Safety and tolerability of a Muse cell-based product in neonatal hypoxic-ischemic encephalopathy with therapeutic hypothermia (SHIELD trial).

Hypoxic-ischemic encephalopathy (HIE), associated with high mortality and neurological sequelae, lacks established treatment except therapeutic hypothermia. Clinical-grade multilineage-differentiating stress-enduring (Muse) cells (CL2020) demonstrated safety and efficacy in nonclinical HIE rat models, thereby leading to an investigator-initiated clinical trial to evaluate CL2020 safety and tolerability in neonatal HIE as a single-center open-label dose-escalation study with 9 neonates with moderate-to-severe HIE who received therapeutic hypothermia. Each patient received a single intravenous injection of CL2020 cells between 5 and 14 days of age. The low-dose (3 patients) and high-dose (6 patients) groups received 1.5 × 106 and 1.5 × 107 cells/dose, respectively. The occurrence of any adverse event within 12 weeks following CL2020 administration was the primary endpoint of this trial. No significant changes in physiological signs including heart rate, blood pressure, and oxygen saturation were observed during or after administration. The only adverse event that may be related to cell administration was a mild γ-glutamyltransferase level elevation in one neonate, which spontaneously resolved without any treatment. All patients enrolled in the trial survived, and normal developmental quotients (≥ 85) in all 3 domains of the Kyoto Scale of Psychological Development 2001 were observed in 67% of the patients in this trial. CL2020 administration was demonstrated to be safe and tolerable for neonates with HIE. Considering the small number of patients, a randomized controlled confirmatory study is warranted to verify these preliminary findings and evaluate the efficacy of this therapy.

ZBP1 activation triggers hematopoietic stem and progenitor cell death resulting in bone marrow failure in mice.

Human bone marrow failure (BMF) syndromes result from the loss of hematopoietic stem and progenitor cells (HSPC), and this loss has been attributed to cell death; however, the cell death triggers, and mechanisms remain unknown. During BMF, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) increase. These ligands are known to induce necroptosis, an inflammatory form of cell death mediated by RIPK1, RIPK3, and MLKL. We previously discovered that mice with a hematopoietic RIPK1 deficiency (Ripk1HEM KO) exhibit inflammation, HSPC loss, and BMF, which is partially ameliorated by a RIPK3 deficiency; however, whether RIPK3 exerts its effects through its function in mediating necroptosis or other forms of cell death remains unclear. Here, we demonstrate that similar to a RIPK3 deficiency, an MLKL deficiency significantly extends survival and like Ripk3 deficiency partially restores hematopoiesis in Ripk1HEM KO mice revealing that both necroptosis and apoptosis contribute to BMF in these mice. Using mouse models, we show that the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) is up-regulated in mouse RIPK1-deficient bone marrow cells and that ZBP1's function in endogenous nucleic acid sensing is necessary for HSPC death and contributes to BMF. We also provide evidence that IFNγ mediates HSPC death in Ripk1HEM KO mice, as ablation of IFNγ but not TNFα receptor signaling significantly extends survival of these mice. Together, these data suggest that RIPK1 maintains hematopoietic homeostasis by preventing ZBP1 activation and induction of HSPC death.

Prognostic heterogeneity and clonal dynamics within distinct subgroups of myelodysplastic syndrome and acute myeloid leukemia with TP53 disruptions.

TP53 aberrations constitute the highest risk subset of myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). The International Consensus Classification questions the blast threshold between MDS and AML. In this study, we assess the distinction between MDS and AML for 76 patients with TP53 aberrations. We observed no significant differences between MDS and AML regarding TP53 genomics. Median overall survival (OS) was 223 days for the entire group, but prognostic discrimination within subgroups showed the most inferior OS (46 days) for AML with multihit allelic state plus TP53 variant allele frequency (VAF) > 50%. In multivariate analysis, unadjusted Cox models revealed the following variables as independent risk factors for mortality: AML (vs. MDS) (hazard ratio [HR]: 2.50, confidence interval [CI]: 1.4-4.4, p = 0.001), complex karyotype (HR: 3.00, CI: 1.4-6.1, p = 0.003), multihit status (HR: 2.30, CI 1.3-4.2, p = 0.005), and absence of hematopoietic cell transplant (HCT) (HR: 3.90, CI: 1.8-8.9, p = 0.0009). Clonal dynamic modeling showed a significant reduction in TP53 VAF with front-line hypomethylating agents. These findings clarify the impact of specific covariates on outcomes of TP53-aberrant myeloid neoplasms, irrespective of the diagnosis of MDS versus AML, and may influence HCT decisions.

Thermal stress, hydration, and salivary and respiratory stress markers in curling players performing a match in the cold.

Curling is a target-based team sport played in a cold environment. The type of stress curling players face during a curling match remains to be determined. In the present study, 16 Japanese curling players performed a practice curling match (six ends lasting 90 min), wherein the following variables were documented: core and skin temperatures, heart rate, thermal sensation and comfort, urine-specific gravity, body fluid loss, salivary cortisol, α-amylase activity, salivary secretory immunoglobulin A (SIgA), and fractionated exhaled nitric oxide (FeNO, a respiratory stress marker). Pre-match resting core temperature was 37.24 ± 0.31°C, which increased up to 37.73 ± 0.41°C during the match (p < 0.001). Facial skin temperatures decreased after the match (all p ≤ 0.015), whereas finger skin temperatures remained unchanged (p ≥ 0.375). Thermal discomfort increased following the match but thermal sensation remained unchanged. Following the match, players lost 0.29 ± 0.15 L body fluid (sweat, respiratory evaporation, and urine), which was nearly compensated by fluid ingestion of 0.22 ± 0.13 L (p = 0.119). Nevertheless, urine-specific gravity increased from 1.021 ± 0.010 to 1.024 ± 0.008 after the match (p = 0.012), with 31% and 50% players being dehydrated at pre- and post-match, respectively. Salivary cortisol decreased (p < 0.001) after the match without changes in salivary SIgA, α-amylase activity, and FeNO (all p ≥ 0.113). Therefore, during a curling match, the core temperature and thermal discomfort increase, whereas the face skin temperature decreases. Additionally, players may undergo dehydration before the match, which could be exacerbated after the match.

Safety and tolerability of a multilineage-differentiating stress-enduring cell-based product in neonatal hypoxic-ischaemic encephalopathy with therapeutic hypothermia (SHIELD trial): a clinical trial protocol open-label, non-randomised, dose-escalation trial.

INTRODUCTION: Neonatal hypoxic-ischaemic encephalopathy (HIE) is an important illness associated with death or cerebral palsy. This study aims to assess the safety and tolerability of the allogenic human multilineage-differentiating stress-enduring cell (Muse cell)-based product (CL2020) cells in newborns with HIE. This is the first clinical trial of CL2020 cells in neonates. METHODS AND ANALYSIS: This is a single-centre, open-label, dose-escalation study enrolling up to 12 patients. Neonates with HIE who receive a course of therapeutic hypothermia therapy, which cools to a body temperature of 33°C-34°C for 72 hours, will be included in this study. A single intravenous injection of CL2020 cells will be administered between 5 and 14 days of age. Subjects in the low-dose and high-dose cohorts will receive 1.5 and 15 million cells per dose, respectively. The primary outcome is the occurrence of any adverse events within 12 weeks after administration. The main secondary outcome is the Bayley Scales of Infant and Toddler Development Third Edition score and the developmental quotient per the Kyoto Scale of Psychological Development 2001 at 78 weeks. ETHICS AND DISSEMINATION: This study will be conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. The Nagoya University Hospital Institutional Review Board (No. 312005) approved this study on 13 November 2019. The results of this study will be published in peer-reviewed journal and reported in international conferences. TRIAL REGISTRATION NUMBERS: NCT04261335, jRCT2043190112.

The 10th Japan Bioanalysis Forum Symposium conference: a report.

The Japan Bioanalysis Forum Symposium was held on 12-14 February 2019 (Yokohama, Japan), in celebration of its 10th anniversary, and over 370 participants from pharmaceutical industries, contractors, academia and regulatory authorities from home and abroad came together in Yokohama. The 3-day symposium particularly aimed to foster collaboration with the scientists surrounding bioanalysts, according to the theme 'Open to the Public.' The symposium also included a broad range of pioneering programs, such as lectures by speakers from DMPK/metabolomics fields, discussions of future bioanalysis and poster presentations by publicly offered presenters as well as the regular ones we had organized. This report summarizes the major topics as a conference report.

Verification of recall criteria for masses detected on ultrasound breast cancer screening.

PURPOSE: Mammography is the only modality for breast cancer screening demonstrated to reduce the mortality rate. However, ultrasonographic screening is already being widely performed as opportunistic screening in Japan. The recall criteria for masses are very important as quality controls. The purpose of this study was to verify these criteria at multiple institutions. METHODS: Screening was performed by five institutions in various regions in Japan. The total number of cases screened at all five institutions was 10,519. RESULTS: The findings that could be concluded to be benign were a cystic pattern and three features of a solid pattern. The cystic pattern was noted in 6512 cases, typical fibroadenoma in 1483 cases, and typical complicated cyst in 70 cases. Only three of these 8065 cases were cancers, so the negative predictive value was 99.9%. The solid pattern with obvious malignant features, i.e., masses with an echogenic halo and/or interruption of the interface and masses with multiple echogenic foci, were noted in 33 cases. Twenty of the 33 cases were malignancy, resulting in a positive predictive value of 66.7%. CONCLUSION: Although some parts of the criteria should be considered further for verification and revision, the current recall criteria are mostly valid.

Impairment of fetal hematopoietic stem cell function in the absence of Fancd2.

Fanconi anemia (FA) results from mutations in the genes necessary for DNA damage repair and often leads to progressive bone marrow failure. Although the exhaustion of the bone marrow leads to cytopenias in FA patients as they age, evidence from human FA and mouse model fetal livers suggests that hematopoietic defects originate in utero, which may lead to deficient seeding of the bone marrow. To address this possibility, we examined the consequences of loss of Fancd2, a central component of the FA pathway. Examination of embryonic day 14.5 (E14.5) Fancd2 knockout (KO) fetal livers showed a decrease in total cellularity and specific declines in long-term and short-term hematopoietic stem cell (LT-HSC and ST-HSC, respectively) numbers. Fancd2 KO fetal liver cells display similar functional defects to Fancd2 adult bone marrow cells, including reduced colony-forming units, increased mitomycin C sensitivity, increased LT-HSC apoptosis, and heavily impaired competitive repopulation, implying that these defects are intrinsic to the fetal liver and are not dependent on the accumulation of DNA damage during aging. Telomere shortening, an aging-related mechanism proposed to contribute to HSC apoptosis and bone marrow failure in FA, was not observed in Fancd2 KO fetal livers. In summary, loss of Fancd2 yields significant defects to fetal liver hematopoiesis, particularly the HSC population, which mimics key phenotypes from adult Fancd2 KO bone marrow independently of aging-accrued DNA damage.

Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii.

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.

Anodal transcranial direct current stimulation over the auditory cortex improved hearing impairment in a patient with brainstem encephalitis.

Transcranial direct current stimulation (tDCS) can alter cortical excitability, and has been effective in treating some neurological disorders. This case report describes the use of tDCS in a 13-year-old female who developed bilateral hearing impairment after brainstem encephalitis when she was 6 years old. Her auditory function was more impaired in her right ear than her left. Anodal stimulation (1 mA) was applied for 10 min to the left auditory cortex once per day for 4 consecutive days to improve her right ear speech discrimination score. Sustained and significant improvement in maximum speech discrimination was observed after the four tDCS treatments. To our knowledge, this is the first case report of improvement in speech discrimination after anodal stimulation of the auditory cortex. These results encourage further studies investigating the beneficial effects of tDCS in patients with hearing impairments.

Structures and reaction mechanisms of the two related enzymes, PurN and PurU.

The crystal structures of glycinamide ribonucleotide transformylases (PurNs) from Aquifex aeolicus (Aa), Geobacillus kaustophilus (Gk) and Symbiobacterium toebii (St), and of formyltetrahydrofolate hydrolase (PurU) from Thermus thermophilus (Tt) were determined. The monomer structures of the determined PurN and PurU were very similar to the known structure of PurN, but oligomeric states were different; AaPurN and StPurN formed dimers, GkPurN formed monomer and PurU formed tetramer in the crystals. PurU had a regulatory ACT domain in its N-terminal side. So far several structures of PurUs have been determined, yet, the mechanisms of the catalysis and the regulation of PurU have not been elucidated. We, therefore, modelled ligand-bound structures of PurN and PurU, and performed molecular dynamics simulations to elucidate the reaction mechanisms. The evolutionary relationship of the two enzymes is discussed based on the comparisons of the structures and the catalytic mechanisms.

Intradiploic ciliated epithelial inclusion cyst of the skull.

A 56-year-old woman presented with a cystic skull lesion in the right temporal bone detected after resection of breast cancer. She underwent resection of the skull tumor for pathological diagnosis and treatment. The tumor was covered with ciliated epithelium and there were no malignant findings. The pathological diagnosis was ciliated epithelial inclusion cyst. Intradiploic inclusion cysts of the skull presenting as a calvarial defect include epidermoid cysts and dermoid cysts, which are clinically difficult to differentiate. Ciliated epithelium lining an intradiploic inclusion cyst is very rare. Surgical resection is essential for a definitive diagnosis and differentiation from a neoplasm.

[A brain metastasis from ampullary carcinoma: a case report].

We report a rare case of brain metastasis from ampullary carcinoma. A 61-year-old woman who had a past history of ampullary carcinoma surgically treated two year previously, presented with symptoms of left visual disturbance and left hemiparesis. Brain magnetic resonance images demonstrated a right occipital lobe tumor with peritumoral edema and midline shift. Gross total resection was carried out. Pathological diagnosis was poorly differentiated adenocarcinoma with compartments of signet-ring cell carcinoma. Metastases outside the abdominal cavity are rare. Brain metastases from ampullary carcinomas are extremely rare and there are few reports about such cases.

[Neuromyelitis optica spectrum disorder: a case report].

Neuromyelitis optica (NMO) is a relapsing inflammatory disease of the central nervous system, usually affecting the optic nerves and the spinal cord. It is presumed to be an antibody-mediated disorder and the target antigen is the water channel aquaporin-4 (AQP4) on astrocyte cell membranes. NMO is a disease caused by astrocyte disorder and is distinct from multiple sclerosis (MS), which is a primarily demyelinating disease caused by oligodendrocyte disorder. In NMO, spinal MRI shows a T2-hyperintense, longitudinally extensive (≥ 3 vertebral segments) spinal cord lesion. The case, which has optic neuritis or transverse myelitis with the presence of AQP4 antibody, is called as NMO spectrum disorder. A 68 year-old woman with a history of hypertension and diabetes mellitus was brought to the former hospital by ambulance with acute onset of tetraparesis. She denied visual acuity disturbance. MRI revealed a T2-hyperintense lesion from C5 to T2 level. Laboratory examination showed the presence of AQP4 antibody and the absence of oligoclonal bands. Low-dose steroid treatment was started after establishing a diagnosis of NMO. She incompletely recovered from disability, although the T2-hyperintense lesion on MRI had almost disappeared six months after the onset. It is important to maintain a high index of suspicion for NMO in cases with a longitudinally extensive spinal cord lesion, because untreated NMO leads to severe disability.

Reversible helix-random coil transition of poly(m-phenylenediethynylene) by a rotaxane switch.

Pendant rotaxane switch-tethering poly(m-phenylene diethynylene) was synthesized by the polyoxidative coupling of a rotaxane containing an axle-terminal m-diethynylbenzene group and an optically active crown ether. The reversible helix-random coil transition of the polymer was successfully performed by the positional switching of the rotaxane wheel.

Structure of N-formylglycinamide ribonucleotide amidotransferase II (PurL) from Thermus thermophilus HB8.

The crystal structure of PurL from Thermus thermophilus HB8 (TtPurL; TTHA1519) was determined in complex with an adenine nucleotide, PO(4)(3-) and Mg(2+) at 2.35 Å resolution. TtPurL consists of 29 α-helices and 28 ß-strands, and one loop is disordered. TtPurL consists of four domains, A1, A2, B1 and B2, and the structures of the A1-B1 and A2-B2 domains were almost identical to each other. Although the sequence identity between TtPurL and PurL from Thermotoga maritima (TmPurL) is higher than that between TtPurL and the PurL domain of the large PurL from Salmonella typhimurium (StPurL), the secondary structure of TtPurL is much more similar to that of StPurL than to that of TmPurL.

Moyamoya disease complicated by Graves' disease and type 2 diabetes mellitus: report of two cases.

The pathogenesis of moyamoya disease remains unknown. Examination of diseases concurrent with moyamoya disease may offer a clue to clarify the pathogenesis. Coexistence of moyamoya disease, Graves' disease, and diabetes mellitus is very rare. We present the first cases in the literature. A 38-year-old man with moyamoya disease and a 43-year-old woman with quasi-moyamoya disease, both concurrent with Graves' disease and type 2 diabetes mellitus, are presented. Both patients underwent antithyroid therapy and revascularization. After normalization of thyroid hormones level and blood glucose level followed by revascularization, the symptoms of cerebral ischemia were improved. The common etiological factors of these diseases are discussed. Genetic and autoimmune factor appeared to be involved in the pathogenesis of the three diseases, which may suggest that these factors play important roles in the pathogenesis of moyamoya disease. Further studies are required to define the pathogenesis of moyamoya disease, especially in cases with comorbidities as in the present patients.

Filtrate of Phellinus linteus Broth Culture Reduces Infarct Size Significantly in a Rat Model of Permanent Focal Cerebral Ischemia.

Phellinus linteus, a natural growing mushroom, has been known to exhibit anti-tumor, anti-inflammatory, anti-allergic and anti-oxidant effects. Aiming to exploit the neuroprotective effects of P. linteus, we evaluated its effects on infarct volume reduction in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion. Filtrate of P. linteus broth culture (various doses), fractionated filtrate (based on molecular weight) or control medium was administered intraperitoneally to rats before or after ischemia induction. Rats were killed at 24 h after the stroke surgery. Cortical and caudoputaminal infarct volumes were determined separately using an image analysis program following staining with 2,3,5-triphenyltetrazolium chloride. Significant cortical infarct volume reductions were found in the pre-treatment groups (30 and 60 minutes before onset of cerebral ischemia) compared with the control group, showing dose dependence. Posttreatment (30 minutes after ischemic onset) also significantly reduced cortical infarct volume. Furthermore, the higher molecular weight (≥12 000) fraction of the culture filtrate was more effective compared with the lower molecular weight fraction. The present findings suggest that P. linteus may be a new promising approach for the treatment of focal cerebral ischemia, with the additional benefit of a wide therapeutic time window since significant infarct volume reduction is obtained by administration even after the ischemic event. Our finding that the higher molecular weight fraction of the P. linteus culture filtrate demonstrated more prominent effect may provide a clue to identify the neuroprotective substances and mechanisms.

Selective transformation of a crown ether/sec-ammonium salt-type rotaxane to N-alkylated rotaxanes.

Versatile functionalization of a crown ether/sec-ammonium salt-type rotaxane was accomplished. The rotaxane underwent reductive N-alkylation with sodium tri(acyloxy)borohydride or sodium tri(acyloxy)borohydride/arbitrary aldehyde in excellent yields. Structural switching based on reversible tert-ammonium/tert-amine conversion by acid and base was demonstrated as a pH-controlled molecular shuttle.