Human RELA dominant-negative mutations underlie type I interferonopathy with autoinflammation and autoimmunity.

Inborn errors of the NF-κB pathways underlie various clinical phenotypes in humans. Heterozygous germline loss-of-expression and loss-of-function mutations in RELA underlie RELA haploinsufficiency, which results in TNF-dependent chronic mucocutaneous ulceration and autoimmune hematological disorders. We here report six patients from five families with additional autoinflammatory and autoimmune manifestations. These patients are heterozygous for RELA mutations, all of which are in the 3' segment of the gene and create a premature stop codon. Truncated and loss-of-function RelA proteins are expressed in the patients' cells and exert a dominant-negative effect. Enhanced expression of TLR7 and MYD88 mRNA in plasmacytoid dendritic cells (pDCs) and non-pDC myeloid cells results in enhanced TLR7-driven secretion of type I/III interferons (IFNs) and interferon-stimulated gene expression in patient-derived leukocytes. Dominant-negative mutations in RELA thus underlie a novel form of type I interferonopathy with systemic autoinflammatory and autoimmune manifestations due to excessive IFN production, probably triggered by otherwise non-pathogenic TLR ligands.

Reduced-intensity conditioning is effective for allogeneic hematopoietic stem cell transplantation in infants with MECOM-associated syndrome.

Mutations in the MECOM encoding EVI1 are observed in infants who have radioulnar synostosis with amegakaryocytic thrombocytopenia. MECOM-associated syndrome was proposed based on clinical heterogeneity. Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for progressive bone marrow failure. However, data regarding allogeneic HSCT for this rare disease are limited. We retrospectively assessed overall survival, conditioning regimen, regimen-related toxicities and long-term sequelae in six patients treated with allogeneic HSCT. All patients received a reduced-intensity conditioning (RIC) regimen consisting of fludarabine, cyclophosphamide or melphalan, and rabbit anti-thymocyte globulin and/or low-dose total body/thoracic-abdominal/total lymphoid irradiation, followed by allogeneic bone marrow or cord blood transplantation from unrelated donors between 4 and 18 months of age. All patients survived and achieved stable engraftment and complete chimerization with the donor type. Moreover, no patient experienced severe regimen-related toxicities, and only lower grades of acute graft-versus-host disease were observed. Three patients treated with low-dose irradiation had relatively short stature compared to three patients not treated with irradiation. Therefore, allogeneic HSCT with RIC is an effective and feasible treatment for infants with MECOM-associated syndrome. Future studies are needed to evaluate the use of low-dose irradiation to avoid risks of other long-term sequelae.

Novel POLE mutations identified in patients with IMAGE-I syndrome cause aberrant subcellular localisation and protein degradation in the nucleus.

BACKGROUND: DNA replisome is a molecular complex that plays indispensable roles in normal DNA replication. IMAGE-I syndrome is a DNA replisome-associated genetic disease caused by biallelic mutations in the gene encoding DNA polymerase epsilon catalytic subunit 1 (POLE). However, the underlying molecular mechanisms remain largely unresolved. METHODS: The clinical manifestations in two patients with IMAGE-I syndrome were characterised. Whole-exome sequencing was performed and altered mRNA splicing and protein levels of POLE were determined. Subcellular localisation, cell cycle analysis and DNA replication stress were assessed using fibroblasts and peripheral blood from the patients and transfected cell lines to determine the functional significance of POLE mutations. RESULTS: Both patients presented with growth retardation, adrenal insufficiency, immunodeficiency and complicated diffuse large B-cell lymphoma. We identified three novel POLE mutations: namely, a deep intronic mutation, c.1226+234G>A, common in both patients, and missense (c.2593T>G) and in-frame deletion (c.711_713del) mutations in each patient. The unique deep intronic mutation produced aberrantly spliced mRNAs. All mutants showed significantly reduced, but not null, protein levels. Notably, the mutants showed severely diminished nuclear localisation, which was rescued by proteasome inhibitor treatment. Functional analysis revealed impairment of cell cycle progression and increase in the expression of phospho-H2A histone family member X in both patients. CONCLUSION: These findings provide new insights regarding the mechanism via which POLE mutants are highly susceptible to proteasome-dependent degradation in the nucleus, resulting in impaired DNA replication and cell cycle progression, a characteristic of DNA replisome-associated diseases.

Clofarabine monotherapy in two patients with refractory Langerhans cell histiocytosis.

BACKGROUND: Better therapeutic options other than conventional chemotherapy for pediatric patients with refractory Langerhans cell histiocytosis (LCH) remain undetermined. CASE: We successfully treated two patients with refractory and risk organ negative LCH with clofarabine (CLO) monotherapy after recurrence. We administered total 23 courses of CLO monotherapy in patient 1 and 4 courses in patient 2. Both patients had distinct clinical manifestations but achieved a durable complete response with acceptable adverse effects of transient myelosuppression. CLO monotherapy was still effective when he had the second recurrent lesion after first completion of CLO in patient 1. We could discontinue prednisolone to control his refractory inflammation of LCH after completing CLO chemotherapy in patient 2. CONCLUSION: Although large-scale studies are warranted, CLO monotherapy could be a therapeutic option for high efficacy and feasibility besides other intensive combination chemotherapies or allogeneic hematopoietic stem cell transplantation for refractory LCH without risk organ involvement in children.

Refractory T-cell/histiocyte-rich large B-cell lymphoma in a patient with ataxia-telangiectasia caused by novel compound heterozygous variants in ATM.

Ataxia-telangiectasia (A-T) is an autosomal recessive chromosomal breakage syndrome caused by mutation of the ATM (A-T mutated) gene, which encodes a protein kinase that has a major role in the cellular response to DNA damage. Approximately, 10% of A-T patients develop lymphoid malignancies. Deaths caused by extreme sensitivity to chemotherapy for malignancy have been reported, and cancer treatment in A-T is extraordinarily difficult, needing careful monitoring and individualized protocols. We report the case of a 12-year-old girl with A-T diagnosed at the age of 3 in association with IgA deficiency and recurrent pulmonary infections. Sanger sequencing revealed compound heterozygosity of the ATM gene, which bore two novel mutations. At the age of 12, she developed stage IV T-cell/histiocyte-rich large B-cell lymphoma. The tumor was resistant to chemotherapy, and she unfortunately died of cardiac insufficiency and multiple organ failure induced by rapid progression of the disease. The treatment approach for children with A-T and advanced-stage B-non-Hodgkin lymphoma must be refined.

Case Report: Infantile-Onset Fulminant Type 1 Diabetes Mellitus Caused by Novel Compound Heterozygous LRBA Variants.

Lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency is a subtype of common variable immune deficiency (CVID). Numerous case reports and cohort studies have described a broad spectrum of clinical manifestations and variable disease phenotypes, including immune dysregulation, enteropathy, and recurrent infections. Although LRBA deficiency is an autosomal recessive primary immunodeficiency resulting in a phenotype similar to CVID, it is a monogenic disease and separate from CVID. Recently, in a report of monogenic primary immunodeficiency disorder associated with CVID and autoimmunity, the most common mutated gene was LRBA. We report the case of a girl who presented with fulminant type 1 diabetes at age 7 months. She later experienced recurrent bacterial infections with neutropenia and idiopathic thrombocytopenic purpura. Clinical genome sequencing revealed compound heterozygosity of the LRBA gene, which bore two novel mutations. A genetic basis should be considered in the differential diagnosis for very young patients with fulminant autoimmunity, and the diagnostic work-up should include evaluation of markers of immunodeficiency.

Identification of pyrogallol from Awa-tea as an anti-allergic compound that suppresses nasal symptoms and IL-9 gene expression.

As the expression level of allergic disease sensitive genes are correlated with the severity of allergic symptoms, suppression of these gene expressions could be promising therapeutics. We demonstrated that protein kinase Cδ / heat shock protein 90-mediated H1R gene expression signaling and nuclear factor of activated T-cells (NFAT)-mediated IL-9 gene expression signaling are responsible for the pathogenesis of pollinosis. Treatment with Awa-tea combined with wild grape hot water extract suppressed these signaling and alleviated nasal symptoms in toluene-2,4-diisocyanate (TDI)-sensitized rats. However, the underlying mechanism of its anti-allergic activity is not elucidated yet. Here, we sought to identify an anti-allergic compound from Awa-tea and pyrogallol was identified as an active compound. Pyrogallol strongly suppressed ionomycin-induced up-regulation of IL-9 gene expression in RBL-2H3 cells. Treatment with pyrogallol in combination with epinastine alleviated nasal symptoms and suppressed up-regulation of IL-9 gene expression in TDI-sensitized rats. Pyrogallol itself did not inhibit calcineurin phosphatase activity. However, pyrogallol suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFAT. These data suggest pyrogallol is an anti-allergic compound in Awa-tea and it suppressed NFAT-mediated IL-9 gene expression through the inhibition of dephosphorylation of NFAT. This might be the underlying mechanism of the therapeutic effects of combined therapy of pyrogallol with antihistamine. J. Med. Invest. 67 : 289-297, August, 2020.

Correction to: the IL1RN Mutation Creating the Most-Upstream Premature Stop Codon Is Hypomorphic because of a Reinitiation of Translation.

The original version of our manuscript, entitled, " The IL1RN mutation creating the most-upstream premature stop codon is hypomorphic because of a reinitiation of translation" unfortunately contained mistakes in Fig. 1a and d legends. The text should read as follows.

Ruxolitinib treatment of a patient with steroid-dependent severe autoimmunity due to STAT1 gain-of-function mutation.

Signal transducer and activator of transcription 1 gain-of-function (STAT1 GOF) mutations are the most common cause of chronic mucocutaneous candidiasis (CMC). We report the effect of oral ruxolitinib, an inhibitor of Janus kinase (JAK) family tyrosine kinases, on the clinical and immune status of a 3-year-old male with steroid-dependent severe autoimmunity due to a STAT1 GOF T385M mutation. The patient's susceptibility to infection improved with antimicrobial prophylaxis and immunoglobulin replacement therapy, but he continued to exhibit severely disabling symptoms of autoimmunity. More than one-third of patients with STAT1 GOF mutations present with autoimmune manifestations, and this patient's mutation was reported to cause CMC with autoimmunity. We analyzed the interleukin (IL)-17A and IFN-γ levels and immunophenotype by flow cytometry before and during treatment with ruxolitinib. The peripheral IL-17A level did not increase, but the IFN-γ level decreased after 4 months of therapy. The STAT1 phosphorylation level decreased significantly upon stimulation of patient cells with IFN-γ. Clinically, cytomegalovirus reactivation occurred, but was controlled. No other adverse effect was noted. We report the potential of JAK1/2 inhibition with ruxolitinib for both CMC and steroid-dependent autoimmunity. However, long-term administration is necessary, as the effect is not sustained after treatment is discontinued.

Prototyping context-aware nursing support mobile system.

A context aware nursing support system to push right information to the right person at the right moment is the key to increase clinical safety under a computerized hospital. We prototyped a system which obtains context from positions of nurses and list of expected clinical procedures. A WoZ test showed that the proposed approach has potential to decrease incidents caused by information delivery error.

Maackiain is a novel antiallergic compound that suppresses transcriptional upregulation of the histamine H1 receptor and interleukin-4 genes.

Kujin contains antiallergic compounds that inhibit upregulation of histamine H1 receptor (H1R) and interleukin (IL)-4 gene expression. However, the underlying mechanism remains unknown. We sought to identify a Kujin-derived antiallergic compound and investigate its mechanism of action. The H1R and IL-4 mRNA levels were determined by real-time quantitative RT-PCR. To investigate the effects of maackiain in vivo, toluene-2,4-diisocyanate (TDI)-sensitized rats were used as a nasal hypersensitivity animal model. We identified (-)-maackiain as the responsible component. Synthetic maackiain showed stereoselectivity for the suppression of IL-4 gene expression but not for H1R gene expression, suggesting distinct target proteins for transcriptional signaling. (-)-Maackiain inhibited of PKCδ translocation to the Golgi and phosphorylation of Tyr(311) on PKCδ, which led to the suppression of H1R gene transcription. However, (-)-maackiain did not show any antioxidant activity or inhibition of PKCδ enzymatic activity per se. Pretreatment with maackiain alleviated nasal symptoms and suppressed TDI-induced upregulations of H1R and IL-4 gene expressions in TDI-sensitized rats. These data suggest that (-)-maackiain is a novel antiallergic compound that alleviates nasal symptoms in TDI-sensitized allergy model rats through the inhibition of H1R and IL-4 gene expression. The molecular mechanism underlying its suppressive effect for H1R gene expression is mediated by the inhibition of PKCδ activation.

Acquisition of chemoresistance to gemcitabine is induced by a loss-of-function missense mutation of DCK.

The anti-tumor activity of gemcitabine (GEM) has been clinically proven in several solid tumors, including pancreatic cancer, biliary tract cancer, urinary bladder cancer, and non-small cell lung cancer. However, problems remain with issues such as acquisition of chemoresistance against GEM. GEM is activated after phosphorylation by deoxycytidine kinase (DCK) inside of the cell; thus, DCK inactivation is one of the important mechanisms for acquisition of GEM resistance. We previously investigated the DCK gene in multiple GEM resistant cancer cell lines and identified frequent inactivating mutations. In this study, we identified two crucial genetic alteration in DCK. (1) A total deletion of DCK in RTGBC1-TKB, an acquired GEM resistant cell line derived from a gall bladder cancer cell line TGBC1-TKB. (2) An E197K missense alteration of DCK in MKN28, a gastric cancer cell line; its acquired GEM resistant cancer cell line, RMKN28, showed a loss of the normal E197 allele. We introduced either normal DCK or altered DCK_E197K into RMKN28 and proved that only the introduction of normal DCK restored GEM sensitivity. Furthermore, we analyzed 104 healthy volunteers and found that none of them carried the same base substitution observed in MKN28. These results strongly suggest that (1) the E197K alteration in DCK causes inactivation of DCK, and that (2) loss of the normal E197 allele is the crucial mechanism in acquisition of GEM resistance in RMKN28.

Repair mechanism of osteochondral defect promoted by bioengineered chondrocyte sheet.

Cell sheet engineering has developed as a remarkable method for cell transplantation. In the field of cartilage regeneration, several studies previously reported that cartilage defects could be regenerated by transplantation of a chondrocyte sheet using cell sheet engineering. However, it remains unclear how such a thin cell sheet could repair a deep cartilage defect. We, therefore, focused on the mechanism of cartilage repair using cell sheet engineering in this study. Chondrocyte sheets and synovial cell sheets were fabricated using cell sheet engineering, and these allogenic cell sheets were transplanted to cover an osteochondral defect in a rat model. Macroscopic and histological evaluation was performed at 4 and 12 weeks after transplantation. Analysis of the gene expression of each cell sheet and of the regenerated tissue at 1 week after transplantation was performed. In addition, green fluorescent protein (GFP) transgenic rats were used as donors (transplanted chondrocyte sheets) or recipients (osteochondral defect models) to identify the cell origin of regenerated cartilage. Cartilage repair was significantly better in the group implanted with a chondrocyte sheet than in that with a synovial cell sheet. The results of gene expression analysis suggest that the possible factor contributing to cartilage repair might be TGFß1. Cell tracking experiments using GFP transgenic rats showed that the regenerated cartilage was largely composed of cells derived from the transplanted chondrocyte sheets.

Quercetin inhibits transcriptional up-regulation of histamine H1 receptor via suppressing protein kinase C-δ/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 signaling pathway in HeLa cells.

It has been reported that the histamine H1 receptor (H1R) gene is up-regulated in patients with allergic rhinitis and H1R expression level strongly correlates with the severity of allergy symptoms. Accordingly compounds that suppress the H1R gene expression are promising as useful anti-allergic medications. Recently, we demonstrated that histamine or phorbol-12-myristate-13-acetate (PMA) stimulation induced the up-regulation of H1R gene expression through the protein kinase Cδ (PKCδ)/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 signaling pathway in HeLa cells expressing H1R endogenously. Quercetin is one of the well-characterized flavonoids and it possesses many biological activities including anti-allergic activity. However, effect of quercetin on H1R signaling is remained unknown. In the present study, we examined the effect of quercetin on histamine- and PMA-induced up-regulation of H1R gene expression in HeLa cells. We also investigated its in vivo effects on the toluene-2,4-diisocyanate (TDI)-sensitized allergy model rats. Quercetin suppressed histamine- and PMA-induced up-regulation of H1R gene expression. Quercetin also inhibited histamine- or PMA-induced phosphorylation of Tyr(311) of PKCδ and translocation of PKCδ to the Golgi. Pre-treatment with quercetin for 3weeks suppressed TDI-induced nasal allergy-like symptoms and elevation of H1R mRNA in the nasal mucosa of TDI-sensitized rats. These data suggest that quercetin suppresses H1R gene expression by the suppression of PKCδ activation through the inhibition of its translocation to the Golgi.

DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells.

Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.

Case of clear cell ependymoma of medulla oblongata: clinicopathological and immunohistochemical study with literature review.

Clear cell ependymoma has been included in the WHO classification of the central nervous system in 1993, after the first report by Kawano et al. Since then, only a few cases have been reported. Most clear cell ependymoma cases reported in the literature so far were located in the supra-tentorial compartment and/or cerebellum, and one case was in the cervical spinal cord. We report a case of clear cell ependymoma whose histological features were sufficient for the diagnosis and was unusually located in the fourth ventricle originating from the medulla oblongata. The tumor showed uniform tumor cells with perinuclear halo, nuclei being centrally located. Most of the tumor cells were arranged as perivascular pseudorosettes, and no ependymal canals or rosettes were evident. Mitotic figures were not frequent. Immunohistochemically, the tumor cells were strongly reactive for glial fibrillary acidic protein and vimentin, and weak and dot-like positive for epithelial membrane antigen. Clear cell change of the tumor cells appeared to be fixation artifact because this feature was not evident in the frozen section.