ABSTRACT
Obstructive sleep apnea (OSA) is quite prevalent during pregnancy and is associated with adverse perinatal outcomes, but its potential influence on fetal development remains unclear. This study investigated maternal OSA impact on the fetus by analyzing gene expression profiles in whole cord blood (WCB). Ten women in the third trimester of pregnancy were included, five OSA and five non-OSA cases. WCB RNA expression was analyzed by microarray technology to identify differentially expressed genes (DEGs) under OSA conditions. After data normalization, 3238 genes showed significant differential expression under OSA conditions, with 2690 upregulated genes and 548 downregulated genes. Functional enrichment was conducted using gene set enrichment analysis (GSEA) applied to Gene Ontology annotations. Key biological processes involved in OSA were identified, including response to oxidative stress and hypoxia, apoptosis, insulin response and secretion, and placental development. Moreover, DEGs were confirmed through qPCR analyses in additional WCB samples (7 with OSA and 13 without OSA). This highlighted differential expression of several genes in OSA (EGR1, PFN1 and PRKAR1A), with distinct gene expression profiles observed during rapid eye movement (REM)-OSA in pregnancy (PFN1, UBA52, EGR1, STX4, MYC, JUNB, and MAPKAP). These findings suggest that OSA, particularly during REM sleep, may negatively impact various biological processes during fetal development.
Subject(s)
Fetal Blood , Fetal Development , Sleep Apnea, Obstructive , Humans , Female , Pregnancy , Fetal Blood/metabolism , Adult , Sleep Apnea, Obstructive/genetics , Fetal Development/genetics , Transcriptome , Gene Expression Profiling , Pregnancy Complications/geneticsABSTRACT
The usefulness of nanotechnology to increase the bioavailability of drugs and decrease their toxicity may be a tool to deal with multiresistant P. aeruginosa (Mr-Pa) respiratory infections. We describe the preparation and the in vivo efficacy and safety of sodium colistimethate-loaded nanostructured lipid carriers (SCM-NLC) by the pulmonary and intramuscular routes. Nanoparticles showed 1-2 mg/L minimum inhibitory concentration against eight extensively drug-resistant P. aeruginosa strains. In vivo, SCM-NLC displayed significantly lower CFU/g lung than the saline and similar to that of the free SCM, even the dose in SCM-NLC group was lower than free SCM. There was no tissue damage related to the treatments. Biodistribution assessments showed a mild systemic absorption after nebulization and a notorious absorption after IM route. Altogether, it could be concluded that SCM-NLC were effective against P. aeruginosa in vivo, not toxic and distribute efficiently to the lung and liver after pulmonary or intramuscular administrations.
Subject(s)
Colistin/analogs & derivatives , Drug Carriers/chemistry , Lipids/chemistry , Lung/microbiology , Nanostructures/chemistry , Pseudomonas aeruginosa/drug effects , Animals , Colistin/administration & dosage , Colistin/adverse effects , Colistin/pharmacology , Female , Inflammation/pathology , Injections, Intramuscular , Lung/pathology , Mice, Inbred BALB C , Microbial Sensitivity Tests , Nanostructures/toxicity , Nanostructures/ultrastructure , Tissue Distribution/drug effects , Toxicity Tests , Treatment OutcomeABSTRACT
Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19-amino-acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD-repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1-binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection.
Subject(s)
Autophagy , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Proteolysis , Amino Acid Motifs , Autophagy-Related Proteins , Carrier Proteins/genetics , HeLa Cells , Humans , Membrane Proteins/genetics , Microtubule-Associated Proteins/genetics , Staphylococcal Infections/genetics , Staphylococcal Infections/metabolism , Staphylococcus aureus/metabolismABSTRACT
Through GWAS studies we identified PATJ associated with functional outcome after ischemic stroke (IS). The aim of this study was to determine PATJ role in brain endothelial cells (ECs) in the context of stroke outcome. PATJ expression analyses in patient's blood revealed that: (i) the risk allele of rs76221407 induces higher expression of PATJ, (ii) PATJ is downregulated 24 h after IS, and (iii) its expression is significantly lower in those patients with functional independence, measured at 3 months with the modified Rankin scale ((mRS) ≤2), compared to those patients with marked disability (mRS = 4-5). In mice brains, PATJ was also downregulated in the injured hemisphere at 48 h after ischemia. Oxygen-glucose deprivation and hypoxia-dependent of Hypoxia Inducible Factor-1α also caused PATJ depletion in ECs. To study the effects of PATJ downregulation, we generated PATJ-knockdown human microvascular ECs. Their transcriptomic profile evidenced a complex cell reprogramming involving Notch, TGF-ß, PI3K/Akt, and Hippo signaling that translates in morphological and functional changes compatible with endothelial to mesenchymal transition (EndMT). PATJ depletion caused loss of cell-cell adhesion, upregulation of metalloproteases, actin cytoskeleton remodeling, cytoplasmic accumulation of the signal transducer C-terminal transmembrane Mucin 1 (MUC1-C) and downregulation of Notch and Hippo signaling. The EndMT phenotype of PATJ-depleted cells was associated with the nuclear recruitment of MUC1-C, YAP/TAZ, ß-catenin, and ZEB1. Our results suggest that PATJ downregulation 24 h after IS promotes EndMT, an initial step prior to secondary activation of a pro-angiogenic program. This effect is associated with functional independence suggesting that activation of EndMT shortly after stroke onset is beneficial for stroke recovery.
ABSTRACT
Bak and Bax are critical apoptotic mediators that naturally localize to both mitochondria and the endoplasmic reticulum (ER). Although it is generally accepted that mitochondrial expression of Bak or Bax suffices for apoptosis initiated by BH3-only homologues, it is currently unclear whether their reticular counterparts may have a similar potential. In this study, we show that cells exclusively expressing Bak in endoplasmic membranes undergo cytochrome c mobilization and mitochondrial apoptosis in response to BimEL and Puma, even when these BH3-only molecules are also targeted to the ER. Surprisingly, calcium was necessary but not sufficient to drive the pathway, despite normal ER calcium levels. We provide evidence that calcium functions coordinately with the ER-stress surveillance machinery IRE1alpha/TRAF2 to transmit apoptotic signals from the reticulum to mitochondria. These results indicate that BH3-only mediators can rely on reticular Bak to activate an ER-to-mitochondria signalling route able to induce cytochrome c release and apoptosis independently of the canonical Bak,Bax-dependent mitochondrial gateway, thus revealing a new layer of complexity in apoptotic regulation.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , Endoplasmic Reticulum/metabolism , Fibroblasts/cytology , Membrane Proteins/metabolism , Mitochondria/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Proteins/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Animals , Apoptosis/drug effects , Bcl-2-Like Protein 11 , Calcium/pharmacology , Calcium Signaling/drug effects , Cell Line , Cytochromes b5/metabolism , Cytochromes c/metabolism , Endoplasmic Reticulum/drug effects , Enzyme Activation/drug effects , Fibroblasts/metabolism , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Mitochondria/drug effects , Mitochondria/enzymology , Protein Serine-Threonine Kinases/metabolism , Protein Transport/drug effects , Stress, Physiological/drug effects , TNF Receptor-Associated Factor 2/metabolismABSTRACT
Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease commonly induced by cigarette smoke. The expression of miRNAs can be altered in patients with COPD and could be used as a biomarker. We aimed to identify a panel of miRNAs in bronchoalveolar lavage (BAL) to differentiate COPD patients from smokers and non-smokers with normal lung function. Accordingly, forty-five subjects classified as COPD, smokers, and non-smokers (n = 15 per group) underwent clinical, functional characterization and bronchoscopy with BAL. The mean age of the studied population was 61.61 ± 12.95 years, BMI 25.72 ± 3.82 Kg/m2, FEV1/FVC 68.37 ± 12.00%, and FEV1 80.07 ± 23.63% predicted. According to microarray analysis, three miRNAs of the most upregulated were chosen: miR-320c, miR-200c-3p, and miR-449c-5p. These miRNAs were validated by qPCR and were shown to be differently expressed in COPD patients. ROC analysis showed that these three miRNAs together had an area under the curve of 0.89 in differentiating COPD from controls. Moreover, in silico analysis of candidate miRNAs by DIANA-miRPath showed potential involvement in the EGFR and Hippo pathways. These results suggest a specific 3-miRNA signature that could be potentially used as a biomarker to distinguish COPD patients from smokers and non-smoker subjects.
ABSTRACT
Psoriasis is a chronic inflammatory skin disease that speeds up the life cycle of skin cells, forming scales and red patches that are itchy and sometimes painful. It is a complex disease of autoimmune origin and genetic predisposition with more than 10 different loci associated. Here we described the production of an iPSC line generated by Sendai Virus (Klf4, Oct3/4, Sox2 and c-Myc) reprogramming of Peripheral Blood Mononuclear Cells (PBMCs) from a Psoriasis patient. The iPSC line generated has normal 46XY karyotype, is free of SeV genome and transgenes insertions, express high levels of pluripotency markers and can differentiate into all three germ layers.
ABSTRACT
A 32 base pair deletion in the C-C chemokine receptor type gene (CCR5-Δ32), the main Human Immunodeficiency Virus (HIV) co-receptor results in a non-functional protein. Individuals homozygous for the CCR5-Δ32 mutation are resistant to HIV infection. Here we report the generation, from pro-erythroblast enriched Peripheral Blood Mononuclear Cells (PBMCs) from a naturally occurring CCR5-Δ32/Δ32 individual, of the fully characterized iPSC line IMEDEAi008-A. The new line has normal karyotype, carry the Δ32 mutation in homozygosity, is free of plasmid integrations, express high levels of pluripotency markers and can differentiate into all three germ layers.
ABSTRACT
Genome editing has become one of the most powerful tools in present-day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator-like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN-F) or membrane receptors (TALEN-M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence-assisted cell sorting (FACS) or magnetic-activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of new TALENs Basic Protocol 2: Genome editing using TALEN-F Alternate Protocol 1: Generation of TALEN-M Support Protocol 1: mRNA in vitro transcription (IVT) of TALEN-T2A-reporter expression vector Alternate Protocol 2: Editing of primary T cells using TALEN-M Basic Protocol 3: Verifying gene editing Support Protocol 2: Rapid expansion protocol for edited T-cells.
Subject(s)
Gene Editing/methods , Transcription Activator-Like Effector Nucleases/metabolism , Cell Proliferation , Cloning, Molecular , Genetic Vectors/metabolism , Humans , Plasmids/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , T-Lymphocytes/metabolism , Transcription, GeneticABSTRACT
Cystic fibrosis (CF) is the main genetic cause of death among the Caucasian population. The disease is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth (meconium ileus), later extending to other organs, generally affecting the respiratory tract. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (cAMP)-dependent, phosphorylation-regulated chloride channel required for transport of chloride and other ions through cell membranes. There are more than 2,000 mutations described in the CFTR gene, but one of them, phenylalanine residue at amino acid position 508 (p.F508del), a recessive allele, is responsible for the vast majority of CF cases worldwide. Here, we present the results of the application of genome-editing techniques to the restoration of CFTR activity in p.F508del patient-derived induced pluripotent stem cells (iPSCs). Gene-edited iPSCs were subsequently used to produce intestinal organoids on which the physiological activity of the restored gene was tested in forskolin-induced swelling tests. The seamless restoration of the p.F508del mutation resulted in normal expression of the mature CFTR glycoprotein, full recovery of CFTR activity, and a normal response of the repaired organoids to treatment with two approved CF therapies: VX-770 and VX-809.
ABSTRACT
The mutation E280A in PSEN1 (presenilin-1) is the most common cause of early-onset familial Alzheimer's Disease (fAD). It presents autosomal dominant inheritance and frequently leads to the manifestation of the disease in relatively young individuals. Here we report the generation of one PSEN1 E280A iPSC line derived from an early-onset patient. OriP/EBNA1-based episomal plasmids containing OCT3/4, SOX2, KLF4, L-MYC, LIN28, BCL-xL and shp53 were used to reprogram oral mucosa fibroblasts. The iPSC line generated has normal karyotype, carry the E280A mutation, is free of plasmid integration, express high levels of pluripotency markers and can differentiate into all three germ layers.
Subject(s)
Alzheimer Disease/genetics , Cell Differentiation , Cellular Reprogramming , Fibroblasts/pathology , Induced Pluripotent Stem Cells/pathology , Mutation , Presenilin-1/genetics , Age of Onset , Alzheimer Disease/pathology , Cells, Cultured , Female , Fibroblasts/metabolism , Heterozygote , Humans , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Middle Aged , PhenotypeABSTRACT
Mucoplysaccharydosis IIIA (MPSIIIA) is the most severe form of Sanfilippo syndrome. Skin fibroblasts from a MPSIIIA compound heterozygous (E447K/R245H) patient were nucleofected with four OriP/EBNA1-based episomal plasmids containing: OCT3/4, SOX2, KLF4, L-Myc, LIN28, BCL-xL and shp53. The two iPSCs lines generated carry both sulfamidase enzyme (SGSH) mutations, are free of plasmid integration, have normal karyotype, express pluripotency-associated markers and are able to differentiate into the three germ layers.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Mucopolysaccharidosis III/metabolism , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Cellular Reprogramming/genetics , Cellular Reprogramming/physiology , Humans , Karyotype , Kruppel-Like Factor 4ABSTRACT
Mucopolysaccharydosis IIIB is the second most frequent form of Sanfilippo syndrome, a degenerative, pediatric lysosomal storage disease (LSD) characterized by severe neurological disorders and death. We have generated two iPSCs lines derived from dermal fibroblast from a MPSIIIB homozygous (P358L) donor. Cells were reprogrammed with OriP/EBNA1-based episomal plasmids containing: OCT3/4, SOX2, KLF4, L-MYC, LIN28, BCL-xL and shp53. Both cell lines are homozygous for the P358L mutation of the α-N-acetylglucosaminidase (NAGLU) gene, have normal karyotype, are free of plasmid integration, express high levels of pluripotency-associated markers and can differentiate into the three germ layers. RESOURCE TABLE: RESOURCE UTILITY: Although the generation of iPSCs has been reported for some lysosomal storage diseases (LSD) in general, and from other mutations of the NAGLU gene in particular (Lemonnier et al., 2011), this is the first time that NAGLU Pro358Leu MPSIIIB-iPSCs lines have been generated and fully characterized demonstrating their quality as iPS cells. RESOURCE DETAILS: Mucopolysaccharidosis IIIB (MPSIII, Sanfilippo syndrome type B) is a pediatric neurodegenerative disorder caused by a deficiency in NAGLU, an enzyme required for lysosomal degradation of heparin sulphate (HS). When the enzyme is absent or malfunctioning, HS accumulates in the cells of several tissues, with devastating effects in the brain and central nervous system. MPSIIIB is inherited in an autosomal recessive manner and presents an incidence between 0.03 and 0.78 cases per 1â¯×â¯105 live births (Fedele, 2015) depending on the country. Currently there is no therapy available. The NAGLU gene was identified in 1996, is located on chromosome 17q21.1 and contains 6 exons. More than 150 NAGLU mutations have been reported, being most of them missense (Valstar et al., 2010). All of them lead to MPSIIIB but, unlike MPSIIIA, none is predominant. The two iPSCs lines described in this report, IMEDEAi005-A and IMEDEAi005-B, (See Table 1) were generated from skin fibroblast obtained from a clinically affected homozygous donor. The mutant allele consists on a Câ¯>â¯T transversion at nucleotide 1073 (1073â¯>â¯T) resulting in a substitution of leucine for proline at codon 358 (Pro358Leu). Skin fibroblasts were reprogrammed to iPSCs by nucleofection with four OriP/EBNA1 (Epstein-Barr nuclear antigen-1) based episomal plasmids encoding 5 reprogramming genes (OCT3/4, SOX2, KLF4, L-Myc, LIN28 and BCL-xL), in addition to a short hairpin RNA against p53. The iPSCs lines showed morphology (Fig. 1A) and growth behaviour typical of human Embryonic Stem Cells (hESC), as well as normal female karyotype (46, XX) (Fig. 1B). After 12 passages, PCR analysis confirmed that both iPSCs lines had completely lost the episomal vectors (Fig. 1C). The identity of iPS cells and their parental fibroblasts was confirmed by STR analysis (Table 2, data not shown) in addition to the identification of the disease-associated mutation in the NAGLU gene by DNA sequencing (Fig. 1D). Regarding the iPSC phenotype, both lines expressed the pluripotency-associated markers: OCT3/4, NANOG, SOX2 and TRA-1-60 (Fig. 1E), and TRA-1-81 quantified by flow cytometry (Fig. 1G), resulting in 88.17% and 83.4% of TRA-1-81 positive cells in IMEDEAi005-A and IMEDEAi005-B respectively. Finally, the differentiation capacity of iPSCs lines was analyzed by embryoid body (EBs) formation. Expression of markers specific of the three germ layers was observed after at least 10â¯days of spontaneous differentiation (Fig. 1F). Mycoplasma analysis was negative for both iPSCs lines (Supplementary Fig. S1). Skin fibroblasts were reprogrammed to iPSCs by nucleofection with four OriP/EBNA1 (Epstein-Barr nuclear antigen-1) based episomal plasmids encoding 5 reprogramming genes (OCT3/4, SOX2, KLF4, L-Myc, LIN28 and BCL-xL), in addition to a short hairpin RNA against p53. The iPSCs lines showed morphology (Fig. 1A) and growth behaviour typical of human Embryonic Stem Cells (hESC), as well as normal female karyotype (46, XX) (Fig. 1B). After 12 passages, PCR analysis confirmed that both iPSCs lines had completely lost the episomal vectors (Fig. 1C). The identity of iPS cells and their parental fibroblasts was confirmed by STR analysis (Table 2, data not shown) in addition to the identification of the disease-associated mutation in the NAGLU gene by DNA sequencing (Fig. 1D). Regarding the iPSC phenotype, both lines expressed the pluripotency-associated markers: OCT3/4, NANOG, SOX2 and TRA-1-60 (Fig. 1E), and TRA-1-81 quantified by flow cytometry (Fig. 1G), resulting in 88.17% and 83.4% of TRA-1-81 positive cells in IMEDEAi005-A and IMEDEAi005-B respectively. Finally, the differentiation capacity of iPSCs lines was analyzed by embryoid body (EBs) formation. Expression of markers specific of the three germ layers was observed after at least 10â¯days of spontaneous differentiation (Fig. 1F). Mycoplasma analysis was negative for both iPSCs lines (Supplementary Fig. S1). In conclusion, we have successfully generated and characterized, for the first time to our knowledge, two human iPSCs lines from a MPSIIIB donor homozygous for the P358L NAGLU mutation. The new lines will complement the existing murine MPS IIIB model, with their potential to be used in a development of a purely human in vitro model of the disease.
Subject(s)
Mucopolysaccharidosis III/genetics , Child, Preschool , Female , Humans , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4ABSTRACT
Cystic Fibrosis (CF) is a monogenic, lethal disease caused by mutations in the cystic fibrosis transmembrane conductance (CFTR) gene. Here we report the production of CF-iPS cell lines from two different p.F508del homozygous female patients (Table 1). Two different primary cell types, skin fibroblasts and keratinocytes, were transfected with retroviral cocktails containing four: c-MYC, KLF4, OCT4 and SOX2 (MKOS) or three: KLF4, OCT4 and SOX2 (KOS) reprogramming factors. Two fibroblast-derived MKOS lines are described in the main text. The lines carry the p.F508del mutation, have a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.
Subject(s)
Cystic Fibrosis/genetics , Induced Pluripotent Stem Cells/metabolism , Animals , Cell Line , Female , Humans , Kruppel-Like Factor 4 , Male , MutationABSTRACT
To characterize the regulation of lymphoid Aiolos transcription factor, we have cloned its promoter. Full promoter and nested deletions were expressed in lymphoid and non-lymphoid cell lines. The minimal promoter activity could be considered as a 172bp upstream from the ATG for Jurkat and HEK293 cells and as a 370bp fragment for U937 cells. Moreover, we have mapped the transcription initiation site. Retardation gels showed binding activity for Ikaros, NFkappaB and AP4 transcription factors and mutations in their binding sites abolish Aiolos promoter activity. Chromatin immunoprecipitation assay revealed that Ikaros, NFkappaB and AP4 are bound to Aiolos promoter. The important function of Ikaros and NFkappaB is underlined by their over expression, which results in the trans-activation of the promoter and drives Aiolos expression in cell lines and in freshly isolated B and T cells, while over expression of a dominant negative Ikaros isoform is able to block Aiolos expression.
Subject(s)
Gene Expression Regulation , Ikaros Transcription Factor/metabolism , Transcription Factors/genetics , Base Sequence , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA Mutational Analysis , Electrophoretic Mobility Shift Assay , Humans , Lymphocytes/metabolism , Molecular Sequence Data , NF-kappa B/metabolism , Promoter Regions, Genetic , Sequence Deletion , Transcription Initiation SiteABSTRACT
Apoptosis of mature T lymphocytes is an essential process for maintaining immune system homeostasis. However, the details of the molecular signaling pathways leading to T cell apoptosis are poorly understood. We used cDNA microarrays containing 15,630 murine genes to study the gene expression profile in T lymphocytes at different time points of IL-2 withdrawal. Comparison of the gene expression profiles revealed that 2% of the genes were affected by cytokine starvation. Interestingly, the apoptotic program rather seems to activate gene expression in the early phase of cell death. On the contrary, transcription was strongly repressed in later stages of apoptosis. Self-organizing map clustering of the 270 differentially expressed transcripts revealed specific temporal expression patterns supporting the idea that IL-2 deprivation triggers a tightly regulated transcriptional program to induce cell death. To validate microarray results, changes in gene expression following IL-2 deprivation were confirmed for selected genes by Northern blot. In addition, the signaling pathways created can explain the molecular events leading to T cell apoptosis, even if the T cell line used in this study might not reflect individual T cell subpopulations expressing different level of IL-2 receptor or IL-2 dependence. Taken together, these results provide novel insights into the temporal regulation of gene expression during T lymphocyte death.
Subject(s)
Apoptosis/genetics , Gene Regulatory Networks , Interleukin-2/metabolism , Repressor Proteins/metabolism , Signal Transduction/genetics , T-Lymphocytes/metabolism , Transcription, Genetic , Animals , Blotting, Northern , Cell Line , Cluster Analysis , Gene Expression Profiling/methods , Interleukin-2/deficiency , Mice , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Reproducibility of Results , T-Lymphocytes/pathology , Time FactorsABSTRACT
Alterations in cell proliferation and cell death are essential determinants in the pathogenesis and progression of several diseases such as cancer, neurodegenerative disorders or autoimmune diseases among others. Complex networks of regulatory factors determine whether cells proliferate or die. Recent progress in understanding the molecular changes offer the possibility of specifically targeting molecules and pathways to achieve more effective and rational therapies. Drugs that target molecules involved in apoptosis are used as treatment against several diseases. Candidates such as TNF death receptor family, caspase inhibitors, antagonists of the p53-MDM2 interaction, NF-kappaB and PI3K pathways and Bcl-2 family members have been targeted as cancer cell killing agents. Moreover, apoptosis of tumor cells can also be achieved by targeting the inhibitor of apoptosis proteins, IAPs, in addition to the classical antiproliferative approach. Disruption of STAT activation and interferon beta therapy have been used as a treatment to prevent the progression of some autoimmune diseases. In models of Parkinson's, Alzheimer's and amyotrophic lateral sclerosis, blocking of Par-4 expression or function, as well as caspase activation, prevents neuronal cell death. Finally, it has been shown that gene therapy may be an encouraging approach for treatment of neurodegenerative disorders.
Subject(s)
Apoptosis/drug effects , Drug Evaluation, Preclinical , Autoimmunity , Humans , Neoplasms/drug therapy , Neoplasms/pathology , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/pathologyABSTRACT
Among the pathogens that affect cystic fibrosis (CF) patients, Pseudomonas aeruginosa is the most prevalent. As a way to fight against this infection, nanotechnology has emerged over the last decades as a promising alternative to overcome resistance to antibiotics in infectious diseases. The goal of this work was to elaborate and characterize lipid nanoparticles for pulmonary delivery of tobramycin. Tobramycin-loaded nanostructured lipid carriers (Tb-NLCs) were prepared by hot melt homogenization technique. In addition, nanoparticles labeled with infrared dye (IR-NLCs) were used to investigate their in vivo performance after pulmonary administration. Tb-NLCs displayed a mean diameter size around 250 nm, high drug encapsulation (93%) and sustained release profile. Tb-NLCs showed to be active against clinically isolated P. aeruginosa. Moreover, Tb-NLCs did not decrease cell viability and were able to overcome an artificial mucus barrier in the presence of mucolytics agents. During the in vivo assay, IR-NLCs were administered to several mice by the intratracheal route using a Penn Century device. Next, the biodistribution of the nanoparticles was analyzed at different time points showing a wide nanosystem distribution in the lungs. Altogether, tobramycin-loaded NLCs seem to us an encouraging alternative to the currently available CF therapies.
Subject(s)
Cystic Fibrosis/drug therapy , Drug Delivery Systems/methods , Nanostructures/administration & dosage , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Tobramycin/administration & dosage , Administration, Inhalation , Animals , Cystic Fibrosis/metabolism , Drug Carriers/administration & dosage , Drug Carriers/metabolism , Female , Humans , Lipids , Mice , Mice, Inbred BALB C , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/physiology , Tobramycin/metabolismABSTRACT
Growth factor deprivation is a physiological mechanism to induce apoptosis. We used an IL-2-dependent murine T cell line to identify proteins that trigger apoptosis. Here we report the identification, the cloning and characterization of ITM2B(s), a protein induced upon IL-2-deprivation. ITM2B(s), which shares the BH3 domain of Bcl-2 family members, is a cytoplasmic and mitochondrial protein. Expression of ITM2B(s) induces apoptosis in IL-2-stimulated cells, but not in IL-4-stimulated cells, while overexpression of the long form of the protein is not able to induce apoptosis. In IL-2-stimulated cells, ITM2B(s) interacts with the antiapoptotic protein Bcl-2, and does not interact with the proapoptotic Bad. Mutation of the critical L and D residues within the BH3 domain abolished the ability of ITM2B(s) to promote apoptosis.
Subject(s)
Apoptosis/physiology , Interleukin-2/pharmacology , Membrane Proteins/physiology , Proto-Oncogene Proteins c-bcl-2/physiology , T-Lymphocytes/metabolism , Adaptor Proteins, Signal Transducing , Alternative Splicing , Amino Acid Sequence , Amino Acid Substitution , Animals , Carrier Proteins/metabolism , Cell Line/drug effects , Cell Line/metabolism , Cloning, Molecular , Gene Expression Profiling , Gene Expression Regulation/drug effects , Macromolecular Substances , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Molecular Sequence Data , Protein Structure, Tertiary , Recombinant Fusion Proteins/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Subtraction Technique , T-Lymphocytes/drug effects , Transfection , bcl-Associated Death ProteinABSTRACT
Many molecules are inducibly localized in lipid rafts, and their alteration inhibits early activation events, supporting a critical role for these domains in signaling. Using confocal microscopy and cellular fractionation, we have shown that the pool of Bad, attached to lipid rafts in proliferating cells, is released when cells undergo apoptosis. Kinetic studies indicate that rafts alteration is a consequence of an intracellular signal triggered by interleukin-4 deprivation. Growth factor deprivation in turn induces PP1alpha phosphatase activation, responsible for cytoplasmic Bad dephosphorylation as well as caspase-9 and caspase-3 activation. Caspases translocate to rafts and induce their modification followed by translocation of Bad from rafts to mitochondria, which correlates with apoptosis. Taken together, our results suggest that alteration of lipid rafts is an early event in the apoptotic cascade indirectly induced by interleukin-4 deprivation via PP1alpha activation, dephosphorylation of cytoplasmic Bad, and caspase activation.