Intractable Diseases Research Articles

Medical genetics and genomic medicine in Japan.

Since 1961, all Japanese citizens have belonged to one of the available medical care insurance systems. This "universal care" system has contributed to the maintenance of health: the life expectancy at birth was 84 years in 2016, and the infant mortality rate (the number of infants dying before reaching 1 year of age) was 2.0 per 1,000 live births, which is one of the lowest rates in the world. The Japanese government initiated the National Program on Rare and Intractable Diseases in 1972. This program has promoted research and expanded support for patients with rare and intractable diseases. Registered patients are eligible for a subsidy scheme that helps to cover medical care costs. Among the 331 diseases that are currently included in this program, more than half of the diseases are Mendelian disorders. The National Program on Rare and Intractable Diseases has fostered research in medical genetics in Japan and many causative genes for Mendelian diseases have been identified by Japanese geneticists. Recently, the Japanese government has determined to support several genomic medicine initiatives including the undiagnosed disease program (Initiative on Rare and Undiagnosed Diseases) and pathogenic variant databases.

Glia-Like Cells from Late-Passage Human MSCs Protect Against Ischemic Stroke Through IGFBP-4.

Stem cell therapy is considered to be a promising future treatment for intractable neurological diseases, although all the clinical trials using stem cells have not yet shown any good results. Early passage mesenchymal stem cells (MSCs) have been used in most clinical trials because of the issues on safety and efficacy. However, it is not easy to get plenty of cells enough for the treatment and it costs too much. Lots of late passage MSCs can be obtained at lower cost but their efficacy would be a big hurdle for clinical trials. If late passage MSCs with better efficacy could be used in clinical trials, it could be a new and revolutionary solution to reduce cost and enhance easier clinical trials. In the present study, it was investigated whether late passage MSCs could be induced into glia-like cells (ghMSCs); ghMSCs had better efficacy and they protected neurons and the brain from ischemia, and insulin-like growth factor binding protein-4 (IGFBP-4) played a critical role in beneficial effect of ghMSCs. ghMSCs were induced from MSCs and treated in in vitro and in vivo models of ischemia. They effectively protected neurons from ischemia and restored the brain damaged by cerebral infarction. These beneficial effects were significantly blocked by IGFBP-4 antibody. The current study demontsrated that late passage hMSCs can be efficiently induced into ghMSCs with better neuroprotective effect on ischemic stroke. Moreover, the results indicate that IGFBP-4 released from ghMSCs may serve as one of the key neuronal survival factors secreted from ghMSCs.

Insights into the biology of fibrodysplasia ossificans progressiva using patient-derived induced pluripotent stem cells.

The demand for development of new drugs remains on the upward trend because of the large number of patients suffering from intractable diseases for which effective treatment has not been established yet. Recently, several researchers have attempted to apply induced pluripotent stem cell (iPSC) technology as a powerful tool for studying the mechanisms underlying the onset of various diseases and for new drug screening. This technology has made an enormous breakthrough, since it permits us to recapitulate the disease phenotype in vitro, outside of the patient's body. Here, we discuss the latest findings that uncovered a mechanism underlying the pathology of a rare genetic musculoskeletal disease, fibrodysplasia ossificans progressiva (FOP), by modeling the phenotypes with FOP patient-derived iPSCs, and that discovered promising candidate drugs for FOP treatment. We also discussed future directions of FOP research.

Glycoengineering of HEK293 cells to produce high-mannose-type N-glycan structures.

Therapeutic proteins are a developing part of the modern biopharmaceutical industry, providing novel therapies to intractable diseases including cancers and autoimmune diseases. The human embryonic kidney 293 (HEK293) cell line has been widely used to produce recombinant proteins in both basic science and industry. The heterogeneity of glycan structures is one of the most challenging issues in the production of therapeutic proteins. Previously, we knocked out genes encoding α1,2-mannosidase-Is, MAN1A1, MAN1A2 and MAN1B1, in HEK293 cells, establishing a triple-knockout (T-KO) cell line, which produced recombinant protein with mainly high-mannose-type N-glycans. Here, we further knocked out MAN1C1 and MGAT1 encoding another Golgi α1,2-mannosidase-I and N-acetylglucosaminyltransferase-I, respectively, based on the T-KO cells. Two recombinant proteins, lysosomal acid lipase (LIPA) and immunoglobulin G1 (IgG1), were expressed in the quadruple-KO (QD-KO) and quintuple-KO (QT-KO) cell lines. Glycan structural analysis revealed that all the hybrid-type and complex-type N-glycans were eliminated, and only the high-mannose-type N-glycans were detected among the recombinant proteins prepared from the QD-KO and QT-KO cells. Overexpression of the oncogenes MYC and MYCN recovered the slow growth in QD-KO and QT-KO without changing the glycan structures. Our results suggest that these cell lines could be suitable platforms to produce homogeneous therapeutic proteins.

Mediators of the homeostasis and effector functions of memory Th2 cells as novel drug targets in intractable chronic allergic diseases.

Allergic patients have life-long chronic inflammatory diseases with repeated relapses and exacerbations. Currently used allergy therapeutics have some limitations, which warrants a search for novel drug targets for allergy treatment. The studies on conventional allergic disease therapeutics have been focused on the pathology of allergy involving effector type 2 helper T cells (Th2). However, it has been suggested that allergen-specific memory Th2 cells are developed after the initial allergen exposure, which may play a critical role in the allergic relapses. Here, we discuss the contribution of memory Th2 cells to allergic diseases and the microenvironmental factors for chronic allergic disease persistence. Since most allergy drugs are prescribed to suppress symptoms of the diseases, targeting the different types of cells or factors contributing to allergic diseases persistence may cure the disease.

Establishment of DHFR-deficient HEK293 cells for high yield of therapeutic glycoproteins

Since the use of protein therapeutics is effective for treating intractable human diseases, the production of biologic therapeutic agents has dramatically increased over the past three decades. The Chinese hamster ovary (CHO) cell lines are the most commonly used host cell expression system for recombinant protein production. High productive and stable clonal cell lines for recombinant protein production have been established from the DHFR-deficient CHO cell using the dihydrofolate reductase/methotrexate (DHFR/MTX) selection methods. Human embryonic kidney 293 (HEK293) cells are alternative host cells widely used for protein production. In most case, however, the cells are used for the transient expression, and there is no gene amplification system in HEK293 cells. In this study, we established a DHFR-deficient HEK293 cell line for the high yield of recombinant proteins. We doubly knocked out DHFR and DHFR2 in the MAN1A1/A2/B1/C1-quadruple knockout HEK293 (QD-KO) cells, using the CRISPR/Cas9 system. The DHFR-deficient QD-KO cells were used to overexpress two proteins, lysosomal acid lipase and the constant fragment of human immunoglobulin G1 by the DHFR/MTX gene-amplification method. This method resulted in a dramatic increase in the two protein expressions in the DHFR-deficient QD-KO cells by increasing MTX concentration. Our system could be adopted in the production of several recombinant proteins including therapeutic proteins.

SAT-LB066 Clinical Characteristics of Incidentally Discovered Functioning Adrenal Tumors: Study of Advancing Care and Pathogenesis of Intractable Adrenal Diseases in Japan (ACPA-J)

Objectives: Pheochromocytoma and paraganglioma (PPGL), Cushing syndrome (CS), bilateral macronodular adrenal hyperplasia (BMAH) and adrenocortical carcinoma (ACC) are rare adrenal tumors. Hence, collaboration of researchers and clinicians from several institutes and constructing patient registry are important to achieve progress in clinical management. We organized a research group and constructed a patient registry system for adrenal tumors named Advancing Care and Pathogenesis of Intractable Adrenal Diseases in Japan (ACPA-J). In the present study, we focused on incidentally discovered adrenal tumors despite the functioning tumors in ACPA-J database, and investigated their clinical characteristics. Subjects and methods: Clinical data of 176 patients with PPGL, 110 patients with CS, 38 patients with BMAH and 45 patients with ACC who were diagnosed 2006-2016 were registered to ACPA-J database by August 2018. Among the patients who have hormone excess confirmed by endocrinological evaluation, we analyzed the clinical characteristics of an incidentaloma (I) group and a non-incidentaloma (non-I) group in which their tumors were found based on signs and symptoms of adrenal disease, and compared those between two groups. Results: The hormone excess was confirmed in 162 patients with PPGL, 110 patients with CS, 38 patients with BMAH and 36 patients with ACC. Of those, 53.2% of PPGL, 36.7% of CS, 76.5% of BMAH and 24.5% of ACC were incidentaloma, respectively. Age in I group was higher in PPGL, CS and ACC compare to that in non-I group. Tumor size was larger in I group than that in non-I group only in ACC. Incidences of metabolic complications (hypertension, glucose intolerance and hyperlipidemia) were not different between two groups in all type of tumor. In PPGL, urine metanephrine levels and normetanephrine levels were not different between two groups. In CS and BMAH, morning plasma (p-) ACTH levels and p-cortisol levels in the morning, the midnight and after dexamethasone suppression test were not different between two groups. A rate of patients with subclinical cortisol excess was 13% in I group and 14% in non-I group in BMAH, whereas that in I group (50%) was higher than that in non-I group (23%) in ACC. Conclusions: The adrenal incidentaloma is defined as the tumor unexpectedly found during work-up for an indication unrelated to suspected adrenal disease. Most of the functioning adrenal tumors accompany signs and symptoms of hormone excess and have to be discovered in the process of work-up for adrenal disease. Our result showed there was not particular difference in characteristics between the incidentalomas and the non-incidentalomas. It was suggested that adrenal functioning tumors could be frequently overlooked. (This research was supported by AMED; JP18ek0109352, the Grant for NCGM; 30-A-1008, Health Labour Sciences Research Grant 2018.) Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

The immunopathology of lung fibrosis: amphiregulin-producing pathogenic memory T helper-2 cells control the airway fibrotic responses by inducing eosinophils to secrete osteopontin.

Fibrosis is defined as excessive deposition of the extracellular matrix (ECM) in the parenchyma of various organs, and sometimes leads to irreversible organ malfunction such as idiopathic pulmonary fibrosis (IPF), a fatal disorder of the lung. Chronic inflammatory stimuli induce fibrotic responses in various organs. Various immune cells, including T helper (Th) cells in the lung, protect the host from different harmful particles, including pathogenic microorganisms. However, the dysregulation of the function of these immune cells in the lung sometimes causes inflammatory diseases, such as lung fibrosis. In this review, we will introduce an outline of the cellular and molecular mechanisms underlying the pathogenic fibrotic responses in the lung. We will also introduce the concept of the "Pathogenic Th population disease induction model," in which unique subpopulations of certain Th cell subsets control the pathology of immune-mediated inflammatory diseases. Finally, we introduce our recent findings, which demonstrate that amphiregulin-producing pathogenic memory Th2 cells control airway fibrosis through the osteopontin produced by inflammatory eosinophils. The identification of this new pathogenic Th cell population supports the concept of "Pathogenic Th population disease induction model", and will provide novel strategies for treating intractable diseases, including lung fibrosis.

Hypoxia-induced disruption of neural vascular barrier is mediated by the intracellular induction of Fe(II) ion

Neural vascular barrier maintains the optimal tissue microenvironment of central nervous system in which neural cells can function normally. In various neural diseases, the decrease in oxygen concentration, hypoxia, of affected tissues is known to accelerate the disease progression through disruption of neural vascular barrier. Therefore, the clarification of mechanisms underlying hypoxia-induced disruption of neural vascular barrier would definitely lead to the establishment of new effective therapies for intractable neural diseases. In the present study, we first found that hypoxia disrupts neural vascular barrier through pathways independent of HIF-1α and HIF-2α. Then, with a specific fluorescence probe for ferrous, Fe(II) ion, we have obtained the interesting data showing that hypoxia increased the intracellular level of Fe(II) ion in endothelial cells of our in vitro model for neural vascular barrier, and that hypoxia-induced disruption of neural vascular barrier could be inhibited by chelating Fe(II) ion in endothelial cells. Furthermore, in the presence of a reducing reagent for reactive oxygen species (ROS), hypoxia could not disrupt the neural vascular barrier despite that the hypoxic increase in intracellular level of Fe(II) ion was confirmed in endothelial cells. These results indicate that hypoxia-triggered increase in the level of intracellular Fe(II) ion and subsequent production of ROS, probably through Fenton reaction, are the essential pathway mediating the disruption of neural vascular barrier under hypoxia.

Research and development on intractable & rare diseases in Japan :Contribution of the National Institute of Public Health to research program management

Research and development on intractable & rare diseases in Japan :Contribution of the National Institute of Public Health to research program management

Long-Term Implantable, Flexible, and Transparent Neural Interface Based on Ag/Au Core-Shell Nanowires.

Neural interfaces enabling light transmittance rely on optogenetics to control and monitor specific neural activity, thereby facilitating deeper understanding of intractable diseases. This study reports the material strategy underlying an optogenetic neural interface comprising stretchable and transparent conductive tracks and capable of demonstrating high biocompatibility after long-term (5-month) implantation. Ag/Au core-shell nanowires contribute toward improving track performance in terms of stretchability (<60% strain), transparency (<83%), and electrical resistance (15 Ω sq-1 ). The neural interface integrated with gel-coated exterior microelectrodes preserves low impedance (1.1-3.2 Ω cm2 ) in a saline solution over the evaluated 5-month period. Besides the use of efficient conductive materials, surface treatment using antithrombogenic polymer tends to prevent the growth of granulation tissue, thereby facilitating clear monitoring of electrocorticograms (ECoG) in a rodent during chronic implantation. The flexible and transparent neural interface pathologically exhibits noncytotoxicity and low inflammatory response while efficiently recording evoked ECoG in a nonhuman primate via optogenetic stimulation. The proposed highly reliable interface can be employed in multifaceted approaches for translational research based on chronic implants.

Phage-Based Artificial Niche: The Recent Progress and Future Opportunities in Stem Cell Therapy.

Self-renewal and differentiation of stem cells can be the best option for treating intractable diseases in regenerative medicine, and they occur when these cells reside in a special microenvironment, called the “stem cell niche.” Thus, the niche is crucial for the effective performance of the stem cells in both in vivo and in vitro since the niche provides its functional cues by interacting with stem cells chemically, physically, or topologically. This review provides a perspective on the different types of artificial niches including engineered phage and how they could be used to recapitulate or manipulate stem cell niches. Phage-based artificial niche engineering as a promising therapeutic strategy for repair and regeneration of tissues is also discussed.

Domesticating the foreign body response: Recent advances and applications.

The foreign body response is an immunological process that leads to the rejection of implanted devices and presents a fundamental challenge to their performance, durability, and therapeutic utility. Recent advances in materials development and device design are now providing strategies to overcome this immune-mediated reaction. Here, we briefly review our current mechanistic understanding of the foreign body response and highlight new anti-FBR technologies from this decade that have been applied successfully in biomedical applications relevant to implants, devices, and cell-based therapies. Further development of these important technologies promises to enable new therapies, diagnostics, and revolutionize the management of patient care for many intractable diseases.

Reactive Amphiphilic Conjugated Polymers for Inhibiting Amyloid β Assembly.

Protein misfolding and aberrant aggregations are associated with multiple prevalent and intractable diseases. Inhibition of amyloid assembly is a promising strategy for the treatment of amyloidosis. Reported here is the design and synthesis of a reactive conjugated polymer, a poly(p-phenylene vinylene) derivative, functionalized with p-nitrophenyl esters (PPV-NP) and it inhibits the assembly of amyloid proteins, degrades preformed fibrils, and reduces the cytotoxicity of amyloid aggregations in living cells. PPV-NP is attached to the proteins through hydrophobic interactions and irreversible covalent linkage. PPV-NP also exhibited the capacity to eliminate Aβ plaques in brain slices in ex vivo assays. This work represents an innovative attempt to inhibit protein pathogenic aggregates, and may offer insights into the development of therapeutic strategies for amyloidosis.

Reactive Amphiphilic Conjugated Polymers for Inhibiting Amyloid β Assembly

AbstractProtein misfolding and aberrant aggregations are associated with multiple prevalent and intractable diseases. Inhibition of amyloid assembly is a promising strategy for the treatment of amyloidosis. Reported here is the design and synthesis of a reactive conjugated polymer, a poly(p‐phenylene vinylene) derivative, functionalized with p‐nitrophenyl esters (PPV‐NP) and it inhibits the assembly of amyloid proteins, degrades preformed fibrils, and reduces the cytotoxicity of amyloid aggregations in living cells. PPV‐NP is attached to the proteins through hydrophobic interactions and irreversible covalent linkage. PPV‐NP also exhibited the capacity to eliminate Aβ plaques in brain slices in ex vivo assays. This work represents an innovative attempt to inhibit protein pathogenic aggregates, and may offer insights into the development of therapeutic strategies for amyloidosis.

Immune Cell-Epithelial/Mesenchymal Interaction Contributing to Allergic Airway Inflammation Associated Pathology.

The primary function of the lung is efficient gas exchange between alveolar air and alveolar capillary blood. At the same time, the lung protects the host from continuous invasion of harmful viruses and bacteria by developing unique epithelial barrier systems. Thus, the lung has a complex architecture comprising a mixture of various types of cells including epithelial cells, mesenchymal cells, and immune cells. Recent studies have revealed that Interleukin (IL-)33, a member of the IL-1 family of cytokines, is a key environmental cytokine that is derived from epithelial cells and induces type 2 inflammation in the barrier organs, including the lung. IL-33 induces allergic diseases, such as asthma, through the activation of various immune cells that express an IL-33 receptor, ST2, including ST2+ memory (CD62LlowCD44hi) CD4+ T cells. ST2+ memory CD4+ T cells have the capacity to produce high levels of IL-5 and Amphiregulin and are involved in the pathology of asthma. ST2+ memory CD4+ T cells are maintained by IL-7- and IL-33-produced lymphatic endothelial cells within inducible bronchus-associated lymphoid tissue (iBALT) around the bronchioles during chronic lung inflammation. In this review, we will discuss the impact of these immune cells-epithelial/mesenchymal interaction on shaping the pathology of chronic allergic inflammation. A better understanding of pathogenic roles of the cellular and molecular interaction between immune cells and non-immune cells is crucial for the development of new therapeutic strategies for intractable allergic diseases.

Integrated radiofrequency array and animal holder design for minimizing head motion during awake marmoset functional magnetic resonance imaging

Marmosets are small New World primates that are posited to become an important preclinical animal model for studying intractable human brain diseases. A critical step in the development of marmosets as a viable model for human brain dysfunction is to characterize brain networks that are homologous with human network topologies. In this regard, the use of functional magnetic resonance imaging (fMRI) holds tremendous potential for functional brain mapping in marmosets. Although possible, implementation of hardware for fMRI in awake marmosets (free of the confounding effects of anesthesia) is not trivial due to the technical challenges associated with developing specialized imaging hardware. Here, we describe the design and implementation of a marmoset holder and head-fixation system with an integrated receive coil for awake marmoset fMRI. This design minimized head motion, with less than 100 μm of translation and 0.5 degrees of rotation over 15 consecutive resting state fMRI runs (at 15 min each) across 3 different marmosets. The fMRI data was of sufficient quality to reliably extract 8 resting state networks from each animal with only 60–90 min of resting state fMRI acquisition per animal. The restraint system proved to be an efficient and practical solution for securing an awake marmoset and positioning a receive array within minutes, limiting stress to the animal. This design is also amenable for multimodal imaging, allowing for electrode or lens placement above the skull via the open chamber design. All computer-aided-design (CAD) files and engineering drawings are provided as an open resource, with the majority of the parts designed to be 3D printed.

Regenerative medicine using stem cells from human exfoliated deciduous teeth (SHED): a promising new treatment in pediatric surgery.

Stem cells from human exfoliated deciduous teeth (SHEDs), being a type of mesenchymal stem cell, are an ideal cell source for regenerative medicine. They have minimal risk of oncogenesis, high proliferative capacity, high multipotency, and immunosuppressive ability. Stem cell transplantation using SHED has been found to have an anti-fibrotic effect on liver fibrosis in mice. SHED transplantation and the bio 3D printer, which can create scaffold-free 3-D images of the liver and diaphragm, provide a new innovative treatment modality for intractable pediatric surgical diseases such as biliary atresia and diaphragmatic hernia.

Vangl2 interaction plays a role in the proteasomal degradation of Prickle2

The PET and LIM domain-containing protein, Prickle, plays a key role in planar cell polarity (PCP) in Drosophila. It has been reported that mutations in the PRICKLE2 gene, which encodes one of the human orthologues of Prickle, are associated with human diseases such as epilepsy and autism spectrum disorder. To develop preventive and therapeutic strategies for these intractable diseases, we studied the regulation of Prickle2 protein levels in transfected HEK293T cells. Prickle2 levels were negatively regulated by a physical interaction with another PCP protein, Van Gogh-like 2 (Vangl2). The Vangl2-mediated reduction in Prickle2 levels was, at least in part, relieved by proteasome inhibitors or by functional inhibition of the Cullin-1 E3 ubiquitin ligase. Furthermore, the expression of Vangl2 enhanced the polyubiquitination of Prickle2. This ubiquitination was partially blocked by co-expression of a ubiquitin mutant, which cannot be polymerised through their Lys48 residue to induce target proteins toward proteasomal degradation. Together, these results suggest that Prickle2 is polyubiquitinated by the Vangl2 interaction in a Cullin-1-dependent manner to limit its expression levels. This regulation may play a role in the local and temporal fine-tuning of Prickle protein levels during PCP signal-dependent cellular behaviours.

Cell Therapies for Parkinson's Disease.

Cell Therapies for Parkinson's Disease.