RESUMO
Accurate serologic evaluation of autoantibodies in patients with autoimmune diseases is critical. In the present study, we established a live cell-based assay for simultaneous detection of multiple autoantibodies in a single serum sample. Autoantibody seropositivity was determined by 3-color flow cytometry using live Chinese hamster ovary cells transiently expressing a target protein of interest fused to enhanced green fluorescent protein and labeled with Alexa Fluor 647 and Hoechst 33342. As a representative example, we applied the strategy for simultaneous detection of 2 recently established biomarkers for central nervous system autoimmune inflammatory demyelinating disorders, anti-aquaporin-4 autoantibody and anti-myelin oligodendrocyte glycoprotein autoantibody, in a single serum sample. This analysis revealed the coexistence of these 2 autoantibodies. We demonstrated that this assay can simultaneously detect 3 different autoantibodies. We propose a quadrant gating strategy of flow cytometry contour plots to clearly distinguish seropositive sera from seronegative sera regardless of the extent of the background signal level or the autoantibody titer. This novel and practical method using a combination of fluorescent proteins and fluorochromes to simultaneously detect multiple autoantibodies improves the efficiency of evaluating serum samples, and therefore provides significant benefits to both the patient and the healthcare professionals performing autoantibody testing.
Assuntos
Autoanticorpos , Animais , Células CHO , Cricetinae , Cricetulus , Citometria de Fluxo/métodos , Humanos , Glicoproteína Mielina-OligodendrócitoRESUMO
The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell. They can be isolated as stage-specific embryonic antigen-3/CD105 double-positive cells. When human fibroblasts were separated into Muse and non-Muse cells and transduced with Oct3/4, Sox2, Klf4, and c-Myc, iPS cells were generated exclusively from Muse cells but not from non-Muse cells. Although some colonies were formed from non-Muse cells, they were unlike iPS cells. Furthermore, epigenetic alterations were not seen, and some of the major pluripotency markers were not expressed for the entire period during iPS cell generation. These findings were confirmed further using cells transduced with a single polycistronic virus vector encoding all four factors. The results demonstrate that in adult human fibroblasts a subset of preexisting adult stem cells whose properties are similar in some respects to those of iPS cells selectively become iPS cells, but the remaining cells make no contribution to the generation of iPS cells. Therefore this system seems to fit the elite model rather than the stochastic model.
Assuntos
Diferenciação Celular , Linhagem da Célula , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Adaptação Fisiológica , Adulto , Animais , Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Antígenos Glicosídicos Associados a Tumores/metabolismo , Linhagem Celular , Células Cultivadas , Derme/citologia , Endoglina , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Antígenos Embrionários Estágio-Específicos/metabolismo , Estresse Fisiológico , TransfecçãoRESUMO
We found adult human stem cells that can generate, from a single cell, cells with the characteristics of the three germ layers. The cells are stress-tolerant and can be isolated from cultured skin fibroblasts or bone marrow stromal cells, or directly from bone marrow aspirates. These cells can self-renew; form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency; and can differentiate into endodermal, ectodermal, and mesodermal cells both in vitro and in vivo. When transplanted into immunodeficient mice by local or i.v. injection, the cells integrated into damaged skin, muscle, or liver and differentiated into cytokeratin 14-, dystrophin-, or albumin-positive cells in the respective tissues. Furthermore, they can be efficiently isolated as SSEA-3(+) cells. Unlike authentic ES cells, their proliferation activity is not very high and they do not form teratomas in immunodeficient mouse testes. Thus, nontumorigenic stem cells with the ability to generate the multiple cell types of the three germ layers can be obtained through easily accessible adult human mesenchymal cells without introducing exogenous genes. These unique cells will be beneficial for cell-based therapy and biomedical research.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Adulto , Animais , Células da Medula Óssea/citologia , Agregação Celular , Diferenciação Celular , Proliferação de Células , Humanos , Transplante de Células-Tronco Mesenquimais , CamundongosRESUMO
Aquaporin-4 (AQP4) has been implicated in cytotoxic brain edema resulting from water intoxication, brain ischemia or meningitis. AQP4 inhibitors suitable for clinical use would thus be expected to help protect against brain edema. Here, we report the effect of inhibitors on water conduction by AQP4 and AQP1 reconstituted into liposomes. Acetazolamide (AZA), an inhibitor of sulfonamide carbonic anhydrase (CA), reversibly inhibits water permeation through AQP4, but not through AQP1. Methazolamide (MZA), another sulfonamide CA inhibitor similar in chemical structure to AZA, shows no significant effect on water conduction by AQP4 or AQP1. Our results thus demonstrate that AZA acts as a reversible inhibitor for AQP4-mediated water conduction and indicate that AZA is specific, at least to some degree, for AQP4. AZA may thus serve as a lead compound for the development of AQP4-specific inhibitors for clinical applications.
Assuntos
Acetazolamida/farmacologia , Aquaporina 4/antagonistas & inibidores , Água/metabolismo , Animais , Aquaporina 1/química , Aquaporina 1/genética , Aquaporina 4/química , Aquaporina 4/genética , Humanos , Cinética , Lipossomos/química , Metazolamida/química , Microscopia Eletrônica de Transmissão , Osmose , Permeabilidade/efeitos dos fármacos , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ácido Valproico/químicaRESUMO
Transient receptor potential (TRP) channels are intrinsic sensors adapted for response to all manner of stimuli both from inside and from outside the cell. Within the TRP superfamily, the canonical TRP-3 (TRPC3) has been widely studied and is involved in various biological processes such as neuronal differentiation, blood vessel constriction, and immune cell maturation. Upon stimulation of surface membrane receptors linked to phospholipase C, TRPC3 mediates transmembrane Ca(2+) influx from outside the cell to control Ca(2+) signaling, in concert with the Ca(2+) release from internal stores. The structural basis of TRP superfamily has, however, been poorly understood. Here we present a structure of the TRPC3 at 15 A resolution. This first 3D depiction of TRP superfamily was reconstructed from 135,909 particle images obtained with cryo-electron microscopy. The large intracellular domain represents a "nested-box" structure: a wireframe outer shell is functionable as sensors for activators and modulators, and a globular inner chamber may modulate ion flow, since it is aligned tandem along the central axis with the dense membrane-spanning core. The transmembrane domain demonstrates a pore-forming property. This structure implies that the TRP superfamily has diversely evolved as sensors specialized for various signals, rather than as simple ion-conducting apparatuses.
Assuntos
Modelos Moleculares , Canais de Cátion TRPC/química , Sítios de Ligação , Linhagem Celular , Membrana Celular/química , Microscopia Crioeletrônica , Humanos , Conformação ProteicaRESUMO
We compared the cytotoxicity of soluble and particulate titanium (Ti), vanadium (V) and nickel (Ni) by biochemical functional analysis and by microscopic morphology with micro-area elemental analysis in vitro using human neutrophils as probes and in vivo in animals. The biochemical analyses of LDH, superoxide anion, TNF-alpha and scanning electron microscopy (SEM) showed that Ni in solution destroys the cell membrane of neutrophils, whereas Ti and V in solution stimulate neutrophils and increase the quantity of released superoxide anions. Fine Ti particles (1-3 microm), which smaller than neutrophils (about 5 microm), were phagocytized by the cells and the results were similar in vivo. These results showed that the cytotoxic effect of Ti particles is size dependent, and that they must be smaller than that of cells. The present study demonstrated that the biochemical functional tests are useful for evaluating the biocompatibility of materials.
Assuntos
Neutrófilos/efeitos dos fármacos , Titânio/toxicidade , Animais , Cátions , Membrana Celular/efeitos dos fármacos , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Neutrófilos/patologia , Neutrófilos/fisiologia , Níquel/toxicidade , Tamanho da Partícula , Próteses e Implantes/efeitos adversos , Ratos , Ratos Wistar , Solubilidade , Superóxidos/metabolismo , Titânio/química , Titânio/farmacocinética , Vanádio/toxicidadeRESUMO
Members of the aquaporin (AQP) family are expressed in almost every organism, including 13 homologues in humans. Based on the electron crystallographic structure of AQP1, the hydrogen-bond isolation mechanism was proposed to explain why AQPs are impermeable to protons despite their very fast water conduction. The mechanism by which AQPs exclude protons remained controversial, however. Here we present the structure of AQP4 at 2.8 A resolution obtained by electron crystallography of double-layered two-dimensional crystals. The resolution has been improved from the previous 3.2 A, with accompanying improvement in data quality resulting in the ability to identify individual water molecules. Our structure of AQP4, the predominant water channel in the brain, reveals eight water molecules in the channel. The arrangement of the waters provides support for the hydrogen-bond isolation mechanism. Our AQP4 structure also visualizes five lipids, showing that direct interactions of the extracellular surface of AQP4 with three lipids in the adjoining membrane help stabilize the membrane junction.