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1.
Clin Transl Sci ; 2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34719115

RESUMO

On October 2020, the US Food and Drug Administration (FDA) approved remdesivir as the first drug for the treatment of coronavirus disease 2019 (COVID-19), increasing remdesivir prescriptions worldwide. However, potential cardiovascular (CV) toxicities associated with remdesivir remain unknown. We aimed to characterize the CV adverse drug reactions (ADRs) associated with remdesivir using VigiBase, an individual case safety report database of the World Health Organization (WHO). Disproportionality analyses of CV-ADRs associated with remdesivir were performed using reported odds ratios and information components. We conducted in vitro experiments using cardiomyocytes derived from human pluripotent stem cell cardiomyocytes (hPSC-CMs) to confirm cardiotoxicity of remdesivir. To distinguish drug-induced CV-ADRs from COVID-19 effects, we restricted analyses to patients with COVID-19 and found that, after adjusting for multiple confounders, cardiac arrest (adjusted odds ratio [aOR]: 1.88, 95% confidence interval [CI]: 1.08-3.29), bradycardia (aOR: 2.09, 95% CI: 1.24-3.53), and hypotension (aOR: 1.67, 95% CI: 1.03-2.73) were associated with remdesivir. In vitro data demonstrated that remdesivir reduced the cell viability of hPSC-CMs in time- and dose-dependent manners. Physicians should be aware of potential CV consequences following remdesivir use and implement adequate CV monitoring to maintain a tolerable safety margin.

2.
Materials (Basel) ; 14(18)2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34576409

RESUMO

The skin protects the body from external barriers. Certain limitations exist in the development of technologies to rapidly prepare skin substitutes that are therapeutically effective in surgeries involving extensive burns and skin transplantation. Herein, we fabricated a structure similar to the skin layer by using skin-derived decellularized extracellular matrix (dECM) with bioink, keratinocytes, and fibroblasts using 3D-printing technology. The therapeutic effects of the produced skin were analyzed using a chimney model that mimicked the human wound-healing process. The 3D-printed skin substitutes exhibited rapid re-epithelialization and superior tissue regeneration effects compared to the control group. These results are expected to aid the development of technologies that can provide customized skin-replacement tissues produced easily and quickly via 3D-printing technology to patients.

3.
Parasit Vectors ; 14(1): 182, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33789729

RESUMO

BACKGROUND: Polo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation. METHODS: To assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites RESULTS: After incubating trophozoites with 5 µM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation. CONCLUSIONS: These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese , Flagelos/fisiologia , Giardia lamblia/enzimologia , Giardia lamblia/fisiologia , /metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Protozoários/genética , Giardia lamblia/genética , Fosforilação , Proteínas de Protozoários/metabolismo , Trofozoítos/crescimento & desenvolvimento
4.
Biomaterials ; 266: 120472, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33120201

RESUMO

Human embryonic stem cells-derived endothelial progenitor cells (hEPCs) were utilized as cell therapeutics for the treatment of ischemic diseases. However, in vivo tracking of hEPCs for predicting their therapeutic efficacy is very difficult. Herein, we developed bioorthogonal labeling strategy of hEPCs that could non-invasively track them after transplantation in hind limb ischemia models. First, hEPCs were treated with tetraacylated N-azidomannosamine (Ac4ManNAz) for generating unnatural azide groups on the hEPCs surface. Second, near-infrared fluorescence (NIRF) dye, Cy5, conjugated dibenzocylooctyne (DBCO-Cy5) was chemically conjugated to the azide groups on the hEPC surface via copper-free click chemistry, resulting Cy5-hEPCs. The bioorthogonally labeled Cy5-hEPCs showed strong NIRF signal without cytotoxicity and functional perturbation in tubular formation, oxygen consumption and paracrine effect of hEPCs in vitro. In hind limb ischemia models, the distribution and migration of transplanted Cy5-hEPCs were successfully monitored via fluorescence molecular tomography (FMT) for 28 days. Notably, blood reperfusion and therapeutic neovascularization effects were significantly correlated with the initial transplantation forms of Cy5-hEPCs such as 'condensed round shape' and 'spread shape' in the ischemic lesion. The condensed transplanted Cy5-hEPCs substantially increased the therapeutic efficacy of hind limb ischemia, compared to that of spread Cy5-hEPCs. Therefore, our new stem cell labeling strategy can be used to predict therapeutic efficacy in hind limb ischemia and it can be applied a potential application in developing cell therapeutics for regenerative medicine.


Assuntos
Células Progenitoras Endoteliais , Animais , Química Click , Modelos Animais de Doenças , Membro Posterior , Humanos , Isquemia/diagnóstico por imagem , Isquemia/terapia , Neovascularização Fisiológica , Células-Tronco , Tomografia
5.
Biomedicines ; 8(11)2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33121085

RESUMO

Despite recent advances in clinical stem cell therapy applications based on human pluripotent stem cells (hPSCs), potential teratoma formation due to the presence of residual undifferentiated hPSCs remains a serious risk factor that challenges widespread clinical application. To overcome this risk, a variety of approaches have been developed to eliminate the remaining undifferentiated hPSCs via selective cell death induction. Our study seeks to identify natural flavonoids that are more potent than quercetin (QC), to selectively induce hPSC death. Upon screening in-house flavonoids, luteolin (LUT) is found to be more potent than QC to eliminate hPSCs in a p53-dependent manner, but not hPSC-derived smooth muscle cells or perivascular progenitor cells. Particularly, treating human embryonic stem cell (hESC)-derived cardiomyocytes with LUT efficiently eliminates the residual hESCs and only results in marginal effects on cardiomyocyte (CM) functions, as determined by calcium influx. Considering the technical limitations of isolating CMs due to a lack of exclusive surface markers at the end of differentiation, LUT treatment is a promising approach to minimize teratoma formation risk.

6.
Antiviral Res ; 184: 104955, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33091434

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is considered as the most significant global public health crisis of the century. Several drug candidates have been suggested as potential therapeutic options for COVID-19, including remdesivir, currently the only authorized drug for use under an Emergency Use Authorization. However, there is only limited information regarding the safety profiles of the proposed drugs, in particular drug-induced cardiotoxicity. Here, we evaluated the antiviral activity and cardiotoxicity of remdesivir using cardiomyocytes-derived from human pluripotent stem cells (hPSC-CMs) as an alternative source of human primary cardiomyocytes (CMs). In this study, remdesivir exhibited up to 60-fold higher antiviral activity in hPSC-CMs compared to Vero E6 cells; however, it also induced moderate cardiotoxicity in these cells. To gain further insight into the drug-induced arrhythmogenic risk, we assessed QT interval prolongation and automaticity of remdesivir-treated hPSC-CMs using a multielectrode array (MEA). As a result, the data indicated a potential risk of QT prolongation when remdesivir is used at concentrations higher than the estimated peak plasma concentration. Therefore, we conclude that close monitoring of the electrocardiographic/QT interval should be advised in SARS-CoV-2-infected patients under remdesivir medication, in particular individuals with pre-existing heart conditions.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , COVID-19/virologia , Miócitos Cardíacos/virologia , Células-Tronco Pluripotentes/citologia , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Amidas/farmacologia , Animais , Antimaláricos/farmacologia , COVID-19/complicações , COVID-19/tratamento farmacológico , Chlorocebus aethiops , Cloroquina/farmacologia , Eletrocardiografia , Citometria de Fluxo , Cardiopatias/complicações , Humanos , Hidroxicloroquina/farmacologia , Microscopia de Fluorescência , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/virologia , Pirazinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Ensaio de Placa Viral
7.
PLoS One ; 15(5): e0232899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392240

RESUMO

Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments.


Assuntos
Técnicas de Cocultura/instrumentação , Células Epiteliais , Células-Tronco Mesenquimais , Nanoestruturas , Animais , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Propriedades de Superfície
8.
Sci Rep ; 10(1): 3939, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127560

RESUMO

Although human induced pluripotent stem cell (hiPSC) lines are karyotypically normal, they retain the potential for mutation in the genome. Accordingly, intensive and relevant quality controls for clinical-grade hiPSCs remain imperative. As a conceptual approach, we performed RNA-seq-based broad-range genetic quality tests on GMP-compliant human leucocyte antigen (HLA)-homozygous hiPSCs and their derivatives under postdistribution conditions to investigate whether sequencing data could provide a basis for future quality control. We found differences in the degree of single-nucleotide polymorphism (SNP) occurring in cells cultured at three collaborating institutes. However, the cells cultured at each centre showed similar trends, in which more SNPs occurred in late-passage hiPSCs than in early-passage hiPSCs after differentiation. In eSNP karyotyping analysis, none of the predicted copy number variations (CNVs) were identified, which confirmed the results of SNP chip-based CNV analysis. HLA genotyping analysis revealed that each cell line was homozygous for HLA-A, HLA-B, and DRB1 and heterozygous for HLA-DPB type. Gene expression profiling showed a similar differentiation ability of early- and late-passage hiPSCs into cardiomyocyte-like, hepatic-like, and neuronal cell types. However, time-course analysis identified five clusters showing different patterns of gene expression, which were mainly related to the immune response. In conclusion, RNA-seq analysis appears to offer an informative genetic quality testing approach for such cell types and allows the early screening of candidate hiPSC seed stocks for clinical use by facilitating safety and potential risk evaluation.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Variações do Número de Cópias de DNA/genética , Genótipo , Teste de Histocompatibilidade , Homozigoto , Humanos , Cariotipagem , RNA-Seq , Transcriptoma/genética
9.
Korean J Parasitol ; 58(6): 675-679, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33412772

RESUMO

MYB2 protein was identified as a transcription factor that showed encystation-induced expression in Giardia lamblia. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of G. lamblia MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of G. lamblia glutamate dehydrogenase were constructed and transfected into Giardia trophozoites. Immunofluorescence assays using anti-HA antibodies indicated that GlMYB2 amino acid sequence #507-#530 was required for the nuclear localization of GlMYB2, and this sequence was named as NLSGlMYB2. We further verified this finding by demonstrating the nuclear location of a protein obtained by the fusion of NLSGlMYB2 and G. lamblia glyceraldehyde 3-phosphate dehydrogenase, a non-nuclear protein. Our data on GlMYB2 will expand our understanding on NLSs functioning in G. lamblia.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Expressão Gênica , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/fisiologia , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Encistamento de Parasitas/genética , Transativadores/genética , Transativadores/metabolismo , Sequência de Aminoácidos , Giardia lamblia/enzimologia , Glutamato Desidrogenase , Gliceraldeído 3-Fosfato , Hemaglutininas , Transativadores/química
10.
Microvasc Res ; 126: 103912, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31433972

RESUMO

Critical limb ischemia is one of the most common types of peripheral arterial disease. Preclinical development of ischemia therapeutics relies on the availability of a relevant and reproducible in vivo disease model. Thus, establishing appropriate animal disease models is essential for the development of new therapeutic strategies. Currently, the most commonly employed model of hindlimb ischemia is the surgical induction method with ligation of the femoral artery and its branches after skin incision. However, the efficiency of the method is highly variable depending on the availability of skilled technicians. In addition, after surgical procedures, animals can quickly and spontaneously recover from damage, limiting observations of the therapeutic effect of potential agents. The aim of this study was to develop a hindlimb ischemia mouse model with similarities to human ischemic disease. To that end, a photochemical reaction was used to induce thrombosis in the hindlimb. After the photochemical reaction was induced by light irradiation, thrombotic plugs and adjacent red blood cell stasis were observed in hindlimb vessels in the light-irradiated zone. Additionally, the photochemically induced thrombosis maintained the ischemic condition and did not cause notable side effects in mice.


Assuntos
Eritrosina , Isquemia/fisiopatologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Trombose/fisiopatologia , Animais , Velocidade do Fluxo Sanguíneo , Modelos Animais de Doenças , Membro Posterior , Isquemia/induzido quimicamente , Luz , Masculino , Camundongos Endogâmicos ICR , Processos Fotoquímicos , Fluxo Sanguíneo Regional , Trombose/induzido quimicamente , Fatores de Tempo
11.
Mater Sci Eng C Mater Biol Appl ; 103: 109729, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31349510

RESUMO

Graphene and its derivatives have seen a rapid rise in interest as promising biomaterials especially in the field of tissue engineering, regenerative medicine, and cell biology of late. Despite its proven potential in numerous biological applications, information regarding the relationship between the different forms of graphene and cell lineages is still lacking partly due to its topical emergence in cellular studies. Herein, we explore the biocompatibility of four types of graphene substrates (chemical vapor deposition grown graphene, mechanically exfoliated graphene, chemically exfoliated graphene oxide, and reduced graphene oxide) with three types of somatic cells (keratinocytes, hepatocytes, endothelial cells) derived from the three germ layers in relation to cell adhesion, proliferation, morphology, and gene expression. The results revealed exceptional cell adhesion for all tested groups but enhanced proliferation and cytoskeletal interconnectivity in graphene oxide and reduced graphene oxide substrates. We were unable to detect any adverse effects in gene expression and survivability during a week of culture. We further show topographic changes to graphene substrates under fetal bovine serum adsorption to better illustrate the actual microenvironment of inhabitant cells. This study highlights the extraordinary synergy between graphene and somatic cells, suggesting the discretionary use of extracellular matrix components for in vitro cultivation.


Assuntos
Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Grafite , Hepatócitos , Células Endoteliais da Veia Umbilical Humana , Queratinócitos , Grafite/química , Grafite/farmacologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo
12.
Nat Commun ; 10(1): 3123, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311935

RESUMO

Since both myocardium and vasculature in the heart are excessively damaged following myocardial infarction (MI), therapeutic strategies for treating MI hearts should concurrently target both so as to achieve true cardiac repair. Here we demonstrate a concomitant method that exploits the advantages of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) and human mesenchymal stem cell-loaded patch (hMSC-PA) to amplify cardiac repair in a rat MI model. Epicardially implanted hMSC-PA provide a complimentary microenvironment which enhances vascular regeneration through prolonged secretion of paracrine factors, but more importantly it significantly improves the retention and engraftment of intramyocardially injected hiPSC-CMs which ultimately restore the cardiac function. Notably, the majority of injected hiPSC-CMs display adult CMs like morphology suggesting that the secretomic milieu of hMSC-PA constitutes pleiotropic effects in vivo. We provide compelling evidence that this dual approach can be a promising means to enhance cardiac repair on MI hearts.


Assuntos
Coração/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Regeneração , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Injeções Intralesionais , Masculino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Ratos , Ratos Endogâmicos F344 , Resultado do Tratamento
13.
Korean J Parasitol ; 57(3): 225-232, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31284344

RESUMO

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1ß, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.


Assuntos
Giardia lamblia/fisiologia , Giardíase/imunologia , Interleucina-17/imunologia , Linfócitos/imunologia , Membrana Mucosa/parasitologia , Animais , Células Cultivadas , Giardíase/genética , Giardíase/parasitologia , Humanos , Imunidade Inata , Interleucina-17/genética , Linfócitos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Membrana Mucosa/imunologia
14.
Korean J Parasitol ; 57(2): 185-189, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31104412

RESUMO

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.


Assuntos
Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Fase G2/genética , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/genética , Regulação para Cima , Antiprotozoários/metabolismo , Afidicolina/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Giardia lamblia/efeitos dos fármacos , Nocodazol/metabolismo , Análise de Sequência de RNA
15.
Korean J Parasitol ; 57(2): 201-206, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31104415

RESUMO

The roles of mast cells in allergic diseases and helminth infections are well known. However, the roles of mast cells in T. gondii infection is poorly understood. This study was focused on the production of pro-inflammatory cytokines (TNF-α, IL-4), chemokines (CXCL8, MCP-1) and nitric oxide (NO) by mast cells in response to soluble lysate of T. gondii tachyzoites. Production of CXCL8 (IL-8), MCP-1, TNF-α and IL-4 were measured by RT-PCR and ELISA. Western blot were used for detection of CXCR-1 and CXCR2. Our results showed that T. gondii lysates triggered mast cells to release CXCL8, MCP-1, TNF-α, IL-4 and to produce NO. This suggests that mast cells play an important role in inflammatory responses to T. gondii.


Assuntos
Misturas Complexas/imunologia , Citocinas/metabolismo , Mastócitos/metabolismo , Óxido Nítrico/metabolismo , Toxoplasma/imunologia , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Reação em Cadeia da Polimerase em Tempo Real
16.
Parasit Vectors ; 12(1): 227, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088539

RESUMO

BACKGROUND: Giardia lamblia, a protozoan pathogen causing diarrheal outbreaks, has characteristic cytoskeletal structures including eight flagella, a median body and a ventral disc. Gamma-giardin is a unique component protein of the cytoskeleton of this protozoan. RESULTS: Through comparative proteomic analysis between different stages of the cell cycle, G. lamblia γ-giardin (Glγ-giardin) was identified as an upregulated protein in the G2-phase. Increased Glγ-giardin expression in G2 was confirmed by western blot and real-time polymerase chain reaction analyses. Knockdown of this protein using a morpholino affected the formation of ventral discs, especially the microribbons of the discs, but exerted little effect on the binding ability of G. lamblia. The number of cells with four nuclei was increased in Glγ-giardin-knockdown cells. Expression of Glγ-giardin was decreased during encystation, in contrast with the G2-phase. CONCLUSIONS: Knockdown experiments demonstrated that Glγ-giardin is a component of the trilaminar structure of the ventral disc. Expression of Glγ-giardin is induced in the G2-phase prior to active cell division, whereas its expression decreases during encystation, a dormant stage of G. lamblia.


Assuntos
Proteínas do Citoesqueleto/genética , Fase G2/genética , Giardia lamblia/citologia , Giardia lamblia/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/química , Citoesqueleto/genética , Técnicas de Silenciamento de Genes , Encistamento de Parasitas , Proteômica , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Regulação para Cima
17.
Colloids Surf B Biointerfaces ; 180: 384-392, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31082776

RESUMO

Precise detection of undifferentiated human pluripotent stem cells (hPSCs) and their entire subsequent elimination are incredibly important in preventing teratoma formations after transplantation. Recently, electrochemical sensing platforms have demonstrated immense potential as a new tool to detect remaining hPSCs in label-free and non-destructive manner. Nevertheless, one of the critical huddles of this electrochemical sensing approach is its low sensitivity since even low concentrations of remaining hPSCs were reported to form teratoma once transplanted. To address this issue, in this study, we report an engineering-based approach to improve the sensitivity of electrochemical sensing platform for hPSC detection. By optimizing the density of gold nanostructure and the matrigel concentration to improve both electro-catalytic property and biocompatibility, the sensitivity of the developed platform toward hESCs detection could reach 12,500 cells/chip, which is close to the known critical concentration of hPSCs (˜10,000 cells) that induce teratoma formation in vivo. Remarkably, the electrochemical signals were not detectable from other types of stem cell-derived endothelial cells (CB-EPCs) even at high concentrations of CB-EPCs (40,000 cells/chip), proving the high selectivity of the developed platform toward hPSC detection. Hence, the developed platform could be highly useful to solve the safety issues that are related with clinical application of hPSC-derived cells.


Assuntos
Eletroquímica/métodos , Ouro/química , Células-Tronco Embrionárias Humanas/citologia , Nanoestruturas/química , Eletrodos , Células Endoteliais/citologia , Sangue Fetal/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Nanoestruturas/ultraestrutura , Compostos de Estanho/química
18.
Stem Cells ; 37(5): 623-630, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30721559

RESUMO

The derivation of human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT) has prompted a re-emerging interest in using such cells for therapeutic cloning. Despite recent advancements in derivation protocols, the functional potential of CHA-NT4 derived cells is yet to be elucidated. For this reason, this study sought to differentiate CHA-NT4 cells toward an endothelial lineage in order to evaluate in vitro and in vivo functionality. To initial differentiation, embryoid body formation of CHA-NT4 was mediated by concave microwell system which was optimized for hESC-endothelial cell (EC) differentiation. The isolated CD31+ cells exhibited hallmark endothelial characteristics in terms of morphology, tubule formation, and ac-LDL uptake. Furthermore, CHA-NT4-derived EC (human nuclear transfer [hNT]-ESC-EC) transplantation in hind limb ischemic mice rescued the hind limb and restored blood perfusion. These findings suggest that hNT-ESC-EC are functionally equivalent to hESC-ECs, warranting further study of CHA-NT4 derivatives in comparison to other well established pluripotent stem cell lines. This revival of human SCNT-ESC research may lead to interesting insights into cellular behavior in relation to donor profile, mitochondrial DNA, and oocyte quality. Stem Cells 2019;37:623-630.


Assuntos
Diferenciação Celular/genética , Células Endoteliais/transplante , Células-Tronco Embrionárias Humanas/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Animais , Membro Posterior/patologia , Membro Posterior/transplante , Humanos , Isquemia/terapia , Camundongos , Técnicas de Transferência Nuclear
19.
Microbiologyopen ; 8(6): e00748, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30318753

RESUMO

Giardia lamblia is a unicellular organism with two nuclei, a median body, eight flagella, and an adhesive disk. γ-Tubulin is a microtubule (MT)-nucleating protein that functions in the γ-tubulin small complex (γ-TuSC) in budding yeast. In this study, G. lamblia γ-tubulin (Glγ-tubulin) was found to bind to another MT-binding protein, namely G. lamblia end-binding protein 1 (GlEB1), via both in vivo and in vitro assays. Hemagglutinin (HA)-tagged Glγ-tubulin localized to the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. The knockdown of Glγ-tubulin expression using an anti-Glγ-tubulin morpholino resulted in a decreased growth rate and an increased failed cytokinesis cells of Giardia. The formation of median bodies was affected, and the central pair of MTs in flagella was frequently missing in the Giardia treated with an anti-Glγ-tubulin morpholino. G. lamblia γ-tubulin complex protein 2 (GlGCP2) and GlGCP3, which are putative components of γ-TuSC, were co-immunoprecipitated with HA-tagged Glγ-tubulin in Giardia extracts. The knockdown of GlGCP2 and GlGCP3 expression also resulted in decreased formation of both the median body and flagella MTs. Knockdown of Glγ-tubulin, GlGCP2, and GlGCP3 expression affected localization of GlEB1 in G. lamblia. In addition, decreased level of GlEB1 caused reduced formation of median body and the central pair of flagella MTs. These results indicated that Glγ-tubulin plays a role in MT nucleation for median body formation and flagella biogenesis as a component of Glγ-TuSC in Giardia and GlEB1 may be involved in this process.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Flagelos/metabolismo , Giardia lamblia/metabolismo , Proteínas de Protozoários/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Flagelos/genética , Giardia lamblia/genética , Ligação Proteica , Proteínas de Protozoários/genética , Tubulina (Proteína)/genética
20.
J Invest Dermatol ; 139(3): 692-701, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30393080

RESUMO

Much of our understanding of human biology and the function of mammalian cells in tissue regeneration have been derived from mechanistically and genetically manipulated rodent models. However, current models examining epidermal wound repair fail to address both the cross-species mechanistic and immunogenic differences simultaneously. Herein, we describe a multifaceted approach intended to better recapitulate human skin recovery in rodent models. First, immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice were intravenously inoculated with human hematopoietic stem cells to become, in essence, humanized, and capable of initiating an adaptive immune response. Next, a chimney-shaped mechanical device was implanted onto the excisional wound site to prevent healing by primary intention (contraction) and expedite cell transplantation. Subsequently, cell therapy was administered by transplanting cord blood-derived endothelial progenitor cells or human pluripotent stem cell-derived endothelial cells into the wound site to examine the regeneration process at a histological level. This study demonstrates human cutaneous repair in a murine model by addressing both the mechanistic and immunogenic differences in the epidermis. We further show human leukocyte recruitment in damaged tissue and improved healing by secondary intention in the transplanted groups, highlighting the need for useful preclinical animal models to better understand leukocyte function in human (tissue repair and) regeneration.


Assuntos
Imunidade Adaptativa/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Pele/lesões , Cicatrização/fisiologia , Ferimentos e Lesões/terapia , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Células Endoteliais/transplante , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Distribuição Aleatória , Regeneração/fisiologia , Ferimentos e Lesões/imunologia
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