RESUMEN
Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.
Asunto(s)
Desarrollo Embrionario , Estratos Germinativos , Células Madre Pluripotentes , Humanos , Diferenciación Celular , Implantación del Embrión , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Estratos Germinativos/citología , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Células Madre Pluripotentes/citología , Interleucina-6/metabolismo , Gástrula/citología , Gástrula/embriología , Amnios/citología , Amnios/embriología , Amnios/metabolismo , Ectodermo/citología , Ectodermo/embriología , Ectodermo/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismoRESUMEN
Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Técnicas Citológicas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Humanos , Factor 4 Similar a Kruppel , Ratones , Mitocondrias/metabolismo , Proteína Homeótica Nanog , Células Madre Pluripotentes/citología , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
The intervillous space of human placenta is filled with maternal blood, and villous trophoblasts are constantly exposed to the shear stress generated by maternal blood pressure and flow throughout the entire gestation period. However, the effects of shear stress on villous trophoblasts and their biological significance remain unknown. Here, using our recently established naïve human pluripotent stem cells-derived cytotrophoblast stem cells (nCTs) and a device that can apply arbitrary shear stress to cells, we investigated the impact of shear stress on early-stage trophoblasts. After 72 h of exposure to 10 dyn/cm2 shear stress, nCTs became fused and multinuclear, and mRNA expression of the syncytiotrophoblast (ST) markers, such as glial cell missing 1, endogenous retrovirus group W member 1 envelope, chorionic gonadotropin subunit beta 3, syndecan 1, pregnancy specific beta-1-glycoprotein 3, placental growth factor, and solute carrier family 2 member 1 were significantly upregulated compared to static conditions. Immunohistochemistry showed that shear stress increased fusion index, human chorionic gonadotropin secretion, and human placental lactogen secretion. Increased microvilli formation on the surface of nCTs under flow conditions was detected using scanning electron microscopy. Intracellular cyclic adenosine monophosphate significantly increased under flow conditions. Moreover, transcriptome analysis of nCTs subjected to shear stress revealed that shear stress upregulated ST-specific genes and downregulated CT-specific genes. Collectively, these findings indicate that shear stress promotes the differentiation of nCTs into ST.
Asunto(s)
Células Madre Pluripotentes Inducidas , Placenta , Femenino , Embarazo , Humanos , Placenta/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Factor de Crecimiento Placentario/metabolismo , Trofoblastos/metabolismo , Gonadotropina Coriónica/farmacología , Gonadotropina Coriónica/metabolismo , Diferenciación CelularRESUMEN
Human pluripotent stem cells (PSCs) express human endogenous retrovirus type-H (HERV-H), which exists as more than a thousand copies on the human genome and frequently produces chimeric transcripts as long-non-coding RNAs (lncRNAs) fused with downstream neighbor genes. Previous studies showed that HERV-H expression is required for the maintenance of PSC identity, and aberrant HERV-H expression attenuates neural differentiation potentials, however, little is known about the actual of function of HERV-H. In this study, we focused on ESRG, which is known as a PSC-related HERV-H-driven lncRNA. The global transcriptome data of various tissues and cell lines and quantitative expression analysis of PSCs showed that ESRG expression is much higher than other HERV-Hs and tightly silenced after differentiation. However, the loss of function by the complete excision of the entire ESRG gene body using a CRISPR/Cas9 platform revealed that ESRG is dispensable for the maintenance of the primed and naïve pluripotent states. The loss of ESRG hardly affected the global gene expression of PSCs or the differentiation potential toward trilineage. Differentiated cells derived from ESRG-deficient PSCs retained the potential to be reprogrammed into induced PSCs (iPSCs) by the forced expression of OCT3/4, SOX2, and KLF4. In conclusion, ESRG is dispensable for the maintenance and recapturing of human pluripotency.
Asunto(s)
Células Madre Pluripotentes/metabolismo , ARN Largo no Codificante/genética , Diferenciación Celular/genética , Células Cultivadas , Reprogramación Celular , Femenino , Silenciador del Gen , Humanos , Factor 4 Similar a Kruppel , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células Madre Pluripotentes/citologíaRESUMEN
Mermithidae is a family of nematodes that parasitize a wide range of invertebrates worldwide. Herein, we report nematodes that were unexpectedly found in three of 486 adult stable flies (Stomoxys calcitrans) captured from three farms (F1, F2, and F3) in different regions of Gifu Prefecture, Japan. We aimed to characterize these nematodes both at the morphological and molecular level. Morphological studies revealed that the nematodes were juveniles of Mermithidae. Phylogenetic analysis based on 18S and 28S rDNA indicated that the mermithids from farms F1 and F2 could be categorized into the same cluster as Ovomermis sinensis and Hexamermis sp., whereas the mermithid from farm F3 clustered with Amphimermis sp. Additionally, these mermithids could be categorized within the same clusters as related mermithids detected in Japan that parasitize various arthropod orders. Our findings suggest that these stable flies may have been parasitized by mermithids already present in the region and that genetically distinct species of mermithids occur across Japan. To the best of our knowledge, this is the first report of mermithids parasitizing adult stable flies in Japan.
RESUMEN
Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.
Asunto(s)
Células Madre Embrionarias/metabolismo , Inestabilidad Genómica , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Metilación de ADN , Células Madre Embrionarias/enzimología , Humanos , Proteoma , TranscriptomaRESUMEN
Forty years have passed since the first pluripotent stem cells (PSCs), mouse embryonic stem cells (ESCs), were established. Since then, several PSCs have been reported, including human ESCs in 1998, mouse epiblast stem cells (EpiSCs) in 2007, induced PSCs (iPSCs) in 2006 and 2007, and naïve human PSCs in 2014. Naïve PSCs are thought to correspond to pre-implantation epiblast cells, whereas conventional (or primed) human PSCs correspond to post-implantation epiblast cells. Thus, naïve and primed PSCs are classified by their developmental stages and have stage-specific characteristics, despite sharing the common feature of pluripotency. In this review, we discuss the current status of PSCs and their use to model human peri-implantation development.
Asunto(s)
Células Madre Pluripotentes/citología , Diferenciación Celular , Desarrollo Embrionario , HumanosRESUMEN
Epigenetic changes are frequently observed in cancer. However, their role in establishing or sustaining the malignant state has been difficult to determine due to the lack of experimental tools that enable resetting of epigenetic abnormalities. To address this, we applied induced pluripotent stem cell (iPSC) reprogramming techniques to invoke widespread epigenetic resetting of glioblastoma (GBM)-derived neural stem (GNS) cells. GBM iPSCs (GiPSCs) were subsequently redifferentiated to the neural lineage to assess the impact of cancer-specific epigenetic abnormalities on tumorigenicity. GiPSCs and their differentiating derivatives display widespread resetting of common GBM-associated changes, such as DNA hypermethylation of promoter regions of the cell motility regulator TES (testis-derived transcript), the tumor suppressor cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), and many polycomb-repressive complex 2 (PRC2) target genes (e.g., SFRP2). Surprisingly, despite such global epigenetic reconfiguration, GiPSC-derived neural progenitors remained highly malignant upon xenotransplantation. Only when GiPSCs were directed to nonneural cell types did we observe sustained expression of reactivated tumor suppressors and reduced infiltrative behavior. These data suggest that imposing an epigenome associated with an alternative developmental lineage can suppress malignant behavior. However, in the context of the neural lineage, widespread resetting of GBM-associated epigenetic abnormalities is not sufficient to override the cancer genome.
Asunto(s)
Reprogramación Celular/genética , Metilación de ADN , Epigénesis Genética , Glioblastoma/patología , Células-Madre Neurales/citología , Animales , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Transformación Celular Neoplásica/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Humanos , Ratones , Ratones Endogámicos NOD , Células Madre Pluripotentes/citología , Trasplante HeterólogoRESUMEN
Several surveys of Sarcocystis infection in sika deer in Japan have shown a high prevalence, but the identification has been unclear because molecular data have been lacking or have been limited to 18S ribosomal RNA gene sequences. Thus, in our previous study based on such sequences, some Sarcocystis isolates from sika deer were not clearly separated from other species in the phylogenetic analysis. In the present study, we therefore characterized sarcocyst isolates from sika deer (Cervus nippon centralis) at the mitochondrial cytochrome c oxidase subunit I gene (cox1). Moreover, we developed a multiplex PCR based on cox1 sequences of all species found, so that we could rapidly identify sarcocysts of these species. Twenty-one sarcocysts from nine sika deer were examined. Five distinct cox1 sequence types, each with a high sequence identity (> 99%), were found, and the sarcocysts could thus be classified into five species. Based on the sequence comparisons and the phylogeny, Sarcocystis spp. of types 1, 3, and 5 are considered to represent three new species, which were most closely related to Sarcocystis silva/Sarcocystis truncata, Sarcocystis entzerothi, and Sarcocystis iberica/Sarcocystis venatoria, respectively. There was a slight uncertainly whether Sarcocystis sp. with type 2 sequences represented a new species or was identical to Sarcocystis tarandi. Type 4 sequences showed 99% identity with those of Sarcocystis pilosa from sika deer in Lithuania and have therefore been assigned to this species. In the multiplex PCR, type-specific fragments were successfully amplified for all five Sarcocystis spp., indicating that this assay may be useful for a rapid identification of sarcocysts of these species.
Asunto(s)
Ciclooxigenasa 1/genética , Complejo IV de Transporte de Electrones/genética , Sarcocystis/clasificación , Sarcocystis/genética , Sarcocistosis/veterinaria , Animales , Secuencia de Bases , Ciervos/parasitología , Genes Mitocondriales/genética , Japón , Lituania , Reacción en Cadena de la Polimerasa Multiplex/métodos , Filogenia , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADNRESUMEN
Currently it is less advanced to understand the pathology of lifestyle disease by using iPS cells because there is partly less direct connection between life style disease and iPS cells. So much more scientists focus on regenerative medicine such as beta cells therapy using iPS cells technologies. It will be indeed a powerful tool to generate beta cells from iPS cells as even in type2 diabetes patients, hyposecretion of insulin from beta cells in pancreas is one of causes. Another reason is complexity of the pathology of life style disease. There are a lot of reasons to cause lifestyle disease. Lifestyle diseases include cancer, chronic liver disease, Type 2 diabetes, heart disease, metabolic syndrome, chronic renal failure, stroke, and obesity. Since obesity is one of major causes of lifestyle diseases, we want to focus on adipogenesis from iPS cells in this review. We analysed and established the differentiation protocol into adipocytes from mouse ES cells and human iPS cells. The other point in this review is the starting pluripotent cells for differentiation. Quality of pluripotent stem cells are one of most critical factors to succeed in getting well-differentiated cells. Recently, we have developed new naive human pluripotent stem cells (PSC),"Reset cells". Naive PSC have more similar to human epibast cells than conventional human PSC. They will be more ideal cells for differentiation because of their hypomethylated status and earlier stage of development.
Asunto(s)
Diferenciación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos , Diabetes Mellitus Tipo 2/terapia , Células Madre Pluripotentes Inducidas/citología , Estilo de Vida , Animales , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Medicina Regenerativa/métodosRESUMEN
Identification of molecular mechanisms involved in generation of different types of adipocytes is progressing substantially in mice. However, much less is known regarding characterization of brown (BAP) and white adipocyte progenitors (WAPs) in humans, highlighting the need for an in vitro model of human adipocyte development. Here, we report a procedure to selectively derive BAP and WAPs from human-induced pluripotent stem cells. Molecular characterization of APs of both phenotypes revealed that BMP4, Hox8, Hoxc9, and HoxA5 genes were specifically expressed in WAPs, whereas expression of PRDM16, Dio2, and Pax3 marked BAPs. We focused on Pax3 and we showed that expression of this transcription factor was enriched in human perirenal white adipose tissue samples expressing UCP1 and in human classical brown fat. Finally, functional experiments indicated that Pax3 was a critical player of human AP fate as its ectopic expression led to convert WAPs into brown-like APs. Together, these data support a model in which Pax3 is a new marker of human BAPs and a molecular mediator of their fate. The findings of this study could lead to new anti-obesity therapies based on the recruitment of APs and constitute a platform for investigating in vitro the developmental origins of human white and brown adipocytes.
Asunto(s)
Adipocitos Marrones/citología , Adipocitos Blancos/citología , Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Factores de Transcripción Paired Box/metabolismo , Adipocitos Marrones/efectos de los fármacos , Adipocitos Marrones/metabolismo , Adipocitos Blancos/efectos de los fármacos , Adipocitos Blancos/metabolismo , Adipogénesis/efectos de los fármacos , Anciano de 80 o más Años , Animales , Diferenciación Celular/efectos de los fármacos , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Factor de Transcripción PAX3 , Fenotipo , Tretinoina/farmacologíaRESUMEN
Tachyzoites of Toxoplasma gondii, an obligate intracellular parasite, actively invade almost all types of nucleated cells. However, T. gondii tachyzoites preferentially infect particular types of animal tissue cells. The mechanism underlying the host cell preference of T. gondii is not yet known. In this study, we found that enzymatic removal of α2,3- but not α2,6-linked sialic acids on the surface of Vero cells decreased T. gondii tachyzoite adhesion or invasion to the treated cells. Although Chinese hamster ovary (CHO) cells express only α2,3-linked sialic acid, a genetically modified CHO cell line constructed by transfection with the α2,6-sialiltransferase gene contains subpopulations with a variety of expression patterns of α2,3- and α2,6-linked sialic acids. When T. gondii tachyzoites were added to the modified CHO cells, the tachyzoites preferentially attached to cells belonging to a subpopulation of cells that highly expressed α2,3-linked sialic acids. Additionally, multiple regression analysis performed to analyse the relationship between the amount of α2,3-linked/α2,6-linked sialic acids and parasite-expressed fluorescence intensity suggested that more tachyzoites adhered to individual α2,3-linked sialic acid rich-cells than to α2,3-linked sialic acid-poor/null cells. The results of confocal laser microscopy confirmed this finding. These results indicate that the host cell preference of T. gondii was, at least partially, affected by the distribution pattern of α2,3-, but almost never α2,6-linked sialic acids on host cells.
Asunto(s)
Células CHO/química , Ácidos Siálicos/química , Sialiltransferasas/genética , Toxoplasma/fisiología , Células Vero/química , Adhesividad , Animales , Línea Celular , Separación Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Citometría de Flujo , Interacciones Huésped-Parásitos , Microscopía Confocal , Neuraminidasa/metabolismo , Análisis de Regresión , Ácidos Siálicos/genética , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
We report a confirmed case of Toxoplasma gondii infection in the lungs of a cow exhibiting respiratory symptoms. At slaughter, white nodules were discovered in lung tissue, accompanied by enlarged hilar lymph nodes. Histological examination revealed the disappearance of alveolar structures in nodular areas, replaced by granulomas containing inflammatory cells. Immunohistochemical staining with anti-T. gondii antibody and nucleotide sequencing of 18S rDNA confirmed T. gondii infection. However, the link between T. gondii and observed symptoms remains unclear. Various factors, including host genetics, underlying diseases, infection route, and exposure level, may contribute to these uncommon symptoms. Although T. gondii infections in cattle are traditionally considered asymptomatic, our study suggests the possible existence of clinical symptoms associated with Toxoplasma infection. Beef cattle are generally not assumed to be a relevant source of human T. gondii infection; however, sporadic transmission by infected edible beef to humans cannot be completely excluded and deserves further studies.
Asunto(s)
Enfermedades de los Bovinos , Toxoplasma , Toxoplasmosis Animal , Bovinos , Toxoplasma/aislamiento & purificación , Toxoplasma/genética , Animales , Toxoplasmosis Animal/parasitología , Toxoplasmosis Animal/patología , Toxoplasmosis Animal/diagnóstico , Enfermedades de los Bovinos/parasitología , Enfermedades de los Bovinos/patología , Pulmón/parasitología , Pulmón/patología , Neumonía/parasitología , Neumonía/veterinaria , Femenino , Granuloma/parasitología , Granuloma/patología , ARN Ribosómico 18S/análisisRESUMEN
The major genetic group of Toxoplasma gondii, known as type I, generally displays high lethality in laboratory Mus musculus (mouse) strains, with few exceptions. However, because rodents are the primary reservoir hosts for T. gondii, if this characteristic manifests in the wild, type I strains would be extinct. Therefore, we hypothesized that populations of wild rodents capable of harboring type I T. gondii asymptomatically exist globally and are not limited to a few localized areas, as previously thought. The strength of mouse resistance to T. gondii is known to depend on the affinity of the mouse-expressed immunity-related GTPases B2 (IRGB2) protein for the T. gondii-expressed rphoptry protein 5B (ROP5B) protein. Therefore, the Irgb2 gene sequences of 12 individuals mice captured at two animal farms in Gifu Prefecture, and on an island in Okinawa Prefecture, Japan were determined, and subjected to a molecular phylogenetic analysis together with those of various mouse strains worldwide. The Irgb2 gene of M. musculus individuals captured on one farm and one island showed diverse sequences. The sequences from two individual mice captured in an animal farm formed a single clade with a wild mouse derived CAST/EiJ strain, known for its exceptional resistance to type I T. gondii lethality. These results suggest that M. musuculus individuals resistant to the Type I T. gondii strain may be present in Japan, in addition to the previously known populations in South Asia, Thailand and India.
RESUMEN
Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.
Asunto(s)
Genoma de Protozoos , Filogenia , Toxoplasma , Toxoplasma/genética , Toxoplasma/clasificación , Humanos , América del Norte , Genoma de Protozoos/genética , Toxoplasmosis/parasitología , China , América Central , Japón , Haplotipos , Variación Genética , Recombinación GenéticaRESUMEN
Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.
Asunto(s)
Reprogramación Celular , Histonas , Células Madre Pluripotentes Inducidas , Factor 4 Similar a Kruppel , Reprogramación Celular/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Histonas/metabolismo , Diferenciación Celular/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Cromatina/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , TranscriptomaRESUMEN
The intracellular parasite Toxoplasma gondii is thought to disseminate throughout the host by circulation of tachyzoite-infected leukocytes in the blood, and adherence and migration of such leukocytes into solid tissues. However, it is unclear whether T. gondii-infected leukocytes can migrate to solid organs via the general circulation. In this study, we developed a real-time quantitative PCR (qRT-PCR) method to determine the rate of infection of peripheral blood mononuclear cells (PBMCs) flowing into and remaining within solid organs in mice. A transgenic T. gondii parasite line derived from the PLK strain that expresses DsRed Express, and transgenic green fluorescent protein-positive PBMCs, were used for these experiments. Tachyzoite-infected PBMCs were injected into mouse tail veins and qRT-PCR was used to measure the infection rates of the PBMCs remaining in the lungs, liver, spleen and brain. We found that the PBMCs in the lungs and liver had statistically higher infection rates than that of the original inoculum; this difference was statistically significant. However, the PBMC infection rate in the spleen showed no such enhancement. These results show that tachyzoite-infected PBMCs in the general circulation remain in the lungs and liver more effectively than non-infected PBMCs.
Asunto(s)
Leucocitos Mononucleares/parasitología , Hígado/patología , Pulmón/patología , Toxoplasma/fisiología , Toxoplasmosis Animal/patología , Animales , Chlorocebus aethiops , ADN Protozoario/aislamiento & purificación , Proteínas Fluorescentes Verdes , Ventrículos Cardíacos/parasitología , Ventrículos Cardíacos/patología , Leucocitos Mononucleares/citología , Hígado/parasitología , Sustancias Luminiscentes , Pulmón/parasitología , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Bazo/parasitología , Bazo/patología , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , Toxoplasmosis Animal/sangre , Toxoplasmosis Animal/parasitología , Células VeroRESUMEN
Canine hepatozoonosis is a worldwide protozoal disease caused by Hepatozoon canis and Hepatozoon americanum and is transmitted by ixodid ticks, Rhipicephalus and Amblyomma spp., respectively. H. canis infection is widespread in Africa, Europe, South America, and Asia, including Japan. The objective of this study was to study the distribution pattern and diversity of H. canis in naturally infected dogs in nine Japanese islands and peninsulas. Therefore, 196 hunting dogs were randomly sampled during the period from March to September 2011 and the ages and sexes were identified. Direct microscopy using Giemsa-stained blood smears revealed H. canis gametocytes in the peripheral blood of 45 (23.6%) dogs. Polymerase chain reaction (PCR) was performed on EDTA-anticoagulated blood, initially with the common primer set (B18S-F and B18S-R) amplifying the 1,665-bp portion of the 18S rRNA gene, and then with the specific primer set (HepF and HepR) amplifying about 660 bp fragments of the same gene. Based on PCR, 84 (42.9%) dogs were positive using the common primer and 81 (41.3%) were positive using the specific primer. The current investigation indicated that all screened areas, except for Sado Island and Atsumi Peninsula, were infected. Yaku Island had the highest infection rate (84.6% in males and 100.0% in females), while Ishigaki Island showed the lowest infection rates (8.3% in males and 17.7% in females). Both sexes were infected with no significant difference. However, diversity of infection among the surveyed islands and peninsulas was significantly different (P < 0.05). Although H. canis has previously been reported in dogs in Japan, the higher infection rate described in the current study and the diversity of infection in a wide range of islands strongly encourage prospective studies dealing with the prevention and treatment of the infection in dogs, as well as control of ticks.