Pompe disease is caused by an inborn defect of lysosomal acid α-glucosidase (GAA) and is characterized by lysosomal glycogen accumulation primarily in the skeletal muscle and heart. Patients with the ...severe type of the disease, infantile-onset Pompe disease (IOPD), show generalized muscle weakness and heart failure in early infancy. They cannot survive over two years. Enzyme replacement therapy with recombinant human GAA (rhGAA) improves the survival rate, but its effect on skeletal muscle is insufficient compared to other organs. Moreover, the patho-mechanism of skeletal muscle damage in IOPD is still unclear. Here we generated induced pluripotent stem cells (iPSCs) from patients with IOPD and differentiated them into myocytes. Differentiated myocytes showed lysosomal glycogen accumulation, which was dose-dependently rescued by rhGAA. We further demonstrated that mammalian/mechanistic target of rapamycin complex 1 (mTORC1) activity was impaired in IOPD iPSC-derived myocytes. Comprehensive metabolomic and transcriptomic analyses suggested the disturbance of mTORC1-related signaling, including deteriorated energy status and suppressed mitochondrial oxidative function. In summary, we successfully established an in vitro skeletal muscle model of IOPD using patient-specific iPSCs. Disturbed mTORC1 signaling may contribute to the pathogenesis of skeletal muscle damage in IOPD, and may be a potential therapeutic target for Pompe disease.
...the refractory cases in our cohort were treated with anti–TNF-α agents, and it successfully induced the remission. ...our study demonstrated that HA20 showed unexpected variation in clinical ...manifestations. ...he was identified as having a heterozygous c.2088+5G>C mutation in TNFAIP3 (Fig E2). At the age of 1 year, colonoscopy revealed the ulceration of rectum and colon. ...he was diagnosed as having nonspecific inflammatory bowel disease and treated with mesalazine.
Duchenne muscular dystrophy (DMD), a severe degenerative skeletal and cardiac muscle disease, has a poor prognosis, and no curative treatments are available. Because decreased autophagy has been ...reported to contribute to skeletal muscle degeneration, therapies targeting autophagy are expected to improve skeletal muscle hypofunction. However, the role of this regulatory mechanism has not been evaluated clearly in DMD cardiomyocytes.
In this present study, we evaluated myocardial fibrosis and its mechanism in mdx mice, a model of DMD, and also evaluated changes in cardiac function.
As assessed by LC3 immunohistochemistry, a small number of autophagosomes were detected in cardiomyocytes of both mdx mice and control wild-type (WT) mice. The number of autophagosomes was significantly enhanced by 4 weeks of isoproterenol-induced cardiac stress in cardiomyocytes of mdx but not WT mice. Simultaneously, isoproterenol increased cardiomyocyte fibrosis in mdx but not WT mice. Administration of chloroquine significantly decreased cardiomyocyte fibrosis in mdx mice, even after isoproterenol treatment. Left ventricle size and function were evaluated by echocardiography. Left ventricular contraction was decreased in mdx mice after isoproterenol treatment compared with control mice, which was alleviated by chloroquine administration.
Heart failure in DMD patients is possibly treated with chloroquine, and the mechanism probably involves chloroquine's anti-inflammatory effects.
Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are T cell subpopulations that possess innate-like properties. We examined the impact of post-hematopoietic stem cell ...transplantation (HSCT) MAIT and iNKT cell recovery on the clinical outcomes of 69 patients who underwent allogeneic HSCT at Kyoto University Hospital. Multivariate analyses identified the absolute number of MAIT cells (< 0.48/μL on day 60 post-HSCT) as the sole independent risk factor for grade I–IV and grade II–IV acute graft-versus-host disease (aGVHD) among patients who underwent bone marrow transplantation; no correlation was observed between post-HSCT iNKT cell recovery and the development of aGVHD. Six of the 15 patients in the MAIT
high
(≥ 0.48/μL) group developed aGVHD, five within the first 30 days post HSCT. In contrast, 13 of the 15 patients in the MAIT
low
(< 0.48/μL) group developed aGVHD, seven after day 30 post HSCT. The overall survival of the MAIT
low
group was slightly shorter than that of the MAIT
high
group. Thus, the post-HSCT recovery of MAIT cells is closely related to the development of delayed onset aGVHD and the outcome of post-HSCT, suggesting its utility for identifying a subset of patients that requires more prolonged and/or intense GVHD prophylaxis.
Objective
To elucidate the genetic background of a patient with neonatal‐onset multisystem inflammatory disease (NOMID) with no NLRP3 mutation.
Methods
A Japanese male child diagnosed as having NOMID ...was studied. The patient did not have any NLRP3 mutation, even as low‐frequency mosaicism. We performed whole‐exome sequencing on the patient and his parents. Induced pluripotent stem cells (iPSCs) were established from the patient's fibroblasts. The iPSCs were then differentiated into monocyte lineage to evaluate the cytokine profile.
Results
We established multiple iPSC clones from a patient with NOMID and incidentally found that the phenotypes of monocytes from iPSC clones were heterogeneous and could be grouped into disease and normal phenotypes. Because each iPSC clone was derived from a single somatic cell, we hypothesized that the patient had somatic mosaicism of an interleukin‐1β–related gene. Whole‐exome sequencing of both representative iPSC clones and the patient's blood revealed a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in diseased iPSC clones. Knockout of the NLRC4 gene using the clustered regularly interspaced short palindromic repeat/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype.
Conclusion
Our findings indicate that the patient has somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing that somatic mutation of NLRC4 causes autoinflammatory symptoms compatible with NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC‐based phenotype dissection for individualized diagnoses.
Background Blau syndrome, or early-onset sarcoidosis, is a juvenile-onset systemic granulomatosis associated with a mutation in nucleotide-binding oligomerization domain 2 (NOD2) . The underlying ...mechanisms of Blau syndrome leading to autoinflammation are still unclear, and there is currently no effective specific treatment for Blau syndrome. Objectives To elucidate the mechanisms of autoinflammation in patients with Blau syndrome, we sought to clarify the relation between disease-associated mutant NOD2 and the inflammatory response in human samples. Methods Blau syndrome–specific induced pluripotent stem cell (iPSC) lines were established. The disease-associated NOD2 mutation of iPSCs was corrected by using a CRISPR-Cas9 system to precisely evaluate the in vitro phenotype of iPSC-derived cells. We also introduced the same NOD2 mutation into a control iPSC line. These isogenic iPSCs were then differentiated into monocytic cell lineages, and the statuses of nuclear factor κB pathway and proinflammatory cytokine secretion were investigated. Results IFN-γ acted as a priming signal through upregulation of NOD2. In iPSC-derived macrophages with mutant NOD2, IFN-γ treatment induced ligand-independent nuclear factor κB activation and proinflammatory cytokine production. RNA sequencing analysis revealed distinct transcriptional profiles of mutant macrophages both before and after IFN-γ treatment. Patient-derived macrophages demonstrated a similar IFN-γ–dependent inflammatory response. Conclusions Our data support the significance of ligand-independent autoinflammation in the pathophysiology of Blau syndrome. Our comprehensive isogenic disease-specific iPSC panel provides a useful platform for probing therapeutic and diagnostic clues for the treatment of patients with Blau syndrome.
Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive inflammatory disease caused by loss-of-function mutations in both alleles of the ADA2 gene. Most patients with DADA2 exhibit ...systemic vasculopathy consistent with polyarteritis nodosa, but large phenotypic variability has been reported, and the pathogenesis of DADA2 remains unclear.
This study sought to assess the clinical and genetic characteristics of Japanese patients with DADA2 and to gain insight into the pathogenesis of DADA2 by multi-omics analysis.
Clinical and genetic data were collected from 8 Japanese patients with DADA2 diagnosed between 2016 and 2019. ADA2 variants in this cohort were functionally analyzed by in vitro overexpression analysis. PBMCs from 4 patients with DADA2 were subjected to transcriptome and proteome analyses. Patient samples were collected before and after introduction of anti- TNF-α therapies. Transcriptome data were compared with those of normal controls and patients with other autoinflammatory diseases.
Five novel ADA2 variants were identified in these 8 patients and were confirmed pathogenic by in vitro analysis. Anti-TNF-α therapy controlled inflammation in all 8 patients. Transcriptome and proteome analyses showed that upregulation of type II interferon signaling was characteristic of DADA2. Network analysis identified STAT1 as a key regulator and a hub molecule in DADA2 pathogenesis, a finding supported by the hyperactivation of STAT1 in patients’ monocytes and B cells after IFN-γ stimulation.
Type II interferon signaling and STAT1 are associated with the pathogenesis of DADA2.
Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton, cornea, ...peripheral nervous system, and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), further modifications are required to improve the robustness, efficacy, and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions, the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70-80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons, glia, melanocytes, and corneal endothelial cells. In addition, cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.
Dried blood spots (DBS) are widely used for screening biomolecular profiles, including enzymatic activities. However, detection of minor proteins in DBS by liquid chromatography–mass spectrometry ...(LC-MS/MS) without pre-enrichment remains challenging because of the coexistence of large quantities of hydrophilic proteins. In this study, we address this problem by developing a simple method using sodium carbonate precipitation (SCP). SCP enriches hydrophobic proteins from DBS, allowing substantial removal of soluble proteins. In combination with SCP, we used quantitative LC-MS/MS proteome analysis in a data-independent acquisition mode (DIA) to enhance the sensitivity and quantification limits of proteome analysis. As a result, identification of 1977 proteins in DBS is possible, including 585 disease-related proteins listed in the Online Mendelian Inheritance in Man.
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