Intravenous immunoglobulin preparations attenuate lysolecithin-induced peripheral demyelination in mice and comprise anti-large myelin protein zero antibody.

Intravenous immunoglobulin (IVIg) has been used to treat inflammatory demyelinating diseases such as chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, and multifocal motor neuropathy. Despite studies demonstrating the clinical effectiveness of IVIg, the mechanisms underlying its effects remain to be elucidated in detail. Herein, we examined the effects of IVIg on lysolecithin-induced demyelination of the sciatic nerve in a mouse model. Mice -administered with IVIg 1 and 3 days post-injection (dpi) of lysolecithin -exhibited a significantly decreased demyelination area at 7 dpi. Immunoblotting analysis using two different preparations revealed that IVIg reacted with a 36-kDa membrane glycoprotein in the sciatic nerve. Subsequent analyses of peptide absorption identified the protein as a myelin protein in the peripheral nervous system (PNS) known as large myelin protein zero (L-MPZ). Moreover, injected IVIg penetrated the demyelinating lesion, leading to deposition on L-MPZ in the myelin debris. These results indicate that IVIg may modulate PNS demyelination, possibly by binding to L-MPZ on myelin debris.

Using the in vitro drug sensitivity test to identify candidate treatments for transient abnormal myelopoiesis.

Approximately 20% of patients with transient abnormal myelopoiesis (TAM) die due to hepatic or multiorgan failure. To identify potential new treatments for TAM, we performed in vitro drug sensitivity testing (DST) using the peripheral blood samples of eight patients with TAM. DST screened 41 agents for cytotoxic properties against TAM blasts. Compared with the reference samples of healthy subjects, TAM blasts were more sensitive to glucocorticoids, the mitogen-activated protein kinase kinase (MAP2K) inhibitor trametinib, and cytarabine. Our present results support the therapeutic potential of glucocorticoids and the role of the RAS/MAP2K signalling pathway in TAM pathogenesis.

Incorporation of Halogenated Amino Acids into Antibody Fragments at Multiple Specific Sites Enhances Antigen Binding.

Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059-152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17-fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.

Pilot study of the combination of sorafenib and fractionated irinotecan in pediatric relapse/refractory hepatic cancer (FINEX pilot study).

BACKGROUND: Preclinical observations suggested a synergistic effect of sorafenib (SFN) and irinotecan (CPT-11) in hepatoblastoma (HB). Thus, we conducted a feasibility study of fractionated CPT-11 combined with SFN to develop a new therapy against relapsed/refractory pediatric hepatic cancer (HC). PROCEDURE: The study was originally designed as a phase I, standard 3+3 dose-finding study to evaluate dose-limiting toxicities (DLTs) for the regimen and the optimal CPT-11 dose in combination with SFN against relapsed/refractory pediatric HC, including HB and hepatocellular carcinoma (HCC). The enrolled patients received SFN at 200 mg/m2 every 12 hours or 400 mg/m2 every 24 hours daily combined with CPT-11 at 20 mg/m2 /day on days 1 to 5 as an initial level 1 dose. RESULTS: Six patients with HB (n = 4) or HCC (n = 2) were enrolled and treated with CPT-11 dose level 1. The median age at enrollment was 8.7 (6.2-16.3) years. All patients received platinum-containing chemotherapy, and five or two patients received CPT-11 or SFN before enrollment, respectively. Regimen toxicities were evaluable in all patients. One of six patients experienced a grade 4 transaminase levels increase, which was defined as a DLT per protocol. Grade 3/4 neutropenia and a grade 3 transaminase level increase occurred in three patients and one patient, respectively. All patients reported grade 1/2 toxicities such as anemia, skin toxicity, gastrointestinal symptoms, and hypoalbuminemia. CONCLUSIONS: Although the study was terminated before determining the maximum-tolerated CPT-11 dose, SFN and CPT-11 at the level 1 dose were concluded to be tolerable in pediatric patients with HC.

Hypofractionated radiotherapy in children with diffuse intrinsic pontine glioma.

BACKGROUND: Overall survival (OS) of patients with diffuse intrinsic pontine glioma (DIPG) is poor, with radiation therapy (RT) the only intervention that transiently delays tumor progression. Hypofractionated RT and re-irradiation at first progression have gained popularity in improving the quality of life of such patients. METHODS: We performed a retrospective review of children with DIPG treated at Kanagawa Children's Medical Center from 2000 to 2018. RESULTS: A total of 24 cases were reviewed. Median age at diagnosis was 6.3 years (1.6-14.0). Twenty patients received RT only once. Thirteen patients received conventionally fractionated RT, and seven patients received hypofractionated RT as up-front RT. Severe toxicities were not observed in patients who received hypofractionated RT. Median OS and time to progression were similar between conventionally fractionated and hypofractionated RT groups.(9.7 [95% confidence interval(CI): 7.1-11.2] versus 11.0[95% CI: 5.2-13.6] months, P = 0.60; 4.2[95% CI: 1.8-8.3] versus 7.1 [95% CI:4.5-8.7] months, P = 0.38). Four patients received re-irradiation at first progression and all patients showed transient neurological improvement and survival more than a year after diagnosis. A 4-year-old boy was re-irradiated 5-and-a-half months after the first re-irradiation; following transient neurological improvement. He survived a further 5 months. CONCLUSION: Hypofractionated RT for children with newly diagnosed DIPG is well tolerated and feasible from the viewpoint of reducing a patient's burden of treatment. Re-irradiation at first progression is suggested to be beneficial.

Factors influencing platelet normalization of transient abnormal myelopoiesis.

BACKGROUND: Abnormal blood cell counts are characteristic of patients with Down syndrome and transient abnormal myelopoiesis (TAM). Although some patients with TAM experience prolonged anemia or thrombocytopenia, hematological factors predicting blood cell count recovery have not been reported yet. The aim of this study was to investigate the factors influencing platelet normalization in TAM. METHODS: A retrospective review of the medical records of 21 patients with TAM admitted to the neonatal intensive care unit at Kanagawa Children's Medical Center between January 2007 and October 2014 was undertaken. RESULTS: In the 16 of 21 patients (76%) experiencing transient thrombocytopenia, a large number of blasts at diagnosis was found to be significantly associated with late platelet recovery (R = 0.669, P < 0.05), and higher platelet counts at diagnosis were significantly associated with later recovery (R = 0.719, P < 0.01). Indeed, a strong positive correlation between blast and platelet counts at diagnosis was found (R = 0.730, P < 0.01). CONCLUSIONS: Our data suggest that high platelet counts at TAM diagnosis might reflect abnormal thrombocyte production from blasts. Thus, physicians should be aware of the possibility of prolonged thrombocytopenia in patients with TAM who exhibit a high platelet and/or blast count at diagnosis.

Exploring disease-specific methylated CpGs in human male genital abnormalities by using methylated-site display-amplified fragment length polymorphism (MSD-AFLP).

The incidence of male reproductive system disorders, especially hypospadias, has been increasing in developed countries since the latter half of the 20th century. Endocrine-disrupting chemicals from the environment are considered to be involved in hypospadias onset through epigenetic alterations. This pilot study aimed to explore disease-specific methylated CpGs in human patient samples using the methylated-site display-amplified fragment length polymorphism (MSD-AFLP) technique developed by our research group [1]. We compared clinical samples from hypospadias and phimosis patients. Foreskin and blood samples were collected from one- to two-year-old patients with hypospadias (N = 3) and phimosis (N = 3) during surgical treatment. MSD-AFLP analysis showed significantly decreased CpG-methylation levels of genes such as MYH11 and increased CpG-methylation levels of genes such as PLA2G15 in hypospadias patients. Hierarchical clustering analysis showed that genes with significantly altered CpG levels were more markedly altered in DNA from blood than from foreskin. Because of the small number of samples, further investigation is necessary to elucidate the association between variations in CpG levels in foreskin and blood DNA and male genital abnormalities. However, our MSD-AFLP method appears to be a useful tool for exploring disease-specific methylated-CpGs in human epidemiological studies.

The Use of Pitch Accent in Word-Object Association by Monolingual Japanese Infants.

This study investigated the lexical use of Japanese pitch accent in Japanese-learning infants. A word-object association task revealed that 18-month-old infants succeeded in learning the associations between two nonsense objects paired with two nonsense words minimally distinguished by pitch pattern (Experiment 1). In contrast, 14-month-old infants failed (Experiment 2). Eighteen-month-old infants succeeded even for sounds that contained only the prosodic information (Experiment 3). However, a subsequent experiment revealed that 14-month-old infants succeeded in an easier single word-object task using pitch contrast (Experiment 4). These findings indicate that pitch pattern information is robustly available to 18-month-old Japanese monolingual infants in a minimal pair word-learning situation, but only partially accessible in the same context for 14-month-old infants.

Dysregulation of schizophrenia-related aquaporin 3 through disruption of paranode influences neuronal viability.

Myelinated axons segregate the axonal membrane into four defined regions: the node of Ranvier, paranode, juxtaparanode, and internode. The paranodal junction consists of specific component proteins, such as neurofascin155 (NF155) on the glial side, and Caspr and Contactin on the axonal side. Although paranodal junctions are thought to play crucial roles in rapid saltatory conduction and nodal assembly, the role of their interaction with neurons is not fully understood. In a previous study, conditional NF155 knockout in oligodendrocytes led to disorganization of the paranodal junctions. To examine if disruption of paranodal junctions affects neuronal gene expression, we prepared total RNA from the retina of NF155 conditional knockout, and performed expression analysis. We found that the expression level of 433 genes changed in response to paranodal junction ablation. Interestingly, expression of aquaporin 3 (AQP3) was significantly reduced in NF155 conditional knockout mice, but not in cerebroside sulfotransferase knockout (CST-KO) mice, whose paranodes are not originally formed during development. Copy number variations have an important role in the etiology of schizophrenia (SCZ). We observed rare duplications of AQP3 in SCZ patients, suggesting a correlation between abnormal AQP3 expression and SCZ. To determine if AQP3 over-expression in NF155 conditional knockout mice influences neuronal function, we performed adeno-associated virus (AAV)-mediated over-expression of AQP3 in the motor cortex of mice and found a significant increase in caspase 3-dependent neuronal apoptosis in AQP3-transduced cells. This study may provide new insights into therapeutic approaches for SCZ by regulating AQP3 expression, which is associated with paranodal disruption.

Synthetic Tyrosine tRNA Molecules with Noncanonical Secondary Structures.

The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity. Here, we report on a method to safely engineer structural variations in this domain utilizing the noncanonical scaffold of tRNAPyl. First, we constructed a naïve hybrid between archaeal tRNAPyl and tRNATyr, which consisted of the acceptor and T stems of tRNATyr and the other parts of tRNAPyl. This hybrid tRNA efficiently translated the UAG codon to 3-iodotyrosine in Escherichia coli cells, when paired with a variant of the archaeal tyrosyl-tRNA synthetase. The amber suppression efficiency was slightly lower than that of the "bench-mark" archaeal tRNATyr suppressor assuming the canonical structure. After a series of modifications to this hybrid tRNA, we obtained two artificial types of tRNATyr: ZtRNA had an augmented D (auD) helix in a noncanonical form and the D and T loops bound by the standard tertiary base pairs, and YtRNA had a canonical auD helix and non-standard interloop interactions. It was then suggested that the ZtRNA scaffold could also support the glycylation and glutaminylation of tRNA. The synthetic diversity of tRNA would help create new tRNA⁻aminoacyl-tRNA synthetase pairs for reprogramming the genetic code.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

BACKGROUND: It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. RESULTS: Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. CONCLUSION: MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes.

The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites. In this study, we applied the codon reassignment technology to achieve the robust and efficient synthesis of chemically functionalized antibodies containing Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) at defined positions. This lysine derivative has a bio-orthogonally reactive group at the end of a long side chain, enabling identification of multiple new positions in Fab-constant domains, allowing chemical conjugation with high efficiency. An X-ray crystallographic study of a Fab variant with o-Az-Z-Lys revealed high-level exposure of the azido group to solvent, with six of the identified positions subsequently used to engineer "Variabodies", a novel antibody format allowing various connections between two Fab molecules. Our findings indicated that some of the created Variabodies exhibited agonistic activity in cultured cells as opposed to the antagonistic nature of antibodies. These results showed that our approach greatly enhanced the availability of antibodies for chemical conjugation and might aid in the development of new therapeutic antibodies.

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli.

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to L-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize L-homoarginine and L-N(6)-(1-iminoethyl)lysine (L-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA(Pyl) CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host's ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to L-homoarginine or L-NIL. The assignment of AGG to L-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.

Long-Term Favorable Course of Aspergillus Endo-, Myo-, and Pericarditis.

Here, we report on a healthy 30-year-old man with no significant medical history, who tested negative for human immunodeficiency virus antigenemia but developed Aspergillus pancarditis. A case of this kind is extremely rare, and to the best of our knowledge, this is the first report of a patient with Aspergillus pancarditis, which generally leads to a very poor outcome, who had a long-term favorable clinical course. A biopsy from the right atrium of hypertrophied atrial septum was essential for obtaining the definitive diagnosis. Long-term administration of an effective antifungal oral agent might account for the patient's favorable outcome.

Spatial Relation Between Left Atrial Anatomical Contact Areas and Circular Activation in Persistent Atrial Fibrillation.

INTRODUCTION: Atrial low-voltage zones (LVZs) may be related to maintenance of atrial fibrillation (AF). The influence of left atrial (LA) contact areas (CoAs) on reentrant or rotor-like sources maintaining AF has not been investigated. METHODS AND RESULTS: Forty patients with persistent AF (PsAF) were analyzed. Three representative CoA regions in the LA (ascending aorta: anterior wall; descending aorta: left inferior pulmonary vein; and vertebrae: posterior wall) were visualized by enhanced CT. Using circular catheters, the LVZs (<0.5 mV) were assessed after restoration of SR, and local activation mapping and frequency domain analyses were performed after induction of AF. Circular activation during AF was visually defined as sites with ≥2 rotations by serial electrograms encompassing >80% of the mean AF cycle length. A pivot was defined as the core of the localized circular activation. Anterior (39/40 patients, 98%), left pulmonary vein antrum (27/40, 68%), and posterior (19/40, 48%) CoAs were identified, and 80% (68/85) of those sites were overlapped by or close (<3 mm) to LVZs. Thirty-six (90%) patients demonstrated circular activation (3.1±1.7 sites/patients) along with significantly higher organized dominant frequencies (6.3 ± 0.5 Hz, regularity-index: 0.26 [0.23-0.41]) within the LA, and the average electrogram amplitude of those pivots was 0.30 mV (0.18-0.52). Of those sites, 55% (66/120) were located at or close to CoA regions. Catheter ablation including of LVZs neighboring CoAs terminated AF in 9 (23%) patients. CONCLUSIONS: External anatomical structures contacting the LA may be related to unique conduction properties in diseased myocardium necessary for PsAF maintenance.

Concentration of neddylation-related molecules in paranodal myelin of the peripheral nervous system.

Neddylation is a reversible post-translational modification in which a small ubiquitin-like molecule called NEDD8 covalently binds to substrate proteins. Although a recent study suggests that neddylation is essential for formation and maintenance of dendritic spines in the brain, the role of this protein modification in the peripheral nerves is wholly unknown. In this study, we demonstrate that neddylation-related molecules, NEDD8 and DCUN1D2 (defective in cullin neddylation 1, domain containing 2), were concentrated at the paranode of peripheral myelin, in addition to the myelinated and unmyelinated Schwann cell bodies. These proteins were localized mainly within larger fibers, but not in fibers with small diameters. Developmental analyses showed that these molecules first appeared at the paranode during later stages of myelination, and this characteristic distribution disappeared in sulfatide-deficient mice in which paranodal axo-glial junctions were disrupted. These results suggest that the myelin paranode may be one of the regions where neddylation occurs within the peripheral nerves.

Microglial phospholipase D4 deficiency influences myelination during brain development.

Phospholipase D4 (PLD4) is expressed in activated microglia that transiently appear in white matter during postnatal brain development. Previous knockdown experiments using cultured microglia showed PLD4 involvement in phagocytosis and proliferation. To elucidate the role of PLD4 in vivo, PLD4-deficient mice were generated and the cerebella were examined at postnatal day 5 (P5) and P7, when PLD4 expression is highest in microglia. Wild type microglia showed strong immunoreactivity for microglial marker CD68 at P5, whereas CD68 signals were weak in PLD4-deficient microglia, suggesting that loss of PLD4 affects microglial activation. At P5 and P7, immunostaining for anti-myelin basic protein (MBP) antibody indicated a mild but significant delay in myelination in PLD4-deficient cerebellum. Similar change was also observed in the corpus callosum at P7. However, this difference was not apparent at P10, suggesting that microglial PLD4-deficiency primarily influences the early myelination stage. Thus, microglia may have a transient role in myelination via a PLD4-related mechanism during development.

Influence of left atrium anatomical contact area in persistent atrial fibrillation-relationship between low-voltage area and fractionated electrogram.

BACKGROUND: Atrial low-voltage areas are suggested to be related to maintenance of atrial fibrillation (AF). The influence of the left atrium (LA) contact area (CoA) has not been investigated. METHODS AND RESULTS: Twenty-two persistent AF patients underwent high-density mapping during AF and sinus rhythm (SR). Three representative CoA regions in the LA (ascending aorta: anterior wall; descending aorta: left inferior pulmonary vein [LIPV]; and vertebrae: posterior wall) were identified. Electrogram analysis of both high dominant frequency (high-DF; >8 Hz) and complex fractionated atrial electrogram (con-CFAE; <50 ms) regions during SR was done. The anatomical relationship between CoA and both the very low-voltage areas (vLVA; <0.2 mV) and high-frequency sources was determined. Forty-seven vLVA (194.4 cm(2)) and 60 CoA (337.0 cm(2)) were documented, and 32 vLVA directly overlapped CoA. The vLVA were preferentially found in the anterior (45%) and posterior (13%) walls of the LA, and in the LIPV (13%), and corresponded to CoA sites. The mean voltage during SR at high-DF sites was significantly lower than that at con-CFAE sites (0.62 vs.1.54 mV; P<0.0001). Seventy-two percent of high-DF sites overlapped CoA, while 54% of con-CFAE did. Furthermore, 44% of high-DF surface area directly overlapped CoA, while only 19% of con-CFAE did. CONCLUSIONS: Very low-voltage regions had a strong association with CoA. Sites with CoA had a higher incidence of fractionated electrograms both during SR and AF.

The neural basis of dishonest decisions that serve to harm or help the target.

We conducted a functional magnetic resonance imaging (fMRI) study to elucidate the neurocognitive mechanisms of harmful and helpful dishonest decisions. During scanning, the subjects read scenarios concerning events that could occur in real-life situations and were asked to decide whether to tell a lie as though they were experiencing those events. Half of the scenarios consisted of harmful stories in which the dishonest decisions could be regarded as bad lies, and the other half consisted of helpful stories in which the dishonest decisions could be regarded as good lies. In contrast to the control decision-making task, we found that the decision-making tasks that involved honesty or dishonesty recruited a network of brain regions that included the left dorsolateral prefrontal cortex. In the harmful stories, the right temporoparietal junction and the right medial frontal cortex were activated when the subjects made dishonest decisions compared with honest decisions. No region discriminated between the honest and dishonest decisions made in the helpful stories. These preliminary findings suggest that the neural basis of dishonest decisions is modulated by whether the lying serves to harm or help the target.