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1.
Haematologica ; 2024 Jun 27.
Article de Anglais | MEDLINE | ID: mdl-38934080

RÉSUMÉ

Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eµ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.

2.
DNA Repair (Amst) ; 135: 103648, 2024 Mar.
Article de Anglais | MEDLINE | ID: mdl-38382170

RÉSUMÉ

DNA damage response (DDR) is a complex process, essential for cell survival. Especially deleterious type of DNA damage are DNA double-strand breaks (DSB), which can lead to genomic instability and malignant transformation if not repaired correctly. The central player in DSB detection and repair is the ATM kinase which orchestrates the action of several downstream factors. Recent studies have suggested that long non-coding RNAs (lncRNAs) are involved in DDR. Here, we aimed to identify lncRNAs induced upon DNA damage in an ATM-dependent manner. DNA damage was induced by ionizing radiation (IR) in immortalized lymphoblastoid cell lines derived from 4 patients with ataxia-telangiectasia (AT) and 4 healthy donors. RNA-seq revealed 10 lncRNAs significantly induced 1 h after IR in healthy donors, whereas none in AT patients. 149 lncRNAs were induced 8 h after IR in the control group, while only three in AT patients. Among IR-induced mRNAs, we found several genes with well-known functions in DDR. Gene Set Enrichment Analysis and Gene Ontology revealed delayed induction of key DDR pathways in AT patients compared to controls. The induction and dynamics of selected 9 lncRNAs were confirmed by RT-qPCR. Moreover, using a specific ATM inhibitor we proved that the induction of those lncRNAs is dependent on ATM. Some of the detected lncRNA genes are localized next to protein-coding genes involved in DDR. We observed that induction of lncRNAs after IR preceded changes in expression of adjacent genes. This indicates that IR-induced lncRNAs may regulate the transcription of nearby genes. Subcellular fractionation into chromatin, nuclear, and cytoplasmic fractions revealed that the majority of studied lncRNAs are localized in chromatin. In summary, our study revealed several lncRNAs induced by IR in an ATM-dependent manner. Their genomic co-localization and co-expression with genes involved in DDR suggest that those lncRNAs may be important players in cellular response to DNA damage.


Sujet(s)
Ataxie-télangiectasie , ARN long non codant , Humains , ARN long non codant/génétique , Altération de l'ADN , Chromatine , Lignée cellulaire , Protéines mutées dans l'ataxie-télangiectasie
3.
J Appl Genet ; 65(1): 95-101, 2024 Feb.
Article de Anglais | MEDLINE | ID: mdl-37917375

RÉSUMÉ

Burkitt lymphoma (BL) is a highly aggressive lymphoma that mainly affects children and young adults. Chemotherapy is effective in young BL patients but the outcome in adults is less satisfactory. Therefore, there is a need to enhance the cytotoxic effect of drugs used in BL treatment. Glutathione (GSH) is an important antioxidant involved in processes such as regulation of oxidative stress and drug detoxification. Elevated GSH levels have been observed in many cancers and were associated with chemoresistance. We previously identified GCLC, encoding an enzyme involved in GSH biosynthesis, as an essential gene in BL. We now confirm that knockout of GCLC decreases viability of BL cells and that the GCLC protein is overexpressed in BL tissues. Moreover, we demonstrate that buthionine sulfoximine (BSO), a known inhibitor of GCLC, decreases growth of BL cells but does not affect control B cells. Furthermore, we show for the first time that BSO enhances the cytotoxicity of compounds commonly used in BL treatment, doxorubicin, and cyclophosphamide. Given the fact that BSO itself was not toxic to control cells and well-tolerated in clinical trials, combination of chemotherapy with BSO may allow reduction of the doses of cytotoxic drugs required to obtain effective responses in BL patients.


Sujet(s)
Lymphome de Burkitt , Glutamate-cysteine ligase , Enfant , Humains , Buthionine sulfoximine/pharmacologie , Buthionine sulfoximine/usage thérapeutique , Glutamate-cysteine ligase/génétique , Glutamate-cysteine ligase/métabolisme , Lymphome de Burkitt/traitement médicamenteux , Lymphome de Burkitt/génétique , Domaine catalytique , Cyclophosphamide/pharmacologie , Doxorubicine/pharmacologie , Glutathion/métabolisme
4.
J Clin Med ; 12(20)2023 Oct 13.
Article de Anglais | MEDLINE | ID: mdl-37892644

RÉSUMÉ

Lymphomas are a group of malignant tumors that originate in the lymphatic system. It is the most common type of blood cancer. It affects the lymph nodes, spleen, bone marrow, blood, and other organs. They can be aggressive or chronic. Hodgkin lymphoma survival rate is 2 in 100,000 people. Young adults aged 20-30 and people over 50 are most often affected. The prognosis of Hodgkin's lymphoma is good, with a survival rate of up to 80 percent. Nevertheless, in 20-30 percent of patients who initially respond to treatment, the disease has a tendency to progress. The positive effect of radiotherapy (RT) on patients' survival rates has been proven in many randomized clinical trials. Although the dose of chest RT has significantly reduced over the years, we still struggle with the long-term complications of post-RT repercussions, mainly because there is no established safe dose of RT affecting the heart. Other complications include earlier onset of coronary artery disease, early and late onset of pericarditis, valve degeneration (predominantly of the left heart), calcification of the aorta and its branches, heart failure, and arrhythmias. One patient can manifest each of the abovementioned complications, as in the present case. That is why choosing the right treatment strategy is crucial.

5.
J Mater Chem B ; 11(36): 8732-8753, 2023 09 20.
Article de Anglais | MEDLINE | ID: mdl-37655519

RÉSUMÉ

Due to the health risks associated with the use of Gd-chelates and the promising effects of using nanoparticles as T1 contrast agents (CAs) for MRI, Mn-based nanoparticles are considered a highly competitive alternative. The use of hybrid constructs with paramagnetic functionality of Mn-based nanoparticles is an effective approach, in particular, the use of biocompatible lipid liquid crystalline nanoparticles (LLCNPs) as a carrier of MnO nanoparticles. LLCNPs possess a unique internal structure ensuring a payload of different polarity MnO nanoparticles. In view of MRI application, the surface properties including the polarity of MnO are crucial factors determining their relaxation rate and thus the MRI efficiency. Two novel hybrid constructs consisting of LLCNPs loaded with hydrophobic MnO-oleate and hydrophilic MnO-DMSA NPs were prepared. These nanosystems were studied in terms of their physico-chemical properties, positive T1 contrast enhancement properties (in vitro and in vivo) and biological safety. LLCNPs@MnO-oleate and LLCNPs@MnO-DMSA hybrids exhibited a heterogeneous phase composition, however with differences in the inner periodic arrangement and structural parameters, as well as in the preferable localization of MnO NPs within the LLCNPs. Also, these hybrids differed in terms of particle size-related parameters and colloidal stability, which was found to be strongly dependent on the addition of differently functionalized MnO NPs. Embedding both types of MnO NPs into LLCNPs resulted in high relaxivity parameters, in comparison to bare MnO-DMSA NPs and also commercially developed CAs (e.g. Dotarem and Teslascan). Further biosafety studies revealed that cell internalization pathways were dependent on the prepared hybrid type, while viability, effects on the mitochondria membrane potential and cytoskeletal networks were rather related to the susceptibility of the particular cell line. The high relaxation rates achieved with the developed hybrid LLCNPs@MnO enable them to be possibly used as novel and biologically safe MRI T1-enhancing CAs in in vivo imaging.


Sujet(s)
Produits de contraste , Oxydes , Imagerie par résonance magnétique , Lipides
6.
Mol Oncol ; 17(11): 2295-2313, 2023 Nov.
Article de Anglais | MEDLINE | ID: mdl-37519063

RÉSUMÉ

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines-K562, ST486, HepG2, and MCF7-which revealed several essential E-boxes and genes. Among them, we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression, and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.


Sujet(s)
Systèmes CRISPR-Cas , Tumeurs , Humains , Systèmes CRISPR-Cas/génétique , Protéines proto-oncogènes c-myc/génétique , Protéines proto-oncogènes c-myc/métabolisme , Lignée cellulaire , Facteurs de transcription/métabolisme , Régulation de l'expression des gènes , Tumeurs/génétique
7.
Genes (Basel) ; 14(4)2023 04 14.
Article de Anglais | MEDLINE | ID: mdl-37107672

RÉSUMÉ

Recently, we have observed two significant pandemics caused by communicable (COVID-19) and non-communicable factors (obesity). Obesity is related to a specific genetic background and characterized by immunogenetic features, such as low-grade systemic inflammation. The specific genetic variants include the presence of polymorphism of the Peroxisome Proliferator-Activated Receptors gene (PPAR-γ2; Pro12Ala, rs1801282, and C1431T, rs3856806 polymorphisms), ß-adrenergic receptor gene (3ß-AR; Trp64Arg, rs4994), and Family With Sequence Similarity 13 Member A gene (FAM13A; rs1903003, rs7671167, rs2869967). This study aimed to analyze the genetic background, body fat distribution, and hypertension risk in obese metabolically healthy postmenopausal women (n = 229, including 105 lean and 124 obese subjects). Each patient underwent anthropometric and genetic evaluations. The study has shown that the highest value of BMI was associated with visceral fat distribution. The analysis of particular genotypes has revealed no differences between lean and obese women except for FAM13A rs1903003 (CC), which was more prevalent in lean patients. The co-existence of the PPAR-γ2 C1431C variant with other FAM13A gene polymorphisms [rs1903003(TT) or rs7671167(TT), or rs2869967(CC)] was related to higher BMI values and visceral fat distribution (WHR > 0.85). The co-association of FAM13A rs1903003 (CC) and 3ß-AR Trp64Arg was associated with higher values of systolic (SBP) and diastolic blood pressure (DBP). We conclude that the co-existence of FAM13A variants with C1413C polymorphism of the PPAR-γ2 gene is responsible for body fat amount and distribution.


Sujet(s)
COVID-19 , Récepteur PPAR gamma , Humains , Femelle , Récepteur PPAR gamma/génétique , Post-ménopause/génétique , Prédisposition génétique à une maladie , Polymorphisme génétique , Obésité/génétique , Protéines d'activation de la GTPase/génétique
8.
Andrology ; 10(8): 1605-1624, 2022 11.
Article de Anglais | MEDLINE | ID: mdl-36017582

RÉSUMÉ

BACKGROUND: Genetic causes that lead to spermatogenetic failure in patients with nonobstructive azoospermia (NOA) have not been yet completely established. OBJECTIVE: To identify low-frequency NOA-associated single nucleotide variants (SNVs) using whole-genome sequencing (WGS). MATERIALS AND METHODS: Men with various types of NOA (n = 39), including samples that had been previously tested with whole-exome sequencing (WES; n = 6) and did not result in diagnostic conclusions. Variants were annotated using the Ensembl Variant Effect Predictor, utilizing frequencies from GnomAD and other databases to provide clinically relevant information (ClinVar), conservation scores (phyloP), and effect predictions (i.e., MutationTaster). Structural protein modeling was also performed. RESULTS: Using WGS, we revealed potential NOA-associated SNVs, such as: TKTL1, IGSF1, ZFPM2, VCX3A (novel disease causing variants), ESX1, TEX13A, TEX14, DNAH1, FANCM, QRICH2, FSIP2, USP9Y, PMFBP1, MEI1, PIWIL1, WDR66, ZFX, KCND1, KIAA1210, DHRSX, ZMYM3, FAM47C, FANCB, FAM50B (genes previously known to be associated with infertility) and ALG13, BEND2, BRWD3, DDX53, TAF4, FAM47B, FAM9B, FAM9C, MAGEB6, MAP3K15, RBMXL3, SSX3 and FMR1NB genes, which may be involved in spermatogenesis. DISCUSSION AND CONCLUSION: In this study, we identified novel potential candidate NOA-associated genes in 29 individuals out of 39 azoospermic males. Note that in 5 out of 6 patients subjected previously to WES analysis, which did not disclose potentially causative variants, the WGS analysis was successful with NOA-associated gene findings.


Sujet(s)
Azoospermie , Protéines Argonaute/génétique , Azoospermie/diagnostic , Azoospermie/génétique , Protéines de liaison au calcium , Helicase , Humains , Immunoglobulines/génétique , Mâle , Protéines membranaires/génétique , Mutation , N-acetylglucosaminyltransferase , Protéines nucléaires/génétique , Nucléotides , Facteurs de transcription , Transketolase/génétique ,
9.
J Tissue Eng Regen Med ; 16(10): 853-874, 2022 10.
Article de Anglais | MEDLINE | ID: mdl-35748158

RÉSUMÉ

The high organ specification of the human heart is inversely proportional to its functional recovery after damage. The discovery of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has accelerated research in human heart regeneration and physiology. Nevertheless, due to the immaturity of iPSC-CMs, they are far from being an representative model of the adult heart physiology. Therefore, number of laboratories strive to obtain a heart tissues by engineering methods by structuring iPSC-CMs into complex and advanced platforms. By using the iPSC-CMs and arranging them in 3D cultures it is possible to obtain a human heart muscle with physiological capabilities potentially similar to the adult heart, while remaining in vitro. Here, we attempt to describe existing examples of heart muscle either in vitro or ex vivo models and discuss potential options for the further development of such structures. This will be a crucial step for ultimate derivation of complete heart tissue-mimicking organs and their future use in drug development, therapeutic approaches testing, pre-clinical studies, and clinical applications. This review particularly aims to compile available models of advanced human heart tissue for scientists considering which model would best fit their research needs.


Sujet(s)
Cellules souches pluripotentes induites , Adulte , Différenciation cellulaire , Humains , Myocarde , Myocytes cardiaques
10.
Kardiol Pol ; 80(7-8): 813-824, 2022.
Article de Anglais | MEDLINE | ID: mdl-35554929

RÉSUMÉ

BACKGROUND: The application of a circulating miR-195 inhibitor could be a helping factor in the in vitro model of human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs). Previously, microRNA-195 (miR-195) expression has been reported to be a negative factor for myogenesis. AIMS: The study aimed to obtain anti-apoptotic and anti-aging effects in in vitro cultured myoblasts and to improve their ability to form myotubes by suppressing miR-195 expression. METHODS: Human wild-type (WT) SkMDS/PC cells incubated with control (nonspecific) miRNA inhibitor and miR-195-inhibited SkMDS/PCs were studied. Functional assays (myotube formation and cell aging), antioxidant, and myogenic gene expression analyses were performed at two time points, at the seventh and eleventh cell passages. RESULTS: Myotube formation was found to be almost 2-fold higher in the miR-195-inhibited SkMDS/PCs population (P < 0.05) compared to WT cells. miR-195 inhibition did not appear to affect cell aging or rejuvenate human SkMDS/PCs. Antioxidant (SOD3 and FOXO) gene expression was augmented in the miR-195-inhibited SkMDS/PCs population, but no positive effect on the remaining antioxidant genes (SOD1, SOD2, and catalase) was observed. A significant increase in MyoD gene expression with a concomitant decrease in MyoG (P < 0.05) was further documented in miR-195- -inhibited SkMDS/PCs compared to WT cells (the eleventh cell passage). CONCLUSIONS: The performed studies may lead to the preconditioning of myogenic stem cells to extend their potential for pro-regenerative activity. The miR-195 inhibitor may serve as a conditioning factor augmenting selective antioxidant gene expression and proliferative potential of SkMDS/PCs, but it does not have an impact on cell aging and/ or apoptosis.


Sujet(s)
Antioxydants , microARN , Différenciation cellulaire/génétique , Humains , microARN/génétique , microARN/métabolisme , Fibres musculaires squelettiques/métabolisme , Muscles squelettiques , Myoblastes/métabolisme , Cellules souches/métabolisme
11.
Am J Stem Cells ; 11(1): 1-11, 2022.
Article de Anglais | MEDLINE | ID: mdl-35295592

RÉSUMÉ

Human primary in vitro cell cultures are among the most challenging procedures in cellular biology laboratory practice. Myoblasts-progenitor of skeletal muscle origin represent a promising therapeutic cell source since the procedure of their isolation is not technically demanding, and the in vitro culture is relatively straightforward. Myoblasts could be considered as the candidates for clinical applications due to their regenerative potential, and as the carriers of therapeutic proteins introduced through genetic modifications. The main goal of this prospective study was to evaluate different myoblasts isolation strategies based on the pre-plating technique and cells density characteristics. Moreover, testing of different myoblast media formulations-both commercially available and in-house made was performed. Our goal was to establish the in vitro protocol of myoblasts culture allowing for preservation of the proliferative potential and desired phenotype. Our results revealed that in culture of myoblasts of human muscle origin, the pre-plate technique and cell density differences did not correlate with changes in the proliferative potential, however it was observed that low density cells maintained expression of the CD56 marker up to the higher passages. Assessment of different types of culture media confirmed the best performance for DMEM based media without Chicken Embryo Extract (CEE) addition. Cells cultured in DMEM+FBS medium revealed high expression of CD56 and CD90 antigens, absence of the hematopoietic markers and presented stable proliferation profile. This finding is in line with guidelines of regulatory agencies recommending removal of the xeno-derived reagents from the manufacturing process of Advanced Therapy Medicinal Products (ATMP). In this study, human myoblasts culture was optimized in vitro under different media conditions. The next approach in assessment of myoblasts propagation for potential clinical applications will be testing of the clinical grade human platelet lysate (hPL) instead of the FBS.

12.
Int J Mol Sci ; 24(1)2022 Dec 26.
Article de Anglais | MEDLINE | ID: mdl-36613797

RÉSUMÉ

Cardiovascular diseases (CVD) is a collective term describing a range of conditions that affect the heart and blood vessels. Due to the varied nature of the disorders, distinguishing between their causes and monitoring their progress is crucial for finding an effective treatment. Molecular imaging enables non-invasive visualisation and quantification of biological pathways, even at the molecular and subcellular levels, what is essential for understanding the causes and development of CVD. Positron emission tomography imaging is so far recognized as the best method for in vivo studies of the CVD related phenomena. The imaging is based on the use of radioisotope-labelled markers, which have been successfully used in both pre-clinical research and clinical studies. Current research on CVD with the use of such radioconjugates constantly increases our knowledge and understanding of the causes, and brings us closer to effective monitoring and treatment. This review outlines recent advances in the use of the so-far available radioisotope markers in the research on cardiovascular diseases in rodent models, points out the problems and provides a perspective for future applications of PET imaging in CVD studies.


Sujet(s)
Maladies cardiovasculaires , Animaux , Maladies cardiovasculaires/imagerie diagnostique , Radio-isotopes , Tomographie par émission de positons/méthodes , Radiopharmaceutiques , Modèles animaux
13.
Int J Mol Sci ; 22(22)2021 Nov 22.
Article de Anglais | MEDLINE | ID: mdl-34830471

RÉSUMÉ

Cardiovascular diseases (CVD), with myocardial infarction (MI) being one of the crucial components, wreak havoc in developed countries. Advanced imaging technologies are required to obtain quick and widely available diagnostic data. This paper describes a multimodal approach to in vivo perfusion imaging using the novel SYN1 tracer based on the fluorine-18 isotope. The NOD-SCID mice were injected intravenously with SYN1 or [18F] fluorodeoxyglucose ([18F]-FDG) radiotracers after induction of the MI. In all studies, the positron emission tomography-computed tomography (PET/CT) technique was used. To obtain hemodynamic data, mice were subjected to magnetic resonance imaging (MRI). Finally, the biodistribution of the SYN1 compound was performed using Wistar rat model. SYN1 showed normal accumulation in mouse and rat hearts, and MI hearts correctly indicated impaired cardiac segments when compared to [18F]-FDG uptake. In vivo PET/CT and MRI studies showed statistical convergence in terms of the size of the necrotic zone and cardiac function. This was further supported with RNAseq molecular analyses to correlate the candidate function genes' expression, with Serpinb1c, Tnc and Nupr1, with Trem2 and Aldolase B functional correlations showing statistical significance in both SYN1 and [18F]-FDG. Our manuscript presents a new fluorine-18-based perfusion radiotracer for PET/CT imaging that may have importance in clinical applications. Future research should focus on confirmation of the data elucidated here to prepare SYN1 for first-in-human trials.


Sujet(s)
Facteurs de transcription à motif basique hélice-boucle-hélice/génétique , Coeur/imagerie diagnostique , Infarctus du myocarde/génétique , Protéines tumorales/génétique , Serpines/génétique , Ténascine/génétique , Animaux , Produits de contraste/pharmacologie , Fluorodésoxyglucose F18/pharmacologie , Fructose bisphosphate aldolase/génétique , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Coeur/effets des médicaments et des substances chimiques , Humains , Imagerie par résonance magnétique , Mâle , Glycoprotéines membranaires/génétique , Souris , Infarctus du myocarde/anatomopathologie , Tomographie par émission de positons couplée à la tomodensitométrie , Rats , Récepteurs immunologiques/génétique , Distribution tissulaire/effets des médicaments et des substances chimiques
14.
Int J Mol Sci ; 22(19)2021 Oct 08.
Article de Anglais | MEDLINE | ID: mdl-34639225

RÉSUMÉ

Current treatment protocols for myocardial infarction improve the outcome of disease to some extent but do not provide the clue for full regeneration of the heart tissues. An increasing body of evidence has shown that transplantation of cells may lead to some organ recovery. However, the optimal stem cell population has not been yet identified. We would like to propose a novel pro-regenerative treatment for post-infarction heart based on the combination of human skeletal myoblasts (huSkM) and mesenchymal stem cells (MSCs). huSkM native or overexpressing gene coding for Cx43 (huSKMCx43) alone or combined with MSCs were delivered in four cellular therapeutic variants into the healthy and post-infarction heart of mice while using molecular reporter probes. Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) performed right after cell delivery and 24 h later revealed a trend towards an increase in the isotopic uptake in the post-infarction group of animals treated by a combination of huSkMCx43 with MSC. Bioluminescent imaging (BLI) showed the highest increase in firefly luciferase (fluc) signal intensity in post-infarction heart treated with combination of huSkM and MSCs vs. huSkM alone (p < 0.0001). In healthy myocardium, however, nanoluciferase signal (nanoluc) intensity varied markedly between animals treated with stem cell populations either alone or in combinations with the tendency to be simply decreased. Therefore, our observations seem to show that MSCs supported viability, engraftment, and even proliferation of huSkM in the post-infarction heart.


Sujet(s)
Cellules souches mésenchymateuses/cytologie , Imagerie moléculaire/méthodes , Myoblastes squelettiques/cytologie , Infarctus du myocarde/anatomopathologie , Myocarde/anatomopathologie , Animaux , Modèles animaux de maladie humaine , Gènes rapporteurs , Humains , Cellules souches mésenchymateuses/métabolisme , Souris , Souris de lignée NOD , Souris SCID , Myoblastes squelettiques/métabolisme , Infarctus du myocarde/métabolisme , Myocarde/métabolisme
15.
Nanomaterials (Basel) ; 11(10)2021 Sep 24.
Article de Anglais | MEDLINE | ID: mdl-34684935

RÉSUMÉ

The biocompatibility of pNiPAM (Poly N-isopropylacrylamide) copolymers has been examined and they did not exert any cytotoxic effects. Their properties and vulnerable temperature characteristics make them candidates for use in medical applications. We synthesized a well-characterized nanoparticles-based cargo system that would effectively deliver a biological agent to human skeletal myogenic cells (SkMCs); among other aspects, a downregulating apoptotic pathway potentially responsible for poor regeneration of myocardium. We confirmed the size of the pNiPAM based spheres at around 100 nm and the nanomeric shape of nanoparticles (NP) obtained. We confirmed that 33 °C is the adequate temperature for phase transition. We performed the dynamics of cargo release. A small amount of examined protein was detected at 10 min after reaching LCTS (lower critical solution temperature). The presented results of the test with BSA (bovine serum albumin) and doxorubicin loaded into nanoparticles showed a similar release profile for both substances. SkMCs incubated with NP loaded with antiapoptotic agent, BCB (Bax channel blocker), significantly diminished cell apoptosis (p < 0.01). Moreover, the lowest apoptotic level was detected in SkMCs treated with camptothecin and simultaneously incubated with pNiPAMs loaded with BCB. Application of nanoparticles loaded with BCB or subjected to BCB alone did not, however, diminish the amount of apparently necrotic cells.

16.
Sci Rep ; 11(1): 19825, 2021 10 06.
Article de Anglais | MEDLINE | ID: mdl-34615887

RÉSUMÉ

Preclinical and clinical studies have shown that stem cells can promote the regeneration of damaged tissues, but therapeutic protocols need better quality control to confirm the location and number of transplanted cells. This study describes in vivo imaging while assessing reporter gene expression by its binding to a radiolabelled molecule to the respective receptor expressed in target cells. Five mice underwent human skeletal muscle-derived stem/progenitor cell (huSkMDS/PC EF1-HSV-TK) intracardial transplantation after induction of myocardial infarction (MI). The metabolic parameters of control and post-infarction stem progenitor cell-implanted mice were monitored using 2-deoxy-18F-fluorodeoxyglucose ([18F]-FDG) before and after double promotor/reporter probe imaging with 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]-FHBG) using positron emission tomography (PET) combined with computed tomography (CT). Standardized uptake values (SUVs) were then calculated based on set regions of interest (ROIs). Experimental animals were euthanized after magnetic resonance imaging (MRI). Molecular [18F]-FHBG imaging of myogenic stem/progenitor cells in control and post-infarction mice confirmed the survival and proliferation of transplanted cells, as shown by an increased or stable signal from the PET apparatus throughout the 5 weeks of monitoring. huSkMDS/PC EF1-HSV-TK transplantation improved cardiac metabolic ([18F]-FDG with PET) and haemodynamic (MRI) parameters. In vivo PET/CT and MRI revealed that the precise use of a promotor/reporter probe incorporated into stem/progenitor cells may improve non-invasive monitoring of targeted cellular therapy in the cardiovascular system.


Sujet(s)
Fluorodésoxyglucose F18 , Imagerie moléculaire , Myoblastes cardiaques/métabolisme , Infarctus du myocarde/métabolisme , Infarctus du myocarde/anatomopathologie , Tomographie par émission de positons couplée à la tomodensitométrie , Cellules souches adultes/métabolisme , Animaux , Modèles animaux de maladie humaine , Échocardiographie , Femelle , Humains , Traitement d'image par ordinateur , Imagerie par résonance magnétique , Mâle , Souris , Souris SCID , Imagerie moléculaire/méthodes , Infarctus du myocarde/imagerie diagnostique , Infarctus du myocarde/étiologie , Polyesters
17.
Antioxidants (Basel) ; 10(8)2021 Jul 27.
Article de Anglais | MEDLINE | ID: mdl-34439451

RÉSUMÉ

Standard sperm evaluation parameters do not enable predicting their ability to survive cryopreservation. Mitochondria are highly prone to suffer injuries during freezing, and any abnormalities in their morphology or function are reflected by a decline of sperm quality. Our work focused on describing a link between the number and the activity of mitochondria, with an aim to validate its applicability as a biomarker of bovine sperm quality. Cryopreserved sperm collected from bulls with high (group 1) and low (group 2) semen quality was separated by swim up. The spermatozoa of group 1 overall retained more mitochondria (MitoTrackerGreen) and mtDNA copies, irrespective of the fraction. Regardless of the initial ejaculate quality, the motile sperm contained significantly more mitochondria and mtDNA copies. The same trend was observed for mitochondrial membrane potential (ΔΨm, JC-1), where motile sperm displayed high ΔΨm. These results stay in agreement with transcript-level evaluation (real-time polymerase chain reaction, PCR) of antioxidant enzymes (PRDX1, SOD1, GSS), which protect cells from the reactive oxygen species. An overall higher level of glutathione synthetase (GSS) mRNA was noted in group 1 bulls, suggesting higher ability to counteract free radicals. No differences were noted between basal oxygen consumption rate (OCR) (Seahorse XF Agilent) and ATP-linked respiration for group 1 and 2 bulls. In conclusion, mitochondrial content and activity may be used as reliable markers for bovine sperm quality evaluation.

18.
Cells ; 10(7)2021 07 16.
Article de Anglais | MEDLINE | ID: mdl-34359973

RÉSUMÉ

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease, causing motor neuron and skeletal muscle loss and death. One of the promising therapeutic approaches is stem cell graft application into the brain; however, an immune reaction against it creates serious limitations. This study aimed to research the efficiency of glial restricted progenitors (GRPs) grafted into murine CNS (central nervous system) in healthy models and the SOD1G93A ALS disease model. The cellular grafts were administered in semiallogenic and allogeneic settings. To investigate the models of immune reaction against grafted GRPs, we applied three immunosuppressive/immunomodulatory regimens: preimplantation factor (PiF); Tacrolimus; and CTLA-4, MR1 co-stimulatory blockade. We tracked the cells with bioluminescence imaging (BLI) in vivo to study their survival. The immune response character was evaluated with brain tissue assays and multiplex ELISA in serum and cerebrospinal fluid (CSF). The application of immunosuppressive drugs is disputable when considering cellular transplants into the immune-privileged site/brain. However, our data revealed that semiallogenic GRP graft might survive inside murine CNS without the necessity to apply any immunomodulation or immunosuppression, whereas, in the situation of allogeneic mouse setting, the combination of CTLA-4, MR1 blockade can be considered as the best immunosuppressive option.


Sujet(s)
Système nerveux central/effets des médicaments et des substances chimiques , Immunosuppression thérapeutique , Immunosuppresseurs/usage thérapeutique , Maladies neurodégénératives/traitement médicamenteux , Cellules souches , Animaux , Système nerveux central/immunologie , Modèles animaux de maladie humaine , Tolérance immunitaire/effets des médicaments et des substances chimiques , Immunosuppression thérapeutique/méthodes , Souris , Maladies neurodégénératives/immunologie , Transplantation de cellules souches/méthodes , Cellules souches/immunologie
19.
Cancers (Basel) ; 13(13)2021 Jun 23.
Article de Anglais | MEDLINE | ID: mdl-34201504

RÉSUMÉ

A hallmark of classical Hodgkin lymphoma (cHL) is the attenuation of B-cell transcription factors leading to global transcriptional reprogramming. The role of miRNAs (microRNAs) involved in this process is poorly studied. Therefore, we performed global miRNA expression profiling using RNA-seq on commonly used cHL cell lines, non-Hodgkin lymphoma cell lines and sorted normal CD77+ germinal centre B-cells as controls and characterized the cHL miRNome (microRNome). Among the 298 miRNAs expressed in cHL, 56 were significantly overexpressed and 23 downregulated (p < 0.05) compared to the controls. Moreover, we identified five miRNAs (hsa-miR-9-5p, hsa-miR-24-3p, hsa-miR-196a-5p, hsa-miR-21-5p, hsa-miR-155-5p) as especially important in the pathogenesis of this lymphoma. Target genes of the overexpressed miRNAs in cHL were significantly enriched (p < 0.05) in gene ontologies related to transcription factor activity. Therefore, we further focused on selected interactions with the SPI1 and ELF1 transcription factors attenuated in cHL and the NF-ĸB inhibitor TNFAIP3. We confirmed the interactions between hsa-miR-27a-5p:SPI1, hsa-miR-330-3p:ELF-1, hsa-miR-450b-5p:ELF-1 and hsa-miR-23a-3p:TNFAIP3, which suggest that overexpression of these miRNAs contributes to silencing of the respective genes. Moreover, by analyzing microdissected HRS cells, we demonstrated that these miRNAs are also overexpressed in primary tumor cells. Therefore, these miRNAs play a role in silencing the B-cell phenotype in cHL.

20.
Antioxidants (Basel) ; 10(6)2021 Jun 15.
Article de Anglais | MEDLINE | ID: mdl-34203726

RÉSUMÉ

The aim of the study was to modify human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) and demonstrate the optimal cell preparation protocol for application in post-infarction hearts. We used conditioned SkMDS/PC culture medium with α-phenyl-N-tert-butyl nitrone (PBN). SkMDS/PCs were cultured under hypoxic conditions and the results were compared to the standard ones. We observed a significant increase of CD-56 positive phenotypic marker the ability to form functional myotubes, increase in the proportion of young cells in cell primary suspensions, and a decrease in the percentage of apoptotic cells among PBN-conditioned cells in normoxia an hypoxia. We also observed significantly higher levels of SOD3 expression; maintained expression of SOD1, SOD2, and CAT; a higher level of BCL2 gene expression; and a rather significant decrease in Hsp70 gene expression in PBN-conditioned SkMDS/PCs compared to the WT population under hypoxic conditions. In addition, significant increase of myogenic genes expression was observed after PBN addition to culture medium, compared to WT population under hypoxia. Interestingly, PBN addition significantly increased the lengths of telomeres under hypoxia. Based on the data obtained, we can postulate that PBN conditioning of human SkMDS/PCs could be a promising step in improving myogenic cell preparation protocol for pro-regenerative treatment of post-infarction hearts.

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