of BR in humans. In absence of liver disease, increased UCB levels are inversely associated to all-cause mortality risk, especially from cardiovascular diseases (CVDs). On the other hand, telomere malfunction is linked to a higher risk of CVDs. To our knowledge, there is no data on whether UCB is linked to telomere length in healthy or diseased individuals In the present study we have observed a relationship between mildly increased serum UCB and telomere length. We used an in vivo approach, assessing telomere length in PBMCs from individuals with GS (n = 60) and matched healthy controls (n = 60). An occurrence of longer telomeres was observed in male individuals chronically exposed to increased UCB, as well as in Gunn rats, an animal model of unconjugated hyperbilirubinaemia. Previously identified differences in immunomodulation and redox parameters in individuals with GS, such as IL-6, IL-1β and ferric reducing ability of plasma, were confirmed and proposed as possible contributors to the occurrence of longer telomeres in GS.
Aging and its aligned loss of muscle mass are associated with higher levels of DNA damage and deteriorated antioxidant defence. To improve the body's overall resistance against DNA damage, ...maintaining a healthy and active lifestyle is desirable, especially in the elderly. As people age, many have to change their residence from home living to an institution, which is often accompanied by malnutrition, depression and inactivity. The current study aimed at investigating the effect of a 6-month progressive resistance training (RT), with or without protein and vitamin supplementation (RTS), or cognitive training (CT), on DNA strand breaks in 105 Austrian institutionalised women and men (65-98 years). DNA damage was detected by performing the single cell gel electrophoresis (comet) assay. Physical fitness was assessed using the chair rise, the 6-min-walking and the handgrip strength test. In addition, antioxidant enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were analysed. Basal DNA damage (lysis) increased significantly after 3 months of intervention in the RT group (T1 - T2 + 20%, P = 0.001) and the RTS group (T1 - T2 + 17%, P = 0.002) and showed a similar tendency in the CT group (T1 - T2 + 21%, P = 0.059). %DNA in tail decreased in cells exposed to H2O2 significantly in the RT (T1 - T2 - 24%, P = 0.030; T1 - T3 - 18%, P = 0.019) and CT (T1 - T2 - 21%, P = 0.004; T1 - T3 - 13%, P = 0.038) groups. Only RT and RTS groups showed significant differences overtime in enzyme activity (RT + 22% CAT-activity T1 - T3, P = 0.013; RTS + 6% SOD-activity T2 - T3, P = 0.005). Contrary to the time effects, no difference between groups was detected for any parameter at any time point. Our results suggest that both CT and RT improve resistance against H2O2 induced DNA damage and that a nutritional supplement has no further protective effect in institutionalised elderly.
Abstract
Localized cutaneous neurofibromas (cNFs) are benign tumors that arise in the dermis of patients affected by Neurofibromatosis Type 1 syndrome, a common disorder driven by alterations in the ...NF1 gene. cNF tumors are heterogenous and comprised of different cell types such as Schwann cells, fibroblasts, and mast cells. While cNFs do not undergo malignant transformation, they carry significant co-morbidities, including itching, pain, and socio-emotional repercussions. There is currently no therapy for cNF beside surgical removal or desiccation, which are typically unable to eliminate all lesions due to their number, with patients developing as many as several hundred cNFs, as well as additional concerns of scarring and potential re-growth. A lack of models to investigate cNFs growth and perform drug screenings has hindered drug discovery and development studies thus far. We set out to develop high throughput screening-compatible patient-derived cNF organoids to perform translational studies. Tumor organoids are promising ex vivo models to recapitulate a patient’s tumor histology, molecular features, and drug responses. To support drug discovery efforts focused on identifying effective systemic therapies for cNF, we have developed an approach to routinely establish and screen cNF tumor organoids. Here we present of our systematic characterization of media conditions together with molecular and functional analysis to validate cNF organoids as a model system. We enrolled n=12 Neurofibromatosis Type 1 syndrome patients in this study and procured cNF tumors that we dissociated to single cells to establish organoids in nine different media compositions. We performed a detailed comparison to the tumor of origin using a combination of immunohistochemistry, flow cytometry, DNA methylation and RNAseq for a subset of samples (n=6 tumors from 5 patients). cNF organoids were successfully established and grown in all cases, regardless of the NF1 alteration present. We determined the optimal medium that had the highest combined correlation (parental vs organoid) across all assays as well as promoted the highest rate of growth ex vivo. We also demonstrated feasibility of screening cNF organoids using our established mini-ring pipeline (Phan et al, 2019; Nguyen and Soragni, 2020; Al Shihabi et al, 2022). In summary, we have optimized conditions for ex vivo growth of cNF organoids that closely recapitulate the molecular and cellular heterogeneity of these tumors as measured by immunohistopathology, DNA methylation, RNAseq and flow cytometry. Our tractable patient-derived cNF organoid platform enables the rapid screening of hundreds of FDA-approved drugs in a patient- and tumor-specific manner.
Citation Format: Huyen T. Nguyen, Emily Kohl, Jessica Bade, Stefan E. Eng, Anela Tosevska, Ahmad Al Shihabi, Jenny J. Hong, Sarah Dry, Paul C. Boutros, Andre Panossian, Sara Gosline, Alice Soragni. A platform for rapid patient-derived cutaneous neurofibroma organoid establishment and screening abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 196.
Localized cutaneous neurofibromas (cNFs) are benign tumors that arise in the dermis of patients affected by neurofibromatosis type 1 syndrome. cNFs are benign lesions: they do not undergo malignant ...transformation or metastasize. Nevertheless, they can cover a significant proportion of the body, with some individuals developing hundreds to thousands of lesions. cNFs can cause pain, itching, and disfigurement resulting in substantial socio-emotional repercussions. Currently, surgery and laser desiccation are the sole treatment options but may result in scarring and potential regrowth from incomplete removal. To identify effective systemic therapies, we introduce an approach to establish and screen cNF organoids. We optimized conditions to support the ex vivo growth of genomically diverse cNFs. Patient-derived cNF organoids closely recapitulate cellular and molecular features of parental tumors as measured by immunohistopathology, methylation, RNA sequencing, and flow cytometry. Our cNF organoid platform enables rapid screening of hundreds of compounds in a patient- and tumor-specific manner.
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•Established patient-derived cutaneous neurofibroma (cNF) organoids from patients with NF1•CNF organoids recapitulate the molecular and cellular features of parental tumors•Identified optimal medium conditions promoting growth while maintaining cNF features•Implemented a high-throughput screening platform to find drugs slowing organoid growth
There is no approved systemic or topical therapy for managing cutaneous neurofibromas (cNFs) in patients with neurofibromatosis type 1, a condition marked by the growth of tens to thousands of benign cNF tumors, which have significant quality-of-life implications. Current models largely focus on Schwann cells, yet cNFs are composed of many different cell types, including fibroblasts and macrophages. Given the genetic and cellular complexity of cNFs, we sought to develop a robust, patient-derived organoid model system that captures the heterogeneity and the molecular profile of the cNF of origin. The cNF organoids are developed in a format compatible with highthroughput screening that can facilitate drug discovery and development efforts to identify therapeutic leads.
Nguyen et al. develop an approach to rapidly establish and screen cutaneous neurofibroma (cNF) organoids. These are benign tumors with no existing systemic therapy, exhibiting significant genetic and cellular heterogeneity. Patient-derived cNF organoids closely recapitulate cellular and molecular features of parental tumors and can be screened for drug discovery.
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