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BackgroundThe highly selective α2 -adrenoreceptor agonist, dexmedetomidine, exerts neuroprotective, analgesic, anti-inflammatory and sympatholytic properties that may be beneficial for perinatal asphyxia. The optimal safe dose for pre-clinical newborn neuroprotection studies is unknown.MethodsFollowing cerebral hypoxia-ischaemia, dexmedetomidine was administered to nine newborn piglets in a de-escalation dose study in combination with hypothermia (whole body cooling to 33.5°C). Dexmedetomidine was administered with a loading dose of 1 μg/kg and maintenance infusion at doses from 10 to 0.6 μg/kg/h. One additional piglet was not subjected to hypoxia-ischaemia. Blood for pharmacokinetic analysis was sampled pre-insult and frequently post-insult. A one-compartment linear disposition model was used to fit data. Population parameter estimates were obtained using non-linear mixed effects modelling.ResultsAll dexmedetomidine infusion regimens led to plasma concentrations above those associated with sedation in neonates and children (0.4-0.8 μg/l). Seven out of the nine piglets with hypoxia-ischaemia experienced periods of bradycardia, hypotension, hypertension and cardiac arrest; all haemodynamic adverse events occurred in piglets with plasma concentrations greater than 1 μg/l. Dexmedetomidine clearance was 0.126 l/kg/h [coefficient of variation (CV) 46.6.%] and volume of distribution was 3.37 l/kg (CV 191%). Dexmedetomidine clearance was reduced by 32.7% at a temperature of 33.5°C. Dexmedetomidine clearance was reduced by 55.8% following hypoxia-ischaemia.ConclusionsDexmedetomidine clearance was reduced almost tenfold compared with adult values in the newborn piglet following hypoxic-ischaemic brain injury and subsequent therapeutic hypothermia. Reduced clearance was related to cumulative effects of both hypothermia and exposure to hypoxia. High plasma levels of dexmedetomidine were associated with major cardiovascular complications.

BackgroundThe highly selective α2 -adrenoreceptor agonist, dexmedetomidine, exerts neuroprotective, analgesic, anti-inflammatory and sympatholytic properties that may be beneficial for perinatal asphyxia. The optimal safe dose for pre-clinical newborn neuroprotection studies is unknown.MethodsFollowing cerebral hypoxia-ischaemia, dexmedetomidine was administered to nine newborn piglets in a de-escalation dose study in combination with hypothermia (whole body cooling to 33.5°C). Dexmedetomidine was administered with a loading dose of 1 μg/kg and maintenance infusion at doses from 10 to 0.6 μg/kg/h. One additional piglet was not subjected to hypoxia-ischaemia. Blood for pharmacokinetic analysis was sampled pre-insult and frequently post-insult. A one-compartment linear disposition model was used to fit data. Population parameter estimates were obtained using non-linear mixed effects modelling.ResultsAll dexmedetomidine infusion regimens led to plasma concentrations above those associated with sedation in neonates and children (0.4-0.8 μg/l). Seven out of the nine piglets with hypoxia-ischaemia experienced periods of bradycardia, hypotension, hypertension and cardiac arrest; all haemodynamic adverse events occurred in piglets with plasma concentrations greater than 1 μg/l. Dexmedetomidine clearance was 0.126 l/kg/h [coefficient of variation (CV) 46.6.%] and volume of distribution was 3.37 l/kg (CV 191%). Dexmedetomidine clearance was reduced by 32.7% at a temperature of 33.5°C. Dexmedetomidine clearance was reduced by 55.8% following hypoxia-ischaemia.ConclusionsDexmedetomidine clearance was reduced almost tenfold compared with adult values in the newborn piglet following hypoxic-ischaemic brain injury and subsequent therapeutic hypothermia. Reduced clearance was related to cumulative effects of both hypothermia and exposure to hypoxia. High plasma levels of dexmedetomidine were associated with major cardiovascular complications.

Aim: To develop standardized diagnostic criteria for 'infection-triggered encephalopathy syndrome (ITES)' and five specific clinical syndromes of ITES., Method: The draft definitions were based on existing criteria, standardized, and discussed by a panel of international experts using nominal group technique over 18 months to achieve consensus. All criteria use the same format: (1) presence of infection/fever; (2) clinical features including encephalopathy; (3) neuroradiological features on magnetic resonance imaging; (4) exclusion of other causes., Results: We first highlighted differences between ITES and infectious and autoimmune encephalitis, which is the most important differential diagnosis. Consensus was achieved to define five specific ITESs: acute encephalopathy with biphasic seizures and late reduced diffusion; acute necrotizing encephalopathy; mild encephalopathy with a reversible splenial lesion; acute fulminant cerebral oedema; and acute shock with encephalopathy and multiorgan failure. Two further conditions that are currently classified as epilepsy syndromes but have similar features to ITES, namely febrile infection-related epilepsy syndrome and hemiconvulsion-hemiplegia-epilepsy syndrome, are also discussed., Interpretation: The consensus definition is expected to improve awareness of this disease concept, provide diagnostic framework, and facilitate future international research and clinical trials., (© 2024 The Author(s). Developmental Medicine & Child Neurology published by John Wiley & Sons Ltd on behalf of Mac Keith Press.)

Abstract: Immunomodulatory drugs (IMiDs) are key drugs for treating multiple myeloma and myelodysplastic syndrome with chromosome 5q deletion. IMiDs exert their pleiotropic effects through the interaction between cell-specific substrates and cereblon, a substrate receptor of the E3 ubiquitin ligase complex. Thus, identification of cell-specific substrates is important for understanding the effects of IMiDs. IMiDs increase the risk of thromboembolism, which sometimes results in fatal clinical outcomes. In this study, we sought to clarify the molecular mechanisms underlying IMiDs-induced thrombosis. We investigated cereblon substrates in human megakaryocytes using liquid chromatography-mass spectrometry and found that thrombospondin-1 (THBS-1), which is an inhibitor of a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13, functions as an endogenous substrate in human megakaryocytes. IMiDs inhibited the proteasomal degradation of THBS-1 by impairing the recruitment of cereblon to THBS-1, leading to aberrant accumulation of THBS-1. We observed a significant increase in THBS-1 in peripheral blood mononuclear cells as well as larger von Willebrand factor multimers in the plasma of patients with myeloma, who were treated with IMiDs. These results collectively suggest that THBS-1 represents an endogenous substrate of cereblon. This pairing is disrupted by IMiDs, and the aberrant accumulation of THBS-1 plays an important role in the pathogenesis of IMiDs-induced thromboembolism., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Introduction: Diagnosing acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) after the first seizure (early seizure/seizures, ES/ESs) is challenging because a reduced apparent diffusion coefficient (ADC) in the cortical or subcortical white matter, often described as having a "bright-tree appearance (BTA)," is usually not observed until secondary seizures (late seizures, LSs) occur. Previous studies have reported hypoperfusion on arterial spin labeling (ASL) within 24 h after ES/ESs in patients with AESD and hyperperfusion within 24 h after LS onset. This study aimed to investigate cerebral blood flow in the hyperacute phase (between ES/ESs and LSs) using ASL in patients with AESD., Methods: Eight ASL images were acquired in six patients with AESD admitted to our hospital from October 2021 to October 2022. ASL findings in the hyperacute phase were investigated and video-electroencephalogram findings obtained around ASL image acquisition in the hyperacute phase were evaluated., Results: Four ASL images were obtained for three patients before LS onset, with three images showing hyperperfusion areas and one image showing hypoperfusion areas. These hyperperfuion regions coincided with BTA on subsequent images of these patients.In one patient, the first ASL image was obtained in the late hyperacute phase and revealed hyperperfusion areas with a slightly abnormal change on diffusion-weighted image (DWI), which were not accompanied by ADC abnormalities. The second ASL image obtained 51 h after the first ASL, and before LS onset revealed more prominent hyperperfusion areas than the first ASL image, which were accompanied by BTA. In another patient, the ASL image obtained 82 h after ES revealed hyperperfusion areas without abnormal change on DWI or ADC., Conclusion: This study revealed that two patients exhibited hyperperfusion regions and another patient exhibited hypoperfusion regions among three patients who underwent ASL imaging during the period from 24 h after ES/ESs to LSs in patients with LSs or cooling initiation in patients without LSs due to early anaesthesia induction (late hyperacute phase). Further prospective studies on cerebral blood flow are required to explore the relationship among the timing of image acquisition, the presence of electrographic seizures, and ASL findings in patients with AESD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kawano, Tokutomi, Kikuchi, Sakata, Sakaguchi, Yokochi, Akita and Matsuishi.)

The present retrospective study aimed to investigate the presence of truncal instability or titubation after the first seizure and second phase in patients with acute encephalopathy with reduced subcortical diffusion (AED). Of the 15 patients with AED who were admitted to our hospital for 3 years and 2 months and had reached developmental milestones for sitting before disease onset, six experienced moderate-to-severe truncal instability while sitting after the first seizure. These patients had a significantly longer first seizure duration and significantly lower GCS scores 12-24 h after the first seizure, as well as significantly higher Tada score and Creatinine and blood glucose levels than those with mild or no truncal instability while in a seated position after the first seizure. Three 1-year-old children with bilateral frontal lobe lesions, particularly in the bilateral prefrontal lobe regions, demonstrated truncal titubation, which has not previously been reported as a clinical feature of AED. Tada score reported to be a predictor of AED prognosis and truncal instability in the sitting position after the first seizure may represent disease severity, but not the specific lesions. Conversely, truncal titubation might be suggestive of bilateral frontal lobe lesions, particularly in patients without severe instability. Further studies on the role of bilateral prefrontal lobe lesions to truncal titubation in patients with AED using more objective evaluation methods, such as stabilometry, are necessary., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kawano, Yae, Sakata, Yokochi, Imagi, Ohbu and Matsuishi.)

Acute encephalopathy with reduced subcortical diffusion (AED), characterised by seizure onset and widespread reduced apparent diffusion coefficient in the cortex/subcortical white matter, is one of the most common acute encephalopathies in children in East Asia. This 14-year single-centre retrospective study on 34 patients with AED showed that therapeutic hypothermia was used for patients with more severe consciousness disturbance after the first seizure or second phase initiation, extrapolating from neonatal hypoxic encephalopathy and adult post-cardiac arrest syndrome. The basal ganglia/thalamus lesions and the Tada score were the poor outcome determinants in the multivariate analysis. The correlation between the worse outcomes and the duration from the first seizure to the initiation of therapeutic hypothermia was observed only in the patients with AED cooled before the second phase. This correlation was not observed in the overall AED population. There was a moderate negative association between the worse outcomes and the duration between the first seizure and the second phase. Therefore, the basal ganglia/thalamus lesions and the Tada score were the outcome determinants for patients with AED. Further investigation is required to examine the efficacy of therapeutic hypothermia in this population while considering the timing of the therapeutic hypothermia initiation and the second phase.

Infarct locations in children with arterial ischemic stroke have primarily been reported to be lobar or in the basal ganglia, and those in patients with Down syndrome (DS) and antiphospholipid syndrome (APS) are typically wide and multiple. No solitary brain stem infarctions have ever been reported in children with DS until now. Here, we report a case of brain stem infarction in a 6-year-old boy with DS who had no cardiac, renal, or intestinal complications. He exhibited ataxic gait and medial longitudinal fasciculus (MLF) symptoms at first presentation. Neuroimaging revealed a localized and isolated lesion in the midbrain. Although he did not satisfy the diagnostic criteria of APS, he showed persistently elevated levels of anticardiolipin antibody (21 U/mL; normal value <10 U/mL). Although he had the risks of a multiple vascular systems disorder, DS, and persistently elevated levels of antiphospholipid antibodies, his lesion was not similar to any of the previously reported cerebral infarctions in DS or in APS. To our knowledge, this is the first report of limited solitary brain stem infarction in a child with DS., (Copyright © 2019. Published by Elsevier B.V.)

Acute disseminated encephalomyelitis (ADEM) is a typically monophasic inflammatory demyelinating disease of the central nervous system with a favorable outcome. However, 2% of ADEM involves acute hemorrhagic leukoencephalitis (AHLE), which is a fulminant and hyperacute variant of ADEM with a poor outcome and high mortality. There are limited case reports of fulminant ADEM including AHLE in children. Herein, we report two pediatric cases of fulminant ADEM. Both cases had a rapid deterioration of consciousness, repetitive seizures, and brain edema on neuroimaging, in addition to atypical neuroradiological findings on magnetic resonance imaging (MRI), a reversible splenial lesion in case 1, and bilateral frontal and occipital cortical lesions in case 2. Both cases were treated with early high-dose methyl-prednisolone and immunoglobulin, while therapeutic hypothermia was also initiated in case 2 after the patient exhibited a decerebrate posture and irregular breathing pattern. Both cases had a favorable outcome. Further case reports on pediatric fulminant ADEM are required to clarify the various clinical types, and to examine the efficacy of various treatment modalities for fulminant ADEM and AHLE in children., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

In a range of animal species, exposure of the brain to general anaesthesia without surgery during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex but include an increase in brain cell death. In humans, attempts to link adverse cognitive development to infantile anaesthesia exposure have yielded ambiguous results. One caveat that may influence the interpretation of human studies is that infants are not exposed to general anaesthesia without surgery, raising the possibility that surgery itself, may contribute to adverse cognitive development. Using piglets, we investigated whether a minor surgical procedure increases cell death and disrupts neuro-developmental and cognitively salient gene transcription in the neonatal brain. We randomly assigned neonatal male piglets to a group who received 6h of 2% isoflurane anaesthesia or a group who received an identical anaesthesia plus 15 mins of surgery designed to replicate an inguinal hernia repair. Compared to anesthesia alone, surgery-induced significant increases in cell death in eight areas of the brain. Using RNAseq data derived from all 12 piglets per group we also identified significant changes in the expression of 181 gene transcripts induced by surgery in the cingulate cortex, pathway analysis of these changes suggests that surgery influences the thrombin, aldosterone, axonal guidance, B cell, ERK-5, eNOS and GABAA signalling pathways. This suggests a number of novel mechanisms by which surgery may influence neural and cognitive development independently or synergistically with the effects of anaesthesia.

The selective α2-adrenoreceptor agonist dexmedetomidine has shown neuroprotective, analgesic, anti-inflammatory, and sympatholytic properties that may be beneficial in neonatal encephalopathy (NE). As therapeutic hypothermia is only partially effective, adjunct therapies are needed to optimize outcomes. The aim was to assess whether hypothermia + dexmedetomidine treatment augments neuroprotection compared to routine treatment (hypothermia + fentanyl sedation) in a piglet model of NE using magnetic resonance spectroscopy (MRS) biomarkers, which predict outcomes in babies with NE, and immunohistochemistry. After hypoxia-ischaemia (HI), 20 large White male piglets were randomized to: (i) hypothermia + fentanyl with cooling to 33.5°C from 2 to 26 h, or (ii) hypothermia + dexmedetomidine (a loading dose of 2 μg/kg at 10 min followed by 0.028 μg/kg/h for 48 h). Whole-brain phosphorus-31 and regional proton MRS biomarkers were assessed at baseline, 24, and 48 h after HI. At 48 h, cell death was evaluated over 7 brain regions by means of transferase-mediated d-UTP nick end labeling (TUNEL). Dexmedetomidine plasma levels were mainly within the target sedative range of 1 μg/L. In the hypothermia + dexmedetomidine group, there were 6 cardiac arrests (3 fatal) versus 2 (non-fatal) in the hypothermia + fentanyl group. The hypothermia + dexmedetomidine group required more saline (p = 0.005) to maintain blood pressure. Thalamic and white-matter lactate/N-acetylaspartate did not differ between groups (p = 0.66 and p = 0.21, respectively); the whole-brain nucleotide triphosphate/exchangeable phosphate pool was similar (p = 0.73) over 48 h. Cell death (TUNEL-positive cells/mm2) was higher in the hypothermia + dexmedetomidine group than in the hypothermia + fentanyl group (mean 5.1 vs. 2.3, difference 2.8 [95% CI 0.6-4.9], p = 0.036). Hypothermia + dexmedetomidine treatment was associated with adverse cardiovascular events, even within the recommended clinical sedative plasma level; these may have been exacerbated by an interaction with either isoflurane or low body temperature. Hypothermia + dexmedetomidine treatment was neurotoxic following HI in our piglet NE model, suggesting that caution is vital if dexmedetomidine is combined with cooling following NE., (© 2017 S. Karger AG, Basel.)

Exposure of the brain to general anesthesia during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex, incompletely understood and may be sexually dimorphic, but include developmentally inappropriate apoptosis, inflammation and a disruption to cognitively salient gene expression. We investigated the effects of a 6h isoflurane exposure on cell death, microglial activation and gene expression in the male neonatal piglet brain. Piglets (n = 6) were randomised to: (i) naive controls or (ii) 6h isoflurane. Cell death (TUNEL and caspase-3) and microglial activation were recorded in 7 brain regions. Changes in gene expression (microarray and qPCR) were assessed in the cingulate cortex. Electroencephalography (EEG) was recorded throughout. Isoflurane anesthesia induced significant increases in cell death in the cingulate and insular cortices, caudate nucleus, thalamus, putamen, internal capsule, periventricular white matter and hippocampus. Dying cells included both neurons and oligodendrocytes. Significantly, microglial activation was observed in the insula, pyriform, hippocampus, internal capsule, caudate and thalamus. Isoflurane induced significant disruption to the expression of 79 gene transcripts, of these 26 are important for the control of transcription and 23 are important for the mediation of neural plasticity, memory formation and recall. Our observations confirm that isoflurane increases apoptosis and inflammatory responses in the neonatal piglet brain but also suggests novel additional mechanisms by which isoflurane may induce adverse neural and cognitive development by disrupting the expression of genes mediating activity dependent development of neural circuits, the predictive adaptive responses of the brain, memory formation and recall., Competing Interests: The authors have declared that no competing interests exist.

We report a case of acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) associated with toxic shock syndrome caused by burns. A one-year-old girl was admitted to our hospital for treatment of severe burns. On day 3, she exhibited a fever, generalized rash and multiple organ failure. She was diagnosed with toxic shock syndrome after burns. She had seizures with fever twice on the same day, followed by secondary seizures on day 8 and transient deterioration of the gross motor functions involved in sitting alone and rolling over. On day 9, MRI diffusion-weighted images showed bright tree appearance (BTA). We conclude that she developed AESD., (Copyright © 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention whereby brief episodes of ischemia/reperfusion of one organ (limb) mitigate damage in another organ (brain) that has experienced severe hypoxia-ischemia. Our aim was to assess whether RIPostC is protective following cerebral hypoxia-ischemia in a piglet model of neonatal encephalopathy (NE) using magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry. After hypoxia-ischemia (HI), 16 Large White female newborn piglets were randomized to: (i) no intervention (n = 8); (ii) RIPostC - with four, 10-min cycles of bilateral lower limb ischemia/reperfusion immediately after HI (n = 8). RIPostC reduced the hypoxic-ischemic-induced increase in white matter proton MRS lactate/N acetyl aspartate (p = 0.005) and increased whole brain phosphorus-31 MRS ATP (p = 0.039) over the 48 h after HI. Cell death was reduced with RIPostC in the periventricular white matter (p = 0.03), internal capsule (p = 0.002) and corpus callosum (p = 0.021); there was reduced microglial activation in corpus callosum (p = 0.001) and more surviving oligodendrocytes in corpus callosum (p = 0.029) and periventricular white matter (p = 0.001). Changes in gene expression were detected in the white matter at 48 h, including KATP channel and endothelin A receptor. Immediate RIPostC is a potentially safe and promising brain protective therapy for babies with NE with protection in white but not grey matter., (© The Author(s) 2015.)

Cooling to 33.5°C in babies with neonatal encephalopathy significantly reduces death and disability, however additional therapies are needed to maximize brain protection. Following hypoxia-ischemia we assessed whether inhaled 45-50% Argon from 2-26h augmented hypothermia neuroprotection in a neonatal piglet model, using MRS and aEEG, which predict outcome in babies with neonatal encephalopathy, and immunohistochemistry. Following cerebral hypoxia-ischemia, 20 Newborn male Large White piglets<40h were randomized to: (i) Cooling (33°C) from 2-26h (n=10); or (ii) Cooling and inhaled 45-50% Argon (Cooling+Argon) from 2-26h (n=8). Whole-brain phosphorus-31 and regional proton MRS were acquired at baseline, 24 and 48h after hypoxia-ischemia. EEG was monitored. At 48h after hypoxia-ischemia, cell death (TUNEL) was evaluated over 7 brain regions. There were no differences in body weight, duration of hypoxia-ischemia or insult severity; throughout the study there were no differences in heart rate, arterial blood pressure, blood biochemistry and inotrope support. Two piglets in the Cooling+Argon group were excluded. Comparing Cooling+Argon with Cooling there was preservation of whole-brain MRS ATP and PCr/Pi at 48h after hypoxia-ischemia (p<0.001 for both) and lower (1)H MRS lactate/N acetyl aspartate in white (p=0.03 and 0.04) but not gray matter at 24 and 48h. EEG background recovery was faster (p<0.01) with Cooling+Argon. An overall difference between average cell-death of Cooling versus Cooling+Argon was observed (p<0.01); estimated cells per mm(2) were 23.9 points lower (95% C.I. 7.3-40.5) for the Cooling+Argon versus Cooling. Inhaled 45-50% Argon from 2-26h augmented hypothermic protection at 48h after hypoxia-ischemia shown by improved brain energy metabolism on MRS, faster EEG recovery and reduced cell death on TUNEL. Argon may provide a cheap and practical therapy to augment cooling for neonatal encephalopathy., (Copyright © 2015. Published by Elsevier Inc.)

Acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) at onset manifests an early seizure (ES) usually lasting more than 30 min. Following ES, some patients exhibit almost clear consciousness with no neurological symptoms, and no MRI abnormality for a few days, which may lead to an initial misdiagnosis of prolonged febrile seizures (PFS). To allow an early diagnosis of AESD, we retrospectively analyzed clinical manifestations, laboratory data, and radiologic and EEG findings in patients with AESD (n=62) having ES of over 30 min, and ones with PFS (n=54), using logistic regression analyses. Multivariate logistic regression analysis revealed that an age below 1.5 years and a Glasgow Coma Scale score of 14 or less than 14 (Japan Coma Scale score of 1 or higher) were high risk factors of developing AESD. We proposed an AESD prediction score system consisting of consciousness level, age, duration of convulsions, enforcement of mechanical intubation, and aspartate aminotransferase, blood glucose and creatinine levels (full score: 9), the mean scores in AESD and PFS being 5.9 and 1.8, which were significantly different (p<0.001). We herein propose a scoring system for differentiating patients with AESD and PFS around the time of ES (score of 4 or more than 4 suggesting AESD), which may contribute to early therapeutic intervention and an improved neurologic outcome., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The relationship between cerebral autoregulation (CA) and the neurotoxic effects of anaesthesia with and without surgery is investigated. Newborn piglets were randomly assigned to receive either 6 h of anaesthesia (isoflurane) or the same with an additional hour of minor surgery. The effect of the spontaneous changes in mean arterial blood pressure (MABP) on the cerebral haemodynamics (oxy- and deoxy-haemoglobin, HbO2 and Hb) was measured using transverse broadband near-infrared spectroscopy (NIRS). A marker for impaired CA, concordance between MABP and intravascular oxygenation (HbD = HbO2 - Hb) in the ultra-low frequency domain (0.0018-0.0083 Hz), was assessed using coherence analysis. Presence of CA impairment was not significant but found to increase with surgical exacerbation. The impairment did not correlate with histological outcome (presence of cell death, apoptosis and microglial activation in the brain).

We describe for the first time an 8-year-old male patient who demonstrated clinically mild encephalitis with a reversible splenial lesion after mumps vaccination. He suffered from transient hallucinations, nuchal rigidity, and inappropriate antidiuretic hormone secretion syndrome. On the 5th day of admission, his head MRI showed symmetrical high-signal-intensity lesions on T2, FLAIR, and diffusion-weighted images in the splenium of the corpus callosum and in the periventricular white matter, while an apparent diffusion coefficient map showed reduced diffusion. The images were not enhanced by gadolinium. Follow-up MRI on the 16th day of admission revealed none of these abnormalities. His serum IgM and IgG antibodies against the mumps virus were positive according to an enzyme immunoassay. Mumps Torii vaccine strain was isolated from the patient's cerebrospinal fluid. Previous reports demonstrated that transient delirious behavior, the syndrome of inappropriate antidiuretic hormone secretion, and good prognosis were the main clinical features of mild encephalitis with a reversible splenial lesion. This case shows that mild encephalitis with a reversible splenial lesion could occur after mumps vaccination., (Copyright © 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Benign convulsions associated with mild gastroenteritis (CwG) are a commonly observed disorder in Asia, especially in infants and seniors. Here, we describe a retrospective study about the clinical features of CwG in 62 children hospitalized at St. Mary's Hospital (Kurume City, Japan) between January 1, 2000 and March 31, 2006, and further evaluate the efficacies of various anticonvulsant treatments for patients with CwG due to either rotavirus or norovirus. Causative diarrheal viruses were detected in 71% of the fecal specimens tested; 30 patients were positive for rotavirus, nine patients were positive for norovirus, two patients were positive for sapovirus, two patients were positive for adenovirus, and one patient was positive for coxackievirus A4. The age of onset for patients with norovirus-positive CwG (16.7+/-2.7 months) was significantly lower than that of patients with rotavirus-positive CwG (23.0+/-8.7 months). The duration of the seizures due to norovirus infection (11.8+/-12.0 h) was significantly longer than that due to rotavirus infection (4.9+/-5.7 h). There were no significant differences between the two groups with regard to the results of blood chemistry analysis, including the concentrations of serum electrolytes, blood glucose levels, and liver function tests. In this preliminary study, the duration of seizures in patients with CwG due to norovirus that was treated with carbamazepine was significantly shorter than the duration of seizures in the patients treated with another anticonvulsant (phenobarbital). Further randomized controlled studies are required to clarify the efficacies of the various anticonvulsants for patients with CwG.

For the treatment of chronic inflammation in the oral cavity, we attempted to develop bioadhesive tablets of bovine lactoferrin (B-LF). Pectin was used as a bioadhesive polymer, and the influence of the degree of esterification and the molecular weight of pectins on the characteristics of B-LF tablets were investigated. Concerning bioadhesive force, a tendency increasing the value according to increase of the esterification of the pectin was confirmed. Sustained release of B-LF from the tablets was observed as the esterification increased, and a possibility for prediction of the time required to release 50% of B-LF by using the equation given by the degree of esterification and the logarithm of the molecular weight was suggested. Pectin cross-linked with Ca(2+) (Ca-PC) was also used for the preparation of the B-LF tablets. Prolonged release of B-LF from the tablets was observed as the Ca(2+) in Ca-PC increased. Our findings suggest that pectin with a high degree of esterification is suitable as a bioadhesive polymer since high bioadhesive force and sustained release are shown. Furthermore, a possibility that the B-LF release could be controlled by adjusting the Ca(2+) concentration in Ca-PC was suggested.

We have previously demonstrated that intestinally infused bovine lactoferrin (bLF) is transported into the blood circulation via the lymphatic pathway, not via the portal circulation. Therefore, in the present study, we further investigated whether intragastrically infused enteric-formulated bLF (EF-bLF) was more efficiently absorbed than bLF from the intestine in adult rats. The rats were randomly divided into three groups: 30 and 300 mg kg(-1) non-enteric-formulated bLF (non-EF-bLF) groups and a 30 mg kg(-1) EF-bLF group. Thoracic lymph was collected from a thoracic lymph duct under general anaesthesia. Bovine lactoferrin was infused into the stomach or duodenal lumen via a needle for a period of over 1 min in a volume of 1 ml kg(-1). The bLF transported into the lymph was assayed quantitatively by double-antibody enzyme-linked immunosorbent assay (ELISA). Following the intragastric administration of bLF, the three groups showed almost the same lymph flow, but the bLF concentration in the lymph fluid in the EF-bLF group increased significantly and peaked 3 h after administration. With intraduodenal administration, the bLF concentration in the lymph fluid of the higher non-EF-bLF group was significantly higher than those of the other groups. The amount of absorbed bLF in the EF-bLF group was, however, about 10 times higher than that in the lower non-EF-bLF group, when it was administered intragastrically. These data show that enteric-formulated bLF is less susceptible to gastric pepsin and is more efficiently absorbed from the intestine than is non-enteric-formulated bLF.

The addition of HP-20 resin to a medium could enhance the growth of Legionella species. Elemental sulfur was isolated as a self-growth-inhibiting substance produced endogenously by Legionella pneumophila from methanol extracts of the resins used to culture the bacterium. Elemental sulfur shows strong growth-inhibiting activity toward all Legionella species tested.

Reporter genes such as firefly luciferase are common tools to monitor gene expression in various systems. As reporter gene represents the expression level of the gene of interest with its enzyme activity, firefly luciferase is most frequently used because its luminescent activity is highly sensitive and less time consumable for assay. However, since firefly luciferase is expressed internally in the cell, lysis of the cell is a critical step, and thus it is difficult to monitor the gene expression level continuously. In this report, we utilized secretive Vargula hilgendorfii luciferase modified to cell surface displayed one by fusing with human EGFR transmembrane sequence. This modified Vargula luciferase was expressed on cell surface without losing its bioluminescent activity. Co-transfection with secretive alkaline phosphatase showed that the behaviors of cell surface displayed Vargula luciferase and secretive alkaline phosphatase are comparable to each other. Furthermore, the luminescence of a single cell expressing cell surface displayed Vargula luciferase can be monitored by using photon counting CCD camera, which indicates that this reporter gene can monitor gene expression in a single cell without cell lysis.

Luciferase of Vargula hilgendorfli is infinitely stable at room temperature in dried state, and its light-emitting reaction is very simple. These unique characteristics of Vargula luciferase have prompted us to engineer chimeric protein, the other moiety chosen for conjugation being streptococcal protein G. A single domain of protein G which binds to IgG of a wide range of species was fused at the N-terminal region of Vargula luciferase. Unexpectedly, we found that the chimeric protein expressed in mammalian COS-1 cells had no IgG-binding ability, probably due to some sort of interaction between the two moieties or some conformational preferences of the IgG-binding domain of protein G when fused to Vargula luciferase. Here we report how we regained the IgG binding of protein G, by the intervention of three alpha-helices of protein A between protein G and luciferase. To our knowledge, the new chimeric protein provides the first reported model of this kind.

We developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for human monocyte chemotactic and activating factor (MCAF), an inflammatory cytokine that plays an important role in the recruitment of blood monocytes to areas of inflammation. The ELISA, which is based on a sandwich method using two newly-developed monoclonal antibodies, could quantitatively detect MCAF in the range between 2.5 pg/ml (50 fg/sample) to 300 pg/ml after incubation for a total of 2 h, and showed no cross-reactivity with various structurally-related IL-8 superfamily proteins. It was not affected by blood or urine components non-specifically, and thus was directly applicable to clinical specimens. When serum and urine samples from healthy subjects were measured, they all turned out to contain detectable levels of MCAF (more than 30 pg/ml). By gel-filtration column chromatography analysis, MCAF in the body fluids was eluted as a single peak at the position corresponding to the molecular weight of 10 kD, suggesting that it exists as a monomer form, free from carrier proteins. The established ELISA here is expected to be effectively used for the further investigations on the relationship of MCAF with various inflammatory diseases.

A plasmid was constructed to express a mouse beta-interferon (IFN-beta) in Escherichia coli under the control of the modified tryptophan (trp) promoter. E. coli carrying the plasmid were cultivated in a minijar fermentor and synthesized up to 2.7 X 10(6) IU/ml of antiviral activity. At the end of the cultivation the cells became elongated and curved.

Recombinant mouse interferon-beta (rMuIFN-beta) produced in Escherichia coli was purified to homogeneity and characterized. The purified protein exhibited a single band of Mr 19,900 on SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions, and also exhibited a single band on native polyacrylamide gel electrophoresis at pH 4.3. The observed molecular weight corresponded to that of the polypeptide moiety of natural MuIFN-beta of Mr 19,700. The amino acid composition and the amino-terminal sequence of the purified rMuIFN-beta were identical to those predicted from cDNA sequence. These results indicate that the purified protein is a nonglycosylated MuIFN-beta, which forms no disulfide-linked dimer and probably exists as a monomeric form.

Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y. (1986) J. Biol. Chem. 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis. We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant. The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit. The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.