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Developing experimental animal models that show clinical symptoms and methods for quantitative and objective evaluation are important for understanding food allergies. Therefore, this study aimed to develop an ovalbumin (OVA)-induced mouse model of food allergy and a useful method to evaluate the symptoms of food allergy. Mice were sensitized via intraperitoneal injection of OVA. Subsequently, local sensitization was performed once weekly by oral administration of OVA. Itching and nasal symptoms were observed after oral administration of the antigen. First, we examined the dose-dependency of the antigen. Symptoms were checked weekly. In order to confirm food allergy symptoms, the effect of histamine H 1 receptor antagonist was examined. Finally, we measured antigen-specific IgE antibody levels in the serum. Scratching behavior, sneezing and nasal rubbing were increased. Both itching and rhinitis symptoms increased steadily, after which, the number of symptoms remained almost constant. No difference was observed between the results of 3- and 5-week-old mice. Cetirizine inhibited these symptoms in a dose-dependent manner. In addition, antigen-specific IgE antibodies were produced in both 3- and 5-week-old mice. This method may be useful for evaluating the symptoms of skin and rhinitis that could not be assessed in the conventional food allergy model and could be induced with a low dose of antigen. In particular, the developed method, which measures the number of itching and nasal symptoms, may enable quantitative, objective, and noninvasive evaluation of food allergy severity. [ABSTRACT FROM AUTHOR]

The Activin/Nodal/ TGF-β signaling pathway plays a major role in maintaining mouse epiblast stem cells (Epi SCs). The Epi SC-maintaining medium, which contains Activin A and bFGF, induces differentiation of mouse embryonic stem cells ( ESCs) to Epi SCs. Here, we show that Activin A also has an ability to efficiently propagate ESCs without differentiation to Epi SCs when combined with a MEK inhibitor PD0325901. ESCs cultured in Activin+ PD retained high-level expression of naive pluripotency-related transcription factors. Genomewide analysis showed that the gene expression profile of ESCs cultured in Activin+ PD resembles that of ESCs cultured in 2i. ESCs cultured in Activin+ PD also showed features common to the naive pluripotency of ESCs, including the preferential usage of the Oct4 distal enhancer and the self-renewal response to Wnt pathway activation. Our finding shows a role of Activin/Nodal/ TGF-β signaling in stabilizing self-renewal gene regulatory networks in ESCs. [ABSTRACT FROM AUTHOR]

CD8+ T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8+ T cell differentiation, T-bet and Eomesodermin (Eomes), have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4), functional characteristics and memory differentiation of CD8+ T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8+ T cells was elevated in chronically infected individuals and highly associated with a T-betdimEomeshi expressional profile. Interestingly, both resting and activated HIV-specific CD8+ T cells in chronic infection were almost exclusively T-betdimEomeshi cells, while CMV-specific CD8+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-betdimEomeshi virus-specific CD8+ T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector) characteristics. The transitional and exhausted phenotype of HIV-specific CD8+ T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART) initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8+ T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8+ T cells to control the viral replication post-ART cessation. [ABSTRACT FROM AUTHOR]

Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3GFP/+ embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3GFP/+ mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease. [ABSTRACT FROM AUTHOR]

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs. [ABSTRACT FROM AUTHOR]

The Hedgehog pathway is activated in various types of malignancies. We previously reported that inhibition of SMO or GLI prevents osteosarcoma growth in vitro and in vivo. Recently, it has been reported that arsenic trioxide (ATO) inhibits cancer growth by blocking GLI transcription. In this study, we analyzed the function of ATO in the pathogenesis of osteosarcoma. Real-time PCR showed that ATO decreased the expression of Hedgehog target genes, including PTCH1, GLI1, and GLI2, in human osteosarcoma cell lines. WST-1 assay and colony formation assay revealed that ATO prevented osteosarcoma growth. These findings show that ATO prevents GLI transcription and osteosarcoma growth in vitro. Flow cytometric analysis showed that ATO promoted apoptotic cell death. Comet assay showed that ATO treatment increased accumulation of DNA damage. Western blot analysis showed that ATO treatment increased the expression of γH2AX, cleaved PARP, and cleaved caspase-3. In addition, ATO treatment decreased the expression of Bcl-2 and Bcl-xL. These findings suggest that ATO treatment promoted apoptotic cell death caused by accumulation of DNA damage. In contrast, Sonic Hedgehog treatment decreased the expression of γH2AX induced by cisplatin treatment. ATO re-induced the accumulation of DNA damage attenuated by Sonic Hedgehog treatment. These findings suggest that ATO inhibits the activation of Hedgehog signaling and promotes apoptotic cell death in osteosarcoma cells by accumulation of DNA damage. Finally, examination of mouse xenograft models showed that ATO administration prevented the growth of osteosarcoma in nude mice. Because ATO is an FDA-approved drug for treatment of leukemia, our findings suggest that ATO is a new therapeutic option for treatment of patients with osteosarcoma. [ABSTRACT FROM AUTHOR]

Human in vitro gametogenesis may transform reproductive medicine. Human pluripotent stem cells (hPSCs) have been induced into primordial germ cell–like cells (hPGCLCs); however, further differentiation to a mature germ cell has not been achieved. Here, we show that hPGCLCs differentiate progressively into oogonia-like cells during a long-term in vitro culture (approximately 4 months) in xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. The hPGCLC-derived oogonia display hallmarks of epigenetic reprogramming—genome-wide DNA demethylation, imprint erasure, and extinguishment of aberrant DNA methylation in hPSCs—and acquire an immediate precursory state for meiotic recombination. Furthermore, the inactive X chromosome shows a progressive demethylation and reactivation, albeit partially. These findings establish the germline competence of hPSCs and provide a critical step toward human in vitro gametogenesis. [ABSTRACT FROM AUTHOR]

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs. [ABSTRACT FROM AUTHOR]

Abstract: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates intestinal immunological homeostasis. However, precise expression patterns of CEACAM1 isoforms remain poorly understood in the intestinal epithelia. Focusing on the small intestinal epithelium of BALB/c mice, we identified three novel splice variants encoding CEACAM1a-2, -2C1, and -4C1 by RT-PCR. CEACAM1a-2, -2C1, and -4C1 demonstrated secretory properties by transfection experiments in vitro. Among them, CEACAM1a-4C1 was the major secreted isoform in vivo due to the soluble/secreted CEACAM1a with a frameshift sequence in the C-terminus, specific for CEACAM1a-2C1 and -4C1. CEACAM1a-4C1 was capable of binding murine hepatitis virus (MHV) and was detected at approximately 120kDa in the small intestinal secretions. Neutralizing effects of the soluble CEACAM1a on MHV infectivity in vitro were demonstrated by using recombinant CEACAM1a-4C1. Our data suggest an intrinsic mechanism operated by free CEACAM1 for surveillance of pathogens and maintenance of homeostasis in the intestine. [Copyright &y& Elsevier]