The use of nanoparticles as a delivery system for a specific antigen could solve many limitations of mucosal vaccine applications, such as low immunogenicity, or antigen protection and stabilization. In this study, we tested the ability of nasally administered chitosan nanoparticles loaded with glycoprotein B of murine cytomegalovirus to induce an immune response in an animal model. The choice of chitosan nanoparticle type was made by in vitro evaluation of sorption efficiency and antigen release. Three types of chitosan nanoparticles were prepared: crosslinked with tripolyphosphate, coated with hyaluronic acid, and in complex with polycaprolactone. The hydrodynamic size of the nanoparticles by dynamic light scattering, zeta potential, Fourier transform infrared spectroscopy, scanning electron microscopy, stability, loading efficiency, and release kinetics with ovalbumin were evaluated. Balb/c mice were immunized intranasally using the three-dose protocol with nanoparticles, gB, and adjuvants Poly(I:C) and CpG ODN. Subsequently, the humoral and cell-mediated antigen-specific immune response was determined. On the basis of the properties of the tested nanoparticles, the cross-linked nanoparticles were considered optimal for further investigation. The results show that nanoparticles with Poly(I:C) and with gB alone raised IgG antibody levels above the negative control. In the case of mucosal IgA, only gB alone weakly induced the production of IgA antibodies compared to saline-immunized mice. The number of activated cells increased slightly in mice immunized with nanoparticles and gB compared to those immunized with gB alone or to negative control. The results demonstrated that chitosan nanoparticles could have potential in the development of mucosal vaccines.
- MeSH
- adjuvancia imunologická MeSH
- aplikace intranazální MeSH
- chitosan * chemie MeSH
- glykoproteiny MeSH
- imunizace MeSH
- imunoglobulin A MeSH
- Muromegalovirus * MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nanočástice * chemie MeSH
- slizniční imunita MeSH
- vakcíny * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Hypothalamic Adult Neurogenesis (hAN) has been implicated in regulating energy homeostasis. Adult-generated neurons and adult Neural Stem Cells (aNSCs) in the hypothalamus control food intake and body weight. Conversely, diet-induced obesity (DIO) by high fat diets (HFD) exerts adverse influence on hAN. However, the effects of anti-obesity compounds on hAN are not known. To address this, we administered a lipidized analogue of an anti-obesity neuropeptide, Prolactin Releasing Peptide (PrRP), so-called LiPR, to mice. In the HFD context, LiPR rescued the survival of adult-born hypothalamic neurons and increased the number of aNSCs by reducing their activation. LiPR also rescued the reduction of immature hippocampal neurons and modulated calcium dynamics in iPSC-derived human neurons. In addition, some of these neurogenic effects were exerted by another anti-obesity compound, Liraglutide. These results show for the first time that anti-obesity neuropeptides influence adult neurogenesis and suggest that the neurogenic process can serve as a target of anti-obesity pharmacotherapy.
Liver sinusoidal endothelial cells (LSECs) play a crucial role in regulating the hepatic function. Endoglin (ENG), a transmembrane glycoprotein, was shown to be related to the development of endothelial dysfunction. In this study, we hypothesized the relationship between changes in ENG expression and markers of liver sinusoidal endothelial dysfunction (LSED) during liver impairment. Male C57BL/6J mice aged 9-12 weeks were fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet (intrahepatic cholestasis) or choline-deficient l-amino acid defined high-fat diet (CDAA-HFD) (non-alcoholic steatohepatitis (NASH)). Significant increases in liver enzymes, fibrosis, and inflammation biomarkers were observed in both cholestasis and NASH. Decreased p-eNOS/eNOS and VE-cadherin protein expression and a significant increase in VCAM-1 and ICAM-1 expression were detected, indicating LSED in both mouse models of liver damage. A significant reduction of ENG in the DDC-fed mice, while a significant increase of ENG in the CDAA-HFD group was observed. Both DDC and CDAA-HFD-fed mice showed a significant increase in MMP-14 protein expression, which is related to significantly increased levels of soluble endoglin (sENG) in the plasma. In conclusion, we demonstrated that intrahepatic cholestasis and NASH result in an altered ENG expression, predominantly in LSECs, suggesting a critical role of ENG expression for the proper function of liver sinusoids. Both pathologies resulted in elevated sENG levels, cleaved by MMP-14 expressed predominantly from LSECs, indicating sENG as a liver injury biomarker.
- MeSH
- acetamidy * MeSH
- dieta s vysokým obsahem tuků škodlivé účinky MeSH
- endoglin metabolismus MeSH
- endoteliální buňky metabolismus MeSH
- intrahepatální cholestáza * MeSH
- matrixová metaloproteinasa 14 MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nealkoholová steatóza jater * patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: By exposing mice carrying a deletion of NADPH oxidase isoform 4, NOX4, specifically in pancreatic β cells (βNOX4-/-) to nutrient excess stimulated by a high-fat diet (HFD), this study aimed to elucidate the role of β-cell redox status in the development of meta-inflammation within the diabetic phenotype. METHODS: The authors performed basic phenotyping of βNOX4-/- mice on HFD involving insulin and glycemic analyses, histochemistry of adipocytes, indirect calorimetry, and cytokine analyses. To characterize local inflammation, the study used caspase-1 activity assay, interleukin-1β immunochemistry, and real-time polymerase chain reaction during coculturing of β cells with macrophages. RESULTS: The phenotype of βNOX4-/- mice on HFD was not associated with hyperinsulinemia and hyperglycemia but showed accumulation of excessive lipids in epididymal fat and β cells. Surprisingly, mice showed significantly reduced systemic inflammation. Decreased interleukin-1β protein levels and downregulated NLRP3-inflammasome activity were observed on chronic glucose overload in βNOX4-/- isolated islets and NOX4-silenced INS1-E cells resulting in attenuated proinflammatory polarization of macrophages/monocytes in vitro and in situ and reduced local islet inflammation. CONCLUSIONS: Experimental evidence suggests that NOX4 pro-oxidant activity in β cells is involved in NLRP3-inflammasome activation during chronic nutrient overload and participates in local inflammatory signaling and perhaps toward peripheral tissues, contributing to a diabetic inflammatory phenotype.
Proteins from the Bcl-2 family play an essential role in the regulation of apoptosis. However, they also possess cell death-unrelated activities that are less well understood. This prompted us to study apoptosis-unrelated activities of the Bax and Bak, pro-apoptotic members of the Bcl-2 family. We prepared Bax/Bak-deficient human cancer cells of different origin and found that while respiration in the glioblastoma U87 Bax/Bak-deficient cells was greatly enhanced, respiration of Bax/Bak-deficient B lymphoma HBL-2 cells was slightly suppressed. Bax/Bak-deficient U87 cells also proliferated faster in culture, formed tumours more rapidly in mice, and showed modulation of metabolism with a considerably increased NAD+/NADH ratio. Follow-up analyses documented increased/decreased expression of mitochondria-encoded subunits of respiratory complexes and stabilization/destabilization of the mitochondrial transcription elongation factor TEFM in Bax/Bak-deficient U87 and HBL-2 cells, respectively. TEFM downregulation using shRNAs attenuated mitochondrial respiration in Bax/Bak-deficient U87 as well as in parental HBL-2 cells. We propose that (post)translational regulation of TEFM levels in Bax/Bak-deficient cells modulates levels of subunits of mitochondrial respiratory complexes that, in turn, contribute to respiration and the accompanying changes in metabolism and proliferation in these cells.
- MeSH
- apoptóza * genetika MeSH
- dýchání MeSH
- lidé MeSH
- mitochondrie genetika metabolismus MeSH
- myši MeSH
- protein Bak * genetika metabolismus MeSH
- protein X asociovaný s bcl-2 genetika metabolismus MeSH
- protoonkogenní proteiny c-bcl-2 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Viral infection may represent a stress condition to the host cell. Cells react to it by triggering the defence programme to restore homeostasis and these events may in turn impact the viral replication. The knowledge about tick-borne encephalitis virus (TBEV) infection-associated stress is limited. Here we investigated the interplay between TBEV infection and stress pathways in PMJ2-R mouse macrophage cell line, as macrophages are the target cells in early phases of TBEV infection. First, to determine how stress influences TBEV replication, the effect of stress inducers H2O2 and tunicamycin (TM) was tested. Viral multiplication was decreased in the presence of both stress inducers suggesting that the stress and cellular stress responses restrict the virus replication. Second, we investigated the induction of oxidative stress and endoplasmic reticulum (ER) stress upon TBEV infection. The level of oxidative stress was interrogated by measuring the reactive oxygen species (ROS). ROS were intermittently increased in infected cells at 12 hpi and at 72 hpi. As mitochondrial dysfunction may result in increased ROS level, we evaluated the mitochondrial homeostasis by measuring the mitochondrial membrane potential (MMP) and found that TBEV infection induced the hyperpolarization of MMP. Moreover, a transient increase of gene expression of stress-induced antioxidative enzymes, like p62, Gclm and Hmox1, was detected. Next, we evaluated the ER stress upon TBEV infection by analysing unfolded protein responses (UPR). We found that infection induced gene expression of two general sensors BiP and CHOP and activated the IRE1 pathway of UPR. Finally, since the natural transmission route of TBEV from its tick vector to the host is mediated via tick saliva, the impact of tick saliva from Ixodes ricinus on stress pathways in TBEV-infected cells was tested. We observed only marginal potentiation of UPR pathway. In conclusion, we found that TBEV infection of PMJ2-R cells elicits the changes in redox balance and triggers cellular stress defences, including antioxidant responses and the IRE1 pathway of UPR. Importantly, our results revealed the negative effect of stress-evoked events on TBEV replication and only marginal impact of tick saliva on stress cellular pathways.
- MeSH
- buněčné linie MeSH
- klíšťová encefalitida * MeSH
- myši MeSH
- peroxid vodíku metabolismus MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- replikace viru MeSH
- viry klíšťové encefalitidy * genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Glial cells expressing neuron-glial antigen 2 (NG2), also known as oligodendrocyte progenitor cells (OPCs), play a critical role in maintaining brain health. However, their ability to differentiate after ischemic injury is poorly understood. The aim of this study was to investigate the properties and functions of NG2 glia in the ischemic brain. Using transgenic mice, we selectively labeled NG2-expressing cells and their progeny in both healthy brain and after focal cerebral ischemia (FCI). Using single-cell RNA sequencing, we classified the labeled glial cells into five distinct subpopulations based on their gene expression patterns. Additionally, we examined the membrane properties of these cells using the patch-clamp technique. Of the identified subpopulations, three were identified as OPCs, whereas the fourth subpopulation had characteristics indicative of cells likely to develop into oligodendrocytes. The fifth subpopulation of NG2 glia showed astrocytic markers and had similarities to neural progenitor cells. Interestingly, this subpopulation was present in both healthy and post-ischemic tissue; however, its gene expression profile changed after ischemia, with increased numbers of genes related to neurogenesis. Immunohistochemical analysis confirmed the temporal expression of neurogenic genes and showed an increased presence of NG2 cells positive for Purkinje cell protein-4 at the periphery of the ischemic lesion 12 days after FCI, as well as NeuN-positive NG2 cells 28 and 60 days after injury. These results suggest the potential development of neuron-like cells arising from NG2 glia in the ischemic tissue. Our study provides insights into the plasticity of NG2 glia and their capacity for neurogenesis after stroke.
- MeSH
- antigeny metabolismus MeSH
- astrocyty metabolismus MeSH
- ischemie mozku * metabolismus MeSH
- mozek metabolismus MeSH
- myši transgenní MeSH
- myši MeSH
- nervové kmenové buňky * metabolismus MeSH
- neuroglie metabolismus MeSH
- oligodendroglie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Venetoclax (VEN), a B-cell lymphoma 2 (BCL2) inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large BCLs, but remissions were generally short, which call for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples, we demonstrated strong synergy between VEN and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments and studies on clones with knockout of expression or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired VEN resistance. Of note, the VEN and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the proapoptotic BCL2L11/BIM and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in VEN resistance. Immunoprecipitation experiments further suggested that the proapoptotic effector BAX belongs to principal mediators of the VEN and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new proapoptotic combination was confirmed in vivo on a panel of 9 patient-derived lymphoma xenografts models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and diffuse large BCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days on/3 days off treatment regimen, which retained the desired antitumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2+ hematologic malignancies irrespective of the BCL2L11/BIM status.
- MeSH
- antitumorózní látky farmakologie terapeutické užití MeSH
- apoptóza účinky léků MeSH
- benzothiazoly MeSH
- bicyklické sloučeniny heterocyklické * farmakologie terapeutické užití MeSH
- chemorezistence * MeSH
- isochinoliny MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- protein bcl-X * metabolismus antagonisté a inhibitory MeSH
- protein BCL2L11 * metabolismus genetika MeSH
- protoonkogenní proteiny c-bcl-2 metabolismus antagonisté a inhibitory MeSH
- sulfonamidy * farmakologie terapeutické užití MeSH
- synergismus léků MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 (RAB31) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.
- MeSH
- extracelulární vezikuly * metabolismus MeSH
- glykolýza * MeSH
- jaterní cirhóza * metabolismus patologie genetika MeSH
- jaterní hvězdicovité buňky * metabolismus patologie MeSH
- játra metabolismus patologie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- Rab proteiny vázající GTP metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.
- MeSH
- cyklin-dependentní kinasy metabolismus genetika MeSH
- embryo savčí metabolismus MeSH
- embryonální vývoj * genetika MeSH
- fenotyp MeSH
- lebka embryologie patologie MeSH
- mentální retardace genetika MeSH
- modely nemocí na zvířatech * MeSH
- mutace genetika MeSH
- myši MeSH
- nervus trigeminus embryologie MeSH
- neurogeneze * genetika MeSH
- obličej embryologie abnormality MeSH
- protein doublecortin MeSH
- rozštěp patra genetika patologie embryologie MeSH
- rozštěp rtu genetika patologie embryologie MeSH
- vývojová regulace genové exprese * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
