Phosphofurin acidic cluster sorting protein 2 (PACS2) plays a vital role in maintaining cellular homeostasis by regulating protein trafficking between cellular membranes. This function impacts crucial processes like apoptosis, mitochondria-endoplasmic reticulum interaction, and subsequently Ca2+ flux, lipid biosynthesis, and autophagy. Missense mutations, particularly E209K and E211K, are linked to developmental and epileptic encephalopathy-66 (DEE66), known as PACS2 syndrome. Individuals with this syndrome exhibit neurodevelopmental delay, seizures, facial dysmorphism, hypotonia, and delayed motor skills.Understanding the impact of these missense mutations on molecular processes is crucial. Studies suggest that E209K mutation decreases phosphorylation, increases the survival time of protein, and modifies protein-protein interaction, consequently leading to disruption of calcium flux and lower resistance to apoptosis induction. Unfortunately, to date, only a limited number of research groups have investigated the effects of mutations in the PACS2 gene. Current research on PACS2 syndrome is hampered by the lack of suitable models. While in vitro models using transfected cell lines offer insights, they cannot fully capture the disease's complexity.To address this, utilizing cells from individuals with PACS2 syndrome, specifically induced pluripotent stem cells (iPSCs), holds promise for understanding phenotypic diversity and developing personalized therapies. However, iPSC models may not fully capture tissue-specific effects of the E209K/E211K mutation. In vivo studies using animal models, particularly mice, could overcome these limitations.This review summarizes current knowledge about PACS2 structure and functions, explores the cellular consequences of E209K and E211K mutations, and highlights the potential of iPSC and mouse models in advancing our understanding of PACS2 syndrome.
- MeSH
- Induced Pluripotent Stem Cells metabolism MeSH
- Humans MeSH
- Mutation, Missense * MeSH
- Mutation MeSH
- Vesicular Transport Proteins * genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.
- MeSH
- Alexander Disease * genetics pathology metabolism MeSH
- Astrocytes * metabolism pathology MeSH
- Cell Differentiation * physiology MeSH
- Glial Fibrillary Acidic Protein * metabolism genetics MeSH
- Induced Pluripotent Stem Cells * metabolism MeSH
- Coculture Techniques MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Mutation MeSH
- Neural Stem Cells metabolism MeSH
- Neurons metabolism pathology MeSH
- Organoids metabolism pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Personalizace a individualizace terapie a diagnostiky pomocí pokročilých in vitro modelů využívajících tkáň pacienta je budoucností klinické medicíny. Tyto modely zahrnují organoidy, tedy trojrozměrné in vitro modely odvozené z dospělých nebo indukovaných pluripotentních kmenových buněk, modely orgánů na čipu a další kombinované modely. Kmenové buňky pro založení těchto kultur lze získat od pacienta za pomocí minimálně invazivních metod a expandovat in vitro pro následná personalizovaná využití. Tento přehledový článek shrnuje charakteristiky jednotlivých přístupů a jejich potenciální využití zejména v transplantační terapii diabetu mellitu a diabetického onemocnění ledvin. Oproti současným metodám transplantace pankreatu a pankreatických ostrůvků, respektive transplantace ledvin, nabízí tyto přístupy řadu výhod. Na závěr jsou prezentována současná klinická využití těchto modelů v prediktivním testování léčebné odpovědi a transplantační terapii s prvními klinickými studiemi využívajícími tyto modely v kauzální léčbě diabetu mellitu.
Personalization and individualization of therapy and diagnostics using advanced in vitro models utilizing patient tissue is the future of clinical medicine. These models include organoids, i.e., three-dimensional in vitro models derived from adult or induced pluripotent stem cells, organ-on-a-chip models, and other combination models. Stem cells for establishing these cultures can be obtained from the patient using minimally invasive methods and expanded in vitro for subsequent personalized applications. This review article summarizes the characteristics of each approach and their potential applications, particularly in transplantation therapy for diabetes mellitus and diabetic kidney disease. Compared with current approaches to pancreas and pancreatic islet transplantation and kidney transplantation, respectively, these approaches offer a number of potential advantages. In conclusion, the current clinical applications of these models in predictive testing of treatment response and transplantation therapy are presented, with the first clinical trials using these models in the causal treatment of diabetes mellitus.
BACKGROUND: Tick-borne encephalitis virus (TBEV) is a significant threat to human health. The virus causes potentially fatal disease of the central nervous system (CNS), for which no treatments are available. TBEV infected individuals display a wide spectrum of neuronal disease, the determinants of which are undefined. Changes to host metabolism and virus-induced immunity have been postulated to contribute to the neuronal damage observed in infected individuals. In this study, we evaluated the cytokine, chemokine, and metabolic alterations in the cerebrospinal fluid (CSF) of symptomatic patients infected with TBEV presenting with meningitis or encephalitis. Our aim was to investigate the host immune and metabolic responses associated with specific TBEV infectious outcomes. METHODS: CSF samples of patients with meningitis (n = 27) or encephalitis (n = 25) were obtained upon consent from individuals hospitalised with confirmed TBEV infection in Brno. CSF from uninfected control patients was also collected for comparison (n = 12). A multiplex bead-based system was used to measure the levels of pro-inflammatory cytokines and chemokines. Untargeted metabolomics followed by bioinformatics and integrative omics were used to profile the levels of metabolites in the CSF. Human motor neurons (hMNs) were differentiated from induced pluripotent stem cells (iPSCs) and infected with the highly pathogenic TBEV-Hypr strain to profile the role(s) of identified metabolites during the virus lifecycle. Virus infection was quantified via plaque assay. RESULTS: Significant differences in proinflammatory cytokines (IFN-α2, TSLP, IL-1α, IL-1β, GM-CSF, IL-12p40, IL-15, and IL-18) and chemokines (IL-8, CCL20, and CXCL11) were detected between neurological-TBEV and control patients. A total of 32 CSF metabolites differed in TBE patients with meningitis and encephalitis. CSF S-Adenosylmethionine (SAM), Fructose 1,6-bisphosphate (FBP1) and Phosphoenolpyruvic acid (PEP) levels were 2.4-fold (range ≥ 2.3-≥3.2) higher in encephalitis patients compared to the meningitis group. CSF urocanic acid levels were significantly lower in patients with encephalitis compared to those with meningitis (p = 0.012209). Follow-up analyses showed fluctuations in the levels of O-phosphoethanolamine, succinic acid, and L-proline in the encephalitis group, and pyruvic acid in the meningitis group. TBEV-infection of hMNs increased the production of SAM, FBP1 and PEP in a time-dependent manner. Depletion of the metabolites with characterised pharmacological inhibitors led to a concentration-dependent attenuation of virus growth, validating the identified changes as key mediators of TBEV infection. CONCLUSIONS: Our findings reveal that the neurological disease outcome of TBEV infection is associated with specific and dynamic metabolic signatures in the cerebrospinal fluid. We describe a new in vitro model for in-depth studies of TBEV-induced neuropathogenesis, in which the depletion of identified metabolites limits virus infection. Collectively, this reveals new biomarkers that can differentiate and predict TBEV-associated neurological disease. Additionally, we have identified novel therapeutic targets with the potential to significantly improve patient outcomes and deepen our understanding of TBEV pathogenesis.
- MeSH
- Cytokines cerebrospinal fluid MeSH
- Adult MeSH
- Encephalitis, Tick-Borne * cerebrospinal fluid metabolism MeSH
- Cells, Cultured MeSH
- Middle Aged MeSH
- Humans MeSH
- Metabolome * physiology MeSH
- Metabolomics MeSH
- Young Adult MeSH
- Neurons * metabolism virology MeSH
- Aged MeSH
- Encephalitis Viruses, Tick-Borne * MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Lipopolysaccharide (LPS)-induced inflammation of lung tissues triggers irreversible alterations in the lung parenchyma, leading to fibrosis and pulmonary dysfunction. While the molecular and cellular responses of immune and connective tissue cells in the lungs are well characterized, the specific epithelial response remains unclear due to the lack of representative cell models. Recently, we introduced human embryonic stem cell-derived expandable lung epithelial (ELEP) cells as a novel model for studying lung injury and regeneration. METHODS: ELEPs were derived from the CCTL 14 human embryonic stem cell line through activin A-mediated endoderm specification, followed by further induction toward pulmonary epithelium using FGF2 and EGF. ELEPs exhibit a high proliferation rate and express key structural and molecular markers of alveolar progenitors, such as NKX2-1. The effects of Escherichia coli LPS serotype O55:B5 on the phenotype and molecular signaling of ELEPs were analyzed using viability and migration assays, mRNA and protein levels were determined by qRT-PCR, western blotting, and immunofluorescent microscopy. RESULTS: We demonstrated that purified LPS induces features of a hybrid epithelial-to-mesenchymal transition in pluripotent stem cell-derived ELEPs, triggers the unfolded protein response, and upregulates intracellular β-catenin level through retention of E-cadherin within the endoplasmic reticulum. CONCLUSIONS: Human embryonic stem cell-derived ELEPs provide a biologically relevant, non-cancerous lung cell model to investigate molecular responses to inflammatory stimuli and address epithelial plasticity. This approach offers novel insights into the fine molecular processes underlying lung injury and repair.
- MeSH
- Cell Line MeSH
- Antigens, CD metabolism MeSH
- Endoplasmic Reticulum * metabolism drug effects MeSH
- Epithelial-Mesenchymal Transition * drug effects MeSH
- Epithelial Cells * drug effects metabolism cytology MeSH
- Cadherins * metabolism MeSH
- Humans MeSH
- Human Embryonic Stem Cells * cytology MeSH
- Lipopolysaccharides * pharmacology MeSH
- Lung * cytology MeSH
- Thyroid Nuclear Factor 1 MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
PURPOSE: This study investigates genes contributing to late-adult corneal dystrophies (LACDs) in aged mice, with potential implications for late-onset corneal dystrophies (CDs) in humans. METHODS: The International Mouse Phenotyping Consortium (IMPC) database, containing data from 8901 knockout mouse lines, was filtered to include late-adult mice (49+ weeks) with significant (P < 0.0001) CD phenotypes. Candidate genes were mapped to human orthologs using the Mouse Genome Informatics group, with expression analyzed via PLAE and a literature review for prior CD associations. Comparative analyses of LACD genes from IMPC and established human CD genes from IC3D included protein interactions (STRING), biological processes (PANTHER), and molecular pathways (KEGG). RESULTS: Analysis identified 14 genes linked to late-adult abnormal corneal phenotypes. Of these, 2 genes were previously associated with CDs in humans, while 12 were novel. Seven of the 14 genes (50%) were expressed in the human cornea based on single-cell transcriptomics. Protein-protein interactions via STRING showed several significant interactions with known human CD genes. PANTHER analysis identified six biological processes shared with established human CD genes. Two genes (Rgs2 and Galnt9) were involved in pathways related to human corneal diseases, including cGMP-PKG signaling, mucin-type O-glycan biosynthesis, and oxytocin signaling. Other candidates were implicated in pathways such as pluripotency of stem cells, MAPK signaling, WNT signaling, actin cytoskeleton regulation, and cellular senescence. CONCLUSIONS: This study identified 14 genes linked to LACD in knockout mice, 12 of which are novel in corneal biology. These genes may serve as potential therapeutic targets for treating corneal diseases in aging human populations.
BACKGROUND: ß-glucans isolated from natural sources have demonstrated pluripotent immunomodulatory potential, making them a promising supportive treatment for the management of recurrent respiratory infections (RRIs) in children. This study aimed to evaluate the effects of a pleuran-based supplement (ß-glucan isolated from Pleurotus ostreatus in combination with vitamin D and zinc) on mucosal immunity -through modulating salivary secretory immunoglobulin A (sIgA) levels -in children with RRIs. METHODS: This monocentric, prospective, open-label pilot study investigated the effect of an orally administered pleuran/vitamin D/zinc supplement (1-2 chewable tablets daily depending on body weight) on the dynamics of sIgA secretion measured in saliva samples collected at three timepoints: at baseline and after 4-6 and 8-10 days. RESULTS: This study included 49 children aged 6-11 years (mean age: 8.2 ± 1.6 years) with a history of one or more of the following conditions in the inclusion criteria: RRIs, allergy, and asthma. After 8-10 days with daily administration of the chewable pleuran/vitamin D/zinc supplement, children exhibited a statistically significant increase in salivary sIgA concentrations compared with baseline (227 ± 211 μg/mL; P = 0.045). No adverse events were observed during the course of the study in relation to the administration of pleuran-based supplement. CONCLUSIONS: We demonstrated the beneficial effects of the short-term administration of a pleuran-based chewable supplement on mucosal immunity through increasing salivatory sIgA levels. This study confirms the favourable safety profile of this pleuran/vitamin D/zinc combination, which could be beneficial for children with acute or recurrent respiratory infections, including children with allergies and/or asthma. Moreover, the significant increases in salivary sIgA concentrations that were observed after a few days of supplementation support the use of pleuran in not only the prevention but also the treatment of acute respiratory infections.
- Publication type
- Journal Article MeSH
Paroxysmální noční hemoglobinurie (PNH) je lionální onemocnění kmenové krvetvorné buňky charakterizované deficitem inhibitorů komplementu na povrchu buněk. Nekontrolovaná aktivace komplementu vede k intravaskulární hemoiýze erytrocytů s vysokým rizikem trombotických komplikací a k selhání kostní dřeně. Anemie a cytopenie v dalších krevních řadách patří společně s hemoglobinurií a vysokou incidencí trombotických komplikací k hlavním příznakům onemocnění. Onemocnění vede k postižení orgánů, zejména k renální insuficienci a plicní hypertenzi. V diagnostice hraje zásadní roii detekce deficitu inhibitorů pomocí průtokové cytometrie. Na PNH je třeba myslet u stavů s negativním Coombsovým testem na hemolytickou anemii, zejména se současnou cytopenií v dalších řadách, u aplastické anemie či při suspekci na myeiodysplastický syndrom a rovněž u nemocných s trombózou, zejména v atypických lokalizacích u mladších nemocných. V léčbě se používají u stavů s opakovanou těžkou hemolýzou inhibitory složky C5 a C3 komplementu. Nemocní s hypoplastickou formou PNH a selháním kostní dřeně jsou indikováni k transliantaci krvetvorných buněk, alternativou u těchto nemocných je podání kombinované imunosuprese.
Paroxysmal nocturnal hemoglobinuria (PNH) represents a clonal disorder of pluripotent hematopoietic stem cell. The disease is characterized by deficiency of complement inhibitors on the cell surface. Uncontrolled activation of complement leads to intravascular hemolysis of red blood cells with high risk of thrombotic complications. The disease is usually connected with chronic kidney disease and pulmonary hypertension. Detection of a lack of inhibitory molecules on cell surface by flow cytometry plays a crucial role in the diagnosis of the disease. PNH should be considered in all patients with Coombs negative hemolytic anemia, especially in those with combination of cytopenia in other cell lines as well as in patients with aplastic anemia or suspected myelodysplastic syndrome. PNH must be also excluded in patients with thrombotic complications, mainly in those with thrombosis occurring in atypical localizations and at a younger age. PNH patients with recurrent episodes of severe intravascular hemolysis are indicated for treatment with inhibitors of C3 or C5 part of complement. Patients with hypoplastic PNH are candidates for hematopoietic stem cell transplantation, an alternative treatment approach to these patients may be combination immune suppression.
The combination of aminophylline and salbutamol is frequently used in clinical practice in the treatment of obstructive lung diseases. While the side effects (including arrhythmias) of the individual bronchodilator drugs were well described previously, the side effects of combined treatment are almost unknown. We aimed to study the arrhythmogenic potential of combined aminophylline and salbutamol treatment in vitro. For this purpose, we used the established atomic force microscopy (AFM) model coupled with cardiac organoids derived from human pluripotent stem cells (hPSC-CMs). We focused on the chronotropic, inotropic, and arrhythmogenic effects of salbutamol alone and aminophylline and salbutamol combined treatment. We used a method based on heart rate/beat rate variability (HRV/BRV) analysis to detect arrhythmic events in the hPSC-CM based AFM recordings. Salbutamol and aminophylline had a synergistic chronotropic and inotropic effect compared to the effects of monotherapy. Our main finding was that salbutamol reduced the arrhythmogenic effect of aminophylline, most likely mediated by endothelial nitric oxide synthase activated by beta-2 adrenergic receptors. These findings were replicated and confirmed using hPSC-CM derived from two cell lines (CCTL4 and CCTL12). Data suggest that salbutamol as an add-on therapy may not only deliver a bronchodilator effect but also increase the cardiovascular safety of aminophylline, as salbutamol reduces its arrhythmogenic potential.
- MeSH
- Albuterol * pharmacology MeSH
- Aminophylline * pharmacology MeSH
- Bronchodilator Agents pharmacology MeSH
- Cell Line MeSH
- Myocytes, Cardiac drug effects metabolism MeSH
- Humans MeSH
- Microscopy, Atomic Force MeSH
- Pluripotent Stem Cells drug effects cytology MeSH
- Arrhythmias, Cardiac * drug therapy MeSH
- Heart Rate drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 Mb tandem duplication of chromosome 17 harbouring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To obtain better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication in cellular homeostasis in CMT1A mouse models and in patient-derived induced pluripotent stem cells differentiated into Schwann cell precursors (iPSC-SCPs). We performed lipidomic profiling and bulk RNA sequencing (RNA-seq) on sciatic nerves of two developing CMT1A mouse models and on CMT1A patient-derived iPSC-SCPs. For the sciatic nerves of the CMT1A mice, cholesterol and lipid metabolism was downregulated in a dose-dependent manner throughout development. For the CMT1A iPSC-SCPs, transcriptional analysis unveiled a strong suppression of genes related to autophagy and lipid metabolism. Gene ontology enrichment analysis identified disturbances in pathways related to plasma membrane components and cell receptor signalling. Lipidomic analysis confirmed the severe dysregulation in plasma membrane lipids, particularly sphingolipids, in CMT1A iPSC-SCPs. Furthermore, we identified reduced lipid raft dynamics, disturbed plasma membrane fluidity and impaired cholesterol incorporation and storage, all of which could result from altered lipid storage homeostasis in the patient-derived CMT1A iPSC-SCPs. Importantly, this phenotype could be rescued by stimulating autophagy and lipolysis. We conclude that PMP22 duplication disturbs intracellular lipid storage and leads to a more disordered plasma membrane owing to an alteration in the lipid composition, which might ultimately lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of patients with CMT1A.
- MeSH
- Cell Membrane * metabolism MeSH
- Charcot-Marie-Tooth Disease * genetics metabolism pathology MeSH
- Gene Duplication MeSH
- Homeostasis * physiology MeSH
- Induced Pluripotent Stem Cells * metabolism MeSH
- Humans MeSH
- Lipid Metabolism * physiology MeSH
- Myelin Proteins * metabolism genetics MeSH
- Mice MeSH
- Sciatic Nerve metabolism MeSH
- Schwann Cells * metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
